Microtox Assay Research Articles

Effect of Dust on the Viability of Vibrio fischeri in the Microtox Test

The standard Microtox test involving the bioluminescent bacterium Vibrio fischeri is a frequently used ecotoxicological bioassay whose EC50 values have been correlated to acute toxicity parameters of vertebrates, to irritancy measures, and to cytotoxicity indices. The aims were to explore the dependence of light output on viable cell number, with the latter estimated with the naked eye using a colorimetric tetrazolium salt method, the effects of dust on the bioluminescence and cell viability, how the viability of the cells is affected after spills, and how spills can be sampled. The lower limit of the linear dynamic range of the light-emitting bacterium was first defined to be 3.7×107 cells/mL, compared with 37×107 cells/mL in the Microtox assay. The effects of dust were then explored in the working range by the method of standard additions by adding 5-, 10-, and 20-mg amounts of Standard Reference Material Urban Dust 1649a. This simulated dust samples collected by a cordless vacuum technique involving a filter cassette. A mass of 20 mg dust totally inhibited the Microtox test at all times (5, 15, and 30 min). Masses of 5 and 10 mg dust lowered the luminescence significantly by 20 and 64%, respectively, after 30 min. However, the viability test was totally inhibited by 5 mg of dust. A spectrophotometric modification of the viability test using a wavelength of 508 nm was developed that was twice as sensitive as the naked eye test, and was as sensitive as the Microtox test. Mechanical shock involved with spilling and sampling bacterial reagent on hard surfaces killed the luminescent bacteria as shown by inhibition of luminescence. The optimum filter cassette for Microtox reagent collection was a 25-mm 1.00-μm PTFE filter in a 25-mm Delrin holder operated at 4.0 L/min, with a Tygon sampling probe.

Chemical contamination and toxicity of sediment from a coastal area receiving industrial effluents in Kuwait.

The Shuaiba coastal area (12.5 x 1.5 km) was examined for contamination with total organic carbon, volatile organic matter, total petroleum hydrocarbons, polycyclic aromatic hydrocarbons, cadmium, chromium, copper, lead, nickel, vanadium, and zinc in sediment; their desorption by aqueous elution; and toxicity to aquatic biota. The pollutants were mainly accumulated in the upstream area facing Mina Al-Ahmadi refinery to Shuaiba harbour. Solid-phase Microtox assays showed severe toxicity, and the LC(50) was negatively correlated with most of the chemical parameters, suggesting that toxicity was the function of collective effects of the pollutants present in sediment. Sea water elutriation showed poor desorption of pollutants from sediment, and the elutriates were not found toxic to Microtox and brine shrimp larvae. Whole sediment suspension in sea water reduced the survival of fingerlings in fish bioassays. Action from Shuaiba Area Authority is required to reduce pollutant accumulation in identified depositional area on the Shuaiba coast by facilitating unrestricted water flow in the area and restricting pollutant discharge at source.

Mycobacterium Diversity and Pyrene Mineralization in Petroleum-Contaminated Soils

Degradative strains of fast-growing Mycobacterium spp. are commonly isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated soils. Little is known, however, about the ecology and diversity of indigenous populations of these fast-growing mycobacteria in contaminated environments. In the present study 16S rRNA genes were PCR amplified using Mycobacterium-specific primers and separated by temperature gradient gel electrophoresis (TGGE), and prominent bands were sequenced to compare the indigenous Mycobacterium community structures in four pairs of soil samples taken from heavily contaminated and less contaminated areas at four different sites. Overall, TGGE profiles obtained from heavily contaminated soils were less diverse than those from less contaminated soils. This decrease in diversity may be due to toxicity, since significantly fewer Mycobacterium phylotypes were detected in soils determined to be toxic by the Microtox assay than in nontoxic soils. Sequencing and phylogenetic analysis of prominent TGGE bands indicated that novel strains dominated the soil Mycobacterium community. Mineralization studies using [(14)C]pyrene added to four petroleum-contaminated soils, with and without the addition of the known pyrene degrader Mycobacterium sp. strain RJGII-135, indicated that inoculation increased the level of degradation in three of the four soils. Mineralization results obtained from a sterilized soil inoculated with strain RJGII-135 suggested that competition with indigenous microorganisms may be a significant factor affecting biodegradation of PAHs. Pyrene-amended soils, with and without inoculation with strain RJGII-135, experienced both increases and decreases in the population sizes of the inoculated strain and indigenous Mycobacterium populations during incubation.

