Bioassays with Vibrio fischeri for the assessment of delayed toxicity

The standardized bioluminescence assay with Vibrio fischeri underestimates the aquatic toxicity of chemicals which interfere with metabolic pathways supporting long term processes like growth and reproduction due to its short incubation time (30 min). Therefore this short term assay was compared with two alternative bioassays with prolonged incubation times using the same test organism: the growth inhibition assay (7 h) and the long term bioluminescence assay (24 h). Two sets of compounds were selected to reflect acute and delayed toxicity. The first group comprised pentachlorophenol, dodecylpyridiniumbromide and 3,4-dichloroaniline and the second nalidixic acid, chloramphenicol and streptomycinsulfate. The effects of compounds with acute toxicity are determined with similar sensitivity in all bioassays. Substances with delayed toxicity show only minor or no toxicities in the standardized short term bioassay but severe effects in both long term bioassays independent of the parameter used. It is concluded that the standardized short term bioluminescence assay exhibits serious limitations for the assessment of aquatic toxicity. The long term bioassays, however, may help to overcome these limitations.     

Toxicity testing with Vibrio Fischeri: A comparison between the long term (24 h) and the short term (30 min) bioassay

The acute bioluminescence inhibition assay using the marine bacterium Vihrio fischeri as the test organism is a widely used short term toxicity test. This paper compares the standard 30 minutes test with a 24 h assay using the same organism and the same test parameter. For that purpose concentration-response relationships were determined for a set of selected substances, reflecting different modes and mechanisms of action.

The results indicate a severe blind spot of the acute biotest: The toxicity of certain specifically acting chemicals is drastically underestimated here, while the chronic biotest gives a more reliable estimate of the toxicity of these substances.     

Ecotoxicological testing with new kinetic Photorhabdus luminescens growth and luminescence inhibition assays in microtitration scale

Miniaturized luminescence and growth inhibition assays in microtitration plates with the terrestric enthomopathogenic nematode symbiont Photorhabdus luminescens (DSMZ 3368T) are presented and compared with standardized tests with Vibrio fischeri (DSMZ 7151/NRRLB-11177) and Pseudomonas putida (DSMZ 50026). Toxicological parameters (EC and G values) of selected reference toxicants (e.g. heavy metals and environmental samples) were obtained at different temperatures and without sodium chloride supplementation. Kinetic data recordings were compared with results of a cuvette test protocol using integral and endpoint calculation. Growth inhibition experiments with reference samples reveal similar or higher sensitivities as for the Vibrio or Pseudomonas spp. The luminescence inhibition assay shows reduced sensitivities to most of the reference samples compared with the V. fischeri standard assay. But G values obtained with other standardized aquatic assay systems with daphnids, algae and growth inhibition assays with V. fischeri and Ps. putida correspond more closely to data observed with Ph. luminescens. The test procedures are easily to perform and to evaluate and seem to be reliable alternatives to the established protocols at low osmolarities. The sample specifity of the toxic responses of the marine and the terrestric strain recommends to employ both assays to determine the toxic potential of environmental samples. This will support to reduce false positive results in future investigations.     

Toxicity testing with luminescent bacteria – Characterization of an automated method for the combined assessment of acute and chronic effects

