A modified Vibrio harveyi mutagenicity assay based on bioluminescence induction

AIMS: Mutagenic pollution of natural environment is currently one of the most serious ecological problems. Therefore, rapid detection of the presence of mutagens is a very important issue. Although many mutagenicity assays have already been described, only a few are suitable for testing samples from natural environment. One of such assays is a microbiological mutagenicity test based on genetically modified Vibrio harveyi strains. The aim of this work was to modify and improve the V. harveyi assay. METHODS AND RESULTS: A series of V. harveyi dark and dim mutants were tested for reversion of their phenotype towards efficient light emission in response to incubation with known mutagens. Luminescence of the A16 strain (luxE mutant) increased significantly after a few hours of such a treatment with various mutagenic agents, revealing a dose-response correlation. Sensitivity of the assay has been determined for different mutagens. CONCLUSIONS: The luminescence-based V. harveyi mutagenicity assay is rapid, sensitive and reveals a dose-response correlation for various mutagens. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay developed in this study is a potentially useful tool in studies on mutagenic pollution of environment, especially marine water.     

Direct addition of cultures of tester bacteria into semi-permeable membrane devices (SPMDs) as a modified procedure for preliminary detection of mutagenic pollution of the marine environment by use of microbiological mutagenicity assays

Mutagenic pollution of the natural environment, including marine waters, is a very serious ecological problem. However, since chemical mutagens usually occur and act at low concentrations, their detection and identification is technically difficult, laborious and time-consuming. Therefore, preliminary detection of mutagenic pollution is commonly based on biological mutagenicity assays. On the other hand, triolein-containing semi-permeable membrane devices (SPMDs) provide a method for concentration of hydrophobic organic contaminants, including a large fraction of the mutagens. Combinations of SPMDs with microbiological toxicity and mutagenicity assays have already been described, but only SPMD-derived extracts, prepared with various organic solvents, were tested in such a way to date. We found that the presence of these solvents could interfere with the Vibrio harveyi bioluminescence-based mutagenicity assay. Moreover, preparation of the extracts from SPMD takes usually at least 48h. Here, we propose a modified procedure, based on direct addition of tester bacteria cultures into SPMD. We found that this procedure is significantly (at least two times) more rapid and several times more sensitive than that based on testing the extracts. This optimization is presented in this report. Moreover, we have performed preliminary studies on samples of marine waters. Positive results (i.e. detection of mutagenic activity) were obtained when test samples came from a region known to be highly contaminated by industrial pollution, while negative results were observed in the case of samples from a region supposed to be of low risk for mutagenic pollution.     

Direct addition of cultures of tester bacteria into semi-permeable membrane devices (SPMDs) as a modified procedure for preliminary detection of mutagenic pollution of the marine environment by use of microbiological mutagenicity assays

Mutagenic pollution of the natural environment, including marine waters, is a very serious ecological problem. However, since chemical mutagens usually occur and act at low concentrations, their detection and identification is technically difficult, laborious and time-consuming. Therefore, preliminary detection of mutagenic pollution is commonly based on biological mutagenicity assays. On the other hand, triolein-containing semi-permeable membrane devices (SPMDs) provide a method for concentration of hydrophobic organic contaminants, including a large fraction of the mutagens. Combinations of SPMDs with microbiological toxicity and mutagenicity assays have already been described, but only SPMD-derived extracts, prepared with various organic solvents, were tested in such a way to date. We found that the presence of these solvents could interfere with the Vibrio harveyi bioluminescence-based mutagenicity assay. Moreover, preparation of the extracts from SPMD takes usually at least 48 h. Here, we propose a modified procedure, based on direct addition of tester bacteria cultures into SPMD. We found that this procedure is significantly (at least two times) more rapid and several times more sensitive than that based on testing the extracts. This optimization is presented in this report. Moreover, we have performed preliminary studies on samples of marine waters. Positive results (i.e. detection of mutagenic activity) were obtained when test samples came from a region known to be highly contaminated by industrial pollution, while negative results were observed in the case of samples from a region supposed to be of low risk for mutagenic pollution.     