Preliminary risk assessment of the wet landscape option for reclamation of oil sands mine tailings: bioassays with mature fine tailings pore water.

Chemical and biological assays have been carried out on the "pore water" that results from the settling of the tailings that accompany bitumen recovery from the Athabasca oil sands. Examination of the nonacidic extracts of pore water by gas chromatography-mass spectroscopy allowed the identification of numerous two- to three-ring polycyclic aromatic compounds (PACs), to a total concentration of 2.6 micrograms/L of pore water. The PACs were biodegraded by microflora naturally present in the pore water. Acute toxicity was associated principally with the acidic fraction (naphthenic acids) of pore water extracts according to the Microtox assay; other work has shown that acute toxicity dissipates fairly rapidly. Both individual PACs and concentrated pore water extracts showed minimal levels of binding to the rat Ah receptor and induced minimal ethoxyresorufin-O-deethylase activity in primary rat hepatocytes, showing an insignificant risk of inducing monooxygenase activity. Taken together with previous work showing negligible mutagenic activity of these extracts, we conclude that it should be possible to develop tailing slurries into biologically productive artificial lakes.

Toxicity Assessment of Atrazine and Related Triazine Compounds in the Microtox Assay, and Computational Modeling for Their Structure-Activity Relationship

The triazines are a group of chemically similar herbicides including atrazine, cyanazine, and propazine, primarily used to control broadleaf weeds. About 64 to 80 million lbs of atrazine alone are used each year in the United States, making it one of the two most widely used pesticides in the country. All triazines are somewhat persistent in water and mobile in soil. They are among the most frequently detected pesticides in groundwater. They are considered as possible human carcinogens (Group C) based on an increase in mammary gland tumors in female laboratory animals. In this research, we performed the Microtox Assay to investigate the acute toxicity of a significant number of triazines including atrazine, atraton, ametryne, bladex, prometryne, and propazine, and some of their degradation products including atrazine desethyl, atrazine deisopropyl, and didealkyled triazine. Tests were carried out as described by Azur Environmental [1]. The procedure measured the relative acute toxicity of triazines, producing data for the calculation of triazine concentrations effecting 50% reduction in bioluminescence (EC50s). Quantitative structure-activity relationships (QSAR) were examined based on the molecular properties obtained from quantum mechanical predictions performed for each compound. Toxicity tests yielded EC50 values of 39.87, 273.20, 226.80, 36.96, 81.86, 82.68, 12.74, 11.80, and 78.50 mg/L for atrazine, propazine, prometryne, atraton, atrazine desethyl, atrazine deisopropyl, didealkylated triazine, ametryne, and bladex, respectively; indicating that ametryne was the most toxic chemical while propazine was the least toxic. QSAR evaluation resulted in a coefficient of determination (r2) of 0.86, indicating a good value of toxicity prediction based on the chemical structures/properties of tested triazines.

Evaluation of Fungal Capacity for Detoxification of Extractives in Scots Pine Sapwood

Ninety wood-inhabiting fungi were screened for their ability to degrade and detoxify acetone extractives in Scots pine (Pinus sylvestris L.) sapwood under solid-state fermentation conditions. Fungal degradation of resin acids and long-chain fatty acids was investigated by high pressure liquid chromatography with evaporative light scattering detection (HPLC-ELSD) and the toxicity of the acetone soluble fraction of wood measured by Microtox assay. Discriminant analysis of data showed different patterns of extractive detoxification for white-rot basidiomycetes and sapstain fungi. The major detoxification skills of basidiomycetes appeared to be related to the highest degradation of resin acids, and particularly dehydroabietic acid. Among them, Ischnoderma benzoinum, Stereum sanguinolentum and Trametes versicolor greatly reduced the sapwood toxicity due to acetone extractives from the EC50 value of 0.08 mg wood ml−1 to levels between 5-10 mg wood ml−1.

Toxicological Evaluation of Surface-water Samples in Sensitised Cultured Fish Cells Compared with the Microtox® Method.

