Ludwig Karbe

Genotoxicity assessment of suspended particulate matter in the Elbe river: comparison of Salmonella microsome test, arabinose resistance test, and umu-test

This study evaluates the applicability of three bacterial short-term genotoxicity test systems to aquatic suspended particulate matter of the Elbe river. This material was sampled in sedimentation vessels after deposition periods of one month. It was extracted in a Soxhlet apparatus with toluene and methanol. Aqueous elutriates were prepared additionally. A solid phase method was developed that enables to incubate bacteria in contact with the particulate material. The test battery consists of two mutagenicity assays (the Ames-test and the Ara-test) and an SOS induction assay (the umu-test). Both mutagenicity assays came to nearly the same assessment of the samples of particulate matter of the Elbe. The quantitative response, however, was higher in the Ara-test. The particulate river material generally induced lower genotoxic potencies in the umu-test than in the mutagenicity assays. This lead to a completely different outcome of the umu-test; 29 out of 35 mutagenic samples were not SOS inducing. No quantitative or rank correlations between the concentrations of anthropogenic contaminants (PAHs, chlorinated hydrocarbons and metals) and the observed effects could be established on a 90% confidence limit. However, there is obvious correspondence between more contaminated regions of the river system and mutagenic effects (Ames- and Ara-test) in the samples from this region, as well as correspondence between low contaminated regions and the absence of mutagenicity. For this reason, the mutagenicity assays appear more favourable to describe the anthropogenic contamination with genotoxins in complex mixtures than the umu-test. The authors recommend the Ara-test for a first genotoxicity screening of complex environmental mixtures. This forward mutagenicity assay is advantageous due to higher effects and lower costs compared to the Ames-test. The development and use of a solid phase version of Ames- and Ara-test revealed the occurrence of a major part of particle-bound mutagens. The hydrophobic nature of these mutagens was also confirmed by the gradually decreasing effects with decreasing lipophilicity of the solvents. The results suggest that the solid phase test and the use of extracts complement each other in detecting mutagens of different lipophilicity. Both versions should be used in order to include a broad variety of compounds.

Induction of DNA strand breaks in rainbow trout Oncorhynchus mykiss under hypoxic and hyperoxic conditions

Abstract Rainbow trout (Oncorhynchus mykiss) were exposed to various degrees of oxygen saturation (3.3–21.1 mg O2/l). After 5 h of exposure, trout were killed and DNA single-strand breaks (DSSB) were determind in the gills by the alkaline unwinding method. Significant increases of DSSB compared to normoxic conditions (11 mg O2/l) were observed under hypoxic and particularly hyperoxic conditions. The highest rate of DSSB occurred when the fish were kept under hypoxic conditions followed by a rapid increase of the oxygen concentration. DSSB formation under these conditions was comparable to that achieved by treatment with 200 μM of the potent carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). These data demonstrate that natural influences, such as varying oxygen concentrations in the water, can be responsible for the induction of DSSB. These findings call into question the usefulness of the measurement of DSSB in fish as an indicator of water pollution by genotoxic chemicals.

In vitro suppression of thymocyte apoptosis by metal-rich complex environmental mixtures: potential role of zinc and cadmium excess

