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Occurrence and estimation of aflatoxin M1 exposure in milk in Serbia Jovana Kos, Jovanka Levi, Olivera uragi, Bojana Koki, Ivana Miladinovi PII: DOI: Reference: To appear in: S0956-7135(13)00517-3 10.1016/j.foodcont.2013.09.060 JFCO 3502 Food Control

Received Date: 26 June 2013 Revised Date: 17 September 2013 Accepted Date: 24 September 2013

Please cite this article as: KosJ., LeviJ., uragiO., KokiB. & MiladinoviI., Occurrence and estimation of aflatoxin M1 exposure in milk in Serbia, Food Control (2013), doi: 10.1016/j.foodcont.2013.09.060. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Occurrence and estimation of aflatoxin M1 exposure in milk in Serbia Jovana Kos1*, Jovanka Levi, Olivera uragi, Bojana Koki, Ivana Miladinovi

Institute of Food Technology, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia

Corresponding author Tel.: +381 21 485 3837; fax: +381 21 450 725. E-mail address: jovana.kos@fins.uns.ac.rs (Jovana Kos)

Abstract

The occurrence of AFM1 was investigated in 150 cows, 10 goats, 5 donkeys, 10 breasts milk and 1 infant formula samples. Analyses were done using Enzyme Linked Immunosorbent Assay

concentrations ranged from 0.01 to 1.2 g/kg. Further, even 129 (86.0%) cows milk samples contained AFM1 in concentration greater than maximum residue levels (MRL) of 0.05 g/kg

was detected in 80.0% goats, 60.0% donkeys and 60.0% of breasts milk samples. Although Serbian Regulation for MRL of AFM1 in milk has been changed and harmonized with EU Regulation in 2011, occurrence of AFM1 in milk in Serbia during 2013 resulted in Regulation changes, and MRL were changed from 0.05 to 0.5 g/kg. On the basis of the obtained concentrations of the AFM1 in cows milk, collected information about average milk intake and mean body weight (bw) for different ages categories, mean ingestion of AFM1 in ng/kg per bw per day were estimated. Obtained results showed that all

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defined by European Union (EU) Regulation. Analysis of other types of milk showed that AFM1

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(ELISA) method. AFM1 was detected in 98.7% of analyzed cows milk samples in

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ages categories, especially children, are exposed with high risk related to presence of AFM1 in milk. There are only a few published data about occurrence of AFM1 in milk in Serbia and none about intake assessment for AFM1. Keywords: Aflatoxin M1, Milk, Serbia, Exposure

Abbreviations: AFM1, Aflatoxin M1; AFB1, Aflatoxin B1; AFs, Aflatoxins; bw, body weight; CRM, certified reference material; EC, European Commission; EFSA, European Food Safety Authority; ELISA, Enzyme Linked Immunosorbent Assay; EU, European Union; FDA, U. S. Food and Drug Administration; IARC, International Agency for Research on Cancer; JECFA, Joint FAo/WHO Expert Committee on Food Additives; LOD, Limit of detection; LOQ, Limit of quantification; Min, Minimum; Max, Maximum; MRL, maximum residue levels; RSDr: relative

under reproducibility; SD, Standard deviation; TDI, tolerable daily intake; UHT, ultra-high temperature; WHO, World and Health Organization.

1. Introduction

Aflatoxins (AFs) are one of the most known and investigated group of mycotoxins which can be found as contaminants in different types of food and feed. AFs are mainly produced by Aspergillus species in agricultural products from tropical and subtropical regions (Decastelli et al., 2007; Ardic et al., 2008). Among approximately 18 identified AFs, aflatoxin B1 (AFB1) is the most common as well as highly toxic, mutagenic, teratogenic and carcinogenic compound (IARC, 1993).

