Ecotoxicity of quinoline and hydroxylated derivatives and their occurrence in groundwater of a tar-contaminated field site.

In the groundwater of a timber impregnation site higher concentrations of hydroxylated quinolines compared to their parent compounds quinoline and isoquinoline were found. Studying the toxicity of parent compounds and metabolites, genotoxicity was found with metabolic activation in the SOS-Chromotest and Ames fluctuation test only for quinoline. An adverse effect on algae was observed only for the parent compounds quinoline and isoquinoline, while in the Daphnia magna immobilization assay most hydroxylated quinoline derivatives showed toxicity. The highest ecotoxic potential was observed in the Vibrio fischeri luminescence-inhibition assay. Comparing experimental EC50-values with QSAR predicted ones, for all compounds apart from isoquinoline and 2(1H)-quinolinone in the V. fischeri test baseline toxicity or polar nacrosis is indicated. In conclusion, the hydroxylation of quinoline leads to a detoxification of the genotoxic potential, while taken additive mixture toxicity and a safety factor into account parent compounds and metabolites are found of ecotoxicological relevance in the groundwater.

Completely automated short-term genotoxicity testing for the assessment of chemicals and characterisation of contaminated soils and waste waters.

GOAL, SCOPE AND BACKGROUND: The umu-test was developed for the detection of effects of chemical mutagens and carcinogens in environmental samples. It is performed according to ISO 13829 with Salmonella choleraesius subsp. chol. (strain TA1535/pSK1002). By automating the entire test, large numbers of toxicants and environmental samples as well as more treatments and parallels can be tested and, additionally, only low sample volumes are needed. In this work, an automated umu-test has been set up by installing a robotic XYZ-platform and a microplate reader inside a cabin. The use of established technical equipment for the automation in combination with a performance according to ISO standards was the essential aim of the approach. After initial preparation, the test is conducted software-controlled, follows the standard and fulfils the validity criteria of the standard procedure. For the optimization of the automated test umu-tests with one concentration of methyl methanesulfonate (MMS) of 166.7 mg/L were carried out. After optimization of incubation and pipetting conditions in the automated test, dose-response curves of various chemicals and environmental samples were assessed. The results of the automated umu-test have been compared with those of the standard manual test. The aim of the study was to show the applicability of an automated test system for the assessment of the genotoxic effects of various chemicals and environmental samples. METHODS: During optimization, tests with 166.7 mg/L of MMS in every well of the microplate are carried out. Chemicals with different physical, chemical and toxicological properties are applied in both test systems. Water samples from different waste water treatment plants, and water extracts of contaminated and uncontaminated soils are assessed in the umu-test. The test is performed in parallel manually according to the standard and automatically using the robotic platform. Dose-response relationships and DLI-values are recorded and compared. RESULTS: The umu-test is applied on a RoboSeq 4204 SE pipetting station (MWG AG, Ebersberg, Germany). The robot is equipped with four holders for disposable tips to avoid undesired mixing of liquids while testing. With this system, it is possible to pipette all liquids. Photometric measurements are performed using a microplate reader. The pipetting station and the photometer are placed in an incubation cabin. According to the standards, exposure and growth of the bacteria are performed at 37 degrees C and the enzyme activity is assessed at 28 degrees C. Since both temperatures can't be adjusted simultaneously in the cabin, the test is performed in deviation from the standard at 33 degrees C. The results show that both the testing of non-volatile substances with strong or moderate genotoxic effects and the testing of water and soil samples in the automatic system work very well. Nevertheless, it is still difficult to characterize volatile chemicals automatically. This is illustrated, e.g. by testing 2- +/- -hydroxyquinoline. In this case, the chemical would not be assessed genotoxically after automatic performance of the umu-test. Sealing of the microplates, for example, avoids the loss of volatile substances, but this step of the procedure can not be performed automatically. Discussion. Only very few studies deal with the automation of bioassays. Eisentraeger et al. (2004) showed the suitability of a miniaturized and automated algae test for the testing of large numbers of environmental samples. Genotoxicity with an automated liquid handling are introduced by White et al. (1996) and Janz et al. (1989). A complete automation including liquid handling, incubation and photometric measurement is a new approach and leads to satisfying results. CONCLUSIONS: Optimization and suitability of the automated test are demonstrated in this study. Induction rates and growth factors do not differ significantly if the incubation temperature and the pipetting mode are optimized. Due to flexible scripting, the newly developed automated test system can also be used to perform other genotoxicity tests. Most results clearly show that genotoxicity tests can be automated completely allowing rapid testing that can be performed over night, for instance. Nevertheless, the test performance has to be optimized step by step depending on the technical characteristics of the automat. In order to overcome these technical problems, detailed knowledge of the hardware and the software of the automat and of the respective genotoxicity test system are needed. During this process of automation, it is very useful to test one genotoxic substance with an identical concentration in every well. RECOMMENDATIONS AND PERSPECTIVES: The automated test system based on the RoboSeq 4204 SE pipetting station (MWG AG, Ebersberg, Germany) still has to be optimized with respect to the testing of volatile compounds. There is a need for removable, gas-tight microplate covers. For non-volatile chemicals and environmental samples, it can be used routinely. Nevertheless, the experiences made during this study can only partly be transferred to other robotic platforms and other bioassays, and automation of bioassays still can be a time-consuming matter.

