Sibylle Maletz

Changes in toxicity and Ah receptor agonist activity of suspended particulate matter during flood events at the rivers Neckar and Rhine — a mass balance approach using in vitro methods and chemical analysis

Background, aim, and scopeAs a consequence of flood events, runoff and remobilized sediments may cause an increase of ecotoxicologically relevant effects from contaminant reservoirs. Aquatic and terrestrial organisms as well as cattle and areas of settlement are exposed to dislocated contaminants during and after flood events. In this study, the impacts of two flood events triggered by intense rain at the rivers Neckar and Rhine (Southern Germany) were studied. Effects in correlation to flood flow were assessed at the river Neckar using samples collected at frequent intervals. River Rhine suspended particulate matter (SPM) was sampled over a longer period at normal flow and during a flood event. Three cell lines (H4L1.1c4, GPC.2D.Luc, RTL-W1) were used to compare Ah receptor agonist activity in different biotest systems. Multilayer fractionation was performed to identify causative compounds, focusing on persistent organic contaminants.Materials and methodsNative water and SPM of flood events were collected at the river Neckar and at the monitoring station (Rheinguetestation, Worms, Germany) of the river Rhine. Water samples were XAD-extracted. SPM were freeze-dried and Soxhlet-extracted using acetone and finally dissolved in dimethyl sulfoxide. Resulting crude extracts were analyzed for cytotoxicity with the neutral red assay. Aryl hydrocarbon receptor (AhR) agonist activity was measured in a set of biological test systems (DR-CALUX, GPC.2D, and ethoxyresorufin-O-deethylase (EROD) assay) and different cell lines. In addition, crude extracts were fractionated using a combined method of multilayer (sequence of acidified silica layers) and carbon fractionation. Fractions from the multilayer fractionation contained persistent organic compounds (polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), polychlorinated biphenyls (PCBs), and some polycyclic aromatic hydrocarbon (PAHs)); fractions from the carbon fractionation were separated into a PCDD/F and a PCB fraction. Dioxin-like activity of multilayer and carbon fractions was determined in the EROD assay and expressed as biological toxicity equivalency concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (bio-TEQs). The calculation of chemical equivalency concentrations (chem-TEQs) and comparison to bio-TEQ values allowed the determination of the contribution of the analyzed persistent compounds to the total biological effects measured.ResultsSoluble compounds in native and extracted water samples resulted in no or minor activity in the toxicity tests, respectively. Filter residues of native water caused increased AhR-mediated activity at the peak of the flood. Activities of SPM of the river Neckar correlated well with the flow rate indicating a flood-dependent increase of toxicity culminating at the peak of flow. River Rhine SPM showed a decrease of activity regarding an SPM sample of the flood event compared to a long-term sample. Excellent correlations with AhR agonistic activity were determined for DR-CALUX and EROD assay, while the GPC.2D assay did not correlate with both other biotests. The activity of persistent dioxin-like acting compounds in multilayer and carbon fractionated PCDD/F and PCB fractions was low if compared to corresponding crude extracts. The congener pattern of PCDD/F revealed that the contaminations mainly originated from products and productions of the chlorine and organochlorine industries.DiscussionNative and extracted water samples could be shown to contain little or no cytotoxic or AhR agonistic compounds. In contrast, particle-bound compounds were shown to be the relevant effect-causing fraction, as indicated by the activities of filter residues of native water and SPM. Compounds other than fractionated persistent PCBs and PCDD/Fs were more relevant to explain AhR-mediated activities of crude flood SPM at both rivers assessed. Biologically detected activities could at least in part be traced back to chemically analyzed and quantified compounds.ConclusionsThe calculation of the portion of persistent PCBs and PCDD/Fs in multilayer fractions causing the high inductions in the EROD assay in combination with chemical analysis provides a suitable tool to assess dioxin-like activity of persistent compounds in SPM sampled over the course of flood events. Depending on the catchment area and annual course of flood events, end points may either indicate an increase or a decrease of activity. In order to determine the ecological hazard potential of mobilized contaminants during flood events, the focus should be set on particle-bound pollutants. Furthermore, PCDD/Fs and PCBs, commonly expected to be the most relevant pollutants in river systems, could be shown to contribute only to a minor portion of the overall AhR-mediated activity. However, they might be most relevant for human exposure when considering persistence and bioaccumulation–biomagnification in the food chain.Recommendations and perspectivesAs a consequence of climate change, flood events will increase in frequency and intensity at least in some regions such as Central Europe. Thus, it is crucial to identify the potential hazard of (re-)mobilized contaminants from reservoirs dislocated via floods and threatening especially aquatic organisms and cattle grazing in flood plains. Since other less persistent compounds seem to be more relevant to explain AhR-mediated activities in flood SPM, nonconventional PAHs and more polar compounds also need to be considered for risk assessment. Effect-directed analysis using broad-range fractionation methods taking into account compounds from polar to nonpolar should be applied for identification of pollutants causing biological effects, thus integrating biological and chemical parameters.

