Diesel particulate extracts in bacterial test systems

The Ames bacterial mutagenicity test system was used to evaluate parameters which may affect the mutagenic activity of diesel particulate extracts. The optimal extraction conditions, extractability of mutagens by simulated biological fluids and the effect of collection method were investigated. The role of solvent was examined by extracting diesel particles with methanol, acetone, cyclohexane, ethyl acetate, n-hexane, dichloromethane, benzene and a benzene-ethanol mixture. Of these, the dichloromethane extract exhibited the highest activity in the Ames test, although methanol yielded the largest extractable mass. Diesel particles were also extracted by dimethyl sulfoxide (DMSO) and four other simulated biological fluids for 48 h at 25, 37, and 45°C to study the effects of temperature. The mutagenic activity of the DMSO extract began to decline at temperatures higher than 37°C after 8 h of incubation. Fetal calf serum was the only simulated biological fluid which eluted mutagenic activity from the particles. No activity was detected in the 0.5% bovine serum albumin, simulated lung surfactant and saline extracts. Diesel particles collected by electrostatic precipitation (ESP) and filtration were studied. The mutagenic activities of both extracts were comparable when expressed as revertants per mg of particle. After the extracts were separated into nine fractions by a solvent partitioning scheme, the majority of the activity was found in the neutral-nonpolar II, neutral polar, strong acid and weak acid fractions. The acid salt fraction from the ESP sample was inactive. These results demonstrate that differences in the extraction conditions can result in differences in the mutagenic activity of diesel particulate extract. Since the mutagens in the extracts are not readily extractable by simulated biological fluids, the question of bioavailability of mutagens in diesel particles must be considered in the final assessment of their potential effects in biological systems and organisms.     

Diesel soot: Mutation measurements in bacterial and human cells

Both hot pipe and dilution chamber samples of the exhaust from a diesel (Oldsmobile 350) engine have been collected, extracted with methylene chloride and those extracts have been tested for mutagenicity in forward mutation assays in human lymphoblasts and S. typhimurium. In the absence of a metabolic activation system, the extract was significantly mutagenic to the bacteria in the range of 0 to 30 μg/ml, but introduced no mutations in human cells at concentrations up to 200 μg/ml under the same conditions of assay medium. However, when assayed in the presence of a postmitochondrial supernatant derived from rat liver, the the soot extracts were significantly mutagenic to both bacteria and human cells in the range of 50–100 μg/ml. Fractionation of the soot extract on the basis of polarity by sequential elution from a silicic acid column permitted concentration of the mutagenic activity in the alkane/toluene eluate, as determined by bacterial assays. Preliminary characterization of this fraction and preliminary studies of pure compounds leads us to suspect the alkyl substituted phenanthrenes as representing at least a significant fraction of the mutagenic activity of this alkane/toluene eluate.     

Mutagenic activity of diesel emission particulate extracts and isolation of the mutagenic fractions

Mutagenic activity in diesel emission particulate extracts was detected by the Salmonella typhimurium/microsome assay. Direct-acting mutagens as well as promutagens requiring metabolic activation were detected. The extracts were fractionated into acidic, basic, and neutral fractions, and the neutral fraction was chromatographed into seven subfractions. Differences in the mutagenic potency of these fractions and subfractions were determined by the Salmonella assay. Fractions containing as yet unidentified compounds, but not polynuclear aromatic hydrocarbons, were found to make a major contribution to mutagenic activity of the extracts.     

Evaporation of phenols from synthetic liquid fuel spills

The environmental impact of a synthetic-fuel spill on a waterway will be determined in part by the rate of dissolution of toxic components into the underlying water and by the rate of removal of those components from the oil by evaporation. In this study, the two film mass transfer model of volatilization was applied to the study of the evaporation of phenols from two synthetic liquid fuels. The rates of evaporation of phenol and alkylated phenols were measured under laboratory conditions. Liquid and gaseous mass transfer coefficients were measured experimentally, and used to calculate Henry's Law coefficients for various phenol-oil pairs from the observed evaporation rates. It was concluded that evaporation rates of phenols from synthetic oils would be significantly less than rates of dissolution in most spills.     

