Toxicological effects of disinfections using sodium hypochlorite on aquatic organisms and its contribution to AOX formation in hospital wastewater

Sodium hypochlorite (NaOCl) is often used for disinfecting hospital wastewater in order to prevent the spread of pathogenic microorganisms, causal agents of nosocomial infectious diseases. Chlorine disinfectants in wastewater react with organic matters, giving rise to organic chlorine compounds such as AOX (halogenated organic compounds adsorbable on activated carbon), which are toxic for aquatic organisms and are persistent environmental contaminants. The aim of this study was to evaluate the toxicity on aquatic organisms of hospital wastewater from services using NaOCl in pre-chlorination. Wastewater samples from the infectious and tropical diseases department of a hospital of a large city in southeast of France were collected. Three samples per day were collected in the connecting well department at 9 a.m., 1 p.m. and 5 p.m. during 8 days from 13 March to 22 March 2001, and a mixture was made at 6 p.m. with the three samples in order to obtain a representative sample for the day. The toxicity test comprised the 24-h EC50 on Daphnia magna and a bioluminescence assay using Vibrio fischeri photobacteria. Fecal coliforms and physicochemical analyses such as total organic carbon (TOC), chloride, AOX, total suspended solids (TSS) and chemical oxygen demand (COD) were carried out. Wastewater samples highlighted considerable acute toxicity on D. magna and V. fischeri photobacteria. However, low most probable numbers (MPN), ranging from <3 to 2400 for 100 ml, were detected for fecal coliforms. Statistical analysis, with a confidence interval of 95%, gave a strong linear regression assessed with r=0.98 between AOX concentrations and EC50 (TU) on daphnia. The identification of an ideal concentration of NaOCl in disinfecting hospital wastewater, i.e. its non-observed effect concentration (NOEC) on algae and D. magna, seems to be a research issue that could facilitate the control of AOX toxicity effects on aquatic organisms. Therefore, it would be necessary to monitor the biocide properties of NaOCl on fecal coliforms at various doses and its toxicity effects on aquatic organisms.     

Toxicity assessment using different bioassays and microbial biosensors

Toxicity assessment of water streams, wastewater, and contaminated sediments, is a very important part of environmental pollution monitoring. Evaluation of biological effects using a rapid, sensitive and cost effective method can indicate specific information on ecotoxicity assessment. Recently, different biological assays for toxicity assessment based on higher and lower organisms such as fish, invertebrates, plants and algal cells, and microbial bioassays have been used. This review focuses on microbial biosensors as an analytical device for environmental, food, and biomedical applications. Different techniques which are commonly used in microbial biosensing include amperometry, potentiometry, conductometry, voltammetry, microbial fuel cells, fluorescence, bioluminescence, and colorimetry. Examples of the use of different microbial biosensors in assessing a variety of environments are summarized.     

Novel approach for assessing heavy metal pollution and ecotoxicological status of rivers by means of passive sampling methods

In order to study the pollution of fluvial ecosystems, it is necessary to analyze not only the levels of chemical contaminants in water, but also those accumulated in the sediment matrix, as well as to assess its ecotoxicological status. Eleven Catalan (Spain) river sections (one sampling point per river) located near urban and industrial areas were sampled during winter of 2009. Water pollutants were collected by using passive samplers as Diffusive Gradient in Thin-Films (DGTs) and Semi-Permeable Membrane Devices (SPMDs). Point water samples were also collected. The concentrations of potentially toxic elements (PTE) in water, filtered water, DGTs and sediment samples were analyzed. Aqueous and organic solvent extracts of sediments samples and organic extracts of SPMDs were performed to assess acute toxicity to Vibrio fischeri by Microtox(?), and chronic toxicity to the green alga Pseudokirschneriella subcapitata. Microtox(?) test was also performed with DGT extracts. The results show that metals content of Catalan river waters are below the freshwater screening US EPA benchmarks, excepting some industrial areas (for Hg, Pb, and Zn). In contrast, sediments levels of some rivers were far above freshwater sediment screening US EPA benchmarks (for Zn, As, Cr, Pb, Ni, Hg, and Mn), particularly in the most industrialized areas. A good correlation was found between toxicity values of extracts (from sediments and DGTs) and PTE levels in sediments. The current results support the suitability of using combined point and passive sampling methods for assessing the chemical and ecotoxicological status of aqueous environments.     

