Selecting a battery of bioassays for ecotoxicological characterization of wastes

This study was conducted in France within the context of waste classification (Hazardous Waste Council Directive 91/689/EEC), and focused on "ecotoxic" property (H14). In 1998, an experimental test strategy was developed to assess ecotoxicological properties of wastes using a battery of six standardized bioassays. This combined direct and indirect approaches integrating two solid-phase tests: emergence and growth inhibition of Lactuca sativa (14 days), mortality of Eisenia fetida (14 days) and four standardized tests performed on water extracts from wastes: growth inhibition of Pseudokirchneriella subcapitata (3 days), inhibition of mobility of Daphnia magna (48 h), inhibition of reproduction of Ceriodaphnia dubia (7 days), inhibition of light emission of Vibrio fischeri (30 min). This study aimed to set up preliminary conclusions on relevancy of this experimental test strategy, based on data obtained since 1998. Results were analyzed from the combined use of Hierarchical Cluster Analysis, Principal Component Analysis and Nonlinear Mapping. These multivariate analyses clearly showed that it was possible to reduce this number of tests without changing the typology of the wastes. A battery of bioassays including one solid phase test and two tests performed on water extracts (L. sativa, V. fischeri and C. dubia) was found as an optimal solution for characterizing the toxicity of the studied wastes. This optimal battery represents a good basis for determining the H14 property.     

A Daphnia magna feeding bioassay as a cost effective and ecological relevant sublethal toxicity test for Environmental Risk Assessment of toxic effluents

Environmental Risk Assessment of chemical products and effluents within EC countries require the use of cost effective standardized toxicity tests that in most cases are restricted to acute responses to high doses. Thus, subtle ecological effects are underestimated. Here we propose a short-term one day Daphnia magna feeding inhibition test as a cost effective and ecological relevant sublethal bioassay. The sensitivity and reliability of the proposed bioassay was tested in the laboratory against standardized bacteria, algae growth, D. magna and fish acute toxicity test by using 16 chemical mixture x water type combinations that included four different water types fortified with four complex chemical mixtures. Water types included ASTM hard water and three selected effluents diluted 1/10 in water to mimic worse field situations that many overexploited arid river ecosystems suffer during summer months when effluents are discharged into them with little dilution. The results obtained denoted a greater sensitivity of the proposed feeding bioassay in 51 out of 65 tests performed with an average sensitivity 50 fold greater than that of the standardized tests. The greater differences were obtained for mixtures that included narcotic chemicals and the lowest differences for those containing pesticides. Furthermore, feeding responses to the studied contaminant mixtures behaved differently to increasing TOC content than those based on bioluminescent bacteria and algae. Increasing TOC coming from sewage treated effluents decrease toxicity to the latter bioassays but increased those of D. magna feeding bioassays. These results empathize the need to include additional bioassays to monitor more accurately and realistically the toxicity of effluents or surface waters dominated by effluent discharges, a quite common situation in America and Mediterranean arid regions.     

Assessment of toxicity reduction after metal removal in bioleached sewage sludge

Sewage sludge can be applied to land to supply and recycle organic matter and nutrients. Trace elements in sludge, however, may accumulate in the soil with repeated sludge applications. Reducing metal content may therefore reduce the adverse effects of sludge application. The objective of this study was to evaluate the efficiency of bioleaching technology in reducing metal content and toxicity as measured by a battery of terrestrial and liquid-phase bioassays. Sludge-soil mixtures simulating the application of sludge to land were tested by means of terrestrial bioassays, barley (Hordeum vulgare L.) seed germination (5 d) and sprout growth (14 d), lettuce (Lactuca sativa) seed germination (5 d), and worm (Eisenia andrei) mortality (14 d). Liquid-phase bioassays, Microtox (Vibrio fischeri, 15 min), lettuce root elongation (L. sativa, 5 d), cladoceran mortality (Daphnia magna, 48 h), and SOS Chromotest (Escherichia coli) were used after elutriation of the sludge. Comparison of the bioassay results (except for D. magna) before and after treatment demonstrated that this bioleaching process reduced both sludge toxicity and metal content. In addition, lower Cu and Zn concentrations found in barley sprouts following treatment supported the assumption that the bioleaching process, by decreasing metal content and bioavailability, reduced sewage sludge toxicity. This study also emphasized the interest of using ecotoxicological bioassays for testing biosolids. In particular, the terrestrial bioassays after simulation of land application and the Microtox test after sludge elutriation proved to be the most appropriate procedures.     

