The endocrine disrupting potential of sediments from the Upper Danube River (Germany) as revealed by in vitro bioassays and chemical analysis

Introduction  

The present study was part of a comprehensive weight-of-evidence approach with the goal of identifying potential causes for the declines in fish populations, which have been observed during the past decades in the Upper Danube River.     

Eine Weight-of-Evidence-Studie zur Bewertung der Sedimentbelastung und des Fischrückgangs in der Oberen Donau

Background and aim Despite intensive and continuous stocking and improvement of water quality since the 1970s, fish populations, especially those of the grayling (Thymallus thymallus), have declined over the last two decades in the upper Danube River (Germany). In order to assess 1) possible links between molecular/biochemical responses and ecologically relevant effects, and 2) if ecotoxicological effects might be related to the decline in fish catches in the upper Danube river, sediment samples and fish were collected at different locations and analyzed using a weight-of-evidence (WOE) approach with several lines of evidence. The objective of the presentation is to introduce the conceptual framework and to review results of the ongoing study. As previously addressed by Chapman and Hollert (2006) a variety of lines of evidence can be used in WOE studies. Briefly, 1) a comprehensive battery of acute and mechanism-specific bioassays was used to characterize the ecotoxicological hazard potential. 2) Histopathological investigations and the micronucleus assay with erythrocytes were applied, analyzing in situ parameters. 3) Diversity and abundance of benthic macroinvertebrates and fish as well as 4) persistent organic pollutants, endocrine disrupting substances, limnochemical parameters and the concentration of heavy metals were recorded. To identify organic contaminants a spotential causes of sediment toxicity assays, 5) effect directed analysis was applied.     

Strategien zur Sedimentbewertung – ein Überblick

Background Being an important determinant in aquatic ecosystems, sediments have gotten more and more into focus of scientific and public discussions. While water quality has been significantly improving during recent years, highly contaminated sediments in many European rivers will still have ongoing impact for several centuries from now. Hence, monitoring and assessment of sediment quality are crucial for national legislation as well as the implementation of the European Water Framework Directive (WFD). Aim On the occasion of the retirement of Prof. Dr. Dr. h.?c. Volker Storch, this article reviews the various concepts of sediment assessment and introduces case studies in sediment toxicology which have been carried out in Heidelberg and surrounding areas. Results and Discussion Initially, the article portrays benefits and drawbacks of chemical analytics and biotest systems. The individual approach has only limited informative value, but combining both perspectives allows for a comprehensive characterization of the state of sediments. As examples of toxicity evaluation based on this strategy, weight-of-evidence studies for tiered investigations and integrated sediment assessment are presented. In addition, a combination of chemical fractionation, bioanalytic investigations and chemical analysis – known as ‘effect-directed analysis’ (EDA) – is discussed. This integrated concept eventually aims at the identification of hazardous substance classes or even of single compounds. Finally, the article raises the issue of sediment mobility as an important parameter for risk analyses of highly contaminated legacy sediments within further WFD implementation. Outlook Using various case studies, the article outlines the potentials of integrated approaches for cause-effect analysis of complex environmental samples within aquatic ecosystems as well as for action programs of management plans dealing with chemically polluted rivers. Effect-directed analysis in particular, but also the combined application of acute and mechanism-specific bioassays together with in-situ investigations, complemented by investigations on sediment mobility, appear promising with regard to comprehensive sediment assessment weight-of-evidence studies.     

The OECD validation program of the H295R steroidogenesis assay: Phase 3. Final inter-laboratory validation study

Background, goals, and scope

In response to increasing concerns regarding the potential of chemicals to interact with the endocrine system of humans and wildlife, various national and international programs have been initiated with the aim to develop new guidelines for the screening and testing of these chemicals in vertebrates. Here, we report on the validation of an in vitro assay, the H295R steroidogenesis assay, to detect chemicals with the potential to inhibit or induce the production of the sex steroid hormones testosterone (T) and 17??-estradiol (E2) in preparation for the development of an Organization for Economic Cooperation and Development (OECD) test guideline.