Characterization of natural organic matter by pyrolysis/GC-MS

Natural organic matter (NOM) in nine water samples selected for the NOM typing project were characterized by pyrolysis in conjunction with gas chromatography and mass spectrometry (GC-MS). NOM samples both isolated by reverse osmosis and evaporation techniques were subjected to high temperature pyrolysis and the products obtained were assigned to one of the four main types: biopolymers carbohydrates, proteinaceous materials, N-acetylamino sugars, and polyhydroxy aromatics. These biopolymers in the water samples were quantified and the quantities were used in characterization. Principal component analysis was employed in revealing the similarities and differences between the samples and between the separation techniques. The results indicate that there are little differences between the samples, and the NOM separated by evaporation preserves most of the organic material compared to the reverse osmosis technique.     

Mutagenic and carcinogenic potency of extracts from diesel related environmental emissions: Simultaneous morphological transformation and mutagenesis in BALB/c 3t3 cells

Particulate extracts from six different environmental emission sources were assayed for genotoxic activity in mouse BALB/c 3T3 clone A31-1 cells. All compounds were tested simultaneously for both transforming and mutagenic (induction of ouabain-resistance) potential with and without exogenous metabolic activation in the form of a 9000 × g postmitochondrial hepatic supernatant fraction from Aroclor-1254 induced Fischer 344 rats. Dichloromethane particulate extracts from the exhaust of two light duty diesel engines (Oldsmobile and Nissan), one heavy duty diesel engine (Caterpillar) and one late model gasoline engine (Mustang II) were assayed in an identical manner to particulate extracts from the emissions of a roofing tar pot and a coke oven. No clear dose-dependent responses were observed, but several of the samples showed significant transforming and mutagenic activity. A qualitative ranking system showed the activity of these particulate extracts for either mutagenesis or transformation was: coke oven = Mustang II gasoline engine > Nissan diesel engine > roofing tar. Particulate extracts from the Oldsmobile diesel engine and the Caterpillar diesel engine showed essentially no activity.     

Fate of oil from two major oil spills: Role of microbial degradation in removing oil from the Amoco Cadiz and IXTOC I spills

The fate of oil was examined in intertidal sediments impacted by the Amoco Cadiz spill in Brittany, France, and in surface waters contaminated by the IXTOC I well blowout in the Bay of Campeche, Mexico. The role of microbial degradation in removing the contaminating hydrocarbons was assessed by laboratory determinations of numbers of hydrocarbon utilizing microorganisms, measurement of microbial hydrocarbon biodegradation potentials, by chemical analyses of samples incubated under controlled conditions and by examination of the evolution of the hydrocarbon mixture in field samples collected after the spill. Analyses included the use of ¹⁴C radiolabelled hydrocarbon tracers, glass capillary-gas chromatography and mass spectrometry. Numbers of hydrocarbon utilizing microorganisms were found to be elevated by several orders of magnitude in oil contaminated samples. Numbers of hydrocarbon utilizing microorganisms appear to provide a useful index of environmental hydrocarbon contamination. However, environmental constraints on microbial processes appear to be most important in determining actual rates of biodegradation rather than numbers of available hydrocarbon utilizing microorganisms. Very high rates of degradation were found in intertidal sediments impacted by the Amoco Cadiz spill. In contrast, rates of degradation in the Gulf of Mexico were found to be much lower, partly due to nutrient limitations and partly due to the formation of large masses of oil-water emulsion or mousse.     

Genetic biomonitoring of an urban population exposed to mutagenic airborne pollutants