A previously described neutral red uptake-inhibition assay on cultured fathead minnow (FHM) fish cells revealed a good correlation with fish toxicity data. The assay was made more sensitive by reducing the number of cells, by using a longer treatment period, and by simultaneous treatment of the cells with sodium dodecyl sulphate and L-buthionine (S,R)-sulphoximine. The fluorimetrically quantified protein content was then used as the endpoint. The results were compared with toxicity data obtained by the Microtox® method with the bacterium, Vibrio fischeri. A series of 82 surface-water samples were investigated. Cell quantitydependent and sample concentration-dependent reductions in total protein content were observed. In all, 32 samples were toxic in the FHM assay. Of the seven samples that were shown to be toxic in the Microtox assay, only three were also toxic in FHM cells. No linear relationship was found for the toxicity results obtained with the two methods. Further research to explain the significance of the many positive responses and to identify possible confounding factors will increase the reliability of the sensitised FHM cell assay.

Temporal ecological assessment of oil contaminated soils before and after bioremediation

Ecotoxicity methods were used to assess different soil and oil combinations before, during and after laboratory bioremediation with associated hydrocarbon analysis. Heavy, medium and light crude oil (API gravity 14, 30, and 55) was spiked (ca. 5% w/w) into two sandy soils in the laboratory having organic carbon concentrations of 0.3 (Norwood) and 4.7% (Norwood/Baccto). The earthworm ( Eisenia fetida) 14-d lethality assay, the modified Microbics Microtox ® Solid-Phase assay, and the 14-d plant seed germination and growth assays using corn, wheat and oats, were spiked and tested during a 360-d laboratory remediation. Eisenia was the most sensitive of the three methods utilized with survival increasing throughout bioremediation with fastest toxicity reduction in the high carbon Norwood/Baccto soils where LC50’s were 100% or greater at the end of 90-d whereas, >150-d were required to achieve a similar result in the low carbon soil. Analysis of the undiluted treatments with oily soil alone showed that earthworm survival was high after 90-d in all high organic carbon soils, and after eight months in the low carbon soils, except for the Norwood soil-light oil treatment, which required 360-d to achieve 100% survival. The Microtox assay was less sensitive with EC50’s 100% or greater observed after 90-d in high carbon soils and after 240-d for all low carbon soils. After bioremediation, no effects on seed germination were observed, although some plant growth inhibition effects remained. There was no direct correlation between total petroleum hydrocarbon concentrations and toxicity.

Color and toxicity removal following tyrosinase-catalyzed oxidation of phenols.

The products of phenol oxidation catalyzed by mushroom tyrosinase (polyphenol oxidase, EC 1.14.18.1) were assessed in terms of their residual color and toxicity. The addition of aluminum sulfate had little effect on the removal of colored products from phenol solutions treated with tyrosinase. Although chitosan was used successfully to remove the color when added before the reaction initiation or after the reaction completion, the required dose of chitosan was lower when it was added after the reaction. In this case, the minimum doses of chitosan required to achieve 90% color removal were proportional to the logarithm of the initial concentration of phenol. The color removal induced by chitosan addition appeared to be the result of chemical interaction followed by a coagulation mechanism. All treated solutions of phenol and chlorophenols, except 2,4-dichlorophenol, had substantially lower toxicities than their corresponding initial toxicities, as measured using the Microtox assay. Chitosan addition significantly enhanced the reduction in toxicity. The toxicities of the phenol solutions treated with tyrosinase were markedly lower than previously reported toxicities of solutions treated with peroxidase enzymes.

Screening Bioavailable Hydrophobic Toxicants in Surface Waters with Semipermeable Membrane Devices: Role of Inherent Oleic Acid in Toxicity Evaluations