The luminescent bacteria test according to EN ISO 11348 is frequently applied in (eco) toxicity testing and is applicable for a huge variety of environmental and industrial samples. A big disadvantage of this method is the very short exposure time, which is expressed in a low sensitivity in regard to substances with a delayed effect. Chronic effects, i.e. interference with cell growth, cannot be assessed with this conventional standard method. The goal of this research was to develop an automated testing system for long term toxicity towards the luminescent bacteria Vibrio fischeri by implementing microtitration-based instrumentation. The optimized method, hereinafter referred to as “kinetic luminescent bacteria test”, can be described as a miniaturized combination of the conventional short-term luminescence inhibition test according to EN ISO 11348 and the Photobacterium phosphoreum growth inhibition test (DIN 38412-37). The validation procedure included the evaluation of six reference compounds (3,4-Dichloroaniline, 3,5-Dichlorophenol, Chloramphenicol, Streptomycin sulfate, Potassium dichromate, Zinc sulfate heptahydrate) and three different endpoints that are acute luminescence inhibition (acute LI) after 30 min, chronic luminescence inhibition (chronic LI) after 24 h and growth inhibition (GI) after 14 h. The optimized method allows the assessment of acute and chronic effects within one test, by what a misinterpretation of the toxicity of substances with delayed bacterial toxicity can be prevented, without abandoning most of the advantages of the conventional short-term test. Therefore, the kinetic luminescent bacteria test is exceptional as an initial screening test for environmental samples or substances with unknown (eco) toxicological characteristics.     

Toxicity of metals and organic chemicals evaluated with bioluminescence assays

The development of a bioluminescent sensor organism (Shk1) that was created for assessing wastewater toxicity was reported several years ago. In order to establish a test battery to better characterize wastewater toxicity, additional luminescent sensor organisms were later created. The present study focused on one promising candidate (PM6), a Pseudomonas spp. strain, because of its high level of luminescence compared to that of other newly created organisms. Using a batch toxicity testing protocol, the toxicity of 7 metals and 25 organic compounds was evaluated with the PM6 and Shk1 assays. Results indicated that the toxicity data of the PM6 and the Shk1 assays were correlated, and no assay appeared to be particularly more sensitive to a group of toxicants than the other assay. The results of the PM6 and Shk1 assays were further evaluated by comparing with the results of the Vibrio fischeri luminescence inhibition assay and activated sludge inhibition assays. Data suggested that PM6 and Shk1 more closely represented activated sludge organisms than V. fischeri. The suitability of using PM6 and Shk1 for assessing wastewater toxicity on activated sludge, both individually and in a test battery, was discussed.     

The toxicity of antibiotic agents to the luminescent bacterium Vibrio fischeri.

Despite their common use the fate and effects of antibiotics in the environment are largely unknown. These compounds may enter the environment through different pathways, resulting in the contamination of waste water or fresh water, where bacteria are most likely the primarily affected organisms. In this paper the toxicity of several drugs, reflecting the most important groups of antibiotics and chemotherapeutics, towards Vibrio fischeri are presented. The chronic bioluminescence inhibition assay with Vibrio fischeri is shown to be sensitive against many of the high volume antibiotics used for veterinary purposes and in aquaculture. Thus the assay may be a valuable tool for an effects assessment and biomonitoring of these xenobiotics. The available data for both parts of the risk assessment procedure--exposure assessment and effects assessment--have to be regarded as insufficient for most antibiotics. When the available data about environmental concentrations of antibiotics are compared with their toxicity towards Vibrio fischeri, direct effects on natural microbial communities are to be expected.     

