Passive Dosing in Chronic Toxicity Tests with the Nematode Caenorhabditis elegans.

In chronic toxicity tests with Caenorhabditis elegans, it is necessary to feed the nematode with bacteria, which reduces the freely dissolved concentration (Cfree) of hydrophobic organic chemicals (HOCs), leading to poorly defined exposure with conventional dosing procedures. We examined the efficacy of passive dosing of polycyclic aromatic hydrocarbons (PAHs) using silicone O-rings to control exposure during C. elegans toxicity testing and compared the results to those obtained with solvent spiking. Solid-phase microextraction and liquid-liquid extraction were used to measure Cfree and the chemicals taken up via ingestion. During toxicity testing, Cfree decreased by up to 89% after solvent spiking but remained constant with passive dosing. This led to a higher apparent toxicity on C. elegans exposed by passive dosing than by solvent spiking. With increasing bacterial cell densities, Cfree of solvent-spiked PAHs decreased while being maintained constant with passive dosing. This resulted in lower apparent toxicity under solvent spiking but an increased apparent toxicity with passive dosing, probably as a result of the higher chemical uptake rate via food (CUfood). Our results demonstrate the utility of passive dosing to control Cfree in routine chronic toxicity testing of HOCs. Moreover, both chemical uptake from water or via food ingestion can be controlled, thus enabling the discrimination of different uptake routes in chronic toxicity studies.

A comparative approach using ecotoxicological methods from single-species bioassays to model ecosystems.

Soft sediments are often hotspots of chemical contamination, and a thorough ecotoxicological assessment of this habitat can help to identify the causes of stress and to improve the health of the respective ecosystems. As an important component of the ecologically relevant meiobenthic fauna, nematodes can be used for sediment assessments, with various assay tools ranging from single-species toxicity tests to field studies. In the present study, microcosms containing sediment were used to investigate direct and indirect effects of zinc on natural nematode assemblages, and acute community toxicity tests considering only direct toxicity were conducted. The responses of the various freshwater nematode species in both approaches were compared with those of Caenorhabditis elegans, determined in standardized tests (ISO 10872). At a median lethal concentration (LC50) of 20 mg Zn/L, C. elegans represented the median susceptibility of 15 examined nematode species examined in the acute community toxicity tests. In the microcosms, Zn affected the nematodes dose-dependently, with changes in species composition first detected at 13 mg Zn/kg to 19 mg Zn/kg sediment dry weight. The observed species sensitivities in the microcosms corresponded better to field observations than to the results of the acute community toxicity tests. Environ Toxicol Chem 2016;35:2987-2997. © 2016 SETAC.

Equilibrium sampling of polychlorinated biphenyls in River Elbe sediments--Linking bioaccumulation in fish to sediment contamination.

Equilibrium sampling can be applied to measure freely dissolved concentrations (cfree) of hydrophobic organic chemicals (HOCs) that are considered effective concentrations for diffusive uptake and partitioning. It can also yield concentrations in lipids at thermodynamic equilibrium with the sediment (clip⇌sed) by multiplying concentrations in the equilibrium sampling polymer with lipid to polymer partition coefficients. We have applied silicone coated glass jars for equilibrium sampling of seven 'indicator' polychlorinated biphenyls (PCBs) in sediment samples from ten locations along the River Elbe to measure cfree of PCBs and their clip⇌sed. For three sites, we then related clip⇌sed to lipid-normalized PCB concentrations (cbio,lip) that were determined independently by the German Environmental Specimen Bank in common bream, a fish species living in close contact with the sediment: (1) In all cases, cbio,lip were below clip⇌sed, (2) there was proportionality between the two parameters with high R(2) values (0.92-1.00) and (3) the slopes of the linear regressions were very similar between the three stations (0.297; 0.327; 0.390). These results confirm the close link between PCB bioaccumulation and the thermodynamic potential of sediment-associated HOCs for partitioning into lipids. This novel approach gives clearer and more consistent results compared to conventional approaches that are based on total concentrations in sediment and biota-sediment accumulation factors. We propose to apply equilibrium sampling for determining bioavailability and bioaccumulation potential of HOCs, since this technique can provide a thermodynamic basis for the risk assessment and management of contaminated sediments.

Bioaccumulation in aquatic systems: methodological approaches, monitoring and assessment.

Bioaccumulation, the accumulation of a chemical in an organism relative to its level in the ambient medium, is of major environmental concern. Thus, monitoring chemical concentrations in biota are widely and increasingly used for assessing the chemical status of aquatic ecosystems. In this paper, various scientific and regulatory aspects of bioaccumulation in aquatic systems and the relevant critical issues are discussed. Monitoring chemical concentrations in biota can be used for compliance checking with regulatory directives, for identification of chemical sources or event-related environmental risk assessment. Assessing bioaccumulation in the field is challenging since many factors have to be considered that can affect the accumulation of a chemical in an organism. Passive sampling can complement biota monitoring since samplers with standardised partition properties can be used over a wide temporal and geographical range. Bioaccumulation is also assessed for regulation of chemicals of environmental concern whereby mainly data from laboratory studies on fish bioaccumulation are used. Field data can, however, provide additional important information for regulators. Strategies for bioaccumulation assessment still need to be harmonised for different regulations and groups of chemicals. To create awareness for critical issues and to mutually benefit from technical expertise and scientific findings, communication between risk assessment and monitoring communities needs to be improved. Scientists can support the establishment of new monitoring programs for bioaccumulation, e.g. in the frame of the amended European Environmental Quality Standard Directive.