Environmental assessment of ester-based lubricants after application.

GOAL, SCOPE AND BACKGROUND: Lubricants based on renewable resources are an environmentally friendly alternative to petrochemical products due to their better ecotoxicological performance and their excellent biodegradability. To improve the technical performance of lubricants, and to reduce friction and wear, the use of additives is nowadays obligatory. The collaborative research center SFB 442 aims at developing environmentally acceptable lubricants that facilitate the avoidance of these additives by transferring their function to suitable coatings. For a complete assessment of the ecological performance of these newly developed lubricants, the whole life cycle including production, application as well as disposal and fate in the environment is studied. In the following study the focus was on the application and its influence on the environmental behavior of the lubricant. The application of lubricants leads, among other things, to the intake of metals due to abrasion from tools, work pieces or mechanical components. Previous examinations indicated a possible influence of metals on the toxicity of eluates prepared from used lubricants (Erlenkaemper et al. 2005). To clarify if the apparent toxicity of used lubricants solely results from the intake of metals, the extractability of these metals from the oil matrix is determined. By combining chemical analyses with bioassays, the bioavailability of metals that are present in the extract is estimated. To further investigate the relevance of metals on toxicity, toxic units (TU) were calculated and related to the results of the bioassays. Interactions between the metals were investigated with aqueous mixtures of metal chlorides and calculations based on the concept of concentration addition and independent action. METHODS: A lubricant mixture was applied to a tribological test bench that simulates real conditions of use and extremely short time load, respectively. Samples were taken at particular times, water soluble fractions (WSF) of these fluids were prepared and dilution series were investigated in several bioassays. Concentration of metals and total organic carbon (TOC) were determined in the eluates. TUs were calculated according to Sprague (1970) and mixture toxicity was calculated according to the concept of concentration addition (Loewe and Muischnek 1926) and independent action (Bliss 1939). RESULTS: Analyses of the metal content of the lubricant and the eluates clearly revealed the availability of the metals in the aqueous extracts. Especially copper, zinc, nickel and chromium were found and their concentrations increased during the time of use. The water extractable fraction, e.g., of copper, rose from 8.8% to 45.3% of the total content in the lubricant after 33.5 hours of use. Tests performed with the algal growth inhibition assay and the luminescence inhibition assay revealed the uptake or absorption by the organisms and, thus, the bioavailability of the metals. The calculation of TUs partly indicated a possible influence of the metals on ecotoxicity of the eluates. Copper reached concentrations equal to or higher than the EC50 value of copper chloride in the growth inhibition assays with algae and Ps. putida as well in the immobilization assay with daphnids. TUs for copper are also larger than 1 for the algal growth inhibition assay. The EL50 values indicated that the luminescence inhibition assay was the most sensitive test system, with values between 4.7% and 9.6%. While the toxicity towards algae and V. fischeri in the growth inhibition assay decreased until both organisms were no longer influenced by the exposure, the EL50 values for the D. magna immobilization assay and the Ps. putida growth inhibition assay decreased with the progressing use of the lubricant. The tested metal salt mixtures showed that Ps. putida, algae and daphnids are the most sensitive organisms with EC50 values below 1 mg/l. DISCUSSION: Although the intake of metals mainly occurred via abrasion of particles, the results revealed the availability of these metals in water. The availability varied for each of the four metals. For both the algal growth inhibition assay and the luminescence inhibition assay, an uptake or absorption of the metals could be demonstrated. The calculated TUs indicated an effect in some bioassays that was not verified in the test itself. The influence of copper on V. fischeri, for example, was not confirmed while the EL50 values for the daphnid bioassay decreased, meaning that the eluates became more toxic with progressing use of the lubricant. The calculations of mixture toxicity based on the concept of concentration addition demonstrated good correlations with the tested metal mixtures, but also a different sensitivity of the organisms. CONCLUSIONS: The results presented here reveal the availability of those metals in water that were taken in during the use of the lubricant in a tribological test bench and, thus, have the possibility of interacting with the organisms. The availability of the metals in the bioassays was proven by chemical analyses. The calculation of TUs and the corresponding EL50 values, however, indicate different availabilities of the metals. The results of the metal salt mixtures show good correlations with calculations of mixture toxicity based on concentration addition. Moreover, the varying sensitivity of the organisms when exposed to eluates or metal mixtures indicates a different bioavailability of the metals and/or the presence of other compounds that exert toxic action. RECOMMENDATIONS AND PERSPECTIVES: For further investigations, the organic oil matrix and its influence on the toxicity have to be taken into account. The toxicity of the eluates may not only be due to metals; additional effects could arise from changes in the lubricant itself.