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standard deviation calculated under repeatability; RSDR, relative standard deviation calculated

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Aflatoxin M1 (AFM1) is the 4-hydroxy derivative of AFB1, formed in liver and excreted into the milk in the mammary glands of both human and lactating animals that have been fed with AFB1 contaminated diet (Fallah et al., 2009; Gurbay et al., 2010). The amount of converted AFB1 from food and feed in AFM1 in milk is influenced by several factors including breed, health, type of diet, milk production, rate of ingestion and digestion, etc (Duarte et al., 2013). Depending on the level of contamination, approximately 0.3-6.2% of AFB1 ingested by livestock is transformed to AFM1 in milk (Ayar et al., 2007). If contaminated feedstuffs are used, AFM1 can be detected in milk 12-24 h after first intake of AFB1, while its concentration decreases to an undetectable level 72 h after the initial intake is stopped (Van Egmond, 1989). Hence, rate of absorption of AFB1 and excretion of AFM1 in milk varies from day to day and from one milking to the next (Duarte et al., 2013). AFM1 shows resistance to heat treatment and mild acidic conditions used in the production of milk and other dairy products (yoghurt, cheese, cream, butter), therefore if milk is

2006; Colak, 2007).

Until 2002, AFM1 was classified in 2B group as a possible carcinogen for human (IARC, 1993). Based on numerous

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regarding

contaminated there is a great possibility of AFM1 appearance in dairy products (Oruc et al.,

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carcinogenic,

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teratogenic,

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genotoxic

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immunosuppressive effect, AFM1 and other AFs (B2, G1 and G2) were reclassified in the first group (IARC, 2002).

Milk has the greatest demonstrated potential for AFs introducing into the human diet since it represents one of the main foodstuffs in human nutrition. Furthermore, infants and young children eat and drink more relative to their size than adults, and due to high intake of milk, children are the most susceptible population to the effects of AFM1 (Erkekoglu et al., 2008).

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Considering that AFM1 was included in first group by carcinogenicity and milk and its derivates are consumed daily, most countries have set up maximum residue levels (MRL) of AFM1 in milk. MRL of AFM1 in milk varies from 0.05 g/kg in European Union (European Commission, 2006b) to 0.5 g/kg established in United States (FDA, 2011). Regulation for MRL of AFM1 in milk in Serbia (Serbian Regulation, 2011) was recently adopted and harmonized with European Union (EU) Regulation. However, presence of AFM1 in milk during January and February 2013 resulted in Regulation changes. During March 2013, relevant authorities changed previously MRL of AFM1 from 0.05 g/kg to 0.5 g/kg (Serbian Regulation, 2013).

Currently, there are more than 200 registered dairies in Serbia, but only 123 of them are active. Other dairies operate in the field of grey economy (nonregistered analytical zone). Big dairies own 90% of total capacity, medium sized 6.0%, whereas small dairy companies only have 4.0% of total capacity. Major milk producers in Serbia are family farms as well as commercial farms

share accounts about 99% of the total milk production in Serbia. Average milk production for the previous years in Serbia has been amounted about 1.6 million tons per year. According to data provided by the Ministry of Agriculture, Forestry and Water Management about 52% were delivered to dairy plants for further processing, while the rest were sold on the green market as processed products from farms or as raw milk (25%), whereas 15% was consumed by household members. The remaining 8.0% of the total milk production was used as livestock feed (Boi et al., 2009).

The prevalence of AFM1 in milk has been reported in many countries (WHO, 2010). However, there is a lack of data regarding AFM1 presence in milk from Serbia. Hence, the aim of this

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both private and state owned (Berkum, 2010). Cow milk is the main milk type produced and its

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report was to investigate the occurrence of AFM1 in different types of milk and to evaluate human exposure to AFM1 in Serbia.

2. Materials and method 2.1. Samples

Present study examined 176 samples of different types of milk. All samples were collected

milk samples were collected during February, March, April and May, respectively. The first group of milk samples included 55 samples of heat-treated milks. Eleven pasteurized, 39 ultra-high temperature (UHT) and 2 organic milk samples were collected from six largest dairy producers in Serbia, while 5 pasteurized milk samples were collected from 5 small dairy producers in Vojvodina (Northern Province of Serbia). From 40 different small milking farms in

included the following samples: 10 goats and 5 donkeys raw milk samples from small milking farms, 10 breast milk samples and 1 sample of infant formula. Breast milk samples were taken

producer, who is the only producer of this type of product in Serbia. Eight pasteurized milk samples from small dairy producers, and 12 pasteurized, 31 UHT and 4 organic milk samples from big dairy producers were a part of fourth group. The heat-treated milk samples from first and fourth group were collected from supermarkets in original packaging and included different types of milk: skimmed, semi-skimmed, full, milk without lactose, organic, milk enriched with vitamins and full milk for coffee. Samples from second and third group were collected in sterile glass bottles. Immediately after collection

