Determination of mycotoxins in biscuits,dried fruits and fruit jams: an assessment of human exposure

ABSTRACT

A reliable, fast and simple method using UHPLC-MS/MS was developed for the determination of aflatoxins B₁ (AFB1), G₁ (AFG1), B₂ (AFB2) and G₂ (AFG2), ochratoxin A (OTA), deoxynivalenol (DON), zearalenone (ZEA), HT-2 toxin and T-2 toxin in crude extracts of biscuits with fruit filling, cookies, dried fruits and fruit jams. The method was successfully demonstrated on 39 samples of biscuits with fruit filling, 34 cookies, 14 dried fruits and 10 fruit jams. The mycotoxins detected in biscuits samples were ZEA, OTA, T-2 and AFB1 with an average concentrations of positive samples of 2.64, 4.10, 8.13 and 1.32 µg kg^?1, respectively; while the mycotoxins detected in jam samples were AFB1, OTA, T-2 and AFB2 with an average concentrations of positive samples of 2.00, 17.7, 4.37 and 1.15 µg kg^?1, respectively. The results showed that the majority of samples were in compliance with relevant regulations. However in eight samples of biscuits and three samples of fig jam the contents of OTA were higher than the existing OTA limits. The combined dietary exposure of selected mycotoxins was estimated for the first time for children, adolescents and adults. The estimated combined dietary exposures were all lower than the proposed value assumed to predict a possible risk scenario.     

The effect of chemical treatment on reduction of aflatoxins and ochratoxin A in black and white pepper during washing

The effect of 18 different chemicals, which included acidic compounds (sulfuric acid, chloridric acid, phosphoric acid, benzoic acid, citric acid, acetic acid), alkaline compounds (ammonia, sodium bicarbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide), salts (acetate ammonium, sodium bisulfite, sodium hydrosulfite, sodium chloride, sodium sulfate) and oxidising agents (hydrogen peroxide, sodium hypochlorite), on the reduction of aflatoxins B₁, B₂, G₁ and G₂ and ochratoxin A (OTA) was investigated in black and white pepper. OTA and aflatoxins were determined using HPLC after immunoaffinity column clean-up. Almost all of the applied chemicals showed a significant degree of reduction on mycotoxins (p?<?0.05). The lowest and highest reduction of aflatoxin B₁, which is the most dangerous aflatoxin, was 20.5%?±?2.7% using benzoic acid and 54.5%?±?2.7% using sodium hydroxide. There was no significant difference between black and white peppers (p?<?0.05).     

Aflatoxins and ochratoxin A and their Aspergillus causal species in Tunisian cereals

ABSTRACT

Occurrence of aflatoxins (AFs) AFB1, AFB2, AFG1, AFG2 and ochra toxin A (OTA) was studied in 65 samples of stored and freshly harvested wheat, barley and maize collected in Tunisia. The mycotoxins were simultaneously extracted and quantified by high performance liquid chromatography. Determination of AF-producing (section Flavi) and OTA-producing Aspergillus species (sections Nigri and Circumdati) was conducted in these samples by species-specific polymerase chain reaction (PCR). Results showed that most of maize samples were contaminated with AFs, data after storage showing lower values than those collected at harvest. All contaminated maize samples contained AFG1 and AFG2, among which 27.78% also had AFB1 and AFB2. This AFs pattern was consistent with the A. parasiticus toxin profile. A. flavus however showed the highest frequency in maize but was also found in barley and wheat where no AFs were detected. In contrast, OTA was neither found in maize nor in barley and only one wheat sample contained OTA. A. niger was the only OTA-producing species detected.     

Aflatoxins in cotton seeds and cotton seed cake from Pakistan

ABSTRACT

A study was conducted to evaluate the natural occurrence of aflatoxins (AFB1, AFB2, AFG1, AFG2) in cotton seeds (n = 110) and cotton seed cake (CSC; n = 110) from Pakistan. All samples were screened by Enzyme-Linked ImmunoSorbent Assay (ELISA). Positive samples were further quantified by IAC-HPLC-FLD. Total contamination frequency and aflatoxins mean levels were 80% and 69 μg/kg in cotton seeds and the corresponding values for cotton seed cake 88% and 89 μg/kg, respectively. Aflatoxin B1was found in all positive samples and co-occurred with AFB2, AFG1, and AFG2. Sixty-four cotton seeds and 71 CSC samples contained aflatoxins levels higher than the ML set for animal feed (20 µg/kg). The results of the present study will help the regulatory authorities to formulate strategies for monitoring aflatoxins in animal feeds.     

