Comparison of the Ceriodaphnia dubia and MicrotoxR inhibition tests for toxicity assessment of industrial and municipal wastewaters

Toxicity tests on effluents from industrial production facilities, municipal and industrial wastewater treatment plants, and stormwater runoff were conducted with the freshwater invertebrate, Ceriodaphnia dubia, and the marine luminescent bacterium, Vibrio fischeri in the Microtox^R test system (Microtox is a registered trademark of AZUR Environmental, Carlsbad, CA.). Percent mortalities of C. dubia in whole effluent, generated in 24- and 48-h exposure periods during the conductance of static-renewal acute and chronic tests were compared with percent reductions in light output by V. fischeri after 15-min exposure periods in the Microtox Inhibition test. A total of 16 effluent and stormwater samples from seven sources were used in tests conducted over a 3-month period. Results of the Microtox Inhibition tests correctly predicted the results of C. dubia tests for all eight nontoxic samples after both 24- and 48-h exposure periods. Of three samples that were toxic to C. dubia within 24 h, the Microtox test also detected toxicity in two of those samples. Results from tests on the remaining five samples showed that while the Microtox Inhibition test indicated the presence of toxic components after 15 min exposure, C. dubia required exposure to potentially toxic samples for 48 h before producing a toxic response. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 375–382, 1999     

Use of four microbial tests to assess the ecotoxicological hazard of contaminated sediments

Three single-species bioassays (Microtox, Selenastrum capricornutum, and Panagrellus redivivus) and a test using microbial communities developed on artificial substrates were used in a series of in situ and laboratory tests evaluating the ecotoxicological hazard of contaminated sediments at two sites on Lake Michigan: Waukegan (Illinois) Harbor and The Chicago Area Confined Disposal Facility Study. In the single-species tests, exposure to elutriates of contaminated sediments significantly inhibited bacterial luminescence, algal photosynthesis, and nematode survival and growth at polluted stations, while elutriates from control stations did not. The battery of three tests is a promising screening tool for in-place pollutants. Protozoan species richness and protozoan phototroph abundance were inhibited by elutriates from contaminated sites, but the abundance of heterotrophic protozoans was enhanced by sediment elutriates from some stations. Microbial community photosynthesis was significantly inhibited by most sediment elutriates, while community respiration was often stimulated; thus, functional responses paralleled the structural changes. Overall, the results of the microbial community tests were consistent with expected patterns of toxicity at the two sites on Lake Michigan. In general, single-species test results agreed with the community bioassays. Although community tests may be more realistic than single-species bioassays in predicting the impact of sediment contamination on actual ecosystems, caution must be exercised in interpreting the results.     

Toxicity analysis of leachates from hazardous wastes via microtox and daphnia magna

One of the most common ways of surface and ground water contamination from hazardous wastes is through its leachates. Toxicity is a meaningful parameter allowing an integrated evaluation of the potential danger of leachates. Two bioassays were considered for measuring toxicity: Microtox and Daphnia magna. The toxicity was measured on leachates obtained, using different leaching procedures, from wastes of a pesticide manufacturing industry and sludge from electroplating wastewater. Twenty-two tests were carried out, measuring IC50 for D. magna and EC50 for Microtox. Both bioassays were compared using three toxicity criteria. The first criterium is classification on toxic/non-toxic at 25 and 50% leachate concentration. The second criterium is classification on percent ranks, and the third on log ranks. Considering these criteria, the agreement between both bioassays is within a 75–85%. It is shown that both, Microtox and D. magna assays could be used as toxicity indicators for the wastes considered. The sensitivity of the bioassays is different depending on leachate composition. In all samples, the leachate concentration of chemicals was measured.     

