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Mycotoxins in fungal contaminated samples of animal feed from western Canada, 1982-1994.

Feed samples from 94 cases involving fungal contamination and suspected mycotoxicosis of farm animals in western Canada were examined during 1982-1994 to assess the incidence of mycotoxins. Samples were analyzed for aflatoxins, ochratoxin A, citrinin, sterigmatocystin, and the fungal estrogen zearalenone. Samples infected with Fusarium fungi were additionally assayed for nivalenol, deoxynivalenol, fusarenone-x, 15-acetyl-deoxynivalenol, diacetoxyscirpenol, HT-2 toxin, and T-2 toxin. Mycotoxins were found in 21 feed samples from 17 cases (18% of the reported cases), generally at levels far below those needed to induce symptoms under laboratory conditions. HT-2 toxin and other type-A trichothecenes were detected in 5 samples, deoxynivalenol and other type-B trichothecenes in 13, ochratoxin A in 5, and citrinin in 2. In 9 cases, symptoms observed in the animals were consistent with the known effects of the mycotoxin(s) found in the particular feed samples.

Animal Feed↗

Determination of the Fusarium mycotoxins nivalenol, deoxynivalenol, 3-acetyldeoxynivalenol, and 15-O-acetyl-4-deoxynivalenol in contaminated whole wheat flour by liquid chromatography with diode array detection and gas chromatography with electron capture detection.

A rapid and sensitive method was developed for simultaneous detection of nivalenol (NIV), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-A-DON), and 15-O-acetyl-4-deoxynivalenol (15-A-DON) in wheat flour. Samples were extracted with acetonitrile-water (84 + 16), and the extract was filtered and purified by a column containing a combination of charcoal, celite, and other adsorbents. For screening analysis, the column eluate was only extracted with ethyl acetate. After evaporation of the solvent, the dried residue was redissolved in acetonitrile-water (2 + 8) and then analyzed by reversed-phase liquid chromatography (LC) with diode array detection. Recoveries of NIV, DON, 3-A-DON, and 15-A-DON from whole wheat flour spiked at 2 levels were 49-55, 92-97, 98-100, and 100-105%, respectively. To quantitate mycotoxin amounts lower than 1 ppm, purified column extracts were evaporated to dryness, derivatized with heptafluorobutyric anhydride, and analyzed by gas chromatography with electron capture detection (GC-ECD). Average recoveries of NIV, DON, 3-A-DON, and 15-A-DON from whole wheat flour spiked at 2 levels, were 45-52, 91-103, 81-85, and 84-92%, respectively. GC-ECD detection limits for all mycotoxins tested at a signal-to-noise ratio of 4:1 were < 30 ng/g. Results of GC-ECD analysis for whole wheat flour samples spiked with mycotoxins at 3 and 10 ppm compared well with results (2.8 and 9.9 ppm) for the same samples analyzed by LC.

Chromatography, Gas↗

Mycotoxins in foods--occurrence, health & economic significance & food control measures.

Mycotoxins of importance in India include aflatoxin, fumonisins, trichothecenes, ergot alkaloids and ochratoxins. The ICMR multicentric study on the occurrence of aflatoxin contamination in risk commodities namely, maize and groundnut showed that 21 per cent of groundnut samples and 26 per cent of maize samples analysed exceeded Indian tolerance limits of 30 micrograms/kg. A study on the aflatoxin intake from maize-based diets in a rural region of Andhra Pradesh showed the intakes to be in the range of 4-100 ng/kg body wt/day. Studies on the occurrence of aflatoxin M1 in milk in the southern and western regions of India indicated levels in the range of 0.05-3.0 micrograms/l. Analysis of feed samples indicated high incidence of aflatoxin B1 contamination in the groundnut cake component. Fumonisins have been shown to occur in Indian maize and sorghum. Studies showed high levels of fumonisins in rain-affected maize and sorghum consumption of which resulted in an outbreak of fumonisin mycotoxicosis in rural regions of the Deccan Plateau. A similar disease outbreak occurred in poultry due to consumption of fumonisin contaminated feed containing rain damaged maize. Biomarkers have been developed for assessing the risk of exposure for two mycotoxins viz., aflatoxin by measurement by ELISA of aflatoxin B1 N7-guanine adduct which has a detection limit of 15.6 pmol aflatoxin B1 N7 guanine; and fumonisin B1 by measurement in urine using HPLC with a detection limit of 8 ng/ml urine. Assessment of the economic implications of aflatoxin contamination showed economic losses resulting in rejection of export consignment of hand-picked-selected (HPS) groundnut and losses in the poultry industry. Approaches for prevention and control of mycotoxin contamination in foods have shown that methods involving the segregation of contaminated or mouldy grains by hand picking and density segregation resulted in a reduction of 70-90 per cent of aflatoxin and fumonisin present in the grains. While harmonization of international regulatory limits, the requirements of food producing countries needs to be recognized and realistic but not idealistic safe limits, need to be proposed.

