Investigations on sediment toxicity of German rivers applying a standardized bioassay battery.

River sediments may contain a huge variety of environmental contaminants and play a key role in the ecological status of aquatic ecosystems. Contaminants adsorbed to sediments and suspended solids may contribute directly or after remobilization to an adverse ecological and chemical status of surface water. In this subproject of the joint research project DanTox, acetonic Soxhlet extracts from three German river sediments from the River Rhine (Altrip and Ehrenbreitstein with moderate contamination) and River Elbe (Veringkanal Hamburg heavily contaminated) were prepared and redissolved in dimethyl sulfoxide (DMSO). These extracts were analyzed with a standard bioassay battery with organisms from different trophic levels (bacteria, algae, Daphnia, fish) as well as in the Ames test and the umuC test for bacterial mutagenicity and genotoxicity according to the respective OECD and ISO guidelines. In total, 0.01% (standard) up to 0.25% (only fish embryo test) of the DMSO sediment extract was dosed to the test systems resulting in maximum sediment equivalent concentrations (SEQ) of 2 up to 50 g l(-1). The sediment of Veringkanal near Hamburg harbor was significantly more toxic in most tests compared to the sediment extracts from Altrip and Ehrenbreitstein from the River Rhine. The most toxic effect found for Veringkanal was in the algae test with an ErC50 (72 h) of 0.00226 g l(-1) SEQ. Ehrenbreitstein and Altrip samples were about factor 1,000 less toxic. In the Daphnia, Lemna, and acute fish toxicity tests, no toxicity at all was found at 2 g l(-1) SEQ. corresponding to 0.01% DMSO. Only when increasing the DMSO concentration the fish embryo test showed a 22-fold higher toxicity for Veringkanal than for Ehrenbreitstein and Altrip samples, while the toxicity difference was less evident for the Daphnia test due to the overlaying solvent toxicity above 0.05% dimethyl sulfoxide (DMSO). The higher toxicities observed with the Veringkanal sample are supported by the PAH and PCB concentrations analyzed in the sediments. The sediment extracts of Altrip and Veringkanal were mutagenic in the Ames tester strain TA98 with metabolic activation (S9-mix). The findings allow a better ecotoxicological characterization of the sediments extensively analyzed in all subprojects of the DanTox project (e.g., Garcia-Kaeufer et al. Environ Sci Pollut Res. doi: 10.1007/s11356-014-3894-4 , 2014; Schiwy et al. Environ Sci Pollut Res. doi: 10.1007/s11356-014-3185-0 , 2014; Hollert and Keiter 2015). In the absence of agreed limit values for sediment extracts in standard tests, further data with unpolluted reference sediments are required for a quantitative risk assessment of the investigated polluted sediments.

Impact of reference geosorbents on oral bioaccessibility of PAH in a human in vitro digestive tract model.

Former studies on human oral bioaccessibility of polycyclic aromatic hydrocarbons (PAH) from natural soil samples using human in vitro digestive tract models (physiologically based extraction tests, PBET) show highly variable results (0-100% of mobilized PAH). Apart from other factors, the type and amount of present geosorbents are assumed to be significant for the degree of desorption/release of PAH into the digestive juice. Therefore, in this study, the reference geosorbents pure quartz sand, Na-montmorillonite clay, Pahokee peat, and charcoal "Sommerhit" were spiked with selected deuterated PAH and employed as single materials in a PBET. Lowest bioaccessibility was determined in charcoal, representing black carbon (0.1 ± 0.1 % for ∑10 PAH-d) in contrast to higher bioaccessibility in peat (6.4 ± 2.2%) and clay (4.8 ± 1.1%). Highest bioaccessibility was determined in sand (26.9 ± 7.5%). The results show a systematic impact of heterogeneous geosorbents on human oral bioaccessibility of PAH and particularly black carbon acting as a very strong geosorbent that reduces human health risk.

Mutagenicity, dioxin-like activity and bioaccumulation of alkylated picene and chrysene derivatives in a German lignite.