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from women who had a mixed diet and only one sample of infant formula was taken from

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Vojvodina, 40 raw milk samples were collected and included in second group. The third group

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during first part of 2013 and classified into four groups. First, second, third and fourth group of

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samples from all groups were transported to the laboratory and analyzed. Sampling was performed according to EU requirements (European Commission, 2006a).

2.2.

Aflatoxin analysis by ELISA

Determination of AFM1 was done by Enzyme Linked Immunosorbent Assay (ELISA) method using I screen AFLAM1 test kit (Tecna S. r. l., Trieste, Italy). Analyses were performed according to the test kits instructions. Procedure is based on binding of free AFM1 from samples and standard solutions to the anti-AFM1 antibodies during first incubation. Any unbound substance is removed in a washing step. A second incubation is performed with an aflatoxinHRP conjugate, which covers all the remaining free binding sites of the antibody. The bound enzyme activity is determined by adding a fixed amount of a chromogenic substrate. The enzyme converts the colorless chromogen into a blue product during the third incubation. The addition of

microplate reader (Thermolabsystem, Thermo, Finland) at 450 nm. The color development is inversely proportional to the AFM1 concentration in the sample. Concentration of AFM1 was

concentrations: 0, 5, 10, 25, 50, 100 and 250 ng/l. Samples with AFM1 concentration greater than 250 ng/l were diluted with sample diluent solution from the test kit and analyzed again. The analytical quality of the ELISA method was assured by the use of certified reference material (CRM) as well as by participation in interlaboratory study. Partially defatted raw lyophilized milk with certified AFM1 content of 0.053 g/kg was used as CRM (MI1142-1/CM, Progetto Trieste, Test Veritas, Padova, Italy) for validation of Iscreen AFLAM1 test kit. The validation parameters (Table 1) were calculated and expressed using European Official Decision

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calculated from calibration curve which was obtained using 7 standards with the following

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the stop reagent leads to a color change from blue to yellow. The absorbance is measured by a

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procedure for screening methods (European Commission, 2002b) and their values were in accordance with recommendations given in Commission Decision (European Commission, 2006a). Further, obtained Z value of 0.67 in interlaboratory study confirmed analytical quality of the results obtained by ELISA method (Ring Test 2013, Mycotoxins, Aflatoxin M1, Test Veritas, Padova, Italy).

2.3.

Sample preparation

Milk samples were prepared according to the manufacturers instructions. Samples were centrifuged at 3000g for 10 minutes. The upper creamy layer was removed by Pasteur pipette and 100 l from the lower phase was used for the analysis.

2.4.

Calculation of estimated daily intake

calculating average milk intake for people in Serbia. The calculation was based on questionnaire examination that included 1500 people of different ages categories and economical status. In

parameters were used: concentrations of the AFM1 in cows milk obtained in the present study, mean intake of milk and mean bw for different ages categories obtained from questionnaire. 3. Results 3.1.

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In this study a total of 176 milk samples were analyzed to determine concentration of AFM1. Obtained results were summarized and shown in Table 2.

