Assessment of estimated daily intake of nitrite by average consumption of processed foods in Korea

This study has been performed to estimate the average daily intake of nitrite used in Korea as a color fixative. The crude estimation of daily intake was calculated based on maximum permitted levels (MPL) and national food disappearance data in 1998. In order to refine estimated daily intake (EDI), daily food consumption nationwide National Health and Nutrition Survey in 1998 and the concentration of nitrite in their permitted foods were applied. The crude EDI of nitrite was 17.85 μg/kg bw/day, representing 25.5% of acceptable daily intake (ADI) assigned by JECFA. The refined average EDI for nitrite was 0.87 μg/kg bw/day, representing 1.25% of ADI. For average consumers of age–sex groups ranged from 0.2 to 4.8 μg/kg bw/day, representing 0.3%–6.9% of the ADI.     

The seasonal variation of aflatoxin M1 in milk and dairy products and assessment of dietary intake in Punjab,Pakistan

Total 372 samples (169 sample from summer and 203 samples from winter) of milk and dairy products were collected from major cities of Punjab, Pakistan, during 2014–2015. The aflatoxin M₁ (AFM₁) levels were analyzed using HPLC, equipped with floresceⁿce detector. The results have showed that AFM₁ was detected in 77 (45.5%) samples of milk and dairy products from summer at the range of LOD to 229.6 ng/L, and 114 (56.1%) samples from winter at a range of LOD to 345.8 ng/L. The highest mean of AFM₁ i.e. 94.9 ± 5.4 ng/L from summer and 129.6 ± 8.4 ng/L from winter was detected in raw milk samples. The results have shown that 37.5% of milk, 32% of UHT milk, 12.5% of powdered milk, 20% of flavored milk, 20% of yogurt and 16% of flavored milk samples from summer were found exceeded the recommended levels of AFM₁ i.e 50 ng/L (European Union permissible limit), as compared to 38.1%, 37.1%, 15.6%, 21.4%, 27.7% and 40% samples of milk, UHT milk, powdered milk, flavored milk, yogurt and flavored yogurt from winter, respectively. The mean level of AFM₁ in samples of milk, UHT milk and powdered milk samples from winter season has significantly (p˂ 0.05) higher as compared to the mean levels in samples from summer season. The dietary exposures of AFM₁ in milk and dairy products were calculated in five different age groups and the highest level of 3.42 and 3.33 ng/L/day in summer and 4.7 and 4.6 ng/L/day in winter were found in 1–5 years of male and female infants, respectively. The results of present study have shown significant levels of AFM₁ in milk and dairy products from Punjab, Pakistan and could pose considerable thereat to the health of local population.     

Assessment of aflatoxin M1 levels in pasteurized and UHT milk consumed in Prishtina,Kosovo

To assess public health hazards associated with the occurrence of AFM1 residues in pasteurized milk and UHT milk a survey was carried out, in Prishtina, capital city of Kosovo. In the present study, a total of 178 samples, 84 pasteurized milk and 94 UHT milk were collected during 6 months (July to December 2013). They were obtained from retail outlets in Prishtina city (Kosovo). The occurrence and concentration range of AFM1 in the samples were investigated by competitive enzyme-linked immunosorbent assay (ELISA) method. There was a high incidence of AFM1 (81.0%) in both pasteurized and UHT milk samples. Eighty three percent (83.3%) of the pasteurized milk samples and seventy eight percent (78.7%) of the UHT milk samples contained AFM1. The positive incidence of AFM1 in the pasteurized milk and the UHT milk samples ranged from 5.16 to 110 ng/L and from 5.02 to 62 ng/L, respectively. AFM1 levels in 18 (21.4%) pasteurized milk samples and 4 (4.2%) UHT milk samples exceeded the maximum tolerable limit of the EC according to the European Union regulation limits of 50 ng/L. AFM1 levels in the samples show that there is a presence of high AFM1 level that constitutes a human health risk in Kosovo. The results of this study imply that more emphasis should be given to the routine AFM1 inspection of milk and dairy products in the Prishtina region.     

