An in vitro/in vivo screening assay as a sensitive tool to assess endocrine disruptive activity in surface water

Abstract

Adult male fathead minnow were exposed for 14 or 28-days under flow-through conditions to undiluted filtered water samples from the rivers Meuse and Rhine in the Netherlands. The experiment included two vessels per treatment each containing 10 fish and samples of five fish were taken after 14 and 28 days. Additional groups were exposed to 17α-ethinylestradiol (EE2) as a reference and untreated drinking water as a negative control. Major endpoints examined included induction of vitellogenin (VTG) synthesis, VTG mRNA activity, hepato-and gonadosomatic indices (HSI and GSI) and gonadal histology. No significant difference was recorded in body weight or mean GSI values between the various treatments. Only exposure to Meuse water resulted in significantly higher HSI means after 14 days. Histological examination showed no apparent effects on gonadal tissue except for eosinophilic blood plasma in fish exposed to Meuse water or EE2. After 14 and 28 days, elevated VTG and VTG mRNA levels were measured in most livers of the fish exposed to Meuse water, but not in the fish exposed to Rhine water. This was confirmed by measuring estrogenic responses in the in vitro ER CALUX® assay. Induction of VTG synthesis proved to be the most sensitive endpoint in the Non Spawning Male Fish Assay for in vivo detection of bio-available estrogenic activity supplementary to a sensitive in vitro assay. The other endpoints examined varied too much and required a higher number of fish or replicates to achieve sufficient power for statistical testing making them less animal friendly.

Determination of endocrine-disrupting chemicals in the liquid and solid phases of activated sludge by solid phase extraction and gas chromatography–mass spectrometry

“…To date, many analytical methods have been developed for detection of EDCs in environmental samples with varied matrices based on either biological [13][14][15] or chromatographic methodology [16][17][18]. Biological techniques can be used to determine the endocrine-disrupting activity in total, whereas chromatographic techniques can identify unknown chemicals and quantify their concentrations individually.…”

A yeast estrogen screen without extraction provides fast, reliable measures of estrogenic activity

“…Matrix effects are a concern with YESne because the yeast is exposed to matrix effects more with this method than in methods that extract estrogenic components from the wastewater, thus removing some of the matrix effect chemicals. However, extraction does not remove all matrix effects, and they cause interference with all methods, especially with liquid chromatography-tandem mass spectrometry (LC-MS) [34,35], gas chromatographymass spectrometry (GC-MS) [20,36], and methods using the human mammalian cell lines [37].…”

“…Most studies with YES measure estrogenic activity that has been captured by liquid-liquid phase extraction [20] or solid phase extraction of 1 to 5 L of a filtered water sample [21,22]. Filtration and solid phase extraction steps require several hours per sample and are subject to sample loss and possible introduction of contaminating estrogenic compounds by the reuse of large flasks and funnels.…”

Lipid rafts, microdomain heterogeneity and inter‐organelle contacts: Impacts on membrane preparation for proteomic studies

“…Matrix effects are a concern with YESne because the yeast is exposed to matrix effects more with this method than in methods that extract estrogenic components from the wastewater, thus removing some of the matrix effect chemicals. However, extraction does not remove all matrix effects, and they cause interference with all methods, especially with liquid chromatography–tandem mass spectrometry (LC‐MS) 34, 35, gas chromatography–mass spectrometry (GC‐MS) 20, 36, and methods using the human mammalian cell lines 37.…”

“…The most salient modification to the Routledge and Sumpter method was the elimination of sample extraction, thereby reducing the need for filtration of a large volume of sample. Most studies with YES measure estrogenic activity that has been captured by liquid–liquid phase extraction 20 or solid phase extraction of 1 to 5 L of a filtered water sample 21, 22. Filtration and solid phase extraction steps require several hours per sample and are subject to sample loss and possible introduction of contaminating estrogenic compounds by the reuse of large flasks and funnels.…”