Identification of active synthetic steroid compounds in impacted river downstream from pharmaceutical industry

The environmental occurrence of emerging pollutants able to disrupt endocrine signalling pathways other than those mediated by estrogen receptors (e.g. corticosteroids, androgens or progestagens) is of recent concern. Recent evidences have suggested that effluents from pharmaceutical industry release drugs into rivers and trigger adverse effect on wildlife. By using in vitro bioassays combined to passive sampling (i.e. Polar organic compound integrative sampler, POCIS), we previously reported the occurrence of estrogenic, glucorticoidic, anti-mineralocorticoidic, progestative compounds and pregnane X receptor (PXR) ligands downstream a pharmaceutical industry release where strong reproductive alteration have been reported in fish. In this study, we report the use of effect directed approach to identify the compounds responsible for these activities. We first assessed mass balance calculation through chemical analyses directed by toxicity profile. Chemical analyses in POCIS crude extract showed the occurrence of high concentrations of dexamethasone, spironolactone, 6-methyl-prednisolone (up to 100 micro g/g of sorbent) that well explained GR and anti-MR activities (up to 100%).Conversely, other biological activities (i.e. estrogenic, PXR-like) were poorly explained by the detected chemicals. Then, sample RP-HPLC fractionation was carried out to unravel the different activities. It allowed the isolation of estrogenic and PXR-like activities from GR, PR and anti-MR. It also revealed the occurrence of MR agonists that were masked by the strong anti-MR activity in the crude extract. In addition, HPLC calibration showed a good fitting between retention times of detected chemicals and several active fractions (e.g. 6-methyl-prednisolone in F11, dexamethasone in F12). Nevertheless, some active fractions were left unexplained by calibration standards suggesting that many active chemicals remain to be identified. Chemical analyses in these fractions are under investigations using LC-HRMS system and results will be presented. Our study demonstrates (1) the usefulness of MBA approach using pre-directed chemical analyses based on toxicity profile (2) the strength of the fractionation to unravel complex mixture effect and to finely isolate active chemicals. Overall, our results underscore the need to increase knowledge on the effects of corticosteroids and progestogens on aquatic organisms for better risk assessment