Methods

A previously optimized and pre-validated protocol was used to assess the potential of 28 chemicals of diverse structures and properties to validate the H295R steroidogenesis assay. These chemicals are comprised of known endocrine-active chemicals and ??negative?? chemicals that were not expected to have effects on the targeted endpoints, as well as a number of test chemicals with unknown modes of action at the level of the steroidogenic pathway. A total of seven laboratories from seven countries participated in this effort. In addition to effects on hormone production, confounding factors, such as cell viability and possible direct interference of test substances with antibody-based hormone detection assays, were assessed. Prior to and during the conduct of exposure experiments, each laboratory had to demonstrate that they were able to conduct the assay within the margin of predefined performance criteria.

Results

With a few exceptions, all laboratories met the key quality performance parameters, and only 2% and 7% of all experiments for T and E2, respectively, were excluded due to exceedance of these parameters. Of the 28 chemicals analyzed, 13 and 14 tested affected production of T and E2, respectively, while 11 and 8 did not result in significant effects on T and E2 production, respectively. Four and six chemicals produced ambiguous results for effects on T and E2 production, respectively. However, four of these cases each for T and E2 were associated with only one laboratory after a personnel change occurred. Significant interference of test chemicals with some of the antibody-based hormone detection systems occurred for four chemicals. Only one of these chemicals, however, significantly affected the ability of the detection system to categorize the chemical as affecting E2 or T production.

Discussion and conclusions

With one exception, the H295R steroidogenesis assay protocol successfully identified the majority of chemicals with known and unknown modes of interaction as inducers or inhibitors of T and E2 production. Thus it can be considered a reliable screen for chemicals that can alter the production of sex steroid hormones. One of the remaining limitations associated with the H295R steroidogenesis assay protocol is the relatively small basal production of E2 and its effect on quantifying the decreased production of this hormone with regard to the identification of weak inhibitors. An initial comparison of the data produced in this study with those from in vivo studies from the literature demonstrated the potential of the H295R steroidogenesis assay to identify chemicals affecting hormone homeostasis in whole organisms. Particularly promising was the lack of any false negatives during the validation and the very low number of false positives (1 out of 28 chemicals for each T and E2).

Perspectives

Based on the results obtained during this validation study and the accordingly revised test protocols, an OECD draft test guideline was developed and submitted to the OECD working group of the national coordinators of the test guidelines program (WNT) for comments in December 2009.     

Der Fischrückgang in der oberen Donau

Background During the last years, a decrease in fish populations has been documented for several river systems in Europe and North America. Since more than 20 years, similar developments have been documented for the upper Danube River between Sigmaringen and Ulm. The grayling (Thymallus thymallus) represents one of the most affected fish species in the upper Danube, and notable decrease has also been recorded for other species such as the European barbel (Barbus barbus). Despite intensive and continuous stocking and improvement of water quality along the Danube since the 1970s, this negative development of fish populations could not be stopped. Aim This article has been designed to give an overview on various investigations into the decline of fish in the upper Danube River. The major focus of this paper is on the presentation of a weight-of-evidence study carried out since 2003 at the Department of Zoology at the University of Heidelberg. Results and discussion During the 1920s, major parts of the upper Danube River were corrected, thus causing dramatic changes in the hydromorphology of the river. The macrozoobenthos, which represents an important source of nutrition for several fish species, depends on the morphological conditions of a river; recent investigations, however, could not show any dramatic changes in macrozoobenthos composition. Earlier investigations of the Danube River primarily focused on water quality, which has, indeed, been continuously improved by extensive modernization of wastewater treatment plants. Contamination by priority organic pollutants in sediments was not given any attention until the mid 1990s, when a clear decline of, e.?g., PAH concentrations were documented. However, results of more recent analyses showed very high PAH concentrations in two sediment samples (Schwarzach Σ 16 PAHs = 26 mg/kg; Öpfingen Σ 16 PAHs = 5 mg/kg). In 2002, the first ecotoxicological investigations were performed at the upper Danube River. The results of a pilot study showed a high ecotoxicological burden for selected sampling sites. As a conclusion of this study, a weight-of-evidence investigation was initiated to make an attempt to identify major pollutants and possible causes for the fish decline. In addition to, e.?g., liver ultrastructure analyses in barbel, the micronucleus test was used as a definite mutagenicity test using erythrocytes and liver samples. The results of the different in vitro tests showed very good correlation well with in situ parameters. Outlook The investigations within this weight-of-evidence study showed that the pollution situation of the Danube is, indeed, significant enough to potentially affect fish populations. However, further investigations are needed to clarify the specific causes for the fish decline. A bioassay-directed fractionation study is underway to identify specific pollutants as well as their sources. This study will also assess the endocrine disrupting potential of Danube sediments via histopathological analyses of gonads of field fish in combination with different in vitro tests. Furthermore, in the planned joint project COMPSEARCH, comparative investigations will evaluate the ecotoxicological hazard potential of different pollution sources and consequences of changes in land use.     