The use of marine bacteria in mutagenicity assays

Mutagenic pollution of environment is a global and important problem. This includes marine environment. Although many mutagenicity assays have been developed, there are specific problems with testing marine water and sediments for mutagenic contamination. One of them is the fact that most of genetically modified strains used in commonly available microbiological mutagenicity assays, like Escherichia coli or Salmonella, survive relatively poorly in marine waters, especially those of higher salinity. Thus, alternative assays have been developed, in which bacteria occurring naturally in marine habitats are employed. These assays, reviewed in this article, appear to be useful in testing not only marine samples but also can be used in other approaches, which involve detection and estimation of the amount of mutagenic compounds.     

Assessment of the Mutagenicity of Sediments from Yangtze River Estuary Using Salmonella Typhimurium/Microsome Assay

Sediments in estuaries are of important environmental concern because they may act as pollution sinks and sources to the overlying water body. These sediments can be accumulated by benthic organisms. This study assessed the mutagenic potential of sediment extracts from the Yangtze River estuary by using the Ames fluctuation assay with the Salmonella typhimurium his (−) strain TA98 (frameshift mutagen indicator) and TA100 (baseshift mutagen indicator). Most of the sediment samples were mutagenic to the strain TA98, regardless of the presence or absence of exogenous metabolic activation (S9 induction by β-naphthoflavone/phenobarbital). However, none of the samples were mutagenic to the strain TA100. Thus, the mutagenicity pattern was mainly frameshift mutation, and the responsible toxicants were both direct (without S9 mix) and indirect (with S9 mix) mutagens. The mutagenicity of the sediment extracts increased when S9 was added. Chemical analysis showed a poor correlation between the content of priority polycyclic aromatic hydrocarbons and the detected mutagenicity in each sample. The concept of effect-directed analysis was used to analyze possible compounds responsible for the detected mutagenic effects. With regard to the mutagenicity of sediment fractions, non-polar compounds as well as weakly and moderately polar compounds played a main role. Further investigations should be conducted to identify the responsible components.     

Detection of mutagenic pollution of natural environment using microbiological assays

One of the most important and serious ecological problems is mutagenic pollution of the natural environment. Therefore, detection of mutagenic compounds in samples taken from natural habitats is of special interest. Microbiological mutagenicity tests seem to be very useful tools for such detection. In this review article, a general view on the tests employing genetically modified bacterial strains designed for detection of low concentrations of mutagenic compounds is presented. Moreover, a comparison of advantages and disadvantages of selected assays, developed early on and more recently, and features of these assays are discussed. It appears that none of the currently available mutagenicity tests is perfect or optimal for all purposes. Thus, a choice for the particular assay must depend on the nature of studies and specific tasks of the experiments to be performed.     

Use of a real time PCR assay for detection of the ctxA gene of Vibrio cholerae in an environmental survey of Mobile Bay

Toxigenic Vibrio cholerae, the etiological agent of cholera, is a natural inhabitant of the marine environment and causes severe diarrheal disease affecting thousands of people each year in developing countries. It is the subject of extensive testing of shrimp produced and exported from these countries. We report the development of a real time PCR (qPCR) assay to detect the gene encoding cholera toxin, ctxA, found in toxigenic V. cholerae strains. This assay was tested against DNA isolated from soil samples collected from diverse locations in the US, a panel of eukaryotic DNA from various sources, and prokaryotic DNA from closely related and unrelated bacterial sources. Only Vibrio strains known to contain ctxA generated a fluorescent signal with the 5' nuclease probe targeting the ctxA gene, thus confirming the specificity of the assay. In addition, the assay was quantitative in pure culture across a six-log dynamic range down to <10 CFU per reaction. To test the robustness of this assay, oysters, aquatic sediments, and seawaters from Mobile Bay, AL, were analyzed by qPCR and traditional culture methods. The assay was applied to overnight alkaline peptone water enrichments of these matrices after boiling the enrichments for 10 min. Toxigenic V. cholerae strains were not detected by either qPCR or conventional methods in the 16 environmental samples examined. A novel exogenous internal amplification control developed by us to prevent false negatives identified the samples that were inhibitory to the PCR. This assay, with the incorporated internal control, provides a highly specific, sensitive, and rapid detection method for the detection of toxigenic strains of V. cholerae.     

