We report results from a systematic one-and-a-half year survey of the estrogenic isoflavones formononetin (FOR), biochanin A (BIO), daidzein (DAI), genistein (GEN), and equol in Swiss midland rivers. FOR was detected in about 90%, the other compounds in 13-56% of the weekly and fortnightly integrated flow proportional samples. Concentrations were mostly in the lower ng/L-range, with a maximum of 524 ng/L and 217 ng/L for equol and FOR, respectively. Due to dilution, concentrations were river discharge dependent with higher numbers in smaller rivers. Total isoflavone loads were in the order of a few kg/y, and occurred mainly during summertime. A complementary river water monitoring campaign throughout the country confirmed the above findings. Circumstantial evidence points to grassland as a major emission source of FOR and BIO (the main compounds in red clover) in surface waters, e.g., their absence in wastewater treatment effluents, better correlations of their loads with grassland areas than with population equivalents, similar isoflavone ratios in river water and grassland runoff. Source apportionment was less clear for DAI, GEN, and equol. The contribution of isoflavones to the total estrogenicity of surface waters is probably small, except maybe in local rural catchments without major anthropogenic activities.

Th is study provides the fi rst broad-scale investigation on the spatial and temporal occurrence of phytoestrogens and mycotoxins in streams in the United States. Fifteen stream sites across Iowa were sampled fi ve times throughout the 2008 growing season to capture a range of climatic and crop-growth conditions. Basin size upstream from sampling sites ranged from 7 km 2 to >836,000 km 2 . Atrazine (herbicide) also was measured in all samples as a frame-ofreference agriculturally derived contaminant. Target compounds were frequently detected in stream samples: atrazine (100%), formononetin (80%), equol (45%), deoxynivalenol (43%), daidzein (32%), biochanin A (23%), zearalenone (13%), and genistein (11%). Th e nearly ubiquitous detection of formononetin (isofl avone) suggests a widespread agricultural source, as one would expect with the intense row crop and livestock production present across Iowa. Conversely, the less spatially widespread detections of deoxynivalenol (mycotoxin) suggest a more variable source due to the required combination of proper host and proper temperature and moisture conditions necessary to promote Fusarium spp. infections. Although atrazine concentrations commonly exceeded 100 ng L −1 (42/75 measurements), only deoxynivalenol (6/56 measurements) had concentrations that occasionally exceeded this level. Temporal patterns in concentrations varied substantially between atrazine, formononetin, and deoxynivalenol, as one would expect for contaminants with diff erent source inputs and processes of formation and degradation. Th e greatest phytoestrogen and mycotoxin concentrations were observed during spring snowmelt conditions. Phytoestrogens and mycotoxins were detected at all sampling sites regardless of basin size. Th e ecotoxicological eff ects from long-term, low-level exposures to phytoestrogens and mycotoxins or complex chemicals mixtures including these compounds that commonly take place in surface water are poorly understood and have yet to be systematically investigated in environmental studies.

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.