Mutagenicity of Airborne Particulates Assessed by Salmonella Assay and the SOS Chromotest in Wrocław,Poland

Abstract

Ambient air particulate matter less than 2.5 μm in aerodynamic diameter (PM_2.5) samples were collected during summer and autumn using a Staplex high-volume air sampler. They were later extracted with dichloromethane in a Soxhlet apparatus. Polyaromatic hydrocarbon (PAH) content in extracts was determined by the high-performance liquid chromatography technique using fluorescence detection, whereas the nitro-PAH content was determined by gas chromatography using mass detection. Four Salmonella typhimurium strains (TA98, TA100, YG1041, and YG1042) were used in assays conducted with and without metabolic activation. The extracts were also tested with the SOS chromotest supplied by Environmental Biodetection Products Incorporated. The obtained results confirmed the Salmonella assay and the SOS chromotest usability for the purpose of atmospheric pollution monitoring within an urban agglomeration. The atmospheric pollution extracts under examination differed among each other regarding total content and percentage of individual compounds, depending on the season of sampling. The highest total PAH content and the highest nitro-PAH content in the tested samples as well as the most extensive range of detected compounds were found in the autumn season (heating season). The highest mutagenicity was noted for PM_2.5 samples collected in autumn. The high values of mutagenicity ratios and induction factors were obtained from assays carried out with and without metabolic activation, which is an argument for the presence of promutagens and direct mutagens. The YG1041 strain proved to be the most effective in detection of mutagenicity of the suspended dust extracts because of its notably high sensitivity to nitro-aromatic compounds. The SOS chromotest was very sensitive to a large spectrum of genotoxic air pollutants and showed a high degree of similarity with the results of the Salmonella assay. In comparison with the frequently used Ames test, the SOS chromotest enables quick analysis of the genotoxic effects of samples using only one tester strain. In addition, its miniaturized design decreases the consumption of tested samples.     

Biodegradability of some antibiotics, elimination of the genotoxicity and affection of wastewater bacteria in a simple test

Most antibiotics and their metabolites are excreted by humans after administration and therefore reach the municipal sewage with the excretions. Only little is known about their biodegradability in aquatic environments. It was recognised that genotoxic substances may represent a health hazard to humans but also may affect organisms in the environment. Therefore, the biodegradability of some clinically important antibiotic drugs (ciprofloxacin, ofloxacin, metronidazole) and hereby the elimination of their genotoxicity was investigated as the first step of an environmental risk assessment using the Closed Bottle test (CBT) (OECD 301 D) and the SOS chromotest. Additionally, to assess toxicity of the antibiotics tested against aquatic bacteria (i) a growth inhibition test (GIT) with Pseudomonas putida was conducted, (ii) a toxicity control was used in the CBT and (iii) the colony forming units (CFUs) were monitored in the test vessels. Worst case concentrations of the antibiotics in hospital effluents were estimated and compared with minimum inhibitory concentrations for susceptible pathogenic bacteria and with the genotoxic potency in the SOS chromotest. Both the concentrations calculated for hospital effluents and the adverse effects in bacteria were in the same order of magnitude. None of the test compounds were biodegraded. The genotoxicity was not eliminated.     

Evaluation of an SOS-Chromotest-based approach for the isolation and detection of sediment-associated genotoxins

A series of experiments was conducted to evaluate an approach advanced by the St. Lawrence Centre (SLC) of Environment Canada for assessing the genotoxic potential of sediments. The SLC method entails the extraction, isolation and solvent exchange of the organic constituents in sediment, and the testing of these solubilized extracts with the SOS Chromotest (Escherichia coli PQ37). A total of five sediments, three variously contaminated by organic compounds and two reference materials certified for persistent organic chemicals, were Soxhlet-extracted. Each of the five extracts was then split, with a portion remaining in crude form and another portion fractionated into two molecular-weight classes of organic contaminants, thus yielding a total of 15 extract samples. The ability of the SOS Chromotest to detect genotoxins in the various organic extracts was evaluated and compared with that of the Ames Fluctuation Assay (Salmonella typhimurium, strain TA100). The intra-laboratory variance associated with the SOS Chromotest was also assessed. Procedural details are presented and results are discussed. The SOS Chromotest results were in good agreement with those of the Ames Fluctuation Assay, especially after metabolic activation. However, the E. coli PQ37 system was slightly more sensitive than the Salmonella assay for detecting genotoxins in the sediment extracts. The SOS Chromotest was also the most discriminating of the two assays, generating SOS-induction factors that were consistent with the organic contamination gradient reported in the sediment samples. The removal of macromolecules from the dichloromethane extracts by size-exclusion chromatography prior to testing enhanced the sensitivity of both test systems. The intra-laboratory variance of the SOS Chromotest ranged from 0.24% to 23.82%, depending on the extract sample. As applied in this study, the SOS Chromotest can serve as a sensitive test for screening the genotoxic potential of uncharacterized sediment extracts. A more sensitive assay would be appropriate, however, as a confirmation for definitive investigations, especially for the detection of direct-acting genotoxins.     