Optimisation of the microbiological mutagenicity assay based on genetically modified Vibrio harveyi strains

Recently, we have developed a novel assay designed for detection of mutagenic pollution of the marine environment. This assay is based on the use of a series of genetically modified strains (named BB7, BB7M, BB7X and BB7XM) of a marine bacterium Vibrio harveyi. Sensitivity of the V. harveyi mutagenicity assay was found to be similar to, or even somewhat higher than, that of the commonly used Ames test. Subsequent studies indicated that this assay may be useful in assessment of mutagenic contamination of the marine environment. Nevertheless, we assumed that improvement of this assay is still possible, and thus we aimed to optimise its procedures. Here we present our research on the optimisation of the V. harveyi mutagenicity assay, which indicated that different tester strains used in this assay give the best results depending upon the experimental conditions employed. Incubation of bacteria in a buffer, rather than in a nutrient broth, containing a mutagen, increased the efficiency of the assay with BB7 and BB7M strains, but had a deleterious effect in the case of BB7X and BB7XM. The latter couple of strains revealed higher mutagenicity in the plate assay, as compared to the liquid medium assay. However, the opposite effect was observed for BB7 and BB7M. Low-dose (1 J m(-2)) UV irradiation, as well as 30 min incubation in 0.1 M CaCl2, had no significant effect on the efficiency of the assay when using BB7 and BB7M, whereas the number of mutagen-induced mutants of BB7X and BB7XM strains increased about two times under these conditions. Our previous experiments indicated that various tester strains revealed different sensitivity to particular mutagens. Thus, a series of strains should be used in the assay. Results presented in this report show that different conditions should be used for two pairs of the tester strains: BB7 and BB7M, and BB7X and BB7XM.     

Fecal Pollution in Coastal Marine Sediments from a Semi-Enclosed Deep Embayment Subjected to Anthropogenic Activities: An Issue to Be Considered in Environmental Quality Management Frameworks Development

Sewage discharge is a major source of pollution in marine environments. Urban wastewaters can directly enter marine environments carrying pathogen organisms, organic loads, and nutrients. Because marine sediments can act as the ultimate fate of a wide range of pollutants, environmental quality assessment in this compartment can help to identify pollution problems in coastal areas. In the present study, characterization of surficial marine sediments allowed assessment of fecal pollution in a semi-enclosed deep embayment that is subjected to anthropogenic activities. Physicochemical parameters and fecal indicators presented a great spatial heterogeneity. Fecal coliform and Clostridium perfringens showed accumulation in an extensive area, not only in proximity to sewage discharge points, but also in sediments at 100 meters depth. Results included herein demonstrated that, in coastal areas, urban wastewater discharge can affect the whole ecosystem through accumulation of fecal matter in bottom sediments. Application of multivariate techniques provided useful information with applicability for management of coastal areas in such complex systems. Environmental implications of wastewater discharge in coastal areas indicate the need to implement and include sediment quality control strategies in legislative frameworks.     

Investigation of bacterial diversity in Brazilian tropical estuarine sediments reveals high actinobacterial diversity

Phylogenetic and statistical analyses of 16S rRNA gene libraries were used for the investigation of actinobacterial communities present in two tropical estuarine sediments (Santos-São Vicente estuary, Brazil). The libraries were constructed from samples collected at the brackish end of the estuary, highly hydrocarbon-contaminated, and at the marine end, uncontaminated. Clones from the marine end of the estuary were all related to sequences from non-cultured Actinobacteria and unidentified bacteria recovered from a wide range of environmental samples, whereas clones from the brackish end were mainly related to sequences from cultured Actinobacteria. Statistical analyses showed that the community recovered from the hydrocarbon-contaminated sediment sample, at the brackish end, was less diverse than the uncontaminated one, at the marine end, and that the communities from the two libraries were differently structured, suggesting that these may have not originated from the same community. The recognition of the spatial pattern of actinobacterial distribution in a natural environment is a first step towards understanding the way these communities are organized, providing valuable data for further investigations of their taxonomic and functional diversity.     

沈阳市城郊表层土壤有机污染评价

城市土壤是保护城市环境的一个重要生态屏障.随着有机污染物的“致癌、致畸、致突变”特性被人们所认知,土壤有机污染也逐渐受到人们的重视.在我国目前缺乏相关环境质量标准的背景下,尝试采用因子分析评价法,对沈阳城郊表层土壤有机污染物进行识别,并对土壤有机污染程度进行定量评价与分级.结果表明,苯并(a)芘、滴滴涕、六氯苯和六六六对土壤污染负荷的贡献率最高,是主要的有机污染因子;利用因子分析评价法,34个城郊表层土壤采样点的有机污染评价综合评分在O-224.62之间,以较轻度污染和中度污染为主,评价结果及分级与实际情况基本相符.在此基础上,分析了各类有机污染物的污染来源,对防治城市土壤污染,进而保障居民的食品安全和饮水安全具有重要意义.     

