Ambrus, Hamilton - Food Safety Assessment of Pesticide Residues (2016)

Food Safety Assessment
of Pesticide Residues
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b2530 International Strategic Relations and China’s National Security: World at the Crossroads
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Food Safety Assessment
Editors
Árpád Ambrus
National Food Chain Safety Office, Hungary
Denis Hamilton
World Scientific
NEW JERSEY • LONDON • SINGAPORE • BEIJING • SHANGHAI • HONG KONG • TAIPEI • CHENNAI • TOKYO
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Published by
World Scientific Publishing Europe Ltd.
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Head office: 5 Toh Tuck Link, Singapore 596224
USA office: 27 Warren Street, Suite 401-402, Hackensack, NJ 07601
Library of Congress Cataloging-in-Publication Data

Names: Ambrus, Á. (Árpád), author. | Hamilton, Denis, author.
Title: Food safety assessment of pesticide residues / Árpád Ambrus
(National Food Chain Safety Office, Hungary), Denis Hamilton.
Description: New Jersey : World Scientific, 2016. | Includes bibliographical references.
Identifiers: LCCN 2016033507 | ISBN 9781786341686 (hc : alk. paper)
Subjects: LCSH: Pesticide residues in food. | Food--Safety measures.
Classification: LCC TX571.P4 A48 2016 | DDC 363.19/2--dc23
LC record available at https://lccn.loc.gov/2016033507
British Library Cataloguing-in-Publication Data

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Copyright © 2017 by World Scientific Publishing Europe Ltd.

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For the patient and supportive spouses and families

of authors and editors
v
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Preface
It was a very pleasant surprise to receive an invitation from Merlyn

Cox to edit a book for publication by Imperial College Press (ICP).
The subject was food safety assessment of pesticide residues, a topic
that has consumed a considerable portion of our working lives. It is
a topic of vital interest to farmers through to consumers and a topic
where scientific progress continues.
The continuously increasing population of the world demands
good quality and safe food in sufficient quantity. In addition,
governments are concerned with assuring security of food supply. To
achieve these goals, the effective protection of crops in the fields and
harvested commodities during storage can only be realized currently
by the use of pesticides, which are an integral part of modern agricul-
tural production. Pesticides are generally toxic substances and their
use should be regulated to protect human health and the environment.
This book leads the reader through the complex process of safety
assessment and regulation of the use of pesticides, including the ver-
ification of the safety of food placed on the market.
Chapter 1 provides the historical background of food safety issues
and development of principles and the process of dietary risk assess-
ment of pesticides.
The following chapters (Chapters 2–7) cover the wide-ranging
activities carried out at international and national levels for the
initial safety assessment of pesticides including the elaboration of
guidelines and guidance documents within the Pesticide Programme
of the Organisation for Economic Cooperation and Development
vii
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viii Preface
(OECD); establishment of national use patterns and safety assess-

ment of pesticides at national or regional level illustrated with the
examples of Australia, European Union and United States; the meth-
ods recommended for collection of food consumption data and the
dietary risk assessment applied at international level; the role of FAO
and WHO and Codex Alimentarius in developing Codex Maximum
Residue Limits, which are used as safety criteria in the Sanitary
and Phytosanitary (SPS) and Technical Barriers to Trade (TBT)
Agreements. But note that maximum residue limits are not safety
limits.
Chapter 8 describes the development and role of scientifically
based pesticide specifications in the national and international main-
tenance of safe and proper use of pesticides. The importance of reg-
ular control of quality of pesticide products is emphasized because
out-of-specification, counterfeit or inferior quality pesticides can pose
health risks even when used according to label instructions.
Growers rely on the label directions approved by their registra-
tion authority for producing food and feed products that comply with
legal pesticide limits. Food manufacturers and retailers are responsi-
ble for the compliance of their marketed products with relevant legal
limits or quality specifications. For this purpose, they should analyse
their purchased materials for comparison with limits and specifica-
tions, taking representative samples (Chapter 9) and evaluating the
analytical results based on their combined uncertainty (Chapter 10).
Official monitoring programmes implemented by governments
aim to verify the compliance of traded food commodities with MRLs
of pesticide residues. A tiered approach is presented for planning
risk based monitoring programmes in Chapter 11, which also gives
some examples for assisting small-scale farms in producing fruits and
vegetables satisfying national or international quality specifications
including MRLs for pesticide residues.
Useful comments and suggestions provided by Kit Chan, Steve
Elison, Coen Graven, Sándor Kemény, Hermine Reich, Bernadette
Ossendorp, Leah Ritter, Pieter Scheelings, András Sebők, Roger
Wood, and the staff of the APVMA and FSANZ are sincerely
acknowledged by the authors of related chapters and the editors.
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Preface ix
Last but not least, we say a big thank you to the authors who have
contributed their special knowledge, technical expertise and valuable
time for preparing this book. It has been a pleasure to work with our
authors, who come from eight different countries in five continents.
Árpád Ambrus and Denis Hamilton

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About the Authors
AMBRUS Árpád is chemical engineer (MSc), Candidate of Chem-

ical Sciences and habilitated university professor. He is an IUPAC
Fellow, member of the Food Safety Subcommittee of the Hungar-
ian Academy of Science (2008–continuing), Joint Meeting on Pes-
ticide Residues (JMPR) FAO Panel (1973–2015). He received the
Silver Cross of Merit award, the IUPAC International Award for
Advances in Harmonised Approaches to Crop Protection Chemistry.
He chairs the Codex Committee on methods of Analysis and Sam-
pling (CCMAS), teaches at the Universities of Debrecen and Szeged,
and supervises six PhD aspirants, published over 80 peer-reviewed
scientific papers. He managed the Hungarian Pesticide Analytical
Laboratories, and the pesticide programme of the FAO/IAEA Train-
ing and Reference Centre, was the deputy director general of Hun-
garian Food Safety Office and retired as chief scientific advisor of the
National Food Chain Safety Office in 2014.
BHULA Rajumati (Raj) was appointed as the Gene Technology

Regulator in 2016. Before that she was the Executive Director for
Scientific Assessment and Chemical Review at the Australian Pesti-
cides and Veterinary Medicines Authority (APVMA). She managed
the chemical review programme of the APVMA and provided tech-
nical advice on residues chemistry, environment and health as part
of the registration of pesticides and veterinary medicines. She has
primarily worked in evaluation of residues data, MRL setting and
pesticide registration and regulation. She was a member of the Aus-
tralian delegation to the Codex Committee on Pesticide Residues
xi
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xii About the Authors
and has been a temporary advisor to the FAO panel of the JMPR.
She has chaired writing groups and contributed to the development
of OECD test guidelines for residues chemistry. She has contributed
to 16 peer-reviewed papers and articles.
BURA László is a diploma-qualified chemical engineer and has a

Masters in chromatography from Technical University of Budapest,
Hungary. Bura is a senior scientific officer at the European Food
Safety Authority, since 2007, in the Pesticides Unit of the Sci-
entific Evaluation of Regulated Products Directorate, where he is
responsible for the assessment of the identity, physical and chemical
properties of pesticide active substances and technical properties of
formulations and analytical methods. He is the secretary of CIPAC
and a member of the FAO panel of the FAO/WHO Joint Meeting on
Pesticide Specifications (JMPS).
CALDAS Dutra Eloisa has a BSc in chemistry and a Masters in

analytical chemistry from University of Brasilia, Brazil (UnB) and
a PhD in agricultural chemistry from the University of California.
She is a full professor at UnB since 1997, and responsible for the
Laboratory of Toxicology, coordinating projects on method develop-
ment and analysis of pesticide residues and contaminants in foods
and dietary exposure assessment, among others. In 2004/2005, was a
post-doctoral fellow at the met@risk unit (Methodologies d’analyse
de risqué alimentaire) at the Institut National de la Recherche
Agronomique, France. Caldas is a member of the FAO panel of the
FAO/WHO JMPR since 1997, and has published over 50 scientific
papers and presented numerous lectures at international meetings.
DOHERTY Michael received his MSc (1990) and PhD (1997)

in marine-estuarine environmental sciences from the University of
Maryland. He is a senior chemist and risk assessor, and has worked for
the U.S. Environmental Protection Agency/Office of Pesticide Pro-
grams/Health Effects Division since 1998. His main responsibilities
have been: evaluation of residue chemistry data submitted to support
pesticide registration and set MRLs; assessing dietary and human
health aggregate risk from use of pesticides; leading risk assessment
December 14, 2016 14:43 Food Safety Assessment of Pesticide Residues 9in x 6in b2668-fm page xiii
About the Authors xiii
teams for in-house and international joint review projects; providing

training to staff, visiting scientists and other regulatory authorities.
He was involved in the preparation of numerous residue chemistry
evaluations and aggregate human health risk assessments for U.S.
EPA. Michael has published six scientific papers.
FARKAS Zsuzsa graduated as a biologist (BSc) at Eötvös Loránd

Science University in 2010 and as a nutritionist (MSc) at Semmel-
weis University in Budapest in 2012. She started to work at the
National Food Chain Safety Office of Hungary, at the Directorate
for Food Safety Risk Assessment in 2013. Since then, she has con-
tributed to the implementation of two international projects: Pilot
Paneu (EFSA) and Baseline (European Commission’s seventh frame
programme). Simultaneously, she has performed her PhD studies at
the Corvinus University of Budapest since 2013, dealing with the
optimization of sampling procedures for testing chemical contami-
nants in food. Zsuzsa had over 10 oral and poster presentations in
Hungarian and international platforms, and was the author or co-
author of eight publications in international, peer-reviewed journals.
HAMILTON Denis has BSc, MSc (University of Queensland),

CChem, is a Fellow of the Royal Australian Chemical Institute
and IUPAC Fellow. He retired in 2009, and was previously Princi-
pal Scientific Officer, Queensland Department of Primary Industries,
Brisbane. His duties included formulation of policy on agricultural
chemistry (pesticides, veterinary drugs and fertilizers) and contam-
inant issues. From 1986 to 2010, Denis participated in JMPR and
chaired the meeting on some occasions. From 2002 to 2010, he par-
ticipated in JMPS and also chaired the meeting on some occasions.
He was the leader of three International Union of Pure and Applied
Chemistry (IUPAC) projects on pesticide residues in food and water
and in 2010 he received the inaugural IUPAC International Award for
Advances in Harmonized Approaches to Crop Protection Chemistry.
Denis has published 37 peer-reviewed scientific papers, and delivered
numerous conference papers and lectures at international training
workshops.
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xiv About the Authors
HUMPHREY Paul has a BSc (Hons.) and a PhD in chemistry

from the University of Adelaide and has had post-doctoral positions
in synthetic chemistry at the University of Bristol and the Univer-
sity of Sydney. He has worked in pesticide residues evaluation at the
Australian Pesticides and Veterinary Medicines Authority (APVMA)
in Canberra for nine years. Paul has contributed to the work of
the annual Joint FAO/WHO Meeting of Pesticide Residues (JMPR)
since 2011 and is on the JMPR roster of experts on pesticide residues.
He is a co-author of over 40 scientific papers.
HORVÁTH Zsuzsanna has BSc in biology and MSc in nutri-

tion from University of Debrecen, Hungary. Currently she is doing
her PhD at Corvinus University of Budapest. Her research topic is
planning risk-based monitoring programmes for pesticides residues.
She works at National Food Chain Safety Office. She participated in
EFSA Pilot-Paneu project for developing and testing methodology
for dietary surveys for risk assessment; and Baseline FP7 project,
where fit-for-purpose sampling plans were developed for different
foodstuffs and contaminants. In 2014 she had a three-month intern-
ship in France, where she studied probabilistic methods of risk assess-
ment. She is the coordinator of the ongoing Hungarian dietary survey
and representative in EFSA Network on Food Consumption Data.
She is author or co-author of eight articles published in scientific
journals.
LINDTNER Oliver studied mathematics at Humboldt University

Berlin, and received his PhD degree at the University of Paderborn.
Since 2003, he works at the Federal Institute for Risk Assessment
(BfR) in Berlin, now as the head of the Unit for Exposure Assess-
ment and Exposure Standardisation. In addition to statistics, he is
experienced in the field of nutrition, dietary exposure assessment,
consumption databases and uncertainty analysis. He leads at BfR
both the KiESEL study aiming to collect food consumption data
for exposure assessments on pesticides among toddlers and children
and the Meal Study, the first total diet study in Germany. Lindtner
is member of the ANS Panel at EFSA, participated in the EFSA
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About the Authors xv
PILOT-PANEU project and serves as a work package leader in the

European TDS Exposure Project.
MACLACHLAN Dugald is director of residues and microbiology

policy in the Exports Division of the Australian Federal Department
of Agriculture. His role involves risk assessment relating to pesticides,
veterinary drugs and contaminants. He leads Australian delegations
to Codex Committee on Residues of Veterinary Drugs in Food as
well as to Codex task forces on antimicrobial resistance and animal
feeding. A particular interest has been in models for predicting pesti-
cide residues in crops and the potential for their transfer from feed to
animal commodities. MacLachlan is a chemist by training and is the
author of 39 peer-reviewed papers and member of the FAO/WHO
JMPR. He has contributed to the drafting of various OECD test
guidelines and guidance documents and to the development of the
OECD MRL calculator.
MARGERISON Sam obtained BSc (Hons) from University of

Canterbury in 1998 and a PhD in Chemistry from the Australian
National University in 2005. He has worked for the APVMA for 12
years, including the last seven years in pesticide residues evalua-
tion. Margerison has participated in two JMPR meetings as an FAO
Temporary Adviser and has prepared or contributed to five residue
evaluations for JMPR.
OCKÉ Marga studied human nutrition at Wageningen University

(MSc), the Netherlands, where she also received her PhD degree.
Since 1990, she works at the Dutch National Institute for Public
Health and the Environment. Her early work was as a nutritional
epidemiologist in the context of large cohort studies. From 2003
onwards, she works as senior scientist and project leader on vari-
ous national and international projects related to food consumption
surveys. Ocké was a member of the EU Menu Working Group with
Advisory Function that advised EFSA on the guidance for national
December 22, 2016 10:51 Food Safety Assessment of Pesticide Residues 9in x 6in b2668-fm page xvi
xvi About the Authors
food consumption surveys. Her scientific interests are: dietary assess-

ment methodology, evaluation of dietary intake and dietary valida-
tion studies. She has published over 30 policy advice reports and 160
scientific papers.
PFEIL Rudolf is a doctor of veterinary medicine and a certified

specialist for laboratory animal science. He has more than 25 years
of professional experience in the experimental investigation of toxico-
logical effects as well as in the regulatory evaluation of pesticides at
the national or international level. Pfeil is head of the unit Toxicology
of Active Substances and their Metabolites at the Federal Institute
for Risk Assessment in Berlin, Germany. He served for more than
13 years as an expert in the Joint FAO/WHO Meeting on Pesticide
Residues (JMPR, WHO Core Assessment Group), has been a mem-
ber in many EC Pesticide Peer Review Meetings and is author or
co-author of more than 40 scientific papers.
ROWLAND Jessudoss has a MS in biology from the University of

Nebraska. Rowland began his career in toxicology and carcinogenesis
at the Eppley Institute for Research on Cancer in Omaha Nebraska.
At present, he is the Deputy Director of the Health Effects Division in
the Office of Pesticide Programs of the U.S. Environmental Protection
Agency. He is responsible for conducting in-depth review, interpreta-
tion and integration of hazard, dietary, occupational and residential
exposure data on pesticide chemicals and ensuring that pesticide
risk assessments adhere to regulatory compliance. Rowland has over
40 years of experience in the design, conduct and review of toxicology
and carcinogenicity studies and technical expertise in hazard identi-
fication and non-cancer and cancer dose–response assessment.
SCHUMACHER David is a senior toxicologist at a German reg-

ulatory agency. He is a food chemist by training and holds a PhD
in natural sciences/toxicology from University of Karlsruhe, Ger-
many. Since 2006 he has been involved in the evaluation of data and
information on the human health toxicological properties of pesticide
active ingredients including the hazard assessment and the derivation
of toxicological reference doses. Additionally, he has been a member
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About the Authors xvii
of EFSA’s Pesticide Peer Review Panel, expert at OECD working

groups on genotoxicity test guidelines and cell transformation assays
and expert in the Joint FAO/WHO Meeting on Pesticide Residues
(JMPR, WHO Part: WHO Core Assessment Group).
SOLECKI Roland Alfred is a biologist and toxicologist

by training and an educated specialist in neuroendocrinology,
toxicopathology and reproductive toxicity. He has professional expe-
rience in the experimental investigation of toxicological effects as well
as in the regulatory evaluation of pesticides at the national or inter-
national level. Solecki is head of the Pesticides Safety Department at
the Federal Institute for Risk Assessment. He served for more than
15 years as a member of the FAO/WHO Joint Meeting on Pesticide
Residues, is author of many peer-reviewed papers in highly ranked
international journals and participated in special working groups of
the EC for regulatory toxicology. He was a member of the several
OECD expert groups and is currently a member of the Scientific
Committee of the European Food Safety Agency.
VALSTA Liisa has a MSc and PhD in Human nutrition from the
University of Helsinki, Finland, and a MSc in Food Science and Tech-
nology (Food Toxicology) from Oregon State University. Since 1991,
she works as a senior scientist at the National Institute for Health
and Welfare (THL) in the Nutrition Unit. Since 2001, she serves
as an Adjunct Professor in Human Nutrition at the University of
Helsinki. In 2009–2014, Valsta worked as a senior scientific officer
at the European Food Safety Authority (EFSA) coordinating the
harmonisation of European dietary surveys through the EFSA EU
Menu initiative. She has published over 100 peer-reviewed and 80
other publications on food composition, human dietary interventions
and dietary and health monitoring.
van der VELDE-KOERTS Trijntje is trained as chemist (BSc)

and since 2000 she holds a position as food safety advisor at the
RIVM (Dutch National Institute for Public Health and the Environ-
ment) in the field of pesticides, biocides and veterinary medicines.
In this position, she has prepared many pesticide residue evaluations
December 14, 2016 14:43 Food Safety Assessment of Pesticide Residues 9in x 6in b2668-fm page xviii
xviii About the Authors
for Draft Assessment Reports for the EU. Trijntje is a Member of

the Residue Chemistry Expert Group of the OECD since 2007 and a
Member of the FAO panel of the Joint Meeting on Pesticide Residues
(JMPR) since 2008. Trijntje is also involved in the preparation of the
Dutch and JMPR models for estimation of pesticide residue dietary
exposure.
WOLTERINK Gerrit (1959) has a MSc in biology and a PhD

in neuropharmacology from Utrecht University, the Netherlands. He
worked 12 years as a neuropharmacologist at Cambridge University
and Utrecht University. Since 2000, he works as a toxicologist at
the Dutch National Institute for Public Health and the Environment
(RIVM) on the toxicological evaluation and human health risk assess-
ment of pesticides, biocides, contaminants, veterinary drugs and food
additives in food and non-food products. He has been a member
of the WHO Panel of the Joint FAO/WHO Meeting on Pesticide
Residues (JMPR, since 2002), the Joint FAO/WHO Expert Com-
mittee on Food Additives (JECFA, 2010–2013) and the Panel on
Plant Protection Products and their Residues (PPR) of EFSA (since
2015). He has published over 50 scientific papers.
YAMADA Yukiko is an international food safety consultant

and advisor to the Ministry of Agriculture, Forestry and Fisheries
(MAFF). She retired from MAFF as the Director-General for Tech-
nological Affairs/Chief Scientific Officer. She has long experience in
food safety risk management, dietary exposure assessment of chem-
icals and related research. She has headed the Japanese Delegation
to many Codex and other international meetings and once was a
vice-chair of the OECD Working Group on Pesticides. She was the
secretary of the Codex Committee on Pesticide Residues and con-
tinues as a member of the FAO Panel of JMPR (2001–2015+) and
the Residue Chemistry Expert Group of OECD (2013–2015+). She
received a PhD degree from Kyoto University, and has published 36
peer-reviewed scientific papers and co-authored 13 books.
YOSHIDA Midori graduated at the faculty of veterinary medicine,

in 1979, and obtained her DVM PhD from Hokkaido University
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About the Authors xix
School of Veterinary Medicine in 1996. She has 35 years of expe-

rience in toxicological pathology and became certificated as a diplo-
mat by Japanese Toxicological Pathology (DJSTP) in 1996, and by
the Japanese College of Veterinary pathologists (JCVP). Yoshida
took part in several international and national expert meetings as an
expert of toxicology, including the FAO/WHO JMPR, Food Safety
commission of Japan. Yoshida currently acts in the board of directors
of three committees: Japanese Society of Toxicologic Pathology, the
Japanese Society of Veterinary Pathology and the Japanese Society of
Toxicology. She is the author/co-author of over 130 scientific papers
and five books.
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List of Tables
2.1 OECD Guidelines for the testing of chemicals, Section 5

pesticide residue chemistry. . . . . . . . . . . . . . . . 30
2.2 Residue guidance documents published

by the Residue Chemistry Expert Group . . . . . . . 31
4.1 Fenitrothion residues in grain and milling fractions

of wheat after a post-harvest treatment. . . . . . . . . 146
4.2 Common toxicity studies and their purposes. . . . . . 155
4.3 Pesticides with unique toxicological profiles and their

mechanisms. . . . . . . . . . . . . . . . . . . . . . . . 171
5.1 Examples of extraction rates of different processed and

derived products in selected countries and conversion
factors derived based on the extraction rates published
by FAO Statistics (FAO, 2000) . . . . . . . . . . . . . 219
5.2 Background information to be collected from survey

participants and non-participants (EFSA, 2014). . . . 228
7.1 Definitions for the purposes of the Codex Alimentarius

related to MRLs for pesticides (Codex Alimentarius
Commission Procedural Manual, 25th edition) . . . . . 274
xxi
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xxii List of Tables
7.2 List of Codex recommendations in the area of pesticide

residues in foods and animal feeds (Codex website on
Codex Standards, updated regularly) . . . . . . . . . 281
8.1 Names and isomer compositions of allethrin

products . . . . . . . . . . . . . . . . . . . . . . . . . . 295
8.2 Isomer composition of cypermethrin compounds
(Compendium, 2015) . . . . . . . . . . . . . . . . . . . 295
8.3 Examples of structural alerts and potential relevant
impurities . . . . . . . . . . . . . . . . . . . . . . . . . 299
8.4 Expression of concentrations of relevant impurities
in glyphosate . . . . . . . . . . . . . . . . . . . . . . . 302
8.5 Water as a relevant impurity . . . . . . . . . . . . . . 309
8.6 Theoretical comparison of the effect of test
temperature-duration combinations on loss of active
ingredient . . . . . . . . . . . . . . . . . . . . . . . . . 320
8.7 Stability specifications for fenthion formulations . . . 322
9.1 Example of material properties and sampling

dimensions . . . . . . . . . . . . . . . . . . . . . . . . 346
9.2 Sources of sampling uncertainty and errors . . . . . . 372
9.3 Examples of sampling steps from field to testing . . . 377
9.4 Sampling guidelines . . . . . . . . . . . . . . . . . . . 380
10.1 Example for the calculations of the average residue

and CV values from four replicate samples . . . . . . 419
10.2 Residues in 10,000 parsley samples of size n obtained
by random sampling from residues in primary samples 420
10.3 Range of CVS values obtained with drawing ‘p’ replicate
samples 10,000 times from normalized residues
in carrot . . . . . . . . . . . . . . . . . . . . . . . . . . 421
10.4 Relationship of the relative 95% range of CV values
and p replicate samples . . . . . . . . . . . . . . . . . 424
10.5 Relative 95% range of CV values calculated
from synthetic lognormal parent populations and
experimental residue values . . . . . . . . . . . . . . . 425
December 14, 2016 14:43 Food Safety Assessment of Pesticide Residues 9in x 6in b2668-fm page xxiii
List of Tables xxiii
10.6 Summary of sampling uncertainties of pesticide residues

in individual crop units or primary sample increments
recommended for practical use . . . . . . . . . . . . . 430
10.7 Summary of estimated sampling uncertainties for crop
groups recommended for practical use . . . . . . . . . 433
10.8 Calculation of typical recovery from test portions spiked
at X (mg/kg) level . . . . . . . . . . . . . . . . . . . . 449
10.9 Illustration of the calculation of CVtyp from replicate
measurements . . . . . . . . . . . . . . . . . . . . . . . 452
11.1 Percentage distribution of residues in median ranges . 478

11.2 Minimum number of samples required to detect at least
one residue above the MRL at the selected violation
rates (βv ) with pre-defined probability (βt ) . . . . . . 481
11.3 Number of lots (n0 ) to be sampled where the total
number of lots (N ) is small . . . . . . . . . . . . . . . 481
11.4 Factors for calculation of expectable highest HR values
with 95% probability . . . . . . . . . . . . . . . . . . . 485
11.5 Relation of fST to the ratio of MRL and STMR . . . 491
11.6 The fnβp corresponding to the number of supervised
trials (N ) . . . . . . . . . . . . . . . . . . . . . . . . . 492
11.7 Recommended number of samples (n) depending on the
calculated weighting factor (F ) with probability levels
(βt %). . . . . . . . . . . . . . . . . . . . . . . . . . . . 503
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List of Figures
Figure 1.1 A treatise on adulterations of food and culinary

poisons (Accum, 1820). . . . . . . . . . . . . 2
Figure 1.2 Risk analysis process. . . . . . . . . . . . . . 8
Figure 1.3 Chemical dietary risk assessment. . . . . . . 9
Figure 4.1 Generation of plant metabolites from

1,2,4-triazole. . . . . . . . . . . . . . . . . . . 123
Figure 4.2 Bixafen residues, means for each group at
each time, in milk during 34 days of a feeding
study on lactating dairy cows at three feed
concentrations. . . . . . . . . . . . . . . . . . 151
Figure 4.3 Residue levels in the tissues from a dairy cow
feeding study with bixafen at the equivalent of
4, 12 and 40 ppm in the animal feed dry weight
for 29 days. . . . . . . . . . . . . . . . . . . . 151
Figure 4.4 The upper tail of the incidence of DDT residues
in 4682 samples of the tissue fat of livestock
from a monitoring program in New Zealand
from July 1990 to June 1994 (JMPR, 1996). 153
Figure 4.5 The main process for toxicological evaluation
of pesticides. . . . . . . . . . . . . . . . . . . 154
Figure 4.6 Process of risk assessment at JMPR. . . . . . 156
xxv
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xxvi List of Figures
Figure 4.7 The process of hazard identification in each

toxicity study. . . . . . . . . . . . . . . . . . 173
Figure 5.1 Fruit supply quantity of selected fruit

categories per capita in different regions of the
world based on food balance sheet data (FAO,
2011). . . . . . . . . . . . . . . . . . . . . . . 204
Figure 5.2 Relationships between food balance sheets,
household budget surveys and national food
consumption surveys (Modified from Ocké
et al., 2016). . . . . . . . . . . . . . . . . . . 205
Figure 5.3 Graphical overview of linkage between food
consumption data and concentration data for
dietary exposure assessments (Boon et al.,
2011) with permission of the ACROPOLIS
project. . . . . . . . . . . . . . . . . . . . . . 221
Figure 5.4 The effect of the starting data on the later
TDS procedures and results. . . . . . . . . . 233
Figure 6.1 GEMS/food consumption 17 cluster diets

(WHO, 2012). . . . . . . . . . . . . . . . . . 245
Figure 6.2 Probabilistic assessment of dietary long-term
exposure to pesticides. . . . . . . . . . . . . . 252
Figure 6.3 Probabilistic assessment of dietary short-term
exposure to pesticides. . . . . . . . . . . . . . 256
Figure 7.1 Elaboration procedure for Codex MRLs and

EMRLs for pesticides. . . . . . . . . . . . . . 279
Figure 8.1 Main heading for TC specification guidelines.

TK has the same headings. . . . . . . . . . . 293
Figure 8.2 Indoxacarb and metalaxyl. . . . . . . . . . . 296
Figure 8.3 Main headings for WG specification guidelines.
Each formulation type has its own list of
physical properties. . . . . . . . . . . . . . . 307
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List of Figures xxvii
Figure 8.4 Thiamethoxam hydrolysis. . . . . . . . . . . 314
Figure 9.1 Example of mass reduction steps. . . . . . . 338

Figure 9.2 Finite and infinite element vegetables. . . . . 342
Figure 9.3 Compositional and distributional heterogeneity
of foods. . . . . . . . . . . . . . . . . . . . . 345
Figure 9.4 Heterogeneity of sugar mixed with cinnamon. 352
Figure 9.5 Whole wheat before and after it is ground to
flour. . . . . . . . . . . . . . . . . . . . . . . 354
Figure 9.6 Distributional heterogeneity in granola. . . . 360
Figure 9.7 Distributional heterogeneity in orange juice. 366
Figure 9.8 Sampling with a tool. . . . . . . . . . . . . . 367
Figure 9.9 Demonstration of the center-of-gravity
principle. . . . . . . . . . . . . . . . . . . . . 369
Figure 9.10 Diagram of fractional shovelling. . . . . . . . 370
Figure 10.1 Distribution of 14 normalized pesticide residues

in cabbage and 6 in strawberry composite
samples (n = 10) derived from independently
treated fields. Each data point on the charts
represents the relative frequency
of residues in bin classes. . . . . . . . . . . . 409
Figure 10.2 Residues measured in crop units taken from
commercially treated fields. (The residues were
measured in whole crop units, e.g. one carrot,
cucumber or bunch of grapes.) . . . . . . . . 411
Figure 10.3 Distribution of CV values in primary samples
(n = 1) and the average residues in composite
samples of size n drawn from a synthetic
lognormal distribution with mean of 1 and CV
of 0.8. . . . . . . . . . . . . . . . . . . . . . . 414
Figure 10.4 Relative 95% range of CV values of residues in
p replicate composite (n = 10) sample sets. . 422
Figure 10.5 Relationship of relative 95% range of CV values
calculated from p replicate random samples
and the number of tested lots. . . . . . . . . 424
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xxviii List of Figures
Figure 10.6 Relative 95% range obtained with taking p

replicate samples from L decision units. . . . 425
Figure 10.7 Variation of values of relative 95% range
of the CV values calculated from two and
four replicate samples from 4, 8, 16, 20 lots
independently selected up to 15 times. . . . . 426
Figure 10.8 Examples of confidence intervals of CVSi values
of crops belonging to root and tuber vegetables 428
Figure 10.9 Cutting segments from large fruits. . . . . . 440
Figure 10.10 Cutting representative portions of large crops. 441
Figure 10.11 Illustration of acceptance and rejection of a
lot based on measured residue (R) values and
combined expanded uncertainty (U ) of the
results. . . . . . . . . . . . . . . . . . . . . . 457
Figure 10.12 Distribution of pesticide residues in composite
samples of size 10. . . . . . . . . . . . . . . . 458
Figure 10.13 Operation characteristics curves for sampling
plans with 1, 2 and 4 replicate samples of size
10. . . . . . . . . . . . . . . . . . . . . . . . . 459
Figure 11.1 Distribution of normalized residues, and the

median and HR values of residues in replicate
samples of size 8 drawn from the parent
population (25,766) of normalized supervised
trial data. . . . . . . . . . . . . . . . . . . . . 479
Figure 11.2 Relationship of fM,n and sample size (n). . . 484
Figure 11.3 Decision tree for the application of tiered
ranking model. . . . . . . . . . . . . . . . . . 488
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Contents
Preface vii
About the Authors xi
List of Tables xxi
List of Figures xxv
1. Introduction to Dietary Risk Assessment of Pesticides 1

1.1 Introduction . . . . . . . . . . . . . . . . . . . . 1
1.2 Food Safety Initiatives by National, Regional
and International Authorities . . . . . . . . . . . 3
1.3 Scientific Developments on Chemical
Risk Assessment . . . . . . . . . . . . . . . . . . 6
1.4 The Dietary Risk Assessment of Pesticides . . . 7
References . . . . . . . . . . . . . . . . . . . . . . . . . 10
2. OECD Guidance Documents and Test Guidelines 13

2.1 Introduction . . . . . . . . . . . . . . . . . . . . 13
2.2 OECD Environment, Health and Safety
Programme . . . . . . . . . . . . . . . . . . . . . 14
2.2.1 OECD Pesticide Programme . . . . . . 14
2.2.2 Harmonization of Data Requirements . 15
2.2.3 OECD Series on Principles of GLP . . . 16
2.3 OECD Test Guideline Programme . . . . . . . . 17
xxix
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2.4 OECDGuidelines and Guidance on Health Effects 19

2.4.1ADME and Toxicokinetics . . . . . . . . 19
2.4.2Acute Toxicity . . . . . . . . . . . . . . 20
2.4.3Repeated-Dose Toxicity in Rodents
and Non-rodents . . . . . . . . . . . . . 21
2.4.4 Carcinogenicity . . . . . . . . . . . . . . 21
2.4.5 Genotoxicity . . . . . . . . . . . . . . . 22
2.4.6 Reproductive Toxicity and Developmental
Toxicity . . . . . . . . . . . . . . . . . . 24
2.4.7 Acute and Repeated-Dose Neurotoxicity
and Delayed Neurotoxicity . . . . . . . . 27
2.4.8 Endocrine Disruption . . . . . . . . . . 28
2.5 OECD Guidelines and Guidance on Pesticide
Residues . . . . . . . . . . . . . . . . . . . . . . 29
2.5.1 Improved Alignment of Data
Interpretation . . . . . . . . . . . . . . . 30
2.5.2 The Livestock Feed Tables . . . . . . . . 32
2.5.3 The OECD MRL Calculator . . . . . . 33
2.6 Conclusions on Residues and Future Directions . 34
References . . . . . . . . . . . . . . . . . . . . . . . . . 35
3. Principles of Safety Assessment of Pesticides

at National Levels 37
3.1 Introduction . . . . . . . . . . . . . . . . . . . . 37
3.2 Data Requirements and Assessment Strategies
Common to Australia, the EU and the USA . . 38
3.2.1 Toxicity Studies . . . . . . . . . . . . . . 39
3.2.2 Residue Chemistry Studies . . . . . . . 41
3.2.2.1 Residue definitions . . . . . . 41
3.2.2.2 MRL determinations . . . . . 43
3.3 Principles of Safety Assessment of Pesticides —
Australia . . . . . . . . . . . . . . . . . . . . . . 44
3.3.1 Legal Framework . . . . . . . . . . . . . 44
3.3.1.1 Regulatory history . . . . . . . 44
3.3.1.2 Current regulatory framework 46
3.3.1.3 Regulatory products . . . . . . 47
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3.3.2 Evaluation of Residues . . . . . . . . . . 48

3.3.2.1 Hazard assessment . . . . . . . 48
3.3.2.2 Assessment of residues and
dietary exposure . . . . . . . . 50
3.3.3 Establishing MRLs . . . . . . . . . . . . 53
3.3.4 Export Trade Considerations . . . . . . 55
3.3.5 Dietary Exposure Calculations . . . . . 57
3.3.6 Public Consultation . . . . . . . . . . . 58
3.3.7 MRL Enforcement and Monitoring . . . 59
3.3.8 International Activities . . . . . . . . . 61
3.4 Principles of Safety Assessment of Plant Protection
Products — European Union . . . . . . . . . . . 62
3.4.1 Legal Framework . . . . . . . . . . . . . 62
3.4.1.1 Regulatory history . . . . . . . 62
3.4.1.2 Regulatory framework and
regulatory scope . . . . . . . . 64
3.4.2 Regulatory Processes . . . . . . . . . . . 66
3.4.2.1 Approval of pesticide active
substances and study
requirements . . . . . . . . . . 67
3.4.2.2 Authorization of plant
protection products
and study requirements . . . . 70
3.4.3 Pre-registration Pesticide Dietary Risk
Assessment within EU . . . . . . . . . . 73
3.4.3.1 Hazard assessment . . . . . . . 73
3.4.3.2 Pre-registration residue
assessment . . . . . . . . . . . 75
3.4.3.3 Pre-registration dietary risk
assessment . . . . . . . . . . . 77
3.4.4 MRL Setting and Import Tolerances . . 79
3.4.5 MRL Enforcement and Monitoring . . . 82
3.4.6 Future Developments . . . . . . . . . . . 84
United States of America . . . . . . . . . . . . . 85
3.5.1 Regulatory Background . . . . . . . . . 85
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3.5.2 Registration Process . . . . . . . . . . . 88

3.5.3 Hazard Assessment . . . . . . . . . . . . 89
3.5.3.1 Hazard characterization and
toxicity profile . . . . . . . . . 89
3.5.3.2 Endpoint selection . . . . . . . 93
3.5.3.3 Point-of-departure selection and
uncertainty factors . . . . . . 94
3.5.4 Residue Chemistry Evaluation . . . . . 97
3.5.4.1 Residue definitions . . . . . . 97
3.5.4.2 MRL determinations . . . . . 98
3.5.4.3 Exposure and risk assessment 101
3.5.5 Enforcement and Monitoring . . . . . . 105
3.5.6 Harmonization with Other Regulatory
Authorities . . . . . . . . . . . . . . . . 106
References . . . . . . . . . . . . . . . . . . . . . . . . . 107
4. Evaluation of Pesticide Residues by FAO/WHO JMPR 113

4.1 Introduction . . . . . . . . . . . . . . . . . . . . 114
4.2 Residue Evaluation . . . . . . . . . . . . . . . . 116
4.2.1 History of JMPR Residue Evaluation . . 116
4.2.2 Metabolism and Environmental Fate . . 119
4.2.2.1 Livestock metabolism . . . . . 120
4.2.2.2 Crop metabolism . . . . . . . 122
4.2.2.3 Environmental fate . . . . . . 124
4.2.2.4 Aerobic soil metabolism . . . . 124
4.2.2.5 Residues in rotational crops . 126
4.2.3 Residue Analysis, Sampling and Sample
Storage . . . . . . . . . . . . . . . . . . 129
4.2.4 Residue Definition . . . . . . . . . . . . 132
4.2.5 Use Pattern . . . . . . . . . . . . . . . . 134
4.2.6 Supervised Residue Trials . . . . . . . . 136
4.2.6.1 The minor crop problem . . . 141
4.2.6.2 Spices . . . . . . . . . . . . . . 142
4.2.7 Food Processing . . . . . . . . . . . . . 143
4.2.8 Livestock and Residues in Meat, Eggs and
Milk . . . . . . . . . . . . . . . . . . . . 147
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Contents xxxiii
4.2.9 Monitoring Data, EMRLs . . . . . . . . 152

4.3 Toxicological Evaluation . . . . . . . . . . . . . . 153
4.3.1 Data Requirements for Toxicological
Evaluation . . . . . . . . . . . . . . . . . 153
4.3.1.1 Toxicity data used for
toxicological evaluation . . . . 153
4.3.2 Importance of Quality and Reliability
Control of Toxicity Data . . . . . . . . . 156
4.3.2.1 Compliance with quality
principles . . . . . . . . . . . . 157
4.3.2.2 Accordance to OECD test
guidelines . . . . . . . . . . . . 157
4.3.2.3 Quality, relevance and utility of
published studies . . . . . . . 157
4.3.3 Main Endpoints for Toxicological
Evaluation . . . . . . . . . . . . . . . . . 159
4.3.3.1 ADME study and kinetics . . 159
4.3.3.2 Acute toxicity, irritation and
sensitization . . . . . . . . . . 161
4.3.3.3 Short-term toxicity . . . . . . 162
4.3.3.4 Long-term toxicity and
carcinogenicity . . . . . . . . . 163
4.3.3.5 Genotoxicity study . . . . . . 164
4.3.3.6 Reproductive and developmental
toxicity studies . . . . . . . . . 166
4.3.3.7 Reproductive toxicity study . 166
4.3.3.8 Developmental toxicity study . 167
4.3.3.9 Neurotoxicity . . . . . . . . . 168
4.3.3.10 Immunotoxicity . . . . . . . . 168
4.3.3.11 Human and epidemiologic data 169
4.3.3.12 Mechanistic data . . . . . . . 169
4.3.4 Considerations on Plant and Animal
Metabolites . . . . . . . . . . . . . . . . 170
4.3.4.1 Process of toxicological
evaluation in risk assessment . 172
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4.3.4.2
Toxicological significance of
species specific lesion . . . . . 180
4.3.5 Current Topics of Toxicological Evaluation
of Chemicals . . . . . . . . . . . . . . . 183
4.3.5.1 3R principles . . . . . . . . . . 183
4.3.5.2 Future methodology for
toxicological evaluation
of pesticides . . . . . . . . . . 184
4.4 Conclusions and Future Directions . . . . . . . . 186
References . . . . . . . . . . . . . . . . . . . . . . . . . 187
5. Towards a Harmonized Food Consumption Survey

Methodology for Exposure Assessment 197
5.1 Introduction . . . . . . . . . . . . . . . . . . . . 197
5.2 Pre-requisites of Pesticide Residue Assessment
Approaches to Food Consumption Data Collection 198
5.2.1 Long-term Exposure (see also Chapter 6 ) 200
5.2.2 Short-term Exposure (see also Chapter 6 ) 201
5.2.3 Statistical Considerations . . . . . . . . 202
5.3 Food Consumption Data Collection Instruments
and Methods . . . . . . . . . . . . . . . . . . . . 203
5.3.1 Sources of National Food Data . . . . . 203
5.3.2 National Food Consumption Surveys . . 206
5.3.3 Dietary Recalls . . . . . . . . . . . . . . 208
5.3.4 Food Records . . . . . . . . . . . . . . . 210
5.3.5 Administration of the Data Collection . 211
5.3.6 Portion Size Estimation . . . . . . . . . 212
5.3.7 Special Considerations in Relation to Data
Collection with Reference to Pesticide
Residue Assessment . . . . . . . . . . . 213
5.4 Food Information . . . . . . . . . . . . . . . . . 213
5.4.1 Food Classification and Description
Systems . . . . . . . . . . . . . . . . . . 213
5.4.2 Handling of Composite Foods . . . . . . 216
5.4.3 Conversion Factors for Consumed Foods
to Raw Agricultural Commodities . . . 217
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Contents xxxv
5.5 Study Organization . . . . . . . . . . . . . . . . 222

5.5.1 Survey Organization and Planning Phase 222
5.5.2 Sampling . . . . . . . . . . . . . . . . . 223
5.5.3 Sample Size . . . . . . . . . . . . . . . . 225
5.5.4 Supporting Non-dietary Survey Data . . 227
5.5.5 Quality Assurance . . . . . . . . . . . . 229
5.6 Data Cleaning and Handling . . . . . . . . . . . 230
5.7 Reporting of Food Consumption Data . . . . . . 231
5.8 Total Diet Studies . . . . . . . . . . . . . . . . . 231
5.9 Future Challenges of Food Consumption Survey
Data Collections . . . . . . . . . . . . . . . . . . 234
References . . . . . . . . . . . . . . . . . . . . . . . . . 234
6. Dietary Exposure and Risk Characterization

for Pesticide Residues in Food 243
6.1 Introduction . . . . . . . . . . . . . . . . . . . . 243
6.2 Food Consumption Data . . . . . . . . . . . . . 244
6.2.1 The GEMS/Food Diets . . . . . . . . . 244
6.2.2 Food Consumption Data for Long-term
Dietary Exposure Estimates . . . . . . . 246
6.2.3 Food Consumption Data for Short-term
Dietary Exposure Estimates . . . . . . . 246
6.3 Pesticide Residue Data . . . . . . . . . . . . . . 247
6.3.1 Pre-registration Residue Data . . . . . . 247
6.3.2 Post-registration Residue Data . . . . . 248
6.3.2.1 Residue data obtained by
monitoring programmes . . . . 248
6.3.2.2 Residue data obtained from
total diet studies . . . . . . . . 249
6.3.2.3 Residue data obtained from
duplicate diet studies . . . . . 251
6.4 Deterministic and Probabilistic Approaches to
Estimate Dietary Exposure to Pesticides . . . . 251
6.4.1 Long-term and Short-term Dietary
Exposure . . . . . . . . . . . . . . . . . 252
6.4.1.1 Long-term dietary exposure . 252
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6.4.1.2 Less-than-lifetime exposures . 253

6.4.1.3 Short-term Dietary Exposure . 254
6.4.2 Cumulative and Aggregate Exposure . . 255
6.5 Harmonization of International Dietary
Exposure Assessment . . . . . . . . . . . . . . . 259
6.6 Risk Characterization of Pesticide Residues
in Food . . . . . . . . . . . . . . . . . . . . . . . 260
6.6.1 Compounds with ADI and/or ARfD . . 261
6.6.2 TTC Approach . . . . . . . . . . . . . . 262
References . . . . . . . . . . . . . . . . . . . . . . . . . 263
7. Importance of Codex Maximum Residue Limits for

Pesticides for the Health of Consumers and
International Trade 269
7.1 What is the Codex Alimentarius Commission? . 269
7.1.1 Importance of the Codex Alimentarius
Commission (CAC) in Food Safety in
Relation to the International Food Trade 269
7.1.2 Statutes of the CAC . . . . . . . . . . . 270
7.1.3 Structure of the CAC . . . . . . . . . . 271
7.1.4 Role of Science and Risk Analysis in the
CAC . . . . . . . . . . . . . . . . . . . . 271
7.2 Establishment of Codex Maximum Residue
Limits (MRLs) for Pesticides, Extraneous
Maximum Residue Limits (EMRLs) and Related
Recommendations . . . . . . . . . . . . . . . . . 273
7.2.1 Codex Definitions of the Terms Related
to Pesticide Residues . . . . . . . . . . . 273
7.2.2 Codex Committee on Pesticide Residues 273
7.2.3 Criteria for Initiating Work on Codex
Recommendations Related to Food Safety 276
7.2.3.1 General criteria . . . . . . . . 276
7.2.3.2 Selection of pesticides for which
MRLs and EMRLs need to be
developed . . . . . . . . . . . . 277
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7.2.4Procedures for Elaborating the MRL and

EMRL and Other Recommendations . . 278
7.2.5 Expression and Specific Issues of MRLs
and EMRLs . . . . . . . . . . . . . . . . 278
7.3 Codex Recommendations Related to Pesticide
Residues in Foods and Feeds Other than MRLs
and EMRLs . . . . . . . . . . . . . . . . . . . . . 280
References . . . . . . . . . . . . . . . . . . . . . . . . . 282
8. Pesticide Specifications and their Methods

for Analysis and Testing 283
8.1 Introduction . . . . . . . . . . . . . . . . . . . . 283
8.2 History . . . . . . . . . . . . . . . . . . . . . . . 286
8.2.1 Origins of JMPS and Its History . . . . 286
8.2.2 Origins of CIPAC and Its History . . . . 287
8.3 CIPAC Methods . . . . . . . . . . . . . . . . . . 288
8.3.1 CIPAC Methods for the Determination
of the Active Ingredient Content . . . . 289
8.3.1.1 Use of the methods . . . . . . 291
8.3.2 Methods for Relevant Impurities . . . . 292
8.4 Technical Materials . . . . . . . . . . . . . . . . 293
8.4.1 Technical Material: Identity . . . . . . . 294
8.4.2 Technical Materials: Purity . . . . . . . 296
8.4.3 Impurities in Technical Materials . . . . 297
8.4.4 Impurity Names . . . . . . . . . . . . . 301
8.4.5 Expression of Relevant Impurities . . . . 302
8.4.5.1 Glyphosate Example (FAO,
2001) . . . . . . . . . . . . . . 302
8.4.6 Reference Material . . . . . . . . . . . . 303
8.4.6.1 Composition of the reference
profile . . . . . . . . . . . . . . 303
8.5 Formulations . . . . . . . . . . . . . . . . . . . . 305
8.5.1 Water as a Relevant Impurity . . . . . . 308
8.5.2 Relevant Impurities and Intended Use . 308
8.6 Equivalence . . . . . . . . . . . . . . . . . . . . . 310
8.7 Physical Properties . . . . . . . . . . . . . . . . 312
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8.7.1 Acidity and pH Range . . . . . . . . . . 312

8.7.2 Persistent Foam . . . . . . . . . . . . . . 314
8.7.3 Dustiness . . . . . . . . . . . . . . . . . 315
8.7.4 Wet Sieve Test . . . . . . . . . . . . . . 315
8.7.5 Suspensibility . . . . . . . . . . . . . . . 316
8.7.6 Pourability . . . . . . . . . . . . . . . . 317
8.7.7 Attrition Resistance . . . . . . . . . . . 317
8.8 Storage Stability . . . . . . . . . . . . . . . . . . 317
8.8.1 Storage Stability at Elevated Temperature 318
8.9 Future Directions . . . . . . . . . . . . . . . . . 321
References . . . . . . . . . . . . . . . . . . . . . . . . . 323
9. Theory and Practice of Sampling for Pesticide

Residue Analysis 327
9.1 Introduction . . . . . . . . . . . . . . . . . . . . 328
9.2 Sampling Terminology . . . . . . . . . . . . . . . 329
9.3 Decision Unit (Lot, Population) . . . . . . . . . 334
9.4 Mass Reduction . . . . . . . . . . . . . . . . . . 335
9.5 Representative Sample . . . . . . . . . . . . . . . 337
9.6 Sampling Correctness . . . . . . . . . . . . . . . 341
9.7 Material Properties and Sampling Dimensions . 341
9.7.1 Finite Element Materials . . . . . . . . 341
9.7.2 Infinite Element or Bulk Materials . . . 342
9.7.3 Sampling Dimensions . . . . . . . . . . . 344
9.7.4 Zero-Dimensional Sampling Model . . . 344
9.7.5 One-Dimensional Sampling Model . . . 347
9.7.6 Two-Dimensional Sampling Model . . . 348
9.7.7 Three-Dimensional Sampling Model . . 348
9.7.8 Packaged Foods in Commerce: Finite or
Infinite? . . . . . . . . . . . . . . . . . . 348
9.8 Pierre Gy’s Theory of Sampling . . . . . . . . . 349
9.8.1 Heterogeneity: Degree to Which a Material
is Not Homogeneous . . . . . . . . . . . 350
9.8.2 Compositional Heterogeneity: Variability
in Composition . . . . . . . . . . . . . . 353
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Contents xxxix
9.8.3 Control Fundamental Sampling Error

by Collecting Sufficient Mass and Reduce
Particle Size . . . . . . . . . . . . . . . . 353
9.8.4 Sampling Constant Varies Greatly in
Different Materials . . . . . . . . . . . . 355
9.8.5 Are We Collecting Sufficient Sample
Mass? . . . . . . . . . . . . . . . . . . . 357
9.8.6 Distributional Heterogeneity is the Spatial
and Temporal Relationship between
Elements . . . . . . . . . . . . . . . . . 358
9.8.7 Control GSE by Collecting Sufficient Mass
and More Increments . . . . . . . . . . . 359
9.8.8 Equal Probability of Selection . . . . . . 361
9.9 Sampling Techniques — Striving
to be Representative . . . . . . . . . . . . . . . . 362
9.9.1 Randomness . . . . . . . . . . . . . . . . 362
9.9.2 Zero-Dimensional Sampling . . . . . . . 362
9.9.3 Systematic Random Sampling . . . . . . 363
9.9.4 One-Dimensional Systematic Sampling . 364
9.9.5 Nugget Effect . . . . . . . . . . . . . . . 364
9.9.6 Two-Dimensional Sampling with Tools . 365
9.9.7 Center of Gravity Rule . . . . . . . . . . 368
9.9.8 Three-Dimensional Sampling and Mass
Reduction . . . . . . . . . . . . . . . . . 368
9.10 Correct vs. Incorrect Sampling . . . . . . . . . . 371
9.10.1 Why, Where, What and When? . . . . . 371
9.11 Combined Estimation of Errors . . . . . . . . . . 375
9.11.1 Sources of Sampling Error . . . . . . . . 375
9.12 Laboratory Sampling Errors . . . . . . . . . . . 378
9.12.1 Dividing the Sample . . . . . . . . . . . 378
9.12.2 Sample Preparation and Processing . . . 378
9.13 Pesticide Residue Sampling Research . . . . . . 379
9.13.1 Collect 10 Increments per Sample ? . . . 379
9.13.2 Pesticide Residues Skewed Distribution
and Variability Factors . . . . . . . . . . 383
9.13.3 Estimates of Minimum Mass . . . . . . 383
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9.13.4 Call for Identification of Factors Affecting

Uncertainty . . . . . . . . . . . . . . . . 384
9.13.5 No Generic Sampling Plan
Can Be Recommended . . . . . . . . . . 384
9.13.6 Large Field to Field Variability due
to Soil, Weather, Environment and
Application Methods . . . . . . . . . . . 384
9.13.7 Twenty-Five Increments per Sample
Provides More Normal Distribution of
Average Residues . . . . . . . . . . . . . 385
9.13.8 Uncertainty for Enforcing MRLs Increased
by 1.2 vs Field Trial Data . . . . . . . . 385
9.13.9 Protocols should be Different for Before
Market Acceptance and After Market
Enforcement . . . . . . . . . . . . . . . 385
9.13.10 GMO and Seed Sampling . . . . . . . . 386
9.14 Standardized Sampling Guidelines . . . . . . . . 386
9.14.1 Integration of TOS and MU . . . . . . . 387
9.14.2 Sample Integrity . . . . . . . . . . . . . 388
9.14.3 Sampling Animal Tissue and Animal
Products . . . . . . . . . . . . . . . . . . 389
9.14.4 Sampling for MRL Development
and Enforcement . . . . . . . . . . . . . 390
9.14.5 Codex Guidelines . . . . . . . . . . . . . 391
9.14.6 Commodity Portion Sampled for MRL
Enforcement . . . . . . . . . . . . . . . 392
9.15 Management Responsibilities . . . . . . . . . . . 393
References . . . . . . . . . . . . . . . . . . . . . . . . . 396
10. Estimation of Uncertainty of Measured Residues

and Testing Compliance with MRLs 405
10.1 Introduction . . . . . . . . . . . . . . . . . . . . 405
10.2 Factors Affecting the Results of Pesticide Residue
Analysis . . . . . . . . . . . . . . . . . . . . . . . 408
10.2.1 Variability of Pesticide Residues within
Fields . . . . . . . . . . . . . . . . . . . 408
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Contents xli
10.2.2 Sources and Calculation of Uncertainty of

Measured Residue Concentrations . . . 410
10.2.3 Characterization of the Distribution
of Pesticide Residues . . . . . . . . . . . 412
10.3 Estimation of Sampling Uncertainty . . . . . . . 415
10.3.1 Methods for Estimation of Uncertainty
of Sampling . . . . . . . . . . . . . . . . 415
10.3.2 Databases Used for Estimation of
Uncertainty of Sampling for Pesticide
Residue Analysis . . . . . . . . . . . . . 416
10.4 Estimation of Sampling Uncertainty Based
on Primary Samples . . . . . . . . . . . . . . . . 417
10.4.1 Effect of Sample Size (n) and Number of
Replicate Samples (p) on Accuracy and
Variability of Estimated Average Residue 418
10.4.2 Effect of Variability of Residues in the
Decision Unit (Sampling Target) . . . . 421
10.4.3 Effect of Number of Lots Sampled and
Replicate Samples Taken . . . . . . . . . 423
on the Results of Supervised Trials . . . . . . . . 426
10.5.1 Calculation of Sampling Uncertainty from
Replicate Composite Samples . . . . . . 426
10.5.2 Calculation of Confidence Limits . . . . 427
10.5.3 Factors Affecting the Estimated Sampling
Uncertainty . . . . . . . . . . . . . . . . 428
10.5.4 Sampling Uncertainty Values Estimated
for Crops and Crop Groups . . . . . . . 429
10.5.5 Practical Use of the Estimated Sampling
Uncertainties . . . . . . . . . . . . . . . 432
10.6 Summary of Sampling Uncertainty for Pesticide
Residue Analysis . . . . . . . . . . . . . . . . . . 434
10.6.1 Methods for the Estimation of Sampling
Uncertainty . . . . . . . . . . . . . . . . 434
10.6.2 Effect of Sample Size on the Uncertainty
of Sampling . . . . . . . . . . . . . . . . 434
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xlii Contents
10.6.3 How Many Lots to be Tested and How

Many Samples To Be Taken? . . . . . . 435
10.6.4 Preconditions for Making Reliable
Decisions based on Uncertainty of
Sampling . . . . . . . . . . . . . . . . . 436
10.7 Effect of Handling of Laboratory Samples on
the Accuracy and Uncertainty of the Results of
Analyses . . . . . . . . . . . . . . . . . . . . . . 436
10.7.1 Sources of Bias . . . . . . . . . . . . . . 436
10.7.2 Variability of Residues in Processed
Samples . . . . . . . . . . . . . . . . . . 438
10.8 Uncertainty and Trueness of Analysis of Residues
in Test Portions . . . . . . . . . . . . . . . . . . 444
10.8.1 Basic Rules of Propagation of Error . . 446
10.8.2 Examples . . . . . . . . . . . . . . . . . 447
10.9 Quality Control of the Determination
of Pesticide Residues . . . . . . . . . . . . . . . . 454
10.9.1 Sampling . . . . . . . . . . . . . . . . . 454
10.9.2 Sample Processing and Analysis . . . . 455
10.10 Use of Combined Uncertainty of Measured
Residues to Verify Compliance with MRLs
and Settling Disputes . . . . . . . . . . . . . . . 456
References . . . . . . . . . . . . . . . . . . . . . . . . . 460
11. Principles of Control of Small-Scale Production

of Fruits and Vegetables and Planning Risk-based
Monitoring Programmes 467
11.1 Introduction . . . . . . . . . . . . . . . . . . . . 467
11.2 Examples for Involvement of Small-Scale Farms in
the Production of Fruits and Vegetables . . . . . 470
11.2.1 Initiative of a Small-Scale Farm in
South-East Asia . . . . . . . . . . . . . 471
11.2.2 Family Farm with Own Packing and
Exporting Capability in Combination with
Contractual Agreement with Other Fruit
Growers in South America . . . . . . . . 472
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Contents xliii
11.2.3 Growers’ Volunteer Cooperation in a

European Country . . . . . . . . . . . . 473
11.2.4 Exporter’s Business Enterprise with
Extended Advisory and Raw Material
Supply Chain in Africa . . . . . . . . . . 474
11.2.5 Exporters Deal with Contracted Farmers
in Africa . . . . . . . . . . . . . . . . . . 475
11.3 Theoretical Background for Elaboration
of Risk-Based Monitoring Programmes . . . . . 477
11.3.1 Field to Field Distribution of Pesticide
Residues . . . . . . . . . . . . . . . . . . 477
11.3.2 Calculation of the Probability of Violation
of MRLs . . . . . . . . . . . . . . . . . . 479
11.3.3 Estimation of the 97.5th percentile
of Residues in Crops Treated with a
Pesticide . . . . . . . . . . . . . . . . . . 483
11.4 Principles of Control of Chemical Substances
in Food . . . . . . . . . . . . . . . . . . . . . . . 485
11.5 Tiered Model for Planning Monitoring
Programmes . . . . . . . . . . . . . . . . . . . . 487
11.6 Practical Application of Tiered Model . . . . . . 490
11.6.1 Tier 1: Data are Available from Supervised
Trials, But There is No Monitoring Data
(Steps 1 and 3) . . . . . . . . . . . . . . 490
11.6.2 Tier 2: Monitoring Data and Established
MRL are Available . . . . . . . . . . . . 496
11.6.3 Evaluation of a Pesticide Shortly After
Registration . . . . . . . . . . . . . . . . 500
11.7 Number of Samples to be Included in Future
Monitoring Programmes . . . . . . . . . . . . . . 502
11.8 Conclusions and Recommendations . . . . . . . . 503
References . . . . . . . . . . . . . . . . . . . . . . . . . 505
12. Future Directions 507
Index 511
b2530_FM.indd 6 01-Sep-16 11:03:06 AM

December 14, 2016 14:42 Food Safety Assessment of Pesticide Residues 9in x 6in b2668-ch01 page 1
Chapter 1
Introduction to Dietary Risk

Assessment of Pesticides
Eloisa Dutra Caldas
Main topics
Food safety initiatives by national, regional and international

authorities
Scientific developments on chemical risk assessment
The dietary risk assessment of pesticides
1.1 Introduction
Humans have always been exposed to hazardous chemicals present
in the diet, including mycotoxins, heavy metals, substances naturally
present in the food or formed during food preparation, and adulter-
ants. Already in the middle of the 18th century, laws were passed in
England to prevent the adulteration of bread and beer with cheaper
ingredients, and to prevent the selling of meat and fish that were
“not wholesome for man’s body” (Roberts, 2001).
In 1820, the chemist Fredrick Accum drew attention to numer-
ous cases of food tampering in a book published in England
called A Treatise on Adulterations of Food, and Culinary Poisons
(Fig. 1.1). The book showed “fraudulent sophistications” of various
food (including bread, beer, wine, milk and tea), drugs, medicines
and other materials, and the methods for detecting them. It was
an immediate success, with a 1,000 copies sold within a month.
Following the first concerns over food adulteration, the industrial
1
2 Food Safety Assessment of Pesticide Residues
Figure 1.1. A treatise on adulterations of food and culinary poisons (Accum,

1820).
revolution and the development of the agriculture put other chem-

icals in the government agenda to guarantee safe food to the
population.
Safety assessment can be described as the scientific understand-
ing and measurement of chemical hazards and chemical exposures,
and ultimately the risks associated with them, and is used syn-
onymously with risk assessment (IPCS, 2009a). In this chapter, an
overview of the first food safety laws and the risk assessment pro-
cedures is presented, and details related to pesticides are given in
Chapters 3 and 4.
Introduction to Dietary Risk Assessment of Pesticides 3
1.2 Food Safety Initiatives by National, Regional

and International Authorities
The first law regulating food in Australia was the Victorian Public
Food Act of 1854, responding to concerns over adulterated foods
and allowing the inspection, seizing and destruction of unwholesome
foods (Richardson and Porter, 2009). In 1991, the National Food
Authority was created, and, in 1995, an agreement between Australia
and New Zealand established a system for the development of joint
food standards, leading to the creation of the Australia New Zealand
Food Authority (ANZFA) in 1996.
In the United States of America, a crucial initiative in the food
safety history was the creation of the Department of Agriculture in
1862, and the setting up of a laboratory for the analysis of food,
soil and fertilizers (FDA, 2014). In 1902, Dr. Harvey Wiley, the
chief chemist of the Department, organized a group of volunteers
to test the effects of the most common food preservatives in use
at the time, including borax, copper sulphate, sulphuric acid, ben-
zoates and formaldehyde, on a group of healthy men, known as the
Poison Squad. This group agreed to consume food containing the
chemicals while their levels in the body were being tracked through
urine and faeces analysis, and any symptoms noted that could be
attributed to those chemicals. The shocking reports of this experi-
ment gained widespread public attention, leading to the publication
of the Food and Drugs Act in 1906. This act prohibited interstate
commerce of misbranded and adulterated foods, drinks and drugs,
and led to creation of the Food and Drug Administration (FDA) as
a law enforcement agency.
In 1937, a dramatic event occurred in the USA and showed
the need to establish drug safety before marketing. It involved the
elixir of sulphanilamide, containing the highly toxic solvent diethy-
lene glycol, which killed 107 persons, including children. This event
led to the publication of the Federal Food, Drug and Cosmetic
(FDC) Act of 1938. In 1954, the Miller Pesticide Amendment to
the FDC Act established the procedures for setting safety limits for
pesticide residues on raw agricultural commodities. This amendment
emphasized that the human health protection should be guaranteed

together with the availability of food at a fair price. In 1958, the Food
Additives Amendment required manufacturers of new food additives
to establish safety. In the same year, the Delaney provision prohib-
ited the approval of any food additive shown to induce cancer in
humans or animals, contradicting the Pesticide Amendment on its
risk–benefit approach. The Delaney provision was the basis for a
recall of cranberry-containing residues of the herbicide aminotriazole
in 1959, an episode known as the “cranberry crisis”.
An important milestone of the dietary risk assessment area was
in 1983 the publication of the report “Risk Assessment in the Federal
Government: Managing the Process”, by the USA National Research
Council, in response to a congressional directive (NRC, 1983). The
report explored the intricate relations between science and policy in
a field of assessment of the risk of cancer and other adverse health
effects associated with human exposure to toxic substances. The work
had three main objectives: (i) to assess the merits of separating the
analytic functions of developing risk assessments from the regulatory
functions of making policy decisions, (ii) to consider the feasibility of
a single organization to conduct risk assessments for all regulatory
agencies and (iii) to developing uniform risk assessment guidelines for
use by all regulatory agencies. While the proposal of a uniform guide-
line in the NRC report concerned only USA agencies, the current
challenge is to develop such guidelines that can be used by national,
regional and international agencies, being one major objective of this
book.
In the United Kingdom, the English surgeon Thomas Wakley
and the physician Arthur Hill Hassall conducted an extensive work
in the 1850s to analyse market samples of food and drink, and con-
cluded that food adulteration was very common and that many of
the adulterated foods were actually poisonous (Dawson, 2014). This
information, coupled with industrial pressure on the government, led
to the development of the United Kingdom Adulteration of Food and
Drugs Act in 1860 and the Sale of Food and Drugs Act in 1875. The
1872 revised Adulteration of Food and Drugs Act made provisions
that led to the foundation of the Society of Public Analysts two years
later. The next major change in UK food law came with the introduc-
tion of the 1984 Food Act, which was further revised in subsequent
years.
In 1893, a local food safety authority in the Netherlands was
established for control of milk, cheese and bread, and the first Dutch
food law was established in 1919. In France, the Food Adulteration
Act was passed in 1905. Cooperation between European countries
started in 1950 and the European Union was established in 1993
by the Treaty of Maastricht. In this same year, the European Com-
mission issued the Directive 93/67/EEC on the principles of risk
assessment of chemicals for humans and the environment. Regulation
No. 178/2002 of the European Parliament and of the Council of 28
January 2002 laid down the general principles and requirements of
food law, established the European Food Safety Authority (EFSA)
and the procedures related to food safety.
In 1956, the first meeting of the FAO/WHO Joint Expert Com-
mittee on Food Additives (JECFA) took place, initiating the activ-
ities in the food safety area at the international level. In 1959, the
Director-General of FAO convened a Panel of Experts on the Use of
Pesticides in Agriculture, which recommended studies to be under-
taken jointly by FAO and WHO on the hazards arising from pesticide
residues in food and feed, on the establishment of principles for set-
ting pesticide tolerances, and on the feasibility of preparing an inter-
national code for toxicological and residue data required to achieve
the safe use of a pesticide. A FAO–WHO joint meeting was held in
Rome in October 1961 to implement this recommendation, but the
first Joint Meeting of the FAO Committee on Pesticides in Agricul-
ture and the WHO Expert Committee on Pesticide Residues (JMPR)
was only held in 1963, when the toxicological properties of a number
of pesticides were studied and a few acceptable daily intakes (ADIs)
established (Ambrus, 2016). In the same year, the FAO–WHO Codex
Alimentarius Commission (CAC) was established, with the purpose
of “protecting the health of the consumers and ensuring fair prac-
tices in the food trade” (CAC, 2015). After a request by the Codex
Committee on Pesticide Residues (CCPR), a Joint FAO/WHO Con-

sultation on Guidelines for predicting the Dietary Intake of Pesticide
Residues was held in 1987 (WHO, 1988)
The International Programme on Chemical Safety (IPCS) was
established in 1980, as a joint venture of the United Nations Envi-
ronment Programme, the International Labour Organization and the
WHO, with the overall objective of establishing the scientific basis
for the assessment of risk to human health and the environment
from exposure to chemicals. One of IPCS’s first publications outlined
the principles for evaluating health risks to progeny associated with
exposure to chemicals during pregnancy (IPCS, 1984).
1.3 Scientific Developments on Chemical

Risk Assessment
In 1954, Lehman and Fitzhugh, of the FDA, suggested that the ADI
for food additives and contaminants should be derived from a no-
observed-adverse-effect-level (NOAEL) in laboratory animals divided
by a safety or uncertainty factor of 100. In 1984, Crump proposed the
use of a benchmark dose (BM or BMD) as an alternative to the use of
the NOAEL to estimate the safe intake level of chemicals by humans.
The author defined the BMD as “a lower statistical confidence limit
for the dose corresponding to a specified increase in level of health
effect over the background level”.
In 1978, Cramer and Ford proposed a strategy for classifying the
substances according to their toxicity based on a decision tree contain-
ing 33 questions requiring a yes or no answer. The classification is com-
bined with intake data to input to each chemical a “protection index”,
or a threshold of toxicological concern (TTC). This approach is useful
to establish priorities and indicate the need for further studies, and was
extensively applied in the food additive area, mainly flavours, and later
for pesticide metabolites by the EFSA and JMPR (see Chapter 6).
Soon, national, regional and international authorities gathered
their best experts with the aim of developing the scientific basis
for the risk assessment, with the USA Environmental Protection
Agency (EPA) leading this process. In 1986, EPA established the
first basis for the assessment of the risk from the exposure to multiple
chemicals, and in 1999 published a guideline for estimating aggregate
exposure. In 2002, the agency published the guidance for estimating
the cumulative exposure to pesticides with the same mechanism of
action. In 2013, EFSA published an opinion over the pesticides that
may be included in the cumulative exposure group having the basis
its toxic action in a target organ.
In 1998, the JMPR published the basis for the establishment
of the acute reference dose (ARfD) for pesticides (JMPR, 1998).
The method for calculating an international estimate of short-term
dietary intake (IESTI; see Chapter 6) was developed by a FAO/WHO
Consultation in 1997, and was first used by the JMPR at its 1999
meeting (JMPR, 1999).
In a project on the Harmonization of Approaches to the Assess-
ment of Risk from Exposure to Chemicals, the IPCS developed a
conceptual framework to evaluate mode of action (MoA) for chemi-
cal carcinogenicity based primarily on the Bradford Hill criteria for
causality (Sonich-Mullin et al., 2001). The MoA framework was later
extended to the adverse outcome pathway (AOP) concept, which
portrays existing knowledge concerning the linkage between a direct
molecular initiating event and an adverse outcome at a biological
level of organization relevant to risk assessment (Ankley et al., 2010).
1.4 The Dietary Risk Assessment of Pesticides

For pesticides, dietary risk assessments are conducted by national
or regional authorities during the registration process for the estab-
lishment of legal maximum residue limits (MRLs) for agricultural
commodities. The residue levels that support the assessment and
MRL setting are obtained from supervised residue trials conducted
with the pesticide applied according to good agricultural practices
(GAP), and the expected residues in food should not pose a health
risk to consumers (see Chapters 3 and 4).
At the international level, risk assessments are conducted by
the JMPR to support the CCPR on their management decisions,
including the establishment of Codex MRLs, to facilitate trade of
safe food (see Chapter 7). Furthermore, risk assessment studies may
be conducted post-registration, using monitoring data to assess the
risks to consumers under a more realistic scenario (Boon et al., 2008,
2015; Caldas et al., 2006).
The chemical dietary risk assessment process, introduced as a
systematic framework by the US National Research Council (NRC,
1983), has evolved rapidly since 2000, mainly regarding the toxicolog-
ical and methodological aspects. Risk assessment is one component
of the risk analysis paradigm, which also includes risk management
and risk communication (Fig. 1.2).
The risk assessment is the scientific part of the risk analysis
process, and establishes the risk as a function of two components:
the hazard and the exposure (Fig. 1.3). The hazard component has
two steps: the hazard identification and the hazard characterization,
which evaluate the hazard potential of the pesticide and are mostly
based on animal data, but can also include information from human
studies (Renwick et al., 2003; IPCS, 2009a). The hazard identifica-
tion determines the nature of the adverse effect that a given pesticide
causes to an organism. The hazard characterization describes quanti-
tatively the severity of this effect through dose–response relationships
and estimates a health-based guidance value by applying a safety
Risk management
Risk assessment regulation and
data analysis and advice
control
-science based- -policy based-
Risk communication
exchange of information
among interested parties
Figure 1.2. Risk analysis process.

Risk = f (hazard & exposure)
Hazard Exposure
1. Hazard identification
2. Hazard 3. Intake
characterization estimation
4. Risk characterization
Is exposure safe?
Figure 1.3. Chemical dietary risk assessment.
factor to the NOAEL found in the most critical study (IPCS, 2009a).
The ADI refers to long-term, lifetime exposure and the ARfD to
short-term exposure, within a time frame of 24 hours (IPCS, 2009b;
see Chapter 4).
In the dietary exposure assessment step (Fig. 1.3; see Chapter 6),
the intake of a pesticide, or a group of pesticides having the same
mechanism of action or effect (cumulative exposure), is estimated by
multiplying the residue level in the food by the amount of the food
consumed per body weight (IPCS, 2009b). It may be estimated for
the general population (all age groups within the population) or may
focus on vulnerable groups like infants, children, pregnant women or
the elderly.
In the risk characterization step, the exposure is compared to
the ADI or ARfD, depending on whether it refers to long-term or
short-term assessment, respectively (IPCS, 2009a).
Depending on the objectives of the assessment and the availabil-
ity of data, two approaches can be used to estimate the exposure
to pesticides. In the deterministic approach, or point estimate, fixed
values of concentration and consumption per body weight, such as
the mean or a given percentile, are used to calculate the intake. In the
probabilistic approach, the concentration and/or consumption vari-

ables are described as distributions, and a statistical model, such as
Monte Carlo, is used to generate an intake distribution and charac-
terize its variability and uncertainty (Kroes et al., 2002; van der Voet
et al., 2015).
In the risk characterization step, the exposure is compared to the
ADI or ARfD, depending on whether it refers to long-term or short-
term assessment, respectively (IPCS, 2009a). When a deterministic
approach is used to estimate exposure, risk may exist when the expo-
sure exceeds guidance value. When the exposure is estimated through
a probabilistic model, a percentile of exposure needs to be chosen by
risk managers for the risk characterization.
The level of uncertainty of the dietary risk assessment of pes-
ticides depends directly on the quality of the toxicological, residue
and food consumption data used in the estimations. The Organisa-
tion for Economic Co-operation and Development (OECD) publishes
guidance documents and test guidelines for testing effects of chemical
on animals, metabolism studies and residue trials to allow a sound
assessment that will support risk managers in their decision and will
facilitate the risk communication to the interested parties, including
the industry and consumers. The OECD guidance and guidelines and
the efforts for harmonization are discussed in Chapter 2.
References1
Accum F. 1820. A treatise on adulteration of food, and culinary poisons. Long-

man, Hurst, Rees, Orme, and Brown in London. Available at: http://pub
licdomainreview.org/collections/a-treatise-on-adulteration-of-food-and-culin
ary-poisons-1820/.
Ambrus Á (ed.). 2016. FAO manual on the submission and evaluation of pesticide
residues data for the estimation of maximum residue levels in food and feed.
3rd edn. FAO Plant Production and Protection Paper. No. 225.
Ankley GT, Bennett RS, Erickson RJ, Hoff DJ, Hornung MW, Johnson RD,
Mount DR, Nichols JW, Russom CL, Schmieder PK, Serrrano JA, Tietge JE
1
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and can be accessed at the websites of the corresponding organizations. Web pages
were accessed during the preparation of this chapter.
and Villeneuve DL. 2010. Adverse outcome pathways: A conceptual frame-

work to support ecotoxicology research and risk assessment. Environmental
Toxicology and Chemistry 29: 730–741.
Boon PE, Van der Voet H, Van Raaij MTM and Van Klaveren JD. 2008. Cumu-
lative risk assessment of the exposure to organophosphorus and carbamate
insecticides in the Dutch diet. Food and Chemical Toxicology 46: 3090–3098.
Boon PE, van Donkersgoed G, Christodoulou D, Crépet A, D’Addezio L,
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Chapter 2
OECD Guidance Documents

and Test Guidelines
Roland Solecki, David M. Schumacher, Rudolf Pfeil,
Rajumati Bhula and Dugald J. MacLachlan
Main topics
OECD Environment, Health and Safety Programme

OECD Test Guidelines Programme
OECD Guidelines and Guidance on Health Effects
OECD Guidelines and Guidance on Pesticide Residues
2.1 Introduction
The activities of the Organisation for Economic Co-operation and
Development (OECD) on Chemical Safety are carried out under the
Environment, Health and Safety (EHS) Programme. The work areas
of the EHS Programme include activities on both pesticides (chemi-
cal or biological products to protect plants, used in agriculture and
related areas) under the OECD Pesticide Programme.
The initial driving force for the development of harmonized
approaches for registration and re-registration of pesticides came
from a workshop held in Washington in 1992. The workshop con-
cluded that a primary goal for OECD should be the sharing of
national review reports, to facilitate recognition of assessments by
member governments, thereby achieving efficiencies in the work of
regulatory authorities. Work sharing was seen as one way to improve
13
risk assessments and to achieve greater consistency in risk assess-

ment outcomes, supported by harmonization of data requirements
and test guidelines, and harmonization of hazard and risk assessment
approaches.
2.2 OECD Environment, Health and Safety

Programme
2.2.1 OECD Pesticide Programme
The OECD Pesticide Programme was established in 1992 to improve
the efficiency and effectiveness of pesticide regulation. The OECD
Working group on Pesticides is set up as a Programme of the Joint
Meeting of the Chemicals Committee and Working Party on Chemi-
cals, Pesticides and Biotechnology. Its main aims were to help OECD
member states to harmonize the data and methods used in testing
and assessing pesticide risks, to share the work of pesticide registra-
tion and re-registration and to help OECD member states reduce
the risks associated with pesticide use. The scope of the Pesticide
Programme included chemical and biological pesticides. One of the
strategic objectives is to further enhance the high level of protection
afforded to human health and to minimize to the extent possible the
levels of risk arising for humans as a consequence of pesticides.
The OECD Pesticide Programme is directed by the Working
Group on Pesticides (WGP), which is composed of representatives
from member governments, international organizations such as Food
and Agricultural Organization (FAO) and World Health Organiza-
tion (WHO), the European Commission, members from the pesticide
industry and representatives from environmental and public interest
groups. The WGP oversees a number of groups such as the Registra-
tion Steering Group (RSG), Biopesticides Steering Group (BPSG)
and the Risk Reduction Steering Group (RRSG).
In recent years, the WGP has increased co-operation in the field
of pesticides with the United Nations’ WHO and FAO, thereby
helping developing countries efficiently establish and manage their
pesticide regulatory systems. This also includes contacts with the
two international bodies that are important in pesticide residue
regulation and trade, the Joint FAO/WHO Meeting on Pesticide
OECD Guidance Documents and Test Guidelines 15
Residues (JMPR) and the Codex Committee on Pesticide Residues

(CCPR).
Today, the OECD Programme on Pesticides and Sustainable
Pest Management Vision for the Future (OECD, 2016a) includes the
core elements of harmonization, sustainable approach, risk reduc-
tion, enhanced communication and partnerships. Here, harmoniza-
tion means the harmonization of regulatory systems and work sharing
to help improve the registration system. This includes:
• the development of harmonized guidelines for the design and con-

duct of tests/studies for regulatory purposes (test guidelines),
• the development of guidance on the use and interpretation of data
generated from tests/studies (guidance documents),
• guidance for sponsors on the preparation of data submissions
(dossiers) according to an agreed data point scheme (OECD, 2005)
and format, and
• guidance on harmonization of hazard assessments to support
national risk assessments.
Member governments, international organizations and industry

working through the WGP have created an international network
that shares information and works collectively to solve common
problems. While work-sharing arrangements are well established at
the regional level (e.g. within the European Union and within the
NAFTA region), the adoption of such working arrangements on a
more global level is challenging.
Since the initiation of the WGP Programme, the various groups
have conducted projects to improve evaluation processes and risk
assessment and risk management procedures for both new and exist-
ing pesticides. There has also been focus on minimizing non-tariff
trade barriers and reducing risks to human health and the environ-
ment arising from use of pesticides.
2.2.2 Harmonization of Data Requirements

In 1994, a survey (OECD, 1994) of ‘Data Requirements for Pesti-
cide Registration in OECD Member Countries’ was published, which
showed that the data requirements for the registration of pesticides
were largely harmonized within the OECD, with most governments

requiring the same or similar studies or data sets.
Out of this survey, a programme of work was established to
undertake projects on the development of data requirements for bio-
logical pesticides; the development of specific test guidelines; the
development of frameworks for hazard assessments for different prod-
ucts, and raising awareness of member government re-registration
and risk reduction programmes. An Expert Group on Pesticide
Residue Chemistry was established in October 2003 to develop har-
monized test guidelines and guidance documents for pesticide residue
chemistry.
2.2.3 OECD Series on Principles of GLP

The Principles of Good Laboratory Practice (GLP) have been devel-
oped to promote the quality, validity and reliability of test data
presented in study reports of non-clinical studies. Such reports are
used to assess the safety of chemicals and chemical products. It is
a supervisory concept covering the organizational process as well
as the conditions under which laboratory studies are planned, per-
formed, monitored, recorded and reported. Its principles are required
to be followed by test facilities carrying out studies for the pur-
poses of assessment of chemicals and other uses relating to the
protection of man and the environment (OECD, 1998). The doc-
uments published in the OECD Series on Principles of GLP and
Compliance Monitoring describe the OECD member states’ com-
mon understanding of the principles of GLP and how to monitor the
compliance.
All studies used in the risk assessment of pesticides in food should
be assessed for adequacy of design and conduct. Compliance with
GLP includes aspects such as the proper care, maintenance and hous-
ing of experimental animals as well as other general study considera-
tions, such as sufficient resources, protocols and written procedures,
characterization of test items and test systems, documentation and
quality assurance (WHO, 2009). To ensure that relevant aspects of a
study are reported, both the study director and the quality assurance
unit need to confirm in writing that the report accurately reflects the
raw data obtained during the performance of the study including all
aspects affecting the quality or integrity of the study.
During JMPR’s assessment of such studies, the compliance with
GLP principles should also be checked (WHO, 2015).
2.3 OECD Test Guideline Programme

The OECD Council adopted a Council Decision in 1981 — on Mutual
Acceptance of Data (MAD) — agreeing that test data generated in
any member country in accordance with OECD Test Guidelines and
OECD Principles of GLP shall be accepted in other member coun-
tries for assessment purposes and other uses relating to the protection
of human health and the environment (OECD, 1981).
Since 1981, in recognition of the advantages of internationally
agreed test methods, OECD member and partner countries have
developed the OECD Guidelines for the testing of chemicals in
order to:
• enhance the validity and international acceptance of test data,

• make the best use of available resources in both governments and
industry,
• avoid the unnecessary use of laboratory animals, and
• minimize non-tariff trade barriers.
The OECD Guidelines for the testing of chemicals are primarily

used in regulatory safety testing and subsequent chemical notifica-
tion and registration. The process of approval and publication of test
methods and guidelines is updated from time to time to keep pace
with progress in science and countries’ regulatory needs. OECD-wide
networks of national coordinators and national experts provide input
from scientists in government, academia and industry. OECD Test
Guidelines (OECD TGs) should not be confused with data require-
ments, which are the prerogative of national authorities (OECD,
2016c).
The OECD TGs Programme provides the supporting struc-
ture for developing new and revising existing OECD TGs. The
responsibilities and various procedures are published in a guidance

document (OECD, 2009b). It describes the structure of the TGs Pro-
gramme, the various responsibilities of those involved in the process
and, in detail, the procedures that are followed. According to this
guidance, National Coordinators (NCs) have a central position in
the Test Guidelines Programme. They submit proposals for new or
revised guidelines (including those suggested by the scientific commu-
nity, industry or non-governmental organizations) and provide com-
ments agreed on at the national level on proposals circulated by the
Secretariat. The Working Group of National Coordinators of the Test
Guidelines Programme (WNT) meets at least once a year to oversee
the TGs Programme and works towards the development of draft
TGs based on a consensus.
A proposed new TG or a request for the revision of an existing
TG should undergo a critical appraisal of its scientific and regula-
tory justification prior to the addition of the project to the pro-
gramme’s work plan. If agreement on a proposal is reached, a draft
Test Guideline is submitted to the WNT for their approval before
it is sent to the OECD Joint Meeting of the Chemicals Committee
and the Working Party on Chemicals, Pesticides and Biotechnology
(OECD JM) for review and endorsement, the Environment Policy
Committee (EPOC) for review and the OECD Council for adoption.
TGs adopted by the OECD Council become effective from the date
of adoption and the Secretariat then arranges for their publication
under the OECD website.
The OECD Guidelines for the testing of chemicals is a collection
of about 150 of the most relevant internationally agreed testing meth-
ods used by government, industry and independent laboratories to
identify and characterize potential hazards of chemicals. OECD test
guidelines are internationally accepted as standards for the design
and conduct of studies required by regulatory authorities for regis-
tration of pesticides. Under the MAD rule, any data generated in
an OECD member country in accordance with the test guidelines
and principles of GLP must be accepted by other OECD members
for the purposes of assessment. Sponsors using the TGs to generate
data for risk assessments can submit the same studies or data sets
to all OECD member governments and partner countries, leading to

the submission of the core set of studies for registration purposes.
TGs are divided into five sections:
• Section 1: Physical Chemical Properties.

• Section 2: Effects on Biotic Systems.
• Section 3: Degradation and Accumulation.
• Section 4: Health Effects.
• Section 5: Other TGs, including Pesticide Residues.
The TGs are available from the OECD website free of charge.
2.4 OECD Guidelines and Guidance on Health Effects

Section 4 of the OECD TGs is most relevant for the toxicological
testing of chemical substances including pesticides. In case more
detailed information is needed, the reader is referred to either the
respective TGs or to general toxicology textbooks (such as Greim and
Snyder, 2008; Hayes and Kruger, 2014; Jacobson-Kram and Keller,
2006). The test guidelines are supported and complemented by spe-
cific OECD Guidance Documents (OECD GD).
In the following, for each relevant endpoint, the aim and the TGs
will be briefly described.
2.4.1 ADME and Toxicokinetics

Data on the absorption, distribution, metabolism and excretion
(ADME) of test compounds inform on the compound’s fate in the
test animals organism. The OECD TG 417 describes in vivo studies
that provide information on mass balance, absorption, bioavailability,
tissue distribution, metabolism, excretion and further toxicokinetic
parameters. Information from OECD TG 417 is important to relate
concentrations or dose levels to the observed effects and to derive
the internal (systemic) dose levels. Often such oral studies, which
are preferably carried out in the rat, are conducted with radioactive
labelled test materials. Additionally, experiments with repeated dose
administration are necessary. Initial estimation of absorption can be
achieved by mass balance determination, but further investigations
such as intravenous (i.v.) administration and biliary excretion studies

might be necessary. Bioavailability can be determined from the com-
parison of plasma–blood kinetics determined in oral and i.v. groups.
The rate and extent of excretion of the administered dose should
be determined by measuring the percent recovered dose from urine,
faeces and expired air. The percent of the total dose in tissues should
at a minimum be measured at the termination of experiment.
2.4.2 Acute Toxicity

The main objectives of acute toxicity testing of pesticides are to
identify the intrinsic toxicity of a chemical substance and to predict
the hazard after a single exposure, using rodents as surrogates for the
human. Furthermore, acute toxicity studies may assist identifying
target organs and species differences in susceptibility and may be
supportive for setting of an acute reference dose.
The only OECD TG for the oral route of acute testing was for
many years the ‘acute oral toxicity test’ (OECD TG 401). It was
deleted in 2002 based on the observation that other methods using
fewer animals were available. As alternative TGs the Acute Oral Tox-
icity — Fixed Dose Procedure (OECD TG 420), the Acute Oral toxi-
city — Acute Toxic Class Method (OECD TG 423) or the Acute Oral
Toxicity: Up-and-Down Procedure (OECD TG 425) are employed.
All of the guidelines involve the administration of a single bolus dose
of test substance to fasted healthy young adult rodents by oral gav-
age, observation for up to 14 days after dosing, recording of body
weight and the necropsy of all animals.
A Guidance Document (OECD No. 24) assists with the choice of
the most appropriate TG while reducing the number of animals used
and avoiding animal suffering (OECD, 2001). Additionally, the GD
provides information on the conduct and interpretation of TGs 420,
423 and 425.
The TG on phototoxicity (OECD TG 432) describes an in vitro
method to evaluate photo-cytotoxicity by the relative reduction in
viability of cells exposed to the chemical in the presence versus
absence of light.
2.4.3 Repeated-Dose Toxicity in Rodents

and Non-rodents
Repeated-dose toxicity studies are conducted to determine adverse
effects induced by chemical substances in mostly rodents, but also
non-rodents. The studies may be of varying duration, generally four
weeks for subacute studies and three months for sub-chronic stud-
ies. Many toxicological parameters (e.g. clinical observations, clinical
pathology, gross necropsy and histopathology) are monitored dur-
ing the study, in order to provide information on target organs or
sites of action, the dose–response relationship, the progression and
reversibility of effects and to identify a no-observed-adverse-effect
level (NOAEL).
OECD TG 407 describes the repeated oral administration of the
compound over 28 days to rodents. Of higher relevance for the assess-
ment of the hazard potential of the active substances is the repeated
dose 90-day oral toxicity study in rodents (OECD TG 408), which
is mostly carried out after initial information on toxicity has been
obtained from acute or repeated dose 28-day toxicity tests. Informa-
tion on the toxicological profile in a second species is gathered in
the repeated dose 90-day oral toxicity study in non-rodents, i.e. dogs
(OECD TG 409). At least eight animals (four female and four male)
should be used for each test group.
The TGs on repeated dose toxicity studies are supplemented by
guidance notes (No. 32), which provide a general guide to the analysis
and evaluation of data from studies involving repeated exposures
of experimental animals to chemicals; and to outline the kind of
information that should be included in a study report to allow an
independent assessment of the toxicity studies (OECD, 2002b).
2.4.4 Carcinogenicity
The objective of the long-term carcinogenicity study is to observe test
animals for a major portion of their life span during or after expo-
sure to various doses of a test substance by an appropriate route
of administration. Laboratory animals are treated for most of the
normal life span of the test species, i.e. 18–24 months for the mouse
and 24 months for the rat. Many toxicological parameters (e.g. body
weight, clinical observations, clinical pathology, gross necropsy and
histopathology) are monitored during such studies, in order to pro-
vide information on target organs, the possible specificity of observed
findings regarding affected species, sex and organs, the dose–response
relationship, the NOAEL for non-neoplastic adverse effects as well as
the respective data for carcinogenic effects.
The carcinogenicity study (OECD TG 451) was intended primar-
ily for use with rats and mice, and for oral administration in both
sexes. Each dose group and concurrent control group should contain
at least 50 animals of each sex and at least three dose levels and
a concurrent control for 18–24 months. The chronic toxicity study
(OECD TG 452) is usually not performed to assess new chemical
substances. However, the combined chronic toxicity/carcinogenicity
study (OECD TG 453) is the most relevant study type to identify car-
cinogenic and the majority of chronic effects, and to determine dose–
response relationships following prolonged and repeated exposure.
A Guidance Document (OECD GD 116) provides additional
information on the design and conduct of studies performed using
OECD TG 451, 452 and 453 (OECD, 2011). Its objective is to assist
users of the TGs to select the most appropriate methodology to assess
the chronic toxicity and carcinogenicity of a test chemical so that par-
ticular data requirements can be met while reducing animal usage if
possible and suitable. It is intended to foster a common approach
among the scientists carrying out such studies and those assessing
them. This contributes to the harmonization activities undertaken
by the OECD and other agencies, such as the WHO.
Further Guidance Notes (OECD GD 35) can be used in the
assessment of such studies and provide mainly guidance on hazard
assessment, but addresses risk assessment aspects only to a certain
extent (OECD, 2002a).
2.4.5 Genotoxicity
Mutagenic or genotoxic properties of chemicals can be assessed with
several test methods.
Mutation is defined as a permanent change in the amount or

structure of the genetic material in a cell. The term mutation
applies both to heritable genetic changes that may be manifested
at the phenotypic level and to the underlying DNA modifications
when known (including specific base pair changes and chromoso-
mal translocations).
The more general terms ‘genotoxic’ and ‘genotoxicity’ apply to
agents or processes that alter the structure, information content or
segregation of DNA, including those which cause DNA damage by
interfering with normal replication processes, or which in a non-
physiological manner (temporarily) alter its replication. Genotox-
icity test results are usually taken as indicators for mutagenic
effects (UN GHS, 2015).
Generally, mutation assays are categorized according to the type

of damage they can detect: changes (1) in the sequence of the DNA
or (2) of the amount or structure of the genetic material in the cell.
The individual available test systems are not capable of detecting all
possible types of damages simultaneously. For a comprehensive eval-
uation, several test systems need to be combined to allow a thorough
assessment.
Additionally, investigations can be performed in vitro or in vivo
in somatic cells or in vivo in germ cells.
The most commonly used in vitro test systems to detect muta-
genic properties are (1) the ‘Ames test’ as a reverse mutation assay
in certain strains of Salmonella typhimurium and Escherichia coli
(OECD TG 471), (2) the chromosomal aberration test (OECD TG
473) or the micronucleus test in mammalian cells (OECD TG 487)
and (3) mutation tests in mammalian cells (HPRT gene mutation
[OECD TG 476] or thymidine kinase gene mutation [OECD TG 490:
mouse lymphoma assay and TK6 assay]).
The cultured cell lines used in routine in vitro testing have only a
limited capacity to metabolize the test compound. To overcome this
limitation — to a certain extent — metabolic activation systems
are used. Usually a preparation from rat liver is used, the so-called
‘S9-mix’. Because of the supplemented co-factors, it is capable of

cytochrome P450-mediated oxidative metabolism reactions. Depend-
ing on specific research questions, S9 mix from different species or
organs may be employed, which might be supplemented with differ-
ent co-factors to support other metabolic reactions (such as those
by glutathione transferases, UDP-glucuronosyltransferases, epoxide
hydrolases or sulfotransferases).
The usual in vivo assay investigates (1) the induction of chromo-
somal aberrations in bone marrow (OECD TG 475) or of micronuclei
in bone marrow or in peripheral lymphocytes (OECD TG 474) or (2)
the induction of mutations in organs of transgenic rodents (OECD
TG 488). One advantage of the in vivo tests over the in vitro tests is
the integration of normal mammalian ADME and the possibility to
select relevant exposure routes. However, for a valid test, sufficient
exposure of the target tissue needs to be ensured.
Several in vivo tests are available that evaluate the chemical’s
mutagenic properties in germ cells. These include the rodent domi-
nant lethal mutation test (OECD TG 478), mammalian spermato-
gonial chromosome aberration test (OECD TG 483) and the mouse
heritable translocation assay (OECD TG 485). The mouse spot test
(OECD TG 484) detects presumed somatic mutations in foetal cells
following transplacental absorption of the test substance. These lat-
ter test systems were conducted regularly in the past, but are used
seldom in modern risk assessment.
The unscheduled DNA synthesis (UDS) test with mammalian
liver cells in vivo (OECD TG 486) identifies substances that induce
DNA repair of DNA containing a region of damage induced by
treatment. The in vivo alkaline single cell gel electrophoresis assay
(alkaline Comet Assay, OECD TG 489) measures increases in DNA
strand breaks in cells prepared from organs of test material-treated
animals.
2.4.6 Reproductive Toxicity and

Developmental Toxicity
Reproductive toxicity and developmental toxicity studies are con-
ducted to identify adverse effects on reproductive physiology and
the development of progeny resulting from exposure to chemical

substances. Laboratory animals (rodents and non-rodents) are used
with rats being the standard species for the one-generation or two-
generation reproductive toxicity study, while rats and rabbits are the
standard species for the developmental toxicity studies.
In reproductive toxicity studies, the effects of a test substance
on the integrity and performance of male and female reproductive
functions or capacity, e.g. effects on oestrus cycle, sexual behaviour,
any aspect of spermatogenesis or oogenesis, or hormonal activity or
physiological response, which would interfere with the capacity to
fertilise, fertilisation itself or development of the fertilised ovum up
to and including implantation, shall be investigated.
The two-generation reproduction toxicity study (OECD TG 416)
is designed to provide general information concerning the effects of a
test substance on reproductive systems, and on the growth and devel-
opment of the offspring. The administration of the substance in grad-
uated doses to several groups of male and female rats is started for
parental animals continued for first generation offspring during their
growth into adulthood, mating and production of a second generation
(until the weaning). The rat is the preferred species for testing. The
studies should include measurements of body weight and body weight
gain of the parents and the offspring, sperm parameters, oestrus cycle
parameters and offspring parameters for developmental landmarks,
clinical daily observations as well as gross necropsy and histopathol-
ogy. A two-generation reproduction toxicity study should provide
information on adverse effects on reproduction, parturition, lacta-
tion, postnatal development including growth and sexual develop-
ment. Additionally, the dose–response relationships for the observed
findings are determined including the NOAELs for effects in parental
animals, offspring and for effects on fertility and reproduction.
The one-generation reproduction toxicity study (OECD TG 415)
for reproduction testing was designed to provide general informa-
tion concerning the effects of a test compound on male and female
reproductive performance but animals are treated and mated only
to produce one offspring generation. In the other aspects, the one-
generation study is similar to the two-generation study.
The extended one-generation reproductive toxicity study (OECD

TG 443) was designed to provide an evaluation of reproductive and
developmental effects that may occur as a result of pre and postna-
tal chemical exposure as well as an evaluation of systemic toxicity
in pregnant and lactating females and young and adult offspring.
In the assay, sexually mature male and female rats of the parental
(P) generation are exposed to graduated doses of the test substance
starting two weeks before mating and continuing through mating,
gestation and weaning of their pups (F1 generation). At weaning,
pups are selected and assigned to cohorts of animals for reproductive–
developmental toxicity testing (cohort 1), developmental neurotox-
icity testing (cohort 2) and developmental immunotoxicity testing
(cohort 3). The F1 offspring receive further treatment with the test
substance from weaning to adulthood.
Regarding the assessment of developmental toxicity, effects on the
progeny, e.g. any effect interfering with normal development, both
before and after birth, can be investigated. This includes morpho-
logical malformations and variations, and functional disturbances as
well as specific reproductive and neurological effects. The aspect of
interference with postnatal development is mainly addressed in the
generational studies described above, whereas the aspect of interfer-
ence with prenatal development is addressed in specific developmen-
tal toxicity studies.
The TG for developmental toxicity testing (OECD TG 414) is
designed to provide information on the effects of prenatal exposure
on the pregnant test animal and on the developing organism. The test
substance is administered to pregnant rats or rabbits at least from
implantation to the day prior to the day of scheduled parturition.
Additionally, to the maternal parameters for body weight and body
weight gain measurements as well as clinical and fertility parameters,
the uterine contents are examined, and the foetuses are evaluated for
external, soft tissue and skeletal changes.
Further Guidance (OECD GD 43) is available on methodologi-
cal aspects and interpretation of data (OECD, 2008). It also covers
the relationship with neurotoxicity testing. The document consti-
tutes an essential supplement to existing OECD TG, which include
the one- and two-generation toxicity study (OECD TG 415 and

416), prenatal developmental toxicity study (OECD TG 414), devel-
opmental neurotoxicity study (OECD TG 426) and the reproduc-
tion/developmental toxicity screening tests (OECD TG 421 and 422).
However, data from other toxicity studies, for example, repeated dose
toxicity studies for systemic toxicity (OECD TG 407, 408 and 409),
may indicate effects on reproductive organs and should be considered
in the assessment as well as existing human data.
The extended one-generation reproductive toxicity study is sup-
plemented with a guidance document (OECD GD 151) (OECD,
2013a) supporting study sponsors and laboratories to plan such stud-
ies and provides details on how they may be conducted (e.g. gath-
ering of key data on the substance to be tested). Nevertheless, the
design of the study will depend upon existing information, regula-
tory requirements and whether or not cohorts have been omitted.
Finally, scientists evaluating the results of such studies for scientific
and regulatory purposes can draw advice on the assessed endpoints
and data interpretation issues not detailed in the TG.
2.4.7 Acute and Repeated-Dose Neurotoxicity

and Delayed Neurotoxicity
A neurotoxic effect is an adverse change in the structure or function
of the nervous system that may result from any chemical exposure.
Such effects may result from single or repeated doses either due
to an agent acting directly on target sites in the nervous system,
or indirectly, by acting on target sites outside the nervous system.
Both single and repeated exposures are possible scenarios for human
exposure. Therefore, the neurotoxicity testing strategy must consider
both situations and should investigate the type, severity and possible
reversibility of the effects.
The neurotoxicity study in rodents (OECD TG 424) was designed
to obtain the information necessary to confirm or to further charac-
terize the potential neurotoxicity of chemicals in adult animals and
includes detailed clinical observations in the home cage and open
field, functional tests including motor activity and neuropathology
using perfusion-fixed tissues. It uses basically the same tests (func-

tional tests and clinical observations) as those recommended in
OECD TGs 407 and 408 but employs a larger sample size than
OECD TG 407, calls for more frequent measurement of functional
tests, requires that observations are conducted without knowledge
of treatment level and allows for a longer exposure period and flexi-
bility in designing neurotoxicity studies so that resource use can be
optimized.
The primary objective of Guidance Document for Neurotoxicity
Testing (OECD GD 20) is to ensure that necessary and sufficient
data are obtained to enable adequate evaluation of the risk of neuro-
toxicity arising from exposure to a chemical (OECD, 2004), whereas
developmental neurotoxicity testing is covered by another Guidance
Document (OECD, 2008) and OECD TG 426.
Delayed neurotoxicity studies were introduced especially for the
testing of organophosphorus substances, in which the test substances
were administered orally in a single dose (OECD TG 418) or during
28 days (OECD TG 419) to domestic hens.
2.4.8 Endocrine Disruption