Distribution of aflatoxins in shelling and milling fractions of naturally contaminated rice

The objective of this study was to determine the distribution of an economically important class of mycotoxins, the aflatoxins, in rice milling fractions. Rice plants grown under field production conditions are frequently infected with types of pathogenic fungi that produce toxic metabolites (mycotoxins). Paddy (seeds) rice from healthy plants in the field was collected and stored on a farm under humid, poorly ventilated conditions. Samples were milled into four fractions (hulls, brown rice, bran and white rice) and analysed for aflatoxins (B₁, B₂, G₁ and G₂) using a validated method. Rice fractions from healthy plants, which contained low levels of aflatoxins (less than 1?µg?kg^?1), were used to determine the efficiency of the extraction method. Seeds stored under poor conditions were found to be contaminated with aflatoxins B₁ and B₂ as were the fractions. The sums of AFB₁ and AFB₂ in stored paddy rice, hulls, brown rice, bran and white rice were 141, 39, 158, 367 and 56?µg?kg^?1, respectively. The ratio of aflatoxin B₁ and B₂ was about 10?:?1. AFG₁ and AFG₂ were less than 1?µg?kg^?1. Thus, brown rice contained 92.9% of the aflatoxins in paddy rice, whereas white rice contained only 27.9%.     

Occurrence of aflatoxins in some Egyptian food crops collected from two coastal regions

Two-hundred-and-forty-one samples representing cereals, legumes, oil seeds and nuts were collected from two coastal regions in Egypt. Mouldy samples were collected from lots stored under different conditions. Forty-eight per cent ((116 samples) of the samples gave bright greenish-yellow fluorescence, whereas 8% (19 samples) contained aflatoxins. Hazel nut, soybean and cottonseed meal samples were free from aflatoxins whilst white corn, yellow corn, peanuts and almonds had relatively higher concentrations of aflatoxins than other crops. Aflatoxin B₁ was present in all contaminated samples whilst G₂ was found only in peanuts. Aflatoxin G₁ was found only in yellow corn, paddy rice and peanut samples. High concentrations of aflatoxins were found in mouldy samples of cereals and legumes which are popular in Egyptian diets. The high concentrations of aflatoxins found in contaminated samples may be due to the method of sampling and to unfavourable storage conditions.     

Increased aflatoxin contamination of dried figs in a drought year

Dried figs (4917 samples) destined for export from Turkey to the European Union were collected between September and December during the very dry crop year of 2007 and tested for aflatoxins B₁, B₂, G₁ and G₂ by immunoaffinity column clean-up and reverse-phase high-performance liquid chromatography (RP-HPLC). While 32% of the samples contained detectable levels of total aflatoxins, 9.8% of them exceeded the European Union limits. Aflatoxin levels were in the range of 0.2–259.46 µg kg^?1 and 2.04–259.46 µg kg^?1 for all samples and samples that exceeded the limits, respectively. A substantial increase in the incidence of aflatoxins was observed in 2007 compared with previous years, most likely due to the drought stress, high temperatures and low relative humidity encountered during the period from January to September of that year. In 2007, the mean temperature was 1–2°C higher, there was 300 mm less total rain, and the mean relative humidity was 10–15% lower than in 2002–06. The average concentration of individual aflatoxins present in the samples was quantified to determine whether the drought conditions promoted certain types of aflatoxins. Among the contaminated samples, aflatoxin B₁ occurred in 97% of the contaminated samples, followed by G₁ in 47%, B₂ in 24%, and G₂ in 6% of samples. Concentrations of individual aflatoxins exhibited great variability among the samples but were not significantly different from those reported in previous studies, which were conducted under conditions without drought and high temperatures.     

