Cross-species bioanalytical assessment of the oxidative stress and xenobiotic metabolism modes of action for environmental water samples - A multi-layered approach to testing and modelling.

Holistic protection goals in environmental hazard and risk assessment, complex pollutant mixtures, and limitations in targeted chemical analysis highlight the need for robust, mechanistically informative bioanalytics to support water quality monitoring. Given the scarcity of high-throughput, non-mammalian in vitro effect-based methods, this study evaluates the suitability of mammalian models as surrogates for aquatic species by investigating interspecies differences in the activation of oxidative stress (Nrf2/Keap1/ARE) and xenobiotic metabolism (AhR/ARNT/XRE) pathways quantified with cellular reporter gene assays. Wastewater treatment plant influent and effluent samples, alongside reference compounds, were analysed in human, mouse, and zebrafish reporter assays. Bioanalytics were complemented by in silico-mediated effect-directed analysis, iceberg-, molecular docking-, and chemical bioavailability-modelling. For Nrf2/Keap1/ARE, high concordance in reporter activity across species was observed in response to environmental samples, whereas the reference compound tert-butylhydroquinone elicited species-/assay-specific differences due to varying ligand affinities for the Keap1 redox sensor. In contrast, metazachlor exposure resulted in conserved activation patterns across species. For AhR/ARNT/XRE, interspecies variability in bioactivity was observed across environmental samples and the reference compound 2,3,7,8-tetrachlorodibenzodioxin, yielding divergent bioequivalent concentration estimates. In silico-mediated effect-directed analysis identified climbazole, daidzein, and thiabendazole as principal aryl hydrocarbon receptor activators, which also displayed species-/assay-specific activity under isolated exposure. Molecular docking confirmed species-dependent receptor-ligand affinities, while bioavailability modelling excluded differential cellular uptake, supporting receptor-mediated mechanisms as key drivers. Collectively, mammalian reporter assays can approximate oxidative stress responses in aquatic species, but limitations remain for xenobiotic metabolism, highlighting the need for species-representative assays and caution when contextually interpreting data from mammalian systems.