Use of dimethyl sulfoxide as solubilization agent in the detection of 2,3,7,8-TCDD by radioimmunoassay

Immunoassays are potentially valuable tools for use in screening environmental samples for a broad range of contaminants, such as polychlorinated dibenzo-p-dioxins (PCDDs). The performance of the radioimmunoassay (RIA) for PCDDs was characterized using 4 solubilization systems: Cutscum, Triton, horse serum, and dimethyl sulfoxide (DMSO). The DMSO based assay appeared to perform best at low 2,3,7,8-TCDD levels. The effects of assay incubation time and hapten storage conditions on the DMSO based assay were assessed. The separation of bound from unbound radioactivity was accelerated without adversely affecting assay performance. Further assay development through the use of an alternative labelled hapten is considered.     

A fluorescence-based screening assay for DNA damage induced by genotoxic industrial chemicals

A rapid screening assay to detect chemically-induced DNA damage resulting from exposure of surrogate DNA to genotoxic compounds is reported. This assay is based on changes in the melting and annealing behavior observed for damaged DNA. Exposure of calf thymus DNA to genotoxic industrial chemicals reduced the extent to which the DNA annealed as measured using a double strand DNA selective fluorescent indicator dye. Formaldehyde, acrolein, crotonaldehyde and bromoethane showed the most prominent effects, chloroacetone and allylamine exhibited lesser effects, and acryrlonitrile showed no statistically significant assay response. The assay response for formaldehyde and crotonaldehyde were measured over the concentration range of 10-100 mM and 50-300 mM, respectively. This assay showed little response for the cytotoxic compounds phenol, cyclohexane and toluene but was sensitive to the effects of DNA damaging compounds such as mitomycin C and glutaraldehyde.     

Reducing bioassay variability by identifying sources of variation and controlling key parameters in assay protocol

Reducing bioassay variability by identifying sources of variation and controlling important parameters in assay protocol was demonstrated in this study. The variability of a bioassay based on a luminescent bacterium was examined as an example. This assay involved the growth of cells, storage at a low temperature, activation, and exposure to a test sample, and the assay response was bioluminescence inhibition. After determining that measurement error was small and negligible, the total assay variability was decomposed in an initial variance components study into between-batch, between-vial, and between-tube variations. Results indicated that between-vial variations accounted for the majority of the total observed variability and that reducing this type of variation would be beneficial. Five parameters in the assay protocol were determined as factors that potentially affected assay variability significantly. A split-plot design was employed to investigate the effects of these factors and some of their interactions on the assay response. One of the five factors, i.e., activation temperature, turned out to have a significant effect. The variance components study was repeated with better control of activation temperature as well as other parameters. Results indicated that the total variability of the bioassay was reduced by approximately 85%.     

Bait-lamina assay as a tool to assess the effects of metal contamination in the feeding activity of soil invertebrates within a uranium mine area

As part of the tier 2 of a site-specific risk assessment, this study was the first reporting an intensive in situ application of the bait-lamina assay; two exposure periods (7 and 14 days) were tested during four seasons in ten different sites, within a uranium mine area and at two different depths. The most contaminated sites (by deposition of sludge from the effluent treatment pond) were discriminated after 14 days of exposure because extremely low percentages of feeding activity were recorded. Previous sub-lethal ecotoxicological assays, already had demonstrated that the habitat function of these soils is compromised. Nevertheless, seasonality has proved to have a significant influence on responses. Thus to strength conclusions about the impact of contaminants, the in situ bait-lamina assay should be performed on different annual seasons, at least for temperate regions. It was also found that some environmental parameters (e.g. soil moisture and litter) can act as confounding factors in the bait-lamina assay.     

Quantitation of paralytic shellfish toxins using mouse brain synaptoneurosomes

A membrane potential assay based on synaptoneurosomes prepared from mouse brain was evaluated further for its utility in estimating saxitoxin and related bioactives. Saxitoxin concentrations quantitated in mussel extracts by the synaptoneurosomal technique correlated well with spiked concentrations in these samples (r2 = 0.995; slope=1.048). Other experiments found that the synaptoneurosomal assay can detect saxitoxin-like bioactives in zooplankton samples and the concentrations measured were consistent with preliminary estimations of saxitoxin equivalents using the [3H] saxitoxin receptor binding technique. Veratrine, a mixture of alkaloids that activate sodium channels, had similar potential as a substitute for veratridine in the synaptoneurosomal assay. The results provide additional evidence that the mouse brain synaptoneurosomal membrane potential assay has excellent capability for quantitation of saxitoxin-like activity in shellfish tissues and may also be applied to zooplankton samples.     