Responses in sediment bioassays used in the Netherlands: can observed toxicity be explained by routinely monitored priority pollutants?

In order to identify the cause of toxicity in sediments and suspended matter, a large number of samples with different degrees of contamination was taken at various locations in The Netherlands. Standard acute bioassays were carried out with the bacterium Vibrio fischeri, the rotifer Brachionus calyciflorus and the anostracan Thamnocephalus platyurus. Chronic standard tests were performed using the water flea Daphnia magna and larvae of the midge Chironomus riparius. Some novel bioassays were performed as well. Most toxic effects observed in standard bioassays with sediments from polluted sediments (class 3 and 4 on a scale of 0-4 according to the Dutch criteria) could be partly explained by toxic concentrations of known persistent priority pollutants, mainly heavy metals and occasionally polycyclic aromatic hydrocarbons. In some of the samples, ammonia toxicity was a confounding factor during testing. Suspended matter from the Meuse river at Eijsden, which may be considered as 'new' sediment (pollution class 2), was moderately to highly toxic in almost all bioassays. This could have been associated with a combination of heavy metals, PAHs and ammonia. At two locations from the Lake IJssel area with no apparent persistent pollution, moderate and strong effects were nonetheless observed in invertebrate tests. This might have been due to agricultural run-off of pesticides, which are not routinely measured in sediments. A few effects on V. fischeri in canals and a small stream could not be explained with standard chemical analysis, but seemed associated with the outlets of sewage water treatment plants and industrial effluents. Additional chemical analysis of pore water samples from five selected sediments yielded more identified substances such as phtalates, decanes, cosanes and fragrances, but it was estimated that their contribution to the effects observed on V. fischeri, D. magna and C. riparius was negligible.     

Identification of organic compounds and ecotoxicological assessment of sewage treatment plants (STP) effluents

An integrated approach combining chemistry and biological methods was conducted to assess the toxicity of seven sewage treatment plant effluents. Solid phase concentration procedures were applied to facilitate the study of organic micro pollutants. A chemical analysis was performed by GC/MS. Organic fraction toxicity was determined by using bioassays such as Daphnia magna and Chlorella vulgaris tests and sub-lethal effects were also evaluated by using Salmonella typhimurium Test (mutagenicity), recombinant yeast screen (estrogenicity), and Oryzias latipes embryo-larval test. More than 49 compounds were detected in the organic fraction due to the various inputs of each effluents. The most frequently detected compounds in the effluents were bisphenol A (BPA), octylphenol (OP), 1,2-benzenedicarboxylic acid, bis(2-ethylhexyl) ester (DEHP) and 1,2-benzenedicarboxylic acid, bis(methylpropyl) ester (DBP). Biological assays showed toxicity effects on D. magna tests in all samples, whereas toxicity on C. vulgaris or S. typhimurium tests were not observed. Estrogenicity and teratogenicity were observed in several samples. The cause-effect relationship could not be established given the high chemical complexity of the effluents and the lack of information available on 70% of the detected compounds subsequent to reviewing various data bases. Nevertheless, due to the high chemical variability revealed by STP effluents, bioassay sets may provide a very useful amount of information for detecting potential toxicity risks.     

