Seasonally different toxicity drivers in a river system revealed by insights from POCIS, bioassays, and suspect...

To determine whether hydrological extremes alter the toxicological modes of action of riverine contaminant mixtures, an integrated framework combining passive sampler, in-vivo and in-vitro bioassays, and high-resolution mass spectrometry-based suspect screening was applied. POCIS were deployed in the Nakdong River of South Korea under contrasting monsoon-driven summer runoff and winter low-flow conditions. Seasonal mixtures differed not only in composition but also in biological mechanisms: summer extracts induced acute toxicity and activation of receptor-mediated endpoints (AR, ER, PPARγ, PXR), whereas winter extracts showed minimal acute toxicity but strong oxidative stress response (Nrf2), consistent with effluent-dominated chronic exposure. Across polarity-based fractions, the polar fraction (F4) accounted for the majority of observed bioactivities. Suspect screening identified 111 chemicals with a clear seasonal differentiation, with pesticides and UV filters prevailing in summer and pharmaceuticals in winter. To address the identification gap in effect-directed analysis, a quantitative potency-balance approach integrating bioanalytical equivalents (BEQ_bio) and chemically predicted equivalents (BEQ_chem) identified telmisartan as a major contributor to PPARγ activity. These findings demonstrate that hydrological regime shifts can drive mechanistic changes in mixture toxicity and highlight the value of potency-based EDA for linking biological effects to causative chemicals.