Mutagenicity of chloroaniline/lignin metabolites in the salmonella/microsome assay

Abstract

Chloroanilines are constituents of many agrochemicals and have been found to be metabolized to succinic acid conjugates, e.g., succinamides and succinimides. The mutagenic potential of five chloroanilines and their succinamides and succinimide derivatives have been tested with two strains of Salmonella typhimurium (TA98 and TA100) with and without rat hepatic micro‐somal fraction. None of the compounds produced a dose response effect with a two‐fold increase in revertants indicating that these compounds are not mutagens or promutagens in these assays.     

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.     

Performance of a thermally desorbable type-tube diffusive sampler for very low air concentrations monitoring

Perkin-Elmer tubes packed with Tenax TA have been tested for the monitoring of low concentrations of aromatic compounds and linear alkanes using diffusive sampling. The uptake rates have been investigated for different exposure doses. A particular hexane and benzene behaviour has been observed: their uptake rates decrease rapidly in the lower exposure doses and so follow an exponential curve. The uptake rates of heavier compounds adopt another tendency: a slow decrease with time following a linear profile. The relative humidity influence has been studied too, and no water perturbation has been recorded.     

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.     

Potential of 2,4-dichlorophenoxyacetic acid conjugates as promutagens in the Salmonella/microsome mutagenicity test

Hepatic S9 preparations from Aroclor 1254 induced rats and 3-methylcholanthrene induced woodchucks were used to investigate, in vitro, the mutagenic potential of five amino acid conjugates of 2,4-Dichlorophenoxyacetic acid (alanine, aspartic acid, leucine, methionine and tryptophan). Five strains of Salmonella typhimurium (TA97, TA98, TA100, TA1535, TA1538) were utilized for this purpose. Dose-response effects producing a two-fold increase of revertants over spontaneous levels were not observed with either S9 preparation indicating that the amino acid conjugates are not promutagens in these assays.     

Potential of 2, 4‐dichlorophenoxyacetic acid conjugates as promutagens in the salmonella/microsome mutagenicity test

Abstract

Hepatic S9 preparations from Aroclor 1254 induced rats and 3‐methylcholanthrene induced woodchucks were used to investigate, in vitro, the mutagenic potential of five amino acid conjugates of 2, 4‐Dichlorophenoxyacetic acid (alanine, aspartic acid, leucine, methionine and tryptophan). Five strains of Salmonella typhimurium (TA97, TA98, TA100, TA1535, TA1538) were utilized for this purpose. Dose‐response effects producing a two‐fold increase of revertants over spontaneous levels were not observed with either S9 preparation indicating that the amino acid conjugates are not promutagens in these assays.     

Ozone artifacts and carbonyl measurements using Tenax GR, Tenax TA, Carbopack B, and Carbopack X adsorbents

Four popular thermally desorbable adsorbents used for air sampling (Tenax TA, Tenax GR, Carbopack B, and Carbopack X) are examined for the potential to form artifacts with ozone (O3) at environmental concentrations. The performance of these adsorbents for the ketone and aldehyde species identified as O3-adsorbent artifacts was also characterized, including recovery, linearity, and method detection limits (MDLs). Using gas chromatography/mass spectrometry, 13 different artifacts were identified and confirmed for both Tenax TA and Tenax GR, 9 for Carbopack B, but none for Carbopack X. Several O3 artifacts not reported previously were identified, including: pentanal, 3-hexanone, 2-hexanone, hexanal, 3-heptanone, and heptanal with Tenax TA; pentanal, 3-hexanone, 2-hexanone, hexanal, and 3-heptanone on Tenax GR; and 1-octene and 1-nonene with Carbopack B. Levels of straight-chain aldehyde artifacts rapidly diminished after a few cycles of adsorbent conditioning/O3 exposure, and concentrations could be predicted using a first-order model. Phenyl-substituted carbonyl artifacts (benzaldehyde and acetophenone) persisted on Tenax TA and GR even after 10 O3 exposure-conditioning cycles. O3 breakthrough through the adsorbent bed was most rapid in adsorbents that yielded the highest levels of artifacts. Overall, artifact composition and concentration are shown to depend on O3 concentration and dose, conditioning method, and adsorbent type and age. Calibrations showed good linearity, and most compounds had reasonable recoveries, for example, 90 +/- 15% for Tenax TA, 97 +/- 23% for Tenax GR, 101 +/- 24% for Carbopack B, and 79 +/- 25% (91 +/- 9% for n-aldehydes) for Carbopack X. Benzeneacetaldehyde recovery was notably poorer (22-63% across the four adsorbents). MDLs for several compounds were relatively high, up to 5 ng. By accounting for both artifact formation and method performance, this work helps to identify which carbonyl compounds can be measured using thermally desorbable adsorbents and which may be prone to bias because of the formation of O3-adsorbent artifacts.     

