Genotoxicity testing of extracts from aflatoxin-contaminated peanut meal, following chemical decontamination.

One of the most important concerns in the decontamination of aflatoxin-containing feed commodities is the safety of the products for food-producing animals and for human consumption of products derived from these animals. A new method, based on the use of florisil and C18 solid phase extraction columns, was developed for the preparation of extracts from decontaminated peanut meal, which allowed testing with in vitro genotoxicity assays without interference of the residual aflatoxin B1. Recovery of degradation products in the extracts was evaluated by the use of radiolabelled [14C]-aflatoxin B1 (AFB1) added to naturally-contaminated peanut meal (3.5 mg AFB1/kg). The meal was treated by a small-scale version of an industrial decontamination process based on ammoniation. Following decontamination, more than 90% of the label could not be extracted from the meal. AFB1 accounted for about 10% of the radiolabel present in the extractable fraction, indicating a total AFB1 reduction of more than 99%. Decontamination of the meal by a number of other small- and industrial-scale ammonia-based processes resulted in similar efficiencies. Application of the extraction procedure resulted in AFB1-rich and AFB1-poor fractions, the latter containing half of the extractable decontamination products but less than 1% of the residual AFB1. Testing in the Salmonella/microsome mutagenicity assay (TA 100, with S9-mix) of the original crude extracts and AFB1-rich fractions prepared from non-treated and decontaminated meal, showed the positive results expected from the AFB1 contents as determined by HPLC analysis. Analysis and testing of the AFB1-poor fractions showed that the various decontamination processes not only resulted in a successful degradation of AFB1 but also did not produce other potent mutagenic compounds. Slight positive results obtained with these extracts were similar for the untreated and treated meals and may be due to unknown compounds originally present in the meal. Results obtained with an unscheduled DNA synthesis (UDS) and Comet assay with rat hepatocytes supported this conclusion. Positive results obtained with the micronucleus assay, using immortalized mouse hepatocytes (GKB), did not clearly reflect the mycotoxin levels and require further examination. It is concluded that the newly developed extraction procedure yields highly reproducible fractions and hence is very suitable for examining the possible formation of less potent degradation products of aflatoxins in short-term genotoxicity tests.

Differences detected in vivo between samples of aflatoxin-contaminated peanut meal, following decontamination by two ammonia-based processes.

A sample of peanut meal, highly contaminated with aflatoxins, has been subjected to decontamination by two commercial ammonia-based processes. The original contaminated and the two decontaminated meals were fed to rats for 90 days. No lesions associated with aflatoxin-induced hepatocarcinogenesis were detected histologically following feeding with the two detoxified meals. There were, however, clear differences between the two meals in respect of growth rates of the rats. In addition, feeding one of the detoxified meals resulted in hepatic abnormalities detected using novel immunohistochemical reagents. Differences between the two detoxified meals were also indicated by the results of studies using meals 'spiked' with [14C]-aflatoxin B1 prior to being subjected to the detoxification processes. The meals differed in the bioavailability of the label. It was concluded that peanut meal where an initial, unacceptable level of contamination with aflatoxins had been reduced by two ammonia-based processes to comparable, acceptable levels, may still have different effects in vivo when incorporated into animal diets.

Absorption, distribution and excretion of aflatoxin-derived ammoniation products in lactating cows.

Peanut meal naturally contaminated with 3.5 mg/kg aflatoxin B1 (AFB1) was spiked with radiolabelled AFB1 (meal 14C-I0) and decontaminated by a small-scale copy of an industrial ammoniation process (meal 14C-I1). During the process 15% of the radioactivity was lost, whereas 90% of the remaining radiolabel could not be extracted from the meal. In the extractable part, AFB1 accounted for 10% of the radiolabel, consistent with a total AFB1 reduction of more than 99%. No degradation products were observed in the extracts. Four lactating cows were fed with a diet containing 15% of either meal 14C-I0 or 14C-I1 for 10 days. On day 9 of this treatment, respectively 23 and 67% of the radiolabel was excreted in the urine and faeces of cows fed meal 14C-I0, as compared with 2 and 101% in the case of cows fed meal 14C-I1. Milk contained respectively 1.35 (meal 14C-I0) and 0.25% (meal 14C-I1) of the radiolabel. Milk samples taken during the equilibrium stage contained respectively 5 and 0.5 ng/ml of AFB1-derived compounds. Aflatoxin M1 (AFM1) accounted for 50-80% of these compounds in the case of milk from cows fed 14C-I0, as compared with 6-20% in the case of 14C-I1. AFB1 to AFM1 carry-over rates for 14C-I0 or 14C-I1 were estimated to be respectively 0.5 and 5.9%. Only liver and kidney samples contained detectable levels of the radiolabel, being respectively 260 and 37 micrograms/kg for cows fed meal 14C-I0, and 10 and 3 micrograms/kg for those fed meal 14C-I1. In the latter case, more than 55% of the radiolabel in the liver could not be extracted, as compared with 90% in the group fed meal 14C-I1. A small part of the extractable radiolabel in the livers of cows fed meal 14C-I0 could be attributed to AFB1 and AFM1 (less than 1% of total radioactivity). In the case of the animals fed 14C-I1 there were indications for the presence of AFB1 and AFM1 (6% of total radioactivity). Decontamination of the highly contaminated (non-radiolabelled) peanut meal by two different industrial ammoniation processes, resulted in a similar reduction of the initial AFB1 levels of 3.5 mg/kg to 15 micrograms/kg. Feeding of diets containing 15% of the non-treated and two treated peanut meals to cows for a period of 10 days, resulted in AFM1 levels in milk of respectively 2.1, 0.04 and 0.07 ng/ml. AFB1 to AFM1 carry-over rates were calculated to be respectively 0.5, 2.0, and 3.6%. It is concluded that the efficient reduction of aflatoxin levels by ammoniation of contaminated peanut meal results in a strong reduction of aflatoxin-related residues in milk and meat of cows, most likely caused by a decreased bioavailability of the degradation products.