Estrogenic effects along the river Saale.

Sediments along the river Saale, one of the main tributaries of the river Elbe, were characterized with the yeast estrogen screen to elucidate possible sources of endocrine-disrupting compounds that might contribute to the downstream contamination of the river Elbe. At two sampling sites, elevated levels of estrogenic activity up to 55,000 ng ethinylestradiol equivalents per kilogram sediment dry weight were detected in the respective sediment extracts. Aliquots of the sediment extracts were analyzed for 4-nonylphenols and natural steroidal estrogens as possible candidates with an estrogenic potential. The maximal concentrations of 4-iso-nonylphenol and estrone were 115 mg/kg dry weight and 20 µg/kg dry weight at the sampling site Luppe, which showed in accordance the highest biological activity. Under consideration of compound concentration and compound specific estrogenic activity the 4-iso-nonylphenols contributed most to the observed estrogenic effect. A strong correlation between the measured estrogenic activity and the concentration of the sediment-associated 4-iso-nonylphenol underlines the relevance of this compound class as a xenoestrogen in the catchment area of the river Saale.

Effect directed analysis and mixture effects of estrogenic compounds in a sediment of the river Elbe.

INTRODUCTION: Endocrine disrupting chemicals (EDCs) are present in the environment and can have serious effects on humans and wildlife. For the establishment of environmental quality guidelines and regulation of EDCs, a better understanding and knowledge of the occurrence and the behavior of environmental EDCs is necessary. The aim of the present study was to comprehensively identify substances that are responsible for the estrogenic effect of an environmental sediment sample taken from the river Elbe/Germany. DISCUSSION: The estrogenic effect of the organic sediment extract was determined using the yeast-estrogen-screen (YES). The sample was fractionated by liquid chromatography (LC) for effect directed analysis. The composition of estrogen-active fractions was further investigated by gas chromatography-mass spectrometry and high-resolution LC-MS analysis. The composition of the environmental sample was rebuilt with pure compounds in order to assess the partition of estrogenic activity caused by the identified compounds. The organic sediment extract showed an estrogenic potential of 1.9 ± 0.4 ng/g ethinylestradiol equivalents in the sediment. The most prominent contaminants with an estrogenic potential were 17ß-estradiol, estrone, and 4-iso-nonylphenols, but other xenoestrogens like bisphenol A and stigmasterol could be found as well. A rebuild of the sample was measured in the YES in order to investigate mixture effects. About 67 % of the observed estrogenic effect in the sediment extract could be explained by a mixture which contained all identified compounds. Chlorophene (o-benzyl-p-chlorophenol)-a widely used antiseptic that was also identified in the sediment extract-has xenoestrogenic properties in the YES that are in the range of other xenoestrogens like 4-n-nonylphenol. This is the first report on chlorophene acting as a xenoestrogen.

Organic carbon source in formulated sediments influences life traits and gene expression of Caenorhabditis elegans.

River water quality is strongly influenced by their sediments and their associated pollutants. To assess the toxic potential of sediments, sediment toxicity tests require reliable control sediments, potentially including formulated control sediments as one major option. Although some standardization has been carried out, one critical issue still remains the quality of sediment organic matter (SOM). Organic carbon not only binds hydrophobic contaminants, but may be a source of mild toxicity, even if the SOM is essentially uncontaminated. We tested two different sources of organic carbon and the mixture of both (Sphagnum peat (P) and one commercial humic substances preparation-HuminFeed(®), HF) in terms of life trait variables and expression profiles of selected life performance and stress genes of the nematode Caenorhabditis elegans. In synchronous cultures, gene expression profiling was done after 6 and 48 h, respectively. The uncontaminated Sphagnum P reduced growth, but increased numbers of offspring, whereas HF did not significantly alter life trait variables. The 6 h expression profile showed most of the studied stress genes repressed, except for slight to strong induction in cyp-35B1 (all exposures), gst-38 (only mixture), and small hsp-16 genes (all exposures). After 48 h, the expression of almost all studied genes increased, particularly genes coding for antioxidative defense, multiple xenobiotic resistance, vitellogenin-like proteins, and genes regulating lifespan. Overall, even essentially uncontaminated SOM may induce several modes of action on the molecular level in C. elegans which may lead to false results if testing synthetic xenobiotics. This contribution is a plea for a strict standardization of the SOM quality in formulated sediments and to check for corresponding effects in other model sediment organisms, especially if using molecular toxicity endpoints.