Toxicity to Daphnia magna and Vibrio fischeri of Kraft bleach plant effluents treated by catalytic wet-air oxidation

Two Kraft-pulp bleaching effluents from a sequence of treatments which include chlorine dioxide and caustic soda were treated by catalytic wet-air oxidation (CWAO) at T=463 K in trickle-bed and batch-recycle reactors packed with either TiO2 extrudates or Ru(3 wt%)/TiO2 catalyst. Chemical analyses (TOC removal, color, HPLC) and bioassays (48-h and 30-min acute toxicity tests using Daphnia magna and Vibrio fischeri, respectively) were used to get information about the toxicity impact of the starting effluents and of the treated solutions. Under the operating conditions, complex organic compounds are mostly oxidized into carbon dioxide and water, along with short-chain carboxylic acids. Bioassays were found as a complement to chemical analyses for ensuring the toxicological impact on the ecosystem. In spite of a large decrease of TOC, the solutions of end products were all more toxic to Daphnia magna than the starting effluents by factors ranging from 2 to 33. This observation is attributed to the synergistic effects of acetic acid and salts present in the solutions. On the other hand, toxicity reduction with respect to Vibrio fischeri was achieved: detoxification factors greater than unity were measured for end-product solutions treated in the presence of the Ru(3 wt%)/TiO2 catalyst, suggesting the absence of cumulative effect for this bacteria, or a lower sensitivity to the organic acids and salts. Bleach plant effluents treated by the CWAO process over the Ru/TiO2 catalyst were completely biodegradable.     

Rapid ecotoxicological bioassay using delayed fluorescence in the green alga Pseudokirchneriella subcapitata

A rapid and simple ecotoxicological bioassay allows quick estimation of the effects of Simazine (CAT) or 3,5-dichlorophenol (3,5-DCP) on the growth of the green alga Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum). The effects of a 15-min exposure to CAT or 3,5-DCP on delayed fluorescence (DF) in P. subcapitata were compared to the results of standard growth inhibition tests involving 72h of exposure to these chemicals at the same concentrations. Integrated DF intensity in the time period from 0.6 to 50s was found to correlate with algal growth inhibition as measured by the standard tests. In addition, the behaviour of DF in this time period exhibited characteristic kinetics indicative of the chemical being tested.     

Seasonal changes in the sensitivity of river microalgae to atrazine and isoproturon along a contamination gradient

A study was undertaken to investigate the environmental impact of herbicides on natural communities of freshwater periphyton and phytoplankton in the river Ozanne and in related nearby water reservoirs, including both pristine and pesticide- (atrazine and isoproturon) contaminated stations. The microalgal toxicity of both herbicides was investigated by short-term studies, using the in vivo fluorescence pattern to perform dose-effect experiments. The taxonomic composition of the communities sampled was assessed by microscopy and by HPLC pigment analysis. The EC50 (periphyton) or EC125 (phytoplankton) values, calculated using in vivo fluorescence endpoints, increased with the herbicide concentration found in the water. In contrast, the structure of the algal communities (periphyton) inhabiting the contaminated stations seemed to be permanently affected when compared to the reference community. A 'memory effect' could be detected, both in herbicide sensitivity and in the structure of periphytic communities that persisted even when peak contaminations had disappeared. This study shows that the response of algal communities is likely to reflect past selection pressures, and suggests that the function and structure of a community could both be modified by the persistent or repeated presence of microcontaminants in natural environments. We could use short-term ecotoxicological tests on freshwater microalgae to assess the effects of past temporary contaminations by agricultural pesticides, and combining this with diversity indices could make it possible to assess the ecotoxicological risk of herbicide contamination even when a complete chemical analysis of the contamination is not feasible.     

The combined toxic effects of nonpolar narcotic chemicals to Pseudokirchneriella subcapitata

This paper presents the toxicity data of 10 nonpolar narcotic chemicals on Pseudokirchneriella subcapitata (green algae) assessed by a new algal toxicity testing technique conducted under air-tight environment. Based on DO production, median effective concentration (EC50) varies from 1.73 mg/L (1-octanol) to 8,040 mg/L (2-propanol). The endpoint of algal growth rate reveals similar sensitivity as that from DO production. Compared to literature data, Pseudokirchneriella subcapitata and Nitrosomonas are apparently more sensitive to nonpolar narcotics than other organisms such as minnow, daphnia, and Tetrahymena pyriformis. Furthermore, good correlations between toxic effects observed from Pseudokirchneriella subcapitata and other aquatic organisms were found. Hence, algal toxicity test can be considered as a surrogate test for estimating the toxicity of nonpolar chemicals to fathead minnow, Microtox, activated sludge, Daphina magna, and Tetrahymena pyriformis. The combined effects of 13 binary mixtures of nonpolar chemicals were investigated using both additive-index method and isobologram analysis. Overall speaking, the joint actions between these chemicals are strictly additive. Model analyses indicate that these compounds act on identical reaction sites or receptors, which verify that these chemicals are of the same toxicity mechanism (narcosis).     

