Effect-based tools for monitoring (xeno)estrogens in surface waters: Variability and reproducibility of sample preparation and five different in vitro assays

Abstract : In vitro bioassays are increasingly used to assess estrogenic activity of environmental water samples and have been suggested as suitable tools for monitoring estrogenic contamination of surface waters. Such assays are of particular use as they measure the overall estrogenic activity of a sample, including the potent steroids 17ß-estradiol (E2) and 17a-ethinyl estradiol (EE2); proposed annual average Environmental Quality Standards (AA-EQS) are 400 and 35 pg/L, respectively. For most routine chemical methods these EQS are below analytical limits of quantification, resulting in difficulties for monitoring E2 and EE2 under the watchlist mechanism of the Water Framework Directive. The use of sensitive in vitro assays could circumvent current detection problems by measuring the total activation potential of estrogenic substances in environmental samples by expressing overall estrogenicity in E2-equivalents (EEQs). It has been shown, however, that different assays lead to different EEQs for the same sample. Reasons for these differences are known, for example sensitivity differences of the assays towards certain substancesbut it remains unclear how to use and interpret bioassay results. Hence the aims of this study are to (1) compare EEQs of reconstituted water samples and extracts assessed by five commonly used bioassays in order to determine the variability and reproducibility of the assays and the sample preparation method (solid phase extraction) and (2) get insights into their validity for environmental monitoring. Initial results for three different bioassays show that inter-day reproducibility of derived EEQs varied between 2.5 and 30%. Comparison of the results from different in vitro assays showed that all assays were able to correctly detect the EEQ of the positive control. Only the ER-CALUX was able to derive EEQs close to the calculated EEQs for the reconstituted samples. The 10x concentration difference between the two reconstituted samples was detected by the ER-CALUX and the T47D-Kbluc. The YES underestimated the estrogenic load of these samples, whereas the T47D-Kbluc overestimated the EEQs of both mixtures. Data from the MELN and GeneBLAzer ER assays are yet to be analyzed. Overall, our findings suggest that in vitro bioassays are comparable to chemical measurements regarding variability and reproducibility of the derived EEQ concentrations.