Deoxynivalenol and zearalenone are among the most prevalent toxins produced by Fusarium spp. They have been investigated in food and feed products for decades but rarely in the environment. We therefore established solid-phase extraction and liquid chromatography-mass spectrometry (LC-MS) methods to quantify these mycotoxins at trace concentrations in aqueous natural samples. In a model emission study, we inoculated a winter wheat field with Fusarium graminearum and subsequently monitored deoxynivalenol and zearalenone in its drainage water. Before during and after harvest in June and July 2007, these toxins were emitted in concentrations from 23 ng/L to 4.9 µg/L for deoxynivalenol and from not detected to 35 ng/L for zearalenone. Simultaneously, in July and August 2007, deoxynivalenol was also detected in a number of Swiss rivers in concentrations up to 22 ng/L and zearalenone was present in several river samples below the method quantification limit. Other mycotoxins might be emitted from Fusarium-infected fields as well, because some of them are produced in similar amounts as deoxynivalenol and zearalenone and exhibit similar or even higher water solubility than deoxynivalenol. The ecotoxicological consequences of the presence of mycotoxins in surface waters remain to be elucidated.

The mycotoxin zearalenone (ZON) is a very potent natural endocrine disrupting chemical, produced by Fusarium graminearum fungi growing on crops such as wheat and maize. Although it is well-investigated in food and feed, very little is known about its environmental fate and behavior. Here, we report the occurrence of ZON on F. graminearum infected wheat and maize fields in crop organs and soil and its emission via drainage water. ZON amounts in the investigated crops and topsoil were between 6.1 and 25.0 and up to 5.6 g/ha, respectively. ZON concentrations in drainage water were in the low nanogram per liter range with a maximum of 35 ng/ L. Cumulated ZON amounts emitted via drainage water ranged from 0.1 to 4.3 mg/ha, depending on the crop cultivated in the respective period. This corresponds to fractions between 0.001 and 0.070% of the initially present ZON amount in the plants. Because of the low concentrations emitted via drainage water, it can be assumed that ZON contributes little if at all to the overall estrogenicity of major surface water bodies. However, in small creeks, mainly fed by agricultural runoff, ZON might be present in environmentally critical concentrations at times of F. graminearum infections.

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In light of the estrogenic potentials and the recent concentration levels found for six phytoestrogens in surface waters, detailed monitoring and assessment of potential input sources are required. An accurate, precise, and sensitive HPLC-MS/MS analytical method incorporating five (13)C 3-labeled internal standards for the quantification of these plant estrogens in various aqueous environmental samples is presented here for the first time. The compounds investigated included biochanin A, daidzein, equol, formononetin, genistein, and coumestrol. The use of [ (13)C 3]biochanin A, [ (13)C 3]daidzein, [ (13)C 3]equol, [ (13)C 3]formononetin, and [ (13)C 3]genistein ensured an accurate quantification of the target analytes unaffected by matrix effects and analyte losses. Absolute method recoveries for all analytes ranged from 63 to 105%, from 63 to 99%, and from 73 to 133%, relative recoveries from 90 to 132%, from 89 to 139%, and from 89 to 115%, method detection levels from 0.5 to 2.7 ng/L, from 0.5 to 2.6 ng/L, and from 0.4 to 11.0 ng/L, and precision from 1 to 19%, from 1 to 16%, and from 1 to 11% in drainage water, river water, and WWTP effluent, respectively. The validated analytical method was applied in investigating the emission of the phytoestrogens via drainage water from a pasture containing 43% red clover ( Trifolium pratense) and in monitoring their occurrence in Swiss surface waters. Isoflavone concentrations ranging from 4 to 157 ng/L and up to 22 ng/L were found in drainage and river water, respectively.