Environmental assessment of ester-based lubricants after application

GOAL, SCOPE AND BACKGROUND: Lubricants based on renewable resources are an environmentally friendly alternative to petrochemical products due to their better ecotoxicological performance and their excellent biodegradability. To improve the technical performance of lubricants, and to reduce friction and wear, the use of additives is nowadays obligatory. The collaborative research center SFB 442 aims at developing environmentally acceptable lubricants that facilitate the avoidance of these additives by transferring their function to suitable coatings. For a complete assessment of the ecological performance of these newly developed lubricants, the whole life cycle including production, application as well as disposal and fate in the environment is studied. In the following study the focus was on the application and its influence on the environmental behavior of the lubricant. The application of lubricants leads, among other things, to the intake of metals due to abrasion from tools, work pieces or mechanical components. Previous examinations indicated a possible influence of metals on the toxicity of eluates prepared from used lubricants (Erlenkaemper et al. 2005). To clarify if the apparent toxicity of used lubricants solely results from the intake of metals, the extractability of these metals from the oil matrix is determined. By combining chemical analyses with bioassays, the bioavailability of metals that are present in the extract is estimated. To further investigate the relevance of metals on toxicity, toxic units (TU) were calculated and related to the results of the bioassays. Interactions between the metals were investigated with aqueous mixtures of metal chlorides and calculations based on the concept of concentration addition and independent action. METHODS: A lubricant mixture was applied to a tribological test bench that simulates real conditions of use and extremely short time load, respectively. Samples were taken at particular times, water soluble fractions (WSF) of these fluids were prepared and dilution series were investigated in several bioassays. Concentration of metals and total organic carbon (TOC) were determined in the eluates. TUs were calculated according to Sprague (1970) and mixture toxicity was calculated according to the concept of concentration addition (Loewe and Muischnek 1926) and independent action (Bliss 1939). RESULTS: Analyses of the metal content of the lubricant and the eluates clearly revealed the availability of the metals in the aqueous extracts. Especially copper, zinc, nickel and chromium were found and their concentrations increased during the time of use. The water extractable fraction, e.g., of copper, rose from 8.8% to 45.3% of the total content in the lubricant after 33.5 hours of use. Tests performed with the algal growth inhibition assay and the luminescence inhibition assay revealed the uptake or absorption by the organisms and, thus, the bioavailability of the metals. The calculation of TUs partly indicated a possible influence of the metals on ecotoxicity of the eluates. Copper reached concentrations equal to or higher than the EC50 value of copper chloride in the growth inhibition assays with algae and Ps. putida as well in the immobilization assay with daphnids. TUs for copper are also larger than 1 for the algal growth inhibition assay. The EL50 values indicated that the luminescence inhibition assay was the most sensitive test system, with values between 4.7% and 9.6%. While the toxicity towards algae and V. fischeri in the growth inhibition assay decreased until both organisms were no longer influenced by the exposure, the EL50 values for the D. magna immobilization assay and the Ps. putida growth inhibition assay decreased with the progressing use of the lubricant. The tested metal salt mixtures showed that Ps. putida, algae and daphnids are the most sensitive organisms with EC50 values below 1 mg/l. DISCUSSION: Although the intake of metals mainly occurred via abrasion of particles, the results revealed the availability of these metals in water. The availability varied for each of the four metals. For both the algal growth inhibition assay and the luminescence inhibition assay, an uptake or absorption of the metals could be demonstrated. The calculated TUs indicated an effect in some bioassays that was not verified in the test itself. The influence of copper on V. fischeri, for example, was not confirmed while the EL50 values for the daphnid bioassay decreased, meaning that the eluates became more toxic with progressing use of the lubricant. The calculations of mixture toxicity based on the concept of concentration addition demonstrated good correlations with the tested metal mixtures, but also a different sensitivity of the organisms. CONCLUSIONS: The results presented here reveal the availability of those metals in water that were taken in during the use of the lubricant in a tribological test bench and, thus, have the possibility of interacting with the organisms. The availability of the metals in the bioassays was proven by chemical analyses. The calculation of TUs and the corresponding EL50 values, however, indicate different availabilities of the metals. The results of the metal salt mixtures show good correlations with calculations of mixture toxicity based on concentration addition. Moreover, the varying sensitivity of the organisms when exposed to eluates or metal mixtures indicates a different bioavailability of the metals and/or the presence of other compounds that exert toxic action. RECOMMENDATIONS AND PERSPECTIVES: For further investigations, the organic oil matrix and its influence on the toxicity have to be taken into account. The toxicity of the eluates may not only be due to metals; additional effects could arise from changes in the lubricant itself.     