Mutagenic activity as a parameter to assess ambient air quality for protection of the environment and human health

Atmospheric pollution has significant effects on maintaining the integrity of ecosystems and on the population's quality of life. Epidemiological studies have clearly associated related health problems, especially respiratory diseases, with exposure to air pollution. Organic compounds adsorbed to the airborne particulate matter are mutagenic in the Salmonella/microsome assay, and a considerable number of them are known to be carcinogenic to rodents. Studies performed at four sites within the urban area of Porto Alegre, capital of the state of Rio Grande do Sul, identified higher mutagenic activity at the sites with heavier vehicle traffic in assays without and with metabolic activation. The responses varied at different seasons of the year, and the highest revertants per cubic meter (rev/m(3)) values were observed in spring for moderately polar compounds, and in summer for non-polar ones. A pilot study was also performed in the region under the influence of a industrial petrochemical area. Most of the sites studied within the industrial area, as compared to others sampled in the nearby environment, presented higher levels of mutagenic activity independent of total suspended particulates (TSP) concentration in the sample. In the urban and industrial regions, the observed mutagenic activities were strongly associated with the presence of polycyclic aromatic hydrocarbons (PAHs). The responses observed in the TA98NR and TA98/1,8-DNP(6) strains suggest the activity of nitrocompounds in both studies. The Salmonella/microsome assay is a sensitive method to define areas contaminated by these compounds, even in samples with TSP values that are consistent with the legal environmental quality standards.     

Immunofluorescent Assay for the Marine Ammonium-Oxidizing Bacterium Nitrosococcus oceanus

Nitrification is one of the important microbiological transformations of nitrogen in the ocean. Traditional enrichment-culture methods for enumerating the autotrophic bacteria which oxidize ammonium to nitrite are very time consuming (months) and are believed to seriously underestimate natural abundances. A fluorescent-antibody assay for a marine ammonium-oxidizing bacterium was developed to provide a rapid and direct means of identifying these microorganisms. Antibodies to Nitrosococcus oceanus were prepared and tested against pure cultures of marine, freshwater, and soil ammonium oxidizers and against bacteria from natural seawater samples. Cell counts of culture samples determined by the fluorescent-antibody assay agreed with hemacytometer and acridine orange counts. Our results demonstrated that the immunofluorescent assay is a powerful tool for the detection of Nitrosococcus in the marine environment.     

溢油事故中渔业资源损失的数值模拟评估模式

海洋溢油污染不仅关系到天然渔业资源、海鸟等生物、海域环境、海岸线生态的破坏,而且对渔业、捕捞业、旅游业都会造成巨大损失,甚至会直接或间接地危害人类的健康。短期来看,一方面石油、燃料油等进入海洋后,对海洋生物资源造成影响;另一方面会危害附近海区的海洋环境,侵害海洋生物以及海鸟赖以生存和栖息的环境。长期来看,持续的海洋污染会导致的生态环境失衡,海洋的生产力也随之下降。溢油事故中的渔业资源损失评估作为追究污染事故责任,尽快恢复海域资源与环境的重要一环需要不断改进创新。为了定量确定海洋溢油事故发生后渔业资源的损失程度,将传统的评估模式与现代科学技术相结合,通过海洋动力学、流体力学、海洋生物学、环境化学等多个学科交叉融合,将流场风场模型、溢油模型、海域调查监测、卫星遥感技术、毒性效应和渔业资源的损失评估方法相结合,形成一种渔业资源损失评估的数值模拟评估模式,以完善溢油事故中渔业资源损失评估体系。在溢油事故现场监测数据的基础上,运用数学计算理论选择相对应的溢油模型,结合具体溢油事故案例的潮流数据和风场数据,模拟海上溢油污染的时空分布情况。采用卫星遥感技术根据不同油品在海水中不同的亮度表现,处理得到溢油油膜信息,与模拟得到的油膜信息进行比对验证,并对模型进行修正,通过模拟得出污染海域油浓度分布与溢油污染范围信息,结合溢油污染对不同海洋生物的毒性效应,得到渔业资源的损失程度,为溢油事故的渔业资源损失评估提供一种思路,为溢油事故发生后的损失评估和事故处理起到一定的参考辅助作用。     