Use of Microtox® and Ceriodaphnia bioassays in wastewater fractionation

Collection system and nonchlorinated secondary effluent samples from a large municipal wastewater system were fractionated using a scheme that included filration, EDTA treatment, C₁₈ solid-phase extraction columns, and air stripping. Microtox required less time than Ceriodaphnia dubia bioassay for determining the toxicity of the numerous test samples generated by the fractionation procedure. Its usefulness was limited to collection system samples, however. Secondary effluent samples, which caused significant mortality of C. dubia, were nontoxic to Microtox. Diazinon was tentatively identified as one of the causative toxicants present. Its LC₅₀ to C. dubia (0.5 μg/L) is within the range of concentrations detected (0.1–0.6 μg/L), whereas the EC₅₀ of diazinon to Microtox is much higher (> 18,000 μg/L).     

Toxicity assessment of industrial effluents from S. Paulo state,Brazil, using short-term microbial assays

Microorganisms have demonstrated several attributes that make them attractive for use in quick screening of effluents and chemicals for toxicity. The aim of this study was to evaluate the acute toxicity and mutagenicity of industrial effluents from São Paulo State using short-term microbial bioassays. Samples of industrial effluents and receiving waters were analyzed for acute toxicity by the Microtox system, a motility test using Spirillum volutans, growth inhibition of Pseudomonas fluorescens, and dehydrogenase assay; for mutagenicity, these samples were analyzed by Salmonella typhimurium (Ames test), Escherichia coli WP₂, and Saccharomyces cerevisiae reversion mutation assays. Among the acute toxicity assays carried out in this study, the Microtox and S. volutans tests showed good sensitivity and general good agreement with the Daphnia similis assay, which demonstrates that these tests are potentially useful as toxicity indicators for the industrial effluents and receiving waters considered. In relation to mutagenicity assays, good results were obtained with the three methods tested. The detection of mutagens in the industrial effluents considered indicates that some constituents of these waste waters discharged in receiving waters can cause adverse biological effects and could be deleterious from a public health standpoint. The data of this research emphasize the importance of acute toxicity and mutagenicity assays as supplementary approaches for a rapid and efficient action in water pollution control, and for evaluation of potential toxic chemical effects.     

Simple two-step sediment extraction procedure for use in genotoxicity and toxicity bioassays

Three simple sediment extraction procedures, using Milli-Q water, DMSO, and methanol, were applied to 16 sediment samples collected from the Thames River, Ontario, Canada. The three extracts were tested for the presence of toxicity and genotoxicity using the Microtox,® Toxi-Chromotest,® and Mutatox® (with and without S9) procedures. Results of the study indicated that the Mutatox genotoxicity screening test was an extremely sensitive procedure responding to chemicals in all three types of extracts. Methanol was found to be more efficient than DMSO in extracting toxic and genotoxic chemicals from these sediments. Procedural details and results are discussed.     

Genotoxicity of some wastewaters in India

Heavy metal concentrations in wastewaters from the industrial estate of Aligarh city (U.P.) India were determined. The analysis of test samples revealed significantly higher levels of Fe, Zn, Cu, Cr, and Ni compared with the pure domestic samples. Raw wastewaters were screened for mutagenic potential using the Ames Salmonella/microsomal test. Mutagenic activity was observed with industrial as well as domestic waste samples. Salmonella typhimurium strains TA102 and TA104 were the most sensitive both in the absence and presence of S9 fraction. A significant decrease in the survival of DNA repair defective Escherichia coli mutants recA, lex A, and polA was observed as compared to their wild-type counterparts in the presence of wastewater. © by John Wiley & Sons, Inc.     