Development of an immuno-immobilized androgen receptor assay (IRA) and its application for the characterization of the receptor binding affinity of different pesticides

Pesticides are synthetic chemicals used not only for improving food and feed production but also for the protection of materials and of human health and well-being. Some of these substances are suspected for adverse effects attributable to an interaction with the endocrine system of vertebrates by mimicking or inhibiting endogenous hormones. One of the biological targets important in this relation is the androgen receptor (AR). To be able to screen environmental samples for the presence of compounds which might interfere with androgen action, we aimed to develop a receptor assay based on recombinant human AR (rhAR). We herein describe an rhAR assay in which the receptor is immobilized in microtiter plates via a specific antibody. The assay can be used for high throughput screening of chemicals spread into the environment. 29 of the most recommended pesticides of the Federal Country Hessen, Germany, were tested for their ability to displace [3H]-DHT bound to the rhAR. This evaluation included the major part of the most common herbicides, insecticides and fungicides and covered three potential groups of endocrine disrupting chemicals. For 28 of the substances evaluated, the relative binding affinity to the rhAR was below 0.1% when compared to dihydrotestosterone (DHT) (100%), only fentinacetate exhibited an affinity of 1.42%. An exchange assay indicated that the binding inhibition was reversible. In consequence, fentinacetate seems to be a hormonally active substance which may be present in vegetables or fish, but also on clothing. We conclude that further investigations on this compound and its metabolites are necessary.     

Genotoxicity assessment of acute exposure of chlorpyrifos to freshwater fish Channa punctatus (Bloch) using micronucleus assay and alkaline single-cell gel electrophoresis

Chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridylphosphorothioate) is one of the organophosphate pesticides widely used in agricultural practices throughout world and irreversible inhibitor of cholinesterase in all animal species. Limited efforts have been made to study acute genotoxic effects of chlorpyrifos (CPF) in different tissues of fish using genotoxic biomarkers. Therefore, the present investigation was aimed to study the induction of DNA damage by CPF in freshwater teleost fish Channapunctatus using micronucleus assay (MN assay) and alkaline single-cell gel electrophoresis (comet assay). The value of LC(50) - 96 h of CPF was determined as 811.98 microgl(-1) for C. punctatus, in a semi-static system and on the basis of LC(50) value three acute concentrations viz., 203, 406 and 609 microgl(-1) were determined. The fishes were exposed to the different concentrations of CPF for 96 h and samplings were done at regular intervals for assessment of the MN frequencies and DNA damage. In general, significant effects (P<0.01) from both concentrations and time of exposure were observed in exposed fishes. It was found that the micronucleus induction was highest on 96 h at all concentrations in the peripheral blood. Similar trend was observed for the DNA damage measured in terms of the percentage of tail DNA in the lymphocyte and gill cells. This study explored the combined use of micronucleus assay and comet assay for in vivo laboratory studies using fresh water fish for screening the genotoxic potential of xenobiotics.     

Development of a real-time polymerase chain reaction assay for the detection of the invasive Mediterranean fanworm, <Emphasis Type="Italic">Sabella spallanzanii</Emphasis>, in environmental samples

The Mediterranean fanworm, Sabella spallanzanii Gmelin 1791, was first detected in the Southern Hemisphere in the 1990s and is now abundant in many parts of southern Australia and in several locations around northern New Zealand. Once established, it can proliferate rapidly, reaching high densities with potential ecological and economic impacts. Early detection of new S. spallanzanii incursions is important to prevent its spread, guide eradication or control efforts and to increase knowledge on the species’ dispersal pathways. In this study, we developed a TaqMan probe real-time polymerase chain reaction assay targeting a region of the mitochondrial cytochrome oxidase I gene. The assay was validated in silico and in vitro using DNA from New Zealand and Australian Sabellidae with no cross-reactivity detected. The assay has a linear range of detection over seven orders of magnitude with a limit of detection reached at 12.4 × 10^?4 ng/μL of DNA. We analysed 145 environmental (water, sediment and biofouling) samples and obtained positive detections only from spiked samples and those collected at a port where S. spallanzanii is known to be established. This assay has the potential to enhance current morphological and molecular-based methods, through its ability to rapidly and accurately identify S. spallanzanii in environmental samples.     

Growth assays with mixed cultures of cyanobacteria and algae assessed by in vivo fluorescence: One step closer to real ecosystems?

A growth toxicity assay with mixed cultures of cyanobacteria and algae using in vivo fluorescence is presented. Test organisms (the green alga Pseudokirchneriella subcapitata and the cyanobacterium Aphanothece clathrata) growing alone and in a mixture were exposed to selected chemicals. P. subcapitata featured a higher sensitivity to toxicants in the presence of A. clathrata compared to the single species assay. On the other hand, growth of a cyanobacterium was not affected by the presence or absence of the green alga. The proposed method seems to be suitable for pre-screening studies of toxicants (algistatic agents, herbicides) applied into the aquatic environment and for the assessment of their impact on natural phytoplankton communities.     