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.     

Comparison of analytical techniques for the determination of aldehydes in test chambers

The level of carbonyl compounds in indoor air is crucial due to possible health effects and the high prevalence of their potential sources. Therefore, selecting a convenient and rapid analytical technique for the reliable detection of carbonyl compound concentrations is important. The acetyl acetone (acac) method is a widely used standard procedure for detecting gaseous formaldehyde. For measuring formaldehyde along with other carbonyl compounds, the DNPH-method is commonly applied. The recommended procedure for measuring volatile organic compounds (VOCs) is sampling on Tenax TA, followed by thermal desorption and GC/MS analysis. In this study, different analytical techniques for the quantification of formaldehyde, pentanal, and hexanal are critically compared. It was found that the acac- and DNPH-method are in very good agreement for formaldehyde. In contrast, the DNPH-method significantly underestimates indoor air concentrations of the higher aldehydes in comparison to sampling on Tenax TA, although both methods are strongly correlated. The reported results are part of the EURIMA-WKI study on levels of indoor air pollutants resulting from construction, building materials and interior decoration.     

A study of 2,4,6-trinitrotoluene inhibition of benzo[a]pyrene uptake and activation in a microbial mutagenicity assay

A number of in vitro and in vivo studies have determined that binary and complex mixtures may interact to produce a toxicity that could not be predicted based on the individual chemicals. The present study was conducted with a binary mixture of model compounds to investigate possible interactions affecting their mutagenicity. The compounds included Benzo[a]pyrene (BAP), a polycyclic aromatic hydrocarbon that is an indirect-acting mutagen of great environmental concern, and 2,4,6-Trinitrotoluene (TNT), a nitro-aromatic compound that is a direct-acting mutagen frequently found as a soil contaminant at munitions sites. This study indicated that a binary mixture of BAP and TNT failed to induce the positive mutagenic response in Salmonella typhimurium strain TA98 characteristic of either compound alone. Spectrofluorometric analysis of BAP, and kinetic analyses of ³HBAP uptake in the presence or absence of TNT using TA98 cells that were treated or untreated with activated rat liver microsomes were performed. In cells preloaded with BAP, cellular BAP fluorescence was rapidly suppressed in the presence of TNT. Mass spectroscopy of BAP and TNT mixtures revealed a number of products, believed to be the result of complexation and nitration, that may account for the antagonistic action of TNT on BAP-induced mutagenicity in TA98 cells. Further, kinetic studies indicated that TNT inhibited the incorporation of BAP into cells.     