Detection of aflatoxin M1 in milk products from China by ELISA using monoclonal antibodies

A rapid and sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) method using monoclonal antibody for measuring aflatoxin M1 (AFM1) in milk and milk products has been described. One monoclonal antibody was isolated and characterized after fusion of myeloma cells with spleen cells isolated from BALB/c mice that had been immunized with AFM1 carboxymethyl oxime conjugated with bovine serum albumin (BSA). Cross-reactivities of the anti-AFM1 monoclonal antibody clone were 100, 13.9, 6.7 and <1% against AFM1, aflatoxin B1 (AFB1), aflatoxin G1 (AFG1) and deoxynivalenol (DON), respectively. Assays of milk samples mixed with AFM1 ranging in concentration from 0.1 to 3.2 ng/ml gave mean ELISA recovery of 98%. The limit of detection concentration of AFM1 was 0.04 ng/ml. AFM1 contamination was measured in 12 samples of raw milk, 15 samples of powdered milk, 104 samples of liquid milk and four cheese samples collected from different supermarkets in Northeast of China. Of 135 milk samples tested, 55 (41%) samples contained AFM1 at levels that ranged from 0.32–0.50 ng/ml, 24 (18%) samples contained 0.16–0.32 ng/ml, and 18 (13%) samples contained 0–0.16 ng/ml; in 38 (28%) samples AFM1 was not detected. The results indicate that the necessary precaution will have to be taken to minimize the AFM1 contamination in milk and milk products from Northeast of China.     

Seasonal effect on aflatoxin M1 contamination in raw and UHT milk from Croatia

Aflatoxin M1 contamination was examined in raw milk (3716) and UHT milk (706) samples collected from farms of eastern Croatia and markets of central Croatia from February to July 2013. A maximal mean AFM1 of 1135.0 ng/L was measured in raw milk in March. The AFM1 levels exceeded the European Union maximum residue permitted amount (EU MRL) in 45.9% raw and 36.2% UHT milk samples in February. In total, AFM1 levels exceeded the EU MRL values in 27.8% of raw and 9.64% of UHT milk samples. A slight decrease in the number of samples exceeding the EU MRLs was recorded in the period March to June. The results showed significant statistical differences between the mean AFM1 concentrations of raw and UHT milk samples collected during February, March, May and June (P < 0.05, all). Also, statistical differences in AFM1 concentrations were found between months for raw and UHT milk (P < 0.001, both). In conclusion, the frequency of control of feed and milk samples should increase and should strive to educate breeders and those involved in milk production about the harmful effect of aflatoxins to animal feed.     

Two year survey on the occurrence and seasonal variation of aflatoxin M1 in milk and milk products in Serbia

The incidence of contamination of aflatoxin M1 (AFM1) in milk and milk products samples collected in Serbia was investigated by using the competitive enzyme linked immunosorbent assay (ELISA) technique. A total of 1438 samples composed of 678 raw milk, 438 heat treated milk and 322 milk product samples that were analyzed during the period of 2013–2014, including all seasons. The AFM1 levels exceeded the European Union maximum residue permitted amount (EU MRL) in 56.3% raw milk, 32.6% heat treated milk and 37.8% of milk product samples. Milk powders had the highest mean AFM1 concentration (0.847 μg/kg) of all types of milk products examined. Mean concentration of AFM1 in raw milk samples during the period of winter in Serbia was 0.358 μg/kg and did not significantly differ from the mean concentrations of 0.375 μg/kg during the spring. However, the AFM1 raw milk concentration in the summer (0.039 μg/kg) and autumn season (0.103 μg/kg) was significantly lower. Seasonal variation of AFM1 concentrations in heat treated milk samples followed the trend observed in raw milk. Mean raw milk AFM1 concentration has dropped down by 10 fold from 0.314 μg/kg in 2013 to 0.035 μg/kg in 2014. The fraction of raw milk samples exceeding the EU MRL has decreased from 62.3% to 11.5% by the end of 2014.     

A survey on the Aflatoxin M1 occurrence and seasonal variation in buffalo and cow milk from Southern Italy

This study evaluates the aflatoxin M1 (AFM1) contamination in 804 samples of raw milk from cow and buffalo, collected randomly in Campania and Calabria regions of Southern Italy over a two years period.The competitive enzyme linked immunosorbent assay (ELISA) method was used to analyze AFM1 in the samples. AFM1 levels result above the CCβ value of 0.004 μg/kg in 51 (12.3%) cow milk samples and in 28 (7.2%) buffalo milk samples. Positive results from screening analysis were confirmed by high performance liquid chromatography with fluorimetric detection (HPLC-FLD) after a procedure of centrifugation, extraction and immunoaffinity column clean-up of milk. Only one cow milk sample exceeded the maximum limit (0.05 μg/kg) set by the European Regulation.The occurrence of AFM1 contamination was significantly (p < 0.05) higher in cold season, particularly fall, than in warm season, principally spring.Our results indicate that feedstuff used in the buffalo and cow farms were not highly contaminated with aflatoxins, determining a good quality of the analyzed milk. Therefore, the AFM1 contamination of the milk does not represent a serious public health problem in both regions in Southern Italy.     