Banana peel: an effective biosorbent for aflatoxins

This work reports the application of banana peel as a novel bioadsorbent for in vitro removal of five mycotoxins (aflatoxins (AFB1, AFB2, AFG1, AFG2) and ochratoxin A). The effect of operational parameters including initial pH, adsorbent dose, contact time and temperature were studied in batch adsorption experiments. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and point of zero charge (pH_pzc) analysis were used to characterise the adsorbent material. Aflatoxins’ adsorption equilibrium was achieved in 15 min, with highest adsorption at alkaline pH (6–8), while ochratoxin has not shown any significant adsorption due to surface charge repulsion. The experimental equilibrium data were tested by Langmuir, Freundlich and Hill isotherms. The Langmuir isotherm was found to be the best fitted model for aflatoxins, and the maximum monolayer coverage (Q₀) was determined to be 8.4, 9.5, 0.4 and 1.1 ng mg^?1 for AFB1, AFB2, AFG1 and AFG2 respectively. Thermodynamic parameters including changes in free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) were determined for the four aflatoxins. Free energy change and enthalpy change demonstrated that the adsorption process was exothermic and spontaneous. Adsorption and desorption study at different pH further demonstrated that the sorption of toxins was strong enough to sustain pH changes that would be experienced in the gastrointestinal tract. This study suggests that biosorption of aflatoxins by dried banana peel may be an effective low-cost decontamination method for incorporation in animal feed diets.     

Occurrence of fumonisins and aflatoxins in cereals from markets of Hebei province of China

Fumonisins B₁, B₂ and B₃ (FB₁, FB₂ and FB₃) and aflatoxins B₁ (AFB₁), B₂ (AFB₂), G₁ (AFG₁) and G₂ (AFG₂) are both major mycotoxins of food concern, because of their wide range of concentration and possible co-occurrence. Therefore, a contamination survey in corn and wheat flour by liquid chromatography–tandem mass spectrometry was carried out. Quantification of fumonisins and aflatoxins was based on internal calibration (by the use of ¹³C₃₄-fumonisin) and external calibration, respectively. Fumonisins were detected in 95% of corn samples and in 7% of wheat flour samples, with the mean level (FB₁?+?FB₂?+?FB₃) of 441?µg?kg^?1 and 0.09?µg?kg^?1, respectively. Low levels of aflatoxins were detected in 37% of the samples with a mean level (B₁?+?B₂?+?G₁?+?G₂) of 0.12?µg?kg^?1. Fumonisins and aflatoxins were not detected in 29% of the samples analysed. Simultaneous occurrence of fumonisins and aflatoxins was observed in 12% of samples.     

Occurrence of certain mycotoxins in corn and corn‐based products and thermostability of fumonisin B1 during processing

A total of 57 samples of corn and corn‐based products collected from various districts of Egypt were analyzed for Fusarium mycotoxins (T‐2, diacetoxyscripenol (DAS( deoxynivalenol (DON) and fumonisin B₁ (FB₁) and aflatoxins. FB₁ was detected in about 80%, 53.85%, 33.3%, and 28.57% of yellow corn, corn meal, white corn, and popcorn samples, respectively. The levels of FB₁ ranged from 10 to 780 μg/kg. T‐2 and DAS were detected in 5% and 10% of yellow corn samples, respectively, and DON was detected in white corn and popcorn samples at levels of 28.8 and 10.1 μg/kg, respectively. Starch samples were found to be free from Fusarium mycotoxins. Baking balady bread at 450°C/min reduced FB₁ to 72.4% while baking Franco bread at 250°C/20 min reduced FB₁ to 57.4%. Boiling of macaroni and corn in water completely removed FB₁ from contaminated samples. On the other side, corn flakes samples were found to be contaminated with aflatoxins B₁ and G₁ at levels ranging from 6 to 10 ppm, whereas 2.9% of samples were contaminated with aflatoxin B₁ > 35 ppm and G1 > 16 ppm.     

Aflatoxins contamination in spices and processed spice products commercialized in Korea

A survey for total aflatoxins (aflatoxins B₁, B₂, G₁, and G₂) was conducted on 88 spices and processed spice products commercialized in Korea. The presence of aflatoxins was determined by high-performance liquid chromatography (HPLC) with fluorescence detector using immunoaffinity column clean-up. Total aflatoxins (AFs) are detected in 12 samples (13.6% of incidence) including seven red pepper powder, two red pepper pastes (Kochujang), two curry and one ginger product. The contamination levels are 0.08–4.45 μg/kg as aflatoxin B₁ and 0.08–4.66 μg/kg as AFs. The liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis on contaminated samples was conducted for the confirmation of detected aflatoxins. The 12 samples which showed aflatoxins by HPLC/FLD were confirmed as aflatoxins by LC–MS/MS.     