Microtox assay for assessment of marine pollution in industrial discharge zone of Kuwait

Twelve major industries in the Shuaiba Industrial Area of Kuwait generate about 30,000 m³/d wastewater, which is discharged to the sea either directly or after treatment. To assess the quality of the Shuaiba bay area, water samples were collected twice a week for six months from seven points of discharge, each representing a source from a major industry. Samples were analyzed for physicochemical characteristics and toxicity using the Microtox assay. Chemical analysis revealed that the contamination of oil was higher at sites 5 and 6 while four heavy metals were evenly distributed at different sites. The Microtox assay indicated that 31 samples out of 378 samples exerted acute to medium levels of toxicity, which were mostly from sites 6 and 7. The episodes of toxicity were greater in the summer months compared to winter months, whereas the pattern of chemical contaminants was not influenced by the seasonal change. © 1997 by John Wiley & Sons, Inc. Environ Toxicol Water Qual 12: 109–115, 1997     

Preliminary comparison of sediment extraction procedures and exchange solvents for hydrophobic compounds based on inhibition of bioluminescence

Two procedures for extracting hydrophobic contaminants from sediment samples collected from various sites in Florida were compared. The sediments, which varied in nature and degree of chemical contamination, were extracted with dichloromethane by shaking (24 h) and sonication (15 min). The dichloromethane extracts obtained were solvent exchanged into either methanol or dimethyl sulfoxide (DMSO). The efficiency of each procedure was evaluated based on the response of Photobacterium phosphoreum in the Microtox assay to the solvent extracts. The 24-h shaking procedure produced significantly higher toxicity than the 15-min sonication procedure for 8 of the 10 sediment samples tested. Comparisons of methanol and DMSO as exchange solvents revealed that there was generally no significant difference between these solvents in terms of toxicity.     

Detecting genotoxic activity in industrial effluents using the SOS Chromotest microplate assay

The SOS Chromotest, a cost-effective short-term bacterial genotoxicity screening assay, was performed to appraise its capacity for detecting the presence of soluble genotoxic activity in industrial effluent samples (organic and inorganic chemical plants, metallurgical plants, pulp and paper mills, municipal wastewater treatment plants). An optimized methodology, based on criteria taking into account β-galactosidase activity (the indicator of SOS gene induction), alkaline phosphatase activity (the indicator of cytotoxicity), and two genotoxicity measurement end points (minimum genotoxic concentration and maximum induction factor), was employed to generate reliable results that overcame the potential interferences inherent to complex wastewaters. Of 48 effluent samples tested, 37 (77%) elicited a significant induction of the Escherichia coli PQ37 SOS response. Effluents from inorganic chemical plants and pulp and paper mills displayed the most potent responses, with and without metabolizing enzymes (S9 mix). In general, chemical data available for some wastewaters supported SOS Chromotest positive responses. The genotoxic activity of whole effluents subjected to a 5-day aeration treatment was as high as that of native (unaerated) samples, suggesting that soluble genotoxicants are relatively recalcitrant to oxidation, although reductions in genotoxic activity did occur. This study indicates that the SOS Chromotest is sufficiently sensitive to screen for the presence of soluble DNA-damaging agents in a wide variety of unconcentrated wastewater samples. © 1996 by John Wiley & Sons, Inc.     

Comparative assessment of the specificity of the brine shrimp and microtox assays to hepatotoxic (microcystin-LR-containing) cyanobacteria

Specific, straightforward, and rapid procedures are required for the detection, identification, and quantification of the potent low molecular weight toxins that are produced by blooms and scums of cyanobacteria (blue-green algae) in waterbodies. Use of the Microtox bioluminescence assay and the brine shrimp (Artemia salina) has been advocated for the initial screening of cyanobacterial blooms for microcystin hepatotoxins. Inhibition of bacterial luminescence in the Microtox assay and brine shrimp mortality were determined with microcystin-containing and nonmicrocystin-containing cyanobacteria. Extraction and fractionation of test samples was undertaken to select and isolate microcystincontaining fractions and reduce interference from other fractions. Maximal inhibition of bacterial luminescence in the Microtox assay occurred with fractions from Microcystis strains and an Anabaena bloom that did not contain microcystins. By contrast, the bioassay of fractions using brine shrimps correlated with the distribution of microcystin-LR in the fractionated Microcystis extracts. © 1994 by John Wiley & Sons, Inc..     