Comparative use of bacterial, algal and protozoan tests to study toxicity of azo- and anthraquinone dyes

Toxicity of two azo dyes (Reactive Orange 16 (RO16); Congo Red (CR)) and two anthraquinone dyes (Remazol Brilliant Blue R (RBBR); Disperse Blue 3 (DB3)) were compared using bacterium Vibrio fischeri, microalga Selenastrum capricornutum and ciliate Tetrahymena pyriformis. The following respective endpoints were involved: acute toxicity measured as bacterial luminescence inhibition, algal growth inhibition, and the effects on the protozoa including viability, growth inhibition, grazing effect and morphometric effects. In addition, mutagenicity of the dyes was determined using Ames test with bacterium Salmonella typhimurium His(-). DB3 dye was the most toxic of all dyes in the bacterial, algal and protozoan tests. In contrast to other dyes, DB3 exhibited mutagenic effects after metabolic activation in vitro in all S. typhimurium strains used. Of the methods applied, the algal test was the most sensitive to evaluate toxicity of the dyes tested.     

Comparison of bioluminescence and nitrification inhibition methods for assessing toxicity to municipal activated sludge

The aim of this study was to compare two alternative toxicity assessment methods to determine wastewater toxicity and predict treatment plant process upsets. The toxicity of two synthetic organic compounds (triclosan and 4-n-nonylphenol), which are commonly detected in municipal wastewater, and municipal and industrial wastewaters with different heavy metals content were evaluated by the nitrification inhibition assay and bioluminescence toxicity test. Comparison between both assays confirmed that Vibrio fischeri is generally more sensitive than autotrophic bacteria, and, if not calibrated, the bioluminescence method tends to overestimate toxic effects on activated sludge biomass. The nitrification inhibition assay appears to predict plant process upsets more accurately. Both methods showed a significant toxicity decrease through treatment that could be partially attributed to the significant heavy metals removal obtained by primary and secondary treatment. A good correlation for the two assays was obtained, as indicated by a high correlation coefficient (r2 = 0.80).     

Use of semi-permeable membrane devices to monitor pollutants in water and assess their effects: a laboratory test and field verification

Uptake of eight pesticides of different classes (organochlorines, synthetic pyrethroids, dinitroanilines, amides) by semi-permeable membrane devices (SPMDs) was studied in a laboratory continuous-flow system. After 20 days of exposure, membrane concentration factors were in the range of 50 000-120 000 for all the analytes except for the amide herbicides propachlor and alachlor, which were not detected in any of the SPMDs. The kinetic data and mathematical models were used to calculate analyte uptake rate constants, SPMD lipid/water partition coefficients, average sampling rates and concentrations of the pesticides in water. To assess the effects of concentrated pesticides, standard bioassays (Salmonella/histidine reversion assay and bioluminescence inhibition in Vibrio fischeri test) were incorporated in the SPMD technique. To test the method in an environmental situation, SPMDs were deployed in polluted sites in Lithuania. Polynuclear aromatic hydrocarbons were the major pollutants detected in the SPMDs deployed in the field. All the SPMD dialysates were highly toxic in the bioluminescence inhibition test but no mutagenic activity was observed in the Salmonella/histidine reversion assay.     

Comparison of bioassays by testing whole soil and their water extract from contaminated sites

The harmful effects of contaminants on the ecosystems and humans are characterised by their environmental toxicity. The aim of this study was to assess applicability and reliability of several environmental toxicity tests, comparing the result of the whole soils and their water extracts. In the study real contaminated soils were applied from three different inherited contaminated sites of organic and inorganic pollutants. The measured endpoints were the bioluminescence inhibition of Vibrio fischeri (bacterium), the dehydrogenase activity inhibition of Azomonas agilis (bacterium), the reproduction inhibition of Tetrahymena pyriformis (protozoon), and Panagrellus redivivus (nematode), the mortality of Folsomia candida (springtail), the root and shoot elongation inhibition of Sinapis alba (plant: white mustard) and the nitrification activity inhibition of an uncontaminated garden soil used as "test organism". Besides the standardised or widely used methods some new, direct contact ecotoxicity tests have been developed and introduced, which are useful for characterisation of the risk of contaminated soils due to their interactive nature. Soil no. 1 derived from a site polluted with transformer oil (PCB-free); Soil no. 2 originated from a site contaminated with mazout; Soil no. 3 was contaminated with toxic metals (Zn, Cd, Cu, Pb, As). In most cases, the interactive ecotoxicity tests indicated more harmful effect of the contaminated soil than the tests using soil extracts. The direct contact environmental toxicity tests are able to meet the requirements of environmental toxicology: reliability, sensibility, reproducibility, rapidity and low cost.     

