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Mycotoxins are natural toxins produced by fungi that may cause adverse health effects thus constituting a public health concern. Deoxynivalenol (DON), a mycotoxin affecting the immune system and causing intestinal disorders, was selected as a priority under the European Human Biomonitoring Initiative (HBM4EU). Urinary total DON levels (tDON) of 1270 participants from six countries were used to characterize the internal exposure of the adult European population and identify the most relevant determinants of exposure. tDON concentrations' P50 and P95 were in the range of 0.41-10.16 µg/L (0.39-9.05 µg/g crt) and 3.25-46.58 µg/L (2.12-33.50 µg/g crt) respectively. Higher tDON levels were observed for (i) male participants from France and Germany, (ii) samples collected in spring and summer, (iii) participants with a lower educational level, (iv) participants living in rural areas, (v) individuals without a job in France and Luxembourg, while in Portugal higher exposure was observed in working individuals, (vi) individuals with higher consumption of cereals and bread. The proportion of individuals with exposure levels exceeding the HBM-GV of 23 µg/L was 12.3 %, ranging from 0.8 % to 20.7 % in the individual countries. This study on mycotoxins exposure has used post harmonized questionnaire data and validated analytical methodologies for analysis and covered countries representing the four geographical regions of Europe, having produced much needed knowledge on the exposure of the European adult population to deoxynivalenol., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The widespread presence of Fusarium mycotoxins in animal feed is a global issue, not only for the health of livestock but also for ensure the safety of food as an end product. High concentrations of zearalenone (ZEN) and deoxynivalenol (DON) have been detected in the diets of Japanese Black (JB) and Holstein Friesian (HF) breeding herds. Consequently, we monitored serum biochemical parameters over a long time in both herds, focusing on anti-Müllerian hormone (AMH) levels and acute-phase inflammation. Additionally, urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and progesterone levels were measured in the HF herd. The JB herd, a ZEN-dominant model with low DON contamination, demonstrated ZEN levels that exceeded the Japanese limit in the purchased total mixed rations (TMR). Conversely, the HF herd, which primary consumes DON-dominant feed with low ZEN contamination, had high DON levels in the dent corn silage. Specifically, the JB herd's TMR contained 1.79 mg/kg ZEN and 0.58 mg/kg DON, whereas the HF herd's silage had 15.3 mg/kg DON (dried sample) and 0.1 mg/kg ZEN. Enzyme-linked immunoassay were used to measure urinary ZEN-DON levels following confirmation through liquid chromatography-tandem mass spectrometry. Urinary ZEN-DON levels measured were significantly correlated ( p < 0.05, r > 0.6) in both herds. In the HF herd, AMH levels increased ( p = 0.01) and serum amyloid A (SAA) levels decreased ( p = 0.02) when contaminated and at the end of the monitoring period. Additionally, urinary ZEN and DON levels were significantly correlated with SAA levels (ZEN: p = 0.00, r = 0.46; DON: p = 0.03, r = 0.33), with an increase in ZEN and DON levels resulting in higher SAA levels. The JB herd showed no significant differences. Additionally, in the HF herd, 8-OHdG/Cre levels increased significantly during major contamination periods ( p < 0.05). Clinical data from the HF herd indicated an increase in mastitis cases and treatment rates during periods of major contamination. Abortion rates in the HF herd decreased from 22.9% (before monitoring) to 8.9% (during the high contamination period) and finally to 1% (at the end of the monitoring period), with corresponding increases in progesterone levels. ZEN-DON contamination adversely affects breeding cattle's productivity, reproductive performance, and health. Therefore, monitoring urinary ZEN-DON is valuable for detecting contaminants and ensuring the safety of food products.

DON and ZEN residues in the blood and urine of dairy cows can be used to predict the outer exposure to DON and ZEN expressed per kilogram diet for a risk evaluation based on comparisons to critical dietary concentrations. This method was used to evaluate the exposure of dairy cows from 12 farms located in Brandenburg, Germany, fed rations with unknown DON and ZEN concentrations (N = 244). The corresponding diet concentrations predicted by different methods from analyzed blood and urine samples varied significantly amongst farms from 0 to 1.6 mg/kg for DON and 0 to 3.0 mg/kg for ZEN at a reference dry matter content of 88% but independently of lactational state (post-partum vs. early lactation). This significant variation was noticed below the critical dietary DON concentration of 5 mg/kg, while the ZEN concentration in one farm exceeded the critical ZEN level of 0.5 mg/kg markedly. Predicted DON concentrations of rations increased with the proportion of maize silage, while the high ZEN concentration found in one farm was most likely related to a higher proportion of sugar beet pulp supposedly highly contaminated by ZEN. Exceeding the critical dietary ZEN concentration and significant variations in DON contents below the critical level was not related to performance, reproductive performance, and health-related traits of cows. For a more consistent evaluation of possible associations between the inner exposure of cows to DON and ZEN, more frequent longitudinal observations of both mycotoxin residue levels and performance and health traits are required., (© 2024. The Author(s).)

The mycotoxin deoxynivalenol (DON) is a frequently found contaminant in cereals and cereal-based products. As a German contribution to the European Joint Programme HBM4EU, we analysed the total DON concentration (tDON) in 24-h urine samples from the German Environmental Specimen Bank (ESB). In total, 360 samples collected in 1996, 2001, 2006, 2011, 2016, and 2021 from young adults in Muenster (Germany), were measured by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) after enzymatic deconjugation of the glucuronide metabolites. tDON was found in concentrations above the lower limit of quantification (0.3 μg/L) in 99% of the samples. Medians of the measured concentrations and the daily excretion were 4.3 μg/L and 7.9 μg/24 h, respectively. For only nine participants, urinary tDON concentrations exceeded the provisional Human biomonitoring guidance value (HBM GV) of 23 μg/L. Urinary tDON concentrations were significantly higher for male participants. However, 24-h excretion values normalized to the participant's body weight did not exhibit any significant difference between males and females and the magnitude remained unchanged over the sampling years with exception of the sampling year 2001. Daily intakes were estimated from excretion values. Exceedance of the tolerable daily intake (TDI) of 1 μg/kg bw per day was observed for less than 1% of all participants. TDI exceedances were only present in the sampling year 2001 and not in more recent sampling years while exceedance of the HBM guidance value was also observed in 2011 and 2021., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The dietary exposure to the mycotoxin deoxynivalenol (DON) can be assessed by human biomonitoring (HBM). Here, we assessed the relation between dietary DON intake and the excretion of its major metabolite DON-15-glucuronide (DON15GlcA) through time, in an everyday situation. For 49 volunteers from the EuroMix biomonitoring study, the intake of DON from each meal was calculated and the excretion of DON and its metabolites was analyzed for each urine void collected separately throughout a 24-h period. The relation between DON and DON15GlcA was analyzed with a statistical model to assess the residence time and the excreted fraction of ingested DON as DON15GlcA (f abs&#95;excr ). F abs&#95;excr was treated as a random effect variable to address its heterogeneity in the population. The estimated time in which 97.5% of the ingested DON was excreted as DON15GlcA was 12.1 h, the elimination half-life was 4.0 h. Based on the estimated f abs&#95;excr , the mean reversed dosimetry factor (RDF) of DON15GlcA was 2.28. This RDF can be used to calculate the amount of total DON intake in an everyday situation, based on the excreted amount of DON15GlcA. We show that urine samples collected over 24 h are the optimal design to study DON exposure by HBM.

Human are exposed to a wide range of mycotoxins through dietary food intake, including processed food. Even most of the mycotoxin exposure assessment studies are based on analysis of foodstuffs, and evaluation of dietary intake through food consumption patterns and human biomonitoring methods are rising as a reliable alternative to approach the individual exposures, overcoming the limitations of the indirect dietary assessment. In this study, human urine samples were analyzed, seeking the presence of deoxynivalenol (DON), ochratoxin A (OTA), zearalenone (ZEA), and their metabolites. For this purpose, 40 urine samples from female and male adult residents in the city of Valencia (Spain) were evaluated by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-ESI-qTOF) after salting-out liquid-liquid extraction. Analytical data showed that 72.5% of analyzed samples were contaminated by at least one mycotoxin at variable levels. The most prevalent mycotoxins were de-epoxy DON (DOM-1) (53%), ZEA (40%), and α-zearalenol (αZOL) (43%), while OTA was only detected in one sample. The mean concentrations in positive samples were DON (9.07 ng/mL), DOM-1 (20.28 ng/mL), ZEA (6.70 ng/mL), ZEA-14 glucoside (ZEA-14-Glc) (12.43 ng/mL), αZOL (27.44 ng/mL), αZOL-14 glucoside (αZOL-14-Glc) (12.84 ng/mL), and OTA (11.73 ng/mL). Finally, probable daily intakes (PDIs) were calculated and compared with the established tolerable daily intakes (TDIs) to estimate the potential risk of exposure to the studied mycotoxins. The calculated PDI was below the TDI value established for DON in both female and male adults, reaching a percentage up to 30%; however, this percentage increased up to 92% considering total DON (DON + DOM-1). On the other hand, the PDI obtained for ZEA and its metabolites were higher than the TDI value fixed, but the low urine excretion rate (10%) considered should be highlighted. Finally, the PDI calculated in the detected positive sample for OTA exceeded the TDI value. The findings of the present study confirm the presence of the studied mycotoxins and their metabolites as some of the most prevalent in urine.