Unlike most TGs described above, which measure adverse effects, the
TGs regarding endocrine disruption place more emphasis on mecha-
nistic information on the effects induced by the test materials. Hence,
mostly individual effects on certain pathways are assessed. Tests are
conducted either in vitro or in vivo.
The OECD initiated a high-priority activity in 1996 to revise
existing and to develop new TGs for the screening and testing of
endocrine disrupting chemicals. Since then, a number of assays have
been developed into test guidelines, which are published as ‘Series on
Testing and Assessment: Testing for Endocrine Disrupters’ (OECD,
2002 et seq. anni).
The available in vitro tests include assays for the detection of
estrogen receptor agonists (OECD TG 455), for the detection of
estrogen receptor agonists and antagonists (OECD TG 457), for
the detection of chemicals with oestrogen receptor binding affinity
(OECD TG 493) and for the detection of chemical effects on steroido-

genesis (OECD TG 456).
The available in vivo assays include short-term screening tests
for oestrogenic properties (uterotrophic bioassay in rodents; OECD
TG 440) and (anti)androgenic properties (Hershberger bioassay in
rats; OECD TG 441).
The screens and tests are contained within the ‘OECD Con-
ceptual Framework for Testing and Assessment of Endocrine Dis-
rupters’, which was revised in 2012. This conceptual framework lists
the OECD TGs and standardized test methods available, under
development or proposed that can be used to evaluate chemicals for
endocrine disruption and is included as an Annex in the Guidance
Document 150 (OECD, 2012). Further information regarding the use
and interpretation of these tests is available in that OECD GD.
2.5 OECD Guidelines and Guidance on

Pesticide Residues
Section 5 of the OECD TG is most relevant for the planning and con-
duct of studies on pesticide residues. The Residue Chemistry Expert
Group (RCEG) commenced work in 2005 on the development of a
series of harmonized TGs (Table 2.1).
For each guideline, content was sourced from existing national
and international guidelines, and areas of commonality and diver-
gence were identified. Then a harmonized TG was drafted that
included all information relevant to the conduct of a particular test
or study. An example of an international guideline considered in this
process is the ‘FAO Guidelines on Pesticide Residue Trials to Pro-
vide Data for the Registration of Pesticides and the Establishment
of Maximum Residue Limits’ (FAO, 1986).
Where differences were identified in one or more source docu-
ments, the guidelines recommend that the study sponsor contact the
relevant authority for further advice or information on that specific
point of difference before commencing the study.
The Overview document ‘Introduction to OECD Test Guide-
lines on Pesticide Residues Chemistry — Section 5 Part A’ includes
Table 2.1. OECD Guidelines for the testing of chemicals, Section 5 pesticide
residue chemistry.
Test
Guideline Date of
Number Topic Publication
501 Metabolism in crops 25 January 2007

502 Metabolism in rotational crops 25 January 2007
503 Metabolism in livestock 25 January 2007
504 Residues in rotational crops (limited field 25 January 2007
studies)
505 Residues in livestock 25 January 2007
506 Stability of pesticide residues in stored 15 October 2007
commodities
507 Nature of the pesticide residues in processed 15 October 2007
commodities high temperature hydrolysis
508 Magnitude of the pesticide residues in 16 October 2008
processed commodities
509 Crop field trial 7 September 2009
brief descriptions of each guideline (OECD, 2013b) while the ‘Guid-

ance Document on Overview of Residue Chemistry Studies’ (OECD,
2009a) provides further explanatory information on the purpose and
use of each TG and guidance document.
2.5.1 Improved Alignment of Data Interpretation

Differences in interpretation of data are major sources of non-
harmonized outcomes between regulators. While drafting the test
guidelines, the RCEG also considered data interpretation issues
alongside study design (see Table 2.2). It was important to deter-
mine if detailed guidance existed for data interpretation, and what
level of guidance was available to data assessors working in regula-
tory authorities. This led to the development of a series of guidance
documents and data reporting templates.
The interpretation of studies leading to different and
non-harmonized outcomes was also identified as a major impediment
to successful work sharing and global joint review outcomes where
all participating members receive the same data package. Although
Table 2.2. Residue guidance documents published by the Residue Chemistry

Expert Group.
Guidance Date of
Number Topic Publication
63 Guidance document on the definition of the July 2009 (revised)

residue
64 Guidance document on overview of residue July 2009 (revised)
chemistry studies
72 Guidance document on pesticide residue August 2007
analytical methods
73 Guidance document on residues in livestock September 2013
96 Guidance document on magnitude of October 2011
pesticide residues in processed commodities
164 Guidance document on crop field trials October 2010
data requirements might differ slightly between regulators, in an ideal

world data interpretation should be consistent.
Each guidance document provides practical examples of how data
generated using the associated TG may be interpreted. Each guid-
ance document reflects ‘best practice’ in data interpretation.
The guidance document on the definition of the residue includes
numerous examples of residue definitions and the basis for the selec-
tion of the definition. Various criteria such as inclusion of metabolites
in a definition, including toxicity considerations, availability of ana-
lytical methods for all components of a definition, definitions already
established and residue definitions for dietary risk assessment are
described.
The ‘Guidance Document on the Magnitude of Pesticide Residues
in Processed Commodities’ includes examples on how to derive pro-
cessing factors for various commodities. It also provides advice on
metabolites which should be taken into account and what to do when
there are different residue definitions for dietary risk assessment and
maximum residue limit (MRL) setting or compliance.
Data interpretation considerations also assisted in the devel-
opment of templates (OECD, 2016b) for data reporting (OECD
Templates #85-1 to #85-7) where prescriptive data entry fields allow

risk assessors to easily locate and transcribe the relevant data into a
report format.
2.5.2 The Livestock Feed Tables

The assessment of pesticide residues in animal feeds and their
transfer to animal commodities is an important consideration that
required further elaboration at an international level. Before publi-
cation of the OECD Livestock Feed Tables in 2006, regulators were
using their own data on the contribution of various feeds to livestock
diets (feed tables). Existing feed tables did not contain information
for all major livestock species, or provided very limited guidance in
terms of feed items for cattle (beef and dairy), sheep, pigs and poultry
(hens and turkeys).
The JMPR was using a very limited table (based on U.S. feed
tables) that did not contain all important commodities that may be
fed to livestock in various regions (Ambrus, 1997). Feed tables are
integral to the consistent estimation of dietary burden or exposure
of the animal to pesticides through feeds.
Livestock production practices around the world were compared
to develop a set of harmonized feed tables that reflected typical pro-
duction diets relevant to livestock of a marketable size. Commodities
or feeds are grouped as forages, roots and tubers, cereal grains and
crop seeds, and by-products or processed commodities and classi-
fied according to carbohydrate concentrate, roughage and protein
concentrate with percentage of dry matter content. Detailed descrip-
tions of the feed commodities allow easy identification and compari-
son to samples taken and analysed in supervised residues field trials.
The comparison of production practices highlighted the fact that
where extensive rearing and free grazing are commonplace, feed com-
modities and associated intakes (percentages in the diet) are vastly
different from regions where livestock are intensively reared in feed-
lots or pens. Sources of feed items vary with seasonal changes, and
in some regions such as the European Union (EU), feeds may be
imported and not locally sourced.
Dietary burden calculations using the feed tables can result in sig-
nificant differences in MRL, for example for cattle raised in Australia
or New Zealand, compared to cattle raised in North America or
Europe. This is highlighted in the JMPR dietary burden calculations
for ametoctradin (FAO, 2013).
In the ametoctradin example, there are three feed commodities
and the percentages in the diet of beef cattle in US–Canada, the EU,
Australia and Japan are calculated. The lowest contribution to the
dietary burden (0.05 ppm) comes from potato culls in US–Canada,
while the highest contribution (116.7 ppm) is from rape forage in
Australia. Hence the highest value is used to determine the animal
commodity MRLs for cattle meat and offal. Similarly, dietary burden
contributions are calculated for dairy cattle to determine the MRL
for milk.
The feed tables and the dietary burden calculations provide a
transparent and harmonized approach for setting animal commodity
MRLs for trade purposes on the basis of residues in livestock feeds.
The information presented by region is useful for regulatory author-
ities, as the methodology associated with use of the feed tables is
harmonized.
The development of a comprehensive table of livestock feeds and
percentages in the livestock diet for US–Canada, the EU, Australia
and Japan by the RCEG and associated guidance has brought atten-
tion to the importance of considering production practices around the
world when setting Codex MRLs for animal commodities. The feed
tables are published in the Guidance Document on Residues in Live-
stock (OECD, 2009a), together with examples of how to calculate
the dietary burden for, beef cattle based on the EU, Australian and
Japanese percentages.
2.5.3 The OECD MRL Calculator

Although sponsors may provide the same residue trial data to a group
of regulators, differences in the approaches for estimating MRLs con-
tributed to the lack of harmonized outcomes. Different methodologies
were used by different regulators including rounding-up of the highest
observed residue value and various statistical approaches such as EU

Methods I and II and the NAFTA MRL Calculator (MacLachlan and
Hamilton, 2010).
The RCEG in 2008 tasked a group of experts sourced from regu-
lators and industry to propose an OECD MRL calculation procedure.
The guiding principles were:
• the procedure must be a practical implementation of sound statis-

tical methods,
• it must be simple to use without requiring extensive statistical
knowledge from a user,
• it should produce a clear and unambiguous MRL proposal for most
residue datasets produced by field trials, and
• it should harmonize the EU and NAFTA procedures as much as
possible.
The result was the OECD MRL Calculator which produces

MRL proposals that target the 95th percentile of the underly-
ing residue distribution. It estimates the MRL as the highest of
(1) mean + 4 × standard deviation, (2) 3 × mean and (3) the highest
residue in the data set of supervised residue trials. The estimated
value is then rounded to produce the MRL proposal. A compre-
hensive explanation for the procedure employed in the calculator is
provided in a white paper published by the OECD (OECD, 2010b)
with a user guide also available (OECD, 2010a).
The OECD MRL calculator is currently utilized by the JMPR
and a number of regulators including those in Australia, the EU and
NAFTA regions.
2.6 Conclusions on Residues and Future Directions

In addition to the already achieved harmonized approaches, there
will be ongoing activity in revising test guidelines and guidance doc-
uments to ensure that best practice in regulatory risk assessment
evolves with changes in technology.
Currently a new guidance document on residues in follow crops
is in preparation, which specifically looks at tiered approaches to risk
assessment, while providing the sponsor or applicant with opportu-

nities to generate additional data post-registration if the residues
profile of a persistent compound is not entirely defined at the time
of approval. This type of approach takes into account the need to
make available safer alternatives to specific older pesticides, while
managing the residue risks through limiting uses in the first instance.
OECD continues to work cooperatively by bringing together gov-
ernments, industry and food safety organizations to deliver outcomes
that are promoted widely and enhance consumer confidence in regu-
latory processes.
References1
FAO, Rome. Appendix IX.
FAO. 1986. Guidelines on pesticide residue trials to provide data for the registra-
tion of pesticides and the establishment of maximum residue limits.
FAO. 2013. Pesticide residues in food. Report 2012, Annex 6. FAO Plant Produc-
tion and Protection Paper 215: 491.
Greim H and Snyder R. 2008. Toxicology and Risk Assessment: A Comprehensive
Introduction. Wiley, Chichester, England.
Hayes AW and Kruger CL. 2014. Hayes’ Principles and Methods of Toxicology.
CRC Press, Boca Raton, FL.
Jacobson-Kram D and Keller KA. 2006. Toxicological Testing Handbook: Prin-
ciples, Applications and Data Interpretation, 2nd edn. CRC Press, Boca
Raton, FL.
MacLachlan DJ and Hamilton D. 2010. Estimation methods for maximum residue
limits for pesticides. Regulatory Toxicology and Pharmacology 58: 208–218.
OECD. 1981. Decision of the council concerning the mutual acceptance of data
in the assessment of chemicals.
OECD. 1994. OECD Series on pesticides no. 1, Data requirements for pesticide
registration in OECD member countries: survey results. OECD/GD (94)47
Environment Monographs No. 77.
OECD. 1998. OECD Principles on good laboratory practice.
OECD. 2001. OECD Series on testing and assessment no. 24. Guidance document
on acute oral toxicity testing.
1
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can be accessed at the websites of the corresponding organizations. Web pages
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and evaluation of chronic toxicity and carcinogenicity studies.
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disrupters.
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for neurotoxicity testing.
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submissions on plant protection products and their active substances (dossier
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Chapter 3
Principles of Safety Assessment

of Pesticides at National Levels
Paul Humphrey1 , Samuel Margerison1 , Trijntje van der Velde-Koerts2 ,
Michael A. Doherty3,∗ and Jessudoss Rowland 3,∗
Main topics
Data requirements and assessment strategies common to Australia,

EU and USA
Toxicity studies
Residue chemistry studies
Principles of Safety Assessment of Pesticides — Australia
Principles of Safety Assessment of Plant Protection Products —
European Union
Principles of Safety Assessment of Pesticides — United States of
America
3.1 Introduction
Prior to being authorized for use, pesticide products or plant
protection products must be evaluated and deemed safe by regulatory
Sections were prepared by:

1
: 3.3; 2 : 3.4; 3 : 3.5
∗
This article was prepared by the authors as part of their official duties for the
U.S. EPA. However, it has not been formally reviewed by the agency, and it does
not necessarily reflect the views of the U.S. EPA.
37
authorities. This chapter presents information regarding pesticide

product or plant protection product assessments as conducted by
Australia, the EU and the USA. Technical experts in toxicology
and residues from governments in Australia, the EU, the USA and
other countries working in the area of food safety contribute to
the Joint Meeting on Pesticide Residues (JMPR) as evaluators.
Australia, the EU, the USA and other countries have a number
of representatives who attend and contribute to the Codex Com-
mittee on Pesticide Residues (CCPR). Experts from Australia, the
EU, the USA and other countries also contribute to the development
of international guidelines through the Organisation for Economic
Co-operation and Development (OECD) and the FAO/WHO. Based
on these international activities, various guidelines and assessments
for authorization of pesticide or plant protection products have been
harmonized on a global scale. The first section of this chapter focuses
on data requirements and assessment strategies common to all three
authorities for toxicity studies and residue chemistry studies. The
next three sections within this chapter focus on topics specific to
Australia, the EU and the USA. Topics include regulatory history,
current legal frameworks, assessment strategies, trade issues and
maximum residue limit (MRL) setting and monitoring. Note that in
the USA, the maximum level of a pesticide residue allowed in a given
commodity is referred to as a ‘tolerance;’ a term which is equivalent
to a MRL in other jurisdictions. For simplicity, this chapter uses the
term ‘MRL’ throughout, regardless of whether or not the jurisdiction
is the USA. In the EU legislation, the acronym MRL is also used for
‘maximum residue level’.
3.2 Data Requirements and Assessment Strategies

Common to Australia, the EU and the USA
Regulatory authorities require data in order to evaluate the safety
of pesticide products or plant protection products for humans, ani-
mals and the environment. These data address physical and chemical
properties, efficacy, toxicity, residues in or on foods and environmen-
tal fate. This section focuses on data requirements and assessment
strategies that are common to all three authorities for toxicity studies
Principles of Safety Assessment of Pesticides at National Levels 39
and residue chemistry studies. Toxicity studies are used to assess the
hazard of a pesticide or plant protection product, while residue chem-
istry studies are used to derive legal standards for pesticide residues
in food (MRLs or tolerance levels) as well as to assess the dietary
exposure to the pesticide. Generally there is a focus, especially with
respect to hazards and residues, on the active ingredients within
products being evaluated for authorization.
The list of studies required to address toxicity and residue chem-
istry is very similar for Australia (APVMA Data Guidelines), the
EU and the USA, and that commonality extends to other evaluating
bodies as well (e.g. JMPR; see Chapter 4). The requirements related
to the design and conduct of the studies are also very similar across
jurisdictions. The work of the OECD (see Chapter 2) has formalized
the international harmonization of study requirements. Australia, the
EU member states and the USA, as regulatory authorities within
OECD member countries, are obliged to accept pesticide registration
submissions conducted to OECD guideline specifications. In addition
to the guidelines available through the OECD, guidelines have been
published by Australia, the EU and by the USA. While there is not
a one-to-one correspondence among the OECD, Australian, the EU
and the USA sets of guidelines, each set of guidelines, as a whole,
addresses a nearly equivalent set of data. Generally, studies are to
be conducted according to established standards for good laboratory
practices (GLP).
3.2.1 Toxicity Studies

Australia, the EU and the USA require studies to assess the toxic
characteristics of a pesticide. These studies cover different exposure
durations, focus on different, specific adverse outcomes (e.g. devel-
opmental effects, reproductive effects, neurotoxic effects and carcino-
genicity) and examine effects across species. The full set of studies
required by each authority is well represented by the set of OECD
toxicology guidelines discussed in Chapter 2.
The toxicology studies are used to derive endpoints, points
of departure and safety factors for use in risk assessment. The
toxicological evaluation of an application for registration of a pes-

ticide or plant protection product with uses in food-producing crops
identifies and quantifies the acute, short-term, intermediate-term and
long-term (chronic) hazards of the chemical. The lowest of the no-
observed-adverse-effect-levels (NOAELs) from studies of appropriate
durations are selected as the critical end points for determination of
toxicological reference values.
Safety factors (called uncertainty factors in the USA) are applied
to the chronic and acute NOAELs to generate the toxicological ref-
erence values, the acceptable daily intake (ADI) and acute reference
dose (ARfD) respectively.
ADI or ARfD (mg/kg bw)

chronic or acute NOAEL (mg/kg bw)
=
safety factor
A safety factor of 100 is usually applied. The default 100-fold

safety factor may be seen to represent the product of an inter-
species factor of 10 and an intra-species factor of 10 that allow for
(1) differences between the average responses in the experimental
animals used in the study identified to derive the point of depar-
ture (POD) and those in average humans and (2) the variability
in responses between average humans and those who are highly
sensitive (IPCS, 1987, 2009). Responses may differ between aver-
age humans and young, old, pregnant, ill and genetically susceptible
people.
The absorption, distribution, metabolism, excretion (ADME)
studies in laboratory animals provide information on the metabolism
of the active substance, which can be used in the interpretation of
the metabolism studies in ruminants and poultry. Data from toxi-
cology studies are also used to set label mandated re-entry intervals
and to provide insights into the toxicological mechanisms of action
in mammals, which can inform the residue definition decision with
respect to potential metabolites of toxicological concern.
3.2.2 Residue Chemistry Studies

Residue chemistry studies are necessary for all pesticides or plant
protection products that are used on food crops or where their use
may result in residues in or on foods. Studies include those designed
to (1) elucidate the metabolism of the active ingredient in crops
and livestock, (2) demonstrate that suitable analytical methods are
available to assess residues in food and feed commodities, (3) depict
the degree of stability of residues in storage, (4) assess the magnitude
of residues in foods and feeds resulting from the use of pesticide
products or plant protection products, (5) assess the magnitude of
residues in foods and feeds resulting from residues left behind in the
soil (rotational crop residues), (6) demonstrate changes in the nature
and magnitude of residues when foods are processed and (7) ascertain
the transfer of residues from feeds to livestock commodities. Within
each of these general areas, there are specific guidelines that delineate
how studies should be conducted, as well as criteria for when certain
studies may be triggered or waived. In addition to the studies that
are generally required, special studies may be requested in order to
better understand the behaviour and magnitude of residues in foods
as consumed.
Residue chemistry data are used to determine appropriate MRLs
in food and feed commodities, the residue definition to enforce those
limits, the residue definition for dietary risk assessment (in combi-
nation with the toxicological data as previously noted) and suitable
residue values for assessing dietary risk.
3.2.2.1 Residue definitions

Residue definitions address the analytes necessary to ensure compli-
ance with pesticide label use directions as well as to ensure pub-
lic safety. As a result, two definitions are considered. The first,
referred to as the tolerance expression (USA) or residue definition
for enforcement (EU and Australia), is for compliance with MRLs.
The second, referred to as the residues of concern (USA) or residue
definition for dietary risk assessment (EU and Australia), is used to
estimate dietary exposure. It is common for the residue definition
for compliance and the residue definition for dietary risk assessment
to be the same. Residue definitions may be specific for a given com-
modity or situation, with different definitions being fairly common
between target crop, rotational crop and livestock commodities.
Residue definitions are established principally by the metabolism
patterns observed in food-producing crops and animals, including
radiolabelled studies in rotational crops and radiolabelled processing
studies, the capabilities of analytical methods and by information
from toxicity studies. In addition, results from supervised field trials,
feeding studies and processing studies may be used to supplement
information from metabolism studies when establishing residue defi-
nitions, especially when metabolism studies show a complex residue
profile.
Particularly for compliance residue definitions, there is a strong
desire to minimize analytical complexity where possible and to select
a single residue that is suitable as a marker of use or misuse of
the pesticide product or plant protection product. Single-component
definitions, and the ability to analyse for a chemical as part of a multi-
residue screen are desirable for compliance residue definitions. A suit-
able marker is a compound that is unique to the active ingredient,
occurs at measurable levels in the commodities of interest and can
be assayed by standard analytical techniques, preferably the multi-
residue methods used by enforcement laboratories. Frequently, the
active ingredient is the residue definition, but a metabolite alone
or in combination with the parent compound is not uncommon. In
cases where the analytical technique converts multiple residues to a
common moiety, a more complex residue definition must be adopted.
For dietary risk assessment, all residues that are expected to con-
tribute significantly to both dietary exposure and hazard are included
in the residue definition. For hazard, this means that the metabolite
is expected to result in the same toxic effect as, and is of similar tox-
icity to, the parent compound. If a metabolite is expected or known
to cause a different toxic effect from that of the parent compound,
then a separate residue definition may be established, with its own
dietary risk assessment (e.g. the metabolites common to the triazole
class of fungicides). For dietary exposure, a residue is considered
for inclusion in the dietary risk assessment definition if it occurs

at greater than 10% of the total radioactive residue in metabolism
studies and at a level of at least 0.01 mg/kg, although those levels
might be significantly lower for a highly toxic compound.
3.2.2.2 MRL determinations

MRLs are generally derived from residue trials (supervised field tri-
als, limited or extensive rotational crop trials, processing studies,
and livestock feeding studies). In order to be considered suitable for
MRL determination, residue trials must reflect the conditions that
result in the highest residues in the analysed matrix. For target crops,
rotational crops and direct treatment to livestock, those conditions
are determined by the pesticide label.
All data from residue studies must be supported by adequate
analytical methods data and storage stability data. Analytical meth-
ods should be able to recover 70–120% of incurred residues, with a
relative standard deviation of not more than 20%.
For non-processed crop commodities, generally referred to as raw
agricultural commodities, MRLs are derived from supervised field
trials conducted according to the worst-case directions on the label,
from a residue perspective. Generally, this translates to the high-
est application rate, applications made at the shortest re-treatment
interval, and harvest occurring at the shortest allowed interval after
the last application (referred to as the pre-harvest interval). Evaluat-
ing authorities allow a 25% variance in the application rate and pre-
harvest interval relative to the proposed label, provided such variance
does not result in unreasonable differences in residue levels. Further-
more, measured residue levels may be adjusted using the principles
of proportionality (see Chapter 4) to account for application rates
that are outside of the 25% variance, provided all other application
parameters are according to label specifications.
The OECD MRL Calculator is used to derive MRLs from super-
vised field trial data (OECD, 2011). If residues are higher at an
interval longer than the minimum PHI on the label, then the higher
residues, coming from samples being harvested in a manner allowed
by the label, are used in the calculator. In the case of supervised
field trials that are not independent, the average (USA) or high-
est (Australia) value across the non-orthogonal trials is used. For
data sets that are completely left-censored (i.e. all values are below
the analytical method’s limit of quantification [LOQ]), an MRL is
typically established at the LOQ. When the residue definition for
enforcement includes multiple components, residues of each compo-
nent are summed for purposes of establishing the MRL. When the
residues of one or more of those components are below their LOQ,
residues are generally assumed to occur at the LOQ.
Frequently the use of a pesticide on agricultural crops results in
residues in livestock feedstuffs, leading to the potential for residues
to transfer into livestock commodities. When residues in feedstuffs
are likely to result in residues in milk, eggs, fat, muscle or edible
offal, Australia, the EU and the USA require that a feeding study be
conducted in order to be able to quantify the transfer of residues from
feedstuffs into livestock commodities. Such feeding studies should
include doses that bracket the expected dietary burden of residues
coming from feedstuffs. The estimated dietary burden is compared
to the results from the feeding study to determine the anticipated
level of residues in livestock tissues, milk and eggs. The resulting
anticipated residue is rounded up to obtain the appropriate MRL for
the animal commodity.
3.3 Principles of Safety Assessment of

Pesticides — Australia
3.3.1 Legal Framework
3.3.1.1 Regulatory history
Before March 1995, the Commonwealth of Australia held respon-
sibility for the evaluation and assessment of selected agricultural
and veterinary (AgVet) chemical products and their clearance for
registration. The states and territories were responsible for the reg-
istration of and control of use for all AgVet chemical products. At
first, the Commonwealth only had informal involvement in the clear-
ance process but from 1 July 1989, the arrangements were put on a
legislative basis.
In July 1991, the Commonwealth, states and territories agreed

to establish the National Registration Scheme for agricultural and
veterinary chemicals, in order to put all assessment and registration
of AgVet chemical products, which had previously been undertaken
independently by the Commonwealth and each of the states and
territories, under one body.
The Agricultural and Veterinary Chemicals (Administration) Act
of 1992 formally established the National Registration Authority for
Agricultural and Veterinary Chemicals (NRA), which subsequently
became the Australian Pesticides and Veterinary Medicines Author-
ity (APVMA) (Ag. Vet. Admin. Act 1992). The regulatory system is
a partnership between the Commonwealth and the states and terri-
tories under which the NRA (now the APVMA) was established as a
Commonwealth statutory authority, with responsibility for the eval-
uation, registration and review of agricultural and veterinary chem-
icals up to their point of sale. The states and territories have kept
responsibility for regulation of products after sale (generally referred
to as ‘control-of-use’).
The APVMA has the functions and powers conferred upon it
by the Administration Act and by the Agricultural and Veterinary
Chemicals Code (AgVet Code) of the participating territories. The
Agricultural and Veterinary Chemicals Code Act of 1994 (Ag. Vet.
Chem. Code Act 1994) contains the AgVet Code as a schedule (AgVet
Code), which in turn empowers the APVMA to evaluate, approve
and register and review active constituents and agricultural and vet-
erinary chemical products (and their associated labels) and to issue
permits and to license the manufacture of veterinary chemical prod-
ucts. It also contains provisions for controls to regulate the supply
of chemical products and provisions ensuring compliance with and
enforcement of the Code.
Among a suite of other legislation administered by the APVMA
are acts and regulations that relate to assessment and collection of
levies, compliance action when there is a suspected offence in relation
to the importation, manufacture or export of AgVet chemicals, and
prosecutions for offences against the AgVet Code or Agricultural and
Veterinary Chemicals Regulations (AgVet Regulations).
3.3.1.2 Current regulatory framework

Before an agricultural or veterinary chemical product can be legally
supplied, sold or used in Australia, it must be registered by the
APVMA. Agricultural products include herbicides, plant growth
regulators, insecticides, fungicides, biocides, vertebrate poisons and
some pest traps and barriers, while veterinary chemicals include vac-
cines, antibiotics, anthelmintics and ectoparasiticides and some vita-
mins and minerals. In addition, a variation to the ingredients of a
product or its use patterns must also be assessed and approved by
the APVMA.
As well as pesticides and veterinary medicines for agricultural
and commercial use, the APVMA registers chemicals that are used
in the household, such as insect sprays, personal insect repellents,
products for treating diseases in home garden plants, pool chemicals
and medicines for companion animals such as dogs, cats and horses.
When an application arrives at the APVMA, the data are exam-
ined by staff from the APVMA to provide advice on for example, the
manufacture, chemistry, toxicology, and the extent of residues that
may be present after the proposed use. External agencies such as the
Department of the Environment (ENV), Food Standards Australia
New Zealand (FSANZ) and external efficacy reviewers provide rele-
vant expert advice to the APVMA. The internal and external advice
will be the basis for the overall decision made by the APVMA.
Gazette notices and web-based communications provide informa-
tion to the public concerning decisions that are made by the APVMA.
The APVMA is a cost-recovered agency. Registrants pay appli-
cation fees to register new products and active constituents, amend a
current registration or apply for a permit. Fees are charged based on
the complexity of evaluation. Fees are also paid each year to renew
the registration of a product. Product registrants also pay an annual
levy, based on the sales of their registered products.
The APVMA, under the AgVet Code, is required to ensure that
any registered pesticide or veterinary chemical product will satisfy
legislatively defined criteria for safety, trade and efficacy.
The safety criteria (AgVet Code, Part 1, Division 1, Section 5A)
require that a registered product ‘will not be an undue hazard to
the safety of people handling the product or using anything con-

taining its residues, will not be likely to have a harmful effect on
human beings, and will not be likely to have an unintended harmful
effect on plants, animals. . . or to the environment’. The safety criteria
therefore cover evaluation of potential adverse effects of pesticides
or veterinary chemicals on the environment, workers handling the
product, bystanders and consumers.
The trade criteria (AgVet Code, Part 1, Division 1, Section 5C)
require that the approved use of a product ‘will not unduly prejudice
trade between Australia and other countries’. In practice, this means
that, for major exported commodities such as cereals, meat and dairy
products, measures are considered to determine whether any residues
arising in those commodities as a result of lawful pesticide use will
comply with the residues standards of importing countries.
The efficacy criteria (AgVet Code, Part 1, Division 1, Section 5B)
require that approved uses of a product will be effective against the
pest, disease or condition the product is intended to treat. A certain
amount of discretion is available to the APVMA regarding applica-
tion of the trade or efficacy criteria, particularly for minor uses or
crops.
It is the safety criteria that provide the ultimate legislative basis
for regulation of the safety of pesticide residues for consumers of
Australian produce.
3.3.1.3 Regulatory products

Approval of active constituents and registration of chemical prod-
ucts is the core process for the APVMA. Information concerning the
quality of the product, human and animal health and safety, efficacy,
environmental safety, likely residues from the proposed use and infor-
mation concerning trade are required for registration of the product.
Once a product is registered, it is approved for the purposes and uses
on the product’s label.
As well as registering products the APVMA issues permits for
uses that are not on a product label (off-label uses). The neces-
sity for a permit often arises for minor crops where registration is
not economically viable for chemical companies. Permits are issued
for research purposes, in emergency situations, or for the use of an

unregistered chemical. Although the data requirements and evalua-
tion processes required for a permit application to be successful are
similar to those of a product application, the requirements may be
less prescriptive.
After a product has been registered, new scientific information
may become available, which suggests that the product is unsafe for
human health, for animal or crop safety or for the environment, may
jeopardize trade or is ineffective. In these cases, chemical reviews
are carried out by the Chemical Review Section of the APVMA
(Chem. Rev.). Chemicals are reviewed according to the level of con-
cern based on advice from the Residues and Trade Section and the
Health Assessment Team of the APVMA, and the Department of
the Environment. The results of reviews may include modifications
to uses of a chemical (e.g. reduced application rates or restricted
application timing), removal from labels of certain uses of the chem-
ical, up to cancellation of all uses of the chemical.
3.3.2 Evaluation of Residues

3.3.2.1 Hazard assessment
The toxicological evaluation of an application for registration of a
pesticide or veterinary chemical with uses in food-producing crops
or animals identifies and quantifies the chronic and acute dietary
hazards of the chemical.
Evaluation of the toxicological data package leads to the selection
of critical end points for chronic and acute toxicity. The lowest of the
NOAELs for acute and chronic studies of animal or human toxicology
are selected as the critical end points for determination of health
standards.
Safety factors are applied to the chronic and acute NOAELs to
generate the health standards, the ADI and ARfD respectively.
ADI or ARfD (mg/kg bw)

Chronic or acute NOAEL (mg/kg bw)
=
Safety factor
In line with most regulators, a safety factor of 100 is usually

applied (see Section 3.2.1). In some cases where there is good tox-
icological information in humans, the inter-species factor of 10 to
account for differences between the average responses in the experi-
mental animals used in the study identified to derive the POD and
those in average humans is not required and an overall safety factor
of 10 is used, e.g. for chlorpyrifos, an ADI of 0.003 mg/kg bw has
been established based on a NOAEL of 0.03 mg/kg bw for plasma
cholinesterase inhibition at the next highest dose in a study with
human volunteers.
An additional factor, giving an overall safety factor greater than
100, may be used if there are concerns regarding the completeness of
the data base, or if the nature of the hazards identified by the tox-
icity testing indicates the need for additional caution. For example,
an additional 10-fold safety factor was incorporated in determining
the ADI for propargite on account of the narrow margin between
the NOAEL and the lowest-observed-adverse-effect-level (LOAEL)
in the critical 20-month rat study.
For the establishment of residue definitions for pesticide active
ingredients, the general procedures of JMPR are followed. Residue
definitions for plant and animal commodities, for both compliance
with MRLs and for dietary risk assessment are established based on
the metabolism patterns observed in food-producing crops and ani-
mals, the observed residues of the parent compound and metabolites
in supervised field residue trials, and with reference to the available
toxicity information for metabolites.
Particularly for compliance residue definitions, there is a strong
desire to minimize analytical complexity where possible. Single-
component definitions, and the ability to analyse for a chemical as
part of a multi-residue screen, are desirable. The APVMA is keen
to harmonize residue definitions with other countries where possible,
for example through the OECD global joint review programme. A
common residue definition among multiple countries means that com-
parison of MRLs in different countries is much easier, and removes
the need for specific analytical methods or reference standards for
individual countries. Similarly, residue definitions established by the
APVMA will where possible be harmonized with Codex residue def-

initions, however this is not always achievable.
3.3.2.2 Assessment of residues and dietary exposure

Plant metabolism studies in crops from each of the five representative
crop groups (leafy crops, root crops, fruits and fruiting vegetables,
pulses and oilseeds, and cereals) are required. If the metabolism in
three crop groups can be shown to be essentially the same, a waiver
may be granted for metabolism studies in the other groups. For
food-producing animals, metabolism studies are typically required
in a ruminant (usually lactating goats) and a poultry (usually lay-
ing hens) species. Studies in a monogastric food-producing mammal
(pigs) may be required if the metabolism in goats and hens differs
significantly from that in rats.
Supervised field residue trials may be conducted in Australia or
overseas, provided the climatic conditions, agricultural practices and
GAP used for the trials are judged to be applicable to the proposed
use pattern. Requirements for the recommended number of trials vary
depending on the significance of the crop in terms of consumption
and trade, ranging from two to four trials, perhaps with support
from residue data in related crops, for a minor crop such as lychees
or spring onions, to 12 trials for a major crop such as wheat.
Given the importance of pastoral and dairy farming in Australia,
both domestically and for export, control of residues of pesticides in
meat and dairy products is critical. As a result, residue data are
also required for animal feeds, including pasture, forage and fodder
fractions of crops grown primarily for human consumption (e.g. cereal
and pulse forages and hays), and by-products of processing raw agri-
cultural commodities (e.g. grape pomace from winemaking).
Use patterns and residue trials in some industries, particularly
wine grapes and cereal grains treated post-harvest, are often designed
by the chemical company with input from the APVMA and the
relevant grower industry group in order to meet specific domestic
and export market requirements, such as MRLs in key importing
countries.
For example, the Australian Wine Research Institute (AWRI)

will often have significant input into the use patterns and residues
trial design for wine grapes. A use pattern in wine grapes might only
allow application up to the end of flowering, even where use of the
same chemical in table grapes is permitted closer to harvest. This
could be intended both to prevent quantifiable pesticide residues in
the wine (which may be a requirement of an importing country), as
well as avoiding tainting of the wine, which can result from some
pesticide residues.
A similar collaborative approach is taken to use pattern and
residue trial design for chemicals applied to stored grains. The
National Working Party on Grain Protection (NWPGP) is a body
comprised of representatives of APVMA, the federal government
Department of Agriculture and Water Resources, and companies
and industry associations involved in grain handling, storage, trade
and export. It provides information, advice and leadership to the
grain industry regarding grain storage and hygiene, legal require-
ments regarding use of grain treatment chemicals, and domestic and
export market standards. For example, the NWPGP, with its detailed
knowledge of residue tolerances in export markets, provided invalu-
able input into the use of spinosad in stored cereal grains, with the
use being restricted to a single application in the supply chain to
ensure compliance with the Codex MRL.
Some reduction in the number of required trials is possible if a
group MRL is sought and data for similar crops in the group are
submitted. Extrapolation to groups or sub-groups is possible and
is a means to establish MRLs for minor crops for which it is not
likely that companies will carry out trials to support their use. As
examples, if data for oranges and lemons are available, a group MRL
can be established for citrus fruit, which covers use on minor citrus
crops, while data for almonds and pistachio can be extrapolated to
the whole tree nuts group.
For major crops, Australian trial data should be provided, which
will reflect the likely residues from application under Australian con-
ditions. These trials can be supported with trial data from overseas
studies. Australian trials should be carried out in various locations
where use is proposed, to reflect the diverse weather conditions across

the continent.
One notable fact relating to residue trial data presented to the
APVMA is the diverse range of crops grown in Australia due to
the wide variety of climatic zones. Crops range from tropical fruits
such as mangoes, custard apples and papaya grown in the Northern
Territory and far north Queensland, through to cool climate veg-
etable (e.g. brassicas) and fruit crops (e.g. raspberries) in Victoria
and Tasmania.
Reference to JMPR evaluated residue data may be made, particu-
larly to support temporary uses under time-limited permit approvals
in minor crops where no Australian residue data exists. Temporary
uses in novel crops such as chia have been supported based on JMPR
data in crops expected to show similar residue behaviour (e.g. canola
or mustard seed (with appropriately conservative temporary MRLs))
pending the generation of local residue data in chia.
Feeding studies (especially in cattle) are of particular importance
in Australia due to the diverse range of feed items grazed by livestock
(especially in times of drought) and the generally higher residue levels
in livestock feeds. Depuration trials, conducted as part of a feeding
study, are often used to estimate half-lives of pesticide residues in
the meat and offal of livestock, to allow for the establishment of an
export slaughter interval (ESI) to meet trade requirements (see below
for further discussion).
Ensuring that exposures to residues in food are below the safe
doses (ADI and ARfD) is an important part of the registration pro-
cess. After the toxicological assessment is carried out to determine
the relevant endpoints, maximum residue limit and dietary exposure
evaluations are performed.
For product and permit applications, the applicant must provide
details of the intended use pattern (amount of the chemical, num-
ber of applications and timing between applications and withholding
period from last application to harvest or to the grazing of a com-
modity by livestock and critical comments). Any critical comments
and restraints should also be supplied.
In general, the types of scientific data which are necessary for

a pesticide residues evaluation, the reasons these data are necessary
and how they are assessed are similar to the reasons described in
Chapter 4, “Evaluation of Pesticide Residues by FAO/WHO JMPR”.
Therefore, discussion here will be largely restricted to matters specific
to the Australian approach to pesticide residues evaluation.
3.3.3 Establishing MRLs

The OECD MRL calculator is used as a guide to the establishment
of MRLs (OECD MRL calculator). If it is decided that an MRL
different from the calculator value should be established, then the
reason for this is noted in the evaluation report. As for JMPR, MRLs
are based on residue data at the critical good agricultural practice
(GAP). Application rates should, in general, be set at the lowest rate
that provides acceptable efficacy (Part 8).
The MRLs established for a particular active constituent, will
sometimes be the same as Codex MRLs for the same active. How-
ever, this will not always be the case, as the Australian GAPs, which
the MRLs accommodate, often differ significantly from the critical
GAPs for which the Codex MRLs were established due, for example,
to different pests and diseases. Codex MRLs are not automatically
adopted by Australia; however, Australia does harmonize residue def-
initions and MRLs with Codex limits where possible. Applications
for individual MRLs to be harmonized with Codex (or other overseas
jurisdictions) can be made on a case by case basis (see the discussion
of tolerances for imported foods below).
The Agricultural and Veterinary Chemicals Code Instrument
No. 4 (MRL Standard) 2012 is a legislative instrument made
under subsection 32(1) of the Agricultural and Veterinary Chemicals
(Administration) Act 1992 (AgVet Admin Act 1992). The APVMA
MRL Standard can be found on the Australian Government ComLaw
website (APVMA MRLs). The APVMA MRL Standard is referenced
by various state laws and the MRLs are used to indicate whether use
of an agricultural or veterinary chemical has been according to the
approved use pattern. As well as listing MRLs for food commodities
(Table 1 in the MRL standard), and residue definitions for chemicals

(Table 3 in the MRL standard), the MRL Standard includes limits
for pesticides in animal feed commodities (Table 4 in the MRL stan-
dard). As described above, animal feed MRLs are established based
on supervised field trial and processing data. The state governments
have responsibility for monitoring standards for livestock feeds.
Food Standards Australia and New Zealand (FSANZ) is a bi-
national body which administers the Australia New Zealand Food
Standards Code, which incorporates a wide range of food safety stan-
dards relating to contaminants, additives and genetically modified
foods as well as pesticide and veterinary medicine residues. MRLs
for food commodities sold in Australia are listed in Schedule 20 of
the Food Standards Code. MRLs entered into the APVMA MRL
Standard, which relate to uses of pesticides or veterinary medicines
in Australian crops, are promulgated into Schedule 20 of the Food
Standards Code (FSANZ MRLs).
Schedule 20 of the Food Standards Code has been adopted by
various state laws so that the MRLs become the maximum concen-
trations of a residue, resulting from the registered use of a pesticide
or veterinary chemical, which are legally permitted in or on food.
Although most aspects of the Food Standards Code are applica-
ble in both Australia and New Zealand, Schedule 20 is an exception.
New Zealand has its own MRL system, reflecting the different agri-
cultural systems in Australia and New Zealand and the fact that New
Zealand has a separate system of pesticide and veterinary medicine
regulation from Australia. Australia and New Zealand operate a
mutual recognition agreement whereby food imported into Australia
from New Zealand, which complies with New Zealand MRLs, is
accepted as complying with Australian standards (and vice versa).
Schedule 20 also incorporates tolerances for pesticide residues
in imported foods, which are regulated directly by FSANZ. Food
importers can apply to FSANZ for establishment of a tolerance for
a chemical–commodity combination not covered by a limit in the
APVMA MRL Standard. These import tolerances will generally be
harmonized with Codex MRLs or MRLs established in the export-
ing country. FSANZ will assess the safety and justification for the
presence of the residues in the imported food, including conducting

dietary exposure assessments. Import tolerances can be established
for chemicals not registered in Australia, provided suitable ADIs and
ARfDs are available, for example via Codex. For example, tolerances
for dinotefuran in imported grapes and cranberries were established
based on U.S. MRLs prior to the registration of dinotefuran for use
(initially in cotton) in Australia. Import tolerances are commonly
established for commodities not grown in significant quantities in
Australia, such as cranberries and tea, or for commodities imported
outside the Australian growing season, such as cherries from Califor-
nia imported during the Australian winter.
3.3.4 Export Trade Considerations

Agricultural trade with other countries is very important for the
Australian economy. For example, approximately two-thirds of the
beef and veal produced in Australia is exported, at a value of over
A$6 billion in 2013/14. Over the five years from 2008/09 to 2012/13,
over 70% of the wheat produced in Australia was exported, at a
value of nearly A$7 billion in 2012/13. Residues in an export com-
modity exceeding the concentration permitted for that commodity in
the importing country, even if complying with Australian standards,
can seriously jeopardize Australian trade. Trade therefore plays an
important part in any considerations made by the Residues and
Trade Section of the APVMA. As discussed earlier, the APVMA is
obliged under the Agvet Code to be satisfied that use of the product
according to the registered use pattern would not unduly prejudice
trade or commerce between Australia and other countries.
Consultation via gazette notices and liaison with grower organi-
zations and other interested bodies takes place before registration of
an agricultural or veterinary chemical product where there is poten-
tial for an increased risk to trade in a major export commodity.
Relevant residue information is summarized in publicly available con-
sultation documents (Public Release Summaries for new active con-
stituents, and Trade Advice Notices for extensions of use of existing
chemicals) where there are trade implications.
The proposed use of a new product or the extension of a regis-

tered product on the following ‘Major export food and feed commod-
ity groups’ (cattle, cattle dairy products, pigs, sheep, goats, poultry
and eggs, cereal grains, citrus fruit, grapes (including dried grapes)
and wine, oilseeds, pome fruit, pulses, stone fruit, sugar and oaten
hay) requires the applicant to submit relevant trade information if
residues in trials are above the LOQ (Part 5B). Impacts on export
trade in commodities derived from the major animal species listed
above from both use of veterinary drugs on the animals, and graz-
ing of the animals on feed from crops treated with pesticides are
considered.
Export intervals (EIs) are an essential tool for the management
of undue prejudice to trade as they assist producers, growers, pro-
cessors and exporters in complying with the import standards of
trading partners when they are lower than Australian MRLs. EIs
may be found on a product label, in advisory information from the
appropriate industry association or in information material provided
by state government departments of agriculture.
EIs will normally be set so that residues in the exported commod-
ity are no greater than the lower of the Codex MRL or the lowest
MRL set by a major trading partner. When a tolerance has not
been established, the target value is the analytical LOQ (typically
0.01 mg/kg).
Scientific data should be submitted to show the depletion of
residues down to the lowest MRL of the major trading partners.
Four types of export interval can be considered:
• The ESI is the minimum time that should elapse after removal of
grazing livestock to clean pasture or feed and slaughter, where the
livestock have been grazing the crop or pasture before the expiry
of the export animal feed interval. For example, for sulfoxaflor, the
depuration phase of the cattle feeding study showed that an ESI
of 14 days was sufficient to ensure that residues of sulfoxaflor in
meat and offal of livestock given treated feed would decline below
the LOQ.
• The export harvest interval is the minimum time that should

elapse between the last application of a pesticide to a crop and
the harvesting of the commodity for export.
• The export animal feed interval is the minimum time that should
elapse between the last application of a pesticide to a crop or
pasture and grazing or harvesting of the crop or pasture as stock
food for animals intended to be slaughtered for export.
• The export grazing interval is the minimum period that should
elapse between the application of a chemical to crop or pasture
and the slaughter of an animal for export, where those animals
have continuously grazed the treated crop or pasture from the
time the chemical was applied.
The export grazing interval is particularly important in wide area

pest treatment such as aerial spraying of insecticides for plague locust
control, where removal of livestock from the treated area prior to
spraying may not be practical, as entire farm properties will generally
be treated in a single operation.
Several options may be available and necessary. For example,
when a treated commodity is fed to animals destined for export and
is itself exported as a commodity, it may be necessary to determine
both an export animal feed interval and an export harvest interval.
An important tool in the management of residues in meat is the
vendor declaration system, which is mandatory for sellers of livestock,
and strongly encouraged for sellers of stockfeeds, and provides infor-
mation to buyers on the residue status of the feedstuff or livestock.
3.3.5 Dietary Exposure Calculations

After the MRL evaluation is carried out, it is necessary to calcu-
late a dietary exposure estimate using the information obtained from
the toxicological and residue evaluations, to ensure that there is no
adverse effect to human health when the produce obtained from the
use pattern is consumed.
Short- and long-term dietary exposures to a chemical are esti-
mated by calculating the National Estimated Daily Intake (NEDI)
and the National Estimated Short Term Intake (NESTI). These

calculations are closely based on the JMPR IEDI and IESTI cal-
culations. The consumption data used are for sub-groups of the pop-
ulation and are provided by FSANZ. The data are based on food
consumption on an as-consumed basis and then calculated as g/kg
bw. It is necessary to include in the calculation all known uses of the
chemical, expected residue levels in the raw commodities and data
showing depletion of residues following washing, peeling, cooking or
other types of processing.
The APVMA ensures that MRLs are established at levels result-
ing in long-term (chronic) and short-term (acute) exposures below
the ADI and ARfD obtained from the toxicological evaluation.
FSANZ reviews the dietary exposure calculation and once it
is satisfied that the risk to public health and safety is accept-
able, FSANZ undertakes public consultation with consumers, pri-
mary producers, importers, state health departments and the World
Trade Organization prior to incorporation of the APVMA deter-
mined MRLs into Schedule 20 of the Food Standards Code.
In Australia, drinking water safety is regulated through the Aus-
tralian Drinking Water Guidelines (ADWG, 2011), a comprehensive
series of health, safety and quality guidelines. For pesticides in drink-
ing water, health-based guidance values are established as required
(e.g. where particular pesticides may be used in drinking water catch-
ments). These limits for pesticides in drinking water are set at 10% of
the ADI for the pesticide, based on a 70 kg adult consuming two litres
of water per day. As a result, when calculating the NEDI for a pesticide,
10% of the ADI is ‘reserved’ for contributions from drinking water.
3.3.6 Public Consultation

As discussed in Section 3.3.4, consultation documents are pub-
lished in relation to registration of products containing new active
constituents (Public Release Summaries), or where use of an existing
active constituent is being extended to include new uses in signifi-
cant export commodities (Trade Advice Notices). These documents
contain summaries of the evaluations of the residue data (as well as
other data such as that relating to environmental effects or toxicol-

ogy). The evaluation reports themselves are not published, however
copies (with confidential commercial information redacted) can be
provided on request.
Extensive consultation is conducted in conjunction with chemical
reviews, with evaluation reports being published via the APVMA
website.
3.3.7 MRL Enforcement and Monitoring

As discussed earlier, state governments in Australia are responsible
for control of the use of agricultural and veterinary chemicals, which
includes enforcement of MRLs. There are a number of residue testing
programmes for monitoring compliance with MRLs in Australia.
The National Residue Survey (NRS) is a federal government
programme operated by the Department of Agriculture and Water
Resources. The NRS has been running since the 1960s, primarily
as a means of facilitating market access for Australian agricultural
products to overseas markets through providing residue monitoring
data. It was initially established following concerns about pesticide
residues in exported meat.
The NRS conducts both random and targeted monitoring
programmes. Random programmes are designed to measure the
occurrence of residues of pesticides, veterinary chemicals and envi-
ronmental contaminants (including previously used organochlorine
pesticides and other persistent organic pollutants such as polychlo-
rinated biphenyls) in sheep, pig and cattle meat, wild caught and
farmed seafood, poultry and game meat (including kangaroo, emu
and wild boar), honey, grains (including cereals, oilseeds, and pulses)
and some horticultural commodities with significant exports such as
pome and citrus fruit. The matrices chosen for analysis in the ani-
mal residue programmes are those expected to contain the highest
residues for a particular class of chemicals (fat for pesticides, kidney
for antibiotics, and liver for heavy metals).
Targeted programmes are concerned with specific or potential
residue problems for sectors of the livestock industry. Examples
include programmes for management of persistent organochlorine
residues in beef from areas with contamination from historical pesti-

cide use, and analyses of beef samples collected at audit for hormone
growth promotants (HGPs) as part of the HGP-free accreditation
programmes supporting exports to the European Union.
The results of the analytical testing are made publicly available
annually in summary form, by commodity–analyte combination.
Violations of the APVMA or FSANZ MRLs, or in some cases,
detections at >0.5× the MRL, may lead to trace-back investigations
to avoid further problems. Where MRLs are not established for a
pesticide–commodity combination, residues of that chemical must
not be detectable in the commodity.
In cases of MRL violations being discovered, state and territory
government food regulators will be alerted (food enforcement author-
ities), who will organize a recall if required. Produce may be seized
and disposed of, more residue testing may be ordered, a property may
be quarantined and the sale of produce may be prevented, until the
produce is found to be safe for consumption and fit for sale in both
domestic and export markets. Legal action may be taken against the
producer if warranted.
The Australian Milk Residue Analysis (AMRA) Survey is an
annual monitoring programme for residues of pesticides, veterinary
chemicals and environmental contaminants in milk (AMRA). The
AMRA survey acts as a quality assurance programme for both the
Australian dairy industry, and countries importing Australian dairy
products. It is funded by the dairy industry and coordinated by the
Victorian state government dairy industry regulator. Around 1,000
raw milk samples are collected from around Australia each year using
both random sampling and stratified random sampling (for target-
ing of residues of particular chemicals only likely to occur in spe-
cific regions or at particular times of year). Analyses are conducted
by laboratories using methods accredited by the National Associa-
tion of Testing Authorities. Compliance with standards is excellent,
with most years finding 100% compliance with Australian MRLs.
As with the NRS, any non-compliances with the MRL standards
are referred to the agriculture department and food safety regu-
lator in the relevant state for trace back procedures or recalls as
required.
The monitoring of imported foods for compliance with Australian

standards is the responsibility of the federal Department of
Agriculture under the Imported Food Inspection Scheme (IFIS).
Consignments of imported food are inspected at a frequency deter-
mined by the risk associated with the particular food and the
compliance history of the importer. General pesticide residue screen-
ing tests are applied to consignments of imported meat, fruit or veg-
etables to determine compliance with the relevant limits in Schedule
20 of the Food Standards Code, while seafood is tested for the antimi-
crobials fluoroquinolones, nitrofurans and malachite green.
Testing is conducted by approved laboratories, with the analyses
arranged and paid for by the importer once a particular consignment
has been selected for inspection. Where a consignment is still under
embargo by the Department of Agriculture, if a pesticide residue is
found in violation of Australian standards, the consignment must be
either brought into compliance with the standard, downgraded (e.g.
used for animal feed), destroyed or re-exported, at the importer’s
expense. If a consignment has been released for sale, the state gov-
ernment food regulators will be alerted, and a recall of the food
conducted if necessary.
The Australian Total Diet Study is conducted approximately
once every two years by FSANZ. It is designed to measure the dietary
exposure of the Australian population to pesticide residues, rather
than as a compliance monitoring programme, so testing is conducted
on ‘table ready’ (i.e. cooked) foods rather than raw produce.
A number of state government departments of agriculture carry
out residue testing programmes for pesticides and other contami-
nants for produce grown or sold in their state, including the Vic-
torian Produce Monitoring Program (VPMP), and targeted residue
monitoring programmes conducted by Biosecurity Queensland.
Industry groups and marketing authorities also conduct targeted
residue programmes, while retail stores have quality assurance pro-
grammes, which may require residue testing.
3.3.8 International Activities

The APVMA has been involved in OECD harmonization activities
since 1998, and has participated in the OECD Global Joint Review
(GJR) programme for new pesticide active substances since 2006.

Over 20 new active constituents have been or are being evaluated as
part of the GJR programme. Sponsors of the pesticide simultaneously
submit a common data dossier to the countries participating in the
review. This programme enables the sharing of the evaluation work-
load, with individual countries leading the evaluation of different data
categories (chemistry, toxicology, residues and environmental safety)
within the review, while the other countries engage in peer review
evaluation. Harmonization of MRLs and residue definitions is more
easily achieved with multiple countries considering the residue data
dossier at the same time. The GJR programme facilitates exports
of agricultural produce, as residue standards can be established in
multiple countries more quickly.
Technical experts in toxicology and residues from APVMA and
other government authorities working in the area of food safety are
currently contributing to the JMPR as evaluators. As discussed ear-
lier, the APVMA makes use of JMPR residue evaluations, particu-
larly as part of its minor use programme. Australia has a number of
representatives who attend and contribute to the CCPR.
Experts from Australia also contribute to the development of
international guidelines through OECD and Food and Agricultural
Organization/World Health Organization (FAO/WHO).
3.4 Principles of Safety Assessment of Plant

Protection Products — European Union
3.4.1 Legal Framework
3.4.1.1 Regulatory history
Within Europe, plant protection product authorizations were con-
ducted under national legislation until 1993. Before 1993, many
European countries had their own legislation on food safety, many
already before World War II. For example, the Netherlands had its
first local food safety authority in 1893 for control of milk, cheese
and bread and the first Dutch food law was established in 1919.
Cooperation between European countries started in 1950 with

a treaty between six countries (Belgium, France, Germany, Italy,
Luxembourg and the Netherlands) on trade in coal and steel. The
European Economic Community (EEC) was established in 1957
by the Treaty of Rome to establish a common (economic) market
between these six countries. The EEC was gradually extended with
six more countries: Denmark, Great Britain and Ireland in 1973,
Greece in 1981 and Portugal and Spain in 1986. The European Union
was established in 1993 by the Treaty of Maastricht between these 12
member states. Cooperation on economic areas was extended with
cooperation on political, legal and security areas (EU, 2007). By 2015
the EU had expanded to 28 member states.
The establishment of the EEC and the EU also led to coopera-
tion between member states in the area of plant protection product
authorization. From 1993 to 2011, plant protection product autho-
rizations were conducted under Directive 91/414/EEC (EEC, 1991).
In 2011, this procedure was modernized and plant protection product
authorizations were conducted under Regulation (EC) No. 1107/2009
(EU, 2009a).
When pesticide use may lead to residues in food or feed, maxi-
mum residue levels (MRLs) are established. Before 1970, European
countries derived their own national MRLs for residues of pesti-
cides in food. Because this frequently led to trade problems between
countries, harmonization of MRLs in the EEC started in the 1970s.
The first EEC MRLs were set in 1976. These EEC MRLs were
established under Council Directives 76/895/EEC, 86/362/EEC,
86/363/EEC and 90/642/EEC. Directives require implementation
into national legislation within a certain time frame and EEC MRLs
replaced or were added to existing national MRLs (partial harmo-
nization). Full harmonization was achieved in 2005, when the Coun-
cil and Parliament of the EU adopted Residue Regulation (EC)
No. 396/2005, implementing that MRLs would be set only at EU level
from 2008 onwards (EU, 2005). Regulations are directly applicable
in all EU member states without implementation into the national
legislation.
Before 2002, the risk assessment and risk management in the

EEC and the EU were performed by the European Commission and
the member states. After 2002, risk management was separated from
risk assessment. The European Commission and member states take
risk management decisions on regulatory issues, including approval
of active substances and setting of MRLs for pesticide residues in
food and feed. The European Food Safety Authority (EFSA) was
set up in January 2002 as an independent source of scientific advice
and communication on risks associated with the food chain. EFSA
is governed by an independent management board, whose members
are mandated to act in the public interest, but do not represent
a government, organization or sector. EFSA’s scientific committee,
panels and units publish scientific opinions or advice to support the
European Commission, European Parliament and EU member states
in taking management decisions. EFSA’s remit covers food and feed
safety, nutrition, animal health and welfare, plant protection, plant
health and environmental safety. Since August 2002, EFSA has been
responsible for the EU peer review of active substances used in plant
protection products. Since 2005, EFSA is involved in giving advice
on setting legal limits for pesticide residues in food.
3.4.1.2 Regulatory framework and regulatory scope

Active substances and plant protection products are authorized for
use under Regulation (EC) No. 1107/2009 (EU, 2009a). The purpose
of this regulation is to ensure a high level of protection of both human
and animal health and the environment and at the same to safeguard
the competitiveness in agriculture by increasing the free movement
and availability of plant protection products within the EU member
states.
The regulation lays down harmonized rules for the approval of
active substances and their plant protection products applicable for
all 28 EU member states. Plant protection products are intended
to be used on live plants or raw agricultural plant commodities
to protect plants or raw agricultural commodities against harmful
organisms (e.g. insecticides, fungicides, nematicides); to influence
the life processes of plants (root hormones, plant growth regulators,
excluding nutrients) or to kill unwanted plants, destroy parts of

plants or inhibit or prevent undesired growth of plants (herbicides,
desiccants).
The scope of Regulation (EC) No. 1107/2009 does not cover prod-
ucts for control of harmful micro-organisms or pest control on any-
thing else than plants or raw agricultural commodities. Preservatives
or disinfectants for control of harmful micro-organisms in food, feed
or drinking water and pesticides for use in livestock premises or food
storage facilities or for use on livestock are not within the scope of this
regulation. For these types of products, other legislation is applica-
ble (e.g. Biocides Product Regulation (BPR) (EC) No. 528/2012, the
Medicinal Products Regulation (EC) No. 726/2004, the feed additive
Regulation (EC) No. 1831/2003 or the food additive Regulation (EC)
No. 1331/2008 and their amendments (EEC, 2003, 2004; EU, 2008,
2012)). These legislations and their evaluations are not discussed
here.
To reduce the risks and impacts of pesticide use on human health
and the environment, the EU has set rules for the sustainable use of
pesticides. Sustainable use is guaranteed by proper training of profes-
sional pesticide users, inspection of application equipment, integrated
pest management (low pesticide input management), information
gathering systems for acute poisoning incidents or chronic poison-
ing developments, minimization of pesticide usage in critical areas
(environmental or health reasons) and prohibition of aerial spraying
(EU, 2009b).
MRLs are established under Regulation (EC) No. 396/2005 (EU,
2005) for all raw agricultural commodities, including plant commodi-
ties, animal feed and animal commodities (meat, edible offal, milk,
eggs and honey). The MRLs apply to the raw agricultural commodi-
ties as defined in Annex I of the Regulation (generally the agricultural
product as traded, for example oranges with peel). The regulation
also contains a list of substances for which no MRLs are required
(Annex IV of the Regulation). For all pesticide–commodity combi-
nations for which no specific MRLs are established, a default MRL of
0.01 mg/kg applies. The MRLs for raw agricultural commodities are
also applicable to processed commodities thereof, taking into account
the change of residues during processing by using processing factors

(Annex VI of the Regulation).
To protect the very young, very low EU-MRLs are set for pro-
cessed foods for infants and young children in Directives 2006/125/
EC and 2006/141/EC and amendments thereof (EU, 2006a, 2006b).
3.4.2 Regulatory Processes

The EU established a two-step process for authorization of plant pro-
tection products. First, the active substance needs to be approved for
use in a plant protection product at EU level by the EU Commission
(COM). Second, every individual plant protection product, contain-
ing that active substance, needs to be authorized for use in a specific
EU member state (MS). A plant protection product can be used in
the EU only if it is scientifically proven that it does not have any
harmful effect on human or animal health or any unacceptable effect
on the environment and that it is effective against the claimed pests.
In the framework of the EU pesticide legislation, different regu-
latory processes can be distinguished:
• Approval of active substances at EU level under Regulation (EC)

No. 1107/2009 (see Section 3.4.2.1);
• Authorization of plant protection products (PPP) at EU member
state level under Regulation (EC) No. 1107/2009. The regula-
tion promotes the mutual recognition process: an authorization in
one EU member state can be recognized upon request in another
EU member state belonging to the zone with comparable agri-
cultural conditions. Four main types of applications can be dis-
tinguished: authorization for one or several member states in a
certain zone, mutual recognition to request authorization in other
member states within a certain zone, minor use authorization or
re-registration of a PPP (see Section 3.4.2.2).
• Setting or amending MRLs at EU level under Regulation (EC)
No. 396/2005. Authorization of a plant protection product can
only be granted, after the MRL that covers the requested use
has been established in Regulation (EC) No. 396/2005 (see Sec-
tion 3.4.4).
Besides these main categories, amendments in the legal conditions for

use, changes in packaging or labelling, changes in the formulation (i.e.
ingredients) of a plant protection product, changes in the production
process or production location of the active substance or plant pro-
tection product must also be assessed and approved at member state
level. Permits for research purposes need to be issued in the member
state where the experiment or trial will be conducted. In emergency
situations demanding quick and effective responses, Regulation (EC)
No. 1107/2009 provides a possibility for a member state to issue an
emergency permit for limited and controlled use for a period not
exceeding 120 days.
Active substances are approved for a maximum period of 10
years. After this period, an active substance renewal programme is
initiated (e.g. AIR-1, AIR-2 and AIR-3). All existing MRLs are peer
reviewed within 12 months after (re-)approval of the active substance
under Article 12 of Regulation (EC) No. 396/2005. This ensures that
approval of the active substance and the legal MRLs are in line with
the most recent scientific knowledge and MRLs reflect the uses autho-
rized in the EU member states as well as requested import tolerances.
Authorizations to place plant protection products on the market
are valid for 10 years and can be renewed. Should the occasion arise,
the member states can decide to withdraw or amend the respective
authorization at any time.
3.4.2.1 Approval of pesticide active substances and study

requirements
The active substance needs to be approved for use in a PPP by
the EU Commission. The procedure for approval consists of several
steps:
• The producer of an active substance, the so-called applicant, pre-

pares a dossier, containing the required test studies in compli-
ance with the data requirements established in Regulation (EC)
No. 283/2013 (EU, 2013a) and a summary report of all studies.
The data requirements are in line with OECD guidelines as dis-
cussed in Chapter 2. This further implies that test studies need to
be conducted according to the Principles of GLP. The applicant

generally consults the so-called rapporteur member state (RMS)
to avoid duplication of tests on vertebrates and to ask advice on
the design of the required studies.
• The applicant applies for EU approval, submitting the dossier to
the RMS of choice. This is the member state responsible for the
initial scientific and technical evaluation of an active substance
dossier.
• The RMS verifies whether the application is admissible by check-
ing the completeness of the submitted dossier against the data
requirements in Regulation (EC) No. 283/2013 (EU, 2013a).
• The RMS prepares a Draft Assessment Report (DAR) based on
the applicant’s dossier and proposed risk assessment. The DAR
contains assessments of physical chemical properties, analytical
methods, intended use, efficacy, human toxicology, residues in food
and feed, environmental fate and behaviour and eco-toxicology. In
addition, the DAR contains an evaluation of potential risks to
consumers of treated agricultural products (dietary risk assess-
ment); to operators applying the pesticide products (professional
users); to workers cultivating and harvesting the treated agricul-
tural products (non-professional users); to bystanders and resi-
dents during and after application of the pesticide product; and to
the environment (including bees, fish, birds and groundwater and
surface water for use as drinking water). Furthermore, the DAR
contains proposals for classification and labelling of the active sub-
stance and MRL proposals for the safe uses identified.
• The EFSA performs peer review of the work done by the RMS,
giving all member states the opportunity to comment on the DAR.
This step in the procedure is intended to ensure consistency in
evaluation. EFSA’s published peer review provides conclusions on
whether the active substance used in a plant protection product
meets the approval criteria as foreseen in the relevant legislative
framework.
• The decision on approval or non-approval of the active substances
is taken at community level by vote in the Standing Committee
on Plants, Animals, Food and Feed (SCoPAFF), a forum in which

risk managers of the EU member states are represented.
• Adoption by the EU Commission;
• Publication in the EU Official Journal in a regulation contain-
ing the conditions or restrictions for approval or reasons for non-
approval of the active substance.
The time between initial application and publication varies

greatly depending on how complex and complete the dossier is. Under
the EU rules of Regulation (EC) No. 1107/2009, it takes approxi-
mately 1.5 years from the date of admissibility to the publication of
a regulation approving the active substance.
Active substances cannot be approved for use in the EU if the
active substance is classified as mutagenic category 1A or 1B, as car-
cinogenic category 1A or 1B unless exposure of humans is negligible
or as repro-toxic category 1A or 1B, unless exposure of humans is
negligible or if the active substance is expected to have endocrine
disrupting properties that may cause adverse effects in humans,
unless exposure of humans is negligible. Negligible exposure means
that the product is used under conditions excluding contact with
humans and where residues in food or feed do not exceed the default
value as indicated in Regulation (EC) No. 396/2005. In addition,
an active substance cannot be approved for use in the EU if it is
considered a persistent organic pollutant (POP), a persistent bio-
accumulative and toxic substance (PBT) or a very persistent and
very bio-accumulative substance (vPvB).
Approval of active substances may be subject to certain con-
ditions or restrictions in relation to aspects of purity of the active
substance, the intended crop and category of users (non-professional,
professional). In exceptional cases, the EU Commission may approve
an active substance in combination with a request for confirma-
tory data, where the data requirements have been amended after
submission of the dossier or to increase confidence in the decision.
All approved substances are listed in Regulation (EC) No. 540/2011
(EU, 2011) and its amendments.
In accordance with Regulation (EC) No. 1107/2009, the

European Commission has to establish a list of substances that are
‘candidates for substitution’. Such a substance would be identified
where:
(a) its ADI, ARfD or AOEL is significantly lower than those of the
majority of the approved active substances within groups of sub-
stances and use categories,
(b) it meets two of the criteria to be considered as a PBT substance,
(c) there are reasons for concern linked to the nature of the crit-
ical effects (such as developmental neurotoxic or immunotoxic
effects),
(d) it contains a significant proportion of non-active isomers,
(e) it is to be classified as carcinogen category 1A or 1B,
(f) it is to be classified as toxic for reproduction category 1A or 1B
or
(g) it is considered to have endocrine disrupting properties that may
cause adverse effects in humans (EU, 2009a).
The European Commission has set up a database where all rele-

vant administrative and legislative information on active substances
can be retrieved, including its regulatory status (e.g. ‘approved’,
‘pending’ or ‘not approved’ for use in the EU) (COM, 2015).
3.4.2.2 Authorization of plant protection products

and study requirements
Plant protection products containing one or more of the (approved)
active substances can be formulated in many ways and used on a vari-
ety of plants and plant products under different agricultural, plant
health and environmental (including climatic) conditions. Granting
authorizations to place plant protection products on the market for
use or trade remains therefore within the remit of individual EU
member states.
Regulation (EC) No. 1107/2009 introduced a zonal application
and assessment for plant protection products to prevent duplica-
tion and to encourage cooperation and harmonization between EU
member states. For this purpose, the EU is divided into three

zones where agricultural, plant health and environmental (includ-
ing climatic) conditions are comparable. Zone A (North) consists
of Denmark, Estonia, Latvia, Lithuania, Finland and Sweden. Zone
B (Centre) consists of Belgium, Czech Republic, Germany, Ireland,
Luxembourg, Hungary, the Netherlands, Austria, Poland, Romania,
Slovenia and the United Kingdom. Zone C (South) consists of Bul-
garia, Greece, Spain, France, Croatia, Italy, Malta and Portugal.
The applicant prepares a dossier containing the required test
studies in compliance with the data requirements established in Reg-
ulation (EC) No. 284/2013 (EU, 2013b). The data requirements are
in line with OECD guidelines as discussed in Chapter 2. This fur-
ther implies that test studies need to be conducted according to the
Principles of GLP.
Applicants need to submit their zonal application simultaneously
in all member states for which an authorization is requested. The
zonal rapporteur (ZRMS) assesses the application in its entirety on
behalf of all concerned Member States (cMS) for which the applica-
tion is intended. The cMS in the zone can comment on the assessment
of the ZRMS and may have specific concerns related to a unique
situation within the borders of that specific cMS.
The ZRMS checks if the active substance(s) in the plant protec-
tion product are approved for use in the EU and checks the com-
pleteness of the submitted dossier against the data requirements in
Regulation (EC) No. 284/2013 (EU, 2013b).
The ZRMS prepares a draft Registration Report (dRR) and eval-
uates the effects and risks of the plant protection product (i.e. the
formulated product with all its ingredients), focusing especially on
the classification and labelling, efficacy, environmental effects, effects
on humans and physical chemical properties of the plant protection
product. Within a period of 12 months after receipt of the appli-
cation, the ZRMS determines whether the plant protection prod-
uct complies with the conditions of authorization as laid down in
Regulation (EC) No. 1107/2009 and whether authorization can be
granted for placing the plant protection product on the market in
the EU member state in question. If the ZRMS requires additional
information, it may extend the initial assessment period for a fur-

ther six months. If the missing information has not been submitted
by the end of that period, the application is considered inadmissible.
Once the authorization is granted, the conditions under which this
authorization is granted are published.
After the ZRMS report has been finalized, the cMS specific eval-
uation follows. A cMS can limit or prohibit the distribution of the
plant protection product within its territory if the product in ques-
tion poses a risk for the health of humans, animals or the envi-
ronment. Once the authorizations are granted, the conditions under
which these authorizations are granted are published.
If a plant protection product has already been authorized in one
EU member state, applicants can request authorization in other EU
member states by means of an application for mutual recognition.
The principle of mutual recognition introduced in Regulation (EC)
No. 1107/2009 enables the authorization holder to place the prod-
uct on the market in another member state, provided the regions
concerned have comparable agricultural, phytosanitary and ecologi-
cal conditions. An individual member state may have member-state-
specific concerns and can limit or prohibit the distribution of the plant
protection product within its territory if the product in question poses
a risk for the health of humans, animals or the environment.
In accordance with Regulation (EC) No. 1107/2009, the
European Commission has established a list of substances that are
‘candidates for substitution’. For plant protection products contain-
ing these active substances, EU member states are required to eval-
uate if they can be replaced (substituted) by other chemical, non-
chemical or natural alternatives.
Minor use crops are crops where plant protection products are
used on a small scale. For such minor uses, it is relatively expensive
to apply for authorization of a plant protection product. To pre-
vent such crops from being devoid in protection, Regulation (EC)
No. 1107/2009 allows that an authorization is extended with minor
uses that are not yet covered by the original authorization. Individual
EU member states are allowed to facilitate the authorization of prod-
ucts for minor uses.
3.4.3 Pre-registration Pesticide Dietary Risk

Assessment within EU
Pre-registration dietary risk assessments are conducted to show that
the use of a plant protection product does not have any harmful
effect on consumers of the treated agricultural products. Depending
on the intended use, pre-registration dietary risk assessments may
be conducted at the time of active substance approval and/or at the
time of plant protection product authorization or upon request of
import tolerances.
• In situations where the active substance is intended to be used on

edible or feed crops, at least one safe use needs to be identified
before the active substance can be authorized for use.
• In situations where the plant protection product is intended to
be used on edible or feed crops, or in case an import tolerance is
requested, it is necessary to consider whether the existing MRL
needs to be modified. If this is the case, an application to amend
the MRL has to be submitted to the European Commission, which
mandates EFSA to perform the dietary risk assessment. MRLs can
only be granted if there is no risk for consumers.
The pesticide residue levels in food must be as low as reasonably

achievable (ALARA principle) and must be safe for consumers. The
EFSA assesses the safety for consumers based on the maximum pes-
ticide residue levels expected in or on agricultural commodities and
their processed products, the toxicity of the pesticide residues and the
different diets of the Europeans. The safety of all consumer groups is
covered including pregnant and nursing women, the unborn, infants
and children, vegetarians and the elderly.
3.4.3.1 Hazard assessment

A more detailed description of the hazard assessment is presented in
Chapters 2 and 4. The toxicological reference values (i.e. an ADI and
an ARfD) are usually derived in the framework of the approval pro-
cess of active substances under Regulation (EC) No. 1107/2009. Tox-
icological reference values are derived from various toxicity studies
conducted on laboratory animals according to OECD guidelines.

Generally, the human toxicology dossier addresses the following
areas: Acute toxicity, short-term toxicity, genotoxicity, long-term tox-
icity, carcinogenicity, reproductive toxicity and neurotoxicity. Each
study is evaluated separately. For each study, if possible, the NOAEL
or benchmark dose (BMD) is derived. The NOAEL is the highest
dose at which the most relevant critical effect (the adverse health
effect that occurs first) is not yet observed. The BMD is the dose
associated with a specified response or level of effect. The NOAEL
or BMD of the most relevant chronic study with the most relevant
animal species is normally used for derivation of the ADI. If a sub-
stance has acute toxic properties, the (sub)acute NOAEL or BMD
is used for derivation of an ARfD. A safety factor of 100 is usually
applied for extrapolation of the overall NOAEL or BMD from labora-
tory animal studies to the relevant reference value (see Section 3.2.1).
Additional safety factors may be applied to account for the use of
a LOAEL instead of a NOAEL, for the short duration of the study,
deficiencies in the database or the nature of the effect and the dose–
response relationship. Subsequently, these reference values (ADI and
ARfD) form the basis of the dietary risk assessment. Because of the
precautionary principle, the EU does not set reference values for a
subset of the population (e.g. for women of childbearing age only).
For ethical reasons, the toxicity assessment of an active sub-
stance or a plant protection product should not be based on tests
or studies involving the deliberate administration of the active sub-
stance or plant protection product to humans with the purpose to
determine a human ‘no observed effect level’ of an active substance.
Similarly, toxicological studies carried out on humans are not used
to lower the safety factors of active substances or plant protection
products.
Regulation (EC) No. 1107/2009 promotes the development of
non-animal test methods in order to produce safety data relevant
to humans and the replacement, restriction or refinement of animal
studies in use. Animal testing needs to be minimized and tests on ver-
tebrates may only be undertaken as a last resort. Duplication of tests
and studies on vertebrates need to be avoided. For the development of
new plant protection products, there is an obligation to allow access

to study results on vertebrates on reasonable terms and to share the
costs of tests and studies on animals. In order to allow applicants to
know what vertebrate studies have been carried out by others, EU
member states keep a list of such studies.
Although the toxicological studies are performed according to
harmonized guidelines, the interpretation of the studies or the con-
clusions on the toxicological reference values may differ between EU
and Codex (CCPR and JMPR).
• Sometimes different toxicological reference values (ADI, ARfD)

are derived within the EU and Codex for the same active sub-
stance because of differing toxicological studies, differing safety
factors or because the severity of the effect is interpreted differ-
ently. Codex (CCPR and JMPR) may use safety factors lower
than 100, while the EU takes 100 as a minimum. The refusal to
use human studies is often the reason why toxicological reference
values (ADI, ARfD) within EU are sometimes lower than those
derived by Codex (CCPR and JMPR).
• The precautionary principle within the EU is the reason why tox-
icological reference values (ADI, ARfD) cannot be set for a sub-
population group. Codex (CCPR and JMPR) may derive an ARfD
for women of childbearing age only, while such an ARfD would be
valid for all adults and children within the EU.
• Sometimes different conclusions are derived on the toxicological
relevancy of metabolites formed in plant or animal commodities
within EU and Codex, resulting in differing residue definitions for
dietary risk assessment.
3.4.3.2 Pre-registration residue assessment

The dossier for approval of active substances or plant protection
products needs to be sufficiently detailed to be able to define any
residue of concern in food or feed. The initial dossier for approval of
the active substance may contain intended use on a limited number
of crops and consequently also the residue information is limited to
those crops. When the use of the active substance is extended later
to more crop types, additional residue studies need to be provided

in the framework of the MRL application to be able to define the
residue of concern in all crops listed in the intended use pattern.
Generally, the dossier needs to reliably predict (a) the residues in
food and feed, including succeeding crops and (b) the residues levels
in food and feed as a result of processing. Generally, the residue
dossier addresses the following areas: metabolism in treated crops,
metabolism in rotational crops, metabolism in livestock, nature of
residues in processed commodities, analytical methods supporting
the submitted studies, analytical methods for enforcement (plant and
animal commodities), stability of residues in (frozen) stored com-
modities, supervised field trials, magnitude of residues in processed
commodities, magnitude of residues in rotational crops, animal feed-
ing studies, product label or draft label detailing the intended use
on specified agricultural crops. Such residue studies are carried out
according to OECD guidelines as described in Chapter 2.
Applicants specify how the plant protection product should be
used in order to ensure efficacy with the minimum quantity of active
substance used. The good agricultural practice (GAP) defines the
dose rate, number of treatments, treatment interval, application
growth stage of the plant and pre-harvest interval for each crop
use. The submitted dossier includes experimental data to define the
residue of concern (for enforcement and dietary risk assessment) and
to estimate the level of the residues following the use of the product
in accordance with the defined GAP.
Residue levels are derived from supervised field trials. These trials
consist of small plots located in the major growing areas within the
EU, where the crop in question normally is cultivated. The cultivation
area within Europe is divided into two regions: (a) Northern and
Central Europe and (b) Southern Europe and the Mediterranean.
At least four independent trials are required for a minor use crop
and eight independent trials are required for a major use crop in
each of these regions. Northern and Central Europe includes Swe-
den, Norway, Iceland, Finland, Denmark, United Kingdom, Ireland,
northern France, Belgium, The Netherlands, Luxembourg, Germany,
Poland, Czech Republic, Slovakia, Austria, Hungary, Switzerland,
Estonia, Latvia, Lithuania, Romania and Slovenia. Southern Europe

and the Mediterranean includes Spain, Portugal, Southern France,
Italy, Greece, Malta, Croatia, Serbia, Bosnia and Herzegovina, For-
mer Yugoslav Republic of Macedonia (FYROM), Turkey, Bulgaria
and Cyprus. Data from other climatic zones (e.g. in the USA) may,
however, in individual cases provide supporting evidence for the eval-
uation of the residue situation in the member states of the EU.
The residue levels in or on food are determined by conducting tri-
als according to the label instructions on the plant protection prod-
uct that are thought to lead to the highest possible residues (critical
GAP). The crops are treated at the latest possible time interval before
harvest at the highest dose rate that is indicated on the label of the
plant protection product. The residues in the agricultural commodi-
ties are analysed by validated analytical methods according to the
agreed residue definition for enforcement and dietary risk assessment.
Although the residue studies are performed according to harmo-
nized guidelines, the estimated residue levels in food or feed may
differ between the EU and Codex (CCPR and JMPR).
• Sometimes different residue definitions are established between the

EU and Codex for the same active substance, resulting in a differ-
ent estimate of the residue levels in food or feed.
• Sometimes different estimates of residue levels in food or feed are
derived within the EU and Codex for the same active substance
because of differing use patterns and/or differing residue studies.
Because of differing critical GAPs for a certain crop (e.g. differing
dose rates or differing pre-harvest intervals) or use on differing
crops, the selected residue data may differ between the EU and
Codex. In addition, the Codex residue dataset is often larger,
because Codex receives residue trials from various countries all
over the world, while the EU generally requires only residue trials
from EU territory or from the importing country.
3.4.3.3 Pre-registration dietary risk assessment

Pre-registration dietary risk assessments are conducted to provide
risk managers with the information whether the intended use of a
plant protection product has a potential to pose a risk for consumers

via consumption of raw or processed commodities from treated crops
or animal commodities derived from livestock fed with treated crops.
Dietary exposure to pesticides is estimated by multiplying food con-
sumption (see below) with the residue present in the food as derived
from the residue assessment (see Section 3.4.3.2). The dietary risk
assessment then consists of comparing the dietary exposure with the
appropriate toxicological reference values as derived from the hazard
assessment (see Section 3.4.3.1).
The long- and short-term dietary risk assessment for adults and
children is performed by utilizing the deterministic pesticide residues
intake model (PRIMo), developed by EFSA. The specific dietary risk
assessment for children is considered necessary because children have
a less varied consumption pattern and a higher caloric consumption
pattern than adults.
The long-term dietary exposure is estimated through the inter-
national estimated daily intake (IEDI) calculations in the PRIMo
model. The residue level used in this model is the median residue from
the supervised field trials (STMR), based on the residue for dietary
risk assessment and based on the residue in the raw edible portion
(citrus fruits and bananas). In the first tier dietary risk assessment,
processing factors are not taken into account, since most diets do
not contain consumption data for processed commodities. However,
if required, more refined calculations have to be performed, which
may include additional information (e.g. processing factors in com-
bination with food consumption of processed products). The IEDI is
calculated for several diets as reported by several EU member states
for various population groups including children as well as from diets
collected by WHO for EU regions. These diets consist of yearly aver-
age consumption values for commodities listed in Regulation (EC)
No. 396/2005. The PRIMo model lists the diets in order of high-
est residue intake as well as the commodities contributing most to
residue exposure.
Single pesticides in drinking water may not exceed 0.1 µg/L
(EEC, 1998). Generally, this limit means that the theoretical median
daily pesticide intake from drinking water is so low (mostly less
than 1% of the ADI) that this is not separately included in the long-
term dietary exposure calculations.
The short-term dietary exposure is estimated through the inter-
national estimated short-term intake (IESTI) calculations in the
PRIMo model. The IESTI calculations are carried out separately for
each commodity as specified by the intended use. The residue level
used in the PRIMo model is the median or highest residue from the
supervised field trials (STMR or HR). The residue (HR or STMR)
is based on the residue for dietary risk assessment and based on the
residue in the raw edible portion (citrus fruits and bananas). Process-
ing factors can be taken into account only if consumption data are
available for the processed commodity. The IESTI is calculated per
commodity and the consumption level represents the highest large
portion for the commodity in question reported by EU member states
for various population groups including children. These consumption
levels usually represent the 97.5 consumption percentile of a certain
food commodity. The PRIMo model lists the commodities in order
of highest residue intake in combination with the originating EU
member state and population group.
Dietary risk characterization involves the process whereby the
estimated intake of residues through all food and water that may
be treated with that pesticide according to the intended use is com-
pared with the toxicological reference values for the pesticide residues.
Chronic toxicity is measured with the ADI and acute toxicity is mea-
sured with the ARfD for all European consumer groups. The estimated
intake of residues may not exceed the value of the ADI for long-term
dietary exposure and ARfD for short-term dietary exposure.
The assessments of EFSA on MRL applications (reasoned opin-
ions) describe the comprehensive scientific evaluation of, and sub-
sequent conclusions from, the consumer exposure assessment and
the risk assessment of pesticide residues resulting from the use of
pesticides.
3.4.4 MRL Setting and Import Tolerances

A MRL is the legally permitted residue concentration in food and
feed. Before a MRL is set in the EU legislation, the dietary risk
assessment has to demonstrate that these residues do not pose an

unacceptable risk to consumers. The MRL represents the highest
residue that is expected if a crop is treated with the pesticide accord-
ing to the label instructions on the plant protection product (GAP).
MRLs provide a mechanism to verify that a produce has only been
treated with pesticides according to authorized agricultural practices,
both for produce treated within the EU and for imported produce.
Any party such as pesticide manufacturers, importers of food or
any other party demonstrating a legitimate interest in health or food
production can request the setting or amendment of an MRL. This
may be for a new use or for a change to the agricultural practice for
an existing use where residues lead to a higher EU MRL.
In order to set an MRL, applicants specify how they intend to use
the plant protection product and they submit residue data as indi-
cated in Section 3.4.3.2. The submitted dossier includes experimental
data to define the marker residue for enforcement and to estimate
the level of the residues in edible or feed crops. The residues in the
raw agricultural commodities are analysed by validated analytical
methods according to the agreed residue definition for enforcement.
The MRL for the raw agricultural commodity is calculated from the
results of all trials in a harmonized way by using the OECD MRL
calculator (OECD, 2011).
Although the same supervised residue trials are used for dietary
risk assessment and for MRL setting, the resulting residues may be
different. In the case of MRL setting, only the marker residue is
analysed in the raw agricultural commodity, while in the case of
dietary risk assessment the marker residue and any toxicologically
relevant metabolites are analysed in the edible portion of the raw
agricultural commodity or a processed commodity.
Equally, if the interested party wishes to import produce into
the EU with residues that may be higher than the EU MRL, an
application to amend the EU MRL needs to be made (import tol-
erance request). In support of such an import tolerance request, the
applicant has to provide the same type of information as for a regu-
lar MRL setting procedure: Information on the GAP in the export-
ing country, experimental data (trials) and any other information
or studies needed to assess consumer safety, including analytical

methods for enforcement. The import tolerance request can only be
granted if a dietary risk assessment has demonstrated that these
residues do not pose an unacceptable risk to consumers. Import tol-
erances may be requested for pesticides authorized in the EU, but
for which different agricultural practices apply to imported commodi-
ties. Alternatively, import tolerances may be requested for pesticides
not authorized for use in the EU, but which nevertheless can be
legitimately used to treat commodities that might be imported into
the EU. Import tolerance applications can be submitted as soon as
all supporting information is available, although there should be an
existing or intended authorization in the exporting country.
In general, an MRL application needs to be submitted to an
EU member state. In many cases, the MRL application would be
submitted and processed at the same time as the relevant application
for authorization of the plant protection product.
The national authorities in the individual EU member states
define how and when the pesticide may be used. That information
is on the label of the plant protection product (i.e. legal instructions
for use). Authorizations are granted on a national basis because local
and environmental conditions and the occurrence of pests may differ
and therefore use of pesticides may differ. The EU member state
evaluates the request and forwards an evaluation report to EFSA.
EFSA provides an opinion on the risk to consumers and the avail-
ability of a suitable monitoring method. As part of the risk assess-
ment, EFSA verifies that the pesticide use (corresponding to the
MRL in question) is safe for consumers by performing a dietary risk
assessment.
On the basis of the EFSA recommendation, the European Com-
mission has to prepare a regulation on the modification of the MRL,
which is presented for an opinion to the SCoPAFF. EU member
states will then decide on the acceptability of the proposed MRL
by qualified majority voting, taking account of EFSA’s opinion and
other relevant factors. The decision of the EU member states is sub-
ject to scrutiny by the European Parliament to ensure compliance
with procedures.
The regulations with the new or modified MRL are published in

the Official Journal as amendment to Regulation (EC) No. 396/2005
(EU, 2005). In 2015, Regulation (EC) No. 396/2005 covered around
1,100 pesticides used in agriculture in or outside the EU. The reg-
ulation also contains a list of substances for which no MRLs are
required. For all pesticide–commodity combinations for which no spe-
cific MRLs are established, a default MRL of 0.01 mg/kg applies. In
case the analytical method is not able to analyse residues as low as
0.01 mg/kg, the default MRL may be set at a higher default level
(e.g. 0.02 or 0.05 mg/kg). In case the default MRL of 0.01 mg/kg
is not safe, a lower default MRL of 0.001 mg/kg may be set (e.g.
fipronil). The MRLs listed in the regulation are now also available
electronically in the European Pesticide database (COM, 2015).
To protect the very young, very low EU-MRLs are set for pro-
cessed foods for infants and young children in Directives 2006/125/
EC and 2006/141/EC and amendments thereof (EU, 2006a,
2006b).
The Codex Alimentarius Commission (CAC) is an international
body that aims to protect the health of consumers and ensure fair
practices in international food trade. The CAC takes decisions on the
proposals derived by JMPR (FAO/WHO) that were agreed in the
CCPR and sets pesticide MRLs once a year. Codex MRLs are non-
statutory levels for the EU. EU MRLs are aligned with the Codex
MRLs (CXLs) and they are adopted in an amendment to Regulation
(EC) No. 396/2005, unless the EU delegation raised a concern or
expressed a reservation during the CCPR. These reservations are
noted in the CCPR report.
3.4.5 MRL Enforcement and Monitoring

Food business operators need to ensure that food and feed complies
with the legal requirements at all stages of production and distribu-
tion. For this purpose, samples are analysed by contract laboratories
to show that the residue levels comply with the legal limits.
National authorities have the obligation to control and enforce
MRLs by taking samples and checking pesticide residues in food
and feed under Regulation (EC) No. 396/2005. Member states also
have the obligation to monitor products from third countries, which

are subject to increased import controls under Regulation (EC)
No. 669/2009 (EU, 2009c).
Member states have set up two types of monitoring programs: (a)
the EU coordinated multi-annual control programme, which defines
the pesticide–commodity combinations that need to be monitored by
random sampling by all member states (e.g. EFSA, 2015a) and (b)
national control programmes, which are mainly risk based and focus
on products that have a higher probability to exceed the legal limits,
taking into account results of previous inspections. Member states
are free to decide on the design of the national control programs:
e.g. number of samples to be taken, type of commodities to be sam-
pled or pesticides to be analysed. Samples for targeted (enforcement)
and random (surveillance) monitoring are taken at different stages
of the food production chain (food stores, food distribution centres,
auctions, transhipments). Around 80,000 samples are analysed every
year in the EU under the two programmes. In 2013, about 11,000 of
the 81,000 samples were processed products.
The EU Commission ensures that the controls are done uni-
formly within the EU member states by ensuring harmonized sam-
pling (EEC, 2002), providing guidance documents for quality control
of analytical methods used for official control, regularly organizing
compulsory proficiency tests for official control laboratories and per-
forming inspections.
When a marketed or imported commodity exceeds the EU MRL
as listed in amendments to Regulation (EC) No. 396/2005, the sam-
pled lot has to be withdrawn from the market. When the pesticide is
not listed in Regulation (EC) No. 396/2005, a default MRL of 0.01
mg/kg applies. Generally, the deterministic EFSA PRIMo model or
a national dietary risk assessment model is used to assess the dietary
risk for a sampled lot exceeding the MRL. Member states have an
obligation to notify the EU Commission of any measures they adopt
that require rapid action and that force the withdrawal from the mar-
ket, or the recall, of food or feed to protect human health. Regulation
(EC) No. 178/2002 outlines a procedure for a Rapid Alert System
for Food and Feed (RASFF). Its purpose is effective notification of
national authorities on a direct or indirect risk to human health deriv-

ing from food or feed. The RASFF warning alerts other EU member
states when pesticide residues are at a worrying level for consumers
so that they can take appropriate action.
3.4.6 Future Developments

An EFSA workshop, co-sponsored by WHO/FAO, was held in 2015
to discuss possible alternatives for the formulas and input parameters
for the IESTI calculations (EFSA, 2015b). This workshop recom-
mended removing the unit weight from the IESTI equations since
unit weights vary significantly between countries and within com-
modities and no single unit weight can be defined. Furthermore, the
workshop recommended using the legal limit (MRL) as the residue
input, rather than the STMR or HR. It was further recommended
combining this residue, where relevant, with a variability factor of
three instead of a variability factor of five or seven to account for
differences in individual units as compared to the composite samples
that are normally measured by enforcement laboratories.
Within the EU, cumulative dietary risk assessment for pesti-
cides and other chemicals is being developed. Grouping of pesti-
cides is based on identification of compounds that exhibit common
adverse outcomes on the same target organ or system. This grouping
methodology is based on the commonality of the effect rather than
on the commonality of the mode of action. The combination of the
effects is based on dose addition, that is, dose addition is also used
for the assessment of mixtures of pesticides with dissimilar modes
of action, provided they produce a common adverse outcome. As a
first step, this methodology was applied to define groups of pesticides
(CAGs), which have an effect on the thyroid and central nervous sys-
tems. Further work includes establishment of CAGs for other organs
and systems (eye, liver, adrenals and on the reproduction and devel-
opment systems) (EFSA 2008, 2009, 2012, 2013a, 2013c).
Harmonized terminology and frameworks for the human risk
assessment of combined exposure to multiple chemicals (‘chemical
mixtures’) are being developed (EFSA 2013b). The Aggregate
and Cumulative Risk of Pesticides: an On-line Integrated Strategy
(ACROPOLIS) project ran during 2010–2014 to improve risk

assessment strategies in Europe. As an output of the project, an inte-
grated on-line software tool (Monte Carlo Risk Assessment, MCRA 8)
was developed to model dietary exposure (ACROPOLIS, 2015). The
probabilistic MCRA 8 model uses a distribution of consumption and
residue concentrations to obtain a distribution of actual dietary expo-
sures for long-term or short-term dietary risk assessment. The model
relies on residue data obtained from random monitoring, total diet
studies or duplicate diet studies. MCRA 8 is further improved for esti-
mation of combined actual exposures to 100 chemicals at a time and
is intended to be used for the dietary cumulative risk assessments that
will be performed by EFSA. The European Commission is investigating
how the results from such analyses can be implemented in EU policy.
The number of different combinations of chemicals that we can get
exposed to in a single day is infinite and an efficient test strategy for
mixtures of chemicals is lacking. The EuroMix project was initiated in
2015 to develop a tiered test strategy for risk assessment of mixtures of
multiple chemicals derived from multiple sources across different life
stages based on existing and new toxicological tests. The contribution
of these chemicals to the combined risk depends on the toxic profile
of the chemical(s) together with the level to which the consumer is
exposed to these chemicals. The main issue in this project is how to
prioritize the thousands of chemicals to be tested, how to test new
chemicals for their synergistic effects and how to group chemicals in a
proper way for appropriate risk assessment allowing refinement wher-
ever needed. The Euromix project will deliver a test strategy and test
instruments that can be used to refine future (aggregate and cumu-
lative) risk assessments of mixtures of chemicals. Ultimately this will
provide information for future risk management decisions on the safety
of chemicals in mixtures (EUROMIX, 2015).

United States of America
3.5.1 Regulatory Background
Regulation of pesticides at the national level in the United States
began with the Federal Insecticide Act of 1910. This law was
enacted in order to protect purchasers and users of insecticides from

fraudulent or non-efficacious products, and it gave the U.S. Depart-
ment of Agriculture (USDA) the authority to establish standards
governing the manufacture of insecticides and fungicides. Concerns
about deleterious health and environmental effects from the use
of pesticides resulted in legislation aimed at protecting the public
as well as non-target plants and animals from pesticide exposures.
In 1947, the Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA) was passed, broadening the labelling requirements for pes-
ticide products and requiring that all pesticide products be registered
prior to interstate or international commerce. In addition, the Fed-
eral Food, Drug, and Cosmetic Act (FFDCA), initially established
in 1938, was amended to require the Food and Drug Administration
(FDA) to set MRLs (referred to as ‘tolerances’ in the U.S. system)
for all pesticide residues in raw agricultural commodities (Section
408, 1954) and in processed foods (Section 409, 1958).
With the establishment of the Environmental Protection Agency
(EPA) in 1970, FIFRA administration was transferred from USDA
to EPA. FIFRA underwent a major revision, including the require-
ment that a pesticide could be registered only if it would not cause
‘. . . unreasonable adverse effects on the environment’. That require-
ment was defined to mean ‘(1) any unreasonable risk to man or the
environment, taking into account the economic, social, and environ-
mental costs and benefits of the use of any pesticide, or (2) a human
dietary risk from residues that result from a use of a pesticide in
or on any food inconsistent with the standard under Section 408 of
the Federal Food, Drug, and Cosmetic Act’. This language clearly
signalled a shift away from product quality to a focus on protec-
tion of the general public and the environment. More recently, the
FFDCA was amended in 1996 with the Food Quality Protection
Act, which among other things specifies that the EPA must ensure
(1) that ‘. . . there is a reasonable certainty that no harm will result
from aggregate exposure to the pesticide chemical residue, including
all anticipated dietary exposures and all other exposures for which
there is reliable information’ and (2) that ‘there is a reasonable cer-
tainty that no harm will result to infants and children from aggregate
exposure to the pesticide chemical residue’. In considering assessment

of pesticides in the USA, it is important to recognize that FIFRA
is a risk–benefit-based statute, which applies to environmental and
human health, whereas FFDCA is a risk-based statue, which applies
only to human health assessments.
Today, responsibility for pesticide safety is shared by EPA, which
registers pesticide products and establishes MRLs in foods and feeds;
FDA, which enforces MRLs in plant commodities; USDA, which
enforces MRLs in meat and poultry commodities; the Occupational
Safety and Health Administration (OSHA), which ensures the safety
of those working with pesticides, including farm workers; and the
Fish and Wildlife Service and the National Oceanic and Atmospheric
Administration, which oversee administration of the Endangered
Species Act.
FIFRA grants the EPA broad authorization to establish or mod-
ify the data deemed necessary for pesticide registration as well as
periodic re-registration. A set of baseline data requirements has been
established based on the proposed uses of a pesticide as well as on the
specific behaviour of that pesticide. In setting data requirements, it
is EPA’s goal ‘. . . to ensure there is sufficient information to reliably
support registration decisions that are protective of human health
and the environment, while avoiding the generation and evaluation of
data that do not materially influence the scientific certainty of a reg-
ulatory decision.’ (EPA Website: Pesticide registration data require-
ments, 3 October 2015). Towards that end, specific studies may be
waived or alternate testing approaches may be used when supported
by sound, defensible scientific rationale. Conversely, additional data
may be required when warranted. The EPA requires that studies be
conducted according to GLP standards. As a member of the OECD,
the U.S. will accept studies conducted according to OECD guidelines,
as well as studies conducted according to the guidelines published by
the EPA. The guideline series 860 (residue chemistry), 870 (health
effects) and 875 (occupational and residential exposure) are the prin-
cipal guidelines for evaluating human health risks from exposure to
pesticides. Relative to a national registration, some residue chemistry
data requirements may be less for special-local-needs registrations or
emergency registrations. In addition to federal registration, many

states require their own registration.
3.5.2 Registration Process
Pesticide registration in the U.S. may take the form of a national
registration (under Section 3 of FIFRA), a state-specific registra-
tion (Section 24(c) of FIFRA), an emergency exemption (Section 18
of FIFRA), or an experimental-use permit (Section 5 of FIFRA).
The Office of Pesticide Programs currently works under a fee-for-
service paradigm, within which applicants pay a service fee which
helps to offset the costs of the registration process. In return, the
applicant is guaranteed a decision date for the application. A regis-
tration application generally includes the service fee, various forms
detailing the request, a statement of the ingredients of the formulated
pesticide product, information about the manufacturing process and
its reliability, proposed labelling for the product and data concerning
potential human health and environmental risks, including studies
regarding residues in foods and feeds. With the exception of an emer-
gency exemption, the public is informed of the registration request
through a Notice of Filing.
After a screening process to ensure that an application pack-
age is complete, the application enters the full review process, with
the allowed amount of time for review depending on the scope of
the application. The review process includes assessment of risks to
human health and the environment, with the former being done
within the Health Effects Division and the latter within the Envi-
ronmental Fate and Effects Division. In addition, the content of the
formulated product is evaluated to ensure that all ingredients, aside
from the active ingredient(s) (i.e. binders, carriers, adjuvants, etc.),
are cleared for use.
Upon completion of the risk assessments, a determination is made
by the risk management groups within the Office of Pesticide Pro-
grams as to whether or not to grant the requested registration and,
in the case of uses where residues may occur on foods or feeds, to
establish MRLs. For new active ingredients or registered active ingre-
dients where there is a substantial change to the components in the
human health risk assessment (e.g. first residential use, first food use,
etc.), the proposed registration decision undergoes a public comment

period. During this time, the public has access to the risk assessment
documents as well as the decision documents (but not to the sub-
mitted studies themselves). A final decision regarding registration is
made after taking into account comments received during the public
comment period.
In addition to the registration process, the U.S. EPA is respon-
sible for periodically reviewing pesticide products to ensure that the
available data and risk assessments used to support registrations are
up to date with current data standards, safety standards and legal
requirements. This re-evaluation (or reregistration) occurs on a time
frame of not greater than 15 years from the date of initial registra-
tion for each pesticide active ingredient. Risk assessments conducted
under the re-evaluation programme are generally available for public
review and comment prior to the EPA making a final registration
review decision.
The USA publishes its MRLs in Title 40 Part 180 of the Code
of Federal Regulations (CFR). Generally, each active ingredient
receives its own paragraph, with sub-parts set aside for (a) gen-
eral MRLs (associated most typically with target commodities),
(b) MRLs for emergency exemptions from a registration, (c) MRLs
with regional registrations, (d) MRLs associated with indirect or
inadvertent residues (e.g. residues in rotational crops, residues from
mosquito adulticide treatments, residues from irrigation with treated
water, etc.), (e) revoked MRLs subject to channel-of-trade provisions
(MRLs that remain in effect until a cancelled product clears channels
of trade) and (f) import MRLs (although these frequently occur in
sub-part (a) with a footnote). With rare exception, a given commod-
ity only appears in one sub-part, since monitoring and enforcement
laboratories would not necessarily know the source of the residue
(e.g. direct treatment vs. rotated crop).
3.5.3 Hazard Assessment

3.5.3.1 Hazard characterization and toxicity profile
Hazard identification is the process of identifying the potential
adverse effects that could occur as a result of various types of
exposure to a particular pesticide. The entire toxicology database

submitted to EPA for a particular pesticide (in support of regis-
tration or re-registration) is considered in the hazard assessments.
EPA’s toxicology data requirements are described in Part 158,
Title 40 of the Code of Federal Regulations (40 CFR Part 158).
For chemicals whose use is likely to result in residues in foods
or feeds, this database typically consists of the following studies:
acute battery (consisting of oral, dermal, and inhalation lethality
studies, dermal and eye irritation studies and a skin sensitization
study, often referred to as the ‘acute 6-pack’); sub-chronic (90-
day) feeding studies in rodents and non-rodents; chronic feeding
studies in rodents and non-rodents; carcinogenicity studies in two
rodent species; prenatal developmental toxicity studies in rodents
and non-rodents; a two-generation reproduction study in rodents;
and acute and sub-chronic neurotoxicity studies in rodents. Other
‘conditionally required’ studies may also be available such as der-
mal penetration, 21-day dermal toxicity, sub-chronic dermal toxic-
ity, sub-chronic inhalation toxicity, acute and sub-chronic delayed
neurotoxicity in hens and a developmental neurotoxicity study in
rodents. Typically, information on relevant metabolites, degrada-
tion products and related compounds is submitted to EPA along
with the studies required to support the pesticide registration or
re-registration. Toxicology data submitted for metabolites or degra-
dates of concern should initially be considered along with the parent
compound.
In addition to the available toxicology studies submitted to EPA,
the available scientific literature for the parent compound, relevant
metabolites and related compounds is also considered during this
process. Open literature articles are evaluated in accordance with
prescribed standards (EPA, 2012).
If a pesticide belongs to a neurotoxic chemical class or has shown
neurotoxic potential, a battery of neurotoxicity screening studies
is required to be submitted to EPA and may include acute, sub-
chronic and developmental neurotoxicity studies in rodents. In the
case of organophosphate chemicals, which cause acetyl cholinesterase
depression (including metabolites or degradation products thereof),
an acute delayed neurotoxicity study in the hen is required. If the

results of this study are determined to be positive, a sub-chronic
neurotoxicity study in the hen is also required. Any neurotoxicity
observed in other submitted toxicity studies is also evaluated and
appropriately documented.
In accordance with the 1996 Food Quality Protection Act
(FQPA), EPA evaluates potential pre and postnatal toxicity on
a case-by-case basis taking into account all pertinent information,
which includes testing of young or developing animals. The rele-
vant studies are prenatal developmental (teratogenicity) studies in
rodents and non-rodents and the two-generation reproduction study
in rodents. Other new studies may also be available for evaluation
such as the developmental neurotoxicity study in rodents.
Sub-chronic studies via the oral, dermal and inhalation routes
are intended to identify effects on organs (liver, kidneys, spleen, lung,
etc.) following daily exposure for up to 90 days.
A two-year feeding study in rat and an 18-month feeding study
in mouse are assessed to determine a chemical’s potential to cause
cancer following long-term exposure.
A battery of studies is assessed to determine a chemical’s poten-
tial for causing mutagenicity or changes in the genetic content of
cells. These tests include Ames Salmonella bacterial point mutation
assay, mouse micronucleus assay, in vitro mammalian point mutation
assay (mouse lymphoma), in vitro and in vivo chromosomal aber-
ration assays and in vitro and in vivo unscheduled DNA synthesis
assays.
The purpose of a hazard and dose–response characterization is to
state and explain conclusions. It provides the reasoning behind selec-
tion of a certain study, or studies, to represent the potential hazard to
humans, and the reasoning for using a study or studies as the basis
for risk assessment. A key feature of characterization is capturing
the points at which choices are made of critical effect or approach, or
the choice is made to obtain more data. This is particularly impor-
tant in cases involving a close call or contending views. Why does
the choice made represent the better view on scientific and public
health grounds? Sometimes a data set is relatively straightforward
and can follow a well-travelled path of assessment, guided by prece-

dent and science policy. Other times, difficult issues are encountered.
In both cases, the choices need to be explained, but the difficult
or debated issues should be highlighted. A concise summary of the
available pesticidal and toxicity mode of action, metabolism and tox-
icokinetic data must be provided. In this regard, are there pertinent
data on other members of the structural class to which the compound
belongs?
Hazard characterization should cover the following areas and be
limited to the most significant and relevant data, conclusions and
uncertainties. The discussion of toxicological effects is a qualitative
description of the chemical’s critical effects. It also describes what is
understood about the underlying basis (i.e. chemical’s mode of toxic
action) for these effects and how the toxicity profile relates to other
chemicals with similar structures.
• What is the intended pesticidal use and what insight does it give
about the mode action and anticipated effects?
• What are the critical effects in terms of human relevance, suffi-
ciency of data? Are they observed in more than one study, more
than one species, both sexes?
• Are the effects different at different exposure durations? Are the
effects cumulative with time?
• Are there additional target organ effects, which were not selected
as endpoints?
• Are there human data available (including epidemiology or
biomonitoring data, etc.)? If so, how do these data relate to the
animal data?
• What species is most biologically or physiologically relevant to
humans?
• Are there other data such as mode of action or similarities with
related agents, or endpoint data on metabolites that are support-
ive? Are there data or studies that are not supportive of human
relevance? How were these considered?
• Are the critical effects typical of other agents with the same mode
of action that have been assessed in the past?
• What, in summary, are the issues that should be highlighted as

difficult or debated and how were they addressed?
3.5.3.2 Endpoint selection

This section summarizes the quantitative analysis, including the
rationale for the selection of toxicological endpoints of concern used
in the risk assessment. The toxicology database should be evaluated
to determine if the studies and data are sufficient (e.g. quality of
data, number and type of endpoints examined, replication of effects,
number of species examined). Sufficient includes data that collec-
tively provide enough information to judge whether a human hazard
could exist within the context of dose, duration, timing and route of
exposure. This includes both human and animal evidence. Once the
database is determined to be sufficient, the toxicology profile must
be assessed to determine critical effects of concern. A critical effect
is one considered the most sensitive for an endpoint from the most
appropriate species. The critical effect can be different for different
exposure scenarios and endpoints.
• Which studies were used to develop the dose-response assessment?