Semipermeable membrane devices (SPMDs) were deployed for 4 weeks in two rivers in Lithuania. The SPMD dialysates were tested in the Microtox assay and, surprisingly, the sample from the relatively clean Ūla River exhibited three times more toxicity than the sample from the Vilnia River receiving discharge from several industrial enterprises and municipal wastewater. The whole dialysates were subjected to bioassay-directed fractionation on silica gel columns. Toxicity testing of each fraction revealed that most of the toxicity was contained in fraction 10, which was eluted with 100% acetone. GC/FID, GC/ECD, and GC/FTIR/MS analysis of the fractions indicated that the major component of this fraction was oleic acid. The oleic acid was most likely the hydrolysis product of methyl oleate, the major impurity of the SPMD triolein. It can be inferred that oleic acid was responsible for the toxicity of this fraction in Microtox, as a threefold difference in the toxicity between the two samples was also marked by a threefold difference in their oleic acid content. Toxicity of unsaturated fatty acids in various tests, including Microtox, has been demonstrated elsewhere. Vilnia fraction 2, which was eluted with 100% hexane, exhibited the most toxicity of the remainder of silica gel fractions. The spectral analysis demonstrated that other toxic fractions contained a number of halogenated compounds and PAHs. In general, SPMDs have proved to be a useful way to screen for hydrophobic toxicants in water. However, sample clean-up procedures to remove oleic acid may be required prior to toxicity testing for the estimation of the true toxic potential of the accumulated pollutants.

Validation of Genetically Engineered Bioluminescent Surfactant Resistant Bacteria as Toxicity Assessment Tools

Bacteria are useful organisms for measuring acute and chronic toxicity. The most popular toxicity tests utilize the inhibition of bioluminescence as an indication of toxicity. An extensive toxicity database on pure chemical compounds has been created using the bioluminescent microorganism, Vibrio fischeri. However, the use of the Microtox assay in applications for environmental samples is not always successful, due to the test organism. Because the genes for bioluminescence have been cloned from V. fischeri, environmentally relevant test strains can be readily constructed. In this study, surfactant-resistant bioluminescent bacterial strains were constructed by transferring a broad host range plasmid containing the bioluminescent genes under the regulation of a constitutive promoter into strains from several bacterial genera. Two test strains, Stenotrophomonas 3664 and Alcaligenes eutrophus 2050, were approximately 400 times more resistant to the nonionic surfactant polyoxyethylene 10 lauryl ether than V. fischeri and are useful for toxicity reduction evaluations of remediation processes which use surfactants for solubilization of hydrophobic pollutants. The use of these strains as alternative test organisms in the Microtox assay was evaluated using nonpolar narcosis as the baseline toxicity mechanisms. The two test strains and V. fischeri indicated linear fits of EC50 values with the octanol/water partition (Kow) for five nonpolar narcotic compounds in acute assays (r2>0.9) with a slope of approximately 1. For all three strains, the y-intercept values were approximately the same, indicating that sensitivity did not vary. These results indicate that the nonpolar narcosis baseline toxicity mechanism may be useful as a general tool to validate the functioning of genetically engineered bioluminescent microorganisms.

Ecotoxicity characterization of dinitrotoluenes and some of their reduced metabolites

In the present study, the toxic effects of 2,4-dinitrotoluene (2,4-DNT), 2,6-dinitrotoluene (2,6-DNT) and a selection of their respective metabolites were examined and compared to 2,4,6-trinitrotoluene (TNT) using the 15-min Microtox ( Vibrio fischen) and 96-h freshwater green alga ( Seienastrum capricomutum) growth inhibition tests. All of the compounds tested were less toxic than TNT. Using the Microtox assay, 2,6-DNT was more toxic than 2,4-DNT and the order of toxicity for 2,6-DNT and its metabolites was: 2,6-DNT ≥ 2A-6NT ⪢> 2,6-DAT; whereas that for 2,4-DNT was: 4A-2NT > 2A-4NT > 2,4-DNT > 2,4-DAT. For the algal test, 2,4-DNT was more toxic than 2,6-DNT and the order of toxicity for 2,4-DNT and its metabolites was: 2,4-DNT > 2,4-DAT ⋟ 4A-2NT = 2A-4NT. The order of toxicity for 2,6-DNT and its reduced metabolites using the algal test was very similar to the Microtox bioassay. These results demonstrate that the reduced metabolites of 2,6-DNT tested in this study were less toxic than that of the parent compound, but certain partially reduced metabolites of 2,4-DNT can be more toxic than the parent molecule. These data put into question the general hypothesis that reductive metabolism of nitro-aromatics is associated with a sequential detoxification process.