Health care industries: potential generators of genotoxic waste

Health care waste includes all the waste generated by health care establishments, research facilities, and laboratories. This constitutes a variety of chemical substances, such as pharmaceuticals, radionuclides, solvents, and disinfectants. Recently, scientists and environmentalists have discovered that wastewater produced by hospitals possesses toxic properties due to various toxic chemicals and pharmaceuticals capable of causing environmental impacts and even lethal effects to organisms in aquatic ecosystems. Many of these compounds resist normal wastewater treatment and end up in surface waters. Besides aquatic organisms, humans can be exposed through drinking water produced from contaminated surface water. Indeed, some of the substances found in wastewaters are genotoxic and are suspected to be potential contributors to certain cancers. The aim of this study was to evaluate the genotoxic and cytotoxic potential of wastewaters from two hospitals and three clinical diagnostic centers located in Jaipur (Rajasthan State), India using the prokaryotic Salmonella mutagenicity assay (Ames assay) and the eukaryotic Saccharomyces cerevisiae respiration inhibition assay. In the Ames assay, untreated wastewaters from both of the health care sectors resulted in significantly increased numbers of revertant colonies up to 1,000–4,050 as measured by the Salmonella typhimurium TA98 and TA100 strains (with and without metabolic activation) after exposure to undiluted samples, which indicated the highly genotoxic nature of these wastewaters. Furthermore, both hospital and diagnostic samples were found to be highly cytotoxic. Effective concentrations at which 20 % (EC₂₀) and 50 % (EC₅₀) inhibition of the respiration rate of the cells occurred ranged between ～0.00 and 0.52 % and between 0.005 and 41.30 % (calculated with the help of the MS excel software XLSTAT 2012.1.01; Addinsoft), respectively, as determined by the S. cerevisiae assay. The results indicated that hospital wastewaters contain genotoxic and cytotoxic components. In addition, diagnostic centers also represent small but significant sources of genotoxic and cytotoxic wastes.     

Urban airborne particulate: genotoxicity evaluation of different size fractions by mutagenesis tests on microorganisms and comet assay

The genotoxic effects of different size fractions of airborne particulate (Total, PM₁₀ and PM_2.5), extracted with acetone or toluene, were evaluated by: the Ames plate test (TA98 and TA100 strains, w/o S9), gene conversion and reversion (w/o endogenous metabolic activation) in the Saccharomyces cerevisiae D7 strain, and the comet assay on human leukocytes. The data on human leukocytes confirm the sensitivity of the comet assay and its applicability to assess genotoxicity in environmental samples. The PM_2.5 fraction of airborne particulate generally shows the highest concentration of DNA-damaging compounds. Genotoxic response, in all the test systems applied, is highly dependent on extraction solvent used. Acetone seems to extract compounds with more similar genotoxic responses in the three test systems used than toluene extracts. Toluene appears to extract air pollutants genotoxic on yeast and leukocytes but is mainly cytotoxic on Salmonella.     