Biomonitoring studies have increased as a consequence of risks and effects to human health on exposure to environmental contaminants, mainly air pollutants. Genetic biomarkers are useful tools for the early assessment of exposure to occupational and environmental pollution. The objective of the present study was to investigate genotoxic effects on people residing and/or working downwind from an oil refinery in southern Brazil and the mutagenic activity of airborne particulate matter (PM10). Samples of peripheral blood and buccal mucosa cells were evaluated using the single-cell gel electrophoresis assay (comet assay) and the micronucleus (MN) assay, respectively. PM10 samples were collected in the target site and the organic matter extraced with dichloromethane was assessed for mutagenic activity in the Salmonella/microsome assay. The exposed group (n = 37) was compared to a reference group (n = 37) of subjects living in an urban area with limited traffic and industrial influence, located far from the main industrial areas. All PM10 organic extracts showed mutagenic positive responses and the effect decreased in the presence of S9 mix indicating that the predominant compounds present were direct-acting mutagens. The responses of YGs strains are consistent with aromatic amines and nitroarenes being present in the PM10 extracts. The group in the area under the influence of the oil refinery (exposed group) showed significantly higher DNA damage in lymphocytes than the reference group. The MN frequencies in buccal mucosa were very low for both groups and no difference between groups was observed. No association was found between age and tobacco smoking habit and level of DNA damages measured by the comet assay. The results indicate that the comet assay was a sensitive tool to detect DNA damage in subjects under the influence of an oil refinery, with marked genotoxic activity in the atmospheric environment.     

Photo-oxidation products of petroleum hydrocarbons in the Eastern Red Sea coastal waters

The level of dissolved oil residues as well as the oxygenated petroleum hydrocarbons in the seawater of the affected area of Jeddah coast, Red Sea was estimated. The dissolved/dispersed petroleum hydrocarbons concentration was found minimal. The minimum level may be attributed to the action of the weathering processes.Selected samples of the dichloromethane extracts of seawater were fractionated, using silica gel columns, into four fractions eluted with solvents of increasing polarity. The fluorescence intensity of these fractions was measured at three different pairs of excitation and emission wavelengths using ultraviolet fluorescence spectrofluorophotometry (UVF). The total fluorescence intensity of the fractions at 280 nm excitation, 327 nm emission wavelengths and 310 nm excitation, 360 nm emission wavelengths exceeds the fluorescence intensity of the whole extract by more than two orders of magnitude. The estimation of higher molecular mass oxygenated compounds in the fourth fractions, read at 380 nm excitation, 430 nm emission wavelengths, indicates rather low concentrations of <1 microg l(-1) hydroxypyrene equivalents. However, the polar fractions (eluted with acetone) account for more than 25% of the total fluorescence intensity of all fractions combined at each pair of wavelengths. The characterization of the products causing the fluorescence particularly the high molecular mass compounds still presents difficulties.Preliminary GC/MS results revealed some evidence for petroleum hydrocarbon oxidation products. Alkyl isobenzofuranones have been found in some seawater extracts. This is an ongoing project to characterize and identify additional compounds from these extracts.     

Mutagenic and carcinogenic potency of extracts of diesel and related environmental emissions: In vitro mutagenesis and oncogenic transformation

Extracts from emissions of four diesel engines, a gasoline engine and three related environmental samples were tested in four in vitro assay systems designed to detect carcinogenic or mutagenic activity of chemicals. Samples from three of four diesel extracts, the gasoline engine, and all three related samples were positive in an enhancement of viral transformation assay. Two diesel samples, the gasoline engine extract and extract from coke oven emissions were positive for mutation induction in Chinese hamster ovary cells. Only the gasoline engine extract and the coke oven sample were positive in a DNA fragmentation assay using alkaline sucrose gradients. Experiments using chemical transformation of Syrian hamster embryo cells as an assay method have not been completed.     

Evaluation of the water genotoxicity from Santos Estuary (Brazil) in relation to the sediment contamination and effluent discharges

The genotoxic activity of water samples collected in 9 different sites within the area of the Santos estuary was preliminary evaluated, and related to previous data on the genotoxicity of sediments and the contents of PAHs in both water and sediment samples. The liquid discharge of a steel mill (coke plant), known to be mutagenic, was chemically analyzed to determine its PAH content. For the water evaluation we employed the Salmonella/microsome assay with the strains TA98 and TA100 with and without S9 mix in the plate incorporation method. The water was filtered with an AP20 membrane before being extracted with XAD4 at natural and acidic pH. The industrial effluent was filtered in 0.45 microm membranes before being extracted with the liquid/liquid method. Both membranes containing the particulate material were extracted using ultrasonication. PAHs were found associated with the suspended particles present in the industrial effluent in accordance with mutagenicity data previously reported. In relation to the estuarine waters, sites 1 and 5 presented low levels of mutagenic activity only in the filtered water (liquid fraction) extracts. At site 3, both the filtered water and particulate solids presented also low mutagenicity. Results show that the mutagenic activity observed in water could not be directly related to the genotoxic activity and PAHs contents of the bottom sediments.     

