Presence of aflatoxin M1 in white and hard cheese samples from Serbia

The level of aflatoxin M1 (AFM1) in 54 samples of white and hard type of cheese produced in Serbia was investigated using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Two methodologies were developed for sample extraction, separately for white and hard type of cheese. The first one involved sample extraction with mixture of acetonitrile/water (86:14, v/v) for white cheese without sample clean-up step. The second method consisted of a sample extraction with dichloromethane/acetone for hard cheese followed by solid-phase extraction (SPE) clean-up by SPE C₁₈ cartridges. The average recoveries of AFM1 from spiked samples at levels of 0.25–0.50 μg/kg, were 73–111% and the precision (RSD) ranged from 7 to 9% with the first method, whilst the average recoveries from samples spiked at levels of 0.125–0.50 μg/kg were 71–80%, with RSD ranged from 4 to 10% for the second method. Due to different matrix effects, the quantification limits were 0.125 and 0.02 μg/kg for white and hard type of cheeses, respectively.Seven samples (13%) exceeded the maximum acceptable level of 0.25 μg/kg has been established for AFM1 in some European countries like Switzerland, France, Austria and Turkey.     

Occurrence and estimation of aflatoxin M1 exposure in milk in Serbia

The occurrence of AFM1 was investigated in 150 cow's, 10 goat's, 5 donkey's, 10 breasts milk and 1 infant formula samples. Analyses were done using Enzyme Linked Immunosorbent Assay (ELISA) method. AFM1 was detected in 98.7% of analyzed cow's milk samples in concentrations ranged from 0.01 to 1.2 μg/kg. Further, even 129 (86.0%) cow's milk samples contained AFM1 in concentration greater than maximum residue levels (MRL) of 0.05 μg/kg defined by European Union (EU) Regulation. Analysis of other types of milk showed that AFM1 was detected in 80.0% goat's, 60.0% donkey's and 60.0% of breasts milk samples.Although Serbian Regulation for MRL of AFM1 in milk has been changed and harmonized with EU Regulation in 2011, occurrence of AFM1 in milk in Serbia during 2013 resulted in Regulation changes, and MRL were changed from 0.05 to 0.5 μg/kg.On the basis of the obtained concentrations of the AFM1 in cow's milk, collected information about average milk intake and mean body weight (bw) for different age's categories, mean ingestion of AFM1 in ng/kg per bw per day were estimated. Obtained results showed that all age's categories, especially children, are exposed with high risk related to presence of AFM1 in milk.There are only a few published data about occurrence of AFM1 in milk in Serbia and none about intake assessment for AFM1.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Levels of aflatoxin M1 in milk,cheese consumed in Kuwait and occurrence of total aflatoxin in local and imported animal feed

A total of 321 milk samples (177 fresh, 105 long-life and 27 powdered milk, and 12 human milk), 40 cheese samples and 84 feed samples were analyzed for aflatoxin M1 (AFM1) and total aflatoxin. The samples were collected randomly during January 2005–March 2007, from Kuwaiti markets. The method used was ELISA technique. Results showed that all fresh milk samples except one were contaminated with AFM1 ranging from 4.9 to 68.7 ng/kg. Eight samples exceeded the EC’s regulatory limit. For the long-life milk samples, the ranges of AFM1 were from below the detection limit to 88.8 ng/kg, with four samples above the action limit of the EC. In the powdered milk samples, AFM1 ranged from 2.04 to 4.14 ng/kg. Of the human milk samples, only five were contaminated, with AFM1 levels ranging from 8.83 to 15.2 ng/kg with a mean 9.7 ng/kg. The cheese samples recorded 80% contamination with AFM1 with a range 23.8–452 and mean of 87.6 ng/kg, with one sample being above the regulatory limit (250 ng/kg) while the feed samples, showed 79.8% contamination with total aflatoxin.     

Estimate of aflatoxin M1 exposure in milk and occurrence in Brazil

The presence of aflatoxin M₁ (AFM₁) was investigated in 125 samples of powdered milk, pasteurized milk and ultra high treated (UHT) milk in the city of São Paulo, and estimates of AFM₁ intake were assessed. The samples were analysed using an immunoaffinity column for cleanup and a HPLC-FLD for determining AFM₁. The quantification limit was 10 ng/kg. AFM₁ was found in 119 (95.2%) at levels ranging from 10 to 200 ng/kg with mean concentration of 31 ng/kg. The average daily intake estimated for AFM₁ was 1 ng/kg bw per day for children and 0.188 ng/kg bw per day for adults.     

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.     

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.     

Assessment of aflatoxin contamination in raw milk and feed in Macedonia during 2013

This work presents the first insight on the aflatoxin M₁ (AFM₁) occurrence in raw milk collected from Macedonia, during the mycotoxin incidence in 2013. The total tested samples number was 3635, collected from February 2013 to January 2014. They were analyzed for AFM₁ with ELISA as screening, and HPLC – fluorescence detection as confirmatory method. The AFM₁ concentrations exceeded the maximum permitted levels in 2.9% of the samples, and the highest detected concentration was 408.1 ng/kg. The follow-up actions including feed sampling revealed aflatoxin B₁ contamination in 31.8% of the samples tested, applying the HPLC-FD method. We estimated the carry-over rate between 0.22 and 3.47%. The experience obtained from the coordinated milk-feed aflatoxin survey could contribute towards increasing awareness for establishing continuous monitoring systems.     