Combination of in vitro bioassays for the determination of cytotoxic and genotoxic potential of wastewater, surface water and drinking water samples

In this study we evaluated genotoxicity and cytotoxicity of native samples of wastewaters (15 samples), surface waters (28 samples) and potable waters (8 samples) with the SOS/umuC assay with Salmonella typhimurium TA1535/pSK1002 and MTT assay with human hepatoma HepG2 cells. The genotoxicity of selected samples was confirmed with the comet assay with HepG2 cells. In the SOS/umuC assay 13 out of the 51 samples were genotoxic: two effluent samples from chemical industry; one sample of wastewater treatment plant effluent; two hospital wastewater samples; three river water samples and four lake water samples. Six samples were cytotoxic for HepG2 cells: both effluent samples of chemical industry, two wastewater treatment plant effluent samples, and two river water samples, however, only the chemical industry effluent samples were genotoxic and cytotoxic, indicating that different contaminants are responsible for genotoxic and toxic effects. Comparing genotoxicity of river and lake water samples with the chemical analytical data of the presence of the residues of pharmaceutical and personal care products (non-steroidal anti-inflammatory drugs, UV filters and disinfectants) in these samples, indicated that the presence of UV filters might be linked to the genotoxicity of these samples. The results showed that the application of the bacterial SOS/umuC assay and mammalian cell assays (MTT and comet assay) with HepG2 cells was suitably sensitive combination of assays to monitor genotoxicity and cytotoxicity of native samples of wastewaters and surface waters. With this study we also confirmed that the toxicity/genotoxicity bioassays should be an integral tool in the evaluation of toxicity of complex wastewaters before the release into environment, as well as for the monitoring of surface water quality, providing data useful in risk assessment.     

Changes in toxicity and genotoxicity of industrial sewage sludge samples containing nitro- and amino-aromatic compounds following treatment in bioreactors with different oxygen regimes

Goals, Scope and Background

From 2005, deposition of organic waste will be banned in Sweden. Likewise, in Germany and Austria, similar bans are being planned, and further countries will probably follow. Thus, there is a need to develop new methods and to refine established techniques for sludge management in the whole of the European Union. For this end, there is also an urgent need for appropriate ecotoxicological approaches to elucidate and assess the hazard potential of sewage sludge. Therefore, the present study was designed to assess the capacity of various established sludge treatment methods using different oxygen regimes to degrade recalcitrant nitro-substituted organic compounds and reduce their toxicity. Sewage sludge samples from a wastewater treatment plant in Sweden (Cambrex Karlskoga AB, industrial area Björkborn) receiving wastewater from industries manufacturing pharmaceutical substances, chemical intermediates and explosives were processed with different sludge treatment methods. Among other treatment methods, bioreactors (for anaerobic and aerobic sludge treatment) were used. In the present investigation, a battery ofin vitro bioassays was employed to compare the cytotoxic and genotoxic potentials of different fractions of sludge samples in order to elucidate whether the treatments were suitable to reduce the toxicity of the sludge.

Methods

In order to investigate the cytotoxicity of the extracts of treated and untreated sludge samples, the acute cytotoxicity test with the permanent cell line RTL-W1 was used. Genotoxicity was tested by means of the comet assay (single cell gel electro-phoresis) with RTL-W1cells, and mutagenicity was assessed with the Ames test using the Salmonella typhimurium strains TA98, TA98NR and TA100. Sludge toxicity was tested in different fractions of organic extracts produced by acetone and hexane extractions. The subsequent clean-up procedure (silica gel chro-matography and elution with hexane and dichloromethane) resulted in two fractions, a lipophilic hexane-fraction and a semi-lipophilic dichloromethane-fraction. For the genotoxicity and mutagenicity tests, these fractions were reunited at equal ratios.