The mycotoxins aflatoxin B 1 (AFB 1 ) and deoxynivalenol (DON) are found worldwide in crops and dietary staples. The prevalence and levels of these contaminants can vary greatly, and data in Bangladeshi food commodities are scarce. To characterize human exposure, we have conducted biomonitoring, analyzing AFM 1 (a metabolite of AFB 1 ) and DON levels in urines of adult cohorts in Bangladesh. Yet, AFM 1 and DON occurrence has not been studied in the very young population of this country. Thus, the same methods, HPLC-FD for AFM 1 and LC-MS/MS for DON analysis, were now applied to determine these biomarkers in urines of infants (n = 49) and young children (n = 105) in Rajshahi and Dhaka district. Overall, AFM 1 and DON detection frequency was 43.5% and 33.4%, with 34.7% and 11.5% in infant and 47.6% and 39.4% in children urines, respectively. The mean AFM 1 levels in all infants (9.1 ± 14.3, max 55.6 pg/mL) and children (8.8 ± 12.9, max 75.3 pg/mL) were not significantly different. The AFM 1 mean level was slightly higher in Dhaka (9.4 ± 12.4) compared to Rajshahi (8.5 ± 13.9 pg/mL) district. The average DON level was about 2-fold higher in infant (3.8 ± 2.9, max 6.8 ng/mL) than children urines (1.6 ± 1.8, max 8.6 ng/mL), and higher in Rajshahi (2.1 ± 2.3 ng/mL) than Dhaka (1.4 ± 1.6 ng/mL) district. The biomarker-based estimated average daily DON intake (29.6 ± 108.3 ng/kg bw in infants and 36.4 ± 81.8 ng/kg bw in children) or the maximum exposure (560 ng/kg bw) do not exceed the current maximum provisional tolerable daily intake value of 1 µg/kg bw for DON, although DON exposure in infants and children is higher than that of Bangladeshi adults. The AFM 1 urine levels in young children are somewhat lower than those found previously in adult cohorts in Bangladesh, but the frequent detection of this biomarker for AFB 1 exposure raises further concerns, also for this vulnerable part of the population. Therefore, continuous surveillance for aflatoxins in Bangladeshi food commodities is clearly required, first to identify major sources of intake and then to reduce exposure.

There are limited data on exposure to mycotoxins in Pakistan. Here, we measured exposure to deoxynivalenol (DON), a common contaminant of wheat, and aflatoxin B 1 (AFB 1 ), a known contaminant of rice, using biomarkers of exposure. Wheat ( n = 195) and rice ( n = 62) samples were analyzed for AFB 1 and DON levels, and the corresponding urinary biomarkers were analyzed in urine samples from a rural population ( n = 264, aged 4-80 years, male 58%) using ultra-sensitive liquid chromatography-tandem mass spectrometry. AFB 1 was detected in 66% of rice (5.04 ± 11.94 µg/kg) and 3% of wheat samples. AFM 1 (hydroxylated form of AFB 1 )was detected in 69% of urine samples, mean 0.023 ± 0.048 ng/mL and DON was detected in 20% of urine samples, mean 0.170 ± 0.129 ng/mL. The maximum probable daily intake for DON derived from the urinary biomarker was 59.8 ng/kg b.w./day, which is below the Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives' tolerable daily intake (1000 ng/kg b.w./day). However, for aflatoxin, the derived margin of exposure (MoE) of (13.2) was well below the safe MoE (10,000) suggested by the European Food Safety Authority. The calculated aflatoxin-associated cancer risk of 0.514/10 5 individuals/year suggests that measures should be taken to reduce the AFB 1 contamination in food, particularly rice, in Pakistan.

Deoxynivalenol is one of the most ubiquitous mycotoxins in the Western diet through its presence in cereals and cereal products. A vast amount of studies indicate the worrying level of exposure to this toxin, while even high percentages of the population exceed the tolerable daily intake. To evaluate and assess dietary exposure, analysis of urinary levels of deoxynivalenol and its glucuronides has been proposed as a reliable methodology. An indirect preliminary method was used based on the cleavage of deoxynivalenol glucuronides through the use of enzymes (β-glucuronidase) and subsequent determination of "total deoxynivalenol" (sum of free and released mycotoxins by hydrolysis). Next, a direct procedure for quantification of deoxynivalenol-3-glucuronide and deoxynivalenol-15-glucuronide was developed. As deoxynivalenol glucuronides reference standards are not commercially available, the indirect method is widely applied. However, to not underestimate the total deoxynivalenol exposure in urine, the direct and indirect methodologies need to be compared. Urinary samples (n = 96) with a confirmed presence of deoxynivalenol and/or deoxynivalenol glucuronides were analysed using both approaches. The indirect method clarified that not all deoxynivalenol glucuronides were transformed to free deoxynivalenol during enzymatic treatment, causing an underestimation of total deoxynivalenol. This short communication concludes on the application of direct or indirect assessment of urinary deoxynivalenol., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Cereal foods are commonly contaminated with multiple mycotoxins resulting in frequent human mycotoxin exposure. Children are at risk of high-level exposure because of their high cereal intake relative to body weight. Hence, this study aims to assess multimycotoxin exposure in UK children using urinary biomarkers. Spot urines ( n = 21) were analyzed for multimycotoxins (deoxynivalenol, DON; nivalenol, NIV; ochratoxin A, OTA; zearalenone, ZEN; α-zearalenol, α-ZEL; β-zearalenol, β-ZEL; T-2 toxin, T-2; HT-2 toxin, HT-2; and aflatoxin B 1 and M 1, AFB 1 , AFM 1 ) using liquid chromatography-coupled tandem mass spectrometry. Urine samples frequently contained DON (13.10 ± 12.69 ng/mL), NIV (0.36 ± 0.16 ng/mL), OTA (0.05 ± 0.02 ng/mL), and ZEN (0.09 ± 0.07 ng/mL). Some samples (1-3) contained T-2, HT-2, α-ZEL, and β-ZEL but not aflatoxins. Dietary mycotoxin estimation showed that children were frequently exposed to levels exceeding the tolerable daily intake (52 and 95% of cases for DON and OTA). This demonstrates that UK children are exposed to multiple mycotoxins through their habitual diet.

Biomarkers for the determination of the dietary exposure to deoxynivalenol (DON) have been proposed in the past but so far no quantification of their use in humans has been carried out. Following a human intervention study with two mycotoxins, namely DON and deoxynivalenol-3-glucoside (DON3G), the renal excretion of these compounds, including their phase II metabolites, was analysed. The purpose was to develop biokinetic models that can be used to determine: (1) the preferred (set of) urinary biomarker(s), (2) the preferred urinary collection period, and (3) a method to estimate the dietary exposure to these mycotoxins. Twenty adult volunteers were restricted in consuming cereals and cereal-based foods for 4 days. At day 3, a single dose of 1 µg/kg body weight of DON or DON3G was orally administered to 16 volunteers; 4 volunteers served as control. All individual urine discharges were collected during 24 h after administration. The metabolism and renal excretion could be described by a biokinetic model using three physiological compartments (gastrointestinal tract, liver, and kidneys). Kinetic analysis revealed a complete recovery of the renal excretion of total DON (mainly DON and its glucuronides) within 24 h after administration of DON or DON3G. The so-called 'reverse dosimetry' factor was used to determine the preferred (set of) biomarker(s) and to estimate the dietary intake of the parent compounds in the future. The fact that DON3G was absorbed and mainly excreted as DON and its glucuronides confirms that DON3G (as well as other modified forms) should be taken into account in the exposure and risk assessment of this group of mycotoxins.

Deoxynivalenol (DON) is a mycotoxin mainly produced by Fusarium graminearum that can contaminate cereals and cereal-based foodstuff. Urinary DON levels can be used as biomarker for exposure assessment purposes. This study assessed urinary DON concentrations in Italian volunteers recruited by age group, namely children, adolescents, adults, and the elderly. In addition, vulnerable groups, namely vegetarians and pregnant women, were included in the study. To determine the urinary DON, its glucuronide and de-epoxydated (DOM-1) forms, an indirect analytical approach was used, measuring free DON and total DON (as sum of free and glucuronides forms), before and after enzymatic treatment, respectively. Morning urine samples were collected on two consecutive days, from six different population groups, namely children, adolescent, adults, elderly, vegetarians and pregnant women. Total DON was measured in the 76% of the collected samples with the maximum incidences in children and adolescent age group. Urine samples from children and adolescent also showed the highest total DON levels, up to 17.0 ng/mg creat . Pregnant women had the lowest positive samples per category (40% for day 1 and 43% for day 2, respectively), low mean levels of total DON (down to 2.84 ng/mg creat ) and median equal to 0 ng/mg creat . Estimation of DON dietary intake reveals that 7.5% of the total population exceeds the TDI of 1 μg/kg bw/day set for DON, with children showing 40% of individuals surpassing this value (male, day 2)., Competing Interests: The authors declare no conflict of interest.