An improved electroelution method for separation of DNA from humic substances in marine sediment DNA extracts

We present a method for the rapid and simple extraction of DNA from marine sediments using electroelution. It effectively separates DNA from compounds, including humic substances, that interfere with subsequent DNA quantification and amplification. After extraction of the DNA from the sediment into an aqueous solution, the crude sample is encased in 2% agarose gel and exposed to an electrical current, which draws the DNA out of the gel into a centrifugal filter vial. After electroelution, the sample is centrifuged to remove contaminants ≤100 000 Da. Recovery of DNA using this method is quantitative and does not discriminate on the basis of size, as determined using DNA standards and DNA extracts from environmental samples. Amplification of DNA is considerably improved due to removal of PCR inhibitors. For Archaea, only these purified extracts yielded PCR products. This method allows for the use of relatively large volumes of sediment and is particularly useful for sediments containing low biomass such as deeply buried marine sediments. It works with both organic-rich and -poor sediment, as well as with sediment where calcium carbonate is abundant and sediment where it is limited; consequently, adjustment of protocols is unnecessary for samples with very different organic and mineral contents.     

Genotoxic, carcinogenic, and teratogenic hazards in the marine environment, with special reference to the Mediterranean Sea.

Genotoxic, carcinogenic, and teratogenic hazards arising out of pollution in the marine environment are discussed in this article, with special reference to the situation in the Mediterranean area. A number of chemical compounds or complex mixtures relevant to marine pollution, either natural or of anthropogenic origin, are tentatively listed, along with protective factors which may play a counteracting role in the same environment. Harmful substances tend to undergo interactions and transformations in seawater, sediments, and marine biota, due to physical, chemical, microbial, or light-mediated mechanisms. Bioaccumulation phenomena in marine organisms may result from food-chain biomagnification processes or from concentration of pollutants by filter feeders. A variety of sources can account for marine pollution by genotoxic, carcinogenic, and teratogenic compounds, but there is a relative paucity of analytical data concerning the Mediterranean. Metabolic transformations of xenobiotics occur in all marine organisms, the biochemical mechanisms in fish being comparable to those which have been extensively investigated in mammals. Induction of metabolic pathways, and especially of the mixed-function oxygenase system, represents the earliest warning signal of exposure to pollutants. Occurrence of neoplastic diseases is documented by experimental and field studies in marine vertebrates as well as in invertebrates. The association with local pollution phenomena has been recognized in several studies, but other etiopathogenetic factors may be also involved, and in some cases tumors have been reported to be unrelated to chemical pollution. Genotoxic agents have been detected by means of suitable techniques in seawater, sediments, and marine organisms. Several studies have investigated the presence of carcinogen-DNA adducts, DNA damage and repair processes, and cytogenetic alterations, such as chromosomal aberrations, sister-chromatid exchanges, and micronuclei, in tissues of marine organisms. However, monitoring of these end-points under field conditions encounters some limitations and problems. Even more fragmentary is the information on teratogenic effects in marine organisms, although interesting test systems have been set up. On the whole, a quite extensive database on all these toxicological issues is already available in the literature, but further studies are warranted for an adequate assessment of genotoxic, carcinogenic, and teratogenic hazards, and possibly counteracting factors in the marine environment, and specifically in the Mediterranean Sea.     

Mutagenicity studies on complex environmental mixtures: selection of solvent system for extraction.

The mutagenic activities in the Salmonella/microsome assay of dichloromethane (DCM) and acetone extracts of complex environmental mixtures were compared. The particulate samples used in the IPCS collaborative study were Soxhlet-extracted twice with DCM followed by a third extraction with acetone. Compared with the mutagenic activity of the first extract, the third (acetone) extract of the urban particulate matter showed a relatively high mutagenic activity. In contrast to this the third extract of the diesel particulate matter contributed very little additional mutagenic activity. Furthermore, 10 filter samples of air particulates from a suburban airport area were collected for comparison of the extraction efficiency of DCM and acetone. Each sample was divided into two samples of identical size followed by extraction with acetone and DCM, respectively. No clear difference in the mutagenic activity of these extracts was observed in strains TA98 and TA98NR. It is concluded that for ambient air particulates (but not emission samples) acetone may extract some mutagenic compounds which are not extracted by DCM. The amount of these additional extractable compounds seems to depend on the composition of the sample. As DCM extracts are better suited for further fractionation and chemical analysis DCM is considered to be the best choice for a general solvent system for extraction of complex environmental mixtures.     

