Inhibition of bioluminescence in a recombinant Escherichia coli

Six toxicants were evaluated for their ability to inhibit bioluminescence in a recombinant Escherichia coli harboring pJE202 that contained the lux (light-producing) genes. With the recombinant E coli. the rank order for toxicity was Zn²⁺ > ethidium bromide > sodium pentachlorophenate > Cu²⁺ > 2,4-dichlorophenoxyacetic acid. The light-producing recombinant E. coli was insensitive to sodium dodecyl sulfate, and was much less sensitive than the Microtox test with the six toxicants. These results do not preclude the use of recombinant bacteria in toxicity testing.     

An assessment of the potential of the microbial assay for risk assessment (MARA) for ecotoxicological testing

Rapid microscale toxicity tests make it possible to screen large numbers of compounds and greatly simplify toxicity identification evaluation and other effect directed chemical analyses of effluents or environmental samples. Tests using Vibrio fischeri (such as Microtox?) detect toxicants that cause non-specific narcosis, but are insensitive to other important classes of contaminants. The microbial assay for risk assessment (MARA) is a 24 h multi-species test that seeks to address this problem by using a battery of ten bacteria and a fungus. But there has been little independent evaluation of this test, and there is no published information on its sensitivity to pesticides. Here, we assess the performance of MARA using a range of toxicants including reference chemicals, fungicides and environmental samples. Mean MARA microbial toxic concentrations and IC₂₀s (20% Inhibitory concentrations) indicate the toxicant concentrations affecting the more sensitive micro-organisms, while the mean IC₅₀ (50% Inhibitory concentration) was found to be the concentration that was toxic to most MARA species. For the two fungicides tested, the yeast (Pichia anomalia) was the most sensitive of the ten MARA species, and was more sensitive than the nine other yeasts tested. The test may be particularly valuable for work with fungicides. Mean MARA IC₅₀s were comparable to values for nine other yeast species and the lowest individual IC₅₀s for each toxicant were comparable to reported IC₅₀s for Daphnia magna, Selenastrum capricornutum and Microtox? bioassays. MARA organisms exhibited more variable sensitivities, with the most sensitive organism being different for different samples, enhancing the likelihood of toxicity detection and giving a toxicity “fingerprint” that may help identify toxicants. The test, therefore, has great potential and would be valuable for ecotoxicological testing of pollutants.     

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.     

Technical methods section ATP-TOX system—a bacterial toxicity screening procedure

When rapidly growing bacterial cells are exposed to low concentrations of toxicants, growth inhibition usually occurs. However, some toxicants not only inhibit bacterial growth but also affect the luciferase activity during ATP determinations. Thus, the observed light output reduction of a test bacterial system is the net result of both bacterial growth and luciferase inhibition. Based on this information, a new toxicity screening test was developed, the ATP-TOX System.     

Inhibition of β-galactosidase biosynthesis in Escherichia coli: Effect of alterations of the outer membrane permeability on sensitivity to environmental toxicants

The use of bacteria as test organisms in rapid ecotoxicity assays is under investigation in several laboratories worldwide. However, little attention has been given to the question of permeability of the outer membrane of gram-negative bacteria to toxicants of environmental interest. We have investigated the effect of physical, chemical, and genetic alterations on the sensitivity of Escherichia coli to environmental toxicants, as measured via inhibition of β-galactosidase biosynthesis. Polymyxin treatment (2 mg/L) was the most promising treatment tested, significantly increasing the sensitivity of wild-type E. coli to pentachlorophenol and sodium dodecyl sulfate. A mutant strain of E. coli (EW1b), with an outer membrane protein alteration (tol C gene), was found to be the most sensitive to hydrophobic compounds and to detergent. EW1b, further sensitized via polymyxin treatment, appears to be a sensitive microorganism for toxicant assay.     

