A fluorescence-based bioassay for aquatic macrophytes and its suitability for effect analysis of non-photosystem II inhibitors

Background, Goals and Scope During the last years the miniaturization of toxicity test systems for rapid and parallel measurements of large quantities of samples has often been discussed. For unicellular algae as well as for aquatic macrophytes, fluorescence-based miniaturized test systems have been introduced to analyze photosystem II (PSII) inhibitors. Nevertheless, high-throughput screening should also guarantee the effect detection of a broad range of toxicants in order to ensure routinely applicable, high-throughput measuring device experiments which can cover a broad range of toxicants and modes of action others than PSII inhibition. Thus, the aim of this study was to establish a fast and reproducible measuring system for non-PSII inhibitors for aquatic macrophyte species to overcome major limitations for use. Methods A newly developed imaging pulse-amplitude-modulated chlorophyll fluorometer (I-PAM) was applied as an effect detector in short-term bioassays with the aquatic macrophyte species Lemna minor. This multiwell-plate based measuring device enabled the incubation and measurement of up to 24 samples in parallel. The chemicals paraquat-dichloride, alizarine and triclosan were chosen as representatives for the toxicant groups of non-PSII herbicides, polycyclic aromatic hydrocarbons (PAHs) and pharmaceuticals and personal care products (PPCPs), which are often detected in the aquatic environment. The I-PAM was used (i) to establish and validate the sensitivity of the test system to the three non-PSII inhibitors, (ii) to compare the test systems with standardized and established biotests for aquatic macrophytes, and (iii) to define necessary time scales in aquatic macrophyte testing. For validation of the fluorescence-based assay, the standard growth test with L. minor (ISO/DIS 20079) was performed in parallel for each chemical. Results The results revealed that fluorescence-based measurements with the I-PAM allow rapid and parallel analysis of large amounts of aquatic macrophyte samples. The I-PAM enabled the recording of concentration-effect-curves with L. minor samples on a 24-well plate with single measurements. Fluorescence-based concentration-effect-curves could be detected for all three chemicals after only 1 h of incubation. After 4–5 h incubation time, the maximum inhibition of fluorescence showed an 80–100% effect for the chemicals tested. The EC50 after 24 h incubation were estimated to be 0.06 mg/L, 0.84 mg/L and 1.69 mg/L for paraquatdichloride, alizarine and triclosan, respectively. Discussion The results obtained with the I-PAM after 24 h for the herbicide paraquat-dichloride and the polycyclic aromatic hydrocarbon alizarine were in good accordance with median effective concentrations (EC50s) obtained by the standardized growth test for L. minor after 7 d incubation (0.09 mg/L and 0.79 mg/L for paraquat-dichloride and alizarine, respectively). Those results were in accordance with literature findings for the two chemicals. In contrast, fluorescence-based EC50 of the antimicrobial agent triclosan proved to be two orders of magnitude greater when compared to the standard growth test with 7 d incubation time (0.026 mg/L) as well as with literature findings. Conclusion Typically, aquatic macrophyte testing is very time consuming and relies on laborious experimental set-ups. The I-PAM measuring device enabled fast effect screening for the three chemicals tested. While established test systems for aquatic macrophytes need incubation times of ≥ 7 d, the I-PAM can detect inhibitory effects much earlier (24 h), even if inhibition of chemicals is not specifically associated with PSII. Thus, the fluorescence-based bioassay with the I-PAM offers a promising approach for the miniaturization and high-throughput testing of chemicals with aquatic macrophytes. For the chemical triclosan, however, the short-term effect prediction with the I-PAM has been shown to be less sensitive than with long-term bioassays, which might be due to physicochemical substance properties such as lipophilicity. Recommendations and Perspectives The results of this study show that the I-PAM represents a promising tool for decreasing the incubation times of aquatic macrophyte toxicity testing to about 24 h as a supplement to existing test batteries. The applicability of this I-PAM bioassay on emergent and submerged aquatic macrophyte species should be investigated in further studies. Regarding considerations that physicochemical properties of the tested substances might play an important role in microplate bioassays, the I-PAM bioassay should either be accompanied by evaluating physicochemical properties modeled from structural information prior to an experimental investigation, or by intensified chemical analyses to identify and determine nominal concentrations of the toxicants tested. The chemicals paraquat-dichloride, alizarine and triclosan were chosen as representatives for the toxicant groups of non-PSII herbicides, PAHs and PPCPs which are often detected in the aquatic environment. Nevertheless, in order to ensure a routinely applicable measuring device, experiments with a broader range of toxicants and samples of surface and/or waste waters are necessary. ESS-Submission Editor: Dr. Markus Hecker (MHecker@Entrix.com)     

