Screening complex effluents for estrogenic activity with the T47D-KBluc cell bioassay: assay optimization and comparison with in vivo responses in fish.

Wastewater treatment plant (WWTP) effluents can contain estrogenic chemicals, which potentially disrupt fish reproduction and development. The current study focused on the use of an estrogen-responsive in vitro cell bioassay (T47D-KBluc), to quantify total estrogenicity of WWTP effluents. We tested a novel sample preparation method for the T47D-KBluc assay, using powdered media prepared with direct effluent. Results of the T47D-KBluc assay were compared with the induction of estrogen receptor-regulated gene transcription in male fathead minnows (Pimephales promelas) exposed to the same effluents. Effluent samples for the paired studies were collected over the course of three months. According to the T47D-KBluc assay, the effluent estrogenicity ranged from 1.13 to 2.00 ng 17β-estradiol (E2) equivalents/L. Corresponding in vivo studies exposing male fathead minnows to 0, 10, 50, and 100% effluent dilutions demonstrated that exposure to 100% effluent significantly increased hepatic vitellogenin (VTG) and estrogen receptor α subunit transcripts relative to controls. The induction was also significant in males exposed to 250 ng E2/L or 100 ng E2/L. The in vitro and in vivo results support the conclusion that the effluent contains significant estrogenic activity, but there was a discrepancy between in vitro- and in vivo-based E2 equivalent estimates. Our results suggest that the direct effluent preparation method for the T47D-KBluc assay is a reasonable approach to estimate the estrogenicity of wastewater effluent.