In a former study, a German lignite extract exhibited toxicity to Danio rerio and Caenorhabditis elegans and was shown to have mutagenic and dioxin-like activity. Besides the comparatively low content of known toxic polycyclic aromatic hydrocarbons (PAH), highly intensive peaks of m/z 274 and m/z 324 were observed during the chromatographic analysis. These compounds are assumed to be alkylated chrysenes and picenes (3,3,7-trimethyl-1,2,3,4-tetrahydrochrysene, 1,2-(1'-isopropylpropano)-7-methylchrysene and an isomer of the latter, 1,2,9-trimethyl-1,2,3,4-tetrahydropicene and 2,2,9-trimethyl-1,2,3,4-tetrahydropicene). These compounds are intermediates in the diagenetic formation of chrysene and picene from triterpenoids. Due to their general high abundance in lignites and the toxicity observed for the lignite extract, the mechanism-specific toxicity and bioavailability of these compounds were investigated in the present study using the approach of effect-directed analysis. After the separation of the compounds from other PAH, their mutagenic activity (Ames Fluctuation test) and dioxin-like activity (EROD activity) were studied. Both, mutation induction factor (up to 2.9±2.7) and dioxin-like activity (Bio-TEQ of 224±75 pg/g; represents the amount (pg) 2,3,7,8-tetrachlorodibenzo-p-dioxin per g coal that would provoke the same toxic effect) were rather low. Bioavailability estimated by the bioaccumulation test with Lumbriculus variegatus was also very limited. Based on the obtained results, the environmental risk of the highly abundant alkylated chrysenes and picenes in lignites is concluded to be low.

Polar polycyclic aromatic compounds from different coal types show varying mutagenic potential, EROD induction and bioavailability depending on coal rank.

Investigations of the bioavailability and toxicity of polycyclic aromatic compounds (PAC) have rarely considered the heterogeneity of coals and the impact of more polar PAC besides polycyclic aromatic hydrocarbons (PAH). Earlier, we investigated the toxicity of eight heterogeneous coals and their extracts. In the present study, the hazard potential with respect to mechanism-specific toxicity of polar fractions of dichloromethane extracts from coals was studied. Polar extract fractions of all coal types except for anthracite induced EROD activity (determined in RTL-W1 cells), independent of coal type (Bio-TEQs between 23 ± 16 and 52 ± 22 ng/g). The polar fractions of all bituminous coal extracts revealed mutagenic activity (determined using the Ames Fluctuation test). No significant mutation induction was detected for the polar extract fractions from the lignite, sub-bituminous coal and anthracite samples, which indicates a higher dependency on coal type for polar PAC here. Additionally, information on bioavailability was derived from a bioaccumulation test using the deposit-feeding oligochaete Lumbriculus variegatus which was exposed for 28 days to ground coal samples. Despite the high toxic potential of most coal extracts and a reduced biomass of Lumbriculus in bituminous coal samples, bioaccumulation of PAH and mortality after 28 days were found to be low. Limited bioaccumulation of PAH (up to 3.6 ± 3.8 mg/kg EPA-PAH) and polar PAC were observed for all coal samples. A significant reduction of Lumbriculus biomass was observed in the treatments containing bituminous coals (from 0.019 ± 0.004 g to 0.046 ± 0.011 g compared to 0.080 ± 0.025 g per replicate in control treatments). We conclude that bioavailability of native PAC from coals including polar PAC is low for all investigated coal types. In comparison to lignite, sub-bituminous coals and anthracite, the bioavailability of PAC from bituminous coals is slightly increased.

Limited waterborne acute toxicity of native polycyclic aromatic compounds from coals of different types compared to their total hazard potential.