Effects of gamma irradiation on aflatoxins

Summary The effects of gamma irradiation on a mixture of aflatoxins B₁, B₂, G₁, G₂ were studied. Standard solutions of A and B were irradiated at 5, 10, and 20 kGy in a solution of water/DMSO (9+1, v/ v) by using a¹³⁷Cs source. The control (0 kGy) and irradiated samples were subjected to RP-HPLC analyses with methanol/water (4+5, v/v) as the mobile phase. Aflatoxin B₁ (AFB₁) was the most radio-sensitive of the four compounds. The radiosensitivity of the other aflatoxins, was in increasing order: G₂, B₂, G₁. Only about 5% of AFB₁ remained after irradiation of solution A at 5 kGy. When the concentration of solution B was increased two-fold, trace amounts of AFB₁ remained after irradiation doses of 10 and 20 kGy. Irradiation was found to be suitable for the destruction of aflatoxins in solution.

---

Einfluß von Gammastrahlung auf die Aflatoxine

Zusammenfassung Der EinfluB von -Strahlen auf die Aflatoxinmischung B₁, B₂, G₁ und G₂ wurde untersucht. Standardlösungen A und B wurden mit -Strahlung von 5; 10; bzw. 20 kGy in der Lösung von Wasser/DMSO (9+1, v/v) unter Verwendung einer¹³⁷Cs-Quelle bestrahlt. Kontroll(0 kGy)-und bestrahlte Proben wurden der RP-HPLC Analyse unter Verwendung von Methanol/Wasser (4+5, v/v) als mobile Phase unterworfen. Aflatoxin B₁ (AFB₁) war die strahlenempfindlichste von den vier, die Strahlenempfindlichkeit der anderen Aflatoxine nahm bei 5 kGy in der Reihenfolge: G₂, B₂, G₁ zu. Nur ca. 5% von AFB₁ ist nach der Bestrahlung üibriggeblie-ben. Wenn sich die Konzentration zweifach erhöht (Lösung B), ist von AFB₁ nur eine Spur nach der 10 kGy- und 20 kGy-Bestrahlung übriggeblieben. Es wurde gefunden, daß die Bestrahlung zur Zerstörung der Aflatoxine in der Lösung geeignet ist.

     

Comparison of homogenization techniques and incidence of aflatoxin contamination in dried figs for export

To determine differences in mean aflatoxin contamination and subsample variance from dry and slurry homogenizations, 10 kg of six different, naturally contaminated dried fig samples were collected from various exporting companies in accordance with the EU Commission Directive. The samples were first dry-mixed for 5 min using a blender and sub-sampled seven times; the remainder was slurry homogenized (1 : 1, v/v) and sub-sampled seven times. Aflatoxin B₁ and total aflatoxin levels were recorded and coefficient of variations (CV) computed for all sub-samples. Only a small reduction in sub-sample variations, indicated by the lower CV values, and slight differences in mean aflatoxin B₁ and total aflatoxin levels were observed when slurry homogenization was applied. Therefore, 7326 dried figs, destined for export from Turkey to the EU and collected during the 2008 crop year, were dry-homogenized and tested for aflatoxins (B_1, B_2, G₁ and G₂) by immunoaffinity column clean-up using RP-HPLC. While 34% of the samples contained detectable levels of total aflatoxins (0.20–208.75 µg kg^?1), only 9% of them exceeded the EU limit of 4 µg kg^?1 in the range 2.0–208.75 µg kg^?1, respectively. A substantial increase in the incidence of aflatoxins was observed in 2008, most likely due to the drought stress experienced in Aydin province as occurred in 2007.     

Aflatoxins contamination of maize in Serbia: the impact of weather conditions in 2015

