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Zearalenone (ZEN) is a mycotoxin that occurs widely in food commodities with particular incidence in cereals. Due to chemical structures similar to the endogenous oestrogen 17-β-estradiol, ZEN and its metabolites exert estrogenic toxicity. Therefore, it is crucial to assess ZEN exposure among the population and biomarker-driven research is a promising method to assess the human exposure. For this reason, ZEN metabolites such as α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), α-zearalenal (α-ZAL), β-zearalenal (β-ZAL), zearalanone (ZAN) (phase I) and the glucuronides ZEN14GlcA, α-ZEL14GlcA and β-ZEL14Glc (phase II) were identified in biological fluids. With a potency factor of 60 relative to ZEN, α-ZEL is the most relevant metabolite in terms of human health. ZEN is characterized by a fast metabolism and excretion, therefore urine is the matrix commonly used to assess the exposure to this mycotoxin and its metabolites. To date, in Portugal, there is a lack of human studies to assess biomarkers of exposure to ZEN. Within the Scope of National Food, Nutrition, and Physical Activity Survey of the Portuguese General Population (2015-2016), 24h-urine samples and non-consecutive dietary assessments (two 24-hour recalls, 8-15 days apart) from 94 participants were included in the present study. Following a salt-assisted matrix extraction, urine samples were analyzed using LC-MS/MS for the simultaneous determination of ZEN, α-ZEL, β-ZEL, α-ZAL, β-ZAL, ZAN and ZEN14GlcA. ZEN and ZEN-14-GlcA were detected in 52% (36/69) and 14% (10/69) of the analyzed samples, with a mean concentration of 1.2 and 6.9 µg/L, respectively. The metabolites α-ZEL, β-ZEL, α-ZAL, β-ZAL, ZAN were not detected in the urine samples. Considering the 24h-urinary volume, the mean dietary excretion of ZEN and ZEN-14-GlcA was 1.5 and 7.8 µg/day, respectively. These data will allow the determination of Probably Daily Intake of zearalenone with more accuracy since it reflects the internal exposure of participants. The present biomonitoring study generates reliable data regarding the exposure of the Portuguese population to ZEN. These data are crucial to perform a more realistic risk assessment, contributing to the knowledge of determinants of this exposure. This research was performed under CESAM (UID/AMB/50017/2013) funded by the Fundação para a Ciência e Tecnologia (FCT), Portugal. The authors also acknowledge the support of MYTOX-SOUTH network. N/A

International audience; Moniliformin (MON) is a mycotoxin with low molecular weight primarily produced by Fusarium fungi and occurring predominantly in cereal grains. Following a request of the European Commission, the CONTAM Panel assessed the risk of MON to human and animal health related to its presence in food and feed. The limited information available on toxicity and on toxicokinetics in experimental and farm animals indicated haematotoxicity and cardiotoxicity as major adverse health effects of MON. MON causes chromosome aberrations in vitro but no in vivo genotoxicity data and no carcinogenicity data were identified. Due to the limitations in the available toxicity data, human acute or chronic health-based guidance values (HBGV) could not be established. The margin of exposure (MOE) between the no-observed-adverse-effect level (NOAEL) of 6.0 mg/kg body weight (bw) for cardiotoxicity from a subacute study in rats and the acute upper bound (UB) dietary exposure estimates ranged between 4,000 and 73,000. The MOE between the lowest benchmark dose lower confidence limit (for a 5% response-BMDL 05) of 0.20 mg MON/kg bw per day for haematological hazards from a 28-day study in pigs and the chronic dietary human exposure estimates ranged between 370 and 5,000,000 for chronic dietary exposures. These MOEs indicate a low risk for human health but were associated with high uncertainty. The toxicity data available for poultry, pigs, and mink indicated a low or even negligible risk for these animals from exposure to MON in feed at the estimated exposure levels under current feeding practices. Assuming similar or lower sensitivity as for pigs, the CONTAM Panel considered a low or even negligible risk for the other animal species for which no toxicity data suitable for hazard characterisation were identified. Additional toxicity studies are needed and depending on their outcome, the collection of more occurrence data on MON in food and feed is recommended to enable a comprehensive human risk assessment.