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

Background, goals, and scopeIn 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 17β-estradiol (E2) in preparation for the development of an Organization for Economic Cooperation and Development (OECD) test guideline.MethodsA 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.ResultsWith 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 conclusionsWith 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).PerspectivesBased 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.

In vitro characterization of the effectiveness of enhanced sewage treatment processes to eliminate endocrine activity of hospital effluents

Occurrence of pharmaceuticals in aquatic ecosystems is related to sewage effluents. Due to the possible adverse effects on wildlife and humans, degradation and removal of pharmaceuticals and their metabolites during wastewater treatment is an increasingly important task. The present study was part of a proof of concept study at a medium sized country hospital in western Germany that investigated efficiency of advanced treatment processes to remove toxic potencies from sewage. Specifically, the efficiency of treatment processes such as a membrane bioreactor (MBR) and ozonation to remove endocrine disruptive potentials was assessed. Estrogenic effects were characterized by use of two receptor-mediated in vitro transactivation assays, the Lyticase Yeast Estrogen Screen (LYES) and the Estrogen Receptor mediated Chemical Activated LUciferase gene eXpression (ER CALUX(®)). In addition, the H295R Steroidogenesis Assay (H295R) was utilized to detect potential disruption of steroidogenesis. Raw sewage contained measurable estrogen receptor (ER)-mediated potency as determined by use of the LYES (28.9 ± 8.6 ng/L, 0.33× concentration), which was reduced after treatment by MBR (2.3 ± 0.3 ng/L) and ozone (1.2 ± 0.4 ng/L). Results were confirmed by use of ER CALUX(®) which measured concentrations of estrogen equivalents (EEQs) of 0.2 ± 0.11 ng/L (MBR) and 0.01 ± 0.02 ng/L (ozonation). In contrast, treatment with ozone resulted in greater production of estradiol and aromatase activity at 3× and greater concentrations in H295R cells. It is hypothesized that this is partly due to formation of active oxidized products during ozonation. Substance-specific analyses demonstrated efficient removal of most of the measured compounds by ozonation. A comparison of the ER-mediated responses measured by use of the LYES and ER CALUX(®) with those from the chemical analysis using a mass-balance approach revealed estrone (E1) to be the main compound that caused the estrogenic effects. Overall, treatment of sewage by use of MBR successfully reduced estrogenicity of hospital effluents as well as substances that are able to alter sex steroid production. However, after ozonation, effluents should undergo further investigations regarding the formation of endocrine active metabolites. The results obtained as part of this study demonstrated applicability of in vitro assays for monitoring of endocrine-modulating potency of treated sewage.