Results and Discussion

The acute cytotoxicity test with RTL-Wl cells revealed a high cytotoxic potential for the semi-li-pophilic DM-fractions of all sludge samples with NR₅₀ values (= effective concentration for 50% cell death in the neutral red test) from 8.9 up to 20 mg sludge d.w./ml medium. A low cytotoxic potential for the hexane fractions of the untreated sludge samples (NR₅₀ 400 to < 400 mg sludge d.w./ml medium) was observed, whereas the hexane fractions of the treated sludge samples showed elevated cytotoxicity increasing further with treatment in the bioreactors. The comet assay indicated that three out of eight of the reunited fractions had a significant genotoxic potential. Whereas the genotoxic potential of one sample treated anaerobically was very high with an induction factor of 11.6, a similar sample (taken from the same anaerobic reactor four months later) and one untreated sample showed lower potentials. The samples treated in another anaerobic bioreactor as well as the samples treated aerobically showed no genotoxic potential. Results indicate that aerobic treatment was basically adequate for reducing the genotoxicity of the sludge, whereas anaerobic treatment was only partly useful for reduction of genotoxicity. The Ames test revealed a very high mutagenic potential for the reunited fractions of the untreated sludge samples with strain TA98 (maximum induction factors (IF_max up to 45) and a relatively high potential for one of the samples treated aerobically (S2, IF_max = 18 (TA98, S9-)), thus documenting the suitability of both anaerobic and aerobic treatments to reduce the mutagenicity of the samples, however, with the aerobic treatment being less effective. Conclusions. Overall, none of the microbiological treatments for wastewater sludge in bioreactors was found to be ideal for general toxicity reduction of the sludge samples. Whereas cytotoxicity of the sludge increased or levelled off in most cases following either treatment, genotoxicity both increased or decreased after anaerobic treatment, depending on the specific sample. However, mutagenicity could generally be reduced by anaerobic treatment and, to a lesser degree, by aerobic treatment. Recommendationsand Perspectives. The complex modification of the diverse damage potentials of sludge sample extracts by use of anin vitro biotest battery following treatment for toxicity reduction in bioreactors showed that considerations of different toxicological endpoints is essential for an adequate hazard assessment. Whereas in the case of cytotoxicity reduction, the reactors proved ineffective, mutagenicity could be reduced significantly at least in some cases in this case study.

     

Contributions of flumequine and nitroarenes to the genotoxicity of river and ground waters

The SOS/umuC assay was performed in conjunction with analytical measurements to identify potential genotoxins in river and adjacent ground waters in the Jialu River basin, China. The major genotoxic activities of the river and adjacent ground waters occurred in the same two fractions (F4 and F11) when assayed using the Salmonella typhimurium strain TA1535/pSK1002. This indicates that ground water near the Jialu River was influenced by the river water. LC-MS/MS analysis indicated that flumequine accounted for 86% and 76% of the genotoxicity in fraction F11 of the river and adjacent ground waters, respectively. When HPLC fractions were tested using the strain NM3009, three fractions showed genotoxic activities for river water sample, while no fractions from ground water samples elicited genotoxic activities. The specific response to the strain NM3009 in one fraction compared with the strain TA1535/pSK1002 suggested the presence of nitroarenes. However, we failed to identify the exact nitroarenes when GC-MS analysis was used to analyze nitroarenes which are well detected in air and soil samples in previous papers.     

The cytotoxic and genotoxic potential of surface water and wastewater effluents as determined by bioluminescence, umu-assays and selected biomarkers

Two bacterial tests employing Photobacterieum phosphoreum (Microtox bioluminescence test) and Salmonella typhimurium TA 1535 pSK1002 (umu-assay) were evaluated to estimate the cytotoxic and genotoxic potential of water samples from the selected rivers in Germany as well as the primary and secondary effluents of some sewage treatment plants. Rainbow trout (Onchorynchus mykiss) were exposed to different concentrations (20-40%) of secondary effluent in the model online aquatic monitoring plant WaBoLu-Aquatox. The toxic potential of water samples from the exposure tanks was determined in two prokaryotic test systems and the biomarkers acethylcholinesterase (AChE) activity in muscle tissue and DNA unwinding assay in liver tissue of fish. Samples from the tested rivers showed no inhibition of the bioluminescence of P. phosphoreum or growth of umu-bacteria. Only primary effluent samples from the treatment plants at the Saale River inhibited the light emission or the growth of test bacteria by more than 20%. The induction ratio of umu-bacteria was in most of the river samples less than the threshold for genotoxicity (IR < 1.5). Only some samples from the Saale River, especially at sites downstream of secondary effluents caused genotoxic responses in the umu-assay. Samples of primary effluents contained the greatest genotoxic potential up to GEUI = 6 which was not detectable in samples of secondary effluents. A concentration range 20-40% secondary effluent inhibited AChE activity in muscle tissue and significantly increased DNA fragmentation in liver tissue of rainbow trout. In contrast, no cytotoxic or genotoxic responses in the umu-assay were caused by water samples. Both bacterial methods can be successfully used to analyse the cytotoxic and genotoxic response of industrial and domestic wastewater and to estimate the effectiveness of sewage treatment units. However, because of their low sensitivity and high susceptibility, they are not reliable as a single test for the detection of cytotoxicity and genotoxicity in surface water. The application of prokaryotic tests systems with biomarkers such as AChE activity and DNA fragmentation in different tissues of test organisms seems to be a useful combination for the assessment of cytotoxic and genotoxic potential in surface water and secondary effluent.     

