Occurrence and mass balance of isoflavones on an experimental grassland field.

Isoflavones and coumestrol (COU) are estrogenic compounds that are naturally produced by plants (e.g., red clover, soybeans). Although these compounds have been extensively studied in food and feed, only little is known about their environmental fate. Therefore, we investigated the occurrence of isoflavones (formononetin, daidzein, equol, biochanin A, and genistein) and COU over 3.5 years in red clover, manure, and soil of a grassland field with and without manure application, as well as their emission via drainage water. Isoflavones were regularly quantified in plant (≤ 15 × 10(6) ng/g(dry weight (dw))), manure (≤ 230 × 10(3) ng/g(dw)), soil (≤ 3.4 × 10(3) ng/g(dw)), and drainage water samples (≤ 3.6 × 10(3) ng/L). In contrast, COU was observed only in manure and soil. Cumulative isoflavone loads emitted via drainage water were around 0.2 × 10(-3) kg/ha/y, which is very little compared to the amounts present in red clover (105-220 kg/ha/y), manure (0.5-1.0 kg/ha/y), and soil (0.1-5.1 kg/ha/y). Under good agricultural practice, no additional emission of isoflavones into drainage water was observed after manure application. With calculated 17ß-estradiol equivalents up to 0.46 ng/L in drainage water, isoflavones can constitute a dominant and ecotoxicological relevant portion of the total estrogenicity in small rural river catchments.

Phytoestrogens and mycotoxins in Iowa streams: an examination of underinvestigated compounds in agricultural basins.

This study provides the first 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 five 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 km2 to > 836,000 km2. Atrazine (herbicide) also was measured in all samples as a frame-of-reference 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%). The nearly ubiquitous detection of formononetin (isoflavone) 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 different source inputs and processes of formation and degradation. The greatest phytoestrogen and mycotoxin concentrations were observed during spring snowmelt conditions. Phytoestrogens and mycotoxins were detected at all sampling sites regardless of basin size. The ecotoxicological effects from long-term, low-level exposures to phytoestrogens and mycotoxins or complex chemicals mixtures including these compounds that commonly rake place in surface water are poorly understood and have yet to be systematically investigated in environmental studies.

Analysis of selected phytotoxins and mycotoxins in environmental samples.

Natural toxins such as phytotoxins and mycotoxins have been studied in food and feed for decades, but little attention has yet been paid to their occurrence in the environment. Because of increasing awareness of the presence and potential relevance of micropollutants in the environment, phytotoxins and mycotoxins should be considered and investigated as part of the chemical cocktail in natural samples. Here, we compile chemical analytical methods to determine important phytotoxins (i.e. phenolic acids, quinones, benzoxazinones, terpenoids, glycoalkaloids, glucosinolates, isothiocyanates, phytosterols, flavonoids, coumestans, lignans, and chalcones) and mycotoxins (i.e. resorcyclic acid lactones, trichothecenes, fumonisins, and aflatoxins) in environmentally relevant matrices such as surface water, waste water-treatment plant influent and effluent, soil, sediment, manure, and sewage sludge. The main problems encountered in many of the reviewed methods were the frequent unavailability of suitable internal standards (especially isotope-labelled analogues) and often absent or fragmentary method optimization and validation.

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.

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 five isoflavones and coumestrol in various solid agroenvironmental matrices using ¹³c3-labeled internal standards.

We developed and validated three different sample preparation and extraction methods followed by HPLC-MS/MS (negative electrospray ionization) analysis for the quantification of estrogenic isoflavones (formononetin, daidzein, equol, biochanin A, and genistein) and coumestrol in red clover, soil, and manure. Plant and manure samples were solid-liquid extracted, whereas soil was extracted with accelerated solvent extraction. Absolute recoveries were between 80 and 93%, 20 and 30%, and 14 and 91% for plant, soil, and manure samples, respectively. Relative recoveries ranged from 75 to 105% for all matrices, indicating that isotope-labeled internal standards (¹³C3-formononetin, ¹³C3-daidzein, ¹³C3-equol, ¹³C3-biochanin A, and ¹³C3-genistein) were capable to compensate for losses during analysis. The limits of detection in red clover, soil, and manure were 3-9 µg/g(dryweight(dw)), 0.6-8.2 ng/g(dw), and 34.2 ng/g(dw) to 17.0 µg/g(dw), respectively. Formononetin was the most dominant compound in red clover plants (up to 12.5 mg/g(dw)) and soil (up to 3.3 µg/g(dw)), whereas equol prevailed in manure (up to 387 µg/g(dw)).

Occurrence and origin of estrogenic isoflavones in Swiss river waters.

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.

Environmental risk assessment for the galenical formulation of solid medicinal products at Roche Basle, Switzerland.

An environmental risk assessment for losses to wastewater from galenical manufacturing at solid medicinal products at F. Hoffmann-La Roche in Basle, Switzerland, was performed based on an annual total materials balance. This balance resulted in a loss factor of 0.2% relative to the sum of all starting materials, which was later confirmed as valid by analysis for 1 specific active pharmaceutical ingredient (API). The initial risk assessment for all 25 different APIs formulated resulted in no evident risk for the wastewater treatment plant, based on biodegradation no-effect data. However, based on acute ecotoxicity data, potential risk to the local receiving water, the River Rhine, was identified from 1 single API, the antibiotic sulfamethoxazole (SMX). A refinement of the risk assessment for SMX, based on chronic ecotoxicity data, or the predicted no-effect concentration (PNEC), and documented sewage works degradability, or the predicted environmental concentration (PEC), led to a significant decrease of the initial PEC/PNEC ratio to well below 1. In view of this refinement, the final conclusion is that the galenical production investigated poses no significant risk to the environment.