Coals contain native polycyclic aromatic compounds (PACs), which include polycyclic aromatic hydrocarbons (PAHs), and heterocyclic aromatic compounds (NSO-PACs) in considerably varying amounts up to 2500 mg/kg. Whereas PAC bioavailability and toxicity from coals are generally considered to be low, few studies have considered potential variations arising from the composition of different coal types including native PAC content. In the present study, fine particles of different coal types exhibiting variable properties were systematically investigated regarding their PAC bioavailability. PAH content reached up to 79 mg/kg EPA-PAH and 865 mg/kg total PAH. Determination of the toxic potential of extracted PACs in bioassays showed inhibition of Caenorhabditis elegans reproduction (up to 94%) and increased mortality of Danio rerio embryos (up to 100%) after exposure to extracts from lignite, sub-bituminous, and bituminous coals. Anthracite extracts showed no effects. Contact assays using whole coal samples revealed no toxicity to D. rerio embryos in any of the coal samples, suggesting low bioavailability of PACs. In contrast, C. elegans reproduction was inhibited by direct coal contact; however, the observed toxicity probably resulted from other coal effects. The results suggest that despite the high toxic potential of PACs present, their bioavailability from different coal types is very limited and independent of coal properties and native PAH content.

Toxic masking and synergistic modulation of the estrogenic activity of chemical mixtures in a yeast estrogen screen (YES).

BACKGROUND, AIM AND SCOPE: Estrogenic and non-estrogenic chemicals typically co-occur in the environment. Interference by non-estrogenic chemicals may confound the assessment of the actual estrogenic activity of complex environmental samples. The aim of the present study was to investigate whether, in which way and how seriously the estrogenic activity of single estrogens and the observed and predicted joint action of estrogenic mixtures is influenced by toxic masking and synergistic modulation caused by non-estrogenic chemical confounders. MATERIALS AND METHODS: The yeast estrogen screen (YES) was adapted so that toxicity and estrogenicity could be quantified simultaneously in one experimental run. Mercury, two organic solvents (dimethyl sulfoxide (DMSO) and 2,4-dinitroaniline), a surfactant (LAS-12) and the antibiotic cycloheximide were selected as toxic but non-estrogenic test chemicals. The confounding impact of selected concentrations of these toxicants on the estrogenic activity of the hormone 17ss-estradiol was determined by co-incubation experiments. In a second step, the impact of toxic masking and synergistic modulation on the predictability of the joint action of 17ss-estradiol, estrone and estriol mixtures by concentration addition was analysed. RESULTS: Each of the non-estrogenic chemicals reduced the apparent estrogenicity of both single estrogens and their mixtures if applied at high, toxic concentrations. Besides this common pattern, a highly substance- and concentration-dependent impact of the non-estrogenic toxicants was observable. The activity of 17ss-estradiol was still reduced in the presence of only low or non-toxic concentrations of 2,4-dinitroaniline and cycloheximide, which was not the case for mercury and DMSO. A clear synergistic modulation, i.e. an enhanced estrogenic activity, was induced by the presence of slightly toxic concentrations of LAS-12. The joint estrogenic activity of the mixture of estrogens was affected by toxic masking and synergistic modulation in direct proportion to the single estrogens, which allowed for an adequate adaptation of concentration addition and thus unaffected predictability of the joint estrogenicity in the presence of non-estrogenic confounders. DISCUSSION: The modified YES proved to be a reliable system for the simultaneous quantification of yeast toxicity and estrogen receptor activation. Experimental results substantiate the available evidence for toxic masking as a relevant phenomenon in estrogenicity assessment of complex environmental samples. Synergistic modulation of estrogenic activity by non-estrogenic confounders might be of lower importance. The concept of concentration addition is discussed as a valuable tool for estrogenicity assessment of complex mixtures, with deviations of the measured joint estrogenicity from predictions indicating the need for refined analyses. CONCLUSIONS: Two major challenges are to be considered simultaneously for a reliable analysis of the estrogenic activity of complex mixtures: the identification of known and suspected estrogenic compounds in the sample as well as the substance- and effect-level-dependent confounding impact of non-estrogenic toxicants. RECOMMENDATIONS AND PERSPECTIVES: The application of screening assays such as the YES to complex mixtures should be accompanied by measures that safeguard against false negative results which may be caused by non-estrogenic but toxic confounders. Simultaneous assessments of estrogenicity and toxicity are generally advisable.