Monitoring methodologies reflecting the long-term quality and contamination of surface waters are needed to obtain a representative picture of pollution and identify risk drivers. This study sets a baseline for characterizing chemical pollution in the Danube River using an innovative approach, combining continuous three-months use of passive sampling technology with comprehensive chemical (747 chemicals) and bioanalytical (seven in vitro bioassays) assessment during the Joint Danube Survey (JDS4). This is one of the world's largest investigative surface-water monitoring efforts in the longest river in the European Union, which water after riverbank filtration is broadly used for drinking water production. Two types of passive samplers, silicone rubber (SR) sheets for hydrophobic compounds and AttractSPETM HLB disks for hydrophilic compounds, were deployed at nine sites for approximately 100 days. The Danube River pollution was dominated by industrial compounds in SR samplers and by industrial compounds together with pharmaceuticals and personal care products in HLB samplers. Comparison of the Estimated Environmental Concentrations with Predicted No-Effect Concentrations revealed that at the studied sites, at least one (SR) and 4-7 (HLB) compound(s) exceeded the risk quotient of 1. We also detected AhR-mediated activity, oxidative stress response, peroxisome proliferator-activated receptor gamma-mediated activity, estrogenic, androgenic, and anti-androgenic activities using in vitro bioassays. A significant portion of the AhR-mediated and estrogenic activities could be explained by detected analytes at several sites, while for the other bioassays and other sites, much of the activity remained unexplained. The effect-based trigger values for estrogenic and anti-androgenic activities were exceeded at some sites. The identified drivers of mixture in vitro effects deserve further attention in ecotoxicological and environmental pollution research. This novel approach using long-term passive sampling provides a representative benchmark of pollution and effect potentials of chemical mixtures for future water quality monitoring of the Danube River and other large water bodies.

RECETOX Faculty of Science Masaryk University Kotlarska 2 61137 Brno Czech Republic

RECETOX Faculty of Science Masaryk University Kotlarska 2 61137 Brno Czech Republic; Global Change Research Institute of the Czech Academy of Sciences Belidla 986 4a 60300 Brno Czech Republic

UBA German Environment Agency Wörlitzer Platz 1 D 06844 Dessau Roßlau Germany

UFZ Helmholtz Centre for Environmental Research Department of Cell Toxicology 04318 Leipzig Germany

UFZ Helmholtz Centre for Environmental Research Department of Cell Toxicology 04318 Leipzig Germany; Environmental Toxicology Department of Geosciences Eberhard Karls University Tübingen Tübingen Germany

UFZ Helmholtz Centre for Environmental Research Department of Effect Directed Analysis 04318 Leipzig Germany

UFZ Helmholtz Centre for Environmental Research Department of Effect Directed Analysis 04318 Leipzig Germany; Goethe University Frankfurt Department of Evolutionary Ecology and Environmental Toxicology Max von Laue Straße 13 60438 Frankfurt Main Germany

University of South Bohemia in České Budějovice Faculty of Fisheries and Protection of Waters Research Institute of Fish Culture and Hydrocenoses Zátiší 728 2 389 25 Vodňany Czech Republic

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