Incidence of aflatoxin M1 in human breast milk in Tehran, Iran

This study examined the exposure of infants to aflatoxin M1 (AFM1) and of lactating mothers to aflatoxin B1 (AFB1), using AFM1 in breast milk as a biomarker for exposure to AFB1. An enzyme-linked immunosorbent assay (ELISA) was modified for the analysis of AFM1 in breast milk samples from 160 women in Tehran, Iran. AFM1 was detected in 157 samples by average concentration of 8.2 ± 5.1 ng/kg (range 0.3–26.7 ng/kg).The concentration of AFM1 in one sample was higher than the maximum tolerance limit accepted by European Union and USA (25 ng/kg), but in 55 samples was higher than the maximum concentration recommended by Australia and Switzerland (10 ng/kg).Logistic regression Analysis failed to show significant correlation between AFM1 and gestational age, education, postnatal age, gender, nationality, clinical condition, the number of family member, the number of children, type and amount of dairy consumption, vegetable, fruits, oil and meat. But it was significant relation to the cereal consumption, also to the height at birth.     

Occurrence of aflatoxin M1 in cheese processed and marketed in Lebanon

Aflatoxin M1 (AFM1) is the principal hydroxylated metabolite of aflatoxin B1 excreted in milk, and subsequently it can be found in a large variety of dairy products thus posing a potential risk to human health when consuming these products. Considering its significant impact on human health, determination of AFM1 levels in dairy products becomes imperative. The present study was undertaken to investigate, for the first time, the levels of AFM1 in various locally processed (halloumi, baladi, shelal, naboulsi, double cream, feta and akkawi) or imported (white and yellow) cheese types available in Lebanese markets. For this purpose a total of 111 cheese samples were randomly collected from local small dairy farms (53 samples), Lebanese leading dairy industries (38 samples) and imported cheeses (20 samples). Aflatoxin M1 was detected in 67.56% out of the 111 samples analyzed in which the levels of AFM1 in 17.33% of the samples were found to exceed the limits (250 ng/kg) allowed by European Commission (EC). Of the analyzed cheese samples, AFM1 levels in 13 cheeses from small local dairy farms were above the limits set by the EC ranging between 255 and 315 ng/kg. None of the cheese samples collected from the Lebanese leading dairy industries or from the imported types exceeded the permissible limit where the highest recorded levels were 77.2 and 3.95 ng/kg, respectively. The results of this study indicate that continuous monitoring of AFM1 levels in commonly consumed cheese in Lebanon should be regularly done. It also shows an alarming situation with respect to cheeses produced from local small farms.     

Risk factors associated with the presence of aflatoxin M1 in raw bulk milk from Argentina

The objectives of this study were to assess aflatoxin M₁ (AFM1) contamination in bulk tank milk, and to further identify the risk factors associated with the presence of AFM1 in raw milk in Argentina. The presence of AFM1 was investigated in 160 bulk tank milk samples collected from farms located in the most important milk production region in Argentina during one year (four seasons). Samples were analysed using immunoaffinity column (IAC) cleanup and UHPLC-MS/MS method for determining AFM1 at low levels of concentrations (LOQ = 0.003 μg L⁻¹). A survey about the potential factors associated with the presence of AFM1 in milk was performed directly in the field through a questionnaire applied to the farmers. Chi-square and logistic regression were performed with presence of AFM1 in milk as dependent variable, and potential risk factors as independent variables. Incidence of AFM1 in raw milk was 38.8% and, in all samples, AFM1 levels were lower than the Southern Common Market (MERCOSUR) Regulation (maximum level accepted = 0.5 μg L⁻¹). Commercial feed consumption (OR = 4.630, P = 0.001), soybean expeller consumption (>0.95 kgDM/cow) (OR = 3.542, P = 0.019), and cotton seed consumption (>1.5 kgDM/cow) (OR = 2.949, P = 0.089) were associated with the incidence of AFM1 in raw milk. Despite the incidence and the level of AFM1 in milk produced and commercialized in Argentina is not a serious problem for public health. The farm breeding intensification and the supplementation with commercial feed, soybean expeller, and cotton seed seems to be the risk factors that impacts on the AFM1 milk contamination. Therefore, Argentina should improve its monitoring program on mycotoxins in animal feed and milk and improve the management practices in farms.     