Confirmation of elemental sulfur toxicity in the Microtox® assay during organic extracts assessment of freshwater sediments

AbstractRecent literature indicates that the elemental sulfur occurring in organic extracts of sediment samples can be toxic to the bacterium Vibrio fischeri, used in standard Microtox® bioassays. This observation was tested by means of the solvent extraction of 14 freshwater sediment samples from rivers tributary to Lake Geneva (Switzerland–France), measuring both Microtox toxicity and the elemental sulfur concentration of the extracts. Aliquots of these sediment extracts were further treated to remove the sulfur by adding acid‐activated copper to the crude extracts; for 18 h in one case, and for 116 h in an other. The results were a significant amount of the observed acute toxicity in the Microtox assay of 81% of sample extracts (n = 42, crude and after cleanup) was due to elemental sulfur, and despite a median decrease of 99.1% of elemental sulfur in the extracts subject to a 116‐h cleanup, sulfur toxicity was not completely excluded for 57% (8/14) of the samples. Clearly, the Microtox methodology needs to be amended to more accurately assess the potential impact of organic pollutants in sediments when solvent extracts are used. This will help to cut down on costly and unnecessary remedial actions.

Metabolism of benzene, toluene, and xylene hydrocarbons in soil

Enrichment cultures obtained from soil exposed to benzene, toluene, and xylene (BTX) mineralized benzene and toluene but cometabolized only xylene isomers, forming polymeric residues. This observation prompted us to investigate the metabolism of 14C-labeled BTX hydrocarbons in soil, either individually or as mixtures. BTX-supplemented soil was incubated aerobically for up to 4 weeks in a sealed system that automatically replenished any O2 consumed. The decrease in solvent vapors and the production of 14CO2 were monitored. At the conclusion of each experiment, 14C distribution in solvent-extractable polymers, biomass, and humic material was determined, obtaining 14C mass balances of 85 to 98%. BTX compounds were extensively mineralized in soil, regardless of whether they were presented singly or in combinations. No evidence was obtained for the formation of solvent-extractable polymers from xylenes in soil, but 14C distribution in biomass (5 to 10%) and humus (12 to 32%) was unusual for all BTX compounds and especially for toluene and the xylenes. The results suggest that catechol intermediates of BTX degradation are preferentially polymerized into the soil humus and that the methyl substituents of the catechols derived from toluene and especially from xylenes enhance this incorporation. In contrast to inhibitory residues formed from xylene cometabolism in culture, the humus-incorporated xylene residues showed no significant toxicity in the Microtox assay.

The Effect of Temperature on the Composition and Relative Toxicity of the Water-Soluble Fraction of Kuwait Crude Oil (Export) in the Seawater

The effect of temperature on the composition of the water-soluble fraction (WSF) of Kuwait crude oil was investigated. The results showed that the WSF consisted of mostly monoaromatic (BTEX) compounds. The total concentration of volatiles in the WSF was about 7.5 mg/1. The increase in the temperature from 15°C to 25°C caused an increase of about 10% and to 35°C did not significantly affect the total concentration. GC-MS analysis of the concentrated extract of the WSF resulted in the identification of nine PAHs and six methylated PAHs. The total concentration of the PAHs ranged from 0.217 mg/1 to 0.634 mg/1. The PAHs increased significantly with increasing temperature. Naphthalene and methylated naphthalenes constituted about 95% of the total PAHs. Microtox assay of the WSF showed that the relative toxicity increased when the temperature was increased from 15° to 25°C. Increasing temperature further to 35°C did not effect the relative toxicity.

Recalcitrant organic compounds (chemical oxygen demand sources) in biologically treated pulp and paper mill effluents: their fate and environmental impact in receiving waters

Most North American pulp and paper mills now biologically treat (biotreat) their liquid effluent. However, treated water still contains effluent‐derived recalcitrant organic material (EROM), measured as chemical oxygen demand (COD), for which emission limits exist in Europe and are being considered in the U.S. Production of microbially resistant, dissolved natural organic material (NOM) typically found in Canadian stream and lake waters occurs slowly under gentle conditions, while mill EROM is generated from lignocellulosics by faster and harsher processes. Similarity of the environmental effects of NOM and pulp and paper mill EROM are examined. Changes occurring over 4 months in biologically treated effluent from two modern Canadian mills and lake NOM when sealed in gas‐ and light‐permeable bags and placed in a pristine Quebec lake are reported. Addition of microbial co‐metabolites significantly improved the dark mineralization of organochlorines surviving mill biological treatment. Mill EROM was light sensitive, nonacutely toxic in the Microtox assay, and similar to NOM in the surrounding lake in most bulk properties. There was no evidence to suggest that placing specific limits on mill EROM (COD) emissions would be environmentally beneficial.