Physicochemical characteristics,mutagenicity and genotoxicity of airborne particles under industrial and rural influences in Northern Lebanon

In this work, the main objectives were to assess the mutagenic and genotoxic effects of fine particulate matter collected in an industrial influenced site in comparison with a non-industrial influenced one (rural site) and to relate the particulate matter (PM) composition to the observed genotoxic effects. At the industrial influenced site, higher concentrations of phosphates, trace metals, and polycyclic aromatic hydrocarbons (PAHs) in particles could be related to the contributions of quarries, fertilizer producer, cement plants, and tires burning. Gasoline and diesel combustion contributions were evidenced in particles collected at both sites. Particles collected under industrial influence showed a higher mutagenic potential on three tested strains of Salmonella typhimurium (TA98, YG1041, and TA102), and especially on the YG1041, compared to particles from the rural site. Furthermore, only particles collected in the vicinity of the industrial site showed a tendency to activate the SOS responses in Escherichia coli PQ37, which is indicative of DNA damage as a result of exposure of the bacteria cells to the action of mutagenic samples. The mutagenicity and genotoxicity of the industrial PM_2.5–0.3 particulates may be attributed to its composition especially in organic compounds. This study showed that proximity of industries can affect local PM composition as well as PM genotoxic and mutagenic potential.     

Mutagenicity evaluation of industrial sludge from common effluent treatment plant

Sludge from common effluent treatment plant (CETP) receiving effluents from textile industries at Mandia Road, Pali, was analyzed to assess the level of mutagenicity. Mutagenicity assay using Salmonella typhimurium tester strains TA 98 and TA 100 gave positive results, thus suggesting presence of genotoxic contaminants in the samples investigated. Further, mutagenic activity of chemical sludge was found to be lesser than that of biological sludge. This result is very surprising and unexpected as it is indicating that some mutagenic compounds are either being formed or certain promutagenic compounds are being converted into stable mutagenic metabolites during the biological treatment of the wastewater effluents. There have been no previous reports giving similar or contrary results. Most of the previous studies have reported effects of single combined sludge.     

Correlations between mutagenic activity of organic extracts of airborne particulate matter, NOx and sulphur dioxide in southern Germany: results of a two-year study

Goals, Scope and Background Among other substances, sulphur dioxide (SO2), nitric oxide (NO) and nitrogen dioxide (NO2) are parameters which are routinely measured to describe basic air quality. Organic extracts of airborne particulate matter contain mutagenic chemical compounds of different origins. The aim of the study was to find correlations between routine monitoring data and mutagenic activity of organic extracts of simultaneously drawn samples.Methods Specimens were collected over a period of two years at 8 sampling sites in south-west Germany. Simultaneously, concentrations of NO, NO2, and SO2 were measured on-line within the framework of the official air monitoring network of Baden-Württemberg, Germany. Dust samples were collected for biotesting using high volume air samplers equipped with glass fibre filters. After sampling was completed, filters were extracted and samples were prepared for biological testing. Mutagenic activity was tested by means of the plate incorporation assay (Ames test) using S. typhimurium TA98 and TA100 tester strains. During the first year of the study, all tests have been performed with and without metabolic activation. Additionally, a series of tests has been performed in parallel with TA98 and TA98NR.Results and Discussion Comparison of Ames test data obtained with and without metabolic activation indicates no statistically significant difference between both methods. Therefore, during the second year of the study, all tests have been performed without metabolic activation. Average yearly activities at the sampling sites were between 1 und 27 Revertants per m³ (Rev/m³). High activities were preferably found at congested sites (Karlsruhe, up to 95 Rev/m³). However, peak values of over 100 Rev/m³ were found in other places where pollution by traffic is significantly lower. The reason for these high level values is not evident. Tests performed using TA98NR tester strain indicate a significant share (average 31%) of compounds requiring activation by nitroreductase for mutagenic activity. Average mutagenic activity can be correlated to routine monitoring parameters. Comparison of averaged data for particular sampling sites indicates significant correlation between nitric oxide and mutagenic activity in TA98 (r²=0.90), while correlation between nitrogen dioxide (0.84) or sulphur dioxide (0.52) and mutagenic activity is weaker. For TA100, correlations are generally weaker than for TA98. Comparison of data for mutagenic activity and routine monitoring data of distant sites being sampled simultaneously shows parallel behaviour.Conclusions Results from this study show that mutagenic activity can be compared to seasonal and local variations of gaseous indicator air pollutants. Tester strain TA98 generally shows the best correlations. Although pollution by particle-bound mutagenic substances is significantly higher during the cold season than during summer on average, mutagenic activity of airborne dust is not a continuous effect. During winter, peak levels as well as low pollution periods can occur. Even during winter time mutagenic activity can reach very low levels typical for summertime. Comparison of results for distant sampling sites where samples have been collected simultaneously indicate that “classical” indicators of air pollution and bacterial mutagenicity of organic extracts from airborne particulate matter are influenced by connected effects. Seasonal trend of mutagenic activity, in particular, is similar to the concentrations of nitrogen oxide. NO is a strong indicator for vehicle exhaust gases. It is concluded that the average mutagenic activity at particular sites can be estimated using NO concentrations as an indicator.     

