Abstract
(Englisch)
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The general objectives of the programme, involving EAWAG, were to examine the occurrence and distribution of endocrine-disrupting effluents across a range of European countries, using existing fish-exposure techniques for the detection of reproductive interference. Those effluents which were shown to be capable of interfering with the fish endocrine system were then analysed for the principal causative agents. Another objective was to collect new information on the impacts of endocrine disrupters on aquatic wildlife. Further objectives were to develop and improve in vitro tools for the rapid detection of endocrine disrupters and to investigate the application of existing in vitro techniques for the direct screening of complex effluents.Using the in vivo fish vitellogenin (VTG) bio-assay, four out of seventeen municipal effluents tested in eight European countries were non-estrogenic, seven were weakly and six were strongly estrogenic, while of the eight industrial effluents tested in eight countries, only two were strongly and one weakly estrogenic, none of these coming from paper mills. COMPREHEND thus demonstrated that estrogenic effluents occur across mainland Europe and that these include both municipal and industrial waste waters. All effluents were additionally tested for estrogenic potency using the yeast estrogen screen (YES) and analysed for steroid hormones and nonylphenol (NP), nonylphenol mono- and -diethoxylate (NP1/2EO). There was no obvious correlation between estrogenic activity (plasma VTG or YES) and NP concentrations. Estrogenic potential expressed as estradiol equivalents, calculated based on measured steroid hormone concentrations on the other hand often matched YES activity. However, the limit of detection and precision of the steroid analysis was strongly influenced by the matrix, yielding uncertainties around 40% close to the detection limit (0.1 - 1 ng/L depending on the matrix and compound).Population studies showed that the numbers of juvenile brown trout were significantly lower in the polluted downstream sites of the River Wyna than in the unpolluted headwaters. However, the observed effect can not be directly linked to endocrine disruption / reduced reproductive success at this time. Exposure of brown trout eggs to river water downstream of sewage effluents resulted in a temperature-independent acceleration of embryonic development to the median eye stage but a delay in the time to hatch. Again it is not possible to link this directly to endocrine disruption at this stage, but it is interesting to note that the Finnish partner also reported a delay in the time to hatch in zebrafish eggs exposed to a municipal wastewater treatment plant effluent.Prior to COMPREHEND, most of the available in vitro assays for estrogenicity testing were based on mammalian receptors, whereas the known impacts of xenoestrogens on wildlife were primarily on fish. EAWAG undertook to investigate a rainbow trout gonad cell line (RTG-2) for estrogen-sensitive genes and their suitability for the development of new in vitro screening tools. The RTG-2 cell line was successfully transfected with an estrogen-responsive reporter gene construct and from this, a new in vitro estrogen screen was developed. The assay showed a similar range of sensitivity as did the mammalian-based systems and, therefore, it is reasonable to use the more amenable mammalian receptor-based assays for environmental monitoring and screening.
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