Deoxynivalenol in Lebensmitteln

Within a joint research project entitled “Analysis and occurrence of importantFusarium toxins (deoxynivalenol and zearalenone) and dietary intake of these toxins by the German consumer”, supported by the German Federal Ministry of Consumer Protection, Food and Agriculture (BMVEL), representative analytical data are generated on the contamination level of foods withFusarium mycotoxins. This paper gives a comprehensive summary concerning the contamination of foods from the German market with deoxynivalenol (DON) in the period from August 2001 to April 2004. More than 4700 food samples (mostly cereals and cereal-containing foods) were purchased from food shops in Germany and analysed for DON by enzyme immunoassay, HPLC, and LC-MS/MS, respectively. All analytical methods were validated through intra- and interlaboratory studies and gave mean recoveries of >80% for each matrix. Although DON was detected with high frequency in all cerealcontaining samples, the mean and median levels were in most products well below the recently established maximum permitted limits in Germany.     

Survey and risk assessment of the mycotoxins deoxynivalenol,zearalenone, fumonisins,ochratoxin A,and aflatoxins in commercial dry dog food

The aim of the present study was to investigate the occurrence of mycotoxins in commercial dog food, as a basis to estimate the risk of adverse effects. Seventy-six dry dog food samples from 27 producers were purchased from retail shops, supermarkets, and specialized pet food shops in Vienna, Austria. The frequency and levels of deoxynivalenol (DON), zearalenone (ZEA), fumonisins (FUM), ochratoxin A (OTA). and aflatoxins (AF) in dry dog food were determined. Mycotoxin analysis were performed by commercial enzyme-linked immunosorbent assay (ELISA) test kits. Confirmatory analyses were done for DON, ZEA, and FUM by high performance liquid chromatography (HPLC) after extract clean-up with immunoaffinity columns. The correlations between ELISA and HPLC results for DON and ZEA were acceptable and indicated that ELISA could be a simple, low cost, and sensitive screening tool for mycotoxins detection, contributing to quality and safety of pet food. DON was the mycotoxin most frequently found (83% positives; median 308 μg/kg, maximum 1,390 μg/kg). ZEA (47% positives, median 51 μg/kg and maximum 298 μg/kg) and FUM (42% positives, median 122 μg/kg and maximum 568 μg/kg) were also frequently detected in dog food. OTA was less frequently found (5%, median 3.6 μg/kg, maximum 4.7 μg/kg. AF were not detected (<0.5 μg/kg) in any sample. The results show that dry dog food marketed in Vienna are frequently contaminated with mycotoxins (DON > ZEA > FUM > OTA) in low concentrations, but do not contain AF. The high frequency of Fusarium toxins DON, ZEA, and FUM indicates the need for intensive control measures to prevent mycotoxins in dog foods. The mycotoxin levels found in dry dog food are considered as safe in aspects of acute mycotoxicoses. However, repeated and long-time exposure of dogs to low levels of mycotoxins may pose a health risk.     

Fusarium toxin contents of maize and maize products purchased in the years 2000 and 2001 in Germany

Samples (n=106) of maize and maize products were analysed for 13 trichothecene toxins and zearalenone (ZON). All 14 toxins examined were detected, although with varying frequency. Cooccurrence of two or more toxins was observed in 96% of samples. The toxins of the scirpenol group scirpentriol, 15-monoacetoxyscirpenol and diacetoxyscirpenol were detected in 14, 27 and 3% of the samples analysed, the toxins of the T-2 group T-2 toxin, HT-2 toxin, T-2 triol und T-2 tetraol were found in 33, 66, 2 and 7%. Toxin content was higher in feeds than in foods (semolina and flour). In food samples, the German regulatory level for DON (500 μg/kg) was not exceeded, three samples of maize flour contained ZON above the regulatory level (50 μg/kg). Presented at the 26^th Mykotoxin-Workshop in Herrching, Germany, May17–19, 2004     