The main aim of this research was to evaluate the toxicokinetic characteristics of fusarenon-X (FX) and its metabolite, nivalenol (NIV), in goats. The amounts of FX and NIV in post-mitochondrial (S-9), microsomal and cytosolic fractions of diverse tissues of the goat were also investigated. FX was intravenously (iv) or orally (po) administered to goats at dosages of 0.25 and 1 mg/kg bw, respectively. The concentrations of FX and NIV in plasma, feces and urine were quantified by liquid chromatography tandem-mass spectrometry (LC-ESI-MS/MS). The concentrations of FX in plasma were quantified up to 8 h with both routes of administration. A large amount of NIV (metabolite) was quantifiable in plasma, urine and feces after both administrations. The C max value of FX was 413.39 ± 206.84 ng/ml after po administration. The elimination half-life values were 1.64 ± 0.32 h and 4.69 ± 1.25 h after iv and po administration, respectively. In vitro experiments showed that the conversion FX-to-NIV mainly occurs in the liver microsomal fraction. This is the first study that evaluates the fate and metabolism of FX in ruminant species., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

For the first time, a comprehensive human intervention study was conducted to unravel the urinary excretion profile and metabolism of the fungal metabolite deoxynivalenol (DON) and its modified form deoxynivalenol-3-glucoside (DON-3-glucoside). Twenty volunteers were restricted in consuming cereals and cereal-based foods for 4 days. At day 3, a single bolus of 1 µg/kg body weight of DON and a single bolus of 1 µg/kg body weight of DON-3-glucoside after a washing-out period of two months was administered, and a 24-h urine collection was performed. The urine was analysed for DON, DON-3-glucoside, 3-ADON, 15-ADON, deepoxy-deoxynivalenol (DOM-1), deoxynivalenol-3-glucuronide (DON-3-glucuronide) and deoxynivalenol-15-glucuronide (DON-15-glucuronide). The urinary biomarker-analysis revealed that DON and DON-3-glucoside were rapidly absorbed, distributed, metabolized and excreted. Sixty-four % of the administered DON and 58% of DON-3-glucoside was recovered in the urine collected within 24 h. DON-15-glucuronide was the most prominent urinary biomarker followed by free DON and DON-3-glucuronide. Moreover, correlations among the presence of DON-15-glucuronide and DON-3-glucuronide were observed (within 24 hours (r = 0.61)). The DOM-1 detected in the urine was higher after the DON-3-glucoside administration. The obtained results are imperative to construct a standardized method to estimate DON-intake by means of urinary biomarkers.

A risk assessment of deoxynivalenol (DON) was recently conducted for the residents in Henan province, China, where wheat as the staple food are highly consumed. A high-throughput sensitive UPLC-MS/MS method following 96-well μElution solid-phase extraction (SPE) were developed and validated for the determination of DON biomarkers in human urine. Isotope labelled internal standard, 13 C-DON, was used for accurate quantification. Urinary samples collected from 151 healthy Chinese aged 2-78 years were processed with and without enzyme hydrolysis to determine total and free biomarkers, respectively. DON, and de-epoxy-deoxynivalenol (DOM-1) to a lesser extent, can be frequently detected in these samples both with and without enzyme hydrolysis. Free DOM-1 was detected at low level in human urine for the first time. Total DON was detected in all samples with a mean concentration at 47.6 ng mL -1 . The mean and median probable daily intakes (PDI) for the whole participants, estimated to be 1.61 μg/kg bw and 1.10 μg/kg bw, both exceeded the PMTDI (1 μg/kg bw/day), indicating a potential risk for the residents in this area, especially for children and adolescents.

A feeding experiment with piglets was performed to examine the efficacy of a wet preservation of Fusarium (FUS)-contaminated maize with sodium sulphite (SoS) based on deoxynivalenol (DON) and zearalenone (ZEN) residue levels in urine, bile and liquor and health traits of piglets. For this purpose, 80 castrated male piglets (7.57 ± 0.92 kg BW) were assigned to four treatment groups: CON- (control diet, with 0.09 mg DON and <0.01 mg ZEN/kg diet), CON+ (diet CON-, wet-preserved with 5 g SoS/kg maize; containing 0.05 mg DON and <0.01 mg ZEN/kg diet), FUS- (diet with mycotoxin-contaminated maize; containing 5.36 mg DON and 0.29 mg ZEN/kg diet), and FUS+ (diet FUS-, wet-preserved with 5 g SoS/kg maize; resulting in 0.83 mg DON and 0.27 mg ZEN/kg diet). After 42 d, 40 piglets (n = 10 per group) were sampled. A clear reduction of DON levels by approximately 75% was detected in all specimens of pigs fed diet FUS+. ZEN was detected in all urine, bile and liquor samples, while their metabolites were only detectable in urine and bile. Additionally, their concentrations were not influenced by SoS treatment. Among the health-related traits, feeding of FUS diets increased the total counts of leukocytes and segmented neutrophil granulocytes irrespective of SoS treatment. SoS treatment increased the total blood protein content slightly with a similar numerical trend in albumin concentration. These effects occurred at an obviously lower level in FUS-fed groups. Moreover, SoS treatment recovered the reduction of NO production induced by feeding diet FUS- indicating an effect on the redox level. As this effect only occurred in group FUS+, it is obviously related to the adverse effects of the Fusarium toxins. In conclusion, treatment of FUS-contaminated maize with SoS decreased the inner exposure with DON as indicated by the lower DON levels in various piglet specimens. However, health-related traits did not consistently reflect this decreased exposure.

Deoxynivalenol (DON), the mycotoxin produced mainly by Fusarium graminearum and found in contaminated cereal-based foodstuff, has been consistently detected in body fluids in adults. Available data in children and adolescents are scarce. This study assessed urinary DON concentrations in children aged 3-9 years ( n = 40) and adolescents aged 10-17 years ( n = 39) in the UK. Morning urine samples were collected over two consecutive days and analysed for free DON (un-metabolised form), DON-glucuronides (DON-GlcA), deepoxy deoxynivalenol (DOM-1), and total DON (sum of free DON, DON-GlcA, and DOM-1). Total DON was detected in the urine of >95% of children and adolescents on both days. Mean total DON concentrations (ng/mg creatinine) were 41.6 and 21.0 for children and adolescents, respectively. The greatest total DON levels were obtained in female children on both days (214 and 219 ng/mg creatinine on days 1 and 2, respectively). Free DON and DON-GlcA were detected in most urine specimens, whereas DOM-1 was not present in any sample. Estimation of dietary DON exposure suggested that 33-63% of children and 5-46% of adolescents exceeded current guidance regarding the maximum provisional tolerable daily intake (PMTDI) for DON. Although moderate mean urinary DON concentrations were shown, the high detection frequency of urinary DON, the maximum biomarker concentrations, and estimated dietary DON exposure are concerning., Competing Interests: The authors declare no conflict of interest. EFSA disclaimer: EFSA is not responsible for the views displayed in the publications and/or in conjunction with the activities for which the grant is used. The funding sponsors had a role in the design of the study; Clear information regarding the required design of the study was given in EFSAs call for proposal. Further details can be found at: .

The Fusarium mycotoxin deoxynivalenol (DON) is a frequent contaminant of cereal-based food and feed. Mammals metabolize DON by conjugation to glucuronic acid (GlcAc), the extent and regioselectivity of which is species-dependent. So far, only DON-3-glucuronide (DON-3-GlcAc) and DON-15-GlcAc have been unequivocally identified as mammalian DON glucuronides, and DON-7-GlcAc has been proposed as further DON metabolite. In the present work, qualitative HPLC-MS/MS analysis of urine samples of animals treated with DON (rats: 2 mg/kg bw, single bolus, gavage; mice: 1 mg/kg bw, single i.p. injection; pigs: 74 µg/kg bw, single bolus, gavage; cows: 5.2 mg DON/kg dry mass, oral for 13 weeks) revealed additional DON and deepoxy-DON (DOM) glucuronides. To elucidate their structures, DON and DOM were incubated with human (HLM) and rat liver microsomes (RLM). Besides the expected DON/DOM-3- and 15-GlcAc, minor amounts of four DON- and four DOM glucuronides were formed. Isolation and enzymatic hydrolysis of four of these compounds yielded iso-DON and iso-DOM, the identities of which were eventually confirmed by NMR. Incubation of iso-DON and iso-DOM with RLM and HLM yielded two main glucuronides for each parent compound, which were isolated and identified as iso-DON/DOM-3-GlcAc and iso-DON/DOM-8-GlcAc by NMR. Iso-DON-3-GlcAc, most likely misidentified as DON-7-GlcAc in the literature, proved to be a major DON metabolite in rats and a minor metabolite in pigs. In addition, iso-DON-8-GlcAc turned out to be one of the major DON metabolites in mice. DOM-3-GlcAc was the dominant DON metabolite in urine of cows and an important DON metabolite in rat urine. Iso-DOM-3-GlcAc was detected in urine of DON-treated rats and cows. Finally, DON-8,15-hemiketal-8-glucuronide, a previously described by-product of DON-3-GlcAc production by RLM, was identified in urine of DON-exposed mice and rats. The discovery of several novel DON-derived glucuronides in animal urine requires adaptation of the currently used methods for DON-biomarker analysis.

Deoxynivalenol (DON) exposure is estimated by the combined measures of urinary DON and DON-glucuronides. In this study, data from single-mycotoxin (SM) and a multimycotoxin (MM) methods were compared for 256 Swedish adult urine samples. Both methods included β-glucuronidase predigestion, immunoaffinity enrichment, and LC-MS/MS. However, the specific reagents, apparatus, and conditions were not identical in part because the MM method measures additional mycotoxins. DON was detected in 88 and 63% of samples using the SM and MM methods, respectively, with the following mean and median concentrations: SM, mean = 5.0 ng/mL, SD = 7.4, range of positives = 0.5-60.2 ng/mL, median = 2.5 ng/mL, IQR = 1.0-5.5 ng/mL; MM, mean = 4.4 ng/mL, SD = 12.9, range of positives = 0.5-135.2 ng/mL, median = 0.8 ng/mL, IQR = 0.3-3.5. Linear regression showed a significant, albeit modest, correlation between the two measures (p = 0.0001, r = 0.591). The differences observed may reflect subtle handling differences in DON extraction and quantitation between the methods.