(Only positive studies, all studies, or some other combination?
What rationale supports the choice? Were certain studies consid-
ered then excluded? Why?)
• Which species were used? Most sensitive, average of all species, or
other?
• Why are certain studies selected to apply to particular exposure
scenarios? If a study has been selected to represent an exposure
scenario in lieu of having a study with exposure that matches the
stage of life, duration or route of concern, what is the impact on
the public health conservativeness of the assessment?
• If human data are available, are they appropriate for use or for
verifying or adjusting the animal dose–response?
• Are there other effects that weigh in the assessment, but that can
be dealt with by addressing the critical effects (e.g. occur at higher
doses)?
• Is the response route-specific (e.g. is the compound absorbed
orally, but not by dermal application)?
• Was a model used to develop the dose–response curve on a study

or group of studies, and, if so, what did it reveal about steepness of
the slope and how was this information considered? What choice
of study or combination of studies was made for a BMD or slope
factor? What rationale supports this choice?
• What, in summary, are the issues that should be highlighted as
difficult or debated and how were they addressed?
3.5.3.3 Point-of-departure selection and uncertainty factors

The dose–response assessment involves identifying a POD for risk
assessment. The POD can be a NOAEL or a LOAEL or a BMD
for the sensitive critical effect. The POD can be used in two ways
in risk assessment: First, it can be divided by uncertainty factors
(UFs) to account for various uncertainties in the data to derive the
reference dose (or RfD). The RfD is defined as an estimate (with
uncertainty spanning perhaps an order of magnitude) of a daily oral
exposure to the human population (including sensitive sub-groups)
that is likely to be without an appreciable risk of deleterious effects
during a lifetime. Second, the POD can be divided by the estimated
human exposure to derive a margin of exposure (MOE), which can be
used to determine whether existing or proposed controls on exposure
of humans meet the ‘reasonable certainty of no harm’ standard.
The RfD process identifies five uncertainty factors (UFs; some-
times described as safety factors) that may be applied to the NOAEL,
LOAEL or BMD to derive an RfD as described below. Although the
default value for each of these factors is 10 times, the exact value of
the uncertainty factor chosen will depend on the quality of the studies
available, the extent of the database, and scientific judgement.
• Inter-species uncertainty factor (UFA ): which is intended to

account for differences between the average responses in the exper-
imental animals used in the study identified to derive the POD and
those in average humans.
• Intra-species uncertainty factor (UFH ): which is intended to
account for the variability in responses between average humans
and those who are highly sensitive. In the USA, an additional
10-fold safety factor to account for potential enhanced suscepti-

bility of infants and children (FQPA safety factor) is required by
that country’s legislation; however, that factor may be reduced
to one, provided that there is sufficient evidence to demonstrate
that potential risks to infants and children are not being underes-
timated.
• Uncertainty factor to extrapolate from sub-chronic to chronic data
(UFS ): when a dose and endpoint of concern are selected from a
sub-chronic study for deriving a chronic RfD.
• Uncertainty factor to extrapolate from the LOAEL to a (surrogate)
NOAEL (UFL ): when a LOAEL is used as the POD for deriving
the chronic RfD (i.e. if no appropriate NOAEL can be identified
in the toxicology database).
• Database uncertainty factor (UFDB ): which is intended to account
for the absence of key data in the database for a given
chemical.
For pesticide chemicals, the statute authorizes EPA to retain a

10-fold FQPA Safety Factor to protect infants and children, taking
into account the potential for pre- and postnatal toxicity and the
completeness of the toxicology and exposure databases. The 10-fold
‘FQPA safety factor’ can be replaced with a different FQPA factor
only if reliable data demonstrate that the resulting level of exposure
would be safe for infants and children.
Risk assessments conducted for food-use chemicals include acute
and chronic dietary exposure scenarios. The acute dietary risk assess-
ment is an estimate of the risk that could result from one day of expo-
sure to pesticide residues in food and water. The chronic dietary risk
assessment is an estimate of the risk that could result from repeated
exposure to pesticide residues in food and water over a lifetime.
Typically, the dose and endpoint selected for the dietary exposure
scenarios is suitable for the general population including infants and
children. However, if necessary, an additional population-specific dose
and endpoint could be selected to account for toxicity targeting a spe-
cific population subgroup (i.e. concern for prenatal toxicity, which is
a potential concern only for women of child-bearing age, the females

13–49 year population sub-group).
An ARfD is defined as the pesticide exposure level at which no
harmful effects are likely to be seen in the most sensitive individu-
als in the population during a single day. Typically, an acute RfD
is established for the general population including infants and chil-
dren. However, an acute RfD can also be established for women of
child-bearing age (female 13–49). In this scenario, a dose and end-
point should be based on in utero effects observed in a developmental
or reproductive animal study following a single oral dose of (expo-
sure to) the pesticide. The acute RfD is used for assessing acute
dietary risk.
The chronic RfD is defined as an estimate (with uncertainty span-
ning perhaps an order of magnitude) of a daily oral exposure to the
human population (including sensitive subgroups) that is likely to be
without an appreciable risk of deleterious effects during a lifetime.
The chronic RfD is used for assessing dietary risk.
PODs and endpoints are also selected to represent non-dietary
exposure scenarios in risk assessments. These scenarios include: inci-
dental oral (hand-to-mouth exposure, especially for children) dermal
and inhalation exposures.
The incidental oral exposure scenario came about as a result of
the FQPA of 1996 to protect infants and children entering pesticide
treated areas and becoming exposed to pesticide products through
hand-to-mouth behaviour. There are several pesticide product uses
that can result in incidental oral ingestion in residential or pub-
lic areas including ingestion of pesticide pellets or granules applied
to lawns and gardens; paint chips containing pesticide residues;
pesticide residues left on turf grass, plants, pets, indoor surfaces
or impregnated materials by hand-to-mouth transfer; actual treated
turf grass, plants and soil; and ingestion of pesticide residues during
swimming.
Dermal and inhalation scenarios represent exposure to work-
ers (e.g. pesticide handlers or applicators) both applying the pes-
ticide product and re-entering areas where the product has been
applied. Depending on the use pattern for the pesticide product,
these scenarios can also represent the general population including

infants and children in residential and public settings.
Depending on the use pattern of the pesticide product (formu-
lation type, methods of application, use rate and frequency) and
the activity patterns of the exposed population sub-group relative to
that use, the potential exposure duration could be short term (i.e.
occurring over a period of 1–30 days), intermediate term (i.e. occur-
ring over a period of one–six months) or long term (greater than one
year). Therefore, a POD and endpoint is selected based on primary
toxic effects observed in an animal study, ideally in young animals,
following oral dosing of the pesticide compound over a comparable
duration (i.e. short or intermediate term).
In accordance with FQPA, when the potential for residential
exposure to the pesticide product exists, aggregate risk assessments
must be performed to consider potential exposure from all sources:
oral, dermal and inhalation. In an aggregate assessment, exposures
from these sources are added together (as appropriate) and are com-
pared to quantitative estimates of hazard (e.g. RfD). In general,
exposures from different routes can only be aggregated if the same
toxic effect(s) were observed in the studies (or when route-specific
studies are not available and route-to-route extrapolation is based
on the same effects).
3.5.4 Residue Chemistry Evaluation

3.5.4.1 Residue definitions
As previously discussed (Section 3.2.2.1), two definitions are typi-
cally established by the EPA. The first, referred to as the tolerance
expression, is for compliance with MRLs and is published in the
U.S. Code of Federal Regulations, Title 40, Part 180. The second,
referred to as the residues of concern or residue definition for dietary
risk assessment, is found in EPA risk assessment documents.
For an enforcement residue definition, the EPA strives to select
a single residue that is suitable as a marker of use or misuse of the
pesticide product. Historically, however, this has not always been
the case, and unnecessarily complex residue definitions may still be
found in the CFR. Generally, U.S. residue definitions for enforcement

of MRLs do not distinguish between various isomers of an active
ingredient even though separate MRL listings may exist (see, for
example, cypermethrin, 40 CFR 180.418).
3.5.4.2 MRL determinations

MRL determinations in the USA commonly follow the principles
addressed in Section 3.2.2.2, including the general requirements for
supervised field trials, processing studies, rotational crop studies and
supporting data (e.g. storage stability and analytical methods). One
area where the policy differs between the U.S. and other bodies is
application of storage stability data. For storage stability, the EPA
will generally correct for up to 30% loss during storage (after correct-
ing for concurrent analytical recovery); however, corrections greater
than 30% may be considered based on the absolute residue levels, the
predictability of the residue loss and on which component(s) of the
residue definition are showing the in-storage losses.
(a) Target Crops: Raw Commodities

For food and feed crops, the U.S. EPA makes extensive use of crop
grouping to extrapolate residue data from a few representative com-
modities to a larger number of commodities within the same group
or sub-group. The crop groupings are defined in the Code of Federal
Regulations (40 CFR 180.41) along with the rules for their imple-
mentation (40 CFR 180.40). The requirements for the number and
geographic distribution of supervised field trials are specified in the
residue chemistry guidelines, and unlike many other jurisdictions, the
U.S. generally requires two samples per trial location.
It is the U.S. EPA’s policy to use the OECD MRL Calculator
(OECD, 2011) to derive MRL levels from supervised field trial data.
Exceptions to the use of the calculator may occur for highly left-
censored data sets or when deemed necessary and appropriate in
order to harmonize with existing MRLs. In implementing the calcu-
lator, the EPA uses the average residue value from each independent
supervised field trial as the input for that trial. Generally, supervised
field trials are considered to be independent if they differ by one or
more attributes that may influence residue levels, including suffi-

cient distance between locations, significant offset between planting
times and differences in crop varieties that are meaningful in terms
of residues (e.g. fruit size, leaf morphology, time to maturity, etc.).
In the case of supervised field trials which the EPA determines are
not independent, the average value across the non-orthogonal tri-
als is used. Generally, data reflecting the shortest PHI on the label
are selected for the calculator. However, if residues are higher at an
interval longer than the minimum PHI on the label, then the higher
residues, being harvested in a manner allowed by the label, are used
in the calculator. For data sets that are completely left-censored (i.e.
all values are below the analytical method’s limit of quantification),
the EPA typically establishes an MRL for residues at the LOQ. In
the special case where there is suitable evidence to demonstrate that
residues will be much less than the LOQ, an MRL is not established
(the EPA terms this a ‘non-food-use’). When the MRL expression
includes multiple components and the residues of one or more of those
components are below their LOQ, residues are typically assumed
to occur at the LOQ and summed for purposes of establishing
the MRL.
(b) Rotational Crops: Raw Commodities

In evaluating rotational crops, the EPA will first use the results from
confined rotational crop studies to determine if plant-back restric-
tions can be set that will result in non-quantifiable residues in rota-
tional crops. If such intervals cannot be set, or if the applicant for
the registration requires plant-back intervals shorter than the above
restrictions, then the EPA requires field rotational crop studies. The
first tier in these studies is a set of limited trials to determine if
residues of interest occur in a non-confined situation. If residues
do accumulate in field rotational crops, then a set of trials equiv-
alent to those required for target crops in terms of numbers and
geographic distribution are generally required. The results of such
trials are used in the same manner as the target crop trials discussed
above to establish MRLs for inadvertent residues in the rotational
crop commodities.
(c) Processed Crop Commodities

For processed forms of raw agricultural commodities, the EPA
requires either processing studies or suitable evidence that residues
of interest will not concentrate to a level above those in the raw com-
modity during processing. There is a set of processed commodities,
defined in the residue chemistry guidelines, that requires processing
studies. In addition to those commodities, applicants for a pesticide
registration may submit studies for other processed commodities if
they wish to have MRLs established for those commodities.
Processing procedures used in studies should mimic common
commercial practices and should use field-incurred residues in the
raw commodity to be processed. It is the EPA’s policy to establish
MRLs for residues in processed commodities only when those residues
will likely be greater than the MRL for the raw commodity. That
determination is made by multiplying the highest average residue
from supervised field trials by the processing factor that results from
a processing study and comparing the result to the MRL from the
raw agricultural commodity. When it is determined that an MRL is
required, the value obtained from the processing factor and highest
average from supervised field trials is rounded up according to the
OECD rounding classes defined in the OECD MRL Calculator to
determine the appropriate MRL level.
(d) Livestock Commodities: Direct Treatment

From a residue chemistry perspective and in terms of study param-
eters, the direct treatment of livestock is analogous to target crop
field trials. Studies for the direct treatment of livestock should be
conducted according to label directions in a manner designed to max-
imize residues in livestock commodities (milk, eggs, fat, muscle and
edible offal). Determination of MRL levels is made using the OECD
MRL Calculator or by expert judgement, depending on the number
of animals used in the studies and the resulting residue levels and
their overall variability.
(e) Livestock Commodities: Transfer from Feedstuffs

In order to establish MRLs in livestock commodities to address
residues that transfer from livestock feeds, the EPA uses data
from livestock feeding studies along with an estimated residue
intake, or dietary burden, to derive anticipated residues in livestock
commodities.
To estimate the dietary burden, the EPA uses an algorithm that
maximizes the residue contributions to the diet from the various
feeds while maintaining a reasonable balance between the carbohy-
drate, protein and roughage components of the animal’s diet. The
estimated dietary burden is compared to the results from the feed-
ing study to determine the anticipated level of residues in livestock
tissues, milk, and eggs. At present, the feeding level nearest to the
dietary burden is used to derive the anticipated residues; however,
the EPA may be adopting a Langmuir-based model, developed by
Health Canada’s Pest Management Regulatory Agency, in the near
future. The Langmuir model uses all of the available information
to produce the anticipated residue, and it accommodates plateau-
ing of residues at increasing dose levels as is often seen in feeding
studies. Regardless of which technique is used, the resulting antici-
pated residue is rounded up according to the OECD rounding classes
defined in the OECD MRL Calculator to obtain the appropriate MRL
for the commodity.
3.5.4.3 Exposure and risk assessment

(f) Dietary
Dietary exposure to pesticide residues is a function of residue lev-
els in food and consumption of that food. The EPA uses a tiered
approach, based on residue data, for assessing dietary exposure. At
the most conservative end of the spectrum, EPA assumes all com-
modities with MRLs bear residues at the level of the MRL (or its
residues-of-concern equivalent). At the most refined end of the spec-
trum, residues from monitoring data or from special studies reflecting
residues ‘at the plate’ and incorporating information about the per-
centage of commodities that are treated are used. Due to the manner
in which the EPA assesses dietary exposure (described below), it is
possible to refine the residue levels used in the risk assessment with-
out underestimating exposure.
For consumption, the USA uses data collected from the What
We Eat in America component of the National Health and Nutrition
Examination Survey, and it is because of the high-quality survey data
that the EPA can use non-MRL residue values and still ensure safety
in its assessments. The surveys are conducted yearly and capture two
days of food and beverage intakes from 7,000–8,000 (or more) individ-
uals made up of a cross section of demographics. The intake data are
compiled across multiple years, resulting in consumption data from
over 20,000 individuals available for modelling dietary exposure. In
order to match foods, reported as-consumed, with residues in com-
modities, the food (e.g. apple pie) is broken down using publicly
available ‘recipe files’ into its component commodities (e.g. apples,
flour, sugar, butter, eggs).
For chronic exposure, average consumption across respondents is
used for each commodity being assessed, under the assumption that
the two-day surveys, when averaged across respondents, provide a
valid reflection of long-term dietary trends within the demographic
group being evaluated. In refining chronic dietary exposure estimates,
average residue levels from supervised field trials or monitoring data
may be used, along with data reflecting the extent to which com-
modities are treated. These refinements are supported by the high
improbability that a consumer will be eating foods consistently con-
taining high-end residue levels and by the ‘dilution’ of residues in
long-term consumption by untreated commodities.
For assessing acute exposure, the distribution of food consump-
tion from the surveys is maintained, allowing for the inclusion of
multiple commodities in the acute assessment. The consumption dis-
tribution can be paired with point-estimates of residues in foods
to obtain an essentially deterministic distribution of daily intakes.
Under this scenario, the EPA uses the estimate at the 95th percentile
of exposure to derive its dietary risk estimate. As a refinement, the
distribution of residue values from supervised field trials or monitor-

ing data can be used to obtain a fully probabilistic distribution of
daily intakes. This approach incorporates the fact that the commodi-
ties consumed during the course of a day will have different relative
amounts of pesticide residues (i.e. not all foods will bear residues
at the high end of their respective residue distributions). Bringing
information about the percentage of commodities that are treated
into the residue distribution allows the consumption of untreated
commodities to be factored into the assessment. The EPA recognizes
that uncertainty in the level of protection afforded by dietary expo-
sure modelling increases as refinements such as monitoring data and
percentage of crop treated are factored into the evaluation. Therefore,
in a refined evaluation, the EPA uses the exposure estimate at the
99.9th percentile to assess risk.
(g) Residential
As previously noted, the FQPA specifies that EPA must ensure that
‘. . . there is a reasonable certainty that no harm will result from
aggregate exposure to the pesticide chemical residue, including all
anticipated dietary exposures and all other exposures for which there
is reliable information’. Therefore, in order to fully assess the safety
of pesticide residues in foods, the EPA must also assess non-dietary
exposure and risk. Most frequently, non-dietary exposures are asso-
ciated with pesticide uses in and around homes, schools, athletic and
recreational fields, etc. As within an agricultural setting, exposures
may occur when handling pesticide products in preparation for appli-
cation, during the application itself and after application. In assessing
these exposures, the EPA assumes that application-related activities
are conducted by adults only, whereas post-application activities may
be for adults as well as children.
Residential exposure assessments are made using a set of algo-
rithms, which have been vetted through the Agency’s FIFRA Science
Advisory Panel. The algorithms take into account such variables as
application rate, the activities involved in various scenarios and the
exposure characteristics of the formulation. As such, the assessments
are geared more towards the product and how it is used, rather than
the chemistry of the active ingredient. However, in order to more accu-

rately assess post-application exposures, the EPA requires chemical-
specific data quantifying the availability of residues on treated foliage
(dislodgeable foliar residues or, for turf, turf transferrable residues).
Dermal and inhalation exposure routes are considered for all life
stages. In addition, oral exposures, from hand-to-mouth activities and
other incidental ingestions, are considered for children.
(h) Aggregate
When the toxicological profile for a given active ingredient shows
that the same toxic effects are occurring at the regulatory points of
departure being used to assess dermal, inhalation and oral exposures,
and those routes of exposure are likely to co-occur, the EPA will esti-
mate aggregate risks from those routes of exposure. The techniques
used to aggregate risks take into account potential differences in the
POD and the uncertainty factors that may exist across the routes
of exposure. Currently, data and models of sufficient quality are not
available to fully integrate chronological exposure patterns for regu-
latory risk assessment. Since points of departure are frequently lower
for exposure durations longer than acute, and residential exposure
scenarios typically are evaluated based on short-term (up to 30 days)
and intermediate-term (up to six months) exposure durations, most
aggregate risk scenarios are adequately addressed by assessing only
short- and intermediate-term exposures. In conducting those assess-
ments, chronic dietary exposure or risk estimates are used as an
estimate of background exposures that co-occur with residential or
other non-dietary exposures. As better data become available, the
EPA will investigate methods for temporally matching dietary and
non-dietary exposures into its aggregate risk assessment strategies.
(i) Cumulative
When the EPA has determined that multiple pesticide compounds
share a common mechanism of toxicity, the agency will conduct a
cumulative risk assessment. Examples of these include assessments
conducted for the organophosphate, N -methyl carbamate, triazine,
chloroacetanilide and pyrethroid classes of chemicals. A common

mechanism of toxicity has been defined to exist when the common
toxic effect(s) occur by the same or essentially the same, sequence
of major biochemical events. Cumulative risk assessments are gen-
erally conducted by selecting an index chemical within the group
that is being assessed, and developing toxicity equivalency factors or
potency factors in order to normalize the dose–response curves across
chemicals. Work is then done to assess exposure to the chemicals in
the group, striving to maintain a similar level of conservatism across
the assessment group.
3.5.5 Enforcement and Monitoring

Enforcement of pesticide MRLs in the USA is under the jurisdictions
of the FDA for crop commodities, fish, dairy products and processed
foods, and the USDA for meat, poultry and certain egg products. The
FDA and USDA work with state agencies to collect samples from
different parts of the country for domestically produced foods and
from ports of entry for imported foods. Available information regard-
ing pesticide use, extent of imported commodities and past results
from monitoring are used to develop annual monitoring plans. Both
FDA and USDA publish the results of their monitoring programs
online.
In a case where a confirmed residue exceeds an established MRL
or action level, or there is a quantifiable residue and no set enforce-
ment level has been established, the lot of food (to the extent that
it is available) is removed from commerce. FDA may issue warn-
ing letter to the growers responsible for the violation and invoke
other sanctions, including seizure or injunction until such time as
the cause of the violation has been corrected. For imported com-
modities, shipments bearing violative residues are refused entry, and
firms may be placed under an Import Alert. The alert may be lifted
once there is sufficient evidence for FDA to have confidence that
future entries will be in compliance with U.S. regulations, including
a minimum of five consecutive non-violative commercial shipments.
On a case-by-case basis, violative food lots may be released into
commerce if it can be demonstrated that the residues have dissi-

pated to compliant levels or, in the case where there is no U.S. MRL,
that residue levels can be demonstrated to be safe according to the
criteria specified in the FFDCA. At this time, the USA will not
adopt residue limits established by Codex or other regulatory author-
ities without conducting its own review; however, in conducting that
review, the EPA will harmonize with existing MRLs to the extent
possible.
3.5.6 Harmonization with Other Regulatory

Authorities
In addition to indicating that a pesticide use has passed a safety
assessment, the existence of an MRL is important as a standard for
trade purposes. Towards that end, the EPA strives to harmonize
pesticide residue limits with those of other regulatory authorities.
Under the requirements for an MRL, the FFDCA, directs EPA to
harmonize U.S. MRL levels with established Codex MRLs. If the
agency does not propose to adopt the Codex level, then rationale
must be provided for public comment as to the reason for departing
from the Codex standard.
As a matter of course, however, many pesticides MRLs are estab-
lished in the USA before MRLs are set through the Codex process. In
order to promote international harmonization, the EPA has been an
active proponent of conducting joint reviews with global review part-
ners. Initially, joint reviews were conducted between the EPA and
Canada’s Pest Management Regulatory Agency as a means to ensure
harmonized review outcomes, especially MRLs, between the USA
and Canada under the umbrella of the North American Free Trade
Agreement (NAFTA). The NAFTA joint reviews have expanded to a
global level. To date, review partners have included Australia, China,
France, Ireland, Mexico, New Zealand and the United Kingdom, in
addition to the USA and Canada.
In addition to the chemical-specific joint review activities, the
USA participates in work being conducted through the OECD,
the FAO of the United Nations and the WHO, as well as other
venues, to develop and promote harmonized data requirements

and evaluation practices. The goal of these activities is to mini-
mize trade barriers resulting from non-harmonized evaluations while
ensuring the efficient use of resources and the continued high-
quality safety assessments currently being conducted by regulatory
authorities.
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4
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b2530_FM.indd 6 01-Sep-16 11:03:06 AM

Chapter 4
Evaluation of Pesticide Residues

by FAO/WHO JMPR∗
Denis Hamilton, Midori Yoshida, Gerrit Wolterink and Roland Solecki
Main topics
Residues (by Denis Hamilton)

History of JMPR residue evaluation
Metabolism and environmental fate
Residue analysis, sampling and sample storage
Residue definition
Use pattern
Supervised residue trials
Food processing
Livestock and residues in meat, eggs and milk
Monitoring data, EMRLs
Toxicology (by Midori Yoshida, Gerrit Wolterink and Roland Solecki)
Data requirements for toxicological evaluation
Main endpoints for toxicological evaluation
Process of toxicological evaluation in risk assessment
Current topics of toxicological evaluation of chemicals
∗
FAO: Food and Agriculture Organization of the United Nations. WHO: World
Health Organization. JMPR: Joint FAO/WHO Meeting on Pesticide Residues.
113
4.1 Introduction
In 1925, apple exports from USA to Britain were interrupted when
arsenic residue levels were found to exceed the limits set by British
food regulations. USA responded by setting tolerances for arsenic in
apples. California introduced the Chemical Spray Residue Act, aim-
ing to control residues of arsenic-based sprays, such as lead arsenate,
on fruits and vegetables and to ensure that consignments of fruit
were not seized in an importing country because of violative residues
(Federighi and Brank, 2001).
Public health concerns and fair practices in the food trade are
still the motivating factors for studies of pesticide residues in food.
The introduction describes historical concerns about contami-
nants and residues in food and early responses to the concerns. Also
introduced is the purpose of toxicological evaluation in risk assess-
ment of pesticides within the scope of the Joint Meeting on Pesticide
Residues (JMPR) by FAO and WHO for consumer risk assessment.
Britain had passed the Food Adulteration Act in 1860 when food
contamination was a public issue. Within a few years, it became
apparent that, without efficient means of detection, the regulations
could not be fully effective (Anon, 1868). After poisoning cases with
adulterated food in Britain in 1900, limits for arsenic content of food,
including fresh fruits, were imposed (Federighi and Brank, 2001).
These were the limits faced by U.S. apple exports.
In 1937, arsenic residues were still of concern. The United States
Department of Agriculture had developed cleaning methods to reduce
arsenic residues on apples before shipment. The Food and Drug
Administration articulated the ‘troublesome problem’ of lead and
arsenic in spray residues on fruits and vegetables. Growers need to use
sufficient lead arsenate to control the insect pests, but the residues
will also be dangerous to consumers if present in sufficient quantity
(Anon., 1937). The procedures of residue evaluation and risk assess-
ment have been designed to handle such troublesome problems. Two
general principles guide the assessment of pesticide residues in food.
First, human exposure should not exceed the acceptable daily
intake (ADI) or the acute reference dose (ARfD) of the pesticide.
Evaluation of Pesticide Residues by FAO/WHO JMPR 115
Second, residue levels should be no higher than necessary when

the pesticide is used as directed by good agricultural practice (GAP),
i.e. the treatment rates and timing are designed to control the pest,
but to leave no more residue in the food commodity than necessary.
Internationally, pesticide residues are evaluated by the JMPR
operating within the Joint FAO/WHO Food Standards Programme,
which lists its first purpose as: ‘protecting the health of the consumers
and ensuring fair practices in the food trade’.
All consumers in different regions of the world are unwillingly
and differently exposed to pesticides via food, depending on their
food intake and the residues in their food. To protect consumers,
human exposure should not exceed safe doses of a pesticide. Based
on these toxicological threshold doses and the consumption data for
a population or subpopulation, maximum residue levels of pesticides
in food are approved as legal maximum residue limits (MRLs), or are
not approved, by (inter)national authorities and Codex Alimentarius
Commission. Accordingly, the competent authorities have to identify
and characterize the adverse effects of each pesticide on human health
appropriately. The International Programme on Chemical Safety
(IPCS) has defined hazard and risk as follows (EHC240, 2009):
Hazard is an inherent property of an agent or situation having

the potential to cause adverse effects when an organism, system
or (sub)population is exposed to the agent. Risk is the probability
of an adverse effect in an organism, system or (sub)population
caused under specified circumstances by exposure to an agent.
Toxicological evaluation is the key process of risk assessment of a

pesticide for humans. Based on various toxicity studies using exper-
imental animals the hazards are characterized, dose–response rela-
tionships are assessed and health-based guidance values are derived.
This is followed by comparison of the toxicological thresholds with
the exposure data in the risk assessment process.
There are two time-dependent types of health-based guidance
values that are used in the assessment of the risk to human health
by chemical exposure including pesticides; the ADI and the ARfD.

The ADI is defined as the amount of pesticide to which humans can
be exposed daily for a lifetime without injury (EHC240, 2009). The
ADI is based on the evaluation of adverse effects that may occur
after acute, short-term or lifetime exposure to chemicals. The effects
may include impairments to reproduction, adverse effects in the next
generations or increased tumour development in the end stage of life.
In chemical regulation control, the specification of no adverse effect
levels is essential for the prediction of human health when exposed
during lifetime, and expressed as ADI. It is noted that the Envi-
ronmental Protection Agency of the United States (USEPA) uses
a chronic reference dose (cRfD) as a maximum exposure level for
long-term effects instead of the ADI.
The second health-based guidance value, the ARfD, was intro-
duced for the evaluation of adverse effects following acute exposure,
i.e. single dose exposure or exposure within 24 hours. The application
of the ADI for acute effects was considered to be overly conservative
and scientifically inappropriate in many cases because endpoints for
setting ADI are generally not based on acute effects. Therefore, for
acute exposure to pesticides, the ARfD was introduced in interna-
tional and national toxicological evaluations. The ARfD is defined as
the amount of pesticide that can be ingested in a period of 24 hours
or less without injury (EHC240, 2009). The process of derivation of
the ADI or the ARfD is described below.
4.2 Residue Evaluation

4.2.1 History of JMPR Residue Evaluation
The unpublished report of Besemer and Pieters (1991) and the first
issue of the FAO Manual (Ambrus, 1997) provided notes on JMPR
history. See also Chapter 7.
In 1961, FAO and WHO convened a meeting in Rome of an
FAO Panel of Experts and a WHO Expert Committee on Pesticide
Residues to ‘consider, among other matters, principles for establish-
ing tolerances for pesticide residues in food’. Its definition of residue
was: ‘A pesticide chemical, its derivatives and adjuvants in or on a
plant or animal. Residues are expressed as parts per million on fresh

weight of the sample’. This meeting explained that the estimation of
a ‘tolerance’ should take into account the range of residues actually
remaining, after GAP, when the food is first offered for consumption.
The meeting also developed the concept of the ‘permissible level’,
calculated from the ADI, the food factor and the average weight of
the consumer. The permissible level was apparently designed to place
an upper limit on the tolerance from a public health perspective. But
the permissible level might also minimize trade restrictions. Although
different countries might establish different tolerances for the same
pesticide on the same food, international trade would not be impeded
as long as the permissible level was not exceeded. Experience has
shown this to be a vain hope; trade risk is not necessarily aligned
with public health risk.
A Food and Agricultural Organization (FAO) Conference on Pes-
ticides in Agriculture in 1962 expressed concern that different residue
tolerances existed between countries: What are the reasons? It rec-
ommended that governments should not adopt residue tolerances
before an international agreement is achieved, which seems rather
impractical.
The Joint FAO/WHO Meeting on Pesticide Residues (JMPR)
was established in 1963 (FAO and WHO, 2000). JMPR would rec-
ommend tolerances for pesticides and environmental contaminants in
specific food products. It would also recommend methods of sampling
and analysis. JMPR would be independent of the Codex Commission.
JMPR members would be appointed in their own right and not as
government representatives and would be eminent independent scien-
tists with expertise in aspects of pesticides, environmental chemicals
and their residues.
In essence, JMPR was to examine matters related to pesticide
residues and to provide scientific advice to FAO, World Health Orga-
nization (WHO) and to the Codex Committee on Pesticide Residues
(CCPR).
The 1963 JMPR noted that ‘permissible levels’ must be calculated
for individual countries or regions because they are based not only on
ADIs, but also on food factors and body weights of consumers.
In the early days of the Codex Committee on Pesticide Residues

and JMPR, tolerances were set on specific foods. CCPR debated
where tolerances would apply: At the point of entry into an importing
country or at the point of consumption?
Tolerances were recommended for foods such as citrus fruit,
strawberries, nuts and whole milk. Comparison of the proposed tol-
erances and the ‘permissible levels’ completed the risk assessment.
JMPR proposed tolerances for residues in raw cereals before milling,
cooking or other processing, i.e. tolerances on food commodities, not
necessarily on foods ready for consumption. Subsequently, the defi-
nition of tolerance no longer referred to the ‘permissible level’.
In 1970, tolerances were recommended for both the commodity
and the edible portion. For example, quintozene tolerance recommen-
dations were:
— Peanuts (whole) 5 ppm.
— Peanuts (kernels) 0.3 ppm.
— Bananas (whole) 1 ppm.
— Bananas (pulp) 0.01 ppm.
Before 1995, a dietary intake assessment was based on a tolerance
or MRL, which was an encouragement to set tolerances or MRLs on
edible portions.
The 1975 JMPR adopted the MRL to replace ‘tolerance’, intro-
duced ‘good agricultural practice’ as part of the MRL definition
and replaced ‘unintentional residue’ and ‘practical residue limit’ with
‘extraneous residue limit’ (see Section 4.2.9).
If the edible portion is different from the commodity of trade, an
MRL expressed on commodity of trade is generally the more practical
for enforcement and a better indicator of GAP compliance. Today,
MRLs are set on the commodity of trade while dietary intake assess-
ments, no longer tied to MRLs, begin with residue levels in edible
portions.
The Codex Classification of Foods and Feeds was developed in the
1980s and was first adopted by the Codex Alimentarius Commission
in 1989 (Joint FAO/WHO Food Standards Programme, 1993). It
provides a name and code number for each commodity, as well as
placing it into a commodity group and explaining which portion of

the commodity is relevant to the MRL and is to be analysed. The
classification is a great international achievement in standardizing
and harmonizing the descriptions of foods and feeds.
As an example of an entry in the classification, ‘strawberry’ is a
member of the ‘berries and other small fruits’ group. The strawberry
code number is FB 0275. ‘The portion to which the MRL applies
(and which is analysed): Whole commodity after removal of caps
and stems’.
Data requirements and assessment methods for pesticide residues
have evolved. The FAO Manual (Ambrus, 2016) summarizes many
years of experience and is very helpful to both data submitters and
residue evaluators.
4.2.2 Metabolism and Environmental Fate

Metabolism studies of a pesticide in crops and livestock generate
data on the nature of the residues occurring in food. The residue may
include the parent compound, metabolites and other transformation
products.
Doses of pesticide labelled with 14 C are administered to livestock
or applied to crops in amounts and with timing that are likely in
practice. 14 C is a carbon isotope with a relative atomic mass of 14; it
emits relatively low-energy β-rays. In the livestock studies, milk and
eggs are collected periodically and after sufficient duration of dosing,
the animal is slaughtered for collection of tissues for analysis.
Feed and food commodities, harvested from the treated crop after
intervals approximating practical use, are examined for the content
and identity of 14 C-containing residues. The 14 C label allows the
observation of the metabolites among all of the natural compounds
derived from the host plant or animal. A molecule with one of its 12 C
atoms replaced by a 14 C behaves in the same way from a chemical
perspective, but the 14 C label allows observation of the behaviour of
the molecule and its breakdown products. Of course, we can observe
only those breakdown products that contain the 14 C label so, in
planning a metabolism study, it is important to incorporate the label
into the molecule in a position that will provide the most information.
Reports sometimes describe metabolites as being identified or

characterized. In the context of metabolism studies, ‘identified’
means that the structure of the metabolite is fully known, and ‘char-
acterized’ means that some properties of the metabolite or metabo-
lites are known (e.g. polarity and solubility), but not the full identity.
The requirements for identification or characterization of a metabo-
lite depend on its concentration in the tissue being analysed and its
percentage of the total residue in that tissue.
4.2.2.1 Livestock metabolism

Pesticide residue evaluation is interested in metabolism in food
producing animals, especially ruminants and poultry. Livestock
metabolism studies should provide answers to questions about pos-
sible residues in meat, milk and eggs:
• What is the composition of the residues in meat, milk and eggs if
the ruminants and poultry consume residue-containing feed?
• How quickly is the dose excreted?
• How much of the dose appears in milk and eggs?
• Is the pesticide or metabolite fat-soluble?
• Is any metabolite identical with another pesticide or the metabo-
lite of another pesticide?
Goats are usually chosen to represent ruminants, and in a typical
goat metabolism study, [14 C] labelled pesticide is administered as a
measured oral daily dose via gelatin capsule to lactating goats for
4–15 days. Milk is collected daily and the animals are slaughtered
for tissue collection within 24 hours of the final dose. The tissues
(muscle, liver, kidney and fat) and the milk are analysed for total
14 C content, i.e. the total radioactive residue or TRR. Components
of the TRR are then identified and characterized where possible.

A comparison of residue levels in the fat tissue with levels in
the muscle tissue gives a measure of fat solubility. Residue levels of
a fat-soluble pesticide may not be the same in different fat depots
within the one animal; ideally the fat from different depots should
be analysed separately, rather than pooled.
CF3
CF3 CF
O
S O
HN CO
OC NH
Flubendiamide
Example. Fat-solubility of flubendiamide (JMPR, 2010c)

Levels of flubendiamide in fat tissues were approximately 12–14 times
the levels in muscle tissues in lactating goat metabolism studies with
[14 C]flubendiamide. The suggestion of fat solubility was supported
by laying hen and lactating cow feeding studies. Flubendiamide was
therefore designated a fat-soluble residue.
Such a designation is important for MRL enforcement. If a sam-
ple of meat is to be analysed for a fat-soluble residue, a portion
of the adhering fat should be sampled for analysis and the residue
concentration is calculated and expressed on the fat. The MRL for a
fat-soluble residue in meat is indicated by the word ‘fat’.
Flubendiamide: MM 0095 Meat (from mammals other than
marine mammals) MRL mg/kg 2 (fat).
In milk, residues of fat-soluble compounds partition into the fat
phase. The distribution of compounds is found by analysis of the two
phases after physical separation of milk into aqueous and fat phases.
Metabolism studies on laying hens are similar in principle to the
goat studies, but eggs are collected for analysis, not milk.
During animal and plant metabolism, pesticides or their metabo-
lites may be conjugated with groups such as glutathione, sulphate or
glucuronic acid. Analytical methods for the residue should include
a hydrolysis step or other means of releasing the residue from the
conjugate if the conjugated residue is included in the defined residue.
4.2.2.2 Crop metabolism

Crop metabolism studies are designed to answer questions such as:
• What is the likely composition of the pesticide residue in harvested

food and feed commodities?
• Will residues appear in the tubers of potatoes after a foliar appli-
cation of pesticide?
• After a foliar application, does the residue remain mostly on the
plant surface?
• After a soil treatment, is the pesticide taken up by the roots of
the crop and is the residue translocated within the plant to reach
seeds, fruits or other edible portion?
The aim of a crop metabolism study is to discover where the

pesticide residues move in the plant and to identify the transforma-
tion products that are formed. The term ‘transformation products’
includes metabolites, products of chemical reaction and photolysis
products.
Transformation product: Chemical species resulting from envi-

ronmental, chemical or metabolic processes on a pesticide
(Stephenson et al., 2006).
Metabolism studies are needed for each type of crop where the
pesticide is to be used. For the purposes of crop metabolism, five
group types are recognized: cereals, fruits, leafy crops, pulses and
oilseeds and root crops.
In a crop metabolism study, crop plants are treated with
[14 C]labelled pesticide with the rate, timing and number of appli-
cations approximating the expected GAP conditions. Fruit, foliage,
grain or other parts of the plant intended for food or feed are sampled
at maturity and analysed for TRR and identification of the residue
components. Combustion of samples and measurement of the 14 C in
the evolved carbon dioxide provides the TRR data. The 14 C data are
usually calculated and expressed as parent compound. Components
of the residue are then identified or characterized.
Cl O
Cl
O
N
N *
O
* N
Difenoconazole
Example. Difenoconazole metabolism in wheat.

Difenoconazole metabolism in wheat provides an example of the
information generated by such a study (JMPR, 2007a). Spring wheat,
in a greenhouse, was foliar sprayed four times with [14 C]triazole
labelled difenoconazole and samples were taken at maturity, 29 days
after the final application, for analysis.
The TRR concentration was much higher in the stalks (54 mg/kg)
than in the grain (1.4 mg/kg) and the composition of the residue was
quite different. Parent difenoconazole was the main component of
the residue in the stalks at 27 mg/kg, but was not identified in the
grain. The main identified component of the residue in the grain was
triazolylacetic acid at 0.28 mg/kg.
Interpretation: Parent difenoconazole is not mobile within the
plant, so residues of difenoconazole occur in parts of the plant directly
sprayed (i.e. the leaves and stalks, but not the grain). Metabolite
1,2,4-triazole and its metabolite triazolylacetic acid are mobile within
the plant and are translocated to the grain. Triazole conjugates with
serine to produce triazolylalanine and triazolylacetic acid (Fig. 4.1).
It is the same story for other triazole fungicides.
JMPR, when reviewing propiconazole, also a triazole fungicide,
explained that the data on propiconazole could not be used to assess
N N COOH N
NH serine N N COOH
N N NH2 N
1,2,4-triazole triazolylalanine triazolylacetic acid
Figure 4.1. Generation of plant metabolites from 1,2,4-triazole.

the toxicity of triazolylalanine because it did not appear to be a non-

ruminant mammalian metabolite (JMPR, 1987a). Animal metabo-
lites can produce their toxic effects, if any, during toxicity testing of
the parent compound. Plant metabolites that are not animal metabo-
lites must be subject to separate toxicity testing.
Identification of plant metabolites that are not also animal
metabolites is an important task in the evaluation of crop metabolism
studies.
4.2.2.3 Environmental fate

From a JMPR perspective, we should ask how the data from envi-
ronmental fate studies can answer questions about residues in food
commodities.
• Does soil metabolism produce new metabolites not identified as

plant metabolites?
• This compound is used at the first-leaf growth stage of root crops;
will residues be present in the soil at harvest time?
• This herbicide controls weeds around fruit trees; does it persist in
the soil and is it taken up through the roots of the fruit trees?
• How do residues from glass-house uses compare with residues from
field uses for this compound, which is susceptible to sunlight pho-
tolysis?
Environmental fate studies relevant to pesticide residues in food

and feed commodities are: aerobic soil metabolism, metabolism in
water–sediment systems, soil photolysis, photolysis in natural pond
water, confined rotational crops and field rotational crops.
4.2.2.4 Aerobic soil metabolism

Soil metabolism studies can tell us about the persistence of a pes-
ticide residue in the soil and the nature of its composition. The
properties of the soil and the test conditions must be measured and
controlled because they will influence the results.
The soil must be characterized by soil type and properties, espe-
cially pH and organic matter content. Dose rate, duration of the test,
temperature and moisture must be under control. The test compound

identity and the position of the 14 C label are necessary. Evolved
14 CO must be captured for analysis.
2
Expected results from a soil metabolism study include:
• Disappearance rate of parent compound;

• Mineralized residue, evolved 14 CO2 ; as percentage of dose.
• Identity of soil metabolites;
• Formation and disappearance rate of each metabolite, indicated by
maximum metabolite concentration and the time when the maxi-
mum occurred;
• Unextracted residue, i.e. 14 C remaining in the soil after solvent
extraction, expressed as parent compound and calculated as per-
centage of dose.
O CN
Cl O
O
Cl
Cypermethrin
Example. Aerobic soil metabolism of cypermethrin (JMPR, 2008b).
[14 C-Cyclopropyl]cypermethrin was dosed into a silty clay loam

soil (pH 6.8, organic matter, 7.9%) at 0.3 mg/kg and incubated for
90 days at 20◦ C and 35% maximum water-holding capacity. By day
90, 75% of the dose was mineralized and 15% remained in the soil
after solvent extraction. Of the parent cypermethrin, 7.3% remained
at day 90 and the composition had changed from a cis:trans ratio
of 40:60 to a ratio of 67:33. DCVA was an identified metabolite,
reaching its maximum concentration of 9.3% of the dose on day 7.
Cl
Cl
COOH
DCVA
After assembling data from other similar experiments on a range

of soils with some variation of test conditions, we can interpret the
results to conclude that cypermethrin disappears relatively quickly
from soils and that its isomer composition changes. The most likely
explanation is that the soil biota hydrolyse the trans-cypermethrin
more quickly than the cis-isomer.
Metabolite DCVA reached its maximum concentration by day 7,
which means it was disappearing more quickly than it was generated
after day 7, so it was also not a persistent residue.
By day 90, most of the dose had been converted to 14 CO2 , i.e. the
portion of the molecule containing the 14 C had been mineralized or
completely broken down. Some of the 14 C was not readily extracted
from the soil, which probably means it had been incorporated into
polymeric natural compounds.
Other soil studies, such as metabolism in water–sediment sys-
tems, are conducted with [14 C]compound in the same general way
as the aerobic soil metabolism studies, but with their own special
conditions.
4.2.2.5 Residues in rotational crops

Crop rotation is the agricultural practice of varying, in a definite
order, the crops grown on the same ground with the intention of
maintaining soil nutrients and minimizing weed, pest and disease
problems. Our concern here is with following crops whether part of
a rotation plan or not.
Cl
NH
O Cl
Boscalid
Can the residues from a pesticide used on one crop remain in the
soil in sufficient quantities to be taken up by a following or rotational
crop when it is planted or sown in that same soil?
If the pesticide is a herbicide with a degree of soil persistence,
the label directions for use will normally provide warnings about
sensitive following crops with advice about the interval of time that
should elapse before planting or sowing.
If the persistent pesticide is a fungicide or insecticide, there may
be no effect on the following crop except the presence of residues in
the feed or food commodity being produced. The presence of residues
has the appearance of a misuse if the pesticide is not approved for use
on the following crop. Where no MRLs may have been established
for the commodities, the illegal residues may pose a trade risk.
Example. Boscalid residues in following crops.
Boscalid is a fungicide with sufficient persistence in the soil to
carry over to a following crop. The joint meeting explained the prin-
ciples adopted for evaluating boscalid residues (JMPR, 2010a).
• For annual crops, estimates of residues from direct treatment

should be added to expected uptake from the soil of residues orig-
inating from previous applications of boscalid.
• Experimental data on residue levels resulting from root uptake
should be extended to commodity groups to cover the many pos-
sibilities of which crops could be following crops. For example,
decisions based on experimental data for celery may be extended
to the stalk and stem vegetables group.
• For permanent crops (e.g. apple or citrus), the evaluation of
residues should rely solely on direct treatment because little con-
tribution is expected from root uptake.
A further complication is that the composition of the residue in

a following crop is not necessarily the same as that resulting from
direct use.
Confined rotational crop studies with [14 C]radiolabelled com-
pounds provide data on the fate of pesticides in this situation.
Labelled compound is applied to the soil and then, after intervals
(e.g. 1, 3 and 12 months of soil metabolism), crops are sown or

planted and grown to maturity for sampling and identification of
the residue components. The representative crops are usually a leafy
vegetable, a root vegetable and a cereal. Such studies are necessarily
‘confined’ because the radioactive material must be under control
and the study director must be able to account for it.
From direct use, the residue, at least in the short term, will
be mainly parent compound. In the longer term, plant metabolism,
chemical degradation and sunlight photolysis may generate one or
more transformation products in sufficient amounts to be included
in the residue definition. Before reaching the rotational crop, the mix-
ture of parent and transformation products will be further changed
by soil metabolism. Finally, only those components of the mixture in
the soil that can be taken up by the roots of the rotational crop and
translocated to the parts that become food and feed will be observed
as the residue in the rotational crop.
O
CF3 O
OCH3
CN O
Cyflumetofen
COOH
CF3
2-trifluoromethylbenzoic acid
Cyflumetofen, an acaricide, provides an example (JMPR, 2014a).

The risk assessment residue definition for plant commodities is the
sum of parent compound and 2-trifluoromethylbenzoic acid expressed
as cyflumetofen, which reflects the composition of the residue from
direct treatment. No parent compound was detected in extracts from
the rotational crops, lettuce, white radish and spring wheat, grown
to maturity. The major component of the residue in the rotational

crops was trifluoroacetic acid (CF3 COOH).
After the residue in rotational crops has been identified, field tri-
als with non-radiolabelled pesticide are needed to quantify the likely
concentrations of residues to be expected in rotational crops under
practical conditions where the pesticide is used at the maximum
application rate permitted. Samples of the harvested food and feed
commodities should be analysed for the residue components identi-
fied in the confined studies. Such field trials also simulate the case
where a recently treated crop has failed for some reason (e.g. a severe
hail storm), and is ploughed to be ready for re-sowing or re-planting
of another crop in the same land.
The information from rotational crop studies is taken into
account in the estimation of maximum residue levels and dietary
intake assessment. The residues are usually undetectable or small
compared with residues from direct treatment. However, a regula-
tory problem occurs when residues appear in commodities from a
crop with no registered uses and no MRLs for that pesticide.
4.2.3 Residue Analysis, Sampling and Sample Storage

Intending registrants develop suitable analytical methods for pesti-
cide residues in raw agricultural commodities, processed food com-
modities, meat, milk and eggs to produce the copious data needed
for registration. The required analytes are parent compound and sig-
nificant transformation products, especially those that are likely to
be included in a residue definition.
We should expect analytical recoveries in the 70–120% range and
limits of quantification (LOQ) of 0.01 mg/kg or better, with sup-
porting validation data. Data on method selectivity, the ability to
separate isomers and to include conjugated residues are necessary in
some cases.
The LOQ of the method should be measured on the whole
method, not just on the final measurement step. From a reviewer
perspective, the lowest concentration where satisfactory analyti-
cal method recoveries are demonstrated is accepted as the LOQ
(Ambrus, 2016, p. 27).
An incurred residue may be more difficult to extract from a plant

or animal sample than is a small amount of pesticide freshly spiked
or fortified into a sample. Validation of an analytical method should
demonstrate that its extraction step efficiently extracts incurred
residues.
Skidmore et al. (1998) recommended that the extraction proce-
dures of analytical methods should be validated on samples contain-
ing incurred radiolabelled residues.
Incurred residue: Residue in a commodity resulting from specific

use of a pesticide, consumption by an animal or environmental
contamination in the field, as opposed to residues from laboratory
fortification of samples (Stephenson et al., 2006).
The radiolabel enables the measurement of incurred residue con-

centration for comparison with the amount extracted. Such sam-
ples, containing radiolabelled incurred residues, are available from
metabolism studies and the opportunity should be taken to test
extraction procedures.
O
O
NH NH
OCH3
Bifenazate
For example, samples of oranges, apples and fat from the

[14 C]bifenazate plant and animal metabolism studies were extracted
and analysed by an HPLC–coulometer method (JMPR, 2006). For
oranges and apples, the bifenazate results from the coulometer
method were about 60% of the results from the 14 C method. For
fat tissue, the two results were quite close.
Analytical methods for enforcement of MRLs in the market place
have different requirements from methods used for generating data
from supervised trials and other pre-registration studies. Ideally, the
components of the enforcement residue definition down to required
LOQ concentrations should be within the capabilities of a multi-

residue method.
Multi-residue method: Analytical method designed to effec-

tively determine a number of pesticide residues simultaneously
Detailed instructions for taking and handling samples from super-

vised field trials are provided in the FAO Manual (Ambrus, 2016).
For commodities in trade, the sampling principle for meat and
poultry products is different from that of plant commodities (Joint
FAO/WHO Food Standards Programme, 1993, pp. 369–386).
A primary sample is a quantity of material taken from a single
place in the lot. Primary samples from a lot are combined to form a
bulk sample, which is reduced to a final sample if too large. The final
sample may become the laboratory sample, or it could be subdivided
into representative portions if more than one laboratory sample is
needed.
Lot: Quantity of material that is assumed to be a single population

for sampling purposes (Stephenson et al., 2006).
For meat and poultry products: When samples are taken from
a lot for enforcement purposes, the lot complies with a Codex MRL
when none of the primary samples contains a residue exceeding the
MRL.
For plant commodities (and dairy products and eggs), the lot
complies with a Codex MRL when a final representative sample,
produced from the primary samples under a controlled procedure
and satisfying a specified minimum sample size, does not contain a
residue exceeding the MRL.
It is not always convenient to analyse residue samples soon after
they are collected. In practice, samples from supervised residue trials
and food processing studies are commonly stored frozen for months
and sometimes for one to two years. Data are required to validate
sample storage (i.e. to provide evidence that the storage conditions
and duration had no substantial effect on the concentration or nature

of the residues).
Storage stability studies may be conducted with representative
commodities. For the purposes of storage stability testing, five plant
sample types have been identified: (1) high water content, (2) high
acid content, (3) high oil content, (4) high protein content and (5)
high starch content. When animal commodity samples are involved,
storage data are needed for animal tissues, milk and eggs.
Parent compound and transformation products that appear in
the residue definitions for enforcement and dietary intake assessment
are the residues to be tested for storage stability.
Conditions and durations where decline exceeds 30% are gener-
ally not acceptable.
Storage testing is quite demanding on the quality of the ana-
lytical method, which must maintain its accuracy and reliability
for the duration of testing, up to two years in some cases. Sam-
ples would normally be analysed at the beginning and end of the
chosen storage duration and perhaps at two or three times during
storage.
Some residues may be severely depleted if the sample is chopped
or homogenized even though residues are quite stable on the sur-
face of a fruit or leaf. Storage of unchopped sample is indicated for
such a residue. Bifenazate is an example of a residue that is suscep-
tible to losses when exposed to chopped sample. Disappearance of
spiked bifenazate residue in chopped potato tuber was so rapid that
it caused difficulty in analytical recovery testing (JMPR, 2006).
4.2.4 Residue Definition

The residue definition is that combination of the pesticide and its
transformation products selected for checking compliance with the
MRL (enforcement residue definition) or for estimating dietary intake
(risk assessment residue definition).
As part of pesticide residue evaluation, suitable residue defini-
tions, usually parent or parent+important transformation products,
are proposed for dietary exposure assessment and for enforcement of
GAP use or checking food commodities in trade.
Example. Dicamba residue definition (JMPR, 2010b)

Definition of the residue for compliance with the MRL for plant
commodities: dicamba
COOH
Cl OCH3
Cl
Dicamba
Definition of the residue for estimation of dietary intake for plant

commodities: sum of dicamba and 5-OH dicamba expressed as
dicamba.
COOH
Cl OCH3
HO Cl
5-OH dicamba
Definition of the residue for compliance with the MRL and for esti-
mation of dietary intake for animal commodities: sum of dicamba
and 3, 6-dichlorosalicylic acid expressed as dicamba.
COOH
Cl OH
Cl
3,6-dichlorosalicylic acid
Points to note:
— In this example, sometimes metabolites are included in the
residue definition and sometimes not, depending on the nature
of the sample and the purpose of the residue definition.
— The phrase ‘expressed as dicamba’ means that the concentration
of a metabolite is multiplied by a molecular mass adjustment
factor (= relative molecular mass of dicamba ÷ relative molecular
mass of metabolite).
The residue evaluator considers a list of factors with influence on the
residue definition.
• Identified plant and animal metabolites and photolysis products;
• Nature of the residues occurring in following or rotational crops;
• Toxicity of identified transformation products;

• Nature of the residues reported in supervised residue trials;
• Capabilities of regulatory analytical methods;
• Fat solubility of the residue;
• Occurrence of conjugates;
• Is a transformation product or analyte also produced by another
pesticide or from some other source?
• Is a transformation product already registered as another pesti-
cide?
• Joint FAO/WHO Expert Committee on Food Additives (JECFA)
marker residue definitions for compounds with veterinary uses.
Ideally, the enforcement residue definition should be a single com-
pound unique to the specific pesticide and amenable to analysis in
a multi-residue method. The enforcement residue definition should
be the same for commodities produced by the transgenic crop and
the non-transgenic crop. In practice, it would be costly and time con-
suming for regulatory laboratories to determine if the test commodity
was transgenic, non-transgenic or a mixture.
For a similar reason, MRLs for commodities that are not eas-
ily distinguished should be the same if at all possible. For example,
barley straw and wheat straw are not readily distinguished and, espe-
cially so, if in the form of chaff.
4.2.5 Use Pattern

The use pattern of a pesticide includes methods of application, appli-
cation rates, timing and restraints and is known as ‘good agricultural
practice’ when it is described in the directions for use on a nationally
registered pesticide label.
The definition of GAP appears in the International Code of Con-
duct on Pesticide Management (FAO and WHO, 2014).
GAP in the use of pesticides includes the officially recommended

or nationally authorized uses of pesticides under actual conditions
necessary for effective and reliable pest control. It encompasses a
range of levels of pesticide applications up to the highest authorized
use, applied in a manner that leaves a residue which is the smallest
amount practicable.
GAP is derived from efficacy trials at the national level. In devel-

oping their products, pesticide companies establish how products
can best be used effectively and safely. The trials aim to determine
efficacy in controlling the pest with minimal or no detriment to
the crop being treated. They should demonstrate optimum applica-
tion rates with current application technology. Efficacy trials should
include the proposed maximum label rate, a lower rate and double
the maximum rate for each pest over at least two seasons and with
adequate pest pressures.
The goal of label directions describing GAP is:
• good pest control with minimum phytotoxicity;
• cost-effective pest control using no more pesticide than necessary;
and
• resulting residues in food and feed commodities no higher than
necessary.
Valid national GAP is central to the estimation of maximum
residue levels.
The GAP for different pests on the same crop is not necessarily
the same. For MRL purposes, we are interested in maximum GAP or
maximum registered uses. At the national level, the maximum reg-
istered use includes the maximum rate of use, maximum number of
applications and the minimum interval between the last application
and harvest (JMPR, 1987b).
At the international level, the highest national registered use is
often described as critical GAP (cGAP), which is used for maxi-
mum residue level estimation if suitable supervised trials data are
available.
Residue levels are generally proportional to application rates
when all of the other parameters of GAP (timing, methods of appli-
cation, etc.) remain the same (MacLachlan and Hamilton, 2011).
Residue levels in trials matching GAP in all but the application rate
may generally be proportionally adjusted and then brought into the
residue evaluation. The benefit is a larger data set supporting the
JMPR recommendations.
The evaluation of residues in soybeans from the use of dicamba
herbicide illustrates the JMPR proportionality approach (JMPR,
2011a). In the U.S., dicamba may be used on soybean crops at

1.12 kg ai/ha1 seven days before harvest. No trials were available
at 1.12 kg ai/ha, but 23 trials had been generated with an applica-
tion rate of 2.24 kg ai/ha. So, the residue levels from the 23 trials
were divided by 2 to produce GAP-equivalent data for evaluation
and recommendations.
Growth stage of the crop is sometimes a better description of
the timing of pesticide application than an interval before harvest.
For example, apple dimpling bug is best controlled by a pesticide
application at the beginning of apple blossom. Such timing cannot
be designated by a pre-harvest interval.
Simple growth stage instructions include: Seed treatment, crop
pre-emergence and three leaves unfolded. Crop growth stage descrip-
tions have been standardized (Meier, 2001). Ideally, growth stage
instructions on labels should align with standardized descriptions.
4.2.6 Supervised Residue Trials

If we use a pesticide according to the label directions, what
residue concentration can we expect in the harvested food or feed
commodity?
Supervised residue trials are designed to answer this question.
Supervised residue trials provide the link between the use pat-
tern, especially the label directions for use, and the residues occur-
ring in the food or feed commodity at harvest. When a sufficient
number of trials are available with a use pattern matching critical
GAP and covering the various commercial crop varieties grown under
the expected range of weather conditions, a likely maximum residue
level can be estimated. The trial data also support estimates for
residue levels expected in edible portions of the food commodity;
these estimates are suitable for dietary exposure or dietary intake
calculations. Maximum residue levels are subsequently converted to
legal maximum residue limits (MRLs) for adoption into regulations.
1
kg ai/ha is kilogram of active ingredient per hectare. To avoid ambiguity in
pesticide application rates, we must specify that the weight refers to the active
ingredient (ai). In some situations it could be the formulated product.
The trials are described as ‘supervised’ because all aspects of

the trials must be under control, observation and recording by a
responsible person, the study director. The detailed information in
the study reports is vital for assessing validity of the data.
Experience shows that a wide spread can be expected for pes-
ticide residue data on feed or food commodities from sets of trials
where application rate, number of applications and timing, including
pre-harvest interval, are identical.
Hamilton et al. (1997) examined the data distributions within
102 sets of trials (identical application and timing within each set)
with a minimum of eight trials per data set and one data point from
each trial. Typically, within a data set the highest residue was three
to five times the median, but it ranged up to 30-fold. A later study
based on the analysis of 1950 supervised trial datasets (25,766 residue
values) selected by the JMPR for estimation of maximum residue
levels revealed that, in about 89% of cases, the maximum residues
in a single dataset were within seven times of the dataset median
(Ambrus et al., 2014).
Within the one trial, between-replicate plots variability is also
substantial. It is not unusual for residues in duplicates to be different
by a factor of 2.
The evaluation of supervised trials must always take into account
such variability within and between trials.
The aim is to estimate a maximum residue level at a convenient
rounded number to cover 95% or more of the residues expected in
practice from the critical GAP use pattern.
Statistical methods have been tried over many years to assist with
calculating maximum residue levels. But the requirements are very
demanding: the data should be a random representative sample from
a single population and extrapolation beyond the range of observed
values is usually necessary. National residue data generation guide-
lines have usually emphasized ‘worst case’ situations or likely high
residue cases rather than random and representative.
JMPR has adopted the Organisation for Economic Co-operation
and Development (OECD) calculator, which estimates the maximum
residue level from a set of residue trials as the highest of (1) mean+4
times standard deviation, (2) 3 times mean and (3) the highest
residue in the data set. It then rounds the estimate to a suitable value
for an MRL. For example, a calculated value of 2.3 mg/kg would be
rounded to 3 mg/kg. The OECD calculator has been helpful, but its
results should always be accepted cautiously, particularly for small
data sets. OECD calculated maximum residue levels are given in the
three worked examples following.
Residues levels are generally so variable that it is impossible to
produce residue levels consistently near the highest residue or the
MRL. When the MRL was assumed as the residue level for estimat-
ing long-term dietary intake, it was an impossible assumption. The
supervised trials median residue (STMR), median of the residues,
one from each supervised trial at critical GAP, is closer to the likely
residue when the pesticide is used according to critical GAP; it is
suitable for estimating long-term dietary intake.
The following three examples illustrate how supervised residue
trial data are interpreted when commodity of trade and edible por-
tion are the same or different and when the residue definitions for
MRL enforcement and for dietary intake calculations are the same
or different.
Example. Commodity of trade=edible portion. Single residue
definition.
In this example, the commodity of trade is the same as the edible
portion and the residue definition for enforcement is the same as for
dietary intake calculations (JMPR, 2014b).
O
N O
CH
Cl C
OCH3
Dimethomorph
OCH3
Dimethomorph is a fungicide with registered uses on grape vines,

with grapes the commodity of trade in this example. It is a mixture
of E- and Z-isomers and its residue definition is: dimethomorph (sum

of isomers).
When grape vines were treated with dimethomorph with the US
critical GAP use pattern in twelve trials in nine different states, the
dimethomorph residues on the grapes were: 0.11, 0.26, 0.41, 0.46,
0.49, 0.55, 0.65, 0.71, 0.75, 0.92, 1.77 and 1.86 mg/kg. The OECD
calculator suggested a maximum residue level of 3 mg/kg (mean +4×
standard deviation).
From this data set, the JMPR estimated a maximum residue
level of 3 mg/kg, an STMR of 0.60 mg/kg and an HR of 1.9 mg/kg.
The STMR and the HR (highest residue in the edible portion of
a commodity) are the median and highest residues of the data set
respectively, and are used in dietary intake calculations.
Example. Commodity of trade=edible portion. Single residue
definition.
The commodity of trade in this example is whole bananas, while the
edible portion is the banana pulp. The MRL is set on whole bananas,
while dietary intake is calculated on the banana pulp.
O
N
N N
S
Buprofezin
Buprofezin is an insect growth regulator with registered uses on

bananas. Its residue definition both for compliance with MRLs and
for estimation of dietary intake is: buprofezin (JMPR, 2012). Bupro-
fezin residues were measured in whole bananas and in banana pulp in
supervised residue trials in USA in 1996 and 2003 when buprofezin
was used at U.S. critical GAP on unbagged bananas: 0.34 kg ai/ha
application rate and harvest one day after the final application.
In the six trials, buprofezin residues in whole bananas were:
0.02, 0.04, 0.05, 0.06, 0.07 and 0.18 mg/kg. The OECD calculator
suggested a maximum residue level of 0.3 mg/kg, but warned of
high uncertainty because of the small dataset. Residues in banana
pulp were measured in five of the six trials; in each case, buprofezin
residues were below the limit of quantification, 0.01 mg/kg.
From the data set for residues on whole commodity, JMPR
estimated a maximum residue level of 0.3 mg/kg for buprofezin on
bananas. Estimates for the STMR and HR, used in dietary intake
calculations and based on the residues in the banana pulp, were both
set as equal to the LOQ, 0.01 mg/kg.
Example. Commodity of trade=edible portion. Two residue defini-

tions.
In this example, the commodities of trade are pome fruits where the
whole commodity is edible. Spirotetramat is an insecticide with regis-
tered uses on pome fruits. Its enforcement residue definition for plant
commodities is: spirotetramat plus spirotetramat enol, expressed as
spirotetramat. The dietary intake residue definition includes four
metabolites: spirotetramat plus the metabolites enol, ketohydroxy,
enol glucoside and monohydroxy, expressed as spirotetramat (JMPR,
2008a).
O
HN
CH3O
O
O
C2H5O
Spirotetramat
Twelve supervised trials on apples and six on pears were avail-

able where spirotetramat had been used in 2005 in nine different
states and provinces in accord with critical GAP in USA and Canada.
Spirotetramat residues, enforcement residue definition, were: apples
0.038, 0.042, 0.051, 0.072, 0.072, 0.077, 0.13, 0.13, 0.14, 0.21, 0.33
and 0.49 mg/kg; pears: 0.075, 0.084, 0.16, 0.17, 0.22 and 0.32 mg/kg.
The apple and pear data were combined to support a pome fruits
maximum residue level estimated at 0.7 mg/kg, a value suggested by
the OECD calculator.
For dietary intake estimations, the residues measured as spirote-

tramat plus four metabolites were: apples 0.073, 0.076, 0.085, 0.11,
0.11, 0.13, 0.17, 0.17, 0.18, 0.37, 0.38 and 0.55 mg/kg; pears: 0.10,
0.16, 0.20, 0.21, 0.26 and 0.37 mg/kg. From the combined apple and
pear dietary intake residue definition data, pome fruits STMR and
HR were estimated as 0.17 and 0.55 mg/kg respectively.
Where possible, JMPR aims to estimate maximum residue levels
for Codex commodity groups. In the spirotetramat example above,
the data for apples and pears produced a recommendation for pome
fruits in preference to individual recommendations for apples and
pears. Codex group MRLs have the advantage that they cover the
MRL needs of minor commodities within the group at minimal
cost.
With sufficient data and when the label directions on a pesticide
specify a crop group, such as citrus trees, that is readily aligned
with a commodity group, such as citrus fruits, proceeding to a group
MRL is relatively straightforward. Commodity group MRLs have
been achieved most readily for citrus fruits, pome fruits, cucurbit
fruiting vegetables and tree nuts.
4.2.6.1 The minor crop problem

In most countries, pesticides are subject to a government registration
or approval process before they can be lawfully used. Governments
typically issue a set of data requirements, which, if fulfilled, should
demonstrate that the pesticide can be safely and effectively used if
label directions are followed.
Safe use includes safety for the user, the environment and for
public health. Effective use means that the pest can be controlled and
the crop is not damaged by the product. The intending registrant,
typically a pesticide company, will take into account the investment
required, including the cost of registration and the data package.
A minor crop is one where the scope for product sales is too
small to justify the investment of developing a full data package. The
anticipated revenue is only sufficient for a small number of supervised
residue trials, to support the setting of an MRL. Some crops, such as
herbs and spices, are classified as very minor, hardly justifying any
data generation.
Minor use crop: crop that is grown on a small area and therefore
uses amounts of pesticides that are too small to justify standard
pesticide registration (Stephenson et al., 2006).
Commodity group MRLs are a very practical way of covering

food and feed commodities from minor crops with acceptable MRLs,
but group MRLs are not always possible. In situations where the
registration of a pesticide for use on a minor or specialty crop is
essential for its viability, the growers themselves or the government
may work with the pesticide company to generate the necessary sup-
porting data.
An example of a minor crop is spring onions. Difenoconazole
residues in spring onions seven days after treatment as described
by the registered use in USA were 2.3, 2.8 and 3.8 mg/kg (JMPR,
2013a). JMPR accepted the three trials as sufficient and estimated
a maximum residue level of 9 mg/kg.
Subsequently, CCPR decided that four trials would be the mini-
mum to support a Codex MRL (CCPR, 2015).
4.2.6.2 Spices
Spices are mostly grown on very small farms in developing countries.
With the number of different spices and the many pesticides, invest-
ment in generating supervised trials data is not generally viable.
CCPR decided that, in the special situation of spices, MRLs sup-
ported by monitoring data would be acceptable.
Spice trade associations from India, USA, Europe and Egypt were
already collecting residue monitoring data (CCPR, 2002). Spices
were regularly tested at the point of entry into importing countries.
JMPR, in 2002, 2004 and 2009, provided guidance on the submission
of monitoring data for residues in spices, now provided in the FAO
Manual (Ambrus, 2016, pp. 63–66).
Monitoring data have different characteristics from supervised

trials data in several respects:
— Pesticide treatments are not known.

— The commodity in trade may be a mixture originating from sev-
eral farms with different pesticide treatments.
— The multi-residue methods used for monitoring may not always
include all of the components of some residue definitions.
Sufficient valid data are required to provide 95% assurance that

at least one sample would exceed the 95th percentile value in the pop-
ulation being sampled. A maximum residue level is then estimated
by rounding up to a value suitable for use as an MRL.
Example. Pirimiphos-methyl residues in spices (JMPR, 2004).
Monitoring data were available for 1,137 samples of the seed sub-
group of spices. Pirimiphos-methyl residues were detected in 16 of
492 samples of anise, ranging from 0.05 to 1.8 mg/kg. JMPR esti-
mated a maximum residue level of 3 mg/kg for the seed subgroup of
spices, based on the anise data.
The median of the 16 detected residues was 0.23 mg/kg and the
high residue was 1.8 mg/kg; these values were available in place of
an STMR and HR for dietary intake calculations. A factor of 0.03
(16 detects from 492 samples) was also provided for use in long-term
intake calculations.
The monitoring data approach for evaluation of spices has been
followed since the early 2000s and, by 2015, Codex MRLs (CXLs)
for spices had been established for 47 compounds.
4.2.7 Food Processing

Many foods are processed before sale and consumption (e.g. wheat
to flour to bread; grapes to juice or wine; oilseeds to vegetable oils;
tomatoes to canned tomatoes and tomato paste; and fruit to dried
fruit). We have questions about the effects of food preparation and
processing on pesticide residues.
• Does the process influence the composition of the residue in the

processed commodity originating from residue in the raw commod-
ity?
• For processes that produce more than one food product or by-
product, which pathways are taken by residues from the raw com-
modity?
• What is the change in concentration of the residue in progressing
from raw commodity to processed food or feed commodity?
Timme and Walz-Tylla (2004) explained that simple hydrolysis

is the most likely degradation mechanism for pesticide residues in
food processing because heating at some stage of the process would
generally inactivate intrinsic enzymes.
Laboratory studies with [14 C]labelled pesticide subjected to suit-
able conditions of pH, temperature and time are conducted to repre-
sent hydrolysis occurring in the processes of baking, brewing, boiling,
pasteurization and sterilization. The 14 C label assists in isolating and
identifying hydrolysis products and accounting for the fate of all of
the starting material. Such laboratory studies are not intended to
simulate commercial food processing; the 14 C studies provide infor-
mation on the identity of compounds to be included in the analyses
of processed commodities from commercial or simulated commercial
food processing.
Example. Hydrolysis of pymetrozine during the conditions of boiling,

baking or brewing (JMPR, 2014c).
After [14 C-pyridine]pymetrozine was subjected to hydrolysis con-
ditions of a pH 5 buffer at 100◦ C for 60 minutes, 57.6% of
the 14 C remained as parent compound with 42.1% hydrolyzed to
3-pyridinecarboxaldehyde.
N N N N
CH NH
O
Pymetrozine
In studies of the fate of pymetrozine in food processes that

include boiling, baking or brewing, 3-pyridinecarboxaldehyde should
be selected as one of the analytes for study.
Concentrations of pesticide residues in processed commodities
may be different from concentrations in the originating raw agricul-
tural commodity (RAC). Processed commodity may have reduced
concentrations if residue is lost by washing and cleaning, or broken
down by heating or other process, or physical separation such as
the peeling of fruit. Increased concentration of residue in processed
commodity may result from drying (e.g. production of dried fruits),
or physical separation such as conversion of oilseed into oil and meal
where fat-soluble residues will preferentially partition into the oil.
For processing studies, residues in the RAC should be incurred
residues. It is not necessary to follow the GAP application rate when
treating a crop for processing studies; in many situations, an exagger-
ated application rate is preferable so that residue levels in processed
commodities are sufficiently high for measurement by the analytical
method.
Processing factor: Residue level of a specific pesticide in the pro-
cessed product divided by the residue level in the starting commod-
ity, usually a RAC. Processing factor = residue level (mg/kg) in
processed product ÷ residue level (mg/kg) in RAC (Stephenson
et al., 2006).
Example. Milling of wheat (JMPR, 2003).

Fenitrothion is registered as a post-harvest storage treatment on
cereal grains as protection against stored products insects. In 1987,
wheat was treated with fenitrothion, stored for one and three months
and then subjected to pilot milling. Fenitrothion residue concentra-
tions were measured in the milling fractions (Table 4.1).
O NO2
CH3O
P
CH3O
S Fenitrothion
Table 4.1. Fenitrothion residues in grain and milling fractions of

wheat after a post-harvest treatment.
Fenitrothion, mg/kg
1-month 3-months’
Commodity Storage Storage Processing Factors
Wheat, whole grain 7.0 7.6

Bran 28 30 4.0, 3.9 mean 3.95
Flour 1.5 2.0 0.21, 0.26 mean 0.235
In this case, much of the residue was on the surface of the grain
and so resided with the bran after milling. The processing factor for
bran was higher than 1, so it was a concentration factor. For flour,
it was less than 1, a reduction factor.
The next step was to calculate STMR and HR values for pro-
cessed commodities (i.e. STMR-P and HR-P values). The STMR
and HR for fenitrothion on cereal grains were estimated as 4.25 and
5.6 mg/kg respectively (JMPR, 2007b).
STMR-P = STMR × processing factor.
STMR-P for flour = 4.25 × 0.235 = 1.00 mg/kg.
STMR-P for bran = 4.25 × 3.95 = 16.8 mg/kg.
HR-P = HR × processing factor.
HR-P for bran = 5.6 × 3.95 = 22.1 mg/kg.
The calculated STMR-P and HR-P values were then used in
dietary intake calculations.
MRLs for raw agricultural commodities also apply to processed
commodities derived from them. For example, an MRL for wheat
also applies to flour, bread, noodles and wheat bran. In the feni-
trothion example, the MRL for cereal grains, which includes wheat,
is 6 mg/kg. Residue levels in flour will be below those for wheat
because the processing factor is less than 1; a similar argument
applies to bread and noodles. But residue levels in wheat bran could
well exceed those in wheat, so a separate MRL is needed for wheat
bran and JMPR has recommended 25 mg/kg as a suitable MRL
for fenitrothion residues occurring from the milling of post-harvest
treated wheat.
For residues arising from a pre-harvest use on wheat, a slightly

different approach is needed. In that case, bulking and blending of
grain from a number of different farms at the storage and milling
facilities will result in dilution and is unlikely to produce grain with
residues exceeding the STMR; a maximum residue level will be esti-
mated accordingly.
In contrast, post-harvest treatment is most likely at the stor-
age facility after the bulking and blending stage and its attendant
dilution. So, the highest residue from the supervised trials is more
influential on maximum residue level estimation.
4.2.8 Livestock and Residues in Meat, Eggs and Milk
Pesticide residues may occur in meat, milk and eggs as a result
of residues in feed materials, which constitute a wide range of
commodities.
Primary animal feeds include legume animal feeds such as alfalfa
fodder; straw, fodder and forage of cereal grains (e.g. maize forage);
and miscellaneous fodder and forage crops (e.g. fodder beet).
Processed commodities used for animal feed include milled cereal
products such as wheat bran; by-products of fruit and vegetable pro-
cessing (e.g. apple pomace) and miscellaneous secondary food com-
modities of plant origin (e.g. cotton seed meal).
Food commodities used as animal feeds include root vegetables
such as potato culls; pulses (e.g. dry beans); and cereal grains (e.g.
maize).
Residues in animal feeds are usually expressed on a ‘dry-weight’
basis. A ‘dry-weight’ basis implies that the commodity is analysed
for pesticide residues as received, that the moisture content is deter-
mined, preferably by a standard method for use on the relevant com-
modity, and that the residue content is then calculated as if it were
wholly contained in the dry matter (Joint FAO/WHO Food Stan-
dards Programme, 1993, p. 328).
When pesticide residues occur in animal feeds, we have some
questions.
• Will the residues occur in primary food commodities of animal
origin (i.e. meat, milk and eggs)?
• At what concentrations will the residues occur in muscle, fat, liver,

kidney, milk and eggs?
• How will the residue be distributed between the fat and non-fat
portions of milk?
Livestock or farm animal feeding studies, usually lactating dairy

cow and laying hen, are designed to answer the questions.
In such a study, groups of animals are dosed daily or twice daily,
usually for 28 days, with pesticide equivalent to anticipated amounts
in the animal feed. Milk or eggs are collected daily for analysis. Ani-
mals are slaughtered within 24 hours of the final dose and muscle,
fat, kidney and liver are collected for analysis.
The livestock metabolism studies have already determined the
nature of the residue in animal commodities. Analytical methods
should be developed and validated for those analytes, including con-
jugates where necessary.
Conjugate: Molecular species produced in living organisms by cova-

lently linking two chemical moieties from different sources.
Example: Conjugate of a pesticide or metabolite with groups such
as glutathione, sulphate, or glucuronic acid making it more soluble
in water and facilitating its compartmentalization within the cell
Residue levels in milk or eggs normally reach a plateau within five

to 10 days, which means that the breakdown and elimination rate of
residues is approximately the same as the dosing rate. In exceptional
cases where a plateau is not reached within 28 days, a longer dosing
regime is needed.
Dosing rates are chosen to cover anticipated intakes of residues
from consumption of feed commodities, including forages and pas-
tures. When data are available on residue levels in feed commodities,
a ‘livestock dietary burden’ can be calculated as a theoretical highest
dose of residues to be expected from the animal diet.
The aim of the livestock dietary burden calculation is to combine
the residue data for feed commodities with specified livestock diets
to estimate an equivalent residue concentration for the whole live-

stock diet. Livestock feeding practices are different from one place
to another because of cultural practices, climate and availability of
feed commodities. Specified diets for beef and dairy cattle, poultry
(broiler, layer and turkey), sheep and pigs have been provided from
four geographical regions USA–Canada, European Union, Australia
and Japan (Ambrus, 2016, pp. 193–204). Additional information is
available in Chapter 2 (the livestock feed tables).
The specified diets include primary animal feeds, by-products of
food processing and food commodities used as animal feeds. For the
purposes of the dietary burden calculation, residue concentrations
are all expressed on a dry weight. Each commodity is expressed as a
maximum percentage of the diet. To make the calculation, we choose
the commodities in descending order of residue concentration, mul-
tiplying each concentration by its percentage of total diet, but only
up to 100% of the diet. A further restraint is the requirement to
observe maximum percentages for commodities of the same group.
For example, in one diet bean seed and pea seed are each specified as
20% of the broiler diet, but they are of the same commodity group,
so we could use only one of them in the calculation. In practice,
an automated spreadsheet, the OECD feed calculator, can make the
calculations for all of the chosen commodities, the various livestock
and the four geographical regions (Ambrus, 2016, p. 282).
The results of the feeding study then produce a direct link
between the livestock dietary burden and the expected residues in
meat, eggs and milk. Studies on dairy cows and laying hens are obvi-
ously quite different in detail but they are similar in principle.
Example. Dairy cow feeding study with bixafen (JMPR, 2013b).

Lactating dairy cows (three cows per group for the two lower doses,
six for the highest) were dosed orally, via capsule, for 29 consecutive
days with bixafen at the equivalents of 4, 12 and 40 ppm in the
dry-weight diet. The unit ‘ppm’ (parts per million) is customarily
used for concentration in feed in animal feeding studies to avoid the
confusion, which could arise between ‘mg/kg’ (mg per kg feed, a
concentration) and ‘mg/kg bw’ (mg per kg of bodyweight, a dose)
(Ambrus, 2016, p. 213). Milk was collected twice daily for analysis
of residues (bixafen and metabolite de-methylbixafen). On day 29,
animals were slaughtered and muscle, fat, liver and kidney were col-
lected for residue analysis. Three animals from the 40 ppm group
were not slaughtered but dosing ceased after day 29 and their milk
was monitored to observe how quickly residues would deplete.
Cl N
N
Cl
HN CHF2
O
F Bixafen
Residue levels in milk reached a plateau after about four days

of dosing with residue levels of approximately 0.025, 0.065 and
0.20 mg/kg in the 4, 12 and 40 ppm dosing groups respectively
(Fig. 4.2). Residue levels in cream were approximately 10× levels
in whole milk and 50× levels in skim milk, demonstrating that the
residue is fat soluble.
The resulting residue levels in the animal tissues as a function of
the concentration in the feed are shown in Fig. 4.3. If, for example,
the livestock dietary burden for bixafen were 10 ppm, we could use
the plot to determine an expected residue of 1.2 mg/kg in liver and
similarly for the other tissues. Such calculations support the estima-
tion of STMRs, HRs and maximum residue levels for meat, milk and
eggs.
Pesticides are registered for direct use on livestock and livestock
housing for control of insects and parasites. Such uses may produce
residues in meat, milk and eggs; suitable supervised trials generate
residue data for evaluation and MRL recommendation. If the same
pesticide has direct uses on livestock and produces residues in feed
commodities, then the source of the higher residues will prevail in the
MRL decision. It would be an unlikely event for the residues from
0.25 Bixafen residues in milk

Stop dosing
Residues, mg/kg
0.2
0.15 4 ppm feed

12 ppm feed
40 ppm feed
0.1
0.05
0
0 5 10 15 20 25 30 35
Days
Figure 4.2. Bixafen residues, means for each group at each time, in milk during
34 days of a feeding study on lactating dairy cows at three feed concentrations.
Dairy cow, feeding study
4Residues in
tissues, mg/kg Liver
Muscle
3 Kidney
Peri-renal fat
2
0
0 5 10 15 20 25 30 35 40 45
Bixafen, ppm in feed
Figure 4.3. Residue levels in the tissues from a dairy cow feeding study with
bixafen at the equivalent of 4, 12 and 40 ppm in the animal feed dry weight for
29 days.
each source to be less than the MRL with the sum exceeding the
MRL.
The direct application is usually specific to a species (e.g. sheep,
while residues in feed could produce residues in any mammalian live-
stock). In this case, if direct application produced the higher residues,
MRLs would be recommended for sheep meat (based on direct use)
and for mammalian meat except sheep meat (residues from feed).
4.2.9 Monitoring Data, EMRLs

DDT, dieldrin, heptachlor and lindane are four insecticidal
organochlorine compounds that were once widely used in crop pro-
tection, but no longer have such uses. They are non-polar fat-soluble
compounds with low soil mobility and high resistance to microbio-
logical attack.
Such compounds are highly persistent in surface soils and they
can become residues in crops grown in that soil and in food-producing
livestock grazing on that soil or on feed sourced from that soil (i.e.
residues that occur are not under the control of GAP).
The JMPR introduced the ‘extraneous maximum residue limit
(EMRL)’ to cover residues of such environmental contaminants in
feed and food commodities. Codex EMRLs have been established
for: aldrin and dieldrin, chlordane, DDT, endrin and heptachlor. In
addition, JMPR (2015c) has recommended EMRLs for lindane in a
number of plant and animal commodities.
Because of the persistence and fat-solubility of these organochlo-
rine compounds, the fat tissue and milk fat of livestock are especially
susceptible to residue accumulation when the animals have environ-
mental exposure to these compounds.
Residue monitoring data are required to support the recommen-
dation of an EMRL. JMPR evaluates the data for relevance, validity
and sufficiency to establish the incidence of residues occurring and to
assess consumer risk. Possible EMRL values are assessed against the
incidence for potential violation rates. The violation rate is described
by the percentage of monitoring samples with a residue exceeding the
selected residue level.
40
35 Incidence of residues as a function of concentration
Incidence
30
25
1.6% of samples
20 exceed 1 mg/kg
15 0.43% of samples
exceed 2 mg/kg
10
0.04% of samples
5 exceed 5 mg/kg
0
0 1 2 3 4 5 6 7
Residue, mg/kg
Figure 4.4. The upper tail of the incidence of DDT residues in 4682 samples of
the tissue fat of livestock from a monitoring program in New Zealand from July
1990 to June 1994 (JMPR, 1996).
The evaluation of DDT in 1996 provides an example of an EMRL

assessment (Fig. 4.4). In that case, the New Zealand data were the
critical population, but the evaluation was supported by analyses on
more than 162,000 samples of meat fat from around the world.
JMPR recommended an EMRL of 5 mg/kg for DDT residues in
mammalian meat (fat). Selection of an EMRL on the basis of the
residue distribution as displayed in Fig. 4.4 is a risk management
decision to be decided by CCPR, as a balance between ‘as low as
reasonably achievable’ and minimizing unnecessary trade disruption.
4.3 Toxicological Evaluation

4.3.1 Data Requirements for Toxicological Evaluation
In this section, the accepted requirements for toxicological data and
the importance of the assessment of the quality, relevance and utility
of all published and proprietary studies are described.
4.3.1.1 Toxicity data used for toxicological evaluation

The main process for toxicological evaluation of pesticides is shown
in Fig. 4.5. Many toxicity studies using experimental animals are
Toxicity data Acute toxicity Reproductive/developmental

toxicity
Repeated toxicity
GLP
Test guidelines TK, Neurotoxicity, Carcinogenicity
MOA, metabolites etc. Genotoxicity
Analysis for toxicity profiles

New
scientific Integrated analysis
evidence in
NOAELs/LOAELs
toxicology
for each toxicity/carcinogenicity
Chronic effect Acute effect
ADI ARfD
Figure 4.5. The main process for toxicological evaluation of pesticides.
evaluated in the toxicological evaluation for setting the ADI and

ARfD. An overview of the required toxicity studies for the evaluation
is listed in Table 4.2.
These toxicity studies may differ with respect to their pur-
pose, experimental design or experimental animals used. The entire
database is aimed at gaining an insight into the toxicological profiles
including toxicological targets, severity of toxicity, interspecies differ-
ences, no-observed-adverse-effect-levels (NOAELs), lowest-observed-
adverse-effect-levels (LOAELs) and mode of action (MOA) for the
observed toxicological effects. Based on this database, the toxicologi-
cal knowledge about the chemical is integrated in order to character-
ize the human health hazard and to determine safe exposure levels for
the entire human population, including susceptible sub-populations.
All toxicity studies as well as other relevant scientific data of
all influences of the different dose levels, toxicokinetics (TK) of pes-
ticide exposure in the tissues, disposition and metabolism within
an organism, and excretion from the body, which are evaluated in
absorption, distribution, metabolism and excretion (ADME) studies
Table 4.2. Common toxicity studies and their purposes.
Study Purpose Species
Oral acute toxicity Acute toxicity, approximate Rat, mouse

study lethal dose
Irritation, sensitization Irritation to skin and eyes, In vitro or rabbit,
skin sensitization guinea pig
Short-term repeated
toxicity study
Toxic effects on target organs Rat, mouse, dog
for short-term exposure
Species differences of
toxicities observed
Long-term repeated
toxicity study,
carcinogenicity
Toxic effects on target organs Rat, mouse
for long-term exposure
Carcinogenicity Rat, mouse
Genotoxic study Genotoxic effects In vitro and in vivo
Reproductive toxicity Toxicity for parents, Rat
study reproduction and offspring
Development toxicity Toxicity for dams, foetus and Rat, rabbit
study embryo
Neurotoxicity study Acute and subacute Rat, hen
neurotoxic effects
Mechanistic studies Toxic effects of metabolites, Rat, mouse, in vitro
mode of action
are necessary for hazard characterization. Although the types of tox-

icity studies requested by legislation are different depending on the
responsible regulatory bodies, basic objectives of each toxicity study
of pesticides as well as of other chemicals, veterinary or medical drugs
and devices are almost common. A short description of each type of
study is presented below. For pesticides, besides national regulatory
bodies, the WHO expert panel of the JMPR provides independent
scientific expert advice to Codex, and has been playing an impor-
tant role in the development of the toxicological evaluation process
of pesticides for over 50 years (Fig. 4.6).
Toxicological evaluation
by WHO group FAO group
Hazard identification
Exposure
Hazard assessment
characterization
Dose-responsibility
extrapolation to humans
Joint
Risk characterization
Figure 4.6. Process of risk assessment at JMPR.
4.3.2 Importance of Quality and Reliability Control

of Toxicity Data
It is crucial for the toxicological evaluation of a substance to have
reliable data that are transparently presented in a study report. As
described in Chapter 2, the quality of toxicity studies is controlled by
two main principles, good laboratory practice (GLP) and test guide-
lines for each toxicity study adopted by OECD or national authorities
since the 1970s.
The question of how to consider different sources of information
such as scientific results published in peer-reviewed literature in addi-
tion to studies conducted according to test guidelines for regulatory
purposes has long been a source of discussion in the public domain as
well as between industry, non-governmental organizations and regu-
latory authorities. Importantly, the criteria taken to judge reliability
and relevance of the studies are considered to influence the decision
about their inclusion into regulatory processes.
Also, JMPR will make transparent in its evaluations the criteria
and approaches used to determine the quality, relevance and utility
of all published and proprietary studies considered. Internal guide-
lines to consolidate the criteria for data inclusion or exclusion with
respect to published and proprietary data sources will be developed
(JMPR, 2015a).
4.3.2.1 Compliance with quality principles

GLP principles or other quality systems of management controls for
toxicity studies of test chemicals were introduced to ensure unifor-
mity, consistency, reliability, reproducibility, quality and integrity of
the data (OECD, 1998). Practically, GLP and other management
systems require transparent quality controls of devices and their stan-
dard manuals used in toxicity studies as well as test facilities. The
GLP principle also controls the qualities of human resources con-
ducting the studies including continuous training and enhancement
of staff quality.
The quality assurance unit (QAU) is independent from test facil-
ity manager and study director. QAU continuously monitors and
audits all studies and related data whether they are conducted in
compliance with the principles of GLP or not. If not, the managers
or study director must respond to the audits. Thus, the QAU plays
an important role in GLP compliance.
4.3.2.2 Accordance to OECD test guidelines

Test guidelines are a set of minimum required specifications for the
testing of chemicals. The OECD test guidelines are internationally
agreed test methods in toxicity studies of pesticides. The guidelines
describe the test and provide information on the method includ-
ing information on the number of animals, route of administration
and the parameters to be assessed. More detailed information on the
OECD test guidelines can be found in Chapter 2.
4.3.2.3 Quality, relevance and utility of published studies

Recently, the importance of a mechanism-based approach in the
extrapolation of the toxicity detected in experimental data to assess
the human health hazard has increased. When considering mecha-
nisms, apart from the observations made in the studies performed
according to guidelines, published information including textbook
or reviewed articles can be useful. Large-scaled and well-designed
epidemiological studies are useful for risk assessment because these
data might directly indicate toxic or carcinogenic risks of chemicals to
humans. However, it should be always carefully considered whether

the published information is suitable for the purpose of risk assess-
ment of chemicals and whether the quality of published data is
acceptable. When published data are used for toxicological evalu-
ation, the following points should be considered:
• Raw experimental data are generally not available in published

studies and therefore cannot be checked. Thus, transparency at
GLP quality level cannot be guaranteed. Several publications have
referred to detailed background data published in separate appen-
dices.
• Although generally reliability comparable to GLP studies can-
not be guaranteed, publications with adequate explanation about
experimental designs (preferably in accordance with OECD test-
guidelines) and publications from journals with review systems are
likely to be suitable for use in the toxicological evaluation.
• Industries that have developed pesticides have sometimes pub-
lished their mechanisms of toxicity in scientific journals, in addition
to the special studies on the mechanisms of action that are directly
submitted for evaluation to the regulatory bodies. In such situa-
tions, the articles may be considered helpful because of scientific
confirmation of the mechanism by peer reviewers of the journal.
• It is very important to check for the specification and formulation
of the test chemical used in a study. Therefore, information on
appropriate preparation and analysis of stability or uniformity of
test materials is required for each toxicity study.
• Publications might be of low reliability if the authors have used test
materials of commercially sold formula or inappropriately prepared
test materials with no description about analysis for stability or
uniformity of test materials. As these factors may greatly affect
the outcome of the study, the relevance of such studies for the
toxicological evaluation of a chemical is questionable.
• Statistical analysis is also an important part of the toxicological
evaluation. However, risk assessors should pay adequate attention
that a statistical significance does not necessarily indicate a toxi-
cological endpoint. The selection of an appropriate statistical test
is part of the study plan to avoid application of incorrect statisti-

cal testing, if the study is finalized. In 2011, EFSA published its
guidance on submission of scientific peer-reviewed open literature
for the evaluation of pesticides that sets out how to identify and
evaluate scientific peer-reviewed open literature (EFSA, 2011).
4.3.3 Main Endpoints for Toxicological Evaluation

Scientific ability and experience of integrating all changes detected
in the study and then accurately choosing appropriate endpoints are
required skills for risk assessors. Practical steps and evaluation of
changes detected in all toxicity studies are introduced in a later sec-
tion, ‘Process of Toxicological Evaluation in Risk Assessment’.
4.3.3.1 ADME study and kinetics

Knowledge of the biological disposition, absorption, distribution,
metabolic biotransformation or elimination (ADME) of a chemical
is a key part of any hazard characterization and risk assessment
(EHC240, 2009). The qualitative characterization of xenobiotic dis-
position is termed pharmacokinetics or toxicokinetics. Toxicants usu-
ally pass through a number of cells (e.g. in the gastrointestinal (GI)
tract) and ultimately the cells of the target organ. The basic unit
of the cell membrane is a phospholipid bilayer. Many toxicants cross
the cell membranes either by passive processes, or by mechanisms
in which the cell provides energy. Active transport is a specialized
system for large-sized compounds to cross membranes rapidly. It is
characterized by movement of chemicals against electrochemical or
concentration gradient, saturability at high substrate concentrations,
selectively for certain structural features of chemicals or competitive
inhibition by chemical cogeners and requirement for expenditure of
energy. The transport system of chemicals plays an important role in
each process of ADME. More detailed information can be obtained
from textbooks of toxicology (Lehman-McKeeman, 2007; Parkinson
and Ogilvie, 2007; Shen, 2007).
Absorption: The process by which toxicants cross body membranes

and enter the bloodstream is referred to as absorption. The GI tract
is the most important site for dietary exposure to a chemical in

food and feed. Solubility is the primary factor affecting absorption.
Insoluble salts and ionized compounds are poorly absorbed, whereas
lipid-soluble substances are generally readily absorbed. A number
of specialized transport systems in the GI tract are involved in the
absorption.
Major parameters of toxicokinetics (TK) data are expressed as
the internal dose in animals based on plasma, serum or blood con-
centrations of the parent chemical or its active metabolites, including
the area under the concentration–time curve (AUC), the peak con-
centration (Cmax), the time of the peak concentration (Tmax) and
the amount of time required for the disappearance of half of the
compound (half-life, T1/2).
Distribution: After entering the blood by absorption a toxicant is
distributed to tissues throughout the body. The rate of distribution
to organs or tissues is determined primarily by blood flow and the
rate of diffusion out of the capillary bed into the cells of a particular
organ or tissue. The penetration of toxicants into cells occurs by
passive diffusion or special transport processes. Binding to plasma
proteins, mainly albumin, is the major site of protein binding. Toxic-
ity is typically manifested by the amount of an unbound xenobiotic,
and differences in plasma protein binding may explain species-specific
differences in the disposition and toxicity of xenobiotics. The liver
and kidney have a high capacity for binding many chemicals. The
compounds with lipophilic nature permit rapid penetration of cell
membranes and uptake by tissues such as storage in body fat. Distri-
bution is influenced by natural barrier systems including blood–brain
barrier, blood–cerebral spinal fluid barrier or blood–testis barrier.
The placental barrier is not a precise anatomical unit like the blood–
brain barrier. In the placenta, xenobiotics transporters differently
expressed in various cells contribute to the barrier system.
Metabolism: Metabolism and biotransformation of toxicants is the
process of converting lipophilic chemicals, which are readily absorbed
into the body to more hydrophilic metabolites that can be excreted
in urine or bile (see below). The metabolism is basically catalysed
by various enzyme systems that can be divided into Phase I

(oxidation, reduction and hydrolysis mechanisms by catabolism in
hepatic enzymes to generally convert foreign compounds to deriva-
tives for Phase II reactions) and Phase II (principally conjugation
or synthesis reactions). Common conjugates include glucuronides,
acetylation products and combinations with glycine. Metabolism of
xenobiotic agents is often complicated, and metabolized xenobiotics
are sometimes more toxic than their parents. In JMPR monographs,
biotransformation pathways of test substances in animals based on
the available information are included.
Excretion: Toxicants are excreted from the body by several routes

such as kidney or faeces. In the kidney, a toxicant filtered at the
glomerulus may remain in the tubular lumen and be excreted with
urine. Toxicants with a high lipid–water partition coefficient are reab-
sorbed efficiently, whereas polar compounds and ions are excreted
with urine. Toxic agents are also excreted from plasma into urine
by passive diffusion through the renal tubule, or by active secretion
through various transporters. Many polar and high-molecular-weight
compounds are excreted into the bile. The proximal convoluted
tubule is the most common site of toxicant-induced injury. For fae-
cal excretion, the biliary route of elimination is the most signifi-
cant source for xenobiotics and their metabolites. Biliary excretion
is regulated predominantly by xenobiotic transporters located on the
canalicular membrane. Biliary excretion is the phenomenon of entero-
hepatic circulation, which means that a compound excreted into bile
enters the intestine, and may be reabsorbed or eliminated with faeces.
4.3.3.2 Acute toxicity, irritation and sensitization

The purpose of acute toxicity studies is to observe the responses of
a test organism to single-dose exposure to pesticides with death as
the major endpoint of the toxicity. Young adult rats are commonly
used. The administration route by gavage is selected because it is
the same exposure route as that of the consumer. The observation
period is usually accepted as 14 days after the single dose treatment.
At termination, all animals are necropsied.
Studies on skin and eye irritation and also on sensitization might

be very important for the evaluation of risks for operators, bystanders
or residents, if they are coming into contact with a pesticide. But
these endpoints may be also helpful for the understanding of irri-
tation or other local adverse dose-dependent effects of the digestive
tract.
4.3.3.3 Short-term toxicity

Short-term toxicity studies are conducted to detect general systemic
toxicity by repeated dose treatment at multiple dose levels. A number
of parameters are evaluated to identify target organs and tissues
for toxicity, observed adverse or no-adverse effect levels and dose–
response relationships of the effects. Although a recovery phase is
not commonly included in a short-term toxicity study of pesticides,
the information about reversibility from the repeated toxicities could
be useful for characterization of hazard. At the evaluation of systemic
toxicity of a chemical, understanding TK and ADME data is very
informative because its toxicity takes place after delivery of chemicals
and their removal from the site of action.
Many authorities request the data of short-term toxicity of two
different species, which are rodent (rat or mouse) and dog. Dura-
tion of the study period is usually 90 days, and young or imma-
ture animals of both sexes are used. Ten rats or four dogs per sex
and per group are usually allocated. Setting multiple dose levels is
necessary to observe dose responsiveness of effects and to determine
a no adverse effect level and the lowest effect level. A four-week tox-
icity study is conducted as a dose finding study for 90 days study. A
short-term toxicity study in mice may be conducted as a range find-
ing study for carcinogenicity study and is applicable for detecting
short-term toxicity. Oral administration in the diet or by gavage is
the common route for pesticides. The one-year dog study is expected
to identify whether the severity of the adverse effect increases with
exposure duration and whether the adverse effect is observed at lower
doses after increased exposure duration. In addition, longer expo-
sure may reveal new toxicities not observed in a study with shorter
duration. Recently, several national authorities eliminated it from

the data requirement lists for registration based on comparison of
toxicities and NOAELs between 13-week and one-year dog studies
(EPA, 2007; Dellarco et al., 2010; EU, 2013).
In a repeated dose toxicity study, various parameters including
continuous monitoring observations such as clinical signs or body
weights, haematology, blood biochemistry, urinalysis or ophthalmol-
ogy could be analysed. At termination, all animals are necropsied for
pathological analysis. Histopathological examination of many organs
and tissues is accepted to be a powerful tool for detection of tox-
icity. For details on experiment designs (species, duration of study,
parameters, etc.) and the toxicological significance of parameters, the
OECD test guideline described in Chapter 2 should be consulted.
4.3.3.4 Long-term toxicity and carcinogenicity

Long-term toxicity–carcinogenicity studies are conducted generally
in rodents to investigate chronic effects and carcinogenicity of the test
compound. A two-year rat study and an 18-month mouse study are
usually required. In combined long-term and carcinogenicity studies,
interim kill (usually at 12 months) is performed. Parameters in the
study are almost identical to those in short-term toxicity studies. For
analysis of carcinogenicity, 50 or more animals per sex are allocated
in each group. Standard experiment designs of (combined) chronic
toxicity and carcinogenicity studies are published in Test Guidelines
451, 452 and 453 by OECD (OECD, 2009a, 2009b, 2009c).
Since the treatment period of the study covers almost the entire
lifetime of rats or mice, a number of age-related non-neoplastic and
neoplastic lesions spontaneously occur in many organs and tissues
in most animals of control and treated groups. Treatment-related
changes observed in a short-term study may often be exacerbated
due to long-term exposure. This is elicited as increased incidence or
severity of the change in many cases, but sometimes it may be masked
by age-related changes. In addition, treatment-related toxicities often
appear as enhancements of spontaneous disease in the target organ
such as increased incidence of chronic progressive nephropathy or
degeneration of the peripheral nerve. Comparison to not only data
in the control group but also historical control data may be neces-
sary for interpretation. Continuous stimulation of the target organ or
tissue by a toxic substance sometimes can result in increased tumour
incidence.
Ageing is known to be associated with unrepaired accumulation
of naturally occurring DNA damages (Freitas and de Magalhães,
2011), some of which might result in spontaneous occurrence of
tumour formation in mammals. If pesticides are considered not geno-
toxic, treatment-related increases in carcinogenicity are interpreted
as a promoting effect for which it is considered acceptable to estab-
lish a threshold for carcinogenicity. In many cases, tumour forma-
tion is related to non-neoplastic changes (e.g. thyroid follicular ade-
noma may be induced by prolonged stimulation of thyroid stimulat-
ing hormone through follicular cell hypertrophy or hyperplasia), and
pesticide treatment may increase the incidence of naturally occur-
ring tumours. Thus, carcinogenicity analysis is basically conducted
to compare incidences, malignancy or early occurrence of tumours
between the control and the treated groups. Tumour growth is a
multi-step development from a precancerous lesion usually diagnosed
as focal hyperplasia to a malignant tumour via a benign type. A dose-
related increase in focal hyperplasia accompanying increased tumour
incidence in a treated group indicates strengthening of carcinogenic-
ity evidence. Increased precancerous lesions only should not be inter-
preted that the chemical is carcinogenic in the carcinogenicity study.
In extrapolation of experimental carcinogenicity to human health
hazard, mechanisms of carcinogenicity are important for hazard char-
acterization. With the development of molecular biology, various
tumour mechanisms in rodents and their relevance to humans have
been clarified (see below). If a pesticide has carcinogenicity, consid-
eration of margin of carcinogenic levels in rodent study to human
exposure level is important for relevance to humans.
4.3.3.5 Genotoxicity study

The differences between genotoxicity and mutagenicity are well
described in the scientific paper by the expert group who updated
WHO/IPCS harmonized scheme in 2009 (Eastmond et al., 2009).