Effect of americium-241 on luminous bacteria. Role of peroxides

The effect of americium-241 (²⁴¹Am), an alpha-emitting radionuclide of high specific activity, on luminous bacteria Photobacterium phosphoreum was studied. Traces of ²⁴¹Am in nutrient media (0.16-6.67 kBq/L) suppressed the growth of bacteria, but enhanced luminescence intensity and quantum yield at room temperature. Lower temperature (4 °C) increased the time of bacterial luminescence and revealed a stage of bioluminescence inhibition after 150 h of bioluminescence registration start. The role of conditions of exposure the bacterial cells to the ²⁴¹Am is discussed. The effect of ²⁴¹Am on luminous bacteria was attributed to peroxide compounds generated in water solutions as secondary products of radioactive decay. Increase of peroxide concentration in ²⁴¹Am solutions was demonstrated; and the similarity of ²⁴¹Am and hydrogen peroxide effects on bacterial luminescence was revealed. The study provides a scientific basis for elaboration of bioluminescence-based assay to monitor radiotoxicity of alpha-emitting radionuclides in aquatic solutions.     

Correlation of field-measured toxicity with chemical concentration and pollutant availability.

Direct field toxicity tests were performed in two Louisiana waterways, Bayous Trepagnier and St. John, on sediments containing organic/heavy metal mixtures. Our approach involved bioluminescent bacterial toxicity assays (using DeltaTox, which qualitatively identifies polluted areas, and Microtox, which quantifies toxicity). Samples were more completely analyzed in our laboratory using inductively coupled plasma atomic emission spectroscopy (ICP-AES) and gas chromatography/mass spectrometry (GC/MS). Results indicate that lead is the primary toxic metal at the sites examined, though concentrations of metals fluctuate due to spatial variation and the dynamic nature of the waterways. Polycyclic aromatic hydrocarbons (PAHs) are the most abundant group of organics measured and appear to contribute to the overall toxic response. DeltaTox located toxic hotspots where there was an average light loss of 53-100%. Toxicity results from both assays agree but are well correlated with concentration measurements only for certain sediment fractions. Overall, the DeltaTox/Microtox approach appears to be rapid and cost effective for on-site hotspot identification, and may increase understanding of hazards associated with heavy metal and organic contaminants in these waterways.     

A bioassay approach to determine the dioxin-like activity in sediment extracts from the Danube River: ethoxyresorufin-O-deethylase induction in gill filaments and liver of three-spined sticklebacks (Gasterosteus aculeatus L.)

Sediment samples from the upper Danube River in Germany have previously been characterized as ecotoxicologically hazardous and contaminants in these sediments may contribute to the observed decline of fish populations in this river section. For the investigation of sediment toxicity there is a need for development, standardization and implementation of in vivo test systems using vertebrates. Therefore, the main objective of this study was to apply and evaluate a recently established fish gill EROD assay as a biomarker in sediment toxicity assessment by using extracts of well characterised sediment samples from the upper Danube River. This to our knowledge is the first application of this novel assay to sediment extracts. Sediments from four different sites along the upper Danube River were Soxhlet-extracted with acetone and dissolved in DMSO. Three-spined sticklebacks (Gasterosteus aculeatus L.) were exposed for 48 h to various concentrations of the extracts, to the positive control beta-naphthoflavone or to the solvent. Measurements of EROD activity in gill filaments and liver microsomes followed the exposure. Concentration-dependent induction of EROD in both gill and liver was found for all sediment extracts. The highest EROD-inducing potency was determined for extracts of sediments from the sites "Opfinger See" and "Sigmaringen" and the EROD activities in gill and liver correlated well. The results from the gill and liver assays were in accordance with in vitro results of previous investigations. The EROD activities measured in the present study corresponded with the concentrations of PAHs, PCBs and PCDD/Fs in the sediment samples derived in a previous study. The sticklebacks in this study were in the reproductive phase and a stronger EROD induction was obtained in the females than in the males. Implementation of the EROD assay in testing of sediment extracts gave highly reliable results which make this assay an ecotoxicologically relevant method for assessment of contamination with Ah receptor agonists in sediments.     

