GHENT UNIVERSITY

FACULTY OF BIOSCIENCE
ENGINEERING
MASTER OF SCIENCE IN ENVIRONMENTAL SANITATION

Academic Year 2016-2017

Environmental Impacts of Pesticides

Practical Report:

Determining the residue of azoxystrobin on apples through liquid

chromatography

ABATO, Jamael
VASQUEZ, Celia
MSc. in Environmental Sanitation

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 Table of contens

1 Introduction .......................................................................................................................................... 3
2 Materials and methods ......................................................................................................................... 4
3 Results and Discussion .......................................................................................................................... 5
4 Conclusion ............................................................................................................................................. 9
5 References .......................................................................................................................................... 10

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1 INTRODUCTION

Azoxystrobin is a broad spectrum fungicide of the class of synthetic compounds called β-

methoxyacrylates. This fungicide can be applied as a foliar spray or a soil-treatment prior to
planting, as it has a residual effect. The mode of action of azoxystrobin is to prevent the respiration
of fungi due to the disruption of electron transport chain, preventing atp synthesis. Azoxystrobin
is effective against all 4 major groups of fungi – the ascomycota, deuteromycota, basidiomycota
and the oomycota. This implies that the fungicide is able to prevent or cure powdery mildew,
downy mildew, rust and and rice blast diseases. Azoxystrobin is approved for use on a wide variety
of crops in the united kingdom, including asparagus, barley, broccoli, calabrese, brussels sprouts,
onions, cabbages, carrots, cauliflowers, collards, combining peas, field beans, kales, leeks, oats,
oilseed rape, potatoes, rye, triticale, vining peas and wheat (accessed from
http://www.agchemaccess.com/azoxystrobin; accessed on 29-04-2017). Azoxystrobin is of low
acute and chronic toxicity to humans, birds, mammals, and bees but is highly toxic to freshwater
fish, freshwater invertebrates, and estuarine/marine fish, and very highly toxic to estuarine/marine
invertebrates (accessed from
https://www3.epa.gov/pesticides/chem_search/reg_actions/registration/fs_pc-128810_07-feb-
97.pdf; accessed on 29-04-2017). Von Stackelberg (2012) reported that the potential for adverse
health effects in human health resulting from exposure to azoxystrobin is low to non-existent.

Purpose of the practical

The purpose of this laboratory experiment is to determine the concentration azoxystrobin

from the acetonitrile extracts of the treated apples. Also, this practical would evaluate the effect of
different process factors applied on the experimental apples on the concentration of azoxystrobin.
Hence, this experiment would also determine which processed factor is likely to be effective in
reducing pesticide residues on azoxystrobin-treated apples.

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2 MATERIALS AND METHODS

For this experiment, the following materials and chemicals were used: 5 apples exposed to
azoxystrobin, 1 blank apple (not treated with azoxystrobin), 1 recovery apple (a blank apple but
exposed to known concentration of azoxystrobin), mixer sorvall type, electric cooking plate,
cooking pot, knife, centrifuge, laboratory weighing scale, electric juicer, 50 ml falcon tubes, 15 ml
pipettes, 10 ml pipettes, 1 ml pipettes, small beakers, volumetric flasks, small glass vials,
acetonitrile, nacl, na3citrate, na2hcitrate, mgso4, miliq water and 10 mg/l azoxystrobin solution.

Apple processing
Five apples exposed to azoxystrobin were processed differently. One apple was peeled
using a knife, one washed with tap water, one juiced, one unpeeled and one cooked. The apple that
was juiced, unpeeled and cooked were never washed with tap water. Also, the core of the apples
(except for the juiced apple) was removed while the stem of the five apples was removed during
processing. For the blank apple and recovery, the core and the stem were also removed but no
processing was applied. After applying all the processing factors to the five experimental apples,
each processed apple was cut into small pieces using a knife. The knife was rinsed with tap water
every time it was used to cut one processed apple. The recovery and blank apple were also cut into
small pieces. After cutting, 10 g of each processed apple was placed separately in a 50 mL falcon
tube. The same was done for the blank and the recovery apple. The falcon tubes were then labeled
with the type of process factor applied to the apples.

