In vitro bioassay tools for the toxicological evaluation of dioxins and dioxin-like compounds in sediments and biota

Since the middle of the 20th century, there has been increasing concern about certain historical contaminants, which due to their potential health effects (e.g. hepatotoxicity, endocrine disruption and infertility) and properties (accumulation in e.g. sediments and tissues) pose a threat to humans and wildlife. Among these contaminants, dioxin-like compounds (DLCs) can be found, including polychlorinated dibenzo p dioxins and dibenzofurans (PCDD/Fs) as well as dioxin-like polychlorinated biphenyls (DL-PCBs). Although emissions of DLCs have been reduced considerably during the past decades, legacy pollution of sediments still constitutes a secondary pollutant source following sediment re mobilizations (e.g. floods or dredging activities). This might cause the majority of the European surface waters not to reach the aims set by the European water framework directive (WFD). It is thus not astonishing that recently, the assessment of sediment and dredged material gained more and more attention and that new concepts have been developed such as clearly defined Environmental Quality Standards (EQS) for sediments and biota. While sediment assessment originally simply based on classical, instrumental methods (e.g. high resolution gas chromatography - high resolution mass spectrometry (HRGC/HRMS)), these methods due to their lacking information on sediment toxicity today are often connected to ecotoxicological and ecological methods. However, those methods often exhibit relatively unspecific endpoints (e.g. growth inhibition) and thus do not allow for a comparison to concentrations measured via HRGC/HRMS. Because many of the toxic effects of DLCs are mediated via the cytosolic aryl hydrocarbon receptor (AhR), and the strengths of activation of the AhR constitutes a specific endpoint of many cell-based in vitro bioassays, these methods are of increasing interest to regulators and risk assessors. Biological equivalent quotients (BEQs) deduced from such assays are directly comparable to toxicity equivalents (TEQs) of HRGC/HRMS analyses. The DioRAMA project is a joint research initiative between the Institute of Environmental Research at the RWTH Aachen University and the Department G3 of the German Federal Institute of Hydrology in Koblenz. Its main goal was to establish in vitro tools for the assessment of DLCs in sediment and biota to improve current risk assessment approaches. Therefore, the present thesis investigated (1) the sensitivity of various in vitro bioassays, the suitability of certain in vitro assays to be used (2) as regulatory tools, (3) to screen the uptake of DLCs by fish and (4) to be used as prioritizing tools for sediment and soil extracts. (4) Finally, the bioavailability of certain DLCs was investigated.A literature study proved the applicability of various in vitro bioassays for the screening of DLCs in a multitude of complex samples, individual compounds and mixtures. To be capable of screening trace contaminants such as DLCs, in vitro bioassays have to be comparably sensitive like instrumental techniques. The literature data reviled that some of the in vitro bioassays approximated the sensitivity limits of chemical analytical methods (~0.1 pM dioxin) and hence suggests the potential suitability of these assays to be used as additionally regulatory tools.To verify this assumption, three in vitro bioassays (RTL-W1 EROD, H4IIE-luc and H4IIE Micro EROD assay) were investigated with regard to their possible implementation into German guidelines for the management of dredged material. Evaluations of intra- and inter-laboratory performance and predicative power of the used bioassays based on extracts of sediments, differently contaminated with DLCs. Except the high sample throughput (RTL-W1 EROD) and the high linear range (H4IIE-luc), the H4IIE Micro EROD assay showed the overall best performance among the three assays and had a similar predictive power like HRGC/HRMS analyses. The H4IIE Micro EROD assay was highly sensitive and showed a satisfying repeatability and cross-laboratory reproducibility, independent of sample complexity. Hence, the H4IIE Micro EROD assay was proven to be highly suitable for the analysis of DLCs and to be used as ptential regulatory tool in the sediment management.In a further study, the uptake of sediment-borne DLCs by common roach, a fish of high ecological relevance, was chemically and bio-chemically investigated using the same differently contaminated sediment samples (see above) as exposure media. Fish was either exposed to black worm - inoculated sediments (dietary eposure) or daily fed with uncontaminated worms. Both chemical and bio-chemical investigations of whole fish extracts predominantly revealed an uptake of sediment-borne DLCs by fish, independent of the sediment DLC contamination degree. BEQs indicated the uptake to be promoted by (1) the suspended matter concentration in the water column and (2) the additional ingestion of feed/sediment (only relevant for the sediment of highest DLC contamination).While this study proved the applicability of the Micro EROD assay for challenging sample matrices such as whole fish homogenates, another study, which chemically and biochemically investigated sediment and soil samples from the river Elbe catchment area, proved the H4IIE Micro EROD assays’ applicability to be used as high throughput screening tool for large sample sets. Samples of highest EROD-inducing potential, even though raw extracts (missing clean-up) were investigated, corresponded well to the via HRGC/HRMS detected contamination hotspots along the river. A H4IIE Micro EROD assay based limit value was deduced from the DLC concentrations of the river Elbe sediment samples and might point towards a future yes/no-decision-level in German guidelines for dredged material. A final study investigated the bioavailabilty of polycyclic aromatic hydrocarbons (PAHs). A tenax desorption experiment was meant to close the gap between instrumental and toxicological results. It was shown that the cumulative concentrations of PAHs desorbing from the four differently contaminated sediments corresponded well to (1) the initial PAH concentrations in sediments, (2) to the effects observed in fish eggs of D. rerio and (3) to a certain extend to RTL-W1 BEQs. This indicated the higher contaminated sediments to pose a potentially higher threat to the aquatic environment.The present findings might contribute to future regulatory decisions in a way that in vitro bioassays such as the Micro EROD assay could be implemented into German guidelines for dredged material to be used as an additional quality measure alongside classically used instrumental analysis and this way could significantly improve current sediment assessment strategies.

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