ABSTRACT

In recent years climate changes recorded in temperate regions of Europe have led to aflatoxin (AF) contamination of maize. Thus, the aim of this study was to investigate the influence of weather conditions on levels of aflatoxin B₁ (AFB1), aflatoxin B₂ (AFB2), aflatoxin G₁ (AFG1) and aflatoxin G₂ (AFG2) in 180 maize samples collected from the main maize-growing regions (Western Ba?ka, North Banat, South Banat and Central Serbia) in Serbia after harvest in 2015. The concentrations of AFs were determined by a validated HPLC method with post-column derivatisation and fluorescence detection (HPLC-FLD). The presence of AFB1, AFB2, AFG1 and AFG2 was detected in 57.2%, 13.9%, 5.6% and 2.8% of maize samples in the concentration ranges of 1.3–88.8 µg kg^–1, 0.60–2.8 µg kg^–1, 1.8–28.5 µg kg^–1 and 2.1–7.5 µg kg^–1 respectively. The recorded smaller amount of precipitation and especially higher air temperatures during the summer of 2015 were favourable for AF production, which resulted in 32.2% and 21.1% of samples being unsuitable for human consumption, since AFB1 and the sum of AFs concentrations were above 5.0 and 10.0 µg kg^–1 respectively. Furthermore, the findings in this study indicate that the microclimate conditions in the investigated regions had a great influence on the contamination frequency of maize with AFs. The highest percentage of samples unsuitable for human consumption, considering both AFB1 and total AFs content were 72.5% and 51.5% respectively from Central Serbia, whilst the lowest percentages of 15.6% and 6.2% respectively were found in Western Ba?ka. These findings confirmed that maize should be continuously monitored in order to protect human and animal health from the harmful effects caused by AFs contamination.     

Aflatoxins and ochratoxin A in stored barley grain in Spain and impact of PCR-based strategies to assess the occurrence of aflatoxigenic and ochratoxigenic Aspergillus spp

Contamination of barley by moulds and mycotoxins results in quality and nutritional losses and represents a significant hazard to the food chain. The presence of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) and ochratoxin A (OTA) in stored barley in Spain has been studied. Species-specific PCR assays were used for detection of Aspergillus flavus, A. parasiticus, A. ochraceus, A. steynii, A. westerdijkiae, A. carbonarius and A. niger aggregate in mycotoxin-positive barley samples at different incubation times (0, 1 and 2 days). Classical enumeration techniques (CFU/g) in different culture media for evaluation of Aspergillus in sections Flavi, Circumdati and Nigri were also used. One hundred and five barley kernel samples were collected in Spanish grain stores from 2008 to 2010, and analyzed using a previously optimized method involving accelerated solvent extraction, cleanup by immunoaffinity column, liquid chromatographic separation, post-column derivatization with iodine and fluorescence detection. Twenty-nine samples were contaminated with at least one of the studied mycotoxins. AFB1, AFB2, AFG1, AFG2, and OTA were detected in 12.4%, 2.9%, 4.8%, 2.9%, and 20% of the samples, respectively. Aflatoxins and OTA co-occurred in 4.8% of the samples. Maximum mycotoxin levels (ng/g) were 0.61 (AFB1), 0.06 (AFB2), 0.26 (AFG1), 0.05 (AFG2), and 2.0 (OTA). The results of PCR assays indicated the presence of all the studied species, except A. westerdijkiae. The PCR assays showed high levels of natural contamination of barley with the studied species of Aspergillus which do not correspond to the expected number of CFU/g in the cultures. These results suggest that a high number of non-viable spores or hyphae may exist in the samples. This is the first study carried out on the levels of aflatoxins and OTA in barley grain in Spain. Likewise, this is the first report on the presence of aflatoxigenic and ochratoxigenic Aspergillus spp. in barley grain naturally contaminated with those mycotoxins using a species-specific PCR approach.     

Prevalence of aflatoxins in blood and urine of Egyptian infants with protein-energy malnutrition

The aim of the present work was to study the presence of aflatoxins in blood and urine of infants with protein-energy malnutrition (PEM). The study was conducted on 60 infants, 30 with kwashiorkor and 30 with marasmus, with 10 age-matched healthy infants studied as a control group. Complete blood count, liver function tests, and determination of the level of aflatoxins (B1, B2, G1, G2, M1, M2, G2a, B3, GM1, P, and aflatoxicol R0) in blood and urine were carried out in all studied infants. Serum aflatoxins were detected in more infants with kwashiorkor (80%) than in those with marasmus (46.7%). The mean serum levels of total aflatoxins, AFB1, AFG1, and AFB2a, were significantly higher in infants with kwashiorkor (p <.001). Aflatoxin B1 (AFB1) was the most commonly detected type. The prevalence of aflatoxin excretion in the urine of infants with kwashiorkor was 80%, a higher value than that in infants with marasmus (46.7%). The mean urinary concentration of total aflatoxins followed the same pattern of distribution (p < .052). There were no significant differences between groups in the mean urinary concentrations of AFB1, AFG1, AFB2a, AFM1, and AFG2a. Aflatoxins were not detected in any of the serum or urine samples of the control group. Aflatoxins are highly prevalent in this study population and show a high degree of correlation with severe PEM.     