Heterocyclic compounds: toxic effects using algae, daphnids, and the Salmonella/microsome test taking methodical quantitative aspects into account.

Heterocyclic aromatic hydrocarbons containing nitrogen, sulfur, or oxygen (NSO-HET), have been detected in air, soil, sewage sludge, marine environments, and freshwater sediments. Since toxicity data on this class of substances are scarce, the present study focuses on possible implications NSO-HET have for ecotoxicity (algae and daphnids) and mutagenicity (Salmonella/microsome test). A combination of bioassays and chemical-analytical quantification of the test compounds during toxicity assays should aid in determination of the hazard potential. Samples of the test concentrations of 14 NSO-HET were taken at the beginning and end of the bioassays; these samples were then quantified by high-performance liquid chromatography. The toxicity potential of the substances was evaluated and compared with the toxicity calculated with the nominal concentrations. Significantly different results were obtained primarily for volatile or highly hydrophobic NSO-HET. The concentration of heterocyclic hydrocarbons can change significantly during the algae and Daphnia test. The EC50 values (effective concentration value: the concentration of a chemical that is required to produce a 50% effect) calculated with the nominal concentrations underestimate the toxicity by a factor of up to 50. Prioritizing the tested compounds according to toxicity, the mutagenic and toxic compounds quinoline, 6-methylquinoline, and xanthene have to be listed first. The greatest ecotoxic potential on algae and daphnids was determined for dibenzothiophene followed by acridine. In the Daphnia magna immobilization test, benzofuran, dibenzofuran, 2-methylbenzofuran, and 2,3-dimethylbenzofuran and also carbazole are ecotoxicologically relevant with EC50 values below 10 mg/L. These substances are followed by indole with a high ecotoxic effect to daphnids and less effect to algae. Only minor toxic effects were observed for 2-methylpyridine and 2,4,6-trimethylpyridine.

Toxicological characterization of 2,4,6-trinitrotoluene, its transformation products, and two nitramine explosives.

The soil and groundwater of former ordnance plants and their dumping sites have often been highly contaminated with the explosive 2,4,6-trinitrotoluene (2,4,6-TNT) leading to a potential hazard for humans and the environment. Further hazards can arise from metabolites of transformation, by-products of the manufacturing process, or incomplete combustion. This work examines the toxicity of polar nitro compounds relative to their parent compound 2,4,6-TNT using four different ecotoxicological bioassays (algae growth inhibition test, daphnids immobilization test, luminescence inhibition test, and cell growth inhibition test), three genotoxicological assays (umu test, NM2009 test, and SOS Chromotest), and the Ames fluctuation test for detection of mutagenicity. For this study, substances typical for certain steps of degradation/transformation of 2,4,6-TNT were chosen for investigation. This work determines that the parent compounds 2,4,6-TNT and 1,3,5-trinitrobenzene are the most toxic substances followed by 3,5-dinitrophenol, 3,5-dinitroaniline and 4-amino-2-nitrotoluene. Less toxic are the direct degradation products of 2,4,6-TNT like 2,4-dinitrotoluene, 2,6-dinitrotoluene, 2-amino-4,6-dinitrotoluene, and 4-amino-2,6-dinitrotoluene. A weak toxic potential was observed for 2,4,6-trinitrobenzoic acid, 2,4-diamino-6-nitrotoluene, 2,4-dinitrotoluene-5-sulfonic acid, and 2,6-diamino-4-nitrotoluene. Octahydro-l,3,5,7-tetranitro-l,3,5,7-tetrazocine and hexahydro-1,3,5-trinitro-l,3,5-triazine show no hint of acute toxicity. Based on the results of this study, we recommend expanding future monitoring programs of not only the parent substances but also potential metabolites based on conditions at the contaminated sites and to use bioassays as tools for estimating the toxicological potential directly by testing environmental samples. Site-specific protocols should be developed. If hazardous substances are found in relevant concentrations, action should be taken to prevent potential risks for humans and the environment. Analyses can then be used to prioritise reliable estimates of risk.

