Fish populations, especially grayling (Thymallus thymallus), have declined over the last 15 years in the upper Danube river between Sigmaringen and Ulm despite both intensive and continuous stocking and improvement of water quality since the 1970´s. In order to assess if ecotoxicological effects might be related to the decline in fish catch at the upper Danube river, sediment samples, suspended matter and waste water from sewage treatment plants were collected at different locations and analyzed in a triad approach including a battery of bioassays, in situ investigations as well as chemical analyses. Freeze-dried sediments were extracted with acetone in a Soxhlet apparatus. In order to investigate the ecotoxicological hazard potential, the following bioassays were used: (1) neutral red assay with RTL-W1 cells (cytotoxicity), (2) comet assay with RTL-W1 cells and Danio rerio (genotoxicity), (3) sediment contact assay with zebrafish embryos (Danio rerio; embryotoxicity), (4) EROD assay with RTL-W1 (dioxin-like activity), (5) CALUX assay with rat hepatoma cells (dioxin-like activity), (6) micronucleus assay (genotoxicity) with RTL-W1 and liver cells from barbel (Barbus barbus) and (7) the Ames test with TA98/TA100 (mutagenicity). Benthic macroinvertebrate diversity and abundance were assessed with family- and species-based bioindices. Histopathological investigations and the micronucleus assay with erythrocytes were applied using barbel caught in the Danube representing in situ parameters. Redoxpotential and pH in situ, AOX, TOC as well as oxygen, nitrite, nitrate and phosphate concentrations were determined. Chemical analyses were performed for heavy metals, PCB, PAH and PCDD/Fs.