Detecting mycotoxins in agricultural commodities

It is difficult to obtain precise and accurate estimates of the true mycotoxin concentration of a bulk lot when using a mycotoxin-sampling plan that measures the concentration in a small portion of the bulk lot. A mycotoxin-sampling plan is defined by a mycotoxin test procedure and a defined accept/reject limit. A mycotoxin test procedure is a complicated process and generally consists of several steps: (a) a sample is taken from the lot, (b) the sample is ground (comminuted) in a mill to reduce particle size, (c) a subsample is removed from the comminuted sample, and (d) the mycotoxin is extracted from the comminuted subsample and quantified. Even when using accepted test procedures, there is variability associated with each step of the mycotoxin test procedure. Because of this variability, the true mycotoxin concentration in the lot cannot be determined with 100% certainty by measuring the mycotoxin concentration in a sample taken from the lot. The variability for each step of the mycotoxin test procedure, as measured by the variance statistic, is shown to increase with mycotoxin concentration. Sampling is usually the largest source of variability associated with the mycotoxin test procedure. Sampling variability is large because a small percentage of kernels are contaminated and the level of contamination on a single seed can be very large. Methods to reduce sampling, sample preparation, and analytical variability are discussed.     

Natural occurrence of mycotoxins in cereals

Besides peanuts and cottonseed, cereal grains are the most important feed and food source that occasionally are naturally contaminated with mycotoxins. The problem of mycotoxins occurring naturally in cereals, especially in corn, has become trouble-some because of changing agricultural technology. The mycotoxin problem in cereals is not restricted to any geographic or climatic region. Toxins are produced on cereals, both in the field and in storage; they involve both the grain and the whole plant. The genera of fungi most involved areAspergillus, Fusarium, Penicillium andClaviceps. Mycotoxins known to occur naturally in cereals include aflatoxins B₁, B₂, G₁ and G₂-as well as aflatoxins M₁ and M₂-ochratoxins A and B, penicillic acid, patulin, ergot, zearalenone, citrinin, T-2, tenuazonic acid, kojic acid and sterigmatocystin. Of these mycotoxins, aflatoxins, patulin, penicillic acid and sterigmatocystin are carcinogens.     

Fungi associated with food and feed commodities from Ecuador

Freshly harvested soybean, rice and corn from farms and corn-based pelleted feeds were collected from ranches from the coastal and mountain regions in Ecuador during 1998, and assessed for fungal contamination. The most prevalent fungi on pelleted feed were Aspergillus flavus and Fusarium graminearum. The prevalent fungi recovered from soybean were F. verticillioides, F. semitectum, Aspergillus flavus and A. ochraceus. In rice, F. oxysporum was the most prevalent toxigenic fungal species recorded, followed by F. verticillioides and A. flavus. In corn, F. verticillioides was the most prevalent fungus isolated in both the coastal and mountain regions, with high isolation frequencies of A. flavus and A. parasiticus at the coast. Based on the toxigenic species recovered, ochratoxin A may pose a contamination risk for soybean. A higher probability of aflatoxin contamination of corn was found in the coastal samples compared to those of the mountain region, while a risk of fumonisin contamination of corn exists in both regions.This revised version was published online in October 2005 with corrections to the Cover Date.     

Natural occurrence of mycotoxins in different spices in Egypt

A total of 120 different samples belonging to 24 kinds of spices collected from different places atAssiut Governorate (Egypt) were examined for the natural occurrence of mycotoxins. TLC analysis of spice extracts revealed the presence of aflatoxins (8–35 μg/kg) in 16 samples of anise, black pepper, caraway, black cumin, fennel, peppermint, coriander and marjoram, sterigmatocystin (10–23 μg/kg) in ten samples of red pepper, caraway, cumin and marjoram and citrinin (8–12 ⧎g/kg) in two samples of black cumin, while ochratoxin A and zearalenone could not be detected.     