Based on prior observations that deoxynivalenol (DON) toxicity is sex-dependent, we compared metabolism and clearance of this toxin in male and female mice. Following intraperitoneal challenge with 1 mg/kg bw DON, the dose used in the aforementioned toxicity study, ELISA and LC-MS/MS analyses revealed that by 24 h, most DON and DON metabolites were excreted via urine (49-86%) as compared to feces (1.2-8.3%). Females excreted DON and its principal metabolites (DON-3-, DON-8,15 hemiketal-8-, and iso-DON-8-glucuronides) in urine more rapidly than males. Metabolite concentrations were typically 2 to 4 times higher in the livers and kidneys of males than females from 1 to 4 h after dosing. Trace levels of DON-3-sulfate and DON-15-sulfate were found in urine, liver and kidneys from females but not males. Fecal excretion of DON and DON sulfonates was approximately 2-fold greater in males than females. Finally, decreased DON clearance rates in males could not be explained by glucuronidation activities in liver and kidney microsomes. To summarize, increased sensitivity of male mice to DON's toxic effects as compared to females corresponds to decreased ability to clear the toxin via urine but did not appear to result from differences in toxin metabolism., Competing Interests: The authors declare no conflict of interest.

Deoxynivalenol (DON) is produced by Fusarium graminearum and is one of the most commonly occurring trichothecenes. Vegetarians are alleged to be a high-risk group for DON exposure due to high intakes of cereals susceptible to the growth of the mycotoxin. This study provides the levels of DON and de-epoxi Deoxynivalenol (DOM-1) in urine analysed by liquid chromatography-mass spectrometry (LC-MS) in UK vegetarians. Over two consecutive days, morning urine samples were collected from 32 vegetarians and 31 UK adult volunteers, and associated food consumption 24 h prior to the sample was recorded. Statistically significant differences between the weight of the UK adults and vegetarians ( t = 3.15. df = 61, p ≤ 0.005 two-tailed) were observed. The mean levels of DON in urine for adults on day 1 was 3.05 ng free DON/mg creatinine, and on day 2 was 2.98 ng free DON/mg creatinine. Even though high mean levels were observed, most adults were within the tolerable daily intake. However, for vegetarians, the mean level of urinary DON on day 1 was 6.69 ng free DON/mg creatinine, and on day 2 was 3.42 ng free DON/mg creatinine. These levels equate to up to 32% of vegetarians exceeding recommended tolerable daily intakes (TDI) of exposure (1 µg/kg b.w./day)., Competing Interests: The authors declare no conflict of interest. EFSA disclaimer: EFSA is not responsible for the views displayed in the publications and/or in conjunction with the activities for which the grant is used. The funding sponsors had a role in the design of the study; Clear information regarding the required design of the study was given in EFSA’s call for proposal. Further details can be found at .

Two experiments were conducted to evaluate potential detoxifying agents on growth of nursery pigs fed deoxynivalenol (DON)-contaminated diets. Naturally DON-contaminated wheat (6 mg/kg) was used to achieve desired DON levels. In a 21-d study, 238 pigs (13.4 ± 1.8 kg BW) were used in a completely randomized design with a 2 × 2 + 1 factorial arrangement. Diets were: 1) Positive control (PC; < 0.5 mg/kg DON), 2) PC + 1.0% Product V (Nutriquest LLC, Mason City, IA), 3) Negative control (NC; 4.0 mg/kg DON), 4) NC + 1.0% Product V, and 5) NC + 1.0% sodium metabisulfite (SMB; Samirian Chemicals, Campbell, CA). There were 6 or 7 replicate pens/treatment and 7 pigs/pen. Analyzed DON was decreased by 92% when pelleted with SMB, but otherwise matched formulated levels. Overall, a DON × Product V interaction was observed for ADG ( 0.05) with a tendency for an interaction for ADFI ( 0.10). As anticipated, DON reduced ( 0.001) ADG and ADFI, but the interaction was driven by even poorer growth when Product V was added to NC diets. Pigs fed NC diets had 10% poorer G:F ( 0.001) than PC-fed pigs. Reductions in ADG due to DON were most distinct (50%) during the initial period. Adding SMB to NC diets improved ( 0.01) ADG, ADFI, and G:F, and improved ( 0.02) ADG and G:F compared to the PC diet. A urinary balance study was conducted using diets 3 to 5 from Exp. 1 to evaluate Product V and SMB on DON urinary metabolism. A 10 d adaptation was followed by a 7 d collection using 24 barrows in a randomized complete block design. Pigs fed NC + SMB diet had greater urinary DON output ( 0.05) than pigs fed NC + Product V, with NC pigs intermediate. Daily DON excretion was lowest ( 0.05) in the NC + SMB pigs. However, degradation of DON-sulfonate back to the parent DON molecule was observed as pigs fed NC + SMB excreted more DON than they consumed (164% of daily DON intake), greater ( 0.001) than pigs fed the NC (59%) or NC + Product V (48%). Overall, Product V did not alleviate DON effects on growth nor did it reduce DON absorption and excretion. However, hydrothermally processing DON-contaminated diets with 1.0% SMB restored ADFI and improved G:F. Even so, the urinary balance experiment revealed that some of the converted DON-sulfonate can degrade back to DON under physiological conditions. While further research is needed to discern the stability of the DON-sulfonate, SMB appears promising to restore performance in pelleted DON-contaminated diets.

Urinary biomarkers of mycotoxin exposure were evaluated in a group of celiac patients ( n = 55) and in a control group of healthy subjects ( n = 50) following their habitual diet. Deoxynivalenol (DON), zearalenone (ZEN), and fumonisin B1 (FB1) were monitored in 105 urinary samples collected from the two groups. Dietary habits were also recorded through compilation of a seven-day weighed dietary diary. Biomarkers of mycotoxin exposure were detected in 21 celiac patients and in 15 control subjects, corresponding to about 34% of total participants. In particular, ZEN was the most detected mycotoxin among all the studied subjects with a total of 19 positive cases. Results did not show a statistically significant difference in mycotoxin exposure between the two groups, and the presence of specific mycotoxins was not related to the intake of any particular food category. Our findings suggest little urgency of specific regulation for gluten free products, although the prevalence of exposure observed in free-living diets of both celiac and healthy subjects underlines the need of a constant surveillance on mycotoxins occurrence at large., Competing Interests: The authors declare no conflict of interest.

Deoxynivalenol (DON) is one of the most commonly occurring trichothecenes, produced mainly by Fusarium graminearum . Little is known about the effect of DON exposure or the levels of DON exposure that occur during pregnancy. The project aimed to provide data on levels of total DON and de-epoxi Deoxynivalenol (DOM-1) in pregnant human urine samples analysed by liquid chromatography-mass spectrometry (LC-MS). Morning urine samples were collected over two consecutive days from 42 volunteers and associated food consumption was recorded for the 24 h prior to the sample. Spearman's rho non-parametric test for correlation was used to assess the data. Levels of DON did not differ significantly between day 1 (mean 29.7 ng/mL urine or 40.1 ng DON/mg creatinine) and day 2 (mean 28.7 ng/mL urine or 38.8 ng DON/mg creatinine ng/mL/day) urine samples. The only significant positive correlation was found between total ng DON/mg creatinine and parity (rho = 0.307, n = 42, p < 0.005 two-tailed) and total ng DON/mg creatinine with baked goods on day 1 (rho = 0.532, n = 42, p < 0.0005 two-tailed). This study provides data on the DON levels in pregnancy in this suburban population and reassurance that those levels are within acceptable limits., Competing Interests: The authors declare no conflict of interest. EFSA disclaimer: EFSA is not responsible for the views displayed in the publications and/or in conjunction with the activities for which the grant is used. The funding sponsors had a role in the design of the study; Clear information regarding the required design of the study was given in EFSAs call for proposal. Further details can be found at http://www.efsa.europa.eu/en/art36grants/article36/gpefsacontam201304.