Heterocyclic Aromatic Hydrocarbons Show Estrogenic Activity upon Metabolization in a Recombinant Transactivation Assay

Heterocyclic aromatic hydrocarbons (hetero-PAHs) are increasingly studied at contaminated sites; especially at former industrial facilities where coal tar-oil was handled, e.g., wood treatment plants, high concentrations of hetero-PAHs are frequently detected in groundwater plumes. In previous studies, fractions of groundwater with high estrogenic activity contained hetero-PAHs and their hydroxylated metabolites. To evaluate this preliminary evidence, selected hetero-PAHs were screened for their estrogenic activity in lyticase yeast estrogen screen (LYES) and ER CALUX. All tested substances were inactive in the LYES. Hetero-PAHs such as acridine, xanthene, indole, 2-methylbenzofuran, 2,3-dimethylbenzofuran, dibenzofuran, dibenzothiophene, quinoline, and 6-methylquinoline were positive in the ER CALUX, with estradiol equivalence factors (EEFs) from 2.85 × 10(-7) to 3.18 × 10(-5). The EEF values of these substances were comparable to those of other xenoestrogens (e.g., alkylphenols or bisphenol A) that are sometimes found in surface water. Chemical analyses revealed that T47Dluc cells could metabolize most of the substances. Among the metabolites (tentatively) identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were hydroxides and their keto tautomers, sulfates, sulfoxides, and N-oxides. Because of their high concentrations measured in groundwater, we conclude that hetero-PAHs and metabolites may be a potential risk and should be the subject of further research.

Biotests for hazard assessment of biofuel fermentation

To meet the increasing demand for energy, development of alternative and renewable energy sources, such as bioenergy, has accelerated during the last decade. In this context, biofuels are one potential replacement for fossil fuels, although their impact on the environment has not been widely studied. Only a few studies are available on toxicity of biofuels and biofuel combustion. Furthermore, for a complete understanding of the environmental impact, the entire life cycle of a biofuel has to be analyzed. This study is an exemplary ecotoxicological investigation of a biomass-to-biofuel production process with respect to the generation of environmentally relevant contaminants either by means of biomass pretreatment or microbial activity. Our aim is the demonstration of the suitability of ecotoxicological biotests as part of a comprehensive hazard assessment of biofuels and related samples or processes. Five ecotoxicological endpoints were assayed to determine the impact of four different biomass pretreatments on process substrates and effluent toxicities. Four different test organisms (bacterium, yeast, fish cell line, and fish embryo) from different trophic levels as well as a combination of acute and mechanism-specific biotests were applied to strengthen the ecotoxicological relevance of this investigation. Biotest results revealed cytotoxic, acute embryotoxic and mutagenic effectiveness, and weak estrogenic activity, with biomass toxicity depending on the mechanism of substrate pretreatment. Open microbial communities (reactor microbiomes) involved in the production process decreased the toxicity considerably to levels of the product n-butyric acid due to degradation of inhibiting by-products, verifying their simultaneous biomass conversion and detoxification potential. Our results demonstrate that ecotoxicological biotests are useful tools for the biofuel industries to gain environmental friendliness as a selling point.

Uptake, Elimination, and Biotransformation of 17α-Ethinylestradiol by the Freshwater Alga Desmodesmus subspicatus

Bioconcentration and transformation of the potent and persistent xeno-estrogen 17α-ethinylestradiol (EE2) by organisms at the basis of the food web have received only little research attention. In this study, uptake, elimination, and biotransformation of radiolabeled EE2 ((14)C-EE2) by the freshwater green alga Desmodesmus subspicatus were investigated. The alga highly incorporated radioactivity following (14)C-EE2 exposure. Up to 68% of the test compound was removed from the medium by D. subspicatus within a rather short time period (72 h C(algae)/C(water): 2200 L/kg wet weight). When the algae were transported to clear medium, a two-stage release pattern was observed with an initially quick elimination phase following slower clearance afterward. Interestingly, D. subspicatus brominated EE2 when bromide was available in the medium, a transformation process demonstrated to occur abiotically but not by algae. The consequence of the presence of more hydrophobic mono- and dibrominated EE2 in the environment remains to be further investigated, as these products were shown to have a lower estrogenic potency but are expected to have a higher bioaccumulation potential and to be more toxic than the mother compound.