Aporrectodea caliginosa, a suitable earthworm species for field based genotoxicity assessment?

There is a growing interest for the application of biomakers to field-collected earthworms. Therefore we have evaluated the usability of native populations of endogeic, widely distributed earthworm Aporrectodea caliginosa in the assessment of soil genotoxicity using the Comet assay. Validation of the Comet assay on earthworm coelomocytes has been established using commercially available Eisenia fetida exposed to copper, cadmium, and pentachlorophenol, along with A. caliginosa exposed to copper in a filter paper contact test. Neutral red retention time (NRRT) assay was conducted on copper exposed and field-collected earthworms. Significant DNA and lysosomal damage was measured using Comet and NRRT assays in native populations of A. caliginosa sampled from the polluted soils in the urban area in comparison to the earthworms from the reference site. The results of this study confirm the employment of A. caliginosa as a suitable species for the in situ soil toxicity and genotoxicity field surveys.     

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.     

Use of protein phosphatase inhibition assay to detect microcystins in Donghu Lake and a fish pond in China

Seasonal variations in the level of total microcystins in water samples collected from Donghu Lake and a fish pond in Wuhan, China, were studied between March 1995 and February 1996 using a protein phosphatase inhibition assay involving a radioactive 32P-labelled substrate. The assay is highly reliable and repeatable, and is probably the most sensitive assay for microcystin detection to date. Results of the survey indicated the presence of microcystins in the water samples, and the concentration of microcystins appeared to be related to the degree of eutrophication and water temperature. There is also a correlative relationship between the quantity of microcystins and the abundance of microcystin-producing cyanobacteria (Anabaena and Oscillatoria) in the water bodies over a year cycle. In the present study, the positive detection of microcystins in water bodies having no signs of algal bloom warns of considerable potential threat of these waters to public health.     

Toxicological effects of marine palytoxin evaluated by FETAX assay

The FETAX (frog embryo teratogenesis assay Xenopus) is considered a useful bioassay to detect health hazard substances. In the study of the marine toxin palytoxin (PTX), FETAX has revealed evident impacts on embryo mortality, teratogenesis and growth at the two highest (370 and 37nM) concentrations used. Significant mortality rates, peaks in the number of malformed embryos and delays in growth were found, while the total sample number fell by about 80% at the end of the assay with the concentrated dose. The histological analysis to evaluate the morpho-functional induced modifications demonstrated damage to the nervous and muscle tissue, a general reduction in the size of the main inner visceral organs and severe injury to the heart structure in some specimens. No inflammatory response was observed.     

Ecotoxicological Assessment of Sediment, Suspended Matter and Water Samples in the Upper Danube River. A pilot study in search for the causes for the decline of fish catches (12 pp)

Goals, Scope and Background Fish populations, especially those of the grayling (Thymallus thymallus), have declined over the last two decades in the upper Danube River between Sigmaringen and Ulm, despite intensive and continuous stocking and improvement of water quality since the 1970s. Similar problems have been reported for other rivers, e.g. in Switzerland, Great Britain, the United States and Canada. In order to assess if ecotoxicological effects might be related to the decline in fish catch at the upper Danube River, sediment, suspended matter and waste water samples from sewage treatment plants were collected at selected locations and analyzed in a bioanalytical approach using a battery of bioassays. The results of this pilot study will be used to decide if a comprehensive weight-of-evidence study is needed. Methods Freeze-dried sediments and suspended particulate matters were extracted with acetone in a Soxhlet apparatus. Organic pollutants from sewage water were concentrated using XAD-resins. In order to investigate the ecotoxicological burden, the following bioassays were used: (1) neutral red assay with RTL-W1 cells (cytotoxicity), (2) comet assay with RTLW1 cells (genotoxicity), (3) Arthrobacter globiformis dehydrogenase assay (toxicity to bacteria), (4) yeast estrogen screen assay (endocrine disruption), (5) fish egg assay with the zebrafish (Danio rerio; embryo toxicity) and (6) Ames test with TA98 (mutagenicity). Results and Discussion The results of the in vitro tests elucidated a considerable genotoxic, cytotoxic, mutagenic, bacteriotoxic, embryotoxic and estrogenic burden in the upper Danube River, although with a very inhomogeneous distribution of effects. The samples taken from Riedlingen, for example, induced low embryo toxicity, but the second highest 17β-estradiol equivalent concentration (1.8 ng/L). Using the fish egg assay with native sediments, a broad range of embryotoxic effects could be elucidated, with clear-cut dose-response relationships for the embryotoxic effects of contaminated sediments. With native sediments, embryotoxicity was clearly higher than with corresponding pore waters, thus corroborating the view that – at least for fish eggs – the bioavailability of particle-bound lipophilic substances in native sediments is higher than generally assumed. The effect observed most frequently in the fish egg assay was a developmental delay. A comparison of our own results with locations along the rivers Rhine and Neckar demonstrated similar or even higher ranges of ecotoxicological burdens in the Danube River. Conclusions The complex pattern of ecotoxicological effects caused by environmental samples from the Danube River, when assessed in an in vitro biotest battery using both acute and more specific endpoints, showed that integration of different endpoints is essential for appropriate hazard assessment. Overall, the ecotoxicological hazard potential shown has indeed to be considered as one potential reason for the decline in fish catches at the upper Danube River. However, based on the results of this pilot study, it is not possible to elucidate that chemically induced alterations are responsible for the fish decline. Recommendations and Perspective . In order to confirm the ecological relevance of the in vitro results for the situation in the field and especially for the decline of the grayling and other fishes, further integrated investigations are required. For linking the weight of evidence obtained by in vitro assays and fish population investigations, the application of additional, more specific biomarkers (e.g. vitellogenin induction, EROD and micronucleus assay) has been initiated in fish taken from the field as well as in situ investigations.     