A multispecies study to assess the toxic and genotoxic effect of pharmaceuticals: furosemide and its photoproduct

Pharmaceutical products for humans and animals, as well as their related metabolites end up in the aquatic environment after use. Recent investigations show that concentrations of pharmaceuticals are detectable in the order of ng/l-mug/l in municipal wastewater, groundwater and also drinking water. Little is known about the effects, and the hazard of long-term exposure to low concentrations of pharmaceuticals for non-target aquatic organisms. This study was designed to assess the ecotoxicity of furosemide, a potent diuretic agent, and its photoproduct in the aquatic environment. Bioassays were performed on bacteria, algae, rotifers and microcrustaceans to assess acute and chronic toxicity, while the SOS Chromotest and the Ames test were utilized to detect the genotoxic potential of the investigated compounds. A first approach to risk characterization was to calculate the environmental impact of furosemide by measured environmental concentration and predicted no effect concentration ratio (MEC/PNEC). To do so we used occurrence data reported in the literature and our toxicity results. The results showed that acute toxicity was in the order of mg/l for the crustaceans and absent for bacteria and rotifers. Chronic exposure to these compounds caused inhibition of growth population on the consumers, while the algae did not seem to be affected. A mutagenic potential was found for the photoproduct compared to the parental compound suggesting that byproducts ought to be considered in the environmental assessment of drugs. The risk calculated for furosemide suggested its harmlessness on the aquatic compartment.     

Mutagenicity of ambient air pollutants collected near aluminum industries

Mutagenicity has been tested in air samples collected in the summer and in the winter near four Norwegian aluminum plants. The samples were separated into a particulate and a volatile fraction and tested for mutagenicity by a quantitative reversion assay which showed that the suspended particles were clearly mutagenic. The volatile part of the air pollutants were cytotoxic to the bacteria and showed only marginal mutagenicity. The particulate fractions were tested more extensively in the Ames Salmonella mutagenicity test, in two laboratories, using the strains TA 98 and TA 100 with and without enzymatic activation (S9). The mutagenicity was relatively high compared to particulate fractions from other areas with industry and dense traffic. The highest mutagenicity was found in TA 100 with enzymatic activation and the lowest in TA 100 without S9. The mutagenicity was influenced by wind speed and direction during sampling. The mutagenic activity was also determined in the nitroreductase deficient strains TA 98NR and TA 98/1.8DNP. A larger reduction in the activity was found compared to samples from other areas, thus indicating a difference in the sample composition.     