The term ‘mutation’ refers to permanent changes in the structure
or amount of the genetic material of an organism that can lead to
heritable changes in its function, and it includes gene mutations as
well as structural and numerical chromosome alterations. The term
‘genotoxicity’ refers to the capability of substances to damage DNA
or cellular components regulating the fidelity of the genome such
as the spindle apparatus, topoisomerases, DNA repair systems and
DNA polymerases and includes all adverse effects on genetic infor-
mation (Eastmond et al., 2009). It should be noted that there are
other mechanisms leading to carcinogenicity and other heritable dis-
eases, but their identification requires additional types of mechanistic
studies. These potentially harmful effects on genetic material may
be mediated directly or indirectly and are not necessarily associated
with mutagenicity. Therefore, it is very important to be aware that
genotoxicity is a broader term than ‘mutagenicity’, which refers to
the capacity to give rise to mutations.
Genotoxicity tests comprise a wide-range of toxicity studies that
identify gene mutations, chromosome aberration and damage to DNA
as early changes of mutation. They are mainly classified into two
groups: (1) Genetic toxicity tests to detect DNA damages including
DNA adduct formation, DNA strand breaks or unscheduled DNA
synthesis, and (2) Mutagenicity tests including gene mutations, chro-
mosome rearrangements or deletions, and loss or gain of segmental
or whole chromosomes, the latter are known as aneuploidy (EHC240,
2009).
The toxicological significance of genotoxicity is the impact it has
on ADI setting in risk characterization. According to current scien-
tific understanding, carcinogenic risks of chemicals that are positive
for genotoxicity have been interpreted to have no threshold and are
not acceptable for ADI setting. Based on the importance, toxicologi-
cal evaluation of genotoxicity is performed using a mechanism based
combination of core battery tests in vitro and in vivo. More detailed
information on the different OECD test guidelines can be found in
Chapter 2.
4.3.3.6 Reproductive and developmental toxicity studies

The purpose of a reproductive toxicity study is to identify and char-
acterize adverse effects of a test substance on the male and female
reproductive system, and on the growth and development of the off-
spring (OECD, 1983, 2001a). Effects on the fertility of the parents are
also important indicators in the toxicological evaluation of endocrine-
disrupting chemicals, if they are interfering with the homeostasis of
sexual hormones. The purpose of a developmental toxicity study is
to provide information concerning the effects of prenatal exposure on
the pregnant test animal and on the developing organism; this may
include assessment of maternal effects as well as death, structural
abnormalities or altered growth in the foetus (OECD, 2001b). Litter-
based analysis is common in reproductive–developmental toxicity
studies in order to avoid influence of genetic background. The expo-
sure period in both reproductive and developmental studies covers
the sensitive period for development (critical period) of each organ.
4.3.3.7 Reproductive toxicity study

A multi-generation study using rats is commonly used to investi-
gate the reproductive toxicity of pesticides. Reproductive ability is
checked at each generation. In the first generation, the treatment
period starts before mating of young adult animals up to the end
of the lactation period in females. Consideration of administration
route is important in view of exposure level in pups. In the second
and third generation, pups start to eat diet from 10 days after birth
when they are still suckling. Therefore, attention to the direct dietary
exposure to the test compound should be paid. A multi-generation
study using rats can be substituted by the Extended One Generation
study, which includes additional modules to investigate the offspring
until adulthood as well as to include developmental neurotoxicity
and developmental immunotoxicity testing in the offspring of the
first generation.
There are endpoints including parental, reproductive or offspring
toxicity in the reproductive studies which are described in detail in
Chapter 2.
4.3.3.8 Developmental toxicity study

Developmental toxicity studies are performed to detect adverse toxic
effects in the embryo and foetus induced by chemicals. Test substance
is usually administered by gavage to rats or rabbits in a developmen-
tal toxicity study. To cover the full developmental period in rats and
rabbits, in the developmental toxicity study treatment starts at the
day of implantation and continues to the day before delivery. (OECD
TG414 — OECD, 2001b).
Endpoints of the developmental toxicity tests are maternal and
foetal effects, such as malformations and variations, implantation
loss or resorption. Variation is defined as less serious findings than
malformation (Chahoud et al., 1999). Harmonizing terminology of
changes in the foetus is important for interpretation of developmen-
tal studies. Scientific societies, such as the Berlin workshop for the
harmonization of terminology for toxicological evaluation, have been
playing important roles in the harmonization of terminology (Solecki
et al., 2003, 2013, 2015; Paumgartten et al., 2009).
For appropriate action of a bioactive substance in the prenatal–
postnatal development period, all organs and tissues have specific
critical periods (windows). These periods are different in humans and
laboratory animals during prenatal and postnatal development and
may be very sensitive for toxic exposure to endogenous or exogenous
substances. Therefore, it is assumed that a single exposure during
a critical period of development may cause harmful foetotoxic or
embryotoxic effects. Especially teratogenic lesions are accepted to
be inducible by single dose exposure at a critical period of pre-
natal development that is relevant for human health, if they are
observed in rats or rabbits. The postnatal period is a critical window
for the development of the several organ systems (e.g. the nervous
system or hormonal effects including disrupting the hypothalamus-
pituitary-gonadal axis). Exposure to exogenous chemicals with estro-
genic activity during this critical postnatal period can disrupt the
axis, resulting in defeminization and anovulation or may induce a
delayed adverse effect that is elicited after maturation or late stage of
life (Takahashi et al., 2013; Ichimura et al., 2015). It is further noted
that at birth the development of the brain has further progressed

in humans than in rodents. However, in contrast to rodents, where
brain development is largely completed at weaning, in the human
this organ continues to develop long after birth.
4.3.3.9 Neurotoxicity
Acute neurotoxicity studies and repeated dose studies (usually three
months) are conducted to detect neurotoxicity. Experimental ani-
mals used for the studies are rats. The experimental design of the
short-term toxicity study is similar to general toxicity study except
for the inclusion of additional parameters for effects on function
and behaviour and morphological and histopathological examina-
tion of the nervous system. In the acute toxicity test, functional
and behavioural tests are conducted. The acute neurotoxicity study
is often the basis for ARfD setting because this is a single exposure
study and a comprehensive examination including a functional obser-
vational battery (FOB) is conducted. If a substance is suspected to
have neurotoxic properties, a developmental neurotoxicity test may
be performed. In a developmental neurotoxicity study, usually per-
formed in rats, the test substance is administered from gestation
day 6 to lactation day 21 (weaning). The effects of treatment on the
animals continue to be investigated into early adulthood (about 10
weeks of age). A developmental neurotoxicity study provides data on
the functional and morphological effects on the developing nervous
system of the offspring that may result from exposure in utero and
during early life. This endpoint is also included as a specific module in
the Extended One Generation Test. Especially for organophosphates
and carbamates, delayed neurotoxicity of these chemicals was tested
in hens (OECD, 1995).
4.3.3.10 Immunotoxicity
Immunotoxicity concerns adverse effects on the immune system
including immunosuppression and allergy or hypersensitivity to
treatment. Although there is no OECD test guideline for immuno-
suppression, immunosuppressive potential of a compound is usually
checked by organ weights, histopathology, subset groups of lympho-

cytes in the immune system or haematology using the general tox-
icity study in rodents. Hypersensitivity is checked by maximization
test using guinea pig (OECD, 1992) or LLNA lymph node assay
in mice (OECD, 2010a, 2010b). The revised OECD TG407 (28-
day repeated dose toxicity study in rodents) (OECD, 2008) and
the OECD TG443 (extended one-generation reproductive toxicity
study — OECD, 2011) provide the opportunity to incorporate more
immunotoxicity parameters in the study design. It should be noted
that currently the investigations into the toxic effects on the immune
system are limited.
4.3.3.11 Human and epidemiologic data

The description below about human data is derived from FAO/
WHO, EHC240 (2009). JMPR has repeatedly considered the use of
human data in pesticides risk assessment, especially when considering
ARfD setting (EHC240, 2009). JMPR noted that human data on the
pesticides, when available, whether from human volunteer studies or
data from other investigations of effects due to human exposure in
the workplace or environment, can be valuable in placing the ani-
mal data in context. However, new human data are now very rarely
generated, because of ethical reasons.
4.3.3.12 Mechanistic data

Mode of action (MOA) describes the key events and process, start-
ing with interaction of a chemical with (a structure in) the cell,
through functional and morphologic changes, resulting in toxicity
or cancer (Sonich-Mullin et al., 2001). The practical MOA approach
was started within the framework analysing the human relevance of
rodent-specific liver tumour development in risk assessment of chem-
icals in the early 2000s (Cohen et al., 2003; Holsapple et al., 2006).
IPCS updated and extended the framework on how to address the
issue of human relevance of a carcinogenic or a non-cancer response
observed in an experimental study using the MOA framework with
a weight of evidence-based approach (Boobis et al., 2006, 2008).
Since then, a broader set of pathways surpassing the MOA has

been launched as adverse outcome pathway (AOP) (OECD, 2013;
Villeneuve et al., 2014a, 2014b). It starts with a molecular initial
event (MIE), in which a chemical interacts with a biological target
(e.g. DNA binding, protein oxidation, or receptor–ligand interaction,
etc.). The MIE triggers one or more key events (KE; e.g. gene activa-
tion, or altered cellular chemistry or tissue development, etc.), which
are sequential and causally connected. The key events ultimately cul-
minate in an adverse outcome (AO) of relevance to human or ecolog-
ical risk assessors (e.g. mortality, disrupted reproduction, cancer or
extinction, etc.). A key event relationship (KER) describes the rela-
tionships between the key events that link the molecular initial event
to the adverse outcome. The development of ‘omics’ technologies (e.g.
transcriptomics, proteomics and metabolomics), in combination with
in vitro test systems, has catalysed our understanding of the effects
exerted by a chemical at the molecular level and our understanding of
potential toxicity pathways that may lead to adverse health effects.
Pesticides sometimes have unique toxicity. Unique toxicities and
their known published mechanisms are listed in Table 4.3.
AChE, acetylcholine esterase; CNS, central nervous system;
PNS, peripheral nervous system; 4-HPPD, 4-hydroxyphenylpyruvate
dioxygenase; TAT, tyrosine transamylase; nAChR, nicotinic acetyl-
choline receptor.
4.3.4 Considerations on Plant and Animal

Metabolites
The toxicological information about metabolites in animals,
livestock, plants or soil is important for residue definition because
consumers are exposed to pesticide residues via plants or livestock
feeding on plant material. JMPR has developed an approach to assess
the toxicological relevance of metabolites and degradant products,
applying a non-testing assessment scheme (WHO, 2015). This scheme
consists of a decision-tree using the threshold of toxicological concern
(TTC) and read-across approaches as tools. The TTC is used to eval-
uate the toxicological relevance of pesticide metabolites for which
Table 4.3. Pesticides with unique toxicological profiles and their mechanisms.
Unique Mechanisms of
Pesticides Toxicity the Toxicity References
Organopho- Inhibition of Inhibition of AChE in the JMPR

sphorus AChE synapses in CNS and PNS, (1998), van
esters leading to overstimulation of Raaj
and car- the postsynaptic receptors. (2001),
bamates EHC240
(2009)
Abamectin Cleft palate in p-Glycoprotein (ABCD1) works Lankas et al.

CF-1 mouse as a main transporter of (1997),
principal barriers for Ceckova-
penetration into the systemic Novotna
circulation against exposure to et al.
GABAegic chemicals such as (2006),
abamectin. CF-1mouse is FSCJ
mixed with genetically (2012)
ABCB1 gene-deficient
sub-population
Mesotrione Ocular Inhibition of 4-HPPD, a key JMPR

toxicity enzyme of the tyrosine (2014d)
induced by catabolic pathway, leading to
increased increased plasma tyrosine
plasma level. Rat is sensitive to ocular
tyrosine toxicity. Male rat also has
level lower catabolism capacity of
TAT than females.
Sulfoxaflor Foetal/neonatal Structure difference of subunit JMPR

forelimb composition between foetal (2011b),
flexure in and adult-type muscle Rasoulpour
rat. The nAChRs. Only rat foetal et al.
abnormali- nAChR but neither rat adult (2012)
ties were nor foetal/adult human
reversible. nAChRs showed
agonist-evoked response to
sulfoxaflor. The prolonged
agonistic activity to foetal rat
nAChR causes muscle
contracture resulting in
forelimb flexure.
there are few or no relevant toxicity data in the context of dietary

risk assessment (Cramer et al., 1978; Munro et al., 1999; Kroes et al.,
2004; EFSA and WHO, 2016).
Read-across is a tool to evaluate a novel compound on the basis
of appropriate toxicological information of compounds considered to
have sufficient structural similarity to the compound in question to
justify (WHO, 2015). More details can be found in the references
cited above.
4.3.4.1 Process of toxicological evaluation in risk assessment

4.3.4.1.1 Hazard identification
Risk assessment forms the scientific basis for risk management
decisions. The relationship between the hazard identification–
characterization process and the exposure assessment in risk assess-
ment is shown in Fig. 4.6. Hazard identification is the first step of
risk assessment, and detects type and nature of adverse effects (=
toxicities) induced by a chemical using all available data including
toxicity studies and human data. The practical process of hazard
identification is summarized in Fig. 4.7. The common processes are
summarized as follows.
The first step is identification of the change induced by the chem-
ical. Increased incidence of alteration in the treated groups compared
to the control group should be nominated in each study.
The second step is to check whether a dose–response relationship
for the effects exists, what the extent of the effect is and whether the
changes are statistically significant. If this is the case, the effects can
be considered treatment related. Among all observed effects, only the
adverse (toxic) effects on the experimental animals should be selected
as appropriate for risk assessment. The selection is often difficult, and
requires scientific expertise. For instance, adverse effects should be
distinguished from adaptive effects, and it should be assessed whether
historical control data are appropriately used. Further details are
described below.
In the third step, it is crucial to identify toxicity targets, char-
acterize toxicological profiles of the test compound and assess toxic
Processes Parameters and dose levels
1. Identification of
change increased
2. Check of dose response/

intensity/ statistically
significance of the changes
3. Integration to clarity targets,

toxicities, to consider MOA Integration
and relevance to humans
4. Confirmation of reliable toxicities
and choose their endpoints
5. Setting LOAEL based on
the most sensitive endpoint
6. Setting NOAEL
Figure 4.7. The process of hazard identification in each toxicity study.
doses based on reliable toxicological data. If the provided toxicology

data are adequate, the MOA may be elucidated and it may be estab-
lished whether the effects are relevant for humans. Integration of
the available toxicological information and subsequently considering
which is the primary step in a MOA pathway are important steps
for the understanding of toxicity of a chemical.
For example, haemolytic anaemia is a diagnosis that has the fol-
lowing characteristics: Decreased haemoglobin, haematocrit or red
blood cells, increased methaemoglobin (which is a causal parame-
ter) and hemosiderin pigmentation in the liver, spleen or monocyte–
macrophage system.
The fourth step is to identify the most sensitive parameters
within each toxicity profile. For example, increased methaemoglobin
formation in blood is considered the most sensitive endpoint in
haemolytic anaemia.
The fifth step is choosing an endpoint observed at the lowest dose
level as the most sensitive endpoint induced by the test chemical. The
dose is the LOAEL. The dose should be converted to or expressed
as the amount per body weight from the concentration or dose in
diet or drinking water. If the endpoint chosen were trivial or minor,
each step from 1 to 5 should be checked to select a more appropriate

endpoint.
The final step is to select the dose below the LOAEL as the
NOAEL). The NOAEL is the highest dose at which adverse effects
are not observed. If the dose-spacing between treatment groups in
a study is large, combining data from more than one study may be
appropriate.
Sometimes a substance induces changes in animals that are not
necessarily adverse, but which could be considered adaptive. Liver
hypertrophy, which is one of the most common effects induced by
chemicals including pesticides, is an example to explain the distinc-
tion between adaptation and adverse effect. To maintain homeostasis
in the whole organism, the liver frequently responds to xenobiotic
exposure by increasing metabolic capacity via nuclear receptor acti-
vation. This will result in hepatocellular hypertrophy and increased
relative liver weight. Such hepatic adaptive responses are potentially
beneficial in the increased capacity of the organism to respond to
chemical-induced stress. However, such adaptive responses have lim-
its to these homeostatic responses, and exceeding status over these
limits should be recognized as adverse (Hall et al., 2012; WHO, 2015).
This interpretation on liver hypertrophy in toxicological evaluation
is commonly accepted by many regulatory authorities. Practically, if
hepatocellular hypertrophy is accompanied with the following alter-
ations these effects should be considered adverse: (1) hepatocellular
degeneration or necrosis with or without accompanying inflamma-
tory reaction, (2) changes indicating damage to biliary tracts, (3)
disruption of fat metabolism, (4) pigmentation and (5) deviation
from typical localization or morphologic features of hypertrophied
hepatocytes.
Evaluations are supported by the use of historical control data,
minor changes and references showing the normal range of alter-
ations; For toxicological evaluation of haematology, blood biochem-
istry and urine analysis data, it is very important to judge whether
related parameters consistently go in the same direction (increase
or decrease) or not. However, lower or higher values are often
encountered in the control group. While in principle the control group
in the study takes precedence over historical control data, the latter
should be used to put the study control data into perspective. In
such cases, historical control data or references showing the normal
range of alterations are informative to prevent misleading judgements
of toxicity. However, the use of historical control data only is not
sufficient to dismiss the adverse effects, but it is one aspect among
others that should be taken into account for the weight of evidence.
In addition, treatment-related changes that are slight, isolated or
not accompanied by typical effects or parameters may be judged as
minor and not considered to be adverse. However, dismissing minor
changes as not-toxicologically relevant should always be justified on
study-by-study basis. A guide on this subject is provided by the
guidance document for WHO monographers and reviewers (WHO,
2015) and the OECD guidance on historical control data.
4.3.4.1.2 Hazard characterization for setting ADI

The ADI concept was introduced in 1957 by the Council of Europe
and later on was taken over by the JMPR. The ADI of a chemical is
the estimate of the amount of a substance in food or drinking water,
expressed on a body weight basis, that can be ingested daily over
a lifetime without appreciable health risks to the consumer on the
basis of all known facts at the time of the evaluation (WHO, 1997).
The ADI is expressed in milligrams of the chemical, as it appears in
the food, per kilogram of body weight.
In hazard characterization, the most relevant adverse effect
observed at the lowest dose exposure should be determined in all
available studies. Practically, during the process for setting NOAEL
in each toxicity study, the toxicological findings observed at LOAEL
are chosen as the most sensitive and reliable endpoints of the test
compound in each toxicity study. Next, comparing all NOAELs used
for toxicological evaluation, the lowest NOAEL is usually deter-
mined the most sensitive NOAEL of the test compound, based
on the most sensitive endpoints. Comparison of NOAELs but not
LOAELs is important for toxicological evaluation of pesticides,
because setting ADI, one of the purposes, is to predict no adverse
effect level on human health when exposed during lifetime described
above. Therefore, ADI should be the applicable level for all (sub)
populations.
When setting ADI, safety factor equal to uncertainty factor
should be considered. The currently routinely applied safety factor
of 100 was introduced in 1954, and consists of two separate 10-fold
factors; one for inter-species differences (animal-human) and one for
human variability (WHO, 1987). Furthermore, the inter-species fac-
tor of 10 can be sub-divided into a value of 4 for differences in kinetics
and a value of 2.5 for toxicodynamic differences (Renwick, 1993). The
factor for intra-species variation may be subdivided into two factors
of 3.16-fold for both kinetics and dynamics (IPCS, 1994) and allows
the use of specific data on a chemical to derive chemical-specific
adjustment factors (CSAF). IPCS (2005) published a guidance doc-
ument on the types and quality of data that could be used to derive
a CSAF.
The safety factor is in most cases 10 × 10 equal to 100. But, if the
ADI is established using human data, the safety factor should be 10.
If any serious, irreversible toxicities with plausibility to humans such
as serious neurotoxicity or malformation are noted at LOAELs or
near LOAELs in the hazard identification process, application of an
additional safety factor should be considered within 10 times at the
maximum level. If an ADI has to be established on a LOAEL based
on the most sensitive endpoint, an additional safety factor should be
also considered within 10 times at the maximum level.
As an alternative to the NOAEL based ADI setting, the bench
mark dose (BMD) concept is also used for toxicological evaluation
(EFSA, 2009). The BMD approach is an alternative way of defining
reference points for risk assessment. Although at present the BMD
method is not routinely used, the scientific supremacy of the BMD
approach compared with the NOAEL method should be an incentive
to apply it at least as a higher-tier or supplementary method when
the critical study for the derivation of a reference value has been
identified (HSE, 2013). The application of the BMD approach might
be considered as a more robust risk estimate with an indication of
the associated uncertainty.
4.3.4.1.3 Hazard characterization for setting ARfD
Prior to 1994, mainly health risks associated with long-term intake

were assessed for pesticide MRL and ADI establishment. The ad hoc
Working Group on Acceptances at the 25th Session of the CCPR
considered the situation in which an ADI is based upon the NOAEL
in a short-term exposure study and requested JMPR (WHO Group)
to develop guidelines for assessing the toxicological significance of
dietary exposure where adverse health effects may result from single
or short-term exposure and to consider the definition of the ADI
when it is based on an adverse health effect following single or short-
term exposure (Codex, 1993; JMPR, 1993). In 1998, the first regu-
lar consideration of all substances with regard to acute toxic alerts
was performed in the WHO panel of the JMPR and a first defi-
nition of the ARfD was published. The 2004 report summarized a
document drafted by a Working Group of the JMPR WHO Core
Assessment Group, which provided a first guidance on the issues to
be considered when determining whether it is necessary to establish
an ARfD on the basis of the hazard profile of a chemical as well
on particular endpoints that may be particularly relevant to acute
effects (Solecki et al., 2005). The ARfD of a chemical was defined
as an estimate of the amount of a substance in food and/or drink-
ing water, normally expressed on a body-weight basis, that can be
ingested in a period of 24 hours or less, without appreciable health
risk to the consumer, on the basis of all the known facts at the time of
evaluation.
The basic principle for ARfD Setting by JMPR is the following
stepwise process, which is described in the JMPR-Guidance (Solecki
et al., 2005):
• Evaluate the total database and establish the toxicological pro-

file for the substance.
The setting of an ARfD should be considered for all pesticides
whose uses may lead to residues in food or drinking water. In the
absence of data to the contrary, all indications of acute toxicity
observed in repeat dose studies should be considered as potentially

relevant to setting of an ARfD.
• Consider the principles for not setting an ARfD.
An ARfD might not be considered necessary, if no findings indica-
tive of effects elicited by an acute exposure are observed at doses up
to about 500 mg/kg bw per day or if no substance-related mortali-
ties are observed at doses up to 1,000 mg/kg bw in single-dose oral
studies. If mortality is the only trigger, the cause of death should be
confirmed as being relevant to human exposures. If an ARfD is not
set, the reasons must be justified and clearly explained.
• Select the appropriate end-points for setting an ARfD.
The most relevant toxicological endpoints for a single (day) expo-
sure in the most relevant or sensitive species should be selected in
the most relevant or adequate study in which these endpoints have
been adequately determined. It is important to identify the NOAELs
for these endpoints, not for the study.
In most cases, endpoints from repeat-dose toxicity studies have
to be used because critical effects of a compound have not been
adequately evaluated in a single-dose study. Particular weight should
be given to observations at the beginning of repeat dose studies.
When using endpoints from repeat dose studies, the evaluator should
check whether effects are likely to occur at the same dose levels also
after acute exposure.
This is likely to be a conservative approach and should be stated
in the evaluation report that refinement is warranted, if an acute
health risk might not be excluded in the first step of risk assessment.
If after consideration of all the endpoints in appropriate available
studies, an ARfD is not set, then the reasons must be clearly justified
and explained.
• Select appropriate safety factors for setting an ARfD.
In determining the appropriate safety factor, it is proposed to
determine whether the database is adequate to support the derivation
of a CSAF and to consider if there is any information to indicate
reduced or increased uncertainty. If not, the 100-fold (or 10-fold)
default should be used. But, whenever a safety factor other than 100
is used, a clear explanation of the derivation of the factor must be

provided.
In 2010, OECD adopted a guidance for the derivation of an ARfD
(OECD, 2010c). It is based on the JMPR Guidance, and should
support a harmonized use of all available data and application of
common scientific principles not only at JMPR, but also in all OECD
countries. Additionally, this guidance proposed the application of an
extended tiered approach for ARfD setting, including a refinement of
exposure and intake assessment, and provided harmonized guidance
on what to do in single special cases, if a single exposure study for a
refinement of the ARfD is considered necessary.
Therefore, this document presents specific guidance how to refine
the exposure calculation for the acute risk assessment (Annex 1),
and how to perform a tailored single exposure study and what are
the minimum parameters, depending on all available data, which
allow the derivation of a NOAEL, LOAEL or benchmark dose for
the most relevant acute effect(s) in the most appropriate species,
but not intended to become a routine data requirement (Annex 2).
The experimental refinement of the ARfD derivation according to an
OECD single exposure study is to consider all available information
on the substance, when the substance is administered orally as a
single exposure in graduated dose levels to groups of experimental
animals, one dose being used per group and a vehicle control group.
Most toxicity should be manifested within 24 hours.
The basic principles for the selection of appropriate endpoints
for setting ARfD are to find adequate endpoints for acute effects
from repeated dose studies, if no effects are observed in single-dose
studies, such as acute neurotoxicity studies in rats. In the guidance
documents, there is specific guidance for the following main end-
points:
• Haematotoxicity.
• Neurotoxicity.
• Endocrine effects.
• Developmental effects.
• Hepatotoxicity and kidney effects.
• Effects on other organs.
A retrospective analysis of ARfD values has examined 198 pes-

ticides, which had been evaluated and peer-reviewed in Europe
between 2000 and 2008. The results of this retrospective analysis
of ARfD values established in the EU in 2008 are quite comparable
to the results of the 2002 analysis by the JMPR (Solecki et al., 2005).
For 48% of all substances, no ARfD was considered necessary because
of the low acute toxicity of these pesticides. The majority of these
ARfD values were based on acute neurotoxicity studies in rats and
developmental toxicity studies in rats or rabbits. In fewer than 10% of
the cases, conservatively established ARfDs were based on repeated
dose toxicity or multi-generation studies and special studies for ARfD
refinement were submitted for 4% of the 198 pesticides.
In a second retrospective analysis (HSE, 2013), 224 pesticides
were evaluated to determine the basis for the derivation of the ARfD
and to determine what degree of consistency was between two organi-
zations and the reasons for any differences. Analyses were performed
on compounds for which both EFSA and the JMPR had set ref-
erence values. The JMPR summary information is available on the
WHO website (JMPR, 2016), together with further details in the
associated toxicological monographs. Fifty-seven active substances
had been considered by both JMPR and EFSA for the derivation
of ARfDs. For 42%, both groups had the same conclusion. JMPR
was more likely than EFSA to conclude that the setting of an ARfD
was not necessary (7 vs. 1). JMPR was also more likely than was
EFSA to base its conclusions on data derived from human studies,
use a CSAF, and to set separate values for women of child-bearing
age. Overall, where there was a difference in the ARfD value, those
concluded by JMPR were normally higher than those of EFSA (70%
of compounds where decisions differed).
4.3.4.2 Toxicological significance of species specific lesion

Some well-known lesions and tumours considered to be species
specific are listed below. References or some explanations are
added to each non-neoplastic and neoplastic lesion. If treatment-
related increases in such diseases or tumour types are observed, a
clear explanation of the species specificity and justification of the
judgement whether or not the lesions are relevant for humans must
be provided in the toxicological evaluation. Each reference below
shows details and good examples for interpretative strategy in risk
assessment.
4.3.4.2.1 Rodent or dog-specific diseases

• Alpha 2U-globulin associated nephropathy and renal tumour in
male rats (Turkstra and van Raaij, 2002).
• Chronic progressive nephropathy (CPN) and associated renal
tumour in aged rats (WHO, 2015).
• Mononuclear cell leukaemia (MNCL) (Synonym: Fisher rat
leukaemia, large granular cell (LGL) leukaemia (Muller, 2005).
• Pheochromocytoma in rats (Pelgrom and van Raaij, 2001).
• Urinary bladder tumour (leiomyosarcoma) in mice.
In a carcinogenicity study of bifenthrin, a pyrethroid insecticide,

in Swiss mice, urinary bladder tumour diagnosed as leiomyosarcoma
was increased, predominantly in males (JMPR, 2009). The data
were peer-reviewed by pathology experts. JMPR concluded that the
increase was not relevant to humans in risk assessment. In the Inter-
national Harmonization of Nomenclature and Diagnostic Criteria for
Lesions in Rats and Mice (INHAND) Project, a joint initiative of
the Societies of Toxicologic Pathology from Europe (ESTP), Great
Britain (BSTP), Japan (JSTP) and North America (STP) to develop
an internationally accepted nomenclature for proliferative and non-
proliferative lesions in laboratory animals, this tumour is termed
mesenchymal proliferative lesion (synonyms: decidual like reaction;
mesenchymal tumour; vegetative lesion) although the aetiology is
unknown (Frazier et al., 2012). This lesion is mouse specific. The
expert group of INHAND commented that this tumour was incor-
rectly diagnosed as leiomyosarcoma in the past (Frazier et al., 2012).
• CAR or PPARα mediated rodent liver tumours
Liver tumour is a most common tumour in rodents induced by

chemicals. Liver tumour induced by constitutive active/androstane
receptor (CAR) activator such as phenobarbital (PB) is considered
to be rodent specific because PB did not induce liver tumour in

CARKO mouse (Yamamoto et al., 2004), and no epidemiological
study revealed that PB could induce liver tumour in humans (Hol-
sapple et al., 2006). Rodent liver tumour by peroxisome proliferator
activated receptor (PPAR α) activation is also accepted as rodent
specific due to no tumour induction of PPAR α knockout mice study
(Holsapple et al., 2006; Mennes and Blaauboer, 2003).
• Thyroid follicular cell tumours secondary induced by liver drug

metabolism enzyme induction
Liver hypertrophy with hepatic drug metabolism enzyme induc-

tion is a common change induced by pesticides in rodents. The
enzyme induction accompanying glucuronidation–sulphoxidation
promotes excess excretion of thyroid hormones from blood, resulting
in decreased T3/T4 and increased TSH production in the pituitary as
negative feedback. Continuous TSH stimulation leads to hypertrophy
and hyperplasia of thyroid follicles, and finally follicular cell tumour.
The absence of thyroxine-binding protein in rats explains why rat is
a sensitive species to promote this tumour. This pathway to increase
the tumour is not relevant to humans (van Raaij, 2002).
Various types of natural occurring changes were reported in
young beagles used for toxicity studies (Sato et al., 2012). Adequate
consideration is necessary to decide whether increased incidence or
severity of these lesions should be treatment related or not in toxi-
cological evaluation because the number per group is very limited.
• Chronic lymphocytic thyroiditis is a common and specific lesion

in dogs (Sato et al., 2012).
• C-cell complex is a change characterized by solid islands of C-cell-
like clear cells seen in the thyroid tissue. It is considered to be
remnants of ultimobranchial bodies formed before differentiation
into follicular and C-cells (Sato et al., 2012).
• Idiopathic canine polyarthritis: A spontaneous arterial disease in
mature and juvenile beagle dogs and companion dogs has many
synonyms (beagle pain syndrome, juvenile polyarteritis syndrome,
necrotizing vasculitis, polyarteritis, periarteritis, idiopathic febrile
necrotizing arteritis, panarteritis, polyarteritis nodosa) depending

on the stage of the disease (Kerns et al., 2001). The heart is the
most affected organ and a serious case was fatal. A reference sug-
gested a possibility of animal models of human immune system–
mediated vasculitis based on morphologic features (Snyder et al.,
1995); however, the aetiology of this disease remains to be deter-
mined yet. A possible relation with treatment might be considered
if their incidences, severities or duration-related enhancements are
increased at the high-dose group in 13-week or one-year dog studies
(JMPR, 2013c).
4.3.5 Current Topics of Toxicological Evaluation

of Chemicals
4.3.5.1 3R principles
The principles of the 3Rs (replacement, reduction and refinement)
are adopted worldwide in many scientific fields using experimental
animals and are embedded in many national and international legis-
lations (National Center for Replacement, Refinement and Reduction
of Animals in Research, 2016). The 3Rs aim to achieve a more ethical
use of animals in scientific research.
Replacement concerns the use of tests in organs, tissues, cells or
subcellular fractions (in vitro testing) or by using mathematical and
computer models (in silico testing) instead of animal tests. Studies
in vertebrates may also be replaced by testing in invertebrates such
as insects or worms.
Reduction of animal use may be achieved by, for instance, opti-
mizing the study design, reducing variation, appropriate use of data
in public literature and the use of new techniques such as imaging.
Examples of this are the OECD Guidelines 423 (acute toxic class
method — OECD, 2002) as alternatives for the classic acute oral and
inhalation toxicity tests in rodents and the extended one-generation
reproductive toxicity study (OECD Guideline 443 — OECD, 2011).
Refinement of studies aims to minimize animal suffering and to
improve animal welfare. Therefore, methods can be implemented to
minimize the pain, distress or lasting harm and to improve housing
and husbandry of the animals. The 3Rs are increasingly seen as a

framework for conducting high-quality science research in the aca-
demic and industrial sectors. To promote the 3Rs and look for better
models or tools that are closer to humans, various approaches have
been and are developed, as described below.
4.3.5.2 Future methodology for toxicological evaluation

of pesticides
4.3.5.2.1 In vitro testing
In vitro methods are based on the use of organs, tissues, cells or
subcellular fractions. These generally are cultured under controlled
conditions in flasks and plates where they can be exposed to chem-
icals and their toxic effect can be measured. Increasingly, human
cells are used since they better predict possible effects on humans.
In particular, in vitro tests for genotoxicity have been used for a
long time. Presently various OECD guidelines on genotoxicity testing
are available using bacteria and yeast cells but also primary mam-
malian cell cultures derived from liver, ovary, bone marrow or blood
or cultured cell lines. In addition, various in vitro tests to study
the irritating and corrosive effects of chemical substances on eye or
skin, in vitro tests investigating the potential hormone-like activity
of chemicals and tests studying the in vitro dermal absorption of
chemicals have been developed and accepted as OECD guidelines.
New in vitro tests are still being developed and, if adopted as valid
alternatives to in vivo studies, may be used in the risk assessment of
pesticides.
4.3.5.2.2 Omics methods

‘Omics’ refers to a field of study in biology, which includes the genome
(genomic), transcription products (transcriptomic), protein products
(proteomic) and metabolic products (metabolomics). Omics aims at
the collective characterization and quantification of pools of biologi-
cal molecules that translate into the structure, function and dynamics
of an organism or organisms (from Wikipedia).
Marx-Stoelting et al. (2015) explained:

‘More advances in omics techniques and molecular toxicology are neces-
sary to provide new strategic perspectives for regulatory toxicology. By
application of modern molecular techniques more mechanistic informa-
tion should be gained to support standard toxicity studies and to con-
tribute to a reduction and refinement of animal experiments required
for certain regulatory purposes. The relevance and applicability of data
obtained by omics methods to regulatory purposes such as grouping of
chemicals, mode of action analysis or classification and labelling needs
further improvement, defined validation and cautious expert judgment.
. . . . . . . . . . . . The aim was to publish a critical overview of the regula-
tory relevance and reliability of omics methods, basic requirements on
data quality and validation, as well as regulatory criteria to decide which
effects observed by omics methods should be considered adverse or non-
adverse. As a way forward, it was concluded that the inclusion of omics
data can facilitate a more flexible approach for regulatory risk assessment
and may help to reduce or refine animal testing.’
4.3.5.2.3 (Q)SAR approach

In silico testing refers to the use of computer models in the eval-
uation of substances. The development of in silico models is based
on the hypothesis that similar compounds should have similar bio-
logical activities and are used to predict how chemical substances
will interact with the body. The models are based on information
from in vitro or in vivo studies. Thus, physiologically based phar-
macokinetic (PBPK) models may predict how a chemical may be
absorbed, distributed, metabolized and excreted in the animal or
human. Quantitative structure–activity relationship (QSAR) models
use the physiochemical properties or molecular features of a chemical
to predict, for instance, whether a chemical will have genotoxic or
carcinogenic properties. Various QSAR models are commercially or
freely available.
4.3.5.2.4 New model for shorter than lifetime

exposure to pesticides
In the current long-term dietary risk assessment of pesticides, the
estimated chronic exposure to pesticide residues in food is compared
with the ADI for that pesticide. However, it was noted that adverse
health effects of pesticides are often induced at similar exposure levels
in short-term and long-term studies in animals. This indicates that
over a wide exposure duration range, the occurrence of adverse health
effects often is not related to the duration of the exposure. Therefore,
it is not useful to derive a short-term ADI, since that is likely to
be similar to the ADI for lifelong exposure. However, presently no
information is available on the exposure level during short periods
(weeks or months) of life. It is conceivable that the ADI is exceeded
in these situations and thus might pose a health concern.
This notion prompted JMPR (2015b) to advise that development
of dietary exposure assessment methods that take into account short-
term toxicity (four weeks) after less-than-lifetime exposures should
be investigated. Special emphasis should be given to commodities for
which exposures at a frequency of two or more times per week are
likely.
4.4 Conclusions and Future Directions

JMPR reviews a wealth of data on the nature, concentrations and
hazards of the residues occurring in food commodities. The infor-
mation gained by the JMPR process is used for dietary exposure
estimates and for proposing MRLs, thus addressing the concerns of
public health and trade.
The JMPR story is a story of continued scientific progress. Mem-
bers bring to the table new ideas and suggestions that have been
tried, some successfully, at the national level. When a new situation
or new demand arises, the ethos of JMPR is to study the background
and history and decide on improvements while remaining faithful to
JMPR principles.
In 1990, a workshop on MRL development was convened in The
Hague prior to the CCPR meeting to consider possible impediments
to national acceptance of Codex MRLs (Codex report, 1990).
One of the questions in the workshop was:
Is there a mechanism for deleting obsolete GAP and MRLs based
upon this GAP from the Codex system?
In the following years, from confused beginnings, the Periodic

Review Program was developed into a streamlined process that keeps
Codex MRLs and JMPR WHO toxicological parameters reasonably
current. JMPR issued data requirements and methods of evalua-
tion for compounds in the Periodic Review Program, while CCPR,
national governments and industry debated and decided on timing,
data availability and future intentions for the programme.
It is a good example of cooperation among all parties to provide
a valid response to a well-founded question.
The strength of JMPR will continue with its ability to apply sci-
entific processes to the task, while having the flexibility and initiative
to handle new situations that regularly arise.
In the future, we might expect the invention of ever more complex
molecules. Perhaps some biologicals will leave residues of concern.
Will we see nanomaterials that influence the physical, chemical and
biological properties of associated pesticides?
JMPR will study each situation carefully and will develop scien-
tific evaluation processes that are practical and acceptable. That is
the JMPR way.
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Chapter 5
Towards a Harmonized Food

Consumption Survey Methodology
for Exposure Assessment
Liisa Valsta, Marga Ocké and Oliver Lindtner
Main topics
Pre-requisites of pesticide residue assessment approaches to food con-

sumption data collection
Food consumption data collection instruments and methods
Food information
Study organization
Data cleaning and handling
Reporting of food consumption data
Total diet studies
Future challenges of food consumption survey data collections
5.1 Introduction
Exposure to pesticides through food consumption is an important
pathway. The general public is also widely aware of this possibil-
ity. Taking all the different foods available, diverging food cultures
present and food handling and processing methods used in the world,
capturing the food consumption information in a comparable way is
a complex effort.
197
Various sources of national food data are available for exposure

assessments. Data from food balance sheets and household budget
surveys are sometimes used as a surrogate for national food con-
sumption data, but the best data to calculate individual long-term
exposure are from individual food surveys. Collecting food consump-
tion data is a time- and resource-demanding effort and needs to be
planned and carried out using high-quality standards at each level of
the process. This chapter focuses on the aspects of food consumption
survey methodology in relation to exposure assessment. In addition,
the total diet study (TDS) methodology is discussed. At the inter-
national level, harmonization of the data collection and processing
methods is critical to increase the comparability and possible pooling
of the data of different geographical regions.
5.2 Pre-requisites of Pesticide Residue Assessment

Approaches to Food Consumption Data Collection
Use of food consumption data in exposure assessment of pesticide
residues can be divided according to source of pesticide residue data.
Three different cases should be differentiated here.
The first situation is in the authorization process. Here the con-
sumption data have to match field trial data provided by appli-
cants. The second situation arises in the case of post-market exposure
assessments based on data of the official food control system. Out of
the food control system, the most important is the food monitoring of
pesticides that intends to give a representative picture of the market
in the respective country or region, such as at the European level.
For instance, food surveillance data did not indicate any chronic risk
in Poland, but acute risks could not be excluded (Struciński et al.,
2015). The third situation where food consumption data will be used
is in the post-marketing situation, but based on total diet study data.
Field trial and food monitoring data are both available at raw
agricultural commodity (RAC) level. For field trial data, it is quite
obvious that RAC level is most appropriate. For food control or
monitoring, this is reasoned by the legislative conditions. Maximum
residue limits (MRLs) for pesticides are set at RAC or at the level of
Towards a Harmonized Food Consumption Survey Methodology 199
commodities of trade, because it is the best way to indicate if good

agricultural practice (GAP) has been followed. Also the MRL residue
definition is chosen for the purpose of checking if GAP has been fol-
lowed. At the same time, this is done assuming that safe foods at the
RAC level will guarantee safe foods produced from these ingredients
and reduce the effort of analysing the whole variety of foods as con-
sumed. Of course, it remains a challenge to find a correct match to
food survey data, where food is reported as consumed (Reich et al.,
2012). RACs are foods as they are coming from agricultural produc-
tion (e.g. wheat grains including husk). Food as consumed considers
weight losses (or in special cases weight gains) due to cooking and
other kitchen preparations (e.g. bread baked from a dough, which is
prepared with flour, a milling product obtained from wheat grains).
Hence, food survey data need to be recalculated and the amounts
from food as consumed transformed to the RAC level to develop
approaches as described by Boon et al. (2009). In case tolerances for
the pesticide are being addressed at another level than RAC (e.g.
in case of processed product like fruit juice), the dietary exposure
assessment needs to be carried out at the level of commodities with
established tolerances (DPR, 2009).
For a TDS, we do not face the need of converting levels in foods to
equivalent levels in RACs, because food is analysed as consumed with
selections based on food survey data. Normally, this will guarantee a
perfect match for later exposure assessment. But a main limitation
of TDS is that only pooled samples are used. Samples need to be
pooled here to reduce the analyses costs for such a high number of
foods representing the whole diet. As a result, from TDS, we get only
concentration data appropriate for long-term exposure.
For instance, as demonstrated by Jensen et al. (2008) and Liu
et al. (2011), pesticide residues can be assessed by deterministic and
probabilistic models but both preferably need individual consump-
tion survey data as an input parameter.
Crépet et al. (2013) demonstrated that food surveys can also be
used for the assessment of mixtures of pesticides without any specific
methodological need, deviating from the assessment in case of single
pesticide residue.
Nevertheless, there might also be cases where different pathways

of exposure have to be taken into account also for pesticide residues.
A model to aggregate dietary exposure and other paths was devel-
oped by Kennedy et al. (2015) and demonstrated using case studies
for residents and bystanders.
The authorization and assessment of pesticide residues are car-
ried out more and more at the regional instead of the national level.
This gives particular importance also to comparable food consump-
tion data and food descriptions at regional level (e.g. at European
level; Verger and Fabiansson, 2008). The heterogeneity in the input
data for exposure of pesticide residues provided by different countries
is well documented (e.g. in Europe; EFSA, 2007) and harmonization
of both food consumption data and conversion factors to RACs are
welcome.
5.2.1 Long-term Exposure (see also Chapter 6 )

Pesticide residues are assessed according to chronic risks and the
exposure is compared to acceptable daily intake (ADI) or similar
health-based reference values. Those values are derived from testing
effects after similar daily dosage over a period that is assumed to
represent life time. Therefore, also food consumption data used to
calculate long-term exposure should relate to those time periods.
This is fulfilled by per capita amounts as reported from national
statistics based on amounts of food produced, imported and exported
in countries. Those data are collected and reported at international
level by the World Health Organization (WHO) Food Safety Pro-
gramme (WHO, 2002). But food production statistics lack the indi-
vidual level to cover specific consumption behaviour as for gender or
age classes. This also applies to data of household budget surveys that
can only provide long-term mean at household but not at individual
level, (see Section 5.3.1).
The best data to calculate individual long-term exposures are
from individual food surveys. For long-term exposure, it is impor-
tant that the survey duration enables calculation of an average over
several days (Boobis et al., 2008). The idea behind this is that for
chronic effects it does not matter whether there is an exceedance
of toxicological reference values on a single day when this is aver-

aged by lower exposure on previous or following days. This means
that it is important to prevent exposure estimates to be biased by
intra-individual variations over time. Short-term surveys could be
compared to longitudinal studies as, for example, done by Riederer
et al. (2011) with the result that the percentage of consumers for sev-
eral foods is underestimated by short-term surveys. Hoffmann et al.
(2002) showed that the use of only two days for intake assessment
compared to 12 days does result in underestimation of low percentiles
and median of, for example, vegetable intake distribution and overes-
timation of high percentiles. Hence, if the number of days is not suf-
ficient to represent a whole year, normally considered to be lifetime,
statistical procedures to extrapolate over time are needed (EFSA,
2012; van Klaveren et al., 2012). Obviously, the order of different
steps in modelling is influencing results when aggregating over more
than one food group (Slob et al., 2010). Several software tools can be
used to facilitate the modelling of pesticide residue exposure. One of
the first tools is the Monte Carlo risk assessment tool (MCRA) hosted
by the National Institute for Public Health and the Environment
of the Netherlands (Rijksinstituut voor Volkgezondheid en Milieu,
RIVM) and including today probabilistic modelling of chronic and
acute risks as well as possibility to assess cumulative risks (van der
Voet et al., 2015). Givens et al. (2007) have discussed the use of lon-
gitudinal studies to estimate lifetime exposure (see also Section 5.6).
5.2.2 Short-term Exposure (see also Chapter 6 )

In many cases, for pesticides, not only a long-term health-based guid-
ance value but also a short-term reference value (acute reference dose,
ARfD) exists. The toxicological values behind this are related to
effects occurring from high exposure due to pesticide residues on a
single eating event or single day (Boobis et al., 2008; Rees and Day,
2000). Therefore, in contrast to the intention for chronic effects, for
exposure assessment of acute effects, the intra-individual variation
over several days needs to be considered (Crossley, 2000).
Already in 2000, Suhre introduced a tiered approach to estimate
acute exposure to pesticides. While for pesticide residues in food
different kinds of data were used within the refinement, the food
consumption was described in all tiers as empirical or parametric
distribution of individual food consumption data. This points out the
high need on quality of the food consumption survey data (Suhre,
2000).
5.2.3 Statistical Considerations

Statistical parameters from food consumption surveys used to esti-
mate deterministic pesticide exposure are average or high percentile
consumption among several individuals (Kroes et al., 2002). Individ-
uals can refer to the total population (mainly for chronic assessments)
as well as to consumers only (for acute and chronic assessment)
(Tucker, 2008). The mean as well as percentiles are often taken from
more than one day and methods are therefore needed to aggregate
over several days for one individual.
Hence, the number of protocol days and the number of individ-
uals participating in the survey mainly influence the accuracy and
precision of the results of the exposure assessment. It is important
to cover intra-individual and inter-individual variability.
The consumption estimates generally depend on whether an indi-
vidual is a consumer of a food item under consideration, the fre-
quency of consumption within the period of interest and the accuracy
of portion size assessment (Kroes et al., 2002).
The best way to consider all the influencing parameters is to use
probabilistic assessments (Lambe, 2002) by separating variability and
uncertainty in the assessment.
The administration of the sampling days is also very important.
With relation to the extrapolation from short to long term, it is
recommended to have non-consecutive consumption days instead of
consecutive days only (Hoffmann et al., 2002). Non-consecutive days
can be defined to have enough time between two days of consump-
tion data to assume that there is no dependence in the consumption
events. This may depend on the food of interest (e.g. it can be consid-
ered that consumption event should not be related to the same weekly
shopping). It is further recommended that possible correlation of food
consumption to specific days of the week has to be controlled by

considering all week days proportionally within a population (Kroes
et al., 2002). Seasonal sampling is obviously influencing results for
chronic as well as for acute exposure of pesticides (Riederer et al.,
2010). It is therefore recommended that the time period of data col-
lection should cover the whole year to follow the seasonal changes in
the diet.
5.3 Food Consumption Data Collection

Instruments and Methods
5.3.1 Sources of National Food Data
Various sources of national food consumption data are available in
countries. Data from food balance sheets and household budget sur-
veys are sometimes used as a surrogate for national food consumption
data.
Food balance sheets are national annual accounts of production
of food, changes in stocks, imports and exports and uses for agricul-
ture and industry. These data give insights into the food supply and
can be expressed as an average value per person of the population.
The main limitation of food balance sheets is that food supply only
provides a crude impression of the potential average consumption.
As an example of food balance sheet data, fruit supply quantities as
g/day per capita in selected fruit categories in different regions of
the world are shown in Fig. 5.1 (FAO, 2011).
In general, food supply data overestimate food consumption con-
siderably. Food and nutrient losses prior to consumption, due to
processing, spoilage, trimming and waste may not be adequately
accounted for; see ‘food available, but not acquired’ in Fig. 5.2. Also,
information on the total average supply per capita does not permit
assessment of differences in consumption by age or sex, nor does it
give insight in the population distribution of food consumption. Since
food balance sheets are expressed in raw commodities, they are used
as a first step in the risk assessment procedure of pesticides (Kroes
et al., 2002).
120
Apples
Bananas
100 Dates
Grapes
Fruit supply quanƟty (g/capita/day)
80
Oranges, Mandarines
Pineapples
60
40
20
0
Africa North America South America Asia Europe Oceania
Figure 5.1. Fruit supply quantity of selected fruit categories per capita in dif-
ferent regions of the world based on food balance sheet data (FAO, 2011).
Household budget surveys and household income and expendi-

ture surveys are conducted at the national level and provide a vari-
ety of information to governments, including consumer price indices,
absolute poverty and food security indicators. Information on food
availability at household level obtained in these surveys may be used
to estimate the average per capita food consumption. In general,
they are cross-sectional surveys conducted over a 12-month period,
the recall period is up to one week and include food acquisition from
purchase, own production and received in-kind (Smith et al., 2014).
The major limitation of household-based surveys is that no infor-
mation is available on the distribution of food consumption among
individual members of the household (i.e. the survey assumes that
children consume as much as adults on a per person basis).
Furthermore, usually the data do not account for outside house-
hold consumption (may underestimate the consumption of some
foods), and for wasted food and food consumed by visitors or pets
(may overestimate the consumption; see Fig. 5.2). In spite of these
Food available but

not acquired
HOUSEHOLD FOOD
FOOD BALANCE BUDGET CONSUMPTION
SHEETS SURVEYS SURVEYS
(per capita) (per member of
(per individual)
household)
NaƟonal food supply Food consumed by
Household food
individuals
acquisiƟon
Out of home food Food not consumed

acquisiƟon (by humans)
Figure 5.2. Relationships between food balance sheets, household budget sur-
veys and national food consumption surveys (modified from Ocké et al., 2016).
limitations, the household-based survey has been used to estimate

dietary intake of pesticides at the national level (Caldas et al., 2006a,
2006b).
Individual-based methods ask individuals to report their con-
sumption of foods and drinks in a certain period of time. National
food consumption surveys intend to do so in study populations
that are representative for a country, or important sub-populations
within a country. Various dietary assessment methods can be used
to collect the food consumption data (see Section 5.3.2). Individual
consumption information provides the possibility for studying spe-
cial subgroups of the population, such as pregnant women, infants,
the elderly or persons in special circumstances, such as hospital
patients. Moreover, national food consumption surveys allow esti-
mation of the population distributions of consumption of foods,
and possible harmful compounds in the diet such as pesticides and
contaminants.
A major limitation of national food consumption surveys is that
the self-reported consumption data are subject to measurement error.
In many national food consumption surveys, an average underesti-
mation of at least 10% of energy intake is observed.
As described above, food balance sheets, household budget survey
and national food consumption data each provide information about
a different level in the chain of food production to consumption. The

results are therefore not directly comparable, as shown in Fig. 5.2.
Only national food consumption surveys allow the estimation of the
population distributions of consumption of foods, intake of energy
and nutrients, and exposure to chemicals like pesticides. For pesticide
dietary intake purposes, the 1997 Geneva consultation recommended
the development of food consumption databases for the entire pop-
ulation (all ages) and children (ages six years and under) (WHO,
1997). Therefore, these types of data will be discussed further in this
chapter.
5.3.2 National Food Consumption Surveys

As explained in the previous section, national food consumption sur-
veys monitor the food consumption of groups of individuals that are
representative of a national population, or important sub-populations
within a country. National food intake surveys provide insight into
the consumption of foods and beverages, the intake of energy and
nutrients and exposure to potentially harmful chemical substances.
Regular repetitions of national food intake surveys show dietary
trends in a population (Ocké et al., 2016). National food consump-
tion surveys differ in target population and study design such as
sampling frame, sampling method, dietary assessment methodology
and supporting non-dietary data (see Section 5.5.4) collected.
Regarding dietary assessment methodology, no single method
is perfect for assessing food consumption in population sur-
veys. Different existing methods each have their advantages and
limitations.
The food record, or food diary, requires the person participating
in the survey to report in real time all the foods and beverages and
their quantities consumed during a specified period (usually seven
days or less; see Section 5.3.4 for more information). The 24-hour
recall method consists of a personal listing of food and beverages
consumed during the 24 hours prior to the recall interview. The inter-
view is assisted by a trained interviewer, who poses questions to the
participant about foods consumed the day before. More information
on this method can be found in Section 5.3.3. A food frequency

questionnaire consists of a structured list of foods or food groups,
and the respondent is asked to estimate the number of times the
food is usually consumed per day, week, month or year. This can be
asked for a typical serving size or questions on portion sizes can be
included in the questionnaire. The 24-hour dietary recall method or
diet records have been recommended as the dietary method of choice
for national food consumption surveys. These methods quantify
short-term intake rather than perceived long-term intakes collected
retrospectively via a food frequency questionnaire. Well-conducted
24-hour dietary recalls and diet records are less biased than food
frequency questionnaires, and thus better able to estimate the pop-
ulation intake distribution (Brussaard et al., 2002).
In the USA, national representative food consumption data are
collected in the dietary part of the NHANES survey, the ‘National
Health and Nutrition Examination Survey’. The dietary part is called
‘What we eat in America’ (USDA, 2013). Diet is assessed with
two non-consecutive 24-hour dietary recalls using the USDA’s auto-
mated multiple-pass method. In addition, targeted questions related
to frequency of consumption of certain foods and beverages are
also included (Ahluwalia et al., 2016). In an inventory that also
included other continents, the 24-hour dietary recall was the most
frequently used method in national food consumption surveys world-
wide (De Keyzer et al., 2015).
National food consumption surveys in Europe are heterogeneous
with respect to dietary assessment methodology and number of days
for which dietary data are collected. This hampers the comparison of
results across countries, for example in dietary exposure assessment
using the data of the Comprehensive European Food Consumption
Database as conducted by European Food Safety Authority (EFSA).
Therefore, EFSA stimulates European Union member states to col-
lect national food intake data in a harmonized way and prepared
guidance for this in 2009 (EFSA, 2009) with an update in 2014
(EFSA, 2014). Moreover, EFSA provides seed money to countries
that conduct food consumption surveys in accordance with the EFSA
guidance. This is called the European Union (EU) Menu project.
The guidance of EFSA was partly based on the results of previ-

ous European research projects and EFSA projects. These include
the ‘European Food Consumption Survey Method’ (EFCOSUM)
Project (Brussaard et al., 2002), the EFCOVAL project (de Boer
et al., 2011), the PILOT-PANEU project (Ambrus et al., 2013),
and the ‘Pilot Studies for Assessment of Nutrient intake and food
Consumption among Kids in Europe’ (PANCAKE) project (Ocké
et al., 2014). For adolescents, adults and the elderly, EFSA rec-
ommends at least two non-consecutive 24-hour dietary recall as
dietary assessment methodology in national food consumption sur-
veys. For children (< 10 years), the recommendation is to use food
records on two non-consecutive days. A 24-hour dietary recall indi-
vidually was considered less appropriate for young children because
the caretaker that has to provide the answers might not have been
with the child for the whole 24 hours (EFSA, 2014). This could
be the case for children that go to kindergartens or to school.
In these cases, the teacher or kindergarten teacher can record
the consumption of the child in the provided food diary. Sec-
tions 5.3.3 and 5.3.4 provide some more details about the two rec-
ommended dietary assessment methods. The Comprehensive Food
Consumption Database of EFSA is a source of information on
food consumption across the EU. It contains detailed data for a
number of EU countries. See http://www.efsa.europa.eu/en/food-
consumption/comprehensive-database for more information.
5.3.3 Dietary Recalls

In a 24-hour dietary recall, a participant recalls all foods and bever-
ages consumed during the past 24 hours or the preceding day. Most
commonly, the recalled day is defined as from when the participant
gets up one day until he or she gets up the next day. Variations to
the time frame or recall sequence are in use, e.g. a 48-hour dietary
recall is used in the Finnish food consumption surveys (Reinivuo
et al., 2010), or a reverse order 24-hour dietary recall in children to
support memory (Baxter, 2009). It is advised that no prior notice is
given to the participant about a possible future interview on personal
food intake. Although notification could help the memory of some

subjects, others might change their usual diet for the occasion.
The ways of administration of the 24-hour dietary recall are
described in Section 5.3.5. The 24-hour recall is often structured
in multiple steps or passes with specific probes to help the respon-
dent to remember all foods consumed throughout the day. The gen-
eral steps are: (1) collect general information about the participant
and the day of recall; (2) for each consumption time: establish the
time, the place and the main food products eaten; (3) describe and
quantify foods reported as eaten; (4) checks for missing and unlikely
high and low consumptions and (5) establish the intake of vitamin
and mineral supplements (Slimani et al., 2011; Conway et al., 2004;
Moshfegh et al., 2008). In the third step of food description, the
relevant specification should be asked per food product (see Sec-
tion 5.4.2). Food quantities are usually assessed by use of household
measures, food models or photographs (see Section 5.3.6 for more
information).
While the recall method depends on the participants’ ability to
remember and adequately describe their diets, this method is not
suitable for children younger than ≈7 years (without help of a care-
taker) and persons with impaired short-term memory. The prevalence
of impaired short-term memory increases above the age of 75 years.
The quality of the 24-hour dietary recall data also depends on the
participants’ knowledge of the foods and recipes consumed. There-
fore, for children between seven and 16 years, it is advised to involve
both the child and the caretaker for a 24-hour dietary recall (EFSA,
2014).
Strengths of the 24-hour dietary recall are that the administra-
tion time can be short, and the respondent burden is relatively small.
In the case of interviewer administration, literacy is not required.
Weaknesses are that respondents’ recall depends on short-term mem-
ory and it is known that omission and intrusions occur; portion size
is difficult to remember and might be mis-estimated and intakes tend
to be underreported.
The 24-hour dietary recall is appropriate for describing the mean
intake of a group. Two or more non-consecutive days provide data on
intra- and inter-individual variation, which allows for the estimation

of the distribution of usual intake for a population.
5.3.4 Food Records

In the food record method, the subject is asked to record all foods and
beverages immediately before or after they are consumed. The food
diary in which the recording is done can be open, semi-open or closed
(EFSA, 2009). A closed form is a pre-coded list of all of the commonly
eaten foods in units of specified portion size. A semi-open form may
be meal-based and pre-structured with a list of foods and amount
options listed, but including sufficient space for other foods. Portion
size estimation can either be through weighing (weighed food record),
or estimated using standard units, household measures or food pho-
tographs (estimated food record). Whereas an open-form includes
no pre-structuring and no list of foods whatsoever, the required time
for food recording and data coding is shorter if the form is more
closed. However, the pre-coded structure and foods might not fit all
participants well, and might influence the recording.
Children below the age of 10 years are unlikely to supply adequate
food records. However, dietary records may be completed by the
parent or caretaker. The food record is often used for national food
consumption surveys, particularly in children. In Europe, it is the
method recommended by EFSA for the youngest age group (<10
years) (EFSA, 2014).
The food record methodology has both strengths and weaknesses.
A food record does not depend on memory and thus has the potential
to be very accurate, particularly if amounts are weighed. However,
the usual eating pattern may be influenced by the recording pro-
cess. In general, respondents must be literate and highly coopera-
tive (Ocké et al., 2016). Keeping a food record, especially a weighed
food record, is a considerable respondent burden. This requirement
may lead to response bias. Moreover, nowadays, no more than 3 or
4 consecutive days of food recording are recommended because of
respondent fatigue. Intakes obtained through food records tend to
be underestimated because of underreporting and or under-eating.
The food record is appropriate for describing the mean intake of a

group. Food recording on two or more non-consecutive days provides
data on intra- and inter-individual variation, which allows for the
estimation of the distribution of usual intake for a population.
5.3.5 Administration of the Data Collection

Various modes of administration of dietary assessment methods exist.
The diet recall was traditionally conducted as a personal inter-
view with open forms, pre-coded questionnaires or tape recorders,
but computer-assisted interviews have become common (e.g. AMPM,
Conway et al., 2004 and GloboDiet; Slimani et al., 2011). With
computer-assisted personal interviewing (CAPI), the interviewer is
guided by automatic response routing to ask tailored questions and
prompts for specific foods. The system thus helps to ensure complete-
ness and consistency across interviewers. Food and recipe databases
are built into the software and can be searched and there are auto-
mated data quality checks. The systematic questions can result in
the interview duration being longer as there may be more steps
for identifying or describing a food. There is also some loss of
flexibility for the interviewer to react appropriately to individual
participants’ answers because of the step-by-step procedure of the
interview.
The interviews can be conducted face-to-face or via telephone.
Validation studies did not show much difference in validity between
these two modes of administration (Livingstone and Black, 2003).
More recently online self-administered 24-hour diet recalls were
developed (e.g. ASA24; Kirkpatrick et al., 2014; MyFood24, Carter
et al., 2015). The computer acts as the interviewer and all foods
are automatically coded so the cost of data collection is significantly
reduced. Many of these systems include automated prompts for items
commonly consumed together and checks for missing drinks or long
time gaps where no foods are reported. They have the advantage
that data collection can be at a time and location convenient to the
respondent. However, subjects are required to be computer literate
and to have access to the Internet. There may also be some loss
of flexibility. An interviewer can tailor the questions to the level of

food knowledge of the respondent and use more descriptive ques-
tions about colour, texture and taste to elucidate details of the types
of foods consumed whereas a pre-programmed computer system
cannot.
Similarly, for diet records, the traditional way of administration
is on paper. The quality of the food recording can be improved by
instructing and training the respondents to record the level of detail
needed to describe adequately the foods and amounts consumed, and
by checking the completed food record in detail with the participant
during an interview. Particularly the level of detail in which foods
are described is difficult to obtain without instruction and check-
ing. Nowadays, online versions of food records and food diary apps
on mobile devices also are available that do not need personnel for
instructions, checking and coding or optical reading.
5.3.6 Portion Size Estimation

Portion size estimation aids have been developed to help participants
estimating the amount of food consumed. These include food pho-
tographs (Nelson et al., 1997), food models (Foster et al., 2008), digi-
tal images of food (Subar et al., 2010) and two-dimensional drawings
(Steyn et al., 2006). Household measures and commercial or standard
units can also be used to give an estimate of the amount of food in
terms of slices, pieces, spoons and cups. Estimation of portion size
is associated with considerable measurement error, which might be
either over or under-estimation (Chambers et al., 2000). Portion size
assessment aids should contain foods and portion sizes appropriate
to the specific eating patterns of a population and should be tested
and validated for the population in which they are intended to be
used. In validation studies addressing errors related to portion size
estimation, perception, conceptualization and memory as psycholog-
ical constructs can be studied (De Keyzer et al., 2011). If no measure
or estimate of the amount of food consumed is collected, then the
average portion sizes derived from previous representative studies
may be used.
5.3.7 Special Considerations in Relation to Data

Collection with Reference to Pesticide
Residue Assessment
As for some pesticides, an ARfD is established only for women
of child-bearing age, consumption data for this sub-population are
needed. Data on this population group are, however, scarce. In case
of missing data, consumption data for the general adult population
can be used as a surrogate for this sub-population (FAO, 2015).
The issues related to combining pesticide residue information
determined in raw commodities with consumption information on
processed, often complex composite foods and dishes, are discussed
in the Sections 5.4.2 and 5.4.3.
5.4 Food Information

5.4.1 Food Classification and Description Systems
To make possible the linkage of foods consumed with correct pesti-
cide residue levels for exposure assessment, the foods reported in a
food consumption survey need to be described accurately. Moreover,
a food grouping system facilitates the reporting of the results. A
common food terminology is the key to be able to obtain reliable
results from dietary exposure assessments. Several food classifica-
tion and description systems have been developed during the past
decades. They are usually reporting-level- and purpose-specific and
therefore several systems exist also in parallel (Ireland et al., 2002;
EFSA, 2010).
Food classification systems are organizations of terms allocating
foods into groups based on similarities from the point of view of the
user (EFSA, 2011). National food classification systems are common
in all continents, but sometimes also regional classifications systems
are in use (De Keyzer et al., 2015). International food classification
systems are necessary both at global level (e.g. the Codex Classifi-
cation of Foods and Animal Feeds, CAC, 1993) and at regional level
(e.g. FoodEx2 classification launched in Europe, EFSA, 2011, 2015).
The classification of foods is highly dependent on the needs of the
user as well as on the food information to be collected. The needs

are different for managing food information (e.g. food composition
vs. pesticide residue data and reporting survey results), although
classification systems may be organized in a way that is useful for
both purposes (EFSA, 2015). Food classification is used in food com-
position databases, in analysing and reporting of food consumption
surveys or dietary exposure assessments of, for example, pesticide
residues or contaminants, and in planning and carrying out total diet
studies. Food classification systems facilitate analysis and reporting
of epidemiological research and are also important in food legislation
and for trade statistics (Greenfield and Southgate, 2003; EFSA, 2011,
2015; European Commission, 2014).
A food classification system depends on the reporting level of the
data collected. Food balance sheet data are built on information of
foods as RACs, while household budget surveys usually included data
at the level of raw foods, foods as purchased and at ingredient level.
On the other hand, for example, in the Codex Classification of Foods
and Animal Feeds, the first separation is between foods and feeds.
Foods of plant and animal origin as well as primary commodities and
processed foods are further separated (CAC, 1993).
Data collected in dietary surveys at individual level provide infor-
mation on food consumption at the level of foods as consumed or as
prepared. Thus, food consumption may be reported at the intake
level, which is not possible either from food balance sheet data or
household budget surveys (EFSA, 2010). Examples of food classi-
fication systems at intake level are, for example, several national
food classification systems for food consumption surveys. Many food
composition databases and classification systems used to compare
international food consumption surveys are built at ingredient or
derivative level (e.g. foods as purchased and raw foods) (EFSA, 2010,
2011, 2015). Most of the food classification systems include some 10
to 25 main food groups, which can be further divided to more detailed
sub-groups (Greenfield and Southgate, 2003; EFSA, 2011).
Food descriptors are a collection of terms recording relevant char-
acteristics of a food. Due to abundance of ways to describe the char-
acteristics of a food, the descriptors are often organized in facets, a
collection of terms of related nature. Such facets are, for example,

the food source from which the food product is derived (e.g. plant or
animal), part of plant or animal or physical state (e.g. liquid or solid)
and type of processing (e.g. raw or drying). Such descriptors facilitate
grouping of foods, comparisons of consumption and help to improve
the accuracy of exposure assessments. Improved and detailed food
description allows the reallocation of foods in parallel food groups,
which may be useful for further analyses and exposure assessments.
Before developments of more systematic thesauri for food descrip-
tion, basic food descriptors have often been included in the food
name. In the 1970s, a comprehensive thesaurus for food description,
named LanguaL, was launched in the USA and has been further
developed and updated in Europe (EFSA, 2010; Ireland and Møller,
2010).
A major undertaking took place in Europe between 2009 and
2011, when EFSA developed, in collaboration with the member
states, a comprehensive food classification and description system
both to cover the needs to classify and describe food in data collec-
tions across different food safety domains (EFSA, 2011). This sys-
tem, named FoodEx2, is a flexible combination of classifications and
food descriptions (EFSA, 2015). The revised and updated FoodEx2
includes altogether eight hierarchies to cover the needs of different
food safety domains, i.e. for exposure assessment purposes (used for
exposure assessments especially of chemical and processing contam-
inants and for classifying and describing food consumption data),
for pesticide residues, zoonoses, feed, veterinary drugs residues and
botanicals. The master hierarchy contains the entire terminology
and is for technical use only. In addition, a reporting hierarchy
exists. The food descriptors are organized in 32 facets, out of which
about 10 facets with several descriptor terms are prioritized in the
harmonized EU menu food consumption data collection (EFSA,
2015).
In the FoodEx2 system, the exposure hierarchy used for food
consumption data is structured in six levels with 21 main food
groups at the top level. It consists of about 4,500 terms (including
hierarchy terms) and about 4,300 reportable terms. The pesticide
hierarchy is based on the Annex I to Commission Regulation (EU)

No 752/2014 (European Commission, 2014). The pesticide hier-
archy consists of 1,802 terms and all the terms are reportable
according to the rules established in the pesticide domain (EFSA
2015).
In addition to classifying and describing foods, also food sup-
plements, which are concentrated sources of nutrients or other sub-
stances (e.g. botanicals with nutritional or physiological effects) need
to be managed and organization of such data requires classifying and
describing their features. Systems to cover this group are also avail-
able (Saldanha et al., 2012; EFSA, 2015).
5.4.2 Handling of Composite Foods

Pesticide residues are mainly determined in raw food items.
Analysing pesticide residues from individual cooked foods (dishes)
appearing in the diets of the participants of food consumption sur-
veys is not viable. Therefore, the disaggregating of complex foods and
recipes with the help of appropriate collection of relevant, at least
general, recipes is needed to enable matching food ingredients with
residue data. To facilitate disaggregation of dishes, recipe databases
are compiled, usually based on recipes from commonly used cook-
books, from recipe archives of the Internet or even based on recipe
collection field work (Reinivuo et al., 2009). For industrially prepared
dishes, recipes may be obtained from the manufacturer. If the origi-
nal recipe of an industrial dish is not available, it can sometimes be
mimicked based on the list of ingredients and the nutrition labelling
information of the food item (Öhrvik et al., 2015).
The challenges to understand the weight changes of a food during
heat treatment go several decades back. Guidelines for calculating
weight yield factors (i.e. the ratio of the weight of the prepared dish
to the weight of the ingredients used; Bognár and Piekarski, 2000),
and tables of yield factors of food constituents have been compiled to
facilitate the disaggregation of composite dishes to raw ingredients as
well as to estimate the weight of prepared dishes from raw ingredients
(Bergström, 1994; Bognár, 2002).
The total cooked weight of a prepared dish or food equals total

uncooked weight multiplied by the weight yield factor of the cooking
method (Reinivuo et al., 2009). Thus, the weight of raw ingredients
representing a cooked dish consumed equals the weight of a cooked
dish divided by the weight yield factor. The European Food Informa-
tion Resource project (EuroFIR AIBSL, www.eurofir.org), has put
forward a standard recipe calculation procedure aiming to calculate
nutrient content of recipes to be used by the European food com-
position database compilers (Reinivuo et al., 2009). Although the
EuroFIR recommended applying weight yield factors at the recipe
level, conversion from edible composite dish to the weight of RACs
would benefit from applying yield factors at ingredient level (i.e.
to take into account the change especially in water content at the
ingredient level). Several European countries announced applying
yield factors at recipe level, Germany and Finland being exceptions
(Reinivuo et al., 2009). Since then, at least Sweden and Norway have
switched to applying weight yield factors of prepared foods at ingre-
dient level, soups being an exception and having the same factor for
all ingredients (Öhrvik et al., 2015).
Weight yield factors for cold dishes (for edible part) are usually
set to 1. For the conversion factors to take into account the edible
part of the RAC (e.g. in case of certain fruit and vegetables; see
Section 5.4.3).
Concerning the need to convert cooked foods to raw ingredients
for analysis of pesticide residues, an exception exists (i.e. the total
diet study process), in which also cooked foods are analysed, but this
takes place only for pooled samples in the TDS procedure.
5.4.3 Conversion Factors for Consumed Foods to Raw

Agricultural Commodities
While pesticide residues are analysed at the level of RACs for human
consumption, a link is needed between the pesticide residue concen-
trations found, and foods and ingredients consumed according to
the national food consumption surveys (Boon et al., 2011). This is
done by applying a set of conversion factors for different steps in
the linking (Boon et al., 2011; Bowman et al., 2013): (1) to convert
cooked foods to uncooked foods (Bergström, 1994; Bognár, 2002)

(for cooked composite foods and dishes, see also Section 5.4.2), (2)
to convert fruits and vegetables to fruits and vegetables including
inedible parts (e.g. with core of an apple, with peel of banana etc.),
(3) to convert processed commodities to originating commodities or
RACs (e.g. fruit juices to respective whole fruits with refuse, peanut
butter to whole raw nuts without the shell, or dried milk or cheese
to fluid milk). Several guidance documents on applying and listing
conversion factors of these kinds are available (Bognar and Piekarski,
2000; Boon et al., 2011; Bowman et al., 2013).
When converting raw edible commodities to RACs to foods, the
extraction rate needs to be taken into account, Extraction rate (%)
indicates in percent terms, the amount of a processed product (e.g.
flour or cheese), obtained from the processing of the originating prod-
uct, which in most cases is a primary commodity (e.g. wheat or
milk, respectively (FAO, 2000). Table 5.1. summarizes some exam-
ples of conversion factors (processing factors) calculated based on the
extraction rates collected from different FAO member states (FAO,
2000). It can be seen that the factors vary between countries and no
harmonized factors are available.
An overview of linkage between food consumption data and con-
centration data for pesticide residue exposure assessment is presented
in Fig. 5.3 published by the ACROPOLIS project (Boon et al., 2011).
The edible part of RAC (eRAC) is presented as the link between
foods consumed and the primary commodities analysed for pesticide
residues. To summarize, the food or ingredient consumed may be
converted to eRAC by taking into account the weight changes during
food preparation. On the other hand, the eRAC consumption may be
converted to RAC level by using processing factors (i.e. conversion
factors between an edible food), for example, cheese and the raw
commodity used to prepare the food (e.g. milk). These kinds of RAC
conversion models are available in several countries and conversion
factors to take all the conversion steps into account have been pub-
lished, e.g. by Boon et al. (2011) and Bowman et al. (2013). Default
processing factors in relation to pesticide residue exposure assess-
ments have also been provided in the U.S. (e.g. in California through
December 14, 2016
Towards a Harmonized Food Consumption Survey Methodology
14:43
Table 5.1. Examples of extraction rates of different processed and derived products in selected countries and
conversion factors derived based on the extraction rates published by FAO Statistics (FAO, 2000).
Food Safety Assessment of Pesticide Residues

Extraction Rates in Use and Provided by Different Countries (%) (FAO, 2000)
Processed and South
Derived Products UK Egypt Africa Canada USA Brazil China India Russia Australia
Flour of wheat 80 82 80 72 74 72 75 90 78 72
Flour of maize 87 94 80 60 59 85 80 80 75 78
Oats, rolled 49 na 60 67 58 46 60 na 55 52
Beet sugar 16 14 na 14 14 na 11 na 12 na
Cane sugar na 10 11 na 11 9 12 11 na 16
Oil of soya beans 18 18 16 17 19 18 15 18 17 17
Oil of sunflower seed 43 50 42 41 41 39 35 36 45 45
Tomato paste na na 30 31 na 25 26 30 na na
Apple juice single strength na na 65 70 72 72 70 60 na 60
Apple juice concentrated na na na 22 22 na na na na na
Pineapple juice single strength na na 25 na na 25 20 20 na 35
9in x 6in
Cream, fresh 11 na 9 25 9 na na na 15 10
Cheese (whole cow milk) 10 25 15 9 13 10 15 na 12 12
(Continued )
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219
page 219
December 14, 2016
220
14:43
Table 5.1. (Continued )

Factors to Convert a Processed or a Derived Product to RAC = 100/Extraction Rate
Processed and South

Derived Products UK Egypt Africa Canada USA Brazil China India Russia Australia
Flour of wheat 1.3 1.2 1.3 1.4 1.4 1.4 1.3 1.1 1.3 1.4
Flour of Maize 1.1 1.1 1.3 1.7 1.7 1.2 1.3 1.3 1.3 1.3
Oats, rolled 2.0 na 1.7 1.5 1.7 2.2 1.7 na 1.8 1.9
Beet sugar 6.3 7.1 na 7.1 7.1 na 9.1 na 8.3 na
Cane sugar na 10.0 9.1 na 9.1 11.1 8.3 9.1 na 6.3
Oil of soya beans 5.6 5.6 6.3 5.9 5.3 5.6 6.7 5.6 5.9 5.9
Oil of sunflower seed 2.3 2.0 2.4 2.4 2.4 2.6 2.9 2.8 2.2 2.2
Tomato paste na na 3.3 3.2 na 4.0 3.8 3.3 na na
Apple juice single strength na na 1.5 1.4 1.4 1.4 1.4 1.7 na 1.7
Apple juice concentrated na na na 4.5 4.5 na na na na na
Pineapple juice single strength na na 4.0 na na 4.0 5.0 5.0 na 2.9
9in x 6in
Cream, fresh 9.1 na 11.1 4.0 11.1 na na na 6.7 10.0
Cheese (whole cow milk) 10.0 4.0 6.7 11.1 7.7 10.0 6.7 na 8.3 8.3
b2668-ch05
page 220
Figure 5.3. Graphical overview of linkage between food consumption data and
concentration data for dietary exposure assessments (Boon et al., 2011) with
permission of the ACROPOLIS project.
A. Recipe data: composition of foods.

B. Conversion ingredients to edible part of RAC (eRAC) considering, for
example, weight changes due to processing.
C. Direct link concentration and consumption data.
D. Conversion RAC as analysed (including inedible parts) to edible eRAC using
processing factors.
the Department of Pesticide Regulations (DPR, 2009)). These fac-

tors are used to take into account any possible increases or decreases
in residues due to processing of RAC, e.g. by washing, cooking, or
freezing. This type of a processing factor is set to 1 for example,
when the residue value is based on the processed food samples (DPR,
2009). Harmonization of the conversion factors would further facili-
tate international comparisons in this area.
In total diet studies, the problem of converting from one state
to another does not arise, because food is analysed as consumed and
selected based on food survey data. The only step where conversion
factors are taken into account is in the sample preparation step,
when estimating the amount of raw materials needed for the food
sample preparations. Normally, the TDS analyses will guarantee a
perfect match for later exposure assessment. A limitation though of
total diet studies is that there are only pooled samples analysed (i.e.
different varieties of same food are mixed up and homogenized before
analyses). In analysing such a high number of foods to reduce cost
and the need to represent the whole diet mean that no details of the
diet of sub-populations are obtained.
5.5 Study Organization

5.5.1 Survey Organization and Planning Phase
Careful planning, adequate resourcing and adequate knowledgeable
personnel of a dietary survey are the cornerstones of a successful
data collection (Ambrus et al., 2013, EFSA, 2014) as in any kind of
large survey (Tolonen, 2013). The objectives of a survey and specific
study questions need to be clear early on, but also a large amount
of practical preparative work needs to be completed to be able to
proceed. Ethical and legal aspects are important and may require a
long time, even months, to get approved depending on the national
rules and practices.
To be able to apply for an approval statement from the local
ethical committee, the survey organization has to provide a detailed
study plan, invitation letter and consent form(s) as well as all ques-
tionnaires to be approved by the ethical committee (Ambrus et al.,
2013; Tolonen, 2013).
The survey budget needs to be carefully estimated and moni-
tored throughout the survey. The budget is determined by many fac-
tors. These include the sample size, duration of the survey, personnel
needed to be recruited and trained, tools for the data collection and
data transfer and other equipment and software needed for the data
management, selected way to organize the study visits (at homes or
in study centres), transportation and accommodation needs during
the field work (Tolonen, 2013).
In case the needed methodologies are not in place in the survey
organization, they need to be planned, tested, piloted and finalized
well in advance before the training of the field work personnel. Com-
pilation of the study protocols is a major task before the piloting and
testing of the data collection methods. In addition, the procedures of
inviting and reminding the subjects about the study visits, methods
related to sampling to assure a nationally representative sample are
important steps of the planning phase. Due to the constantly decreas-
ing participation rates in most of the countries, any possible efforts
increasing the participation rate are important to be considered in
the planning phase (EFSA, 2014). Any method available to encour-
age subject participation — a convincing invitation letter, flexible
data collection appointment schedule, an SMS to remind about the
visit, friendly and professional survey personnel, local media coverage
of the survey or an ethically appropriate incentive depending on the
local rules and regulations — is valuable and needs to be planned in
advance (Ambrus et al., 2013; Tolonen, 2013; EFSA, 2014). Keep-
ing the burden for participants as low as possible, and procedures
as pleasant as possible is also important to obtain high response
rates.
The training of the field work personnel is a very important part
of the survey preparations and quality assurance plan of the project.
It needs to be tailored based on the methodologies applied and the
background knowledge of the personnel to be trained. When dietary
software is used for food consumption data collection, the focus needs
to be on getting enough deep knowledge of the national food list,
practical use of the dietary software, the databases built into the
software and supporting materials (e.g. portion size estimation tools)
used in the food consumption data collection and as well as practical
training of dietary interviews and meeting with the survey subjects
(EFSA, 2014). Evaluation of the field staff performance also during
the actual data collection phase is useful from a quality assurance
point of view.
5.5.2 Sampling
The optimal sampling frame of a dietary survey depends on national
conditions, population-group under consideration and other data
sources to be matched with dietary protocol data. Within the

PILOT-PANEU-project financed by EFSA for harmonizing and
establishing food survey methodologies in European countries, all
pilot countries applied different sampling strategies. For instance, in
Bulgaria, sampling was adapted to different age groups by using the
census as well as a list of registered general practitioners recruit-
ing adults and elderly and lists of schools for recruitment of adoles-
cents (Ambrus et al., 2013). In the PANCAKE project (also financed
by EFSA), stratified random sampling from National Registers was
used by participating countries (Ocké et al., 2012). Although this
is the preferred sampling frame, experience learned that sufficient
time should be allotted to obtain permission for usage. Moreover, it
was expected that a national population register may not be possible
to be used in all European countries (Ocké et al., 2012). To avoid
bias introduced by different percentage of non-responders in relevant
sub-populations, a cluster sampling was applied and non-response by
the primary person of the cluster was replaced by another person in
the cluster with similar socio-demographic and geographic charac-
teristics, rather than by a randomly drawn additional sample of the
national register (Ocké et al., 2012).
Based on PILOT-PANEU and PANCAKE surveys, EFSA has
set recommendations on how to conduct a dietary survey (EFSA,
2014). There it is stated that all age-groups between three month
and 74 years should be covered by dietary surveys and preferably
sampled from national population registers (EFSA, 2014). Infants
younger than three months and elderly above 75 years are excluded
for practical reasons. The sampling is recommended to be done in a
way that results in proportional distribution of participants in the
sample corresponding to that of the national population. Important
criteria to describe representativeness are geographical regions within
countries, rural or urban areas, age classes, gender and education or
socio-economic status. Normally, not all of these stratifications can
be fully controlled during the survey. This is pointing out the need
for random sampling within strata, aiming for a high response rate
and trying to avoid systematic drop-out of population groups that
are more difficult to be reached or less motivated to participate.
In the risk communication to the consumer, it would also be

very valuable to assess the impact of different diets like vegetarians
or organic diet to the pesticide exposure (Lu et al., 2006; Oates and
Cohen, 2011), which means that people following such diets should
be identifiable in the survey and represented by appropriate sample
sizes. Similarly, risk assessment for vulnerable groups is of particular
relevance. In most cross-sectional surveys, those sub-populations as
pregnant or lactating women, vegetarians or ethnic minorities are
included. However, the sampling size does mostly not allow a sep-
arate assessment. In the EFSA Comprehensive Food Consumption
Database, the highest number within countries is 59 (1.4%, Denmark)
for breastfeeding or pregnant women and 287 (2%, Germany) for veg-
etarians. This could be overcome by oversampling of the respective
part of the total population.
Even, if the inclusion of ethnic minorities is highly recommended
(Ocké et al., 2012), this is quite challenging and expensive because
often language barriers have to be overcome by translating all ques-
tionnaires, protocols and study material and recruiting multilingual
interviewers.
In general, it can be stated that sampling and non-response is
driving much the quality of survey results. Estimates based on dietary
surveys can easily be biased by non-representative study populations.
To assess possible bias, it is recommended to monitor non-responders
(EFSA, 2014).
5.5.3 Sample Size

The sample size of a food survey determines the precision of the esti-
mates of statistical parameters describing the consumption amount
of respective populations. Depending on the objective of the assess-
ment, a different sample size is required. In the case of pesticides
mean consumption within a population is of interest for chronic risks
and 97.5th percentile of consumers only for acute risks (WHO, 1997).
For comparison of mean consumption between different sub-groups
of the population or over time, other criteria for minimum sample
sizes has to be chosen than for estimation of high or low percentiles
of food consumption within a population. Also when the objective

is to calculate percentage of population eating a specific food (per-
centage of consumers), different methods have to be used to derive
minimum sample size. For pesticide risk assessment, it should be
kept in mind that also precise estimates of the 97.5th percentile of
consumers only is needed. This is especially difficult for pesticides
that are restrictively used on specific foods with low percentages of
consumers in the population. For these pesticides, sample sizes of
the food survey might be too small to derive precise estimates and
confidence intervals are very broad.
All power calculation methods to derive appropriate sample sizes
depend on variability within the population. The higher the variabil-
ity, the higher is the needed sample size to reach a given precision of
the estimates. In the EFCOSUM project, a number of 1000 individ-
uals was considered to be enough to estimate mean fat intake with
a precision of 5% (Brussaard et al., 2002; EFCOSUM, 2002). But it
was also shown that already for a broad food group like fruits and
vegetables a sample size of 2000 would be needed to assess mean
intake with the same precision (EFCOSUM, 2002). Because assess-
ment of pesticide intake is normally calculated at the more detailed
food group level, it is obvious that variability and resulting sample
sizes to be considered are higher or precision of estimates decreases.
Some statistical consequences due to small sample sizes are described
by Tucker (2008).
The samples sizes derived by power calculations are net sample
sizes after non-response. The gross sample size to be invited for the
survey should also take into account expected response rates. To cal-
culate minimum sample size needed, the process should be started
with checking all criteria of representativeness and identify the small-
est stratum. For this stratum, the sample size has to be determined
to estimate relevant statistical parameters with required precision.
Based on sample size, other strata can be chosen proportionally and
summed up to total sample size needed.
In EFSA’s Comprehensive European Food Consumption
Database (www.efsa.europa.eu), a sample size between 250 and 2,495
is included for surveys only addressing children of different age
groups. For adults, minimum of 410 and maximum of 13,926 individ-

uals are included in dietary surveys of this database and 1,751–4,134
for surveys addressing children and adults.
The general recommendation on minimum net sample size given
by EFSA is 260 in each of the six age-groups from three months
up to 74 years, but inclusion of more than the minimum number of
subjects in the study is strongly recommended (EFSA, 2014).
The necessity and advantages of a food consumption survey at
the European level as a whole have been discussed several times.
Volatier et al. (2002) listed the additional considerations to the sam-
ple size when data are collected in different countries to be represen-
tative at the European level.
5.5.4 Supporting Non-dietary Survey Data

Supporting non-dietary survey data means all other information that
is needed in the data analysis or later for the exposure assessment.
This includes socio-demographic background information, anthropo-
metrics (e.g. weight) and other background information related to
food consumption (e.g. special conditions affecting diet or special
diet pattern (e.g. vegetarian diet, slimming diet)). Data on body
weight is essential to calculate pesticide exposure per kg of body
weight. The minimum set of background information recommended
by EFSA for the harmonized EU menu methodology is summarized
in Table 5.2 (EFSA, 2014).
It has been shown that in many studies the non-responders of a
survey differ from their background compared to the population par-
ticipating in the survey. Those not participating are more often men,
from younger age groups or oldest age groups, are more often from
lowest socio-economic groups or low educated, unemployed and single
(Jousilahti et al., 2005, Lundberg et al., 2005, Tolonen et al., 2005,
Torvik et al., 2012, Demarest et al., 2013). Therefore, in addition to
the background information obtained from the subjects participat-
ing in the food consumption survey, it is important to be able to
analyse the possible non-response bias of a survey. Therefore, col-
lection of background information is recommended also from those
refusing to participate in the main data collection. The age and sex
Table 5.2. Background information to be collected from survey participants and

non-participants (EFSA, 2014).
To be To be
Collected Collected
From From Non-
Information Participants Participants Comment
Data collection date X X

Who provided the answers X X If the subject cannot
answer personally
Who checked the data at site X
Special circumstances affecting X
the data collection
Participant ID X (X)a
Sex X X Available from the
sampling frame
Date of birth X May be obtained from
the sampling frame
Age X X Available from the
sampling frame
Size of household X
Region, area or city of X X May be obtained from
residence the sampling frame
Education level X X In the case of children,
define the education
level of the parents;
May be obtained from
the sampling frame
Employment status X In the case of children,
define the employment
status of the parents
Professional category X In case of children, the
professional category
of the respondent
Ethnicity X Self-defined
Special conditions X
Special diet pattern X Possible to use multiple
codes if more than one
type is applicable
Weight X Self-reported (if >10
years old) or measured
Height X If body mass index
(BMI) to be estimated
Reason for not participating X If feasible
Question on food habits X If feasible
a
The combining of non-participants information to morbidity and mortality
statistics may provide additional insight to the non-participant bias of the survey,
if ethically approved by the country.
of the non-participants are often available from the sampling frame.