Animal Diseases↗

Microbial detoxification of mycotoxin deoxynivalenol.

Deoxynivalenol (DON) is a trichothecene secondary metabolite produced by Fusarium species infecting cereal crops. As a mycotoxin, DON causes losses in livestock production and poses a health risk to humans consuming contaminated cereal products. DON also acts as a virulence factor, facilitating the colonization of host plants by Fusarium spp. Enzymatic detoxification of mycotoxins in feed additives and genetically modified crops is a promising approach for the reduction of mycotoxin contamination of feeds and food. A prerequisite for the development of biotechnological strategies for DON detoxification is the availability of genes encoding suitable enzymatic activities. With the goal of isolating microbial cultures that can be used as a source of such activities, we screened 1285 microbial cultures from farmland soil, cereal grains, insects and other sources for DON transformation under aerobic conditions. One mixed culture transformed DON into two chromatographically separable products. The main product of the transformation was purified and its structure was elucidated by mass spectroscopy, (1)H-NMR, (13)C-NMR and proton-proton and carbon-proton correlated NMR spectroscopy. The structure of this product was determined to be 3-keto-4-deoxynivalenol. The DON-transforming mixed culture survived and retained its transforming activity during a starvation period of six months at 20 degrees C. Transformation of DON was suppressed by low concentrations of glucose and high concentrations of tryptone and yeast extract. Cell-free supernatants obtained either by filtration through a 0.22 microm membrane filter or by centrifugation did not exert DON-transforming activity. Trichothecenes 15-acetyl-DON, 3-acetyl-DON and fusarenon-X were also transformed.

Aerobiosis↗

In vitro transformation of the Fusarium mycotoxins deoxynivalenol and zearalenone by the normal gut microflora of pigs.

The biotransformation of the Fusarium mycotoxins deoxynivalenol and zearalenone by the normal bacterial gut flora of pigs was examined in this in vitro study. For that purpose, suspensions of intestinal contents (duodenum, jejunum, caecum, colon, rectum) of porcine origin were incubated anaerobically with deoxynivalenol (DON) or zearalenone (ZEA). DON and ZEA were degraded by the flora of the caudal segments (caecum, colon, rectum) of the gut--particularly the colon content--whereas the microorganisms of the cranial segments (duodenum, jejunum) exhibited no transforming activity. DON was showed to be deepoxidated, ZEA was hydrolyzed to alpha-zearalenol and an unknown metabolite. The transformation of DON was correlated with a loss of cytotoxicity, which could be demonstrated in the MTT(3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium++ + bromide)-cell-culture assay using swine kidney cells as target cells. The results of the study presented here correspond with the data found in in vivo studies. On the basis of these findings one could conclude that this in vitro method seems to be well suited to the study of the transformation of mycotoxins by the microflora of the gut. The in vitro study is cheaper than a feeding trial, and the preliminary information on the metabolism of mycotoxins obtained in such studies is helpful in designing feeding trials more clearly. Besides the simple and fast handling, reproducibility and the protection of the animals studied are further advantages of this in vitro method. In connection with the MTT-cell-culture assay, additional information about the cytotoxic potential of the bacterial transformation products can be obtained.

Animals↗

The mycotoxins citrinin, gliotoxin, and patulin affect interferon-gamma rather than interleukin-4 production in human blood cells.