Distribution of aflatoxins in shelling and milling fractions of naturally contaminated rice

The objective of this study was to determine the distribution of an economically important class of mycotoxins, the aflatoxins, in rice milling fractions. Rice plants grown under field production conditions are frequently infected with types of pathogenic fungi that produce toxic metabolites (mycotoxins). Paddy (seeds) rice from healthy plants in the field was collected and stored on a farm under humid, poorly ventilated conditions. Samples were milled into four fractions (hulls, brown rice, bran and white rice) and analysed for aflatoxins (B(1), B(2), G(1) and G(2)) using a validated method. Rice fractions from healthy plants, which contained low levels of aflatoxins (less than 1?μg?kg(-1)), were used to determine the efficiency of the extraction method. Seeds stored under poor conditions were found to be contaminated with aflatoxins B(1) and B(2) as were the fractions. The sums of AFB(1) and AFB(2) in stored paddy rice, hulls, brown rice, bran and white rice were 141, 39, 158, 367 and 56?μg?kg(-1), respectively. The ratio of aflatoxin B(1) and B(2) was about 10?:?1. AFG(1) and AFG(2) were less than 1?μg?kg(-1). Thus, brown rice contained 92.9% of the aflatoxins in paddy rice, whereas white rice contained only 27.9%.     

High performance liquid chromatographic determination of aflatoxins in chilli,peanut and rice using silica based monolithic column

A simple and rapid high performance liquid chromatographic with fluorescence detection method for the determination of the aflatoxin B₁, B₂, G₁ and G₂ in peanuts, rice and chilli was developed. The sample was extracted using acetonitrile:water (90:10, v/v%) and then purified by using ISOLUTE® multimode solid phase extraction. After the pre-column derivatisation, the analytes were separated within 3.7 min using Chromolith® performance RP-18e (100–4.6 mm) monolithic column. To assess the possible effects of endogenous components in the food items, matrix-matched calibration was used for the quantification and validation. The recoveries of aflatoxins that were spiked into food samples were 86.38–104.5% and RSDs were <4.4%. The method was applied to the determination of aflatoxins in peanut (9), rice (5) and chilli (10) samples. Liquid chromatography–tandem mass spectrometry analysis using triple quadruple analyser and operated in the multiple reaction monitoring modes on the contaminated samples was performed for confirmation.     

Occurrence of aflatoxins in selected spices in Poland

The aim of this study was to determine the contamination of aflatoxins B₁, B₂, G₁, G₂ in some selected spices (n = 84) in the Kuyavian-Pomeranian province of Poland. Aflatoxins were found in 63.1 % of the analysed samples. The presence of these compounds was confirmed in all the samples of pepper, nutmeg and turmeric. The maximum content for the sum of aflatoxins B₁, B₂, G₁, G₁ exceeding the acceptable level (10 μg/kg) was 16.91 μg/kg in one sample of nutmeg and 12.1 μg/kg in one sample of pepper, whereas in one sample of nutmeg the maximum content was equal to the regulatory limit. The lowest degree of contamination was found in black pepper.     

Aflatoxins and heavy metals in animal feed in Iran

The occurrence of aflatoxin (aflatoxin B₁, aflatoxin B₂, aflatoxin G₁ (AFG₁) and aflatoxin G₂ (AFG₂)) and heavy metal (Pb, Cd, As and Hg) contamination was determined in 40 industrially produced animal feed samples which were collected from the southwest of Iran. The results indicated that 75% of samples were contaminated by four aflatoxins and the level of AFB₁ and sum of aflatoxins were higher than the permissible maximum levels in Iran (5 and 20 µg kg^?1, respectively) in all feed samples. A positive correlation was found between four types of aflatoxins in all the tested samples (p < 0.01) and the positive correlation between AFG₁ and AFG₂ was significant (r² = 0.708). All feed samples had lead concentrations lower than the maximum EU limit, while 5%, 17% and 42.5% of feed samples had As, Cd and Hg concentrations higher than the maximum limits, respectively.