Toxicity of binary mixtures of reactive toxicants

This study evaluated the toxicity of binary mixtures of reactive toxicants using the Microtox test. Greater than additive effects were quite frequently observed (18%) among chemicals with different mechanisms of toxicity, and some of them were severely synergistic. The concentration-addition model, therefore, may not be appropriate for estimating the multiple toxicity of mixtures containing reactive toxicants. The slope of a chemical's concentration-response curve was found to play an important role in determining the mode of joint actions, which could be related to the complex joint action identified earlier. The results of this study have been summarized into several relationships that can be used to estimate the potential risks for mixtures with unknown toxicity. © 1996 by John Wiley & Sons, Inc.     

Toxicity of zinc in three microbial test systems

A feasibility study on the potential use of three bacterial test systems on the toxicity screening of zinc is presented. In this investigation, the toxicity screening procedures included, were the Microtox test using a luminescent halophyte bacterial strain, Photobacterium phosphoreum, a motility test employing Spirillum volutans, and a growth zone inhibition test using Bacillus cereus as the test organism. The EC₅₀ value of zinc has been found to be 1.35 mg/L with the Microtox test under optimum test conditions (T₁₅o_ct_15min). However, the toxic response of zinc was significantly dependent upon the test temperature and incubation time. It decreased at higher temperatures and increased with longer incubation periods. In the case of the motility test, the minimum effective concentration (90%) value of zinc was 3.00 mg/L at optimum assay conditions (T₂₈o_ct_60min) while the toxicity of zinc in the growth zone inhibition procedure was found to be 2.25 mg/L at 30°C after 18 h incubation. Overall, the study showed that the Microtox test was the most sensitive screening procedure followed by the growth zone inhibition test, and the motility test was least sensitive among the three test systems. The growth zone inhibition procedure was the simplest of all the systems. © 1996 by John Wiley & Sons, Inc.     

Comparison of cell-free and whole cell luminescence assays in toxicity testing

The effects of eight organic and eight inorganic compounds on light emission by two cell-free bioluminescent systems were compared to the effects of these substances on light emission by whole cells of Photobacterium phosphoreum (Microtox® strain). Crude luciferase preparations from the firefly (Photoniuspyralis) and Vibrio (Photobacterium) fischeri were exposed to the toxicants at or near the Microtox EC₅₀ values for 0.25, 5, or 15 min and then reacted with the necessary reagents for luminescence (ATP for the firefly reaction; NADH, FMN and n-decylaldehyde for the bacterial extract). The light emission values, relative to controls, were then compared to those obtained after exposure of P. phosphoreum cells to the toxicants for the same duration. The three systems were found to differ in both response time and sensitivity. The firefly extract was found to be most sensitive to inorganics while whole cells of P. phosphoreum were most sensitive to organics after 0.25 min of exposure. However, after 5 and 15 min both systems were nearly equivalent in their sensitivity to the test toxicants. The V. fishceri cell extract was found to be extremely sensitive to three of the inorganics tested at 0.25 minutes, but was either unaffected or stimulated by the remaining inorganic and organic compounds at all times examined. Our results indicate that the sensitivity of detection of certain toxic compounds may be optimized by selection of a particular luminescent system.     

The effect of toxic discharges on ATP content in activated sludge

ATP measured by the luciferin-luciferase bioluminescence assay was used to examine the effect of toxic substances on whole microbial communities in activated sludge mixed liquor samples. The response of the microorganisms to toxicants is rapid using ATP reduction as the criterion. The sensitivity of the mixed populations to various toxicant types (e.g., organic material and heavy metals) is lower than when using single species toxicity tests such as the Microtox bioassay. The differences in sensitivity is considered a function of acclimatization, modification of the toxicant by the waste physicochemical environment, and the predominance of less sensitive organisms than those used in the Microtox bioassay (Photobacterium phosphoreum). ATP bioluminescence is, however, considered an important rapid test utilizing natural waste treatment microorganisms in determining the toxicity of wastes discharged to sewer. It can detect whether wastewater will have an effect on the biodegradation capability of the resident population of microrganisms. © 1997 by John Wiley & Sons, Inc. Environ Toxicol Water Qual 12: 23–29, 1997     

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.     