Heavy metal toxicity to Lemna minor: studies on the time dependence of growth inhibition and the recovery after exposure

Environmental concentrations of toxic substances are not necessarily constant but fluctuate over time. Periods of intense exposure might be followed by episodes with a relatively low or no exposure, in principle allowing exposed organisms to recover from toxic injury. The growth reproduction assay with the limnic vascular plant Lemna minor allows for convenient studies on the time dependence of the aquatic toxicity of chemicals. Here we report on a study with four priority metals (Zn, Cu, Ni, Cd). Aims of the study were to determine the impact of the exposure duration on the observed toxicities and to determine the potential for recovery. The bioconcentrations of the test metals were recorded during the exposure in order to analyse, whether changes in the internal concentrations are a governing factor for the dynamics of toxicity. After an exposure of 7 days, Cd and Cu showed the highest toxicity to Lemna (EC50's of 1.9 and 9.7 microM respectively), while Ni and Zn had a slightly lower toxicity (EC50's of 56.3 and 46.1 microM respectively). Additionally, Zn showed a severely delayed toxicity and the exposed plants did not recover even 7 days after the exposure had ended. This is in sharp contrast to the other test metals, for which a considerable recovery was observed. These results indicate the necessity to more thoroughly consider the dynamics of toxicity, instead of recording toxic effects only after a constant exposure over a fixed time.     

Toxicity assessment of collected fractions from an extracted naphthenic acid mixture

Recent expansion within the oil sands industry of the Athabasca Basin of Alberta, Canada has led to increased concern regarding process-affected wastewaters produced during bitumen extraction. Naphthenic acids (NAs) have been identified as the primary toxic constituents of oil sands process-affected waters (OSPW) and studies have shown that with time, microbial degradation of lower molecular weight NAs has led to a decrease in observed toxicity. As earlier studies identified the need for an "unequivocal demonstration" of lower molecular weight NAs being the primary contributors to mixture toxicity, a study was initiated to fractionate an extracted NA mixture by molecular weight and to assess each fraction's toxicity. Successful molecular weight fractionation of a methylated NA mixture was achieved using a Kugelrohr distillation apparatus, in which fractions collected at higher boiling points contained NAs with greater total carbon content as well as greater degree of cyclicity. Assays with Vibrio fischeri bioluminescence (via Microtox assay) revealed that the lowest molecular weight NAs collected had higher potency (EC50: 41.9+/-2.8 mg l(-1)) than the highest molecular weight NAs collected (EC50: 64.9+/-7.4 mg l(-1)). Although these results support field observations of microbial degradation of low molecular weight NAs decreasing OSPW toxicity, it is not clear why larger NAs, given their greater hydrophobicity, would be less toxic.     

Ozonation of oxytetracycline and toxicological assessment of its oxidation by-products