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Occurence of AFM1 in different type of milk

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order to estimate mean ingestion of AFM1 in ng/kg per body weight (bw) per day following

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Recent data about average milk intake in Serbia are not available, therefore we approached

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As can be seen, all examined samples from first group were contaminated with AFM1. Further, only 1 UHT and 2 organic milk samples from first group had AFM1 concentration lower than 0.05 g/kg. Even 52 (94.6%) of analyzed samples from first group had AFM1 concentration greater than MRL defined by EU Regulation (European Commission, 2006b) and Serbian Regulation that was valid at the time of examination (Serbian Regulation, 2011). Analysis of 40 raw milk samples collected from small milking farms showed that the greatest percentage of samples (62.5%) had AFM1 concentration between 0.05 and 0.50 g/kg. Furthermore, 30 (75.0%) and 5 (12.5%) samples from second group were unsuitable for human consumption according to EU and new Serbian Regulation (Serbian Regulation, 2013), respectively. Out of 26 samples of different types of milk from third group in 4 goats milk samples AFM1 concentration was greater than 0.05 g/kg. Further, 4, 3 and 6 samples of goats, donkeys and breast milk samples were contaminated with AFM1 in concentration range from 0.005 to 0.05

Concentrations of AFM1 exceeded MRL defined by EU Regulation even in 45 (81.8%) milk samples, while 2 (3.63%) samples were not in accordance with new Serbian Regulation (Serbian Regulation, 2013).

Since cows milk covers 99.0% of total milk consumption in Serbia (Boi et al., 2009), results obtained for that type of milk were additionally analyzed and summarized in Table 3. Only in 2 (1.30%) out of 150 samples AFM1 was not detected. Further, concentrations of AFM1 in 100% of pasteurized and 91.3% of UHT milk samples exceeds MRL defined by EU Regulation, while 1 (4.5%) pasteurized milk sample had AFM1 concentration greater than MRL defined in new Serbian Regulation. Analysis of samples from small milking farms showed that among 13 pasteurized and 40 raw milk samples, 100% and 75.0% were not suitable for human

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g/kg, respectively. AFM1 was detected in all analyzed samples from fourth group.

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consumption according to EU Regulation, and 38.5% and 12.5% according to Serbian Regulation, respectively. Comparing AFM1 levels in organic and others cows milk, it was found that organic milk samples were contaminated in a lower frequency. One (16.7%) out of six organic milk samples exceeded European MRL. On the other hand, the highest concentrations of AFM1 were founded in raw (0.90 g/kg) and pasteurized (1.20 g/kg) milk samples from milking farms.

3.2.

Estimated daily intake of AFM1

After questionnaire examination of 1500 people of different ages categories we obtained results in terms of average body weight (bw) and average intake of milk (Table 4). Further, we also investigated what kind of milk is most commonly used in Serbia. Only 10 (0.67%) out of 1500 examinees do not drink cows milk, while 6 of them drink other milk types (soy and goat) and 2

Since cows milk is mainly used (99.3%) and 98.7% of analyzed cows milk samples were contaminated with AFM1, exposure of human regarding AFM1 was investigated. Two samples (1.33%) of raw cows milk with AFM1 concentration lower than 0.005 g/kg were not included in this estimation. Table 4 represents data regarding calculated ingestion of AFM1 in terms of minimum (0.01 g/kg), average (0.21 g/kg) and maximum (1.2 g/kg) AFM1 concentration obtained in this study.

4. Discussion Hot and dry weather conditions described in our previous study (Kos et al., 2013) could be the possible reason for high contamination frequency of AFM1 in milk from Serbia. Prolonged

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have allergic reaction on cows milk.

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drought noted during maize growing season 2012 was favorable for Aspergillus moulds growth and AFs productions. Presence of AFs was detected in 68.5% of examined maize samples in the concentration range from 1.01 to 86.1 g/kg (Kos et al., 2013). Occurrence of AFs in maize and feed were also confirmed in our second report (Levi et al., 2013). AFs were analyzed in 302 maize, 49 maize silage and 27 complete feed samples and detected in 69.9%, 38.7% and 37.0% of analyzed samples, respectively. In Serbia, maize is mainly (80.0%) used as a component of animal feed (Maslac, 2011), therefore if maize is contaminated there is a great possibility of AFM1 appearance in milk and milk products.