Screening of aflatoxin M1 occurrence in selected milk and dairy products in Terengganu,Malaysia

The study was conducted to screen the occurrence of aflatoxin M₁ (AFM₁) in 53 selected milk and dairy product samples (11 liquid milk, 12 powdered milk, 8 3-in-1 beverages, 6 condensed sweetened milk, 2 evaporated milk, 7 cultured milk drink, 5 yogurt and 2 cheese samples). These samples were purchased from selected markets in Terengganu, Malaysia in January 2014 based on a questionnaire survey among 212 respondents on the types and brands of milk and dairy products that were frequently consumed. Based on the responses, 53 milk and dairy products were purchased and the competitive enzyme-linked immune-absorbent assay (ELISA) method was used to determine the level of AFM₁ in the samples. Of 53 samples, 19 samples were positive with AFM₁ (35.8%) ranging from 3.5 to 100.5 ng/L. Although 4/53 (7.5%) of the tested samples had the contamination level greater than the European Commission (EC) limit (>50 ng/L), the contamination levels were still below the Malaysia Food Regulation 1985 limit (less than 500 ng/L). This study provided a pioneering data on the occurrence of AFM₁ in milk and dairy products in Malaysia.     

Variation of aflatoxin M1 contamination in milk and milk products collected during winter and summer seasons

Total 221 samples of milk and milk products were collected during winter (November 2011–February 2012) and 212 samples were collected during summer (May–August 2012) from central areas of Punjab, Pakistan. The samples were analyzed for the presence of aflatoxin M₁ (AFM₁) with a validated HPLC method equipped with florescence detector. The results revealed that from winter season almost 45% samples of milk and milk products were found to be contaminated with AFM₁ i.e. 40% of raw milk, 51% of UHT milk, 37% of yogurt, 60% of butter and 43% of ice cream samples and 27, 24, 25, 34 and 17% of samples were found above the recommended limit for AFM₁, respectively. However, from summer season 32% samples of milk and milk products were found to be contaminated i.e. 36% of raw milk, 31% of UHT milk, 29% of yogurt, 40% of butter and 24% of ice cream and 23, 23, 18, 20 and 5% of samples were found above the permissible limit for AFM₁, respectively. The levels of contamination in winter milk and milk product samples were significantly higher (α ≤ 0.05) than in summer season. The occurrence of AFM₁ in milk and milk products were higher, demanding to implement strict regulations and also urged the need for continuous monitoring of milk and milk products in order to minimize the health hazards.     

Analysis of aflatoxin M1 and M2 in commercial dairy products using high-performance liquid chromatography with a fluorescence detector

Aflatoxin M₁ (AFM₁) and M₂ (AFM₂) in commercial dairy products were analyzed by high-performance liquid chromatography (HPLC) with a fluorescence detector (FLD). To ensure an accurate analysis, two derivatization methods, bromination and aflatoxin–trifluoroacetic acid derivatization (ATD), were compared. The limits of detection (LODs) of the bromination method were 124.42–151.73 ng/kg, and the recovery rates were between 64 and 102%. The detection rates and concentration levels of AFM₁ were 6–74% and 14.48–270.94 ng/kg, respectively. AFM₁ was detected in 74% of milk powder samples and 36% of ice cream samples. The mean values of AFM₁ in milk powder and ice cream samples were 270.94 and 33.16 ng/kg, respectively. In the case of AFM₂, the detection rates were 2–10%, and the concentration levels were 20.62–55.67 ng/kg in milk and milk powder. Among milk and milk powder samples, ultra heat-treated (UHT) milk had lower AFM₁ contamination levels than pasteurized milk.     

The occurrence and effect of unit operations for dairy products processing on the fate of aflatoxin M1: A review

Mold contamination has challenged the safety of feed production and processing because of its undeniable role in the spoilage and the possible consequent toxicity impact on human health and the economy. Aflatoxin M₁ (AFM₁) is a hepatocarcinogenic derivative of aflatoxin B₁ excreted into the milk after ingestion of feed contaminated by certain molds. Because of the important role of dairy products, especially milk in the human diet, there is a huge concern about the presence of AFM₁ in milk and dairy products. In this article, the occurrence of AFM₁ and the fate of AFM₁ during processing of milk and dairy products, such as yoghurt and cheeses, since 1996 until today, was reviewed. The evaluation of mechanisms by which AFM₁ is affected by each processing step is of major importance to provide useful and accurate information to develop risk assessment studies and risk management strategies.     