Deoxynivalenol (DON) is a frequent mycotoxin contaminant in cereal crops worldwide and can cause adverse health effects in exposed animals and humans. Since DON contamination in Bangladeshi food is unexplored, we conducted a biomonitoring study to assess DON exposure in the Bangladeshi population and compare it with that of German adults. In total 214 urines were collected, n=164 in Bangladesh and n=50 in Germany. In Bangladesh rural and urban residents of Rajshahi district provided urines in two seasons (n=69 in summer, n=95 in winter, with 62 participants enrolled in both periods). Urinary DON and its de-epoxy metabolite DOM-1 were measured by a previously validated sensitive LC-MS/MS method. In Bangladeshi urines, DON was detectable in 27% (range 0.16-1.78ng/mL) in summer and 31% (range 0.16-1.21ng/mL) in winter season. There was no significant difference at the mean DON level between season (summer 0.17±0.25ng/mL and winter 0.16±0.18ng/mL) and region (rural or urban residents). The metabolite DOM-1 was not detected in any urine from Bangladesh. In contrast, DON and DOM-1 were detected in 100% (range 0.16-38.44ng/mL) and 40% (range 0.10-0.73ng/mL), respectively, of the German urines. The mean DON level in German urines (9.02±6.84ng/mL) was about 53-fold higher than that found in Bangladeshi samples. This indicates a low and high dietary DON exposure among the adult population in Bangladesh and Germany, respectively. The biomarker concentrations found and published urinary excretion rates for DON then served to calculate the daily mycotoxin intake in both cohorts: the mean DON intake in Bangladesh being 6ng/kg b.w., and in Germany a mean of 268 and maximum intake of 975ng/kg b.w., values lower than the provisional maximum tolerable daily intake of 1μg/kg b.w. set by the WHO/JECFA., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Evaluation of environmental risk factors in the development of autism spectrum disorder (ASD) is needed for a more complete understanding of disease etiology and best approaches for prevention, diagnosis, and treatment. A pilot experiment in 54 children (n = 25 ASD, n = 29 controls; aged 12.4 ± 3.9 years) screened for 87 urinary mycotoxins via liquid chromatography-tandem mass spectrometry to assess current exposure. Zearalenone, zearalenone-4-glucoside, 3-acetyldeoxynivalenol, and altenuene were detected in 9/54 (20%) samples, most near the limit of detection. No mycotoxin/group of mycotoxins was associated with ASD-diagnosed children. To identify potential correlates of mycotoxin presence in urine, we further compared the nine subjects where a urinary mycotoxin was confirmed to the remaining 45 participants and found no difference based on the presence or absence of mycotoxin for age (t-test; p = 0.322), gender (Fisher's exact test; p = 0.456), exposure or not to selective serotonin reuptake inhibitors (Fisher's exact test; p = 0.367), or to other medications (Fisher's exact test; p = 1.00). While no positive association was found, more sophisticated sample preparation techniques and instrumentation, coupled with selectivity for a smaller group of mycotoxins, could improve sensitivity and detection. Further, broadening sampling to in utero (mothers) and newborn-toddler years would cover additional exposure windows.

Four diets contaminated with 1.1 to 5.0 mg/kg deoxynivalenol (DON) and 0.4 to 2.4 mg/kg zearalenone (ZEA) were fed to four groups of six growing Large White pigs. Urine samples were collected after 3 to 4 days and again after 6 to 7 days on the diets. On each sampling day, half of the animals were sampled in the morning, after an 8-h fast, and the other half were sampled in the afternoon, after 7 h of ad libitum access to feed. The urinary concentrations of DON, DON-glucuronide, DON-3-sulphate, de-epoxy-DON, as well as of ZEA, ZEA-14-glucuronide, α-zearalenol and α-zearalenol-14-glucuronide, analysed using LC-MS/MS, were used to calculate urinary DON and ZEA equivalent concentrations (DONe and ZEAe). The urinary concentration of DONe (P < 0.001), but not of ZEAe (P = 0.31), was lower in the fasted than that in the fed animals. The urinary DONe/creatinine and ZEAe/creatinine ratios were highly correlated with DON and ZEA intake per kg body weight the day preceding sampling (r = 0.76 and 0.77; P < 0.001). The correlations between DON intake during the 7 h preceding urine sampling in the afternoon and urinary DONe/creatinine ratio (r = 0.88) as well as between mean ZEA intake during 3 days preceding urine sampling and urinary ZEAe/creatinine ratio (r = 0.84) were even higher, reflecting the plasma elimination half-time of several hours for DON and of more than 3 days for ZEA. ZEAe analysed in enzymatically hydrolysed urine using an ELISA kit was highly correlated with the LC-MS/MS data (r = 0.94). The urinary DONe and ZEAe to creatinine ratios, analysed in pooled urine samples of several pigs fed the same diet, can be used to estimate their exposure to DON and ZEA.

The aim of this study was to determine the levels of selenium, T-2 toxin, and deoxynivalenol (DON) contamination in Kashin-Beck disease (KBD) areas and provide information for understanding the high prevalence of KBD in Qinghai Province. A total of 183 subjects were chosen in a KBD-prevalent county (Guide County) and a non-KBD county (Huangzhong County) in Qinghai Province, northwestern China, and the samples of wheat flour, soil, drinking water and blood, urine, and hair of children were collected from these residents. The selenium concentrations from all these sources were determined using atomic fluorescence spectrophotometry. The levels of T-2 toxin and DON contamination in the wheat flour samples were assayed using HPLC-MS/MS. The average selenium content in the soil, drinking water, and wheat flour samples from KBD areas were 26.93 ± 10.06 μg/kg, 0.097 ± 0.038 μg/L, and 9.50 ± 7.17 μg/kg, respectively. Among these, the selenium levels in the drinking water and wheat flour samples from the KBD endemic county were significantly lower than those from the non-KBD county. For the selenium nutrient status, only the hair selenium concentration of children from the KBD endemic county was significantly lower than that from the non-KBD county. The contents of T-2 toxin in all wheat samples were below the detection limit (0.4 μg/kg). The levels of DON contamination in wheat flour samples from KBD and non-KBD children's households within the KBD endemic county were relatively higher, with average levels of 302 ± 49 and 280 ± 48 μg/kg, respectively. The DON level of wheat flour samples from the children's households in the non-KBD county was significantly lower than that from the KBD endemic county. These results suggest that the lower selenium status in Guide County still remains. While the selenium nutritional status of the local children has improved to some extent, partly due to the introduction of food produce from nonlocal areas. DON contamination in the wheat flour may be involved in the fluctuating high prevalence rates of KBD in children in the KBD endemic Guide County in Qinghai Province.

A direct, fast and sensitive LC-MS/MS method was developed to measure biomarkers for mycotoxin exposure in human urine. In total, 32 biomarkers were quantitatively or semi-quantitatively measured in 32 urine samples of Belgian volunteers using two injections. All urine samples contained deoxynivalenol-15-glucuronide, the major detoxification metabolite of deoxynivalenol, in the ng/mL range. Also deoxynivalenol-3-glucuronide and de-epoxy-deoxynivalenol-glucuronide were present in, respectively, 90 and 25% of the samples, while deoxynivalenol was detected in 60% of the samples, in lower concentrations. Deoxynivalenol glucuronides were the major biomarkers for deoxynivalenol exposure. Ochratoxin A was detected in 70% of the samples in pg/mL. Citrinin and/or dihydrocitrinone were detected in 90% of the samples, also in concentrations of pg/mL. The presence of ochratoxin A and citrinin was confirmed by a second method using sample cleanup by immunoaffinity columns, followed by LC-MS/MS. Our data show that humans are much more exposed to citrinin than realized before and suggest further work on citrinin exposure in relation with ochratoxin A exposure, as both mycotoxins are nephrotoxic.

Mycotoxins are important food contaminants responsible for health effects such as cancer, nephrotoxicity, hepatotoxicity or immunosuppression. The assessment of mycotoxin exposure is often based on calculations combining mycotoxin occurrence data in food with population data on food consumption. Because of limitations inherent to that approach, the direct measurement of biomarkers of exposure in biological fluids has been proposed as a suitable alternative to perform an accurate mycotoxin exposure assessment at individual level. For this reason, the BIOMYCO study was designed to assess mycotoxin exposure in Belgian adults and children using urinary biomarkers of exposure. Morning urine was gathered in a representative part of the Belgian population according to a standardised study protocol, whereby 155 children (3-12 years old) and 239 adults (19-65 years old) were selected based on random cluster sampling. These urine samples were analysed for the presence of 33 potential biomarkers with focus on aflatoxins, citrinin (CIT), fumonisins, trichothecenes, ochratoxin A (OTA), zearalenone and their metabolites using two validated LC-MS/MS methods. Nine out of the 33 analysed mycotoxins were detected whereby deoxynivalenol (DON), OTA, CIT and their metabolites were the most frequently detected. Deoxynivalenol-15-glucuronide was the main urinary DON biomarker and was found in all urine samples in the ng/mL range. Furthermore deoxynivalenol-3-glucuronide was quantified in 91% of the urine samples collected from children and in 77% of the samples collected from adults. Deoxynivalenol was detected in 70% and 37% of the samples of children and adults respectively. For the first time deepoxy-deoxynivalenol-glucuronide was detected in children's urine (17%). In the samples collected by adults, the prevalence was 22%. Whereas all these mycotoxins contaminated the urine samples in the ng/mL range, CIT and OTA were present in much lower concentrations (pg/mL). OTA contaminated 51% and 35% of the samples collected by children and adults respectively. CIT and its metabolite were present in 72% and 6% of children's urine, whereas they contaminated 59% and 12% of adult's urine. Finally, α-zearalenol and β-zearalenol-14-glucuronide were found in respectively one and two samples from adults. The exposure to DON, OTA and CIT was compared between subgroups and urinary mycotoxin concentrations differed significantly among age and gender. Based on the urinary levels, the daily intake of DON and OTA was estimated and evaluated whereby, depending on the used method, 16-69% of the population possibly exceeded the tolerable daily intake for DON and 1% for OTA. The BIOMYCO study is the first study whereby a multi-toxin approach was applied for mycotoxin exposure assessment in adults and children on a large-scale. Moreover, it is the first study that described the exposure to an elaborated set of mycotoxins in the Belgian population. Biomarker analysis showed a clear exposure of a broad segment of the Belgian population to DON, OTA and CIT. The risk assessment based on these data indicates a potential concern for a number of individuals whereby young children need special attention because of the relatively higher food intake per kg body weight., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The trichothecene mycotoxin deoxynivalenol (DON) is a contaminant of crops worldwide and known to cause adverse health effects in exposed animals and humans. A small survey reported the presence of DON in maize samples in Bangladesh, but these data are insufficient to assess human exposure, and also, biomonitoring data are still scarce. The present study applied biomarker analysis to investigate the DON exposure of pregnant women in Bangladesh. Urine samples were collected from pregnant women living in a rural (n = 32) and in a suburban (n = 22) area of the country. Urines were subjected to enzymatic hydrolysis of glucuronic acid conjugates and to immunoaffinity column clean-up prior to LC-MS/MS analysis of DON and its de-epoxy metabolite DOM-1. The limits of detection (LOD) for DON and DOM-1 in urine were 0.16 ng/mL and 0.10 ng/mL, respectively. DOM-1 was not detected in any of the urines, whilst DON was detectable in 52% of the samples at levels ranging from 0.18-7.16 ng/mL and a mean DON concentration of 0.86 ± 1.57 ng/mL or 2.14 ± 4.74 ng/mg creatinine. A significant difference in mean urinary DON levels was found between the rural (0.47 ± 0.73 ng/mL) and suburban (1.44 ± 2.20 ng/mL) cohort, which may be related to different food habits in the two cohorts. Analysis of food consumption data for the participants did not show significant correlations between their intake of typical staple foods and DON levels in urine. The biomarker concentrations found and published urinary excretion rates for DON were used to estimate daily mycotoxin intake in the cohort: the mean DON intake was 0.05 µg/kg b.w., and the maximum intake was 0.46 µg/kg b.w., values lower than the tolerable daily intake of 1 µg/kg b.w. These first results indicate a low dietary exposure of pregnant women in Bangladesh to DON. Nonetheless, further biomonitoring studies in children and in adult cohorts from other parts of the country are of interest to gain more insight into DON exposure in the population of Bangladesh.

