Overview of results from the WaterTox intercalibration and environmental testing phase II program: Part 1, statistical analysis of blind sample testing.

There is an urgent need to evaluate the presence of toxicants in waters used for human consumption and to develop strategies to reduce and prevent their contamination. The International Development Research Centre undertook an intercalibration project to develop and validate a battery of bioassays for toxicity testing of water samples. The project was carried out in two phases by research institutions from eight countries that formed the WaterTox network. Results for the first phase were reported in the special September 2000 issue of Environmental Toxicology. Phase II involved toxicity screening tests of environmental and blind samples (chemical solutions of unknown composition to participating laboratories) using the following battery: Daphnia magna, Hydra attenuata, seed root inhibition with Lactuca sativa, and Selenastrum capricornutum. This battery was also used to assess potential toxicity in concentrated (10x) water samples. Results are presented for a set of six blind samples sent to the participating laboratories over a 1-year period. Analyses were performed for each bioassay to evaluate variations among laboratories of responses to negative controls, violations of test quality control criteria, false positive responses induced by sample concentration, and variability within and between labs of responses to toxic samples. Analyses of the data from all bioassays and labs provided comparisons of false positive rates (based on blind negative samples), test sensitivities to a metal or organic toxicant, and interlaboratory test variability. Results indicate that the battery was reliable in detecting toxicity when present. However, some false positives were identified with a concentrated soft-water sample and with the Lactuca and Hydra (sublethal end-point) tests. Probabilities of detecting false positives for individual and combined toxic responses of the four bioassays are presented. Overall, interlaboratory comparisons indicate a good reliability of the battery.

Overview of results from the WaterTox intercalibration and environmental testing phase II program: part 2, ecotoxicological evaluation of drinking water supplies.

Because of rapid population growth, industrial development, and intensified agricultural production increasing amounts of chemicals are being released into the environment, polluting receiving water bodies around the world. Given the potential health risk associated with the presence of toxicants in water sources used for drinking yet the scarcity of available data, there is a need to evaluate these waters and develop strategies to reduce and prevent their contamination. The present study examined the applicability of a battery of simple, inexpensive bioassays in environmental management and the relevance of the test results in establishing the toxicological quality of water sources and drinking water within the framework of the eight-country WaterTox Network, sponsored by the International Development Research Centre, Ottawa, Canada. Seventy-six samples were collected from surface and groundwater sources and seven samples from drinking water treatment plants. Each sample was tested with a core battery of bioassays (Daphnia magna, Hydra attenuata, and Lactuca sativa root inhibition tests) and a limited set of physical and chemical parameters. In addition, three labs included the Selenastrum capricornutum test. When no toxic effects were found with the battery, samples were concentrated 10x using a solid-phase extraction (SPE) procedure. Nonconcentrated natural water samples produced a toxic response in 24% of cases with all three core bioassays. When all bioassays are considered, the percentage of raw samples showing toxicity with at least one bioassay increased to 60%. Of seven treated drinkingwater samples, four showed toxicity with at least one bioassay, raising the possibility that treatment processes in these instances were unable to remove toxic contaminants. The Daphnia magna and Hydra attenuata tests indicated a high level of sensitivity overall. Although only three of the eight countries used S. capricornutum, it proved to be an efficient and reliable bioassay for toxicity assessment.

A modified SOS-Chromotest procedure to test for genotoxicity and cytotoxicity in sediments directly without extraction.

A modified SOS-Chromotest bioassay using a chromogenic pad (pad procedure) was developed to test for genotoxicity in sediments directly without extraction. This test is based on the de novo synthesis of beta-galactosidase enzyme by a genetically-engineered E. coli strain PQ37. In the bioassay, an exponential growth phase antibiotic-containing culture of the test bacterium is introduced into a series of tubes with the first tube containing 0.1 gram of sediment. Serial dilutions are then made and the tubes of sediment plus bacterial culture are incubated at 37 degrees C for four hours, followed by placing a drop of each mixture on a chromogenic pad and additional incubation for 20 hours at 37 degrees C. The solid particulates are then washed off with tap water and positive (genotoxic) activity is noted by the presence of a distinctive blue colour on the pad. The SOS-Chromotest pad procedure may be best used as a relative measure of genotoxicity by comparing results to a reference sample. In addition it can also determine sediment cytotoxicity by comparing samples spiked with a genotoxic standard (i.e., 4-nitroquinoline-N-oxide). Preliminary results suggest that this new bioassay is highly sensitive, consistent and discriminating.