Toxicity-directed fractionation of effluents using the bioluminescence of Vibrio fischeri and gas chromatography/mass spectroscopy identification of the toxic components

A procedure is presented for fractionating and identifying the dominant acute toxicants in effluents using the Microtox test to evaluate toxicity. Initial characterization of chemical and physical properties of the major toxicants was performed, and showed that the dominant toxicants were lipophilic. Samples containing lipophilic components were then fractionated by column chromatography, the fractions tested for toxicity, and toxic components identified. These toxicants were quantified in the effluent and a toxicity evaluation was performed as a material balance in toxicity. Three effluents were studied and found to contain only a few (2–4) toxicants or groups of related compounds. An effluent from a pharmaceutical industry contained a drug precursor and a drug constituent as dominant toxicants, and the combined toxicity of the two compounds, accounted for 87% of that of the effluent. A contribution from the interaction of the two compounds was observed. An effluent from a forest product industry contained two unsaturated fatty acids and an unsaturated fatty acid amide as dominant toxicants, and almost the total toxicity of the sample (97%) was accounted for by the three compounds with a contribution from the interaction of the three. The acute toxicity of a textile industry effluent was dominated by two unsaturated fatty acids and two tridecanols, and these compounds accounted for 84% of the toxicity of the original effluent. A mixture of aliphatic hydrocarbons was found in a toxic fraction, but was not further characterized, although the hydrocarbons may have contributed to the toxicity of the sample. © 1996 by John Wiley & Sons, Inc.     

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).     

The relative sensitivity of microtox®, daphnid,rainbow trout,and fathead minnow acute lethality tests

Relative sensitivity and correlations between the Microtox® test and three commonly used acute lethality bioassays (i.e., rainbow trout, fathead minnow, Daphnia) are reviewed and discussed. All relevant data available for comparison were separated and evaluated based on chemical groupings. Generally, Microtox was more sensitive than or as sensitive as the acute lethality tests for pure individual organics, but was less sensitive to most inorganics. Microtox was not as sensitive as acute lethality tests to effluents or leachates with a high component of insecticides, herbicides, inorganics, pharmaceuticals or textiles, or highly lipophilic contaminants. As the complexity and toxicity of industrial effluents increased, the correlations and sensitivity of Microtox increased, with a corresponding decrease in data variability. Additionally, limitations of the available data are that (1) comparisons of relative sensitivity varied with the compounds and organisms tested; (2) there was a lack of standardization in approach and a failure to provide adequate details on the origin of the cited toxicity data, which confused and obscured comparisons; and (3) published reviews commonly failed to identify fish species and test details for cited data.     

The effect of ionic solutes on EC50 values measured using the microtox test

The Microtox test uses the marine luminescent bacterium, Photobacterium phosphoreum. To provide the organism with osmotic protection, sodium chloride (2.0%) is usually added to freshwater samples before analysis. This procedure is shown to cause a dramatic lowering of measured toxicity where ionic compounds are the major toxicants present. The reason for this is the consequent variation in ionic strength upon addition of NaCl rather than formation of less-toxic chloro complexes. Data are given for the toxicants Zn, Cd, Cu, NH₄⁺, and phenol. It was found that measurements on aqueous samples could be made without adjustment provided that the salinity was in the range of 1.0–7.0%. Freshwaters can also be analyzed without adjustment of ionic strength by adding sucrose (20.4%) to provide the necessary osmotic protection. It is therefore recommended that the ionic strength of aqueous samples should not be altered as part of the test and that receiving waters should be used as the diluent wherever possible.     

Toxicity of binary mixtures of organic chemicals

Toxicity of binary mixtures of organic chemicals was evaluated using the Microtox test, in order to derive a more realistic approach for predicting mixture toxicity. Both additive and less than additive effects were found from binary mixtures of nonreactive toxicants. Concentration addition can be expected between two nonreactive chemicals, provided that the requirement of parallel dose—response curves is fulfilled. The study also identified the conditions for a complex—type joint action to take place. Finally, experimental isobolograms were analyzed based on a bivariate toxicity model to provide a rational estimation of model parameters. The isobolograms and the additive indices generated by this study can be used to predict the combined toxic effects from various organic mixtures. © by John Wiley & Sons, Inc.     