Detection of Aircraft Deicing/Antiicing Fluid Additives in a Perched Water Monitoring Well at an International Airport

Tolyltriazoles have recently been shown to be the primary agents in aircraft deicing/antiicing fluids (ADAFs) responsible for acute Microtox activity. However, little is known about the fate and effect of these compounds in the environment resulting from airport deicing activities. This research communication describes the first evidence that constituents within ADAFs, other than glycols, are present in subsurface water samples from a major North American airport at environmentally significant concentrations. These concentrations are approximately 25 times higher than the reported EC50 values in Microtox assays.

Comparison of toxicities and mechanism of action ofn‐alkanols in the submitochondrial particle and theVibrio fischeribioluminescence (Microtox®) bioassay

AbstractMany studies on the toxicity ofn‐alkanols have been conducted and quantitative structure‐activity relationships (QSARs) established comparing bioassay toxicity data to carbon number. The results typically indicate increasing toxicity with increasingn‐alkanol chain length, but often higher homologues are not assessed, where toxicity no longer increases with carbon number—a phenomenon commonly called the cutoff effect. The mode of toxic action of these compounds has been designated narcosis I; however, the specific mechanism is unknown and widely debated. This study compares results for two commonly used bioassays, the submitochondrial particle (SMP) and the Microtox® bioassay, for the homologous series ofn‐alkanols ranging from methanol (C1‐OH) to stearyl alcohol (C18‐OH). Analysis of dose‐response curve slopes indicates that the SMP assay exhibits a general mechanism of toxicity, whereas the Microtox assay exhibits this general mechanism to short‐chain alkanols, but subsequently switches to specific interaction with the higher alkanols. This specific interaction is likely competitive inhibition of the bacterial luciferase. Comparison of the toxicities in these assays with octanol/water partition coeffients (Kow), the results of whole‐organism tests, and a bacterial luciferase assay further substantiate this claim and indicate that the SMP is a better model of toxicity in whole organisms.

Ecotoxicological Assessment of Ashes and Particulate Matter from Fluidized Bed Combustion of Coal

Toxicity of ash and particulate matter from the gases generated in coal fluidized bed combustion (FBC) has been determined by the Microtox assay according to the standard leachate procedure. Results are compared with the polycylic aromatic hydrocarbon (PAH) content of the particulate matter, which was determined by fluorescence spectroscopy. Although PAHs are considered highly toxic compounds, the low ecotoxicity values obtained could be explained by the low solubility of the compounds in water. The Microtox assay may underestimate the toxicity of water-insoluble compounds unless they are previously extracted with an organic solvent. Nevertheless, this type of assay can be very useful for measuring the potential toxicity of residues when exposed to water sources such as rain water and the risk of the components being dissolved and transported by runoff water.

Discharge of disinfected wastewater in recipient aquatic systems: fate of allochthonous bacterial and autochthonous protozoa populations.

The discharge of disinfected effluents affects the bacterivorous ability of protozoa and the effect depends on the disinfectant applied. Chlorine provokes a decrease in the number of protozoa and a delay in the bacterivorous ability. The discharge of ozonated and peracetic acid-treated wastewater provokes only an initial slight decrease in bacterivorous ability. No correlation was found between toxicity values detected using the Microtox assay and the effect of disinfected effluents on freshwater protozoa population. After the disinfection processes, recipient systems (fresh and marine water) have different effects on the survival of Escherichia coli populations discharged to them. The effect of the freshwater recipient system is less negative than the effect provoked by sea-water, and the differences detected depend on the disinfection treatment applied. The wastewater bacterial population as a whole is able to grow after discharge of disinfected wastewater to receiving waters. However, in the absence of predation or competition, the recipient systems exert selection, with rod-shaped bacteria predominating.