Development and validation of a new fluorescence-based bioassay for aquatic macrophyte species

Bioassays with unicellular algae are frequently used as ecotoxicological test systems to evaluate the toxicity of contaminated environmental samples or chemicals. In contrast, aquatic macrophyte test systems are still rarely used as they are laborious to handle because species exhibit distinct ecological requirements. The aim of this study was to establish a fast and reproducible measuring system for aquatic macrophyte species to overcome those limitations for use. Thus, a newly developed pulse-amplitude modulated chlorophyll fluorometer (Imaging-PAM) was applied as an effect detection in short-term bioassays with aquatic macrophyte species. This multiwell-plate-based measuring device enables the incubation and measurement of up to 24 samples in parallel. The Imaging-PAM was used (i) to establish and validate the sensitivity of the test systems to three Photosystem II (PSII) inhibitors (atrazine, prometryn, isoproturon), (ii) to compare the test systems with established biotests for macrophytes and (iii) to define necessary time scales in aquatic macrophyte testing. The results showed that fluorescence-based measurements with the Imaging-PAM allow rapid and parallel analysis of large amounts of aquatic macrophyte samples and of toxicants effects of the PSII inhibitors tested on aquatic macrophytes. Measurements revealed a good correlation between obtained median effective concentrations (EC50s) for the new and the established biotest systems. Hence, the Imaging-PAM measuring device is a promising tool to allow fast chemical effect screening for high amounts of samples with little time and material and thus offers scope for high-throughput biotesting using aquatic macrophyte species.     

Quantitative structure-activity relationships for toxicity and genotoxicity of halogenated aliphatic compounds: wing spot test of Drosophila melanogaster

Halogenated aliphatic compounds were evaluated for toxic and genotoxic effects in the somatic mutation and recombination test employing Drosophila melanogaster. The tested chemicals included chlorinated, brominated and iodinated; mono-, di- and tri-substituted; saturated and unsaturated alkanes: 1,2-dibromoethane, 1-bromo-2-chloroethane, 1-iodopropane, 2,3-dichloropropene, 3-bromo-1-propene, epibromohydrin, 2-iodobutane, 3-chloro-2-methylpropene, 1,2,3-trichloropropane, 1,2-dichloroethane, 1,2-dichlorobutane, 1-chloro-2-methylpropane, 1,3-dichloropropane, 1,2-dichloropropane, 2-chloroethymethylether, 1-bromo-2-methylpropane and 1-chloropentane. N-methyl-N-nitrosourea served as the positive and distilled water as the negative control. The set of chemicals for the toxicological testing was selected by the use of statistical experiment design. Group of unsaturated aliphatic hydrocarbons were generally more toxic than saturated analogues. The genotoxic effect was observed with 14 compounds in the wing spot test, while 3 substances did not show any genotoxicity by using the wing spot test at 50% lethal concentration. The highest number of wing spots was observed in genotoxicity assay with 1-bromo-2-chloroethane, 1,2-dichloroethane, 1,2-dibromoethane and 1-iodopropane. Nucleophilic superdelocalizability calculated by quantum mechanics appears to be a good parameter for prediction of both toxicity and genotoxicity effects of halogenated aliphatic compounds.     