Bestimmung von Deoxynivalenol und Deepoxy-Deoxynivalenol in Milch

A HPLC method with UV/diode array detection for the determination of deoxynivalenol (DON) and deepoxy-deoxynivalenol (DOM-1) in milk was developed. Milk was incubated with β-glucuronidase and then defatted. After purification by immunoaffinity chromatography, DON and DOM-1 were separated on a C18 reversed phase column with acetonitril/water (10/90) as the mobile phase and detected at 218 nm. Limits of quantification were 1 μg/l for both toxins, with mean recoveries (1–10 μg/l) of 97% (DON) and 84% (DOM-1), respectively. Milk samples (pasteurized, UHT; n=32) from German retail shops were analysed by this method. Neither DON/DOM-1 nor their glucuronides were found in any sample. These results are consistent with published studies indicating that in lactating cows, DON and DOM-1 are mostly eliminated through urine, and that the carry-over into milk is negligible.     

Need to determine the relative developmental risks of Fusarium mycotoxin dexoynivalenol (DON) and Benomyl (BEN) in wheat

The wheat supply is known to have levels of deoxynivalenol (DON) from fungal infections. The fungal infections can be suppressed with benomyl (BEN). Under certain exposure laboratory conditions DON and BEN are developmental toxins. Since use of BEN may result in concurrent dietary exposure to both compounds, evaluation of the relative developmental risks is needed. The dietary NOAELs for pup decreased body weight for DON and BEN are 0.375 mg/kg/day in mice and 5 mg/kg/day in rat, respectively. Human exposure to DON can be estimated from surveys of wheat grain and finished products which gave a mean DON concentration of 0.86 ppm. The tolerance level for BEN in grain is 0.2 ppm. Presence of DON and BEN in wheat at these concentrations would not impact wheat consumption. A modified margin of exposure (modMOE) was developed using “a”; kg (food) /kg (body weight) as the constant for wheat consumption. The modMOE for DON is 28/a and that for BEN is 1667/a. Comparative relative risks can be expressed as the ratio of BEN risks to DON risks (60). Treatment with BEN at the most efficacious time decreased DON levels to 34% of controls, and decreased the comparative risk ratio from 60 to 18. The potential for fungicides to decrease mycotoxin health risks is important in the safety assessment for foodstuffs. This approach improves on current practices for food safety assessment by including the expected reductions in risks from naturally occurring mycotoxins in the regulatory analysis.     

Effects of long-term storage on Fusarium toxin concentrations in wheat--sources of error of the analytical results

The concentrations of the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) of a heavily contaminated wheat grain batch were followed over a period of 1 year by taking samples 15 times every 28 days. The air temperature and relative humidity at the top of the wheat batch ranged between 7 and 22 degrees C and 44 and 55%, respectively, and corresponded to a variation in the moisture content of the wheat grain between 11.5 and 12.3%. None of these fluctuations were related to ZON and DON concentrations, which varied between 0.46 and 0.66 and 15.0 and 19.5 mg/kg DM. Therefore, the data were used to analyse the error sources for the analytical results. It was found that the variance proportions due to sampling and sample preparation plus analysis were not similar for DON and ZON. The variance proportion due to sampling was found to be 0.62 for ZON, which corresponded to a variance proportion of 0.38 due to sample preparation plus analysis. In contrast, the latter variance proportion for DON was estimated to be 1.0 and consequently completely superimposed the sampling error. It is concluded that long-term storage of contaminated wheat grain does not affect the concentrations of DON and ZON considering the measured fluctuations in ambient temperature, relative humidity and moisture content of the grain. Therefore, no degradation of DON and ZON occurred during the storage of wheat for a period of one year under ambient conditions. The effects of sampling and sample preparation plus analysis on the final analytical results are different for DON and ZON and require further consideration.     

