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.     

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.     

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.     

我国不同区域水源水拉森指数比较分析

目前远距离调水已经成为解决区域水资源不足的一项重要措施。但在水源切换时,切换前后水源水的腐蚀性如果存在显著差异,可能会因腐蚀加剧而出现"黄水"或"红水"现象。因此,了解不同区域、不同类型水源的腐蚀性差异,对于科学合理地进行水资源调配具有重要意义。拉森指数(LR)是目前评价水质对铁质管网腐蚀程度的常用方法。本研究以14个城市的26个水源水为研究对象,对模拟常规工艺处理后的滤后水进行了水质分析,并计算出其拉森指数。结果表明,相对于湖库水和地下水,河流水的拉森指数偏高,有44%的河流水拉森指数大于1,具有较强的腐蚀倾向。按区域分华东和中南地区、按流域分黄河和珠江流域的水源水拉森指数平均值大于1,水源切换条件下应关注其腐蚀性情况。     

Evaluation of chlordimeform and degradation products for mutagenic and DNA-damaging activity in Salmonella typhimurium and Escherichia coli

The mutagenic activity of chlordimeform and two of its breakdown products, 4-chloro-o-toludine and 4-chloro-N-formyl-o-toluidine were determined with five histidine dependent strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA98, TA100) and five tryptophan dependent strains of E. coli WP2 (WP2, WP2uvrA, WP67, CM611, CM571) with and without rat liver microsomal enzymes. 4-chloro-o-toluidine increased the number of the reversions of the S. typhimurium strain TA1535 more than two fold over spontaneous at the concentration of 400 micrograms/plate. The results of the DNA repair tests in the Salmonella TA1538/TA1978 and E. coli multirepair deficient systems showed that both breakdown products were active in inducing damage not repaired in at least one repair deficient strain while chlordimeform itself was inactive.     

Polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) and 2,3,7,8-TCDD equivalents (TEQs) in sediment from the Hyeongsan River, Korea

Sediment, pore water and water samples from the Hyeongsan River, Korea were analyzed for several classes of halogenated aromatic hydrocarbons (HAHs) and their dioxin-like activities were evaluated using the in vitro H4IIE-luc bioassay. Polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and biphenyls (PCBs) were detected in sediments from all six sampling locations with mean concentrations of 2.8 x 10(2) pg/g, 190 pg/g, and 61.4 ng/g, dw, respectively. Polycyclic aromatic hydrocarbons (PAHs) were predominated by 4-6 ring compounds with concentrations in the range of 5.30-7680 ng/g, dw. Chemical profiles of target analytes in sediment and water samples revealed that there was a gradient of concentrations along the river from upstream to downstream, which suggested that the primary source was a wastewater reservoir adjacent to a sewage treatment plant (STP). TEQs derived by summing the product of concentrations of individual congeners by their respective relative potencies (REPs or TEFs) ranged from 4.3 x 10(-1) to 1.1 x 10(3) pg/g, dw. Raw Soxhlet extracts from all six sampling locations induced significant dioxin-like responses in the H4IIE-luc bioassay. TCDD-EQs derived from H4IIE bioassay ranged from 7 x 10(-3) to 1.5 x 10(3) pg/g, dw, which were significantly correlated with TEQs (r2 = 0.994, p < 0.05). Among the three Florisil fractions tested, PCDD/Fs in fraction (F2) induced the greatest magnitude of response (range: 24-83%-TCDD-max.) in the H4IIE-luc assay. Comparison of the TEQ and TCDD-EQ suggested little non-additive interaction between fractions and AhR-active and inactive compounds. Concentrations of individual congeners as well as TEQs and TCDD-EQs suggest inputs from the industrial center waste stream in the Hyeongsan River.     