Azoxystrobin QuEChERS extraction

The azoxystrobin was then extracted from the processed apples. First, each falcon tube
containing the processed apple (and the blank apple) was pipetted with 15 mL acetonitrile and
shaken for 3 minutes. Acetonitrile was used as the extracting solvent in this experiment. After this,
the tubes were brought to a mixer (sorvall type) to homogenize the apple and the solution in the
tube. Every time the mixer was used for the next processed apple, the mixer was washed with tap
water and wiped with tissue paper. For the recovery apple, 10 mg/L of azoxystrobin solution was
added prior to the pipetting of acetonitrile. After mixing, each falcon tube containing the mixed
apple was added with 1.5 g NaCl, 1.5 g Na3Citrate, and 750 g Na2HCitrate. The tube was then

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shaken for 2 minutes after which 6 g of MgSO4 was then added and shaken for another 2 minutes.
The added salts in this experiment will push the fungicide from the apple to the acetonitrile, hence
extracted from the apple. After this, the tubes were then centrifuged for 5 minutes to settle the
apple residues at the bottom. After centrifuging, a visible supernatant (the acetonitrile containing
the fungicide) was found on the upper layer of the mixture. Finally, 1 mL of the supernatant was
pipetted from each tube and placed separately in a 10 mL volumetric flask. The flask was then
added with 9 mL MiliQ water. Then, sample was obtained from each flask and placed into small
glass vials. The vials were then covered and labeled with the group number and the name of
processing factor from which the sample was obtained. The samples were then analyzed through
liquid chromatography.

3 RESULTS AND DISCUSSION

The concentrations of the extracted azoxystrobin (mg/L) from the processed apples, blank
apple and from the recovery apple as measured through liquid chromatography was provided in
the class. However, these concentrations were based from a sample in the vial that was originally
obtained from 1 mL acetonitrile extract which was diluted with 9 mL MiliQ water to reach a
volume of 10 mL. Hence, the provided concentrations were multiplied with 10 to account for the
dilution that was done. Then, these concentrations were then converted in mg/kg using Equation
1 below. For Group 3, these concentrations are presented in Table 1 below.

𝑚𝑔
𝑚𝑔 𝑒𝑥𝑡𝑟𝑎𝑐𝑡𝑒𝑑 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 ( )𝑥 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑎𝑑𝑑𝑒𝑑 𝑎𝑐𝑒𝑡𝑜𝑛𝑖𝑡𝑟𝑖𝑙𝑒 (𝐿)
Equation 1. 𝑒𝑥𝑡𝑟𝑎𝑐𝑡𝑒𝑑 𝑎𝑧𝑜𝑥𝑦𝑠𝑡𝑟𝑜𝑏𝑖𝑛 ( )= 𝐿
𝑥 1000
𝑘𝑔 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑝𝑟𝑜𝑐𝑒𝑠𝑠𝑒𝑑 𝑎𝑝𝑝𝑙𝑒 (𝑔)

Where: volume of added acetonitrile = 0.015 L

weight of processed apple = 10 g
extracted concentration = concentrations in the extract (considering dilution)

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Table 1. Concentration of the extracted azoxystrobin (mg/L and mg/kg) from the processed apples, blank apple and
recovery apple.

Tested Apple Extracted azoxystrobin Extracted azoxystrobin

(mg/L) (mg/kg)
Cooked 7.3939 11.0909
Juiced 0.7293 1.0940
Peeled 0.5879 0.8819
Washed 0.4788 0.7182
Unpeeled 1.5504 2.3256
Blank 0.0051 0.008
Recovery 0.1136 0.170