Effects of long-term storage on Fusarium toxin concentrations in wheat - sources of error of the analytical results

The concentrations of the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) of a heavily contaminated wheat grain batch were followed over a period of 1 year by taking samples 15 times every 28 days. The air temperature and relative humidity at the top of the wheat batch ranged between 7 and 22°C and 44 and 55%, respectively, and corresponded to a variation in the moisture content of the wheat grain between 11.5 and 12.3%. None of these fluctuations were related to ZON and DON concentrations, which varied between 0.46 and 0.66 and 15.0 and 19.5 mg/kg DM. Therefore, the data were used to analyse the error sources for the analytical results. It was found that the variance proportions due to sampling and sample preparation plus analysis were not similar for DON and ZON. The variance proportion due to sampling was found to be 0.62 for ZON, which corresponded to a variance proportion of 0.38 due to sample preparation plus analysis. In contrast, the latter variance proportion for DON was estimated to be 1.0 and consequently completely superimposed the sampling error. It is concluded that long-term storage of contaminated wheat grain does not affect the concentrations of DON and ZON considering the measured fluctuations in ambient temperature, relative humidity and moisture content of the grain. Therefore, no degradation of DON and ZON occurred during the storage of wheat for a period of one year under ambient conditions. The effects of sampling and sample preparation plus analysis on the final analytical results are different for DON and ZON and require further consideration.     

Deoxynivalenol in commercial beer — screening for the toxin with an indirect competitive ELISA

Antibodies specific for deoxynivalenol (DON) were prepared by immunizing rabbits with a deoxynivalenol-hemiglutaryl-HSA conjugate. The antibody showed cross-reactivity with DON, 15a-acetyl-DON (15-Ac DON), and 3a-acotyl-DON (3-Ac DON) of 100%, 216%, and 260%, respectively. No cross-reactivity was observed against 20 further trichothecenes of the A-, B-, and C-type. An indirect competitive ELISA procedure was set up for the detection of DON in liquid matrices occurring in the brewing process, Including beer. ELISA was sensitive down to 3.8 ng/mL. Detection limit for DON in tenfold diluted beer was 50 ppb. Recovery of the toxin in spiked beer was 45%, 77%, and 83% at toxin levels of 50; 500; and 1,000 ppb, respectively. ELISA results corresponded quite well with HPLC data. 196 commercial beers from several German breweries, including 36 gushing-positive samples, were assayed for DON. Screening revealed toxin levels from not detectable to 569 ppb. 69% of the gushing beers had marked levels of DON. 37% of the gushing-negative beer samples contained DON in detectable amounts with a maximum content of 172 ppb. Wheat beers had significantly higher DON-contents than beers derived from barley. Concentration of the toxin was significantly higher in gushing beers than in nongushing beers from both cereal sources.     

Zearalenon in Lebensmitteln

The research project “Methods of Analysis and occurrence of important Fusarium toxins (deoxynivalenol and zearalenone) as well as the intake of these toxins by the German consumer” supported by the Federal Ministry of Consumer Protection, Food and Agriculture (BMVEL) is processed by the institutions mentioned above. This work represents a comprehensive summary of the contamination of food by zearalenone (ZEA).     

Intake estimates for trichothecene toxins of the population in Southwest Germany in 1998 and in 1999

The intake of theFusarium toxins deoxynivalenol (DON), nivalenol (NIV), HT-2 and T-2 toxin (HT-2, T-2), 3-, 15-acetyldeoxynivalenol (3-, 15- ADON), and fusarenon-X (FUS-X) was calculated for adults, children and babies, for an area of southwest Germany and two years (1998, 1999). Estimates were based on consumption data of bread and pasta by both adults and children and of infant food by babies, reported for the German population in a study on behalf of the European Union, and on toxin contents of a total of 208 samples of these commodities. No exceeding of the tolerable daily intake (TDI) of DON, NIV and the sum of HT-2 and T-2, as stated by the EU, was found for adults (70 kg body weight (BW)) and for babies (10 kg BW), independent of year and level of consumption. For children (20 kg BW) the intake of DON exceeded the TDI in 1998 for high, and in 1999 for both mean and high consumers. For both years the intake of the sum of HT-2 and T-2 was below the TDI following mean but above this value following high consumption. The intake of NIV was far below the TDI for both levels of ingestion. The daily intake of each of the three toxins 3-, 15- ADON and FUS-X was below 0.03, 0.11 and 0.05 μg/kg/BW for adults, children and babies, respectively. Presented at the 27^th Mykotoxin-Workshop, Dortmund. Germany, June 13–15, 2005     