Furthermore, Serbian Regulation (Serbian Regulation, 2010a) for MRL of AFs in maize intended for animal consumption was adopted during 2010. However, harmonization with EU Regulation (European Commission, 2002a) was not complete and there are noticeably differences that exist between these two Regulations (Table 5). According to European Regulation maize intended for

of AFs. Also, the main difference is observed in allowed MRL of AFs in feed materials. The obtained results in the present study are comparable to those performed in some other countries which have indicated high levels of AFM1 in milk samples. The greatest numbers of published studies are from Mediterranean and Middle East, where the environmental conditions may favor the occurrence of AFs in agricultural products and than AFM1 in milk (EFSA, 2010). Numerous studies from Iran, India, Thailand, Brazil, Syria and Kenya indicate great risk related to the presence of AFM1 in milk and milk products (Duarte et al., 2013). Several published studies from Europe indicate low level of AFM1 in milk. Recent study from neighboring Croatia showed that 98.4% of raw milk samples collected during 2009 had AFM1 concentration lower than 0.05 g/kg (Bilandi et al., 2010). Furthermore, among 196 milk samples collected during

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animal consumption had to be tested on AFB1, while Serbian Regulation defines control of sum

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2009-2010 in Greece (Tsakiris et al., 2013) and 40 samples in Portugal during 2011 (Duarte et al., 2013), only 1.0% and 5.0% of samples exceeded European MRL, respectively. Results from Italy (Meucci et al., 2010) and Spain (Cano-Sancho et al., 2010) showed that all analyzed samples were in accordance with EU Regulation. It should be noted that there is still no published data from Europe regarding occurrence of AFM1 in milk from 2012-2013 period, which was characterized with favorable weather conditions for Aspergillus species growth and AFs production, especially in Southeast Europe.

Presence of AFM1 in milk from Serbia was published in only few reports. PolovinskiHorvatovi et al., (2009) have shown that none of 70 analyzed cows milk samples were contaminated with AFM1 in concentration greater than 0.05 g/kg. In the same year Jankovi et al. (2009) analyzed 23 milk samples and in 3 (13.0%) samples concentration of AFM1 was higher than 0.05 g/kg. Lower contamination frequency of AFM1 in previous reports from

AFs in maize and other feed material in Serbia in previous years (Koki et al., 2010; Mati, et al., 2010; Kos et al., 2013).

Published studies regarding presence of AFM1 in organic milk give contradictory information in terms of lower or higher levels of AFM1 in comparison to conventional milk (Ghidini et al., 2005; Vallone et al., 2006). Organic milk samples analyzed in present study were taken from the only producer of that type of milk in Serbia. More rigorous control of feed material and raw milk before processing could be the possible explanation for better obtained results in comparison to conventional cows milk samples. There are a several data regarding occurrence of AFM1 in other types of milk (except cows). Previous studies confirmed our results that milk from others animals are very often less

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Serbia in comparison to the obtained results in this study can be explained with the absence of

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contaminated with AFM1 than cows milk, because of the different digestive apparatuses and mechanism of AFB1 assimilation of animals and for different feeding used in cows breeding (Barbiroli et al., 2007; Hussain et al., 2010; Fallah et al., 2011). Also, it should be noted that data about occurrence of AFM1 in donkeys milk are very limited.

Since AFs and AFM1 from contaminated food can be transported to breast milk and cause development of several diseases in newborn child (Gurbay et al., 2010), breast milk samples were included in this study. Analysis of 10 breast milk samples showed that determined AFM1 concentration in 6 samples ranged from 0.006 to 0.022 g/kg. Although, founded AFM1 concentrations in breast milk were lower than 0.025 g/kg which is allowed MRL for milk and infant formula intended for infant and young children (Serbian Regulation 2010b; European Commission, 2006b), it should be pointed that any level of AFM1 represent risk to infant since milk is their main food. Breast milk remains the best source of nutrition for infants and World

life (Dowling et al., 2012). However, when breast-feeding is not possible or enough, many different infant formulas are available for infants and young children. In Serbia there is only one milk industry that produces infant formulae. Analysis of infant formulae sample showed that concentration of AFM1 was in accordance with Serbian and EU Regulations (Serbian Regulation 2010b, European Commission, 2006b). Unfortunately, great numbers of infant and young children in Serbia immediately after breast-feeding start to drink cows milk. Possible reasons for early introduction of cows milk are economic situation in Serbia and/or traditional diet. Early intake could provoke a variety of allergic reactions (Ivakhnenko et al., 2013) and could represent great risks related to AFM1.