Mycotoxin producing moulds may contaminate numerous agricultural commodities either before harvest or during storage. A varied diet consisting of different foods may therefore be contaminated with a range of mycotoxins. The aim of the present study was to study concurrent exposure to mycotoxins through urinary multi-biomarker analysis, as well as its possible associations with the diet. Urinary samples from 252 adults, participating in the Swedish national dietary survey Riksmaten 2010-11, were collected together with a 4-day diet record. Concurrent mycotoxin exposure was studied using a multi-biomarker LC-MS/MS method. The results revealed that exposure to mycotoxins is common and concurrent exposure to more than one toxin was found in 69% of the study population. However, when comparing the number of toxins detected with the reported consumption data it was difficult to distinguish food patterns which would indicate an increased risk of exposure to many mycotoxins simultaneously. This is the first study to investigate concurrent mycotoxin exposure and urinary levels of fumonisin B1 (FB1), fumonisin B2 (FB2), nivalenol (NIV), ochratoxin A (OTA), zearalenone (ZEA), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL) and de-epoxydeoxynivalenol (DOM-1) among adults in Sweden., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A duplicate diet study was designed to explore the occurrence of 15 Fusarium mycotoxins in the 24 h-diet consumed by one volunteer as well as the levels of mycotoxins in his 24 h-collected urine. The employed methodology involved solvent extraction at high ionic strength followed by dispersive solid phase extraction and gas chromatography determination coupled to mass spectrometry in tandem. Satisfactory results in method performance were achieved. The method's accuracy was in a range of 68%-108%, with intra-day relative standard deviation and inter-day relative standard deviation lower than 12% and 15%, respectively. The limits of quantitation ranged from 0.1 to 8 µg/Kg. The matrix effect was evaluated and matrix-matched calibrations were used for quantitation. Only deoxynivalenol (DON) was quantified in both food and urine samples. A total DON daily intake amounted to 49.2 ± 5.6 µg whereas DON daily excretion of 35.2 ± 4.3 µg was determined. DON daily intake represented 68.3% of the established DON provisional maximum tolerable daily intake (PMTDI). Valuable preliminary information was obtained as regards DON excretion and needs to be confirmed in large-scale monitoring studies.

Deoxynivalenol (DON) is a trichothecene mycotoxin regularly occurring in cereals. Rats are often used to study toxicokinetics of DON and related compounds, yet only about 30 % of the administered dose is typically recovered. Recently, it was reported that DON is partly metabolised to previously undetected DON- and deepoxy-DON (DOM) sulfonate in rats and tentative structures were proposed. The present work describes the production and characterisation of DON-, DOM- and DON-3-glucoside (D3G) sulfonates of three different series; the development and validation of liquid chromatography tandem mass spectrometry (LC-MS/MS)-based methods for determination of DON, DOM, D3G and their sulfonates in rat faeces and urine; and application of the methods to samples from a DON and D3G feeding trial with rats. In addition to previously produced DON sulfonates (DONS) 1, 2 and 3, D3G sulfonates 1, 2 and 3; and DOM sulfonates (DOMS) 2 and 3 were synthesised, purified and characterised. The developed methods showed apparent recoveries of all investigated compounds between 68 and 151 % in faeces and between 48 and 113 % in urine. The recovery of DON, D3G and their metabolites from faeces and urine of rats (n = 6) administered in a single dose of 2.0 mg/kg b.w. DON or the equimolar amount of D3G was 75 ± 9 % for the DON group and 68 ± 8 % for the D3G group. DON-, DOM- and D3G sulfonates excreted in faeces accounted for 48 and 47 % of the total amount of administered DON and D3G. Urinary excretion of sulfonates was <1 %. In both treatment groups, DONS 2 was the major metabolite 0-24 h after treatment, whereas DOMS 2 was predominant thereafter. The developed methods can also be used for investigation of DON (conjugate) sulfonate formation in other animal species.

Scope: In this study, the exposure of a German population (n = 101) to mycotoxins was assessed using an LC-MS/MS urinary multibiomarker approach. Food consumption of the participants was documented with a food frequency questionnaire to correlate mycotoxin exposure with individual nutritional habits., Methods and Results: The presence of 23 urinary biomarkers including trichothecenes (deoxynivalenol (DON), DON-3-glucuronide (DON-3-GlcA), T-2 toxin, HT-2 toxin (HT-2, HT-2-toxin-4-glucuronide (HT-2-GlcA), fumonisins (fumonisin B1, fumonisin B2), aflatoxins (aflatoxin B1, aflatoxin G2, aflatoxin B2, aflatoxin M1), zearalenone and derivatives (zearalanone, α-zearalenol, β-zearalenol, zearalenone-14-O-glucuronide, zearalanone-14-O-glucuronide, α-zearalenol-14-O-glucuronide/β-zearalenol-14-O-glucuronide), ochratoxin A, ochratoxin alpha, enniatin B and dihydrocitrinone was evaluated using a validated, sensitive "dilute and shoot"-LC-MS/MS method applying Scheduled MRM(TM) technology. Six mycotoxins and urinary metabolites were detected (DON, DON-3-GlcA, zearalenone-14-O-glucuronide, T-2 toxin, enniatin B, and dihydrocitrinone) in 87% of the samples in single- or co-occurence. Only DON and DON-3-GlcA were detectable in quantifiable amounts. A provisional mean daily intake of 0.52 μg DON/kg body weight was calculated. No statistical evidence for the correlation of staple food intake and urinary biomarker concentration could be determined., Conclusion: The results of this study suggest a low everyday exposure of the investigated German population to mycotoxins, but reveal peak exposures above the widely accepted tolerable daily intake to DON in parts of the population., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this pilot survey human urine samples were analyzed for presence of 15 mycotoxins and some of their metabolites using a novel urinary multi-mycotoxin GC-MS/MS method following salting-out liquid-liquid extraction. Fifty-four urine samples from children and adults residents in Valencia were analyzed for presence of urinary mycotoxin and expressed in gram of creatinine. Three out of 15 mycotoxins were detected namely, HT-2 toxin, nivalenol and deoxynivalenol (DON). 37 samples showed quantifiable values of mycotoxins. Co-occurrence of these contaminants was also observed in 20.4% of assayed samples. DON was the most frequently detected mycotoxin (68.5%) with mean levels of 23.3 μg/g creatinine (range: 2.8-69.1 μg/g creatinine). The levels of urinary DON were used to carry out an exposure assessment approach. 8.1% of total subjects were estimated to exceed the DON provisional maximum tolerable daily intake (PMTDI) (1 μg/kg b.w.). Two out of 9 exposed children exceeded the DON PMTDI thus, making them the most exposed based on the urinary results., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Plants can metabolize the Fusarium mycotoxin deoxynivalenol (DON) by forming the masked mycotoxin deoxynivalenol-3-β-D-glucoside (D3G). D3G might be cleaved during digestion, thus increasing the total DON burden of an individual. Due to a lack of in vivo data, D3G has not been included in the various regulatory limits established for DON so far. The aim of our study was to contribute to the risk assessment of D3G by determination of its metabolism in pigs. Four piglets received water, D3G (116 μg/kg b.w.) and the equimolar amount of DON (75 μg/kg b.w.) by gavage on day 1, 5 and 9 of the experiment, respectively. Additionally, 15.5 μg D3G/kg b.w. were administered intravenously on day 13. Urine and feces were collected for 24 h and analyzed for DON, D3G, deoxynivalenol-3-glucuronide (DON-3-GlcA), deoxynivalenol-15-GlcA (DON-15-GlcA) and deepoxy-deoxynivalenol (DOM-1) by UHPLC-MS/MS. After oral application of DON and D3G, in total 84.8±9.7% and 40.3±8.5% of the given dose were detected in urine, respectively. The majority of orally administered D3G was excreted in form of DON, DON-15-GlcA, DOM-1 and DON-3-GlcA, while urinary D3G accounted for only 2.6±1.4%. In feces, just trace amounts of metabolites were found. Intravenously administered D3G was almost exclusively excreted in unmetabolized form via urine. Data indicate that D3G is nearly completely hydrolyzed in the intestinal tract of pigs, while the toxin seems to be rather stable after systemic absorption. Compared to DON, the oral bioavailability of D3G and its metabolites seems to be reduced by a factor of up to 2, approximately., (Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Scope: This study assessed deoxynivalenol (DON) exposure in children from three geographic locations within Tanzania, over three time points in 1 year, using a urinary biomarker of exposure., Methods and Results: A total of 166 children aged 6-14 months were studied at a maize harvest and followed up twice at 6-month intervals. On two consecutive days, morning urine was collected from each child and urinary DON was measured using an LC-MS method, with and without β-glucuronidase hydrolysis in order to assess free DON (fDON) and glucuronide DON. Overall, urinary DON increased significantly along with the three visits (geometric mean 1.1, 2.3, and 5.7 ng/mL, at visits 1, 2, and 3, respectively, p < 0.01). fDON was 22% of urinary total DON. Urinary DON excretion rate was 74% in village Kikelelwa based on food DON level and food consumption. Assuming 360 mL of urine excreted per day, 10, 19, and 29% of children at visits 1, 2, and 3, respectively, exceeded the provisional maximum tolerable daily intake of 1000 ng/kg b.w./day., Conclusion: Young children in Tanzania are chronically exposed to DON due to eating contaminated maize, although exposure levels varied markedly by region and season., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Dispositions of deoxynivalenol (DON) in rats and chickens were investigated, using a radiotracer method coupled with a novel γ-accurate radioisotope counting (γ-ARC) radio-high-performance liquid chromatography ion trap time-of-flight tandem mass spectrometry (radio-HPLC-IT-TOF-MS/MS) system. 3β-(3)H-DON was chemically synthesized and orally administrated to both sexes of rats and chickens as single or multiple doses. The results showed that DON was widely distributed and quickly eliminated in all tissues. The highest concentration was found in the gastrointestinal tract at 6 h post-administration. Substantially lower levels were detected in the kidney, liver, heart, lung, spleen, and brain. Three new metabolites were identified tentatively as 10-deoxynivalenol-sulfonate, 10-deepoxy-deoxynivalenol (DOM-1)-sulfonate, and deoxynivalenol-3α-sulfate. Deoxynivalenol-3α-sulfate was a major metabolite in chickens, while the major forms in rats were DOM-1 and DON. Additionally, a higher excretion rate in urine was observed in female rats than in male rats. The differences in metabolite profiles and excretion rates, which suggested diverse ways to detoxify, may relate to the different tolerances in different genders or species.