Quality assessment of bed sediments of the Po River (Italy)

Comprehensive and contemporary evaluations of physical, chemical and toxicological endpoints have been performed on bed sediments of the Po River, the major Italian watercourse. Two extensive sampling campaigns were conducted in summer and winter low-flow conditions. Composite sediment samples were collected from ten reaches of the main river: the first was located in the upper region (ambient control), and the others downstream of the confluences of nine principal tributaries. The two sampling programs were paralleled by contemporary investigations on the macroinvertebrate community. The particle-size composition along the Po River showed a relatively uniform distribution of fine sand, a progressive downstream decrease of coarse sands and a corresponding increase of fine materials. The levels of polychlorinated biphenyls (PCB), polycyclic aromatic hydrocarbons (PAH), extractable organo halides (EOX), Cd, Cr, Cu, Hg, Ni, Pb and Zn were determined in sediment fine particles (< 63 microm), and showed marked changes across the ten river reaches. Their longitudinal trends, as those of organic carbon and total nitrogen, were very similar and largely independent of the survey season. Sediment quality benchmarks were used to evaluate sediment chemistry, and, although the overall level of contamination was from moderate to low, the reaches located downstream of the tributaries Dora Riparia, Dora Baltea, Lambro and Oglio were considered to be at risk. Sediments were tested for toxicity on Oncorhynchus mykiss, Ceriodaphnia dubia, Raphidocelis subcapitata and Vibrio fischeri. The toxicity tests were conducted both with sediment extracts and whole samples. Sediment extracts showed toxic potentials that were consistent with the spatial distribution of contaminants. Whole-sediment toxicity showed moderate/low effects which also included false positives and negatives. Alterations of the macroinvertebrate community were found for many kilometers downstream of Dora Riparia, and with a seasonal dependence, also in other reaches of the Italian river. Principal component analysis (PCA) was used to describe the longitudinal and temporal changes of the Po River, and allowed the selection of the most useful and discriminating indicators.     

Combined toxicity effects of MTBE and pesticides measured with Vibrio fischeri and Daphnia magna bioassays

Methyl-tert-butyl ether (MTBE), a fuel oxygenate that is added to gasoline, commonly contaminates aquatic systems, many of which are already contaminated with pesticides. The toxic effects (EC(50) value) of several pure pesticides (Diuron, Linuron, Dichlofluanid, Sea nine, Irgarol and tributyltin (TBT)) were measured and compared with the EC(50) value of the pesticide mixed with MTBE, using the Vibrio fischeri and Daphnia magna acute toxicity assays. The interaction between chemicals was evaluated in terms of the effects of mixing on the EC(50) value (i.e. the concentration (mg/L) of a compound or mixture that is required to produce a 50% change in a toxic response parameter) and the time required to generate the toxic response. Presence of MTBE enhanced the EC(50) value of several pesticides (Diuron, Dichlofluanid, TBT and Linuron) and/or the toxic response manifested more rapidly than with pure pesticides. Toxicity enhancements were quite substantial in many cases. For example, the presence of MTBE increased the toxicity of Diuron by more than 50% when tested with the V. fischeri assay (5, 15 and 30 min exposure). Also, the toxic response manifested itself within 5 min whereas without the MTBE the same response arose in 30 min. Presence of MTBE increased the toxicity of Dichlofluanid by 30% when measured with the D. magna assay. Toxicities of only two pesticides (Sea nine and Irgarol) were not raised by the presence of MTBE.     

