Human-impacted rivers often contain a complex mixture of organic micropollutants, including pesticides, pharmaceuticals and industrial compounds, along with their transformation products. Combining chemical target analysis for exposure with in vitro bioassays for effect assessment offers a holistic view of water quality. This study targeted the River Elbe in Central Europe, known for its anthropogenic pollution exposure, to obtain an inventory of micropollutant contamination during base flow and to identify hotspots of contamination. We identified tributaries as sources of chemicals activating the aryl hydrocarbon receptor quantified with the AhR-CALUX assay, including historically contaminated tributaries and a newly identified Czech tributary. Increased neurotoxicity, detected by differentiated SH-SY5Y neurons' cytotoxicity and shortened neurite length, was noted in some Czech tributaries. A hotspot for chemicals activating the oxidative stress response in the AREc32 assay was found in the middle Elbe in Germany. An increase in oxidative stress inducing chemicals was observed in the lower Elbe. While effect-based trigger values (EBT) for oxidative stress response, xenobiotic metabolism and neurotoxicity were not exceeded, estrogenicity levels surpassed the EBT in 14% of surface water samples, posing a potential threat to fish reproduction. Target analysis of 713 chemicals resulted in the quantification of 487 micropollutants, of which 133 were active in at least one bioassay. Despite this large number of bioactive quantified chemicals, the mixture effects predicted by the concentrations of the quantified bioactive chemicals and their relative effect potency explained only 0.002-1.2% of the effects observed in the surface water extracts, highlighting a significant unknown fraction in the chemical mixtures. This case study established a baseline for understanding pollution dynamics and spatial variations in the Elbe River, offering a comprehensive view of potential chemical effects in the water and guiding further water quality monitoring in European rivers.

Biology Centre Czech Academy of Sciences Institute of Hydrobiology Ceské Budejovice 370 05 Czech Republic

Department Exposure Science Helmholtz Centre for Environmental Research UFZ Permoserstr 15 Leipzig 04318 Germany

Department of Cell Toxicology Helmholtz Centre for Environmental Research UFZ Permoserstr 15 Leipzig 04318 Germany

Department of River Ecology Helmholtz Centre for Environmental Research UFZ Brückstr 3a Magdeburg 39114 Germany

Department of Shelf Sea System Ecology Alfred Wegener Institut Helmholtz Zentrum für Polar und Meeresforschung Kurpromenade 201 Helgoland 27498 Germany

Environmental Toxicology Department of Geosciences Eberhard Karls University Tübingen Schnarrenbergerstr 94 96 Tübingen 72076 Germany

GEOMAR Helmholtz Centre for Ocean Research Kiel Wischhofstraße 1 3 Kiel 24148 Germany

Institute of Carbon Cycles Helmholtz Centre Hereon Max Planck Straße 1 Geesthacht 21502 Germany

Institute of Ecology Evolution and Diversity Goethe University Max von Laue Str 13 Frankfurt am Main 60438 Germany

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