If not, this information would be useful to be collected, for example,
through a non-response questionnaire or short telephone interview.
Also additional background information about reasons preventing
participation and an indicator question on food habits may be infor-
mative (Table 5.2). It is noteworthy, though, that some countries do
not allow asking any additional questions from the non-responders
because of privacy laws, which need to be taken into account in the
planning phase of the survey (EFSA, 2014; Tolonen, 2013). The back-
ground information may be collected by mailed or web-administered
questionnaires or by personal interviews. Certain core information
may be available from the sampling frame (EFSA, 2014).
5.5.5 Quality Assurance

The objective of quality assurance is to ensure that the quality of
the data is high and, if applicable, the results across different inter-
viewers or field work staff are comparable. An important part of
the quality assurance is also the training of the survey staff (Tolo-
nen, 2013). Since national food consumption surveys usually have a
long period of recruitment of participants and of data collection (one
year or longer), it is important to monitor quality issues regarding
recruitment and data collection during the process and practices be
corrected if possible and necessary (Ocké et al., 2012). For example,
during the field work, insight into the distribution across the days of
the week can be useful to adjust and improve the distribution of the
days of the week during the remaining field work period. Or insight
into the quality of the interviewer can be used in additional training
of the interviewers and personal feedback.
It is advised to prepare a quality assurance protocol before start-
ing a national food consumption survey. The protocol should describe
quality controls with regard to study organization, sampling of the
study population and representativeness, quality of the interviews or
instructions and checks, any data entry, cleaning of the dietary data,
interpretation of the dietary data and other data and performance of
any devices. For each quality aspect, the aim, probable responsible
person or organization and quality indicator should be indicated. An
example of a quality protocol is available in the PANCAKE supple-

mentary materials (Ocké et al., 2012).
5.6 Data Cleaning and Handling

After data collection, data cleaning is an important phase that can
start after the first data are received, simultaneously with the survey
until its end.
In the collected food consumption data and other information
collected (e.g. weight and socio-demographic background data), it
is advised to check for missing, impossible and improbable data,
extreme values or outliers. It may be useful to screen the obtained
distributions of variables of interest and to create cut-off criteria for
outliers to better identify any suspect data (EFSA, 2014).
A general impression of the quality of the food consumption
data can be obtained by comparison of reported energy intake and
expected energy intake based on height, weight and level of physical
activity (at the individual or the population level). Formulas to esti-
mate expected energy intake and cut-off values for improbable energy
intake data have been published (Schofield, 1985; Black, 2000).
After the food consumption data are checked, further handling
includes conversion to ingredients and raw agricultural products and
subsequent linkage to databases with concentrations of pesticides in
foods. After linkage is performed, statistical analyses can be con-
ducted. In these analyses, survey weights can be used to make the
data representative for the target population.
When chronic pesticide exposure is of interest, and food con-
sumption is measured on a few days, exposure per kg body weight
per day should be calculated and then usual intake modelling should
be applied. Various tools to model usual intake exist. These include
the Nusser method, NCI method, MSM method, MCRA tool and
SPADE (van Klaveren et al., 2012).
An alternative to linking the food consumption data to databases
with concentrations of pesticides in foods is to conduct chemical
analysis in foods directly. This approach is adopted in total diet
studies. The principles of total diet studies are given in Section 5.8.
5.7 Reporting of Food Consumption Data

Collecting food consumption data is a time- and resource-demanding
effort and needs to be carried out using high-quality standards at
each level of the process. It is, therefore, highly recommended that
the results would be available also to other interested users both
nationally and internationally. The most useful minimum informa-
tion to be reported includes, for example, the specifications of the
survey setting, sampling frame and method and inclusion and exclu-
sion criteria, responsible institutions for the data, details of methods
and tools used for the data collection, participation rate, descrip-
tion of the subjects, seasons and days of the week covered, food
classification and description system used, method of identification
of misreporters as well as other background information collected
(e.g. anthropometrics and socio-demographic background data). The
reporting of survey data in general and food consumption survey
data specifically have been the focus of recent guidance documents
in Europe (Tolonen, 2013), (EFSA, 2014).
From the perspective of exposure assessment (e.g. of pesticide
residues), it is important to report the daily food intake at the level of
food weight (grams of food) per day and in relation to the individual
body weight (if possible) (i.e. grams of food per kg body weight per
day). Further, it is important not only to report the mean and median
consumption of the total population, but also high percentiles, per-
centage of consumers, and mean, median and high percentiles for
consumers only.
5.8 Total Diet Studies

TDS are a specific method to collect food concentration data focused
not on food surveillance but on exposure assessment. At least for
non-volatile pesticides, the TDS approach is generally considered an
appropriate method (Vin et al., 2014) and hence some pesticides were
also prioritized at the Fifth International Workshop on TDS (WHO,
2015). Hence, several national TDS already have dealt with pesticide
residues (Egan, 2013; FSANZ, 2011; Mercier et al., 2014; Nougadère
et al., 2012).
A joint working group of EFSA, WHO and Food and Agricultural

Organization (FAO) in 2011 set out three criteria for TDS. The
first of the three criteria is to be representative of the whole diet,
which means that more than 90% of the foods contributing to
total mean food consumption should be analysed within a TDS
(EFSA/FAO/WHO, 2011b). To fulfil this criterion, information on
food consumption in the respective population is a prerequisite for
a TDS and individual food survey data are recommended for that
(Vannoort, 2013).
When establishing a TDS food list for analyses of pesticide
residues, similar principles have to be applied as for other substances.
Ideally, a food survey used to define the food list should be able to
differentiate between different age-groups and for specific diets like
vegetarian diet or organic diet that might be related to pesticide
residue level in the foods (Akhandaf et al., 2015). Further it will
be important to separate consumption due to regional or seasonal
aspects (Charrondiere, 2013).
Even if data from food surveys of individuals are considered to
be most appropriate for planning a TDS, food balance sheets or
GEMS/Food Regional Diets or data from household budget sur-
veys also are used (EFSA/FAO/WHO, 2011a). Figure 5.4 shows the
advantages of different kinds of consumption data used in planning of
a TDS. Whereas data from GEMS/Food Regional Diets are available
for all and from household budget surveys for most countries, food
consumption survey data of individuals are more expensive and not
available in all countries (Spungen, 2015). Food consumption survey
data, on the other hand, do have the advantage that they are mostly
reported as food as consumed and need not to be transformed for use
in a TDS programme. The joint working group of EFSA, WHO and
FAO divided TDS used for screening purposes and for use in refined
exposure assessments (EFSA/FAO/WHO, 2011b). Because in the
case of screening, only a limited number of broad food categories are
selected for analyses food balance sheets or household budget data
might be detailed enough for defining the food list. In the case of a
ALTERNATIVE INPUT DATA NEEDED FOR STARTING A TDS
FOOD BALANCE HOUSEHOLD BUDGET FOOD CONSUMPTION

SHEETS SURVEYS SURVEYS
(per capita) (per member of household) (per individual)
- Available for all countries - Available for most countries - Not available for all
countries
- Missing informaƟon on high - Missing informaƟon on high
consumers and variaƟon consumers and variaƟon - InformaƟon on high
between sub-populaƟons within households consumers, specific diets and
variaƟon between sub-
- Not food as consumed - Not food as consumed
populaƟons
- More appropriate for - More appropriate for
- Food as consumed
screening TDS screening TDS
- Appropriate for refined TDS
ANALYSIS OF FOODS AND TDS RESULTS

Increasing possibility to improve accuracy of results and apply modelling
techniques
FOOD SAMPLING SCHEMA

Increasing possibility to add sampling details and to focus on sub-populaƟons
Figure 5.4. The effect of the starting data on the later TDS procedures and
results.
more refined TDS, the food lists will be adapted to the specific needs
of groups of substances (Lindtner et al., 2015).
The second criterion of the TDS definition is the analyses of food
as consumed (EFSA/FAO/WHO, 2011b). Because pesticide residues
are affected by kitchen preparation as peeling and cooking, TDS has
a particular importance for refinement of long-term risk assessments
(Vin et al., 2014).
The third criterion is related to a main limitation of the TDS
approach (i.e. the loss of variability due to pooling of samples
(EFSA/FAO/WHO, 2011b)). Therefore, it has to be noted that TDS
is appropriate only for chronic risk assessment of pesticides. Acute
risk assessments need data from food monitoring programs and there-
fore both approaches ideally complement each other.
5.9 Future Challenges of Food Consumption

Survey Data Collections
Future data collection methods should focus on methods that have
a small participant burden or that are attractive to participants, so
that response rates increase. The use of modern technologies (e.g.
mobiles) might help for this purpose, although for some population
groups, it might not be appropriate. Examples of promising data
collection methods are online 24-hour dietary recalls, devices that
automatically take pictures of all food consumed, and applications
for food records using barcodes and food pictures for portion sizes, or
automatic recognition of photographed consumed food and volumes.
These promising technologies need to be extensively tested in the
general population and important subgroups of the population and
validated positively before they can be incorporated in food con-
sumption surveys. At the moment, various interesting developments
and validation studies with modern technologies are on-going (Ngo
et al., 2009; Martin et al., 2014; Long et al., 2010; Illner et al., 2012).
At the international level, harmonization of the data collection meth-
ods would further increase the comparability and possible pooling of
the data of different geographical regions (EFSA, 2014).
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Chapter 6
Dietary Exposure and Risk

Characterization for Pesticide
Residues in Food
Eloisa Dutra Caldas and Trijntje van der Velde-Koerts
Main topics
Introduction
Food consumption data
Pesticide residue data
Deterministic and probabilistic approaches to estimate exposure to
pesticides
Risk characterization of pesticide residues in food
6.1 Introduction
Dietary exposure to pesticides is estimated by multiplying food con-
sumption with the residue present in the food. It may cover the gen-
eral population or focus on vulnerable groups, estimated for a single
pesticide or a group of compounds with the same mechanism of action
or same effect (cumulative dietary exposure), and may be combined
with other routes (aggregate exposure). The estimates may refer
to long-term (chronic, lifetime) or short-term (acute) exposure and
may be derived through deterministic or probabilistic approaches.
More recently, a less-than-lifetime exposure has also been considered.
To characterize the risk from the exposure, the long-term dietary
intake is compared with the acceptable daily intake (ADI) and the
243
short-term dietary intake is compared with the acute reference dose

(ARfD).
This chapter presents and discusses the data used to estimate the
dietary exposure to pesticides, the methods used in the estimations
and in the characterization of the risk from the exposure.
6.2 Food Consumption Data

Food consumption information may be obtained at a population
level from food production statistics, which represent foods available
for consumption by the whole population, typically in the form as
produced. However, a more refined data can be obtained through
food consumption surveys at the household or individual levels. The
methods for collecting individual consumption data are discussed in
Chapter 5. In this chapter, we will discuss the GEMS/Food Diets,
which are based on food production statistics, and used for esti-
mating long-term exposure to pesticides by the Joint Meeting on
Pesticide Residues (JMPR).
6.2.1 The GEMS/Food Diets

Population-based methods provide gross annual estimates of the
national availability of food commodities based on food supply and
utilisation data, and may be used to calculate the average per capita
availability of raw and semi-processed agricultural commodities. Such
data is provided by the World Health Organization (WHO) Global
Environment Monitoring System/Food Contamination Monitoring
and Assessment Programme (GEMS/Food) to be used by the JMPR
and other expert bodies when conducting chronic dietary exposure
assessment at international level.
The GEMS/Food Diets are based on data derived from the FAO
Food Balance Sheets (FBS) and Food Supply Utilisation Account
(SUA), and reflect what is produced in a country plus what is
imported, minus what is exported and wasted, and then divided
by the number of inhabitants. The five GEMS/Food Regional Diets
(Middle Eastern, Far Eastern, African, Latin American and Euro-
pean), introduced in 1989 (WHO, 2003), were based on the FAO
Dietary Exposure and Risk Characterization for Pesticide Residues 245
Figure 6.1. GEMS/Food Consumption 17 Cluster Diets (WHO, 2012).
data for the period of 1990–1994 and were in place and used by
the JMPR for the long-term exposure assessment for pesticides until
2005, when they were replaced by the 13 GEMS/Food Consumption
Cluster Diets. The 13 cluster diets were developed based on average
FAO SUA data from 183 countries for the period 1997–2001 using
19 marker foods to group the countries into the clusters (Ambrus,
2016). In 2012, WHO introduced a new method to cluster the FAO
data, based on the data from 2002 to 2007 and similarities between
dietary patterns in 179 countries (Sy et al., 2013). This method
resulted in the 17 GEMS/Food Consumption Cluster Diets (Fig. 6.1),
which have been used by the JMPR since 2013.
The major limitations of using food supply and utilisation data
to estimate dietary exposure are that they actually reflect food avail-
ability rather than food consumption, and no differentiation can be
made to specific population groups. In spite of the many uncertainties
and limitations, they represent the best available source of data for
international comparison and are adequate for predicting long-term
intake of pesticides, given the inherent uncertainties of the other
parameters of the dietary risk assessment process.
6.2.2 Food Consumption Data for Long-term

Dietary Exposure Estimates
For deterministic dietary exposure approaches using the GEMS-food
diets, the average daily consumption is estimated by dividing the
yearly available amount of a particular commodity within a country
by 365 and then by the number of inhabitants within the country.
Information on food availability at household level may be used
to estimate the average per capita food consumption, however as
no information is available on the distribution of food consumption
among individual members of the household, it assumes that all indi-
viduals within a household consume the same amount of a given food.
Furthermore, the data do not account for outside household con-
sumption (may underestimate the consumption of some foods), and
for wasted food and food consumed by visitors (may overestimate the
consumption). In spite of these limitations, household budget survey
has been used to estimate dietary intake of pesticides at the national
level (Caldas et al., 2006a, 2006b).
For household or individual based methods, the average daily
consumption is derived from a distribution of food consumption val-
ues for individual persons within the survey, whereby all the persons
within the selected population group are taken into account (con-
sumers and non-consumers) (IPCS, 2009).
For probabilistic dietary exposure approaches, the individual con-
sumption data are used on a kg food per person per day or kg food per
kg bw per day basis, and the distribution of these consumption data
is used to generate a dietary exposure distribution (EFSA, 2012a).
6.2.3 Food Consumption Data for Short-term

Dietary Exposure Estimates
Food consumption data for short-term dietary exposure are prefer-
ably derived from individual based methods. For deterministic
dietary exposure approaches, the so-called large portion is obtained
from the 97.5 percentile (P97.5) of a distribution of food consumption
values, or preferentially food consumption per body weight (bw), for
a particular raw or semi-processed food commodity for consumers

only (WHO, 1997; EFSA, 2011).
For probabilistic dietary exposure approaches, the individual con-
sumption data are used on a kg food per person per day or kg food per
kg bw per day basis, and the distribution of these consumption data
is used to generate a dietary exposure distribution (EFSA, 2012a).
6.3 Pesticide Residue Data

Dietary exposure to pesticides can be assessed before a pesticide has
been authorized for use (pre-registration) or when the pesticide is
used and is actually present in the food supply (post-registration).
Pre- and post-registration pesticide residue data have a different ori-
gin and consequently the concentration levels are also different.
6.3.1 Pre-registration Residue Data

Pre-registration residue data are obtained from supervised residue
trials conducted according to good agricultural practice (GAP),
which are the instructions for use specified on the pesticide prod-
uct label. In general, the trials are conducted by the manufacturer
of the pesticide product as part of the registration process, but they
may also be conducted by governments, farmer associations or other
parties. The supervised residue trials need to be conducted accord-
ing to Organisation for Economic Co-operation and Development
(OECD) guidelines and guidance (Chapter 2), and residue data are
obtained from composite samples of raw commodities: At least 12–24
units per sample for large to medium-sized commodities, and at least
1–2 kg per sample for small-sized commodities (Ambrus, 2016). Pre-
registration residue data are also used by the JMPR for the assess-
ments at international level after the registration has been granted
by national authorities (see Chapter 4).
Residue data from supervised trials are used to estimate max-
imum residue limits (MRLs) as well as supervised trials median
residues (STMR) and highest residues (HR). STMR and/or HR can
be set at zero if the pesticide application is conducted early in the
growing season and/or before harvested parts have been formed, and
if results from metabolism studies and/or supervised field trials at

exaggerated dose rates confirm the zero residues (Chapters 3 and 4).
The STMR is used for deterministic long-term dietary exposure
estimates, while the STMR and HR are used for deterministic short-
term dietary exposure estimates (JMPR, 2015; EFSA, 2011). The
distribution of the results obtained from all acceptable independent
trials may be used for probabilistic long-term and short-term dietary
exposure estimates (EFSA, 2012a).
In case consumption data are available for semi-processed or
processed commodities, the pre-registration residue results from the
raw agricultural commodity (RAC) may be corrected for changes in
residue levels during processing (such as peeling, cooking, milling
and juicing). A processing factor is defined as the ratio between the
residue level of a specific pesticide in the processed product and the
residue level in the starting commodity, usually a RAC. Process-
ing studies are conducted, generally, by the pesticide manufacturer
according to OECD guidelines and guidance (OECD, 2008). The
residue results in the RAC are then multiplied by the median or best
estimate processing factor to get STMR-P and HR-P values for use
in the dietary exposure estimations (see Chapter 4 for the JMPR
procedure). The distribution of all individual processing factors may
be used for probabilistic long-term and short-term dietary exposure
estimates (EFSA, 2012a).
6.3.2 Post-registration Residue Data

Post-registration residue data are obtained from monitoring, total
diet studies or duplicate diets.
6.3.2.1 Residue data obtained by monitoring programmes
Various countries have established pesticide monitoring programmes,
which are conducted mostly to enforce the established tolerances
(MRLs) (EFSA, 2015a, Brazil, 2015; DAWR, 2014; USFDA, 2015).
Sampling for pesticide monitoring is mostly done in raw commodities
and data are obtained from composite samples collected according
to the Codex protocol (Codex, 1999). Domestic foods are typically
collected in food stores and food distribution centres, and imported
foods are collected at the point of entry in the country. Therefore,

monitoring data represent the actual residues that can be expected
in the foods as traded and residue levels are often much lower than
those in the pre-registration supervised trial samples. Reasons for
that include: (a) crops have been treated at lower dose rates than
permitted on the pesticide product label; (b) crops have been har-
vested at longer pre-harvest intervals than stated on the label; (c)
degradation of the residues may have occurred during transport and
storage and (d) the composite monitoring sample may consist of a
mixture of treated and untreated food commodities.
Data from targeted sampling in monitoring programmes are
collected when an exceeding of the MRL is suspected based on pre-
vious inspection data. Random data are the preferred data for post-
registration dietary exposure assessment as they are expected to be
representative for the food available in commerce. The pesticide and
commodities included in the programme, the number of samples anal-
ysed and the origin of the samples vary among countries and regions,
and are defined based on different factors, including costs, monitor-
ing history data and the importance of each commodity in the dietary
pattern. In 2013, the European Union (EU) monitored 80,000 samples
for pesticide residues, but these represented only 12 food commodities
(EFSA, 2015a). In USA, the results of the pesticide monitoring pro-
gramme conducted in the 2012 fiscal year covered 5,523 analyses of over
100 commodities, which was 79% of imported food (USFDA, 2015).
Random monitoring is conducted on a limited number of pro-
cessed commodities (e.g. wine and juice). In case consumption data
are available for semi-processed commodities and no monitoring
results are available for these commodities, the residue results from
the raw commodity may be corrected for changes in residue levels
during processing by using the processing factors obtained at pre-
registration or from other sources.
6.3.2.2 Residue data obtained from total diet studies

Over 50 countries around the world are conducting TDS, generally
at least once every three to four years; the USFDA has been the
most consistent, by conducting a TDS at least once a year since
1961 (WHO, 2015). TDS are distinct from regulatory monitoring in

that they determine pesticide residues not in the raw commodity,
but in foods that are prepared table-ready for consumption. The
sampled foods are washed, peeled and/or cooked before analysis,
simulating typical consumer handling. TDS foods are sampled as
‘market baskets’ comprising samples of different foods that repre-
sent the average consumer’s diet within a country. In addition to
being analysed for pesticide residues, TDS foods may also be anal-
ysed for contaminants, nutrient elements and industrial chemicals
(EFSA/FAO/WHO, 2011; Moy and Vannoort, 2013).
Since foods are prepared as they would be consumed prior to
analysis, residue levels in the TDS samples are often much lower
than those in the monitoring samples because processing may reduce
pesticide residues significantly.
A TDS needs to be planned carefully, taking into account knowl-
edge of residue concentration data from monitoring, consumption
patterns for relevant population groups (e.g. small children) and mar-
ket shares for food commodities. If resources are limited, TDS may
necessitate pooling of samples and/or analysis of a limited number
of samples per food commodity. This affects identification of those
food commodities with pesticide residues, statistical precision and/or
representativeness of sampling (WHO, 2015).
TDS sampling design may vary among countries, but there have
been efforts worldwide to harmonize the approaches (EFSA/FAO/
WHO, 2011). A coverage of 80–95% of food in the diet is often tar-
geted for inclusion in the TDS food list, selected using consumption
pattern (e.g. food consumed at more than 1 g/day per person or by
at least 10% of the consumers).
Within the U.S., foods (market baskets) are collected from super-
markets, grocery stores and fast food restaurants four times each
year, once in each of four regions of the country. Each region consists
of three cities that differ every year. The survey includes about 300
different foods in every city, a list that is updated from time to time
to reflect changes in eating patterns in the country (USFDA, 2015).
In a TDS conducted in 14 cities in Japan, Tsukakoshi (2011)
found a high intra-city variance, which was greater than the inter-city
variance for all of the food groups studied. The authors suggested
that grouping food samples collected from different stores in the same
city can improve the representativeness of the results.
6.3.2.3 Residue data obtained from duplicate diet studies

In duplicate diet studies, the participants consume their ordinary diet,
but they save a duplicate portion of all food and drinks consumed
within a timeframe of 24 hours. Collected food items are weighed,
non-edible parts removed and samples homogenized, resulting in sev-
eral pooled food samples per day for analysis (Kroes et al., 2002;
EFSA, 2011). Hence, consumption and residue data information are
obtained within the study for each participant. Duplicate diet stud-
ies are expensive and are often limited in number of participants and
samples. Because of these limitations, they are more useful for looking
at average dietary exposure rather than at high-end dietary exposure,
and are particularly useful for estimating dietary exposure of a par-
ticular well-defined population subgroup, such as children (Lu et al.,
2010) and small communities (Melnyk et al., 2014).
6.4 Deterministic and Probabilistic Approaches to

Estimate Dietary Exposure to Pesticides
Deterministic approaches for estimating the dietary exposure to pes-
ticides are generally applied during pre-registration using residue data
obtained from supervised field trials, and are also used by the JMPR.
Deterministic models use a single-point estimate for both consump-
tion and the residue, such as the mean, median, 97.5 percentile or
a maximum value. The major advantages of this approach are the
simplicity of the calculation, it is easily understood by risk managers
and can be easily communicated to the interested parties. However,
the estimates tend to over-estimate the actual dietary exposure and
only provide limited information to risk managers (Kroes et al., 2002).
Although limited, a deterministic estimation is important as an initial
diagnostic of a risk situation, and indicates the need for generating
additional data for a refinement of the assessment.
Probabilistic approaches are generally used post-registration
using residue data obtained from monitoring, total diet studies
or duplicate diet studies. Probabilistic models use a distribution
95P
X Mean concentration
Food consumption of the pesticide, mg/kg
modeled distribution, kg/kg bw Usual intake, mg/kg bw
Figure 6.2. Probabilistic assessment of dietary long-term exposure to pesticides.
of consumption data and residue concentration data to obtain a

distribution of actual dietary exposures, normally using Monte Carlo
techniques (Fig. 6.2). There are a variety of software tools avail-
able for conducting probabilistic dietary exposure assessment of pes-
ticides and other chemicals, including the DEEM-FCID/Calendex
model used by USEPA (2015a), and the Monte Carlo risk assessment
(MCRA) used by the Netherlands (Van der Voet et al., 2015).
The main advantages of the probabilistic approach are that it
provides different scenarios of dietary exposure to risk managers to
help on their decisions, results can be associated with a quantita-
tive measure of uncertainty, and the impact of each parameter in
the assessment can be evaluated (sensitivity analysis) (Kettler et al.,
2015). However, the models are more complicated to perform, and
therefore more time-consuming, and the results may be more difficult
to communicate to risk managers and to the public due to different
dietary exposure scenarios that can be generated. Furthermore, prob-
abilistic methods can only be used when the raw data are available,
which limits their application under certain circumstances. It is the
method of choice to be used for cumulative and aggregate (dietary)
exposure assessment.
6.4.1 Long-term and Short-term Dietary Exposure

6.4.1.1 Long-term dietary exposure
In a deterministic assessment, the long-term dietary exposure, or
estimated daily intake (EDI), may be estimated by multiplication
of the average food consumption level (Cf in kg food/bw) with the
average (or median) pesticide residue concentration in the food (Rf in
mg/kg). The overall dietary exposure (EDI in mg/kg bw) is obtained
by summation of all intakes from foods (up to n foods) containing

the pesticide residue (Eq. (6.1)).
n

EDI = (Rf × Cf ) (6.1)
1
The average residue concentration is thought to represent the long-

term average of actual residue concentrations. The average consump-
tion is obtained by taking into account consumers and non-consumers
of a food by a given population. The median residue value of super-
vised residue trials (STMR) is used by the JMPR and by most
national authorities during pre-registration. Post-registration assess-
ments are generally conducted using the average residues found in
monitoring studies.
Probabilistic long-term dietary exposure assessments gener-
ally are conducted only post-registration, mostly using monitoring
residue data. Estimates of the actual long-term dietary exposure dis-
tribution may be obtained from statistical modelling of the 24-hour
consumption (usual consumption) multiplied by the average residue
concentration, to estimate the usual intake (Van der Voet et al.,
2015) (Fig. 6.2). Different exposure scenarios, such as the 95 per-
centile of the intake distribution, can be selected to characterize the
dietary risk.
6.4.1.2 Less-than-lifetime exposures

Experience has shown that potency of toxicological effects is often
similar over a wide range of exposure durations ranging from 4 to
104 weeks in laboratory animals. This suggests that in those cases
the manifestation of adverse effects is not related to the duration of
exposure. Current dietary risk assessment procedures do not consider
possible exposure scenarios where the time-weighted average dietary
exposure during more than one day but less than lifetime (up to
four weeks) is greater than the lifetime average dietary exposure. As
it is not known whether the ADI is exceeded is such a situation,
dietary exposure models need to be developed that take into account
short-term dietary exposure over a four-week period.
Special emphasis needs to be given to frequent dietary exposure

during the entire lifetime, seasonal dietary exposure during the entire
lifetime or frequent dietary exposure during a short period in life (e.g.
at infancy, at childhood, or in a nursing home; JMPR, 2015).
6.4.1.3 Short-term dietary exposure

Short-term dietary exposure assessments focus on the high end of the
residue and food consumption distributions. In a deterministic assess-
ment, mostly conducted during pre-registration, the intake may be
estimated multiplying the large portion food consumption by a high
pesticide residue concentration in those foods. Each food commodity
is assessed individually, since it is considered highly unlikely that the
same person consumes a large portion of more than one food on the
same day, whereby each food contains a high residue of the same
pesticide. The highest residue concentration from supervised field
trials (HR, in mg/kg) is thought to represent the short-term high
actual residue concentration. The large portion food consumption
(LP, in kg food/person) is obtained by taking the 97.5th percentile
of a ‘consumer-only’ distribution of the food in question.
The JMPR calculates the international estimated short-term
intake (IESTI) for both children (up to six years old) and adult
populations considering four different cases (Eqs. (6.2)–(6.5); JMPR,
2003). Large portion (LP, in kg food/person), body weight (bw, in
kg/person) and unit weight (unit weight of the edible portion, U e in
kg, or unit weight of the raw agricultural commodity, URAC in kg)
information used in the equations was provided by Codex member
countries. The equations can also be used by national authorities
during the pre-registration of a pesticide.
In Case 1, the residue in a composite sample (raw or processed)
reflects the residue level in a meal-sized portion of the commodity
URAC is below 0.025 kg. It also applies to meat, edible offal and eggs
and for grains, oilseeds and pulses treated post-harvest.
Case 1:
LP × HR
IESTI = (6.2)
bw
In Case 2a, Ue is less than the LP and in case 2b, Ue is greater than
the LP. In both cases, a variability factor (ν) of 3 is applied to the
composite residue to estimate the residue level in a high-residue unit.
This factor is defined as the residue level in the 97.5th percentile unit
divided by the mean residue level for the lot.
Case 2a:
Ue × HR × v + [(LP − Ue ) × HR]
IESTI = (6.3)
bw
Case 2b:
LP × HR × v
IEST = (6.4)
bw
Case 3 allows for the likely bulking and blending of processed
commodities such as flour, vegetable oils and fruit juices, and the
STMR is used as residue level. It also applies to milk, grains, oil
seeds and pulses treated pre-harvest.
Case 3:
LP × STMR
IESTI = (6.5)
bw
Probabilistic short-term dietary exposure integrated by Monte
Carlo sampling combines random draws of consumption patterns
(individual-days) with random values sampled from the residue con-
centration distribution for each relevant food (Fig. 6.3). Variability
factor can also be included in the model (Van der Voet et al., 2015).
The outcome is a distribution of actual short-term dietary exposures.
As for the long-term exposures, and different scenarios (percentiles)
can be selected to characterize the dietary risk (e.g., 99.9P; Fig. 6.3).
6.4.2 Cumulative and Aggregate Exposure

Exposure to pesticide is traditionally performed for a single com-
pound at a time and for one pathway at a time (e.g. dietary expo-
sure). However, people may be exposed to more than one chemical
via the diet and other pathways, because a food may contain more
than one chemical, people eat combinations of foods that contain
different chemicals or are exposed to chemicals through more than
one pathway (e.g. food or drinking water and residential or occupa-
tional) or through more than one route (oral intake, dermal contact
99.9P
X Actual intake distribuƟon,

Food consumpƟon PesƟcide concentraƟon
(mg/kg bw)
DistribuƟon (kg/kg bw) distribuƟon (mg/kg)
Figure 6.3. Probabilistic assessment of dietary short-term exposure to pesticides.
or by inhalation). If these compounds act by the same mechanism

and produce the same toxicological effect, the conventional way of
estimating exposure to pesticides and other chemicals may lead to an
underestimation of the health risk (Boon et al., 2008). Cumulative
aggregate exposure models take into account combined exposure to
multiple chemicals (‘mixtures’) via multiple pathways (Meek et al.,
2011). In general, the cumulative and/or aggregate assessments are
conducted using probabilistic models.
Cumulative dietary exposure scenarios take into consideration
that dietary exposure could occur to multiple pesticides that act by
the same mechanism. A common mechanism of toxicity is identified
when two or more pesticides (or other chemicals) cause a common
toxic effect by the same, or essentially the same, sequence of major
biochemical events, interpreted as mode of action (USEPA, 2002;
Boobis et al., 2008). Such scenarios assume that individual effects
are dose-additive and that there are no synergistic or antagonistic
effects. Cumulative dietary exposure could be limited to pesticides,
but could also be wider interpreted as cumulative dietary exposure
to various chemicals across various regulatory frameworks like pesti-
cides, biocides, veterinary medicines and food contact materials.
A cumulative aggregate exposure assessment begins with the
identification of a group of chemicals that induce a common toxic
effect by a common mechanism of toxicity. During the hazard
characterization phase, the various endpoints associated with the
common mechanism of toxicity are identified, as well as the test
species/sex that might serve as a uniform basis for determining
relative potencies among the chemicals of interest. A screening-level
assessment may be conducted to identify the exposure pathways
that contribute most to the overall exposure, and identify a subset

of common mechanism chemicals to be included in the cumulative
assessment (cumulative assessment group, CAG) (USEPA, 2002).
In the United States of America, the USEPA considers cumulative
aggregate exposures to pesticides under the Food Quality Protec-
tion Act of 1996, and the assessment is currently conducted for
five groups of pesticides that share a common mechanism of tox-
icity: organophosphates, N -methyl carbamates, triazines, chloroac-
etanilides and pyrethrins/pyrethroids (USEPA, 2015b).
Once a CAG is defined, a dose–response analysis is performed on
each CAG member to determine its toxic potency for the common
toxic effect in relation to an index chemical. Potency normalization
approaches have been used for dioxins and other aryl hydrocarbon
receptor agonists (toxic equivalent), polycyclic aromatic hydrocar-
bons and pesticides (relative potency factor, RPF) (IPCS, 2009;
USEPA, 2015a). The RPF of a pesticide p is defined as the ratio
between the toxicity endpoint (Tox; benchmark dose [BMD], no-
observed-adverse-effect-level [NOAEL]) of the index compound (IC)
and the toxicity endpoint of the pesticide p (Eq. (6.6)). The RPFs are
used to convert the concentration of all chemicals in the CAG into
equivalents of the index chemical. For dietary cumulative exposure
to pesticides, if a sample contains more than one pesticide within a
CAG, the cumulative residue in the sample, expressed as the index
compound (CRIC ) is estimated according to (Eq. (6.1)), where R is
the residue concentration of pesticide p:
ToxIC
RPF = (6.6)
Toxp
p1
p2
p3
CRIC = Rp1 × RPFp1 + Rp2 × RPFp2 + Rp3

×RPFp3 (6.7)
Other approaches to conduct cumulative exposure assessment to

chemicals include the hazard index, the cumulative risk index, the
reference point index and the combined margin of exposure (Boobis
et al., 2008).
The WHO International Programme on Chemical Safety (IPCS)
developed a framework for cumulative aggregate risk assessment
designed to aid risk assessors in identifying priorities for risk manage-
ment of a wide range of applications where co-exposures to multiple
chemicals are expected. It is based on a hierarchical approach that
involves consideration of exposure and hazard at all four tiers, with
each tier being less conservative and more accurate and data intensive
than the previous one (Meek et al., 2011).
Within the EU, cumulative dietary risk assessment for pesti-
cides and other chemicals is being developed. Grouping of pesti-
cides is based on identification of compounds that exhibit similar
toxicological properties in a specific organ or system (i.e. common
adverse outcomes on the same target organ/system). This group-
ing methodology is based on the commonality of the effect rather
than on the commonality of the mode of action. A combination of
the effects is based on dose addition irrespective of the modes of
action, provided they produce a common adverse outcome. As a first
step, this methodology was applied to define groups of pesticides
(CAGs), which are toxic to the thyroid and central nervous systems,
and can be expanded to other organs/systems (eye, liver, adrenals
and on the reproduction and development systems) (EFSA, 2013a,
2013b).
Currently, harmonized terminology and frameworks for the
human risk assessment of combined exposure to multiple chemicals
are being developed (EFSA, 2013c). Aggregate and Cumulative Risk
of Pesticides, an on-line integrated Strategy (ACROPOLIS) project
was implemented in 2010–2014 to improve risk assessment strate-
gies in Europe (ACROPOLIS, 2015). The EuroMix project was ini-
tiated in 2015 to develop a tiered test strategy for risk assessment of
mixtures of multiple chemicals derived from multiple sources across
different life stages based on existing and new toxicological tests
(EUROMIX, 2015).
Various studies on cumulative dietary risk assessment have been

conducted worldwide, mainly to organophosphorus and carbamate
pesticides (Caldas et al. (2006a) in Brazil, Boon et al. (2008) and
Bosgra et al. (2009) in the Netherlands; Jensen et al. (2009) in Den-
mark and Blaznik et al. (2015) in Slovenia). Other cumulative assess-
ment groups include the dithiocarbamates (Caldas et al., 2006b), the
anti-androgenic pesticides vinclozolin, procymidone and prochloraz
(Müller et al., 2009), the endocrine disruptors epoxiconazole, prochlo-
raz, procymidone and tebuconazole (Jensen et al., 2013) and the
triazoles (Boon et al., 2015).
6.5 Harmonization of International Dietary

Exposure Assessment
The actual consumption data, residue data and exposure pathways
are specific for a certain region, because consumption habits and
authorized pesticide uses differ between countries. However, even
with these particularities, harmonization of the methods used in the
exposure assessment should be sought by national, regional and inter-
national organizations.
Given that the design of food consumption studies can have a
critical impact on any dietary exposure assessment, harmonization of
study design should be achieved to the extent possible (Chapter 5).
Ideally, food consumption surveys for dietary exposure should be
based on individual dietary records of a least two non-consecutive
days, whereby also the individual body weights are recorded. The
food consumption survey needs to be conducted in such a way that
consumption values are a faithful reflection of the consumption habits
within a population (urban, rural, gender, age) throughout the year
(seasonal crop commodities, all days in the week). Especially the less
frequent consumed (seasonal) commodities and consumption habits
of minority groups within a population are underrepresented in the
current food consumption surveys.
EU has harmonized the deterministic long-term dietary expo-
sures within the region using the Pesticide Residues Intake Model
(PRIMo), where the consumption data for several EU countries have
been listed, as well as the GEMS/Food cluster data representing EU

countries. The National Estimated Daily Intake (NEDI) is calculated
for each country and population group and the highest NEDI is used
to characterize the dietary risk (EFSA, 2015b).
Short-term dietary intake calculations are based on the highest
large portion of a particular commodity within the countries rep-
resented in the model (e.g. PRIMo for EU or the JMPR IESTI
for Codex Members). Each large portion is derived from a national
food consumption survey rather than a worldwide food consumption
survey.
The PRIMo model for short-term dietary intake currently used
by EU is similar to the JMPR IESTI (Eqs. (6.2)–(6.5)), with the
exception of the variability factor, as the values 5 or 7 are used
depending on the crop (EFSA, 2015b). An international workshop
was organized in 2015 by EFSA to revisit the deterministic short-
term dietary intake equations (EFSA, 2015c). This workshop rec-
ommended replacing the variability factors currently used by EU
to the value of 3, to harmonize with the JMPR approach. Other
recommendations included to remove the unit weight (U ) from the
equations, since they vary significantly and no single unit weight can
be defined for a crop, and use the MRL as residue input, rather
than the STMR or HR. These recommendations were acknowledged
by the 2015 JMPR. However, the meeting agreed that the outcome
of the current and proposed equations need to be compared before
considering changes in the current approach (JMPR, 2015). One rec-
ommendation of the workshop that may be adopted by the JMPR as
the data became available was to define the LP as 97.5th percentile
of consumption per body weight of a consumer-only distribution,
instead of the 97.5th percentile of consumption on a kg/person basis,
as currently used.
6.6 Risk Characterization of Pesticide

Residues in Food
Within the risk assessment process, the risk characterization step
may be defined as the qualitative and, wherever possible, quantitative
determination, including attendant uncertainties, of the probability

of occurrence of known and potential adverse effects of an agent in
a given organism, system, or (sub)population, under defined expo-
sure conditions (IPCS, 2004). In this step, the estimated exposure is
compared with the acceptable levels of exposure, ADI and/or ARfD.
All registered pesticides have an ADI established and for some
having a relevant acute toxicity, also an ARfD. However, some com-
pounds produced during the pesticide metabolism in the plant or
during processing may not be covered by the pesticide ADI/ARfD,
and a different approach should be used to include these compounds
in the risk characterization.
6.6.1 Compounds with ADI and/or ARfD

ADI and ARfD are most commonly based on the most sensitive end-
point and the corresponding point of departure value (e.g. NOAEL or
BMD after exposing laboratory animals to the chemical in question).
Moreover, to arrive at the ADI for long-term intake or the ARfD for
short-term dietary intake, safety or uncertainty factors are applied
not only to take into account differences between animals and humans
(inter-species factor) and among humans (intra-species factor), but
also to account for the use of a LOAEL instead of a NOAEL, for
the short duration of the study, deficiencies in the database or the
nature of the effect and the dose–response relationship (see Chapters
2 and 4).
A long-term dietary risk assessment is necessary in all cases, while
a short-term dietary risk assessment is needed only when an ARfD has
been allocated. In the risk characterization step of the risk assessment
process, the estimated intakes after long-term or short-term dietary
exposures are compared with the acceptable levels of dietary expo-
sure of the pesticide (ADI or ARfD). In a cumulative dietary expo-
sure assessment, the cumulative dietary intake, estimated using the
cumulative residue normalized for the index compound (Eq. (6.7)) is
compared with the ADI or ARfD of the index compound.
In the deterministic long-term dietary exposure approach, the
EDI for a defined population group in each country or within a group
of countries in a region is compared to the ADI and the country popu-

lation group with the highest dietary exposure is used to characterize
dietary risk.
In the deterministic short-term dietary exposure approach, the
risks are characterized separately for each food commodity. The esti-
mated pesticide intake over a 24-hour period for a particular food
commodity (IESTI) for defined population groups in each country
or within a group of countries is compared to the ARfD and the
population group or country with the highest dietary exposure is
used to characterize dietary risk for that commodity.
In the risk characterization, the relevant question to be answered
is: Is the dietary exposure of this population to this pesticide or
group of pesticides safe? A public health concern may exist when the
intake exceeds the ADI and/or the ARfD. In this case, it is important
that the risk assessor acknowledge the uncertainties involved in the
parameters used in the estimations, and identifies the possible refine-
ments in the assessments, including the availability of residue and/or
consumption data in processed commodity, and more appropriate
data for ADI or ARfD setting. Furthermore, probabilistic assessment
may be used to estimate the proportion of the population group pos-
sibly at risk, and provide to the risk manager relevant information
for decisions that may be needed to reduce the dietary exposure,
including not approval or cancel the approved use of the pesticide in
the crop(s) that contributed most to the dietary exposure.
6.6.2 TTC Approach

Residues of pesticides in foods often comprise not just the active sub-
stance (parent compound) but also degradation/metabolism prod-
ucts formed or taken up from soil in treated crops or formed during
processing of the raw agricultural commodities. Where such products
are also formed in the laboratory test animal species used to derive
the ADI and ARfD for the parent compound, it is assumed that
their hazard will have been addressed in the hazard assessment of
the parent compound. When they are not formed or formed at low
levels, additional hazard assessment of these products is necessary
(EFSA, 2012b).
In such cases, a threshold of toxicological concern (TTC)

approach is followed (EFSA, 2012a; JMPR, 2013).
• As a first step, the dietary exposure to the degrada-

tion/metabolism products is estimated, either based on actual
measured values in the food crop or based on ratios between par-
ent and degradation/metabolism products in relevant metabolism
studies.
• As a second step and where appropriate, the TTC can be derived
by computational methods, involving the separate or sequen-
tial use of (quantitative) structure–activity relationship and read-
across in the prediction of genotoxicity and developmental toxicity.
• As a third step and where appropriate, relative toxic potencies are
determined for the degradation/metabolism product in relation to
the parent compound.
• As a fourth step, these relative toxic potencies are applied to the
calculated dietary exposure of the degradation/metabolism prod-
uct and these are added to the estimated dietary exposure of the
parent compound for comparison with the reference values of the
parent compound (ADI, ARfD).
If the TTC approach is not appropriate, comprehensive toxicity test-

ing is needed to derive separate toxicological reference values for the
degradation/metabolism products.
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b2530_FM.indd 6 01-Sep-16 11:03:06 AM

Chapter 7
Importance of Codex Maximum

Residue Limits for Pesticides
for the Health of Consumers
and International Trade
Yukiko Yamada
Main topics
What is the Codex Alimentarius Commission?

Establishment of Codex maximum residue limits (MRLs) for
pesticides, extraneous maximum residue limits (EMRLs) and related
recommendations
Codex recommendations related to pesticide residues in foods and
feeds other than MRLs and EMRLs
7.1 What is the Codex Alimentarius Commission?

7.1.1 Importance of the Codex Alimentarius
Commission (CAC ) in Food Safety in Relation
to the International Food Trade
The World Trade Organization (WTO) was formed for trade
facilitation and offers a forum for governments to negotiate
trade agreements and to settle trade disputes. Among the WTO
Agreements, the Agreement on the Application of Sanitary and
Phytosanitary Measures (SPS Agreement) (WTO, 1995a) and
Agreement on Technical Barriers to Trade (TBT Agreement) (WTO,
269
1995b) are related to international food trade. The SPS Agreement

applies to all food safety measures ‘which may, directly or indirectly,
affect international trade of food’ (Article 1.1). This agreement
explicitly states in its Annex A Definitions that the standards,
guidelines and recommendations established by the CAC relating to
food additives, veterinary drug and pesticide residues, contaminants,
methods of analysis and sampling, and codes and guidelines of
hygienic practice are regarded as the international standards,
guidelines and recommendations for food safety.
The SPS Agreement describes the need for scientific principles
and sufficient scientific evidence (Article 2.2); using Codex
‘standards, guidelines or recommendations’ (Article 3.1); and risk
assessment as a basis (Article 5.1). These requirements apply not
only to all the members of WTO but also to the CAC.
However, the SPS Agreement allows introducing or maintaining
measures resulting in a higher level of protection than would be
achieved by measures based on the Codex standards, guidelines
or recommendations, if there is a scientific justification, or as a
consequence of the level of health protection a member of WTO
determines to be appropriate. Such measures shall not be inconsistent
with the provisions of the agreement (Article 3.3).
7.1.2 Statutes of the CAC

The CAC was established by the Food and Agriculture Organization
of the United Nations (FAO) and the World Health Organization
(WHO) in 1963. It gained more importance after the SPS Agreement
and TBT Agreement came into effect in 1995.
The Statutes of the CAC were first adopted in 1961 by the 11th
Session of the FAO Conference and in 1963 by the 16th Session of
the World Health Assembly, and revised in 1966 and 2006. The term
‘Codex Alimentarius’ means ‘food code’ in Latin. The purpose of
the CAC is spelled out in Article 1 of the Statutes and includes,
inter alia, (a) protecting the health of the consumers and ensuring
fair practices in the food trade; and (b) promoting coordination of
all food standards work undertaken by international governmental
Importance of Codex Maximum Residue Limits 271
and non-governmental organizations. Its major task is to develop

and establish standards, codes of practice, guidelines and other
recommendations for foods and feeds moving in international trade.
The Rules of Procedure of the CAC were adopted in 1963 at
the First Session of the CAC and amended many times since then.
These Statutes and Rules along with principles, definitions and
procedural issues related to Codex work are contained in the Codex
Alimentarius Commission Procedural Manual (FAO, 2016 et seq.
anni). The Procedural Manual has been updated after each session of
the Commission and is the ‘must-have’ document for any participants
of Codex sessions.
7.1.3 Structure of the CAC

The supreme body of the Codex Alimentarius is the Commission,
which has met annually, since 2003, alternately in Rome and Geneva
and is supported by the chairperson, three vice-chairpersons, the
Executive Committee and the Codex Secretariat located in the FAO
Headquarters in Rome. The Executive Committee consists of the
chairperson, vice-chairpersons, coordinators (countries) of the six
regions and seven further members elected from seven geographic
locations. Under the CAC, there are four categories of subsidiary
bodies: general subject committees, commodity committees, regional
coordinating committees and ad hoc task forces. For each Codex
committee, a host country is appointed by the CAC. Its subsidiary
bodies and their host countries are shown on the last page of the
Procedural Manual. The number of subsidiary bodies may change
because ad hoc working groups are established as necessary, and
dissolved upon completion of the work assigned to them.
The Codex Committee on Pesticide Residues (CCPR) is
responsible for issues related to pesticide residues in foods and feeds
moving in international trade.
7.1.4 Role of Science and Risk Analysis in the CAC

As stipulated in the SPS Agreement, any decisions of the CAC
in the area of food safety shall be based on scientific principles
while implication of proposals for Codex members’ economic interests

should also be considered during their elaboration (see Section 7.2.4).
In 1995, the CAC agreed on the ‘Statements of Principle
Concerning the Role of Science in the Codex Decision-Making
Process and the Extent to Which Other Factors Are Taken into
Account’. The first of the four statements specifies that the
recommendations of CAC shall be based on the principle of sound
scientific analysis and evidence. In 2001, the CAC agreed on the
criteria for the consideration of the other factors referred to in the
second statement of principle (i.e. other factors than science).
Further to the science-based approach, the work of Codex related
to food safety shall follow the risk analysis framework. The CAC first
implemented risk analysis in 1993 and, since then, risk analysis has
been the solid basis of any food safety related work of the CAC,
including work on pesticide residues. The CAC in 1997 established
the ‘Statements of Principle Relating to the Role of Food Safety Risk
Assessment’. The CAC adopted the ‘Definitions of Risk Analysis
Terms Related to Food Safety’ (1997; amended in 1999, 2003, 2004)
and the ‘Working Principles for Risk Analysis for Application in
the Framework of the Codex Alimentarius’ (2003). Since then, a
number of recommendations were developed on risk analysis for use
by specific Codex committees dealing with food safety (contained in
the Procedural Manual) and for use by governments in specific food
safety areas (available on the Codex website).
The ‘Statements of Principle Relating to the Role of Food Safety
Risk Assessment’ emphasize the importance of basing food safety
related Codex decisions and recommendations on risk assessment
and sound science; functional separation of and interaction between
risk assessment and risk management; and presentation of risk
characterization in readily understandable and useful form.
In 2007, the CAC first adopted the ‘Risk Analysis Principles
Applied by the Committee on Pesticide Residues’ and subsequently
issued a revision in 2014. An annex on the Proportionality Concept
was adopted in 2013. It contains risk assessment policy, risk
assessment and the role of the Joint FAO/WHO Meeting on Pesticide
Residues (JMPR), risk management and the role of CCPR, and risk
communication. Under the risk assessment policy, the decisions made

by the CCPR are included as requests for the JMPR to implement,
such as group MRLs, MRLs for fat-soluble pesticides in milks
and mammalian meats, MRLs for spices on a basis of monitoring
data, setting EMRLs, proportionality concept and MRLs for minor
crops. These are explained in detail in Chapter 4. The general
aspect section (Section 1) and risk management section (Section 5)
explain the criteria for selecting pesticides for evaluation by JMPR
and elaboration procedures for MRL and EMRL setting. They are
explained in Sections 7.2.3 and 7.2.4. The risk analysis principles
applied by the CCPR are continuously evolving and incorporate
the new procedures developed and recommended by the JMPR and
adopted by CAC. The latest version of the risk analysis principles is
included in the CAC Procedural Manual published regularly (FAO,
2016 et seq. anni).
7.2 Establishment of Codex Maximum Residue Limits

(MRLs) for Pesticides, Extraneous Maximum
Residue Limits (EMRLs) and Related
Recommendations
7.2.1 Codex Definitions of the Terms Related
to Pesticide Residues
There are a number of terms specific to the work on pesticide residues
in foods and feeds, such as MRLs, EMRLs, good agricultural practice
(GAP) and so on. The definitions of these terms were developed by
the CAC and contained in the Procedural Manual (see Table 7.1).
7.2.2 Codex Committee on Pesticide Residues

The CCPR is one of the Codex general subject committees and
is responsible for developing recommendations related to pesticide
residues in foods and feeds in international trade. It was hosted by the
Netherlands from 1966 to 2007 and then has been hosted by China
since 2007. The CCPR first met in 1966, and since then, it has met
once each year except 1971, 1973 and 1976. Its terms of reference
are described below. The most important task of the CCPR is to
Table 7.1. Definitions for the purposes of the Codex Alimentarius related to
MRLs for pesticides (Codex Alimentarius Commission Procedural Manual, 25th
edition).
Term Definition
Pesticide Any substance intended for preventing, destroying, attracting,

repelling or controlling any pest including unwanted species of
plants or animals during the production, storage, transport,
distribution and processing of food, agricultural commodities
or animal feeds or which may be administered to animals for
the control of ectoparasites. The term includes substances
intended for use as a plant growth regulator, defoliant,
desiccant, fruit thinning agent or sprouting inhibitor and
substances applied to crops either before or after harvest to
protect the commodity from deterioration during storage and
transport. The term normally excludes fertilisers, plant and
animal nutrients, food additives and animal drugs.
Pesticide Any specified substance in food, agricultural commodities or
residues animal feed resulting from the use of a pesticide. The term
includes any derivatives of a pesticide, such as conversion
products, metabolites, reaction products and impurities
considered to be of toxicological significance.
Codex The maximum concentration of a pesticide residue (expressed
maximum as mg/kg), recommended by the CAC to be legally permitted
limit for in or on food commodities and animal feeds. MRLs are based
pesticide on GAP data and foods derived from commodities that
residues comply with the respective MRLs are intended to be
(MRL) toxicologically acceptable.
Codex MRLs, which are primarily intended to apply in
international trade, are derived from estimations made by the
Joint Meeting on Pesticide Residues (JMPR) following:
(a) toxicological assessment of the pesticide and its residue;
and
(b) review of residue data from supervised trials and
supervised uses including those reflecting national good
agricultural practices. Data from supervised trials
conducted at the highest nationally recommended,
authorized or registered uses are included in the review. In
order to accommodate variations in national pest control
requirements, Codex MRLs take into account the higher
levels shown to arise in such supervised trials, which are
considered to represent effective pest control practices.
(Continued)
Table 7.1. (Continued)
Term Definition
Consideration of the various dietary residue intake estimates

and determinations both at the national and international
level in comparison with the acceptable daily intake (ADI),
should indicate that foods complying with Codex MRLs are
safe for human consumption.
Good The nationally authorized safe uses of pesticides under actual
agricultural conditions necessary for effective and reliable pest control. It
practice in encompasses a range of levels of pesticide applications up to
the use of the highest authorized use, applied in a manner which leaves
pesticides a residue which is the smallest amount practicable.
(GAP) Authorized safe uses are determined at the national level and
include nationally registered or recommended uses, which
take into account public and occupational health and
environmental safety considerations.
Actual conditions include any stage in the production, storage,
transport, distribution and processing of food commodities
and animal feed.
EMRLa A pesticide residue or a contaminant arising from
environmental sources (including former agricultural uses)
other than the use of the pesticide or contaminant substance
directly or indirectly on the commodity. It is the maximum
concentration of a pesticide residue that is recommended by
the CAC to be legally permitted or recognized as acceptable
in or on a food, agricultural commodity or animal feed. The
concentration is expressed in milligrams of pesticide residue
or contaminant per kilogram of the commodity.
a
This definition was included in the Codex Alimentarius, Second Edition,
Volume 2 (Joint FAO/WHO Food Standards Programme, 1993) and transcribed
in the FAO Plant Production and Protection Paper ‘Submission and evaluation of
pesticide residues data for the estimation of maximum residue levels in food and
feed’ (Ambrus, 2016). However, this definition is not included in the Procedural
Manual.
establish Codex MRLs for foods and feeds moving in international

trade.
Terms of Reference of the CCPR (FAO, 2015 et seq. anni).
(a) To establish maximum limits for pesticide residues in specific

food items or in groups of food;
(b) To establish maximum limits for pesticide residues in certain

animal feeding stuffs moving in international trade where this is
justified for reasons of protection of human health;
(c) To prepare priority lists of pesticides for evaluation by the
JMPR;
(d) To consider methods of sampling and analysis for the
determination of pesticide residues in food and feed;
(e) To consider other matters in relation to the safety of food and
feed containing pesticide residues and
(f) To establish maximum limits for environmental and industrial
contaminants showing chemical or other similarity to pesticides,
in specific food items or groups of food.
Information on the dates, place and agenda of each session of CCPR

can be found on the Codex website along with working documents
for the session, that can be downloaded free of charge.
7.2.3 Criteria for Initiating Work on Codex

Recommendations Related to Food Safety
7.2.3.1 General criteria
The elaboration of Codex standards including MRLs and EMRLs,
codes of practice, guidelines and other recommendations follows
the eight-step or five-step procedure spelled out in the Procedural
Manual. In most of the cases, the committees concerned propose
new work for approval of the CAC. In some cases, the CAC itself
decides to start new work.
According to the Procedural Manual, to initiate elaboration of
a standard (including an MRL for pesticide) or related text, the
relevant Codex committee should consider its terms of reference,
the priorities established by the Commission in the Strategic Plan
for a period of five years, the relevant outcomes of the critical
review conducted by the Executive Committee and the prospect of
completing the work within a reasonable period of time. Any proposal
should be assessed against the criteria set out in the Procedural
Manual.
The general criterion is the consumer protection from the point

of view of health, food safety, ensuring fair practices in food
trade and taking into account the identified needs of developing
countries. Among the five criteria applicable to general subjects,
the following criteria relate to MRLs for pesticide in foods and
feeds: diversification of national legislations and apparent resultant
or potential impediments to international trade; scope of work; and
establishment of priorities between the various sections of the work
and consideration of the global magnitude of the problem or issue.
7.2.3.2 Selection of pesticides for which MRLs and EMRLs

need to be developed
The ‘Risk Analysis Principles Applied by the Committee on Pesticide
Residues’ state the criteria (nomination requirements, prioritization
criteria and scheduling criteria) for selection of pesticides for
inclusion in the Codex Priority Lists for JMPR evaluation. Three
different cases are described for new pesticides, new uses of
previously reviewed pesticides and other evaluations. The nomination
requirements for new pesticides are: (1) an intention to register the
pesticide for use in a Codex member; (2) the foods or feeds proposed
for consideration should be traded internationally; (3) there is a
commitment by the member or observer to provide supporting data
for the review of the pesticide in response to the JMPR ‘data call-in’;
(4) the use of the pesticide is expected to give rise to residues in or
on a food or feed moving in international trade; (5) the pesticide has
not been already accepted for consideration and (6) the nomination
form has been completed.
Pesticides that have not been reviewed toxicologically for more
than 15 years or not having a significant review of adopted MRLs
for 15 years are subject to so-called ‘periodic review’ by JMPR.
The Priority Lists are first considered by the working group
of CCPR and then finalized by the plenary of CCPR. Established
Priority Lists are attached to the report of each session of CCPR and
subject to approval for new work by the CAC. The establishment
of agendas of JMPR is the responsibility of the Joint FAO/WHO
Secretaries. It should be noted that the JMPR is a scientific advisory

body and independent and separate from the Codex system.
7.2.4 Procedures for Elaborating the MRL and EMRL

and Other Recommendations
There are two different elaboration procedures for Codex standards,
guidelines and other recommendations: eight-step uniform procedure,
and five-step accelerated procedure. Codex committees can decide
to omit Steps 6 and 7 of the uniform procedure where there is
unanimous agreement on a proposal while only the CAC decides to
use the accelerated procedure. The Procedural Manual states that at
Steps 3, 5, 6 and 8, all aspects, including possible implications of the
proposal for their economic interests, can be considered or submitted
in comments.
The elaboration of MRLs and EMRLs follows the same
procedures as summarized in Fig. 7.1. Specific to MRL and EMRL
setting is that (1) the recommendations for MRLs and EMRLs after
each JMPR session are distributed by the Codex Secretariat in a
circular letter to the Codex Contact Point of each Codex member,
and interested international organizations for comment at Step 3;
(2) when JMPR recommends withdrawal of existing MRLs under
the periodic review but there is a commitment to provide necessary
data to support such MRLs, they will be held for, at the longest,
four years in the Codex system and (3) if a Codex member does
not agree with toxicological or other recommendations of JMPR, the
member has an opportunity to send their concern to JMPR in the
concern form contained in Annex A or B of CCPR Risk Analysis
Principles.
7.2.5 Expression and Specific Issues of MRLs

and EMRLs
Currently there are a number of notes used after numeric values of
MRLs to explain the respective MRL or EMRL as follows:
Codex Alimentarius
Commission (CAC)
Approval Codex Committee

Joint FAO/WHO Meeting on Pesticide
on Pesticide Residues Residues
(JMPR) Priority List of Pesticide
(CCPR)
for Evaluation by JMPR
Agenda prepared by the Development of
JMPR Secretaries the Priority Lists1
Interaction
ADI/ARfD
Maximum Residue Levels CCPR Comments
CAC
recommended as at Step 5
Maximum Residue Limits Discussion of Discussion of
Comments MRLs at Step 4 MRLs at Step 5
Extraneous Maximum at Step 3
Residue Limits Comments Comments
at Step 5/8 at Step 6
CAC CCPR
Codex MRLs Discussion of Discussion of
Codex EMRLs MRLs at Step 8 Comments MRLs at Step 7
or 5/8 at Step 8
Figure 7.1. Elaboration procedure for Codex MRLs and EMRLs for pesticides.
Note 1: Only Codex Members (i.e. governments) have the right to propose
chemicals for inclusion in the Priority Lists. For proposing a chemical, the criteria
for the prioritization process of compounds for evaluation by JMPR contained in
Section IV of the Codex Procedural Manual should be consulted.
(*) At or about the limit of determination;

(fat) The MRLs apply to the fat of meat (only after the MRLs for
fat soluble pesticides for meat);
Po The MRL accommodates post-harvest treatment of the
commodity;
PoP The MRL accommodates post-harvest treatment of the
primary food commodity (only after the MRLs for processed
commodities);
E The MRL is based on extraneous residues.
For a compound used both as pesticide and veterinary medicine,

it is possible that different MRLs are recommended by JMPR (as
pesticide) and JECFA (as veterinary medicine), or by JMPR from
uses on feed crops or from use on ectoparasites of livestock. If such
a case occurs, the higher value will prevail in Codex. If a compound
is used for control of ectoparasites and evaluated by JMPR, the
recommended MRL will have a note to indicate that the MRL
accommodates external animal treatment.
7.3 Codex Recommendations Related to

Pesticide Residues in Foods and Feeds
Other than MRLs and EMRLs
The CAC has adopted the recommendations, other than MRLs and
EMRLs, on a basis of work by the CCPR (Table 7.2).
The Codex Classification of Foods and Animal Feeds is intended
primarily to ensure the uniform nomenclature of foods of plant
and animal origin and animal feeds for expressing MRLs and
EMRLs; and secondarily to classify foods into groups or sub-groups
for the purpose of establishing group MRLs. The classification is
not a mere botanical or zoological classification. Other factors are
also taken into consideration, such as morphology (such as fruits,
root and tuber, leafy vegetables, cereal grains, tree nuts, etc.),
other similar characteristics and residue potential. The classification
covers primary plant commodities, primary animal commodities,
with scientific names where relevant, and processed commodities
of plant and animal origin. The document contains information on
the portion of commodity to which the MRL applies and which is
analysed for consistent checking of compliance of a food or feed with
the relevant MRL.
There are a number of recommendations on methods of analysis
and sampling as shown in Table 7.2 for checking the compliance with
Codex MRLs.
All the recommendations in Table 7.2 can be downloaded free of
charge from the Codex website.
Table 7.2. List of Codex recommendations in the area of pesticide residues

in foods and animal feeds (Codex website on Codex Standards, updated
regularly).
Recommendation Reference
Recommendations for Codex members

MRLs for pesticides in foods and animal feeds
EMRLs in foods and animal feeds
Codex Classification of Foods and Animal CAC/MISC-4
Feeds
Recommended methods of sampling for the CAC/GL 33-1999
determination of pesticide residues
Guidelines on good laboratory practice in CAC/GL 40-1993
pesticide residue analysis
Analysis of pesticide residues: portion of CAC/GL 41-1993
commodities to which Codex MRLs apply
and which is analysed
Guidelines on the use of mass spectrometry CAC/GL 56-2005
(MS) for identification, confirmation and
quantitative determination of residues
Guidelines on estimation of uncertainty of CAC/GL 59-2006
results
Principles and guidance on the selection of CAC/GL 84-2012
representative commodities for the
extrapolation of MRLs for pesticides to
commodity groups
Information document for Codex members
Information document on the application of
the guidance to facilitate the establishment
of MRLs for pesticides for minor crops
Recommendations for the CCPR (other than procedural matters)
Working principles for risk analysis for Procedural Manual,
application in the framework of the Codex 25th Ed., Section IV Risk
Alimentarius Analysis
Definitions of risk analysis terms related to
food safety
Risk analysis principles applied by the Codex
Committee on Pesticide Residues
References1
Ambrus Á. (ed). 2016. FAO Manual on the Submission and Evaluation of Pesticide
Residues Data for the Estimation of Maximum Residues Levels in Food and
Feed. 3rd ed., FAO Plant Production and Protection Paper. 225.
FAO. 1993. Joint FAO/WHO Food Standards Programme. Codex Alimentarius,
2nd ed., Vol. 2, Pesticide Residues in Food. FAO, Rome.
FAO. 2016 et seq. anni. Joint FAO/WHO Food Standards Programme. Codex
Alimentarius Commission Procedural Manual, 25th ed., FAO, Rome.
World Trade Organization. 1995a. Agreement on the Application of Sanitary and
Phytosanitary Measures, WTO, Geneva.
World Trade Organization. 1995b. Agreement on Technical Barrier to Trade,
WTO, Geneva.
1
The FAO/WHO Food Standards Programme and World Trade Organization
publications cited in this chapter are freely available and can be accessed at the
Chapter 8
Pesticide Specifications and their

Methods for Analysis and Testing
Denis Hamilton and László Bura
Main topics
History
CIPAC methods
Technical materials
Formulations
Equivalence
Physical properties
Storage stability
Future directions
8.1 Introduction
Pesticide specifications provide an objective description of a pesti-
cide. Further, methods for analysis and testing allow samples of the
pesticide to be checked for compliance with the specifications.
Buyers and sellers use the specifications to agree on the qual-
ity of the material being traded. Regulatory agencies rely on the
specifications as the quality guideline for registered products in the
marketplace.
Farmers should expect that registered pesticides, when used
according to label instructions, will be effective and safe for the
283
user, the crop and the environment. They should also expect pes-
ticide products to be consistent in quality. Food and Agricultural
Organization (FAO) specifications for agricultural pesticides play an
important role in meeting those expectations.
The users of pesticides for vector and public health pest control
programmes should also expect safety, efficacy and consistency when
pesticides are used as directed. World Health Organization (WHO)
specifications for public health pesticides play an important role here.
For many years, FAO and WHO have published specifications
for pesticides with agricultural uses and public health uses. For a
particular pesticide, specifications are first published for the technical
material, followed by specifications for the formulations.
In the early 1990s, the need for more transparency in the devel-
opment of specifications was recognized. Also, there was a need to
link the specifications to the composition of the technical material
used in toxicology testing that supported the acceptable daily intake
(ADI) for that pesticide. To facilitate harmonization of specification
development, in 2002, the first edition of the FAO/WHO Manual was
published. The manual has continued to evolve to reflect experience
and progress in scientific and technological developments in pesti-
cides, formulations, testing and in data assessment. FAO and WHO
took the opportunity to harmonize their processes so that the same
pesticide used for agricultural and public health purposes would have
the same specifications supported by the same test methods.
The FAO/WHO Joint Meeting on Pesticide Specifications
(JMPS) was established with its first meeting at FAO, Rome in 2002.
The JMPS reviewed data and draft specifications from manufacturers
or other proposers and, when satisfied that requirements had been
fulfilled and specifications were suitable, recommended publication
by FAO and WHO.
The Collaborative International Pesticides Analytical Council
(CIPAC) publishes analytical and test methods for pesticides after
thorough testing. FAO and WHO specifications rely on CIPAC
methods.
Technical materials of the same compound manufactured by two
different processes from different starting materials are unlikely to
Pesticide Specifications and their Methods for Analysis and Testing 285
have the same purity and are unlikely to contain the same impurities.
The reference material is the one that has been used in the toxicity
and ecotoxicity tests. JMPS has developed an equivalence determi-
nation procedure to decide if the other materials are equivalent or
not to the reference material.
The Manual (FAO/WHO, 2010, p. 37) explains the nature of
specifications:
A specification should not require judgement to be exercised by the

buyer, so the clauses in it should describe quantifiable parameters
and provide limits for them.
Each property (e.g. suspensibility) in a specification clause must

be clearly defined and a suitable value or range of values prescribed.
The clause must specify a standardized test method.
Physical properties of formulations are very important for the
safe and effective use of the pesticide. Several physical property spec-
ifications and test methods are available for each formulation type.
Examples of physical properties are dustiness, wettability, suspensi-
bility and emulsion stability.
Storage stability specifications are designed to ensure that prod-
ucts will still be of good quality even after an extended time between
manufacture and sale. Analytical and test methods are studied
collaboratively to ensure that different laboratories can achieve com-
parable results on replicate samples.
The International Code of Conduct on Pesticide Management
(FAO and WHO, 2014, Clause 4.2) suggests that ‘each country
should possess or have access to facilities to verify and exercise con-
trol over the quality of pesticides offered for sale or export’. Such
testing facilities are needed to check registered products that may
be out of specification but testing is also necessary to identify and
remove fake or counterfeit products from the marketplace.
These illegal pesticides range from careful copies, where the con-
tents match the ingredients stated on the label, to poor quality fakes.
Counterfeit products pose unwarranted risks to the user, to the envi-
ronment and to the consumer of food commodities where counterfeit
products have been used. They also pose risks to the treated crops.
It is not always straightforward to identify counterfeit products by
simply observing the container, the label and the product. Labora-
tory work may be needed. Clause 6.1.13 of the International Code of
Conduct (FAO and WHO, 2014) recognizes that governments should
detect and control the illegal trade in pesticides.
FAO and WHO pesticide specifications and robust analytical and
test methods provide the means to achieve the required control.
8.2 History
8.2.1 Origins of JMPS and Its History
During the 1990s, the FAO Group on Pesticide Specifications, in its
work on international quality standards for agricultural pesticides,
assisted FAO in its aim of ensuring that available pesticide products,
especially in developing countries, were of the required quality and
that they were adequately packaged and labelled. At the same time,
WHO had similar aims for public health pesticides.
FAO and WHO undertook a process of harmonisation, where
pesticide technical grade specifications would be common to FAO and
WHO and the data supporting those specifications would be assessed
jointly. They recognized that formulations developed for agricultural
uses and for public health uses may not necessarily be the same.
Methods of analysis and testing would be the same, with sub-
stantial reliance on CIPAC methods.
The process would depend on establishing a reference profile for
an active ingredient consisting of its impurity profile, toxicology pro-
file and ecotoxicology profile (i.e. a clear link would be drawn between
the hazards of the material and its composition).
Other technical materials of the same active ingredient, but from
different sources or different manufacturing processes would then be
compared with the reference profile for ‘equivalence’.
The first FAO/WHO JMPS was held in June 2002 at FAO Head-
quarters in Rome. Special mention should be made of Gero Vaagt and
Morteza Zaim from FAO and WHO respectively, who envisaged the
harmonized approach and proceeded to modernize the processes of
data evaluation. They worked diligently and constructively to make it

happen. Alan Hill (Central Science Laboratory, Ministry of Agricul-
ture and Fisheries, UK) also deserves special recognition for his work
in preparing a working manual and a training manual. He chaired the
JMPS and kept it on track through its early years.
The pesticide industry has been supportive of the process and
has been willing to provide data, including commercial-in-confidence
data.
FAO and WHO have organized regional training courses based
on the training manual to introduce JMPS procedures to partici-
pants from many countries. JMPS has continued to develop, refine
and modify its processes as new issues have come to light. Its success
may be seen in the wide acceptance of FAO and WHO pesticide spec-
ifications and the adoption at national level of the JMPS methods of
data evaluation in the establishment of pesticide specifications.
8.2.2 Origins of CIPAC and Its History

The need for robust analytical and test methods for pesticide
products was recognized in the 1950s. R de B Ashworth of the UK
Plant Pathology Laboratory at Harpenden convinced a number of
European countries to join the UK in a collaborative programme of
developing standardized methods for analysis and testing of pesticide
formulations.
A committee of official analytical chemists was established as the
CIPAC which worked closely with chemists in industry to develop
and test suitable methods.
CIPAC Handbook Volume 1 was published in 1970 as a manual of
analytical methods, physical property tests, reagents and preparation
of pure pesticides for use as standards. CIPAC has periodically pub-
lished further handbooks with new methods of analysis and testing,
as well as with revised methods. CIPAC has a systematic programme
of revising the published methods.
The Association of Official Analytical Chemists (AOAC), now
AOAC International, in the USA, was doing similar work on methods
for pesticide formulations and the two organisations agreed in
1974 on mutual recognition of methods developed and successfully
tested by the other. CIPAC agreed on mutual recognition also with

American Society for Testing & Materials (ASTM) International in
2001.
From early times, FAO recognized the need for reliable and robust
test and analysis methods, which led to close cooperation between
FAO and CIPAC. This close cooperation has been joined by WHO
and continues through JMPS. Analytical methods, supporting FAO
and WHO specifications, for the the determination of active ingredi-
ents in technical and formulated pesticides must be collaboratively
tested and approved by CIPAC or AOAC.
Analytical methods for the determination of relevant impurities,
or for the determination of isomer ratio as part of an identity test,
must be peer (independent laboratory) validated.
8.3 CIPAC Methods

CIPAC methods are investigated collaboratively in accordance with
internationally accepted rules for inter-laboratory studies.
The methods chosen are selected on the basis of their general
applicability to as many formulations of the active ingredient as pos-
sible. CIPAC methods for the determination of the active substance
content are developed for the determination of one single compound
and the applicability to mixtures of that compound with other pes-
ticides or for the determination of the chemical in another type of
formulation should always be demonstrated. Caution is needed when
applying a method to formulations or mixtures other than for which
it originally was developed. In general, validation data are considered
formulation specific; however, it is recognized that manufacturers
may produce a number of very similar formulations and it may be
possible to use a single method for analysing them.
Analytical methods for active ingredient content commonly rely
on gas–liquid chromatography (GLC) or high-performance liquid
chromatography (HPLC). Many stationary GLC phases and HPLC
packing materials are available on the market. Those specified in the
methods have been investigated collaboratively and proven to give
good results. If they are not available for any reason, alternative
phases or packing materials of same polarities or comparable prop-

erties may be used, but in every case the performance of substitutes
should be checked.
The preparation and use of reagents (RE) used in CIPAC meth-
ods are published in CIPAC Handbook E (pp. 245–382). CIPAC mis-
cellaneous techniques (MT) are published in CIPAC F, H, J, K, L,
M and N.
Precision data are presented in terms of repeatability limit (r)
and reproducibility limit (R) and are calculated by the International
Organization for Standardization (ISO) method ‘Precision of the test
methods — Determination of repeatability and reproducibility by
inter-laboratory tests’ (ISO, 1986).
Repeatability is the closeness of agreement obtained with the same

operator in the same laboratory. Reproducibility is the closeness
of agreement between laboratories. Repeatability limit r = 2.8∗ σr ,
reproducibility limit R = 2.8∗ σR .
8.3.1 CIPAC Methods for the Determination

of the Active Ingredient Content
Before undergoing a collaborative trial, usually a method has already
been validated in a laboratory. The validation method should address
linearity of response for the analyte (and internal standard, if appro-
priate) in the method, an estimate of the precision of the procedure,
a demonstration of its accuracy and evidence of selectivity in the
presence of other components in the formulations.
For methods where the cross-applicability of validation is
claimed, the evidence on the following points should be available:
• The formulations should contain the same (or very similar) co-
formulants; any qualitative change in co-formulants should be
checked for potential interference.
• The formulations should not differ markedly in physico-chemical
properties (e.g. pH).
• The concentrations of active ingredients in the analytical solutions

must remain within the demonstrated linearity ranges.
• Any changes in relative co-formulant concentrations should not
yield significant interference.
The development of a CIPAC method is normally divided into

three parts:
1. A small scale trial involving three to five laboratories is conducted

to ensure that the method is suitable for a large-scale trial. If the
results of the small-scale trial suggest that the method is unsuitable,
the method must either be modified or a different method chosen.
The pilot trial would then have to be repeated. Small-scale trials,
however, are not mandatory, only recommended for the identification
of possible shortcomings of the method.
2. Full-scale international collaborative trial: The organisation of a

full-scale trial is announced via a CIPAC information sheet and con-
ducted according to the CIPAC criteria. The report on the full-scale
collaborative trial and the results of the trial are presented and eval-
uated at an annual CIPAC technical meeting.
Laboratories participating in a collaborative trial are usually
those likely to use the test method under consideration. In practice,
only a limited number of laboratories are likely to be willing to take
part from all over the world. The recommended minimum number
of participating laboratories in a collaborative study is eight, as a
minimum of eight valid data sets is required.
After the completion of the analytical part, the results are sent
to the organizing laboratory. Statistical treatment of results is based
on the procedure described in International Standard ISO 5725 (ISO,
1986) and IUPAC Recommendations on the Harmonization of Col-
laborative Analytical Studies (Horwitz, 1988).
3. Decision, publication
Decisions whether the reproducibility and repeatability of the
methods are satisfactory depend on individual circumstances. The
approach to determine acceptability is to compare the reproducibility
relative standard deviation of the study RSDR (Exp.) with the the-
oretical RSDR (Calc.) calculated from the Horwitz curve (Horwitz,
1982):
RSDR (Calc.)% = 2(1−0.5 log c)
where c is the concentration of the analyte as a decimal fraction (e.g.

for 100% concentration, c = 1).
If RSDR (Exp.) as determined from the collaborative study is not
larger than RSDR (Calc.) calculated for the relevant concentration,
the method should be acceptable.
An accepted method will receive the status ‘provisional’ and the
method will be made publicly available on the CIPAC website in
the form of a ‘pre-published method’. After a certain time period
(normally one year), if there are no objections to its performance, the
method will be promoted to ‘full’ and published in the next CIPAC
handbook. After a method has been published in a handbook, it will
disappear from the pre-published method scheme.
8.3.1.1 Use of the methods

Initially r and R were intended to be used as criteria to determine
whether a difference between two single test results can be ascribed
to random fluctuations. A difference larger than r or R is suspect, and
may justify the conclusion that there exists a systematic difference
between the two test results, or may justify additional investigation.
Thus r and R may be called ‘critical differences’, to be applied to
a pair of test results respectively obtained under repeatability and
reproducibility conditions.
An example of using these values in a laboratory is comparing
the absolute difference between the two results with the repeatabil-
ity limit r. When the difference is smaller than r, the final quoted
result is the average of the two results and this should not dif-
fer from that declared in the FAO specification by more than the
appropriate tolerance given in the specification. The tolerances in
FAO specifications refer to the average analytical result obtained and
take into account manufacturing, sampling and analytical variations.
When the absolute difference exceeds the appropriate limit, then the
laboratory should have in place procedures for obtaining the final
quoted result (for example by producing one or two further results
and deriving the final quoted result according to ISO 5725).
For the situations when new formulations are introduced after the
collaborative study of a particular compound has been completed,
and time and financial constraints make it improbable that new col-
laborative studies will be conducted for one formulation only, CIPAC
has introduced a procedure for the extension of scope of an analyti-
cal method. This covers the application of a standardized method to
a different matrix or concentration range from those for which the
method originally was accepted. Adoption of a method without any
testing would be incompatible with the principles of standardisation;
therefore, this procedure is aiming to prevent important formulations
from remaining without standardized methods.
8.3.2 Methods for Relevant Impurities

Following the request made by the FAO and the WHO to consider
independent laboratory validations of analytical methods for the
determination of relevant impurities defined in FAO/WHO specifica-
tions in the scope of its activities, CIPAC decided that the method
validation and development should be handled in principle as the
CIPAC methods for substances. The validation should be conducted
as a validation study with a minimum of four independent labora-
tories. The laboratories chosen to conduct the trials must not have
been involved in the method development and in its subsequent use.
Provided this criterion is met, two of the laboratories chosen to con-
duct the trial may belong to the applicant’s organisation. In contrast
to a CIPAC full trial the validation criteria should be met by each
participating laboratory. The methods, the results and the statisti-
cal evaluation thereof are evaluated and possibly adopted at CIPAC
meetings. Adopted methods, if necessary with remarks from CIPAC,
are made available on the CIPAC website. Neither ‘provisional’ nor
‘full’ status for analytical methods for the determination of relevant
impurities in technical material or formulations is given.
TECHNICAL MATERIAL TC
1 Description
2 Active ingredient
2.1 Identity tests
2.2 ...... [COMMON-NAME] content
3 Relevant impurities, if required

3.1 RELEVANT IMPURITY A
3.2 Water
4 Physical properties, if required

4.1 Acidity, Alkalinity
Figure 8.1. Main heading for TC specification guidelines. TK has the same
headings.
8.4 Technical Materials

The Manual (FAO/WHO, 2010, p. 260) defines technical material
(TC) and technical concentrate (TK).
TC: A material resulting from a manufacturing process comprising the
active ingredient, together with associated impurities. This may contain
small amounts of necessary additives.
TK: A material resulting from a manufacturing process comprising the
active ingredient, together with associated impurities. This may contain
small amounts of necessary additives and appropriate diluents.
Figure 8.1 provides the main headings for technical materials

specifications.
One way to understand the concept of the TK is to imagine that
the manufactured product is not purified to TC grade by removing
water, solvents or salt counter-ions if they are necessary or acceptable
constituents in the formulation to be produced.
Propamocarb hydrochloride is prepared as a TK (FAO, 2013a).
Theoretically a TC could be prepared, but it is very hygroscopic,
readily absorbing moisture from the atmosphere. In this example, a
TK is more practical than a TC.
N NH O
O
Propamocarb
CH3
+N N+ CH3
Paraquat
Paraquat is an example of a technical material containing small

amounts of necessary additives. Because paraquat has been the vehi-
cle for suicide attempts in a number of countries, additives to the
technical material have been chosen to minimize such attempts and
also to reduce accidental oral ingestion (Baltazar et al., 2013).
The current FAO specification (FAO, 2008a) requires paraquat
dichloride TK to contain an effective emetic. The TK may also con-
tain added dyes and stenching agents. The emetic must be more
quickly absorbed than the paraquat and be quick acting. The pur-
pose of the dyes and stenching agents is to warn and inhibit people
from deliberate or accidental oral ingestion.
8.4.1 Technical Material: Identity

Nomenclature is a difficult area, but it does need to be correct. Docu-
ments should be very precise about which compound is being studied.
Some compounds are mixtures, which is a further complication. Man-
ufacturers provide the following information to confirm the identity
of the pesticide compound.
ISO common name; International Union of Pure and Applied
Chemistry (IUPAC) chemical name; Chemical Abstracts Service
(CAS) chemical name, CAS Registry number; CIPAC number; syn-
onyms including trade names; structural formula, molecular formula;
and relative molecular mass (formerly molecular weight).
The various allethrin isomer mixtures are a good example of the
care needed in precise identification (Table 8.1). Various commer-
cially produced mixtures of isomers are available. In some cases they
Table 8.1. Names and isomer compositions of allethrin products.

allethrin: 8 stereoisomers, 4 pairs
H H
of enantiomers
d -allethrin:
COO COO
1R,trans;1R O 1R,trans;1S O
[1R,trans ;1R] + [1R,trans ;1S] +
[1R,cis;1R] + [1R,cis;1S], approx
H
ratio 4:4:1:1 H
bioallethrin: COO COO
[1R,trans ;1R] + [1R,trans ;1S] 1R,cis;1R O 1R,cis;1S O
approx ratio 1:1

d-trans allethrin = bioallethrin
esbiothrin:
[1R,trans ;1R] + [1R,trans ;1S]
approx ratio 1:3
S -bioallethrin: [1R,trans;1S]
Table 8.2. Isomer composition of cypermethrin compounds (Compendium,

2015).
Cypermethrin Alpha-cypermethrin Zeta-cypermethrin

CAS: 52315-07-8 CAS: 67375-30-8 CAS: 52315-07-8
Isomer CIPAC 332(%) CIPAC 454(%) CIPAC 733(%)
1R, cis−R 14 — 3
1S, cis−S 14 — 22
1R, cis−S 11 50 22
1S, cis−R 11 50 3
1R, trans −R 14 — 3
1S, trans−S 14 — 22
1R, trans −S 11 — 22
1S, trans−R 11 — 3
are the same isomers but in different ratios (e.g. bioallethrin and
esbiothrin).
Cypermethrin isomer mixtures present similar complications.
The isomer compositions of cypermethrin, alpha-cypermethrin and
zeta-cypermethrin are summarized in Table 8.2. Companies have
developed specific cypermethrins with enhanced concentration of the
more biologically active isomers or mixtures.
COOCH3
O COOCH3
COOCH3 N
Cl N N
N
O OCH3
Indoxacarb O OCF3
Metalaxyl
Figure 8.2. Indoxacarb and metalaxyl.
CAS registry numbers are good identifiers of chemical

compounds, but a CAS number is not necessarily unique to each
compound (e.g. the same number is used for the cypermethrin and
zeta-cypermethrin mixtures).
A further complication is that sometimes the ISO common name
includes an inactive isomer, and sometimes not. For example, the
ISO name ‘metalaxyl’ includes the active and inactive isomers; the
ISO name ‘indoxacarb’ is only the active S-isomer (Fig. 8.2).
Biopesticides that are living organisms are not chemicals and
do not have CAS numbers. However, they can be assigned CIPAC
numbers.
8.4.2 Technical Materials: Purity

The minimum purity specified for the majority of TCs falls in the
950–990 g/kg range. Maximum purity is not specified for TCs because
higher purity should generally maintain or improve quality.
However, the minimum purity for a minority is outside of that
range, with some examples below 900 g/kg. For example, the min-
imum purity of lambda-cyhalothrin (FAO, 2013b) is 810 g/kg. The
presence of other cyhalothrin isomers accounts for the low value for
purity; the specified minimum for total cyhalothrin is 900 g/kg.
With increased purity of the technical material and the conse-
quent decrease of impurity levels, the influence of the impurities on
mammalian toxicity or on other important properties is reduced.
For a TK, a minimum and a maximum concentration of active
ingredient are specified.
8.4.3 Impurities in Technical Materials

The impurities in technical materials may consist of: Starting materi-
als and intermediates of the synthesis, by-products of synthesis such
as isomers of the intended product, solvents and water and impurities
in starting materials. In addition, impurities may be produced if the
active ingredient breaks down during storage.
For botanical pesticides (chemicals with pesticidal activity pro-
duced naturally within a plant; Stephenson et al., 2006), impurities
arise from other plant components that are extracted together with
the desired active substance. Impurities may also be introduced dur-
ing harvest and production. For example, aflatoxins may occur in
azadirachtin insecticide as a result of Aspergillus spp infecting the
neem seed at harvest (FAO, 2006a).
Some biopesticides are biological agents with pesticidal activity
(e.g. Bacillus thuringiensis; Stephenson et al., 2006). The most likely
impurities are contaminating micro-organisms, metabolites (toxins),
cellular parts and spores.
JMPS requires data on the composition of technical materials
including maximum manufacturing limits for impurities occurring
at 1 g/kg and above. Also required are analyses on five commercial
batches of the technical material with identification and quantitative
data on all impurities present at 1 g/kg and above, accounting for at
least 98% of the material.
JMPS also requires data on those impurities occurring at less
than 1 g/kg and proposed as relevant impurities.
A relevant impurity (FAO/WHO, 2010, p. 256) is defined as:
A by-product of the manufacture or storage of a pesticide, which,

compared with the active ingredient, is toxicologically significant to
health or the environment, is phytotoxic to treated plants, causes
taint in food crops, affects the stability of the pesticide or causes
any other adverse effect.
Previous experience suggests that some types of impurity, even

occurring at lower concentrations than 1 g/kg, need more attention
because of likely toxicity or other hazard. The term ‘structural alert’
means that some structural moieties within the active ingredient

draw our attention to the possible occurrence of certain potential
relevant impurities. Examples are shown in Table 8.3. Adequate
information should be available on the occurrence or absence of the
potential impurity above an acceptable detection limit.
In 1976 in Pakistan, workers using malathion for mosquito reduc-
tion in the malaria control programme suffered poisoning (Baker
et al., 1978). Worker safety practices were not ideal and exposure
mainly occurred through excessive skin contact with spray liquid.
Workers were using water-dispersible powders (probably wettable
powders, WPs) containing approximately 50% malathion and 50%
carrier.
CH3S S COOC2H5
P
CH3O
O
Isomalathion COOC2H5
CH3O S COOC2H5
P
CH3O
S
COOC2H5
Malathion
The analysis of samples from the three brands being used revealed
that isomalathion was present at 0.68%, 4.6% and 8.8%, all expressed
as percentage of malathion content. Workers who had used the third
brand had the most severely depressed red-cell cholinesterase activity
(a sign of organophosphorus poisoning).
Isomalathion is a contaminant of malathion TC at the time of
manufacture. The WHO specification allows a maximum of 4 g/kg in
the TC (WHO, 2003). Isomalathion is also produced during storage
and during formulation. Isomalathion is more toxic than malathion,
but it also potentiates the toxicity of malathion so that the relatively
low toxic malathion becomes toxic.
The relative safety of malathion in mammals is explained by
its rapid hydrolysis by carboxylesterases. Impurities such as isoma-
lathion that inhibit carboxylesterase activity will increase the toxicity
of malathion (i.e. will potentiate the toxicity of malathion).
Table 8.3. Examples of structural alerts and potential relevant impurities.
Structural Alert Potential Relevant Impurity
OR
Cl
Cl O Cl
Cl
Trichlorophenoxy- Cl Dioxins Cl O Cl
NHR
Cl
Dichloroanilino- Cl Tetrachloro-azobenzene
N N
Cl Cl
Cl Cl
S S
C2H5O OC2H5
P P
O,O-diethyl ester of phosphorothioic Sulfotep C2H5O O OC2H5
acid
S
C2H5O
P R
C2H5O O
NO
NH N
Secondary amine R2 R1 N -nitroso- R2 R1
Cl
Cl Cl
Cl Cl
Chlorinated benzene compounds Hexachlorobenzene Cl

R1 R1
Cl Cl Cl Cl
Cl R2 Cl R2
Cl R3
WHO no longer has specifications for malathion WP formula-

tions, but the specifications for the malathion emulsifiable concen-
trate (EC), which may be dispersed in water for spraying, allows an
isomalathion maximum of 0.8 % of the malathion content, which is
far below the 4.6 and 8.8% in the offending products used in 1976.
Control of pesticide quality through pesticide specifications
should reduce the future occurrence of cases such as that with
malathion in 1976.
The relevant impurities in copper oxychloride and other copper-
based fungicides depend on the source of the copper. FAO speci-
fications for lead, arsenic and cadmium impurities in copper-based
fungicides are based on the use of clean copper in their manufacture.
The maximum limits for arsenic, lead and cadmium are set at 0.01%,
0.05% and 0.01% respectively of the copper content (FAO, 1991).
When scrap copper is recovered from recycled electrical and elec-
tronic equipment, the solder and metalwork may contribute lead and
cadmium to the recovered product. The specifications are intended
to keep such contamination out of quality products.
S
NH O N
N
O O
Oxamyl
The secondary amine group in oxamyl, a systemic insecticide

and nematicide, suggested the possibility of N -nitrosamine forma-
tion if nitrite were present in the starting materials or reaction mix-
tures during oxamyl manufacture. The primary manufacturer, who
had supplied the data for FAO review, had taken precautions to
minimize contamination. Possible N -nitrosamines were not detected
(LOQ 0.1 mg/kg) in five batches of oxamyl (FAO, 2008b).
Because none was reported as detected in the data provided
for review, the JMPS decided not to establish a specification for
a nitrosamine relevant impurity. A note to the specification points
out that other manufacturing processes may produce nitrosamines,
which, if present in measurable concentrations, would require control

by a specification for a relevant impurity.
O Cl
O
N N
N
Cl Cl
Prochloraz
In a similar example, the presence of a 2,4,6-trichlorophenoxy

moiety in prochloraz suggested the possibility of dioxin contami-
nation (FAO, 2009a). Available data showed that no dioxin was
detected in the product under consideration. The result was that
a specification for the relevant impurity was not needed but an alert
to the possibility was given in a note attached to the specification.
8.4.4 Impurity Names

Pesticides themselves have common names issued by ISO. The impu-
rities have no official common names. The full systematic name is
suitable for very simple compounds, but is quite unwieldy for more
complex compounds, which need trivial names for discussion, data
tables and the like.
CH3O S COOC2H5
P
CH3O
O
COOC2H5
Malaoxon
Mostly, trivial names for compounds related to the active ingre-

dient are chosen so as to be quite unambiguous. For example, the
impurity in many organophosphorus pesticides where the P=S is
replaced by P=O is named by replacing ‘thion’ with ‘oxon’, as hap-
pens for malaoxon as an impurity in malathion.
S
CH3O
P
CH3O O NO2
Parathion-methyl
O
CH3S
P
CH3O O NO2
S-methyl-parathion-methyl
The trivial name ‘S-methyl parathion’ has been used for an impu-
rity in parathion-methyl, but it is a confusing name because it sounds
like a derivative of parathion. Parathion is a diethyl ester.
‘S-methyl-parathion-methyl’ is clearer.
8.4.5 Expression of Relevant Impurities

8.4.5.1 Glyphosate Example (FAO, 2001)
Sometimes the concentrations of impurities are expressed as a con-
centration in the whole product and sometimes as a concentration
related to the active ingredient. Two impurities in glyphosate illus-
trate these two cases (Table 8.4).
N -nitrosoglyphosate (NNG) was identified as a relevant impurity
and maximum limits were set for glyphosate technical materials and
Table 8.4. Expression of concentrations of relevant impurities in glyphosate.
Formaldehyde,
N -nitrosoglyphosate, Expressed as g/kg
Glyphosate Expressed as mg/kg of the Glyphosate
Material of Whole Product Acid Content
Glyphosate acid TC Maximum 1 mg/kg Maximum 1.3 g/kg

Glyphosate acid TK Maximum 1 mg/kg Maximum 1.3 g/kg
Glyphosate isopropylamine salt TK Maximum 1 mg/kg Maximum 1.3 g/kg
Glyphosate soluble concentrates SL Maximum 1 mg/kg Maximum 1.3 g/kg
Glyphosate water soluble granules SG Maximum 1 mg/kg Maximum 1.3 g/kg
formulations. The same absolute limit of 1 mg/kg was set for tech-
nical grade and formulations because additional nitroso compound
may be generated in steps subsequent to the glyphosate synthesis
(i.e. during conversion to a salt and preparation of granules). Free
nitrites in the water or nitrogen oxides in the hot air drying the
granules can add to the load of nitroso compound.
Formaldehyde was also identified as an impurity at the
glyphosate synthesis stage, but is not generated in later stages, so
the limit for formaldehyde content can be expressed as a constant
ratio to the glyphosate content in the specifications for formulations.
8.4.6 Reference Material

8.4.6.1 Composition of the reference profile
The reference profile of a technical grade material (TC) describes the
composition and impurity profile of that material. It will usually be
the material with the most comprehensive set of toxicological and
ecotoxicological studies.
The intention is for FAO and WHO pesticide specifications to
describe the material associated with the hazard profile used in
the risk and safety assessments. For example, the ADI should be
based on toxicology testing with material described in the reference
profile.
The reference profile is supported by extensive data produced
by the company that developed the compound. The package of data
includes
manufacturing pathway, starting materials and conditions,

composition of the technical grade material, including manufac-
turing quality control limits for active ingredient and impurities,
identity and chemical and physical properties and
toxicological and ecotoxicological summaries.
An impurity profile, a toxicological profile and an ecotoxicologi-

cal profile, produced from the data package, constitute the reference
profile.
In the first instance, the pesticide specification relates directly

to a pesticide with a composition and properties described in the
reference profile. Subsequently, products from other manufactur-
ers or manufacturing processes may be added to the specifica-
tion, but this should take place after a process of ‘equivalence
determination’ between the subsequent product and the reference
profile.
FAO specifications carry a prefatory paragraph explaining:
This specification is based on an evaluation of data submitted

by the manufacturers whose names are listed in the evaluation
reports. It should be applicable to TC produced by these manufac-
turers. The specification may not be appropriate for TC produced
by other manufacturers. (Paraphrased for brevity and to delete
specific details.)
Specifications are initially established on technical material

related to the reference profile. It is important to add clauses about
impurities only for those that have some influence on quality or safety
(i.e. relevant impurities). Clauses for non-relevant impurities may
artificially exclude products that are quite satisfactory.
‘The specification may not be appropriate for TC produced by other

manufacturers.’
TCs from other manufacturers may be produced in different

manufacturing processes with different starting materials, where the
impurity profiles are unlikely to be identical with the reference profile.
The specifications are not appropriate for controlling the composi-
tion and properties of these TCs until they have been evaluated and
additional relevant impurities, if necessary, are added to the specifi-
cations, or other properties are amended as needed.
N N
O O
CN CH3O
COOCH3
Azoxystrobin
Sometimes changes are made to the specification to accommodate

the product of a second manufacturer. The reference azoxystrobin TC
(FAO, 2009b) was described as ‘off-white powder’, while the second
TC was described as ‘yellowish powder’. The description in the spec-
ifications was changed to ‘. . . an off-white to light brown powder . . .’
to accommodate both.
8.5 Formulations
A formulation is a pesticide preparation supplied by a manufacturer
for practical use (Stephenson et al., 2006). It usually consists of an
active ingredient and inert ingredients. Inerts may be water, solvents,
fillers, surfactants or other substances serving a specific purpose. Sur-
factants are chosen to improve emulsification, dispersion and appli-
cation properties.
Numerous formulation types have been developed; it is conve-
nient to use the two-letter code published by CropLife International
(CropLife International, 2008) to describe each formulation.
Examples:
EC Emulsifiable concentrate. A liquid, homogeneous formulation
to be applied as an emulsion after dilution in water.
FS Flowable concentrate for seed treatment. A stable suspension
for application to the seed, either directly or after dilution.
WP Wettable powder. A powder formulation to be applied as a
suspension after dispersion in water.
The Manual (FAO/WHO, 2010, pp. 71, 118) classifies formula-
tions into solids and liquids and how they are to be diluted and
prepared for application. As well, several miscellaneous products are
described. Here are some examples.
Solids for direct use Example: GR granules.