Acute toxicity of hardboard mill effluents to different bioindicators

The risk assessment of two kinds of wastewaters from a hardboard mill were determined by acute toxicity tests. Three different bioindicators—water fleas from a local stream (Daphnia pulex), local fish hatchery specimens (Salmo gairdneri), and a bacterial test (Vibrio fischeri Microtox test) were considered. The three tests showed a wide range of sensitivities and presented different but compatible LC₅₀/EC₅₀ values, with S. gairdneri being the most sensitive. The obtained toxicity values could serve as a reference to evaluate the toxicity of wastewater from this type of industries. © 1997 by John Wiley & Sons, Inc. Environ Toxicol Water Qual 12: 39–42, 1997     

Source Apportionment of Particulate Matter in the U.S. and Associations with Lung Inflammatory Markers

Size-fractionated particulate matter (PM) samples were collected from six U.S. cities and chemically analyzed as part of the Multiple Air Pollutant Study. Particles were administered to cultured lung cells and the production of three different proinflammatory markers was measured to explore the association between the health effect markers and PM. Ultrafine, fine, and coarse PM samples were collected between December 2003 and May 2004 over a 4-wk period in each city. Filters were pooled for each city and the PM samples were extracted then analyzed for trace metals, ions, and elemental carbon. Particle extracts were applied to cultured human primary airway epithelial cells, and the secreted levels of interleukin-8 (IL-8), heme oxygenase-1, and cyclooxygenase-2 were measured 1 and 24 h following exposure. Fine PM sources were quantified by the chemical mass balance (CMB) model. The relationship between toxicological measures, PM sources, and individual species were evaluated using linear regression. Ultrafine and fine PM mass were associated with increases in IL-8 (r² = .80 for ultrafine and r² = .52 for fine). Sources of fine PM and their relative contributions varied across the sampling sites and a strong linear association was observed between IL-8 and secondary sulfate from coal combustion (r² = .79). Ultrafine vanadium, lead, copper, and sulfate were also associated with increases in IL-8. Increases in inflammatory markers were not observed for coarse PM mass and source markers. These findings suggest that certain PM size fractions and sources are associated with markers of lung injury or inflammation.     

Bioassay assessment of impacts of tar sands extraction operations

This report describes the application of ten bioassays (battery of tests approach) to waters and sediments collected from the tar sands area of northern Alberta, Canada. In this study there were three main goals: (1) to establish the presence and sources of toxicants in these northern waters and sediments, (2) to determine if their presence is related to sediment composition, and (3) to establish which of the various sediment extracting procedures used was most efficient in producing responses in the bioassays used. Results of these investigations indicated that there were two obvious sources of toxicants/genotoxicants, the tar sands extracting area and streams passing through tar sands or oil shales and at least two unknown sources. Based on the techniques used in this study, there does not appear to be a strong sediment structure–toxicant response relationship. Extraction procedure results were variable and are discussed in detail in the report. © by John Wiley & Sons, Inc.     

Effect-directed investigation and interactive effect of organic toxicants in landfill leachates combining Microtox test with RP-HPLC fractionation and GC/MS analysis

Landfill leachates contain a large amount of unknown harmful compounds derived from domestic and industrial sources. A toxicity effect-directed approach was used to identify biologically active compounds in three landfill leachate samples (S1–S3) by combining the Microtox test with reverse-phase high performance liquid chromatography (RP-HPLC) fractionation and gas chromatography/mass spectrometry (GC/MS) analysis. Organic toxicants were recovered from coarse fractions only in S1 and in S2. Fine fractionation exhibited a somewhat different toxicity pattern in S1 and S2. GC/MS analysis positively identified Bisphenol A (BPA) and 4-t-butylphenol (4-t-BP) in both samples, N-ethyltoluenesulfoneamide (NETSA) was detected only in S1. However, their concentrations were not high enough to be responsible for the observed toxicity in original samples. A synergistic effect among detected organic compounds (BPA, NETSA, and 4-t-BP) was demonstrated. Each compound present at 1/7 of its individual EC₅₀, might lead to undesirable mixture toxicity, which indicated that interactive effects may, to a certain extent, play a role in landfill leachates with complex matrices. The results from further hydrophobicity analysis and estrogen receptor (ER) competitive binding assays of fraction 13 of both samples gave evidence that some possible toxicants that failed to be identified by GC/MS might be endocrine disrupting chemical(s) (EDC) with a log K _ow range of 3.5–3.7 in both samples.     