Human exposures to mycotoxins through dietary intake are a major health hazard and may result in various pathophysiological effects. Although Thailand is a country at increased risk due to its climatic conditions, no comprehensive dataset is available to perform proper exposure assessment of its population with regard to mycotoxins. Therefore, this pilot study was conducted to investigate and evaluate the exposure levels of major mycotoxins (aflatoxin B₁, ochratoxin A, fumonisins, zearalenone and trichothecenes). Sixty first-morning urine samples were collected from healthy volunteers who live in the Bangkok metropolitan area and surrounding provinces (Pathumthani, Nonthaburi, Samutprakarn and Samutsakorn). Urine samples were analysed by a LC-MS/MS-based multi-biomarker method following a so-called 'dilute and shoot' approach. Results generally indicated low mycotoxin exposures in most individuals through the determination of the four biomarkers that were detected in urine samples, i.e. aflatoxin M₁, ochratoxin A (OTA), as well as the deoxynivalenol (DON) metabolites DON-3-glucuronide and DON-15-glucuronide in 10 of 60 individuals. The maximum concentrations were used to estimate the daily intake confirming that none of the individuals exceeded the tolerable daily intake (TDI) of DON (maximum 26% of TDI) or OTA (maximum 22% of TDI). However, the maximum exposure of aflatoxin B₁, estimated to be 0.91 µg (kg bw)⁻¹ day⁻¹, should raise some concerns and suggests further studies utilising a more sensitive method. Low exposure to Fusarium toxins was also confirmed by the absence of zearalenone, α-zearalanol, β-zearalanol and zearalenone-14-glucuronide as well as T-2 toxin, HT-2 toxin, nivalenol and free DON. This is the first multi-mycotoxin biomarker study performed in Southeast Asia.

Subsistence farmers are exposed to a range of mycotoxins. This study applied novel urinary multi-mycotoxin LC-MS/MS methods to determine multiple exposure biomarkers in the high oesophageal cancer region, Transkei, South Africa. Fifty-three female participants donated part of their maize-based evening meal and first void morning urine, which was analysed both with sample clean-up (single and multi-biomarker) and by a 'dilute-and-shoot' multi-biomarker method. Results were corrected for recovery with LOD for not detected. A single biomarker method detected fumonisin B1 (FB1) (87% incidence; mean±standard deviation 0.342±0.466 ng/mg creatinine) and deoxynivalenol (100%; mean 20.4±49.4 ng/mg creatinine) after hydrolysis with β-glucuronidase. The multi-biomarker 'dilute-and-shoot' method indicated deoxynivalenol-15-glucuronide was predominantly present. A multi-biomarker method with β-glucuronidase and immunoaffinity clean-up determined zearalenone (100%; 0.529±1.60 ng/mg creatinine), FB1 (96%; 1.52±2.17 ng/mg creatinine), α-zearalenol (92%; 0.614±1.91 ng/mg creatinine), deoxynivalenol (87%; 11.3±27.1 ng/mg creatinine), β-zearalenol (75%; 0.702±2.95 ng/mg creatinine) and ochratoxin A (98%; 0.041±0.086 ng/mg creatinine). These demonstrate the value of multi-biomarker methods in measuring exposures in populations exposed to multiple mycotoxins. This is the first finding of urinary deoxynivalenol, zearalenone, their conjugates, ochratoxin A and zearalenols in Transkei., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Bio-monitoring of human exposure to mycotoxin has mostly been limited to a few individually measured mycotoxin biomarkers. This study aimed to determine the frequency and level of exposure to multiple mycotoxins in human urine from Cameroonian adults. 175 Urine samples (83% from HIV-positive individuals) and food frequency questionnaire responses were collected from consenting Cameroonians, and analyzed for 15 mycotoxins and relevant metabolites using LC-ESI-MS/MS. In total, eleven analytes were detected individually or in combinations in 110/175 (63%) samples including the biomarkers aflatoxin M1, fumonisin B1, ochratoxin A and total deoxynivalenol. Additionally, important mycotoxins and metabolites thereof, such as fumonisin B2, nivalenol and zearalenone, were determined, some for the first time in urine following dietary exposures. Multi-mycotoxin contamination was common with one HIV-positive individual exposed to five mycotoxins, a severe case of co-exposure that has never been reported in adults before. For the first time in Africa or elsewhere, this study quantified eleven mycotoxin biomarkers and bio-measures in urine from adults. For several mycotoxins estimates indicate that the tolerable daily intake is being exceeded in this study population. Given that many mycotoxins adversely affect the immune system, future studies will examine whether combinations of mycotoxins negatively impact Cameroonian population particularly immune-suppressed individuals., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In this pilot survey the levels of various mycotoxin biomarkers were determined in third trimester pregnant women from eastern Croatia. First void urine samples were collected and analysed using a "dilute and shoot" LC-ESI-MS/MS multi biomarker method. Deoxynivalenol (DON) and its metabolites: deoxynivalenol-15-glucuronide and deoxynivalenol-3-glucuronide were detected in 97.5% of the studied samples, partly at exceptionally high levels, while ochratoxin A was found in 10% of the samples. DON exposure was primarily reflected by the presence of deoxynivalenol-15-glucuronide with a mean concentration of 120 μg L(-1), while free DON was detected with a mean concentration of 18.3 μg L(-1). Several highly contaminated urine samples contained a third DON conjugate, tentatively identified as deoxynivalenol-7-glucuronide by MS/MS scans. The levels of urinary DON and its metabolites measured in this study are the highest ever reported, and 48% of subjects were estimated to exceed the provisional maximum tolerable daily intake (1 μg kg(-1) b.w.)., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