Mycoflora and mycotoxins natural occurrence in corn from entre rios province, Argentina

Corn samples were collected in 1999 from three departments of Entre Réos province, Argentina, and were surveyed for mould contamination and natural occurrence ofFusarium mycotoxins, ochratoxin A and aflatoxins.Fusarium verticillioides was the most prevalent fungal species recorded at all departments. Zearalenone, deoxynivalenol and ochratoxin A were not found in any samples. Only one of the 52 corn samples analysed was contaminated with aflatoxin B₁ (17 μg/kg). Fumonisin B₁ was found in 58 % of samples (range of positive samples: 47– 3,347 μg/kg), fumonisin B₂ in 33.0 % (range of positive samples: 23–537 μg/kg) and fumonisin B₃ in 25.0 % (range of positive samples: 24–287 μg/kg) of them. This is the first report on the natural occurrence of mycotoxins in corn from Entre Ríos province, Argentina. Levels of fumonisins were lower than detected in other Argentinian provinces.     

A survey on the occurrence of mycotoxins in wheat and maize from western Romania

Samples of wheat (n = 25) and maize (n = 30) for animal consumption, collected in 1997 after harvest from western Romania, were analyzed by enzyme immunoassays for mycotoxin contamination. Toxins analyses included deoxynivalenol (DON), 3-acetylDON, 15- acetylDON, fusarenone X (FX), T-2 Toxin (T-2), diacetoxyscirpenol (DAS), zearalenone (ZEA), fumonisin B1 (FB1), aflatoxin B1 (AFB1), ochratoxin A (OA), and citrinin (CT). DON and acetylDONs were the major contaminants in wheat (100%) and maize (46%). Median values for DON, 3-acetylDON, and 15-acetylDON were 880 μg kg-1, 66 μg kg- 1, and 150 μg kg-1 in wheat, and 890 μg kg-1, 180 μg kg-1, and 620 μg kg- 1 in maize, respectively. Additionally, 3,15-diacetylDON was detected in some samples by HPLC-EIA analysis. All samples were negative for FX (<150 μg kg-1). T-2 was found in wheat (n = 6) and maize (n = 1) at levels between 13 and 63 μg kg- 1. DAS (2.6 μg kg-1) was found in one maize sample. ZEA occurred in all wheat and in four maize samples, median values were 10 μg kg-1 and 250 μg kg-1, respectively. One maize sample contained FB1 (140 μg kg-1). All samples were AFB1-negative (<4 μg kg-1). OA was found in one wheat sample (37 μg kg- 1), CT was found in one maize sample (580 μg kg- 1). This first reported natural occurrence of a range of mycotoxins in Romanian feeding stuff shows that DON and acetyl DONs may be present at levels which may affect animal production. This revised version was published online in June 2006 with corrections to the Cover Date.     

Natural occurrence of toxigenic fungi and mycotoxins in rice bran

Thirty four samples of rice bran, of which 9 were from raw (untreated) rice (RR) and 25 from parboiled rice (PbR) were collected from commercial rice mills in and around Madras and analysed for storage mycoflora and mycotoxins. Fungi of the Aspergillus flavus group were present in 29 of the 34 samples (8 from RR and 21 from PbR) in quantities ranging from <1–432 thousand propagules/g, though not always as the dominant mycoflora. Fungal numbers were usually higher in RR than in PbR samples.Five of the 9 RR samples and 6 of the 25 PbR samples were positive for aflatoxins. Among 29 isolates of A. flavus obtained from the bran samples, 16 isolates –6 from RR bran and 10 from PbR bran — were found to be toxigenic in vitro. Some isolates of A. candidus also seemed to produce aflatoxin and other fluorescent substances.     

Natural occurrence of mycotoxins and mycoflora of argentinien popcorn

Argentina is the first popcorn exporting country worldwide. In 1997-1998 harvest season, 40,000 ha were sown with a production of 125,000 tons; 120,000 tons of this production were exported to more than 40 countries. The objectives of this study were to isolate and to identify the fungi present in this cereal and to assess the occurrence of mycotoxins in freshly harvested popcorn in Buenos Aires province, Argentina, in 1999. All popcorn samples showed fungal contamination. A total of 4,211 isolates were recovered from popcorn kernel samples. The prevalent species isolated wasFusarium verticillioides followed byPenicillium funiculosum, F. graminearum andAlternaria alternata. No aflatoxins, zearalenone, deoxynivalenol or ochratoxin A were detected in the 42 popcorn simples. All samples but one were contaminated with fumonisins (FB) in a range in ug/kg of (not detected-529) for FB₁; (not detected-216) for FB₂ and (not detected-103) for FB₃. Fumonisin contamination levels in Argentinean popcorn were lower than observed in flint corn. No significant differences in fungal and fumonisins contamination levels were observed in the different tested hybrids.     