Contribution for tier 1 of the ecological risk assessment of Cunha Baixa uranium mine (Central Portugal): II. soil ecotoxicological screening

This study presents the first ecotoxicological data concerning the soils of the area surrounding the Cunha Baixa uranium mine. Our main goal was to categorise soils from the area based on their toxicity profiles using a battery of cost- and time-effective bioassays (elutriate approach - Microtox and Daphnia acute tests; whole-soil approach - Microtox and avoidance assays with Eisenia andrei), as a part of tier 1 of an ongoing Environmental Risk Assessment. No acute toxicity was found for any of the 10 sites/soils using Microtox or Daphnia. On the contrary, the behavioural response of E. andrei was found to be an extremely sensitive endpoint, allowing the discrimination of highly to moderately toxic soils based on their toxicity profiles (as a function of soil concentration). Soils exhibiting highest toxicity corresponded to areas subjected to runoffs or sludge deposition from the aquatic effluent, while non-toxic soils were farthest to the mine. Data obtained in avoidance assays strengthen the previous evaluation of risks based on chemical data and supported decisions about proceeding for tier 2.     

Evaluation of water column and sediment toxicity from an abandoned uranium mine using a battery of bioassays

Uranium mining activities in Cunha Baixa, Mangualde (Portugal), were extensive between 1967 and 1993, with high production of poor ore. Ore exploitation left millions of tons of tailings in the surrounding area, close to human houses. Contamination of the area (water and soil compartment) presently represents a serious hazard to humans and wildlife. The aim of this work was to evaluate the acute toxicity of water and sediments from a pond that floods a uranium mine pit, in two periods (spring and autumn). High contents of metals were found in water samples (chiefly Mn, Fe, Al, U, Sr). A battery of assays was applied to screen the acute toxicity of the different compartments using algae, crustaceans and dipterans. Results showed that the sediments were non-toxic, unlike the superficial water. Water toxicity was higher in the autumn, when the effluent was more acidic, compared to spring. In the water toxicity assays, the relative sensitivity of the test species used was Daphnia longispina>Pseudokirchneriella subcapitata>Daphnia magna. The present study is part of the chemical and ecotoxicological characterisation of the aquatic compartment performed in the Tier 1 of the Ecological Risk Assessment of the Cunha Baixa mining area.     

Toxicity assays: a way for evaluating AOPs efficiency

The technical feasibility and performance of photocatalytic degradation of aqueous methomyl (50 mg/L) have been studied at pilot scale in two well-defined systems of special interest because natural-solar UV light can be used: heterogeneous photocatalysis with titanium dioxide and homogeneous photocatalysis by photo-Fenton. The pilot plant is made up of compound parabolic collectors specially designed for solar photocatalytic applications. Experimental conditions allowed pesticide disappearance, degree of mineralisation and toxicity achieved in the two photocatalytic systems to be compared. Total disappearance of methomyl is attained by photo-Fenton in 60 min and by TiO2 in 100 min. Hundred percent of nitrogen and sulphur are recovered as ammonium and sulphate. By contrast, complete mineralisation of total organic carbon (TOC) is not achieved even after quite a long time (more than 300 min). Three different bioassays (Vibrio fischeri, Daphnia magna and a Microalga) have been used for testing the progress of toxicity during treatment. All remained toxic down to very low-pesticide concentrations and in some bioassays were still toxic after total disappearance of the pesticide. Only if treatment is maintained throughout enough mineralisation (i.e. TOC disappearance), the toxicity is reduced to below the threshold (EC50%).     

A new method for evaluating biological safety of environmental water with algae, daphnia and fish toxicity ranks

