Toxicology evaluation of Atlantic Canadian seafood processing plant effluent

The purpose of this study was to carry out an acute aquatic toxicity assessment on select effluent samples from Atlantic Canadian seafood processing plants. Raw effluent acute aquatic toxicity for the flatfish and salmon effluents was assessed using the acute lethality test and Microtox? test. The effectiveness of dissolved air flotation treatment (DAF) in removing acute toxicity from these effluents was evaluated using the Microtox? test. The salmon effluent failed the acute lethality test using rainbow trout while the flatfish effluent showed acute toxicity in the Microtox? test with a 50% inhibiting concentration (IC₅₀) of 38.84%. Subsequent treatment by DAF of the flatfish and salmon effluents increased IC₅₀ values by 20% and 26% respectively. The findings of this study indicate that all of the processing effluents sampled showed characteristics that could potentially degrade effluent receiving waters and acute toxicity was demonstrated in the two raw finfish effluents. Application of DAF treatment was successful in significantly increasing Microtox? IC₅₀ values, thereby reducing acute toxicity, but failed to entirely remove acute toxicity. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2010.     

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.     

Photobacterium phosphoreum toxicity bioassay. I. Test procedures and applications

This review of Photobacterium phosphoreum toxicity bioassay, commonly known as the Microtox^TM tests, consists of two parts. The first part, I. Test Procedures and Applications, is a selective literature review which describes the principle, methodology, experimental conditions and procedures, and the applications of the Microtox^TM test. It includes a list of relevant references and tables showing comparisons of Microtox test results with those of various other acute toxicity assays for selected compounds and complex industrial effluents. The second part of this review deals with toxicity data compilation.     

Toxicity evaluation of metal plating wastewater employing the Microtox assay: a comparison with cladocerans and fish

The relative sensitivity of the Microtox assay is closely related to the type of toxicant, and hence its utility in biomonitoring effluents is better evaluated on a case-by-case basis. The Microtox assay, employing the marine bacterium Vibrio fischeri, was evaluated for its applicability in monitoring metal plating wastewater for toxicity. The results of the Microtox assay after 5, 15, and 30 min of exposure, were compared with data obtained from conventional whole effluent toxicity testing (WET) methods that employed Daphnia magna, Ceriodaphnia dubia, and the fathead minnow (Pimephales promelas). The Microtox assay produced notably comparable EC50 values to the LC50 values of the acute fathead minnow toxicity test (< 0.5 order of difference). The Spearman's rank correlation analyses showed that the bacterial assay, regardless of exposure duration, correlated better with the acute fish than the daphnid results (p < 0.05). These observations were consistent to other studies conducted with inorganic contaminants. The relative sensitivity of the 30-min Microtox assay was within the range of the two frequently used acute daphnid/fish toxicity tests. In conclusion, the Microtox assay correlated well with the acute fathead minnow data and is well suited for toxicity monitoring for these types of industrial wastes.     

Toxicity of different industrial effluents in Taiwan: a comparison of the sensitivity of Daphnia similis and Microtox

Industrial effluents are known to exhibit toxicity toward different aquatic organisms. In Taiwan management of these discharges still relies on chemical and physical and physical characteristics of water, although various standard method for assessing aquatic toxicity have been proposed by the Taiwan Environmental Protection Administration. In this study we examined the toxicity and compared the sensitivity of different types of industrial effluents using two proposed toxicity tests: the Daphnia similis acute toxicity test and the Microtox acute assay (Vibrio fischeri). Results showed that electroplating effluents were the most toxic of all the effluents tested, followed by acrylonitrile manufacturing, pulp/paper, and tannery effluents. The EC50 of an electroplating effluent for D. similis and V. fischeri (15 min) was as low as, respectively, 2.9% and 3.9% of the whole effluent. The other effluents were not acutely toxic to either organism tested. However, the tests exhibited different sensitivity toward various discharges. Only the electroplating and acrylonitrile manufacturing effluents had effects on both organisms. These results indicate the importance of the incorporation of aquatic toxicity tests into the management scheme for treated wastewaters.     

A multispecies toxicity assessment procedure using flow microcalorimetry: Comparison with other toxicity evaluation methods

The acute toxicity of various contaminants and effluents towards a heterogenous aerobic culture has been determined using flow microcalorimetry. The toxicity of these contaminants and effluents was also evaluated towards a marine bacteria (type Photobacterium phosphoreum, Microtox test), a green algae (Selenastrum capricornutum, inhibition of growth) and a small crustacean (Daphnia magna, inhibition of mobility). The 50% (IC₅₀) or 20% (IC₂₀) inhibitory concentrations were obtained for each toxicity test, and compared to each other. The results of the present study suggest that, under proper conditions, a mixed aerobic culture could yield toxicity estimates comparable to those obtained from a combination of single species tests.     