The OECD Validation Program of the H295R Steroidogenesis Assay for the Identification of In Vitro Inhibitors and Inducers of Testosterone and Estradiol Production. Phase 2: Inter-Laboratory Pre-Validation Studies (8 pp)

Background, Goals and Scope In response to concerns that have been raised about chemical substances that may alter the function of endocrine systems and result in adverse effects on human health, an OECD initiative was undertaken to develop and validate in vitro and in vivo assays to identify chemicals that may interfere with endocrine systems of vertebrates. Here we report on studies that were conducted to develop and standardize a cell-based screening assay using the H295R cell line to prioritize chemicals that may act on steroidogenic processes in humans and wildlife. These studies are currently ongoing as part of the ‘Special Activity on the Testing and Assessment of Endocrine Disruptors’ within the OECD Test Guidelines Program to review, develop, standardize, and validate a number of in vitro and in vivo toxicological assays for testing and assessment of chemicals concerning their potential to interact with the endocrine system of vertebrates. Study Design Six laboratories from five countries participated in the pre-validation studies. Each laboratory tested the effects of three model chemicals on the production of testosterone (T) and estradiol (E2) using the H295R Steroidogenesis Assay. Chemicals tested were well described inducers or inhibitors of steroidogenic pathways (forskolin, prochloraz and fadrozole). All experiments were conducted in 24 well plates following standard protocols. Six different doses per compound were analyzed in triplicate per plate. A quality control (QC) plate was run in conjunction with the chemical exposure plate to account for inter-assay variation. Each chemical exposure was conducted two or three times. Results All laboratories successfully detected increases and/or decreases in hormone production by H295R cells after exposure to the different model compounds and there was good agreement in the pattern of response for all groups. Forskolin increased both T and E2 while fadrozole and prochloraz decreased production of both hormones. All chemicals affected hormone production in a dose-dependent manner with the exception of fadrozole which caused maximum inhibition of E2 at the two least concentrations tested. Some inter-laboratory differences were noted in the alteration of hormone production measured in chemically exposed cells. However, with the exception of the production of T measured at one laboratory in cells exposed to forskolin, the EC₅₀s calculated were comparable (coefficients of variation 34–49%) for all hormones. Discussion and Perspectives The results indicated that the H295R Steroidogenesis Assay protocol was robust, transferable and reproducible among all laboratories. However, in several instances that were primarily related to one laboratory there were unexplained minor uncertainties related to the inter-laboratory hormone production variation. Based on the findings from this Phase 2 prevalidation study, the H295R Steroidogenesis Assay protocol is currently being refined. The next phase of the OECD validation program will test the refined protocol among the same group of laboratories using an extended set of chemicals (∼30) that will include positive and negative chemical controls as well as a broad spectrum of different potential inducers and inhibitors of steroidogenic pathways. Submission Editor: Dr. Carsten Brühl (bruehl@uni-landau.de)