Occurrence of zearalenone on Fusarium graminearum infected wheat and maize fields in crop organs, soil, and drainage water.

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.

Quantification of zearalenone in various solid agroenvironmental samples using D6-zearalenone as the internal standard.

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.

Fusarium mycotoxins: overlooked aquatic micropollutants?

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 microg/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.

Determination of the cross-reactivities for alpha-zearalenol, beta-zearalenol, zearalanone, alpha-zearalanol, and beta-zearalanol on three commercial immunoaffinity columns targeting zearalenone.

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 alpha-zearalenol, beta-zearalenol, zearalanone, alpha-zearalanol, and beta-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 1-39%. 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.

Quantification of six phytoestrogens at the nanogram per liter level in aqueous environmental samples using 13C3-labeled internal standards.

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.

Quantification of estrogenic mycotoxins at the ng/L level in aqueous environmental samples using deuterated internal standards.

Because of their pronounced estrogenicity, resorcyclic acid lactones (RALs) are of concern in aqueous environments even at the low ng/L level. Therefore, we developed an accurate, precise and sensitive HPLC-MS/MS method to detect these mycotoxins in different aqueous environmental samples. The compounds investigated included zearalenone (ZON), alpha- and beta-zearalenol, zearalanone as well as alpha- and beta-zearalanol. The use of isotope labelled internal standards (in this case deuterated RAL-analogues) ensured an accurate quantification of the target analytes, independent of matrix compounds interfering with the analytes during ionisation and analyte losses occurring during sample preparation. Sample enrichment was carried out by solid-phase extraction (SPE) using Supelclean Envi-18 cartridges. Absolute method recoveries for all analytes ranged from 95 to 108%, 70 to 102%, and 76 to 109%, method detection limits from 0.5 to 2.1 ng/L, 0.4 to 1.1 ng/L, and 0.8 to 12.4 ng/L and precision from 3 to 14%, 2 to 13% and 4 to 16% in drainage water, river water and wastewater treatment plant (WWTP) effluent, respectively. The method was applied to verify the emission of RALs from a Fusarium graminearum infested crop field into the drainage system. Zearalenone was present in drainage water in concentrations up to 30 ng/L. So far, none of the other five investigated compounds have been detected.