Chemical and biological profiles of sediments as indicators of sources of contamination in Hamilton Harbour. Part II: bioassay-directed fractionation using the Ames Salmonella/microsome assay

Bottom sediment and suspended sediment samples from Hamilton Harbour (western Lake Ontario) and from a major tributary were profiled using a bioassay-directed fractionation approach. Sample extracts were fractionated using an alumina/Sephadex gel clean-up procedure to afford non-polar aromatic fractions which were characterized using chemical analyses and the Ames/microsome bacterial assay in Salmonella typhimurium strains YG1025 with the addition of oxidative metabolism (S9), and YG1024 without S9. Non-polar aromatic fractions of selected samples were separated by normal phase HPLC into 1-min fractions which were subjected to bioassay analyses. The bioassays using strain YG1025+S9, a TA100-type strain, were performed to assess genotoxicity arising from the presence of polycyclic aromatic hydrocarbons (PAH). Fractions which exhibited mutagenic activity contained PAH with molecular masses of 252, 276 and 278 amu; these fractions contained over 80% of the genotoxicity attributable to PAH. Individual compounds identified using Gas Chromatography-Mass Spectrometry analyses in these active fractions included benzo[a]pyrene, indeno[cd]pyrene and dibenz[a,h]anthracene. The YG1025+S9 mutagenic activity profiles were similar for all samples. Mutagenic activity profiles generated using strain YG1024-S9, a TA98-type strain sensitive to compounds characteristic of mobile source emissions, were very different. The mutagenic activities in strain YG1024-S9 were greatest for harbour-suspended sediment samples collected from sites impacted by a major tributary. Suspended sediments collected near areas known to contain high levels of coal tar-contamination in the bottom sediments contained higher levels of genotoxic PAH than suspended sediments collected from other areas of the harbour.     

Application of semipermeable membrane device (SPMD) to assess air genotoxicity in an occupational environment

Semipermeable membrane device (SPMD) is a passive sampler that sequesters lipophilic contaminants, mimicking the bioconcentration in the fatty tissue of organisms. This study was designed to assess the use of SPMD and biological tests (Comet assay and Ames test) for air monitoring. For this purpose an occupational environment with expected polycyclic aromatic hydrocarbons (PAHs) contamination (coke plant) was selected for a case study. The SPMDs were deployed in five occupational contaminated sites and in a control site. The SPMD dialysates were chemically analysed and examined for in vitro DNA-damaging activity in human cells (Jurkat) by Comet assay and for mutagenicity with the Ames test (TA98 strain, w/o S9). Total suspended particulates were also collected and analysed (GC–MS). No biological effect of SPMD extract was revealed in the control site. On the other hand, air samples collected with SPMDs within the coke plant showed variable degrees of genotoxic and mutagenic activity. The highest effects were associated with the highest PAH level recovered in the SPMDs extracts and in particulate samples.Results obtained support the sensitivity of biological tests associated to SPMD sampling for evaluating the health risk of potentially contaminated work environments highlighting the usefulness of SPMDs for environmental air quality monitoring.     

Approach for detecting mutagenicity of biodegraded and ozonated pharmaceuticals, metabolites and transformation products from a drinking water perspective

Many pharmaceuticals and related metabolites are not efficiently removed in sewage treatment plants and enter into surface water. There, they might be subject of drinking water abstraction and treatment by ozonation. In this study, a systematic approach for producing and effect-based testing of transformation products (TPs) during the drinking water ozonation process is proposed. For this, two pharmaceutical parent substances, three metabolites and one environmental degradation product were investigated with respect to their biodegradability and fate during drinking water ozonation. The Ames test (TA98, TA100) was used for the identification of mutagenic activity present in the solutions after testing inherent biodegradability and/or after ozonation of the samples. Suspicious results were complemented with the umu test. Due to the low substrate concentration required for ozonation, all ozonated samples were concentrated via solid phase extraction (SPE) before performing the Ames test. With the exception of piracetam, all substances were only incompletely biodegradable, suggesting the formation of stable TPs. Metformin, piracetam and guanylurea could not be removed completely by the ozonation process. We received some evidence that technical TPs are formed by ozonation of metformin and piracetam, whereas all tested metabolites were not detectable by analytical means after ozonation. In the case of guanylurea, one ozonation TP was identified by LC/MS. None of the experiments showed an increase of mutagenic effects in the Ames test. However, the SPE concentration procedure might lead to false-positive results due to the generation of mutagenic artefacts or might lead to false-negative results by missing adequate recovery efficiency. Thus, these investigations should always be accompanied by process blank controls that are carried out along the whole ozonation and SPE procedure. The study presented here is a first attempt to investigate the significance of transformation products by a systematic approach. However, the adequacy and sensitivity of the methodology need to be further investigated. The approach of combining biodegradation and ozonation with effect-based assays is a promising tool for the early detection of potential hazards from TPs as drinking water contaminants. It can support the strategy for the evaluation of substances and metabolites in drinking water. A multitude of possible factors which influence the results have to be carefully considered, among them the selectivity and sensibility of the mutagenicity test applied, the extraction method for concentrating the relevant compounds and the biocompatibility of the solvent. Therefore, the results have to be carefully interpreted, and possible false-negative and false-positive results should be considered.     