Solids for dispersion Example: WG water dispersible gran-
ules.
Solids for dissolution Example: SG water soluble granules.
Liquids, simple solutions Example: UL ultra-low volume liquids.
Liquids, for dispersion Example: EC emulsifiable concentrates.
Liquids, suspensions Example: SC aqueous suspension con-
centrates.
Liquids, multi-character Example: SE aqueous suspo-emulsions.
Devices. Example: MC mosquito coils.
Each formulation type has its own set of specification guidelines,
but many guidelines are common across related formulations.
The specification requirements of WG, water dispersible granules,
will provide an example of what to expect in most specifications
(Fig. 8.3).
A formulation specification has five sections. Sections 1–3 are
similar to those of the TC.
Section 4, setting the specifications on formulation physical prop-
erties, is very important. The list of required physical properties
depends on the type of formulation.
Each clause of the physical property specifications will specify a
limit value and a test method.
Example. 4.2 Wettability (MT 53.3).
The formulation shall be completely wetted in five seconds with-
out swirling.
CIPAC Method MT 53.3 ‘Wetting of wettable powders’ describes
the reagents, test procedure and how to interpret the observations
for the wettability specification. Note that the method designed for
wettable powders has been adapted to water dispersible granules.
The specified time (five seconds) in this example is derived from
observations of the manufactured product but should also match
what is expected of a ‘good’ product.
Example. 4.6 Persistent foam (MT 47.3).
There shall be a maximum of 10 ml after one minute.
WATER DISPERSIBLE GRANULES WG
1 Description
2 Active ingredient
2.1 Identity tests
2.2 ...... [COMMON-NAME] content
3 Relevant impurities
3.1 By-products of manufacture or storage,
if required
3.2 Water
4 Physical properties
4.1 Acidity, Alkalinity
4.2 Wettability
4.3 Wet sieve test
4.4 Degree of dispersion
4.5 Suspensibility
4.6 Persistent foam
4.7 Dustiness
4.8 Flowability
4.9 Attrition resistance
5 Storage stability
5.1 Stability at elevated temperature
Figure 8.3. Main headings for WG specification guidelines. Each formulation

type has its own list of physical properties.
CIPAC Method MT 47.3 ‘Determination of the foaming of sus-

pension concentrates’ explains the procedure and interpretations for
testing the foaming properties of formulations. Note that the method
designed for suspension concentrates has been adapted to other for-
mulations such as water-dispersible granules.
The specified volume, in this case 10 ml, depends on measure-
ments on the product and should be what is expected of a ‘good’
product.
Specifications for storage stability are included in Section 5 of the

specifications, and describe the temperature, conditions and duration
of the accelerated storage test. The CIPAC test method MT 46.3 pro-
vides guidance on storage performance because many formulations
that perform poorly in the accelerated test do not have satisfactory
shelf lives (at least two years) in hot or temperate climates (Dobrat
and Martijn, 2000).
See also: 8.8. Storage stability.
Example. 5.1 Stability at elevated temperature (MT 46.3).
After storage at 54 ± 2◦ C for 14 days, the determined average
active ingredient content must not be lower than 97% relative to the
determined average content found before storage and the formulation
shall continue to comply with the clauses for pH range (4.1) and wet
sieve test (4.3).
In this example, after the formulation is held at 54◦ C for 14
days under the conditions of CIPAC MT 46.3, ‘Accelerated storage
procedure’, a small decline in active ingredient to 97% of the start-
ing value is permitted. The stored material must still comply with
selected physical property specifications.
8.5.1 Water as a Relevant Impurity

Relevant impurities (FAO/WHO, 2010, p. 256) are those by-products
of manufacture or storage that are, compared with the active ingre-
dient, deleterious in some way (see more information on relevant
impurities in Section 8.4.3). In some situations, water is a relevant
impurity and its maximum concentration is limited by specification
(Table 8.5).
If one mole of water reacts with one mole of pesticide, a small
amount of water will hydrolyse much larger amounts of pesticide.
For example, the molecular mass of dimethoate is 229.3, so 2 grams
of water would hydrolyse 25.5 g of dimethoate (229.3 × 2/18).
8.5.2 Relevant Impurities and Intended Use

Glyphosate herbicide may be applied over-the-top to genetically engi-
neered glyphosate-tolerant cotton plants for weed control. Sometimes
cotton leaves have suffered damage (Patent, 2005), which has been
Table 8.5. Water as a relevant impurity.
Water, Relevant
Impurity
Specification
Compound (Maximum) Reason
Dimethoate TC 2 g/kg (1) Stability of dimethoate

(2) Adverse effect on EC
preparation
Etofenprox TC 5.0 g/kg Adverse effect on EC

preparation
Fosetyl-aluminium TC 7 g/kg Hydrolysis produces phosphite
ion — may be phytotoxic
Bacillus thuringiensis 50 g/kg Detrimental to product quality
subspecies israelensis, of biological pesticide
strain AM65-52 WG
attributed, at least in part, to the presence of an impurity in the

glyphosate: N -(phosphonomethyl)iminodiacetic acid (PMIDA). The
patent describes methods for mitigating the effects of PMIDA and
its salts.
O
HO
P NH COOH
HO
Glyphosate
O COOH
HO
P N COOH
HO
PMIDA
Where glyphosate is used as a non-selective herbicide, PMIDA

would not be classified as a relevant impurity. The PMIDA would
just contribute in a very minor way to the herbicidal activity of the
glyphosate formulation.
However, in glyphosate products designed for use on glyphosate-
tolerant cotton, PMIDA would likely be classified as a relevant impu-
rity. Whether an impurity is classified as relevant or not may depend
also on the intended uses of the product.
8.6 Equivalence
The International Code of Conduct on Pesticide Management (FAO
and WHO, 2014, Article 2) defines equivalence broadly as
‘the determination of the similarity of the impurity and toxico-

logical profile, as well as of the physical and chemical properties,
presented by supposedly similar technical material originating from
different manufacturers, in order to assess whether they present
similar levels of risk’.
In practice, determination of equivalence by the JMPS involves a

comparative assessment of the impurity and toxicological profiles, as
well as other data. The comparison is between the proposed technical
material and the reference profile technical material.
The evaluation aims to answer the crucial question, ‘Does the
proposer technical material pose a greater hazard than the reference
technical material?’
The greater hazard could be of the same nature, but with higher
potency or it could be of a different nature (e.g. the proposer TC
might exhibit skin irritation), which did not occur with the reference
material.
A proposer TC may be of higher purity than the reference mate-
rial and may show lower toxicity. The proposer TC would then be
equivalent to the reference material provided that none of its prop-
erties was defective in comparison with the reference.
Equivalence determination (FAO/WHO, 2010, pp. 27–30) has
been developed to make as much use of the chemical data as pos-
sible. In a Tier 1 equivalence determination, the composition of the
proposer technical material is compared closely with the composition
of the reference technical material.
The data requirements for a Tier 1 equivalence determination
consist of:
• identity and physical and chemical properties of the active

ingredient,
• manufacturing pathway and recent batch analysis data,
• composition of the technical grade material, including manufac-

turing quality control limits for active ingredient and impurities
and
• results of mutagenicity testing.
A Tier 2 determination relies on the package of toxicological and eco-

toxicological testing. Tier 2 is pursued when the Tier 1 determination
is inconclusive and cannot reach a clear conclusion of equivalence or
a clear conclusion of non-equivalence.
OCH3
O N N
O O
S
NH N N
COOCH3
Tribenuron-methyl
For tribenuron-methyl equivalence evaluation (FAO, 2011a), a

package of Tier 1 data included information on the manufacturing
process, manufacturing limits for active ingredient and impurities, 5-
batch analysis data and mutagenicity testing (bacteria, in vitro). The
Tier 1 data were sufficient for a decision that the new TC was equiva-
lent to the tribenuron-methyl reference TC supporting the reference
profile.
N NO2
N
NH
Cl CH2 N
Imidacloprid
Imidacloprid provides an example where Tier 1 evaluation was

inadequate for equivalence determination (FAO, 2013c). When a sec-
ond manufacturer of imidacloprid proposed a TC for equivalence
determination, it was found that the synthetic pathway of manufac-
ture and the impurity profile were quite different from those of the
reference profile. Consequently, the evaluation moved to the toxicol-

ogy studies available under Tier 2.
8.7 Physical Properties

Numerous physical properties have been introduced to control the
safety, stability and behaviour of pesticide formulations. CIPAC test
methods for physical properties are available to check if a product
complies with its specification. Each specification clause states which
test method is to be used.
The idea is to devise a laboratory test method that produces
results relevant to the safe and effective use of the pesticide.
In some situations, physical properties such as particle size dis-
tribution may be important in the manufacturing phase but, if not
important in the product usage phase or, if already captured by an
existing clause, they would not be included in FAO or WHO speci-
fications. For example, particle size distribution may be under some
control by a ‘dustiness’ clause.
Just a few of the available physical properties are discussed here.
8.7.1 Acidity and pH Range

Acidity and pH range are different properties. Acidity is measured
by titration and is calculated and expressed as sulfuric acid, H2 SO4
equivalents. The pH of a water-based formulation or a mixture of
sample (1 g) with water (100 ml) is measured with a pH meter and
glass electrode. The pH range in the specification is the acceptable
range for a quality product.
NH O
O
Propoxur
A specification for maximum acidity applies to the propoxur tech-

nical material, TC (WHO, 2005). It is not a matter of stability of
the propoxur. Propoxur is formulated into water-based aerosols (AE)
and the presence of acid would likely corrode the aerosol can. In this
case the purpose of the specification is to protect the container.
A specification for pH range is a useful control for active ingre-
dients that are susceptible to hydrolysis at some pHs. A formulation
should be designed to keep within a pH range of active ingredient
stability.
Cl Cl
O
N
N O
O
Mefenpyr-diethyl
Mefenpyr-diethyl is a safener (a chemical that reduces toxicity of

a herbicide to a specific crop plant) and is always formulated with a
herbicide (FAO, 2011b). Mefenpyr-diethyl is sensitive to hydrolysis at
high and low pH, so a specification for pH range of the EW (emulsion,
oil in water) formulation is essential. Furthermore, a herbicide to be
co-formulated with mefenpyr might also be unstable at some pHs.
The pH specification for the formulation should accommodate both
compounds.
Thiamethoxam is stable to hydrolysis except at high pH, so why
would a specification for pH range be needed (FAO, 2014)?
Hydrolysis of thiamethoxam produces nitrous oxide (Fig. 8.4)
and even a small amount of hydrolysis would be enough to pressurize
the sealed containers for liquid formulations such as the suspension
concentrate (SC) and flowable concentrate for seed treatment (FS).
So, the pH range is required for the liquid formulations, but is not
necessary for thiamethoxam solid formulations such as the water dis-
persible granules (WG).
N O N O
Cl Cl
N N N N
S N2O + S
Thiamethoxam N Nitrous oxide O

NO2
Figure 8.4. Thiamethoxam hydrolysis.
8.7.2 Persistent Foam

When a spray tank is being filled or when the spraying liquid is being
agitated, generation of excess foam is hazardous to the operator and
the environment.
The test method relies on a glass stoppered graduated 250 ml
cylinder. A sample is mixed with water in a standard and repro-
ducible way and the volume of foam produced is recorded. The
accepted maximum value in specifications is 60 ml, but if readily
achievable, a smaller value will become the specification. If the com-
pound is typically co-formulated with other compounds, the situation
is not so predictable and the maximum 60 ml foam is accepted as the
specification.
NH N
Cyprodinil
A good policy is to set specifications within reasonably tight lim-

its, but with some practical latitude. For persistent foam, the Manual
(FAO/WHO, 2010, p. 50) gives 60 ml as a maximum. The proposed
maximum value of 60 ml for persistent foam in the emulsifiable con-
centrate (EC) formulation of cyprodinil, a fungicide, was questioned
(FAO, 2009c). The justification in this case was that cyprodinil is
coformulated with other fungicides, so to cover the variety of possi-
bilities, the maximum 60 ml should be specified.
8.7.3 Dustiness
The user will be exposed if dust is released into the air when a pesti-
cide container is opened or when the pesticide is being measured out
ready for preparation and application. The dustiness specification
applies to granular formulations, which, ideally, should not release
dust.
In the test method, a sample of the granular formulation is
allowed to fall through a tube. The amount of dust can be measured
in the air by means of a light beam or, alternatively, by collection on
a filter disc and subsequent weighing.
The ratings from the test are: (1) nearly dust-free, (2) essentially
non-dusty and (3) dusty. Category 2 (essentially non-dusty) is the
most common rating in FAO and WHO specifications. Category 3 is
not acceptable.
When oxamyl granules (GR) were examined for dustiness, the
rating was ‘essentially non-dusty’ (FAO, 2008b). Because of the acute
toxicity of oxamy, the concern was to tighten the specification as far
as possible. Normally ‘essentially non-dusty’ means dust of 12–30 mg
of dust are released from 30 g of sample in test procedure MT 171.
For oxamyl granules, ‘essentially non-dusty’ means a maximum of
18 mg of dust, which was achievable in practice.
8.7.4 Wet Sieve Test

Blockage of the filters in spraying machinery or blockage of spray
nozzles themselves is a nuisance, resulting in poor and uneven appli-
cation to the crop. Finding where the blockage has occurred and rec-
tifying it may result in additional exposure of the operator through
direct contact of spray liquid on hands.
The wet sieve test aims to control the content of insoluble mate-
rial that could block filters or nozzles (FAO/WHO, 2010, p. 51). It
applies to any of the formulations, liquid or solid, that contain solid
particles intended for dispersion into water before use. In the test,
a sample of formulation is dispersed into water and the mixture is
transferred to a 75 µm sieve and rinsed through with water. The
weight of material retained on the sieve is measured and expressed

as a percentage of the sample weight.
OCONHCH3
Carbaryl
When the carbaryl specifications were reviewed in 2006, the old

specification for wet sieve test on the wettable powder (WP) limited
the residue to 2% on a 45 µm sieve (FAO, 2007). The question was
raised: could that limit be reduced to 1% with the now required
75 µm sieve? The manufacturer explained that 1% may be possible,
but carbaryl in co-formulations with other active ingredients may not
meet the 1%. The specification for wet sieve test on carbaryl WP was
set at ‘maximum: 2% retained on a 75 µm test sieve’.
8.7.5 Suspensibility
Suspensibility is the property that enables a homogeneous suspension
of insoluble particulate matter to be maintained.
Suspensibility is important for those formulations delivering the
active ingredient as water-insoluble particulate matter to be sus-
pended homogeneously in the spray mixture. The most important
formulations here are the SC, the water-dispersible granule (WG)
and the WP.
In the test, a suspension of the sample in water of specified hard-
ness is formed in a glass graduated cylinder and allowed to stand
for a standard time at a controlled temperature. Then the top nine-
tenths of the liquid are drawn off by suction leaving one-tenth for
analysis and calculation of percentage suspensibility. A minimum of
60% suspensibility is expected for a good formulation.
A proposed minimum suspensibility specification of 50% for
lambda-cyhalothrin WGs was questioned because the normally
acceptable minimum is 60%. In this case, further information sug-
gested that the lower value would be acceptable (FAO, 2013b).
8.7.6 Pourability
The viscosity of a suspension concentrate (SC) formulation helps
to keep the particles in suspension during storage. However, if the
formulation is too viscous, it is difficult to pour and measure.
The test method measures the remainder of a sample poured
from a glass measuring cylinder under standardized conditions. It
applies to SC formulations and other similar viscous liquid formu-
lations. For SC formulations, pourability specifications range from 3
to 9%.
Pourability is a property of the formulation itself. How well it
pours from a commercial container depends on the characteristics of
the container–formulation combination.
8.7.7 Attrition Resistance

When granular formulations are moved, stacked or transported the
granules suffer attrition, i.e. particles or dust are generated as the
granules lose small amounts of material. The dust creates additional
occupational exposure for the user and could also reduce efficacy of
the product.
The laboratory method for measuring attrition resistance of gran-
ules simulates possible attrition by rolling a sample of dust-free gran-
ules with glass marbles in a sealed bottle under controlled conditions
for a fixed number of rotations. Fines and dust are then removed on
a 125 µm sieve and the residual granules are weighed for calculation
as percentage of the original sample weight, expressed as percentage
attrition resistance.
Minimum attrition resistance values in specifications for granules
(GR), WGs and water-soluble granules (SG) are mostly in the 98–
99% range, but with occasional values at 85–90%.
8.8 Storage Stability

The labels of perishable goods, particularly foods, often display ‘use
by’ or ‘best before’ dates. Pesticide products cannot be stored forever,
so ‘expiry’ dates on their labels have been suggested for them also.
A WHO Committee pointed out that an artificial disposal prob-

lem may arise if expiry dates are too conservative (WHO Expert
Committee, 2001). Also, if users perceive that expiry dates are some
measure of when diminished performance is expected, they may be
tempted to use higher application rates. But an expiry date may
have been set because of increased user risk from toxic impurities
produced during storage and increased usage rates would exacerbate
the risks.
The Manual (FAO/WHO, 2010, p. 255) defines ‘release date’.
Release date : The date from which the supplier guarantees a

shelf-life of at least two years, unless stated otherwise, under actual
conditions of storage in the area where the technical grade active
ingredient or formulation is to be marketed.
The Manual explains that formulations are intended to remain

within specification for two years after the release date if stored in
good conditions in the original unopened container. If a product has
been stored in adverse conditions or for too long, one possibility is
analysis and testing to determine if it is still suitable for use.
Labels should explain the consequences of adverse conditions or
storage for an excessive interval (loss of effectiveness, increased user
hazard or poor physical properties).
8.8.1 Storage Stability at Elevated Temperature

Temperature is an important factor that influences storage stability
of pesticide products.
Neuenschwander (1992) examined the actual temperatures expe-
rienced by products under a variety of climatic conditions. In the
normal course of events, products are exposed to ambient conditions
including the daily temperature cycle. The package and container
are important. He showed that, for polyethylene jugs in a carton
box under the tropical sun, the product did not get warmer than air
temperature. For a black painted steel drum, the product reached a
temperature 11◦ C above ambient. These are extreme conditions; the

label would normally provide a warning such as ‘Store in the closed
original container in a cool, well-ventilated area. Do not store for
prolonged periods in direct sunlight’.
Real-time testing at 30◦ C is adequate for tropical areas, while
20◦ C is suitable for temperate climates (Neuenschwander, 1992).
The accelerated storage procedure, MT46.3, requires storage of
the test sample at 54◦ C for 14 days (FAO/WHO, 2010, pp. 63–64).
The specification guideline draws attention to products that may be
affected by such a high temperature and provides alternative tem-
peratures and durations: four weeks at 50◦ C, six weeks at 45◦ C, eight
weeks at 40◦ C, 12 weeks at 35◦ C or 18 weeks at 30◦ C.
Almost always, the storage stability specification has required
the conditions ‘54 ± 2◦ C for 14 days’ and the test result of active
ingredient content not lower than 95% relative to the content before
storage. However, some specifications require 97% while others have
lesser requirements (e.g. 80%).
Experience has shown that, if a product passes the accelerated
storage test at 54◦ C for 14 days, it is expected to be stable in storage
for at least two years (Neuenschwander, 1992).
The Arrhenius equation provides the theory for the influence of
temperature on reaction rate (Moore, 1958).
Ea
ln(k) = − + ln(A) (8.1)
R×T
where k is the reaction rate constant, Ea is the activation energy of
the reaction, R is the universal gas constant, T is absolute tempera-
ture and A is a constant.
The results of the accelerated storage test have been interpreted
as: A 5% loss of active ingredient after 14 days at 54◦ C predicts a
similar loss of active ingredient at practical shelf storage temperature
after two years.
We can calculate Ea /R from the rates of reaction at two tem-
peratures. In this case, T1 = 54 + 273 = 327 and, assuming storage
temperature is 15◦ C, T2 = 15 + 273 = 288.
Table 8.6. Theoretical comparison of the effect of test temper-

ature-duration combinations on loss of active ingredient.
Calculated Calculated % Loss

Test Test Reaction of Active Ingredient
Temperature Duration Rate, Days−1 During the Test
50 ± 2◦ C 4 weeks 2.49 × 10−3 7.0 ± 1.3%

45 ± 2◦ C 6 weeks 1.56 × 10−3 6.6 ± 1.2%
40 ± 2◦ C 8 weeks 9.67 × 10−4 5.4 ± 1%
35 ± 2◦ C 12 weeks 5.89 × 10−4 5.0 ± 1%
30 ± 2◦ C 18 weeks 3.53 × 10−4 4.5 ± 1%
Rates of reaction: k1 = 0.05/14 = 3.57 × 10−3 and

k2 = 0.05/730 = 6.85 × 10−5 (units, day−1 ).

k1 Ea T1 − T2
ln = × (8.2)
k2 R T1 × T2

Ea 3.57 × 10−3 327 − 288
= ln ÷ = 9547 (8.3)
R 6.85 × 10−5 327 × 288
If T2 = 288 and k2 = 0.05/730 = 6.85 × 10−5 , for a reaction with
Ea /R = 9547, what should be observed under the alternative times
and temperatures of the test (Table 8.6)? Note: The ranges for %
loss are calculated from the test temperature ranges ±2 C.
With allowance for the assumptions of a 15◦ C shelf storage tem-
perature and an Ea of 9547 × R, the calculations suggest that the
various temperatures and times of the accelerated storage test will
give similar results for the stability of active ingredients.
When tribenuron-methyl water-dispersible granules (WG) were
subjected to the accelerated storage test (FAO, 2011a), more than
5% degraded at 54◦ C in 14 days, but no more than 4% at 35◦ C in 12
weeks. The above calculations suggest that such variations between
the tests are to be expected. The active ingredient concentration was
not significantly degraded when the product was stored in practical
conditions for two years in a tropical climate.
While the chemical stability of the active ingredient should be
revealed in the accelerated storage test, the effect on physical prop-
erties of formulations is not so predictable (e.g. if the temperature
were to reach the melting point of an active ingredient in a SC even

for a short time, poor suspensibility would be expected). Increased
water solubility of the active ingredient at the elevated temperature
of the test could create the conditions for particle size growth in an
SC, also contributing to poor suspensibility.
Niclosamide provides an example where storage at 54◦ C causes
changes in physical properties, whereas storage at 40◦ C does not.
Niclosamide is used for controlling snails and to remove unde-
sirable fish from commercial fish ponds before restocking (FAO,
2004). The accelerated storage test for niclosamide olamine SC relies
on the 40◦ C and eight weeks conditions because at 54◦ C crystal
growth occurs, which adversely affects suspension properties. Under
field conditions in tropical climates such problems have not been
observed.
NO2
Cl O
NH
Cl
OH
Niclosamide
The specifications for niclosamide emulsifiable concentrate (EC)

and WP include the 54◦ C storage test. The EC is a solution and the
opportunities for particle size growth in WP do not occur.
The nature of the formulation may influence the stability of some
active ingredients. For fenthion (FAO, 2006b), liquid formulations
appear more stable than the solid formulations (Table 8.7).
8.9 Future Directions

The term ‘biopesticide’ includes natural substances and living organ-
isms (Czaja et al., 2015). The living organisms include microbial
pesticides (fungi, bacteria and viruses) and invertebrates (e.g. preda-
tory insects). Specifications and hazard description for this category
of biopesticide need more than an adaptation of the processes for
synthetic chemicals.
Table 8.7. Stability specifications for fenthion formulations.
Specification
Active % Remaining, Accelerated
Ingredient Formulation Storage Test
Fenthion DP Dustable powder ≥ 80

WP Wettable powder ≥ 90
UL Ultra-low volume liquid ≥ 95
EC Emulsifiable concentrate ≥ 95
EW Emulsion, oil in water ≥ 95
For living organism pesticides, we might expect the development

of DNA techniques for:
• identification of active ingredient and
• identification of impurity pathogens and measurement of their con-
centrations.
Toxic metabolites will require identification and control.

Specifications will be needed for genetically engineered (GE)
crops that produce their own pesticide for protection from insect,
fungal, microbial, etc. attack.
Specification guidelines and suitable procedures for hazard
assessment are needed for these new situations. For example, the
accelerated storage test depends on the theoretical effect of temper-
ature on a chemical reaction rate. How could it apply to a living
organism?
For some old compounds, manufacture may transfer from the
pioneering company owning the patent to generics companies with
modified methods of production. Specifications may be different
with respect to impurity profiles or isomer composition (Verger and
Boobis, 2013). Perhaps, no longer will there be material with the orig-
inal reference profile on the market and a new reference profile will
be needed. At the Codex Committee on Pesticide Residues (CCPR)
meeting in 2013, it was agreed that when a company owning the
pesticide patent was no longer interested in commercialization, an
interested member country could submit a data dossier to maintain
support of Codex maximum residue limits (MRLs) needed for the
international food trade. It would be an opportune time to revise

the reference profile and bring the specifications up to date also.
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and Teeters WR. 1978. Epidemic malathion poisoning in Pakistan malaria
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Baltazar MT, Dinis-Oliveira RJ, Guilhermino L, de Lourdes Bastos M, Duarte
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salicylate with low mammalian toxicity and effective herbicidal activity. Pest
Mgmt Sci. 69: 553–558.
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2008.
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M and Ludwicki JK. 2015. Biopesticides — towards increased consumer
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FAO. 2006b. FAO specifications and evaluations for agricultural pesticides.
Fenthion.
Carbaryl.
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1
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tions for pesticides. November 2010 — second revision of the First Edition.
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and drugs. Anal Chem. 54: 67A.
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rative studies. Resulting from the IUPAC Workshop on the Harmonization of
Collaborative Analytical Studies, Geneva, Switzerland, 4–5 May 1987. Pure
Appl. Chem. 60: 855–864.
ISO. 1986. International Standard ISO 5725. Second edition 1986-09-15.
Moore WJ. 1958. Physical Chemistry, 3rd edn. Longmans, Green and Co. London
and New York, p. 546.
Neuenschwander E. 1992. Storage temperatures and product temperatures under
extreme conditions. CIPAC Symposium, Zurich.
Patent. 2005. Mitigating necrosis in transgenic glyphosate-tolerant cotton plants
treated with herbicidal glyphosate formulations. U.S. provisional patent
application Ser. No. 60/659,001, filed Mar. 4, 2005 and U.S. provisional appli-
cation Ser. No. 60/713,948, filed 1 September 2005.
relating to pesticides (IUPAC recommendations 2006). Pure Appl. Chem. 78:
2075–2154.
Verger PJP and Boobis AR. 2013. Re-evaluate pesticides for food security and
safety. Science 341: 717–718.
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teenth report of the WHO Expert Committee on Vector Biology and Control.
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Propoxur.
b2530_FM.indd 6 01-Sep-16 11:03:06 AM

Chapter 9
Theory and Practice of Sampling

for Pesticide Residue Analysis
Jo Marie Cook and Árpád Ambrus
Main topics
Introduction
Sampling terminology
Decision unit (lot, population)
Mass reduction
Representative sample
Sampling correctness
Material properties and sampling dimensions
Pierre Gy’s Theory of Sampling (TOS)
Sampling techniques — striving to be representative
Correct versus incorrect sampling
Combined estimation of errors
Laboratory sampling errors
Pesticide residue sampling research
Standardized sampling guidelines
Management responsibilities
327
9.1 Introduction
All measurements begin with a sampling process, unless the entire
material is analysed. For pesticide residue analysis, sampling begins
with the collection of a portion of food, feed or soil from a crop field
or commercial lot but continues even when the laboratory sample is
chopped, ground or blended. Many steps in the process from field to
instrumental analysis are sampling steps where the theory of sam-
pling (TOS) tells us that errors must be minimized. This is often not
recognized by the laboratory processing the sample. The contribution
of sampling and laboratory processing to overall analytical error and
uncertainty are frequently not estimated. Significant efforts are made
to measure, validate and quality control the analytical error, without
regard or interest in the determination of all the errors contributing
to the uncertainty in a test measurement.
Pesticide residue sampling seeks to answer the question, ‘What
is the concentration of pesticide in a material?’ In the context of
this book, the material may be a pesticide formulation, food, feed,
soil or other agriculture-related substance. The vast universe of nat-
ural crops and processed commodities provides an infinite variety of
challenges to the sampler. The large volume of the material to be
sampled, ranging from agricultural fields and cargo ships to man-
ufacturing facilities and packaged product in warehouses magnifies
these challenges. The focus of this chapter will be on the process
of sampling and the minimization of variability and errors due to
sampling.
Pesticides contribute significantly to the yield of agricultural
crops and are carefully regulated to ensure that residues do not
present a health risk. The Codex Alimentarius Commission (CAC)
under the Food and Agriculture Organization of the United Nations
(FAO) and the World Health Organization (WHO), and most gov-
ernments such as Australia, Canada, China, European Union, Japan,
New Zealand and the United States, govern the levels of pesticide
residues allowable in foods. This topic was discussed in more detail
in Chapter 7. Thousands of samples are collected every year for pes-
ticide residue analysis to protect the consumer, enforce regulations,
Theory and Practice of Sampling for Pesticide Residue Analysis 329
support registration and conduct research. In 2008, scientists were

asked to estimate and report the uncertainty in analytical mea-
surements of pesticide residues (JCGM, 2008); however sampling or
sample processing steps were not included. The estimation of ana-
lytical measurement uncertainty in pesticide residue analysis is now
widely accepted. Since that time, multiple authors have called for the
inclusion of sampling and sample processing error in the estimation
of measurement uncertainty.
How much variability or uncertainty can be attributed to sam-
pling and laboratory processing? This is a question to be answered
in Chapter 10. However, it is not enough to blindly measure the
variability in the sampling steps of the analytical process without
clearly understanding the sources of sampling error and minimizing
them wherever possible. Without care to assure the sample repre-
sents the true composition of the target material or lot, analytical
measurements and the decisions made based on these measurements
may be very inaccurate. The sampling of foods for pesticide residue
analysis has been studied extensively by multiple authors and several
guidelines have been written. TOS, developed by Pierre Gy (1998,
2004a, 2004b, 2004c, 2004d, 2004e, 2004f), was refined and applied
to statistical process control by Pitard (1993), and further utilized in
sampling studies by others. TOS focuses on the process of sampling
and strives to minimize variability and bias due to sampling. Much
of this chapter is devoted to sampling theory, strategies to minimize
sampling errors and the integration of TOS with the extensive body
of research in sampling for pesticide residues. The work mentioned
above and many additional references and recommended readings are
provided at the end of this chapter.
9.2 Sampling Terminology

Various sampling books, articles and guidelines use the same terms
differently and different terms are used for the same thing (Thiex
et al., 2015a, 2015b). Some of these discrepancies will be noted but
it will be impossible to identify all of them. Emphasis will be placed
on terminology used in the International Union of Pure and Applied
Chemistry (IUPAC) (Stephenson et al., 2006), Codex (Codex, 1999,

2004, 2009), FAO Manual (Ambrus, 2016c) and TOS (Pitard, 1993;
Gy, 1998). Most importantly, the word ‘sample’ requires a descriptor
to identify the particular step in its lifecycle. For example, a ‘primary
sample’ may consist of one or more ‘increments’ from a single position
in a crop field, a ‘consignment’ or a ‘lot’. The ‘laboratory sample’ may
consist of multiple increments or a ‘composite sample’. Laboratory
processing results in a ‘test sample’ from which an ‘analytical test
portion’ is extracted and an analytical test ‘aliquot’ is injected on
an instrument. Those involved in analytical testing and sampling
are encouraged to study sampling in more detail but take care to
understand how terms are being used in each reference.
Characteristic: A property that helps to identify or differentiate

between items within a given lot (Codex, 2004).
Sampling: Procedure used to draw or constitute a sample (Codex,

2004).
Sample: A set composed of one or several items (or a portion
of matter) selected by different means in a quantity of matter
(population) and intended to provide information on a given char-
acteristic of that matter and to form a basis for a decision con-
cerning the matter or process, which produced it (Codex, 2004).
The word ‘sample’ needs to be qualified as the type of sample. Terms

used for the same purpose are included in parentheses:
• Aggregate sample (composite sample): Sample made up of set pro-

portions of other samples (Stephenson et al., 2006).
• Aliquot: Known amount of a homogeneous material, assumed to be
taken with negligible sampling error (Stephenson et al., 2006).
• Analytical sample (test sample): The material prepared for analysis
from the laboratory sample, by separation of the portion of the
product to be analysed and then by mixing, grinding, fine chopping,
etc., for the removal of analytical portions with minimal sampling
error (Codex, 1999).
• Analytical portion (test portion): A representative quantity of

material removed from the analytical sample, of proper size for
measurement (Codex, 1999).
• Bulk sample (multiple increment or multiple item sample): Com-
bined and well mixed aggregate of the primary samples taken from
a lot (Codex, 1999).
• Composite sample (aggregate sample): Combined primary samples
or increment samples such as 24 fruits. Composite sample may
also refer to combined replicate samples or combined samples from
replicate trials (Stephenson et al., 2006). Note that samples from
different trials should NOT be combined in formal residue trials.
• Final sample: Bulk sample or, if too large, prepared from the bulk
sample by a method of mass reduction (Codex, 2004) (see Sub-
Sample).
• Grab sample (specimen): A non-random sample selected for a given
reason such as convenience (Pitard, 1993).
• Increment sample: Individual portion (unit) of material collected
by a single operation of a sampling device from bulk materials or
large units (Stephenson et al., 2006).
• Laboratory sample: The sample sent to, or received by the labora-
tory. The laboratory sample may be the whole or a part of the bulk
sample (Codex, 1999).
• Population in statistics: The totality of individual observations
about which inferences are to be made, existing anywhere in the
world or at least within a definitely specified sampling area limited
in space and time (Sokal and Rohlf, 1995).
• Primary sample (increment, item, gross sample): One or more
units taken from one position in a lot (Codex, 1999). Primary
sample may also refer to one or more increments or units ini-
tially taken from a population or lot. Portions may be combined
(composite or aggregate sample) or kept separate (gross sample)
(Stephenson et al., 2006) (see bulk sample).
• Random sample: Sub-set of a sampling population that is arrived
at by selecting units such that each possible unit has a fixed and
determinate probability of selection (Stephenson et al., 2006).
• Replicate sample(s): Additional samples collected under comparable

conditions at any point in the sampling process (GOODSamples,
2015).
• Representative sample: A sample in which the characteristics of
the lot from which it is drawn are maintained and each of the
items or increments of the lot has been given the same probability
of entering the sample (Codex, 2004).
• Specimen (grab sample): Portion taken under conditions such that
the sampling variability cannot be assessed (IUPAC, 1990).
• Purposive selection: Operator chooses the units of the lot to select.
These are specimens (Pitard, 1993).
• Sub-sample (Secondary Sample): 1. Portion of the sample obtained
by selection or division; 2. Individual unit of the lot taken as part of
the sample; 3. final unit of multistage sampling (Stephenson et al.,
2006).
• Split samples: Aliquot taken of a laboratory sample or test sample
or the sample is otherwise subdivided (Stephenson et al., 2006).
• Test portion (analytical portion): Sub-sample, of proper size for
a chemical analysis or other test, removed from the test sample
• Test sample (analytical sample): Homogenous sample, prepared
from the laboratory sample by mixing, grinding, blending, fine-
chopping, etc. from which test portions are removed for analysis
with minimal sampling error (Stephenson et al., 2006).
Increment (item): Quantity of material drawn at one time from

a larger quantity of material to form a sample (Codex, 2004).
Individual portion (unit) of material collected by a single operation
of a sampling device from bulk materials or large unit (Stephenson
et al., 2006).
Commodity: Primary food commodity: Product in or nearly in

its natural state intended for processing into food for sale to the
consumer or as a food without further processing. It includes
irradiated primary food commodities and products after removal
of certain parts of the plant or parts of animal tissue (Ambus,

2016c, p. 153).
Primary feed commodity: The product in or nearly in its natural
state intended for sale to: (a) the stock farmer as feed which is used
without further processing for livestock animals or after silaging
or similar farm processes; (b) the animal feed industry as a raw
material for preparing compounded feeds (Ambrus, 2016, p. 153).
Processed food: Product resulting from the application of physi-
cal, chemical or biological processes to a primary food commodity
(Ambrus, 2016c, p. 52).
Raw agricultural commodity (RAC): Part of a crop used as a food
or feed commodity directly from the harvested crop without pro-
cessing (Stephenson et al., 2006). The same as primary food com-
modity (Ambrus, 2016c, p. 52).
Secondary food commodity: Primary food commodity which has

undergone simple processing, such as removal of certain portions,
drying, husking and comminution, which do not basically alter the
composition or identity of the product (Ambrus, 2016, p. 154).
Consignment: A quantity of some commodity delivered at one time

consisting in either a portion of a lot or a set of several lots
(Codex, 2004).
Lot (batch): A defined quantity of some commodity manufac-

tured or produced under conditions, which are presumed uniform
(Codex, 2004). A specific identified portion of a batch, having uni-
form character and quality within specified limits; or, in the case of
a product produced by continuous process, it is a specific identified
amount produced in a unit of time or quantity in a manner that
assures its having uniform character and quality within specified
limits (USCFR, 2016a).
Batch: Quantity of material that is known or assumed to be

produced under uniform conditions. A lot may consist of one or
more batches (Stephenson et al., 2006).
9.3 Decision Unit (Lot, Population)

What is the analyte of interest and in what material? What questions
need to be answered about the analyte? At what level do we want to
know the concentration? Do we want to know the concentration of
carbaryl in an individual apple (a single serving size); the distribution
of carbaryl from apple to apple in a grove; the average concentration
of carbaryl in each tree; the average concentration of carbaryl in the
grove as a whole or the distribution of carbaryl across multiple field
trials? The statistician might call the apple, the tree or the grove the
population. Regulators often refer to the apples harvested from a par-
ticular grove on a particular day as the lot. Combining the concepts
of population or lot with the question to be answered, GOODSamples
(2015) coined the terminology ‘decision unit’ to describe that mate-
rial (population or lot) from which a sample will be taken, analyses
conducted, inferences drawn from these analyses and a regulatory,
risk assessment or other decision made. This concept was further
described by Ramsey (2015a, 2015b).
Decision unit (lot, population): The material from which a sample

is collected and to which an inference is made (GOODSamples,
2015).
Three essential data quality objectives have been identified (USEPA,

2000; Ramsey and Hewitt, 2005): What is the question? What is the
population? What is the desired confidence? GOODSamples calls
these the sample quality criteria (SQC).
SQC: A series of statements that clarify programme’s technical

and quality needs to support defencible decisions including state-
ment of the question to be answered, definition of the decision
unit and the desired confidence in the inference (GOODSamples,
2015).
A clear specification of the decision unit and SQC is critical to the

creation of a ‘fit for purpose’ sampling protocol. It is important that
the laboratory also clearly understands the SQC and the sampling
protocol to assure that laboratory sample handling and processing
will maintain the integrity and representativeness of the decision unit.
The all-important role of management in the coordination and com-
munication among all parties involved in the sampling, analysis, use
of the data and decision-making process is discussed as a conclusion
to this chapter.
The sampling of food and feed commodities for the analysis of
pesticide residues occurs for various reasons such as determining the
maximum pesticide residue from a field trial; surveying fresh, pro-
cessed and market basket commodities to assess consumer exposure
rates; monitoring of the food supply to provide statistical data for
risk assessment; enforcing maximum residue limits (MRLs) of prod-
uct in channels of trade; controlling quality of manufactured foods
and feeds; certifying products for import and export, identifying
environmental contaminants or drift; conducting pesticide misuse
investigations, and conducting experimental tests for new agricul-
tural chemicals and others. While the principles of representative
sampling remain the same, each type of sampling and the sampling
plan is different due to the sampling objectives and confidence needed
in the data.
9.4 Mass Reduction

In the context of pesticide residue analysis and assuming that we are
not going to collect the entire decision unit for analysis, a sample
is a small amount of material taken from the decision unit, which
will be used to conduct analytical testing and estimate the pesticide

concentration in the decision unit. This is sometimes referred to as
mass reduction.
Mass reduction: The sole aim of sampling is to reduce the mass

of a lot without significantly changing its other properties (Gy,
1998). The process of selecting a smaller mass from a larger mass,
not to be confused with comminution or particle size reduction
(GOODSamples, 2015).
Gy (2004b) tells us, ‘The sole purpose of sampling is to reduce pro-

gressively the mass of a lot to that of an assay portion, light enough
(small enough) to be submitted integrally to analysis.’ Brown et al.
(2009) tell us that ‘The most important merit of any mass reduction
method is the ability to deliver an unbiased split of material with
the smallest possible variation in repeated runs, that is, best possible
accuracy and precision’. Each mass reduction step introduces some
error and the possibility of bias.
Bias: The difference between the expectation of the test result or

measurement result and the true value. It is the total systematic
error as contrasted to random error (Codex, 2009).
A representative sample is critically important to assure that the

analytical result will reflect the true average quantity of pesticide
in the decision unit. Analytical results, along with an estimate of
the variability associated with all mass reduction sampling, sample
processing and analytical testing, can be used to draw inferences
about the material from which it was extracted and make scientific
and defensible decisions. This assumes that a small, representative
mass has been collected from the decision unit in a manner that
assures it has the same characteristics as the decision unit with regard
to the analyte of interest within an allowable error. We will discuss
this further in Section 9.11.
Inference: The process of estimating a concentration or character-

istic about a larger amount of material from the analysis of one
or more samples (GOODSamples, 2015).
Comminution: A crushing, grinding or pulverizing stage that

diminishes the fragment size of a lot, sample or increment (Pitard,
1993).
9.5 Representative Sample

The word ‘representative’ is used in so many different ways that it is
necessary to further describe it for food and feed sampling.
Ramsey said that it is an operational word and ‘Assessing rep-
resentativeness can only be accomplished in the context of the ques-
tion the data are supposed to address.’ Ramsey goes on to conclude
that ‘A representative sample is one that answers a question about
a population (lot, decision unit) with a certain confidence’. And
‘It is a result of careful planning and proper design’ (Ramsey and
Hewitt, 2005). Note the emphasis on the process of sampling and
SQC. A statistical analysis of uncertainty may be very misleading if
the sampling was conducted in a manner that misrepresents the deci-
sion unit. Gy (1998) states that ‘a sample is representative when it is
taken by a selection method that is both accurate and reproducible’.
And he goes on to explain that the sample ‘is characterized by the
absence of bias and an acceptable variance’. Pitard (1993) tells us
that ‘Representativeness is the exclusive property that synthesizes
both accuracy and precision’.
As a single, randomly selected increment could be greatly dif-
ferent from the average of all the increments in the decision unit,
it is never possible to collect a single grab sample or specimen (e.g.
one apple from the tree, one handful of parsley, an ounce of deli
cheese, one bore sample of soil) and make the assumption that it is
representative of the decision unit as a whole. Peterson et al. (2004)
compared many different field and laboratory sampling techniques,
Figure 9.1. Example of mass reduction steps.

Note: Mass of five large crops making up one bulk or laboratory sample, such as
watermelon, jackfruit etc., may be over 50 kg. Figure 9.1 demonstrates how mass
reduction begins with the collection of the primary sample increments at one of
many different stages in the field to table continuum (agricultural field, shipping
dock, manufacturing firm, distribution centre, retail outlet, etc.) This results in
a composite or bulk sample comprising multiple primary samples or increments.
The composite sample is shipped to the laboratory, and when received becomes
the laboratory sample. The laboratory receives the sample and may conduct addi-
tional mass reduction such as sub-sampling, which should be avoided wherever
possible because it can introduce significant error or even bias. (For details see
Section 9.7) The laboratory may clean, peel or otherwise prepare the sample and
often performs comminution to reduce the particle size of the laboratory sample,
which results in a test or analytical sample. A test portion is extracted and an
exceptionally small test aliquot is introduced to an analytical instrument such as
a liquid chromatograph or gas chromatograph mass spectrometer.
demonstrating the extremely large errors generated by grab sampling

and insisted that grab sampling must be discontinued. Gy (2004b)
tells us that a grab sample is merely a specimen, collected without
regard to sampling correctness or probabilistic increment selection.
If it is a purposive sample, selected by a regulatory officer to fulfill
a given purpose, such as a food taken from the refrigerator of an ill

person, it should be labelled as such.
Sample correctness: Sample selection is correct when all the

constituent elements of the lot have an equal probability of being
taken into the sample and the increments and the sample are
not altered in any way (Gy, 1998). Sampling is correct when it
gives all elements, in the batch to be sampled, a uniform proba-
bility of being selected (Pitard, 1993). A condition achieved when
bias is controlled to a negligible level. The major sources of bias
include increment delimitation error (IDE), increment extraction
error (IEE) and increment weighting error (IWE) (GOODSam-
ples, 2015).
Probabilistic selection: A probabilistic selection is correct when the

selecting probability is uniformly distributed among all units mak-
ing up the lot and nil for material that does not belong to the lot
(Pitard, 1993). Selection is probabilistic when all the constituent
elements of the lot to be evaluated have a non-zero probability
of being taken into the sample (Gy, 1998). Selection is non-
probabilistic when certain constituent elements of the lot to be
evaluated have a zero probability of being taken into the sample
(Gy, 1998).
We will come to find out that a representative sample is the collection

of a number of increments selected from the decision unit, where
each of the fragments or items of the decision unit has the same
probability of being selected.
Fragment (particle): Compact unit belonging to the lot. During

the selection process, this unit is assumed to be indivisible. As
fragments become smaller, the term ‘particle’ is preferred; however,
both words have the same meaning irrespective of the size (Pitard,
1993).
Item (individual, unit, element): An actual object on which a set of

observations may be made, and which is drawn to form a sample
(Codex, 2004).
Unit (item, element): The smallest discrete portion in a lot, which

should be withdrawn to form the whole or part of a primary sample
(Codex, 1999).
Sufficient mass of multiple, randomly selected increments should be

collected from the same decision unit and combined to form a mul-
tiple increment or composite sample. For example, if a package of
bagged lettuce was collected from a retail grocery and found to
contain a non-approved pesticide residue or a residue exceeding the
MRL, the regulatory agency could say that the specific bag of lettuce
contained an unapproved pesticide. However, it could not say any-
thing about the other bags of lettuce available for purchase at the
store even if from the same lot. Conversely, and more important for
food safety professionals, if no pesticide residues were found on the
lettuce sample, the regulatory agency could not infer that there were
no pesticide residues on the lettuce in the other bags in the store. In
order for a sample to represent the lot, it should be a composite of
multiple increments, randomly selected from the lot.
Despite all efforts to develop sampling plans that represent the
decision unit, every analytical result will have an associated sampling
and analytical error. All too often the reported uncertainty in the
analytical results is derived from analytical error only with no con-
sideration for the error inherent in the procedures used from sampling
to withdraw a test portion for extraction. In many cases, the sampling
error is much greater than the analytical error. Non-representative
sampling can lead to inaccurate, biased analytical results, inaccurate
assumptions and non-defensible decisions. Without a clear knowledge
of the possible deviation from the true value in any measurement, it
is impossible to make accurate decisions.
9.6 Sampling Correctness

Today’s reader will immediately recognize a similarity between Gy’s
representative sample and the measurand plus the measurement
uncertainty, but there is an important difference. Gy’s work seeks to
assure accuracy, minimize error and eliminate bias. It is not enough to
simply measure the sampling variance. The variability (error, uncer-
tainty) in the sampling should be reduced as much as possible. An
analytical measurement cannot be expressed in its totality without
the variance associated with it, and a result with an unidentified bias
can be very misleading. Bias in sampling is very difficult to detect,
much less quantify. If only the best looking and largest fruits or veg-
etables are collected from the most convenient portions of the plant,
the estimate of total pesticide application may be very high compared
to the average of the entire harvest. If some particles or portions of
the decision unit (volatiles, oils, skin, peel, seed, precipitate, crumbs,
etc.) are preferentially excluded and not selected in the primary sam-
pling or laboratory processing, but contain high amounts of pesticide,
falsely low residues will be reported. If small and medium-size parti-
cles are collected but larger particles are excluded from the sample, the
amount of pesticide may be biased either high or low, depending on the
concentration of the pesticide in the excluded particles. Sampling in a
manner where the bias is controlled to an acceptably low level is called
‘sampling correctness’. Information regarding both the accuracy and
precision enables the user to make more reliable decisions.
It is important to determine the mass and number of increments
that should be collected from a particular decision unit in order to
make the desired determination about the entire decision unit. It
is even more important to assure that all increments are correctly
sampled so that all particles and portions of the decision unit have
an equal probability of being selected.
9.7 Material Properties and Sampling Dimensions

9.7.1 Finite Element Materials
Fresh grown commodities often exist as distinct, individual items
(citrus, pome and tropical fruits or root, bulb, Brassica, fruiting or
Figure 9.2. Finite and infinite element vegetables. This figure shows a variety of
fresh vegetables, many of which are finite element materials such as winter squash
and heads of cabbage. As the element size decreases, the choice of finite vs. infinite
element material sampling depends on the scale at which the sample is collected.
If a bunch of leafy greens such as spinach or parsley are seen as a single element,
then bunches can be collected separately, but in the field, at the time of har-
vest, leafy greens may demonstrate many of the characteristics of infinite element
materials.
even heads of leafy vegetables) that can be separately collected and

counted. These are known as finite element materials. Separate ele-
ments of the material may be randomly identified and selected as
increments of a multiple increment or composite sample.
Element (fragment, particle): The constituent elements of a lot

of material are the smallest elements that can be considered to
be immutable in the physical, chemical and mechanical conditions
of sampling. Solid constituent particles = fragments (liquids and
gases = molecules and ions) (Gy, 1998).
Finite element materials: Materials composed of elements that
can be individually identified and individually selected at random
(GOODSamples, 2015).
Particle: Very small fragment.
9.7.2 Infinite Element or Bulk Materials

When sampling commodities composed of smaller elements, frag-
ments or particles (e.g. nuts, grains, seeds, spices, processed foods,
animal feeds, soils), specific items cannot be collected. These are

known as infinite element or bulk materials.
Infinite element materials: Materials composed of elements that

cannot be individually identified nor individually selected at ran-
dom (GOODSamples 2015).
Identifying and accounting for all the sources of variability in infinite

element materials is often complex. While universal to all materi-
als, Gy’s theories are essential to infinite element particulate and
liquid materials. Gy’s theories are also critical in laboratory pro-
cessing where a finite element sample (24 zucchini or squash, eight
watermelons, 12 heads of lettuce) is chopped, sliced or blended and
transformed into a TOS infinite element material. Increments from
these materials must be collected as groups of fragments and parti-
cles, which are removed using some type of tool or a technique such as
shovelling or scooping. Each individual portion extracted is called an
increment (e.g. a scoop of nuts, a drum thief sample of honey, a core
sample of soil, a scoop of irrigation pond water). The tool used to
extract an increment may not reach and select all portions of the bulk
material (e.g. the scoop of nuts may only reach down into half the
bin; the drum thief may not reach the bottom of the barrel of honey;
the core soil sample may exclude large clumps and let very fine sands
fall out; the scoop of water may push light weight or oily materials
aside). The very action of extracting an increment can change the
temporal or spatial variability within the infinite element material.
Scooping up a grain may cause finer fragments to fall to the bottom.
Opening a closed container may release volatile flavours. When col-
lecting bulk materials, it becomes very important to correctly collect
the primary sample using procedures that will provide access to all
portions of the bulk material and select increments with appropriate
tools that represent all the fragments in the same proportions as they
are present in the lot. We’ll see that the shape of the increments, the
number of increments and the total mass collected are critical factors
in sampling of bulk materials.
Bulk materials: Units that are individually too large to be taken

as primary samples (such as drums, cheeses, etc.). The units
(increments) are created with a sampling device (Codex 1999).
9.7.3 Sampling Dimensions

TOS identifies four sampling dimensions — zero, one, two or three —
in terms of the prominent and available sampling dimensions of the
lot and the shape of the increments that are collected or extracted.
These are not to be confused with, but are related to, the three phys-
ical dimensions of length, width and height. Gy found the identifica-
tion of the sampling dimension so critical, that in his 2004 articles
commemorating 50 years of TOS, he devoted parts I, II and III to
zero and one dimensional, qualitative and quantitative sampling (Gy,
2004b, 2004c, 2000d).
Sampling dimensions (Gy, 1998; Pitard, 1993; GOODSamples,

2015):
• Zero: Material made up of non-ordered finite elements that are
independent from one another
• One: Material made up of ordered elements in time or space.
Sample by taking a cut or slice of one dimension.
• Two: Material of unordered elements, which is small enough
that a sampling tool may reach all portions such as a bag of
grain. Sample by taking a slice of the entire depth or two dimen-
sions.
• Three: Material which is so large that it cannot be sampled with-
out breaking it up into one- or two-dimensional units.
9.7.4 Zero-Dimensional Sampling Model

Gy defines lots made up of a large number of non-ordered
finite elements that are independent from one another as zero-
dimensional sampling materials. There is no specific tool needed
to collect these increments. Each element or item is an increment.
Figure 9.3. Compositional and distributional heterogeneity of foods. (a) Shows

beans and peas as separate commodities. The kidney beans and the chick peas
might be counted separately and be considered finite element materials but it
would be very difficult to collect a primary sample by selecting individual split
peas or lentils. Each of the kidney beans has a little different size, weight, colour,
composition and residue level. This is known as compositional heterogeneity. (b)
Shows the beans and peas mixed together into a soup mix. Each bean and pea
retains its own compositional heterogeneity and now a scoop or primary sample
or increment of the mix will also have distributional heterogeneity because the
smaller split peas and lentils will segregate to the bottom of a bag. In (c), when
collecting an increment, perhaps with a sampler of a diameter similar to the jar
in the picture, proportionally more of the kidney beans will be excluded from
the increment then the smaller lentils because as they approach the lip of the
jar they fall away more frequently. A larger sampling tool diameter would lead
to a more representative sampling of the larger kidney beans and to sampling
correctness.
Conventional statistics may be applied if zero-dimensional elements

have approximately uniform masses (+/−20%). Each individual item
may be its own decision unit and analysed in its entirety. For exam-
ple, to determine the distribution of persistent pesticide in carrots,
100 carrots might be collected randomly and analysed separately.
Each of the items from the crop field is separate and individual
from the others. A sampling plan, which identifies the field of carrots
as the decision unit, specifies how many carrots will be collected
at random from the field. Of course, the primary sampling process
must still be representative and the rule of equal probability still
applies. Each and every carrot would have to be available for collec-
tion and no carrots could be more desirable to collect than others.
Table 9.1. Example of material properties and sampling dimensions.
Sampling
Dimension Elements Increment Technique Examples
Zero (0) Finite Individual Select each individual Apples, apple

element element sauce in jars
One (1) Finite Individual Periodic selection in Apples moving in
element time or distance packing house
One (1) Infinite Cross- Periodic selection in Apple sauce in
section time or distance production
Two (2) Finite Transform Periodic selection in Apples unloaded
to 1 time or distance from truckloads
or shipping
containers
Two (2) Finite Individual Random selection by Apples or jars of
element case and position in apple sauce in
case cases
Two (2) Infinite Increment Probe, drum thief, Apple sauce in 20
of entire coring litre drums
depth
Three (3) Finite or Transform Move the material or Apples in
infinite to 1 select samples while container ship
it is being off or truck
loaded.
Three (3) Finite or Transform Splitting or fractional Apple sauce in
infinite to 2 shovelling 300 litre vat
Note: This table provides some examples of dimensional lots. There is no

prominent sampling dimension for lots comprised of finite element materials as
each increment is collected as an individual item. These are known as zero-
dimensional lots.
If a lot is available as a long stream with one-dimension being very large
compared to the other two, such as a material on a conveyor belt, it is known
as one-dimensional lot as the increments are selected by taking slices of the
material from the elongated dimension. The width and depth of the material
must be entirely available for extraction of each increment.
If material is available in a bag, bin, drum or maybe even a truck or field, where
the increments can be extracted by probing to the bottom of the container, then
the width and length of the lot are the most important dimensions but the depth
of the container is not as critical because the entire depth of the lot is accessible.
These are known as two-dimensional lots as the increments must be chosen in a
representative way across the length and width of the sampled surface.
Huge piles of grain, protein powder or contaminated soil, ship loads of shrimp
and vats of processed food are too large to be sampled across the entire length,
width and depth of the material. These are known as three-dimensional lots and
they are so large that they can only be sampled by transforming them to a zero-,
one- or two-dimensional lot.
Each individual finite element item is then the increment to be sam-

pled. Error may be reduced and confidence in the test result increased
by assuring an appropriate random selection protocol and collecting
more carrots for each multiple increment composite sample.
Much of the research and empirical testing of sampling plans for
pesticide residues were developed for these finite element decision
units. These types of commodities contain objects that can be indi-
vidually selected at random. However, it is still important to identify
and account for the sources of heterogeneity, even in finite element
materials (e.g. small and large tomatoes, vegetables growing close
to the ground and others high on the plant, oranges growing inside,
outside, high and low on the tree, root crops growing in variable soil
conditions) in order to assure representative sampling.
Gy tells us that if the elements in a finite element material are
entirely independent of each other and very similar to each other
in physical characteristics, perhaps by about 20%, then they may
be treated using traditional statistics. Even the casual observer will
realize that this assumption is frequently not correct for food com-
modities. It is important to identify the degree of heterogeneity in
materials sampled to assure use of appropriate sampling plans.
9.7.5 One-Dimensional Sampling Model

Gy refers to a series of ordered units in either time or space as one-
dimensional sampling materials. If the decision unit were carrots that
are ready for harvest, primary samples could be taken at uniform
intervals during the entire harvesting process, thus assuring that all
carrots are represented in the sample. How could we do this? Gy tells
us that the best way to accomplish this would be to select increments
as they are moving, perhaps on a conveyer belt (ordered units in
space) or perhaps in bushel baskets as they move from the field to
the truck (ordered units in time). When materials are moving in a
single or thin layer in a continuous stream, the elements (fragments,
items) of the lot are all available and increments can be collected
at regular time intervals throughout the entire harvest. The field
of carrots, which is transformed to a long stream of carrots, is a
one-dimensional material. It may be static (long, thin layered row of
harvested carrots) or moving as described above. One-dimensional

materials are very long in one axis compared to a relatively uniform
(±20%) cross-section of material. The sampling of the material is
controlled primarily by taking thin increments of the total width
and depth (one dimension) along the length, which should be quite
large compared to the other two.
9.7.6 Two-Dimensional Sampling Model

Large bags (feed, flour, sugar) or drums (honey, syrup) and other
semi-large containers are two-dimensional materials. Any lot that
may be sampled using a probe that will reach the bottom of the
container is being sampled using two-dimensional sampling. There
are very specific methods for using these tools. The intention is to fill
the probe with an elongated cross-section of the lot. Two dimensional
sampling is discussed in much more detail in Section 9.9 along with
the proper use of sampling tools.
9.7.7 Three-Dimensional Sampling Model

Decision units made up of very large units of infinite element materials
(e.g. piles of protein powder, cargo containers of flour, silos of grain)
are three-dimensional sampling materials. They must be transformed
in some way that all the material will be equally available for selection.
A traditional and inexpensive but often biased method is to divide up
the material into equal fractions, small enough to sample as two dimen-
sions. For example, large machinery can be used to move the material
into smaller piles and then random piles are selected for sampling.
The preferred method, but not as readily available, is to move the
material along a conveyor belt and extract increments intermittently.
In the laboratory, smaller bags of grains or feeds may be split with
equipment known as riffle splitters, which will be discussed later.
9.7.8 Packaged Foods in Commerce: Finite or

Infinite?
Food safety professionals are often faced with commodities that are
being moved in commerce. This is often a special case of finite element
material (product packaged in bags, boxes, cans or jars) presented in

a three-dimensional lot (e.g. truck load, warehouse of pallets, ship-
ping container) that must be split into fractions and then increments
selected from each fraction. The sampling plan depends on the dimen-
sion at which the material is sampled, the definition of the decision
unit and the heterogeneity of the material. The heterogeneity of the
material is usually unknown unless sampling validations have been
conducted. For this reason, replicate sampling is encouraged in order
to have some measure of the variability in the sampling. It is already
known that the heterogeneity can vary with different commodity
and analyte combinations, which makes this challenge even more
complicated. Sample collections at the farm and manufacturing level,
where product is accessible and may be collected while in motion with
periodic selection of increments is preferred. Collection at points of
loading or off-loading is also a better option than at the point where
packages are in a static position or only a few items are available
at a terminal retail outlet. Minimizing the sources of error and TOS
should always be a consideration when developing these types of
sampling plans.
9.8 Pierre Gy’s Theory of Sampling

Pierre Gy began publishing his work on sampling of particulate
materials in 1954, introducing TOS, while working in the mining
industry. Three important publications, including an English trans-
lation of Sampling for Analytical Purposes (Gy, 1998), were pub-
lished from 1982 to 1998. Since that time, Gy’s work has evolved
and been adopted by statisticians and scientists in many different
fields including food and feed due to its universal laws, concepts and
equations. Pitard (1993) published an important reference and guide
to sampling with a second edition in 1993; Pierre Gy’s Sampling
Theory and Sampling Practice, which further describes the theory
and practice of Gy’s principles as universal to all sampling projects.
Smith (2001) published a primer to TOS for those needing a general
introduction to the terminology and theory. In the 1970s, while Gy
was working on sampling theory in the mining industry. Codex and
pesticide manufacturers such as Ciba Geigy and Shell were studying

the distribution of pesticide residues on food crops. Work on pesticide
residue sampling was conducted in parallel with little recognition of
Gy’s TOS, despite the complementary nature of the studies. How-
ever, in the 1990s, due in large part to the English translations of
Gy’s work and the work of Pitard, Esbensen et al. (2009, 2010, 2012,
2014) and many others realized the universal applicability of Gy’s
TOS and began to integrate these concepts into sampling for food
and feed and soil matrices, all of which are of particular importance
to pesticide residue analysis.
TOS is both elegant and simplistic in its ability to identify core
properties of materials that lead to sampling errors; however, it is
frequently not intuitive. TOS concepts, on first introduction, require
some study and reflection, but it is well worth the effort. This chapter
will only introduce the concepts of TOS. For those writing sampling
plans, preparing samples in the laboratory or interpreting the data
to make decisions, it is important to become more familiar with the
theory and practice of TOS.
Pitard (1993) integrated sampling theory with concepts of sta-
tistical process control and total quality management. However, he
cautions that one cannot have reliable, unbiased and accurate test
measurements without identifying and minimizing the seven major
sources of sampling variability. Recognizing the heterogeneity in the
material to be sampled and devising a plan to minimize sampling
errors that may result from the selection of a small portion of a
heterogeneous material is the foundation of TOS.
9.8.1 Heterogeneity: Degree to Which a Material

is Not Homogeneous
Heterogeneity is the degree to which the elements (constituents, frag-
ments or particles) of a material are different from each other.
Heterogeneity: A lot is heterogeneous relative to a given character-

istic if the characteristic is not uniformly distributed throughout
the lot (Codex, 2004). The condition of a lot under which all the
elements are not strictly identical (Pitard, 1993).
Homogeneous: A lot is homogenous relative to a given character-
istic if the characteristic is uniformly distributed according to a
given probability law throughout the lot (Codex, 2004). The con-
dition of a lot under which all the elements are strictly identical
(Pitard, 1993).
Nearly all materials are heterogeneous. If a material were entirely

homogeneous, every particle or molecule of that material would have
exactly the same physical and chemical composition. Every element
would have the same composition as the average of the entire lot
and thus there would be no sampling challenges. But the real world
is much more interesting and thus we have to approach sampling
with more information about the heterogeneity of the materials. Even
materials that we describe as homogeneous are not so if examined in
more detail. Crystalline sugar and fine milled wheat flour may seem
very homogenous until examined under the microscope or filtered
through a fine sieve. In terms of TOS, only highly pure liquids and
gases approach homogeneity. As all materials, even pure liquids, are
impure to some small degree, it is safe to say that all foods, feeds
and environmental samples are definitely heterogeneous, to a great
degree. It is for this reason that scientists need to be careful when
using the word ‘homogeneous’ because it has a special meaning in
both sampling at the macro level and in the mass reduction sampling,
which occurs in the laboratory.
In TOS, Gy (1998) describes two primary types of heterogeneity:
Compositional (or constitutional) and distributional (spatial or tem-
poral). Each material has differing amounts of compositional and
distributional heterogeneity that give rise to variability in the incre-
ments collected from the same material. This variability results in
sampling error, which is usually expressed as relative standard devi-
ation or the coefficient of variation (CV) of replicate samples taken
using the same sampling process. However, errors introduced due
to bias or incorrect sampling may not be directly measurable. Both
types of errors should be recognized and minimized in order collect

a truly representative sample.
Compositional (constitutional) heterogeneity (CH): The hetero-

geneity that is inherent to the composition of each fragment or
particle making up the lot (Pitard, 1993).
Distributional heterogeneity (DH): The heterogeneity that is inher-
ent to the manner in which separate and distinct particles or units
are scattered or spread out within the lot (Pitard, 1993).
Figure 9.4. Heterogeneity of sugar mixed with cinnamon. A close-up of sugar in

the background shows crystalline differences. When adding cinnamon to sugar,
it would seem easy to mix until there is a very homogeneous mixture but the
powdered cinnamon segregates together and away from the crystal sugar to some
extent, even after extensive mixing. If we were to look even closer, we would find
that the cinnamon is still small brown particles among the differently shaped
sugar crystals. Even small clumps of sugar tend to rise to the top with mixing.
These are two different types of distributional heterogeneity. This tendency of
different powders to separate must be carefully controlled in manufactured dry
ingredients such as powdered drink mixes.
9.8.2 Compositional Heterogeneity: Variability

in Composition
CH, as its name suggests, is the variability in the concentration of the
analyte of interest from element to element in a material. An example
of CH is the difference in concentration of a pesticide from kernel
to kernel in a bushel of wheat. This intrinsic CH is a fundamental
property of the material making up the lot. It cannot be reduced to
zero. It was this characteristic that led Gy to describe the sampling
error that arises from compositional heterogeneity as fundamental
sampling error (FSE) because it can be reduced but can never be
totally eliminated.
9.8.3 Control Fundamental Sampling Error

by Collecting Sufficient Mass and Reduce
Particle Size
When all other errors have been eliminated, FSE still exists because it
is an intrinsic characteristic of the elements making up the material.
Minkkinen (2004) says it is the minimum error of an ideal sampling
procedure. Gy tells us that the relative variance or CV2 of the fun-
damental sampling error may be expressed as shown in the following
equation:

1 1
CVFSE = Cd
2 3
− (9.1)
Ms ML
CVFSE = coefficient of variation or relative standard deviation of the
fundamental error
C = sampling constant (specific to analyte and commodity)
d = diameter of largest elements (cm) (95% limit of the size distri-
bution)
Ms = mass of multiple increment composite sample collected (g)
ML = mass of the lot from which the composite sample is col-
lected (g).
If the mass of the lot is very large compared to the mass of the
composite sample, ML may be dropped from the equation, which
leads to the simplest form of Gy’s equation for fundamental variance:
Cd3
CV2FSE = (9.2)
Ms
(a) (b)
(c) (d)
Figure 9.5. Whole wheat before and after it is ground to flour. Reducing particle
size does not necessarily reduce heterogeneity. Whole wheat grains are actually
very uniform in size and shape but when ground to flour, the outside layer, fibre-
containing bran separates from the inside protein and carbohydrate layer. Bran
peeling for a grain can be seen in (a). When grinding to make flour, the particle
size is considerably reduced but the bran and the endosperm separate, forming
two distinct types of particles of different size, shape and composition, which
leads to considerable distributional heterogeneity and an increase in compositional
heterogeneity. As the wheat is ground to finer particle size in (b) and (c), the
darker brown specs of bran become more apparent. What is more difficult to
show in pictures is the tendency of the bran specs to separate from the white flour
and the way the flour clumps together forming little mounds instead a uniform,
smooth flowing material as in (d).
In relative terms, if the particle size of the material is reduced or the

mass of the composite sample is increased, then the error due to CH
can be reduced.
When primary samples are taken from a lot, there is usually no
possibility of reducing the particle size so sufficient mass must be
collected. However, if the particle size of the laboratory sample can

be reduced in a representative manner, with all the elements of the
laboratory sample represented in the same proportions as they were
present in the lot, then a smaller mass test sample can represent the
sampled lot. This is the principle used when laboratories crush, grind
or comminute to prepare analytical test samples and extract a very
small analytical test analytical portion.
9.8.4 Sampling Constant Varies Greatly in

Different Materials
The constant C describes the characteristics of the material in the
lot and consists of four factors: the shape of the particles (f ), the
uniformity of the particle sizes (g), the degree to which the analyte
of interest is associated with the particles (β) and the concentration
of the analytes of interest and the density of the critical particles (c),
as shown in the following equation:
C = f gβc (9.3)
f : the shape parameter varies from 0 ≤ f ≤ 1 with spherical
materials approximately 0.5 and flakes and needles nearer to 0.1. It
is the ratio of the volume of the sampled particles of a certain char-
acteristic dimension to the volume of a cube of the same dimension.
g: the size range parameter varies from 0 ≤ f ≤ 1, where uniform
particle size equals 1 and wide size distributions may be 0.25. Cereals
are approximately 0.75.
β: the liberation parameter ranges from 0 ≤ f ≤ 1. If the critical
components are completely separated from the other particles β = 1,
but if the analytes of interest are totally associated β = 0.
x
L
β= (9.4)
d
The liberation factor, β, is an empirical correction factor and may
be estimated for particulate materials if the critical component may
be liberated with particle size reduction as shown in Eq. (9.4) where
x = 0.5; d is the diameter of the critical particles and L = 95% distri-
bution size of the particles when at least 85% of the critical particles
are liberated. (Note that the constant C changes if the material is

ground or crushed (Minkkinen, 2004).) Far too many materials do
not fit these ideal conditions and it has been suggested that x may
vary from 0.5 to 1.5 (Eurachem/Citac, 2007).
c = the constitutional parameter (c = g/m3 ) is related to ana-
lyte concentration and densities of all the components. Despite its
units, c is not directly related to density. If enough information is
available, such as in some reference materials, c may be estimated
using Eq. (9.5) where αL = the average concentration of the criti-
cal component in the lot, α = the concentration of the analyte in
the critical particles, ρc = the density of the critical particles and
ρm = the density of the matrix or diluent particles.
2
1 − ααL αL
c= αL ρc + 1 − ρm (9.5)
αα α
The constitutional parameter, c, varies greatly in different materials
but increases as the analyte concentration decreases, so sampling
constants for residue analyses are higher than those for higher con-
centration component analyses. However, there is no generalized
relationship that can be used to estimate the effect of analyte
concentration on the value of the sampling constant for different
commodities and analytes.
It is useful to know what factors affect the sampling constant
but much more difficult to accurately determine it. If we have a uni-
form material of a well-characterized particle size and density and an
analyte that can be measured with sufficient accuracy and precision,
it might be possible to estimate c by performing multiple analyses
of the material with different particle sizes and use Eq. (9.1) to esti-
mate c. This involves extraction of 50–100 elements or small compos-
ites (≥10 increments each) for one- and two-dimensional materials,
respectively, and individually determining their physical attributes
and concentration of critical analytes. For well-characterized materi-
als, such as standard reference materials, especially those with well-
characterized particle size, shape and analyte concentration, c can be
experimentally determined. If the individual parameters cannot be
determined, c can be empirically estimated if DH is minimized and

there are no other significant sources of error.
Note : It is important not to extrapolate the value of Gy’s sampling
constant from one material to another without sufficient verification.
It is clear from the parameters of the constant, c, that as variabil-
ity in particle shape and size increases and the analyte is more tightly
bound to the particles, the sampling constant and FSE increases.
While it may be outside of the scope of many sampling situations to
calculate c, it is clear that collecting larger sample sizes or decreas-
ing the fragment size by chopping, milling, blending or other forms of
comminution may reduce the variability due to CH. Unfortunately,
if not carefully controlled, it is possible is introduce error during
comminution processing. While still applicable in a relative sense,
laboratory processed sample slurries of fresh fruits and vegetables
are more difficult to characterize than ground dry materials (Ambrus
et al., 2015).
Caution : Not all samples will obey these simple relationships. FSE
is only one source of sampling error and may be one of the smaller
contributions to overall sampling error in complex food matrices.
In order to accurately estimate FSE for any material, all other
sources of error must be controlled. The following references will
provide the user with more details in the proper use of these equa-
tions and the concerns of some researchers (Gy, 1998; Pitard, 1993;
Geelhoed, 2011).
9.8.5 Are We Collecting Sufficient Sample Mass?

How can we determine if sufficient mass is being collected during
primary sampling? Pitard (1993) and Maestroni et al. (2000) have
described a method for estimating sufficient mass as seen in Eq. (9.6).
Assuming we are unable to determine c, we can estimate if the mass
collected is sufficient by analysing 30–50 composite samples of a given
mass (Ms1 ) and determine the average concentration and variance
of the critical analyte and then repeat the analysis with composite
samples of at least 10 times the mass (Ms2 ). It is important that
the entire composite sample be analysed. If sufficient mass has been

collected, the following ratio of variance to concentration times mass
will fall between 0.5 and 2. Perfect homogeneity would yield a ratio
of 1. This relationship could also be used to demonstrate sufficient
test portion mass.
 S2 
a2
1
Ms1
0.5 ≤  Ss12 ≤2 (9.6)
a2
2
Ms2
s2
s2 = variance expressed as standard deviation;

a = concentration of critical analyte;
M = total mass of sample analysed;
Ms2 ≥ 10 × Ms1 .
9.8.6 Distributional Heterogeneity is the Spatial

and Temporal Relationship between Elements
DH is the variability in the location of the elements (fragments, par-
ticles) of a material in relationship to each other. This can happen
in space, time or both. An example of DH is the tendency of smaller
or denser particles to fall to the bottom or oils and gases to rise
to the top of a material, which leads to segregation error. Another
example of DH is the sticking or grouping together of elements in the
material, which leads to grouping error. DH nearly always exists to
some degree, and is altered with physical manipulation of the mate-
rial. Mixing may reduce or increase DH. DH leads to a sampling
error referred to as grouping and segregation error (GSE), which is
a measure of the extent to which the elements of a primary sample
vary in location and with time.
Gy tells us that DH is proportional to CH and if all the elements
are identical, then CH = DH. As DH increases, so does CH because
the variability in concentration of the analyte of interest from element
to groups of elements will increase and therefore DH is always less
than CH (Gy, 2004b):
1+YZ
DHL = CHL (9.7)
1+Y
Y = grouping parameter where Y ≥ 0 that characterizes the size

of groups, which, when sampling lot L, will become the increments.
The smaller the increment size, the smaller Y and also DHL .
Y = 0 when the increments are made up of a single element and
DHL = CHL .
Z = segregation parameter. It is a dimensionless segregation param-
eter characterizing the type of distribution of the constituents within
the lot 1 ≥ Z ≥ 0.
Z = 1 when completely segregated.
Z = 0 when elements are distributed completely randomly (i.e. no
relationship between location and concentration).
9.8.7 Control GSE by Collecting Sufficient Mass

and More Increments
There are four primary ways to reduce GSE — more mass, more
increments, mixing and reduced particle size. The first two are of
most importance for primary samples because most lots are too large
to mix or reduce particle size. But as soon as the laboratory sample
is manipulated by crushing, grinding or mixing, FSE and GSE are
changed, and it is possible to increase GSE or even worse, intro-
duce bias. For example, if a box of granola cereal is mixed, the small
crumbs or dense nuts may segregate to the bottom as seen in Fig. 9.6.
If a grain is milled, the kernel and the husk may separate, creating
two very different elements as seen in Fig. 9.5, with distinctly differ-
ent compositions (increased FSE), which may segregate according to
their density (increased GSE). If cinnamon is added to a jar of sugar,
increased mixing will reduce the tendency of the spice to form pock-
ets of brown powder as seen in Fig. 9.4. Distributional heterogeneity
may approach zero in the case of zero-dimensional finite elements
that are totally independent of each other but DH exists and is often
underestimated in sampling on infinite element materials and can
lead to significant undetected bias.
As explained above, DH is related to CH. Thus, if you collect
a larger mass of material for each increment, you reduce both FSE
and GSE because you collect more of the different sized groups and
Figure 9.6. (DH) in granola. DH might be referred to as the ‘granola effect’.

In granola, the crunchy sweet chunks of oats and honey form large groups and
the crumbs fall easily to the bottom of any container along with smaller seeds
while nuts and raisins seem to hold their own throughout the mix. In the figure,
a plastic bag of granola was mixed and set down for the picture, gently resting
against a glass jar. When the bag was turned over, the crumbs were easily seen at
the bottom of the bag. When mixed and repeated again several times the smaller
elements of the granola would always find their way to the bottom of the bag. It
only took a few moments. The action of mixing frequently increases GSE rather
than reducing DH.
fragments of the lot. If you collect more individual increments of mass

large enough to select the largest groups, you have a better chance of
collecting all the different types of groups and minimize GSE. If you
collect more increments, you reduce GSE because you also collect
more of the different groups. This is assuming, of course, that each
of the fragments of the sample have equal probability of selection
and are not excluded by the sampling technique. If you were able to
move, separate or mix the sample without increasing GSE, you could
reduce the segregation error.
While the FSE and GSE, introduced during collection and trans-
portation of the composite primary sample, cannot be changed, mass
reduction of the entire laboratory sample to a test sample by mixing
and comminution (e.g. chopping, grinding, blending) may reduce the
laboratory processing FSE and GSE introduced when selecting the

analytical test portion. Because the entire laboratory sample is usu-
ally available and small enough to be thoroughly mixed, the particle
size can be reduced and representative increments collected before
segregation occurs. If you reduce the particle size, you reduce the
size of the groups and thus their difference from each other and con-
sequently reduce both FSE and GSE when selecting the analytical
test portion.
9.8.8 Equal Probability of Selection

Gy (2004b) tells us that correct sampling errors occur when ‘selec-
tion with uniform probability’ and ‘errors result from the two forms
of heterogeneity of the material (CH and DH)’. (see previous discus-
sion and definitions in Section 9.5) Thus we need to be aware of the
heterogeneity in the lot and collect, with equal probability of selec-
tion, the appropriate mass and number of increments to minimize
FSE and GSE.
Conversely, increment selection is non-probabilistic when certain
constituent elements of the lot to be evaluated have a zero probabil-
ity of being taken into the sample (Gy, 1998). This concept requires
some discussion because it is either all too often ignored or incorrectly
assumed. Much of the work of Gy is dedicated to the identification
of errors generated when elements of the lot are not given an equal
probability of selection. These types of errors may be difficult to
detect unless the sampler closely examines the material in the lot and
understands the proper selection and use of tools for selecting incre-
ments. All protocols should be specific about methods used to assure
equal probability of selection and samplers should be well trained
in their implementation. Quality control measures such as, periodic
evaluation of multiple composite samples collected from the same
lot and regular oversight of sampling operations should be in place.
Sampling records should include a description of how the primary
sample was collected including the method of increment selection.
Photographs of the lot and the increment selection process can be
very valuable documentation.
9.9 Sampling Techniques — Striving

to be Representative
9.9.1 Randomness
As mentioned previously, equal probability of selection and sampling
correctness are not possible if all portions of the lot are not avail-
able for selection. There are many different methods of assuring ran-
domness and some are even systematic randomness. Some inspectors
carry a portable random number generator device with them. Smart
phone random number generator applications are available for free.
The distribution of the analytes of interest in the materials of inter-
est or heterogeneity has an important bearing on the choice of the
sampling technique. Simple randomness and the collection of more
replicates will not assure a perfect sampling plan if the different ele-
ments don’t have an equal probability of being sampled or if the
sampling method excludes or overlooks a significant portion of the
material. It is important to inform samplers that simply walking
up to a field or grove or warehouse and collecting whatever seems
appropriate is not really random and can introduce bias, even if the
samplers are doing their best to be objective in their selections. The
brightest and largest and most available green pepper or strawberry
will always try to jump right into the sampling bag.
Assuming sample correctness, quality control, such as replicate
sampling, may be conducted to estimate the uncertainty due to sam-
pling. Data users should be aware of the sampling uncertainty and
determine if the data is precise enough for their purposes. If not, TOS
provides several strategies for reducing the sampling error. Uncer-
tainty in sampling for pesticide residues will be further discussed in
Chapter 10.
9.9.2 Zero-Dimensional Sampling

For sampling zero-dimensional materials, simple random sampling
can be employed. For example, divide the lot into sections and then
let a random number generator choose which sections will be sam-
pled. If sampling boxes, bins or drums in a warehouse, the pallets and
packages can be assigned numbers and those to be sampled selected
randomly from several different locations. Of course, if there are mul-

tiple tomatoes in the box, selection of tomatoes from different loca-
tions within the box is advised. In an agricultural field, an imaginary
grid can be drawn of the field and the choice of sampling locations
chosen randomly. In groves of trees, divide each tree into quarters
by drawing an imaginary grid from north to south and east to west
and then subdividing the quarters into top, middle and bottom and
perhaps even inside and outside of the limbs. Each randomly selected
tree might be sampled in a different randomly selected position. The
mass and number of increments collected for a primary composite
sample is determined by the heterogeneity of the lot. Considerable
data is available for the uncertainty when using Codex food sampling
guidelines (Codex, 1999).
9.9.3 Systematic Random Sampling

There are sampling plans for agricultural fields that divide the
acreage into 100 units and choose an increment every 10 units from
a randomly chosen starting point. In a systematic random method,
an ‘M’ or ‘Z’ pattern is drawn across the field and then increments
are taken every few units from a randomly chosen starting point,
thus assuring that any patterns in spraying are represented (Ramsey
and Argyraki, 1997). Sometimes sampling plans suggest that the
edges are eliminated due to propensity for overspray. Ramsey and
colleagues studied the uncertainty in different methods of field sam-
pling, between sampler variations, designed a reference sampling site
that could be used to validate sampling methods and made a pro-
posal for sampling proficiency testing. They found that a minimum
of eight duplicate samples were needed to measure sampling uncer-
tainty (Ramsey et al. 1997, 1999, 2001, 2010, 2011; Squire et al.,
2000; Lee and Ramsey, 2001; Boon et al., 2007; Lyn et al., 2007a,
2007b).
In routine sampling plans, periodic replicates could provide the
data needed to assess uncertainty. However, as we have stressed
before, an acceptable repeatability does not assure lack of bias. Only
a good understanding of the factors affecting heterogeneity of the
analyte in the material of interest can enable the design of accurate

sampling plans.
9.9.4 One-Dimensional Systematic Sampling

For one-dimensional materials, increments are sampled at regular
time intervals or regular distances where product is spread out in
a long narrow row and increments are selected every few feet. The
increments are either combined into composite samples or tested indi-
vidually and graphed on a variogram. Tools used in one-dimensional
sampling can be very sophisticated and are often automated and
combined with on-line sensors. The tools used must, however, com-
ply with TOS principles by slicing through the entire width and
depth of the material and provide an equal probability of selection
for all the elements. Automated sampling may introduce some of the
same errors as two-dimensional tools. Manufacturing facilities can
use systematic incremental sampling and graph the results using var-
iograms. The regular and systematic testing of incoming ingredients
or manufactured products while in motion is one of the most repre-
sentative methods of sampling. Peterson and Esbensen (2005) showed
that systematic sampling has much lower variance than purely ran-
dom sampling. A study of these results can be used to detect vari-
ations in the manufacturing process or seasonal changes as well as
identify the point at which a product does not meet specifications
and the volume that needs to be rejected. The study of variograms
is outside the scope of this chapter but very useful information can
be found in the works of Gy and others (Gy, 1998, 2004b, 2004d;
Pitard, 1993; Peterson and Esbensen, 2005; Esbensen et al., 2009,
2010, 2012).
9.9.5 Nugget Effect

Most samplers will be familiar with the difficulty in sampling for
aflatoxins because they are only present in small portions of the
grain or peanuts. The presence of a nugget of material, which has a
high concentration of the analyte of interest in an otherwise analyte-
free material, continues to be a challenge for all sampling methods.
TOS would advise us to transform the product to a one-dimensional

material and conduct systematic periodic testing but if the incre-
ments are not large enough or frequent enough, the infrequent nugget
might not be detected. Systems of on-line, continuous monitoring are
one of the only effective methods for detecting this type of extreme
compositional heterogeneity.
9.9.6 Two-Dimensional Sampling with Tools

Bags of grains and barrels of liquids such as honey are sampled using
probes or ‘thieves’. One probe design contains an outer cylinder with
holes and an inner, movable solid cylinder. The probe is pushed to the
bottom of the bag or drum and then the inner cylinder is removed to
allow the particles to fill the sampling probe, which is then removed.
Several increments are extracted with the probe and combined to
form a composite sample. If the tool does not provide equal prob-
ability of selection, this leads to an increment delimitation error. If
the tool does not collect or retain all of the material, this leads to
an increment extraction error. If the sampling tool cuts, crushes or
contaminates the material, this leads to increment preparation error,
which is incorrect sampling (Gy, 2004b).
Many samplers make the mistake of thinking that liquids are
uniform and do not recognize the need to sample to the bottom of
the tank or to assure that floating material is also collected pro-
portionately. As described for orange juice in Fig. 9.7, a liquid with
low-density layer (e.g. oil on water, cream on milk), suspended solids,
gradient layers of immiscible liquids and insoluble material may be
exceptionally difficult to sample properly. When sampling liquids, it
is difficult to get a cross-section of all the material as any attempt to
scoop will push dispersed solids aside. When the material includes
volatile chemicals, very special techniques are needed to assure that
important components of the sample do not escape to the air. It may
be necessary to collect both gas and liquid samples.
Example 1. If you are sampling honey from drums that have arrived
in a container ship, you must be able to choose from any of the 75,
200-litre drums of honey, those from the front, middle and back of
Figure 9.7. Distributional heterogeneity in orange juice. The figure shows the
multiple components of orange juice. It may seem easy to sample until the signif-
icant GSE is identified. Orange juice is a dynamic, multiple component mixture,
which changes with time and location, and contains pulp, which tends to fall to
the bottom; peel oil, which tends to rise to the surface; foam, which sits on the
top and gradually dissolves, important flavor volatiles that evaporate and juice,
which is a slurry of suspended particles.
the cargo container. Once you have access to the drums of honey,
you will need to use some form of selection to choose which drums
to sample. You could number each one of the drums in a regular
pattern and then use a random number generator to choose which
drums to sample. As you know, the number of drums sampled should
be related to the heterogeneity of honey from barrel to barrel and
not because there is a standard protocol for sampling honey and it
indicates you should choose three drums. Suppose you want to know
if three drums provide an adequate and representative sample. You
could choose three drums randomly and take one or more increments
from each drum and combine them into a multiple increment com-
posite sample. You could then select three drums again, randomly
and repeat the process. Three replicate composite samples are even
Figure 9.8. Sampling with a tool. This figure is a simplified diagram demonstrat-
ing some of the sources of error in two-dimensional sampling. A case, bag, drum,
bin or small truckload might be sampled by inserting a cylinder-like tool. The
smaller particles (yellow) that have segregated at the bottom may not be sampled
proportionately despite the best designed tool. Most probes are designed for fairly
small particle sizes (orange) such as grains, liquids or dry feeds. If the material
has larger particles (green), they may be excluded from the tool often enough
to be inadequately represented in the increments and ultimately the composite
sample. This may introduce another sampling bias. A rule of thumb is that the
opening of the sampling tool should be about three times the length of the largest
particles.
better. The difference in the concentration of analyte in the replicate

composite samples will give you an indication of the error in the
sampling procedure (assuming the laboratory and analytical errors
are relatively low). You also need to be able to extract honey from all
positions in that drum. This is a two-dimensional sampling material
and a probe-type sampler might be used. A tool blank might be part
of the quality control to assure it doesn’t contribute any contamina-
tion. The drum may have a 30 cm thick solid layer of crystalline honey
at the top of the drum with a liquid layer beneath and solid impurities
coating the bottom of the drum. The tool must be able to extract all
elements of the honey with equal probability. The challenge of equal
probability of selection is far from simple. However, at the processing

plant, the drums of honey are heated, emptied, stirred and sometimes
filtered before being packaged into smaller units. This might be the
ideal time to select several appropriate size increments.
9.9.7 Center of Gravity Rule

It may be convenient to visualize the proper extraction of an incre-
ment with a tool by remembering that all the particles of the material
that have their centre of gravity located within the volume of the
increment extractor tool should be included in the increment. Those
elements whose centre of gravity lies outside of the increment volume
should not be extracted. If the elements’ centre of gravity are located
within the volume of the extractor, they should remain as part of
the increment and be extracted and added to the multiple incre-
ment composite sample. This is true for both one-dimensional sam-
pling where a cross-section of moving particles might be extracted
or when using a sample thief, trier or auger to extract product
from a bag or drum. However, Pitard (1993) tells us that ‘in many
instances it is just impossible to perform a correct delimitation’ of
two-dimensional lots.
9.9.8 Three-Dimensional Sampling and

Mass Reduction
When faced with a three-dimensional sampling material, the only
accurate way to sample is to move the material. For very large bulk
materials, it may be necessary to reduce the mass in several steps.
Fractional shovelling, rotary splitting and stationary splitting are
commonly used. Peterson et al. (2004) conducted a comprehensive
comparison of many different mass reduction techniques and the riffle
splitter was the only device found representative. Unfortunately, rif-
fle splitting is not applicable to non-flowing materials such as ground
meats and thick liquids. See Pitard (1993) for a more detailed descrip-
tion of the principles, designs and use of all the commonly used mass
reduction procedures.
Figure 9.9. Demonstration of the center-of-gravity principle. This figure uses

the soup mix from Fig. 9.3 to demonstrate the principle of centre of gravity. In
picture (a), the soup mix is falling into a jar whose opening is only about two
times the length of the kidney beans. Bean 1 is easily falling into the jar while
bean 2’s centre of gravity is outside the lip of the jar and will not be collected.
Bean 3 is probably not going to make it into the jar either. The large kidney
beans falling away can take the chick peas with it but the smaller split peas and
lentils can easily make their way into the sample. (b) is a glass with an opening
approximately three times the length of the kidney beans and the competition
for entry into the glass is much less than seen in (a).
It is easy to realize from Fig. 9.10 that there must be better,

more representative ways to mass reduce large decision units. There
are several types of splitters, which can be utilized in the laboratory
for dry powders, granular and other free-flowing materials. A rotary
splitter mechanically splits the laboratory sample into hundreds of
increments and is the recommended method. A stationary splitter
contains a hopper where the material is loaded and falls past grated
splitters into separate pans. It is less expensive but not quite as effec-
tive and prone to operator error. A technique known as coning and
quartering is not recommended. AAFCO’s ‘Guidelines for Preparing
Laboratory Samples’ and ISO Guide 6498 provide useful information
on the use of these mass reduction techniques (AAFCO, 2008; ISO,
2012).
Figure 9.10. Diagram of fractional shovelling. This figure graphically demon-

strates the fractional shovelling technique, which can be used with some success
if executed properly. In this example, a large mound of primary ingredient, a
protein powder, has been off-loaded from a container ship to a warehouse and it
needs to be tested for melamine. As we know, melamine can be very hazardous
in small quantities but this powder may have come from multiple sources and
so a few probes of the ‘mound’ of material is not sufficient to assure regulators
that it is contaminant free. Of course, it would be better if it was converted to
a one-dimensional sampling material but it was not possible in this case. Using
a shovel (usually mechanical) material of equal size is alternately added to each
of five or more smaller piles until the entire mound is separated. Then one of the
piles is chosen at random for testing. Two or more mass reductions will probably
take place before increments are extracted for a composite sample.
Rules for Tools

• Equal probability of selection from very large to fine particulates;
• Equivalent size increments produced;
• Do not contaminate the sample;
• Do not adsorb or change the analyte of interest;
• Easy to clean between uses or disposable;
• Maintained in proper working order;
• Trained operator who understands operation and limitations;
• Simple and reliable tools lead to less operator error.
9.10 Correct vs. Incorrect Sampling

Scientists often state that ‘the degree of heterogeneity is the deter-
mining factor of sampling error’ (Horwitz, 1990). Heterogeneity def-
initely affects sampling error from FSE and GSE and these may
be unavoidable but sampling incorrectness can contribute greatly to
error, especially to sampling bias. There is variability introduced by
any sampling technique. Sources of error are categorized in Table 9.2.
When sampled with an equal probability of selection, the variability
results from correct sampling error. When samples are taken in a
non-probabilistic manner, the rule of equal probability and sample
integrity are not upheld and incorrect sampling errors occur. These
can be generated by incorrect sampling designs, equipment or meth-
ods. Gy, Pitard and others describe errors that arise when the sample
was taken incorrectly and avoidable errors were introduced. These
errors have been described in the previous sections. All the errors
that can occur with the collection of the primary sample can also
occur at the laboratory level. The same principles of TOS apply
but at a smaller material scale. Codex makes a distinction between
sample preparation and sample processing.
Sample preparation: Includes actions taken to prepare the analyti-

cal sample from the laboratory (bulk) sample, such as reducing the
size of a large bulk sample by sub-sampling or removing foreign
materials and parts of the sample material that are not analysed
and may include washing, peeling, cooking, etc. so that foods are
prepared as for normal consumption (FAO/WHO, 2009).
Sample processing: Includes physical operations performed to pre-

pare a well-mixed or homogeneous matrix to form the analytical
sample, from which the test portions for the analysis are taken
(FAO/WHO, 2009).
9.10.1 Why, Where, What and When?

GOODSamples (2015) cautions that the decision unit to be sampled
must be clearly defined and the sampler must understand the purpose
Table 9.2. Sources of sampling uncertainty and errors.
Correct Sampling Errors Properties of the Material being Sampled

Fundamental sampling error Errors due to constitutional heterogeneity
(FSE)
Grouping and segregation Errors due to distributional heterogeneity
error (GSE)
Point selection errors One dimensional sampling
Long-range point selection Variations, trends over time or across space
error
Periodic point selection error Variations in periodic levels over time or across space
Correct or Incorrect
Sampling Errors May or May not be Avoidable
Materialization errors One- and two-dimensional sampling
Increment delimitation error The shape of the increment prevents equal
probability that all the elements may be selected
Increment extraction error Not all of the elements with their centre of gravity
within the increment are extracted and included
Preparation and
processing errors One-, two- or three-dimensional sampling
Sample preparation error Sample heterogeneity or integrity errors introduced
as part of mass reduction and compositing, as well
as due to cleaning, peeling, husking, shelling, etc.
performed to obtain the portion of the commodity
which is to be analysed.
Sample processing error Sample heterogeneity or integrity errors introduced
due to mass reduction of the laboratory sample to
prepare a more homogeneous analytical sample
such as comminution
Incorrect Sampling Errors Avoidable Errors
Increment preparation error Sample integrity deviations such as contamination,
physical and chemical changes, shipping, storing,
exposure to temperature or light human error, etc.
Increment weighting error Incorrect assignment of weights when combining
increments of different mass
Gross errors Deliberate or accidental improper sampling; Sample
does not meet the intended purpose
Note: Gy (1996, 2004b) introduced the concept of correct and incorrect sampling error,
including both the variability that is unavoidable (uncertainty) and the variability that
arises from improper sampling practices as errors. Other authors have expanded upon
and further defined the concepts of correct and incorrect error and how it should be
properly integrated into the estimate of total measurement uncertainty (Pitard, 1993;
Eurachem/CITAC, 2007; Esbensen and Wagner, 2014; Wagner and Esbensen, 2015). The
table attempts to summarize the primary sources of error identified by these authors,
while also conveying the understanding that, no matter how well designed a sampling
procedure (e.g. increment extraction, sample processing) any mass reduction step may
introduce variability into the combined measurement uncertainty.
for sampling and have spent sufficient time to identify the decision
unit correctly. Collecting the wrong sample for the intended purpose
would be an example of a gross error. If regulatory surveillance is
intended to identify violations in imported products, each lot should
be sampled separately. As product lot numbers are often small and
difficult to see, this may take some time. The goal is to determine the
average residue concentration in a specific lot. In operations where
the product is moving, the designation of the beginning and end of
the lot must be specified and attainable. (For example: If multiple
trucks from different farms are being off-loaded, it would be desir-
able to know the beginning and end of the load from a particular
truck, the owner of the commodity and the origin (field, production
facility etc.)). If the intent is to know the average concentration of
pesticide in a field of strawberries, it is not representative to col-
lect three punnets from the roadside stand. If the goal is to know
the residue exposure from a single serving, many smaller increment
samples might be collected and the distribution of residues within
the lot determined. If the intent is to know the average exposure to a
pesticide throughout the country, the scale of the experiment is much
larger and samples would need to be collected from different locations
across the country. Partnered with a representative primary sample
are records containing a clear definition of the decision unit and an
accurate description of the primary sample and how it was collected.
Example 2. The U.S. Pesticide Data Program.
Why: The United States Pesticide Data Program (PDP) was designed
to provide the U.S. Environmental Protection Agency (EPA) with
residue data which EPA combines with consumption data to deter-
mine dietary exposure. For their dietary risk assessment models, EPA
needs to convert the data to single serving exposure. EPA evaluates
the risk of a particular pesticide for both individual and cumula-
tive exposures from multiple foods and water. The non-detects are
just as important to EPA as the detections. The lower the analytical
detection limit, the lower the possible estimated exposure value. EPA
also uses the top 90% detections in their models for all of the data
available. If there is no PDP data, they use the highest residue found
in the field residue trials, which is usually much higher than actual
dietary exposure levels.
Decision Unit: The challenge for PDP is to develop a sampling
plan, which will represent the single-serving pesticide residue expo-
sure to specific pesticide and commodity combinations in the popu-
lation across the entire U.S.
Where: Ten to 12 states were chosen to participate in PDP based
on their laboratory experience with pesticide residue analysis, state
agriculture production and location in the four different geographic
census regions of the country. The number of samples collected is
weighted based on state population data. Each state collects samples
monthly and is also assigned two to three commodities to analyse.
Samples are collected at distribution centres and terminal markets
that service grocery stores, restaurants, schools and hospitals. The
dates and sites where samples are collected are chosen randomly by
U.S. Department of Agriculture statisticians in quarterly sampling
plans. The larger distribution centres are sampled more often than
the smaller ones. The metrics used to develop the PDP sampling
plan are revisited every few years and adjusted as needed.
What: PDP analyses about 14 food commodities at any given
time with commodities rotating in and out of the programme
throughout the year. Each commodity is screened monthly for one
to two years. Important, highly consumed commodities are retested
(e.g. apples, bananas, tomatoes) every 5–10 years. The commodities
tested are recommended by EPA based on their particular risk assess-
ment needs. Fresh, processed and baby foods are analysed. Sample
weights are approximately 0.5–2.2 kg (or more for large commodities
such as watermelon) collected from a single case or location in an
effort to closer represent single serving exposure. It is very impor-
tant to EPA that they understand the weight collected in order to
determine the possible exposure to a single serving. (For example, if
five tomatoes were collected and the average residue was 0.1 µg/kg,
and only one of the tomatoes was positive, the single serving residue
concentration would be 0.5 µg/kg.) The samples are cleaned and
peeled in a manner to represent the manner in which the food is
usually eaten.
What: Pesticide residues analysed in the program are chosen

based on EPA needs for risk assessment, established U.S. and foreign
MRLs for the commodity and amenable to multiresidue analysis.
When data is needed for certain pesticides that must be analysed
by single analyte methods, special arrangements have been made to
conduct these analyses, usually in EPA laboratories. Each commod-
ity is analysed by multi-residue methods for 100 or more pesticides.
The numbers of pesticides screened in each commodity is increasing
every year.
When: Each month, each state collects their assigned number
of samples per commodity and ships them to the state laboratory
analysing that commodity. About 14 commodities and 60 samples per
commodity are collected each month. Each commodity is collected
for one to two years for a total of up to 1,400 samples per commodity.
Most commodities are tested for two years to account for seasonal
and year-to-year variations.
9.11 Combined Estimation of Errors

9.11.1 Sources of Sampling Error
Table 9.2 lists sources of the sampling error. Gy’s FSE and GSE have
already been discussed. In a systematic sampling of one-dimensional
materials, the material may vary with time or distance. If there
are variations over time or space such as trends, this is a long-
range point selection error. If there are variations in the particu-
lar time or distance chosen for the sample selection, this is periodic
point selection error. This may be a true and measurable manufac-
turing or material variability and not a limitation in the manner
in which the increments were selected. This is actually important
information.
Gy introduced three materialization errors: delimitation, extrac-
tion and preparation errors, which were discussed in Section 9.8.
These errors arise when a tool is used to extract an increment and
the elements of the material are not selected, retained or are changed
by the sample extraction. The process of taking a sample with a
piece of equipment, whether the slice of sample in a one-dimensional
process or the extraction of increments with a probe sampler in a

two-dimensional process, will lead to some errors, which might be cor-
rect and unavoidable or incorrect errors. If the sampling introduces
some alteration of the sample such as contamination or degradation,
this is an avoidable incorrect sampling error. There are also multiple
ways that the sample might be damaged during sampling or con-
taminated by the sampling process. Sampling blanks are important
quality control when these types of errors can occur.
Some complicated sampling plans may call for weighting some
increments (or units) as more important than others. These are
either mathematical adjustments made to the data based on assump-
tions from prior information or adjustments to the numbers of incre-
ments or composite samples to collect. Errors from weighting incor-
rectly lead to increment weighting error. For example, weighing may
be based on previous testing that indicates clay is more contam-
inated than sandy soil and so more clay increments are collected
and the concentration in sandy soil is estimated. In another type
of weighting, census data might be used to determine how many
samples are collected in each region of the country and assumptions
drawn about the pesticide exposure across the entire population.
See Example 9.2.
Some of the many possible sampling steps from field to analysis
are shown in Table 9.3. The error in the final analytical test result
includes the error from all the ‘sampling’, which occurs from the
initial selection of the increments from the lot to the completion
of the extraction and instrumental analysis. As the errors due to
sampling for pesticide residues can be large, estimated to range from
20 to over 100% (Ambrus and Soboleva, 2004; Farkas et al., 2015a),
they can contribute significantly to the total error, even given errors
as high as 25% for pesticide residue analytical test measurements.
The combined uncertainty of measured pesticide residues, expressed
as a relative standard deviation, (CVR ) is equal to the square root
of the sum of the squares of the primary sampling (CVS ), sample
processing (CVSP ) and analytical testing uncertainties (CVA ) as seen
in Eq. (9.8) (Farkas et al., 2015b). Methods for estimating this error
Table 9.3. Examples of sampling steps from field to testing.

Sampling Step Description Sampling Considerations
PRE–LABORATORY
Sampling plan or Procedure for obtaining Predefine DU* and sampling
protocol representative sample purpose, mass and number
of increments
Decision unit, lot, Material to be sampled and Record description,
batch analysed identification numbers
Primary sample Single increment, item or Record mass and method of
unit selection
Composite or Multiple increments Representative, randomly
aggregate combined selected
Replicate sample Another sample collected Increments selected
from the same DU using randomly from different
the same protocol locations
Prepared sample Sample after removal of Processed portion or
outer leaves, shells, husks, commodity
etc in the field Defined by purpose or
regulation
Sub-sample Portion of the primary or Not representative May
prepared sample such as introduce bias and error
alternate quarters
Split sample Equal portions of the Use multiple increment sam-
composite sample pling; May introduce error
LABORATORY
Laboratory sample Sample received by the Maintain sample and analyte
laboratory after packaging integrity and chain of
and shipment custody
Laboratory split Equal portions of laboratory Use multiple increment
sample sample sampling
May introduce error
Laboratory A portion of the laboratory Not representative May
sub-sample sample such as alternate introduce bias and error
quarters
Laboratory prepared Bulk sample after cleaning, Defined by purpose,
sample peeling, pitting, coring, regulation
shelling, husking, sieving, Error cannot be determined
etc
Test (analytical) Laboratory sample, Adds error
sample (Laboratory processed by comminution May introduce bias
processed sample) (cutting, chopping, May increase GSE
splitting, drying, milling)
Test (analytical) Mass taken for extraction or Multiple increment sampling
portion other testing
TESTING or ANALYSIS
Instrumental portion Mass or volume taken for Validation required
instrumental analysis
∗
DU: decision unit
will be discussed in Chapter 10.

CVR = (CV2S + CVS p2 + CV2A ) (9.8)
9.12 Laboratory Sampling Errors

9.12.1 Dividing the Sample
The fragility of many pesticide compounds and the dynamic chemical
compositions of most foods and feeds provide a challenging array of
opportunities for sampling error, many of which can jeopardize the
integrity of the laboratory sample. Subs or splits taken from the
composite sample, the laboratory sample or the test samples should
be selected in a representative manner with sufficient mass and incre-
ments. If the correct mass and number of increments were collected
for the primary sample and only half of that primary sample was
selected for laboratory preparation and processing, then TOS would
predict that FSE and GSE could both increase. At each step in labo-
ratory preparation and processing, error may be minimized by taking
multiple increments instead of a single grab samples. Most labora-
tories don’t recognize the opportunity to minimize errors in these
steps of the operation much less validate how much they contribute
to overall uncertainty.
9.12.2 Sample Preparation and Processing

In Table 9.2, the sample preparation and processing errors were sepa-
rated from the increment preparation error because of the large num-
ber of laboratory sample handling procedures that can increase GSE
or compromise the sample integrity. These include errors introduced
due to packaging, shipping, receiving and storing samples as well
as cleaning, peeling, husking, shelling and other sample preparation
steps that may occur before or after arrival at the laboratory. Labo-
ratory processing errors include chemical and physical changes such
as exposure to heat or light, drying, comminution and cryogrinding.
Great care should be taken when preparing and processing that the
integrity and representativeness of the lot is retained.
9.13 Pesticide Residue Sampling Research

Pesticide residue sampling has long been an important field of study
for chemists and statisticians. The Codex, EU and EPA guidelines
specify the weight and number of increments for different types of
pesticide residue primary sampling but much of this work was done
for finite element materials. See Table 9.4 for a list of guidelines.
It has always been recognized that larger numbers of increments
and weights of samples would result in lower uncertainty and higher
confidence in test measurements but, in view of practical limitations
of increasing the sample mass and size, it was more desirable to
collect data from a wider variety of crops and field trial locations and
conditions in order to be able to quantify the world-wide variability
of residue levels. Some of the work that has been done to define the
mass and number of increments for different types of pesticide residue
analysis are summarized below.
9.13.1 Collect 10 Increments per Sample ?

Ambrus (1979) studied pesticides in two finite element foods and
three pesticides, apples (phosphamidon) and tomatoes (mancozeb
and zineb). In this early work, he recognized that it was not possible
to collect 80–100 samples for all testing. He studied the effect of
the number of primary samples or increments per laboratory sample
on field trial data. He determined that residues are not normally
distributed but skewed positive, vary with position on the plant and
from field to field. He found little difference between the variabil-
ity of laboratory samples consisting of 10 or 20 apples or tomatoes.
Thus, in many cases, you will see recommendations for the collection
of at least 10 increments or units per sample. In 1996, using the
apples data previously studied and also in soil cores (dieldrin) and
100 g primary samples of butter bean (p, p -DDT), Ambrus (1996)
reported the relative uncertainty for random samples of size 5, 10
and 25 increments per primary sample was 40%, 30% and 20%. This
work helped to support the recommendations of Codex (1999) for
the number of increments per primary sample.
Table 9.4. Sampling guidelines.