Scientific fields are always changing, and it is important to determine the directions in which they are heading as plans are made for future research activities and research results transition to applications and policies. The MycoKey project, an EU-funded Horizon 2020 project, includes a series of "Roundtable Discussions" to gather information from leading researchers inside and outside the project on directions in which the field of mycotoxicology is trending. This presentation includes a summary of the roundtable discussion on priorities and critical issues in chemical detection and monitoring of mycotoxins. The goal was to identify key elements that could impact when and how methods are used to identify mycotoxins and to increase food safety. The discussion was managed by using the Nominal Group discussion Technique (NGT). The NGT provides for equal input from all participants and is well-known as a process for generating a large number of ideas, while also providing a mechanism for ranking them. The discussion was focused on four following key questions: oto identify scientific and technological innovations which hold promise for development/improvement in the future (question 1); oto identify and prioritize critical elements making methods effectively applicable in -industry for autocontrol and HACCP (question 2), -developing countries with limited analytical capabilities (question 3), -official control with particular reference to multi-mycotoxin methods (question 4). The rankings and the total list of responses provided a rich and detailed context from which particular ideas and general trends could be extracted. Test kits, usually antibody based, were one major focus of the discussions because of their many favorable features, e.g., cost, speed, and ease of use. Most important was considered the degree to which antibody-based diagnostics have become an acceptable standard in many practical applications. Much of the discussion was on how to improve these kits and to speed the detection of mycotoxins in locations where access to a well-equipped analytical chemistry laboratory is neither timely nor cost-effective. The second area of focus for this discussion was multi-mycotoxin detection protocols and challenges remaining before these protocols become methods of choice for regulated mycotoxins. This second point mirrors the first, through the increasing importance of technically highly sophisticated multi-mycotoxin detection protocols. These protocols need more standardization and cross-laboratory validation, but are the future for many official regulatory controls, especially as the number of toxins that are regulated increases. Multi-mycotoxin assays are being developed at a large number of locations and international inter-laboratory validation and comparability of results are needed to establish credibility of results across both scientific and regulatory boundaries. Common critical factors for analyses in academic, industrial, and governmental laboratories and developing countries were speed, expense, accuracy/reliability, data management, and, particularly in developing countries, operator training. Finally, forging a partnership between scientists and well-placed communications experts was recognized as an essential step to communicating risks while retaining overall confidence in the safety of the food supply and the integrity of the food production chain. This work was supported by H2020- MycoKey-(E.U.3.2-678781)

Mycotoxin analytical methods in food matricesinclude rapid screening and confirmation techniques. The amount of publications covering rapid and sensitive on-site tests suitable for non-laboratory simultaneous determination of several analytes in various matrices is constantly rising. Development of both quantitative (ELISA, FLISA, FPIA, immunosensors) and qualitative (lateral flow, membrane-based flow-through) systems for (multi)mycotoxin detection is one of the fast-growing trends. Developments of various recognition elements as well as novel sensitive labels are currently ongoing in order to design a reliable and rapid test-system. Natural receptors such as antibodies are most widely used, but also their synthetic analogues and engineered elements can be employed. Working with complicated matrices or designing of a regenerable test-system can only be realized through the use of stable molecularly imprinted polymers (MIPs) or synthetic peptides. Whereas scFv and scAb fragments or aptamers cannot stand an aggressive environment, they provide high specificity, which is important for analysis of structurally-related compounds. Different labels can be used in immunochemical methods depending on the sample matrix, starting from enzymes (as the most sensitive label) to colloidal gold (widely used). Colloidal semiconductor nanocrystals or quantum dots (QDs) have emerged as a new class of fluorescent labels for biomedical diagnostics, molecular imaging and chemical analysis. The unique optical properties of QDs enable the simultaneous detection of multiple analytes on one single spot provided their conjugates are labeled with QDs which are fluorescent in different parts of the spectrum. Different strategies can be employed for QDs hydrophilization (such as encapsulation with amphiphilic polymer, silica and liposomes) performing different functionalities on the surface and therefore ensuring different conjugation techniques to synthesize QD-labeled immunoreagents. This presentation will give an insight into current trends and innovations in immunochemical methods for (multi)mycotoxin rapid screening. Examples of rapids tests developed in the frame of the Mycokey EU project will be given.