Acute toxicity evaluation of olive oil mill wastewaters: A comparative study of three aquatic organisms

Acute toxicity of olive mill wastewaters (traditional and continuous processes) collected from different regions of Portugal was evaluated using three test species (Vibrio fischeri formerly Photobacterium phosphoreum, Thamnocephalus platyurus, and Daphnia magna) and correlated with several physical and chemical parameters. Acute toxicity of these effluents, expressed in LC₅₀ or EC₅₀, ranged from: 0.16 to 1.24% in Microtox test, 0.73 to 12.54% in Thamnotoxkit F test, and 1.08 to 6.83% in Daphnia test. These values reflect the high toxicity of the olive mill wastewaters to all test species. Statistical analysis of the results shows a high correlation between the two microcrustacean bioassays. Microtox test did not correlate significantly with the other bioassays used. A significative correlation (p≤0.05) could also be established between L(E)C₅₀ obtained in the microcrustacean tests and some physicochemical parameters of the effluent. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 263–269, 1999     

Potential genotoxicity of sediments from the Great Lakes

Thirty-eight organic extracts of sediment samples collected from 28 sites in three Great Lakes priority areas (Grand Calumet River, Buffalo River, and Saginaw River) were evaluated with the new activated Mutatox Genotoxicity Assay. This in vitro procaryotic assay used rat hepatic S9 for exogenous metabolic activation and a dark mutant strain of the luminescent bacterium Photobacterium phosphoreum for detection of environmental DNA-damaging substances (genotoxins). A genotoxic response was indicated when the test chemical restored the luminescent state in bacteria; the degree of light increase identified the relative genotoxicity of the sample. Within the three priority areas sampled, 27 sites showed evidence of genotoxins, 23 of 28 sites (82%) were designated genotoxic; 4 were suspect (14%), and 1 was negative (3%). Assay sensitivity to known progenotoxins arylamines (2-aminoanthracene and 2-aminofluorene) and polycyclic arylhydrocarbons [benzo(α)pyrene and pyrene] in complex sediment mixtures was ?1 μg per cuvette. The activated Mutatox Assay was a sensitive, specific, predictive, and short-term test for detecting the presence of genotoxins in complex environmental sediments.     

Toxicity evaluation using bioassays in rural developing district 063 Hidalgo,Mexico

One of the oldest and biggest irrigation districts that use wastewater in Mexico is the Rural Developing District 063 (RDD-063). The water supplies of this district are the industrial and domestic discharges of Mexico City and its metropolitan area. RDD-063 production of grains, vegetables, and fruits as well as cattle products supplies the Mexico City market, so it is important to evaluate the chemical risks to which this population is exposed. Fourteen sampling sites were selected for collection of water, soil, and sediment samples in March, July, and October 1993. Bioassays with Daphnia magna, Spirillum volutans, and Panagrellus redivivus were used to estimate acute toxicity and genotoxicity in water samples, sediment pore water, and Milli-Q water extracts of soil. Acute toxicity effects were detected in March and October, while in July genotoxicity responses were found. The results demonstrate that RDD-063 had serious ecological damage, and its waters, soils, and sediments represent a hazard to animal and human health. Bioconcentration and persistence of toxic substances in drinking and irrigation water is a major problem that requires a long-term solution. © 1996 by John Wiley & Sons, Inc.     

