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Occurrence and estimation of aflatoxin M1 exposure in milk in Serbia Jovana Kos, Jovanka Lević, Olivera Đuragić, Bojana Kokić, Ivana Miladinović 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., LevićJ., ĐuragićO., KokićB. & MiladinovićI., 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 Lević, Olivera Đuragić, Bojana Kokić, Ivana Miladinović

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 cow´s, 10 goat´s, 5 donkey´s, 10 breasts milk and 1 infant formula samples. Analyses were done using Enzyme Linked Immunosorbent Assay

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

was detected in 80.0% goat´s, 60.0% donkey´s 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 µg/kg. On the basis of the obtained concentrations of the AFM1 in cow´s milk, collected information about average milk intake and mean body weight (bw) for different age’s categories, mean ingestion of AFM1 in ng/kg per bw per day were estimated. Obtained results showed that all

AC C

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

TE D

(ELISA) method. AFM1 was detected in 98.7% of analyzed cow´s milk samples in

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mutagenic. are exposed with high risk related to presence of AFM1 in milk.. AFs are mainly produced by Aspergillus species in agricultural products from tropical and subtropical regions (Decastelli et al. ultra-high temperature. Aflatoxins. WHO. Keywords: Aflatoxin M1. Serbia. There are only a few published data about occurrence of AFM1 in milk in Serbia and none about intake assessment for AFM1. Food and Drug Administration. maximum residue levels. relative standard deviation calculated M AN U SC RI PT . especially children. aflatoxin B1 (AFB1) is the most common as well as highly toxic. 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. LOQ. Limit of quantification. Joint FAo/WHO Expert Committee on Food Additives. UHT. 1993). CRM. IARC. bw. AFs. Milk. Among approximately 18 identified AFs. RSDr: relative under reproducibility. Standard deviation. World and Health Organization. Minimum. JECFA. TDI. European Union. RSDR. AC C EP TE D standard deviation calculated under repeatability. European Commission. Max. LOD. Ardic et al. ELISA. tolerable daily intake. body weight. Aflatoxin M1. EFSA. 2008). teratogenic and carcinogenic compound (IARC. Exposure Abbreviations: AFM1. EU.. FDA. SD.ACCEPTED MANUSCRIPT 2 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 age’s categories. 1. Enzyme Linked Immunosorbent Assay. European Food Safety Authority. 2007. certified reference material. EC. Aflatoxin B1. Limit of detection. AFB1. S. Maximum. U. MRL. International Agency for Research on Cancer. Min.

2013). children are the most susceptible population to the effects of AFM1 (Erkekoglu et al. AFM1 and other AFs (B2. etc (Duarte et al. 2002)..ACCEPTED MANUSCRIPT 3 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 Aflatoxin M1 (AFM1) is the 4-hydroxy derivative of AFB1. G1 and G2) were reclassified in the first group (IARC.. AFM1 can be detected in milk 12-24 h after first intake of AFB1. 1993). 2007). cheese. Based on numerous EP studies TE D regarding contaminated there is a great possibility of AFM1 appearance in dairy products (Oruc et al. AFM1 was classified in 2B group as a possible carcinogen for human (IARC. SC teratogenic.. 2010). 1989). cream. If contaminated feedstuffs are used.2% of AFB1 ingested by livestock is transformed to AFM1 in milk (Ayar et al. 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. while its concentration decreases to an undetectable level 72 h after the initial intake is stopped (Van Egmond. 2013). 2007). infants and young children eat and drink more relative to their size than adults. therefore if milk is 2006. Hence. Depending on the level of contamination. type of diet. 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.3-6. RI PT genotoxic and immunosuppressive effect. AFM1 shows resistance to heat treatment and mild acidic conditions used in the production of milk and other dairy products (yoghurt. milk production.. and due to high intake of milk. rate of ingestion and digestion. M AN U carcinogenic. Furthermore. Milk has the greatest demonstrated potential for AFs introducing into the human diet since it represents one of the main foodstuffs in human nutrition. AC C .. The amount of converted AFB1 from food and feed in AFM1 in milk is influenced by several factors including breed. Colak. health. Until 2002. Gurbay et al. 2009.. approximately 0. 2008).. butter).

2010). 2013). Currently.0% of total capacity.5 µg/kg established in United States (FDA.0%. Average milk production for the previous years in Serbia has been amounted about 1. there are more than 200 registered dairies in Serbia. 2010). MRL of AFM1 in milk varies from 0.05 µg/kg to 0. Cow milk is the main milk type produced and its M AN U SC RI PT . Other dairies operate in the field of grey economy (nonregistered analytical zone).0% of the total milk production was used as livestock feed (Božić et al. However. The remaining 8. there is a lack of data regarding AFM1 presence in milk from Serbia. 2011) was recently adopted and harmonized with European Union (EU) Regulation. 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%). According to data provided by the Ministry of Agriculture. 2006b) to 0.. medium sized 6. most countries have set up maximum residue levels (MRL) of AFM1 in milk. During March 2013.5 µg/kg (Serbian Regulation.ACCEPTED MANUSCRIPT 4 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 Considering that AFM1 was included in first group by carcinogenicity and milk and its derivates are consumed daily. 2011). presence of AFM1 in milk during January and February 2013 resulted in Regulation changes. 2009). relevant authorities changed previously MRL of AFM1 from 0. However. Big dairies own 90% of total capacity. but only 123 of them are active.6 million tons per year. The prevalence of AFM1 in milk has been reported in many countries (WHO.05 µg/kg in European Union (European Commission. the aim of this AC C EP TE D both private and state owned (Berkum. whereas small dairy companies only have 4. Major milk producers in Serbia are family farms as well as commercial farms share accounts about 99% of the total milk production in Serbia. Regulation for MRL of AFM1 in milk in Serbia (Serbian Regulation. Hence. whereas 15% was consumed by household members.

while 5 pasteurized milk samples were collected from 5 small dairy producers in Vojvodina (Northern Province of Serbia). Samples from second and third group were collected in sterile glass bottles. Eleven pasteurized. 2. The third group M AN U SC during first part of 2013 and classified into four groups. organic. third and fourth group of RI PT . milk without lactose. and 12 pasteurized. All samples were collected milk samples were collected during February. Breast milk samples were taken producer.1. Eight pasteurized milk samples from small dairy producers. 31 UHT and 4 organic milk samples from big dairy producers were a part of fourth group. Samples Present study examined 176 samples of different types of milk. respectively. full. April and May. who is the only producer of this type of product in Serbia. The first group of milk samples included 55 samples of heat-treated milks. second. From 40 different small milking farms in included the following samples: 10 goat´s and 5 donkey´s raw milk samples from small milking farms. 39 ultra-high temperature (UHT) and 2 organic milk samples were collected from six largest dairy producers in Serbia. First. Materials and method 2. semi-skimmed. The heat-treated milk samples from first and fourth group were collected from supermarkets in original packaging and included different types of milk: skimmed. March. milk enriched with vitamins and full milk for coffee. 10 breast milk samples and 1 sample of infant formula. Immediately after collection AC C EP from women who had a mixed diet and only one sample of infant formula was taken from TE D Vojvodina.ACCEPTED MANUSCRIPT 5 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 report was to investigate the occurrence of AFM1 in different types of milk and to evaluate human exposure to AFM1 in Serbia. 40 raw milk samples were collected and included in second group.

Any unbound substance is removed in a washing step. A second incubation is performed with an aflatoxinHRP conjugate. Italy) for validation of I´screen AFLAM1 test kit. The addition of microplate reader (Thermolabsystem.2. Thermo. Italy). The bound enzyme activity is determined by adding a fixed amount of a chromogenic substrate. 2.. Padova. The enzyme converts the colorless chromogen into a blue product during the third incubation. The color development is inversely proportional to the AFM1 concentration in the sample. Sampling was performed according to EU requirements (European Commission. Samples with AFM1 concentration greater than 250 ng/l were diluted with sample diluent solution from the test kit and analyzed again. Concentration of AFM1 was concentrations: 0. Test Veritas. 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. 2006a). Trieste. 5. The validation parameters (Table 1) were calculated and expressed using European Official Decision AC C EP calculated from calibration curve which was obtained using 7 standards with the following TE D the stop reagent leads to a color change from blue to yellow. Procedure is based on binding of free AFM1 from samples and standard solutions to the anti-AFM1 antibodies during first incubation. The absorbance is measured by a M AN U SC RI PT . 100 and 250 ng/l.053 µg/kg was used as CRM (MI1142-1/CM. Progetto Trieste. which covers all the remaining free binding sites of the antibody. 25. 50. l. Aflatoxin analysis by ELISA Determination of AFM1 was done by Enzyme Linked Immunosorbent Assay (ELISA) method using I ‘screen AFLAM1 test kit (Tecna S. Finland) at 450 nm. 10. Analyses were performed according to the test kits instructions. r.ACCEPTED MANUSCRIPT 6 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 samples from all groups were transported to the laboratory and analyzed.

4. Test Veritas. In parameters were used: concentrations of the AFM1 in cow´s milk obtained in the present study. Mycotoxins. Aflatoxin M1. AC C Occurence of AFM1 in different type of milk EP order to estimate mean ingestion of AFM1 in ng/kg per body weight (bw) per day following TE D Recent data about average milk intake in Serbia are not available. The calculation was based on questionnaire examination that included 1500 people of different age’s categories and economical status. The upper creamy layer was removed by Pasteur pipette and 100 µl from the lower phase was used for the analysis. obtained Z value of 0.1. Padova. 3. Samples were centrifuged at 3000g for 10 minutes. 2. 2002b) and their values were in accordance with recommendations given in Commission Decision (European Commission. mean intake of milk and mean bw for different age’s categories obtained from questionnaire. 155 156 157 158 In this study a total of 176 milk samples were analyzed to determine concentration of AFM1. Results 3. Calculation of estimated daily intake calculating average milk intake for people in Serbia. Sample preparation Milk samples were prepared according to the manufacturer´s instructions. therefore we approached M AN U SC RI PT . 2006a).ACCEPTED MANUSCRIPT 7 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 procedure for screening methods (European Commission. Italy). Further.67 in interlaboratory study confirmed analytical quality of the results obtained by ELISA method (Ring Test 2013. 2.3. Obtained results were summarized and shown in Table 2.

AFM1 was detected in all analyzed samples from fourth group. concentrations of AFM1 in 100% of pasteurized and 91. results obtained for that type of milk were additionally analyzed and summarized in Table 3. 2013).0%) and 5 (12.30%) out of 150 samples AFM1 was not detected. Only in 2 (1. while 2 (3. only 1 UHT and 2 organic milk samples from first group had AFM1 concentration lower than 0. 2013). 3 and 6 samples of goat´s.5%) pasteurized milk sample had AFM1 concentration greater than MRL defined in new Serbian Regulation. 2011).05 Concentrations of AFM1 exceeded MRL defined by EU Regulation even in 45 (81. 2006b) and Serbian Regulation that was valid at the time of examination (Serbian Regulation. 2009).0% were not suitable for human AC C EP TE D µg/kg.8%) milk samples. Since cow´s milk covers 99. Even 52 (94. respectively. donkey´s and breast milk samples were contaminated with AFM1 in concentration range from 0. Further..3% of UHT milk samples exceeds MRL defined by EU Regulation.05 and 0. all examined samples from first group were contaminated with AFM1. Further.ACCEPTED MANUSCRIPT 8 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 As can be seen. while 1 (4.5%) had AFM1 concentration between 0. 30 (75.6%) of analyzed samples from first group had AFM1 concentration greater than MRL defined by EU Regulation (European Commission. 100% and 75.005 to 0. respectively. Analysis of 40 raw milk samples collected from small milking farms showed that the greatest percentage of samples (62. Furthermore. Out of 26 samples of different types of milk from third group in 4 goat´s milk samples AFM1 concentration was greater than 0. Analysis of samples from small milking farms showed that among 13 pasteurized and 40 raw milk samples.05 µg/kg.50 µg/kg. Further. 4.0% of total milk consumption in Serbia (Božić et al.5%) samples from second group were unsuitable for human consumption according to EU and new Serbian Regulation (Serbian Regulation. M AN U SC RI PT .05 µg/kg.63%) samples were not in accordance with new Serbian Regulation (Serbian Regulation.

Discussion Hot and dry weather conditions described in our previous study (Kos et al. 3. Table 4 represents data regarding calculated ingestion of AFM1 in terms of minimum (0. it was found that organic milk samples were contaminated in a lower frequency.21 µg/kg) and maximum (1.2. we also investigated what kind of milk is most commonly used in Serbia. exposure of human regarding AFM1 was investigated.005 µg/kg were not included in this estimation.67%) out of 1500 examinees do not drink cow´s milk. 2013) could be the possible reason for high contamination frequency of AFM1 in milk from Serbia.ACCEPTED MANUSCRIPT 9 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 consumption according to EU Regulation.5% according to Serbian Regulation. M AN U SC RI PT .2 µg/kg) AFM1 concentration obtained in this study.01 µg/kg). Estimated daily intake of AFM1 After questionnaire examination of 1500 people of different age´s categories we obtained results in terms of average body weight (bw) and average intake of milk (Table 4).. average (0.20 µg/kg) milk samples from milking farms.5% and 12. Only 10 (0. On the other hand. Two samples (1.3%) and 98. and 38.90 µg/kg) and pasteurized (1. Further.7% of analyzed cow´s milk samples were contaminated with AFM1. Comparing AFM1 levels in organic and others cow´s milk. respectively. Prolonged AC C EP TE D have allergic reaction on cow´s milk. the highest concentrations of AFM1 were founded in raw (0. One (16.7%) out of six organic milk samples exceeded European MRL. 4.33%) of raw cow´s milk with AFM1 concentration lower than 0. while 6 of them drink other milk types (soy and goat) and 2 Since cow´s milk is mainly used (99.

5% of examined maize samples in the concentration range from 1. Occurrence of AFs in maize and feed were also confirmed in our second report (Lević et al. 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.4% of raw milk samples collected during 2009 had AFM1 concentration lower than 0. Serbian Regulation (Serbian Regulation. 2011). 2010a) for MRL of AFs in maize intended for animal consumption was adopted during 2010.1 µg/kg (Kos et al. 2010).05 µg/kg (Bilandžić et al. 38.01 to 86.7% and 37. However. In Serbia. Furthermore.. AFs were analyzed in 302 maize. therefore if maize is contaminated there is a great possibility of AFM1 appearance in milk and milk products.0% of analyzed samples. The greatest numbers of published studies are from Mediterranean and Middle East. maize is mainly (80. India. among 196 milk samples collected during AC C EP TE D animal consumption had to be tested on AFB1. Thailand. respectively. 2013).. harmonization with EU Regulation (European Commission. 49 maize silage and 27 complete feed samples and detected in 69. where the environmental conditions may favor the occurrence of AFs in agricultural products and than AFM1 in milk (EFSA. 2010). while Serbian Regulation defines control of sum M AN U SC RI PT . Recent study from neighboring Croatia showed that 98. Numerous studies from Iran.ACCEPTED MANUSCRIPT 10 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 drought noted during maize growing season 2012 was favorable for Aspergillus moulds growth and AFs productions.9%. Several published studies from Europe indicate low level of AFM1 in milk.. Syria and Kenya indicate great risk related to the presence of AFM1 in milk and milk products (Duarte et al. 2013). Also. Brazil. 2002a) was not complete and there are noticeably differences that exist between these two Regulations (Table 5). 2013).0%) used as a component of animal feed (Maslac. the main difference is observed in allowed MRL of AFs in feed materials. Presence of AFs was detected in 68.. Furthermore. According to European Regulation maize intended for of AFs.

respectively. only 1. 2013) and 40 samples in Portugal during 2011 (Duarte et al. (2009) have shown that none of 70 analyzed cow´s milk samples were contaminated with AFM1 in concentration greater than 0. 2013).0% of samples exceeded European MRL. Results from Italy (Meucci et al. Vallone et al.. Matić. et al.. It should be noted that there is still no published data from Europe regarding occurrence of AFM1 in milk from 2012-2013 period. There are a several data regarding occurrence of AFM1 in other types of milk (except cow´s).. PolovinskiHorvatović et al. 2010. In the same year Janković et al. Presence of AFM1 in milk from Serbia was published in only few reports. Previous studies confirmed our results that milk from others animals are very often less AC C EP TE D Serbia in comparison to the obtained results in this study can be explained with the absence of M AN U SC RI PT ..0%) samples concentration of AFM1 was higher than 0. More rigorous control of feed material and raw milk before processing could be the possible explanation for better obtained results in comparison to conventional cow´s milk samples. 2010. Kos et al.05 µg/kg. 2013). Lower contamination frequency of AFM1 in previous reports from AFs in maize and other feed material in Serbia in previous years (Kokić et al.0% and 5.. (2009) analyzed 23 milk samples and in 3 (13...05 µg/kg. which was characterized with favorable weather conditions for Aspergillus species growth and AFs production.. 2005. Organic milk samples analyzed in present study were taken from the only producer of that type of milk in Serbia.. 2010) and Spain (Cano-Sancho et al. 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. especially in Southeast Europe.. 2006). 2010) showed that all analyzed samples were in accordance with EU Regulation.ACCEPTED MANUSCRIPT 11 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 2009-2010 in Greece (Tsakiris et al.

. because of the different digestive apparatuses and mechanism of AFB1 assimilation of animals and for different feeding used in cow´s breeding (Barbiroli et al. European Commission. Analysis of 10 breast milk samples showed that determined AFM1 concentration in 6 samples ranged from 0. many different infant formulas are available for infants and young children.025 µg/kg which is allowed MRL for milk and infant formula intended for infant and young children (Serbian Regulation 2010b.. 2012). Analysis of infant formulae sample showed that concentration of AFM1 was in accordance with Serbian and EU Regulations (Serbian Regulation 2010b.. Early intake could provoke a variety of allergic reactions (Ivakhnenko et al.022 µg/kg. 2006b). 2013) and could represent great risks related to AFM1. 2011).006 to 0. it should be pointed that any level of AFM1 represent risk to infant since milk is their main food. great numbers of infant and young children in Serbia immediately after breast-feeding start to drink cow´s milk. Also.. However.. when breast-feeding is not possible or enough. Fallah et al. AC C EP TE D and Health Organization (WHO) recommends exclusive breast-feeding for the first two years of M AN U SC RI PT . 2010.ACCEPTED MANUSCRIPT 12 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 contaminated with AFM1 than cow´s milk. Unfortunately. Hussain et al. founded AFM1 concentrations in breast milk were lower than 0. Breast milk remains the best source of nutrition for infants and World life (Dowling et al.. 2007. Possible reasons for early introduction of cow´s milk are economic situation in Serbia and/or traditional diet. European Commission. breast milk samples were included in this study. 2010). In Serbia there is only one milk industry that produces infant formulae. 2006b). it should be noted that data about occurrence of AFM1 in donkey´s 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. Although.

estimated intakes of AFM1 for adults were expressed for minimum. et al..2 ng/kg bw/day as tolerable daily intake (TDI) of AFM1.2 ng/kg bw/day only for children from 1 to 5 years. 2009).08 ng/kg bw/day in Brazil (Shundo. For obtained minimum concentration of AFM1. and 3. AC C EP TE D currently calculated daily intake of AFM1 in Serbia are higher than daily intake of AFM1 in M AN U SC RI PT .26. Based on the results shown in Table 4 it could be noted that calculated ingestion for obtained average and maximum concentrations for all age´s categories are significantly higher than TDI. that results from JECFA did not include data from Serbia..39 to 7.01 ng/kg bw/day in France (Leblanc.. The most of the previously estimated intakes of AFM1 through milk consumption in some countries were calculated for adults. 0.. and some of these are following: 0. 2007). Further. they concluded that daily exposure even with concentration lower than 1 ng/kg bw contributed to the risk of liver cancer. Different intake of milk and bw for different age´s categories influenced significant differences in daily ingestion of AFM1. and from 2.26 ng/kg bw/day in Morocco (Zinedine et al. 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). et al. Although International expert committees (JECFA. average and maximum obtained concentration of AFM1 and those values ranged from 0.ACCEPTED MANUSCRIPT 13 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 Kuiper-Goodman (1990) expressed 0. Obtained ingestion of AFM1 in this study greater than TDI value (0.42 to 1.18 ng/kg bw/day. It should be pointed.2 ng/kg bw/day) calculated by Kuiper-Goodman (1990) indicate high risk related to AFM1 since this toxin has carcinogenic. In the present study.02 to 0. 2010).305 ng/kg bw/day in Spain (Cano-Sancho et al. 2001) did not specify TDI for AFs. respectively. 2005).06. 0. 0. From the showed data it can be stated that other five world regions. calculated ingestion is higher than 0.

& Con.. Sert. 1596–1599. gene mutation. infants RI PT .ACCEPTED MANUSCRIPT 14 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 teratogenic. Ayar. This study was supported by the Ministry of Education. Food and Chemical Toxicology. (2008). AC C EP TE D 6. Conclusion Obtained results indicate high contamination frequency of AFM1 in examined cow´s milk samples. Journal of Food Safety. H. 27. Science and Technological Development of the Republic of Serbia.. A. D. M. 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. Determination of aflatoxin B1 levels in deep-red ground pepper (isot) using immunoaffinity column combined with ELISA. Project No. M.. genotoxic and immunosuppressive effect and could cause DNA damage. 5. A. 7. 199–207. (2007). chromosomal anomalies and cell transformation (IARC. 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. & Durmaz.H. Atasever.. Karakaya. A study on the occurrence of aflatoxin in raw milk due to feeds. Acknowledgements M AN U SC concern regarding human exposure to AFM1 from February to May 2013. III 46012. 2002). Y. Furthermore. 46. References Ardic..

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Validation parameters I´screen AFLAM1 test kit LOD 1.97 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 .54 RSDR 4.ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT Table 1.50 LOQ 5.00 RSDr 3.

50 - Average±SD 0.25 Min-Max 0.7) 26 (83.0) 4 (40.08-0.60) 37 (97.5) 1 (8.ACCEPTED MANUSCRIPT 2 3 Small Farms Farms 4 AC C EP TE D Small Pasteurized 8 No: Number of samples Range of concentration: number (percentage) Average±SD: Average concentration (µg/kg) ± standard deviation (µg/kg) Min-Max: minimum and maximum concentrations (µg/kg) M AN U Big Big Pasteurized Raw Goat Donkey Breast milk Infant formula Pasteurized UHT Organic 5 40 10 5 10 1 12 31 4 2 (5.07.005-0.08±0.05-0.18±0.27±0.02-0.10 0.04 0.5) 4 (40.06-0.05 0.0.0) 5 (12.006-0.04±0.40 0.22±0.02 0.08-0.0) 4 (40.03 0.22 0.0.36 0.0) 25 (62.005.9) 1 (25.02.20 0.90 0.03±0.0) 3 (60.41 0.09 0.0) 2 (40.02 0.01-0.09 0.30) 1 (12.70 0. Occurence of AFM1 in different types of milk Sample Group Producers No category Big Pasteurized 10 UHT 38 1 Organic 2 < 0.85 SC .00) 2 (20.0) 6 (60.1) 3 (75.005 - Range of concentration (µg/kg) 0.02±0.01±0.035 0.20 0.008-0.50 10 (100) 1 (2.0) 5 (16.11 0.006 0.0.35 0.5) Table 2.022 0.0) 7 (87.005-0.4) 2 (100) - > 0.5) RI PT 4 (80.0) - 1 (20.45±0.20±0.67±0.16-1.08 0.19±0.0) 11 (91.0) 1 (100) - 8 (20.24 0.

005 0.00) 8 (20.08-1.0.30) 19 (12.5) 5 (12.70) 63 (91.5) UHT 69 6 (8.005-0.3) Organic 6 5 (83.05 0.70 0.05-0.ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT Table 3.7) Small Pasterized 13 8 (61.50) 5 (38.02-0. Occurence of AFM1 in cow´s milk Sample Range of concentration (µg/kg) Producers No category < 0.41 0.0) 25 (62.20 .20 0.30) Min-Max 0.005.7) No: Number of samples Range of concentration: number (percentage) Average±SD: Average concentration (µg/kg) ± standard deviation (µg/kg) Min-Max: minimum and maximum concentrations (µg/kg) > 0.01-0.5) 11 (7.3) 1 (16.50 Big Pasterized 22 21 (95.50 1 (4.01-1.5) Total 150 2 (1.90 0.5) Farms Raw 40 2 (5.7) 118 (78.08 0.06-0.

39 0.6 Minimum ingestion of AFM1 ng/kg bw/day male 0.8 13.49 female 6.21 female 0.18 Average ingestion of AFM1 ng/kg bw/day male 6.11 0.23 0.34 1.43 0.02 3.02 Average ingestion of AFM1 calculated for obtained mean concentration of AFM1 (0.51 0.21 µg/kg) Minimum ingestion of AFM1 calculated for obtained minimum concentration of AFM1 (0.ACCEPTED MANUSCRIPT Table 4.80 female 35.42 0.44 0.11 female 0.18 2.45 2.03 AC C EP TE D M AN U SC > 55 89 71 0.02 0.09 0.17 .30 0.01 µg/kg) Maximum ingestion of AFM1 calculated for obtained maximum concentration of AFM1 (1.06 0.90 3.96 0.03 0.4 7.69 2.42 0.56 Maximum ingestion of AFM1 ng/kg bw/day male 36.20 µg/kg) RI PT 2.26 0. Estimate of AFM1 human exposure Age´s 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 0.31 0.8 10.32 0.44 0.26 1.22 0.86 0.

2010a AFs 0.005 0.05 Complete feedstuffs for dairy animals (mg/kg) 0.ACCEPTED MANUSCRIPT Table 5. Comparison of Serbian and EU Regulation All feed Regulation Toxin materials (mg/kg) European Commission.02 Serbian Regulation. 2002a AFB1 0.01 AC C EP TE D M AN U SC RI PT .

022 3.1 0.160 0.032 0. 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 (µg/kg) per day bw/day European Union 0.023 6.5 0.290 0.20 Middle East 0.10 Africa 0.8 0.120 0.042 0.360 12 0.002 0.005 0.ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT Table 6.058 Far East 0.6 0. 2001) .002 *results are taken from Joint Expert Committee on Food Additives (JECFA.11 Latin America 0.

7% cow´s.0% goat´s milk samples exceeded MRL Calculated average intakes of AFM1 were above than recommended value for TDI AC C EP TE D M AN U SC RI PT .0% donkey´s and 60.0% breast milk was contaminated AFM1 concentration in 86. 60.0% cow´s and 40. 80.0% goat´s.ACCEPTED MANUSCRIPT • • • • Presence of AFM1 was examined in 176 different types of milk 98.