Antibiotic formulation effluents are well known for their difficult elimination by traditional bio-treatment methods and their important contribution to environmental pollution due to its fluctuating and recalcitrant nature. In the present study the effect of ozonation on the degradation of oxytetracycline (OTC) aqueous solution (100mgl(-1)) at different pH values (3, 7 and 11) was investigated. Ozone (11mgl(-1) corresponds the concentration of ozone in gas phase) was chosen considering its rapid reaction and decomposition rate. The concentration of oxytetracycline, chemical oxygen demand (COD), biochemical oxygen demand (BOD) and BOD5/COD ratio were the parameters to evaluate the efficiency of the ozonation process. In addition, the toxic potential of the OTC degradation was investigated by the bioluminescence test using the LUMIStox 300 instrument and results were expressed as the percentage inhibition of the luminescence of the marine bacteria Vibrio fischeri. The results demonstrate that ozonation as a partial step of a combined treatment concept is a potential technique for biodegradability enhancement of effluents from pharmaceutical industries containing high concentration of oxytetracycline provided that the appropriate ozonation period is selected. At pH 11 and after 60min of ozonation of oxytetracycline aqueous solutions (100 and 200mgl(-1)) the BOD5/COD ratios were 0.69 and 0.52, respectively. It was also shown that COD removal rates increase with increasing pH as a consequence of enhanced ozone decomposition rates at elevated pH values. The results of bioluminescence data indicate that first by-products after partial ozonation (5-30min) of OTC were more toxic than the parent compound.     

A comparison of five rapid direct toxicity assessment methods to determine toxicity of pollutants to activated sludge

Five rapid direct toxicity assessment methods were used in three European partner countries to determine the toxicity of single toxicants, mixed toxicants and real industrial wastes. The final aim was to protect microbial degradation of organic wastes in biological treatment processes and hence enhance the quality of treated effluents to be discharged to the environment. Nitrification inhibition, Respirometry, Adenosine triphosphate luminescence and Enzyme inhibition were tested utilising activated sludge as the testing matrix. The Vibrio fischeri toxicity test was used as a surrogate to compare the various microbial bioassays. The IC50 (toxicant concentration eliciting a 50% inhibitory effect) was determined for a number of pollutants including single toxicants Cd, Cr, Cu, Zn, 3,5-dichlorophenol, toluene and linear alkylbenzenesulphonate (LAS); a standard mixture of metals and LAS; a standard mixture of organics and LAS, and 16 industrial effluents. The V. fischeri bioassay was also chosen in order to assess quality control of toxicant preparation during testing in the different laboratories of the partner countries. Comparisons of sensitivity, cost of implementation, cost per test, relevance, and ease of use were made. The most sensitive bioassays were V. fischeri and Nitrification inhibition, however, this depended in the main on the pollutant and mixtures tested. It is recommended that during assessment of wastewater toxicity a suite of tests be used rather than reliance on one particular test.     

Toxicity evaluation of reactive dyestuffs, auxiliaries and selected effluents in textile finishing industry to luminescent bacteria Vibrio fischeri

The toxicity of 17 selected process effluents, 11 reactive dyestuffs and 8 auxiliaries from a textile dyeing and finishing mill in Ayazaga, Istanbul, Turkey was evaluated by bioluminescence test using bacteria Vibrio fischeri in LUMIStox 300. The EC20 and EC50 for auxiliaries, the EC20 for dyestuffs were determined. For selected process effluents GL-values, the dilution level at which a wastewater sample causes less than 20% inhibition, were examined. Our results demonstrate that the toxicity assessment with luminescent bacteria is effective and of practical use for chemicals applied in textile finishing industry with the limitation of the deep dark-colored dye bath samples and for the related effluents. Inhibition effects of numerous dyestuffs as well as auxiliaries to luminescent bacteria differed considerably with a range 5-600 mg l(-1) for EC20 and 9-6930 mg l(-1) for EC50, respectively. Among 17 effluents, I sample exhibited high toxicity (GL = 100), 7 showed moderate toxicity (GL = 12-32), and 9 had a GL-value <10 indicating a low or no toxicity.     

Toxicity monitoring and classification of endocrine disrupting chemicals (EDCs) using recombinant bioluminescent bacteria

A recombinant bioluminescent Escherichia coli, DPD2794, containing the recA promoter region fused to luxCDABE originating from Vibrio fischeri was used for detecting genotoxicity caused by endocrine disrupting chemicals (EDCs) to cells. As well, several other recombinant bioluminescent bacteria, including TV1061, which is sensitive to protein damage (grpE::luxCDA BE), DPD2511, sensitive to oxidative damage (katG::luxCDABE), and DPD2540, sensitive to membrane damage (fabA::luxCDABE), were used for evaluating other possible modes of toxicity. It was found that the recombinant bacteria could monitor the toxic effect, not estrogenic effect, due to the presence of various EDCs through the measurement of bioluminescence (BL) and cell growth rate, which depend upon the type of toxicity occurring and which of the four strains was used. In addition, it was found that the damage caused by EDCs can be classified into several groups upon their mechanisms of toxic action.     