Exposure to molds diminishes the numbers of T-helper type 1 (Th1) cells in the peripheral blood of children and is a risk factor for the development of allergic diseases (results of LARS: Leipzig Allergy Risk Children Study, Mueller et al. 2002). We hypothesized that mycotoxins are responsible for this effect and therefore investigated the influence of citrinin, gliotoxin, and patulin on human peripheral blood mononuclear cells (PBMC). CD3/CD28-stimulated PBMC of healthy donors were incubated for 24 h with the mycotoxins in serial dilutions and triplicates. Vitality and proliferation were tested using the MTT assay and T-cell function by the expression of cytokines (ELISA, intracellular cytokine staining, and real-time polymerase chain reaction (RT-PCR) for interferon-gamma (IFN-gamma) and interleukin-4 (IL-4). The cytokine secretion was inhibited at concentrations 2-130 times lower compared to vitality (ELISA versus MTT assay). The strongest inhibition of cytokine expression was found for IFN-gamma: 8.3 microg/mL citrinin, 34.2 ng/mL gliotoxin, and 64.8 ng/mL patulin caused a 50% inhibition of the IFN-gamma release (50% inhibitory dose, ID(50)). For IL-4 release the corresponding ID(50) values were 21.6 microg/mL citrinin, 82.8 ng/mL gliotoxin, and 243.2 ng/mL patulin. Furthermore, 3 ng/mL patulin caused a significant increase of IL-4 but a significant suppression of IFN-gamma. On the mRNA level, after 24 h an unaltered or enhanced IL-4 was observed compared to a reduced IFN-gamma expression. Using a method of intracellular cytokine staining, we were able to show that the described effects are caused by a reduction of the number of IFN-gamma-producing T lymphocytes rather than by a reduced functional capacity of the single cell. We suggest that mycotoxins primarily cause stronger inhibition of IFN-gamma-producing Th1 cells, which may lead to T-cell polarization toward the Th2 phenotype and may raise the risk for the development of allergies.

Anti-Bacterial Agents↗

Structure/function studies of T-2 mycotoxin with a monoclonal antibody.

A BALB/C murine monoclonal antibody (mAb) specific for the trichothecene mycotoxin T-2 was previously generated. The anti-T-2 antibody, designated HD11, can detect T-2 in the nanogram range employing an enzyme-linked immuno-sorbent assay. The HD11 antibody at a concentration of 1 microgram/ml completely protected against the T-2-induced cytotoxicity of the human epidermoid carcinoma cell lines Hep-2 and KB. Fine specificity analysis was performed using 10 structurally related T-2 metabolites to inhibit the specific binding of HD11 to T-2 mycotoxin. The results suggest a binding specificity of the protective HD11 antibody for the bulky hydrophobic alkyl side chain at position R5 and the acetyl groups at positions R2 and R3 of the T-2 mycotoxin molecule. HD11 anti-T-2 mAb, which bound to the T-2 metabolite acetyl T-2 (with a substituted acetyl group at R1 position), efficiently neutralized its in vitro cytotoxicity. On the other hand, the cytotoxicity of T-2 metabolites neosalaniol and 3' OH HT-2, both of which lack the alkyl side chain at position R5 and which did not bind to HD11, was unaffected by HD11.

Antibodies, Monoclonal↗

The role of intestinal microflora in the metabolism of trichothecene mycotoxins.

The role of faecal and intestinal microflora on the metabolism of trichothecene mycotoxins was examined in this study. Suspensions of microflora obtained from the faeces of horses, cattle, dogs, rats, swine and chickens were incubated anaerobically with the trichothecene mycotoxin, diacetoxyscirpenol (DAS). Micro-organisms from rats, cattle and swine completely biotransformed DAS, primarily to the deacylated deepoxidation products, deepoxy monoacetoxyscirpenol (DE MAS) and deepoxy scirpentriol (DE SCP). By contrast, faecal microflora from chickens, horses and dogs failed to reduce the epoxide group in DAS and yielded only the deacylation products, monoacetoxyscirpenol (MAS) and scirpentriol (SCP), in addition to unmetabolized parent compound. Intestinal microflora obtained from rats completely biotransformed DAS to DE MAS, DE SCP and SCP; and T-2 toxin to the deepoxy products, deepoxy HT-2 (DE HT-2) and deepoxy T-2 triol (DE TRIOL). Rat intestinal microflora also biotransformed the polar trichothecenes, T-2 tetraol and scirpentriol, to their corresponding deepoxy analogues. Deepoxy T-2 toxin (DE T-2) was synthesized from T-2 toxin and demonstrated to be 400 times less toxic than T-2 toxin in the rat skin irritation bioassay and non-toxic to mice given 60 mg/kg ip, demonstrating that epoxide reduction is a significant single step detoxification reaction for trichothecene mycotoxins.