Direct sediment toxicity testing procedure using sediment-chromotest kit

A semiquantitative direct sediment toxicity testing procedure using the Sediment-Chromotest was developed. This procedure has advantages over many other toxicity bioassays. It is simple, quick, and reliable, and does not require special instrumentation. The direct sediment toxicity testing procedure involves the use of serial dilutions of solid phase suspensions mixed with stressed bacteria (Escherichia coli) and a cocktail containing the specific inducer for the chromogenic enzyme and other essential factors. The assay is based on the ability of toxicants to inhibit the de novo synthesis of an inducible enzyme, beta-galactosidase. The amount of de novo synthesized enzyme is determined by a colorimetric reaction. This new procedure measures the presence of bioavailable toxicants in sediments, suspended sediments, soils, and other solid wastes directly without extraction and concentration. © by John Wiley & Sons, Inc.     

Ecotoxicological evaluation for the screening of areas polluted by mining activities

The aim of this study was to evaluate the applicability of three bioassays representing multiple trophic levels, for the preliminary ecotoxicological screening of sediments from sites contaminated by mining activities. Of the bioassays used in this study, the ostracod test was the most responsive. Vibrio fischeri luminiscence inhibition assay was less sensitive to the toxicants in the sediments than the phytotoxicity assays. The general trend observed was an increase in toxicity values measured by the bioassays with increasing metal mobilization in sediment samples. Therefore, the test battery can be used as a rapid and sensitive tool to evaluate the heavy metal contamination in sediments.     

Comparison of respiration and luminescent tests in bacterial toxicity assessment

The toxicity assessment of chemicals on complex microbial communities are generally preferred to toxic tests conducted with pure cultures of bacteria. The purpose of this study is to compare the “Respiration” test studying metabolic criteria of mix bacterial populations and the “Microtox” test using lyophilized cultures of luminescent bacteria. The inhibition of oxygen consumption was compared with the inhibition of the bioluminescence of Photobacterium phosphoreum. In the first part of this study, the methodology of the “Respiration” test and the “Microtox” test was improved to optimize their performance in toxicity assessment. In the second part, toxicity of an organic chemical, the 3,5-Dichlorophenol, and an inorganic chemical, copper sulfate, was evaluated with the two tests. Results were compared and characteristics of the testing methods are discussed according to their sensitivity, reproducibility, representativity and ease of execution. The repeatability study for the “Respiration” test and the “Microtox” test gives variation coefficients less than 15% and 10% respectively. The variation coefficients concerning the reproducibility study are found to be 15% or 18% for the “Respiration” test, 5% or 28% for the “Microtox” test, depending on the tested toxicant. These two toxicity tests are easy to perform. But the “Microtox” test offers the advantage of being more sensitive and much faster. On the other hand, the “Respiration” test which was conducted on a microbial community is probably more representative of microflora in rivers.     

Peroxidation levels in Eschericia coli as a potential toxicity marker: Effect of nickel and methyl-parathion

The effects of nickel and methyl-parathion were studied in subinhibiting concentrations (40 and 62.5 μg/mL, respectively) on the levels of cellular peroxidation in Escherichia coli. These effects were evaluated using the thiobarbituric acid (TBA) test and the determination of lipofuscin-like substances. Both toxicants caused an increase in cellular peroxidation levels, although at the concentrations used, nickel was shown to be a better inducer than methyl-parathion. The TBA test gave more significant differences than the determination of lipofuscin-like substances when the control cultures were compared to those containing the toxicant. This finding reveals that the TBA test could be used as a toxicity marker in E. coli for the development of ecotoxicity biotests. © 1994 by John Wiley & Sons, Inc..     

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