Mutatox test: a new test for monitoring environmental genotoxic agents

In this study, Yamaska River water and Milli-Q water and organically extracted sediment extracts were used to evaluate the sensitivity of a new genotoxicity screening test, the Mutatox test. Also in this study, the samples were tested for acute and chronic toxicity using the following screening test procedures: Microtox, Daphnia magna, Ceriodaphnia reticulata and ATP-TOX Systems. The Mutatox test is based on the use of a dark mutant strain of Photobacterium phosphoreum and is sensitive to chemicals which are (1) DNA damaging agents (2) DNA intercalating agents, (3) DNA synthesis inhibitors and (4) direct mutagens. In this study, the Mutatox test was found to be a simple-to-perform sensitive procedure which added greater scope to the battery of tests approach. Preliminary indications are that this procedure may prove to be one of the more responsive and valuable tests in the 'battery of tests' approach to environmental screening.     

Relative sensitivity of one freshwater and two marine acute toxicity tests as determined by testing 30 offshore E & P chemicals

Acute toxicity of 30 offshore E & P (Exploration and Production) chemicals was measured using the three standard test organisms Daphnia magna (freshwater cladoceran), Acartia tonsa (marine copepod) and Skeletonema costatum (marine diatom alga). Test chemicals included 20 water-soluble and 10 (partially) non-soluble products. For 22 out of the 30 chemicals, the difference in sensitivity between the three tests varied within one order of magnitude. A very good correlation was found between the two marine tests (r = 0.96, P < 0.01, n = 30), and a correlation coefficient of r = 0.78 (P < 0.01, n = 30) was found between D. magna and both A. tonsa and S. costatum, individually. When the comparison of D. magna and A. tonsa sensitivity was based only on the water-soluble chemicals, a significantly higher correlation was obtained (r = 0.84, n = 20), indicating that the sample preparation method used for the (partially) non-soluble chemicals (the water accommodated fraction (WAF) method) induces additional variation between tests performed with different test media. (Partially) non-soluble chemicals are characterised by phase separation or precipitation at the concentrations used for testing. In a WAF-based test, each test concentration/exposure level is prepared separately, and following mixing and separation, only the water phase is used for testing. Toxicity is related to the amount of substance originally added to the mixing vessels. For 25 of the 30 chemicals, D. magna was found to be less sensitive than the marine copepod by a factor >2. The generally higher sensitivity of the marine toxicity tests compared to the Daphnia test emphasise the importance of using marine data for environmental hazard classification as well as for environmental risk assessment purposes.     

Ecotoxic Effect of Photocatalytic Active Nanoparticles (TiO2) on Algae and Daphnids (8 pp)