References Guidance Document Sampling Information
OECD, 2009 OECD test guideline 509: Directions for carrying out magnitude of
Crop field trials residue crop field trials, no. of trials,
locations, sampling procedures, RAC,
commodity to analyse, field size, mass,
# increments. . . (based on FAO
Manual)
OECD, 2007 OECD test guideline 505: Directions for carrying out feeding
Residues in livestock studies, no. of animals, portion of
tissue, milk, eggs, development and
enforcement of MRL.
Ambrus, FAO Manual 3rd ed., International guidelines for the
2016c Submission and establishment of maximum residue
evaluation of pesticide levels, data required for JMPR
residues data for the evaluations including sampling and
estimation of sample processing, mass, no. of
maximum residue increments. Also RAC, portion of
levels in food and feed commodity and commodity group
tables, consumption data.
USEPA, U.S. EPA, Residue U.S. guidelines for conducting magnitude
1996 chemistry, test of residue field trials including no. of
guidelines, OPPTS trials, locations, sampling procedures,
860.1500 series mass, no. of increments.
USCFR, U.S. code of federal U.S. data requirements for conducting
2016b regulations, residue testing for pesticide residue registration
chemistry data including product chemistry,
requirements toxicological, ecological, environmental,
residue testing but details are found in
the EPA 860 guidelines.
USCFR, U.S. code of federal U.S. data requirements for enforcement of
2016c regulations, tolerances pesticide residue tolerances. 180.41
and exemption from gives crop group tables with
tolerances for representative crops required in field
pesticides in foods trial testing.
USFDA, U.S. FDA, Pesticide Describes the portion of sample to be
1999 analytical manual, tested for enforcement of tolerances.
Vol. 1, 102 B
USFDA, U.S. FDA, Investigations Chapter 4. guidance for collection of
2014 operations manual samples for regulatory enforcement of
tolerances including, RAC, commodity,
sample mass, # increments. (based
largely on Codex 1999)
(Continued )

USDA, 2014 USDA National Residue Annual testing plans: numbers of

program for meat and poultry animals, types of tissue, sample
mass.
Codex 1993a The FAO/WHO 1993 Codex Original commodity classification
classification of foods and upon which commodity groups
animal feeds, 2nd edition are based.
Codex, 1999 CAC/GL 33/1999, Includes tables describing the
Recommended methods of primary food, nature of the
sampling for the primary sample to collect,
determination of pesticide minimum laboratory sample
residues for compliance with weight/# increments.
MRLS,
EU, 2002 EU Commission Directive EC directive based on CAC/GL
2002/63/EC, Establishing 33/1999.
community methods of
sampling for the official
control of pesticide residues
in and on products of plant
and animal origin
Codex, 2004 CAC/GL 50-2004, General Guide for sampling of foods being
Guidelines on Sampling tested for compliance including
attribute sampling and
acceptance testing.
Eurachem/ M.H. Ramsey and S.L. R. Theory of sampling, sources of
Citac 2007 Ellison (eds), error, quality control,
Eurachem/EUROLAB/ measurement of uncertainty,
CITAC/Nordtest/AMC multiple examples.
Guide: Measurement
uncertainty arising from
sampling: a guide to methods
and approaches
DS, 2013 Dansk standard DS 3077, General theory of sampling and
Representative sampling — examples, detailed fly ash
horizontal standard example.
Nordtest, C. Gron, J.F. Hansen, Expands upon Eurachem/Citac
2007 B. Magnusson, A. 2007 with multiple examples.
Nordbotten, M. Krysell,
K.J. Andersen, U. Lund,
Uncertainty from sampling —
Nordtest 604
GOOD U.S. FDA Cooperative U.S. general theory of sampling of
Samples, Agreement with APHL, foods and feeds for regulatory
2015 AAFCO and AFDO, organizations based on Pierre
GOODSamples: guidance on Gy’s TOS.
obtaining defensible samples
(Continued )

AAFCO, Association of American Feed Comprehensive feed sampling

2014 Control Officials, Feed guide with detailed 2D bulk
Inspector’s Manual, Fifth materials sampling including
Edition, Published by description and use of tools,
Inspection and Sampling mass, no. of increments.
Committee
AAFCO, Association of American Feed Feed sampling of two-dimensional
2008 Control Officials, Guidelines materials such as dry feeds
for preparing laboratory including recommendations for
samples mass reduction and tool choice,
use and maintenance.
NELAC, FMSO-Vol. 1&2-2008, General Accreditation body requirements
2008 requirements for field for environmental sampling.
sampling and measurement
organizations
ISO, 1986 ISO 7002-1986-12-15, Definitions, Standards for
Agricultural food products — accreditation such as 17025,
Layout for a standard method multiple specific standards for
of sampling from a lot sampling for inspections by
attributes or variables.
ISO, 2003 ISO 11648-1,2, Statistical General sampling guide, only
aspects of sampling from bulk partially adopts theory of
materials sampling.
Australia, Australia New Zealand Food Online regulations governing
2015 Standards Code Australia’s and New Zealand’s
MRLs.
Canada, Health Canada Maximum Online database specifying
2015 residue Limits for Pesticides Canada’s pesticide MRLs.
China, 2014 China’s Maximum residue English translation of China’s
Limits for Pesticides in Food MRLs, prepared by the U.S.
Dept. of Agriculture.
Japan, Imported Foods Inspection Details for imported food methods
2015a&b Services of sampling and inspection
requirements and MRLs.
Schedule 3 table gives mass, #
increments
New Ministry of primary industries MRL legislation around the world.
Zealand
2015
Note: This table summarizes several guidelines developed for sampling for pesti-
cide residues, especially for magnitude of residue field trials and pesticide residue
regulatory enforcement. A column of annotations has been added to help the user
identify the guidelines that might be the most useful for their purpose.
9.13.2 Pesticide Residues Skewed Distribution

and Variability Factors
Ambrus (2000) studied the distribution of selected pesticide residues
in apples, kiwi, potatoes and butter beans. He confirmed the non-
normal, skewed positive distribution and found that 299, 120 and 59
random primary samples were needed to estimate the 99th, 97.5th
and 95th percentile of the residues at the 95% confidence level. The
distribution of residues was not significantly influenced by size, shape
and density of plants and nature of active ingredient or mode of
application of pesticides. A ‘variability factor’, the ratio of the 97.5th
percentile of residue to the mean residue, was introduced to aid expo-
sure safety assessments. A ‘default variability factor of 3 was recom-
mended for the estimation of residue levels in high-residue units in
the international estimate of short term intake calculations where
unit weights exceed 25 g’ but specific variability factors are still used
when data are available (Hamilton et al., 2004; FAO, 2013). Ambrus
(1979) also advised that stratified random sampling might be applied
if positions of low-, medium- and high-pesticide deposits are identi-
fied. This work helps to identify significant sources of heterogeneity,
which should be taken into account in sampling protocols.
Variability factor: The factor applied to the composite residue to

estimate the residue level in a high-residue unit, defined as the
residue level in the 97.5th percentile unit divided by the mean
residue level for the lot (Ambrus, 2016, p. 128).
9.13.3 Estimates of Minimum Mass

Ambrus and Lantos (2002), while working to identify the uncertainty
in decline studies, described a method for estimating the contribu-
tions to primary composite sampling, sample processing and sample
test analysis, utilizing a methodology to estimate the minimum mass
of a single increment for a specified confidence. This estimate of the
appropriate mass has been discussed as one of the most important
factors in controlling sampling variability.
9.13.4 Call for Identification of Factors

Affecting Uncertainty
Ambrus and Soboleva (2004) studied 8,844 crop-analyte test mea-
surements taken from 19 field trials and 57 different lots for medium-
sized crops. The average residue levels and coefficients of variation
had large lot-to-lot differences but there was no significant differ-
ence in the CVs of different crop and analyte lots and the CVs
vs residue level for an individual lot are normally distributed. So,
while the residues are not normally distributed, the relative vari-
ability is approximately normally distributed depending on the size
of the sample. The uncertainty of sampling medium-size crops for
pesticide residues of 5, 10, and 25 increment composite samples was
estimated to be 37%, 25% and 16% respectively, which were similar
to the values reported in 1996. This work clearly called for the iden-
tification of factors affecting sampling uncertainty and the inclusion
of sampling uncertainty in reporting of pesticide residue data. This
is, of course, also advised in TOS but it should also be noted that
much of the reported data on the mass and number of increments
for pesticide residue analysis is based on finite element crops.
9.13.5 No Generic Sampling Plan

Can Be Recommended
Farkas et al. (2014) studied carrots as well as parsley, describing the
mass of the parsley increments as a ‘handful’ with 10 increments per
composite sample weights ranging from 63 to 207 g; however, the
residue uncertainties for primary and n = 10 increment composite
samples was 0.678 and 0.21, respectively. It was shown that the dif-
ference in CVs is inversely proportional to the square root of the
number of replicate samples but no optimum generic sampling plan
was recommended.
9.13.6 Large Field to Field Variability due to Soil,

Weather, Environment and Application Methods
The work of Ambrus et al. (2006, 2009, 2016) focused on the uncer-
tainty of sampling in multiple crops and situations. Uncertainty will
be discussed in Chapter 10, but this work also informs us of sample
criteria, which may be the source of the greatest heterogeneity. Farkas

et al. (2015a) stated that ‘elaboration of statistically based sampling
plans for control of food requires information on the distribution of
residues within a lot or batch of a commodity and how this distribu-
tion varies between lots’. Residues have been found to vary within trees
and plots and between fields and are thought to be affected by soil,
environment, weather and application methodologies. To compensate
for large (80%) field-to-field variability, Horvath et al. (2014) recom-
mended that 15 or more field trials be used to set MRLs. Ambrus et
al. (2014a) found that the variability in residues was independent of
chemical structure, residue level and pre-harvest interval.
9.13.7 Twenty-Five Increments per Sample Provides

More Normal Distribution of
Average Residues
In later work, Farkas et al. (2015a) stated that ‘There is no optimum
for sample size and number of lots to be tested for estimation of
sampling uncertainty’. Taking a minimum of six replicate composite
samples from at least 8–12 lots is recommended to obtain a relative
95% range of sampling uncertainty within 50%.
9.13.8 Uncertainty for Enforcing MRLs Increased

by 1.2 vs Field Trial Data
Farkas et al. (2015a) reported field trial studies covering 106 crops
from at least 10 different countries. This data estimates uncertainty
of sampling for many different crops and crop groups and can be
used in planning sampling protocols to meet the needs of the user.
For enforcing MRLs, these uncertainties are increased by a factor
of 1.2 to account for larger variations in normal growing conditions
compared to field trials.
9.13.9 Protocols should be Different for

Before Market Acceptance and
After Market Enforcement
As mentioned previously, it is very important to design a sampling
plan to fit the purpose. Farkas et al. (2015b) explained that different
sampling plans are needed for testing compliance with MRLs of pes-
ticide residues on commodities before (include sampling uncertainty)
and after marketing (based on measurand + laboratory uncertainty).
9.13.10 GMO and Seed Sampling

While not specifically related to pesticide residues, the sampling chal-
lenges addressed for these other food issues may be informative to
the reader. Genetically modified organisms (GMO) and the produc-
tion of genetically modified foods have been a concern, especially
in the EU. Regulations such as EC/1830/2003 require traceability,
labelling and thresholds for unintentional contamination of GMOs
(EU, 2003). Heterogeneity of GMOs in raw food materials presents
real challenges in sampling. Sampling of grains, pure ingredients and
final products is most ideally handled at the manufacturing level,
where full access to the product enables continuous monitoring and
quality control (Gilbert, 1999; Kay and Paoletti, 2002). Attribute
sampling is still regularly practiced and studied for GMO (USDA,
1995; Koblinsky and Bertheau, 2005). The need for further evaluation
and study of these strategies, the identification of appropriate mass,
increments and sampling interval have been identified by Paoletti
(Paoletti et al., 2003, 2006). Seed testing also has specific regula-
tions on sampling (ISTA, 2016). The emergence of GMOs has led to
increased interest in the evaluation of seed sampling plans (Remund
et al., 2001; Paoletti et al., 2003).
9.14 Standardized Sampling Guidelines

Much work has been done to develop standardized field trial sam-
pling protocols and to study the variability of results so that MRLs
might be established. Subsequent work has recommended sampling
protocols to enforce these MRLs. CODEX and other professional
organizations have published guidelines for sampling that focus on
the number of units and quantity of material, which constitutes a
sample such as the OECD Guideline for testing of chemicals — Crop
Field Trials, #509 (OECD 2009; the EPA 860.1500 Field trial guide-
lines (USEPA, 1996); Codex CAC/GL 33 Recommended methods of
sampling for the determination of pesticide residues for compliance

with MRLs (Codex, 1999), CAC/GL 50-2004 General Guidelines
on Sampling (Codex, 2004) and the U.S. FDA Pesticide Analytical
Manual (USFDA, 1999) (see Table 9.4).
While there is considerable data supporting these guidelines,
especially for finite element food products (apples, cabbages, etc.),
they represent agreed upon standardized sample sizes and may not
provide the most representative sample possible. They do not provide
comprehensive guidance to avoid bias, assure accuracy or minimize
error. To avoid any argument on the regulatory actions taken based
on these samples, the MRLs refer to the average residue concentra-
tion in the sample taken, complying with the minimum sample size
(number of primary samples) and mass specified in the guidelines. It
is important to recognize the variability inherent in these sampling
approaches and sampling’s contribution to measurement uncertainty,
which will be discussed in Chapter 10.
Recognizing the vast complexity of food commodities and the
inherent challenges in collecting representative samples, some impor-
tant guidelines, which reference TOS, were published including
Eurachem/CITAC Measurement uncertainty arising from sampling
(Eurachem/CITAC, 2007); A Nordtest handbook for sampling plan-
ners on sampling quality assurance and uncertainty estimation
(Nordtest, 2007) and the Danish standard 3077:2013, Representative
sampling — horizontal standard (DS, 2013). With this chapter we hope
to bring these works together and provide the reader with resources
from both that should be considered in planning sampling protocols.
9.14.1 Integration of TOS and MU

In 2014, Esbensen and Wagner (2014) called for integration of TOS
with measurement uncertainty (MU) saying that ‘The responsibility
of the TOS is to deliver a representative analytical aliquot for analysis
with documentable minimum total sampling errors because of com-
petent command of the entire lot-to-aliquot sampling process, while
all errors characterizing the analytical processes are validated by a
comprehensive MUanalysis estimation’. The most critical difference
between conventional methods of measurement of uncertainty and

TOS is the active minimization of sampling error, especially in regard
to incorrect sampling bias, which can never be completely measured,
is inconstant and cannot be estimated using conventional statistics.
Incorrect sampling errors must be eliminated as it is not possible
to apply a correction because this is not a systematic and pre-
dictable effect. Sampling bias can introduce an error that is very
significant and yet unmeasurable and perhaps even undetectable.
Esbensen and Wagner point out that MU, as prescribed in the guide
to the expression of uncertainty in measurement (JCGM, 2008) and
EURACHEM/CITAC, 2007 guides, do not adequately account for
and control errors due to primary sampling, laboratory blending,
splitting, subsampling and selection of the test portion prior to anal-
ysis. They encourage the utilization of TOS to minimize the errors
attributed to sampling prior to analysis. If sampling is not conducted
in a manner that truly represents all the components of the sample,
the analytical result may be biased or unnecessarily imprecise and
yet these errors may never be evident and lead to a MU that is
inaccurate and misleading. Integration of TOS in sampling for foods
and feeds is discussed further in a series of papers published in 2015
(see Recommended Reading).
9.14.2 Sample Integrity

The guidelines caution to avoid contamination such as the collection
of control crops before the treated ones. Crops are not washed except
for gentle brushing to remove dirt such as for potatoes and other
root crops. To prevent degradation of the commodity or pesticide of
interest, shipping frozen or cool with dry ice packs is recommended
although some pesticides such as fumigants may need to be anal-
ysed immediately. Prior knowledge of the stability of the chemicals
and their possible interactions with the crops of interest is necessary
to adequately design the sampling plans for field studies. Analyti-
cal test method validation, metabolism and storage stability studies
conducted under the OECD guidelines will inform the laboratory of
the proper laboratory handling and storage. For example, grinding
and blending crops can accelerate degradation of the active ingredi-

ent. Some methods prescribe frozen storage of the whole crop and
analysis immediately after processing such as grinding, horizontal
chopping or blending.
Codex and EU guidelines do not permit cutting or otherwise
dividing large crops in the field. With caution for cleanliness and
avoidance of cross contamination, shelling nuts, removing beans from
pods and other common field harvesting practices are allowed. Sub-
sampling, where different parts of the crop will be analysed (e.g.
pulp and peel), may occur in the laboratory. Weighing of separate
commodity portions may be necessary for crops where multiple por-
tions are analysed. The practice of quartering very large commodities
such as melons and cabbage and then selecting alternate quarters for
analysis is still practised in many laboratories but this form of sample
size reduction has been shown to contribute additional uncertainty
to the laboratory processing stage of the analysis and may affect the
stability of residues (Omeroglu et al., 2013). It is important to con-
sult available research and regulatory guidelines before sub-sampling.
Validation and estimation of uncertainty and verification of analyte
integrity is recommended.
9.14.3 Sampling Animal Tissue and Animal Products

Extensive animal studies are conducted to determine the fate of
pesticides in animals, animal commodities and animal by-products.
Animal tissues, animal by products (e.g. fat, liver, and kidney) and
animal products (e.g. eggs, milk) are tested to determine pesticide
residue levels resulting from animals feeding on treated commodities.
OECD (2007) guidelines for testing of residues in livestock recom-
mend the testing of 20 animals, five each for four different evenly
spaced time periods. Tissues taken include skeletal muscle (0.5 kg),
perirenal, subcutaneous and back fat (0.5 kg), liver (0.4 kg) and
kidney (0.2 kg). Milk (0.5 L) and eggs (3) are collected every 3–4
days in the morning and evening. Samples from different animals
are not combined. Animals are treated at elevated dosage levels.
Ambrus (2016c) cautions that studies must be conducted long enough
to determine the half-life of the chemical in the animal. Sometimes

processed food studies are also conducted. For enforcement of MRLs
in meat, the MRL applies to the whole commodity, except for fat
soluble pesticides where the MRL applies to a portion of carcass fat.
For eggs, the MRL applies to whole egg whites and yolks combined
after removal of shell.
The USDA (2015) publishes the national residue program sam-
pling plans annually. In 2014, they proposed to collect 1,100 import
and 6,200 domestic samples, which are divided into three tiers: sched-
uled sampling, suspect animals noted during inspections and suspect
animals identified due to previous violative findings. Sample weights
are approximately 1 kg muscle and 0.5 kg each of kidney and liver.
Their testing includes multi-residue testing for pesticides and antibi-
otics (USFR, 2012). The guidelines reviewed recommended testing of
multiple animals and multiple tissue types but the sampling instruc-
tions did not specify multiple increments from multiple positions on
the carcass or organ (USDA, 2014).
9.14.4 Sampling for MRL Development

and Enforcement
The OECD field trial sample sizes are based on the FAO Manual
2002, which has since been updated and published as the 3rd edition
(Ambrus, 2016). The FAO Manual 2016 and the OECD guidelines
published in 2009 provide similar information about sampling.
The FAO Manual contains more details about the entire process
of pesticide residue studies to establish MRLs by the JMPR. The
section on sampling and analytical methods provides some general
directions for variations in sampling due to different study purposes
including (1) crop metabolism studies may require the distribution of
pesticides in the edible and non-edible commodity portions; (2) crops
harvested early in development (baby spinach and leafy greens) should
both be sampled; (3) rotational studies may contain both human food
and animal feed commodities to be sampled; (4) samples in livestock
metabolism studies may include milk, eggs and multiple tissues; (5)
dietary risk assessment may require testing of the edible portion or
single serving units; (6) in surveillance and monitoring of foods in the

channels of trade, the sampling method including number of incre-
ments and total weight must be specified. Codex has separate guide-
lines (CAC/GL-33) for enforcement of MRLs (Codex, 1999).
9.14.5 Codex Guidelines

CAC/GL 33-1999, Recommended Methods of Sampling for the
Determination of Pesticide Residues for Compliance with MRLs
(Codex, 1999), describes the minimum number of units (increments)
to be collected for plant, egg, dairy, meat and poultry samples. While
specific about the number of units and total laboratory sample size,
the guidance does not account for heterogeneity differences in sam-
ples and often subject sampling to practicality saying ‘primary sam-
ples should be combined and mixed well, if practicable, to form the
bulk sample’. No specific instructions are given for sub-sampling at
the laboratory. For meat, poultry and some other products packaged
or in bulk, which can be assumed to be well mixed, the collection of a
single sample (grab) is recommended. The 2002/63/EC (EU, 2002),
from the European Communities is based on CAC/GL 33-1999. Sam-
pling recommendations in the U.S. Food and Drug Administration’s
Investigator Operations Manual also closely match these recommen-
dations (USFDA, 2014).
CAC/GL 50-2004, General Guidelines on Sampling (Codex,
2004) takes into account some of heterogeneity parameters and vary-
ing characteristics of decision units defined in TOS such as the
objective for sampling, qualitative vs quantitative characteristics,
isolated vs continuous lots and bulk vs finite element samples. The
standard distinguishes sampling methods for homogeneous decision
units (uniform to a particular characteristic within a given probabil-
ity) from heterogeneous decision units but this standard is intended
for control of food safety parameters such as ingredients and micro-
biological contamination. The standard states the guidelines do not
cover control of non-homogeneous goods, homogeneous goods where
the measurement error is not negligible (larger than one third of sam-
pling error). As the analytical measurement variability in pesticide
residues has been estimated to be up to 25% around 0.1 mg/kg

residue levels (1 sd. of spike recoveries), and the sampling variability
is minimum 20% (Ambrus et al., 2014b; Farkas et al., 2014, 2015a),
the standard does not apply to most pesticide residue analyses. The
standard does, however, address many important concepts of defen-
sible samples including representative sampling, random selection of
increments, multiple increment sampling, the estimation of errors to
include sampling according to the equation and acceptance criteria
for the characteristic (attribute) of interest. While is it always impor-
tant to incorporate the tenets of TOS for any sampling protocol,
the establishment of defined sampling procedures and quality control
together with documented regulatory specifications based on these
procedures allows growers, manufacturers, suppliers and regulators
to share and compare data.
9.14.6 Commodity Portion Sampled for

MRL Enforcement
Among other things, the FAO Manual, 3rd edition (Ambrus, 2016c),
Appendix VI contains detailed recommendations on sample process-
ing and the portion of commodity to analyse for enforcement of
MRLs. These recommendations are largely based on Codex 1993
recommendations although ordered quite differently. As described
before, the RAC, in the form that is moved through trade, is sam-
pled from field trials, additional cleaning, preparing, processing and
analysis occurs and data derived from these tests is used to develop
the MRLs. Table VI provides a commodity grouping, which is only
in part based on the Codex Commodity Groups, and the portion of
the RAC to which MRLs apply. Some MRLs may include additional
instructions for the portion that may apply such as nuts without the
shell or peas without the pod.
In the USA, the EPA provides some brief descriptions of RAC to
be tested for MRL enforcement in the 40 CFR 180.1 (USCFR, 2016c)
but recognizing the limited guidance, the U.S. FDA, which is tasked
with enforcement of MRLs in the U.S. provides more instructions.
The FDA Pesticide Analytical Manual, Vol. 1,102 B (USFDA, 1999)
further describes the portion of sample to be tested for enforcement

of MRLs.
9.15 Management Responsibilities

Management must recognize the critical connection between accurate
and precise sampling and the quality of the data generated from these
samples. Regulatory and food safety officials make decisions that
could critically compromise the quality of our food supply if based
on faulty data. Management must ensure policies and procedures are
in place to provide for quality sampling whether conducted by entities
under their supervision or provided by a separate organization. Man-
agement must budget sufficient resources for representative sampling
including staff, equipment and training. Ultimately, it is the decision
makers, those scientists, regulators, manufacturing plant managers,
importers or exporter, and others, who must use the data to make
decisions. They must take an active interest in sampling, develop a
working knowledge of sampling theory and practice, ask educated
questions and make informed decisions about all aspects of the data
provided to them.
Sampling should not be an additional or secondary task. Sam-
pling takes considerable time from the development and study of
sampling protocols to identification of sampling sites, site planning,
travel to and from the site to selection of the samples, packaging
and shipment of samples. Samplers must be given the time, train-
ing and resources such as vehicles, laptops, collection tools and
packaging materials to assure carefully selected, well documented
and representative samples. The practice of sampling for pesticide
residues requires staff who have an understanding of the procedures
needed to assure analyte and sample integrity. If samples arrive with
poor documentation, are frequently lost or are not even the correct
commodity, one needs to consider what else might be wrong with
these samples. Without a recognition of the importance of sampling,
all the money spent on analysing the samples is compromised.
Laboratories are often not directly responsible for sampling and
do not demand a place at the table when sampling decisions are
made. These same labs will spend hundreds of thousands of dollars

on analytical equipment and recruit the most talented, well edu-
cated scientists available. They attend numerous professional meet-
ings where all the intricacies of their analyses are discussed with
little attention paid to the primary sample and just a small amount
of attention to the processing of the laboratory sample, mostly in
regard to the recovery and stability of the analytes. One of the most
important steps that management can take to improve sampling is
to join together with laboratory technicians and samplers to define
why samples are being taken, what commodities or materials must
be tested, what information is needed and how decisions will be made
from the data generated. Collaborations among all parties who will
generate or use the data derived from sampling should agree upon
the necessary precision and accuracy, the appropriate procedures,
the resources needed to provide both the samples and the analytical
testing and what actions will be taken based on the data.
Sampling plans should first establish the purpose for sampling.
Specifics such as who, when, where and what types of samples to col-
lect, together with the size and number of increments and total mass,
should be specified. Protocols should also describe the procedures
to be used together with the appropriate equipment. As manage-
ment becomes familiar with sampling theory and practice, they will
begin to understand the importance of appropriate, well-maintained
sampling tools and equipment. There are always compromises that
need to be made based on cost and benefit, but when sampling pro-
tocols are understood and agreed upon, decisions may be made in
an educated and defensible manner with all parties understanding
how the sampling will affect the quality of the data and factor this
information into their decisions.
Sampling is not a simple process of picking up something for the
laboratory to analyse. Samplers need to have training in sampling
theory that is appropriate to the complexity of their duties. Even the
best written protocol will need to be modified to meet the realities
faced by samplers in the field. Without an adequate background in
TOS and a voice at the table when protocols are written, samplers
may unintentionally introduce bias into the sample that they do not
even recognize and can never be detected by simple analytical mea-

surements in the lab. Not only do samplers need to have field decision
making skills but they need to have a strong foundation in the scien-
tific method and be able to adequately describe any deviations from
protocol that may have been made. These field notes are a valuable
part of the analytical data record and can help decision makers to
qualify a sample as preliminary, suspect or highly suspect if a problem
is detected. Perhaps even more important, a clear description of the
decision unit and how the sample was taken can help decision mak-
ers decide if a decision unit is within acceptable levels. Inadequate
sampling may often underestimate the concentration of a residue in
the decision unit. Food safety agencies frequently share data in an
effort to find the source of a problem. Without confidence in how
the sample is collected and a clear idea of its relevance to the deci-
sion unit, multiple confirmational samples, additional analyses and
valuable time must be spent to corroborate a finding. Management
needs to understand the value of sampler training and the savings
this investment provides when they have verifiable data and can make
crucial decisions with confidence.
Management and laboratory staff need to fully understand
objectives of the sampling in order to maintain the integrity of
the analytical process from the decision unit to the autosampler
vial. For example, laboratories may need to develop efficient ways
to handle larger samples because processing smaller samples may
not adequately represent the decision unit. In another example, par-
ticulate bulk samples such as grains or animal feed may need to
be sub-sampled using specialized equipment such a riffle splitters.
Management needs to understand the necessity and provide for the
acquisition, use and maintenance of this type of sampling apparatus.
Cryogenic sample processing may be necessary to preserve analytes
or provide a small, uniform sample size. Sampling theory not only
applies to the primary sample taken directly from the decision unit,
but all the subdividing, chopping, cutting, comminution and sam-
pling that occurs in the laboratory up to and including the selection
of the analytical sample. From the moment that a laboratory sample
is processed, it becomes particulate matter and joins the realm of
TOS. In our discussions of TOS, the importance of sampling cor-

rectness, adequate mass and multiple increments will become clear.
Much as laboratories operate under a system of good laboratory
practices and accreditation, management should establish a system
of oversight and process quality control that assures reliable and
scientifically defensible sampling and laboratory sample processing.
These procedures are the foundation upon which all other analyti-
cal work stands. The use of process validation together with routine
replicate sampling, blanks and contamination checks provide man-
agement with valuable quality control data needed to determine the
error due to all aspects of the analytical process from the collection of
the sample to laboratory mass reductions to analysis. Wouldn’t it be
wonderful if sampling operations could also operate under a system
of verification and constant process improvement?
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b2530_FM.indd 6 01-Sep-16 11:03:06 AM

Chapter 10
Estimation of Uncertainty
of Measured Residues and Testing
Compliance with MRLs
Zsuzsa Farkas, Jo Marie Cook and Árpád Ambrus
Main topics
Factors affecting the results of pesticide residue analysis

Estimation of sampling uncertainty
Estimation of sampling uncertainty based on primary samples
Estimation of sampling uncertainty based on the results of supervised
trials
Summary of sampling uncertainty for pesticide residue analysis
Effect of handling of laboratory samples on the accuracy and uncer-
tainty of the results of analyses
Uncertainty and trueness of analysis of residues in test portions
Quality control of the determination of pesticide residues
Use of combined uncertainty of measured residues to verify compli-
ance with MRLs and settling disputes
10.1 Introduction
It is generally accepted that any measurement result cannot be better
than the sample that is analysed (Ramsey and Ellison, 2007; Sam-
pling and Sample Handling Working Group, 2015) and the results
405
cannot be properly interpreted without information on their associ-

ated uncertainty (Ellison and Williams, 2007; ISO, 2005).
Uncertainty: Parameter associated with the result of a measure-

ment that characterizes the dispersion of the values that could rea-
sonably be attributed to the quantity of the measurand (Stephenson
et al., 2006).
Though most analysts and users of analytical data are aware of

these fundamental facts, the progress is slow in practice to minimize
the error and eliminate bias in the results of pesticide residue analysis
considering the whole process from sampling to reporting the results.
There are a number of scientific reports available indicating
potential undesirable effects of sample mass reduction by quarter-
ing, chopping or grinding of the laboratory sample on the stabil-
ity of some analytes and variability of the residue concentration in
small test portions taken from the comminuted sample material for
extraction. See the Recommended Reading section in the reference
list for more details. Method validation guidelines (available in the
Recommended Reading section in the reference list) specify the need
for tests to verify the efficiency of extraction, homogeneity (well
mixed condition) of analytical sample, stability of analytes during
the storage of samples, purified extracts and analytical standard
solutions. Nevertheless, even recently published papers report the
reproducibility of the analyses based on recovery studies carried out
with spiked test portions, which can only provide information from
the point of adding standard mixture to the test portion of a few
grams. It should be noted that neither recovery studies, nor pro-
ficiency tests provide information on the efficiency and accuracy of
size reduction of the laboratory sample, which may include multi-step
procedures.
Chapter 9 described the theory and practice of sampling and con-
sidered the sampling as a continuum including all steps from selecting
an appropriate sampling plan, through collecting natural crop units
or single increments, mass reduction to a test portion, small enough
Uncertainty of Sampling and Sample Processing 407
to be submitted as a whole to analysis. It provides guidance for

improving accuracy and minimizing variability and bias.
The objects or targets of sampling for pesticide residue analysis,
called decision units in Chapter 9 (EURACHEM Guide calls it sam-
pling target) (Ellison and Williams, 2007), are heterogeneous both in
terms of composition (residue concentrations are most likely differ-
ent in individual crop units) and distribution (spatial on the treated
plants among plants and within the peel and pulp of fruits). Conse-
quently, to prepare a test portion of a few grams that may represent,
for instance, several tons of fruits grown in an orchard requires full
attention to all steps of the process by everybody involved in their
implementation.
The measured residue concentrations are subject to random, sys-
tematic and gross errors, which may be present in every step. The
potential presence of the latter two in the sampling operations and
the possibilities to reduce them were discussed in Chapter 9.
This chapter provides guidance for the estimation of the
uncertainty, reflecting the random errors, of individual steps of the
determination of pesticide residues, and the use of the combined
uncertainty of the measured residue values for making decisions on
the compliance of the sampled lot or batch with legal limits or spe-
cific quality standards of food products. Typical systematic and gross
errors, which might affect the results, will be mentioned but not dis-
cussed in detail.
Random error is a component of measurement error that in repli-

cate measurements varies in an unpredictable manner (ISO, 1994).
Systematic error is a component of measurement error that in
replicate measurements remains constant or varies in a predictable
manner (ISO, 1994).
Gross errors refer to unintentional or unpredictable errors while
generating the analytical results. Errors of this type invalidate the
measurement (WHO, 2009). They are so serious that there is no
alternative to abandoning the experiment and making a completely
fresh start (Miller and Miller, 2010).
10.2 Factors Affecting the Results of Pesticide

Residue Analysis
10.2.1 Variability of Pesticide Residues within Fields
Uniform deposition is impossible to achieve during the application of
pesticides. The variability of residues in or on the crops is inevitable.
It may result from many sources, including biotic and abiotic factors
(Dubus et al., 2003). Initial deposit can be affected by application
technique, spray technology (i.e. sprayer type, settings, nozzle type),
growth stage and canopy structure. For example, fruits at the top and
on the outside regions are likely to receive more deposits than those
which are inside the canopy. (see Recommended Reading). Note that
post-harvest treatments on fruits and vegetables also produce large
variability.
Besides initial deposition, dissipation processes also determine
the level of residues in or on crops that can be affected by factors
such as weather, microclimate or crop growth. Wash-off caused by
rain or irrigation can also result in significant losses of residues (Smith
and MacHardy, 1984; Frank et al., 1985, 1987).
On the other hand, the relative variability of residues is practi-
cally not affected by the mean value of the residue, the time between
pesticide application and sampling (ageing of the residue), the vol-
ume and application rate used for foliar application, or the chem-
ical and physical properties of the active ingredient or formulation
(Ambrus, 2000). The similarity of the distributions of residues deriv-
ing from different fields is illustrated in Fig. 10.1 (Ambrus et al.,
2014).
Statistical analysis of experimental data revealed that variability
caused by inaccuracies of analytical methods does not significantly
contribute to the overall variability (uncertainty of the measured
residue) (Hill and Reynolds, 2002), which means that the main con-
tributor to the variability must be the difference of residue values
between individual crop units within a decision unit.
25 12
10
20
Relative frequency [%]

8
15
6
10
4
5
2
0 0
0.5 1 1.5 0.4 0.9 1.4 1.9
Normalized residues in strawberry Normalized residues in cabbage
Figure 10.1. Distribution of 14 normalized pesticide residues in cabbage and 6

in strawberry composite samples (n = 10) derived from independently treated
fields. Each data point on the charts represents the relative frequency of residues
in bin classes.
Composite sample: Combined increment samples, or combined

replicate samples, or combined samples from replicate trials. Pre-
ferred term to bulk sample which is ambiguous (Horwitz, 1990).
Normalized residues: The individual residues making up one data
set are divided by their average value.
The variability of pesticide residues was extensively studied. In

the experiments (Recommended Reading), 100–300 primary crop
units were taken mainly from commercially cultivated fields in 14
countries and analysed separately with multi-residue procedures for
detecting all residues present in the samples. Over 21,000 individual
residue values measured in root and leafy vegetables, small-, medium-
and large-size fruits comprised of 182 data sets, including the combi-
nations of 46 pesticides and 20 crops. The characteristic features of
these data sets are summarized in Section 10.2.3.
Because of numerous factors mentioned above, which can influ-
ence the distribution of pesticide residues in and on crops, individual
samples taken from treated fields or monitoring show high variability
(on average, their relative standard deviation, also called coefficient

of variation (CV), is 80–110%) (Ambrus et al., 1996; Lentza-Rizos
and Balokas, 2001; Harris, 2000; Hill, 2000). Occasionally outstand-
ingly high values (10–12 times the median of the dataset) of residues
in individual samples of fresh fruits and vegetables can occur (Harris,
2000; Harris et al., 2000).
Examples (Ambrus, 2006; Horváth et al., 2013) for the variability
of pesticide residues measured in individual crop units taken from
random positions in treated fields can be seen in Fig. 10.2.
10.2.2 Sources and Calculation of Uncertainty of

Measured Residue Concentrations
When correctly sampled, the major source of the variability of the
measured residues in replicate primary and composite samples is the
inherent compositional and distributional (spatial) heterogeneity of
the materials to be examined.
The random variation of the results is called the combined uncer-
tainty of the measurement, which is expressed as the standard devi-
ation or relative standard deviation (CV). The latter expression
provides the advantage of making the variations of the measurand
present in samples at different concentrations comparable.
The uncertainty of the measured residue concentration is com-
prised of the uncertainty of sampling (CVS ), reduction of the large
bulk or laboratory sample (CVSS ), sample processing, including com-
minution of laboratory sample with chopping, grinding and with-
drawing the test portion for extraction (CVSp ), and analysis of
residues present in the test portion (CVA ) (Ambrus, 2004).
According to the basic rule of error propagation, the combined
uncertainty of the measurement result (CVR ) can be expressed as:

CVR = CV2S + CV2SS + CV2Sp + CV2A (10.1)
When the uncertainty of the laboratory phase of the analysis
(CVL ) incorporates the subsampling and sample processing together
Alphamethrin in grape
1.4
1.2
Residues [mg/kg]
1
0.8
0.6
0.4
0.2
0
0 20 40 60 80 100 120
Random sampling posiƟon
Pirimiphos-methyl in cucmber
1.4
1.2
Residues [mg/kg]
1
0.8
0.6
0.4
0.2
0
0 20 40 60 80 100 120
Cyproconazole in carrot
0.4
0.4
0.3
Residues
0.3
0.2
0.2
0.1
0.1
0.0
0 20 40 60 80 100 120

Figure 10.2. Residues measured in crop units taken from commercially treated
fields. (The residues were measured in whole crop units, e.g. one carrot, cucumber
or bunch of grapes.)
with the uncertainty of the analysis, it can be expressed as

CVL = CV2SS + CV2Sp + CV2A (10.2)
The uncertainty of sampling can be calculated as:

CVS = CV2R − CV2L (10.3)
Each component of Eq. (10.2) can be further subdivided if neces-
sary for identification of the major source of uncertainty, as will be
indicated in the following sections.
The contribution of individual components to the combined
uncertainty of the results varies depending on the sample size, the
procedures applied for subsampling, efficiency of sample processing,
the analytical method used and the concentration of the residue to
be measured. Under optimum conditions for the performance of each
step, in case of analysis of pesticide residues in fruits and vegetables,
the sampling, subsampling and sample processing and analyses of
extracts may contribute, on an average, to 71%, 6% and 23% of total
variance of measured residues, respectively (Ambrus et al., 2011).
10.2.3 Characterization of the Distribution

The characteristic features of the distribution of pesticide residues in
fruits and vegetables were evaluated by Horváth et al. (2013).
The relative standard deviation (CV) calculated from the
residues measured in primary samples taken from one field or lot
provides only one estimate for the true variability of residues in the
treated crops. Repeated sampling would result in different residue
distributions. The best estimate of the typical spread of residues in
primary samples is the average of CV values (0.8) calculated from
residues detected in unit crops taken from multiple field trials. Small
samples of size 5–10 substantially underestimate by 5–23% the CV
of the parent population having a true CV of 0.80.
Point to note: The correct calculation would be based on the
square root of the average variance of residues. However, it cannot
be applied because of the large differences of the average residues in
various datasets.
To obtain an estimate of CV value within a few percent (2–3%)

of the true CV value, a minimum of 300 random samples must be
taken (Ambrus, 2009) as shown in Fig. 10.3.
Primary sample: Collection of one or more increments or units

initially taken from a population. Note that portions may be com-
bined (composited or bulked sample) or kept separate (Horwitz,
1990).
Parent population: Elements of the decision unit (sampling target).
The gamma, lognormal and Weibull distributions fitted on the

experimental data overestimated the level of residues at the low con-
centration range and underestimated over 98th percentiles. On an
average, the best estimate was obtained with lognormal distribution.
The underestimation is attributed to the fact that the residues are
very scattered at the high concentration range and the parametric
fitting software’s cannot properly represent them. Consequently, the
variability of residues in composite samples drawn from synthetic log-
normal distributions is somewhat underestimated and larger variabil-
ity may be expected in case of sampling field-treated commodities.
The variability of residues in natural units of marketed commodi-
ties may be covered with a lognormal distribution having a CV value
of 1.1. The larger variability of residues in market samples compared
to field samples may be attributed to variability resulting from the
large-scale commercial application of pesticides compared to the lim-
ited sampling areas applied in the referred studies and supervised
residue trials and the potential mixing of lots that received different
treatments.
Drawing 1000 random composite samples of size 5, 10, 25, 100,
120 and 300 from a lognormal distribution with µ = 1 and σ = 0.8,
the variances of their components follow the central limit theorem
(Eq. (10.4)) indicating its applicability for the skewed distribution of
pesticide residues:
Vi
Vn = (10.4)
n
40
35 n=4
30 n=10
25 n=20
20 n=50
15 n=100
10 n=300
0
0 0.5 1 1.5 2
CV
25
n=1
20 n=4
n=10
15 n=20
n=50
10
0
0 0.5 1 1.5 2 2.5
Residues [mg/kg]
Figure 10.3. Distribution of CV values in primary samples (n = 1) and the
average residues in composite samples of size n drawn from a synthetic lognormal
distribution with mean of 1 and CV of 0.8.
Notes: The scale of upper chart is larger to better show the nature of the residue
distribution. The maximum residue in primary samples was 9.15 mg/kg. It is not
shown on the chart.
where Vi is the variance of residues in individual crop units making

up a composite sample of size n, and Vn is the variance of aver-
age residues in composite samples. The distribution of the average
residues in composite samples of size n is also shown in Fig. 10.3.
10.3 Estimation of Sampling Uncertainty

10.3.1 Methods for Estimation of Uncertainty
of Sampling
When taking a sample in order to determine the residue content in a
certain crop, a small amount is collected from a much larger quantity.
In an ideal situation, the small amount reflects the composition of
the whole, but actually it never perfectly does. To be able to make
correct decisions, the end-user of the result of the analysis must be
aware of information on uncertainty arising from all possible sources,
including sampling uncertainty.
Not so long ago, uncertainty of sampling was practically ignored
and a much larger focus was on uncertainty coming from analyti-
cal phases of the measurement. By now, it has become well known
that the uncertainty of sampling is often the main contributor to
the combined uncertainty of the measurement result (Ambrus, 2006;
Lyn et al., 2002, 2003, 2007a). Recognizing the importance of this
uncertainty led to the development of sampling protocols for different
materials (ISO, 2003; CAC, 1999, 2004) and strategies for the esti-
mation and investigation of measurement uncertainty arising from
sampling.
There are two main approaches: One is drawing random samples
from primary residue populations with computer modelling and the
other is the so-called duplicate method.
The empirical method was initially applied to determine the dis-
tribution of pesticide residues in soil (Taylor et al., 1985; Kratochvil,
1985) and fruits (Ambrus, 1979). Applying random sampling with
replacement for taking composite samples of various sizes, the uncer-
tainty of sampling was determined based on the residues measured
in primary samples. Ambrus concluded (Ambrus, 1996; Ambrus and
Soboleva, 2004) that the typical sampling uncertainties expressed as
CV are about 0.16–0.2, 0.25–0.3 and 0.37–0.4 for pesticide residues
in composite samples of size 25, 10 and 5, respectively. These find-
ings were confirmed for root and tuber vegetables (CV=0.2, n = 10)
based on the much larger primary residue data sets (Farkas et al.,
2014).
The ‘duplicate method’ elaborated by the Eurachem-EUROLAB-

CITAC-Nordtest Working Group on Uncertainty from Sampling and
published in a guide (Ramsey and Ellison, 2007) and a review (Ram-
sey and Thompson, 2007), is now widely used (Lyn et al., 2007b,
2007c; Kuselman, 2008; Reiter et al., 2011). It requires taking dupli-
cate samples from the target and duplicate analysis of the samples.
The results are statistically evaluated with the analysis of variance
(ANOVA). The guide recommends taking duplicate samples from
at least eight sampling targets, which was confirmed by Lyn and
co-workers, who have found that a minimum of eight different lots
(targets) are needed to get uncertainty values with acceptable con-
fidence levels when taking duplicate samples (Lyn et al., 2007b). In
addition, the Nordtest handbook for sampling planners on sampling
quality assurance and uncertainty estimation was prepared, which
follows the same principles but intended to be rather more practical
(Grøn et al., 2007).
10.3.2 Databases Used for Estimation of

Uncertainty of Sampling for Pesticide
Residue Analysis
The results of studies on within field distribution of residues in pri-
mary crop units, described briefly in Section 10.2.1, provided a unique
database for estimation of sampling uncertainty applying replicate
random sampling with replacement described in Section 10.4.
FAO/WHO JMPR evaluated the supervised residue trials data
for estimation of maximum residue levels, median and high residues
(see Chapter 4). In some of the supervised residue trials, replicate
samples were taken from the single-trial plots in order to get more
precise information on the average pesticide residue concentration in
or on crops. The data derived from the replicate sampling of super-
vised trial plots were collected from the JMPR reports between 1997
and 2010 (FAO, 1997–2010). It contained 12,087 replicate sample
sets (duplicate samples in >99.95% of the cases) and 25,876 indi-
vidual residue values. The database comprised 706 pesticide–crop
combinations formed from 66 different pesticides and 107 different

crops. It must be noted that supervised trials are carefully planned
and implemented under strict conditions on small experimental plots
with particular attention to the uniform pesticide application and
treatment of crops. Furthermore, the sizes of samples taken are usu-
ally larger (Ambrus, 2016) than specified for regulatory control of
residues (CAC, 1999; EC Directive, 2002), for testing the compli-
ance of products with MRLs. Consequently, the residues measured
in composite samples may show less variability than those present
after commercial application. These conditions have to be taken into
account when the residue data are used for estimating the uncertainty
of sampling.
In the studies of Farkas and co-workers, supervised residue trial
data was used for the estimation of sampling uncertainty, where repli-
cate composite samples were available. Modelling was used for the
calculation of confidence intervals of estimated sampling uncertainty
values (Farkas et al., 2015a, 2015b).

on Primary Samples
The sampling uncertainties were estimated by Farkas et al. (2014)
applying an Excel macro for drawing random samples with replace-
ment from the experimental data sets described in Section 10.2.1.
For the validation of the sampling procedure, composite samples
were drawn randomly 10,000 times with replacement from the parent
population of natural numbers ranging from 1 to 120. Each compos-
ite sample consisted of 10 randomly selected numbers. The average
occurrence of the randomly selected numbers corresponded to the
theoretically expected 833 (120 ∗ 833 = 99, 960), with a CV of 0.035.
It was considered acceptable in view of the calculated CV10 of 0.253
for random composite samples of size 10 drawn from primary sample
population with the typical CVprim of 0.8. The relatively minor vari-
ation due to random sampling would have no effect on the outcome
of the estimated sampling uncertainties.
10.4.1 Effect of Sample Size (n) and Number of

Replicate Samples (p) on Accuracy and
Variability of Estimated Average Residue
Composite samples of size n of 5, 10 and 25 were taken randomly
1000–10,000 (N ) times with replacement by applying the validated
MS Excel macro from each of the residue populations consisting of
120 primary samples and from the combined ‘normalized’ data sets of
parsley and carrot primary residue populations. In all cases described
below, the random sampling was performed with replacement with-
out specifically mentioning it.
Sample size (n): The number of units, or quantity of material,

constituting the sample (CAC, 1999).
Drawing N random samples of size n was repeated p times inde-

pendently from the same parent population representing the situa-
tion when replicate samples are taken from the same lot. The average
residues in composite samples (Rn ) were calculated from the mass of
individual primary samples (gi ) and the concentration of the residues
(ci ) measured in the primary samples (Eq. (10.5)).
n
i=1 ci × gi
Rn = n (10.5)
i=1 gi
The residues in random composite samples were arranged in a
matrix consisting of p columns and N rows, containing the p repli-
cates and the number of composite samples taken N times, respec-
tively. Residue values being in one row made up one replicate sample
set. Table 10.1 shows for example the first two rows of the N × p
matrix for p = 4 replicate samples.
The uncertainty of sampling, based on p replicate composite
samples, was expressed with the relative standard deviations of the
residues in one replicate sample set (p). The CV value was calculated
from the relative differences with range statistics for sample sets with
≤ 10 data points applying the following equation:
Rmax − Rmin
CVR = (10.6)
R̄ × d2
Table 10.1. Example for the calculations of the average residue and CV values
from four replicate samples
Residues in
Random Composite Samples Relative
Sample # Average Difference Calculated
#=1→N 1 2 3 4 Residues of Residues CV
1 0.989 0.953 0.918 0.73 0.898 0.289 0.14

2 0.97 1.147 1.077 0.975 1.042 0.17 0.082
where Rmax , Rmin and R̄ are the maximum, minimum and mean
residue values, d2 is a factor depending on the number of residue
data points (n) in one data set. The values of d2 are: 1.128, 1.693,
2.059, 2.326, 2.534, 2.704, 2.847, 2.970 and 3.078 for n = 2, 3 · · · 10,
respectively (Anderson, 1987).
For larger sample sizes the CV = SD/R̄ was calculated with the
usual equation of the standard deviation:
2

Ri − R̄
SD = (10.7)
p−1
The standard deviations estimated with range statistics and with
Eq. (10.7) are somewhat different and the two estimates should not
be compared directly. Consequently, the range statistics were used
only for small number of replicate samples in this study.
The average residue (R̄) in replicate samples and the average CV
(CV) in N sample sets were calculated as their arithmetic mean.
The 95% range of the average residues and their CV values were
calculated as the difference between the 97.5th (P 0.975) and 2.5th
(P 0.025) percentiles of the values obtained in N sample sets. The
relative 95% range of CVi values (CIr0.95 ) was calculated from N
sample sets as:
CVP 0.975 − CVP 0.025
CVr0.95 = (10.8)
CVi
where CVi is the average of N CVi values.
The relationship of sample size and the average residues in com-
posite samples were studied by drawing N = 10, 000 random samples
Table 10.2. Residues in 10,000 parsley samples of size n obtained

by random sampling from residues in primary samples.
Residues in Samples
of Size ‘n’ (mg/kg)
Sample
Pesticide Size (n) P0.025 Average P 0.975 CV
Azoxystrobin 1a 0.0027 1.35

5 0.0008 0.0027 0.0076 0.6
10 0.0011 0.0027 0.0056 0.42
25 0.0016 0.0027 0.0045 0.27
Difenoconazole 1a 0.113 0.36
5 0.0795 0.1127 0.1496 0.16
10 0.0889 0.1128 0.1387 0.11
25 0.0979 0.1131 0.1297 0.07
Linuron 1a 0.0683 0.271
5 0.0533 0.0681 0.0855 0.12
10 0.0575 0.0684 0.0802 0.08
25 0.0612 0.0683 0.0758 0.05
a
Average residue in primary samples calculated with Eq. (10.5).
of size n from various parent populations. The results obtained are

illustrated with some examples in Table 10.2.
The results indicate that repeatedly drawn composite samples
of size ≥ 5 provide an unbiased estimate of the average residue of
the parent population. Additional modelling, however, revealed that
N ≥ 1, 000 is sufficient to obtain an estimate for the mean value
within 1%.
On the other hand, the variability of residues in primary samples
or sample increments of the parent populations is underestimated
when composite samples of size 5–10 are drawn repeatedly two or
four times from the same parent population.
The average CV calculated from replicate samples at or above 8
approached the true CV of the parent population (Table 10.3). The
larger the sample size the closer the average CV was to the true CV
value. A previous study (Ambrus, 2006) indicated that in case of a
single sample n ≥ 300 was required to obtain an estimate of the true
CV value within 2–3%.
Table 10.3. Range of CVS values obtained with draw-

ing ‘p’ replicate samples 10,000 times from normalized
residues in carrot.
Calculated CVS
Values
Sample Replicate
Size (n) Samples (p) P 0.025 Average P 0.975
5 1 0.265
2 0 0.193 0.545
8 0.118 0.246 0.445
30 0.18 0.259 0.365
10 1 0.19
2 0 0.146 0.408
8 0.086 0.18 0.298
30 0.136 0.187 0.245
25 1 0.12
2 0 0.094 0.264
8 0.057 0.114 0.184
12 0.07 0.117 0.175
30 0.088 0.119 0.154
Point to note: For small samples, the square root of pooled vari-
ances provides an unbiased estimate of the standard deviation and
the CV value derived from it.
10.4.2 Effect of Variability of Residues in the

Decision Unit (Sampling Target)
The CV of residues in primary crop units derived from various pesti-
cide crop combinations varied greatly (0.11–1.44 with an average of
0.8). The effect of the variability of residues in primary samples on
the estimated sampling uncertainty was studied with drawing 10,000
random samples of size 10 from synthetic lognormal datasets with
µ = 1 and CV values of 0.11, 0.18, 0.25, 0.26, 0.36, 0.40, 0.55, 1.06
and 1.44. These data sets represent the actual variability of residues
observed in primary samples derived from field trials. The random
sampling procedure was repeated 4, 8, 12, 20 and 30 times. The rela-
tive difference of the 95% range of CVi (CIr0.95 ) values was calculated
with Eq. (10.8). The results are shown in Fig. 10.4.
2.0
1.8
1.6
1.4
CIr0.95
1.2
1.0
0.8
0.6
0.4
0 5 10 15 20 25
Number of replicate samples
0.11 0.18 0.25 0.36 0.55 0.79 1.06 1.42
3.0
2.5
2.0
y = 3.8738× -0.583
CIr0.95
1.5 R² = 0.9939
1.0
0.5
0.0
0 5 10 15 20 25
Number of replicate samples (p)
Figure 10.4. Relative 95% range of CV values of residues in p replicate composite

(n = 10) sample sets.
The results indicate that:
• the relative 95% range (CIr0.95 ) of the average CV value is within

3% for four replicate samples, which is practically independent
from the variability of the residues in the parent population (CV
from 0.11 to 1.42), which means that the inferences made based
on modelling of sampling uncertainty are generally applicable;
• the precision of the estimation of sampling uncertainty up to six
to eight replicate samples is substantially increasing, however over
eight replicate samples the gain is negligible;
• since the difference in CIr0.95 values are relatively small, based

on their averages, the tendency of the relationship between the
number of replicate samples and the relative 95% range can be
described with an equation CIr0.95 = 3.87p−0.583 with the coeffi-
cient of determination (R2 ) of 0.9939.
10.4.3 Effect of Number of Lots Sampled and

Replicate Samples Taken
Random samples of size 10 were taken 10,000 times from a parent
population (µ = 1; CV = 0.8) representing the typical average vari-
ability of residues in primary samples. The random sampling was
repeated up to 20 times to represent 20 independent decision units.
Replicate random samples of 2, 4, 6 and 8 were taken from each par-
ent population, and the CV values of sample sets (N = 10, 000) were
calculated as described in Section 10.4.1 and shown in Table 10.1.
The results are presented in Fig. 10.5.
The relationship of the relative 95% range of CV values obtained
from p replicate samples drawn from L independent lots are summa-
rized Table 10.4.
The larger exponents in case of two and four replicate samples
reflect the larger uncertainty of CV values estimated based on small
number of replicate samples.
The effect of number of lots tested and number of replicate
samples taken were also studied using the primary residue val-
ues (100–320 per experimental field) measured in 182 independent
crop-pesticide combinations representing independent decision units
(lots, L).
In this case, N = 10, 000 random samples of size 10 were drawn
from each of the 182 primary residue populations.
Data sets of 2, 4, 8, 16, 32, 64 and 128 were selected randomly,
without replacement from the 182 decision units consisting of 10,000
samples of size 10, 2 and 4 times (p). The CVp=2 and CVp=4 value
of the two and four replicates was calculated with Eq. (11.6). The
relative 95% range of the average CVp=2 and CVp=4 values of the 2,
4, 8, 16, 32, 64, 128 and 182 decision units are presented in Fig. 10.6.
1.8
1.6
p2
1.4
p4
1.2
CIr0.95
p6
1
0.8 p8
0.6
0.4
0.2
0
0 5 10 15 20 25
Number of decision units
Figure 10.5. Relationship of relative 95% range of CV values calculated from p

replicate random samples and the number of tested lots.
Table 10.4. Relationship of the rel-

ative 95% range of CV values and p
replicate samples
p CIr0.95 R2
2 2.3349∗ L−0.513 0.9874

4 1.4771∗ L−0.507 0.9955
6 1.187∗ L−0.501 0.997
8 1.0135∗ L−0.502 0.9981
The CIr0.95 values calculated from the synthetic lognormal parent

populations and from the experimental residue values (Table 10.5)
clearly indicate that experimental data result in somewhat larger
variability than those obtained from synthetic lognormal distribution
(µ = 1; SD = CV = 0.8).
Consequently, interferences related to sampling uncertainty
should only be drawn from the experimental data populations.
2.5
2
CIr 0.95
1.5
p2
p4
1 y = 2.4679x-0.439
R² = 0.981
y = 1.4195x-0.442
0.5 R² = 0.9825
0
0 50 100 150 200
Number of lots sampled
Figure 10.6. Relative 95% range obtained with taking p replicate samples from
L decision units.
Table 10.5. Relative 95% range of CV values calculated from synthetic log-
normal parent populations and experimental residue values.
Calculated 95% range of CV Values
Log-norm, p = 2 Experim. p = 2 Log-norm, p = 4 Experim. p = 4

CIr0.95 = 2.34∗ CIr0.95 = 2.47∗ CIr0.95 = 1.48∗ CIr0.95 = 1.42∗
L L−0.513 L−0.439 L−0.507 L−0.442
2 1.64 1.82 1.04 1.42

4 1.15 1.34 0.73 1.04
6 0.93 1.12 0.6 0.77
8 0.8 0.99 0.51 0.64
12 0.65 0.83 0.42 0.57
16 0.56 0.73 0.36 0.47
20 0.5 0.66 0.32 0.42
The variability of calculated values of relative 95% range based

on 15 consecutive random sampling from L = 4, 8, 16 and 20 com-
posite sample data sets (n = 10) obtained from the 182 decision
units of experimental residue data ranged between 3.8 and 5.5%
(Fig. 10.7) indicating that the modelling resulted in robust estimates
1.8
1.6
1.4
1.2
CIr 0.95
p2 L4
p2 L8
1
p2 L16
0.8 p2 L20
0.6
0.4
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Serial number of repeated sampling
Figure 10.7. Variation of values of relative 95% range of the CV values calculated
from two and four replicate samples from 4, 8, 16, 20 lots independently selected
up to 15 times.
which were minimally affected by the concentration level and distri-

bution of residues in the lots from which the samples were taken.
These results reinforce the previous ones that show that the relative
95% range is practically independent from the variability (CV1 ) of
residue concentrations in the parent population.

on the Results of Supervised Trials
10.5.1 Calculation of Sampling Uncertainty from
Replicate Composite Samples
The database derived from supervised residue trials contained repli-
cate samples, therefore the CV values of residues measured in one
replicate sample set (indicated as CV1 ) were calculated with range
statistics (Eq. (10.6)). Because the d2 values are different, the CV
values were calculated separately for replicate sample sets consisting
of two, three or four residue values, and their average for each com-
modity was calculated afterwards. The average of the CVR1 values
calculated from Ni trials with different pesticides was considered as

the characteristic sampling uncertainty of a single (i) commodity
(further referred to as CVRi ).

CVR1
CVRi = i (10.9)
Ni
Samples collected in supervised trials typically consist of 12 large
crops, and 12 medium or small-sized ones or sample increments
(Ambrus, 2016). To account for the difference between the sample
size in supervised trials and official control of commodities (CAC,
1999), the CVSi values calculated for each (i) crop from supervised
√ √
trials were adjusted with a factor of 1.095 (f1 = 12/ 10) for
√ √
medium and small-sized crops and a factor of 1.55 (f2 = 12/ 5 =
1.55) for large-sized crops (large-sized fruits, Brassica vegetables and
cucurbits).
The characteristic sampling uncertainty, CVS , of a crop group
consisting of i crops is calculated from the pooled variances of the
adjusted CVSi values of the crops belonging to one group. All CVSi
values were taken into account.

df i ×CV2Si
CVS = (10.10)
df i
10.5.2 Calculation of Confidence Limits

The approximate confidence intervals for the estimated sampling
uncertainty were calculated based on the Chi-square distribution:

df∗CV2S
LCL = (10.11)
χ0.975

df∗CV2S
UCL = (10.12)
χ0.025
where df, the degree of freedom, is the number of replicate sample
sets of a crop or a crop group, and χ0.975 and χ0.025 are the tabu-
lated values for 97.5th and 2.5th percentiles of the χ2 , distribution,
respectively (MedCalc). In those cases, where χ2 values were not
0.0 0.1 0.2 0.3 0.4 0.5 0.6
Potato /104/
Sugar beet root /51/
Carrot /39/
Radish (root) /28/
Turnip /9/
Beetroot (root) /8/
Chives (fresh) /7/
Figure 10.8. Examples of confidence intervals of CVSi values of crops belonging

to root and tuber vegetables (Number of samples sets (k) is given in brackets).
The darker parts indicate the upper confidence limit.
available to the corresponding df, they were calculated with linear

interpolation.
As the values of relative 95% range obtained from experimental
data showed very small variation (Fig. 10.7), the fitted equation of
CIr0.95,i = 2.4679k−0.439 with R2 = 0.981 (Fig. 10.6) was used to
estimate the confidence intervals around the average CVS , calculated
from N trials, because the supervised trial data sets contained mainly
duplicate samples.
The absolute ranges were calculated by multiplying the values of
relative 95% range with the estimated average CVSi and CVS values.
The upper confidence limit was calculated proportionally to UCL-
CVS that obtained from Chi2 distribution to take into account its
asymmetrical nature.
Examples of CVSi values and their confidence intervals can be
seen in Fig. 10.8. The size of the confidence intervals inversely pro-
portional to the number of replicate sample sets. It is increasing with
decreasing number of sample sets used for calculation of CVSi .
10.5.3 Factors Affecting the Estimated

Sampling Uncertainty
Supervised residue trials are carried out under circumstances differ-
ent from those of normal agricultural conditions (Ambrus, 2016). The
pre-harvest interval (interval between last application and harvest)
is the shortest, the permitted dose and the application frequency are
the highest prescribed in the registration or use permit documents.

Farkas and co-workers examined the possible difference between the
uncertainty of sampling estimated from supervised residue trials and
samples taken from fields treated according to normal farming prac-
tice. Based on the results they recommended a factor of 1.2 to account
for the larger variability that can occur in case of residues mea-
sured in field samples, which must be incorporated in the estimated
sampling uncertainty values for practical use. It was also declared
that the sampling uncertainty cannot be defined if the decision unit
includes crops of different origin (Farkas et al., 2015b).
10.5.4 Sampling Uncertainty Values Estimated

for Crops and Crop Groups
Based on the database of replicate samples derived from 12,087
independent supervised residue trials, the sampling uncertainty was
determined for 107 individual crops (Table 10.6) and 24 different
crop groups (Table 10.7).
Because of the large variability of residues in raw agricultural
commodities (e.g. fresh fruits and vegetables), the sampling uncer-
tainty cannot be determined reliably from a few measurements.
Underestimation of sampling uncertainty may have severe conse-
quences in testing compliance of commodities with legal limit (Sec-
tion 10.10) before offering them for sale. Therefore, the upper con-
fidence limits of the estimated average sampling uncertainties are
also indicated in Tables 10.6 and 10.7 and can be used in case of
pre-marketing self-control and testing the residue levels in raw agri-
cultural commodities used for further processing including cleaning
packing etc. It is the responsibility of the decision maker to choose
whether to use the sampling uncertainty values (CVSprim ) or the
upper confidence limit of the CVSprim values (UCLprim ). Both of
tabulated values were corrected with the recommended factor of 1.2.
Point to note: as the MRLs of pesticide residues refer to the aver-
age residue in laboratory sample satisfying the minimum sample size
and mass requirements, therefore the sampling uncertainty should not
be considered in testing compliance with MRLs.
Table 10.6. Summary of sampling uncertainties of pes-

ticide residues in individual crop units or primary sam-
ple increments recommended for practical use.
Crop Name Na CVSprim UCLprim
Foods
Almond 60 0.49 0.60
Apple 636 0.65 0.70
Artichoke 8 0.20 0.35
Avocado 10 1.5 2.5
Banana 47 0.82 1.0
Barley (grain) 63 0.59 0.73
Basil (dry) 9 0.46 0.77
Basil (fresh) 12 0.22 0.34
Bean 72 0.81 0.98
Bean (dry) 137 1.4 1.6
Beetroot (root) 8 0.76 1.3
Bermuda grass (forage) 8 0.46 0.77
Bermuda grass (hay) 8 0.46 0.77
Blackberry 15 0.50 0.77
Blueberry 74 0.56 0.68
Broccoli 205 0.91 1.0
Brussels sprout 22 1.1 1.5
Cabbage (head) 398 1.1 1.2
Cantaloupe 64 1.0 1.3
Carrot 39 1.3 1.6
Cauliflower 73 1.0 1.2
Celery 267 0.60 0.67
Cherry 189 0.69 0.78
Coffee (dry bean) 18 1.7 2.5
Cotton (undelinted seed) 21 1.7 2.3
Cotton seed 130 1.0 1.2
Cranberry 55 0.43 0.53
Cucumber 111 0.89 1.1
Egg plants 11 0.42 0.67
Endive 16 0.26 0.40
Grape 494 0.79 0.86
Grapefruit 99 0.92 1.1
Hop (cones, dried) 87 0.69 0.82
Hops (fresh) 17 0.85 1.3
Japanese apricot 8 0.37 0.65
Leaf lettuce 769 0.92 0.99
Lemon 195 0.75 0.85
(Continued)
Lima beans 15 1.1 1.7

Lima bean (dry) 13 1.1 1.7
Mandarin 36 1.1 1.5
Mango 32 0.54 0.72
Melon 74 0.94 1.1
Mint leaves 18 0.56 0.82
Mustard green 345 0.47 0.51
Nectarine 9 0.50 0.84
Onion (bulb) 44 1.3 1.7
Onion (green) 29 1.0 1.4
Orange 328 0.69 0.76
Paddy rice (husked grain) 62 0.54 0.66
Papaya 13 1.2 1.9
Pea (dry) 68 0.83 1.0
Pea (edible podded) 18 1.2 1.7
Pea (succulent seeds) 36 0.93 1.2
Peach 409 0.99 1.1
Peanut 52 0.51 0.65
Pear 316 0.66 0.73
Pecan 39 0.64 0.84
Pepper 503 1.1 1.2
Pineapple 21 0.93 1.3
Plum 344 1.0 1.2
Potato 104 0.55 0.65
Radish 61 0.74 0.91
Radish (root) 28 0.83 1.1
Rape 16 0.62 0.93
Rape (seed) 23 0.66 0.92
Raspberry 22 0.60 0.84
Rice (grain) 64 0.40 0.49
Sorghum (grain) 27 0.50 0.68
Soya bean 13 0.79 1.2
Soybean (dry) 128 1.1 1.3
Spinach 304 0.80 0.88
Squash 77 1.1 1.4
Strawberry 215 0.98 1.1
Sugar beet root 51 1.2 1.5
Sugar cane 15 2.0 3.1
Sunflower seed 21 1.0 1.5
Tea (fresh) 52 2.1 2.7
(Continued)
Tomato 624 1.0 1.2

Turnip 9 0.60 1.0
Wheat (fresh grain) 33 0.46 0.61
Wheat (grain) 86 0.865 1.039
Animal feeds
Alfalfa (forage) 48 1.43 1.8
Alfalfa (hay) 64 1.1 1.3
Almond (hull) 238 0.72 0.81
Barley fodder (hay and straw) 93 0.85 1.0
Barley straw 85 0.68 0.81
Bean (green) plant 40 1.2 1.6
Bean forage 31 0.37 0.49
Bean hay 14 1.1 1.7
Beetroot (top) 8 0.34 0.59
Corn forage 247 1.0 1.1
Cotton gin trash 58 0.70 0.88
Maize straw 110 1.1 1.2
Oat foliage 9 0.46 0.77
Pea hay 28 0.67 0.91
Peanut (hay) 133 1.1 1.3
Peanut fodder 13 0.77 1.2
Peanut hulls 89 0.43 0.52
Rice (shoot panicle) 16 0.30 0.44
Rice (straw) 66 1.1 1.3
Rye straw 12 1.0 1.6
Sorghum (fodder) 72 0.98 1.2
Soybean (forage) 184 0.61 0.69
Soybean (hay) 199 0.70 0.79
Sugar beet (top) 261 0.85 0.95
Wheat (forage) 191 0.68 0.77
Wheat (straw) 353 0.70 0.77
a
Number of replicate sample pairs used for estimation of
CVSprim and UCLprim .
10.5.5 Practical Use of the Estimated

Sampling Uncertainties
CVSprim and UCLprim values indicated in Tables 10.6 and 10.7
are the estimated sampling uncertainty expressed in terms of
primary samples. The CVn values corresponding to the appropriate
Table 10.7. Summary of estimated sampling uncertainties for crop groups rec-
ommended for practical use.
Small-sized fruitsb 768 0.96 1.1

Medium-sized fruits 2139 0.52 0.57
Large-sized fruits 560 0.78 0.62
Medium-sized vegetables 1211 1.1 1.2
Bush berries 171 0.98 1.1
Legume vegetables 211 1.1 1.2
Brassica vegetables 698 1.5 1.1
Cucurbits 337 1.4 1.1
Leafy vegetables 1872 0.87 0.92
Root and tuber vegetables 256 0.89 1.0
Stalk and stem vegetables 276 0.59 0.69
Pulses 346 1.2 1.4
Cereal grains 340 0.62 0.71
Grasses, for sugar or syrup production 15 2.9 4.4
Tree nuts 101 0.57 0.72
Oilseeds 247 0.98 1.1
Seeds for beverages and sweets 22 1.6 2.6
Legume forage and fodder 288 0.83 0.96
Straw, hay (of legume feeds) 523 0.88 0.91
Cereal forage, fodder and straw 1176 0.86 0.97
Grass forage 19 0.65 1.1
Grass hay 18 0.46 0.75
Dried herbs 99 0.67 0.84
By-products for animal feed 391 0.66 0.75
a
Number of replicate sample pairs used for estimation of CVSprim and UCLprim .
sample sizes (n) intended to be used for premarket self-control

can be calculated by applying the central limit theorem CVn =
√
CV1 / n. For example, when having a composite sample of orange
from 12 fruits, according to Table 10.6, the CVS value will be
√
0.69/ 12 = 0.20.
CVSprim and UCLprim values for crops calculated from fewer than
eight sample sets (N ) are not given in Table 10.6, because estimates
based on fewer than eight trials may be very inaccurate and their use
is not recommended. In cases of crops when the sampling uncertainty
was estimated from 8 to 20 sample sets, the results should be handled
with care. In these cases, further refinement is needed for the certain
crop or crop group when more information will be available. It is
the duty of the risk manager, or other responsible person to decide

concerning the use of the sampling uncertainty value that belongs to
an individual crop or to choose the more reliable CVSprim or UCLprim
value given for the corresponding crop group.
In case of crops or commodities that are not covered by the esti-
mated typical sampling uncertainties, it is recommended to take a
minimum of six replicate samples from at least eight lots to obtain a
relative 95% range of sampling uncertainty within 50%.
10.6 Summary of Sampling Uncertainty for Pesticide

Residue Analysis
Because of the large variability of pesticide residues, uncertainty of
sampling is often the main contributor to the combined uncertainty of
the measured residues. The main findings of recent studies regarding
the uncertainty of sampling are summarized hereunder.
10.6.1 Methods for the Estimation of

Sampling Uncertainty
According to the studies of Horváth et al. (2013) and Farkas et al.
(2014), drawing random samples from primary residue populations
with computer modelling provides comparable estimates for CVS val-
ues to those obtained with taking composite samples from treated
fields. However, the number of composite samples required to cover
the likely variability of residues within several fields is much lower
than would be required if residues would be measured in primary
samples.
The CVn of composite samples can be calculated from the CV1
of primary samples according to the central limit theorem, and the
CVSprim values summarized in Tables 10.6 and 10.7 can be converted
for different sample sizes (Ambrus and Soboleva, 2004).
10.6.2 Effect of Sample Size on the Uncertainty

of Sampling
The uncertainty of sampling unambiguously depends on the size of
the composite sample. The findings indicate that for pesticide residue
control, taking more than 25 primary samples from medium and large
crops to make up a composite sample is not practical, because over
a sample size of 25, the gain of reducing sampling uncertainty is
marginal. On the other hand, processing and properly comminuting
large amount of material is very difficult in the laboratory, and it
would increase the uncertainty of sample processing and the cost of
determination of pesticide residues.
10.6.3 How Many Lots to be Tested and How Many

Samples To Be Taken?
Horváth and co-workers found that lognormal distribution is the best
for describing the distribution of pesticide residues, however, it may
underestimate the true variability of residues measured in field sam-
ples (Horváth et al., 2013). Examining the effect of the number of
tested lots on the estimated sampling uncertainty from synthetic
parent populations and field samples confirmed this finding; both
modelling methods showed the same tendency regarding the rela-
tive 95% range of estimated sampling uncertainty values, but the
modelling made from the synthetic generated lognormal distribution
resulted in lower values than those made from field samples (Farkas
et al., 2014, 2015a).
It should be pointed out that the results regarding relative 95%
range of sampling uncertainty values were practically independent
from the CV value of the parent residue population.
The relative 95% range of estimated sampling uncertainty rapidly
decreases with the increasing number of sampled lots up to eight
lots, but over 50 lots, the gain is marginal. Between 8 and 20 tested
lots, the confidence intervals are relatively large. This conclusion is
in accordance with the ISO Standard (ISO, 1986). It recommends a
minimum of 20 lots to be tested for the determination of the uncer-
tainty of sampling of bulk materials with as many replicate composite
samples as possible.
Replicate composite samples taken repeatedly from a single field
may give substantially different estimates for the sampling uncer-
tainty. However, for over 12 replicates, the gain in 95% range of
estimated sampling uncertainty gets marginal (Farkas et al., 2014).
There is no optimum for sample size and number of lots to be

tested for the estimation of sampling uncertainty unless the cost of
the experiment is taken into account. The same way as there is no
single optimum solution for preparing sampling plans. It is the task of
the risk managers working for food and feed manufacturers, traders
and government authorities to find the right balance between the
number of samples included in the sampling plans for controlling
quality of the produce or testing compliance with legal limits and
the cost of sampling and analysis as well as the economic loss due to
lower selling price of non-compliant or rejected lots.
10.6.4 Preconditions for Making Reliable Decisions

based on Uncertainty of Sampling
The applicability and limitations of the use of the estimated sampling
uncertainties are summarized in Section 10.5.5. It is emphasized that
reliable decisions can only be made, if the samples are taken accord-
ing to the principles of theory and practice of sampling described in
Chapter 9. The samples should be taken by well-trained sampling
officers provided with the necessary tools and information on the
sampling target (decision unit) and the objectives of the analysis
of pesticide residues based on the sample. It is the responsibility of
the owner of the commodity to be sampled to separate and clearly
identify the parts of the sampling target that have different origin
and, consequently should be sampled separately.
10.7 Effect of Handling of Laboratory Samples on

the Accuracy and Uncertainty of the
Results of Analyses
10.7.1 Sources of Bias
The concentration of pesticide residues may change during
a. shipping the samples to the laboratory,
b. storing the samples before analyses,
c. preparation and processing of samples,
d. storage of analytical standard solutions, and
e. extraction, cleanup and instrumental analysis.
To avoid or limit the loss of residues during shipping and storage,

the time between sampling and delivery to the laboratory should be
kept as short as possible. The properly packed samples should be
stored in a cool environment, for instance by placing blue ice or dry
ice around it. Upon arrival at the laboratory, the not deep-frozen
samples should be prepared without delay and placed in deep-freezer
at or below –20◦ C until further processing. Information on storage
stability of residues in representative matrices is available from the
technical documentation provided by the petitioner for registration
or authorization of the pesticide. In addition, detailed information
can be found in the JMPR Evaluations (FAO, 1997–2015) of those
pesticide residues that are included in the Codex Alimentarius pro-
gramme (see Chapter 7).
Losses due to room temperature and dry ice cryogenic comminu-
tion have been shown to range from highly significant to negligible
depending on the analyte — commodity combination (Hill et al.,
2000; Fussel et al., 2007a, 2007b; El-Bidaoui et al., 2000). The use of
blending in the presence of dry ice has been suggested to prevent or
reduce losses for pesticides such as dithiocarbamates, chlorothalonil,
captan, captafol, chlozolinate, folpet, dichlorvos, dicofol, dichloflu-
anid, etridiazole and iprodione but losses in processing with dry ice
cryomilling still vary with commodity (Fussel et al., 2002, 2007b;
Alder et al., 2000).
Depending on the objectives of the analyses, the portions of lab-
oratory samples to be analysed are separated from the rest of the
sample material. This process may involve, for instance, removing the
withered outer leaves of leafy vegetables, rinsing root vegetables to
remove adhering soil, removing stones from peach and mango or peb-
bles and other foreign materials from soil samples. It is a very impor-
tant process as it may significantly change the measured residue level
and produce biased results. The above operations are called sample
preparation according to the Codex and JMPR terminology, and
they should be clearly distinguished from sample processing, which
includes comminuting the portion of the sample to be analysed to
obtain a statistically well-mixed, so-called homogeneous, matrix from
which the test portions are withdrawn for analyses. The 2010 revision
of Codex GL-1993 provides a detailed instruction on the preparation

of samples for testing compliance with MRLs (CAC, 2010).
Sample preparation: The procedure used, if required, to convert the

laboratory sample into the analytical sample, by removal of parts
(soil, stones, bones, etc.) not to be included in the analysis (CAC,
1993).
The first of the two processes, which may be required to convert
the laboratory sample into the test sample is the removal of parts
that are not to be analysed, if required (SANCO, 2013).
Sample processing: The procedure(s) (e.g. cutting, grinding,

mixing) used to make the analytical sample acceptably homoge-
neous with respect to the analyte distribution, prior to removal of
the analytical portion. The processing element of preparation must
be designed to avoid inducing changes in the concentration of the
analyte (CAC, 1993).
The second of two processes which may be required to con-
vert the laboratory sample into the test sample. The process of
homogenization, comminution, mixing, etc., if required (SANCO,
2013).
10.7.2 Variability of Residues in Processed Samples

The inherent variability of pesticide residues among discrete elements
(primary samples) of the laboratory sample is called compositional
heterogeneity (CH) and the difference in residue concentrations
within portions of individual crop units also adds distributional het-
erogeneity (DH), for example, different residue concentrations in the
peel and pulp of fruits, outer and inner leaves of cabbage or head
lettuce. The CH and DH result in inevitable variation of average
residues in test portions taken from the comminuted laboratory sam-
ple. In addition to heterogeneity, the sub-sampling of large crops (e.g.
watermelon, jackfruit) will further increase the variability of average
residues in test portions.
According to the sampling guidelines (CAC, 1999; European

Commission 2002) a minimum of five large crops shall be taken
randomly from the decision unit (sampling target) to make up one
laboratory sample. For instance, the total mass of samples of head
cabbage, watermelon and jackfruit can be 25–75 kg. That amount of
material cannot be handled in a regular pesticide residue laboratory,
consequently the mass of sample has to be reduced. The selection of
one or two units might introduce significant gross error and should
not be done. The size reduction should be made by cutting segments
of possibly the same mass in longitudinal directions to represent the
same or very similar mass surface ratio as it is in the original crops
(Fig. 10.9). Cutting slices is not permitted as it would change signif-
icantly the surface mass ratio leading to biased results if the residues
are not uniformly distributed within the crops. Fig. 10.10 illustrates
the ways for proper sample size reduction.
Omeroglu et al. (2013) studied the uncertainty of mass reduction
of large crops. Each of the five fruits making up the laboratory sample
were cut into six longitudinal segments and analysed independently.
One segment was selected from each fruit with repeated random sam-
pling without replacement, and the weighted average residue in six
segments was calculated.
The relative uncertainties were 17 and 21% for field-treated jack-
fruits and cucumbers, respectively, and 7% for post-harvest treated
papaya. The results indicate that the variability resulting from sub-
sampling, even if it is correctly performed, may be a significant com-
ponent of the combined uncertainty of the results, and cannot be
neglected.
The concept of sampling constant proposed by Ingamells and
Switzer (1973) was used to determine homogeneity of processed lab-
oratory sample (Ambrus et al., 1996; Maestroni et al., 2000; Tiryaki
and Baysoyu, 2006). Ingamells’ constant (KSp ) is the weight of a test
portion (m) from a well-mixed material where the relative sampling
uncertainty (CVSp = Sa /a) of the critical analyte concentration (a)
is held to 1% at 68% confidence:
KSp = m × CV2Sp (10.13)
Figure 10.9. Cutting segments from large fruits. Upper: jackfruit 16.5 kg; lower:
papaya 2.75 kg. For jackfruit and similarly large fruits, each fruit might need to
be divided into 12 segments to obtain sample mass that can be comminuted in a
blender (approximate mass of reduced sample would be about 7 kg). One segment
is selected from each fruit for further processing.
If the material is well mixed, then the KSp is constant for small
(msm ) and large (mlg ) test portions; therefore:
mL × CV 2lg = msm × CV 2sm (10.14)
msm
s2lg = s2sm × (10.15)
mlg
The well-mixed condition of the comminuted laboratory sample
can be verified by analysing large and small test portions repeat-
edly (mlg ≥ 10msm ). Since the determination of slg and ssm is rel-
atively imprecise, Wallace and Kratochwill suggested to test with
one sided F -test at 90% or lower confidence the null hypothesis,
namely that the two sides of Eq. (10.15) are not different (Wallace
and Kratochwill, 1987). If the test indicates that the difference is
Figure 10.10. Cutting representative portions of large crops.
not significant, the comminuted sample can be considered statis-

tically well mixed. Then the uncertainty of sample processing can
be calculated from the replicate (≥ 7) measurements of small and
large test portions (CVL ) of treated crops and concurrent recovery
studies (CVA ) carried out with untreated sample materials. Note: If
subsampling is involved in the sample processing, then the repeated
analyses shall be carried out with subsamples obtained from different
segments of the large crops or portions of bulk material reduced with
appropriate technique.

CVSp = CV2L − CV2A (10.16)
The test should preferably be carried out (Ambrus et al., 1996; Mae-
stroni et al., 2000) with sample matrices containing stable residues
in well-detectable concentration. Alternately, the surface of sample
material can be treated with a test substance, and then the sam-
ple is processed either at ambient temperature or under cryogenic
conditions.
Maestroni et al. (2000) spiked the surface of samples with radi-
olabelled chlorpyrifos and measured the 14 C activity. Scintillation
counting is quick and robust using labelled analyte, and the uncer-
tainty of analysis is <2%, which makes it negligible compared to that
of the processing. This method is an excellent validation tool. How-
ever, validations using spikes may not capture all the heterogeneity
of incurred, more tightly bound residues. Furthermore, using radiola-
belled compounds is not readily accessible in routine pesticide residue
laboratories. Therefore, applying a mixture of stable compound and
several others of unknown property in known concentrations for sur-

face treatment is a practical option (Ambrus et al., 2016), enabling
to test the efficiency of sample processing and stability of analytes
at the same time.
Maestroni et al. (2000) and Fussell et al. (2007a) demonstrated
between-commodity variations for the same pesticides. In order to
obtain the same between-analytical test portion variability for room
temperature and dry ice cryogenic processing 110 g vs. 5 g, respec-
tively, was needed for tomatoes, but 5 g of oranges were sufficient
for either method. Microscopic evaluations of room temperature
comminuted broccoli, corn, oranges, oysters and squash showed sig-
nificant differences in the physical characteristics in different com-
modities (Tiryaki and Baysoyu, 2006; Ambrus et al., 2016).
The variability of residues in test portions is inversely propor-
tional to the square root of the mass of test portions extracted and
proportional to the third power of the diameter of the largest particles
in the comminuted sample matrix. Assuming that the mass of the
laboratory sample (ML ) is much larger than the mass of test portion
(Ms or mTp ), Eq. (9.1) in Chapter 9 describes the same relationship
as Eq. (10.13):

3
2 Cd KSp
CVSp = , CV Sp = 2 (10.17)
Ms mTp
The first part of Eq. (10.17) provides a relationship of CVSp to the
diameter of the largest particles (more precisely the upper 95th per-
centile of the diameters of the particles in the comminuted matrix
according to Gy’s theory) that is included in the overall constant
K in the second part of Eq. (10.17). This relationship to diameter is
important in the reduction of heterogeneity when milling laboratory
samples.
Maestroni et al. (2000) compared comminution using choppers,
blenders and dry ice for 1, 25, 50 and 250 gram test sample sizes.
The efficiency of laboratory sample comminution was shown to be
dependent on the equipment, method and commodity, but it was
independent of the analyte. It was also recommended that comminu-
tion should reduce peel to <0.5 mm sizes to keep CVSp < 0.10 in
case of 25 g test portion. Double blending and blending with dry

ice provided two- to four-fold and two- to three-fold improvements,
respectively. Apple and tomato laboratory sample processing uncer-
tainty was reduced six-fold by double blending with dry ice. Note,
however, that double blending may result in substantial loss of labile
analytes. Provided that the processing conditions remain the same,
if the mass of test portion is decreased tenfold, the CVSp will be
3.2 times higher (Eq. (10.17)). Therefore, testing the efficiency of
the sample processing should be included in the method validation.
Reduced test portion mass should only be used after verifying that
the resulted CVSp is preferably <0.3CVA , or the combined uncer-
tainty of determination of residues (CVL ) remains acceptable for the
purpose of the analyses.
As seen in Eqs. (9.1) and (10.17), the variability of residues in
test portions is not only related to the mass of the test portion but
the diameter of the largest particles. This relationship is particularly
important when significantly reducing the size of the test portion.
Extensive work is ongoing in this area, applying cryomilling, gen-
erally performed at reduced temperatures, using either dry ice or
liquid nitrogen, to reduce the particle size to around 350 µm. Results
of Saha, Li, Gooding, and their co-workers, presented at the annual
meetings of the American Chemical Society in 2013, 2014 and at the
13th IUPAC International Congress of Pesticide Chemistry, revealed
that similar CVL can be obtained with 0.05–0.2 g test portions after
cryomilling as with 2.5–5.0 g test portion processed with usual labo-
ratory mills such as vertical cutter mixer (Lehotay and Cook, 2015;
Lehotay et al., 2015b). Riter et al. (2015) expanded upon this work
with an inter-laboratory assessment of the two-stage dry ice/micro
milling. Preliminary results like these demonstrate the value of micro-
milling and high-throughput testing.
While useful in general terms, Gy’s equation for FSE, as seen in
Eqs. (9.1) and (10.17), is only applicable to samples where the par-
ticles of the laboratory sample are very uniform in size, shape and
composition. Foods are frequently not uniform enough in composition
to assume that there is a simple, direct relationship between the par-
ticle size diameter (d3 ) cubed and the mass (Ms ) of the sample. The
constant C can change significantly and unpredictably with particle

size reduction. Additionally, contributions from distributional het-
erogeneity and other sampling errors are not accounted for by this
equation. There are significant differences in the heterogeneity of the
wide variety of pesticide — commodity combinations. For each new
application, validation studies with incurred residues are needed to
demonstrate that sufficient mass is comminuted at each stage of the
laboratory processing and that no additional variability or bias is
introduced.
10.8 Uncertainty and Trueness of Analysis of

Residues in Test Portions
The uncertainty of the determination of pesticide residues in test
portions depends on the method used. Most of the steps of the anal-
ysis affect both the uncertainty, the trueness and accuracy of the
measured residue concentration and the magnitude of their effects
can usually be determined by the same tests. The CAC/GL 59-2006
provides a detailed list of sources of systematic and random errors
in preparation of the test portions and in analyses of samples (CAC,
2006).
Accuracy: Closeness of agreement between the result of a measure-

ment and the (conventional) true value of the measurand (CAC,
2009).
Trueness: The closeness of agreement between the average of an
infinite number of replicate measured quantity values and a ref-
erence quantity value. Note: Measurement accuracy should not be
used for ’measurement trueness’ and vice versa (CAC, 2009).
Systematic error: Component of measurement error that in repli-
cate measurements remains constant or varies in a predictable
manner (CAC, 2006).
Bias is the total systematic error as contrasted to random error.
There may be one or more systematic error components contribut-
ing to bias. A larger systematic difference from the accepted ref-
erence value is reflected by a larger bias value (CAC, 2006).
The fitness of the method for intended purposes is judged

based on its performance characteristics determined initially during
method validation (OECD, 2007). They may be continuously refined
and expanded to include additional sample materials and pesticide
residues (CAC, 1993), based on the data of regular internal quality
control and specific tests required by the method validation proto-
cols. Full validation experiments should be performed on at least
one raw agricultural commodity (RAC) from each of the relevant
representative commodity groups (SANCO, 2013) (high water con-
tent, high acid content and high water content, high sugar and low
water content, high oil content and very low water content, high oil
content and intermediate water content, high starch and or protein
content and low water and fat content; meat (muscle) and seafood,
milk and milk products, eggs, fat from food of animal origin, difficult
and unique commodities). The number of commodity groups tested
can be adjusted to the intended use of the method. The scope of the
method can always be extended later on.
The extraction efficiency is regarded as a key element of the per-
formance characteristics of the method, as it may significantly influ-
ence the accuracy of the analytical results. Poor extraction efficiency
can be a major source of bias in a method. However, it cannot be
checked by traditional recovery studies carried out with samples for-
tified shortly before analysis. Where data are available, the efficiency
of the sample extraction steps used in the analytical methods can be
compared with radiolabel measurements on residue components in
samples from the metabolism studies (Ambrus, 2016).
Once the efficiency of extraction is verified, the precision and
trueness of the methods are preferably determined with ≥ 10 repli-
cate recovery tests performed with all analytes included in the scope
of the methods and representative commodities, which do not contain
the analytes of interest in detectable concentration (blank sample).
If blank sample is not available, the recovery tests may be performed
with thoroughly homogenized sample materials containing the ana-
lyte(s) in known concentration. The test portions can be spiked with
mixtures of pesticide analytical standards, which can be well sepa-
rated and quantitatively determined with the available instruments.
Recovery: Proportion of the amount of analyte, present in or added

to the analytical portion of the test material, which is extracted and
presented for measurement (Thompson et al., 1999).
Barwick and Ellison (2000) prepared detailed guidance on use

of validation data for estimation of uncertainty. Some examples for
the most frequently occurring situations are given in the following
sections.
10.8.1 Basic Rules of Propagation of Error

Detailed description of principles is given in EURACHEM/CITAC
Guide (Ellison and Williams, 2012). The relevant parts are summa-
rized hereunder in a simplified form.
(a) The results of calculation are obtained with the linear combina-
tion of measured values
Y = C1 P ± C2 Q ± C3 R · · · (10.19)
The random error of the result is calculated as:

S(y(xP,Q,R ) = (C1 × sP )2 + (C2 × sQ )2 + (C3 × sR )2 . . .
(10.20)
where SP , SQ and SR are the standard deviations of the mea-
surements of P , Q and R.
The systematic error is calculated as
∆y = C1 × αP + C2 × βQ + C3 × γR . . . (10.21)
where αp , βQ and γR are the systematic errors of P , Q and R
measured components.
(b) The final result is obtained with multiplication or division:
k×P
y= (10.22)
Q×R
sP
CVP = (10.23)
Y
The random error is calculated as

CVY = k × CV2P + CV2Q + CV2R (10.24)
The systematic error is calculated as

α β γ
∆y/y = + + (10.25)
P Q R
The systematic errors can be either positive or negative and may
compensate each other, therefore the sign must be included in
Eq. (10.25).
10.8.2 Examples
Example 1. Determination of trueness.
The trueness of the result can be simply calculated as the arith-
metic mean, Q̄, of n, replicate recovery tests carried out with blank
sample material. Ideally a trueness study should be performed with
≥ 25 replicates (ISO, 1994). However, for practical reasons, Barwick
and Ellison (2000) suggests ≥ 10, and the FAO Manual (Ambrus,
2016) requires the analyses of minimum five replicates at each of the
minimum two fortification levels together with two control samples.
Before calculation of the average recovery, the recovery values
obtained shall be checked for outliers with Grubbs test (ISO, 1994).
The test statistics are given by
Qn − Q̄ Q̄ − Q1 Qn − Q1
G highest = , Glowest = , G =
s s s
(10.26)
where Q1 or Qn are the suspect value, or Q1 and Qn are both suspect
values (G ), and s is the standard deviation calculated with all data.
The critical values can be obtained, for instance, from the ISO 5725
standard or from the Internet (Grubbs test). According to ISO 5725,
when the test statistics calculated is smaller than the critical value
at 95 level the suspect value is not an outlier. In cases where the
statistics calculated is between the 95% and 99% critical values, the
suspect value is a straggler though kept in the data set. Finally, if
the tests statistics is higher than the critical value at 99% level, the
suspect value is to be considered as an outlier.
Point to note: The critical values should be selected for two-sided
test, as given by ISO5725 (ISO, 1994).
For the ith analyte, the recovery is calculated from the recovered
concentration (Ci,rec ) and the spiked concentration (Cspike ):
Cirec C̄irec
Qi = or Q̄i = (10.27)
Cspike Cspike
Many laboratories include several hundreds of residues in the scope

of their multi-residue methods. To reduce the cost, it is advisable to
perform the recovery tests with standard mixtures including as many
analytes (h) as can be quantitatively determined together. Typical
recoveries can be conveniently calculated for subgroups of compounds
with single factor ANOVA using the built-in algorithm in MS Excel.
To identify the subgroups, a simple statistical test, the least signifi-
cant difference (lsd) is calculated (Miller and Miller, 2010):

2
lsd = t0.05,h(n−1) × s (10.28)
n
where s2 is the within sample estimate of the expected variance of

the typical recoveries:

Vi
s= (10.29)
h
The calculation is illustrated, with ordered average recovery values,
in Table 10.8 with h = 9 and n = 3 replicate measurements. In
reality, n should preferably be ≥ 5 and h can be unlimited. The lsd,
once computed, permits to tell whether any pair of means in the
recovery values are significantly different or not (Sokal and Rohlf,
1995).
The calculated lsd is 0.097. If the difference between the recov-
eries of neighbouring compounds, arranged in increasing order, is
less than the lsd, then we can assume that their average recover-
ies are statistically not different. It can be seen from the average
results that the mean recoveries of methamidophos and permethrin
are significantly different from lindane and p, p -DDE, respectively.
Therefore, these compounds could be excluded from the calculation
of typical recovery.
Table 10.8. Calculation of typical recovery from test

portions spiked at X (mg/kg) level.
Replicate
Analyte (h) Measurements (n) Mean VAR
Methamidophos 0.70 0.77 0.73 0.732 0.007

Lindane 0.80 0.83 0.87 0.833 0.001
Endosulfan 0.85 0.90 0.93 0.894 0.002
Azinphos-methyl 0.83 0.92 0.95 0.898 0.004
Propargite 0.87 0.93 1.00 0.933 0.004
Chlorpyrifos 0.87 1.00 1.00 0.956 0.006
Ethion 0.87 1.02 1.03 0.972 0.008
p, p -DDE 0.95 0.97 1.03 0.983 0.002
Permethrin 1.10 1.10 1.10 1.100 0.000
Average 0.004
Points to note:
• The lower recovery of methamidophos can be explained with its

practically unlimited water solubility. However, there is very lit-
tle difference in the physical-chemical properties of p,p –DDE and
permethrin. Since the lsd test is not very powerful, the pro-
fessional judgement of the analysts is always required for the
correct interpretation of the results of statistical tests. There-
fore, the analyst may decide to include p,p -DDE in the calcu-
lation of typical recovery of 0.946. The methamidophos may be
grouped together with other highly water-soluble residues such as
dimethoate, acephate, etc.
• It is not necessary that the same number of recovery tests is avail-
able for each analyte.
• If many analytes are included in the test mixture, it is quite likely
that several sub-groups can be formed, and their typical recoveries
and their random variations can be calculated for each sub-groups.
• If the typical recoveries of compounds in one sub-group are not sig-
nificantly different at different spike levels, the typical recovery,Q,
should also be calculated for properly selected analyte concentration
range for either individual compounds or sub-groups of h com-
pounds based on recovery tests performed at around the limit of
quantification, at the expectable highest residue level (or at estab-

lished MRLs) and intermediate concentrations to cover residues
present in the samples. The numerical calculation is the same as
shown above.
Example 2. Uncertainty of recovery values.

The uncertainty is calculated as the standard deviation of n
repeated measurements excluding the identified outliers. If n is ≤3,
the standard deviation should be calculated with Eq. (10.6).
Before the typical uncertainty applicable for all members of a
subgroup is calculated, the homogeneity of variances of recoveries
of individual compounds (variances are statistically not different)
has to be verified with Cochran test, which compares the maximum
observed variance to the sum of variances of the dataset (ISO, 1994).
The test statistics is:
Vmax
C= (10.30)
Vi
If the calculated C is smaller than the critical value, than we
can assume that the variances observed are coming from the same
population. The critical values can be taken from statistical hand-
books, ISO5725-2 or the Internet (Cochran test). For the eight com-
pounds showing similar recoveries in Table 10.8, the calculated C
value (0.304) is smaller than the critical value (0.4775) at α = 0.05
significance level. Therefore, a typical repeatability CVr value can be
calculated.
Point to note: The number of replicate measurements within one
subgroup should be the same. However, if h is large a few missing
value may be permitted (ISO, 1994). When n is different for various
analytes, for instance, the Bartlett test can be used (Bartlett test).
The uncertainty of the recovery of those residues that do not
fit in the subgroup shall be calculated separately, and the typical
uncertainty shall be calculated without them.
Once the homogeneity of variances is confirmed, the typical
uncertainty of the recoveries (uQ ) carried out at the same spike level
is calculated from the pooled variances of recoveries of compounds
being in the subgroup:

df 1 × V1 + df 2 × V2 + · · · df h × Vh
uQ = (10.31)
df i
where the dfi is the degree of freedom of the calculated variances (Vi )
for the individual compounds.
When the recovery tests are carried out at different spike concen-
trations, then the variance is concentration dependent. Therefore, the
Cochran test is carried out with CV2 values. If it indicates that they
may come from the same population, then the typical uncertainty
shall be calculated from the corresponding CV values (Barwick and
Ellison, 2000):

df 1 × CV21 + df 2 × CV22 + · · · df h × CV2h
CVp =
df 1 + df 2 + · · · .df h

df i × CV2i
= (10.32)
df i
The pooled CVP will characterize the repeatability or reproducibil-
ity of the procedure depending on the conditions under which the
recovery tests have been performed. The typical uncertainty, utyp , of
the recovery studies is calculated by multiplying the CVp with the

typical recovery, Q. The degree of freedom is equal to: df i .
Example 3. Long-term reproducibility of the results.
It can be calculated from the results of repeated analyses of

retained test portions taken from the homogenized sample materials
in which residues were measured. Calculation is illustrated with an
example in Table 10.9.
The Cochran test is performed with the CV values. It gives only
approximate estimate because the Cochran test is for variances and
the CV2 are not unbiased estimate of variances except in very large
samples. The test statistic (0.623), calculated using Eq. (10.30) is
smaller than the critical tabulated value of 0.78. Consequently, the
suspect CV of 0.443 is not an outlier, and CVtyp is calculated from
Table 10.9. Illustration of the calculation of

CVtyp from replicate measurements.
Residues
Measured (mg/kg)
No of Tests 1st 2nd CVaR CV2
1 0.06 0.1 0.443 0.196

2 0.35 0.34 0.026 0.001
3 0.6 0.46 0.234 0.055
4 0.75 0.75 0 0
5 1.19 1.55 0.233 0.054
6 1.57 1.41 0.095 0.009
Average CVR = CVtyp 0.1718
a
Calculated using Eq. (10.6).
the six replicate measurements. The degree of freedom of the corre-

sponding standard deviation is six.
The results of the continued reanalyses of replicate test portions
can be used alone or combined with those obtained during the initial
validation to get a more robust estimate of the reproducibility of the
results obtained with the method.
Example 4. Estimation of uncertainty of reported concentra-
tion of an analyte corrected for the recovery of the method.
Measured residue is adjusted with a single recovery value obtained
from blank sample
The measured concentration is R0 and the recovery (Q) is 0.85
with a reproducibility CVrec and CVA value of 12%. The reported
result is:
R = R0 /Q (10.33)

CVR = CVA + CVrec = 2 × CV2A = 0.1697
2 2
(10.34)
The result is reported with expanded uncertainty: R ± 2 × CVR =
R ± 0.34∗ R0 .
The average recovery is determined under reproducibility
conditions.
The measured residue value was divided by the average recovery

obtained from 15 replicate tests under reproducibility condition. The
uncertainty of the reported result is:

CV2A 0.122
CVR = CV2A + = 0.122 + = 0.1239 (10.35)
15 15
The result reported with expanded uncertainty: R ± 0.24∗ R0 .

Note that adjusting the measured value with an average recovery
obtained with ≥ 15 replicate tests only slightly increases the uncer-
tainty of the reported results (12 ↔ 12.4%) in contrast to applying
the result of a single recovery test (17%). If the recovery of the ana-
lyte has to be taken into account, the average recovery should be
determined and used.
No blank sample is available.
The recovery of the analyte is determined with spiking (Cspike )
the test portion containing the analyte (C0 ), and the measured con-
centration in the spiked test portion is Crec . The typical CVA value
is 0.12.
The recovery calculated from n replicate tests is:
C̄rec − C̄0 δ
Q= = (10.36)
Cspike Cspike
The calculation is performed in two steps:

(i) sδ = s2rec + s20 (10.37)
sδ
(ii) CVδ = (10.38)
δ

CVQ = CV2δ + CV2spike (10.39)
Under normal conditions CVδ CVspike and CVQ ∼ CVδ because

adding 100 µl–1 ml to the test portion with Hamilton syringe has an
approximate relative uncertainty of ≤0.01.
Example 5. Calculation of reported concentration of

dimethoate and its metabolite omethoate.
The reported value is the sum (CDT ) of the measured dimethoate

and omethoate concentrations expressed as dimethoate and it is
calculated as:
CDT = CD + k × CO (10.40)
where CD is the measured concentration of dimethoate, CO is the

measured concentration of omethoate and k is a factor for molecular
weight correction.
The precision study carried out previously resulted in relative
standard deviations of CVD and CVO .
sD = CD × CVD ; sO = CO × CVO (10.41)

uDT = sDT = (CD × CVD )2 + (k × C O × CVO )2 (10.42)
The reported result is: CDT ± 2uDT .
10.9 Quality Control of the Determination

10.9.1 Sampling
Inherently large within field variation of pesticide residues in crop
units or single increments characterized by a typical average CV of
0.8, according to Eq. (10.1), results in the theoretically expected
CVs of 0.37, 0.26 and 0.16 of the average analyte concentrations
in composite samples of size 5, 10 and 25, respectively. Drawing
1,000 times, two and four replicate random samples from parent
population of samples of size 10 yielded minimum and maximum
relative residue ranges of 0–0.95 and 0.062–1.38, respectively. The
results clearly indicate that the reliability of sampling cannot be
verified based on replicate random samples. The only way of obtain-
ing reliable representative samples is to follow the guidance given in
Section 9.10.
10.9.2 Sample Processing and Analysis

The number of possible combinations of pesticide residues and sam-
ple materials is practically infinite. Methods can only be validated
with representative members of crop groups and all residues, which
are in the scope of the methods following the guidelines for method
validation (Alder et al., 2000; Magnusson and Örnemark, 2014;
SANCO, 2013). The validation protocol should include testing the
stability of analytes in all phases of the laboratory operations and
the uncertainty of sample processing.
The efficiency of sample processing may vary from sample to
sample, therefore it should be regularly tested by spreading a small
portion of the homogenized laboratory sample on a Petri dish and
visually observing the particle sizes. In addition, retained test por-
tions, containing analyte(s) in well detectable concentration(s), should
be blindly re-analysed a few weeks later. If the results of the original
analysis (R0 ) and the re-analysis (R1 ) are within the critical range
(ISO, 1994), then one can conclude that the reproducibility of method
is consistent with that obtained during validation of the method:
|R1 − R0 | ≤ 2.8 × CVL × R̄ (10.43)
where R̄ is the average of R0 and R1 and CVL is the reproducibility
of the method established during method validation with a minimum
of 15 measurements repeated under reproducibility conditions. For
three and four replicate measurements, the factor will be 3.3 and 3.6,
respectively.
In order to test the stability of analytes in test portions dur-
ing long-term storage, several test portions, containing one or more
well-detectable stable compound(s), can be withdrawn from the com-
minuted laboratory sample and spiked with analytical standard mix-
tures of known concentration. One portion is analysed at the time
of the sample preparation, while the others are stored in refrigerator
or freezer and re-analysed at various time intervals. Based on the
results, the reproducibility of the measurement can be tested with
the stable compound and the storage stability of the others can be
tested by comparing the original results to the new ones.
10.10 Use of Combined Uncertainty of Measured

Residues to Verify Compliance with
MRLs and Settling Disputes
The maximum residue limits are defined as the legally permissible
maximum residue concentration of pesticide residues in the appro-
priate portion of commodities (CAC, 2010) making up the composite
samples of specified mass and size (CAC, 1999; European Commis-
sion, 2002) at the time when the product is first offered for sale.
The term ‘pesticide residue’ includes all residue components that are
defined for enforcement purposes by the JMPR (Chapter 4) or the
responsible regulatory agencies (Chapter 3).
This definition excludes the uncertainty of sampling from the
control of compliance of marketed commodities. The uncertainty
of the laboratory phase of the determination of pesticide residues
(Eq. (10.2)) shall be estimated according to the principles summa-
rized in Sections 10.2.2. and 10.9.
It follows from the definition of MRL that during the official
control, the average residue of any single random composite or bulk
sample satisfying the minimum requirements of sampling standards
should comply with the MRL. On the other hand, before the product
is placed on the market or exported, it should be verified that all
parts or at least high percentage of the lot in terms of the minimum
mass of composite (bulk) sample (CAC, 1999; EC Directive, 2002)
contain residue at or below the corresponding MRL. To do that, the
contribution of sampling to the combined uncertainty of the results
(Eq. (10.1)) should be considered.
The general principles of conformity assessment are described in
the guide prepared by the Joint Committee for Guides in Metrology
(JCGM, 2012) and the basic situation is depicted in Fig. 10.11.
Conformity assessment: Activity to determine whether specified
requirements relating to a product, process, system, person or body
are fulfilled (JCGM, 2012).
The product is rejected if R−U > MRL, and accepted if R+U ≤
MRL. For the central cases, decision can only be made if the decision
rules are defined. In the absence of a decision rule for such cases, the
conclusion must be in doubt.
Valid rejection
False rejection?
False acceptance?
MRL
Valid acceptance
Figure 10.11. Illustration of acceptance and rejection of a lot based on measured

residue (R) values and combined expanded uncertainty (U ) of the results.
The problem is that the guidance documents and regulations

on conformity assessment do not specify what kind of uncertainty
shall be taken into account, which may often lead to dispute between
sellers and buyers. It should be agreed between the trading partners
in advance.
The regulatory agencies generally follow the JCGM guidance and
reject a lot if the average residue in laboratory sample exceeds the
maximum limit beyond reasonable doubt taking into account the
correction for recovery, if required, and measurement uncertainty.
For pesticide residues, many agencies follow the Codex recommen-
dation and do not correct the results with the analytical recovery.
The European Union defined a default value of 50% for the com-
bined expanded uncertainty of the measured residues, which should
be taken into account in deciding on the compliance of tested lot. It
means that the sampled lot will be considered non-compliant if the
measured residue is ≥2MRL.
With this decision rule, the value of the measurand is above the
MRL with at least 97.5% confidence (SANCO, 2013).
Considering the distribution of pesticide residues in composite
samples of size 10 (Fig. 10.12), it is obvious that if, the true residue
content of the lot is equal to the MRL (1 mg/kg in the given example)
12 120%
MRL= 1 mg/kg
10 100%
Cumulative frequency
8 80%
6 60%
4 40%
2 20%
0 0%
0 1 2 3 4
Residue in composite samples [mg/kg]
Figure 10.12. Distribution of pesticide residues in composite samples of size 10.