Fate of glutaraldehyde in hospital wastewater and combined effects of glutaraldehyde and surfactants on aquatic organisms

Glutaraldehyde (GA), an aliphatic dialdehyde disinfectant, and surfactants, one of the major components of detergents, are widely used in hospitals in order to eliminate pathogenic organisms causing nosocomial infectious diseases. After their use, disinfectants and surfactants reach the wastewater network together. The discharge of chemical compounds from hospital activities into wastewater is also a well-known problem, causing pollution of water resources and constituting an ecological risk for aquatic organisms. In this study, the chemistry and toxicology of GA and surfactant mixtures were reviewed in order to estimate their fate in aquatic ecosystems. Furthermore, their joint effects on aquatic organisms were experimentally assessed in the laboratory. A simple model of the additive joint action of toxicants was used to determine combined acute toxicity effects on the bacteria luminescence and Daphnia mobility of three mixtures containing GA at 1.5 x EC50 24 h [in mg/L] on Daphnia and anionic, cationic and nonionic surfactants at twice their critical micellar concentration (CMC). The mixture of GA and a cationic surfactant gave an EC50 30 min on Vibrio fischeri of 0.158%, with a concentration of 0.04 mg GA/L and 1.04 mg CTAB/L, which provided an additive action. The interaction between GA and an anionic surfactant on V. fischeri produced an antagonistic joint action with an EC50 30 min of 3.95%, containing 1.06 mg GA/L and 33.2 mg SDS/L. A synergistic action with an EC50 30 min of 8.4% on V. fischeri was observed for the mixture containing GA and a nonionic surfactant. Antagonistic interactions were observed for the joint action between GA and the surfactants studied on Daphnia. The mixture of GA and CTAB was more toxic (EC50 24 h=0.02%) than the two other mixtures (EC50 24 h GA+SDS=6%; EC50 24 h GA+TX 100=10%). This study provides new data on the toxicity of certain hospital pollutants entering the aquatic environment and detected in surface and groundwaters. It is necessary to study the joint effects of GA and surfactant mixtures following chronic and sublethal standard bioassays in order to estimate the contribution of the additive joint action models in assessing the environmental risk of hospital wastewater (HW).     

An evaluation of mixed species in-situ and ex-situ feeding assays: the altered response of Asellus aquaticus and Gammarus pulex

Mixed species feeding assays were undertaken with pollution sensitive (Gammarus pulex) and tolerant (Asellus aquaticus) macro-invertebrates during August 2003 and April 2004. The purpose of this study was to establish if a test animals' response is comparable during in-situ and ex-situ toxicity tests. Seven test sites were established along an undisclosed stream, which received leachate discharge from an unlined, disused UK landfill site. Sampling points A-B were upstream of the contamination, C was adjacent to the influx and D-G were downstream of the leachate discharge (at 100 m intervals). During the in-situ and ex-situ tests, 2-week-old male laboratory bred A. aquaticus and G. pulex were used as test animals. The animals were transplanted to the seven sampling points for the duration of the in-situ tests, whilst water samples from each site were returned to the laboratory for ex-situ testing. The results show that the animals' mortality and feeding rates followed similar trends during the in-situ and ex-situ tests, however, the animals' response was amplified during the in-situ tests. It was also observed that the effects were greater in April, compared to August that may be attributed to a higher frequency of rainfall during spring, which could have flushed a greater proportion of the contaminant load from the waste mass and as a consequence, higher levels of pollution may have leached into the stream from the landfill site. The study, therefore, concludes that in-situ toxicity tests are a more precise monitoring technique, in comparison to ex-situ assays.     