Background, Aim and Scope Due to their large potential for manifold applications, the use of nanoparticles is of increasing importance. As large amounts of nanoparticles may reach the environment voluntarily or by accident, attention should be paid on the potential impacts on the environment. First studies on potential environmental effects of photocatalytic TiO2 nanoparticles have been performed on the basis of widely accepted, standardized test systems which originally had been developed for the characterization of chemicals. The methods were adapted to the special requirements of testing photocatalytic nanoparticles. Materials and Methods: Suspensions of two different nanoparticles were illuminated to induce their photocatalytic activity. For testing, the growth inhibition test with the green alga Desmodesmus subspicatus and the immobilization test with the daphnid Daphnia magna were selected and performed following the relevant guidelines (algae: ISO 8692, OECD 201, DIN 38412-33; daphnids: ISO 6341, OECD 202, DIN 38412-30). The guidelines were adapted to meet the special requirements for testing photocatalytic nanoparticles. Results: The results indicate that it is principally possible to determine the ecotoxicity of nanoparticles. It was shown that nanoparticles may have ecotoxicological effects which depend on the nature of the particles. Both products tested differ in their toxicity. Product 1 shows a clear concentration-effect curve in the test with algae (EC50: 44 mg/L). It could be proven that the observed toxicity was not caused by accompanying contaminants, since the toxic effect was comparable for the cleaned and the commercially available product. For product 2, no toxic effects were determined (maximum concentration: 50 mg/L). In the tests with daphnids, toxicity was observed for both products, although the concentration effect-curves were less pronounced. The two products differed in their toxicity; moreover, there was a difference in the toxicity of illuminated and non-illuminated products. Discussion: Both products differ in size and crystalline form, so that these parameters are assumed to contribute to the different toxicities. The concentration-effect curves for daphnids, which are less-pronounced than the curves obtained for algae, may be due to the different test organisms and/or the differing test designs. The increased toxicity of pre-illuminated particles in the tests with daphnids demonstrates that the photocatalytic activity of nanoparticles lasts for a period of time. Conclusions: The following conclusions can be drawn from the test results: (I) It is principally possible to determine the ecotoxicity of (photocatalytic) nanoparticles. Therefore, they can be assessed using methods comparable to the procedures applied for assessing soluble chemicals. - (II) Nanoparticles may exert ecotoxicological effects, which depend on the specific nanoparticle. - (III) Comparable to traditional chemicals, the ecotoxicity depends on the test organisms and their physiology. - (IV) The photocatalytic activity of nanoparticles lasts for a relevant period of time. Therefore, pre-illumination may be sufficient to detect a photocatalytic activity even by using test organisms which are not suitable for application in the pre-illumination-phase. Recommendations and Perspectives: First results are presented which indicate that the topic 'ecotoxicity and environmental effects of nanoparticles' should not be neglected. In testing photocatalytic nanoparticles, there are still many topics that need clarification or improvement, such as the cause for an observed toxicity, the improvement of the test design, the elaboration of a test battery and an assessment strategy. On the basis of optimized test systems, it will be possible to test nanoparticles systematically. If a potential risk by specific photocatalytic particles is known, a risk-benefit analysis can be performed and, if required, risk reducing measures can be taken.     

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.     

Applicability of the bioluminescence inhibition test in the 96-well microplate format for PAH-solutions and elutriates of PAH-contaminated soils

The use of conventional plastic microplates for a miniaturised luminescent bacteria test may result in an underestimation of the toxicity for poorly water soluble highly adsorbing toxicants such as PAHs. In this study, the suitability of microplates for testing elutriates of PAH-contaminated soils was investigated. The LUMIStox test was performed as the standard test in the miniaturised format using contaminated soil elutriates and aqueous solutions of four selected PAHs (viz. naphthalene (NAP), acenaphthene (ACE), fluorene (FLU), and phenanthrene (PHE)). For the aqueous PAH-solutions, we observed reduced light inhibition values for the miniaturised bioassay when using black microplates made of polypropylene (PP) and polystyrene (PS) compared to the standard LUMIStox test. This phenomenon was most likely due to adsorption of toxicants to the microplate surfaces with PAHs of lower water solubility being significantly more affected; however, after minimizing the exposure of samples to plastic surfaces, polystyrene microplates revealed equivalent performance (>80% 'relative' light inhibition) to the standard glass cuvette test system. For soil elutriates, black microplates again exhibited slightly lower light inhibition values while white plates made of PS and Barex resulted in a pronounced overestimation of toxicity for a coloured soil elutriate. In general, microplates were applicable for testing elutriates of PAH-contaminated soils. In cases where samples are coloured or turbid, the application of black microplates is recommended.     