Based from the used extraction procedure and from liquid chromatograph analysis of the
apple extracts, washing and peeling processes resulted to lower concentrations of azoxystrobin in
the apple extracts (in mg/L or mg/kg). Washing seemed to remove most of the azoxystrobin
residues from the treated apple than peeling the apple (although the difference in concentration is
not large enough). The result contradicted various claims that peeling apples is the most effective
way to remove the pesticides on the apple. In this experiment, we obtained lower concentration of
azoxystrobin in washed apple extract than in the peeled apple extract because during processing,
the apple was washed thoroughly for several minutes (the way how the experimenter washes the
apple she eats). Although, the differences on how the extraction procedure was done per processed
apple and the slight differences in the amount of added salts in the processed apple can also be
considered as contributing factors for obtaining such results. On the other hand, extracts from
cooked and unpeeled apples yielded higher azoxystrobin concentrations (in mg/L or mg/kg). This
implied that the two processing techniques were less effective than the others in removing
azoxystrobin residues from the treated apple (at least from our group’s results). Based from our
results, washing is the most effective processing factor that can reduce azoxystrobin residues from
the apple followed by peeling, juicing, unpeeling and lastly cooking. To verify and compare our
results to the other groups, their outputs are presented in Table 2 below.

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Table 2. Concentration of the extracted azoxystrobin (in mg/L and in mg/kg) on the extracts of the tested apples
from the other groups.

Group Extract Processed apples

concentration Cooked Juiced Peeled Washed Unpeeled Blank Recovery

1 mg/L 2.0059 0.9343 5.5280 9.2659 8.0764 0.0019 0.1142

mg/kg 3.0089 1.4015 8.2920 13.8989 12.1146 0.00285 0.1713
2 mg/L 1.7309 1.0831 0.5363 0.3102 2.6364 0.0062 0.0858
mg/kg 2.5964 1.6247 0.8045 0.4653 3.9546 0.0093 0.1287
4 mg/L 1.6961 0.6883 0.1714 0.1831 0.9996 0.0318 0.0939
mg/kg 2.5442 1.0325 0.2571 0.2747 1.4994 0.0477 0.14085
5 mg/L 2.7594 1.9110 25.6243 21.7116 18.2900 0.2095 0.31425
mg/kg 4.1391 2.8665 38.4365 32.5674 27.4351 0.3164 0.4746

Based on the results presented in Table 2, different process technique worked better in the
other groups. For group 1, juicing the apple yielded the lowest azoxystrobin concentration in the
extract while washing the apple resulted to higher fungicide residue in the extract. For group 2,
washing the apple resulted to the lowest azoxystrobin concentration while unpeeling it lead to the
highest fungicide residue in the extract. For group 4, peeling and washing resulted to lower
concentrations of azoxystrobin in the analyzed extracts (this is almost similar with our findings)
while unpeeling the apple resulted to much higher pesticide residues. For group 5, juicing seemed
to be the most effective processing technique since it generated the least azoxystrobin
concentration in the extract while peeling was the least effective since it resulted to the highest
azoxystrobin residue in the extract. The result of the last group seems to be strange because peeling
the apples should not result to a residue higher than unpeeling it. Again, these differences in the
results can be attributed to how accurate and careful this practical exercise was done by each group
and possible occurrence of contamination in the processed apples. Some errors could occur when
the experiment was performed. A graphical representation of the obtained azoxystrobin extract
concentrations is presented in Figure 1 below.

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 Azoxystrobin in apple extract
45,00
40,00

Concentration mg/Kg
35,00
30,00
25,00
20,00
15,00
10,00
5,00
0,00
Cooked Juice Peeled Washed Unpeeled
Tested apple

G1 G2 G3 G4 G5

Figure 1. Concentration of azoxystrobin (mg/kg) in the processed apples.

The efficiency of the extraction method used in the experiment was evaluated by determining the
method recovery (%) using Equation 2 below. Concentrations used for this equation are in mg/kg.

𝑐𝑜𝑛𝑐.𝑖𝑛 𝑟𝑒𝑐𝑜𝑣𝑒𝑟𝑦 𝑎𝑝𝑝𝑙𝑒−𝑐𝑜𝑛𝑐.𝑖𝑛 𝑏𝑙𝑎𝑛𝑘 𝑎𝑝𝑝𝑙𝑒

Equation 2. 𝑅𝑒𝑐𝑜𝑣𝑒𝑟𝑦 (%) = 𝑥 100
𝑡ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑎𝑑𝑑𝑒𝑑 𝑎𝑧𝑜𝑥𝑦𝑠𝑡𝑟𝑜𝑏𝑖𝑛

The concentration of the extracted azoxystrobin for the blank and recovery apple are shown
in Table 1. In the experiment, 10 µL of a 10 mg/L of azoxystrobin solution was added to the falcon
tube containing the 10 g of recovery apple. These data were used to calculate the theoretical
concentration of the added azoxystrobin (in mg/kg) using Equation 3 below.