Novel approach for assessing heavy metal pollution and ecotoxicological status of rivers by means of passive sampling methods

In order to study the pollution of fluvial ecosystems, it is necessary to analyze not only the levels of chemical contaminants in water, but also those accumulated in the sediment matrix, as well as to assess its ecotoxicological status. Eleven Catalan (Spain) river sections (one sampling point per river) located near urban and industrial areas were sampled during winter of 2009. Water pollutants were collected by using passive samplers as Diffusive Gradient in Thin-Films (DGTs) and Semi-Permeable Membrane Devices (SPMDs). Point water samples were also collected. The concentrations of potentially toxic elements (PTE) in water, filtered water, DGTs and sediment samples were analyzed. Aqueous and organic solvent extracts of sediments samples and organic extracts of SPMDs were performed to assess acute toxicity to Vibrio fischeri by Microtox(?), and chronic toxicity to the green alga Pseudokirschneriella subcapitata. Microtox(?) test was also performed with DGT extracts. The results show that metals content of Catalan river waters are below the freshwater screening US EPA benchmarks, excepting some industrial areas (for Hg, Pb, and Zn). In contrast, sediments levels of some rivers were far above freshwater sediment screening US EPA benchmarks (for Zn, As, Cr, Pb, Ni, Hg, and Mn), particularly in the most industrialized areas. A good correlation was found between toxicity values of extracts (from sediments and DGTs) and PTE levels in sediments. The current results support the suitability of using combined point and passive sampling methods for assessing the chemical and ecotoxicological status of aqueous environments.     

Determination of radionuclide levels in rainwater using ion exchange resin and γ-spectrometry

The evaluation of radioactivity accidentally released into the atmosphere involves determining the radioactivity levels of rainwater samples. Rainwater scavenges atmospheric airborne radioactivity in such a way that surface contamination can be deduced from rainfall rate and rainwater radioactivity content. For this purpose, rainwater is usually collected in large surface collectors and then measured by γ-spectrometry after such treatments as evaporation or iron hydroxide precipitation. We found that collectors can be adapted to accept large surface (diameter 47 mm) cartridges containing a strongly acidic resin (Dowex AG 88) which is able to quantitatively extract radioactivity from rainwater, even during heavy rainfall. The resin can then be measured by γ-spectrometry. The detection limit is 0.1 Bq per sample of resin (80 g) for ¹³⁷Cs. Natural ⁷Be and ²¹⁰Pb can also be measured and the activity ratio of both radionuclides is comparable with those obtained through iron hydroxide precipitation and air filter measurements. Occasionally ²²Na has also been measured above the detection limit. A comparison between the evaporation method and the resin method demonstrated that 2/3 of ⁷Be can be lost during the evaporation process. The resin method is simple and highly efficient at extracting radioactivity. Because of these great advantages, we anticipate it could replace former rainwater determination methods. Moreover, it does not necessitate the transportation of large rainwater volumes to the laboratory.     

Development of extraction method of pharmaceuticals and their occurrences found in Japanese wastewater treatment plants

In this study, occurrence of 66 PPCPs (pharmaceuticals and personal care products) in liquid and solid phases of sewage sludge was elucidated. The extraction methods for the PPCPs from sludge were newly developed employing Pressurized Liquid Extraction (PLE) and Ultrasonic Solvent Extraction (USE). As an appropriate method, PLE using water (pH2), PLE using methanol (pH4), and USE using mixture of methanol and water (1/9,v/v, pH11) was found most effective because total recovery of most of the PPCPs indicated 40 to 130%. The developed extraction method with previously developed method for liquid phase analysis was applied to field survey at wastewater treatment plants (WWTPs) in Japan. 56 compounds were detected from the primary sludge and 61 compounds were detected from the excess sludge. The concentration was ranged between several ng/g and several μg/g. Solid-water distribution coefficient (Log K_d) ranged between 0.9 L/kg (Caffeine) and 3.7 L/kg (Levofloxacin) for primary sludge and between 1.4 L/kg (Sulpirid) and 4.3 L/kg (Mefenamic acid) for excess sludge.     