Assessment of methods for evaluating the treatability of sewage and the effects of industrial discharges

Five tests, based on methods currently in use for measuring the inhibition of rates of respiration, nitrification, and growth of bacteria, are evaluated for use as screening tests for assessing the treatability of settled sewage and toxicity of industrial effluent. A treatability factor is derived for each test for a series of settled sewages and a toxicity factor for industrial effluents by comparison with control standards. The Microtox test gives greater sensitivity, repeatability, and precision, but possible problems in the interpretation of the results warrant further comparison with the remaining tests, all of which show reasonable promise.     

Chemical speciation and toxicity of metals assessed by three bioluminescence-based assays using marine organisms

Metal toxicity is a function of the biology of the target organism and the chemical speciation of the metal. The toxicity of 11 metals was assessed with three cell-based bioassays based on marine organisms: the bacterium Photobacterium phosphoreum of the Microtox bioassay, an environmental strain of P. phosphoreum, and photocytes isolated from the brittlestar Ophiopsila californica. Metal speciation was calculated for three commonly used media: NaCl-based Microtox bioassay medium, artificial seawater glycerol, and artificial seawater. Decreased bioluminescence was considered a proxy for cell toxicity. In all three assays the elements Cd and Hg exhibited similar speciation as well as similar toxicity profiles. The element Cu was toxic in all three assays despite different metal speciation for the P. phosphoreum bioassay. The element Ag was toxic to both bacterial strains but not to photocytes despite a similar chemical speciation for all three assays. In general, the Microtox bioassay was sensitive to all metals (except Pb), whereas the photocytes were the least sensitive to the metals. The heightened response of the Microtox bioassay probably resulted from a combination of the limited complexing power of the medium and the greater sensitivity of the bacterial strain.     

Acute toxicity assessment of industrial effluents with a microplate-based Hydra attenuata assay

The acute toxicity potential of ten industrial plants located in Toyama Prefecture (Japan) was appraised with a microplate-based assay developed with the freshwater cnidarian Hydra attenuata. Three measurement end points (LC₅₀, EC₅₀, and TC or “threshold concentration”) were determined based on specific morphological changes displayed by Hydra under conditions of progressive intoxication. Four effluents were shown to be lethal toward Hydra while eight induced sublethal toxicity responses, LC₅₀s varied from 18.8 to > 100% v/v, while EC₅₀s ranged from 15 to > 100% v/v. Similarly, lethal and sublethal TCs ranged from 17.7 to > 100% v/v and from 8.8 to > 100% v/v, respectively. Statistical analyses performed on all toxicity data for the ten effluents confirmed that the sublethal end points (EC₅₀s and sublethal TCs), previously unreported to assess complex wastewaters, proved to be more sensitive than the lethal end points (LC₅₀s and lethal TCs). This was also reflected by lethality to sublethality ratios, which ranged from 1 to 2.6 (LC₅₀/EC₅₀ comparisons) and from 1.1 to 5.4 (LC₅₀/TC comparisons) within a 96 h exposure period. Similar statistical analyses undertaken on 24, 48, 72, and 96 h toxicity data failed to show any significant time-related differences, thereby suggesting that an exposure time as short as 24 h would not diminish test sensitivity. Since Hydra displayed an apparent increase in sensitivity toward a few effluents with time of exposure, however, we would nevertheless recommend a 96 h time frame for this microtest. A correlation was also observed between conductivity and Hydra responses, highlighting a possible link to the presence of toxic metal ions. Based on our study, this simple and cost-effective microassay appears valuable as a (sub)lethal toxicity screening tool for effluents. Additional studies are planned with chemicals and other environmental matrices to better circumscribe its scope of usefulness. © 1997 by John Wiley & Sons, Inc. Environ Toxicol Water Qual 12: 53–60, 1997     

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.     

Ecotoxicological study of Lithuanian and Estonian wastewaters: selection of the biotests,and correspondence between toxicity and chemical-based indices

The toxicity of industrial and urban wastewater (WW) samples collected in Lithuania and Estonia was evaluated by using a suite of biological tests comprising the Algaltoxkit F with Selenastrum capricornutum, the Charatox with Nitellopsis obtusa, Daphtoxkit F with Daphnia magna, Thamnotoxkit F with Thamnocephalus platyurus, Protoxkit F with Tetrahymena thermophila and the Microtox with Vibrio fischeri. The Charatox and Thamnotoxkit F tests showed highest relative sensitivity, responding to 80-90% of samples, respectively, and both expressed good discrimination capacity between samples. Principal Component and pairwise correlation analysis allowed to select test-battery consisting of Charatox, Thamnotoxkit and Microtox. The WW toxicity was evaluated by means of cumulative indices such as average toxicity (AvTx) and two indices derived from the PEEP-index (Environ. Toxicol. Water Qual. 8 (1993) 115). In addition to these integrated evaluations of test-battery response, WW toxicity was evaluated according to the most sensitive test (MST) in the battery. The linear regression analysis between cumulative toxicity indices and chemical-based indices (derived from comparison of WW chemical concentrations and their respective maximum allowable concentration) revealed positive linear relationships (r(2)=0.7-0.8), while toxicity evaluation based on the MST was less positively related with chemical analysis data (r(2)=0.5-0.6). Although better coincidence between the toxicity and chemical-based assessments was achieved when information from all tests in the battery was assembled, the prediction of toxicity from chemical data was still limited. In search of suitable test-battery for the screening of certain type of WWs, a preliminary study comprising excessive suite of tests might be useful.     

