Modification of the umu-assay (ISO 13829) accounting for cytotoxicity in genotoxicity assessment: a preliminary study.

In this study, the umu-assay (ISO 13829) was modified by addition of the resazurin-reduction assay to assess the cytotoxic potential of toxins. The aim was to develop a test system that was capable of examining both genotoxicity and cytotoxicity on the basis of the metabolic health of the cells so as to provide a better assessment of the negative influence of toxic effects on the evaluation of genotoxicity. The test was established and validated with mitomycin C (MMC), 1,3-dinitropyrene (1,3-DNP), 4-nitroquinoline-1-oxide (4-NQO) and chloramphenicol (CHL) as toxins with known responses in the umu-assay. The results indicate that the modified umu-assay was able to reveal genotoxic responses towards MMC, 1,3-DNP and 4-NQO up to 1µg/ml. Further, the assay was able to determine the cytotoxicity of CHL, MMC and 4-NQO. Hence, the modified umu-assay was proven to be a sensitive test for determining the cytotoxicity and genotoxicity of toxins, with results that were comparable with literature data. Moreover, the modified umu-assay showed that cytotoxic concentrations that cause >50% inhibition of dehydrogenase activity (DHA) can result in unreliable computation of genotoxicity.

Assessing the importance of dietborne cadmium and particle characteristics on bioavailability and bioaccumulation in the nematode Caenorhabditis elegans.

To investigate the impact of multiple cadmium exposure pathways on bioavailability and bioaccumulation, Caenorhabditis elegans were exposed to either dissolved cadmium or to both particulate (dietborne) and dissolved cadmium. The dietborne metal exposure scenarios included evaluations of two bacteria species (Escherichia coli and Arthrobacter globiformis), varying bacteria concentrations, artificial particles (with different surface functional groups or coated with lipopolysaccharides), as well as a mixture of both bacteria and artificial particles. Total cadmium concentration in the nematodes was used as a proxy for total metal accumulation. Internal cadmium availability was determined using the cadmium-responsive gene-1 (cdr-1) transcript level, as quantified by real-time polymerase chain reaction. Particle-associated exposure was the predominant contributor to cadmium bioavailability and bioaccumulation; however, no clear relationship between cdr-1 expression and cadmium body burden was observed. The quality and quantity of particles, rather than the total particle-associated metal load, was of primary importance in regulating cadmium uptake and accumulation. Nematodes exposed to cadmium-contaminated bacteria displayed significantly higher levels of cdr-1 expression relative to artificial particles. Furthermore, C. elegans displayed a statistically significant difference in cadmium accumulation patterns between the biotic diet, abiotic particles, and aqueous phase. Waterborne exposure caused an increase in total cadmium body burden following inhibition of the P-glycoprotein transport system in nematodes. This may suggest that cadmium taken up by the aqueous phase is detoxified preferentially by excretion.

Reproduction of the estuarine and marine amphipod Corophium volutator (Pallas) in laboratory for toxicity testing.

The acute whole sediment bioassay with the estuarine and marine amphipod Corophium volutator (Pallas) is broadly used within Europe. Hitherto, the test is carried out with field-collected animals. In order to provide a more standardized and continuously available test organism reproduction and growth experiments were performed for a period of one year in laboratory under simulated summer conditions (light/dark 16:8 at 15, 19, and 23 degrees C). C. volutator was the first time reproduced successfully in laboratory for several generations and independent from its natural life cycle also in winter. The females produced two to three broods with a mean number of 96 offspring. A mean growth rate of 0.07 mm per day was determined at 15 degrees C. The reproduction and growth experiments provide consequently the essential base for the development of a chronic toxicity test with C. volutator.

Impact of particle size and light exposure on the effects of TiO2 nanoparticles on Caenorhabditis elegans.

The increasing use of engineered nanoparticles in industrial and consumer products leads to a release of the anthropogenic contaminants to the aquatic environment. To obtain a better understanding of the environmental effects of these particles, the nematode Caenorhabditis elegans was used to investigate the organism-level effects and in vivo molecular responses. Toxicity of bulk-scale (∼160 nm) and nanoscale (21 nm) titanium dioxide (TiO2 ) was tested under dark and light conditions, following ISO 10872. The expression of sod-3, a mitochondrial superoxide dismutase, was quantified as an indicator for oxidative stress induced by the photocatalytically active material. Particle sizes were estimated using dynamic light scattering and scanning electron microscopy. Although both materials agglomerated to a comparable secondary particle size of 300 nm to 1500 nm and were ingested into the intestine, only nanoscale-TiO2 significantly inhibited reproduction (lowest-observed-effect-concentration [LOEC]: 10 mg/L). Light exposure induced the production of reactive oxygen species (ROS) by nanoscale-TiO2 and increased toxicity of the nanomaterial from a median effect concentration of more than 100 mg/L to 53 mg/L. No evidence was found for inner cellular photocatalytic activity of nanoscale-TiO2 . Therefore, oxidative damage of the membranes of intestinal cells is suggested as a potential mode of action. Results highlight the importance of primary particle size and environmental parameters on the toxicity of TiO2 .