A bioassay approach to determine the dioxin-like activity in sediment extracts from the Danube River: ethoxyresorufin-O-deethylase induction in gill filaments and liver of three-spined sticklebacks (Gasterosteus aculeatus L.)

Sediment samples from the upper Danube River in Germany have previously been characterized as ecotoxicologically hazardous and contaminants in these sediments may contribute to the observed decline of fish populations in this river section. For the investigation of sediment toxicity there is a need for development, standardization and implementation of in vivo test systems using vertebrates. Therefore, the main objective of this study was to apply and evaluate a recently established fish gill EROD assay as a biomarker in sediment toxicity assessment by using extracts of well characterised sediment samples from the upper Danube River. This to our knowledge is the first application of this novel assay to sediment extracts. Sediments from four different sites along the upper Danube River were Soxhlet-extracted with acetone and dissolved in DMSO. Three-spined sticklebacks (Gasterosteus aculeatus L.) were exposed for 48 h to various concentrations of the extracts, to the positive control beta-naphthoflavone or to the solvent. Measurements of EROD activity in gill filaments and liver microsomes followed the exposure. Concentration-dependent induction of EROD in both gill and liver was found for all sediment extracts. The highest EROD-inducing potency was determined for extracts of sediments from the sites "Opfinger See" and "Sigmaringen" and the EROD activities in gill and liver correlated well. The results from the gill and liver assays were in accordance with in vitro results of previous investigations. The EROD activities measured in the present study corresponded with the concentrations of PAHs, PCBs and PCDD/Fs in the sediment samples derived in a previous study. The sticklebacks in this study were in the reproductive phase and a stronger EROD induction was obtained in the females than in the males. Implementation of the EROD assay in testing of sediment extracts gave highly reliable results which make this assay an ecotoxicologically relevant method for assessment of contamination with Ah receptor agonists in sediments.     

The mutagenic activity of thirty polycyclic aromatic hydrocarbons (PAH) and oxides in urban airborne particulates

Data are presented on the mutagenicity of an organic extract of a composite sample of urban air particulates and of thirty PAH compounds in such samples, including four quinone derivatives, isolated quantitatively by thin-layer chromatography and identified by fluorescence or other spectral techniques. Mutagenic activity was determined by the Ames assay, using histidine auxotrophs of Salmonella typhimurium strains TA98, TA100, TA1537 and TA1538. All compounds were dissolved in dimethyl sulfoxide (DMSO) which was the least toxic of eight organic solvents tested.The mutagenic activity of a benzene extract of suspended particulates from the air of Hamilton, Ontario was significantly greater with strain TA98 than with strain TA100, suggesting the presence of more frame-shift acting mutagens. The mutagenic response of this extract was similar with and without S-9 activation.Mutagenic tests on the 30 PAH compounds indicated that only the benzo(a)pyrene quinones were direct acting mutagens. All of the chemical compounds were tested with and without S-9 activation. The following PAH showed unequivocal mutagenic activity, with S-9 activation: benz(a)anthracene, benzo-(a)pyrene, benzo(ghi)perylene, benzo(rst)pentaphene, benzophenanthrene, chrysene, 1, 2, 3, 4-dibenzanthracene, 2, 3, 6, 7-dibenzanthracene, and 3-methyl cholanthrene. The quinones of 1, 6-; 3, 6-; and 6, 12-benzo(a)pyrenes showed weak mutagenic activity but 3, 6-benzo(a)pyrene elicited also a photodynamic response.     