Deoxynivalenol (DON) is a mycotoxin primarily produced by Fusarium fungi, occurring predominantly in cereal grains. Following the request of the European Commission, the CONTAM Panel assessed the risk to animal and human health related to DON, 3-acetyl-DON (3-Ac-DON), 15-acetyl-DON (15-Ac-DON) and DON-3-glucoside in food and feed. A total of 27,537, 13,892, 7,270 and 2,266 analytical data for DON, 3-Ac-DON, 15-Ac-DON and DON-3-glucoside, respectively, in food, feed and unprocessed grains collected from 2007 to 2014 were used. For human exposure, grains and grain-based products were main sources, whereas in farm and companion animals, cereal grains, cereal by-products and forage maize contributed most. DON is rapidly absorbed, distributed, and excreted. Since 3-Ac-DON and 15-Ac-DON are largely deacetylated and DON-3-glucoside cleaved in the intestines the same toxic effects as DON can be expected. The TDI of 1 μg/kg bw per day, that was established for DON based on reduced body weight gain in mice, was therefore used as a group-TDI for the sum of DON, 3-Ac-DON, 15-Ac-DON and DON-3-glucoside. In order to assess acute human health risk, epidemiological data from mycotoxicoses were assessed and a group-ARfD of 8 μg/kg bw per eating occasion was calculated. Estimates of acute dietary exposures were below this dose and did not raise a health concern in humans. The estimated mean chronic dietary exposure was above the group-TDI in infants, toddlers and other children, and at high exposure also in adolescents and adults, indicating a potential health concern. Based on estimated mean dietary concentrations in ruminants, poultry, rabbits, dogs and cats, most farmed fish species and horses, adverse effects are not expected. At the high dietary concentrations, there is a potential risk for chronic adverse effects in pigs and fish and for acute adverse effects in cats and farmed mink.

Development, validation and one-site testing of immunoassays for rapid mycotoxin detection is one of the priority tasks of the MycoKey project (http://www.mycokey.eu/), an EU multidisciplinary project, aimed to develop smart solutions to reduce the major occurring mycotoxins in economically important food and feed chains. In this framework, a multiplex dipstick immunoassay based method for the simultaneous quantitative determination of major Fusarium toxins, namely deoxynivalenol (DON), zearalenone (ZEA), and fumonisins (FUM) in cereals (wheat, barley and maize) is under development. The dipstick format is based on an indirect competitive approach. The DON/ZEA/FUM prototype is based on a nitrocellulose lateral flow device using a reader to enable quantitative determination of mycotoxin contamination in cereal extracts. Three test lines (mycotoxin-BSA conjugates) and one control line are located on the strip membrane, whereas labeled antibodies were freeze-dried within a microwell. The prototype strip test development included the following steps: definition of coating process and detection reagent formulation parameters; definition of assay parameters like incubation time, migration time, volume sampling to reach minimum performance requested in term of LoD (limit of detection), LoQ (limit of quantification); development of a sample preparation protocol allowing satisfactory mycotoxin recoveries. The total time of analysis is 25 minutes including pre analytical treatment. Preliminary results showed the DON/ZEA/FUM prototype to be compatible with the minimum acceptable performances, and to be suitable for its application to the analysis of real samples containing the target mycotoxins at levels close to EU regulatory levels.

An overview of liquid chromatographic methods, mainly employing fluorescence detection together with sample pre-treatment methods, is presented for the determination of the toxic group of fumonisin mycotoxins in various matrices.

The glucosinolate profile of black radish (Raphanus sativus L. niger) based dietary supplements has been investigated by HPLC-PDA, LC-ESI-MS/MS and LC-APCI-MS/MS systems. Optimization of the MS/MS parameters and LC conditions was performed using sinigrin reference standard and rapeseed certified reference material (BC190) respectively. An LC-ESI-MS/MS system was used to detect (screen) and identify the naturally occurring intact glucosinolates (GLs). The intact GLs identified were then desulfated and quantified on an HPLC-PDA system as desulfo-glucosinolates (DS-GLs). Prior to quantification, the DS-GLs were identified using an APCI-MS/MS. The HPLC-PDA method performance criteria were evaluated using glucotropaeolin potassium salt. The validated method was applied for the analysis of six dietary supplements. In total, six glucosinolates were identified and quantified in the dietary supplements; glucoraphasatin (0.2-0.48mg/g), glucosisaustricin (0.37-0.91mg/g), glucoraphenin (0.84-1.27mg/g), glucoputrajivin (0.14-0.28mg/g), glucosisymbrin (0.70-0.99mg/g) and gluconasturtiin (0.06-0.12mg/g). Glucoraphenin was the most abundant glucosinolate in all samples.

The present study evaluated the nutritional value of sesame oil cake (SOC) in rainbow trout fry (initial body weight of 1.42 g) in a growth trial performed for 45 feeding days at 15 ± 1 °C. A series of five isonitrogenous (380 g digestible protein kg-1 dry matter (DM)) and isoenergetic (18.1 MJ digestible energy kg-1 DM) diets were formulated in which the digestible SOC protein progressively replaced 0%, 13%, 26%, 39% and 52% of the digestible protein of a high quality fish meal (D0-D52). Growth rate of fry significantly improved in fish fed SOC diets compared to the fishmeal control diet (D0) whatever the SOC inclusion level. This positive effect on growth was mainly related to a marked improvement of voluntary feed intake. The decrease of feed efficiency observed with increasing SOC was entirely explained by the reduction of DM and energy digestibilities with SOC incorporation. Nitrogen retention efficiency (nitrogen gain/nitrogen intake) was high (40–41%) and significantly reduced only in fish fed D52 (37%). Our results suggest that SOC can be a suitable protein source for a carnivorous fish and replace at least half of the fishmeal protein (without amino acid supplementation) without growth reduction in rainbow trout fry.