Biacore biosensor immunoassay for 4-nonylphenols: assay optimization and applicability for shellfish analysis

A rapid Biacore biosensor immunoassay of 4-nonylphenols was developed. Two types of antibodies were used in the study: polyclonal antibodies with high cross-reactivity towards technical 4-nonylphenol and a monoclonal antibody very specific to 4-n-nonylphenol. 9-(p-Hydroxyphenyl)nonanoic acid was immobilized onto surface of a sensor chip. The best assay sensitivity was achieved using a flow rate of 50 microl min(-1) and injection time of 2 min. For the assay incorporating monoclonal antibodies a limit of detection 2 ng ml(-1) for 4-n-nonylphenol was achieved. With polyclonal antibodies one order lower sensitivity was observed for 4-nonylphenols. High background level of calibration curve for technical 4-nonylphenol was decreased by using IgG fraction of polyclonal antibodies in combination with lower amount of immobilised 9-(p-hydroxyphenyl)nonanoic acid. Sensitivity of the assay was improved by using a chip with a new derivative on a surface-N-aminobutyl [2-(4-hydroxyphenyl)ethylamine] (limit of detection--5 ng ml(-1)). Applicability of the developed assays to ecological monitoring was checked in experiments using shellfish samples. 4-n-Nonylphenol from spiked samples was extracted into hexane followed by clean-up on NH2 SPE columns. Calibration curves generated for cockles, mussels and oyster samples were identical (limit of detection about 10 ng g(-1)) whereas for scallop samples a slight decrease (about 5-10%) of absolute response was observed. In the assay using the monoclonal antibody specific to 4-n-nonylphenol 31 shellfish samples were found to be negative. Results obtained with polyclonal antibodies indicated that two scallop samples contained a quantity of 4-nonylphenols. The developed biosensor assay could be applied for shellfish analysis as a preliminary screening method.     

On the cytotoxicity of some microbial volatile organic compounds as studied in the human lung cell line A549

The cytotoxicity of 13 microbial volatile organic compounds (MVOC) was studied using a human lung carcinoma epithelial cell line A549 in a colony formation assay and two colorimetric assays: the microculture tetrazolium assay (MTT assay) and the cellular protein assay (methylene blue-MB assay). For comparison, two known cytotoxic substances: the non-volatile mycotoxin gliotoxin and the mono-functional alkylating agent methyl methanesulfonate (MMS) were studied. Concentration-response curves for each agent were established and the IC50 value (concentration resulting in 50% inhibition of colony growth or absorbance) was estimated. There are differences in toxicity levels between the MVOC tested and gliotoxin and MMS. The most toxic MVOC was 1-decanol which was as effective as MMS in all test systems. 1-decanol was about 10-fold more toxic than the other MVOC. All MVOC tested were more than 1000-fold less toxic than gliotoxin.     

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.     

Urban airborne particulate: genotoxicity evaluation of different size fractions by mutagenesis tests on microorganisms and comet assay

The genotoxic effects of different size fractions of airborne particulate (Total, PM₁₀ and PM_2.5), extracted with acetone or toluene, were evaluated by: the Ames plate test (TA98 and TA100 strains, w/o S9), gene conversion and reversion (w/o endogenous metabolic activation) in the Saccharomyces cerevisiae D7 strain, and the comet assay on human leukocytes. The data on human leukocytes confirm the sensitivity of the comet assay and its applicability to assess genotoxicity in environmental samples. The PM_2.5 fraction of airborne particulate generally shows the highest concentration of DNA-damaging compounds. Genotoxic response, in all the test systems applied, is highly dependent on extraction solvent used. Acetone seems to extract compounds with more similar genotoxic responses in the three test systems used than toluene extracts. Toluene appears to extract air pollutants genotoxic on yeast and leukocytes but is mainly cytotoxic on Salmonella.     

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).     

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.