To Örebro University

Goals, Scope and Background. Fish populations, especially thoseof the grayling (Thymallus thymallus), have declined over thelast two decades in the upper Danube River between Sigmaringenand Ulm, despite intensive and continuous stocking and improvementof water quality since the 1970s. Similar problems havebeen reported for other rivers, e.g. in Switzerland, Great Britain,the United States and Canada. In order to assess if ecotoxicologicaleffects might be related to the decline in fish catchat the upper Danube River, sediment, suspended matter andwaste water samples from sewage treatment plants were collectedat selected locations and analyzed in a bioanalytical approachusing a battery of bioassays. The results of this pilotstudy will be used to decide if a comprehensive weight-of-evidencestudy is needed.

Methods. Freeze-dried sediments and suspended particulate matterswere extracted with acetone in a Soxhlet apparatus. Organicpollutants from sewage water were concentrated usingXAD-resins. In order to investigate the ecotoxicological burden,the following bioassays were used: (1) neutral red assaywith RTL-W1 cells (cytotoxicity), (2) comet assay with RTLW1cells (genotoxicity), (3) Arthrobacter globiformis dehydrogenaseassay (toxicity to bacteria), (4) yeast estrogen screen assay(endocrine disruption), (5) fish egg assay with the zebrafish(Danio rerio; embryo toxicity) and (6) Ames test with TA98(mutagenicity).

Results and Discussion. The results of the in vitro tests elucidateda considerable genotoxic, cytotoxic, mutagenic, bacteriotoxic,embryotoxic and estrogenic burden in the upper DanubeRiver, although with a very inhomogeneous distribution of effects.The samples taken from Riedlingen, for example, inducedlow embryo toxicity, but the second highest 17β-estradiol equivalentconcentration (1.8 ng/L). Using the fish egg assay with nativesediments, a broad range of embryotoxic effects could beelucidated, with clear-cut dose-response relationships for theembryotoxic effects of contaminated sediments. With nativesediments, embryotoxicity was clearly higher than with correspondingpore waters, thus corroborating the view that – atleast for fish eggs – the bioavailability of particle-bound lipophilicsubstances in native sediments is higher than generally assumed. The effect observed most frequently in the fish egg assay was a developmental delay. A comparison of our own results with locationsalong the rivers Rhine and Neckar demonstrated similaror even higher ranges of ecotoxicological burdens in theDanube River.

Conclusions. The complex pattern of ecotoxicological effectscaused by environmental samples from the Danube River, whenassessed in an in vitro biotest battery using both acute and morespecific endpoints, showed that integration of different endpointsis essential for appropriate hazard assessment. Overall, theecotoxicological hazard potential shown has indeed to be consideredas one potential reason for the decline in fish catches atthe upper Danube River. However, based on the results of thispilot study, it is not possible to elucidate that chemically inducedalterations are responsible for the fish decline.

Recommendations and Perspectives. In order to confirm the ecologicalrelevance of the in vitro results for the situation in thefield and especially for the decline of the grayling and otherfishes, further integrated investigations are required. For linkingthe weight of evidence obtained by in vitro assays and fishpopulation investigations, the application of additional, morespecific biomarkers (e.g. vitellogenin induction, EROD and micronucleusassay) has been initiated in fish taken from the fieldas well as in situ investigations.