Natural occurrence of Fusarium mycotoxins in field samples from the 1992 Wisconsin corn crop.

Analysis of 98 moldy corn samples collected in Wisconsin between November 1992 and January 1993 for Fusarium toxins by various immunochemical assays revealed overall average mycotoxin concentrations of 305.6, 237.7, and 904.3 ng/g for type A trichothecenes (TCTCs), deoxynivalenol (DON)-related type B TCTCs (total DON), and zearalenone (ZE), respectively. A small portion (5.1%) of the samples was found to be contaminated with high levels ( > 1 microgram/g) of type A TCTCs and total DON during the whole survey. Over 40% of the samples had 100 to 1,000 ng of total DON per g, while 17% of the samples had the same levels of type A TCTCs. The analytical data were consistent with those from mycological examinations for the samples in which various toxic Fusarium spp., including F. sporotrichioides, F. poae, and F. graminearum, were found. The samples received in November 1992 had relatively low concentrations of toxin; the average levels of type A TCTCs and total DON were 9.9 and 79 ng/g, respectively. The toxin concentrations became progressively higher in the samples received in December. The average levels for the type A TCTCs and total DON increased to 920 and 335 ng/g, respectively. However, the levels of ZE were higher in the samples collected earlier. The average levels for samples collected in November and late December were 1,195 and 242 ng/g, respectively. Analysis of selected samples by high-performance liquid chromatography monitoring with an enzyme-linked immunosorbent assay revealed that T-2 toxin, HT-2 toxin, diacetoxyscirpenol, neosolaniol, and T-2 tetraol (T-2-4ol) were common in these samples. Statistical analysis revealed a weak correlation between the levels of total type A TCTCs and total DON in the samples (r = 0.18, P = 0.09), but a strong correlation between the levels of ZE and total type B TCTCs (r = 0.75, P < 0.0001) was found. The mycotoxin levels of total type A TCTCs, total DON-related type B TCTCs, and ZE in the cobs (5.2, 3.9, and 21 micrograms/g, respectively) were considerably higher than those in the kernels (1.0, 0.5, and 0.5 microgram/g, respectively). The type A toxin levels increased from a range of 14 to 35 ng/g to a range of 110 to 538 ng/g after the moldy corn samples were held at 5 degrees C for 8 days in the laboratory.     

Fumonisin mycotoxins in human hair

This study shows for the first time the accumulation of fumonisin mycotoxins in human hair of population clusters exposed to contaminated maize, and thus the feasibility of human hair analysis for the assessment of past fumonisin exposure. Composite hair samples were obtained from the Bizana, Butterworth and Centane districts within the Transkei region of the Eastern Cape Province of South Africa. Following methanol extraction and strong anion exchange clean up, the fumonisins FB₁, FB₂ and FB₃ were detected using high performance liquid chromatography coupled to electrospray ionization-mass spectrometry (HPLC-ESI-MS). Hair from Centane and Butterworth showed mean levels of FB₁ of 26.7 and 23.5 μg kg^?1 hair, respectively. FB₂ was only detected in hair from Centane and in one sampling point in Butterworth, with mean levels of 6.5 and 5.7 μg kg^?1 hair, respectively. Hair samples from Bizana, on the other hand, were found to contain higher levels of FB 1 (mean 33.0 μg kg^?1 hair) and FB 2 (mean 11.1 μg kg^?1 hair). No samples contained more than trace levels of FB 3 . Recoveries from spiked hair samples using this method ranged from 81% to 101%, demonstrating the applicability of hair analysis in assessing human exposure to fumonisin mycotoxins.     