Highly Sensitive Quantitative PCR for the Detection and Differentiation of Pseudogymnoascus destructans and Other Pseudogymnoascus Species

White-nose syndrome is a fungal disease that has decimated bat populations across eastern North America. Identification of the etiologic agent, Pseudogymnoascus destructans (formerly Geomyces destructans), in environmental samples is essential to proposed management plans. A major challenge is the presence of closely related species, which are ubiquitous in many soils and cave sediments and often present in high abundance. We present a dual-probe real-time quantitative PCR assay capable of detecting and differentiating P. destructans from closely related fungi in environmental samples from North America. The assay, based on a single nucleotide polymorphism (SNP) specific to P. destructans, is capable of rapid low-level detection from various sampling media, including sediment, fecal samples, wing biopsy specimens, and skin swabs. This method is a highly sensitive, high-throughput method for identifying P. destructans, other Pseudogymnoascus spp., and Geomyces spp. in the environment, providing a fundamental component of research and risk assessment for addressing this disease, as well as other ecological and mycological work on related fungi.     

Multivariate statistical approach to the temporal and spatial patterns of selected bioindicators observed in the North Sea during the years 1995–1997

A comprehensive database, containing biological and chemical information, collected in the framework of the bilateral interdisciplinary MARS project (”biological indicators of natural and man-made changes in marine and coastal waters”) during the years 1995–1997 in the coastal environment of the North Sea, was subjected to a multivariate statistical evaluation. The MARS project was designated to combine a variety of approaches and to develop a set of methods for the employment of biological indicators in pollution monitoring and environmental quality assessment. In total, nine ship cruises to four coastal sampling sites were conducted; 765 fish and 384 mussel samples were analysed for biological and chemical parameters. Additional information on the chemical background at the sampling sites was derived from sediment samples, collected at each of the four sampling sites. Based on the available chemical data in sediments and black mussel (Mytilus edulis) a pollution gradient between the selected sites, was established. The chemical body burden of flounder (Platichthys flesus) from these sites, though, did not reflect this gradient equally clear. In contrast, the biological information derived from measurements in fish samples displayed significant a regional as well as a temporal pattern. A multivariate bioindicator data matrix was evaluated employing a factor analysis model to identify relations between selected biological indicators, and to improve the understanding of a regional and temporal component in the parameter response. In a second approach, applying the k-means algorithm on the data matrix, two significantly different clusters of samples, characterised by the current health status of the fish, were extracted. Using this classification a temporal, and in the second order, a less pronounced spatial effect was evident. In particular, during July 1996, a clear sign of deteriorating environmental conditions was extracted from the biological data matrix. Received: 20 June 1999 / Received in revised form: 8 November 1999 / Accepted: 8 November 1999     

The Salmonella mutagenicity assay in a surface water quality monitoring program based on a 20-year survey

Since 1979, the Environmental Agency of S?o Paulo State in Brazil, CETESB, has been using the Salmonella mutagenicity assay to assess the quality of natural waters. This paper is a compilation of data obtained during the last 20 years from more than a thousand samples. Potencies up to 30,000 revertants/l were observed in 137 positive samples. The Salmonella typhimurium strain TA98 was more sensitive than TA100; 79% of the mutagenicity was detected by this strain, regardless of the presence of S9-mix. A classification of the mutagenic response was proposed to facilitate in the dissemination of the information to the public. The classification was low, moderate, high and extreme for samples with mutagenic potency (revertants/l equivalent) of < 500, 500-2500, 2500-5000 and > 5000, respectively. As a result of this effort to standardize methodologies, compile and classify the mutagenic effect of water pollution, in 1998, the Salmonella mutagenicity assay was officially and systematically included in the S?o Paulo State Water Quality Monitoring Program. This assay has proven to be a useful tool in the identification of important pollution sources. Correction and prevention actions in Water Pollution Control Programs were generated as a result.