Toxicity of organomercury compounds: bioassay results as a basis for risk assessment.

A former mercury plant, where mercury salts and organomercurials for pesticide use were produced, caused soil contamination in high concentrations. Typical organomercurial products included ethylmercury, phenylmercury, methoxyethylmercury and ethoxyethylmercury compounds. Risk assessment of these sites must be carried out before any major clean-up processes can be planned. A sensitive speciation technique for the various organomercury species in environmental matrices is a prerequisite for toxicity investigations. In this connection, a high-performance liquid chromatography-atomic fluorescence spectrometry (HPLC-AFS) technique has been developed to differentiate between and determine the presence of eight organomercury compounds in environmental samples. Using this technique, methylmercury, ethylmercury and phenylmercury and some unknown organomercury species were found in soil samples collected from the sites of an old mercury products producing plant. With regard to risk assessment, it is necessary to assess the toxicity of the organomercurials. As different microbial metabolic pathways react differently to mercury and its compounds, batteries of bioassays are, therefore, useful to evaluate the toxicity of pollutants. To describe the toxicity and genotoxicity of MeHg+, MeOEtHg+, EtHg+, EtOEtHg+ and PhHg+, p-tolymercury chloride, nitromersol and Hg2+ six bioassays were used: resazurin reduction method, Spirillum volutans test, nematode toxicity assay Panagrellus redivivus, Toxi-Chromotest and SOS-Chromotest. A ranking of the toxicity of the organomercurial is shown. The SOS-Chromotest indicated genotoxicity for 5-7 organomercurials.

River evaluation using ecotoxicological and microbiological procedures.

One of the many problems researchers and managers face is to try and interpret data collected from various river studies. There is an underlying awareness of the incompleteness of most data bases due to a variety of problems outside of those concerning sample processing.In this study we have attempted, by using the battery of tests approach (12 tests) and the examination of different types of samples such as water, Milli-Q water extracted sediments, and organic solvent extracted sediments, to explore the implication of spacial and temporal sampling programmes.The results obtained were not unexpected: there were spatial and temporal influences, sometimes so great that it would appear that you were examining samples from different parts of the country. However, data arising from solvent extracted sediments were found to be much less influenced by temporal sampling than by spatial variation. Another interesting and important observation was that sediment composition appeared to have no relationship to toxicant content.In this study, the first field application of the Mutatox test (genotoxicity) was carried out and it was found to be a very responsive test in all three types of samples. This test shows great promise as laboratory studies indicate it is sensitive to many of the chemicals which trigger the Ames test.

Mutatox test: a new test for monitoring environmental genotoxic agents.

In this study, Yamaska River water and Milli-Q water and organically extracted sediment extracts were used to evaluate the sensitivity of a new genotoxicity screening test, the Mutatox test. Also in this study, the samples were tested for acute and chronic toxicity using the following screening test procedures: Microtox, Daphnia magna, Ceriodaphnia reticulata and ATP-TOX Systems. The Mutatox test is based on the use of a dark mutant strain of Photobacterium phosphoreum and is sensitive to chemicals which are (1) DNA damaging agents (2) DNA intercalating agents, (3) DNA synthesis inhibitors and (4) direct mutagens. In this study, the Mutatox test was found to be a simple-to-perform sensitive procedure which added greater scope to the battery of tests approach. Preliminary indications are that this procedure may prove to be one of the more responsive and valuable tests in the 'battery of tests' approach to environmental screening.

The presence of bacterial virus in groundwater and treated drinking water.

Ten raw urban well water samples and twelve water samples collected from distribution lines after the well waters were treated were examined for bacteriological and coliphage/bacteriophage populations. The raw well waters were found to contain <1/100 ml total coliforms and fecal streptococci, but they all contained varying concentrations of coliphage and bacteriophage. The treated waters were all found to have <1 total coliforms and fecal streptococci per 100 ml with the exception of one treated water sample from Community C. However, even though the treated water samples contained free and total chlorine levels varying from 0.05 to 1.5 ppm, they were all found to contain usually greater amounts of coliphage and bacteriophage than the raw well waters.

Assessment of toxicant activity in sediments by the ECHA biocide monitor.