Animals↗

The effect of ADH and insulin on active sodium transport across frog skin in the presence of alternariol mycotoxin.

1. The effect of vasopressin and insulin on active sodium transport across frog skin in the presence of internal alternariol mycotoxin was studied, using the short-circuit technique. 2. Vasopressin stimulates sodium transport across frog skin by decreasing the resistance to sodium entry into the epithelial cells, thus partially removing the inhibition on the short-circuit current due to the action of Alternariol mycotoxin. 3. Even insulin which is known to increase the short-circuit current by a different mechanism, determines a rapid reversal effect on the inhibition due to Alternariol. 4. These data confirm the different action of the two hormones on active sodium transport across frog skin, and furthermore are indicative of an inhibition of transepithelial sodium transport by Alternariol mycotoxin probably via the sodium pump.

Animals↗

Degradation of trichothecene mycotoxins by aqueous ozone.

The degradation of ten trichothecene mycotoxins by aqueous ozone was monitored by liquid chromatography-ultraviolet-mass spectrometry (LC-UV-MS). Saturated aqueous ozone ( approximately 25 ppm) degraded these mycotoxins to materials that were not detected by UV or MS. At lower levels (approximately 0.25 ppm) of aqueous ozone, intermediate products were observed. On the basis of UV and MS data, it is proposed that the degradation begins with attack of ozone at the C9-10 double bond with the net addition of two atoms of oxygen. The remainder of the molecule appears to have been left unaltered. The oxidation state at the allylic carbon 8 position had a significant effect on the ease of reaction, as determined by moles of ozone required to effect oxidation. The amount of ozone required to effect oxidation to intermediate products and subsequent degradation followed the series allylic methylene (no oxygen) < hydroxyl (or ester) < keto. Ozonation was also sensitive to pH. At pH 4-6, all mycotoxins studied degraded readily; at pH 7-8 the degree of reactivity was dependent upon the carbon 8 oxidation state; at pH 9, there was little or no reaction. Structures for some of the intermediate products are proposed.

Chromatography, Liquid↗

A novel colorimetric yeast bioassay for detecting trichothecene mycotoxins.

A novel colorimetric microbial bioassay for toxicity has been developed; it shows particular sensitivity to trichothecene mycotoxins. The assay uses inhibition of expression of beta-galactosidase activity within the yeast Kluyveromyces marxianus as a sensitive toxicity indicator, cultures remaining yellow, rather than turning deep green-blue, in the presence of X-gal, a chromogenic substrate. The assay is conducted in standard microtitre plates, permitting small volumes (160 microl) and many replicates, and can be scored either automatically by a plate-reader, or by eye. Factors likely to affect the efficacy of the bioassay, including carbon source, solvents, inoculum cell density, and the use of membrane-modulating agents (MMAs), were assessed. Polymyxin B nonapeptide was the most effective toxicity-enhancing MMA tested, enabling the trichothecene mycotoxin, verrucarin A, to be detected at a concentration of about 1 ng/ml. The assay's reproducibility was examined using polymyxin B sulfate, a cheaper MMA, and another trichothecene mycotoxin, T2 toxin: reproducibility and sensitivity were better for the beta-galactosidase X-gal endpoint than for an alternative chromogenic toxicity indicator, the respiratory substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).

Biological Assay↗

Real-time PCR detection of toxigenic Fusarium in airborne and settled grain dust and associations with trichothecene mycotoxins.