Comparative study of estrogenic potencies of estradiol, tamoxifen, bisphenol-A and resveratrol with two in vitro bioassays

This study was undertaken to compare the sensitivity of two in vitro screening test methods and to determine the accuracy of predicted response to spiked laboratory water samples. A newly developed enzyme-linked receptor assay (ELRA) and a widely used yeast estrogen screen (YES) assay were selected to evaluate the estrogenic responses. Four natural, pharmaceutical, xenobiotic or phytobiotic chemicals: 17beta-estradiol (E2), tamoxifen, bisphenol-A and resveratrol were examined, and 17beta-E2 was used as a positive control. 17beta-E2 can strongly induce estrogenic response in both test systems, however, ELRA was found to be more sensitive to 17beta-E2 with a detection limit of 0.07 microg/l compared to 0.88 microg/l in YES assay. Similar results were obtained for bisphenol-A and resveratrol, and their estrogen potencies relative to E2 (100%) determined by ELRA were at least 5.6 times greater than produced by YES assay. ELRA was unable to distinguish the anti-estrogen tamoxifen and YES assay is also poor at distinguishing. Comparison of response to spiked laboratory water samples show that ELRA can give accurate determination to all four chemicals with recoveries among 70-120%, while YES can only give accurate determination to 17beta-E2 and bisphenol-A with recoveries among 69-112%. The comparative results provide evidence that ELRA is more suitable for rapid screening estrogenic potency of the environmental samples. Combination of ELRA and mammalian cellular assay will constitute an advantageous test to specify agonistic or antagonistic effects.     

An integrated approach to the toxicity assessment of Irish marine sediments: application of porewater Toxicity Identification Evaluation (TIE) to Irish marine sediments

An integrated approach to the ecotoxicological assessment of Irish marine sediments was carried out between 2004 and 2007. Phase I Toxicity Identification Evaluation (TIE) of sediment porewaters from two sites on the east coast of Ireland were conducted. Initial Tier I screening of three Irish sites identified the need for TIE after significant toxicity was observed with Tisbe battagliai and the Microtox assay at two of the assayed sites (Alexandra Basin and Dunmore East). Porewaters classified as toxic were characterised using four manipulations, ethylenediaminetetraacetic acid (EDTA) chelation, sodium thiosulphate addition, C(18) Solid Phase Extraction (SPE) and Cation Exchange (CE) SPE. Prior to initial testing, and TIE manipulations, all porewater samples were frozen at -20 degrees C for several months until required. After initial Tier I testing Alexandra Basin porewater was classified as highly toxic by both assays while Dunmore East porewater only warranted a TIE with T. battagliai. Results of TIE manipulations for Alexandra Basin porewater and the Microtox Basic test were inconclusive. The toxicity of the porewater in this assay was significantly reduced after freezing. Three experimental episodes were conducted with one month between each for the Alexandra Basin porewater. After each month of freezing the baseline toxicity was further reduced in the Microtox assay, therefore it was not possible to draw accurate conclusions on the nature of the active contaminants in the sample. However, toxicity to T. battalgiai did not change after storage of the porewater. The C(18) and CE SPE decreased the toxicity of Alexandra Basin porewater to the copepod indicating that both organic and cationic compounds (e.g. metals) were active in the sample. Dunmore East porewater was assayed with T. battalgiai and again a combination of organic and inorganic compounds were found to be partly responsible for the observed toxicity (C(18), CE SPE and EDTA reduced toxicity). Results from these TIEs provide insight into the complexity of interpreting marine TIE data from porewater studies where mixtures of unknown substances are present.     

The effect of suspended particles coated by humic acid on the toxicity of pharmaceuticals, estrogens, and phenolic compounds

The sorption characteristics of 10 organic chemicals, categorized as pharmaceuticals, estrogens and phenols, onto synthetic suspended particle (i.e., alumina) coated with humic acid were investigated according to their octanol-water partition coefficient (K(ow)). Chemical analyses were performed with gas chromatography and mass spectrometry (GC/MS) and high performance liquid chromatography (HPLC). The effects of particles on the toxicity reduction were evaluated using bioassay tests, using Daphnia magna and Vibrio fisheri for phenols and pharmaceuticals, and the human breast cancer cell MCF-7 for estrogens. Sorption studies revealed that 22 and 38% of octylphenol and pentachlorophenol, respectively, were removed by suspended particle, whereas 2,4-dichlorophenol was not removed, which was directly proportional to the logK(ow) value. Similar to the sorption tests, suspended particles significantly reduced the acute toxicities of octylphenol and pentachlorophenol to D. magna and V. fisheri (p<0.01), but there was no significant difference in the toxicity of 2,4-dichlorophenol to D. magna (p=0.8374). Pharmaceuticals, such as ibuprofen, gemfibrozil and tolfenamic acid, showed no discernible sorption to the suspended particle, with the exception of diclofenac, which revealed 11% sorption. For estrogens, such as estrone, 17beta-estradiol and 17alpha-ethynylestradiol, the results indicated no reduction in the sorption test. This may be attributed to the polar interaction by functional groups in sorption between pharmaceuticals and estrogens and suspended particles. In the bioassays, presence of suspended particles did not significantly modify the toxicity of pharmaceuticals (regardless of their K(ow) values) to D. magna, V. fisheri or E-screen.     