A survey of aflatoxin M1 of raw cow milk in China during the four seasons from 2013 to 2015

In the present study, a total of 1550 raw cow milk samples were collected from Southern, Northern, Northeast, and Western regions of China during the four seasons from 2013 to 2015. Samples were analyzed for aflatoxin M1 (AFM1) using high performance liquid chromatography (HPLC). In 2013, AFM1 was detected in 21% of 366 raw cow milk samples with levels ranging between 0.01 and 0.24 μg/L. In 11.7% of samples, AFM1 levels were >0.05 μg/L, which is the legal limit in the European Union. The mean and median of positive samples were 0.069 ± 0.052 μg/L and 0.056 μg/L, respectively. In 2014, AFM1 was detected in 28.5% of 624 raw cow milk samples, with levels ranging from 0.01 to 0.25 μg/L. Of these samples, 7.7% had AFM1 levels exceeding 0.05 μg/L, with a mean of 0.042 ± 0.039 μg/L and median of 0.028 μg/L. AFMI was detected in 14.1% of 560 raw cow milk samples in 2015, with levels ranging from 0.01 to 0.144 μg/L. In 1.8% of these samples, AFM1 levels were above 0.05 μg/L, with a mean of 0.026 ± 0.024 μg/L and median of 0.017 μg/L. Our results demonstrate that AFM1 levels of the samples did not exceed the legal limit of 0.5 μg/L in China, the United States, and Codex Alimentarius Commission. Geographically, AFM1 contamination was more predominant in raw cow milk samples from Southern China than in those from other regions, with a higher number of samples containing AFM1 levels above 0.05 μg/L in 2013, 2014, and 2015. AFM1 levels were higher in autumn than in the other seasons during the entire study period. According to our survey, AFM1 contamination has been well-controlled in China during recent years; however, some samples still exceeded the European Union (EU) legal limit. Better prevention and management of aflatoxins in both feed and milk should be considered especially in Southern regions of China and in autumn.     

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.     

Transfer of aflatoxin M1 from milk to ripened cheese in three Italian traditional production methods

Robiola and Primosale, two fresh cheeses, and Maccagno, an hard-type cheese, were produced using milk that was naturally and artificially contaminated with aflatoxin M₁ (AFM₁) at the levels of 10, 50 and 200 ng/l. Concentrations of AFM₁in milk and cheeses were determined by liquid chromatography and fluorimetric detection, coupled with immunoaffinity column extraction. In the Robiola production method, AFM₁ levels in whey ranged between 30% and 65% of the total amount of the toxin present in the milk, while Primosale and Maccagno, that share the same rennet based cheesemaking procedure, showed an higher percentage of AFM₁ partitioning to whey.For each cheese-making method, the concentration of AFM₁ on fresh matter was higher in the cheese compared to the original milk. The fresh cheeses showed a concentration factors of 1.43 and 2.20 for Primosale and Robiola, respectively, whereas the Maccagno cheese showed a value of 6.71. For all the production methods considered, when using milk not exceeding the maximum acceptable level of 0.05 μg AFM₁/kg set by EU, the resulting cheese also complied with current Italian recommendations for AFM₁ contamination (450 ng AFM₁/kg).     

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.     

A modified lateral flow immunoassay for the detection of trace aflatoxin M1 based on immunomagnetic nanobeads with different antibody concentrations

A modified lateral flow immunoassay (two-step assay) was developed to detect trace aflatoxin M1 (AFM1) in raw milk. In contrast to conventional LFIA, two kinds of immunomagnetic nanobeads (IMNBs) were used. One IMNB with high antibody concentration was used to capture AFM1 in the test sample, whereas the other IMNB with low antibody concentration was used to elucidate the results of the test. Critical factors, such as antibody concentration of IMNBs and size of IMNBs, were investigated. The two-step assay exhibited an ideal sensitivity to screen trace AFM1 in milk samples without extra sample pretreatment. The cutoff value of the naked eye was 0.02 μg/L and satisfied the European Union's maximum limit of AFM1 in raw milk, heat-treated milk, and milk used to manufacture milk-based products and even in baby foods. With the same antibody, sensitivity was enhanced approximately 25 and 50 times when compared with conventional IMNB-based LFIA and gold-based LFIA, respectively. Corresponding results of 13 raw milk samples were obtained between this two-step assay and referenced enzyme-linked immunosorbent assay.     

Seasonal prevalence level of aflatoxin M1 and its estimated daily intake in Pakistan

Aflatoxin M₁ level was measured in 520 milk samples during autumn, winter, spring and summer seasons of 2013–14 in five districts of Southern Punjab-Pakistan. Analyses were performed by using enzyme linked immuno sorbent assay (ELISA) method. Aflatoxin M₁ was found positive in 93% milk samples in the range of 0.001–0.26 μg/l while 53% samples were found to exceed the European Union maximum Limit for aflatoxins M₁ i.e. 0.05 μg/l. The seasonal prevalence level of AFM₁ was found in the order of winter > spring > autumn > summer. Comparing the aflatoxin M₁ level during different day times, morning milk was found 37–50% more contaminated than evening milk. The estimated daily intake (EDI) of aflatoxins M₁ during different seasons of year for various age groups was found in the range of 0.22–5.45 ng/kg/day. Infants were found in the highest risk group while adults were in the lowest. The results of the study indicate that people of Pakistan are at high risk of health issues related with aflatoxins M₁.