Use of enzyme-linked immunosorbent assay to screen for aflatoxins,ochratoxin A,and deoxynivalenol in dry pet foods

The objective of this study was to perform a market survey on dry pet foods using enzyme-linked immunosorbent assay (ELISA) to detect total aflatoxins (AFs), ochratoxin A (OTA), and deoxynivalenol (DON). Pet food products (n?=?58) marketed for dogs, cats, birds, and rabbits were tested in duplicate with ELISA, and results above the limit of quantitation were confirmed using liquid chromatography tandem mass spectrometry (LC-MS/MS). OTA was detected in one product (rabbit food) and AFs were detected in two products (one dog treat and one bird treat). In contrast, DON was detected in the majority (74%) of products tested. Bird and rabbit products were the most affected by DON, with levels above 0.5 μg/g in 50 and 80% of samples, respectively. One rabbit sample tested positive for both OTA and DON. Overall, the findings of this study revealed a low incidence of AFs and OTA in commercial pet food. Although DON was detected in numerous products, the levels were well below those associated with acute toxic effects.     

Intracellular Expression of a Single Domain Antibody Reduces Cytotoxicity of 15-Acetyldeoxynivalenol in Yeast

15-Acetyldeoxynivalenol (15-AcDON) is a low molecular weight sesquiterpenoid trichothecene mycotoxin associated with Fusarium ear rot of maize and Fusarium head blight of small grain cereals. The accumulation of mycotoxins such as deoxynivalenol (DON) and 15-AcDON within harvested grain is subject to stringent regulation as both toxins pose dietary health risks to humans and animals. These toxins inhibit peptidyltransferase activity, which in turn limits eukaryotic protein synthesis. To assess the ability of intracellular antibodies (intrabodies) to modulate mycotoxin-specific cytotoxocity, a gene encoding a camelid single domain antibody fragment (V_HH) with specificity and affinity for 15-AcDON was expressed in the methylotropic yeast Pichia pastoris. Cytotoxicity and V_HH immunomodulation were assessed by continuous measurement of cellular growth. At equivalent doses, 15-AcDON was significantly more toxic to wild-type P. pastoris than was DON. In turn, DON was orders of magnitude more toxic than 3-acetyldeoxynivalenol. Intracellular expression of a mycotoxin-specific V_HH within P. pastoris conveyed significant (p = 0.01) resistance to 15-AcDON cytotoxicity at doses ranging from 20 to 100 μg·ml⁻¹. We also documented a biochemical transformation of DON to 15-AcDON to account for the attenuation of DON cytotoxicity at 100 and 200 μg·ml⁻¹. The proof of concept established within this eukaryotic system suggests that in planta V_HH expression may lead to enhanced tolerance to mycotoxins and thereby limit Fusarium infection of commercial agricultural crops.     

Grundfutterqualität in Sachsen: Ergebnisse der mykotoxikologischen Untersuchungen an Silomais der Jahre 1997–2003

Maize for silage purposes is investigated on Zeralenone (ZEA) and Deoxynivalenol (DON) in Saxony since 1997. About 45 samples from various regions of Saxony are analyzed every year. The occurrence of DON and ZEA was proved for 20 to 90% of all samples. Most of the samples showed both of these toxins. The quantity of the detected toxins was generally very low, even in so called “Fusarium years” only 20% of samples exceeded the lowest border of orientation values for critical dietary concentrations in cattle feed. We observed a higher contamination with DON in samples from the region “Erzgebirge” and clear higher contents of ZEA in the region “Sächsisches Heideland”. Correlation between toxin content and other parameters like cultivation, pre-fruit etc. were not observed, probably because of masking these effects by variety and location.     

