Complex mixtures of micropollutants, including pesticides, pharmaceuticals and industrial chemicals emitted by wastewater effluents to European rivers may compromise the quality of these water resources and may pose a risk to ecosystem health and abstraction of drinking water. In the present study, an integrated analytical and bioanalytical approach was applied to investigate the impact of untreated wastewater effluents from the city of Novi Sad, Serbia, into the River Danube. The study was based on three on-site large volume solid phase extracted water samples collected upstream and downstream of the untreated wastewater discharge. Chemical screening with liquid chromatography high resolution mass spectrometry (LC-HRMS) was applied together with a battery of in vitro cell-based bioassays covering important steps of the cellular toxicity pathway to evaluate effects on the activation of metabolism (arylhydrocarbon receptor AhR, peroxisome proliferator activated receptor gamma PPARγ), specific modes of action (estrogen receptor ERα, androgen receptor AR) and adaptive stress responses (oxidative stress, inflammation). Increased effects, significantly changed contamination patterns and higher chemical concentrations were observed downstream of the wastewater discharge. A mass balance approach showed that enhanced endocrine disruption was in good agreement with concentrations of detected hormones, while only a smaller fraction of the effects on xenobiotic metabolism (<1%) and adaptive stress responses (0-12%) could be explained by the detected chemicals. The chemical and effects patterns observed upstream of the discharge point were fairly re-established at about 7 km downstream, demonstrating the enormous dilution capacity of this large river.

Australian Rivers Institute School of Environment Griffith University Southport QLD 4222 Australia; The University of Queensland National Research Centre for Environmental Toxicology Brisbane QLD 4108 Australia

Australian Rivers Institute School of Environment Griffith University Southport QLD 4222 Australia; UFZ Helmholtz Centre for Environmental Research Cell Toxicology 04318 Leipzig Germany; Eberhard Karls University Tübingen Environmental Toxicology Center for Applied Geosciences 72074 Tübingen Germany; The University of Queensland National Research Centre for Environmental Toxicology Brisbane QLD 4108 Australia

Masaryk University Research Centre for Toxic Compounds in the Environment Kamenice 753 5 62500 Brno Czechia

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

UFZ Helmholtz Centre for Environmental Research Effect Directed Analysis 04318 Leipzig Germany; RWTH Aachen University Institute for Environmental Research Aachen Germany

University of Novi Sad Faculty of Sciences Trg Dositeja Obradovica 2 21000 Novi Sad Serbia

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