Concerns have been raised about exposure to mycotoxin producing fungi and the microbial volatile organic compounds (MVOCs) they produce in indoor environments. Therefore, the presence of fungi and mycotoxins was investigated in 99 samples (air, dust, wallpaper, mycelium or silicone) collected in the mouldy interiors of seven water-damaged buildings. In addition, volatile organic compounds (VOCs) were sampled. The mycotoxins were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (20 target mycotoxins) and quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). Morphological and molecular identifications of fungi were performed. Of the 99 samples analysed, the presence of one or more mycotoxins was shown in 62 samples by means of LC-MS/MS analysis. The mycotoxins found were mainly roquefortine C, chaetoglobosin A and sterigmatocystin but also roridin E, ochratoxin A, aflatoxin B1 and aflatoxin B2 were detected. Q-TOF-MS analysis elucidated the possible occurrence of another 42 different fungal metabolites. In general, the fungi identified matched well with the mycotoxins detected. The most common fungal species found were Penicillium chrysogenum, Aspergillus versicolor (group), Chaetomium spp. and Cladosporium spp. In addition, one hundred and seventeen (M)VOCs were identified, especially linear alkanes (C9-C17), aldehydes, aromatic compounds and monoterpenes.

The present experiment investigated whether lysine (Lys) utilization efficiency is affected by dietary protein level in rainbow trout when Lys is first limiting in the diet. Eighteen isoenergetic (22.9 MJ digestible energy/kg dry matter (DM)) Lys-deficient diets containing 250, 450 or 650 g crude protein/kg DM and between 6 and 20 g Lys/kg DM were prepared. Wheat gluten, and a crystalline amino acid (AA) mixture corresponding to 47% of the total dietary nitrogen, were used as the principal dietary AA sources. L-Lys.HCl was supplemented at the expense of a dispensable AA mixture. Each diet was fed twice a day to apparent satiety to one group of 70 fry for 30 days. Whole-body protein accretion and Lys gain were determined by the comparative slaughter technique and used to evaluate Lys utilization efficiency above maintenance. Increasing the dietary Lys concentration resulted in linear increases of Lys gain, protein gain and voluntary feed intake. Lys gain or protein accretion increased linearly with increasing dietary Lys intake at all three protein levels and there was no significant difference amongst the regressions, indicating that Lys utilization efficiency was not affected by protein level. Therefore, all data were pooled to calculate a single linear regression for all treatments. Lys was utilized with an efficiency of 71 (SE 3) % above maintenance and 117 (SE 13.6) g Lys intake were required for 1 kg protein accretion in trout fry. Finally, increasing the protein level in isoenergetic diets led to a dramatic decrease of voluntary feed intake in trout, suggesting that fish regulate their feed consumption to achieve a certain target protein deposition and not on the basis of energy intake, at least at marginal Lys intake in wheat gluten-based diets.

An automated headspace solid phase microextraction method followed by GC-MS analysis was used to evaluate and compare the in vitro production of microbial volatile organic compounds (MVOCs) on malt extract agar, plasterboard and wallpaper. Five fungal strains were isolated from the walls of water-damaged houses and identified. In addition, four other common molds were studied. In general, MVOC production was the highest on malt extract agar. On this synthetic medium, molds typically produced 2-methylpropanol, 2-methylbutanol and 3-methylbutanol. On wallpaper, mainly 2-ethylhexanol, methyl 2-ethylhexanoate and compounds of the C8-complex such as 1-octene-3-ol, 3-octanone, 3-octanol and 1,3-octadiene were detected. The detection of 2-ethylhexanol and methyl 2-ethylhexanoate indicates an enhanced degradation of the substrate by most fungi. For growth on plasterboard, no typical metabolites were detected. Despite these metabolite differences on malt extract agar, wallpaper and plasterboard, some molds also produced specific compounds independently of the used substrate, such as trichodiene from Fusarium sporotrichioides and aristolochene from Penicillium roqueforti. Therefore, these metabolites can be used as markers for the identification and maybe also mycotoxin production of these molds. All five investigated Penicillium spp. in this study were able to produce two specific diterpenes, which were not produced by the other species studied. These two compounds, which remain unidentified until now, therefore seem specific for Penicillium spp. and are potentially interesting for the monitoring of this fungal genus. Further experiments will be performed with other Penicillium spp. to study the possibility that these two compounds are specific for this group of molds.