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and Health Organization (WHO) recommends exclusive breast-feeding for the first two years of

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Kuiper-Goodman (1990) expressed 0.2 ng/kg bw/day as tolerable daily intake (TDI) of AFM1. Based on the results shown in Table 4 it could be noted that calculated ingestion for obtained average and maximum concentrations for all ages categories are significantly higher than TDI. For obtained minimum concentration of AFM1, calculated ingestion is higher than 0.2 ng/kg bw/day only for children from 1 to 5 years. Different intake of milk and bw for different ages categories influenced significant differences in daily ingestion of AFM1.

Although International expert committees (JECFA, 2001) did not specify TDI for AFs, they concluded that daily exposure even with concentration lower than 1 ng/kg bw contributed to the risk of liver cancer. Further, JECFA (2001) established intake of AFM1 for five regions based on the regional consumption of milk, assuming bw (60 kg) and weighted mean concentrations of AFM1 in milk obtained in various monitoring programs (Table 6). It should be pointed, that results from JECFA did not include data from Serbia. From the showed data it can be stated that

other five world regions.

The most of the previously estimated intakes of AFM1 through milk consumption in some countries were calculated for adults, and some of these are following: 0.08 ng/kg bw/day in Brazil (Shundo, et al., 2009), 0.01 ng/kg bw/day in France (Leblanc, et al., 2005), 0.305 ng/kg bw/day in Spain (Cano-Sancho et al., 2010), and 3.26 ng/kg bw/day in Morocco (Zinedine et al., 2007). In the present study, estimated intakes of AFM1 for adults were expressed for minimum, average and maximum obtained concentration of AFM1 and those values ranged from 0.02 to 0.06, 0.42 to 1.26, and from 2.39 to 7.18 ng/kg bw/day, respectively. Obtained ingestion of AFM1 in this study greater than TDI value (0.2 ng/kg bw/day) calculated by Kuiper-Goodman (1990) indicate high risk related to AFM1 since this toxin has carcinogenic,

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currently calculated daily intake of AFM1 in Serbia are higher than daily intake of AFM1 in

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teratogenic, genotoxic and immunosuppressive effect and could cause DNA damage, gene mutation, chromosomal anomalies and cell transformation (IARC, 2002).

5. Conclusion

Obtained results indicate high contamination frequency of AFM1 in examined cows milk samples. Since milk represents one of the main foodstuffs in human diet in Serbia there was a

and children represent the most susceptible population to the effects of AFM1 since they drink more milk relative to their size. In order to avoid risk related to presence of AFM1 in milk there is a need for permanent control and harmonization of Serbian Regulations with EU Regulations in terms of AFM1 in milk and AFs in feed.

This study was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, Project No. III 46012.

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6. Acknowledgements

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Zinedine, A., Gonzlez-Osnaya, L., Soriano, J. M., Molt, J. C., Idrissi, L., & Maes, J. (2007).

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Table 1. Validation parameters Iscreen AFLAM1 test kit LOD 1.50 LOQ 5.00 RSDr 3.54 RSDR 4.97 Recovery 107.6 LOD: limit of detection (ng/kg) LOQ: limit of quantification (ng/kg) RSDr: relative standard deviation calculated under repeatability conditions RSDR: relative standard deviation calculated under reproducibility conditions

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2 3

Small Farms Farms

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Small Pasteurized 8 No: Number of samples Range of concentration: number (percentage) AverageSD: Average concentration (g/kg) standard deviation (g/kg) Min-Max: minimum and maximum concentrations (g/kg)

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Pasteurized Raw Goat Donkey Breast milk Infant formula Pasteurized UHT Organic

5 40 10 5 10 1 12 31 4

2 (5.00) 2 (20.0) 2 (40.0) 4 (40.0) 1 (100) -

8 (20.0) 4 (40.0) 3 (60.0) 6 (60.0) 5 (16.1) 3 (75.0) -

1 (20.0) 25 (62.5) 4 (40.0) 11 (91.7) 26 (83.9) 1 (25.0) 7 (87.5)