The cumulative frequency curve crosses the MRL=1 mg/kg at 53.72%. Conse-
quently, the residues in about 46% of the samples will be above the MRL.
during the pre-marketing self-control, and the measured residue is

compared to the MRL of 1 mg/kg, wrong decision will be made
in about 46% of the cases, because the residues in other compos-
ite random samples may exceed the MRL. Therefore, if the owner
wants to assure that the commodity would conform to the MRL
with high probability an action limit (AL) lower than the MRL
should be selected taking into account the combined uncertainty of
the results including the sampling uncertainty. The AL, often called
‘guard banding’ (Ellison and Williams, 2007) should not be exceeded
by residues in random samples taken from the targeted lot.
The action limit depends on the sample size, number of replicate
samples tested and the level of risk considered acceptable by the
owner for getting the product rejected as a result of official control.
Equations (10.44) and (10.45) describe the basic relationship
between AL and MRL:
MRL = AL + k × CVR × AL (10.44)
MRL
AL = (10.45)
1 + kCV R
The value of k depends on the targeted compliance level (Pc ) or the

acceptable violation rate (Pv = 1 − Pc ) agreed between the buyer
and seller (decision rule). For fruits and vegetables, the distribution
of residues in composite samples of size ≥25 approximates normal
distribution. In this case the k is equal to the corresponding stan-
dard normal variate (Z). Normal distribution of the results can also
be assumed for the analyses of well mixed liquid samples such as
milk, wine, refined oil, etc. In this cases, it can be expected that the
CVS << CVA .
In other cases, lognormal distribution describes best the relative
frequency distribution of pesticide residues (Horváth et al., 2013)
and the residue values after log-transformation follow normal dis-
tribution. Details of the calculation are described by Farkas et al.
(2015b).
Figure 10.13 depicts, for example, the operation characteristic
curves obtained following different sampling plans.
100
90
80
Probability of Accepting Lot (%)
70
60
50
40
30
20
10
0
0 1 2 3
Lot Pesticide Concentration (mg/kg)
(1) 1x10 primary samples, AL < 0.3 mg/kg
Figure 10.13. Operation characteristics curves for sampling plans with 1, 2 and
4 replicate samples of size 10. The figure shows that 8% of the lot may con-
tain residue above the 1 mg/kg MRL even if four replicate independent random
samples were analysed and each had residues ≤0.95 mg/kg.
Calculation of OC curves for many practical situations revealed

that an AL of 0.3MRL would assure over 99% compliance of the
sampled fruit and vegetables commodities (sample size >≥ 10) in
> 95% of the cases, provided that the sampled commodity was a
single lot.
Point to note: The inevitable uncertainty of the measured residues
shown with the above examples dictates the preventive actions of
responsible food manufacturers or retail companies to set acceptance
limits for the purchased raw materials, often called ‘private stan-
dards’, below the official MRLs, in order to assure that their prod-
ucts after processing or offering directly for sale would comply with
the legal limits. Such a limit does not negate the validity of Codex
Standards, but reflects the responsibility of operators along the food
chain.
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Chapter 11
Principles of Control
of Small-Scale Production
of Fruits and Vegetables
and Planning Risk-based
Monitoring Programmes
Zsuzsanna Horváth and Árpád Ambrus
Main topics
Examples for involvement of small-scale farms in the production

of fruits and vegetables
Theoretical background for elaboration of risk-based monitoring
programmes
Principles of control of chemical substances in food
Tiered model for planning monitoring programmes
Number of samples to be included in future monitoring programmes
Conclusions and recommendations
11.1 Introduction
There is an increasing demand worldwide for sufficient, good quality
and safe food:
• Consumers want tasty, nutritious and safe products.

• Trade partners want good quality, safe food that is delivered on
time and at the correct temperature.
467
• Regulators at local, state, national and international level want:

— safe food that complies with legal limits,
— consumers protected from harmful products and
— sustainable farming practices that produce commodities of
marketable quality and do not breach quarantine regulations
or adversely affect farm workers or the environment.
Farmers and growers of raw agricultural commodities want to

harvest and sell their products safely to have sufficient income for
maintaining a reasonable living standard of their family.
Quality and safety of food are now dictated by consumer prefer-
ences. Retailers and supermarkets want to meet this demand. They
now set product specifications and establish new quality and safety
standards in production and post-harvest handling processes that
need to be met by suppliers. No longer can farmers just grow what
they like, or what government authorities advise them to grow. Farm-
ers should respond to consumer demands regarding taste, variety,
size, colour and presentation, but they cannot comply with these
demands alone. Therefore, to achieve the above goals, a close coop-
eration of all stakeholders of the food chain is necessary according to
the principles of good agriculture practice (GAP).
GAP are “practices that address environmental, economic and

social sustainability on-farm processes, and result in safe and qual-
ity food and non-food agricultural products” (FAO 2003)
The main elements of GAP on farm include: food safety, quality,

quarantine, environment and sustainability, worker health and safety,
food security and avoidance of bio-terrorism and allergens.
All of these elements are backed up by a management process and
documentation that includes a traceability system. The principles of
GAP have been developed for over 11 groups of the agricultural pro-
duction and the relevant specific critical control points are defined by
quality control and certification systems such as the GLOBALG.A.P.
(2015) (previously EUREPGAP).
Principles of Planning Risk-based Monitoring Programmes 469
The production of high-quality and safe food requires the appro-

priate control of the whole process, including proper selection of the
raw materials and agricultural commodities, application of good crop
management, storage, manufacturing and processing practices, which
can be achieved with the establishment of the “hazard analysis and
critical control points” in the HACCP programme specific for the
given production.
HACCP: A system which identifies, evaluates and controls hazards

that are significant for food safety (FAO 1997).
As a general rule, the growers and food manufacturers are respon-

sible for the control of their production based on suitable food safety
and quality criteria to verify compliance of their products along the
production chain.
In addition, the effectiveness of the regulatory measures and self-
control systems employed by the producers should be verified by
implementing national monitoring programmes.
The basic questions for which answers should be given by the
competent government authorities are:
• Is there a health risk of consumers derived from the consumed

food?
• Are the marketed commodities complying with the legal limits?
The major objectives of monitoring programmes are therefore to

obtain information on the compliance of the commodities with legal
limits and to provide data for the dietary exposure assessment of
consumers to various residues and contaminants.
Due to the very large number of agricultural products, not all of
them can be tested with equal frequency. The ongoing monitoring
programmes in Australia, European Union and USA, for example,
are described in Chapter 3. The commodities to be included in the
programmes may be selected taking into consideration, among oth-
ers, the dietary importance of the food item, toxicity and concen-
tration of residues occurring at harvest in supervised trials, prior
information from monitoring programmes on the frequency and level
of detectable residues in the marketed commodities, and the required

intensity of protection of plants and stored products from pests and
diseases under specific environmental and climatic conditions.
A tiered approach, which can be used for prioritizing crop–
pesticide combinations in planning risk-based monitoring pro-
grammes, depending on the available information, is described in
this chapter. It has been used in Hungary for planning annual pro-
grammes for monitoring pesticide residues in raw agricultural com-
modities. The principle of second tier may also be used as part of an
early warning sampling plan to assure safe export of fresh fruits and
vegetables.
It should be noted and emphasized that even the most extensive
pesticide residue control programme, unless every single lot is sam-
pled and analysed, cannot guarantee the compliance with legal limits
and safety of agricultural products. It can only be achieved by the
application of integrated pest management with the use of constant
and high-quality pesticides and complying with other provisions of
GAP.
Farmers, as managers of natural-resource-based production sys-
tems, face many and varied biological, technical and socio-economic
issues. Only large farms and enterprises have the resources to employ
professional specialists for managing plant production and protec-
tion, at current technological level, and producing food that meets
consumers’ expectations. Small-scale farms can be competitive only if
they combine their resources and or utilize reliable advisory systems
providing them the necessary know-how.
A few successful practical arrangements, which can be used, for
example, to promote the integration of small farms in high-quality
fruit and vegetable production, are described in the next section.
11.2 Examples for Involvement of Small-Scale Farms

in the Production of Fruits and Vegetables
In many areas of the world, the majority of the farmers are not
literate and that has restrained their ability in taking initiatives for
uplifting their productivity and quality, and their ability in effectively
linking their produce with markets. Individual farms below a certain
size do not have the necessary technical know-how or cannot afford

to employ an expert adviser. There are several options, proven to
work well in practice, which provide the framework to grow crops
and meeting the market demands profitably. They may be adapted
to local conditions noting their advantages and limitations.
11.2.1 Initiative of a Small-Scale Farm in South-East

Asia
A knowledgeable, determined and target-oriented owner entered into
carambola production and expanded his activities from local sale
to solid export based on the implementation of basic principles of
GLOBALG.A.P. with the assurance of meeting hygienic standards
and sustainable production.
The owner noticed certain shortfalls in the production system,
including that the farmers lacked:
• The ability and resources for producing on larger scale to achieve
viable commercial and or export production;
• The knowledge of correct usage of agro-chemicals. (These farm-
ers have been brought up to think that increased dose rate, more
frequent application would give higher yield.);
• The cohesiveness of a carambola fraternity that individual farmers
could benefit in learning from each other and coordinating their
production activities that could promote their harvest and logistics
management;
• The motivation to participate in the scheme.
In trying to resolve these shortcomings, the K-Farm GAP Pro-
gram was started with a group of carambola farmers. The main ele-
ments of the program are briefly described hereunder.
Recognizing that the post-harvest use of pesticides in carambola
is an issue, the farmers were persuaded to follow a different pesticide
application programme that included dosage and frequency of the use
of a new list of recommended pesticides. The farmers were taught and
made to understand the biological, chemical and production ratio-
nale why the new chemical application made better sense. The K-
Farm promised the carambola farmers market accessibility instead
of higher farm gate prices. Under the GAP programme, the farmers
began to produce their crop commercially, as contracted suppliers,

under a brand that they played a big part in, and felt to own it. To
assure continual input of technical knowledge and information for
a sustained carambola production, the owner developed very close
linkages with the agriculture research agencies and universities as
well as, with chemical companies. A very close and transparent rela-
tionship was developed with the European buyers so that they could
fully appreciate the work done by the farmers.
The essence of the K-Farm Programme is founded on the ele-
ments of GAP blended with common sense practices and one that
gives a commercial viability to the carambola farmers. The farmers
support the programme because they appreciate the benefits of a
sustained commercial access to the market rather than taking their
chances in a speculative market of supply and demand.
The sustainability of the K-Farm GAP Program rests on the
commercial linkage between the farmers and retailers. The farmers
benefit from the continual investment of technical knowledge and
information; they also benefit financially from more economic usage
of agrochemical inputs and better control of the qualities of their
crops. The company K-Farm benefits from the commercial trust of
the farmers. Finally, the retailers benefit from a credible supplier who
can comply with their requirements.
In this programme, the application of GAP is not coerced onto
anybody, but is enthusiastically welcomed by the farmers. The costs of
bringing the technical knowledge and information is compensated in
the commercial transactions of the carambola exports to a dedicated
buyer. It is a GAP Program that gives the small and rural carambola
farmer a chance to venture into the mainstream supply chain.
11.2.2 Family Farm with Own Packing and Exporting

Capability in Combination with Contractual
Agreement with Other Fruit Growers in South
America
Members of a family specialized in growing apples realized that
the major profit is absorbed by the interim phases of getting their
product to the export market. They decided to establish a system

enabling the direct export of their fruits. They produced apples under
the strict control of trained family members, including among others,
pruning of trees, plant protection and soil fertility management. They
built a modern facility for cold storage, sorting, packing and shipping
their fruits mainly to Europe. The computerized traceability system,
based on the barcodes placed on each package, would enable field
identification and checking the history of technological operations
during the growing season if any complaint would be raised regarding
their products.
To increase their capacity, neighbouring farms were contracted to
produce apples according to the strict specification and technologi-
cal advice of the specialists of the main farm. Analysis of pesticide
residues was carried out by a contracted laboratory in relatively few
samples because the technical management was confident that their
fruits grown under strictly controlled conditions would comply with
the most stringent requirements of their export partners.
11.2.3 Growers’ Volunteer Cooperation in a

European Country
Farmers growing vegetables under plastic cover and in greenhouses
with areas of 0.1–0.5 ha realized that they could not sell their prod-
ucts alone because the logistic centre where they could sell their
products required a certificate of GLOBAL G.A.P. Owners of about
80 farms decided to form a cooperative for coordinated production
and marketing of their products. The main governing principles of
the joint operation were:
• An experienced agronomist was employed who was in charge of

advising on:
— application of fertilizers and pesticides,
— selection of propagation materials and
— supervision of implementation of integrated pest management;
• The quantity and variety of various vegetables were decided by
the annual meeting of the owners, based on the market demand
(representative of wholesale market was present at the meeting)

and recommendation of the agronomist;
• For a small establishment, the pesticides were applied by special-
ized groups trained in safe and proper use of pesticides with cal-
ibrated equipment (larger farms could maintain their equipment
and apply the pesticides);
• The harvested crops were sorted and packed according to quality
requirements of the market in a common store of the cooperative
at the end of the day and the packed fresh commodities were put
on the market next morning;
• The pesticide residue contents of the harvested crops were tested
based on a random sampling programme including the minimum
number of samples required by the manager of the wholesale
market;
• Farms producing crops with residues above maximum residue limit
(MRL) were excluded from the joint marketing of the products or
they had to pay deterring fine in case of serious negligence or
disregarding the recommendation of the agronomist, and
• The cost of the management of cooperative and joint expenses were
paid proportionally to the value of jointly marketed commodities.
11.2.4 Exporter’s Business Enterprise with Extended

Advisory and Raw Material Supply
Chain in Africa
Safe Food, one of the major exporters of fresh fruits and vegeta-
bles, established a country-wide network of suppliers of all kinds of
basic materials (seeds, propagation materials, fertilizers, pesticides,
etc.), application and cultivation equipment, agronomists’ advisory
service, pest and disease forecast system and local or district col-
lection points for purchasing the harvested crops. The retailers of
the company sell only agrochemicals and equipment with certified
and controlled quality. Advice on their proper use is available free
of charge for the farmers at the time of purchase. Records are kept
on buyer, goods purchased, etc. At the time of harvest, the growers
can sell their products complying with quality specification at the
collection points and receive premium price (about 10% higher than
the average market price) provided that they present the certificate
of the regional member of the advisory service confirming that the
relevant use recommendations were followed. The product purchased
can be traced back to farm level.
The products provided for sale by the company as well as those
purchased from the growers are randomly checked for various qual-
ity parameters, pesticide residues and chemical contaminants by the
company’s own laboratory.
There are no contractual obligations between the growers and
the company. The whole system is operated by market forces and it
is based on the mutual interest of the stakeholders. It is claimed that
there is sufficient supply of agricultural commodities to fulfill needs of
the export contracts of the company as the growers are motivated by
the premium price paid to them if they comply with the company’s
preconditions.
This system has the definite advantage over contractual arrange-
ments between buyers and sellers based on pre-fixed price, but
its smooth functioning assumes a well-organized complex supply
chain of raw materials, advisory network and laboratory control
facility.
11.2.5 Exporters Deal with Contracted Farmers

in Africa
Some vegetables are grown in over 65,000 small and medium-sized
farms of 0.5–10 ha. The crop can be harvested 8–10 times within a
year. The major exporters established supply chains including collec-
tion centres, cooled stores, sorting and packing facilities. The growers
are contracted annually to supply specified quantity of crops at a
fixed price.
The export companies employ agronomists for advising the grow-
ers and controlling the storage and use of pesticides. However, they
cannot keep close contact with each farmer due to shortage of time,
and they have no means to discipline the farmers if they do not follow
the recommendations.
Fertilizers and pesticides are not provided by the export compa-

nies. The growers may buy them from the locally available retailers,
which sell them without any quality certificate. Many of the retail-
ers are not technically competent in handling the chemicals, much
else giving advice on their effective usage and safety handling. For
compensation of inferior biological efficacy, the farmers increase the
dosage rate leading to high residue levels. Furthermore, they often
use active substances that are not authorized in the targeted export
market.
The harvested vegetables are transported to the collection centres
of the exporters where the crops are pre-sorted and cleaned from
foreign materials. The pre-cleaned crop is transported to the main
packing houses within 12 hours where it is stored in cold rooms until
the final sorting, washing and packing in labelled boxes are carried
out. The packed product is ready to be airfreighted during the night
to the importing county.
The exporters exercise utmost care about the hygienic handling
of the harvested crops but pay much less attention to promoting the
principles of good farming practice.
The system has many positive features, but there are some
shortcomings, which should be noted and possibly improved if it
is intended to be used in other situations:
• The appropriate technological advice does not always reach all

farmers;
• As the price is fixed in advance at the lowest acceptable level, the
farmers are tempted to sell their products to other agents at higher
price in case of shortage of the crop;
• Agrochemicals of verified quality are not made available; the farm-
ers are tempted to purchase cheaper illegal products of low or
questionably quality leading to violation of MRLs;
• The analysis of pesticide residues in harvested crops is occasional
and the sampling is not representative.
11.3 Theoretical Background for Elaboration

of Risk-Based Monitoring Programmes
11.3.1 Field to Field Distribution of Pesticide
Residues
The among-fields distribution of pesticide residues in composite sam-
ples was analysed and evaluated based on the supervised trial results
reported by the FAO/WHO JMPR between 1997 and 2011 (Ambrus
et al., 2014). It is pointed out that supervised trials are carried out
under strictly controlled conditions on small plots. Consequently,
the within plot distribution of residues is likely more uniform than
within large fields treated with the pesticide; therefore, the average
residue in composite samples may properly represent the magnitude
of residues following the application of the pesticides. Subsequently
the database was considered suitable for the evaluation of the among
fields distribution of pesticide residues.
Those datasets were selected, which contained a minimum of five
residue values and less than 50% of them were below the limit of
quantitation (LOQ).
Limit of quantitation: Lowest concentration of a pesticide residue

in a defined matrix where positive identification and quantitative
measurement can be achieved using a specified analytical method
Each data set consisted of one residue value from independent

trials conducted according to the critical GAP and selected by the
Joint Meeting on Pesticide Residues (JMPR) for estimation of maxi-
mum residue levels to be used to establish MRLs by the Codex Com-
mittee on Pesticide Residues (CCPR) (see Chapter 4). The 25,766
trials, representing a wide range of practical conditions, comprised
1950 pesticide–crop combinations.
The average magnitude of residues in the datasets varied at a
large extent. In order to make the spread of residues in various data
sets comparable for characterizing the distribution of residues, the

individual residue values making up one dataset were divided by
their average (e.g. iprodione residues in eight cherry samples).
The median (M) of residues was used as a reference point for
characterizing the spread of residues in individual datasets, as it was
not influenced by the values below LOQ being present at < 50%.
Each data set was considered to be a sample taken from the parent
population of residues derived from supervised trials. If an additional
set of composite samples were taken from the same treated plots, they
would most likely contain different residues as can be seen from the
variability of residues in replicate samples used for the estimation
of sampling uncertainty in Chapter 10. The individual residues (Ri )
being in one data set were sorted into their corresponding median
ranges: R < M; M ≤ R < 3M; 3M ≤ R < 4M; 4M ≤ R < 5M;
5M ≤ R < 6M; 6M ≤ R < 7M and R ≥ 7M. The percentage of
occurrence of 25,766 residues in median ranges and their cumulative
frequency are summarized in Table 11.1.
Table 11.1. Percentage distribution of residues in median ranges.
Percentage of Data Sets in the Median (M) Ranges

3M ≤ R 4M ≤ R 5M ≤ R 6M ≤ R
R<3M < 4M < 5M < 6M < 7M 7M ≤ R
Cumulative % 54.50 71.61 78.58 85.92 88.68 100.00
The R ≤ M range contained about 50% of all residue values.

The residues in the M < R ≤ 3M range were present only in about
4.21% of the cases, indicating that the typical spread of residues are
larger than 3M (see Fig. 11.1). The results indicate that residues in
different fields receiving the same or similar treatments vary within
wide range: 55, 72, 79, 86 and 89% of the 25,766 residues values
were, respectively, within three, four, five, six and seven times the
median value of the corresponding dataset. Consequently, if we find a
supervised trial residue dataset within, for instance, the ≤3M range,
it indicates that probably the high residues were not included in the
sample, and there is high probability (∼45%) for underestimation of
the highest expectable residue.
18
16
14
12
10
8
Supervised Trial Data
6 HR
4 Median
2
0
0 2 4 6 8 10 12
18
P0.05 of median
16 P0.975 of supervised trial data
14
12
10
Supervised Trial
8 Data
HR
6
4 Median
2
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
Figure 11.1. Distribution of normalized residues, and the median and HR values
of residues in replicate samples of size 8 drawn from the parent population (25766)
of normalized supervised trial data.
11.3.2 Calculation of the Probability of

Violation of MRLs
Let βv the probability that a random sample contains a residue above
the allowed limit (e.g. MRL). Then the percentile of samples at or
below the limit, βp , is 1 – βv . The number of random samples (n)
required for finding at least one value above a selected percentile (βp)
of the parent population (e.g. residues in samples taken from apple
orchards registered for supplying fruits for a packing house) with a
specified probability level (βt ) can be calculated with the following
equations based on the binomial distribution:
lg(1 − βt1 )
βt1 = 1 − βpn or n = (11.1)
lgβp
The probability to find at least two samples with residues above

the selected percentile (βp = 1 − p) is:

n 0 n 1
βt2 = 1 − p (1 − p)n − p (1 − p)n−1 (11.2)
0 1
or expressing with βp:

n
βt2 = 1 − βpn − (1 − βp )1 (βp )n−1 (11.2a)
n−1
Points to note:
The above equations provide correct information if the following
preconditions are satisfied:
• All orchards, fields or greenhouses to be controlled are accounted
for in advance;
• The products harvested from the individual orchards represent
individual lots;
• The lots to be sampled are randomly selected (each of them has
equal chance to be chosen) without the prior knowledge of the
owner;
• The lots to be sampled should be selected by drawing random
numbers without replacement; that is one lot shall be sampled
only once;
• The number of lots to be sampled (N ) is much larger than the
number of samples (n) calculated with Eq. (11.1);
• When the number of samples (n) calculated with Eq. (11.1) is ≥
0.1N, the number of samples to be taken (n0 ) should be calculated
with Eq. (11.3) (CAC, 1999).
n
n0 = (11.3)
1 + (n − 1)/N
The violation rate (βv ) is equal to 1 − βp . For example, the number
of samples required for detecting a residue above the MRL at least in
one sample at various violation rates is given in Table 11.2 for large
N and in Table 11.3 for cases where the number of lots to be tested
is relatively small and the violation rate is fixed at βv = 2%.
Table 11.2. Minimum number of samples required to detect

at least one residue above the MRL at the selected violation
rates (βv ) with pre-defined probability (βt ).
βt [%] 80 90 95 98 99 99.9
βv [%] Number of Samples to be Taken
0.1 1609 2302 2995 3911 4603 6905

1 161 230 299 390 459 688
2 80 114 149 194 228 342
2.5 64 91 119 155 182 273
5 32 45 59 77 90 135
10 16 22 29 38 44 66
Table 11.3. Number of lots (n0 ) to be sampled where the total

number of lots (N ) is small.
N 120 150 200 1000 5000

βv = 2%
βt [%] n n0
80 80 49 53 58 75 79
90 114 59 66 73 103 112
95 149 67 75 86 130 145
98 194 75 85 99 163 187
99 228 79 91 107 186 219
99.9 342 90 105 127 256 321
n: Number of samples that would be required in case of large

number of lots.
βt %: The probability of finding at least one lot that contains
residue above the MRL if the expected violation rate is 2%.
The results obtained with the analysis of n or n0 samples can be

interpreted and used in different ways:
(a) If we took 149 random samples (Table 11.2) from a population

of apple orchards with similar characteristics (e.g. apples treated
with pesticides according to the registered use patterns (dosage
rate, pre-harvest interval etc.), and did not find residues above
the MRLs, we can state with 95% probability that less than 2%
of apple lots could contain residues above the MRL.
(b) If a residue above the MRL was detected only in one sample,
we can state with 80% probability, based on Eq. (11.2), that no
more than 2.0% of apple lots contain residues above the MRL.
(c) For a regular monitoring programme, we plan to take 45 samples
from a given commodity, we will have only 60% chance to find
one sample, which contains residue above the MRL if 98% of
all lots comply with the MRLs (βv = 2%), calculated from Eq.
(11.1) and not shown in the table.
(d) If we have to verify compliance with an MRL in 99.5% of the
lots with 99% confidence, then we have to plan to take random
samples from 919 lots.
The number of samples calculated with Eqs. (11.1) and (11.2)

is independent from the number of decision units (N ) of the same
characteristics to be tested. For instance, crops treated according to
use recommendations and grown in one growing season. However,
the sampling plan shall be prepared for each growing season inde-
pendently.
It can be seen that the number of samples to be taken and anal-
ysed is rapidly increasing, when low violation rate shall be verified
with increasing probability. It is the responsibility of the risk man-
agers to decide what level of control should be employed taking into
account among others:
• The targeted compliance level (βp = 1 − βv ) and probability (βt )

of its verification;
• The risk that the owner of the commodity is willing to take when
marketing a commodity containing residues above the MRL or the
maximum acceptable residue concentration specified by the buyer
(sometimes called ’private standard’);
• The technical level of the crop production and protection;
• Availability and application of registered or authorized pesticides
in good and constant quality (see Chapter 8);
• The commitments of the growers to comply with use recommen-

dation of pesticides, that is they grow their crops according to the
principles of GAP and
• The laboratory capacity available.
11.3.3 Estimation of the 97.5th percentile of Residues

in Crops Treated with a Pesticide
The deterministic calculation of acute exposure to pesticide residues
is based on the highest residue (HR) value of residue data set
obtained from the supervised trials selected by the JMPR (Chapter
4) or regulatory agencies (Chapters 3 and 6). The estimated exposure
aims to cover 97.5th percentile of likely residue, which occur follow-
ing the application of pesticides according to critical GAP. One data
set, typically representing 5–8, occasionally larger number of trials, is
only one sample taken from the population of residues deriving from
the application of pesticides. If another set of samples were taken or
additional trials were conducted, different residue values would be
measured in the samples taken.
To study the relationship between the median residues of data
sets and the 97.5th percentile of the parent population of residues in
composite samples derived from supervised trials, random samples of
size (n) from 4 to 32 were drawn 10,000 times with replacement from
the normalized parent population of 25,766 supervised trial residue
data with characteristic parameters of mean: 1; SD: 0.974; median:
0.823; P0.95: 2.465; P0.975: 3.009; max: 9.601.
Sample size (n): The number of units, or quantity of material,

constituting the sample (CAC 1999).
The distributions of the normalized residues, their median and

the HR values obtained in samples of size 8 are shown in Fig. 11.1.
It indicates that the median values have a symmetrical
distribution close to normal, while the HR values spread over the
whole residue range of supervised trials as can be expected. Based
on one sample, consisting of residue values from N supervised tri-
als, it cannot be decided with reasonable confidence how does the
8.0
7.5
P0.05med
7.0
6.5
y = 10.233x-0.228
fM,n
6.0 R² = 0.9909
5.5
5.0
4.5
4.0
0 5 10 15 20 25 30
Number of residue values in a dataset
Figure 11.2. Relationship of fM,n and sample size (n).
experimental HR value relate to the 97.5th percentile of the parent

population of residues. The larger the size of the sample the better
is the chance to obtain an HR value close to the 97.5th percentile of
the parent population.
In order to get an estimate for the potentially highest HR values
from different sample sizes with 95% probability, the ratio of the
known 97.5th percentile of the parent population (P 0.975R = 3.009)
and the 5th percentile of the medians (P 0.05M,n ) in random samples
were calculated for each sample size:
P 0.975R
fM,n = (11.4)
P 0.05M,n
The relationship of fM,n and the size of random samples drawn
from the normalized parent population is shown in Fig. 11.2 and
described with Eq. (11.5).
fM,n = 10.233 × n−0.228 (coefficient of determination R2 = 0.9909)

(11.5)
The expectable highest residue HRP 0.975 is calculated as:
HRP 0.975 = fM,n × STMR (11.6)
Table 11.4. Factors for calcu-

lation of expectable highest HR
values with 95% probability.
n fM,n,0.975 n fM,n,0.975
3 8.0 10 6.1
4 7.5 11 5.9
5 7.1 12 5.8
6 6.8 13 5.7
7 6.6 14 5.6
8 6.4 15 5.5
9 6.2 16 5.4
The factors calculated with Eq. (11.5) for sample sizes ranges from 3
to 16 are given in Table 11.4. The calculated factors approximately
correspond with the experimental distribution of supervised trial
data (Table 11.1), which indicates that 86% of the residue values
(for all data sets consisting of ≥ 5 residue values) were < 7M.
The expectable variation of residues in small data sets (N ≤ 4)
is larger, reflected by the factors of 7.5 and 8.0.
11.4 Principles of Control of Chemical

Substances in Food
Potentially harmful substances in food can be distinguished whether
they are used on purpose for the production of food (e.g. additives,
pesticides, veterinary drugs) or contaminate the food regardless of
the producers and manufacturer’s intention, such as naturally occur-
ring substances (e.g. mycotoxins) or industrial pollutants (e.g. PCBs,
heavy metals). Utilization of intentionally used substances in food,
such as pesticides, are strictly regulated, and their concentration is
limited by establishing MRLs. However, monitoring programmes or
targeted field surveys are needed to verify the effectiveness of the
regulatory measures and adherence to specified use conditions in
practice.
Monitoring programme: Sampling and analyses of pesticide

residues in biological and environmental samples taken according
to prearranged schedules (Stephenson et al., 2006).
Targeted field survey: Samples are taken from randomly selected
fields known to be treated with the pesticide of interest.
The major goals of monitoring pesticide residues in raw

agricultural commodities are to verify, shortly after the registration
of a pesticide, that the use patterns were correctly defined, and MRLs
are not exceeded if the pesticides were used according to the label
instructions. Confirmation that following the specified use patterns
would provide proper protection of crops and leads to residues below
the MRL can be done by taking samples from fields of known pesti-
cide treatment history as part of targeted field surveys.
Regulatory agencies have not specified acceptable violation rates
(βv % = 100 − βp %). Random monitoring programmes, including all
marketed commodities, may provide information on the level of com-
pliance with MRLs under practical conditions. Compliance close to
100% (e.g. βp = 99.9%) cannot be verified with high probability
as it would require the analyses of very large number of samples
(Table 11.2), which are not feasible under normal circumstances.
The BASELINE Consortium (Ambrus et al., 2013), carried out
research within the 7th Framework Programme of the European
Union, recommended 98% compliance (βv ≤ 2%) as food safety
performance objectives for chemical contaminants and pesticide
residues. The application of this criterion is recommended for plan-
ning monitoring programmes.
Due to the practically infinite combinations of crops and pes-
ticides, it is not possible to sample all commodities marketed and
analyse their pesticide residue content. The basic information that
may be considered for ranking commodity–pesticide combinations
in planning monitoring programmes are, for instance, the results of
supervised trials; acute reference dose (ARfD); historical monitor-
ing data derived from national monitoring programmes or reported
by other countries; food intake data; notifications published by the
Rapid Alert System for Food and Feed (RASFF) operated by the
European Commission; and the kinds and amounts of pesticides sold

in the country.
The tiered model, which is presented hereunder, facilitates weigh-
ing the importance of certain raw agricultural commodity pesticide
combinations taking into account the information available at the
time of registration of a pesticide, and the results of previous monitor-
ing programmes. Though the ranking is based on the frequency and
level of selected pesticide residues occurring in a specific commod-
ity, it is recommended that samples are analysed with multi-residue
methods including as many residue components as technically possi-
ble, in addition to using specific single analyte methods if required.
11.5 Tiered Model for Planning Monitoring

Programmes
For the first tier of the model, supervised trial residue data, MRLs,
ARfD, if relevant, and national or WHO GEMSFood cluster diet are
the starting points. These data should be available at the national
registration authority for each pesticide for different crops from the
dossiers submitted to support national evaluation, or from the JMPR
Reports (FAO, 1997–2015).
For the second tier, the available historical monitoring or tar-
geted field survey data can be taken into account together with food
consumption data. The risk ranking based on supervised trials can
be refined with the use of monitoring data.
The decision tree, shown in Fig. 11.3, assists planning monitoring
programmes in different situations and helps to use the model.
Explanation for Fig. 11.3.

Detailed calculations are shown in Section 11.6.
Step 1:
Review the results of supervised residue trials (ST):
1. ST: R? Would detectable residue remain in the harvested crop?

• No detectable residue is expected in or on treated crop → no
need to test.
Figure 11.3. Decision tree for the application of tiered ranking model.
ST: supervised trial; R: residue; ESTIe : estimated short term intake with 95%
probability level, A%: percentage of area expected to be treated with a given
pesticide from the total cultivated area of the commodity; M ?: are results of
monitoring programmes available?
FMRL , FM0 : weighting factors.
FaM , Fast : factor indicating short term intake concern.
• MRL=LOQ*, this situation may occur when:

— No detectable residue can be expected with analytical
methods available at the time of the registration of the
pesticide, and the pesticide is used according to the label
instruction;
— The registration authority does not want the compound
to be used, therefore the MRL is set at LOQ. Residues
may occur in crops if the product is illegally used or in

commodities imported from countries where the product is
authorized. Therefore, the commodities concerned could be
included in the monitoring programme.
• R > LOQ, res. (residues) expected: The use of compound
results in detectable residues in treated commodities → go to
step 2.
Step 2: the targeted use and the treated area can be taken into
account:
2. A%? Is the percentage of the area expected to be treated with a
given pesticide from the total cultivated area of the commodity
known?
• If treated area < 2%, calculate the likely maximum residue lev-
els based on principles described in the first tier of the model.
Then, calculate the estimated short term intake (ESTIe ) with
likely highest residues (HRP 0.975 ). In this case, if the pesticide
has no ARfD, we can consider that there is no acute risk, thus
there is no need to include the pesticide in the monitoring
programme.
— If ESTIe < ARfD, the risk is considered very low →no need
to test;
— If ESTIe ≥ ARfD → initiate targeted field surveys, because
finding treated crops would have very low probability in case
of random sampling including all marketed commodities;
• If the treated area is larger than 2% or not known → go to
step 3.
Step 3: historical monitoring data are considered:
3. M? Are the results of previous monitoring programmes available?
• Yes; is the proportion of R ≥ MRL larger than 2%?
— If yes, initiate targeted field survey;
— If no, apply second tier of the model and calculate combined
weighing factor (FM 0 ) and factor reflecting the acute risk
(FaM ), if there is an ARfD for the pesticide. Choose the
larger one as FT2 .
◦ FT2 ≤ 10 →no need to test,

◦ FT 2 > 10 → include the pescticide–commodity combina-
tion in random monitoring programme,
◦ if FT2 ≥ 100 → initiate targeted field survey.
• No monitoring data are available, apply first tier of the model
and evaluate supervised trial residue data. Calculate FMRL and
Fast (if applicable)
— if Fast ≥ 100 → initiate targeted field surveys,
— if Fast < 100, choose the larger of Fast or FMRL .
if Fast or FMRL < 10? → no need to test,
Fast or FMRL ≥ 10? rank the commodity–pesticide com-
bination for inclusion in monitoring programme based on
Fast or FMRL .
11.6 Practical Application of Tiered Model

11.6.1 Tier 1: Data are Available from Supervised
Trials, But There is No Monitoring Data
(Steps 1 and 3)
The first tier uses the data from supervised trials conducted before
the registration of a pesticide. Therefore, it is applicable for newly
approved pesticides or for those where former monitoring data is not
available.
Two different factors (FMRL and Fast ) are calculated at this stage.
FMRL reflects the uncertainty of estimation of the maximum residue
level based on the limited supervised trial data. Fast indicates the
potential acute exposure in relation to the established ARfD.
The weighting factor, used for ranking the commodity–pesticide
combination, FTI , is the higher of the FMRL or Fast .
Calculation of FMRL
FMRL = fST + fnβp (11.7)
The fST is calculated from the ratio of the MRL and STMR based on
the cumulative frequency of residues in median ranges (Table 11.1).

fST = 100 − P% (11.8)
Table 11.5. Relation of fST to the

ratio of MRL and STMR.
MRL–STMR Ratio Σ P% fST
>7M 100 0
6M ≤ R < 7M 89 11
5M ≤ R < 6M 86 14
4M ≤ R < 5M 79 21
3M ≤ R < 4M 72 28
< 3M 55 45
The corresponding factors are shown in Table 11.5.

The fnβp is to accommodate the uncertainty of estimation of
MRLs as a result of limited number of supervised trials.
fnβp = 0.5(100 − βt %) (11.9)
The βt is calculated with Eq. (11.1) from the number of super-
vised trials (N ) assuming that 95% of the residues are included
(βp = 0.95) in the MRL according to the underlying principles of
OECD MRL calculator (OECD, 2011).
An adjusting factor of 0.5 is applied to give proportional weight
for the number of trials and the MRL–STMR ratio.
The calculated weighting factors for various N are shown in
Table 11.6.
Calculation of Fast
It is calculated only if ARfD has been established.
ESTIe
Fast = % (11.10)
ARfD
The calculation of ESTI depends on the size of the commodity.
It is described in Chapter 6. WHO’s ‘Template for the evaluation of
the acute exposure’ (WHO, 2014) can be applied for the calculation
of ESTIe . In this case, insert the HRP 0.975 and the corresponding
national food consumption data of the given commodity, if available,
in the template. In other cases, the intake figures included in the
template are used.
The template gives the % exceedance of the maximum ARfD
which is equal to Fast factor.
Table 11.6. The fnβp corresponding

to the number of supervised trials (N ).
Percentile: βp = 0.95
N βt % 100 − βt % fn0.95
3 14 86 43
4 19 81 40
5 23 77 38
6 26 74 37
7 30 70 35
8 33 66 33
9 37 63 32
10 40 60 30
15 32 68 34
25 47 53 27
Since the estimation of HR based on few trials is very uncertain

(Horváth et al., 2014), the multiplying factors given in Table 11.4
are used for calculation of the highest expectable residues, based on
N trials, with 95% probability with Eq. (11.6) (HRP 0.975 = fM,n ×
STMR).
Points to note:
• Carefully follow the Manual when values are changed in the WHO
IESTI template (WHO, 2014).
• It is recognized that the HR P 0.975 would provide an overestimate for
ESTI in most of the cases, however initially a conservative esti-
mate is considered appropriate for the assessment of short-term
intake because of the potential serious consequences of its underes-
timation. The initial estimate should be refined based on the results
of targeted field surveys.
Example 1. No residue expected in treated crop.
The 2015 JMPR evaluated abamectin and agreed for the follow-
ing residue definition for abamectin in plant commodities for enforce-
ment and dietary risk assessment: Avermectin B1a.
The critical GAP for potato is 2 × 21 g ai/ha and 14 days PHI.

Thirteen potato trials conducted with three to six applications at
GAP and at 6 × 112 g ai/ha no abamectin residues were detected
in potato tubers (< 0.005 mg/kg). As no residue was detected at
exaggerated rates, the meeting estimated an HR of 0 mg/kg.
As no residue is expected in treated potato, there is no need
to include avermectin B1a, which can only be detected with a spe-
cific single-analyte method, in the monitoring of pesticide residues in
potato.
Example 2. MRL of 0.01* is in effect in the targeted export market.
The 2008 JMPR established an ARfD of 0.02 mg/kgbw/day, and
recommended an MRL of 0.5 mg/kg with a PHI of seven days for
sweet peppers expressed as dimethoate and an HR of 1.3 mg/kg for
the sum of dimethoate and the 10 times more toxic omethoate.
In Happyland, the use of dimethoate is authorized based on the
JMPR recommendation. However, dimethoate residues are not per-
mitted in one of its major export partner, which set the MRL at the
LOQ (0.01* mg/kg). Safe Food wanted to assure that its product
would comply with the export market provisions, therefore the retail-
ers of company (see Sec. 11.2.4) were instructed to advise farmers not
to use dimethoate in sweet peppers intended to be exported.
Therefore, with the involvement of its advisory service, a random
sampling programme including sampling of 114 fields was initiated
providing 90% probability (βt ) of compliance (Table 11.2).
Example 3. Area treated is known.
Abamectin obtained an experimental clearance for use in veg-
etables in Sunnyland based on the critical GAPs evaluated by the
2015 JMPR (PHI=3 days, MRL=0.07 mg/kg for sweet peppers). In
order to verify the applicability of use patterns considered by the
JMPR, the registration authority issued permit for the import of the
product sufficient for protection of vegetables grown on about 200
ha (<1% of total growing area). The imported product was provided
to the growers’ cooperatives (see Section 11.2.3) located at various
locations in the country on the condition that the product would be
used in green peppers grown in greenhouse, all pesticide applications

would be recorded and the intended use and harvest time would be
reported.
Altogether 600 greenhouses were included in the targeted sampling
programme and 120 samples were taken from randomly selected fields
during the two harvesting periods within a year. Based on Eq. (11.3),
the compliance of ≥ 98% of the harvested crops could be verified with
95% confidence, provided that none of the samples contained residue
above the MRL of 0.07 mg/kg.
Example 4. Supervised trial data are only available, no ARfD.
The residues in artichoke derived from supervised field trials per-

formed with pesticide X were: 1.1, 1.2, 1.6, 2.0, 2.2, 2.4, 2.5 and
2.8 mg/kg. Establishing ARfD was not necessary. The registration
authority of Sunnyland established an MRL of 6 mg/kg applying the
OECD calculator (OECD, 2011).
Application of decision tree (Fig. 11.3):
— Step 1: We can conclude that residues are expected and MRL was
set above the LOQ. → go to Step 2.
— Step 2: Proportion of the treated area is not known. → go to
Step 3.
— Step 3: Monitoring data is not available → apply first tier.
Taking into account the STMR value of 2.1 mg/kg, the

MRL/STMR ratio is < 3, the corresponding fST , from Table 11.5, is
45.
The fnβp for N = 8 residue data is 33 from Table 11.6. For other
number of residue data not included in the table fnβp should be
calculated with Eq. (11.9).
FMRL = 45 + 33 = 78
Include artichoke in random monitoring programme with num-

ber of samples decided according to the principles described in
Section 11.7.
Example 5. Supervised trial data are only available, ARfD estab-

lished.
Tebuconazole in lettuce.
Based on supervised trials residue data (N = 8): 3.2, 2.3, 1.4, 1.3,
0.65, 0.44, 0.23, 0.18 mg/kg), the 2008 JMPR estimated maximum
residue level, STMR and HR of 5, 0.975 and 3.2 mg/kg, respectively.
The 2015 JMPR established an ARfD = 0.3 mg/kgbw/day
Following the decision tree (see Example 4), first tier was
applied:
(a) Calculation of FMRL = fST + fnβp :
Determine fST : 6M> ratio of MRL and STMR ≥5, from

Table 11.5: fST = 14.
Determine fn0.95 : number of supervised trials in this dataset
is N = 8. Choose the corresponding factor from Table 11.6:
fn0.95 = 33.
Calculate combined weighting factor with Eq. (11.7):
FMRL = 14 + 33 = 47
(b) Calculation of Fast :

First calculate the estimated likely highest residue (HRP 0.975 ):
From Table 11.4: N = 8 → fP 0.975 = 6.4.
The HRP 0.975 = STMR × fP 0.975 = 0.975 × 6.4 = 6.24 mg/kg.
Calculate ESTIe with HRP 0.975 .
In this case, national consumption data is not available, thus the
WHO IESTI calculation template is used after entering HRP 0.975
of 6.24 mg/kg in the template.
The calculated short-term intake amounts to 50% of the ARfD
for general population (Fast = 50).
(c) Choose the higher of FMRL and Fast : Fast > FMRL :
Fast = 50 → Random monitoring of residues in lettuce is recom-
mended. Take samples according to the principles described in
Section 11.7.
11.6.2 Tier 2: Monitoring Data and Established MRL

are Available
This procedure is intended for ranking the importance of testing
residues of pesticides, which were used ≥ 3 years and monitoring
data are available for that period. The weighting factor, FT 2 , indi-
cating the importance of testing a given pesticide residue in a selected
commodity, is calculated by taking into account the combined effect
of the number of residue data (n) derived from monitoring pro-
grammes, the frequency (fp ) of occurrence of residues (in FM 0 factor;
see Eq. (11.11)) and the potential for acute intake problem based on
ratio of the ESTI and ARfD: FaM .
(1) Calculation of combined weighting factor:

FM0 = (fm + fp ) (11.11)
Determine fm from the number of monitoring data, n, for the
examined commodity–pesticide combination:
Probability of detection of residues (βt ) above the 98th per-
centile, the recommended performance objective, from n samples
is calculated with Eq. (11.1).
The weighting factor is
fm = (100 − βt ) (11.12)
Point to note: if n is larger than 220, the factor is negligible and
can be taken into account with ‘0’.
Determine fp , from the frequency of occurrence of detectable
residues:
n
Ri × MRL−1
fp = 100 i=1 (11.13)
n
where Ri − s are the measured (and not the reported) residues in
samples derived from the monitoring programmes. If Ri < LOQ,
then Ri is counted with 0.
Points to note:
(a) Where there is no MRL, replace its value with 0.01 mg/kg (see
Step 1 MRL=LOQ*) in the calculation of fp .
(b) Where the frequency of residues >MRL is larger than 2% in

the tested samples, a specific targeted survey may be initiated
with at least 40–60 samples for the given pesticide commod-
ity combination, taking the samples at harvest in randomly
selected sites known to be treated with the pesticide.
(2) Acute exposure of consumers:
ESTIM
FaM = (11.14)
ARfD
The ESTI is calculated from the largest residue detected in sam-

ples derived from monitoring programmes taking into account the
definition of residues for dietary intake calculations.
Points to note:
• The residue definition for enforcement and dietary risk assessment
purposes can be different.
• If the residue definition for enforcement and dietary risk assess-
ment purposes are different, multiply the residues measured
according to residue definition for enforcement purposes with
the conversion factor, if available, to obtain the total significant
residue for calculation of ESTI.
• If conversion factor had not been established by the JMPR or
responsible regulatory agency earlier, estimate the factor based on
the ratios of residues measured in supervised trials according to
the two residue definitions in consultation with residue and tox-
icological experts. The estimated factor shall be approved by the
responsible regulatory agency.
• If appropriate conversion factor could not be estimated, depending
on the nature of toxicity of residue components and their likely
concentration level relative to that used for enforcement purposes,
if justified, initiate targeted field survey with the determination of
all residue components included in the definition for dietary risk
assessment.
Weighting factor: FT 2 is the larger of FM0 and FaM .
Example 6. Tebuconazole residues in apple

MRL=1 mg/kg; ARfD=0.3 mg/kgbw/day.
The definition of residue for enforcement and dietary risk assess-

ment for plant and animal commodities: tebucoazole.
Tebuconazole was looked for in 635 samples during the 4-year
monitoring programme. Of the 635 samples, 24 contained tebucona-
zole residue ≥LOQ=0.01 and the residues were < LOQ in 631 sam-
ples.
The detected residues, mg/kg, in rank order were: 0.96, 0.59,
0.21, 0.13, 0.09, 0.08 (3), 0.06 (3), 0.05, 0.03, 0.03, 0.02 (9) and 0.01.
Following the decision tree (Fig. 11.3) second tier is applied:
(a) In view of n > 220, fm is practically 0.

(b) The frequency of occurrence is calculated with Eq. (11.13) insert-
ing 0 in cases where the residue was below LOQ and noting that
the MRL=1 mg/kg:
fp = 100∗ (2.68 + 631∗ 0)/635 = 0.42
FM 0 = 0 + 0.42 = 0.42
(c) FaM is calculated with the highest residue (0.96 mg/kg) detected
applying the WHO IESTI template: FaM = 20.
Random monitoring of residues in apple is recommended includ-

ing tebuconazole in the multi residue procedure applied for detecting
residues in apple.
Example 7. Different residue definition for enforcement and risk

assessment: spirotetramat in sweet corn.
The JMPR established an ADI of 0–0.05 mg/kg/bw/day and an

ARfD of 1 mg/kg/bw and defined the residues as follows: residue for
enforcement in plant commodities: spirotetramat plus spirotetramat
enol, expressed as spirotetramat; residue in plant commodities for
dietary intake: spirotetramat plus the metabolites enol, ketohydroxy,
enol glucoside, and monohydroxy, expressed as spirotetramat.
LOQ=0.01 mg/kg.
In supervised trials conducted according to GAP the residues

[mg/kg] after seven days of treatment in sweetcorn were:
Australia:
Spirotetramat
and enol 0.056 0.056 0.1 0.12 0.12 0.24 0.4
Spirotetramat
+4 metabolite 0.12 0.12 0.18 0.18 0.18 0.3 0.62
Canada: (Average of two replicate samples)
Spirotetramat
and enol 0.04 0.061 0.235 0.48 0.545
Spirotetramat
+4 metabolite 0.071 0.125 0.31 0.6 0.695
The JMPR estimated a maximum residue level of 1.5 mg/kg,
and for dietary risk assessment, an STMR residue of 0.31 mg/kg
and an HR of 0.75 mg/kg measured in one of the replicate samples.
The JMPR did not recommend factor for calculation of residues for
dietary intake calculation from the residues measured in monitoring
programmes.
The compound was registered in Sunnyland based on the recom-
mendation of the JMPR.
Spirotetramat residues were determined in 67 sweet corn sam-
ples taken within the last four years of monitoring programme. The
spirotetramat + enol residues (mg/kg) detected were in rank order:
2.1, 1.2, 1.1, 0.9, 0.5 (3),0.4 (3), 0.33(5), 0.15 (3), 0.05 (4),0.04 (8),
<LOQ (37).
(a) βt is calculated with Eq. (11.1): βt % = 74.2,

fm = 100 − 74.2 = 25.8
(b) fp is calculated with Eq. (11.13): fp = 10.6.

(c) FM 0 = fm + fp = 84.8 = 85.
(d) Calculation of FaM .
Conversion factor was not estimated by the JMPR. However, the

residues determined according to the residue definitions for dietary
intake (Crisk ) and enforcement (Cenforc ) can be used for calculation

of the conversion factor. The regression of Crisk over Cenforc gave a
linear relationship: Crisk = 1.23 × Cenforc + 0.041 with coefficient of
regression, R2 = 0.9785.
Applying the regression equation, the maximum residue was cal-
culated (2.6 mg/kg) for the estimation of ESTI.
ESTI calculated with WHO template for all variants of sweetcorn
resulted in ESTI of 0–1% of ARfD. FaM = 1.
Since residues above the MRL occurred only in one sample
(1.5%), random monitoring of residues in sweetcorn is recommended
taking samples according to the principles described in Section 11.7.
11.6.3 Evaluation of a Pesticide Shortly After

Registration
This procedure is the combination of the first and second tier and
intended for ranking the importance of testing pesticide residues
shortly after registration where monitoring data are available from
k < 3 years.
The selected weighting factor, FT 1−T 2 , indicating the importance
of testing a given pesticide residue in a selected commodity, is cal-
culated taking into account the factor derived from supervised trials
(on the first tier, FT 1 ) and the results of available monitoring data
(on the second tier, FT2 ) in proportion:
Combined weighting factor:
FT1−T2 = ((3 − k) × FT1 + k × FT2 )/3 (11.15)
where FT1 (the larger of Fast and FMRL ) and FT2 (the larger of
FM0 and FaM ) are the appropriate factors from the first tier and the
second tier, and k is the number of years from which monitoring data
are available.
Example 8. Abamectin in lettuce.
Based on the results of supervised field trials (FAO/WHO JMPR
Report 2015). (N = 8): 0.007, 0.011, 0.019, 0.020, 0.035, 0.045,
0.047 and 0.097 mg/kg. The JMPR estimated a maximum residue
level, STMR and HR of 0.15 mg/kg, 0.0275 g/kg and 0.097 mg/kg
for respectively.
ARfD=0.003 mg/kg/bw.
Monitoring data is available on two-year usage of the pesticide
in head lettuce.
Number of monitoring data: 110, R > MRL 0.91%.
Highest residue observed in a sample: HRM = 0.34 mg/kg.
I. Calculation of FT1 the larger of FMRL and Fast factors
(1) Calculation of FMRL = fST + fnβp :
Determine fST : MRL/STMR=5.4 from Table 11.5: fST = 14.
Determine fn0.95 : Number of supervised trials in this data set
is N = 8. Choose the corresponding factor from Table 11.6:
fn0.95 = 33.
Calculate combined weighting factor with Eq. (11.7):
FMRL = 14 + 33 = 47
(2) Calculation of Fast :
Determination of likely maximum residue level (HRP 0.975 ):
n = 8 → fP 0.975 = 6.4 from Table 11.4.
The HRP 0.975 = STMR × fP 0.975 = 0.0275 × 6.4 = 0.176 mg/kg.
Calculate ESTIe and Fast by entering into the WHO template
the HRP 0.975 .
The calculated ESTI (Fast ) values for general populations and
children are 40% and 130% of the maximum ARfD, respectively.
The higher value is chosen: Fast = 130.
FMRL < Fast → FT 1 = Fast in Eq. (11.15)
II. Calculation of FT 2 — the larger factor of FM 0 and FaM
(1) Combined weighting factor: FM0 = (fm + fp ) Determine fm:
fm = (100 − βt %) = 100 − 89 = 11,
where probability of detection of residues above the 98th per-
centile from N = 110 samples is βt , calculated with Eq. (11.1).
Determine fp : The fp = 13.1 was calculated from monitoring
data with Eq. (11.13) assuming zero residues if R < LOQ was
reported.
Calculate FM 0 : FM0 = Fm + fp = 24.1.
→ According to the decision tree the FaM has to be calculated
in the next step.
(2) Calculation of FaM :

Use the WHO template for the calculation of ESTI by entering
the HRM .
Highest residue in monitoring data: HRM = 0.34 mg/kg.
FaM = 80% of maximum ARfD for general population.
FaM = 260% of maximum ARfD for young children (use the
higher FaM ).
FM0 < FaM → FT2 = FaM in Eq. (11.15).
III. Calculation of combined weighting factor with Eq. (11.15):

FT1−T2 = ((3 − k) × FT1 + k × FT2 )/3 = ((3 − 2)∗ 130 + 2∗ 260)/3 =
216.6 → 217.
Since FT1−T2 > 100 targeted sampling programme is recom-
mended to verify if the established use patterns are applicable under
the particular growing conditions.
11.7 Number of Samples to be Included in Future

Monitoring Programmes
The number of samples that can be taken annually as part of the
monitoring programmes depends on the laboratory testing capacity
and financial resources available. The presented model provides sup-
port for planning the random monitoring of pesticide residues in mar-
keted commodities taking into account the potential risk of violation
of MRLs and or acute exposure exceeding the ARfD. It is not aimed
to use for special cases such as the occurrence of an extraordinary
pest or active substances applied with special use permit.
In case of a large-scale production, the pesticide−commodity
combinations, which indicate the risk of short-term intake problem or
residues exceeding the MRL over 2% of the samples should preferably
be included in the random monitoring programme and tested with
about 95% probability of finding defective lots.
Where the potential risk from pesticide residues is lower, then
lower probability of detection may be acceptable, which can be
achieved with the analysis of fewer samples.
In the ideal case, the number of random samples to be taken
depending on the weighting factors is shown in Table 11.7.
Table 11.7. Recommended number of

samples (n) depending on the calcu-
lated weighting factor (F ) with prob-
ability levels (βt %).
F n βt %
≥ 100 149 95
≥ 75 115 90
≥ 50 100 87
≥ 40 60 70
≥ 30 45 60
≥ 20 30 45
≥ 15 15 26
≥ 10 10 18
< 10 0 0
Alternatively, the weighting factor (F ) can also be directly used

for ranking the importance of the analysis of various commodity–
pesticide combinations. Where the sufficient testing capacity or finan-
cial resources are not available, the most critical commodity pesticide
combinations should be given the priority and allocating the number
of samples to other commodities proportionally to their weight.
In order to clarify specific problematic situations, targeted field
surveys are recommended, where sampling of treated commodities
should also be based on random selection of sites. Since the pesticide
treatment history is known in these cases, if limited laboratory test-
ing capacity is available, lower probability of detection of defective
lots may be acceptable taking a minimum of 40–60 samples.
11.8 Conclusions and Recommendations

The potential combinations of pesticide residues and food com-
modities are practically infinite. It is impossible and unnecessary to
include all of them equally in the monitoring programmes. Primar-
ily, the risk of violation of MRLs or exposure exceeding the ARfD
shall be taken into account in prioritizing the commodities and the
number of samples to be taken. In addition, the need for protection of
crops (pest pressure), expected pesticide use, pesticide sales, export
market MRLs and capability of multi-residue methods can be taken

into account.
The two-tiered model for planning monitoring programmes
presented in this chapter makes best use of all available relevant
information, and assists the risk managers to design the sampling
plan suitable for the particular circumstances. It also provides the
necessary flexibility for selecting appropriate probability for making
decisions.
Though the plan ranks commodities in view of selected pesticide
residues, it is assumed that the samples taken are analysed with
multi-residue procedures. The methods should be supported with
effective quality control measures enabling the quantification of the
uncertainty of all steps of the whole determination process and ver-
ification of the reliability of the results (see details in Section 10.7).
In order to reveal potential risks, the methods applied should cover
as many residues as technically possible.
For processing the large number of residue data, tailor made data-
processing software should be employed for recording the essential
information related to the samples and calculating FM0 and FaM for
each commodity–pesticide combination automatically.
It is important to recognize that determination of the compliance
of a commodity with 100% probability is an unreachable goal in
practice. In case of premarket control of commodities, if the residue
content is compared to the maximum legal limits, there is a high
probability of making a wrong decision. For assuring the compliance
at targeted percentage of the product, a lower level than the MRL,
a so-called action limit (AL) is recommended to be applied. When
using the AL as a reference point, the combined uncertainties of sam-
pling, sample processing and analysis should be taken into account
to ensure the compliance with legal limits according to the decision
rules agreed by the trading partners.
The weighting factors calculated in tier 2 can also be used to
evaluate the results of analyses of samples taken before the commod-
ity is placed on the market or exported. In this case the appropriate
action limits shall be inserted in Eq. (11.13) instead of the MRL.
The AL shall be decided according to the MRLs prevailing on the
targeted export market, or the acceptable maximum residue levels

(sometimes called private standards) specified by the buyer, and the
decision rules defining the acceptance criterion agreed by the trading
partners. Details for application of these principles are provided in
Section 10.10.
Especially, when the limited laboratory capacity enables taking
only lower number of samples than given in Table 11.7, a default
AL of 0.3 MRL may be inserted in Eq. (11.13). If the calculated FM0
exceeds 100, it provides an early warning for the potential exceedance
of MRL in the given commodity. Under such situation, an increased
frequency of sampling may be warranted to identify non-compliant
lots and reduce the number of lots rejected jeopardizing the reputa-
tion of the supplier of the commodity. Simultaneously, the growers
should be advised to carefully control or avoid applying those pesti-
cides which remain on crops at unusually high concentration under
the particular weather conditions.
References1
Ambrus Á, Horváth Zs, Farkas Zs, Szabó IJ, Dorogházi E and Szeitzné-Szabó M.
2014. Nature of the field-to-field distribution of pesticide residues, Journal of
Environmental Science and Health 49: 229–244.
Ambrus Á, Valero A, Farkas Zs, Horváth Zs, Szabó IJ and Braun S. 2013. Rec-
ommended sampling schemes to test for chemical contaminants and micro-
organisms, BASELINE Reports D6.7 and D6.9.
CAC. 1999. Recommended method of sampling for the determination of pesticide
residues for compliance with MRLs, CAC/GL 33-1999 rev 2.
FAO. 2003. Development of a framework for good agricultural practices.
FAO. 1997–2015. Pesticide residues in food- Reports of the Joint FAO/WHO
Meeting on Pesticide Residues. FAO Plant Production and Protection Paper
Nos. 145, 148,153, 163, 167, 172, 176, 178, 183, 187, 193, 196, 200, 211, 215,
219, 221, 223. FAO Rome.
GLOBALG.A.P. 2015. General regulations crop rules V.5.0.
Horváth Zs, Sali J, Zentai A, Dorogházi E, Farkas Zs, Kerekes K, Szeitzné-Szabó
M, and Ambrus Á. 2014. Limitations in the determination of maximum
1
The FAO, OECD and WHO publications cited in this chapter are freely available
and can be accessed at the websites of the corresponding organizations. Web pages
residue limits and highest residues of pesticides, Journal of Environmental

Science and Health, Part B 49: 143–152.
OECD. 2011. OECD MRL calculator: user guide. OECD Series on pesticides,
No. 56.
relating to pesticides. Pure and Applied Chemistry 78: 2075–2154.
WHO. 2014. Template for the evaluation of acute exposure (IESTI).
Chapter 12
Future Directions
Árpád Ambrus and Denis Hamilton
When we look to past history as a guide to the future, we see

steady scientific and administrative progress in the practices related
to assessment and control of pesticide residues in food. But we also
see that, from time to time, the steady progress is punctuated by
public controversies about residues in food that have stimulated spe-
cial responses by government and industries.
Various aspects of food safety and food security will remain
among the first priorities of governments, industry managers and
the general public. Integrated pest management with efficient and
sustainable use of pesticides will continue to be an integral part of
modern agricultural production.
New types of pesticide active substances including those with
reduced risk characteristics, natural products and biopesticides will
be developed. Their introduction and marketing will be increasingly
linked with novel formulation, including nano particles and advanced
application technologies designed to enhance the biological efficacy,
while reducing exposure to applicators, bystanders and the environ-
ment.
Further research on mode of action for toxic effects in mammals
will assist the interpretation of toxicity data and refine extrapola-
tions from test animals to humans. Many in vitro (literally ‘in glass’)
tests on cells, tissues and the like are already being used to examine
biological effects of pesticides. We may expect new in vitro tests to
be developed and validated for employment in support of existing in
vivo tests and ultimately as alternatives.
507
Biopesticides that consist of microbial pesticides or contain living

organisms have different characteristics from chemical pesticides and
will require different guidelines. Relevant impurities are more likely
to be biological entities rather than chemical and a different approach
will be needed. Do such pesticides leave detectable residues and are
analytical and test methods available to check if such a pesticide has
been used or not? Proof of such use may be needed, for example, to
allow export of food commodities from a quarantine area.
Farmers, tempted to purchase counterfeited products offered at
lower prices, may not realize the substantial differences from the
original products. The quality control of new formulations is a very
challenging task and may require increasing the capabilities of testing
laboratories. Its scope should include routine testing of the physical
and chemical properties of formulations, in addition to determining
the active ingredient content, which is not sufficient alone. Verifying
the authenticity of traditional and especially of novel formulations
might exceed the capabilities of some official testing laboratories.
Therefore, the technical assistance of original manufactures would
be needed.
To facilitate practical realization of the benefits of their new
active substances and formulations, pesticide manufacturers would
also be more involved in developing country-specific label instructions
for the efficient application conditions suitable for local technological
level and environmental conditions and training of local extension
staff.
Organisation for Economic Co-operation and Development
(OECD) working groups will continue revising test guidelines and
guidance documents to ensure that best practice in regulatory
risk assessment evolves addressing the challenges arising from the
introduction of novel products. Furthermore, current test meth-
ods will be refined based on the scientific developments in related
areas, such as involving in vitro tests, computer modelling of
structure–activity relationship and optimization of animal experi-
ments. The cooperation of government, industry and food safety
organizations will enhance the consumer confidence in regulatory
processes.
Future Directions 509
Realizing that safety of produced raw agricultural commodities

and compliance of pesticide residue levels with legal limits or quality
requirements of food manufacturers and distributors originate in
the quality of the pesticides and the condition of use of pesticides
and handling of the harvested crops, national government agencies
and interested private companies will extend their practical advisory
activities and introduce incentives for assisting farmers in implement-
ing the principles of good agricultural practice in the use of pesticides.
Risk-based national pesticide residue monitoring and targeted
sampling programmes will be implemented to verify the efficiency of
the provisions made to promote the safe and efficient use of pesticides
aiming also to satisfy the intended market requirements.
The resource and time requirements of collection of correctly
taken representative samples will be recognized as the primary basis
of correct management decisions, which would be made taking into
account the relevant combined uncertainty of the results of the anal-
yses of pesticide residues present in the samples when they are com-
pared to the applicable residue limits.
It is not possible to predict when and where public controver-
sies about pesticide residues erupt, but when they occur they can
be disruptive for farmers, industry and trade. A strategic approach
is needed, where people in government and industry have a good
knowledge of the situation and ensure that correct procedures are
followed. They also should be able to solve the problems underlying
the public concern and minimize any disruptions.
Public controversies have sometimes highlighted gaps in the reg-
ulatory systems. In response, the authorities search for answers from
existing data or they may require a different type of study in future
data requirements. We may expect this again in the future.
Codex MRLs, elaborated by the Codex Committee on Pesti-
cide Residues (CCPR), are the international criteria for pesticide
residues that divide food commodities between those that are legally
fit for human consumption and for international trade and those that
are not.
The Joint Meeting on Pesticide Residues (JMPR), as the inde-

pendent scientific advisory body to CCPR, will continuously con-
sider all relevant scientific information and will refine its procedures
to make best use of that information for evaluation of maximum
residue levels and estimation of dietary intakes. In order to facilitate
general acceptance of Codex MRLs, the exposure assessment proce-
dures applied by the JMPR and national authorities will be harmo-
nized as far as possible. Further on, the GEMS/Food database will be
continuously updated with national data, obtained with reliable and
transparent food consumption surveys. The new food consumption
data will be regularly incorporated into the database used by the
JMPR for estimation of dietary intakes of pesticide residues.
December 14, 2016 14:43 Food Safety Assessment of Pesticide Residues 9in x 6in b2668-index page 511
Index
A adverse outcome pathway (AOP), 7,

absorption, 19, 24, 40, 154, 159–160 170
absorption, distribution, metabolism advisory network, 475
and excretion (ADME), 40, 154 aerobic soil metabolism, 124–126
acceptable daily intake (ADI), 5, 40, aflatoxins, 297, 364
114, 200, 243, 284 age groups, 9, 224, 232
accelerated storage procedure, 308, aggregate exposure, 7, 86, 103, 243
319 aggregate risk, 97, 104, 258
accuracy, 132, 202, 289, 336, 341, 387, aggregate sample, 330–331
405–406, 418, 444 Agreement on the Application of
acidity, 312–313 Sanitary and Phytosanitary
acidity and pH range, 312 Measures (SPS Agreement), 269
ACROPOLIS, 258 agro-chemicals, 471
action limit (AL), 458, 504 aliquot, 330, 332, 387
active constituent, 45–47, 53, 55, 58, allergy, 168
62 allethrin, 294
active ingredient, 39, 41, 49, 88, 98, ambient temperature, 441
104, 286, 288–289, 293, 297, 302, American Society for Testing &
305, 310, 319, 408, 508 Materials (ASTM), 288
active substance, 21, 40, 62, 64, aminotriazole, 4
66–70, 72, 74–75, 77, 262, 476, 502, analyte integrity, 377, 389
507 analytical methods, 31, 41, 43, 68, 76,
active transport, 159 121, 129–130, 133–134, 148, 287,
acute exposure, 116, 178, 201, 203, 292, 390, 408, 445, 488
243, 490, 497 analytical portions, 330, 332, 355, 377
acute reference dose (ARfD), 40, 114, analytical recoveries, 129
244, 486, 489–490 analytical sample, 330–331, 338,
acute toxic alerts, 177 371–372, 377, 395, 406, 438
acute toxicity, 20, 48, 74, 79, 155, aneuploidy, 165
161, 168 animal products, 389
additives, 293–294 animal tissue, 333, 389
adverse effect, 8, 165–166, 168 animal welfare, 183
adverse health effects, 170 apple pomace, 147
511
512 Index
application rates, 43, 48, 53, 134–135, buprofezin, 139

318 buyers, 457
arithmetic mean, 419
arsenic residue, 114 C
arterial disease, 182 14
C label, 119, 125
Arthur Hill Hassall, 4
candidates for substitution, 70, 72
Arrhenius equation, 319
carbamates, 168
Association of Official Analytical
Chemists (AOAC), 287 carcinogenic risks, 157, 165
authorization process, 198 carcinogenicity, 7, 21, 39, 74, 155,
average recovery, 447–448, 453 162–164, 181
azadirachtin, 297 CAS, 296
CAS Registry number, 294
B catabolism, 161, 171
central limit theorem, 413, 433
Bacillus thuringiensis, 297, 309
centre of gravity, 368
baking, 144–145
characterization, 8, 10, 79, 89, 91,
baking, brewing, boiling,
120, 159, 162, 165, 172, 184
pasteurization, 144
chemical name, 294
Bartlett test, 450
chemical substances, 19, 21, 184, 206,
batch, 297, 300, 310, 333–334, 339,
467, 485
377, 385, 407
Chi-square distribution, 427
behavioural tests, 168
benchmark dose (BMD), 6, 74, 94, children, 3, 9, 66, 73, 75, 78, 86, 95,
176, 179, 257 103, 204, 208, 250, 254, 501
bias, 336–341, 351, 359, 362–363, 367, cholinesterase activity, 298
371, 377, 387–388, 394, 407 chopping, 332, 360, 389, 395, 406, 410
bile, 160 chronic reference dose (cRfD), 116
binomial distribution, 479 chronic toxicity, 24, 79, 163
bioallethrin, 295 CIPAC Handbook, 287
biopesticides, 296, 321 CIPAC methods, 283, 286
biotransformation, 160–161 CIPAC number, 294, 312
bixafen, 149–151 clinical signs, 163
bladder tumour, 181 cluster diet, 245, 487
blank sample, 445, 447, 453 cluster sampling, 224
blood–brain barrier, 160 Codex Alimentarius, 270–271, 274,
botanical pesticides, 297 281, 437
brain development, 168 Codex Alimentarius Commission
bran, 146 (CAC), 5, 82, 115, 118, 269
bread, 143, 146 Codex Classification of Foods and
breastfeeding women, 225 Animal Feeds, 118, 214, 280–281
brewing, 145 Codex Committee on Pesticide
bulk materials, 331–332, 342–344, Residues (CCPR), 15, 38, 117, 271,
368, 435 323, 509
bulk sample, 131, 331, 338, 371, 391, Codex general subject committees,
395, 409, 456 273
Index 513
Codex maximum limit for pesticide conjugated residue, 121, 129

residues (MRL), 51, 54, 82, 106, conjugates, 133
274 conjugation, 161
Codex Secretariat, 278 consignment, 330, 333
Collaborative International Pesticides consumer, 202, 225–226
Analytical Council (CIPAC), 284, controlled quality, 474
308 conversion factors, 217–218
collaborative trial, 289 copper oxychloride, 300
combined estimation of errors, 327, core battery tests, 165
375 correct sampling error, 361, 371–372
combined margin of exposure, 258 cotton seed meal, 147
combined uncertainty, 376, 405, 407, counterfeit products, 285
410, 415, 434, 439, 456, 458, 509 criteria for the prioritization, 279
commercial application of pesticides, critical end points, 40, 48
413 critical GAP, 135, 140, 477, 483, 493
comminuted sample, 406, 441 critical values, 447, 450
comminution, 337–338, 357, 369, 372, crop groups, 50, 429, 455
377–378, 438, 442 crop metabolism, 122, 124
commodity groups, 56, 127, 141, 281, crop rotation, 126
445 CropLife International, 305
commodity of trade, 118, 138 cryomilling, 437, 443
common adverse outcome, 84, 258 cumulative aggregate exposure, 256
common mechanism of toxicity, 104, cumulative assessment groups, 259
256–257 cumulative dietary exposure, 261
common name, 294, 296, 301 cumulative exposure, 7, 9, 257–258,
compliance residue definition, 41–42, 373
49 cumulative frequency, 458
compliance with MRLs, 41, 49, 59, cumulative risk assessment, 85, 104
97, 139, 386, 391, 405, 429, 438, 456 cypermethrin, 295
composite foods, 213, 218
composite sample, 84, 247–248, 254, D
330–331, 338, 340, 353–354, 357, 24-hour dietary recall method, 207
361, 363, 366–368, 378, 384, 409, data cleaning, 197, 230
414–418, 420, 426, 433–435, 454, data quality, 185
456–457, 477–478, 483 data requirements, 14–15, 17, 31, 38,
compositional, 410 67, 71, 87, 90
compositional (constitutional) DDT, 152–153
heterogeneity (CH), 345, 351–354, decision rule, 457, 459
365, 372 decision tree, 487–488, 494, 495
computer models, 183, 185 decision unit, 334–337, 340, 345,
computer-assisted personal 347–349, 371, 377, 391, 395, 407,
interviewing (CAPI), 211 413
concurrent recovery, 441 default value, 457
confined rotational crop studies, 127 delimitation error, 339
conformity assessment, 456 depuration trials, 52
514 Index
deterministic and probabilistic efficacy trials, 134–135

models, 199 efficiency of extraction, 406, 445
deterministic approach, 9–10, 251 efficiency of sample processing, 412,
deterministic calculation, 483 442, 455
developmental effects, 179 EFSA Comprehensive Food
developmental immunotoxicity, 166 Consumption Database, 225
developmental neurotoxicity, 166, 168 elderly, 205, 208
developmental studies, 166 element, 340–348, 350–353, 355,
developmental toxicity, 24, 166–167, 358–359
180, 263 embryotoxic effects, 167
dieldrin, 152 emetic, 294
dietary assessment, 205 emulsifiable concentrate (EC), 305,
dietary burden, 44, 101 321
dietary exposure, 102–103, 160, 177, endocrine disruption, 28–29
186 endocrine effects, 179
dietary exposure assessment, 9, 259 endpoint, 93, 159, 161
dietary intake assessment, 118, 132 endpoints for acute effects, 179
dietary risk assessment, 41–42, 49, 73, energy intake, 230
76–77, 80, 95 enforcement, 3, 41, 44, 59, 82, 98,
dietary survey, 214, 222, 224 105, 121, 130, 132, 385, 390, 392,
dimethoate, 308 456, 492, 497
dimethomorph, 138–139 enterohepatic circulation, 161
dioxins, 299 environmental contaminants, 117
disease forecast system, 474 environmental fate, 119, 124
distribution of residues, 373, 383, 416 epidemiological studies, 157
distributional heterogeneity (DH), equal probability of selection, 360–362
345, 351–354, 358–359, 366, 372 equipment, 65, 222, 300, 348, 371,
DNA damages, 164–165 375, 393, 442, 474
dose–response relationship, 8, 115, equivalence determination, 285, 310
172 error, 205, 212, 327–328, 340, 396, 406
drinking water, 58, 68 error propagation, 410
dry ice, 437, 442–443 estimated short term intake, 488–489
dry-weight, 147, 149 estimation of uncertainty, 281, 415,
duplicate samples, 363, 416, 428 452
duration of the exposure, 186 etofenprox, 309
dustiness, 285, 315 estrogenic activity, 167
ethnic minorities, 225
E EU menu, 207, 227
early warning sampling plan, 470 European Food Safety Authority
ecotoxicology profile, 286 (EFSA), 5, 64, 207
ectoparasites of livestock, 280 European Union, 5
edible part of RAC (eRAC), 218, 221 Excel macro, 417
edible portion, 78, 118, 122, 136, 139, excretion, 19, 161, 182
254, 390 executive committee, 271
effective use, 141, 285, 312 expanded uncertainty, 452, 457
Index 515
expiry dates, 318 food description, 200, 209

export intervals, 56 food diary, 206, 212
export slaughter interval (ESI), 52 food frequency questionnaire, 207
exposure to multiple chemicals, 7, food groups, 207, 214, 250
84–85, 199, 256, 258 food list, 232–233
extension of scope of an analytical food models, 209
method, 292 food preparation, 143
extraction procedures, 130 food processing, 144, 149
extraneous maximum residue limit food record, 206, 210, 212, 234
(EMRL), 152, 269 food safety risk assessment, 272
FoodEx2, 213, 215
F formaldehyde, 302–303
1996 Food Quality Protection Act formulation, 158, 283–284, 286
(FQPA), 91 formulation physical properties, 306
fair practices in the food trade, 5, formulation types, 305
114–115, 270 fosetyl-aluminium, 309
FAO Manual, 116, 119, 131, 142, 380, fragment, 339, 342, 347, 357, 360
390, 447 frequency of consumption, 207
FAO specifications, 291, 304 full validation, 445
FAO/WHO Joint Expert Committee functional observational battery
on Food Additives (JECFA), 5 (FOB), 168
fat tissue, 120–121, 130, 152 fundamental sampling error (FSE),
fat-soluble, 120, 152 353, 372
fat-soluble pesticides, 273, 279
fat-soluble residue, 121 G
feeding studies, 52, 90 gene mutations, 165
fenitrothion, 145–146 general population, 9
fenthion formulations, 322 genotoxicity, 23, 164–165, 184
final sample, 331 genetically engineered (GE) crops,
finite element materials, 341–348, 322
379, 384 glass-house uses, 124
flour, 146 Global Joint Review (GJR), 62
flowable concentrate, 305, 313 GLOBALG.A.P., 468
fodder, 147 glucuronides, 161
foetotoxic, 167 glyphosate, 302, 303
following crops, 126–127 glyphosate-tolerant cotton, 308
food acquisition, 204 good agricultural practice (GAP), 7,
food adulteration act, 114 53, 115, 118, 135, 199, 247, 273,
food and agriculture organization, 270 275, 509
food as consumed, 199, 232 good laboratory practices (GLP), 16,
food balance sheets, 203 39, 156, 396
food classification, 213–214 grab sample, 331–332, 333–338, 378,
food consumption, 10, 214 391
food consumption data, 215 granules, 317
food consumption survey, 217, 227 grinding, 406, 410
516 Index
gross error, 373, 407, 439 I

gross sample, 228, 331 identification, 32, 84, 120, 124, 165,
grouping and segregation error 172, 231, 250, 256, 281, 290, 322,
(GSE), 358–361, 366, 371–372, 375, 344, 384, 393, 473
377–378 identity, 294
growth stage, 124, 136 illegal residues, 127
Grubbs test, 447 immunotoxicity, 168
guard banding, 458 import tolerance, 54, 67, 73, 80
guidelines, 270–271, 379 impurities, 285, 288
guidelines of hygienic practice, impurity names, 301
270 impurity pathogens, 322
impurity profile, 286, 303, 322
H increased user risk, 318
HACCP, 469 incorrect sampling errors, 371
haematotoxicity, 179 increment, 331–332, 337–349, 356,
haemolytic anaemia, 173 359–370, 372
increment delimitation error, 365, 372
harmonization, 10, 63, 70, 106, 198,
increment extraction error (IEE),
200, 221, 234
339, 365, 372
harmonization of terminology,
increment weighting error, 372, 376
167
incurred residues, 43, 130, 444
hazard, 39–40, 115, 186
independent laboratory validations,
hazard characterization, 155, 159,
292
175, 177
individual level, 214
hazard identification, 172–173
individual residue, 409, 416, 478
health-based guidance, 9
inert ingredients, 305
hepatotoxicity, 179
infants, 205, 224
heptachlor, 152 inference, 331, 334–336
heterogeneity, 345, 347, 349–354, infinite element material, 342–343,
358–363, 366, 371–372, 383, 346, 348, 359
386 ingredient, 214, 216
hexachlorobenzene, 299 intake data, 207
highest residue (HR), 43, 78, 483, integrated pest management, 470
498, 501 internal quality control, 445
historical control data, 164 International Code of Conduct, 285,
homogeneity of variances, 450 310
homogeneous, 330, 350–352, 371–372, international collaborative trial, 290
379, 391 international estimate of short-term
Horwitz curve, 291 dietary intake (IESTI), 7
household budget surveys, 232 International Organization for
human data, 169 Standardization (ISO), 289
human health hazard, 154 International Programme on
hydrolysis, 161 Chemical Safety (IPCS), 6, 115
hydrophilic metabolites, 160 inter-individual variability, 201
hypersensitivity, 168 International Standard ISO 5725, 290
Index 517
interspecies differences, 154 liver tumour, 169

intra-individual variations, 201 livestock feed tables, 32
intra-species variation, 176 livestock metabolism, 120
intrinsic enzymes, 144 lognormal distribution, 413
in silico testing, 183 long-term exposure, 199–200
in vitro testing, 183 long-term storage, 455
in vivo studies, 185 longitudinal studies, 201
isomalathion, 300 loss of residues, 437
lot, 131, 331–332, 456–457
J lowest-observed-adverse-effect-level
JMPR Evaluations, 437 (LOAEL), 49, 154
JMPR members, 117
Joint FAO/WHO Consultation, 6 M
Joint FAO/WHO Expert Committee magnitude of residues, 41, 76, 477
on Food Additives (JECFA), 134 major crop, 50–51
Joint FAO/WHO Meeting on malathion, 298, 300–301
Pesticide Residues (JMPR), 272 malformations, 167
management responsibilities, 327, 393
K manufacturing limits, 297, 311
K-Farm Programme, 472 manufacturing pathway, 303
key event relationship (KER), 170 margin of exposure (MOE), 94
kidney, 120 marker, 97
kidney effects, 179 market acceptance, 385
mass reduction, 327, 331, 335–338,
L 368–372, 382, 396
label directions, 127, 135 material properties, 327, 346
laboratory animals, 6 materialization errors, 372
laboratory capacity, 483, 505 maximum registered uses, 135
laboratory sample, 330–331, 438–440 maximum residue level, 136–140, 147,
laboratory sampling errors, 327, 378 489, 495
lactating dairy, 148 maximum residue limit (MRL), 7, 38,
lactating women, 225 273, 335
lambda-cyhalothrin, 296, 316 measurement error, 205
LanguaL, 215 measurement uncertainty (MU), 329,
large-size fruits, 409 341, 387, 415, 458
laying hen, 148 mechanisms of toxicity, 158
leafy vegetables, 409, 437 median, 478, 483
legume animal feeds, 147 median ranges, 478
limit of determination, 278 median residue, 138, 247, 253, 483
limit of quantification, 44, 56, 99, 140 medium-sized fruits, 433
lindane, 152 metabolism, 42, 49–50, 160
linearity of response, 289 metalaxyl, 296
lipophilic chemicals, 160 method selectivity, 129
liver, 160, 173 method validation, 443, 445
liver hypertrophy, 174 methods for enforcement, 130
518 Index
methods of analysis, 270, 280, 286 non-consecutive consumption days,

milk fat, 152 202
Miller Pesticide Amendment, 3 non-dietary data, 206
mineralized residue, 125 non-participants, 229
minimum mass, 383 non-responders, 224
minor crop, 141–142 noodles, 146
minor use crop, 72, 76, 142 normal distribution, 459
mode of action (MoA), 7, 154 ‘normalized’ data sets, 418
monitoring data, 8, 113, 142, 249, normalized residues, 479, 483
486–502 Nugget Effect, 364–365
monitoring programmes, 199, 482, Number of Lots Sampled, 423
485–486
Monte Carlo, 10, 201, 252 O
Monte Carlo risk assessment tool obsolete GAP, 186
(MCRA), 201 OECD MRL calculator, 33, 43, 98,
mosquito coils, 306 100, 491
multi-residue, 49 OECD pesticide programme, 13
multi-residue methods, 131, 143, 448 OECD test guidelines, 157
multiple increment sampling, 331, OECD test guidelines programme, 13
340, 342, 347, 353, 366, 368, official control, 427, 458
377–378, 392 Omics methods, 184
mutagenic, 22 one-dimensional sampling, 346–347,
mutagenicity, 164 364, 368, 370, 372
Mutual Acceptance of Data (MAD), organic diet, 225
17 Organisation for Economic
Co-operation and Development
N (OECD), 10
N -nitrosamine, 300 organochlorine compounds, 152
N -nitrosoglyphosate (NNG), 302 organophosphates, 168
National Estimated Daily Intake origins of JMPS and its history, 286
(NEDI), 57, 260 oxamyl granules (GR), 315
National Estimated Short Term
Intake (NESTI), 58 P
national food consumption surveys, 97.5th percentile, 225, 255, 261, 383,
205–206, 229 483
national population register, 224 paraquat, 294
natural crop units, 406 parent compound, 42, 49, 91, 119,
neoplastic lesion, 163, 180 122, 129, 262
neurotoxicity, 26–27 parent population, 412, 418, 420
NHANES survey, 207 particle, 339
Niclosamide, 321 participant burden, 234
no observed effect level, 74 participation rates, 223
no-observed-adverse-effect-levels particle size reduction, 336, 355
(NOAELs), 154 peak concentration, 160
nomenclature, 294 percentage of consumers, 201
Index 519
periodic review program, 187 procedural manual, 272

permissible level, 117–118 processed commodities, 144–145, 248,
persistent foam, 314 262
Pesticide Data Program, 373–375 processed food, 333, 342, 390
pesticide monitoring programmes, 248 processing factor, 145–146, 218,
pesticide products, 68, 86 248–249
Pesticide Residues Intake Model processing studies, 43
(PRIMo), 259 production of fruits and vegetables,
pesticide specifications, 283 470
pH range, 308 propamocarb hydrochloride, 293
phenobarbital (PB), 181 proportionality, 135
physical property specifications, 308 protecting the health, 270
phytotoxic, 309 public health concern, 262
phytotoxicity, 135 public health risk, 117
Pierre Gy, 329, 349 published studies, 157
pilot trial, 290 purposive selection, 332
Pirimiphos-methyl residues, 143 pymetrozine, 144
placental barrier, 160
plant protection product, 38, 40 Q
point estimate, 9, 251 quality assurance, 16, 61, 223, 229,
point of departure (POD), 40 416
pooled CVP , 451 quality assurance unit (QAU), 157
population, 330–331, 334 quality principles, 157
portion size, 202, 207, 209, 212, 234 quantitative structure–activity
post-registration, 8, 253 relationship (QSAR), 185
poultry, 120
pourability, 317 R
practical residue limit, 118 3Rs (replacement, reduction and
pre-harvest interval (PHI), 43, 136 refinement), 183
pre-registration, 253 radiolabelled compounds, 127, 441
precision data, 289 random error, 326, 407, 444, 446
pregnant women, 205, 225 random samples, 331, 379, 454, 458
primary animal feeds, 147, 149 random sampling, 223, 331, 362, 474,
primary crop units, 421 489
primary feed commodity, 333 range finding study, 162
primary sample, 131, 338, 340, range of the average residues, 419
343–345, 421, 423 range statistics, 418
prioritizing, 470, 503 raw agricultural commodity (RAC),
probabilistic assessment, 262 3, 65, 80, 86, 100, 129, 146, 248, 333
probabilistic dietary exposure, 246 re-treatment interval, 43
probabilistic model, 10, 251, 256 recipe data, 221
probabilistic modelling, 201 recommended number of samples, 503
probabilistic selection, 338–339, 361, recovery, 98, 132, 162, 394, 406, 441,
371 445–452
probability level, 479, 488 reference dose (RfD), 94
520 Index
reference point index, 258 retained test portions, 451, 455

reference profile, 312, 323 risk analysis, 8, 271–272, 277–278
refinement of the assessment, 251 risk assessment, 1–2, 4, 6–8, 10, 258,
registry, 296 260
regulatory agencies, 456 risk characterization, 9–10, 260, 262
regulatory authorities, 38, 106 risk communication, 8, 10, 225
relative differences, 418 risk management, 8, 64, 88, 153, 172,
relative potency factor (RPF), 257 258, 272
relevant impurities, 288, 292, 297, risk managers, 10, 251, 262, 434, 436
299, 302, 308 risk ranking, 487
release date, 318 role of science, 271
reliability of sampling, 454 root vegetables, 147, 437
renal tumour, 181 rotational crop(s), 42, 76, 89, 99, 124,
repeat dose studies, 178 128
repeatability limit, 289 rules of propagation of error, 446
repeated-dose toxicity, 21 ruminants, 40, 120
replicate sample, 285, 331–332, 351,
384, 409, 416, 418–420, 422–423, S
425–429, 434, 458, 478 safe use, 5, 68, 73, 141
replicate sampling, 396 safety factor, 9, 39–40, 49, 74–75,
representative commodities, 98, 132, 176, 178
281, 445 sample, 117, 129–130, 330, 347, 483,
representative commodity groups, 445 486–487, 496
representative crops, 128 sample mass, 357, 379, 406, 440
representative sample, 131, 137, 327, sample preparation, 221, 371, 378,
332, 336–339, 341, 352, 377, 387 437–438, 455
representative sampling, 332, 345, 347 sample processing, 329, 336, 371–372,
representativeness, 224, 250, 335, 337, 376, 380, 383, 392, 395, 410, 435,
379 438, 441, 455
reproducibility limit, 289 sample quality criteria (SQC),
reproducibility of the analyses, 406 334–335, 337
reproductive toxicity, 24 sample size reduction, 389, 439
reproductive toxicity study, 166 sample size(s), 222, 225–226, 412,
residential exposure, 87, 103 428–429, 483–484
residue analysis, 113, 150 sample storage, 113, 131
residue chemistry, 38, 97–98, 100 sampling, 128–129, 223
residue control, 470 sampling constant, 353, 355–357, 439
residue definition, 40–41, 113, 170, sampling correctness, 327, 338–339,
492–499 341, 345, 362, 396
residue definitions for dietary risk sampling device, 331–332, 344
assessment, 41, 75 sampling dimensions, 341, 344–348
residue definition for enforcement, sampling errors, 350, 375–378, 444
41–42, 44, 77, 138, 497–498 sampling frame, 206, 229
residue monitoring data, 59, 142, 152 sampling mass, 340–341, 353–355,
response bias, 210, 227 357–358, 363
Index 521
sampling method, 206, 362, 364, 391 specimen, 331–332, 337–338

sampling plan, 362–363, 406, 459, 482 spirotetramat, 140–141
sampling principle, 131 split sample, 332, 377
sampling procedure, 330, 353, 367, spray residues, 114
372, 392, 417, 421 stability of analytes, 406, 442,
sampling protocols, 383, 387, 393, 415 455
sampling target, 407, 413, 416, 421, stability at elevated temperature,
436, 439 308, 318
sampling tool, 343–345, 348, 361, stable compound, 441, 455
364–365, 367–368, 370, 382, 394 standard units, 210
sampling uncertainty, 362, 372, standardized test method, 29, 285
384–385, 405, 415, 417, 421, 424, starting materials, 297, 303
426, 428–429, 432, 434–435, 439, statistical analysis, 158, 337, 408
458, 478 statistical methods, 34, 137
scientific advice, 64, 117 stenching agents, 294
scope of the methods, 445, 455 sterilization, 144
secondary sample, 332 STMR, 255, 260, 484
seasonal sampling, 203 storage stability, 43, 98, 132, 285,
secondary food commodity, 333 308, 317
segments, 439, 441 storage stability specifications,
selected percentile, 479–480 285
sellers, 57, 283, 457, 475 storage stability studies, 132, 388
semi-processed agricultural stratifications, 224
commodities, 244 structural alert, 297, 299
sensitivity analysis, 252 structural similarity, 172
sensitization, 90, 161–162 structure–activity relationship, 185,
settling disputes, 405, 456 263, 508
short-term exposure, 9, 155, 177, 201, study populations, 205, 225
256 sub-sample, 331–332, 377, 395
short-term surveys, 201 sub-sampling, 338, 412, 441
shorter than lifetime exposure, 185 Sulfotep, 299
single eating event, 201 supervised field trial, 42–44, 54, 78,
single increments, 406, 454 98, 102
size reduction, 336, 355, 389, 406, 439 supervised residue trials, 7, 139, 247,
skin and eye irritation, 162 487
small-scale farms, 467, 470 supervised trials, 486–487, 490
soil metabolites, 125 supervised trials median residue
soil type, 124 (STMR), 138, 247
solid constituent particles, 342 supply chain, 51, 472, 474–475
sources of bias, 339, 436 surfactants, 305
specialty crop, 142 survey duration, 200
species, 162, 178 suspensibility, 316
specification, 43, 116, 157, 209, 231, systematic random sampling,
283, 285, 288, 291, 293, 298, 304, 363
306, 312, 364, 392, 468, 473 systemic toxicity, 26, 162
522 Index
T toxicological evaluation, 40, 48, 58,

target crops, 43, 98–99 113, 153, 159, 165, 172, 181,
target organ, 7, 20–22, 84, 92, 155, 183–184
162, 164, 258 toxicological profiles, 154, 171–172,
target population, 206, 230 303, 310
targeted field surveys, 485–486 toxicological relevance of metabolites,
targeted sampling, 249, 494, 502, 509 170
TDS sampling design, 250 toxicological significance, 163, 165,
Technical Barriers to Trade (TBT 177, 180, 274
Agreement), 269 toxicological thresholds, 115
technical concentrate (TK), 293–294 trade names, 294
technical material (TC), 283–285, 293 trade restrictions, 117
technical materials: purity, 296 trade risk, 117, 127
teratogenic lesions, 167 transfer of residues, 41, 44
test guidelines, 10, 13, 156–157, 163, transformation products, 110, 122,
380, 508 128–129, 132–134
test portion, 331, 358, 361, 388 transgenic crop, 134
test sample, 330, 332, 360, 378 treatment-related changes, 163, 175
tetrachloro-azobenzene, 299 triazolylalanine, 123–124
Theory of Sampling (TOS), 328–330, tribenuron-methyl, 311
344, 349–378, 381 trichlorophenoxy, 299
thiamethoxam, 313 trichlorophenoxy moiety, 301
Thomas Wakley, 4 trueness, 444, 447
three-dimensional sampling, 346, 348, TSH stimulation, 182
368, 372 tumour incidence, 164
threshold of toxicological concern two-dimensional sampling, 346, 348,
(TTC), 263 365, 367, 372
thyroid, 84, 164, 182 typical recovery, 448–449, 451
thyroxine-binding protein, 182 typical uncertainty, 450–451
tiered model, 467, 487
Tier 1 equivalence determination, 310 U
Tier 2 determination, 311 uncertainty, 103, 176, 202, 252, 388,
tolerance, 5, 38, 73, 80, 98, 114, 478, 490–491, 504
117–118, 199, 248, 291, 380 uncertainty factors, 40, 94, 104, 176,
total diet study (TDS), 61, 108, 198, 261
217, 249–250 underestimation, 201, 205, 256, 413,
total radioactive residue (TRR), 43, 429, 478, 492
120 unextracted residue, 125
total sampling errors, 387 unintentional residue, 118
toxic equivalent, 257 unique toxicological profiles, 171
toxicants, 159–161 unit, 340, 395
toxicity data, 153, 156, 172, 507 unit weight, 84, 254, 260, 383
toxicity targets, 172 United Kingdom adulteration of food
toxicokinetics (TK), 19, 154, 159 and drugs act in 1860, 4
toxicological endpoint(s), 93, 158, 178 urine analysis data, 174
Index 523
USA environmental protection week days, 203

agency, 7 Weibull distribution, 413
USA National Research Council, 4 weight of evidence, 169, 175
use pattern, 46, 50–52, 57, 134, 139, weighting factor, 488, 490–491, 495,
481, 486, 493, 502 497, 500, 502–503
usual consumption, 253 well-mixed material, 439
usual intake, 210–211 wettability, 285, 306
wet sieve test, 315–316
V wettable powder, 298, 305, 316
variability, 10, 40, 84, 94, 101, 137, WHO specifications, 284, 288, 292,
176, 202, 226, 233, 329, 336, 312, 315
341–343, 350, 353, 357–358, 376, WHO template, 500
384–385, 387, 392, 406, 408, 412, women of child-bearing age, 96, 180,
418, 421, 424, 434, 438, 478 213
variability factor, 84, 255, 383 World Trade Organization (WTO),
variability of residues, 408, 413 269
vegetarians, 73, 225
vertical cutter mixer, 443 X
veterinary uses, 134 xenobiotics, 160–161
Victorian Public Food Act of 1854, 3
violation of MRLs, 476, 479, 503 Z
violation rate, 152, 459, 480–482, 486 zero-dimensional sampling,
vulnerable groups, 9, 225, 243 344–347
zonal application, 70
W
water as a relevant impurity, 308–309
water-soluble residues, 449

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Food Safety Assessment

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Library of Congress Cataloging-in-Publication Data

British Library Cataloguing-in-Publication Data

Copyright © 2017 by World Scientific Publishing Europe Ltd.

Desk Editors: Herbert Moses/Mary Simpson

Typeset by Stallion Press

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For the patient and supportive spouses and families

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It was a very pleasant surprise to receive an invitation from Merlyn

(OECD); establishment of national use patterns and safety assess-

Árpád Ambrus and Denis Hamilton

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b2530_FM.indd 6 01-Sep-16 11:03:06 AM

About the Authors

AMBRUS Árpád is chemical engineer (MSc), Candidate of Chem-

BHULA Rajumati (Raj) was appointed as the Gene Technology

xii About the Authors

BURA László is a diploma-qualiﬁed chemical engineer and has a

CALDAS Dutra Eloisa has a BSc in chemistry and a Masters in

DOHERTY Michael received his MSc (1990) and PhD (1997)

About the Authors xiii

teams for in-house and international joint review projects; providing

FARKAS Zsuzsa graduated as a biologist (BSc) at Eötvös Loránd

HAMILTON Denis has BSc, MSc (University of Queensland),

xiv About the Authors

HUMPHREY Paul has a BSc (Hons.) and a PhD in chemistry

HORVÁTH Zsuzsanna has BSc in biology and MSc in nutri-

LINDTNER Oliver studied mathematics at Humboldt University

About the Authors xv

PILOT-PANEU project and serves as a work package leader in the

MACLACHLAN Dugald is director of residues and microbiology

MARGERISON Sam obtained BSc (Hons) from University of

OCKÉ Marga studied human nutrition at Wageningen University

xvi About the Authors

food consumption surveys. Her scientiﬁc interests are: dietary assess-

PFEIL Rudolf is a doctor of veterinary medicine and a certiﬁed

ROWLAND Jessudoss has a MS in biology from the University of

SCHUMACHER David is a senior toxicologist at a German reg-

About the Authors xvii

of EFSA’s Pesticide Peer Review Panel, expert at OECD working

SOLECKI Roland Alfred is a biologist and toxicologist

van der VELDE-KOERTS Trijntje is trained as chemist (BSc)

xviii About the Authors

for Draft Assessment Reports for the EU. Trijntje is a Member of

WOLTERINK Gerrit (1959) has a MSc in biology and a PhD

YAMADA Yukiko is an international food safety consultant

YOSHIDA Midori graduated at the faculty of veterinary medicine,

About the Authors xix

School of Veterinary Medicine in 1996. She has 35 years of expe-

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2.1 OECD Guidelines for the testing of chemicals, Section 5

2.2 Residue guidance documents published

4.1 Fenitrothion residues in grain and milling fractions

4.2 Common toxicity studies and their purposes. . . . . . 155

4.3 Pesticides with unique toxicological proﬁles and their

5.1 Examples of extraction rates of diﬀerent processed and

5.2 Background information to be collected from survey