Fumonisin mycotoxins in human hair

This study shows for the first time the accumulation of fumonisin mycotoxins in human hair of population clusters exposed to contaminated maize, and thus the feasibility of human hair analysis for the assessment of past fumonisin exposure. Composite hair samples were obtained from the Bizana, Butterworth and Centane districts within the Transkei region of the Eastern Cape Province of South Africa. Following methanol extraction and strong anion exchange clean up, the fumonisins FB₁, FB₂ and FB₃ were detected using high performance liquid chromatography coupled to electrospray ionization-mass spectrometry (HPLC-ESI-MS). Hair from Centane and Butterworth showed mean levels of FB₁ of 26.7 and 23.5 μg kg^-1 hair, respectively. FB₂ was only detected in hair from Centane and in one sampling point in Butterworth, with mean levels of 6.5 and 5.7 μg kg^-1 hair, respectively. Hair samples from Bizana, on the other hand, were found to contain higher levels of FB 1 (mean 33.0 μg kg^-1 hair) and FB 2 (mean 11.1 μg kg^-1 hair). No samples contained more than trace levels of FB 3 . Recoveries from spiked hair samples using this method ranged from 81% to 101%, demonstrating the applicability of hair analysis in assessing human exposure to fumonisin mycotoxins.     

Natural occurrence ofAlternaria mycotoxins in the grain and chaff of cereals

Samples of wheat and rye heads with evident black discoloration were collected in 1986 and 1987. Four fungal genera colonized such heads:Alternaria, Cladosporium, Drechslera, andEpicoccum. In chaff of wheat (19%) and rye (10%) samples alternariol was present in amounts up to 1.8mg/kg, and alternariol methyl ether up to 0.51 mg/kg. In 1 out of 21 wheat samples alternariol was also present in kernels (0.59mg/kg).     

Simultaneous occurrence of fumonisin B1 and other mycotoxins in moldy corn collected from the People's Republic of China in regions with high incidences of esophageal cancer.

A total of 31 corn samples collected from households in the counties of Cixian and Linxian of the People's Republic of China, where high incidences of esophageal cancer have been reported, were analyzed for fumonisin B1 (FB1), aflatoxin, and total trichothecene mycotoxins. High levels of FB1 (18 to 155 ppm; mean, 74 ppm) were found in 16 of the samples that showed heavy mold contamination. FB1, at lower levels (20 to 60 ppm; mean, 35.3 ppm), was also found in 15 samples, collected from the same households, that did not show any visible mold contamination. The levels of aflatoxin in the samples were low (1 to 38.4 ppb; mean, 8.61 ppb). High levels of total type-A trichothecenes were also found in the moldy corn samples (139 to 2,030 ppb; mean, 627 ppb). Immunochromatography of selected samples revealed that these samples contained T-2 toxin, HT-2 toxin, iso-neosolaniol, monoacetoxyscirpenol, and several other type-A trichothecenes. The concentration of total type-B trichothecenes in 15 moldy corn samples was in the range of 470 to 5,826 ppb (mean, 2,359 ppb). High levels (3.7 to 5.0 mg/g) of FB1 were produced in corn in the laboratory by five Fusarium moniliforme strains isolated from the moldy corn. These fungi were also capable of forming various nitrosamines (5 to 16 micrograms per flask) in the presence of nitrate and precursor amines.(ABSTRACT TRUNCATED AT 250 WORDS)     

First survey on the natural occurrence of Fusarium mycotoxins in Bulgarian wheat

Wheat for human consumption (140 samples) was collected after harvest from all regions of Bulgaria. The 1995 crop year was characterized by heavy rainfall in the spring and summer months. The internal mycoflora of wheat samples was dominated by Fusarium spp. and Alternaria spp., and storage fungi were rarely present. The samples were analysed for contamination with Fusarium mycotoxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), T-2 Toxin (T-2), diacetoxyscirpenol (DAS), and zearalenone (ZEA), using enzyme immunoassay methods. DON and ZEA were the predominant toxins, with a contamination frequency of 67% and 69%, respectively. The average levels of these toxins in positive samples were 180 g/kg (DON) and 17 g/kg (ZEA), maximum concentrations were 1800 g kg^–1 and 120 g kg^–1, respectively. Acetyl derivatives of DON, namely 3-AcDON and 15-AcDON, were found in 2.1 % and 0.7% of the samples, at at maximum level of about 100 g kg^–1. Only one sample was positive for T-2 (55 g/kg), DAS was not detected. This is the first report about the natural occurrence of a range of Fusarium mycotoxins in wheat for human consumption in Bulgaria.Abbreviations 3-AcDON 3-acetyldeoxynivalenol - 15-AcDON 15-acetyldeoxynivalenol - DAS diacetoxyscirpenol - DON deoxynivalenol - EIA enzyme immunoassay - T-2 T-2 toxin - ZEA zearalenone     