Occurrence of organochlorine insecticides, PCBs and PCB congeners in waters and sediments of the Ebro River (Spain)

Complex mixtures extracted from air filters exposed for 24 h in two sessions (27 July and 02 August 1991) and at two locations (Merced, downtown, and Pedregal de San Angel, south-west) in Mexico City were analysed. The organic extracts were from airborne particles equal or smaller than 10 microns (PM10), and from total suspended particles (TSP). These organic extracts were assayed in the somatic mutation and recombination test (SMART) in wings of Drosophila melanogaster using two different crosses as well as in the Salmonella/microsome assay using strain TA98 with and without S9 fraction. The presence of polycyclic aromatic hydrocarbons (PAH) in the extracts was determined by gas chromatography. The genotoxic activities observed in the two test systems were comparable with the indirect mutagens producing greater response than the direct mutagens. The quantities of particulate matter as well as the genotoxic activities were higher on 02 August than on 27 July 1991 for both locations. The amounts of airborne particles and the resulting genotoxic activities were higher at Merced than at Pedregal. In both biological systems, PM10 were more genotoxic than TSP. These results demonstrate the sensitivity of the Drosophila wing SMART-which is an in vivo eukaryotic genotoxicity assay-as a biological monitor of environmental pollution related to airborne particles.     

长江、嘉陵江(重庆段)源水有机提取物的致突变活性及其季节变化

为了确定并比较重庆主城区段长江、嘉陵江源水有机提取物的致突变性及其季节变化规律，分别于春、夏、冬季采用GDX－120大孔树脂，对位于城区上游、城区中段、城区下游以长江、嘉陵江源水的5个水厂的进厂水进行了有机物的浓缩提取。提取物的致突变活性采用经典的Ames试验平板掺入法评估，测试菌株为TA98及TA100，同时做加与不加S9的比较。结果显示，嘉陵江及长江源水的有机提取物均有不同程度的致突变活性。嘉陵江源水明显大于长江源水，城区中段源水明显大于上游段及下游段源水。多数断面显示平水期致突变活较为显著并且移码型致突变性大于碱基置换型致密变性。研究结果提示，城市污染源已导致长江、嘉陵江源水具备致突变活性，控制两江沿岸的各种水污染源已成为当务之急。     

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.     

Characterizing dissolved organic matter and evaluating associated nanofiltration membrane fouling

Natural organic matter (NOM) characteristics were determined for three ground waters exhibiting different water quality conditions. The water quality of the three feed waters collected at various water table depths was characterized by XAD-8/-4 resin adsorption, high performance size exclusion chromatography with ultraviolet and dissolved organic carbon (DOC) detections, and Fourier transform infrared spectroscopy (FTIR) to determine NOM fractionation, molecular weight, and NOM functional groups, respectively. Systematic studies were conducted to identify potential NOM foulants in ground water for nanofiltration (NF) membrane fouling. The results show that the hydrophobic fraction of NOM in all of the samples was significantly high (71-93%) compared to the hydrophilic (1.7-22.6%) and transphilic (5.3-6.6%) fractions. However, insignificant flux-decline (less than 5%) was observed for the highest DOC (36.9 mg l(-1)) and hydrophobic NOM (93%) containing groundwater compared to the other lesser DOC and hydrophobic NOM containing ground waters. This is presumably due to either higher fractions of hydrophilic and transphilic NOM or inorganic interactions that may be major foulants. Based on FTIR, aromatic foulants were observed at 1662 cm(-1) (CO-NH2 or CO conjugated with aromatic rings) for the fouled NF membrane with the relatively low DOC source waters. The contact angle of the clean membrane (52 degrees ) decreased with fouling up to 42-47 degrees for fouled membranes with the various samples.     

Mutagenicity tests with gallic and tannic acid in the Salmonella/mammalian microsome assay

Gallic acid, tannic acid mixture and a purified fraction of tannic acid were evaluated for possible mutagenic activity in three strains of Salmonella typhimurium, TA98, TA100, and TA1535. These chemicals were not mutagenic either before or after activation with rat and woodchuck microsomal and cytosolic enzymes. However, tannic acid mixture and tannic acid fraction both gave a significantly (p = 0.05) dose-related reduction in the number of the revertant colonies, compared to the normal spontaneous revertants with no apparent toxic effects in the background lawn. With an agar diffusion assay, the chemicals exhibited toxic effects at 5000 micrograms/disc.     

Evaluation of chlordimeform and degradation products for mutagenic and DNA‐damaging activity in salmonella typhimurium and escherichia coli

Abstract

The mutagenic activity of chlordimeform and two of its breakdown products, 4‐chloro‐o‐toludine and 4‐chloro‐N‐formyl‐o‐toluidine were determined with five histidine dependent strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA98, TA100) and five tryptophan dependent strains of E. coli WP₂. (WP₂, WP₂uvrA, WP67, CM611, CM571) with and without rat liver microsomal enzymes. 4‐chloro‐o‐toluidine increased the number of the reversions of the S. typhimurium strain TA1535 more than two fold over spontaneous at the concentration of 400 μg/plate.