Ozone Artifacts and Carbonyl Measurements Using Tenax GR,Tenax TA,Carbopack B,and Carbopack X Adsorbents

Abstract

Four popular thermally desorbable adsorbents used for air sampling (Tenax TA, Tenax GR, Carbopack B, and Carbopack X) are examined for the potential to form artifacts with ozone (O₃) at environmental concentrations. The performance of these adsorbents for the ketone and alde-hyde species identified as O₃-adsorbent artifacts was also characterized, including recovery, linearity, and method detection limits (MDLs). Using gas chromatography/mass spectrometry, 13 different artifacts were identified and confirmed for both Tenax TA and Tenax GR, 9 for Carbopack B, but none for Carbopack X. Several O₃ artifacts not reported previously were identified, including: pentanal, 3-hexanone, 2-hexanone, hexanal, 3-heptanone, and heptanal with Tenax TA; pentanal, 3-hexanone, 2-hexanone, hexanal, and 3-heptanone on Tenax GR; and 1-octene and 1-nonene with Carbopack B. Levels of straight-chain aldehyde artifacts rapidly diminished after a few cycles of adsorbent conditioning/O₃ exposure, and concentrations could be predicted using a first-order model. Phenyl-substituted carbonyl artifacts (benzalde-hyde and acetophenone) persisted on Tenax TA and GR even after 10 O₃ exposure-conditioning cycles. O₃ breakthrough through the adsorbent bed was most rapid in adsorbents that yielded the highest levels of artifacts. Overall, artifact composition and concentration are shown to depend on O₃ concentration and dose, conditioning method, and adsorbent type and age. Calibrations showed good linearity, and most compounds had reasonable recoveries, for example, 90 ±15% for Tenax TA, 97 ±23% for Tenax GR, 101 ±24% for Carbopack B, and 79 ±25% (91 ±9% for n-aldehydes) for Carbopack X. Benzeneacetaldehyde recovery was notably poorer (22–63% across the four adsorbents). MDLs for several compounds were relatively high, up to 5 ng. By accounting for both artifact formation and method performance, this work helps to identify which carbonyl compounds can be measured using thermally desorbable adsorbents and which may be prone to bias because of the formation of O_3- adsorbent artifacts.     

Detection of hospital wastewater genotoxicity with the SOS chromotest and Ames fluctuation test

The genotoxic potential of Rouen University Hospital wastewater was evaluated by the SOS chromotest (on Escherichia coli PQ37) and Ames fluctuation test on Salmonella typhimurium strains TA 98 and TA 100 without metabolic activation. The samples were taken during the hospital maximal activity period (8:00 a.m. to 6:00 p.m.) over three one-week periods of the year. The simultaneous use of SOS chromotest and Ames fluctuation test allows us to carry out a preliminary screening of the hospital wastewater and to gain some insight by which mechanism the genotoxic compounds act. Out of a total of 18 daytime unconcentrated samples tested, 10 (55%) are positive in at least one assay. The two tests have different sensitivity. Indeed, nine genotoxic samples (50%) are detected by the Ames test, and four (22%) by the SOS chromotest. Distribution and intensity of the genotoxic response are different at the three periods. In order to explain this phenomenon, the influence of the rain levels is discussed. This work showed that the hospital wastewater samples tested were overall genotoxic, the response intensity being inflected by the pluviometry. Efforts are now under way to try to identify one or several genotoxic compounds in order to take precautionary measures to limit their release in hospital wastewaters.     

Halogenated diphenyl ethers identified in avian tissues and eggs by GC/MS

Chlorinated and brominated diphenyl ether compounds (DPEs) have been detected by gas Chromatography/ mass spectrometry (GC/MS) in eggs and carcasses of fish-eating birds from Louisiana, Michigan, Ohio, Rhode Island, Texas, Virginia, and Ontario, Canada. Three of these compounds were quantified; the highest concentration of 0.90 ppm tetrachloro DPE was found in a common tern carcass ( ) from Rhode Island. This is the first time that the halogenated DPEs have been reported in avian tissues. The significance of these residues is unknown.     