Fast and sensitive aflatoxin B1 and total aflatoxins ELISAs for analysis of peanuts,maize and feed ingredients

Aflatoxins are a group of carcinogenic compounds produced by Aspergillus fungi that can grow on different agricultural crops. Both acute and chronic exposure to these mycotoxins can cause serious illness. Due to the high occurrence of aflatoxins in crops worldwide fast and cost-effective analytical methods are required for the identification of contaminated agricultural commodities before they are processed into final products and placed on the market. In order to provide new tools for aflatoxin screening two prototype fast ELISA methods: one for the detection of aflatoxin B₁ and the other for total aflatoxins were developed. Seven monoclonal antibodies with unique high sensitivity and at the same time good cross-reactivity profiles were produced. The monoclonal antibodies were characterized and two antibodies showing IC₅₀ of 0.037 ng/mL and 0.031 ng/mL for aflatoxin B₁ were applied in simple and fast direct competitive ELISA tests. The methods were validated for peanut matrix as this crop is one of the most affected by aflatoxin contamination. The detection capabilities of aflatoxin B₁ and total aflatoxins ELISAs were 0.4 μg/kg and 0.3 μg/kg for aflatoxin B₁, respectively, which are one of the lowest reported values. Total aflatoxins ELISA was also validated for the detection of aflatoxins B₂, G₁ and G₂. The application of the developed tests was demonstrated by screening 32 peanut samples collected from the UK retailers. Total aflatoxins ELISA was further applied to analyse naturally contaminated maize porridge and distiller's dried grain with solubles samples and the results were correlated with these obtained by UHPLC-MS/MS method.     

Effect of seasonal variations and lactation times on aflatoxin M1 contamination in milk of different species from Punjab, Pakistan

During October 2009 to September 2010, aflatoxin M₁ (AFM₁) levels were analyzed by HPLC-FLD in 356 milk samples of different lactating species (buffalo, cow, goat, sheep and camel) from Punjab (Pakistan). Recoveries of AFM₁ ranged from 92 to 97% and the limit of detection (LOD) was 0.004 μg/L. For all lactating species the mean concentration of AFM₁ was significantly higher in winter season than in summer (p < 0.05). The results showed that 55, 56, 32, 58 and 27% of winter milk samples of buffalo, cow, goat, sheep and camel exceeded the EU maximum limit (0.05 μg/kg), compared with 38, 33, 21, 36 and 14% of summer milk samples, respectively. For all lactating species the mean concentration of AFM₁ was significantly higher in morning milks than in evening milks (p < 0.05). The percentage of morning milk samples exceeding the EU maximum limit was 72, 67, 69, 71 and 44% for buffalo, cow, goat, sheep and camel, while for evening milks percent non compliant rates were 39, 30, 18, 33 and 25%, respectively. The level of AFM₁ tended to be higher in animal species fed mainly on concentrate mixtures (buffalo and cow) than in other species grazing on fresh greens.     

Detoxification of aflatoxin M1 in yoghurt using probiotics and lactic acid bacteria

Probiotics and lactic acid bacteria (LAB) are widely used in food fermentation and preservation. This study was carried out to assess the potential of five strains of LAB and bifidobacteria to remove aflatoxin M1 (AFM1) from yoghurt. The stability of the AFM1 complexes formed with them in both viable and non-viable (heat- or acid-treated) forms was assessed by repetitive aqueous extraction. Strains with high ability in removing AFM1 were selected to study its ability to remove AFM1 from spiked milk during yoghurt production and storage. Three treatments from yoghurt that produced from spiked milk were studied. Treatment A was the control using fermented yoghurt culture (Streptococcus thermophilus and Lactobacillus bulgaricus). Treatment B was fermented by 50% yoghurt culture (S. thermophilus and L. bulgaricus) and 50% Lactobacillus plantrium. Treatment C was fermented by yoghurt culture (S. thermophilus and L. bulgaricus) and 50% Lactobacillus acidophilus. The samples were collected during different storage (5 °C) times (1, 3, 5 and 7 days) to determine the ability of the stains to reduce AFM1. The results indicted that there were significant differences (P < 0.05) between the strains in their ability to reduce AFM1 in MRS broth media in the viable stage, heated stage and acid treatment. L. plantrium was the highest strain capable of removing AFM1. Yoghurt fermented by 50% yoghurt culture (S. thermophilus and L. bulgaricus) and 50% L. plantrium recorded the highest reduction in the level of AFM1 at the end of storage period. The results revealed the possibility of using some strains of LAB and bifidobacteria in detoxification of AFM1-contaminated foods.     