Natural occurrence of aflatoxin andAlternaria mycotoxins in oilseed rape from Catalonia (Spain): Incidence of toxigenic strains

During an investigation of the mycoflora on oilseed rape, the predominant fungal species present in 20 samples collected from Catalonia (Spain) wereAlternaria alternata (Fries) Keissler,Penicillium spp. andAspergillus flavus. None of the 20 samples analyzed presented contamination byAlternaria mycotoxins (tenuazonic acid, alternariol, alternariol methyl ether, altertoxin I and altertoxin II). Only aflatoxin B₁ was detected in 1 of the 20 samples analyzed, with a concentration of 0.25 ppb. Of the 40Aspergillus flavus strains isolated from oilseed rape samples, only 3 revealed aflatoxigenic capacity. None of thePenicillium spp. isolated from oilseed rape samples revealed mycotoxigenic capacity (citreoviridin, griseofulvin, citrinin, patulin and penicillic acid).     

Mycoflora and mycotoxins of peanut (Arachis hypogaea L.) seeds in Egypt. 1-sugar fungi and natural occurrence of mycotoxins

Sixty-four species and 2 varieties which belong to nineteen genera of fungi were identified from 40 peanut seed samples collected from different places in Egypt by using a dilution-plate method on glucose-Czapek's medium. The most frequent genera were Aspergillus (21 species & 2 varieties), Penicillium (16 species) and Fusarium (6 species). A. flavus, A. fumigatus, A. niger, P. chrysogenum and F. oxysporum were the most common fungal species. Forty seven percent of the samples proved to be toxic to brine shrimp (Artemia salina) larvae. Thin-layer Chromatographic analysis revealed that peanuts were contaminated by aflatoxins (11 samples), thrichothecene-toxins (10 samples), zearalenone (one sample) and citrinin (one sample). We believe that this is the first report of the natural occurrence of zearalenone and trichothecene-toxins in peanuts.     

Simultaneous and rapid detection of six different mycotoxins using an immunochip

Mycotoxins are highly toxic contaminants in food, animal feed, and commodities. The study has developed an immunochip for quantifying the concentrations of six mycotoxins: aflatoxin B1, aflatoxin M1, deoxynivalenol, ochratoxin A, T-2 toxin, and zearalenone, which were added to drinking water. The complete antigens (Ags) of the mycotoxins were contact printed and immobilized onto agarose-modified glass slides with 12 physically isolated subarrays, based on the reaction of both diffusion and covalent bond. The optimal concentration of each antigen and antibody (Ab) was obtained using an Ag-Ab immunoassay. Based on the indirect competitive immunoassay for the simultaneous detection of six mycotoxins in one single chip, six standard curves with good logistic correlation (R(2)>0.97) were respectively plotted. The working ranges (0.04-1.69, 0.45-3.90, 20.20-69.23, 35.68-363.18, 0.11-1.81, and 0.08-7.47 ng/mL, respectively) were calculated, as well as the median inhibitory concentrations (0.31±0.04, 1.49±0.21, 34.54±1.30, 134.06±11.75, 0.49±0.05, and 1.54±0.22 ng/mL, respectively), when six mycotoxins were detected simultaneously. Finally, the recovery rates in drinking water generally ranged from 80% to 120% on the same chip, with an intra-assay coefficient of variation lower than 15%. We successfully established an immunochip for simultaneous detection of six mycotoxins within 4h, with advantages of using minimal samples and being visually semiquantitative with our naked eyes. In summary, the method could be developed on one single chip for detecting multiple contaminants in actual samples.     

Natural occurrence of mycotoxins other than aflatoxin in Africa,Asia and South America

This is a compilation of data on the occurrence of mycotoxins other than aflatoxin in foods and feeds from Africa, Asia and South America with 75 references.