Effects of multiwalled carbon nanotubes and triclocarban on several eukaryotic cell lines: elucidating cytotoxicity, endocrine disruption, and reactive oxygen species generation

To date, only a few reports about studies on toxic effects of carbon nanotubes (CNT) are available, and their results are often controversial. Three different cell lines (rainbow trout liver cells (RTL-W1), human adrenocortical carcinoma cells (T47Dluc), and human adrenocarcinoma cells (H295R)) were exposed to multiwalled carbon nanotubes, the antimicrobial agent triclocarban (TCC) as well as the mixture of both substances in a concentration range of 3.13 to 50 mg CNT/L, 31.25 to 500 μg TCC/L, and 3.13 to 50 mg CNT/L + 1% TCC (percentage relative to carbon nanotubes concentration), respectively. Triclocarban is a high-production volume chemical that is widely used as an antimicrobial compound and is known for its toxicity, hydrophobicity, endocrine disruption, bioaccumulation potential, and environmental persistence. Carbon nanotubes are known to interact with hydrophobic organic compounds. Therefore, triclocarban was selected as a model substance to examine mixture toxicity in this study. The influence of multiwalled carbon nanotubes and triclocarban on various toxicological endpoints was specified: neither cytotoxicity nor endocrine disruption could be observed after exposure of the three cell lines to carbon nanotubes, but the nanomaterial caused intracellular generation of reactive oxygen species in all cell types. For TCC on the other hand, cell vitality of 80% could be observed at a concentration of 2.1 mg/L for treated RTL-W1 cells. A decrease of luciferase activity in the ER Calux assay at a triclocarban concentration of 125 μg/L and higher was observed. This effect was less pronounced when multiwalled carbon nanotubes were present in the medium. Taken together, these results demonstrate that multiwalled carbon nanotubes induce the production of reactive oxygen species in RTL-W1, T47Dluc, and H295R cells, reveal no cytotoxicity, and reduce the bioavailability and toxicity of the biocide triclocarban.

Tox-Box: securing drops of life - an enhanced health-related approach for risk assessment of drinking water in Germany

This article introduces ‘Tox-Box’, a joint research project designed to develop a holistic approach towards a harmonized testing strategy for exposure- and hazard-based risk management of anthropogenic trace substances in drinking water to secure a long-term drinking water supply. The main task of the Tox-Box consortium is to enhance the existing health-related indicator value concept (German: GOW-Konzept - Gesundheitlicher Orientierungswert) through development and prioritization of additional end point-related testing strategies for genotoxicity, neurotoxicity, germ cell damage, and endocrine effects. In this context, substance-specific modes of action will be identified and characterized. Toxicological data collected by the 12 Tox-Box subprojects will be evaluated and weighted to structure a hierarchical testing strategy for an improved risk assessment. A technical guidance document for exposure and hazard-based risk management of anthropogenic trace substances in drinking water will eventually be prepared.ZusammenfassungDieser Artikel stellt das Verbundprojekt “Tox-Box” vor, das einen ganzheitlichen Ansatz für eine harmonisierte Teststrategie eines Expositions-bezogenen und Gefahren-basierten Risikomanagements von anthropogenen Spurenstoffen in Trinkwasser entwickeln und somit einen Beitrag zur langfristigen Sicherung der Trinkwasserversorgung leisten soll. Die Hauptaufgabe des Tox-Box-Konsortiums ist die Weiterentwicklung des bestehenden GOW-Konzeptes (Gesundheitlicher Orientierungswert) durch Erforschung und Priorisierung zusätzlicher Endpunkt-bezogener Teststrategien für Gentoxizität, Neurotoxizität, Keimzellschädigung und endokrine Effekte. In diesem Kontext werden zudem Substanz-spezifische Wirkmechanismen identifiziert und charakterisiert. Im Anschluss werden die toxikologischen Daten aus den 12 Teilprojekten evaluiert und gewichtet um eine hierarchische Teststrategie für eine verbesserte Risikobewertung zu erstellen. Zum Abschluss des Projektes wird eine technische Richtlinie für ein Expositions-bezogenes und Gefahren-basiertes Risikomanagement von anthropogenen Spurenstoffen im Trinkwasser erstellt.