Evaluation of beta-naphthoxyacetic acid for mutagenic activity in the Salmonella/mammalian microsome assay

Beta-naphthoxyacetic acid (BNOA) is used as a plant growth regulator on tomatoes and strawberries. It is the active ingredient in Blossom-Set and Berry-Set, two plant hormone sprays for fruit-set. The mutagenic activity of BNOA was evaluated in four strains of Salmonella typhimurium (TA97, TA98, TA100 and TA1535) in the presence and absence of liver microsomal and cytosolic enzymes derived from Aroclor induced rats. BNOA did not produce any significant increase (p less than 0.05) in the reversion of any of the four tester strains in the standard plate incorporation assay. Results of the agar overlay toxicity tests indicates that the chemical shows toxic effects at concentrations above 500 micrograms/plate. It was concluded that under the conditions of these tests, BNOA did not exhibit any mutagenic activity.     

Investigating the sources of the mutagenic activity found in a river using the Salmonella assay and different water extraction procedures

In the routine São Paulo state (Brazil) surface water quality-monitoring program, which includes the Salmonella microsome mutagenicity assay as one of its parameters, a river where water is taken and treated for drinking water purposes has repeatedly shown mutagenic activity. A textile dyeing facility employing azo-type dyes was the only identifiable source of mutagenic compounds. We extracted the river and drinking water samples with XAD4 at neutral and acidic pH and with blue rayon, which selectively adsorbs polycyclic compounds. We tested the industrial effluent, raw, and treated water and sediment samples with YG1041 and YG1042 and compared the results with the TA98 and TA100 strains. The elevated mutagenicity detected with YG-strains suggested that nitroaromatics and/or aromatic amines were causing the mutagenicity detected in the samples analyzed. Positive responses for the blue rayon extracts indicated that mutagenic polycyclic compounds were present in the water samples analyzed. The mutagen or mixture of mutagens present in the effluent and water samples cause mainly frameshift mutations and are positive with and without metabolic activation. The Salmonella assay combined with different extraction procedures proved to be very useful in the identification of the origin of the pollution and in the identification of the classes of chemical compounds causing the mutagenic activity in the river analyzed.     

New Evidence for the presence of arsenocholine in shrimps (Pandalus borealis) by use of pyrolysis gas chromatography — atomic absorption spectrometry/mass spectrometry

An analytical method for the structure elucidation of quarternary organoarsenic compounds in trace amounts in fish and crustaceans has been developed in this laboratory. The two major organoarsenicals found in shrimps have been separated by ion-exchange chromatography and their stucture has been studied by the use of pyrolysis gas chromatography. Their degradation in the pyrolyzer unit has been studied by the use of atomic absorption spectrophotometer or mass spectrometer as detectors. A comparison of the analytical data of these substances and data from synthetic reference substances show complete agreement. The structures of reference compounds have been confirmed by use of nuclear magnetic resonance spectrometry and fast atom bombardment (FAB) mass spectrometry. It is demonstrated that both of the organoarsenic compounds in shrimps have a trimethylarsonio moiety e.g. the compounds have a quarternary structure. Furthermore, the more basic organoarsenic compound has a mass spectrometric fragmentation pattern similar to synthetic arsenocholine and acetylarsenocholine. Treatment of arsenobetaine with hot base produces trimethylarsineoxide in a high yield (96%). Arsenocholine, however, remains unchanged during this treatment. Treatment with sodium benzenethiolate does not result in chemical demethylation, which shows that this method together with gas chromatography-mass spectrometry is not feasible for confirmation of its structure.     

Identification of nonionic detergents by GC/CI-MS: I. A complementary method or an attractive alternative to GC/EI-MS and other methods?

Tertiary octylphenol and lauryl alcohol ethoxylates (with one to six oxyethylene groups) have been detected in the effluent from the primary clarifiers of wastewater treatment plant, using their chemical ionization (CI) mass spectra. The GC/CI-MS appears to be a very suitable method for the selective identification of these compounds.     