Inhalation of immunomodulating mycotoxins produced by Fusarium spp. that are commonly found in grain dust may imply health risks for grain farmers. Airborne Fusarium and mycotoxin exposure levels are mainly unknown due to difficulties in identifying Fusarium and mycotoxins in personal aerosol samples. We used a novel real-time PCR method to quantify the fungal trichodiene synthase gene (tri5) and DNA specific to F. langsethiae and F. avenaceum in airborne and settled grain dust, determined the personal inhalant exposure level to toxigenic Fusarium during various activities, and evaluated whether quantitative measurements of Fusarium-DNA could predict trichothecene levels in grain dust. Airborne Fusarium-DNA was detected in personal samples even from short tasks (10-60 min). The median Fusarium-DNA level was significantly higher in settled than in airborne grain dust (p < 0.001), and only the F. langsethiae-DNA levels correlated significantly in settled and airborne dust (r(s) = 0.20, p = 0.003). Both F. langsethiae-DNA and tri5-DNA were associated with HT-2 and T-2 toxins (r(s) = 0.24-0.71, p < 0.05 to p < 00.01) in settled dust, and could thus be suitable as indicators for HT-2 and T-2. The median personal inhalant exposure to specific toxigenic Fusarium spp. was less than 1 genome m(-3), but the exposure ranged from 0-10(5) genomes m(-3). This study is the first to apply real-time PCR on personal samples of inhalable grain dust for the quantification of tri5 and species-specific Fusarium-DNA, which may have potential for risk assessments of inhaled trichothecenes.

Air Microbiology↗

Dietary intake of some important mycotoxins by the Swedish population.

To estimate the intake of some mycotoxins from food in Sweden, approximately 600 samples were collected and analysed for aflatoxins, ochratoxin A, patulin and trichothecenes. Intakes were calculated for average and high consumers among adults and children and compared with the tolerable daily intake (TDI) of the respective toxin. Mycotoxin levels in the food samples were generally below the European/national maximum limits. However, high levels of aflatoxins were found in some samples of Brazil nuts and pistachios. The intake of ochratoxin A, patulin and trichothecenes was found to be below the temporary, TDI values (tTDI) proposed for these toxins by international expert groups, although the intake of trichothecenes (expressed as T-2 toxin equivalents) in children with a high consumption of cereals was close to the tTDI for T-2 toxin. Since there is to date no established numerical tTDI for aflatoxins, such a value was estimated for use within the project. The calculated intake of aflatoxins in high consumers exceeded this tTDI by a factor of two. In conclusion, the exposure to mycotoxins in Sweden did not generally, give rise to any major health concerns in the present study. However, the high levels of aflatoxins in certain commodities emphasize the need for preventive measures and improved control of toxin levels in these food items. Furthermore, the need for regulatory levels for trichothecenes in cereal products should be evaluated.

Adolescent↗

Effects of the mycotoxins citrinin and ochratoxin a on hepatic mixed-function oxidase and adenosinetriphosphatase in neonatal rats.

The effects of citrinin, ochratoxin A, or a combination of the two mycotoxins on the hepatic monooxygenase system and on hepatic and renal adenosinetriphosphatase (ATPase) activities were examined in neonatal rats exposed to a single treatment of one or both toxins. Animals received (po) 25 mg/kg citrinin, 1 mg/kg ochratoxin A, or 25 mg/kg citrinin plus 1 mg/kg ochratoxin A within 24 h of birth. Pups were killed 12 d later. Citrinin or ochratoxin A alone did not affect hepatic ATPase. Renal oligomycin-sensitive Mg2+-ATPase was inhibited to the same degree by ochratoxin A and the combination treatment. A synergistic effect of the two mycotoxins was observed on renal Na+-K+-ATPase. Significant effects, due to the mycotoxin interaction, were also observed on cytochrome P-450 content, NADPH-dependent dehydrogenase, and NADPH-cytochrome c reductase.

Adenosine Triphosphatases↗

[The significance of the mycotoxins desoxynivalenol, zearalenone and ochratoxin A for agricultural domestic animals].

Mycotoxins are metabolic products of mycotoxins which have various chemical structures and show various toxic effects. Deoxynivalenol (vomitoxin) is an important economic factor in pig production due to growth depression and suppression of the immune system. Previous studies have shown that the 1-ppm limit in the sole feed for pigs should not be exceeded. Studies of methods of detoxification have as yet not produced conclusive results. Zearalenone has an tolerable effect and may lead to fertility disturbances on the oestrogen production in pigs and can cause remarkable economic damage even in the ppb range. Recommendations of upper limits cannot be made on the basis of the available results. The kidney toxin ochratoxin A is of importance in pig and poultry breeding and--due to its accumulation in the tissue--represents a possible source of danger to man. Since a possible carcinogenic effect of the toxin cannot be excluded, its content in animal rations should be kept as low as possible. For ruminants mycotoxins as a whole do not represent a particular source of danger as these substances can be degraded or converted by rumen flora.

