Bioassays and biochemical biomarkers for assessing olive mill and citrus processing wastewater toxicity

The toxicity and the biochemical effects of olive mill wastewater and citrus processing wastewater were evaluated using acute toxicity bioassays (Gammarus pulex and Hydropsyche peristerica) and biochemical biomarkers (acetylcholinesterase [AChE] and glutathione S‐transferase [GST]). The bioassays indicated toxic properties of olive mill and citrus processing wastewaters. The 24 h LC₅₀ values of olive mill wastewater ranged from 2.64% to 3.36% for G. pulex and 3.62% to 3.88% for H. peristerica, while the LC₅₀ of citrus processing wastewater was 25.26% for G. pulex and 17.16% for H. peristerica. Based on a five‐class hazard classification system applied for wastewaters discharged into the aquatic environment, olive mill wastewater and citrus processing wastewater were classified as highly toxic and toxic, respectively. Results of the biochemical biomarkers showed that both agroindustrial effluents at increasing sublethal wastewater concentrations could cause inhibition of the AChE and induction of the GST activities. These first results showed that both species as well as their AChE and GST activities have the potential to be used as indicators and biomarkers for assessing olive mill and citrus processing wastewaters quality. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2011.     

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     

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.     

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.     

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.     

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.     

Toxicity of elemental sulfur in sediments

Elemental sulfur occurs naturally in marine and limnic sediments. Elemental sulfur, brought in solution in aqueous media by using organic solvents such as methanol as carrier solvent, was toxic in a bacterial luminescence test, known as the Microtox test. Previously, it has been shown that the toxicity in the luminescence test of whole sediments also was correlated to i.a. elemental sulfur using multivariate statistical analysis. Organic solvent extracts of sediments obtained in receiving waters of effluents from a pulp and paper mill was toxic in the luminescence test, and using a toxicity evaluation procedure, the toxic substance was identified as octameric cyclic sulfur, S₈. The substance dominated the toxicity in extracts of both a contaminated sediment and a sediment from a control area. Since the toxicity in the Microtox test of aqueous solutions of S₈ decreased upon storage, a conversion process of the toxic form was indicated. Acute toxicity of S₈ was not limited to the luminescent bacteria in the Microtox test, but was observed in tests with fish larvae if tested with the transient form of elemental sulfur. Tests of acute toxicity with zebra fish and perch larvae were responsive to elemental sulfur. Probably, the toxic form of elemental sulfur is the single cyclic octamer, that due to low aqueous solubility, binding to particulate sediment material or aggregation is converted into a nontoxic form. Acute toxic effects may occur in sulfur containing sediments of varying redox potentials or where elemental sulfur deposits are turbated. © 1998 John Wiley & Sons, Inc. Environ Toxicol Water Qual 13: 217–224, 1998     

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.     

Water and sediment ecotoxicity studies in Temuco and Rapel River Basin,Chile

Five samples were collected from the Rapel River Basin near the city of Rancagua and three from the city of Temuco. Three of the samples were collected from raw drinking water supplies. The following bioassays were performed on some or all of the samples: Microtox; Microtox solid phase test; SOS-Chromotest with and without S9; Toxi-Chromotest; Sediment-Chromotest; Panagrellus redivivus percent survival and percent maturation; submitochondrial reverse electron transfer and forward electron transfer tests; Daphnia magna 24 h acute toxicity; ECHA biocide monitor, and the competitive immunoassay tests for benomyl, metolachlor, atrazine, and triazines. All the sampling sites were positive for the presence of genotoxicants requiring S9 activation while three sites also indicated the presence of direct-acting genotoxicants/mutagens (−S9). Also, all the sites were positive for the presence of pesticides. In some samples there was 100% inhibition of P. redivivus maturation. Details and discussion on the implication of the results are presented. © 1996 by John Wiley & Sons, Inc.     

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 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.     

Toxicity estimation of treated coke plant wastewater using the luminescent bacteria assay and the algal growth inhibition test

Among several bioassays, the Scenedesmus subspicatus chlorophyll fluorescence test and the Photobacterium phosphoreum bioluminescence assay were selected to examine their applicability for toxicity evaluation of changes in coke plant effluent quality. In addition to the ecotoxicological parameters, a chemical analysis of dissolved organic carbon (DOC), ammonia-N, and nitrate-N was performed. It was demonstrated that the toxicity values obtained from the bioassays give a first indication on potential hazard and successful treatment. Decreasing toxicity values went along with decreasing DOC or ammonia-N values, but the sensitivity toward DOC reduction was higher. Sensitivity toward pure compounds was assessed by comparing the EC₅₀ values of the luminescent bacteria assay with the corresponding EC₅₀ values of the algal bioassay. The data showed a poor correlation between the two bioassays. © by John Wiley & Sons, Inc.     