The application of bioassays as indicators of petroleum-contaminated soil remediation

Bioremediation has proven successful in numerous applications to petroleum contaminated soils. However, questions remain as to the efficiency of bioremediation in lowering long-term soil toxicity. In the present study, the bioassays Spirotox, Microtox, Ostracodtoxkit F, umu-test with S-9 activation, and plant assays were applied, and compared to evaluate bioremediation processes in heavily petroleum contaminated soils. Six higher plant species (Secale cereale L., Lactuca sativa L., Zea mays L., Lepidium sativum L., Triticum vulgare L., Brassica oleracea L.) were used for bioassay tests based on seed germination and root elongation. The ecotoxicological analyses were made in DMSO/H2O and DCM/DMSO soil extracts. Soils were tested from two biopiles at the Czechowice oil refinery, Poland, that have been subjected to different bioremediation applications. In biopile 1 the active or engineered bioremediation process lasted four years, while biopile 2 was treated passively or non-engineered for eight months. The test species demonstrated varying sensitivity to soils from both biopiles. The effects on test organisms exposed to biopile 2 soils were several times higher compared to those in biopile 1 soils, which correlated with the soil contaminants concentration. Soil hydrocarbon concentrations indeed decreased an average of 81% in biopile 1, whereas in biopile 2 TPH/TPOC concentrations only decreased by 30% after eight months of bioremediation. The bioassays were presented to be sensitive indicators of soil quality and can be used to evaluate the quality of bioremediated soil. The study encourages the need to combine the bioassays with chemical monitoring for evaluation of the bioremediation effectiveness and assessing of the contaminated/remediated soils.     

The comparison of rapid bioassays for the assessment of urban groundwater quality

Groundwater is a complex mixture of chemicals that is naturally variable. Current legislation in the UK requires that groundwater quality and the degree of contamination are assessed using chemical methods. Such methods do not consider the synergistic or antagonistic interactions that may affect the bioavailability and toxicity of pollutants in the environment. Bioassays are a method for assessing the toxic impact of whole groundwater samples on the environment. Three rapid bioassays, Eclox, Microtox and ToxAlert, and a Daphnia magna 48-h immobilisation test were used to assess groundwater quality from sites with a wide range of historical uses. Eclox responses indicated that the test was very sensitive to changes in groundwater chemistry; 77% of the results had a percentage inhibition greater than 90%. ToxAlert, although suitable for monitoring changes in water quality under laboratory conditions, produced highly variable results due to fluctuations in temperature and the chemical composition of the samples. Microtox produced replicable results that correlated with those from D. magna tests.     

Physicochemical substance properties as indicators for unreliable exposure in microplate-based bioassays

In the last years many efforts were made to transform standardized algal test protocols into low-cost microplate assays. While advantages were pointed out frequently, limitations are not systematically addressed, thus hindering a widespread utilisation. In this study a group of organic substances with a wide distribution of volatility (log K_AW from −6.53 to −2.13) and lipophilicity (log K_OW from 1.26 to 4.92) was investigated with respect to the influence of these physicochemical properties on their algal toxicity in different assays. Therefore the EC₅₀ values were determined with a microplate assay based on ISO 8692 protocol and the results were compared with those of an established algal growth inhibition test conducted in air tight glass vessels. Using the ratio of the EC₅₀ values, a clear connection between biological response and volatility as well as lipophilicity of test substances could be detected. Chemicals with a log K_OW higher than 3 or a Henry coefficient log K_AW higher than −4 were identified as less effective in the microplate assay than in the comparative assay. The loss in nominal concentration due to physicochemical properties could be shown to contribute to this using HPLC analysis. Consequently, when using microplate assay’s one should be aware that lipophilic and volatile chemicals might be underestimated in their toxicity, which could be indicated from evaluating related physicochemical properties modelled from structural information prior to an experimental investigation.     

A Strategy for the Evaluation of Sensory and Pulmonary Irritation Due to Chemical Emissions from Indoor Sources

Abstract

Sensory and pulmonary irritation are physiological responses to chemical exposure which result in characteristic, measurable changes in respiratory activity in mice. A standard method has been applied to the estimation of sensory irritation associated with a specific chemical exposure. This method has been correlated with human responses to these chemicals. Symptoms associated with chemical irritants are consistent with complaints due to problems with indoor air quality, which may include eye and upper respiratory tract irritation, headaches, and nausea. A stepwise strategy for assessing the contribution of indoor products to sensory and pulmonary irritation is discussed in the current paper. The strategy includes product emissions testing using dynamic environmental chambers, the selection of suspected irritants for respiratory irritation testing, respiratory irritation testing of individual compounds and representative mixtures using synthesized atmospheres, and the evaluation of test data to determine those compounds which may contribute to sensory and pulmonary irritation in humans. The current strategy is being applied to evaluate carpet system materials and their constituent chemicals.     