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.     

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     

Bioassays for the Evaluation of Landfill Leachate Toxicity

This article reviews the application of bioassays for assessing the toxicity hazard posed by landfill leachate discharged to an aquatic environment. Landfill leachate is a complex mixture of chemicals; thus it is difficult to assess the risk posed to aquatic wildlife using standard chemical identification techniques, such as gas chromatography–mass spectroscopy (GC-MS). From this review it is clear that toxicity testing, using species that represent the different trophic levels, is a superior way to predict the risk posed by discharge than chemical analysis. Previous studies assessed leachate toxicity using bacteria, algae, plants, invertebrates, fish, and genotoxicity. Studies showed that leachate exhibits a wide range of toxicities to the species tested. Ammonia, alkalinity, heavy metals, and recalcitrant organics were identified to be the cause of adverse responses from the test organisms. Concentrations of these chemicals were found to depend upon the types of waste landfilled. As part of this review, Slooff analysis was applied to published results to calculate the sensitivity of test species. It was concluded that Lemna minor and Thamnocephalus platyurus were the most sensitive tests and, Vibrio fischeri (Microtox) was the least sensitive test available. Little is known about the sensitivity of each species to the different types of waste that might have been landfilled. A battery of tests needed for a more accurate assessment of landfill leachate is proposed. Some of the more common tests have been replaced by more sensitive tests that produce more relevant results for the industry and regulators.     

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.     

A Test Battery Approach for the Ecotoxicological Evaluation of Estuarine Sediments

The purpose of this study was to evaluate the overall sensitivity and applicability of a number of bioassays representing multiple trophic levels, for the preliminary ecotoxicological screening (Tier I) of estuarine sediments. Chemical analyses were conducted on sediments from all sampling sites to assist in interpreting results. As sediment is an inherently complex, heterogeneous geological matrix, the toxicity associated with different exposure routes (solid, porewater and elutriate phases) was also assessed. A stimulatory response was detected following exposure of some sediment phases to both the Microtox and algal bioassays. Of the bioassays and endpoints employed in this study, the algal test was the most responsive to both elutriates and porewaters. Salinity controls, which corresponded to the salinity of the neat porewater samples, were found to have significant effects on the growth of the algae. To our knowledge, this is the first report of the inclusion of a salinity control in algal toxicity tests, the results of which emphasise the importance of incorporating appropriate controls in experimental design. While differential responses were observed, the site characterised as the most polluted on the basis of chemical analysis was consistently ranked the most toxic with all test species and all test phases. In terms of identifying appropriate Tier I screening tests for sediments, this study demonstrated both the Microtox and algal bioassays to be more sensitive than the bacterial enzyme assays and the invertebrate lethality assay employing Artemia salina. The findings of this study highlight that salinity effects and geophysical properties need to be taken into account when interpreting the results of the bioassays.     

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     

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.     

Photobacterium phosphoreum toxicity bioassay. II. Toxicity data compilation

This review of Photobacterium phosphoreum toxicity bioassay, commonly known as the Microtox tests, consists of two parts. This second part (Toxicity Data Compilation) is a listing of all published and certain unpublished Microtox toxicity data for single organic compounds, including some salts and organometallic compounds. This listing consists of three tables: the first provides the reference source, toxicity value(s), chemical formula, name, the Chemical Abstracts Service (CAS) and Registry of Toxic Effects of Chemical Substances (RTECS) accession numbers, and molecular weight of each compound. At present, this table contains approximately 500 entries, which are ordered by the compounds' molecular formula. Two additional tables, ordered accordingly, are intended to provide quick access through cross-references with the CAS and RTECS numbers, respectively.     

ATP-TOX system—a new,rapid, sensitive bacterial toxicity screening system based on the determination of ATP

A new toxicity screening test, based on the inhibition of bacterial growth and luciferase activity by toxicants was developed. In the ATP-TOX System, chemical toxicity was found to be time-dependent and increased with increasing exposure time up to 5 hours. Three organisms were evaluated in this study: E. coli K-12 PQ37, Pseudomonas fluorescens and Salmonella typhimurium. E. coli K-12 PQ37 was found to be the most sensitive organism. It was also shown that P. fluorescens was more sensitive to toxicants when grown in minimal medium than in nutrient broth, suggesting that nutrients may have a protective effect on the bacterium. In comparative studies using selected toxic chemicals the ATP-TOX System was found to be more sensitive than the Spirillum volutans test and comparable to the Microtox test. Toxicant activity in sediment samples was found to be time dependent and increased with increasing exposure time in both ATP-TOX (E. coli) System and Microtox. The ATP-TOX System is complementary to the Microtox test as it also provides indications of low grade toxicant activity which is only manifested in actively growing cells over several life cycles. Thus, the ATP-TOX System appears to be an ideal screening test for sediment toxicity. The data indicate that the ATP-TOX System is sensitive, rapid, reproducible, economical and has great potential in applied studies.