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4 (80.0) 5 (12.5) 1 (8.30) 1 (12.5)

Table 2. Occurence of AFM1 in different types of milk Sample Group Producers No category Big Pasteurized 10 UHT 38 1 Organic 2

< 0.005 -

Range of concentration (g/kg) 0.005-0.05 0.05-0.50 10 (100) 1 (2.60) 37 (97.4) 2 (100) -

> 0.50 -

AverageSD 0.220.11 0.200.09 0.030.02 0.670.35 0.190.20 0.080.09 0.020.02 0.010.006 0.450.22 0.180.10 0.040.03 0.270.25

Min-Max 0.06-0.40 0.08-0.41 0.01-0.04 0.16-1.20 0.005- 0.90 0.008-0.24 0.005-0.035 0.006-0.022 0.07- 0.70 0.02- 0.36 0.02-0.08 0.08-0.85

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Table 3. Occurence of AFM1 in cows milk Sample Range of concentration (g/kg) Producers No category < 0.005 0.005-0.05 0.05-0.50 Big Pasterized 22 21 (95.5) UHT 69 6 (8.70) 63 (91.3) Organic 6 5 (83.3) 1 (16.7) Small Pasterized 13 8 (61.5) Farms Raw 40 2 (5.00) 8 (20.0) 25 (62.5) Total 150 2 (1.30) 19 (12.7) 118 (78.7) No: Number of samples Range of concentration: number (percentage) AverageSD: Average concentration (g/kg) standard deviation (g/kg) Min-Max: minimum and maximum concentrations (g/kg)

> 0.50 1 (4.50) 5 (38.5) 5 (12.5) 11 (7.30)

Min-Max 0.06-0.70 0.02-0.41 0.01-0.08 0.08-1.20 0.005- 0.90 0.01-1.20

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Table 4. Estimate of AFM1 human exposure Ages categories Body weight (kg) male 1-5 5-15 15-25 25-55 14 38 74 90 female 14 37 55 69 Intake of milk per day (l) male 0.44 0.42 0.44 0.21 female 0.43 0.32 0.11 0.18 Average ingestion of AFM1 ng/kg bw/day male 6.45 2.34 1.26 0.49 female 6.26 1.86 0.42 0.56 Maximum ingestion of AFM1 ng/kg bw/day male 36.8 13.4 7.18 2.80 female 35.8 10.6 Minimum ingestion of AFM1 ng/kg bw/day male 0.31 0.11 female 0.30 0.09 0.02 0.03 0.03

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> 55 89 71 0.22 0.23 0.51 0.69 2.90 3.96 0.02 Average ingestion of AFM1 calculated for obtained mean concentration of AFM1 (0.21 g/kg) Minimum ingestion of AFM1 calculated for obtained minimum concentration of AFM1 (0.01 g/kg) Maximum ingestion of AFM1 calculated for obtained maximum concentration of AFM1 (1.20 g/kg)

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Table 5. Comparison of Serbian and EU Regulation All feed Regulation Toxin materials (mg/kg) European Commission, 2002a AFB1 0.02 Serbian Regulation, 2010a AFs 0.05

Complete feedstuffs for dairy animals (mg/kg) 0.005 0.01

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Table 6. Daily intake of AFM1 in milk in the five regional diets Weighted mean Milk intake AFM1 AFM1 intake Region (kg/day) in milk ng/person ng/kg (g/kg) per day bw/day European Union 0.290 0.023 6.8 0.11 Latin America 0.160 0.022 3.5 0.058 Far East 0.032 0.360 12 0.20 Middle East 0.120 0.005 0.6 0.10 Africa 0.042 0.002 0.1 0.002 *results are taken from Joint Expert Committee on Food Additives (JECFA, 2001)

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Presence of AFM1 was examined in 176 different types of milk 98.7% cows, 80.0% goats, 60.0% donkeys and 60.0% breast milk was contaminated AFM1 concentration in 86.0% cows and 40.0% goats milk samples exceeded MRL Calculated average intakes of AFM1 were above than recommended value for TDI

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