Development of enzyme-immunoassays based on egg yolk antibodies for the detection of mycotoxins

Enzyme-linked immunosorbent assays (ELISA) proved to be a fast and simple method for the detection of mycotoxins and other undesired contaminants in food and feed. The present study is focused on the optimisation and exploitation of the egg yolk antibody technology in order to develop competitive ELISAs for the detection of mycotoxins in cereals. Due to its importance as one of the most relevant Fusarium mycotoxins, the trichothecene deoxynivalenol (DON) was selected as representative. Chickens were immunised with different protein conjugates performing varying booster intervals. The antibodies were isolated by the poly(ethylene glycol) precipitation method according to Polson. By use of these antibodies an indirect competitive ELISA was developed for the detection of DON. First investigations of naturally contaminated wheat samples showed a good correspondence with results obtained by GC-ECD when calibration in blank wheat extracts was performed.     

Exposure of oocytes to the Fusarium toxins zearalenone and deoxynivalenol causes aneuploidy and abnormal embryo development in pigs

Fungi of the Fusarium species can infect food and feed commodities and produce the mycotoxins zearalenone (ZEA) and deoxynivalenol (DON). Since both toxins have been reported to reduce fertility, the mechanisms of ZEA and DON on inhibition of oocyte maturation were examined. Pig oocytes were matured in the presence of ZEA (a mycotoxin with estrogenlike activity), 17beta-estradiol, and DON (all 3.12 micromol/L). Zearalenone, 17beta-estradiol, and DON inhibited oocyte maturation and caused approximately 34% of the oocytes to form an aberrant spindle. Different ratios of ZEA:DON did not lead to a more severe inhibition of oocyte maturation. Both mycotoxins caused abnormal formation of the meiotic spindle. The developmental competence of oocytes matured in the presence of mycotoxins was further investigated after in vitro fertilization. Presence of ZEA (3.12 micromol/L) during maturation reduced the percentages of oocytes that cleaved and formed a blastocyst to about 12%, compared with 25% of control oocytes. Maturation in the presence of equimolar concentrations of DON was not compatible with development. The ploidy of blastomeres from blastocysts derived from mycotoxin-exposed oocytes was analyzed with fluorescent in situ hybridization. All blastocysts, even those from the control group, contained at least one blastomere with abnormal ploidy, but the variation in the percentages of aneuploid blastomeres was significantly larger in embryos from oocytes exposed to mycotoxins. It is concluded that ZEA and DON can lead to abnormal spindle formation, leading to less fertile oocytes and embryos with abnormal ploidy, and that the effects of ZEA and DON are not synergistic.     

Fusarium toxins in cereals — Results from eight German Federals States in 2000

A total of 353 cereal samples from 8 German Federal States were analysed for Fusarium toxins in the 2000' survey. The level of mycotoxin contamination of the samples was higher than in the previous year due to higher moisture during flowering in some regions. Concerning deoxynivalenol (DON), the prevailing mycotoxin in all the samples, we have to consider, that 24% of the samples as well as the average concentration exceed the advisory level of 500 μg/kg. The application of Fusarium active fungicides reduced the DON level from 963 μg/kg (samples without treatment) to 630 μg/kg (all samples from treated and untreated fields). It has to be emphasised that the number of nivalenol (NIV) positive samples increased in comparison to the previous year from 9% to 17%. In addition, an increase in the level of NIV was detected, the maximum concentration in winter wheat was 3480 μg/kg. Cereals were rather moderately contaminated with zearalenone (ZEA), more than 80% of the samples contained less than 50 μg/kg.     