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.     

Genotoxicity detected in wild mice living in a highly polluted wetland area in south western Spain

A field study was carried out in the south of the Iberian Peninsula in an industrial area in the neighbourhood of Huelva city, SW Spain, and in a natural area (Do?ana National Park) for comparison, to estimate the genetic risk induced by environmental pollution in wild mice. Genotoxic effects in a sentinel organism, the Algerian mice (Mus spretus) free living in the industrial area were compared with animals of the same species living in the natural protected area. The single cell gel electrophoresis, or Comet assay, was performed as a genotoxicity test in peripheral blood of mice. Our results clearly show that mice free living in the contaminated area bear a high burden of genetic damage as compared with control individuals. The results suggest that the assessing of genotoxicity levels by the Comet assay in wild mice can be used as a valuable test in pollution monitoring and environmental conservation.     

Chemical and biological profiles of sediments as indicators of sources of contamination in Hamilton Harbour. Part II: bioassay-directed fractionation using the Ames Salmonella/microsome assay

Bottom sediment and suspended sediment samples from Hamilton Harbour (western Lake Ontario) and from a major tributary were profiled using a bioassay-directed fractionation approach. Sample extracts were fractionated using an alumina/Sephadex gel clean-up procedure to afford non-polar aromatic fractions which were characterized using chemical analyses and the Ames/microsome bacterial assay in Salmonella typhimurium strains YG1025 with the addition of oxidative metabolism (S9), and YG1024 without S9. Non-polar aromatic fractions of selected samples were separated by normal phase HPLC into 1-min fractions which were subjected to bioassay analyses. The bioassays using strain YG1025+S9, a TA100-type strain, were performed to assess genotoxicity arising from the presence of polycyclic aromatic hydrocarbons (PAH). Fractions which exhibited mutagenic activity contained PAH with molecular masses of 252, 276 and 278 amu; these fractions contained over 80% of the genotoxicity attributable to PAH. Individual compounds identified using Gas Chromatography-Mass Spectrometry analyses in these active fractions included benzo[a]pyrene, indeno[cd]pyrene and dibenz[a,h]anthracene. The YG1025+S9 mutagenic activity profiles were similar for all samples. Mutagenic activity profiles generated using strain YG1024-S9, a TA98-type strain sensitive to compounds characteristic of mobile source emissions, were very different. The mutagenic activities in strain YG1024-S9 were greatest for harbour-suspended sediment samples collected from sites impacted by a major tributary. Suspended sediments collected near areas known to contain high levels of coal tar-contamination in the bottom sediments contained higher levels of genotoxic PAH than suspended sediments collected from other areas of the harbour.     

Optimisation of a microbial bioassay for contaminated soil monitoring: bacterial inoculum standardisation and comparison with Microtox assay

This work represents the first step to set up a toxicity testing procedure and to evaluate the sensitivity of the test microorganism to several classes of environmental pollutants. First, three different techniques were employed to standardise the microbial inoculum, then two different toxicity assessment protocols have been compared: Microtox and a dehydrogenase (DHase) activity inhibition test. The main goal was the optimisation of a microbial bioassay based on the dehydrogenase activity (DHase) inhibition in Pseudomonas fluorescens bacterial strain ATCC 13525. Triphenyl tetrazolium chloride (TTC) was used as electron acceptor and its reduction produces Triphenyl formazane (TPF). The P. fluorescens DHase inhibition bioassay was investigated for being a reliable and rapid method for assessing toxicity. The optimisation of the operating conditions resulted in a repeatable bioassay. Then, P. fluorescens and Vibrio fischeri sensitivity were firstly compared by testing Zn++, one of the reference compounds for Microtox test. In addition, other compounds (Ni++, Cd++, Cu++, phenol) were also tested with both bioassays. A high statistical significance of data was obtained with the logistic curve. The present work has demonstrated that P. fluorescens is as sensitive as Microtox culture (V. fischeri), for some of the metal ions. With reference to organic compounds, the lower sensitivity of P. fluorescens to phenol makes its use difficult in organic polluted samples.     