Animal Feed↗

The mycotoxin deoxynivalenol affects nutrient absorption in human intestinal epithelial cells.

Deoxynivalenol (DON) is a mycotoxin belonging to the tricothecene family that has many toxic effects in animals, including diarrhea and weight loss. Using the human epithelial intestinal cell line HT-29-D4 as an in vitro model, we studied the effect of DON on the uptake of different classes of nutrients, including sugars, amino acids and lipids. At low concentrations (below 10 micro mol/L), DON selectively modulated the activities of intestinal transporters: the D-glucose/D-galactose sodium-dependent transporter (SGLT1) was strongly inhibited by the mycotoxin (50% inhibition at 10 micro mol DON, P < 0.05), followed by the D-fructose transporter GLUT5 (42% inhibition at 10 micro mol/L, P < 0.001), active and passive L-serine transporters (30 and 38% inhibition, respectively, at 10 micro mol/L, P < 0.05). The passive transporters of D-glucose (GLUT) were slightly inhibited by DON (15% inhibition at 1 micro mol/L, P < 0.01), whereas the transport of palmitate was increased by 35% at 10 micro mol/L DON (P < 0.001). In contrast, the uptake of cholesterol was not affected by the mycotoxin. At high concentrations (100 micro mol/L), SGLT1 activity was inhibited by 76% (P < 0.01), whereas the activities of all other transporters were increased. The selective effects of DON on intestinal transporters were mimicked by cycloheximide and deoxycholate, suggesting that inhibition of protein synthesis and induction of apoptosis are the main mechanisms of DON toxicity in intestinal cells.

Amino Acids↗

Expression of a truncated form of ribosomal protein L3 confers resistance to pokeweed antiviral protein and the Fusarium mycotoxin deoxynivalenol.

The contamination of important agricultural products such as wheat, barley, or maize with the trichothecene mycotoxin deoxynivalenol (DON) due to infection with Fusarium species is a worldwide problem. Trichothecenes inhibit protein synthesis by targeting ribosomal protein L3. Pokeweed antiviral protein (PAP), a ribosome-inactivating protein binds to L3 to depurinate the alpha-sarcin/loop of the large rRNA. Plants transformed with the wild-type PAP show lesions and express very low levels of PAP because PAP autoregulates its expression by destabilizing its own mRNA. We show here that transgenic tobacco plants expressing both the wild-type PAP and a truncated form of yeast L3 (L3delta) are phenotypically normal. PAP mRNA and protein levels are very high in these plants, indicating that L3delta suppresses the autoregulation of PAP mRNA expression. Ribosomes are not depurinated in the transgenic plants expressing PAP and L3delta, even though PAP is associated with ribosomes. The expression of the endogenous tobacco ribosomal protein L3 is up-regulated in these plants and they are resistant to the Fusarium mycotoxin DON. These results demonstrate that expression of an N-terminal fragment of yeast L3 leads to trans-dominant resistance to PAP and the trichothecene mycotoxin DON, providing evidence that both toxins target L3 by a common mechanism.

Amino Acid Sequence↗

Wild rice as fermentation substrate for mycotoxin production.

Many cereal grains have been studied for their suitability as substrates for the fermentative production of mycotoxins. However, except for aflatoxin, wild rice has not been investigated. Hence, five mold cultures known to produce the mycotoxins ochratoxin-A, penicillic acid, patulin, vomitoxin, and zearalenone were grown on wild rice under varying conditions of moisture and temperature to determine whether this grain would serve as a suitable substrate for toxin production. Under appropriate fermentation conditions, good yields of ochratoxin-A and moderate amounts of patulin were obtained, but only small amounts of penicillic acid, vomitoxin, and zearalenone were elaborated. An extract from a sample of naturally molded wild rice contained 0.8 microgram of patulin per g of rice. The predominating mold was identified as Aspergillus clavatus. Under identical cultural conditions, this isolate and a known patulin-producing strain of A. clavatus yielded approximately equivalent amounts of the mycotoxin.

Aspergillus↗