The results of the DNA repair tests in the Salmonella TA1538/TA1978 and E. coli multirepair deficient systems showed that both breakdown products were active in inducing damage not repaired in at least one repair deficient strain while chlordimeform itself was inactive.     

Leaching properties of some degradation products of alachlor and metolachlor

Once in soil, pesticides undergo degradation processes that give rise to a complex pattern of metabolites. Those presenting a significant percentage of formation, genotoxic and leaching properties may pose a threat to human health associated with the consumption of drinking water. The aim of this study is to assess the hazard potential of some metabolites that may occur in ground water. 2,6-diethylaniline, 2-chloro-2',6'-diethylacetanilide, 2-hydroxy-2',6'-diethylacetanilide, metabolites of alachlor and 2-ethyl-6-methylaniline, metabolite of metolachlor, were chosen for their genotoxic properties. Under laboratory conditions, these metabolites showed DT50 = 1-5 days and Koc = 45-357. Their leaching potential, calculated according to Gustafson, is very low and, therefore, they should not be regarded as contaminants of ground waters. Aged residue leaching studies as well as preliminary studies on well waters seem to confirm these findings.     

Sources and emission of greenhouse gases in Danube Delta lakes

Production of methane and carbon dioxide as well as methane concentrations in surface waters and emissions to the atmosphere were investigated in two flow-through lake complexes (Uzlina–Isac and Puiu–Rosu–Rosulet) in the Danube Delta during post-flood conditions in May and low water level in September 2006. Retained nutrients fuelled primary production and remineralisation of bioavailable organic matter. This led to an observable net release of methane, particularly in the lakes Uzlina, Puiu and Rosu in May. Input from the Danube River, from redbuds and benthic release contributed to CH₄ concentrations in surface waters. In addition to significant river input of CO₂, this trace gas was released via aerobic remineralisation within the water column and in top sediments. Emission patterns of CO₂ widely overlapped with those of CH₄. Generally, greenhouse gas emissions peaked in the lake complex adjacent to the Danube River in May due to strong winds and decreased with increasing hydrological distance from the Danube River. Intense remineralisation of organic matter in the Danube Delta lakes results in a net source of atmospheric greenhouse gases.     

Organochlorine and organophosphorus pesticide residues in the Atoya river basin, Chinandega, Nicaragua

The hydrographic basin of the Atoya river, located in the Department of Chinandega, one of the main cotton producing regions in Nicaragua, is intensively contaminated by pesticides. Samples of river waters and sediments, as well as strategically selected wells have been analyzed to study variations in the concentrations of organochlorine and organophosphorus pesticide residues between the dry season (November-April) and the rainy season (May-June). Generally, higher concentrations of pesticides have been detected in the river waters and sediments in the dry season. DDT, DDD, DDE compounds and toxaphene are the most frequent organochlorine residues found in the water and sediment samples, while endrin, aldrin, dieldrin and lindane are mainly found in the waters of rivers and wells. Organophosphorus compounds were rarely detected. However, residues of ethion, methyl-parathion and ethyl-parathion were found in high concentrations in some river and well water samples. Generally, organochlorine compounds tend to accumulate in the fine grain-size fractions, rich in organic matter, except DDE, which concentrates basically in the coarse grain-size fractions.     

Ocean pollution from land-based sources: East China Sea, China

The environment of East China Sea (ECS) has been faced by huge stresses from anthropogenic activities and population growth in the Yangtze River drainage basin and the areas along the coasts. Improper use of natural resources and short-term economic objectives have resulted in severe environmental degradation in a fairly short time frame and the degradation has now reached a level where the health and well being of the coastal populations are threatened. The main pollutants are inorganic nitrogen, phosphate, oil hydrocarbons, organic matters and heavy metals. Nutrients cause eutrophication of the coastal waters and the estuarine area and very often stimulate the occurrence of red tides. The environmental pollution of Yangtze River basin directly impact on the state of the marine environment in the ECS. The ecosystem stability is maintained by a steady water discharge from the river, that mixes with the marine salty water in the estuary, and the sediment loads from the river that balance ocean erosion in the delta and its adjacent coastal area. The large-scale water transfer and dam constructions in the Yangtze River basin will change this basis. For the ECS the challenge is to reverse the negative processes taking place and to restore ecosystem balance. The main challenge is to integrate socioeconomic and environmental decision making in order to promote sustainable development. A better understanding of the driving forces in society that cause these environmental pressures is required in order to overcome these obstacles. International cooperation may be an important contributor to the progress and in particular provide access to financial, technological, scientific and human resource assistance.     