Salmonella/microsome mutagenicity assays of exhaust from diesel and gasoline powered motor vehicles

Motor vehicle exhaust from prechamber injection diesel and gasoline powered passenger cars, sampled during US FTP 1973 test cycles and comprising both particulate matter and compounds condensable at ambient temperature, has been assayed for mutagenicity in the Salmonella/microsome test. Mutagenic components were to a large extent active in the absence of the mammalian microsomal preparation. The mutagenicity of both particulate matter and condensate from diesel exhaust and condensate from gasoline exhaust was decreased in the presence of the microsomal preparation whereas the mutagenicity of particulate matter from gasoline exhaust was enhanced by microsomal activation. A comparison between the investigated diesel and gasoline exhaust samples shows that the mutagenic effect in the Salmonella test of the diesel exhaust is more than ten times higher than that of the gasoline exhaust. Fractionation with respect to polarity indicates that the mutagenic components mainly are distributed in neutral aliphatic, aromatic, and oxygenated fractions. Tests for mutagenic monofunctional nitroarenes by an anaerobic assay indicate that such compounds at most are marginally present in the exhaust samples as compared with their presence in airborne particulate matter collected in an urban environment.     

基于受体作用理论的类雌激素效应测试方法

以17β雌二醇为标准参考物质,对基于受体作用理论的环境激素分析方法实验条件进行了重构和优化,建立了环境类雌激素分析方法。结果表明:用营养缺陷型培养基30℃恒温振荡66 h培养时间进行重组基因酵母的复苏与增殖,20℃～35℃水浴样品与酵母混合液培养3～6 h暴露,优先选用机溶剂处理法释放β-半乳糖苷酶,35℃恒温水浴进行酶活测试反应,采用反应终止后2.5 h 420 nm波长进行分析测试,同时实际样品的分析,加测不少于一个的稀释水样效应值,可使方法更加科学高效、准确度高,应用于各类实际样品分析。     

上海城市河岸带对降雨径流氮垂直去除研究

在上海市城市河岸带人工绿地建设了微区径流场,进行了5次模拟降雨径流实验,研究了人工绿地岸带下渗流中氮浓度和去除率的时空变化。结果表明：人工绿地岸带对垂直下渗流中氮有显著的净化作用,且主要集中于土壤的0~30 cm以内,TN和NH4+去除率在399%和398%以上,NO3-+NO2-去除率除11月为负值外,其余月份均在100%以上;在30~60 cm深度,由于土壤氮的析出导致径流中氮浓度增加,去除率降低;而60~90 cm深度的去除率增加。径流场内下渗流中氮浓度具有明显的水平空间变化,随距入水端距离的增加,30 cm深度下渗流中氮浓度先上升后下降;人工绿地岸带对下渗流中氮的净化作用均随淹水时间的延长呈降低趋势,且季节变化明显,在10月和4月具有较高的去除率,0~30 cm内TN、NH4+和NO3-+NO2-的去除率可分别达635%、891%和416%以上     

Mutagenic properties of PM2.5 air pollution in the Padana Plain (Italy) before and in the course of XX Winter Olympic Games of "Torino 2006"

PM2.5 is one of the most important aspects of environmental health. This air pollutant is breathable and it is implicated in several chronic adverse health effects such as the decrease of respiratory functionality and cancer. Several in vitro bioassays are able to predict the mutagenic/carcinogenic activity of the environmental pollutants and mixtures of them. In this study PM2.5 air pollution was daily monitored in three cities located in the Northern part of Italy and the mutagenic properties of the PM2.5 organic extracts were also assessed. Samplings lasted 14 months and cover the period of the Winter Olympic Games of "Torino 2006". In this work, the levels of PM2.5, its mutagenic properties (detected with Salmonella typhimurium assay), the role of the Olympic Games as environmental factor and some meteorological data are discussed. The mean concentration of PM2.5 measured in Torino was 45.4 (+/-30.6) microg/m(3), in Pavia 37.6 (+/-25.6) microg/m(3), in Verona 43.1 (+/-28.5) microg/m(3). Findings of the monthly pool bioassay were in Torino 107 (+/-104) net revertans/m(3), in Pavia 108 (+/-89) net revertans/m(3), in Verona 128 (+/-109) net revertans/m(3). The Olympic Games period data show that PM2.5 pollution and its load of mutagenic potential are different and partially independent phenomena. The Olympic Games had not a great impact on the PM2.5 pollution. The exclusive PM2.5 gravimetric analysis shows a potential human risk if compared with the latest international guide values but it does not describe exhaustively the human health risk associated to the presence of this particular air pollutant. Moreover, the chemical and biological activity qualification of the PM organic extracts as a whole, can instead improve the knowledge.