QSAR modeling for predicting mutagenic toxicity of diverse chemicals for regulatory purposes

The safety assessment process of chemicals requires information on their mutagenic potential. The experimental determination of mutagenicity of a large number of chemicals is tedious and time and cost intensive, thus compelling for alternative methods. We have established local and global QSAR models for discriminating low and high mutagenic compounds and predicting their mutagenic activity in a quantitative manner in Salmonella typhimurium (TA) bacterial strains (TA98 and TA100). The decision treeboost (DTB)-based classification QSAR models discriminated among two categories with accuracies of >96% and the regression QSAR models precisely predicted the mutagenic activity of diverse chemicals yielding high correlations (R ²) between the experimental and model-predicted values in the respective training (>0.96) and test (>0.94) sets. The test set root mean squared error (RMSE) and mean absolute error (MAE) values emphasized the usefulness of the developed models for predicting new compounds. Relevant structural features of diverse chemicals that were responsible and influence the mutagenic activity were identified. The applicability domains of the developed models were defined. The developed models can be used as tools for screening new chemicals for their mutagenicity assessment for regulatory purpose.

     

Photochemical mutagen formation from aerobically treated night-soil effluent and the contributive substances

Effluents from a night-soil treatment plant, where night-soil was aerobically treated by an activated sludge process, were irradiated with a UV lamp excluding short wavelengths less than 300 nm as a model of exposure to sunlight and the mutagenicities of the ethylacetate extracts from the irradiated effluents were assayed using Salmonella typhimurium TA98. The extracts exhibited mutagenicity toward S. typhimurium TA98 in the absence of rat liver S9 fraction only when the effluents were fortified with nitrite ion (more than 6 ppm) by over aeration or by artificial addition of nitrite, indicating that a limiting factor for mutagen formation is nitrite ion concentration. Nine organic-N-containing compounds as models of the organic components in the effluent were also irradiated and direct-acting potent mutagens were found to be produced from such compounds having indole moiety as indole, oxindole, tryptophan and tryptamine.     

Evaluation of sample recovery of malodorous livestock gases from air sampling bags, solid-phase microextraction fibers, Tenax TA sorbent tubes, and sampling canisters

Odorous gases associated with livestock operations are complex mixtures of hundreds if not thousands of compounds. Research is needed to know how best to sample and analyze these compounds. The main objective of this research was to compare recoveries of a standard gas mixture of 11 odorous compounds from the Carboxen/PDMS 75-microm solid-phase microextraction fibers, polyvinyl fluoride (PVF; Tedlar), fluorinated ethylene propylene copolymer (FEP; Teflon), foil, and polyethylene terephthalate (PET; Melinex) air sampling bags, sorbent 2,b-diphenylene-oxide polymer resin (Tenax TA) tubes, and standard 6-L Stabilizer sampling canisters after sample storage for 0.5, 24, and 120 (for sorbent tubes only) hrs at room temperature. The standard gas mixture consisted of 7 volatile fatty acids (VFAs) from acetic to hexanoic, and 4 semivolatile organic compounds including p-cresol, indole, 4-ethylphenol, and 2'-aminoacetophenone with concentrations ranging from 5.1 ppb for indole to 1270 ppb for acetic acid. On average, SPME had the highest mean recovery for all 11 gases of 106.2%, and 98.3% for 0.5- and 24-hr sample storage time, respectively. This was followed by the Tenax TA sorbent tubes (94.8% and 88.3%) for 24 and 120 hr, respectively; PET bags (71.7% and 47.2%), FEP bags (75.4% and 39.4%), commercial Tedlar bags (67.6% and 22.7%), in-house-made Tedlar bags (47.3% and 37.4%), foil bags (16.4% and 4.3%), and canisters (4.2% and 0.5%), for 0.5 and 24 hr, respectively. VFAs had higher recoveries than semivolatile organic compounds for all of the bags and canisters. New FEP bags and new foil bags had the lowest and the highest amounts of chemical impurities, respectively. New commercial Tedlar bags had measurable concentrations of N,N-dimethyl acetamide and phenol. Foil bags had measurable concentrations of acetic, propionic, butyric, valeric, and hexanoic acids.