Sources and emission of greenhouse gases in Danube Delta lakes

Production of methane and carbon dioxide as well as methane concentrations in surface waters and emissions to the atmosphere were investigated in two flow-through lake complexes (Uzlina–Isac and Puiu–Rosu–Rosulet) in the Danube Delta during post-flood conditions in May and low water level in September 2006. Retained nutrients fuelled primary production and remineralisation of bioavailable organic matter. This led to an observable net release of methane, particularly in the lakes Uzlina, Puiu and Rosu in May. Input from the Danube River, from redbuds and benthic release contributed to CH₄ concentrations in surface waters. In addition to significant river input of CO₂, this trace gas was released via aerobic remineralisation within the water column and in top sediments. Emission patterns of CO₂ widely overlapped with those of CH₄. Generally, greenhouse gas emissions peaked in the lake complex adjacent to the Danube River in May due to strong winds and decreased with increasing hydrological distance from the Danube River. Intense remineralisation of organic matter in the Danube Delta lakes results in a net source of atmospheric greenhouse gases.     

The Mutagenic Activity of Particulate Organic Matter Collected with a Dilution Sampler at Coal-Fired Power Plants

The Deep Creek Lake Study of 1983 provided an opportunity to obtain emission samples from coal-fired power plants with a dilution sampler for mutagenicity testing. Stack and ambient samples of particulate matter were collected with a dilution sampler at three coal-fired power plants in West Virginia. Samples were sequentially extracted with cyclohexane (CX), dichloromethane (DCM) and acetone (ACE) and tested for mutagenicity in the Ames Salmonella/microsome assay using TA98 (-S9). For the stack samples, the CX, DCM and ACE fractions constituted 1.0, 0.7 and 98.1 percent of the total extractable organic material (EOM), respectively, compared to 28.5, 7.4 and 64.1 percent for the ambient samples. In contrast, the mutagenic activity of the organic fractions was concentrated in the CX and DCM fractions.

The cyclohexane- and dichloromethane-soluble fractions of the stack samples from all locations exhibited mutagenicity when tested in the plate incorporation assay. No significant response was observed with the acetone fraction. When tested with Kado's modification of the preincubation assay, the acetone-soluble fraction did exhibit mutagenic activity comparable to that of the other fractions when expressed in units of revertants per milligram of particular matter. Chemical analyses of one of the acetone-soluble fractions indicated that half of the mass was sulfuric acid while the remainder consisted of C, H and O. More than 30 peaks were detected in the high pressure liquid chromatogram of this fraction.

Although little mutagenic activity was detected in the polar ACE fraction of the diluted stack emissions samples with this single bioassay, in view of the large mass of this fraction, further investigation of the chemical composition and genotoxic activity of this fraction would be prudent.     

Evaluating the fish safety level of river water and wastewater with a larval medaka assay

A larval medaka (Oryzias latipes) assay was proposed to evaluate the fish safety level of river waters and wastewaters. Organic toxicants were 10-100 times concentrated with adsorption cartridges from 4 l of river water or 1-10 times concentrated from 400 ml of wastewater. Toxicity of these concentrated solutions was determined by exposing 48-72 h post-hatch age larvae for 48 h. The method effectively revealed a variation of the median lethal concentration ratio (LCR50) from 13 to >100 in 125 river water samples, and from <1 to >10 in five typical wastewater samples. Ayase River, which takes water mostly from agricultural or household discharge, showed significantly (P<0.001) lower LCR50 than Sagami River that takes natural water as the source. Safety Levels in both Sagami River and Ayase River were influenced by the irrigation activity, LCR50 at some sites showing a seasonal-specific decrease in winter. Pollution from pulp and paper industries contributed to the low LCR50 in several tributaries of Ayase River. Required little manpower in sampling, pretreatment and testing, the proposed larval medaka assay was proved as an efficient tool for screening those high risk sites for priority management.