Sampling plans for the determination of aflatoxin B1 in large shipments of animal feedstuffs.

Incremental samples (50, 100, and 500 g) were systematically collected from large shipments of copra meal pellets, copra cake, and palm kernel cake to study the distribution of aflatoxin B1 and evaluate adherence of distribution to the model, CV(2)is (EQ) = A + B/Mis (where CVis = coefficient of variation of the true concentration of aflatoxin B1 within the incremental samples; Mis = mass of the incremental samples; and A and B are constants). Also evaluated was the distribution of aflatoxin B1 among 1 kg composite samples, produced both by random combination of existing incremental samples and by collection of 1 kg composite samples (composed of 10 x 100 g increments) from additional batches of copra meal pellets and cottonseed cake. The efficiency of selected sample preparation (grinding and subdivision) procedures was compared, culminating in the development and description of a variety of sampling plans. The coefficient of variation (CV) among incremental samples varied from 0 to 38%, and was independent of incremental sample size. No significant difference (F-test, 5% significance level) was found between the efficacy of 4 sample preparation methods when these methods were applied to the commodities described above. Various sampling plans were evaluated with estimated CVs from 4.0 to 12.5%, for the aflatoxin B1 content of the composite samples.

Uptake of aflatoxin B1 and T-2 toxin by two mycotoxin bioassay microorganisms: Kluyveromyces marxianus and Bacillus megaterium.

Uptake of aflatoxin B1 (AFB1) and trichothecene T-2 toxin from growth medium by mycotoxin bioassay strains of Klutyveomyces marxianus and Bacillus megaterium was assessed by incubating, washing, and sonicating the cells, extracting samples with chloroform, and analysing the extracts by a combination of high-performance thin-layer chromatography (HPTLC) and fluorescence densitometry. Using cultures of K. marxianus, the entire AFB1 dose was recovered and no AFB1 metabolites were detected. Less than 1% of the AFB1 was recovered from the cells, and AFB1 did not inhibit growth. Methanol in the incubation medium had no significant effect on the levels of AFB1 associated with K. marxianus cells. The entire dose of T-2 toxin was also recovered from K. marxianus cultures, and no metabolites were detected; again, less than 1% of T-2 toxin was cell-associated, but growth was completely inhibited. AFB1 partially inhibited the growth of B. megaterium; approximately 12% of the dose could not be recovered, and no AFB1-related metabolites were detected. Methanol increased the levels of recoverable AFB1 associated with B. megaterium cells. In the case of T-2 toxin, around 8% of the dose was not recovered, and no metabolites were detected; growth of B. megaterium was stimulated. These results suggest irreversible binding of both toxins, or derivatives of them, to the cells of B. megaterium.

A colorimetric technique for detecting trichothecenes and assessing relative potencies.

We tested a novel colorimetric toxicity test, based on inhibition of beta-galactosidase activity in the yeast Kluyveromyces marxianus, for sensitivity to a range of mycotoxins. A variety of trichothecene mycotoxins could be detected. The order of toxicity established with this bioassay was verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin > acetyl T-2 toxin > neosolaniol > fusarenon X > T-2 triol > scirpentriol > nivalenol > deoxynivalenol > T-2 tetraol. The sensitivity of detection was high, with the most potent trichothecene tested, verrucarin A, having a 50% effective concentration (concentration of toxin causing 50% inhibition) of 2 ng/ml. Other mycotoxins (cyclopiazonic acid, fumonisin B1, ochratoxin A, patulin, sterigmatocystin, tenuazonic acid, and zearalenone) could not be detected at up to 10 micrograms/ml, nor could aflatoxins B1 and M1 be detected at concentrations up to 25 micrograms/ml. This test should be useful for trichothecene detection and for studies of relevant interactions-both between trichothecenes themselves and between trichothecenes and other food constituents.

Determination of aflatoxins in groundnut meal by high-performance liquid chromatography: a comparison of two methods of derivatisation of aflatoxin B1.