In this study, an innovative approach to evaluate biological safety of environmental water with toxicity ranks was proposed. Widely used species, algae (Selenustrum capricornutum), daphnia (Daphnia magna) and fish (Oryzias latipes larvae) belonging to three trophic levels in aquatic ecosystem, were selected and combined as a test set to measure the bio-toxicity of water sample. Maximum exposure concentrations for algae, daphnia and fish test were respectively designed as 10-, 50- and 50-fold of river water based on a simplification of conventional toxicity extrapolation method EU Directive EEC/93/67. A novel assessment index "safety score" of 1, 2, 3 and 4 with 1 being the safest was established for normalizing the toxicity effects. Safety score was determined according to the highest exposure concentration where adverse ecotoxicological effects could not be observed, and a triangle figure was designed to visually describe the safety scores of three toxicity tests. Finally, in order to conveniently evaluate the biological safety of environmental water, an integrated assessment index "bio-safety rank" (BSR) was established and determined according to the safety scores of the three tests, and with the index BSR, water sample could be ranked as A, B, C or D with A being the safest. It was shown that the proposed new method was effective for screening and evaluating the biological safety of river water in case studies.     

Assessing chemical toxicity with the bioluminescent photobacterium (Vibrio fischeri): a comparison of three commercial systems

The inhibition of light emitted by the bioluminescent bacterium, Vibrio fischeri, is the basis for several toxicity bioassays. The inhibitory effects of 81 chemicals, after 5 min contact time, were studied at eight concentrations using reagents from three commercial assay systems (ToxAlert 10, Microtox and LUMIStox). Solubility in water was the limiting factor in determining the selection of chemicals for study. The effective nominal concentrations (EC) resulting in 20, 50 and 80% inhibition were determined using Ln dose/Ln gamma plots and the results obtained for each system were compared by linear regression. The chemical concentrations producing 10-90% inhibition extended over 9 orders of magnitude and ranged from a minimum of 0.001 ppm to a maximum of 1,000,000 ppm. The toxicity of many chemicals was apparently related to their pH in solution and at high chemical concentrations, to osmotic imbalance. The fact that the same operator tested the same solutions simultaneously on three different systems reduced sources of error and variability and improved the consistency and reliability of the results. Only five compounds gave EC 50s that varied more than three-fold between assays. These data provide comparisons of toxicity that have not been previously available and demonstrate that, when used under standardised conditions, these bioluminescence-based toxicity assays produce very similar results.     

Chemical and ecotoxicological assessment of polycyclic aromatic hydrocarbon--contaminated sediments of the Niger Delta, Southern Nigeria

The extent of environmental contamination and sources of polycyclic aromatic hydrocarbons (PAHs) compounds to sediments of the Niger Delta, Nigeria were assessed using combined chemical analysis and toxicity bioassay techniques. Concentrations of two- to six-ring PAHs of molecular mass 128-278 and toxicity to Vibrio fischeri and Lemna minor are considered in this investigation. Levels of the sum of the 16 USEPA priority pollutant PAHs varied from 20.7 to 72.1 ng/g dry weight. Sediment PAH levels were highest in samples collected from Delta Steel located at the outskirts of Warri, and Quality control centre, Ughelli West; with total PAH concentrations of 72.1 and 67.5 ng/g dry weight, respectively. The overall levels of PAHs in this study are low compared to other regions and reveal moderate PAHs pollution in the sediments of the Niger Delta. Two- and three-ring aromatic hydrocarbons predominated in almost all the sediments, which indicate a petrogenic origin. The sediment total PAH (PAHtot) concentration, normalized to organic carbon content (OC), ranged from 120.2 to 1.99 ng PAHtot/mg OC; and showed distinctively that the sedimentary organic matter of the sample from Delta Steel is highly contaminated with PAHs, and had a value of 120.2 ng PAHtot/mg OC. The toxicity bioassays indicated that the sample collected from Warri Refinery Area (SDWRR) was the most toxic to V. fischeri, with an EC50 value of 0.45 mg sediment equiv./mL test medium; and samples from Ogunu (SDOGN) and Warri Refinery area (SDWRR) showed high toxicity to L. minor, with percent inhibitions of 42.6% and 33.67%, respectively, after 7 days of exposure. The total PAH concentrations showed no correlation with toxicity bioassays, and thereby implied that chemical analysis of PAHs cannot be an indicator of sediment toxicity.     