Sediment contact tests as a tool for the assessment of sediment quality in German waters.

A sediment contact test (SCT) battery consisting of five ecotoxicological test systems was applied to 21 native freshwater sediments characterized by a broad variety of geochemical properties and anthropogenic contamination. Higher plants (Myriophyllum aquaticum), nematodes (Caenorhabditis elegans), oligochaetes (Lumbriculus variegatus), zebrafish embryos (Danio rerio), and bacteria (Arthrobacter globiformis), representing various trophic levels and exposure pathways, were used as test organisms. The test battery detected sediment toxicity caused by anthropogenic pollution, whereas the various tests provided site-specific, nonredundant information to the overall toxicity assessment. Based on the toxicity pattern derived from the test battery, the sediments were classified according to a newly proposed classification system for sediment toxicity assessment. The SCT-derived classification generally agreed well with the application of consensus-based sediment quality guidelines (SQGs), especially with regard to sediments with high toxic potential. For sediments with low to medium toxic potential, the SQGs often underestimated the toxicity that was detected by the SCTs, underpinning the need for toxicity tests in sediment quality assessment.

Interlaboratory comparison of a standardized toxicity test using the nematode Caenorhabditis elegans (ISO 10872).

A ring test was carried out within the standardization process of ISO 10872 to evaluate the precision of the toxicity test for the nematode Caenorhabditis elegans. Eight different laboratories tested aqueous solutions of the reference substance benzylcetyldimethylammonium chloride as well as native sediments and soils for toxic effects on the growth and reproduction of C. elegans. Validity criteria were met in all laboratories. Average median- and low-effect concentrations were determined to be 15.1 mg L(-1) (EC50) and 8.7 mg L(-1) (EC10) for growth and 7.5 mg L(-1) (EC50) and 3.8 mg L(-1) (EC10) for reproduction of C. elegans, with ECx values showing a high degree of reproducibility (CV(R) : <21% and <11% for EC10 and EC50, respectively) and repeatability (CV(r) : <20% and <7% for EC10 and EC50, respectively). The toxic effects of the sediments and soils revealed by the different laboratories were well related to each samples' degree of chemical contamination. Moreover, the effects showed an acceptable reproducibility (CV(R) : 5-33% and 0-28% for growth and reproduction, respectively) and repeatability (CV(r) : 3-13% and 0-12% for growth and reproduction, respectively). The present study confirms that the toxicity test with C. elegans according to ISO 10872 is a reliable and precise tool to assess the toxicity of aqueous media, freshwater sediments, and soils.

Effects of phenanthrene on lemna minor in a sediment-water system and the impacts of UVB.

The objective of this study was to evaluate if the Lemna-bioassay is appropriate to test contaminated sediments. A mixture of sand was spiked with phenanthrene to investigate whether sediment-bound pollutants can affect the plants via direct contact of the roots or the underside of the leaves. After 24h of equilibration for sorption/desorption processes, the test was carried out in the sediment-water mixture, and another test was performed with the aqueous phase which was separated from the sediment. The growth inhibition of Lemna was nearly the same in both tests. Hence it follows that the toxicant is adsorbed from the aqueous phase via the underside of the leaves and sediment bound phenanthrene is not bioavailable. Polycyclic aromatic hydrocarbons are known to show photoinduced toxicity to plants in the presence of UV which is a result of photosensitization reactions in the plant and photomodification to more toxic and better soluble photoproducts. Photoinduced toxicity could be observed in the water phase during UVB treatment, whereas the presence of suspended sediment probably lowered the UV intensity, resulting in a lower growth inhibition.

Application of a sediment quality triad and different statistical approaches (Hasse diagrams and fuzzy logic) for the comparative evaluation of small streams.

In order to evaluate the ecotoxicological contamination of 12 aquatic sites in streams within the catchment area of the Neckar River, a sediment quality triad (SQT) approach was applied. In contrast to Chapman's original triad approach, not only sediments, but also surface waters were examined. In brief, to obtain a comprehensive insight into the potential ecotoxicological hazard, both acute toxicity and more specific effects such as mutagenic, genotoxic, teratogenic, dioxin- and estrogen-like responses were recorded. Different statistical methods (ranking, cluster analyses, Hasse diagram techniques, and fuzzy logic) for the evaluation and presentation of the SQT data were compared with respect to the needs of environmental decision-making. Results document advantages and disadvantages of the methods applied for the evaluation and classification of data within the complex data matrixes. The ranking procedure presented as well as the Hasse diagram technique seem to be suitable tools to elucidate the pattern of the ecotoxicological load. However, these classification methods require expert knowledge to interpret the ranking results with respect to decision-making in environmental sciences and policy. In contrast, fuzzy logic allows both to develop site-specific expert systems in order to assess the ecotoxicological burden and to give insight into the pattern of the contamination.