Biological and chemical analysis of the toxic potency of pesticides in rainwater.

A newly developed method for measuring the integrated esterase inhibiting potency of rainwater samples was applied in practice, and the results are compared to the toxic potency calculated from concentrations of 31 organophosphate (OP) and carbamate pesticides, out of a total of 66 chemically analyzed pesticides. In addition, the general toxic potency of the rainwater samples was evaluated in a microtiter luminescence assay with Vibrio fischeri bacteria. Rainwater samples were collected over four consecutive 14-day periods in both open and wet-only samplers. The esterase inhibiting potency of the open rainwater samples (expressed as ng dichlorvos-equivalents/l) corresponded well with the chemical analyses of the rainwater samples collected by both types of samplers (r = 0.83-0.86). By far, the highest esterase inhibiting potency was found in a sample collected in an area with intense horticultural activities in June, and was attributed to high concentrations of dichlorvos, mevinphos, pirimiphos-methyl and methiocarb. The esterase inhibiting potency of this sample was equivalent to a dichlorvos concentration of 1380 ng/l in the rainwater, which is almost 2000 times higher than the maximum permissible concentration (MPC) of dichlorvos set for surface water in Netherlands. Maximum individual concentrations of dichlorvos and pirimiphos-methyl even exceeded the EC50 for Daphnia, suggesting that pesticides in rainwater pose a risk for aquatic organisms. Not all responses of the luminescence-assay for general toxicity could be explained by the analyzed pesticide concentrations. The bio-assays enable a direct assessment the toxic potency of all individual compounds present in the complex mixture of rainwater pollutants, even if they are unknown or present at concentrations below the detection limit. Therefore, they are valuable tools for prescreening and hazard characterization purposes.     

Automated color correction method for Vibrio fischeri toxicity test. Comparison of standard and kinetic assays.

We demonstrate in this study that the toxicity of solid and highly colorful samples can be measured with kinetic bioassay using luminescent bacterium Vibrio fischeri. The Flash assay, named after the test protocol, is performed with a tube luminometer. In this method, each sample acts as a reference for itself, and therefore, the color correction is possible with minimal hands-on-time. The bacteria are dispensed into the sample and the signal is recorded continuously. The maximum signal received after immediately dispensing is compared to the signal after an incubation period. With many chemicals, the toxic effects are obtained after a very short contact time. However, different chemicals have different modes of toxicity. Thus, kinetic data from sample analyses after 15 or 30 min for this bacterium gives an additional dimension for obtaining reliable results. The performance of the test was compared to the standardized photobacteria test protocol with reference chemicals. The repeatability of the test was excellent. The coefficient of variation was normally below 1% with 10 replicates.     

Toxicity characterisation of sediment porewaters collected from UK estuaries using a Tisbe battagliai bioassay

The lethal toxicity of sediment porewater isolated from samples collected from six United Kingdom estuaries was tested using a Tisbe battagliai bioassay. A selection of Phase 1 toxicity identification evaluation (TIE) procedures was then used to characterise the substances responsible for the measured toxicity. In samples collected in 2000, cationic metals, ammonia, organic compounds were identified as the cause of toxicity in the most toxic sample collected. Some of the toxicity remained uncharacterised. It was shown that the toxicity in samples collected from the same locations in 2001 was due to ammonia. The successful application of the T. battagliai bioassay demonstrates that it is a suitable assay for marine sediment porewater TIE and that combined with simple characterisation procedures allow an initial assessment to be made on the type of substances responsible for the measured toxicity.