The ECHA Biocide Monitor, a dipstick procedure originally designed to test the effectiveness of in situ biocides, was evaluated as a possible environmental toxicity assessment procedure. The dipstick procedure was applied to 94 sediment extracts which were also tested by three well documented toxicant screening procedures; Microtox, Daphnia magna and Spirillum volutans. In these samples the Daphnia magna test proved to be the most sensitive indication of toxicant activity (63 of 94) with the ECHA Biocide Monitor easily being the second most sensitive procedure with 27 of 94 sediments indicating the presence of toxic substances. Results of the investigation are detailed.

Incidence of coliphage in potable water supplies.

Samples of drinking water from different sources in greater Cairo, Egypt, and bottled drinking water were tested for total coliform, fecal coliform, and coliphage populations. Of the 147 samples tested, 4 samples were positive for both total coliforms and coliphage, 65 samples were negative for total coliforms, fecal coliforms, and coliphage, and 78 samples were positive for coliphage and negative for total coliforms and fecal coliforms. The incidence of coliphage in these potable water supplies reflects the probability of human pathogenic virus survival in these waters also.

Enumeration of Klebsiella spp. in cold water by using MacConkey-inositol-potassium tellurite medium.

MacConkey-inositol-potassium tellurite agar was field tested for its ability to selectively enumerate Klebsiella species from the waters of the Saint John River Basin, which include fresh and marine waters. Water temperature varied from 1 to 6 degrees C during the survey period. Results of the study indicated that 77% of the typical colonies on MacConkey-inositol-potassium tellurite medium were Klebsiella species, but the total Klebsiella population enumerated was greatly underestimated.

Sensitivity of Legionella pneumophila to sunlight in fresh and marine waters.

Studies were carried out to assess the sunlight sensitivity of Legionella pneumophila suspended in fresh and marine waters. Comparison studies on sunlight sensitivity of lake water bacteria, Pseudomonas aeruginosa, Escherichia coli and Streptococcus faecalis, were also undertaken. The effects of full sunlight and polyacrylic-screened sunlight were monitored in the study. Results indicate that L. pneumophila cells are slightly more sensitive to sunlight in seawater than in fresh water. Enumeration of sunlight-stressed bacteria in fresh water was found to be dependent on the medium used, and the following order of sensitivity to sunlight, from least to most sensitive, was noted: natural lake water bacteria, L. pneumophila, P. aeruginosa, E. coli, and S. faecalis.

Incidence of Legionella organisms in selected Ontario (Canada) cities.

The distribution of Legionella pneumophila in water inside buildings was examined by means of culture methods. Cooling tower sumps and condenser valves harboured the organism at the highest frequency and in the highest concentrations. The bacterium was also frequently isolated from potable water systems, including hot and cold mixed taps, drinking water fountains and showers. When water quality parameters were examined, only elevated pH, total particulate nitrogen and alkalinity were correlated with the occurrence of L. pneumophila. Survival of the organism in water was increased at slightly basic pH and lower temperatures. The proliferation of the organism in water within buildings is probably due to a number of interrelated environmental factors that influence its survival and growth.

The fate and impact of oil and oil-dispersant mixtures in freshwater pond ecosystems: introduction.

Oil and oil-dispersant mixtures were added to the surface waters of a series of man-made ponds. The fate of the oil and dispersant (Norman Wells crude and Corexit 9527 respectively) were studied as well as the impact of the added chemicals on the ponds' ecosystems. Elements of the ecosystems studied include bacteria, fungi, phytoplankton, periphyton , proto- and mesozooplankton , zoobenthos and surface insects. In addition a number of water quality parameters were regularly monitored. Comparisons were made between oil-treated and control ponds, as well as oil-dispersant treated and oil and/or control ponds. This paper describes the experimental set up and provides a summary of the findings reported in the following five papers.

Study of long term effects of oil and oil-dispersant mixtures on freshwater microbial populations in man made ponds.

In this paper, the results of a 19 month investigation of microbial communities subjected to the effects of oil and oil plus dispersant additions in man made ponds are reported. Microbial biomass estimations by ATP (adenosine triphosphate) and microscopic procedures using epifluorescence indicated that oil and oil plus dispersants had little or no effect on these parameters, and any effect noted was stimulatory. However, detailed examination of specific populations indicated that oil and oil plus dispersant additions were stimulatory for short periods of time to the populations studied. Seven days after the oil and dispersant additions to the ponds, no mutagenic or toxic activities to bacteria were noted.