Are sediments a risk? An ecotoxicological assessment of sediments from a quarry pond of the Upper Rhine River

PurposeContaminated sediments are an important exposure pathway for the aquatic fauna in the Rhine River. We applied bioassays with the aim to characterize the ecotoxicological hazard potential of sediments of an oxbow lake of the Rhine River, especially to fish. Potential effects on fish and water flea were evaluated indirectly by applying in vitro and in vivo bioassays in the laboratory. Results were compared with those of the official German risk assessment of dredged sediments.Materials and methodsSediments taken from 13 sites along a 600-m transect line were tested for acute toxicity to water flea (Daphnia magna immobilization test), teratogenicity, and embryotoxicity (sediment contact test with Danio rerio), as well as for cytotoxicity (neutral red retention assay with RTL-W1 cells) and estrogenic effects (lyticase-assisted yeast estrogen screen (L-YES) assay). The tests were conducted using pore water, organic extracts, or native sediments. Spatial patterns of the measured effects were also assessed.Results and discussionVirtually all samples induced estrogenic, teratogenic, embryotoxic, and cytotoxic effects, but no acute toxicity on D. magna was observed. Cytotoxicity was in accordance with previous studies on the Rhine, Neckar, and Danube Rivers. Estrogenic effects were in the range of estradiol equivalent (EEQ) values detected in UK estuaries. Although sediment contact tests with D. rerio embryos showed virtually no mortality, sublethal effects were common. Some of the effects increased with increasing distance to the main channel.ConclusionsThe test with D. magna is, along with bacteria and algae toxicity assays, an important part of the German standard risk assessment for sediments. However, it failed to identify the ecological hazard of our sediment samples to fish. Our results indicate that adverse effects on fish are possible and suggest the need for revising risk assessment procedures in order to address the risk for this important organism group in aquatic ecosystems.

Investigation of potential endocrine disrupting effects of mosquito larvicidal Bacillus thuringiensis israelensis (Bti) formulations.

Bti is successfully used as a biological control agent for mosquito control. It has proven to be ecological friendly, and thus, is used in ecologically sensitive habitats. Recent investigations of groundwater in Germany have detected estrogenic activity in five consecutive groundwater wells in a region where Bti is applied. Therefore, it was suspected that this compound can act as an environmental xenoestrogen. In the present study, five Bti formulations as well as the active ingredient, VectoBac® TP (TP), were investigated regarding their estrogenic activity using the LYES and ER CALUX® assays. Furthermore, their steroidogenesis disruption properties were studied using the H295R Steroidogenesis Assay. Additionally, field samples from a Bti application area as well as samples from an artificial pond were examined. Three of the Bti formulations and the active ingredient TP showed significant estrogenic activity in the LYES (up to 52 ng·l(-1) estradiol equivalents (EEQ) in the 18-fold concentration) and/or the ER CALUX® (up to 1 ng·EEQ·l(-1) in the 18-fold concentration). In the H295R significant but weak effects with no dose-response-relationship on the production of estradiol, and 21-hydroxyprogesterone (WDG) as well as testosterone (TP) by H295R cells could be observed. The field samples as well as the samples from the artificial pond showed no significant increase of estrogenic activity after application of TP or WDG in the ER CALUX®. With the exception of the controlled laboratory experiments with direct application of Bti to the utilized in vitro test systems the present study did not reveal any significant effects of Bti on endocrine functions that would indicate that the application of Bti could cause adverse endocrine effects to organisms in aquatic ecosystems. Instead, our results support previous studies that the use of Bti products against mosquitos would be safe even for sensitive habitats such as conservation areas.