Zearalenone (ZON) is known as a very potent, naturally occurring estrogenic mycotoxin. It is one of the most prevalent mycotoxin produced as a secondary metabolite by Fusarium species growing on cereals such as wheat and corn. It has been studied extensively in food and feed products for decades but only rarely and somewhat by chance in the environment. We therefore elucidated its agro-environmental fate and behavior by conducting a series of field studies and monitoring campaigns. Specifically, ZON was investigated in plants, soils and drainage waters from wheat and corn fields artificially infected with Fusarium graminearum. In addition, manure, sewage sludge and surface waters were analyzed for ZON. Three main input pathways of ZON onto soil could be identified: i) wash-off from Fusarium-infected plants (in the order of 100 mg/ha), ii) plant debris remaining on the soil after harvest (up to few g/ha), and iii) manure application (in the order of 100 mg/ha). Our results show that these input sources altogether caused the presence of several g/ha of ZON in topsoil. Compared to this, ZON emission by drainage water from Fusarium-infected fields was generally low, with maximum concentrations of 35 ng/l and total amounts of a few mg/ha. Due to dilution, ZON concentrations dropped below environmental relevance in larger surface water bodies. However in small catchments dominated by runoff from agricultural land, ZON might substantially contribute to the estrogenicity of such waters. Apart from ZON, other natural toxins monitored in this study, such as the mycotoxin deoxynivalenol or the estrogenic phytoestrogen formononetin, emitted to and occurred in surface waters at considerably higher amounts. To date their ecotoxicological effects are largely unknown.

Because of its pronounced estrogenicity, zearalenone may be of concern not only in the aqueous but also in the terrestrial environment. Therefore, we developed several analytical methods to quantify zearalenone in different solid matrices of agroenvironmental relevance (i.e., plant organs, soil, manure, and sewage sludge). The use of D(6)-zearalenone as the internal standard (IS) was essential to render the analytical method largely matrix-independent because it compensated for target analyte losses during extract treatment and ion suppression during ionization. Soil and sewage sludge samples were extracted with Soxhlet, whereas plant material and manure samples were extracted by liquid solvent extraction at room temperature. Absolute recoveries for zearalenone were 70-104% for plant materials, 105% for soil, 76% for manure, and 30% for sewage sludge. Relative recoveries ranged from 86 to 113% for all matrices, indicating that the IS was capable to largely compensate for losses during analysis. Ion suppression, between 8 and 74%, was in all cases compensated by the IS but influenced the method quantification levels. These were 3.2-26.2 ng/g(dryweightdw) for plant materials, 0.7 ng/g(dw) for soil, 12.3 ng/g(dw) for manure, and 6.8 ng/g(dw) for sewage sludge. Plant material concentrations varied from 86 ng/g(dw) to more than 16.7 microg/g(dw), depending on the organ and crop. Soil concentrations were between not detectable and 7.5 ng/g(dw), depending on the sampling depth. Zearalenone could be quantified in all manure samples in concentrations between 8 and 333 ng/g(dw). Except for two of the 85 investigated sewage sludge samples, zearalenone concentrations were below quantification limit.

Immunoaffinity extraction has become increasingly important as a sample preparation and cleanup method in mycotoxin analysis. In this study, the antibody specificities of 3 commercial immunoaffinity columns (IACs) targeting zearalenone (ZON) were compared for -zearalenol, -zearalenol, zearalanone, -zearalanol, and -zearalanol. The recoveries of ZON and its 5 analogs were determined in triplicate when extracted from 10 mL circumneutral river water samples spiked with 20 ng analyte individually or in a mixture. The analytes were analyzed by means of electrospray ionization liquid chromatography/tandem mass spectrometry using deuterated internal standards for quantitation. Recoveries ranged from 69 to 115% for all analytes with relative standard deviations of 139%. Cross-reactivities for the analogs were >80% when applied both individually and in a mixture. No significant competition effects were observed when the compounds were applied as a multianalyte mixture well below the stated IAC capacities. The results obtained here demonstrate that all IACs tested are highly cross-reactive towards the 5 ZON derivatives and may be applied for their simultaneous extraction or cleanup.