𝑚𝑔
Equation 3. 𝑇ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑐𝑜𝑛𝑐. 𝑜𝑓 𝑎𝑑𝑑𝑒𝑑 𝑎𝑧𝑜𝑥𝑦𝑠𝑡𝑟𝑜𝑏𝑖𝑛 ( 𝑘𝑔 ) =
𝑚𝑔
𝑐𝑜𝑛𝑐.𝑜𝑓 𝑎𝑑𝑑𝑒𝑑 𝑎𝑧𝑜𝑥𝑦𝑠𝑡𝑟𝑜𝑏𝑖𝑛 ( )𝑥 𝑣𝑜𝑙.𝑜𝑓 𝑎𝑑𝑑𝑒𝑑 𝑎𝑧𝑜𝑥𝑦𝑠𝑡𝑟𝑜𝑏𝑖𝑛 (𝐿)
𝐿
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑝𝑟𝑜𝑐𝑒𝑠𝑠𝑒𝑑 𝑎𝑝𝑝𝑙𝑒 (𝑔)
𝑥 1000

𝑚𝑔
10 𝑥 0.0001 𝐿 𝑔
Applying Equation 3, 𝐿
10 𝑔
𝑥 1000 = 0.1 𝑘𝑔 is the theoretical concentration of

the added azoxystrobin. This was used to calculate the recovery of the method using Equation 2.

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 Therefore, using Equation 1, the recovery of our method is 162.8% and calculated as
𝑔 𝑔
0.170 − 0.008
𝑘𝑔 𝑘𝑔
follows: 𝑔 𝑥 100 = 162. 8%.
0.1
𝑘𝑔

The method recovery of the other groups was calculated using their own blank and
recovery apple extract concentrations (mg/kg). The results are presented in Table 3 below. As can
be seen in the table below, the recovery method by all groups except group 4 are greater than
100%. Group 4’s recovery seems to be ideal and makes sense as compared with the other groups.
However, a using QuEChERS sample preparation methods for extracting pesticides from fruits
and vegetables have shown that a recovery more than 100% is possible and this can be attributed
to the pH of the extract (Lehotay et al., 2010).

Table 3. Method recovery (%) of all groups.

Group Method recovery (%)

1 168.45
2 119.40
3 162.75
4 93.15
5 160.35

4 CONCLUSION

Based from our results, peeling and washing the azoxystrobin-exposed apples are the most
effective methods that can be done to reduce the azoxystrobin residue on the apple. Consuming
unpeeled apple would exposed the consumer to higher concentration of pesticide residue. It is
therefore recommended that when eating apple, it should be washed thoroughly or peeled.
Moreover, the QuEChERS extraction method showed very high recovery for the investigated
pesticide. Although the calculated recovery for this extraction method must be verified and
checked, since most groups showed a recovery of more than a 100%, this might imply that the
extraction technique used is very efficient in azoxystrobin recovery from the apples. Therefore,
this can be a very useful technique for extracting pesticides from crops or fruits for analysis
purposes.

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5 REFERENCES

Lehotay S, Son K, Kwon H, Koesukwiwat W, Fu K, Mastovska E & Leepipatpiboon J. (2010).

Comparison of QuEChERS sample preparation methods for the analysis of pesticide residues in
fruits and vegetables. J. Chromatogr. A, 1217 (2010) 2548.)

Von Stackelberg K. (2012). A systematic review of the evidence for adverse human health effects
from exposure to azoxystrobin in the environment.

http://www.agchemaccess.com/azoxystrobin
https://www3.epa.gov/pesticides/chem_search/reg_actions/registration/fs_pc-128810_07-feb-
97.pdf

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