Sediment contaminants and microtox toxicity tested in a direct contact exposure test

Limnic and brackish water sediments were tested in a modified contact exposure bioluminescence test, the Microtox test. A variety of chemical constituents were analyzed in the sediments such as metals, chlorinated pesticides, and polychlorinated biphenyls. Sulfur in the common elemental form and pore water hydrogen sulfide were also analyzed. The measured effect in the Microtox toxicity test was correlated with the various chemical parameters to determine the origin of the toxic effect. Based on multivariate data anlaysis, a group of metals including Cu, Zn, Pb, and Cd were correlated positively with the Microtox toxicity tested in the direct contact test. Similarly, but to a lesser extent sulfur, hydrogen sulfide, and the pesticides p,p′-DDT and p,p′-DDD were also correlated. Other pesticides and all the analyzed polychlorinated biphenyls were poorly, if at all, correlated with the toxicity of the samples. In a comparison with the Microtox toxicity of the pure compounds, it was found that, of the analyzed and tested compounds, Zn, Pb, Cu, and elemental sulfur were present in amounts high enough to produce an effect in the test system. This calculation was, however, based on the assumption (unrealistic) that the total amount of a compound in the sediment was available in the test. On the other hand, the metals Cd, Cr, and Ni were found at concentrations of a few percent or less of their EC₅₀ concentrations in the Microtox test of the sediment, γ-Hexachlorocyclohexane and p,p′-DDT were also far less than the concentration required to give an effect in the test system. The pore water content of hydrogen sulfide was also too small to affect the test organism at the EC₅₀ dilution and, similarly, the fraction of the toxicant in the remaining aqueous phase in the sediment after separation of the pore water. Thus only the three metals Zn, Pb, and Cu, and elemental sulfur, were found in concentrations that would give an effect in the test system (0.68–398 times the effect), provided that the substances were available for the organisms. Consequently, elemental sulfur, Zn, Pb, and Cu were indicated as causing the effect in the Microtox test of sediments and not a series of other metal ions, nor tested chloropesticides or chlorinated biphenyls. © 1996 by John Wiley & Sons, Inc.     

Mutagenicity and genotoxicity of PM2.5 issued from an urbano‐industrialized area of Dunkerque (France)

Epidemiological studies have demonstrated the link between chronic exposure to particulate matter (PM), especially particles with an aerodynamic diameter lesser than 2.5 µm (PM_2.5), and lung cancer. Mechanistic investigations focus on the contribution of the various genotoxicants adsorbed onto the particles, and more particularly on polycyclic aromatic hydrocarbons or nitroaromatics. Most of the previous studies dealing with genotoxic and/or mutagenic measurements were performed on organic extracts obtained from PM_2.5 collected in polluted areas. In contrast, we have evaluated genotoxic and mutagenic properties of urbano‐industrial PM_2.5 (PM) collected in Dunkerque (France). Thermally desorbed PM_2.5 (dPM) was also comparatively studied. Suspensions of PM and dPM (5–50 µg per plate) were tested in Salmonella tester strains TA98, TA102 and YG1041 ± S9mix. Significant mutagenicity was observed for PM in YG1041 ± S9 mix. In strain TA102 – S9mix, a slight, but not significant dose–response increase was observed, for both PM and dPM. Genotoxic properties of PM and dPM were evaluated by the measurement of (1) 8‐OHdG in A549 cells and (2) bulky DNA adducts on A549 cells and on human alveolar macrophages (AMs) in primary culture. A dose‐dependant formation of 8‐OHdG adducts was observed on A549 cells for PM and dPM, probably mainly attributed to the core of the particles. Bulky DNA adducts were observed only in AMs after exposure to PM and dPM. In conclusion, using relevant exposure models, suspension of PM_2.5 induces a combination of DNA‐interaction mechanisms, which could contribute to the induction of lung cancer in exposed populations. Copyright © 2010 John Wiley & Sons, Ltd.