Evaluation of DGT as a metal speciation tool in wastewater

This paper aims to evaluate the performance of the diffusive gradient in thin film technique (DGT) as a speciation tool for metals in wastewater. The validity of metal sampling by DGT in wastewater was checked. DGT was used in parallel with the Daphnia magna acute toxicity test in order to obtain information on the speciation of copper and cadmium in diluted and spiked filtered wastewater (raw and treated) from two treatment plants. Combining the chemical (DGT) and the biological methods (D. magna toxicity test) allowed metal to be fractionated into inorganic, labile organic and inert organic metal. Copper was mainly found as inert organic complexes, whereas the major part of cadmium was found to be labile organic complexes. The proportion of inert organic copper complexes was higher in the presence of treated wastewater than in raw wastewater. The use of restricted gels in DGT devices discriminated more labile organic cadmium than labile organic copper, indicating that cadmium weak ligands have more complex structures than copper weak ligands. In our experimental conditions (i.e. a high metal to ligand ratio), DGT, even equipped with restricted gels, was able to accumulate labile organic complexes. This result highlights that the ecotoxicological interpretation of DGT measurement should be considered carefully. DGT is a reliable tool to assess the chemical characteristics of metals (i.e. reactivity) in wastewater, but it does not ensure that only inorganic metal is measured.     

Toxicological assessment of indium nitrate on aquatic organisms and investigation of the effects on the PLHC-1 fish cell line

Indium nitrate is mainly used as a semiconductor in batteries, for plating and other chemical and medical applications. There is a lack of available information about the adverse effects of indium compounds on aquatic organisms. Therefore, the toxic effects on systems from four trophic levels of the aquatic ecosystem were investigated. Firstly, the bacterium Vibrio fischeri, the alga Chlorella vulgaris and the cladoceran Daphnia magna were used in the toxicological evaluation of indium nitrate. The most sensitive model was V. fischeri, with a NOAEL of 0.02 and an EC(50) of 0.04 mM at 15 min. Although indium nitrate should be classified as harmful to aquatic organisms, it is not expected to represent acute risk to the aquatic biota. Secondly, PLHC-1 fish cell line was employed to investigate the effects and mechanisms of toxicity. Although protein content, neutral red uptake, methylthiazol metabolization, lysosomal function and acetylcholinesterase activity were reduced in cells, stimulations were observed for metallothionein levels and succinate dehydrogenase and glucose-6-phosphate dehydrogenase activities. No changes were observed in ethoxyresorufin-O-deethylase activity. To clarify the main events in PLHC-1 cell death induced by indium nitrate, nine modulators were applied. They were related to oxidative stress (alpha-tocopherol succinate, mannitol and sodium benzoate), disruption of calcium homeostasis (BAPTA-AM and EGTA), thiol protection (1,4-dithiotreitol), iron chelation (deferoxiamine) or regulation of glutathione levels (2-oxothiazolidine-4-carboxylic acid and malic acid diethyl ester). The main morphological alterations were hydropic degeneration and loss of cells. At least, in partly, toxicity seems to be mediated by oxidative stress, and particularly by NADPH-dependent lipid peroxidation.     

TOXICITY TESTING OF GROUNDWATER QUALITY

Groundwater is an important resource in the UK, with 45% of public water supplies in the Thames Water region derived from subterranean sources. In urban areas, groundwater has been affected by anthropogenic activities over a long period of time and from a multitude of sources. At present, groundwater quality is assessed using a range of chemical species to determine the extent of contamination. However, analysing a complex mixture of chemicals is time-consuming and expensive, whereas the use of an ecotoxicity test provides information on (a) the degree of pollution present in the groundwater and (b) the potential effect of that pollution. Microtox^TM, Eclox^TM and Daphnia magna microtests were used in conjunction with standard chemical protocols to assess the contamination of groundwaters from sites throughout the London Borough of Hounslow and nearby Heathrow Airport. Because of their precision, range of responses and ease of use, Daphnia magna and Microtox^TM tests are the bioassays that appear to be most effective for assessing groundwater toxicity. However, neither test is ideal because it is also essential to monitor water hardness. Eclox^TM does not appear to be suitable for use in groundwater-quality assessment in this area, because it is adversely affected by high total dissolved solids and electrical conductivity.