This study was conducted to investigate mycotoxin exposure in children (n=220, aged 1.5-4.5years) from high mycotoxin contamination regions of Cameroon and to examine the association between the mycotoxin levels (in total 18 analytes) and several socio-demographic factors and anthropometric characteristics. A cross-sectional study was conducted in six villages in Cameroon with 220 children. Mycotoxins and their metabolites were detected in 160/220 (73%) urine samples. There were significant differences in the mean contamination levels of ochratoxin A (p=0.01) and β-zearalenol (p=0.017) between the two agro-ecological zones investigated. Likewise significant differences were observed in the mean levels of aflatoxin M1 (p=0.001) across the weaning categories of these children. The mean concentration of aflatoxin M1 detected in the urine of the partially breastfed children (1.43ng/mL) was significantly higher (p=0.001) than those of the fully weaned children (0.282ng/mL). Meanwhile, the mean concentrations of deoxynivalenol (3.0ng/mL) and fumonisin B1 (0.59ng/mL) detected in the urine of the male children was significantly (p value 0.021 for deoxynivalenol and 0.004 for fumonisin B1) different from the levels detected in the urine of female children; 0.71ng/mL and 0.01ng/mL for deoxynivalenol and fumonisin B1 respectively. In this study, there was no association between the different malnutrition categories (stunted, wasting and underweight) and the mycotoxin concentrations detected in the urine of these children. However, there is sufficient evidence to suggest that children in Cameroon under the age 5 are exposed to high levels of carcinogenic substances such as fumonisin B1, aflatoxin M1 and ochratoxin A through breastfeeding. To the best of our knowledge, this is the first report of its kind carried out in West Africa to determine multi-mycotoxin exposure in infants., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

This study reports on the detailed investigation of human deoxynivalenol (DON) and zearalenone (ZEN) in vivo metabolism through the analysis of urine samples obtained from one volunteer following a naturally contaminated diet containing 138μg DON and 10μg ZEN over a period of four days. Based on the mycotoxin intake and the concentrations of mycotoxin conjugates in urine, a mass balance was established. The average rates of DON excretion and glucuronidation were determined to be 68 and 76%, respectively. The investigation of formed glucuronides revealed DON-15-glucuronide as main conjugation product besides DON-3-glucuronide. Furthermore, for the first time in human urine a third DON-glucuronide was detected and the fate of ingested masked DON forms (3-acetyl-DON and DON-3-glucoside) was preliminary assessed. The mean excretion rate of ZEN was determined to be 9.4%. ZEN was mainly present in its glucuronide form and in some samples ZEN-14-glucuronide was directly determined 3-10h after exposure. For the first time concrete figures have become available for the excretion pattern of DON and ZEN-glucuronides throughout a day, the comparison of total DON in 24h and first morning urine samples and the urinary excretion rate of total ZEN in humans following exposure through naturally contaminated food. Therefore, valuable preliminary information has been obtained through the chosen experimental approach although the study involved only one single individual and needs to be confirmed in larger monitoring studies. The presented experiment contributes to a better understanding of human DON and ZEN in vivo metabolism and thereby supports advanced exposure and risk assessment to increase food safety and examine the relationship between these mycotoxins and potentially associated chronic diseases in the future., (Copyright © 2013 Michael Sulyok. Published by Elsevier Ireland Ltd.. All rights reserved.)

Direct determination of urinary mycotoxins is a better approach to assess individual's exposure than the indirect estimation from average dietary intakes. In this study, a new analytical method was developed and validated for simultaneous analysis of aflatoxin B1, deoxynivalenol, fumonisin B1, ochratoxin A, zearalenone and T2 toxin and their metabolites in pig urine. In total 12 analytes were selected. A salting-out assisted liquid-liquid extraction procedure was used for sample preparation. High performance liquid chromatography/tandem mass spectrometry was used for the separation and detection of all the analytes. The extraction recoveries were in a range of 70-108%, with the intra-day relative standard deviation and inter-day relative standard deviation lower than 25% for most of the compounds at 3 different concentration levels. Meanwhile the method bias for all the analytes did not exceed 20%. The limits of quantification ranged from 0.07ngmL(-1) for ochratoxin A to 3.3ngmL(-1) for deoxynivalenol. Matrix effect was evaluated in this study and matrix-matched calibration was used for quantification. The developed method was also validated for human urine as an extension of its application. Finally, the developed method was applied in a pilot study to analyze 28 pig urine samples. Deoxynivalenol, aflatoxin B1, fumonisin B1 and ochratoxin A were detected in these samples., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Over the past 20 years, exposure to mycotoxin producing mold has been recognized as a significant health risk. Scientific literature has demonstrated mycotoxins as possible causes of human disease in water-damaged buildings (WDB). This study was conducted to determine if selected mycotoxins could be identified in human urine from patients suffering from chronic fatigue syndrome (CFS). Patients (n = 112) with a prior diagnosis of CFS were evaluated for mold exposure and the presence of mycotoxins in their urine. Urine was tested for aflatoxins (AT), ochratoxin A (OTA) and macrocyclic trichothecenes (MT) using Enzyme Linked Immunosorbent Assays (ELISA). Urine specimens from 104 of 112 patients (93%) were positive for at least one mycotoxin (one in the equivocal range). Almost 30% of the cases had more than one mycotoxin present. OTA was the most prevalent mycotoxin detected (83%) with MT as the next most common (44%). Exposure histories indicated current and/or past exposure to WDB in over 90% of cases. Environmental testing was performed in the WDB from a subset of these patients. This testing revealed the presence of potentially mycotoxin producing mold species and mycotoxins in the environment of the WDB. Prior testing in a healthy control population with no history of exposure to a WDB or moldy environment (n = 55) by the same laboratory, utilizing the same methods, revealed no positive cases at the limits of detection.

Deoxynivalenol (DON) is a potent mycotoxin produced by Fusarium molds and affects intestinal nutrient absorption and barrier function in experimental and farm animals. Free DON and the plant metabolite DON-3-β-d-glucoside (D3G) are frequently found in wheat and maize. D3G is stable in the upper human gut, but some human intestinal bacteria release DON from D3G in vitro. Furthermore, some bacteria derived from animal digestive systems degrade DON to a less toxic metabolite, deepoxy-deoxynivalenol (DOM-1). The metabolism of D3G and DON by the human microbiota has not been fully assessed. We therefore conducted in vitro batch culture experiments assessing the activity of the human fecal microbiota to release DON from D3G. We also studied detoxification of DON to DOM-1 by the microbiota and its potential effect on urinary DON excretion in humans. Fecal slurry from five volunteers was spiked with DON or D3G and incubated anaerobically (from 1 h to 7 days), and mycotoxins were extracted into acetonitrile. Mycotoxins were detected in fecal extracts and urine by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The fecal microbiota released DON from D3G very efficiently, with hydrolysis peaking after 4 to 6 h. The fecal microbiota from one volunteer transformed DON to DOM-1. Urine from the same volunteer also contained DOM-1 (4.7% of DON), whereas DOM-1 was not detectable in urine from other volunteers. Our results confirm that the fecal microbiota releases DON from its glycosylated form, hence increasing the toxic burden in exposed individuals. Furthermore, this is first evidence that the human fecal microbiota of one volunteer detoxifies DON, resulting in the appearance of DOM-1 in urine.

The aim of this study was to examine the effects of a control diet (CON, 0.25 mg DON/kg diet) or a Fusarium toxin-contaminated diet (FUS, 4.49 mg DON/kg diet) without and with humic substances (HS) (CON-HS and FUS-HS, 0.23 and 4.56 mg DON/kg diet, respectively) on piglets during a 5-week growth trial starting after weaning (6.7 ± 0.9 kg live weight, n = 20/group). Feed intake was significantly reduced by feeding the FUS containing diets by approximately 21% compared with the CON diet irrespective of HS supplementation. The decrease in live weight gain paralleled the feed intake depression and amounted to approximately 26%. Feeding the FUS diet was clearly reflected by the DON levels in blood. While only traces of DON with median concentrations of 3 ng/ml (2-5 ng/ml) and 2 ng/ml (0-3 ng/ml) were detected in piglets fed the CON and CON-HS diets, respectively, significantly higher levels of 22.5 ng/ml (7-30 ng/ml) and 23.5 ng/ml (15-32 ng/ml) were found in piglets fed the FUS and FUS-HS diet, respectively. The urinary excretion of DON and its metabolite de-epoxy-DON as percentage of DON intake was not significantly influenced by HS supplementation and amounted to 24.1 and 20.2% for groups FUS and FUS-HS, respectively. In conclusion, the tested HS preparation cannot be recommended as a DON inactivating feed supplement for pigs.

Deoxynivalenol-3-β-D-glucoside (D3G), a plant metabolite of the Fusarium mycotoxin deoxynivalenol (DON), might be hydrolyzed in the digestive tract of mammals, thus contributing to the total dietary DON exposure of individuals. Yet, D3G has not been considered in regulatory limits set for DON for foodstuffs due to the lack of in vivo data. The aim of our study was to evaluate whether D3G is reactivated in vivo by investigation of its metabolism in rats. Six Sprague-Dawley rats received water, DON (2.0 mg/kg body weight (b.w.)) and the equimolar amount of D3G (3.1 mg/kg b.w.) by gavage on day 1, 8 and 15, respectively. Urine and feces were collected for 48 h and analyzed for D3G, DON, deoxynivalenol-glucuronide (DON-GlcA) and de-epoxy deoxynivalenol (DOM-1) by a validated LC-tandem mass spectrometry (MS/MS) based biomarker method. After administration of D3G, only 3.7±0.7% of the given dose were found in urine in the form of analyzed analytes, compared to 14.9±5.0% after administration of DON, and only 0.3±0.1% were detected in the form of urinary D3G. The majority of administered D3G was recovered as DON and DOM-1 in feces. These results suggest that D3G is little bioavailable, hydrolyzed to DON during digestion, and partially converted to DOM-1 and DON-GlcA prior to excretion. Our data indicate that D3G is of considerably lower toxicological relevance than DON, at least in rats., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)