Effect of suspended solids concentration and filament type on the toxicity of chlorine and hydrogen peroxide to bulking activated sludge

Toxic inhibition of three communities of filamentous microorganisms by chlorine and hydrogen peroxide was investigated in relation to the total suspended solids concentration of bulking acitvated sludges. The filament communities studied were dominated by type 1701, Sphaerotilus natans, and the combination of type 021N and Thiothrix spp., respectively. Activity of filamentous and gross biomass was assessed separately using the malachite green-INT (MINT) test. The effectiveness of toxicants was characterized in terms of the concentration required to inhibit 50% of microbial dehydrogenase activity (IC₅₀). IC₅₀s were found to be proportional to total suspended solids concentration. Values for filamentous biomass were generally lower than those for gross biomass. Relative sensitivity (ratio of gross IC₅₀ to filamentous IC₅₀) did not vary significantly over the practical range of total suspended solids concentraton. This parameter was unaffected by the nature of the toxicant in S. natans and type 021N/Thiothrix dominated sludges, but was toxicant specific in type 1701 dominated sludge. Overall, S. natans and type 1701 were the least sensitive of the filament types studied. Bulking caused by these organisms has been successfully cured by toxicant addition in numerous past instances. It is therefore expected that type 021N, Thiothrix, and most other types of filamentous bulking can also be controlled by this technique.     

Enzyme biosynthesis versus enzyme activity as a basis for microbial toxicity testing

A toxicity assay based on inhibition of β-galactosidase biosynthesis was compared to a similar assay based on inhibition of β-galactosidase activity. In both tests, Escherichia coli grown in a minimal medium was exposed to isopropyl-β-thiogalactoside to induce β-galactosidase biosynthesis. The induction step preceded contact of cells with the toxicant in the enzyme activity assay, whereas in the enzyme biosynthesis test, the inducer was added following contact of cells with the toxicant. Relative sensitivity was judged on the basis of responses to the heavy metals Hg²⁺, Cu²⁺, and Cd²⁺, and the organics 3,4-dichlorophenol, formaldehyde, Hydrothol, phenol, sodium dodecyl sulphate, and toluene. Comparison of these results to the IC₅₀s achieved with other microbial systems, Daphnia bioassay, and fish bioassay indicates that the enzyme activity test was moderately sensitive to heavy metals but was insensitive to organic toxicants. The test based on inhibition of enzyme biosynthesis was sensitive to both heavy metals and toxic organics.     

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.     

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.     

A comparison of techniques used to extract solid samples prior to acute toxicity analysis using the microtox test

To date, there has been no widely accepted or standardized procedure for preparing leachates of solid samples for acute toxicity bioassays such as Microtox. Leaching procedures reported in the literature were evaluated for use with nonspecific environmental sample submissions. Using toxic environmental samples, two experiments were conducted to compare the effect that varying selected parameters would have on the effectiveness of the leaching procedure in removing toxicants from a solid sample matrix. Parameters that were varied included the type of toxicant, aqueous solvent system used to leach the sample, ratio of solid:liquid, type of mixing action, length of extraction time, and method of removing suspended solids from the leachate. Results from these experiments demonstrated that unacceptable variations of measured toxicity of a solid sample occurs with simple modifications of the method. The most dramatic shifts in 15 min EC₅₀ values were observed between vacuum filtering (EC₅₀ mean = 55.8%) and centrifuging (EC₅₀ mean = 22.3%) samples to clarify leachates. This and other significant interactions, between sample contaminant and aqueous system, suggest that no standard procedure will give readily interpretable results for different types of contamination. Investigators should carefully consider the application of the assay (e.g., the detection of toxicants using a sensitive method, or modeling the leaching of toxicants under specific environmental conditions) before selecting a leaching procedure. © 1996 by John Wiley & Sons, Inc.     

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.     

Probability distributions of toxicant sensitivity for freshwater rotifer species

Probability distributions of species sensitivities describe variation among species in response to toxicants. These distributions indicate what proportion of species assemblage is expected to suffer adverse effects upon toxicant exposure. Sensitivity distributions demonstrate how representative model species used in toxicity tests are of entire species assemblage. Despite theoretical importance in ecological risk assessment, few probability distributions were characterized for aquatic animals. We characterized sensitivity distributions for nine freshwater rotifer species to pentachlorophenol and cadmium. Endpoints were 24 h mortality and 30 min in vivo enzyme activity. The 95% confidence limits for mean PCP LC50s were 1.3–183 and 33–187 μg L⁻¹ for cadmium. A log toxicant concentration plot vs rank species susceptibility was constructed for each toxicant. A Kolmogorov–Smirinov goodness of fit test indicated these distributions did not differ significantly from normality, with R²s ranging 0.75–0.89. A species' sensitivity rank commonly changed three or four ranks with toxicant or endpoint change. Across toxicants and endpoints, Asplanchna girodi was the least sensitive species with mean percentile rank 78 and Trichocerca pusilla was most sensitive with rank 25 percentile. The Rotifer species most commonly used in toxicity tests, Brachionus calyciflorus, ranked 72nd percentile, placing it among the least sensitive rotifer species. Tenth centile for rotifer exposure to PCP is 2.8 and 17.2 μg L⁻¹ for cadmium. These values are near both toxicants' lower 95% confidence limit for all nine rotifer species. Toxicity assessments based only on B. calyciflorus with PCP LC50 of 160 μg L⁻¹ and cadmium LC50 of 270 μg L⁻¹ were not protective of most sensitive rotifer species by more than an order of magnitude. Sensitivity distributions characterization is likely to define toxicant concentrations which are harmful for rotifer assemblages better than any single species toxicity test. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 361–366, 1999     