Acute toxicity bioassay using Daphnia obtusa as a test organism

The introduction of the cladoceran Daphnia obtusa as a test organism in toxicity bioassays was considered. Development of bioassay included strain isolation, culture conditions, and toxicity test adaptation. Acute toxicity tests for Cr(VI), Mn(II), Cu(II), Cd(II), and Hg(II), phenol, sodium dodecyl sulfate, methanol, ethanol, and paraquat at 24 and 48 h of exposure show a sensitivity within the range reported for other cladocerans or fish. Sensitivity to carbaryl was higher than that found for fish. These results and the spread distribution of the species in the South of South America makes the bioassay a possible good option for the monitoring of toxicants in freshwater systems in the region. © 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).     

Ecotoxicological effects of larvicide used in the control of Aedes aegypti on nontarget organisms: Redefining the use of pyriproxyfen

The continued widespread use of larvicides in Aedes aegypti control programs is still a necessary strategy, since there are no apparent efficient vaccines against arboviruses. However, chemical approaches may affect nontarget organisms and produce detrimental effects to environmental health. Therefore, the aim of this study was to conduct toxicity testing for pyriproxyfen at different concentrations using Daphnia magna and Artemia salina as model organisms to evaluate the ecotoxicological parameters. This study describes the toxicological effects of pyriproxyfen on both microcrustaceans, which are widely used in bioassays because of their sensitivity to changes in hydrosphere. Data demonstrated that the calculated EC_50-48h value of pyriproxyfen was 2.5 μg/for D. magna and A. salina; the no-observed-effect concentration (NOEC) and the lowest-observed-effect concentration (LOEC) of pyriproxyfen were found to be 0.63 and 1.25 μg/L for Artemia salina and Daphnia magna, respectively. In chronic toxicity and reproduction tests on D. magna, a calculated CL_50-7day (lethality on 50% of daphnids after 7 days of chronic test) and an EC_50-21day (50% reduction in the reproductive output of parental daphnids after 21 days of exposure) higher than 1.25 μg/L pyriproxyfen were observed. The time of first reproduction was significantly increased in D. magna after exposure to environmentally relevant concentrations of pyriproxyfen, but other reproduction parameters were not markedly altered. Environmental risk assessment revealed that pyriproxyfen is highly toxic for both branchiopods. Data demonstrated that pyriproxyfen may produce adverse effects on the aquatic ecosystem at concentrations required to control Ae. aegypti.     

A new culture-free microbiotest for routine detection of cyanobacterial toxins

The Thamnotoxkit F^TM was evaluated for detecting cyanobacterial toxins. This is a 24 hour bioassay using, as test organisms larvae of the freshwater anostracan crustacean Thamnocephalus platyurus hatched from cysts. Freeze-dried Microcystis aeruginosa samples from freshwaters throughout Hungary were tested. Good correlation was obtained between the results of intraperitoneal mouse bioassays and Thamnotoxkit F^TM assays. The LC₅₀'s for purified microcystins- LR, -RR, and -YR ranged from 0.1 to 2.27 μg per mL, respectively in the Thamnotoxkit assay. The LC₅₀ of laboratory cultures of Cylindrospermopsis raciborskii isolated from samples from Lake Balaton, was 0.574 mg lyophilized material per mL, in a four-laboratory trial. Although not developed originally for detecting cyanobacterial toxins, the Thamnotoxkit F^TM has good potential as an initial screening procedure for toxicity assessment of cyanobacterial scums and blooms in freshwaters. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 466–472, 1999     

Comparison of toxicity detected by five bioassays during bioremediation of diesel fuel-spiked soils