Integral assessment of estrogenic potentials of sediment-associated samples

GOAL, SCOPE AND BACKGROUND: Exogenic endocrine-active substances are also called 'Endocrine Disrupting Chemicals' (EDC). They imitate or hinder the function of natural endogenic hormones or disturb the synthesis or the metabolism of hormones or of hormone receptors. The Enzyme-Linked Receptor Assay (ELRA) can detect estrogenic and anti-estrogenic effects at the level of receptor binding and is a useful tool for the integrative detection of contaminant effects. Although the test system has been used repeatedly in sediment assessments, the questions have remained concerning how it responds to variations in the physico-chemical matrix. For some bioassays, the salinity of the sample is a critical factor. This is especially relevant when testing wastewater samples or when sediment-associated samples in the tidal reaches of rivers are tested. Sediments in the tidal reaches of rivers change their salinity several times a day. Against this background, it would be beneficial to have a test procedure of known salinity tolerance. On account of this, the salinity tolerance of the ELRA was tested, assessed with reference substances at several salinity levels, and compared with the E-Screen method and a Yeast Estrogen Screen (YES), which are also frequently applied in environmental testing. The aim of this paper was to explore when the salinity limits within these test procedures are applicable. The trials should reveal the working range to be expected, characterize the salinity-dependent variations in sensitivity of the test, and provide options for methodological adjustments to improve the stability against increased salinity. METHODS: The ELRA was carried out with the human Estrogen Receptor alpha. (ER) using the same principle like a competitive immunoassay based on ligand-protein interaction. However, an essential difference is the use of a physiologically relevant receptor instead of an antibody as a linking protein. The ELRA measures the competition of sample estrogens and anti-estrogens against estradiol supplied as a BSA-coating conjugate for the binding site of dissolved ER. Estradiol or xeno-estrogen binding is quantified by a biotynilated anti-ER antibody and the subsequent measurement of peroxidase activity by a streptavidin-POD-biotin complex. The E-Screen was performed with the human breast cancer cell line MCF-7, which expresses the estrogen receptor constitutively. Cell proliferation depends on binding of estrogens or xeno-estrogens with the receptor. After incubation, estrogen-dependent cell growth was measured by sulforhodamin B staining. The YES was performed with a recombinant yeast strain, transfected with a receptor and a reporter plasmid bearing the estrogen receptor and a vitellogenin gene fused with the reporter gene lacZ. Estrogen or xeno-estrogen-dependent gene induction was measured indirectly by LacZ activity. The salinity levels were simulated in varying concentrations with NaCl from 0 to 40 per thousand or Artificial Sea Water (ASW) from 0 to 32 per thousand. RESULTS: The study characterized the factor 'salinity' for the prospective application fields of the ELRA. With reference substances such as 17-beta-estradiol, the ELRA showed classical sigmoidal concentration-effect relations in a range from 0.05 to 100 microg/l under physiological conditions. After a methodological adjustment to compensate decreasing receptor-binding affinity of estrogens and xeno-estrogens at higher salinity levels, the ELRA became applicable under salinity conditions up to concentrations of 20.5 per thousand. In tests, the ELRA reached under the influence of salinity a mean limit of detection of 0.062 microg/l 17-beta-estradiol. The mean relative inter-test error was around 11%. Above concentrations of 20.5 per thousand there is a risk of false negative assessment. Compared with the E-Screen method using the MCF7 cell line and the yeast estrogen test system (YES), the ELRA shows a lower sensitivity to 17-beta-estradiol. In the E-Screen, the cell proliferation was strongly reduced by sodium chloride induced cytotoxicity. In comparison with the E-Screen, the salinity tolerance of the YES and YAS methods is significantly higher. DISCUSSION: Despite adaption, total salinity tolerance could not be achieved with the ELRA. Freshwater samples were generally appraisable. Higher salinity levels above 20.5 per thousand would tend towards false negative results. The low inter-test error of 11% makes the ELRA suitable for the detection of estrogenic and anti-estrogenic potentials of single substances, substance mixtures, and of environmental samples. CONCLUSIONS: The ELRA is very fast and reproducible, it can be used for high-throughput screening in a microplate format at low cost, it is robust to microbial contamination, and is less susceptible to cytotoxic interferences than cell culture methods. RECOMMENDATIONS AND PERSPECTIVES: In their established form, the YES and the E-Screen methods are not applicable for liquid phase testing at higher salinity conditions. The salinity-adapted test version of the ELRA described here shows a broader working range for samples. Native water samples of more or less brackish origin or high-salinity effluent samples are testable. Results of tests with sediment associated samples of different salinity will be subject of a forthcoming publication.     