Excessive amounts of heavy metals (e.g. Zn, Cu, Mn, Cr) are accumulated in river bottom sediments (RBS), being available to humans and animals along food chains. Increased exposure of mammals to certain metals (Cr, Cu) induces immunosuppresion, due to DNA damage and decreased survival of lymphoid cells. By contrast, excess of Zn and Cd causes inhibition of apoptosis thus suggesting increased survival of genetically mutated cells and higher cancer risks in exposed populations. Rat thymic lymphocytes represent a well-established model for apoptosis testing. The primary goal of our study was to assess the degree of apoptosis modulation with a number of RBS extracts differing in their metal contents. A series of freshly deposited RBS was collected at nine sampling stations along the Elbe River. All sediments were rich in Fe, Mn and Zn. The contents of Cu, Cr, Ni, Cd, Hg, Pb and As were much lower and interrelated. The short-term cytotoxicity of aqueous sediment extracts was assessed, using the following criteria: total cell counts; incidence of apoptosis and necrosis (morphological detection by fluorescent microscopy); and nuclear chromatin decay (by DNA flow cytometry). RBS extracts produced both apoptosis and necrosis of thymocytes. High contents of zinc and other heavy metals in the samples correlated with decreased thymocyte apoptosis (r= -0.543 to -0.608, P <0.01). The rates of thymocyte damage showed a distinct dependence on the time and region of sampling. Apoptosis modulation was also tested with pure salts of Mn(II), Zn(II), Cu(II), Cr(III) and Cd(II), at the test concentrations of 1, 10 and 100 microM. Cu(II) and Cr(III) proved to induce marked dose-related apoptosis, whereas Zn(II) ions caused significant suppression of apoptosis. These effects were similar to those trends observed with metal-rich sediments. In the present study. DNA flow cytometry proved to be a less sensitive index of cell death than morphological assay of apoptosis and/or necrosis. In summary, inhibition of lymphocyte apoptosis by RBS extracts and pure metals is associated with excess of zinc and, probably, cadmium. The proposed model of lymphoid cell apoptosis is a promising tool for screening cytotoxic effects of complex environmental samples.

The use of the Salmonella BA9 forward mutation assay in sediment quality assessment: mutagenicity of freshly deposited sediments of the River Elbe

The aim of the present study was to investigate whether bacterial mutagenicity assays can be applied in sediment quality assessment. The arabinose-sensitive forward mutation strain Salmonella typhimurium BA 9 was added to freshly deposited sediments of the River Elbe, collected during March, April, and May 1994. Up to twelve locations were sampled each month and mutagenicity was determined employing the solid phase and sequentially prepared toluene- and methanol-extracts of the sediments. Mutagenicity was detected at all sites within the solid sediments without addition of S9-mix; however, the addition of mammalian enzymes (S9) enhanced the mutagenic effect. In contrast, mutagenicity of toluene extracts containing the lipophilic fraction of the sediment samples was higher in the absence of S9-mix; peaks of mutagenic activity in these samples were observed at Dessau (mouth of the River Mulde) and close to the city of Hamburg (Bunthaus). Similar results were obtained with methanolic extracts of the sediments, although the effects were usually lower in comparison to the corresponding toluene extracts. These results show that the mutagenicity assays are capable of assessing water/sediment contamination and reveal that the mutagenicity detected in sediments reflects local industrial activities as well as hydrologic conditions.

Environmental Risks of Mining Metalliferous Muds in the Atlantis II Deep, Red Sea

After two companies were awarded a 30-year license for the exploration and exploitation of metalliferous sediments in the Atlantis II Deep (Red Sea) in 2011, we herewith present conclusions and recommendations derived from an environmental risk assessment, the Metalliferous Sediment Atlantis II Deep (MESEDA) study, conducted in the period 1977–1981. For economic reasons, this program was discontinued before final report delivery and fell dormant for 30 years. The effects of environmental disturbances of the benthic and the near-bottom water layer habitats in and around the mining site deserve further and more modern risk assessments. We examine the relevance of our 1981 recommendations and of subsequent publications to the extended period of resource extraction planned for this century and recommend more up-to-date risk assessment investigations and evaluations.

Sediment mutagenicity testing: development of substance specific bacterial strains for the detection of mutagenic aromatic nitrogen compounds and oxidative mutagens

Abstract The arabinose resistance forward mutation assay was chosen for the development of bacterial strains in order to get specific mutagenic responses. Special strains of Salmonella typhimurium were constructed which show an elevated expression of nitroreductase and O-acetyltransferase. They were shown to be highly sensitive to mutagenic nitro-compounds (e.g. 1-nitropyrene and 1,8-dinitropyrene) and, after metabolic activation by rat liver S9-mix, also to mutagenic amino-compounds (2-aminoanthracene). Furthermore, strains of Escherichia coli with reduced expression of antioxidative enzymes (catalase and superoxide dismutase) were constructed. However, they were only moderately sensitive to oxidative mutagens such as quinones, nitrogen compounds, and the herbicide paraquat, because, in contrast to the Salmonella strains used, they build up a complete gram-negative cell wall. For this reason, the Escherichia strains were further genetically altered in order to make their cell wall penetrable to lipophili...