Biodegradation of petroleum contaminants is an effective and generally inexpensive approach for reducing their concentrations in soils. However, little information is available on the toxicological status of contaminated soils and the fate of target hydrocarbons following bioremediation. Four texturally distinct soils were contaminated with diesel fuel and bioremediated in microcosms at 22°C, with moisture contents of 85% of soil water holding capacity, and nitrogen (N) and/or phosphorus (P) nutrient amendments. The progress of bioremediation was monitored using chemical and toxicological analyses. Soil toxicity was measured using five short-term bioassays: seed germination, red blood cell hemolysis, solid-phase Microtox, SOS-chromotest, and Toxi-chromotest. Reductions in target compound concentration were not always predictive of reductions in soil toxicity. Conflicting trends were indicated by the toxicity test results. For example, total petroleum hydrocarbon analysis revealed decreased hydrocarbon concentrations in all four soils following bioremediation but seed germination and seedling emergence data indicated increased soil toxicity. In contrast, the Microtox test data indicated decreased toxicity in two of the four soils. These results suggest that measurements of target contaminant concentrations should be complemented with several different soil toxicity bioassays, particularly when evaluating the ability of bioremediation to reduce the adverse effects of contaminants in soil. © 1998 John Wiley & Sons, Inc. Environ Toxicol Water Qual 13: 117–126, 1998     

Ecotoxicity and Environmental Risk Assessment of Larvicides Used in the Control of Aedes aegypti to Daphnia magna (Crustacea,Cladocera)

Dengue transmitted by mosquitoes of the genus Aedes, species aegypti, is a major public health concern in Brazil. The chemical control of the mosquito larvae has been performed with the larvicide temephos since 1967. However, vector resistance was reported to temephos in several Brazilian states, and the Ministry of Health ordered the replacement of this larvicide by diflubenzuron (DFB), an inhibitor of chitin synthesis. Both insecticides are diluted in water with larvae and are able to reach aquatic environments in which they subsequently adversely damage nontarget organisms. The aims of this study were to (1) determine the acute toxicity (EC50) and environmental risk (RQ) of DFB and temephos to the microcrustacean Daphnia magna, and (2) evaluate the chronic toxicity (no-observed-effect concentration [NOEC] and lowest-observed-effect concentration [LOEC]) of these larvicides to D. magna. The experiments were performed according to a completely randomized design. The estimated 48-h EC₅₀ of temephos was 0.15 μg/L (lower limit = 0.1 and upper limit = 0.2 μg/L) and the 48-h EC₅₀ of DFB was 0.06 μg/L (lower limit = 0.03 and upper limit = 0.1 μg/L). RQ values were 4.166.7 to DFB and 6.666.6 to temephos. NOEC and LOEC values were respectively 2.5 and 5 ng/L for DFB, and respectively 6.2 and 12.5 ng/L for temephos. Thus, temephos and DFB are classified as highly toxic to Daphnia magna and pose a high environmental risk to this species. Mortality of D. magna was observed at concentrations lower than those used in the field to control A. aegypti larvae.     

Estimating the toxicity of chlorinated organic compounds using A multiparameter bacterial bioassay

A battery of microbial tests that included contemporary measurement of specific growth rate, intracellular ATP level, and respiration rate proved capable of distinguishing among toxicants that acted primarily as (a) catabolic inhibitors, (b) uncouplers, and (c) inhibitors of biosynthetic processes. The concentration of chlorinated phenols and ethanes that produced a 50% reduction in specific growth rate (ECk₅₀) provided the most useful single-parameter indication of relative chemical toxicity generated within the test battery. Among the chlorinated ethanes tested, toxicity resulted from nonspecific chemical interactions with bacterial membranes, as indicated by correlation between toxicity (ECk₅₀) and measures of lipophilicity. The ECk₅₀ value for 1,2-dichloroethane was 8.5 × 10^?3M as compared to 1.7 × 10^?4M for the more hydrophobic pentachloroethane. Intermediate ECk₅₀ values were obtained for 1,1,1-trichloroethane and 1,1,2,2-tetrachloroethane. For a comparable degree of lipophilicity, the chlorinated phenols were about an order of magnitude more toxic than chlorinated ethanes. ECk₅₀ values for Spirochaeta aurantia at pH 7 ranged from 1.3 × 10^?3M for 2-chlorophenol to 9.4 × 10^?6M for penta-chlorophenol. There was indirect evidence that the toxicity of chlorinated phenols arises from both specific and nonspecific chemical interactions with bacterial membranes. The test battery proved capable of distinguishing among the relative toxicities of the chemicals tested at least as well as other bioassay procedures including Microtox and Daphnia-based tests. However, ECk₅₀ was less sensitive than parametric measures of chemical toxicity generated via other procedures.     

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