Cytotoxic effects of DON and T-2 toxin on plant cells

The effect of deoxynivalenol (DON) and T-2 toxin on mitotic index (MI) and relative division rate (RDR) in actively dividing onion (Allium cepa L.) root-tip cells was studied. Both these toxins resulted in decline of mitotic activity which was inversely proportional to the concentrations of these toxins. T-2 was more effective resulting in 59% RDR value at 2.5 ppm whereas DON treated root cells had 78% RDR at the same concentration as compared to respective sets of controls.     

Comparison of three immunological methods for detecting staphylococcal enterotoxins from food

AIMS: Immunologically based assays for the detection of staphylococcal enterotoxins are numerous. These techniques include radio immunosorbent assays and enzyme-linked immunosorbent assays (ELISA), some of which are available as commercial kits. The purpose of this study was to compare the performances of three commercial immunoassays. METHODS AND RESULTS: Two automated detection systems, VIDAS SET bioMèrieux, VIDAS SET2 bioMérieux and an ELISA method, TRANSIA PLATE Staphylococcal Enterotoxins Diffchamb were compared for detecting different quantities of purified staphylococcal enterotoxins (A, B, C2, D and E) added to food. CONCLUSIONS: VIDAS SET2 had a greater specificity (100%) and sensitivity than VIDAS SET and TRANSIA PLATE Staphylococcal Enterotoxins. More precisely, VIDAS SET2 could detect <0.5 ng g(-1) of toxins A and B, <1 ng g(-1) of toxins C2 and E and 1 ng g(-1) of toxins D and E. SIGNIFICANCE AND IMPACT OF THE STUDY: Because staphylococcal food poisoning (resulting from ingestion of low levels of staphylococcal enterotoxins) is one of the most common forms of foodborne illness there is a need for specific and sensitive methods for detecting these enterotoxins. VIDAS SET2 appears to be suitable for detecting staphylococcal enterotoxins from food.     

The hemolytic activity of deoxynivalenol and T-2 toxin.

The hemolytic effects of deoxynivalenol (DON) and T-2 toxin (T-2) individually on rat erythrocytes were studied at different concentrations. Sodium azide was used as an enzyme inhibitor to prevent T-2 toxin metabolism. The concentration of T-2 was controlled by GC-MS and no decrease of the toxin was found during the time of the experiment. In spite of the much higher toxicity of T-2 toxin to eucaryotic cells, DON and T-2 showed similar lytic activity toward erythrocytes at high and low concentrations. Neither of these toxins at a concentration of 130 micrograms/ml, produced significant hemolysis even after 11 hr incubation. This finding suggests that there is a threshold level for both T-2 and DON, below which the lytic reaction does not occur. An additional hemolysis test was conducted in the presence of mannitol, glutathione, ascorbic acid, alfa-tocopherol, and histidine. The assay demonstrated that all the compounds inhibited to some extent the hemolytic reaction of the toxins. It is suggested that DON and T-2 exert their toxicity on procaryotic cells in three different ways: by penetrating the phospholipid bilayer and acting at the subcellular level, by interacting with the cellular membranes, and by free radical mediated phospholipid peroxidation. Most probably, more than one mechanism operates at the same time.     

Distinct effects of equal levels of Fusarium toxins (Deoxynivalenol) in growing pigs. Possible reasons

The mycotoxin deoxynivalenol (DON, vomitoxin) is well documented to cause reduced feed consumption (FC) and weight gain (WG) rates in sensitive animal species (e.g. swine). In establishing feeding guidelines and diagnoses of mycotoxin related diseases it is important to establish a “dose and response” factor-but a simple relationship is not always present for DON and other Fusarium toxins. Possible reasons for these findings (e.g. experimental design and/or role of toxin source) are discussed with reference to previous research.