S9 metabolism integrated CALUX® assay reveals invisible endocrine activities of sediments from two eutrophic freshwater lakes in China

Endocrine-disrupting compounds (EDCs) are of increasing concern due to their prevalence in aquatic ecosystems and potential harm to aquatic life and human health. The in vitro chemically activated luciferase gene expression (CALUX®) assay is widely used to detect endocrine active chemicals (EACs) but its limited metabolic capacity may lead to underestimation or overestimation of test samples depending on metabolic (in)activation. This study evaluated the induced rat liver-derived S9 fraction, a widely used external metabolic activation system, at three protein concentrations (0.01, 0.05, and 0.1 mg/mL) for its cytotoxicity and metabolic performance in the CALUX® assay targeting estrogenic (ERα) and androgenic (AR) receptor, using four reference chemicals: benzo[a]pyrene, tamoxifen, dihydrotestosterone, and flutamide. Subsequently, twelve sediment samples from Taihu and Yangcheng Lakes in China were assessed for endocrine potential using the CALUX® assay with and without S9-activation. In the S9 metabolism-incorporated CALUX® assay, 0.01 mg/mL was identified as the optimal S9 concentration. Additionally, sediments from Taihu and Yangcheng Lakes exhibited cytotoxicity, estrogenic and (anti-)androgenic activities, with and without S9-activation. Notably, S9 supplementation generally reduced cytotoxicity, estrogenic and androgenic activities, while anti-androgenicity remained unchanged. This study highlights the importance of incorporating metabolic activity into the CALUX® assay for effective monitoring of sediment quality in comprehensive environmental risk assessments.