Hazard characterization and identification of a former ammunition site using microarrays, bioassays, and chemical analysis.

More than 100,000 tons of 2,4,6-trinitrotoluene were produced at the former ammunition site Werk Tanne in Clausthal-Zellerfeld, Germany. The production of explosives and consequent detonation in approximately 1944 by the Allies caused great pollution in this area. Four soil samples and three water samples were taken from this site and characterized by applying chemical-analytical methods and several bioassays. Ecotoxicological test systems, such as the algal growth inhibition assay with Desmodesmus subspicatus, and genotoxicity tests, such as the umu and NM2009 tests, were performed. Also applied were the Ames test, according to International Organization for Standardization 16240, and an Ames fluctuation test. The toxic mode of action was examined using bacterial gene profiling assays with a battery of Escherichia coli strains and with the human liver cell line hepG2 using the PIQOR Toxicology cDNA microarray. Additionally, the molecular mechanism of 2,4,6-trinitrotoluene in hepG2 cells was analyzed. The present assessment indicates a danger of pollutant leaching for the soil-groundwater path. A possible impact for human health is discussed, because the groundwater in this area serves as drinking water.

Influence of laboratory storage on the organic contaminant content and water-extractable ecotoxicological potential of soil samples.

At present there is a high level of uncertainty about whether or not soil samples, which are required to be toxicologically characterized, could be preserved without affecting their toxicological characteristics. In the existing DIN/ISO standards for soil investigations, there is no documentation on the storage of soil samples after collection for (eco-)toxicological investigations. Furthermore, procedures for receiving justifiable and verifiable results from soil samples are quite vague. Therefore, the stability and changes in the biological effects of different soil contaminants were investigated by varying the storage conditions and the storage times of the soil. The limitations of storing soils could therefore be subsequently outlined. Recommendations for the optimization of storage conditions for back-up soil samples were made. These recommendations have the capability of finding entrance into DIN/ISO standards.

Electrical discharge machining: occupational hygienic characterization using emission-based monitoring.

Hazardous potential in industrial environments is normally assessed by means of immission-based sampling and analyses. This approach is not adequate, if effects of specific technical adjustments at machine tools or working processes on hygienic parameters should be assessed. This work has focused on the optimization of a manufacturing process (electrical discharge machining, EDM), with regard to risk reduction assessment. It is based on emission analyses rather than immision analyses. Several technical EDM parameters have been examined for their influence on air-based emissions. Worktools and workpieces used have a strong influence on aliphatic compounds and metals but not on volatile organic compounds (benzene, toluene, ethylene-benzene and xylene (BTEX)) and polycyclic aromatic hydrocarbons (PAHs) in air emissions. Increasing the dielectric (mineral oil) level above processing location decreases BTEX, chromium, nickel and PAH emissions. Aliphatic compounds, in contrast, increase in air emissions. EDM current used has a positive relationship with all substances analyzed in air emissions. Indicative immission concentrations, as can be expected under EDM conditions, are estimated in a predictive scenario. The results of this characterization give rise to an important conclusion in that risk assessment so far has been using incorrect parameters: total aliphatic compounds. Maximum level of chromium is reached long before limit values of aliphatic compounds are exceeded. Because of the fact that metals, like chromium, also have a higher hazardous potential, metal analysis should be introduced in future risk assessment. This experimental approach, that captures total emission of the electrical discharge machine, and is not solely based on immission values, has lead to a better understanding of the production process. This information is used to extract recommendations regarding monitoring aspects and protection measures.

Light-emitting diodes for the illumination of algae in ecotoxicity testing.

Ecotoxic effects of chemicals, preparations, and environmental samples are routinely quantified in standardized algae growth inhibition tests using microalgae like Desmodesmus subspicatus. The intention of this investigation was to prove the suitability of light-emitting diodes (LEDs) as an alternative to tubular discharge lamps in algae growth inhibition tests. This was approached by demonstrating that growth of algae was similar with both illuminating devices. Growth factors of growth controls were determined using LEDs. Growth curves of different light intensities of LEDs were compared with those of tubular discharge lamps. By testing certain chemicals with both illumination methods comparable results for E(r)C50 values were obtained. It could be shown that small-scale construction of incubators using LEDs is possible and could be advantageous, especially for the illumination of algae in 96-well microplate growth inhibition assays.