Levels of aflatoxin M1 in different types of milk collected in Serbia: Assessment of human and animal exposure

The level of aflatoxin M1 (AFM1) in 50 milk samples collected from February to June 2013 from Serbian market or domestically produced was determined using simple non-specific sample preparation method based on solid phase extraction (Oasis HLB, Waters) and ultra-high performance liquid chromatography with heated electrospray ionization triple quadrupole mass spectrometry (UHPLC/HESI-MS/MS). The range of detection was between < LOD and 1.44 μg/kg with mean value of 0.30 μg/kg. Thirty-eight samples (76%) exceed the maximum level of 0.05 μg/kg sets by EU. The highest level of 1.44 μg/kg was found in raw sample of domestically produced milk while the lowest one in organic produced milk. The evaluation of the exposure degree of AFM1 through the milk consumption by the average Serbian citizen was estimated at levels of 1.420, 0.769 and 0.503 ng/kg bw/day during February, April and May, respectively. Estimation of the corresponding concentration of AFB1 in feedstuffs was evaluated as 18.75 μg/kg. The calculated hazard index of 7.1, 3.8 and 2.5 for February, April and May, respectively, was higher than 1 indicated serious risk of AFM1 to Serbian consumers. This work presents the first insight in the occurrence of AFM1 in milk collected in Serbia as well as mycotoxin intake through milk consumption by Serbian adult population.     

Novel monoclonal antibody-based sensitive enzyme-linked immunosorbent assay and rapid immunochromatographic strip for detecting aflatoxin M1 in milk

Monoclonal antibody (mAb) that is specific to AFM1 was generated from the hybridoma cell line, 10F3C10, which was obtained by the fusion of mouse NS1 myeloma cells with the spleen cells of mouse that had been immunized with AFM1-bovine serum albumin (BSA). The 10F3C10 mAb is belong to the immunoglobulin G1 isotype. Both competitive direct and indirect enzyme-linked immunosorbent assay (ELISA) was utilized to characterize the mAb for AFM1. The concentrations of AFM1, AFB1 and AFG1 that caused 50% inhibition (IC₅₀) of the binding of AFM1-horseradish peroxidase (AFM1-HRP) to the antibody were found to be 0.022, 0.310 and 2.12 ng/mL, respectively. The immunochromatographic strip (immunostrip) assay with mAb-gold nanoparticle conjugates as a detection marker exhibited a visual limit of detection of 0.1 ng/mL for AFM1 and the analysis took a total of 10 min. Closely examining 17 milk-based samples using cdELISA revealed that four were slightly contaminated with AFM1 at concentrations from 0.002 to 0.054 ng/mL. All milk samples were negative in the immunostrip test because the levels of contaminant were below the detection limit of the strip. Notably, the presented cdELISA and immunostrip methods are highly sensitive methods for detecting AFM1 in milk.     

Assessment of aflatoxin M1 contamination in the milk of four dairy species in Croatia

Aflatoxin M1 (AFM1) concentrations were measured in bulk cow milk samples from eastern Croatia, and in cow, goat, sheep and donkey bulk milk samples from other parts of Croatia during the period July–September 2013. AFM1 levels in milk were measured in the ranges (ng/L): cow 3.65–162.3 (eastern Croatia) and 2.69–44.9 (other regions of Croatia); goat 2.78–40.8; sheep 2.11–5.87; donkey 3.43–10.4. The concentration of AM1 exceeded the EU MRL in 6.7% of cow milk samples from eastern Croatia. The highest level measured was 162.3 ng/L. AFM1 levels exceeded the LOQ value (23.2 ng/L) in only 59 samples of cow milk and two samples of goat milk of the total 402 samples analysed. A significant difference was found between the mean AFM1 concentrations of cow milk from eastern and other regions of Croatia (P < 0.05). The elevated AFM1 levels in cow milk from eastern Croatia indicate the use of contaminated supplementary feedstuff in some farms during the study period.