Effect-directed analysis with high-throughput fractionation and a 4-tiered bioassay battery for identification of toxic drivers in WWTP effluent

• High-throughput fractionation EDA with LC-HRMS and 4-tiered bioassay battery • Whole-effluent activity in ER, AhR and Nrf2 bioassays, active fractions for ER • Four pharmaceuticals were identified on confidence level 2 • Ecotoxicity predictions selected 75 compounds of potential concern • 60% of selected effluent compounds were detected downstream release point This study developed, validated, and applied a high-throughput effect-directed analysis (EDA) using a 4-tiered bioassay battery to assess wastewater treatment plant (WWTP) and recipient river water. The EDA was validated using spiked pure water, river water, effluent and influent samples from a WWTP with compounds having estrogen receptor (ER), androgen receptor (AR), aryl hydrocarbon receptor (AhR) or Nrf2 activity. During the proficiency testing, the effluent of that WWTP was confirmed to contain ethinylestradiol (EE2) (0.010 ppb), bisphenol A (BPA) (0.150 ppb) and nimodipine (0.002 ppb), and the influent contained BPA (0.152 ppb) and androstenedione (0.090 ppb). When applied to effluent from another WWTP, bioassays for the whole effluent extract detected activity for ER, AhR, and Nrf2 but not AR. Fractionation revealed ER-active fractions, with potentially four predicted ER-binding compounds, though they were unlikely to fully explain the detected activity. This potentially suggest low concentrations of another present ER agonist (0.00038 ppb<x<0.006 ppb E2 equivalents). Suspect screening of the natural wastewater effluent sample identified 38 compounds at confidence level 2 and 277 at confidence level 3.1. Of the level 2 compounds within ER-active fractions, four pharmaceuticals (benzonatate, promethazine N -oxide, solpecainol, and hoquizil) were identified. Among 121 compounds identified at confidence level 2 and 3.1, 82% persisted in WWTP influent, while 60% were found downstream in river water and only 1 compound was detected upstream, indicating effluent discharge influences micropollutant distribution. This paper highlights the importance of establishing and improving methods for EDA of water with relevance for environmental and human exposure using a variety of bioanalytical endpoints.