Mutagenicity and genotoxicity assessment of industrial wastewaters

The genotoxicity of industrial wastewaters from Jajmau (Kanpur), was carried out by Ames Salmonella/microsome test, DNA repair-defective mutants, and Allium cepa anaphase–telophase test. Test samples showed maximum response with TA98 strain with and without metabolic activation. Amberlite resins concentrated wastewater samples were found to be more mutagenic as compared to those of liquid–liquid extracts (hexane and dichloromethane extracts). The damage in the DNA repair defective mutants in the presence of Amberlite resins concentrated water samples were found to be higher to that of liquid–liquid-extracted water samples at the dose level of 20 μl/ml culture. Among all the mutants, polA exhibited maximum decline with test samples. Mitotic index (MI) of root tip meristematic cells of A. cepa treated with 5, 10, 25, 50, and 100 % (v/v) wastewaters were significantly lower than the control. Complementary to the lower levels of MI, the wastewaters showed higher chromosomal aberration levels in all cases investigated.     

Polycyclic organic material (POM) in urban air. Fractionation,chemical analysis and genotoxicity of particulate and vapour phases in an industrial town in Finland

Polycyclic organic material (POM) was collected by high-volume sampling on filter and on XAD-2 resin from the air of a small industrial town in Finland. Concurrent chemical analysis and the assays for genotoxic activity were performed on the particulate and the vapour phases of ambient air POM and their chemical fractions. Furthermore, correlations between seasonal meteorological parameters and POM concentrations were studied to reveal characteristic POM profiles for various emission sources. The range of total POM concentrations varied from 115 to 380 ng m⁻³ in late spring and from 17 to 83 ng m⁻³ in early winter. No direct correlation of ambient POM was seen with the temperature, but rather with the wind direction from various emission sources. Especially the low molecular weight compounds were associated with wind direction from industrial sources. Genotoxic activity, as detected by the Ames Salmonella/microsome test and the SCE assay in CHO cells, was found not only in the paniculate phase samples but also in the vapour phase. The polar fractions of some of the samples showed genotoxic activity, and also direct mutagenicity was observed with both the assay systems; these facts support the significance of compounds other than conventional polycyclic aromatic hydrocarbons (PAH) in the samples.     

A level change in mutagenicity of Japanese tap water over the past 12 yr

A relative comparison study of mutagenicity in Japanese tap water was conducted for 1993 and 2005 surveys. It intended to assess the effects of advanced water treatment installations to water works, improvement of raw water quality and improvement of residual HOCl concentration controlling. Sampling points (taps) were the same in both surveys. The results of 245 samples obtained by the Ames Salmonella mutagenicity test (Ames test) were analyzed. The Ames tests were conducted by using Salmonella typhimurium TA98 and TA100 strains with and without exogenous activation (S9). With the exception of TA100-S9, the other conditions needed no discussion as a factor in the mutagenicity level change. The average mutagenicity in 1993 and 2005 under the conditions of TA100-S9 were 2600 and 1100 net revertant L⁻¹, respectively. This indicated that the mutagenicity level of Japanese tap water decreased during the 12-yr period. Particularly a remarkable decrease in mutagenicity was observed in the water works where the advanced water treatments were installed during the 12-yr period. The advanced water treatments were effective in decreasing the mutagenicity of tap water. Mutagenicity also decreased in the water works with conventional water treatments; the improvement of residual HOCl concentration controlling was also considered to be effective in decreasing the mutagenicity of tap water.