Hazard assessment of chemicals in soil

Based on a stepwise (tiered) approach, degradation, adsorption and leaching tests as well as various effect tests (using plants, microorganisms and animals) are recommended for the testing of environmental chemicals. If, after the tests of tiers 1 and 2, the results of a monospecies-effect-test (including a safety factor) are within the range of the predicted exposure, the ecotoxicological hazard should be determined using a terrestrial model ecosystem. Some of the tests for the proposed strategy were selected from practical experience in testing environmental chemicals in the laboratory, and some on the basis of a comprehensive literature review.     

Genotoxic assessment on river water using different biological systems

This paper reports genotoxicity and toxicity data in water samples collected in Sinos River, an important water course in the hydrographic region of Guaíba Lake, Rio Grande do Sul State, south of Brazil. This river is exposed to intense anthropic influence by numerous shoes, leather, petrochemical, and metallurgy industries. Water samples were collected at two moments (winter 2006 and spring 2006) at five sites of Sinos River and evaluated using in vitro V79 Chinese hamster lung fibroblasts (cytotoxicity, comet assay and micronucleus test) and Allium cepa test (toxicity and micronucleus test). Comet and micronucleus tests revealed that water samples collected exerted cytotoxic, toxic, genotoxic and mutagenic effects. The results showed the toxic action of organic and inorganic agents found in the water samples in all sites of Sinos River, for both data collections. The main causes behind pollution were the domestic and industrial toxic discharges. The V79 and A. cepa tests were proved efficient to detect toxicity and genotoxicity caused by complex mixtures. This study also showed the need for constant monitoring in sites with strong environmental degradation caused by industrial discharges and urban sewages.     

A comparative study on characterization of textile wastewaters (untreated and treated) toxicity by chemical and biological tests

Toxicity of textile wastewaters (untreated and treated) and their ingredient chemicals was quantified in terms of their chemical characteristics, fish (Gambusia affinis) mortality and end point growth responses of duckweed (Lemna aequinoctialis) in short-term bioassays. Other parameters of fish bioassay were erythrocyte morphology and its counts. Despite of a definite correlation between data of biological tests (LC/EC(50) values) with that of chemical tests, biological tests were found to be relatively more sensitive to both wastewaters and ingredient chemicals. Amongst all the examined parameters of test organisms, fish RBCs (morphology and counts) sensitivity to pollutants in the wastewaters was usually maximum and therefore, their study should be included in the routine fish bioassay. Other advantage of biological test such as on Lemna is even detection of eutrophic potential of wastewaters, as noted at their higher dilutions. The ingredient chemicals (major) contributing maximum toxicity to textile dye wastewater were, acids (HCl and H(2)SO(4)), alkali (Na(2)O SiO(2)), salt (NaNO(2)) and heavy metal (Cu), whereas dyes (4) were relatively less toxic.