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     

Rapid toxicity assessment using esterase biomarkers in Brachionus calyciflorus (rotifera)

We have developed biomarkers of sublethal toxicity in the freshwater rotifer Brachionus calyciflorus based on the reduction of enzyme activity. Esterase and phospholipase A2 activity was quantified in single rotifers using image analysis and a fluorescence detection system. Esterase activity was localized in the gut and phospholipase A2 activity in the corona of females. Quantitation of enzyme activity demonstrated that toxicant stress reduced activity in a dose-dependent manner. Concentration-response relationships are described for 10 compounds representing a variety of toxicant classes and NOECs are reported. Esterase and phospholipase A2 activities were generally less sensitive end points than reproduction NOECs, but usually were more sensitive than LC₅₀s. Since in vivo enzyme activity can be assessed in 1 h, these biomarkers will be useful where rapid results are important. The cost of performing in vivo enzyme inhibition tests is substantially less than traditional whole animal tests because these require three times more person-hours to execute. Obtaining test animals by hatching cysts, their sensitivity to toxicants, and the rapid results make the rotifer esterase and phospholipase A2 tests good candidates for inclusion in a test battery for rapid toxicity assessment. © 1994 by John Wiley & Sons, Inc..     

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.     

ISTA13--international interlaboratory comparative evaluation of microbial assay for risk assessment (MARA)

The microbial assay for risk assessment (MARA) is an innovative system based on an array of 11 different microbial species freeze-dried in a 96-well micro-titer plate. Developed for testing the toxicity of chemicals, mixtures, and environmental samples, the assay employs species of a taxonomically diverse range. In addition to 10 prokaryotic species, a eukaryote (yeast) is included in the range. The MARA's innate scope of a multi-dimensional test allows determination of toxicity based on a unique assay fingerprint or index, numerically expressed as the mean microbial toxic concentration (MTC). The most significant potential of the test is in the additional inference that can be conveyed to the toxicity evaluation because of the presence of each of the constituent species. The performance of MARA was evaluated to ascertain its capability and potential scope in an intralaboratory trial. Sensitivity to toxicants and different environmental samples was assessed. Evaluation included comparison with other tests; namely Microtox, invertebrate (Daphnia magna and Thamnocephalus platyurus) microbiotests, and respiration- and nitrification-inhibition tests. MARA's performance was further assessed with the implementation of an international interlaboratory trial. This involved the participation of 13 laboratories ranging from academic establishments to regulatory agencies. The results of the testing will be presented with assessment of the extent of variability and specific assay components. The trial evaluation indicated that performance of the assay was satisfactory and the results were within the acceptable range. MARA is a robust multispecies assay offering scope for toxicity assessment of a diverse range of samples.     

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).     

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.     

Using Proales similis (Rotifera) for toxicity assessment in marine waters

There is a need to develop more animal species for assessing toxicity in marine environments. Cyst‐based toxicity tests using invertebrates are especially fast, technically simple, cost‐effective, and sensitive to a variety of toxicants. Over the past 30 years, a variety of toxicity endpoints have been measured using the marine rotifer Brachionus plicatilis hatched from cysts, including mortality, reproduction, ingestion, swimming, enzyme activity, and gene expression. A consensus has developed that the most ecologically relevant toxicity measurements should be made using more than one species. Furthermore, it has been noted that the rotifer species toxicant sensitivity distribution is much broader than which endpoint is measured. This implies that toxicity should be measured with the simplest, fastest, least expensive test available on as many species as feasible. If a battery of test species is to be used to estimate toxicity, diapause egg‐based toxicity tests that do not require culturing of test animals will be key. In this paper, we describe how diapause eggs of a new marine rotifer, Proales similis, can be produced, stored and hatched under controlled conditions to produce animals for toxicity tests. Methods are described for quantifying the toxicity of copper, mercury and cadmium based on mortality, ingestion, reproduction, and diapause egg hatching endpoints. We found that reproduction and ingestion endpoints were generally more sensitive to the metals than mortality or diapause egg hatching. When the copper sensitivity of P. similis was compared to Brachionus manjavacas and B. plicatilis using an ingestion test, similar EC50s were observed. In contrast, the B. rotundiformis ingestion EC50 for copper was about 4× more sensitive. Although diapause egg hatching was not the most sensitive endpoint, it is the most ecologically relevant for assessing sediment toxicity. Our discovery of diapausing eggs in the P. similis life cycle has created a conundrum. We have not observed males or sex in P. similis populations, which is a direct contradiction to the orthodox view of the monogonont rotifer life cycle. Work is needed to clarify how diapause egg production is accomplished by P. similis and whether sexual reproduction is involved.     

Application of a toxicity identification evaluation for a sample of effluent discharged from a dyeing factory in Hong Kong

A first toxicity identification evaluation (TIE) was conducted in three phases using the Microtox test to identify the major toxicant(s) in effluent discharged from a dyeing plant in Hong Kong. In Phase I toxicity characterization indicated that anions were likely to be the major toxicants for the entire effluent. In Phase II concentrations of sulfite and other anions in the original and the anion exchange resin-treated effluent samples were determined by ion chromatography. Anions, which were found in the effluent at comparatively high concentrations and were suspected of being responsible for the toxicity to luminescent bacteria, were selected for further study in Phase III. Investigation in Phase III using the spiking and mass balance approaches confirmed that the sulfite ion was the major toxicant in the effluent.     

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.     

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.     

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.     

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     

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.