Tiered genotoxicity testing of enriched river water samples using zebrafish in vitro and in vivo models: A Joint Danube Survey 4 case study

• Geno- and embryo-toxicity of Danube water samples were assessed in zebrafish models. • ZFL cells showed higher sensitivity than the SOS/ umuC and embryo assays. • ZFL cells proved to be a sensitive early-warning model for sublethal genotoxicity. • There is a high heterogeneity in data on genotoxic potential of detected substances. • QSAR predictions partly matched experimental findings but revealed major data gaps. The increasing complexity of aquatic pollution, dominated by diverse and often uncharacterized chemical mixtures, challenges traditional monitoring approaches. In this study, we assessed the genotoxic potential of surface water samples collected during the Joint Danube Survey 4 (JDS4) using large-volume solid-phase extraction (LVSPE) combined with a comprehensive battery of bioassays. Twenty-three enriched water samples from the Danube River and its major tributaries were evaluated for genotoxicity using a tiered testing strategy comprising the SOS/ umuC assay, zebrafish liver (ZFL) cell-based assays (cytotoxicity, comet assay, cell cycle), and zebrafish embryo assays. While no genotoxicity was detected in the prokaryotic SOS/ umuC assay, ZFL assays revealed significant DNA damage in 16 out of 23 samples, with notable genotoxicity observed in samples from the middle Danube section. In contrast, no teratogenic effects were observed in zebrafish embryo assays at concentrations up to REF100. These findings demonstrate the superior sensitivity of ZFL cells compared to both prokaryotic and in vivo embryo models. The study also highlights a critical gap in available genotoxicity data for detected substances, emphasizing the need for standardized databases and testing frameworks. Overall, our results support zebrafish-based in vitro assays as effective tools for effect-based monitoring, providing early warnings of genotoxic pollution in complex aquatic environments.