Identification of mutagens in freshwater sediments by the Ames-fluctuation assay using nitroreductase and acetyltransferase overproducing test strains.

Extracts of sediments from an area of concern in the Elbe river basins (Spittelwasser creek) were analyzed with the Ames-fluctuation test and in parallel with gas chromatography/mass spectrometry for compound identification. The standard test strains TA 98 and TA 100 showed mutagenicity mainly in medium-polar fractions of the sediment extracts. PAHs contribute to the overall mutagenic potential of the sample. Especially, cyclopenta[c,d]pyrene that was previously not defined as a priority hazardous substance has to be considered as well. The addition of metabolically competent test strains, which overexpress nitroreductase and acetyltransferase (e.g., YG1041 and YG1042) to the test battery, increased significantly the sensitivity of the Ames test for medium polar to polar genotoxins. The increased mutagenicity that was found in these bacterial strains indicates the presence of nitroarenes and/or aromatic amines. In fact, a number of heterocyclic and nitrogen-substituted aromatic compounds were identified in the sediments of the Spittelwasser creek of which methyl parathion, 1-naphthylamine, and N-phenyl-2-naphthylamine are mutagenic.

Using meiofauna to assess pollutants in freshwater sediments: a microcosm study with cadmium.

The direct and indirect effects of Cd on benthic communities were assessed in a freshwater microcosm study over a period of seven months (218 d). Cadmium was regarded as a model substance to evaluate the usefulness of small-scale laboratory microcosm with microscopic fauna. In particular, effects on the meiofauna community, an ecologically important but rather neglected benthic component, were investigated. In addition, some microfaunal parameters (protozoan abundance and microbial activity) were determined. The sediment was spiked with nominal Cd concentrations of 10, 100, and 1,000 mg/kg dry weight. Because of the strong binding of Cd to sediment particles, measured Cd pore-water concentrations never exceeded 129.5 ± 40.7 µg/L. At 1,000 mg/kg dry weight, the abundances of the two dominant meiofauna taxa, nematodes and oligochaetes, were significantly reduced throughout the present study. Regarding nematodes, species of bacterivorous taxa (Daptonema, Eumonhystera) decreased, whereas species of predacious and omnivorous taxa (Mononchus, Dorylaimus, and Ironus) increased in dominance in microcosms of the highest Cd concentration. Transient effects on microfauna were observed, especially in the first half of the present study, with a reduction in microbial activity and protozoan abundance. However, in microcosms receiving the highest Cd concentration, the abundance of the flagellate Euglena mutabilis increased significantly toward the end of the present study. The results of the present study support the use of small-scale microcosms with natural meiofauna communities as a suitable tool to assess the impact of pollutants in freshwater sediments.

Gene expression profiling to characterize sediment toxicity--a pilot study using Caenorhabditis elegans whole genome microarrays.

BACKGROUND: Traditionally, toxicity of river sediments is assessed using whole sediment tests with benthic organisms. The challenge, however, is the differentiation between multiple effects caused by complex contaminant mixtures and the unspecific toxicity endpoints such as survival, growth or reproduction. The use of gene expression profiling facilitates the identification of transcriptional changes at the molecular level that are specific to the bio-available fraction of pollutants. RESULTS: In this pilot study, we exposed the nematode Caenorhabditis elegans to three sediments of German rivers with varying (low, medium and high) levels of heavy metal and organic contamination. Beside chemical analysis, three standard bioassays were performed: reproduction of C. elegans, genotoxicity (Comet assay) and endocrine disruption (YES test). Gene expression was profiled using a whole genome DNA-microarray approach to identify overrepresented functional gene categories and derived cellular processes. Disaccharide and glycogen metabolism were found to be affected, whereas further functional pathways, such as oxidative phosphorylation, ribosome biogenesis, metabolism of xenobiotics, aging and several developmental processes were found to be differentially regulated only in response to the most contaminated sediment. CONCLUSION: This study demonstrates how ecotoxicogenomics can identify transcriptional responses in complex mixture scenarios to distinguish different samples of river sediments.

Nematode communities in contaminated river sediments.

Nematode communities of eight sites from three river catchments were investigated in terms of the genera composition, feeding types, and life-history strategists. The sampling sites showed a gradient of anthropogenic contamination with heavy metals and organic pollutants being important factors in differentiating the sites. Nematode community structure was related to sediment pollution and the hydro-morphological structure of the sampling sites. Heavily contaminated sites were characterized by communities with high relative abundances of omnivorous and predacious nematodes (Tobrilus, c-p 3; Mononchus, c-p 4), while sites with low to medium contamination were dominated by bacterivorous nematodes (Monhystera, Daptonema; c-p 2) or suction feeders (Dorylaimus, c-p 4). The relatively high Maturity Index values in the heavily polluted sites were surprising. Nematodes turned out to be a suitable organism group for monitoring sediment quality, with generic composition being the most accurate indicator for assessing differences in nematode community structure.