A comparison has been made between pre-column and post-column derivatisation of aflatoxin B1 (AFB1) during estimation by high-performance liquid chromatography of this toxin in groundnut meal. The effect of the use of different derivatisation reagents on the quantification of aflatoxin B2 (AFB2) has also been evaluated. Both AFB1 and AFB2 were analysed at eight levels of artificial contamination. Five replicate analyses were carried out at each level on both groundnut meal extract (acetone-water, 85:15, v/v) and extraction solvent alone. A statistical evaluation of the results gave limits of detection of 1.1 and 0.3 micrograms/kg for AFB1 and AFB2 respectively, using pre-column derivatisation compared with 1.5 and 0.8 micrograms/kg for the post-column method. Recoveries of over 90% from the spiked groundnut meal extracts were achieved for both derivatisation methods.

Design of sampling plans for mycotoxins in foods and feeds.

The control of the occurrence of mycotoxins in foods and feeds requires effective surveillance and quality control procedures which facilitate the identification and control of the mycotoxin problem respectively. Surveillance and quality control procedures involve a sequence of sampling, sample preparation, and analysis steps; and the integrity of the data produced by these procedures will be determined by the effectiveness of these steps. It is imperative that the sampling step is performed as accurately as possible so that the sample collected is representative of the batch of food or feed under investigation. Needless to say, the collection of a biased sample will completely invalidate the resultant analytical data. Most attempts to develop effective sampling protocols have focused upon the aflatoxins, since the majority of current regulations are concerned specifically with this group of mycotoxins. However, the design of effective sampling protocols has been severely hindered by the highly skewed distribution of the aflatoxins in foods and feeds. Studies already performed indicate that representative samples of commodities, composed of large particles (e.g., corn and oilseed kernels) should be 10 kg in weight, at least, and composed of approximately one hundred incremental samples. Similar studies have indicated that samples of oilseed cakes and meal, however, should be composed of fifty incremental samples which afford a composite sample of approximately 5 kg in weight.

Development and evaluation of analytical methodology for the determination of aflatoxins in palm kernels.

A rapid, simple and reproducible method for the simultaneous estimation of aflatoxins AFB1, AFB2, AFG1 and AFG2 in palm kernel samples has been developed by optimizing the sample preparation, solvent extraction, sample clean-up and quantification procedures. The aflatoxins are extracted from a slurried palm kernel sample with an acetone-water (80 + 20, v/v) mixture and the crude extract is cleaned up by solid-phase extraction using a phenyl bonded phase cartridge. The extract is passed through the cartridge with a water-methanol (93 + 7) mixture. Subsequent elution of the aflatoxins retained on the cartridge is achieved with a 3 ml aliquot of chloroform. The aflatoxin content of the eluate is quantified using a bi-directional high-performance thin-layer chromatography procedure. A critical evaluation of the proposed method was carried out by statistical comparison with the British Standard Method. The proposed procedure was shown to be more efficient and precise. Consistent recoveries of over 90% were achieved from spiked palm kernel extracts and detection limits were found to be 3.7, 2.5, 3.0 and 1.3 micrograms kg-1 for AFB1, AFB2, AFG1 and AFG2 aflatoxins, respectively.

Mycotoxins in food.

The presence of mycotoxins in a wide range of foodstuffs can lead to many different toxic conditions in both man and domestic animals. The major fungi responsible for producing these toxins are species of Aspergillus, Penicillium, Fusarium and Alternaria, although other genera are involved as well, for example Claviceps, Diplodia and Arthrinium. An overview is given of the major mycotoxins responsible for illnesses following ingestion of contaminated foods, with particular emphasis on the effects produced in humans. Compounds discussed include the aflatoxins, cyclopiazonic acid, tenuazonic acid, the trichothecenes, zearalenone, wortmannin, fumonisins B1 and B2, patulin, ochratoxin A, diplodiatoxin and diplosporin.

Analytical methodology for the determination of aflatoxins in peanut butter: comparison of high-performance thin-layer chromatographic, enzyme-linked immunosorbent assay and high-performance liquid chromatographic methods.

High-performance thin-layer chromatography (HPTLC) was applied to the separation and quantification of aflatoxin in 300 jars of "crunchy" peanut butter. A critical evaluation of the proposed HPTLC method has been carried out by statistical comparisons with a commercially available enzyme-linked immunosorbent assay (ELISA) kit and a high-performance liquid chromatographic (HPLC) method. The statistical tests indicated that whilst the distributions of the data sets obtained with each method were similar, the HPLC method was found to be biased. Over-all results indicated that the HPTLC method gave more consistent data, relatively lower standard deviations and lower coefficients of variation. The ELISA kit was found to be less precise than the HPTLC and HPLC methods and prone to some loss of sensitivity caused by matrix interference.

Biogenic sulfur and the acidity of rainfall in remote areas of Canada.

Sulfur released from any given natural or anthropogenic source carries an isotopic signature that can be used to trace its flow through the environment. Measurements of the concentration and isotopic composition of sulfur in weekly bulk precipitation samples collected over a 4-year period at a remote location in northern Ontario were recorded. The long-term isotopic data and the measurement on the production and release of dimethyl sulfide from boreal wetlands show that biogenic sources can account for up to 30 percent of the acidifying sulfur burden in the atmosphere in remote areas of Canada. The data suggest that significant biological reemission of anthropogenic sulfur is occurring. The role of this process in the continuing acidification of the environment for years to come must be a matter of concern.