Impact of dredging in a shallow coastal lagoon: Microtox Basic Solid-Phase Test, trace metals and Corophium bioassay

The aim of this work was to measure survival of the amphipod Corophium insidiosum and luminescence inhibition in the marine bacterium Vibrio fisheri on surface sediment samples collected from a shallow coastal lagoon (Pialassa Baiona, northern Adriatic Italian coast) before execution of dredging operations to deepen the main inner channel of the lagoon and restore the water circulation. Trace metal (Cd, Cu, Cr, Hg, Ni, Pb) concentrations, grain size and organic carbon matter content as loss of ignition were also measured. Toxicity testing with V. fisheri was carried out according to the Microtox Basic Solid-Phase Test (BSPT) protocol. The preliminary outcomes of this work show that: (a) the investigated area can be categorised as moderately degraded; (b) there is no evident spatial pattern in sediment toxicity and trace metal concentrations; (c) Microtox responses are not biased by sediment characteristics such as silt, clay and organic matter content.     

Comparison of the sensitivity of different toxicity test endpoints in a microalga exposed to the herbicide paraquat

The use of herbicides constitutes the principal method of weed control but the introduction of these compounds into the aquatic environment can provoke severe consequences for non-target organisms such as microalgae. Toxic effects of these pollutants on microalgae are generally evaluated using phytotoxicity tests based on growth inhibition, a population-based parameter. However, physiological cellular endpoints could allow early detection of cell stress and elucidate underlying toxicity mechanisms. Effects of the herbicide paraquat on the freshwater microalga Chlamydomonas moewusii were studied to evaluate growth rate and cellular parameters such as cellular viability and metabolic activity assayed by flow cytometry and DNA damage assayed by the comet assay. Sensitivity of growth and parameters assayed by flow cytometry were similar, showing a significant effect in cultures exposed to a paraquat concentration of 0.1 microM or higher, although in cultures exposed during 48 h to 0.05 microM, a significant stimulation of cellular fluorescein fluorescence was observed, related to cellular metabolic activity. After only 24 h of herbicide exposure significant DNA damage was observed in microalgal cells exposed to all paraquat concentrations assayed, with a 23.67% of comets in cultures exposed to 0.05 microM, revealing the genotoxicity of this herbicide. Taking into account the results obtained, comet assay provides a sensitive and rapid system for measuring primary DNA damage in Chlamydomonas moewusii, which could be an important aspect of environmental genotoxicity monitoring in surface waters.     

Extent of sonochemical degradation and change of toxicity of a pharmaceutical precursor (triphenylphosphine oxide) in water as a function of treatment conditions

The degradation of triphenylphosphine oxide (TPPO) in water, a toxic compound typically found in effluents from the pharmaceutical industry, by means of ultrasonic irradiation at 20 kHz has been investigated with emphasis on the effect of various parameters on conversion and acute toxicity. Experiments were carried out at liquid volumes of 50 and 80 ml, electric power outputs of 125, 187.5 and 250 W, initial TPPO concentrations of 10, 100 and 350 mg/L and temperatures of 5, 20, 35, 50 and 70 degrees C. TPPO conversion was found to increase with increasing power output and decreasing initial concentration and temperature. Measurements of dissolved total carbon showed that liquid-phase degradation by-products were more stable to ultrasonic irradiation than TPPO. Addition of t-butanol as a radical scavenger at a concentration of 1000 mg/L nearly completely suppressed TPPO degradation. Conversely, addition of radical promoters (Fe(2+) ions or H(2)O(2)) had a positive effect on degradation. