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Aflatoxin B1 occurrence in maize sampled from Croatian farms and feed factories during 2013
Food Control 40 (2014) 286e291
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Aflatoxin B1 occurrence in maize sampled from Croatian farms and feed factories during 2013 b Jelka Pleadin a, *, Ana Vuli c a, Nina Persi a, Mario Skrivanko , Brankica Capek c, Zeljko Cvetni ca a b c
Croatian Veterinary Institute Zagreb, Savska 143, 10000 Zagreb, Croatia Croatian Veterinary Institute, Veterinary Institute Vinkovci, Josipa Kozarca 24, 32100 Vinkovci, Croatia Ministry of Agriculture Republic of Croatia, Planinska 2a, 10000 Zagreb, Croatia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 October 2013 Received in revised form 6 December 2013 Accepted 17 December 2013
The aim of the study was to determine the level of aflatoxin B1 (AFB1) in maize sampled from farms and feed factories situated in Northern, Central and Eastern Croatia during 2013, following the occurrence of cow milk AFM1 contamination. Maize samples (n ¼ 633) were analysed using Enzyme-Linked Immunosorbent Assay (ELISA) as a screening method and High Performance Liquid Chromatography Tandem Mass Spectrometry (LCeMS/MS) as a confirmatory method. Mean AFB1 value found in maize coming from all investigated regions equalled to 81 mg/kg, with the maximal value of 2072 mg/kg found in maize obtained from Eastern Croatia. The observed contamination might have arisen on the grounds of extremely hot (>98%) and dry (<2%) weather witnessed from May to September 2012 during the maize growth and harvesting period, which might have favoured AFB1 production and consequently the contamination of dairy cattle feeds. In order to prevent the adverse effects of AFB1 on humans and animals, and also to reduce losses in agricultural production, systematic monitoring and further investigations of AFB1 contamination are necessary. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Aflatoxin B1 Maize Occurrence Farms and feed factories Croatia
1. Introduction Mycotoxins are toxic secondary metabolites produced by various genera of fungi that contaminate crops and processed food and feed worldwide. As livestock production plays a significant role in high-quality food production, contamination of cereals by a number of mycotoxins has raised an ever increasing concern. If animals eat feed contaminated with mycotoxins, the mycotoxin residua could affect both animal and human health (Chelkowski, 1998; Horn, 2005). Aflatoxins are known to be produced by Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius, often contaminating cereals along the line of their growth, harvest, storage, transport and processing (Bryden, 2007). Aflatoxin B1 (AFB1) represents a highly toxic, mutagenic, teratogenic and carcinogenic compound that exhibits an immunosuppressive activity and acute and chronic toxicity in both humans and animals (Eaton & Gallagher, 1994; Massey, Stewart, Daniels, & Ling, 1995; Meggs, 2009). It is the most potent hepatocarcinogen known in mammals, classified by the International Agency for Research on Cancer (IARC) under Group 1 as carcinogenic to humans (IARC, * Corresponding author. Tel.: þ385 16123626; fax: þ385 16123670. E-mail address: [email protected] (J. Pleadin). 0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2013.12.022
2002). Its metabolite aflatoxin M1 (AFM1), known as the “milk toxin” as it is excreted in milk of farm animals fed on AFB1contaminated feedstuffs, is also cytotoxic, but less mutagenic and genotoxic than AFB1, its carcinogenic potency in sensitive species being about one order of magnitude lower than that of AFB1 (Prandini et al., 2009). As the presence of AFB1 in food and feed can be dangerous for human health and represents a huge economic problem, the main source of AFB1 exposure thereby being cereals and cereal-based products (Scudamore & Patel, 2000), monitoring of conditions under which the latter are produced becomes very important. Literature data have shown that AFB1 can be found in a wide range of feedstuffs prior to harvesting, depending on the regional differences and climate prevailing on the harvest site; significant quantities of this toxin can be found in maize as well (Reddy et al., 2009). Co-occurrence of mycotoxins, particularly aflatoxins, is also an issue of special concern, as complementary toxicity may be expected (Bryden, 2012). A number of chemical, biological and physical methods have been investigated and employed in an attempt to inactivate aflatoxins in contaminated food- and feedstuffs (Rustom, 1997). Factors that promote fungal infection and AFB1 production in cereals are inoculum availability, weather conditions and pest
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infestation during crop growth, maturation, harvesting and storage (Lopez-Garcia & Park, 1998, chap. 9). Its incidence in food and feed is therefore relatively high in tropical and subtropical regions (Hell, Cardwell, & Poehling, 2003). Given that it has been proven that in cows fed on contaminated feed AFB1 gets to be converted into AFM1 and subsequently excreted into their milk, concerns about the entry of mycotoxins into the food chain through meat, eggs, milk and dairy products have been raised (carryover effects) (Markov et al., 2013). Based on the increased AFM1 concentration observed in dairy cattle milk, the aim of this study was to investigate AFB1 levels in maize originating from different farms and feed factories in Northern, Central and Eastern Croatia; namely, the maize the cattle was fed on was suspected to be the main source of contamination. 2. Materials and methods 2.1. Samples From February to September 2013, a total of 633 maize samples (genus 2012) had been sampled from different farms and feed factories situated in Croatia. Maize sampling and preparation of the test samples were performed in full line with ISO 6497:2002 and ISO 6498:1998, respectively. The samples were divided into three groups based on their origin in the following manner: Group one: 76 samples coming from Northern Croatia; Group two: 97 samples coming from Central Croatia, and Group three: 460 samples coming from Eastern Croatia (i.e. the region having the highest milk production of them all). The prepared test portions were ground into a fine powder having a particle size of 1.0 mm using an analytical mill (Cylotec 1093, Tecator, Sweden) and stored at þ4 C prior to AFB1 analysis. 2.2. Apparatus and chemicals The ELISA method was performed using an auto-analyzer ChemWell 2910 (Awareness Technology, Inc, USA). The HPLC (LC) system (1260 Infinity, Agilent Technologies, Santa Clara, USA) consisted of a degasser, a binary pump, an auto-sampler and a column compartment, and was coupled with a 6410 QQQ-mass spectrometer (MS) (Agilent Technologies, Santa Clara, USA). AFB1 standards employed to the effect of analytical methods’ validation were provided by SigmaeAldrich Chemie GmbH (Steinheim, Germany). All other chemicals used for AFB1 extraction and analysis were either of an analytical (ELISA) or of an HPLC grade (LC-MS/ MS). 2.3. ELISA method 2.3.1. Extraction procedure Maize grains were prepared using 5 g of the homogenized sample supplemented with 25 mL of 70%-methanol and shaken vigorously head-over-head on a shaker for 3 min. The extract was then filtrated (Whatman, black ribbon); in the next step, 1 mL of the obtained filtrate was diluted with an appropriate volume of deionized water. 2.3.2. Performance of the assay AFB1 concentration was determined using the ELISA method that made use of RidascreenÒ kits provided by R-Biopharm (Darmstadt, Germany); the procedure was carried out in full line with the instructions of the kit manufacturer. Each kit contains a micro-titer plate with 96 wells coated with antibodies against AFB1, aqueous AFB1 standard solutions (0, 1, 5, 10, 20, and 50 mg/L), peroxidase-conjugated AFB1, substrate/chromogen (urea peroxide),
287
a stop-reagent (1 N-sulphuric acid), and a washing buffer (10 mMphosphate buffer, pH ¼ 7.4). The obtained AFB1 concentrations were calculated from a six-point calibration curve taking thereby the applied dilution factor into due account, and then corrected for the recovery value. 2.3.3. Validation procedure Parameters necessary for the validation of the ELISA method were determined using the control maize samples (blank samples analyzed earlier). The limit of detection (LOD) was calculated from the mean value obtained with ten blank maize samples plus three standard deviations. The recovery rate was determined at four different levels (2, 5, 10 and 50 mg/kg) by virtue of spiking the control samples with the prepared standard AFB1 working solution (100 mg/L) adopted for in-house use (six replicates per concentration level per day). For the determination of intermediate precision, the same steps were repeated on two additional occasions by two independent analysts within a three- month period and under the same analytical conditions. Determination of trueness was performed by virtue of analysis of eight replicates of certified maize reference material (CRM) in AFB1 range of 4.49e11.54 mg/kg (T04209QC, Fapas, Sand Hutton, York). 2.4. LCeMS/MS method 2.4.1. Sample preparation To 25 g of the sample, 100 mL of the extraction solution (ACN/ H2O ¼ 80/20) were added. The mixture was shaken for 2 h on a horizontal shaker and afterwards filtrated (Whatman, black ribbon). One mL of the obtained filtrate was diluted with 3 mL of ultrapure water, mixed and filtrated through 0.45 mm-RC filter. Forty mL of the sample were injected into the LC system. 2.4.2. LCeMS/MS conditions LC separation was performed on XBridge BEH C18 columns (150x4.6, particle size 2.5 mm) at the flow rate of 0.80 mL/min and the temperature of þ40 C. The mobile phase consisted of the constituent A (0.1%-formic acid dissolved in water) and the constituent B (acetonitrile). A gradient elution program was employed as follows: 0e3 min: 90%-A, 18 min: 10%-A, 18.1 min: 90%-A, with the post-run time of 4 min and the injection volume of 40 mL. The conditions under which the mass spectrometry was performed were as follows: electro-spray ionization, positive polarity, capillary voltage of 6 kV, source temperature of þ350 C, nebulizer operating pressure of 45 psi, and the gas flow rate of 9 L/min. The mass spectrometer was operated in the multiple reaction monitoring mode, the protonated molecular AFB1 ion at m/z ¼ 313 being the precursor ion. Two product ions at m/z ¼ 285 and m/z ¼ 241 were monitored. The quantification was performed during the most intense transition (m/z 313 / 285) by virtue of extrapolation from six-point calibration curves. 2.4.3. Validation procedure LCeMS/MS validation was carried out according to the Commission Decision 2002/657/EC using an alternative approach of matrix-comprehensive in-house factorial design validation. The software used for the factorial design and calculation was InterVal Plus (quo data, Gesellschaft für Qualitätsmanagement und Statistik GmbH, Dresden, Germany). Within the frame of the validation process, decision limit (CCa), detection capability (CCb), precision, recovery, repeatability and in-house reproducibility of the method were studied. The validation process started with the factorial design where five factors were set at two levels. The factors in question were as follows: the analyst (A/B), the extraction time (2 h/3 h), the matrix (maize/barley), extract storage modality (þ4 C
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overnight/no storage) and RC filters producers (Agilent Technologies/Phenomenex). In order to validate the method, 8 runs, each consisting of samples spiked at 6 concentration levels, were completed on different days using factor combinations produced by the software. In total, 48 measurements were performed. Within each run, blank samples were fortified at six concentration levels: 2.5, 5, 7.5, 15, 30, and 60 mg/kg. In addition, a blank matrix sample and a fortified matrix sample were included into each run. 2.5. Statistical analysis
Table 2 Results of validation of LCeMS/MS employed to determine the presence of AFB1 in maize. Concentration (mg/kg)
sra (mg/kg)
RSD (%)
swrb (mg/kg)
RSD (%)
Recovery (%)
2.5 5.0 7.5 15 30 60
0.43 0.44 0.47 0.57 0.86 1.56
17.2 8.9 6.2 3.8 2.9 2.6
0.43 0.44 0.47 0.57 0.88 1.63
17.3 8.9 6.2 3.8 2.9 2.7
101.1 100.5 100.3 100.1 100.0 99.9
a
Statistical analysis was performed using Statistica Ver. 10.0 software (StatSoft Inc. 1984e2011, USA), with a statistical significance set at 95% (p ¼ 0.05). 3. Results and discussion 3.1. Validation study Literature has shown that the methods of choice for mycotoxin determination are mainly based on the use of enzyme-linked immunosorbent assay (ELISA), thin liquid chromatography (TLC), high performance liquid chromatography (HPLC) and recently liquid chromatography with mass spectrometry (LCeMSn) (Bryden, 2012; Krska et al., 2008; Pleadin et al., 2012, 2013). In this investigation, the determination of AFB1was carried out using the validated ELISA method as a quantitative screening method, while in samples containing AFB1in levels higher than the MPL, the mycotoxin presence was confirmed using LCeMS/MS (as recommended under the Directive 2002/32/EC). ELISA validation resulted in LOD value of 1.1 mg/kg. The mean recovery and the intermediate precision were determined to be 91.0 and 92.8%, respectively, with acceptable mean relative standard deviations of 5.8 and 7.4%, respectively (Table 1). The mean AFB1 concentration obtained in CRM along the line of trueness ascertainment was 9.8 mg/kg, which is acceptable given the established criterion (defined under the Commission Decision 2002/657/ EC). LCeMS/MS validation resulted in CCa and CCß values of 5.86 and 6.70 mg/kg, respectively, and in the mean recovery of 100.3% (Table 2). Judging by the obtained validation results, the applied ELISA and LCeMS/MS methods are capable of reliable and efficient determination and confirmation of AFB1presence in maize samples. AFB1 concentrations obtained using the ELISA method, which was employed with the analysis of each and every sample under investigation, are presented and discussed below. Representative LCeMS/MS chromatograms are presented in Fig. 1. 3.2. AFB1 level in maize The levels of AFB1 determined in each Croatian region are shown in Table 3. AFB1 was detected in 38.1% of maize samples, with 28.8% of the samples containing the mycotoxin in levels higher than the MPL (20 mg/kg) (Commission Directive 2003/100/ Table 1 Recovery and intermediate precision obtained during the course of validation of ELISA method employed to determine the presence of AFB1 in maize. Spiked AFB1 (mg/kg)
Mean recovery (%)
CV (%)
Intermediate precision (%)
CV (%)
2 5 10 50
85.4 90.7 92.2 95.5
6.1 5.7 6.3 4.9
88.5 93.2 93.6 95.9
8.4 7.3 7.1 6.7
b
sr e Repeatability standard deviation. swr e In-house reproducibility standard deviation.
EC). The highest percentage of samples containing the mycotoxin in concentrations higher than the MPL was observed in Eastern Croatia (36.5%). This region is the Croatian leader in grain production, as well as in farming and milk production. The maximal overall AFB1 level detected in the samples under analysis was 2072 mg/kg; the level in question is over 100 folds higher than the maximal permitted level stipulated for maize used as a feed component. In Central and Northern region, very high AFB1 levels were determined as well, with maximal observed values of 1728 and 945 mg/kg, respectively, but the number of samples coming from these regions that contained AFB1 in levels over the MPL was significantly lower. The results of the analysis of variance (ANOVA) revealed statistically significant differences (p < 0.05) in AFB1 concentrations found in samples coming from the same region, as well as between the investigated regions. In comparison to the results yielded by earlier extensive research, which revealed the maximal AFB1 level in maize grown in Europe and the Mediterranean region to be 311 mg/kg (Binder, Tan, Chin, Handl, & Richard, 2007), AFB1 levels observed in this study are significantly higher, pointing towards an extremely high maize contamination that took place in Croatia during 2013. The obtained values are also much higher than the levels documented in the same study in 1291 feed grain samples sourced from Asia and Oceania, which contained AFB1 in the maximal concentration of 457 mg/kg. The same goes for the AFB1 levels documented in maize in some North African countries (Zinedine & Mañes, 2009). The mean AFB1 levels ranging from 0.9 mg/kg to maximal 154.13 mg/kg have lately been observed in Iran (Karami-Osboo, Mirabolfathy, Kamran, Shetab-Boushehri, & Sarkari, 2012). The latest research in this part of Europe, performed in Serbia by Kos, Mastilovic, Jani c Hajnal, & Sari c (2013) during 2012, showed aflatoxin presence in 137 (68.5%) samples, in concentrations ranging from 1.01 to 86.1 mg/kg (mean, 36.3 mg/kg). As the authors concluded, weather changes seen in previous years might be liable for aflatoxin-related issues, so that further reductions in maize yield can be expected in the future; the same may be assumed to apply for Croatia as well. AFB1 contamination of feed in such huge concentrations has never been seen in Croatia before. The analyses of AFB1 presence conducted over the last decades have shown only sporadic ap pearances of this toxin in the imported maize (Segvi c Klari c, Cvetni c, Pepeljnjak, & Kosalec, 2009). 2012 research conducted by the Croatian Food Agency, running under the title “A study of the incidence of mycotoxins in feedstuffs and feed mixtures in Croatia” (HAH, 2012), which included 20 largest animal feed-producing Croatian companies and provided the analysis of a total of 300 samples, showed the presence of AFB1 in 20% of the samples in the concentration range of 1e10 mg/kg, that is to say, in concentrations significantly lower than those obtained in this study. Given that elevated mycotoxin concentrations are usually associated with humidity and temperature as the critical factors for mould formation and thus also mycotoxin production (Bryden,
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Fig. 1. Representative LCeMS/MS-MRM chromatograms descriptive of AFB1 presence in maize samples: a) the blank sample spiked with AFB1 at 60 mg/kg for matrix matched calibration curve; b) naturally contaminated maize sample containing AFB1 at the same level.
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Table 3 AFB1 levels determined in maize samples originating from three Croatian regions using ELISA and LCeMS/MS.
No of samples Positive samples over totala (%) Averageb (mg/kg) Max (mg/kg) Min (mg/kg) Median (mg/kg) SD (mg/kg) Over MPLc (%)
Easter
Central
North
All samplesd
460 39.6 165 2072 1.2 32.0 324 36.5
97 27.8 34 1728 1.1 1.1 206 12.4
76 42.1 44 945 1.3 1.3 192 10.5
633 38.1 81 2072 1.1 17.5 240 28.8
a
Percentage of positive samples over total in which AFB1 was detected. For samples containing AFB1 higher than the MPL (>20 mg/kg), the results of both analytical methods were taken into account. c Percentage of samples having the AFB1 content higher than the MPL. d Summary of AFB1values obtained across the three regions. b
2012; Pleadin et al., 2013), the results obtained by this study could possibly be attributed to these factors as well. High levels of AFB1 maize contamination seen in Croatia during 2013 might have mainly been associated with weather conditions, given that the Croatian Meteorological and Hydrological Institute recorded the year 2012 as extremely warm (>98%) and dry (<2%), characterised by a very low average rainfall. Such conditions were extremely favourable for the formation of moulds and AFB1 in maize. This study represents the first report on high AFB1 maize contamination of cereals coming from regions known to be major Croatian producers of cereals and milk. Such a contamination might also be influenced by other environmental conditions that enhance AFB1 production during maize growing and storage seasons, as reported by earlier studies (Abbas et al., 2002; Garrido, Hernández Pezzani, & Pacin, 2012; Kos et al., 2013). Physical grain damage can also affect hygroscopic characteristics of maize during storage, thus leading to a higher incidence of mould production coming as a result of increased kernel moisture content. It is well known that a decrease in moisture content increases the probability of mechanical kernel damage, the broken kernels subsequently representing a favourable substrate for mycotoxin-producing fungi growth. Data have shown that drying helps avoiding fungal activity during storage, therefore the kernels should be properly stored, so as to keep their moisture content at 13% or less (Prandini et al., 2009). The true scope of mycotoxin contamination risk that can ever more hardly be contained to a single area should not be ignored, especially in view of feed market globalisation. Should such a contamination remain widespread, the use of contaminated grains shall become more frequent, especially when it comes to the production of feed mixtures and shall, indirectly, lead to food chain contamination. In order to prevent future cases of high cereal contamination, it is necessary to implement the HACCP (Hazard Analysis and Critical Control Points) system (Bryden, 2007), and to improve agricultural, crop production and crop storage practices based on the results of screening and confirmatory analyses that allow for an unambiguous detection of contaminating compounds. Continuous implementation of annual feed monitoring programs that imply the analysis of the targeted number of representative samples for different mycotoxins, and monitoring of risk factors such as weather conditions and other factors identified as important for mycotoxin formation, has become imperative. Proper grain storage, mould growth control and decontamination of feed using different procedures, so as to inhibit the production of AFB1 and prevent its toxic effects on human and animal health, should be practiced on a regular basis. To prevent future aflatoxin contamination outbreaks, improper farming practices favouring contamination of feed and food should be improved (Prandini et al., 2009).
4. Conclusions The study pointed towards an extremely high contamination of Croatian maize used as farm animal feed. Such a contamination could be associated with extremely hot and dry weather seen during 2012 that enhanced the production of AFB1 in maize. Due to the significant health risk for humans and animals associated with the presence of AFB1 in food and feed, it is very important to establish adequate control measures through well defined national monitoring programs, which shall “keep an eye” on weather conditions during the production year. Application of each and every measure capable of preventing feed and consequently food contamination and possible adverse effects of this toxin on human and animal health, as well as economic losses, is of the outmost importance for the agricultural industry. References Abbas, H. K., Williams, W. P., Windham, G. L., Pringle, H. C., Xie, W., & Shier, T. W. (2002). Aflatoxin and fumonisin contamination of commercial corn (Zea mays) hybrids in Mississippi. Journal of Agricultural and Food Chemistry, 50, 5246e 5254. Binder, E. M., Tan, L. M., Chin, L. J., Handl, J., & Richard, J. (2007). Worldwide occurrence of mycotoxins in commodities feeds and feed ingredients. Animal Feed Science and Technology, 137, 265e282. Bryden, W. L. (2007). Mycotoxins in the food chain: human health implications. Asia Pacific Journal of Clinical Nutrition, 16, 95e101. Bryden, W. L. (2012). Mycotoxin contamination of the feed supply chain: implications for animal productivity and feed security. Animal Feed Science and Technology, 173, 134e158. Chelkowski, J. (1998). Distribution of Fusarium species and their mycotoxins in cereal grains. In K. K. Sinha, & D. Bhatnagar (Eds.), Mycotoxins in agricultural and food safety (pp. 45e64). New York, USA: Marcel Dekker. Commission Directive 2003/100/EC of 31 October 2003 amending Annex I to directive 2002/32/EC of the European Parliament and of the Council on undesirable substances in animal feed. Official Journal of the European Union. L 285/33. Commission of the European Communities. (2002). Commission Decision 2002/ 657/EC of 12th August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Official Journal of the European Communities. L 221. Directive 2002/32/EC of the European Parliament and of the Council of 7 May 2002 on undesirable substances in animal feed. Official Journal of the European Communities. L 140/10. Eaton, D. L., & Gallagher, E. P. (1994). Mechanisms of aflatoxin carcinogenesis. Annual Review of Pharmacology and Toxicology, 34, 135e172. Garrido, C. E., Hernández Pezzani, C., & Pacin, A. (2012). Mycotoxins occurrence in Argentina’s maize (Zea mays L.), from 1999 to 2010. Food Control, 25, 650e665. HAH (Croatian Food Agency). (23 October 2012). Mycotoxins in feed. Scientific opinion, adopted by. HAH Scientific Committee. http://www.hah.hr/english/ panels.php. Hell, K., Cardwell, K. F., & Poehling, H. M. (2003). Relationship between management practices fungal infection and aflatoxin for stored maize in Benin. Journal of Phytopathology, 151, 690e698. Horn, B. W. (2005). Ecology and population biology of aflatoxigenic fungi in soil. In K. A. Hameed (Ed.), Aflatoxin and food safety (pp. 95e116). 6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487-2742: Tylor & Francis group/CRC Press. International Agency for Research on Cancer (IARC). (2002). Aflatoxins. In IARC Monograph on the evaluation of carcinogenic risks to humans (Vol. 82) (pp. 171e 300). Lyon, France: IARC/World Health Organization. Karami-Osboo, R., Mirabolfathy, M., Kamran, R., Shetab-Boushehri, M., & Sarkari, S. (2012). Aflatoxin B1 in maize harvested over 3 years in Iran. Food Control, 23, 271e274. Kos, J., Mastilovi c, J., Jani cHajnal, E., & Sari c, B. (2013). Natural occurrence of aflatoxins in maize harvested in Serbia during 2009e2012. Food Control, 34, 31e34. Krska, R., Schubert-Ulrich, P., Molinelli, A., Sulyok, M., McDonald, S., & Crews, C. (2008). Mycotoxin analysis: an update. Food Additives and Contaminants, 25, 152e163. Lopez-Garcia, R., & Park, D. L. (1998). Management of mycotoxin hazards through post-harvest procedures. In D. Bhatnagar, & K. K. Sinha (Eds.), Mycotoxins in agriculture and food safety. New York: Marcel Dekker. Massey, T. E., Stewart, R. K., Daniels, J. M., & Ling, L. (1995). Biochemical and molecular aspects of mammalian susceptibility to aflatoxin B1 carcinogenicity. Proceedings of the Society for Experimental Biology and Medicine, 208, 213e227. Markov, K., Pleadin, J., Bevardi, M., Vah ci c, N., Sokoli c-Mihalek, D., & Frece, J. (2013). Natural occurrence of aflatoxin B1, ochratoxin A and citrinin in Croatian fermented meat products. Food Control, 34, 312e317. Meggs, W. J. (2009). Epidemics of mold poisoning past and present. Toxicology of Industrial Health, 25, 571e576.
J. Pleadin et al. / Food Control 40 (2014) 286e291 Pleadin, J., Sokolovi c, M., Persi, N., Zadravec, M., Jaki, V., & Vuli c, A. (2012). Contamination of maize with deoxynivalenol and zearalenone in Croatia. Food Control, 28, 94e98. Pleadin, J., Vah ci c, N., Pers, N., Sevelj, D., Markov, K., & Frece, J. (2013). Fusarium mycotoxins’ occurrence in cereals harvested from Croatian fields. Food Control, 32, 49e54. Prandini, A., Tansini, G., Sigolo, S., Filippi, L., Laporta, M., & Piva, G. (2009). On the occurrence of aflatoxin M1 in milk and dairy products. Food and Chemical Toxicology, 47, 984e991. Reddy, K. R. N., Abbas, H. K., Abel, C. A., Shier, W. T., Oliveira, C. A. F., & Raghavender, C. R. (2009). Mycotoxin contamination of commercially important agricultural commodities. Toxin Reviews, 28, 154e168.
291
Rustom, I. Y. S. (1997). Aflatoxin in food and feed: occurrence, legislation and inactivation by physical methods. Food Chemistry, 59, 57e67. Scudamore, K. A., & Patel, S. (2000). Survey for aflatoxins, ochratoxin A, zearalenone and fumonisins in maize imported into the United Kingdom. Food Additives and Contaminants, 17, 407e416. Segvi c Klari c, M., Cvetni c, Z., Pepeljnjak, S., & Kosalec, I. (2009). Co-occurrence of aflatoxins, ochratoxin A, fumonisins, and zearalenone in cereals and feed, determined by competitive direct enzime-linked immunosorbent assay and thin-layer chromatography. Arhiv Za Higijenu Rada I Toksikologiju, 60, 427e434. Zinedine, A., & Mañes, J. (2009). Occurrence and legislation of mycotoxins in food and feed from Marocco. Food Control, 20, 334e344.
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Aflatoxin B1 occurrence in maize sampled from Croatian farms and feed factories during 2013 b Jelka Pleadin a, *, Ana Vuli c a, Nina Persi a, Mario Skrivanko , Brankica Capek c, Zeljko Cvetni ca a b c
Croatian Veterinary Institute Zagreb, Savska 143, 10000 Zagreb, Croatia Croatian Veterinary Institute, Veterinary Institute Vinkovci, Josipa Kozarca 24, 32100 Vinkovci, Croatia Ministry of Agriculture Republic of Croatia, Planinska 2a, 10000 Zagreb, Croatia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 October 2013 Received in revised form 6 December 2013 Accepted 17 December 2013
The aim of the study was to determine the level of aflatoxin B1 (AFB1) in maize sampled from farms and feed factories situated in Northern, Central and Eastern Croatia during 2013, following the occurrence of cow milk AFM1 contamination. Maize samples (n ¼ 633) were analysed using Enzyme-Linked Immunosorbent Assay (ELISA) as a screening method and High Performance Liquid Chromatography Tandem Mass Spectrometry (LCeMS/MS) as a confirmatory method. Mean AFB1 value found in maize coming from all investigated regions equalled to 81 mg/kg, with the maximal value of 2072 mg/kg found in maize obtained from Eastern Croatia. The observed contamination might have arisen on the grounds of extremely hot (>98%) and dry (<2%) weather witnessed from May to September 2012 during the maize growth and harvesting period, which might have favoured AFB1 production and consequently the contamination of dairy cattle feeds. In order to prevent the adverse effects of AFB1 on humans and animals, and also to reduce losses in agricultural production, systematic monitoring and further investigations of AFB1 contamination are necessary. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Aflatoxin B1 Maize Occurrence Farms and feed factories Croatia
1. Introduction Mycotoxins are toxic secondary metabolites produced by various genera of fungi that contaminate crops and processed food and feed worldwide. As livestock production plays a significant role in high-quality food production, contamination of cereals by a number of mycotoxins has raised an ever increasing concern. If animals eat feed contaminated with mycotoxins, the mycotoxin residua could affect both animal and human health (Chelkowski, 1998; Horn, 2005). Aflatoxins are known to be produced by Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius, often contaminating cereals along the line of their growth, harvest, storage, transport and processing (Bryden, 2007). Aflatoxin B1 (AFB1) represents a highly toxic, mutagenic, teratogenic and carcinogenic compound that exhibits an immunosuppressive activity and acute and chronic toxicity in both humans and animals (Eaton & Gallagher, 1994; Massey, Stewart, Daniels, & Ling, 1995; Meggs, 2009). It is the most potent hepatocarcinogen known in mammals, classified by the International Agency for Research on Cancer (IARC) under Group 1 as carcinogenic to humans (IARC, * Corresponding author. Tel.: þ385 16123626; fax: þ385 16123670. E-mail address: [email protected] (J. Pleadin). 0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2013.12.022
2002). Its metabolite aflatoxin M1 (AFM1), known as the “milk toxin” as it is excreted in milk of farm animals fed on AFB1contaminated feedstuffs, is also cytotoxic, but less mutagenic and genotoxic than AFB1, its carcinogenic potency in sensitive species being about one order of magnitude lower than that of AFB1 (Prandini et al., 2009). As the presence of AFB1 in food and feed can be dangerous for human health and represents a huge economic problem, the main source of AFB1 exposure thereby being cereals and cereal-based products (Scudamore & Patel, 2000), monitoring of conditions under which the latter are produced becomes very important. Literature data have shown that AFB1 can be found in a wide range of feedstuffs prior to harvesting, depending on the regional differences and climate prevailing on the harvest site; significant quantities of this toxin can be found in maize as well (Reddy et al., 2009). Co-occurrence of mycotoxins, particularly aflatoxins, is also an issue of special concern, as complementary toxicity may be expected (Bryden, 2012). A number of chemical, biological and physical methods have been investigated and employed in an attempt to inactivate aflatoxins in contaminated food- and feedstuffs (Rustom, 1997). Factors that promote fungal infection and AFB1 production in cereals are inoculum availability, weather conditions and pest
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infestation during crop growth, maturation, harvesting and storage (Lopez-Garcia & Park, 1998, chap. 9). Its incidence in food and feed is therefore relatively high in tropical and subtropical regions (Hell, Cardwell, & Poehling, 2003). Given that it has been proven that in cows fed on contaminated feed AFB1 gets to be converted into AFM1 and subsequently excreted into their milk, concerns about the entry of mycotoxins into the food chain through meat, eggs, milk and dairy products have been raised (carryover effects) (Markov et al., 2013). Based on the increased AFM1 concentration observed in dairy cattle milk, the aim of this study was to investigate AFB1 levels in maize originating from different farms and feed factories in Northern, Central and Eastern Croatia; namely, the maize the cattle was fed on was suspected to be the main source of contamination. 2. Materials and methods 2.1. Samples From February to September 2013, a total of 633 maize samples (genus 2012) had been sampled from different farms and feed factories situated in Croatia. Maize sampling and preparation of the test samples were performed in full line with ISO 6497:2002 and ISO 6498:1998, respectively. The samples were divided into three groups based on their origin in the following manner: Group one: 76 samples coming from Northern Croatia; Group two: 97 samples coming from Central Croatia, and Group three: 460 samples coming from Eastern Croatia (i.e. the region having the highest milk production of them all). The prepared test portions were ground into a fine powder having a particle size of 1.0 mm using an analytical mill (Cylotec 1093, Tecator, Sweden) and stored at þ4 C prior to AFB1 analysis. 2.2. Apparatus and chemicals The ELISA method was performed using an auto-analyzer ChemWell 2910 (Awareness Technology, Inc, USA). The HPLC (LC) system (1260 Infinity, Agilent Technologies, Santa Clara, USA) consisted of a degasser, a binary pump, an auto-sampler and a column compartment, and was coupled with a 6410 QQQ-mass spectrometer (MS) (Agilent Technologies, Santa Clara, USA). AFB1 standards employed to the effect of analytical methods’ validation were provided by SigmaeAldrich Chemie GmbH (Steinheim, Germany). All other chemicals used for AFB1 extraction and analysis were either of an analytical (ELISA) or of an HPLC grade (LC-MS/ MS). 2.3. ELISA method 2.3.1. Extraction procedure Maize grains were prepared using 5 g of the homogenized sample supplemented with 25 mL of 70%-methanol and shaken vigorously head-over-head on a shaker for 3 min. The extract was then filtrated (Whatman, black ribbon); in the next step, 1 mL of the obtained filtrate was diluted with an appropriate volume of deionized water. 2.3.2. Performance of the assay AFB1 concentration was determined using the ELISA method that made use of RidascreenÒ kits provided by R-Biopharm (Darmstadt, Germany); the procedure was carried out in full line with the instructions of the kit manufacturer. Each kit contains a micro-titer plate with 96 wells coated with antibodies against AFB1, aqueous AFB1 standard solutions (0, 1, 5, 10, 20, and 50 mg/L), peroxidase-conjugated AFB1, substrate/chromogen (urea peroxide),
287
a stop-reagent (1 N-sulphuric acid), and a washing buffer (10 mMphosphate buffer, pH ¼ 7.4). The obtained AFB1 concentrations were calculated from a six-point calibration curve taking thereby the applied dilution factor into due account, and then corrected for the recovery value. 2.3.3. Validation procedure Parameters necessary for the validation of the ELISA method were determined using the control maize samples (blank samples analyzed earlier). The limit of detection (LOD) was calculated from the mean value obtained with ten blank maize samples plus three standard deviations. The recovery rate was determined at four different levels (2, 5, 10 and 50 mg/kg) by virtue of spiking the control samples with the prepared standard AFB1 working solution (100 mg/L) adopted for in-house use (six replicates per concentration level per day). For the determination of intermediate precision, the same steps were repeated on two additional occasions by two independent analysts within a three- month period and under the same analytical conditions. Determination of trueness was performed by virtue of analysis of eight replicates of certified maize reference material (CRM) in AFB1 range of 4.49e11.54 mg/kg (T04209QC, Fapas, Sand Hutton, York). 2.4. LCeMS/MS method 2.4.1. Sample preparation To 25 g of the sample, 100 mL of the extraction solution (ACN/ H2O ¼ 80/20) were added. The mixture was shaken for 2 h on a horizontal shaker and afterwards filtrated (Whatman, black ribbon). One mL of the obtained filtrate was diluted with 3 mL of ultrapure water, mixed and filtrated through 0.45 mm-RC filter. Forty mL of the sample were injected into the LC system. 2.4.2. LCeMS/MS conditions LC separation was performed on XBridge BEH C18 columns (150x4.6, particle size 2.5 mm) at the flow rate of 0.80 mL/min and the temperature of þ40 C. The mobile phase consisted of the constituent A (0.1%-formic acid dissolved in water) and the constituent B (acetonitrile). A gradient elution program was employed as follows: 0e3 min: 90%-A, 18 min: 10%-A, 18.1 min: 90%-A, with the post-run time of 4 min and the injection volume of 40 mL. The conditions under which the mass spectrometry was performed were as follows: electro-spray ionization, positive polarity, capillary voltage of 6 kV, source temperature of þ350 C, nebulizer operating pressure of 45 psi, and the gas flow rate of 9 L/min. The mass spectrometer was operated in the multiple reaction monitoring mode, the protonated molecular AFB1 ion at m/z ¼ 313 being the precursor ion. Two product ions at m/z ¼ 285 and m/z ¼ 241 were monitored. The quantification was performed during the most intense transition (m/z 313 / 285) by virtue of extrapolation from six-point calibration curves. 2.4.3. Validation procedure LCeMS/MS validation was carried out according to the Commission Decision 2002/657/EC using an alternative approach of matrix-comprehensive in-house factorial design validation. The software used for the factorial design and calculation was InterVal Plus (quo data, Gesellschaft für Qualitätsmanagement und Statistik GmbH, Dresden, Germany). Within the frame of the validation process, decision limit (CCa), detection capability (CCb), precision, recovery, repeatability and in-house reproducibility of the method were studied. The validation process started with the factorial design where five factors were set at two levels. The factors in question were as follows: the analyst (A/B), the extraction time (2 h/3 h), the matrix (maize/barley), extract storage modality (þ4 C
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overnight/no storage) and RC filters producers (Agilent Technologies/Phenomenex). In order to validate the method, 8 runs, each consisting of samples spiked at 6 concentration levels, were completed on different days using factor combinations produced by the software. In total, 48 measurements were performed. Within each run, blank samples were fortified at six concentration levels: 2.5, 5, 7.5, 15, 30, and 60 mg/kg. In addition, a blank matrix sample and a fortified matrix sample were included into each run. 2.5. Statistical analysis
Table 2 Results of validation of LCeMS/MS employed to determine the presence of AFB1 in maize. Concentration (mg/kg)
sra (mg/kg)
RSD (%)
swrb (mg/kg)
RSD (%)
Recovery (%)
2.5 5.0 7.5 15 30 60
0.43 0.44 0.47 0.57 0.86 1.56
17.2 8.9 6.2 3.8 2.9 2.6
0.43 0.44 0.47 0.57 0.88 1.63
17.3 8.9 6.2 3.8 2.9 2.7
101.1 100.5 100.3 100.1 100.0 99.9
a
Statistical analysis was performed using Statistica Ver. 10.0 software (StatSoft Inc. 1984e2011, USA), with a statistical significance set at 95% (p ¼ 0.05). 3. Results and discussion 3.1. Validation study Literature has shown that the methods of choice for mycotoxin determination are mainly based on the use of enzyme-linked immunosorbent assay (ELISA), thin liquid chromatography (TLC), high performance liquid chromatography (HPLC) and recently liquid chromatography with mass spectrometry (LCeMSn) (Bryden, 2012; Krska et al., 2008; Pleadin et al., 2012, 2013). In this investigation, the determination of AFB1was carried out using the validated ELISA method as a quantitative screening method, while in samples containing AFB1in levels higher than the MPL, the mycotoxin presence was confirmed using LCeMS/MS (as recommended under the Directive 2002/32/EC). ELISA validation resulted in LOD value of 1.1 mg/kg. The mean recovery and the intermediate precision were determined to be 91.0 and 92.8%, respectively, with acceptable mean relative standard deviations of 5.8 and 7.4%, respectively (Table 1). The mean AFB1 concentration obtained in CRM along the line of trueness ascertainment was 9.8 mg/kg, which is acceptable given the established criterion (defined under the Commission Decision 2002/657/ EC). LCeMS/MS validation resulted in CCa and CCß values of 5.86 and 6.70 mg/kg, respectively, and in the mean recovery of 100.3% (Table 2). Judging by the obtained validation results, the applied ELISA and LCeMS/MS methods are capable of reliable and efficient determination and confirmation of AFB1presence in maize samples. AFB1 concentrations obtained using the ELISA method, which was employed with the analysis of each and every sample under investigation, are presented and discussed below. Representative LCeMS/MS chromatograms are presented in Fig. 1. 3.2. AFB1 level in maize The levels of AFB1 determined in each Croatian region are shown in Table 3. AFB1 was detected in 38.1% of maize samples, with 28.8% of the samples containing the mycotoxin in levels higher than the MPL (20 mg/kg) (Commission Directive 2003/100/ Table 1 Recovery and intermediate precision obtained during the course of validation of ELISA method employed to determine the presence of AFB1 in maize. Spiked AFB1 (mg/kg)
Mean recovery (%)
CV (%)
Intermediate precision (%)
CV (%)
2 5 10 50
85.4 90.7 92.2 95.5
6.1 5.7 6.3 4.9
88.5 93.2 93.6 95.9
8.4 7.3 7.1 6.7
b
sr e Repeatability standard deviation. swr e In-house reproducibility standard deviation.
EC). The highest percentage of samples containing the mycotoxin in concentrations higher than the MPL was observed in Eastern Croatia (36.5%). This region is the Croatian leader in grain production, as well as in farming and milk production. The maximal overall AFB1 level detected in the samples under analysis was 2072 mg/kg; the level in question is over 100 folds higher than the maximal permitted level stipulated for maize used as a feed component. In Central and Northern region, very high AFB1 levels were determined as well, with maximal observed values of 1728 and 945 mg/kg, respectively, but the number of samples coming from these regions that contained AFB1 in levels over the MPL was significantly lower. The results of the analysis of variance (ANOVA) revealed statistically significant differences (p < 0.05) in AFB1 concentrations found in samples coming from the same region, as well as between the investigated regions. In comparison to the results yielded by earlier extensive research, which revealed the maximal AFB1 level in maize grown in Europe and the Mediterranean region to be 311 mg/kg (Binder, Tan, Chin, Handl, & Richard, 2007), AFB1 levels observed in this study are significantly higher, pointing towards an extremely high maize contamination that took place in Croatia during 2013. The obtained values are also much higher than the levels documented in the same study in 1291 feed grain samples sourced from Asia and Oceania, which contained AFB1 in the maximal concentration of 457 mg/kg. The same goes for the AFB1 levels documented in maize in some North African countries (Zinedine & Mañes, 2009). The mean AFB1 levels ranging from 0.9 mg/kg to maximal 154.13 mg/kg have lately been observed in Iran (Karami-Osboo, Mirabolfathy, Kamran, Shetab-Boushehri, & Sarkari, 2012). The latest research in this part of Europe, performed in Serbia by Kos, Mastilovic, Jani c Hajnal, & Sari c (2013) during 2012, showed aflatoxin presence in 137 (68.5%) samples, in concentrations ranging from 1.01 to 86.1 mg/kg (mean, 36.3 mg/kg). As the authors concluded, weather changes seen in previous years might be liable for aflatoxin-related issues, so that further reductions in maize yield can be expected in the future; the same may be assumed to apply for Croatia as well. AFB1 contamination of feed in such huge concentrations has never been seen in Croatia before. The analyses of AFB1 presence conducted over the last decades have shown only sporadic ap pearances of this toxin in the imported maize (Segvi c Klari c, Cvetni c, Pepeljnjak, & Kosalec, 2009). 2012 research conducted by the Croatian Food Agency, running under the title “A study of the incidence of mycotoxins in feedstuffs and feed mixtures in Croatia” (HAH, 2012), which included 20 largest animal feed-producing Croatian companies and provided the analysis of a total of 300 samples, showed the presence of AFB1 in 20% of the samples in the concentration range of 1e10 mg/kg, that is to say, in concentrations significantly lower than those obtained in this study. Given that elevated mycotoxin concentrations are usually associated with humidity and temperature as the critical factors for mould formation and thus also mycotoxin production (Bryden,
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Fig. 1. Representative LCeMS/MS-MRM chromatograms descriptive of AFB1 presence in maize samples: a) the blank sample spiked with AFB1 at 60 mg/kg for matrix matched calibration curve; b) naturally contaminated maize sample containing AFB1 at the same level.
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Table 3 AFB1 levels determined in maize samples originating from three Croatian regions using ELISA and LCeMS/MS.
No of samples Positive samples over totala (%) Averageb (mg/kg) Max (mg/kg) Min (mg/kg) Median (mg/kg) SD (mg/kg) Over MPLc (%)
Easter
Central
North
All samplesd
460 39.6 165 2072 1.2 32.0 324 36.5
97 27.8 34 1728 1.1 1.1 206 12.4
76 42.1 44 945 1.3 1.3 192 10.5
633 38.1 81 2072 1.1 17.5 240 28.8
a
Percentage of positive samples over total in which AFB1 was detected. For samples containing AFB1 higher than the MPL (>20 mg/kg), the results of both analytical methods were taken into account. c Percentage of samples having the AFB1 content higher than the MPL. d Summary of AFB1values obtained across the three regions. b
2012; Pleadin et al., 2013), the results obtained by this study could possibly be attributed to these factors as well. High levels of AFB1 maize contamination seen in Croatia during 2013 might have mainly been associated with weather conditions, given that the Croatian Meteorological and Hydrological Institute recorded the year 2012 as extremely warm (>98%) and dry (<2%), characterised by a very low average rainfall. Such conditions were extremely favourable for the formation of moulds and AFB1 in maize. This study represents the first report on high AFB1 maize contamination of cereals coming from regions known to be major Croatian producers of cereals and milk. Such a contamination might also be influenced by other environmental conditions that enhance AFB1 production during maize growing and storage seasons, as reported by earlier studies (Abbas et al., 2002; Garrido, Hernández Pezzani, & Pacin, 2012; Kos et al., 2013). Physical grain damage can also affect hygroscopic characteristics of maize during storage, thus leading to a higher incidence of mould production coming as a result of increased kernel moisture content. It is well known that a decrease in moisture content increases the probability of mechanical kernel damage, the broken kernels subsequently representing a favourable substrate for mycotoxin-producing fungi growth. Data have shown that drying helps avoiding fungal activity during storage, therefore the kernels should be properly stored, so as to keep their moisture content at 13% or less (Prandini et al., 2009). The true scope of mycotoxin contamination risk that can ever more hardly be contained to a single area should not be ignored, especially in view of feed market globalisation. Should such a contamination remain widespread, the use of contaminated grains shall become more frequent, especially when it comes to the production of feed mixtures and shall, indirectly, lead to food chain contamination. In order to prevent future cases of high cereal contamination, it is necessary to implement the HACCP (Hazard Analysis and Critical Control Points) system (Bryden, 2007), and to improve agricultural, crop production and crop storage practices based on the results of screening and confirmatory analyses that allow for an unambiguous detection of contaminating compounds. Continuous implementation of annual feed monitoring programs that imply the analysis of the targeted number of representative samples for different mycotoxins, and monitoring of risk factors such as weather conditions and other factors identified as important for mycotoxin formation, has become imperative. Proper grain storage, mould growth control and decontamination of feed using different procedures, so as to inhibit the production of AFB1 and prevent its toxic effects on human and animal health, should be practiced on a regular basis. To prevent future aflatoxin contamination outbreaks, improper farming practices favouring contamination of feed and food should be improved (Prandini et al., 2009).
4. Conclusions The study pointed towards an extremely high contamination of Croatian maize used as farm animal feed. Such a contamination could be associated with extremely hot and dry weather seen during 2012 that enhanced the production of AFB1 in maize. Due to the significant health risk for humans and animals associated with the presence of AFB1 in food and feed, it is very important to establish adequate control measures through well defined national monitoring programs, which shall “keep an eye” on weather conditions during the production year. Application of each and every measure capable of preventing feed and consequently food contamination and possible adverse effects of this toxin on human and animal health, as well as economic losses, is of the outmost importance for the agricultural industry. References Abbas, H. K., Williams, W. P., Windham, G. L., Pringle, H. C., Xie, W., & Shier, T. W. (2002). Aflatoxin and fumonisin contamination of commercial corn (Zea mays) hybrids in Mississippi. Journal of Agricultural and Food Chemistry, 50, 5246e 5254. Binder, E. M., Tan, L. M., Chin, L. J., Handl, J., & Richard, J. (2007). Worldwide occurrence of mycotoxins in commodities feeds and feed ingredients. Animal Feed Science and Technology, 137, 265e282. Bryden, W. L. (2007). Mycotoxins in the food chain: human health implications. Asia Pacific Journal of Clinical Nutrition, 16, 95e101. Bryden, W. L. (2012). Mycotoxin contamination of the feed supply chain: implications for animal productivity and feed security. Animal Feed Science and Technology, 173, 134e158. Chelkowski, J. (1998). Distribution of Fusarium species and their mycotoxins in cereal grains. In K. K. Sinha, & D. Bhatnagar (Eds.), Mycotoxins in agricultural and food safety (pp. 45e64). New York, USA: Marcel Dekker. Commission Directive 2003/100/EC of 31 October 2003 amending Annex I to directive 2002/32/EC of the European Parliament and of the Council on undesirable substances in animal feed. Official Journal of the European Union. L 285/33. Commission of the European Communities. (2002). Commission Decision 2002/ 657/EC of 12th August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Official Journal of the European Communities. L 221. Directive 2002/32/EC of the European Parliament and of the Council of 7 May 2002 on undesirable substances in animal feed. Official Journal of the European Communities. L 140/10. Eaton, D. L., & Gallagher, E. P. (1994). Mechanisms of aflatoxin carcinogenesis. Annual Review of Pharmacology and Toxicology, 34, 135e172. Garrido, C. E., Hernández Pezzani, C., & Pacin, A. (2012). Mycotoxins occurrence in Argentina’s maize (Zea mays L.), from 1999 to 2010. Food Control, 25, 650e665. HAH (Croatian Food Agency). (23 October 2012). Mycotoxins in feed. Scientific opinion, adopted by. HAH Scientific Committee. http://www.hah.hr/english/ panels.php. Hell, K., Cardwell, K. F., & Poehling, H. M. (2003). Relationship between management practices fungal infection and aflatoxin for stored maize in Benin. Journal of Phytopathology, 151, 690e698. Horn, B. W. (2005). Ecology and population biology of aflatoxigenic fungi in soil. In K. A. Hameed (Ed.), Aflatoxin and food safety (pp. 95e116). 6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487-2742: Tylor & Francis group/CRC Press. International Agency for Research on Cancer (IARC). (2002). Aflatoxins. In IARC Monograph on the evaluation of carcinogenic risks to humans (Vol. 82) (pp. 171e 300). Lyon, France: IARC/World Health Organization. Karami-Osboo, R., Mirabolfathy, M., Kamran, R., Shetab-Boushehri, M., & Sarkari, S. (2012). Aflatoxin B1 in maize harvested over 3 years in Iran. Food Control, 23, 271e274. Kos, J., Mastilovi c, J., Jani cHajnal, E., & Sari c, B. (2013). Natural occurrence of aflatoxins in maize harvested in Serbia during 2009e2012. Food Control, 34, 31e34. Krska, R., Schubert-Ulrich, P., Molinelli, A., Sulyok, M., McDonald, S., & Crews, C. (2008). Mycotoxin analysis: an update. Food Additives and Contaminants, 25, 152e163. Lopez-Garcia, R., & Park, D. L. (1998). Management of mycotoxin hazards through post-harvest procedures. In D. Bhatnagar, & K. K. Sinha (Eds.), Mycotoxins in agriculture and food safety. New York: Marcel Dekker. Massey, T. E., Stewart, R. K., Daniels, J. M., & Ling, L. (1995). Biochemical and molecular aspects of mammalian susceptibility to aflatoxin B1 carcinogenicity. Proceedings of the Society for Experimental Biology and Medicine, 208, 213e227. Markov, K., Pleadin, J., Bevardi, M., Vah ci c, N., Sokoli c-Mihalek, D., & Frece, J. (2013). Natural occurrence of aflatoxin B1, ochratoxin A and citrinin in Croatian fermented meat products. Food Control, 34, 312e317. Meggs, W. J. (2009). Epidemics of mold poisoning past and present. Toxicology of Industrial Health, 25, 571e576.
J. Pleadin et al. / Food Control 40 (2014) 286e291 Pleadin, J., Sokolovi c, M., Persi, N., Zadravec, M., Jaki, V., & Vuli c, A. (2012). Contamination of maize with deoxynivalenol and zearalenone in Croatia. Food Control, 28, 94e98. Pleadin, J., Vah ci c, N., Pers, N., Sevelj, D., Markov, K., & Frece, J. (2013). Fusarium mycotoxins’ occurrence in cereals harvested from Croatian fields. Food Control, 32, 49e54. Prandini, A., Tansini, G., Sigolo, S., Filippi, L., Laporta, M., & Piva, G. (2009). On the occurrence of aflatoxin M1 in milk and dairy products. Food and Chemical Toxicology, 47, 984e991. Reddy, K. R. N., Abbas, H. K., Abel, C. A., Shier, W. T., Oliveira, C. A. F., & Raghavender, C. R. (2009). Mycotoxin contamination of commercially important agricultural commodities. Toxin Reviews, 28, 154e168.
291
Rustom, I. Y. S. (1997). Aflatoxin in food and feed: occurrence, legislation and inactivation by physical methods. Food Chemistry, 59, 57e67. Scudamore, K. A., & Patel, S. (2000). Survey for aflatoxins, ochratoxin A, zearalenone and fumonisins in maize imported into the United Kingdom. Food Additives and Contaminants, 17, 407e416. Segvi c Klari c, M., Cvetni c, Z., Pepeljnjak, S., & Kosalec, I. (2009). Co-occurrence of aflatoxins, ochratoxin A, fumonisins, and zearalenone in cereals and feed, determined by competitive direct enzime-linked immunosorbent assay and thin-layer chromatography. Arhiv Za Higijenu Rada I Toksikologiju, 60, 427e434. Zinedine, A., & Mañes, J. (2009). Occurrence and legislation of mycotoxins in food and feed from Marocco. Food Control, 20, 334e344.