Comparative studies on algal toxicity testing using fluorometric microplate and Erlenmeyer flask growth-inhibition assays.

Two fluorometric microplate algae growth-inhibition assays with a liquid volume of 2 mL and 200 microL per well are presented, and comparative studies on the toxicity of chemicals are carried out with Erlenmeyer flask assays. The test procedures are in accordance with the standards ISO 8692 (DIN 38412 L9 and EN 28692), OECD 201 and DIN 38412 L33. By testing four toxicants several times laboratory internal repeatability is proven. Statistical evaluation demonstrates that the results obtained with both the 24-well and the 96-well microplates are nearly identical with the results of the Erlenmeyer flask assay. Therefore, the microplate growth-inhibition assays can be applied for the testing of a wide range of chemicals and environmental samples if some methodical aspects are taken into account. Apart from that, there is a strong need for harmonization if the dependency of the EC values on the toxicological endpoint is considered.

Assessment of the influence of use on ecotoxicological characteristics of synthetic ester lubricants.

Synthetic ester lubricants need optimisation about their technical and their ecotoxicological characteristics. To determine the ecotoxicological potential the required examinations can be based on the procedure for a risk assessment of chemicals. At present risk classification of lubricant oils is carried out with new oil fluids that are normally prepared before application in aqueous bioassays. In order to improve the ecotoxicological characteristics of some lubricant oils, the quality of the preparation method has been optimised. The resulting preparation protocol leads to aqueous extracts of the oil fluids that can be tested using biological assays. The extent of the changes of the chemical composition caused by the use as well as the ecotoxicological effects caused by additives have to be taken into consideration. For this reason various used lubricants are tested in addition to new oil fluids. In this work various lubricant samples were examined with standardised bacterial growth assays with Vibrio fischeri and Pseudomonas putida, luminescence inhibition assay with V. fischeri, survival assay with Daphnia magna and algal growth inhibition assay with Scenedesmus subspicatus. The chemical characterisation of the aqueous extracts included the determination of pH, conductivity, heavy metals, the content of dissolved organic carbon, inorganic anions and the content of phosphorus. The results emphasize the thesis that environmentally acceptable lubricants can undergo a change of their ecotoxicological potential during the use. Some of the substances that are normally added to base fluids in order to enhance the applicability of the oils may possess a high toxicological potential.

Determination of urease activity in soils by carbon dioxide release for ecotoxicological evaluation of contaminated soils.

A method for the quantification of urease enzyme activity has been set up, which is based on the quantification of carbon dioxide set free into the head space of gastight vessels. The method can be applied for ecotoxicological characterisation of contaminated soil samples besides other methods like soil respiration measurements or nitrification inhibition tests. The sieved soil sample can be incubated under nearly natural conditions with an adjusted water content of about 50% of the water holding capacity. Ammonia or urea do not need to be extracted, since carbon dioxide release is correlated to urease activity. Thus carbon dioxide release is a direct result of urease activity which can be measured in the head space using gastight syringes and gaschromatographic equipment. The urease activity is determined by comparing the carbon dioxide release of incubation vessels with and without urea supply. The applicability of this method has been demonstrated by experiments with N-(n-butyl)phosphoric triamide (NBPT), copper ions and zinc ions as known inhibitors of urease activity.

Biodegradability testing of synthetic ester lubricants--effects of additives and usage.

The optimised biodegradability test system "O2/CO2 Headspace Test with GC-TCD" is used for the assessment of synthetic ester lubricants. The effects of both additives and usage on biodegradability are examined and discussed. Ester based cutting fluids and hydraulic fluids with and without additives are used under defined conditions at machine tools and hydraulic and plain bearing test benches. The lubricants are characterised additionally with respect to kinematic viscosity, acidity and elemental composition. Furthermore, a formulated mineral oil is characterised before and after usage at an hydraulic test bench. The results clearly show that the mineral oil is far less biodegradable than the ester oils and that their biodegradability is not affected by usage. Biodegradability of the ester oils is mainly depending on the characteristics of the base fluids and not affected by the additives. Antioxidants are influencing stability respectively biodegradability indirectly, since they prevent oxopolymerisation effects. Other effects of usage on biodegradation are not detected. In this context, the antioxidants ensure ready biodegradability and have a positive effect on the environmental fate of synthetic ester lubricants.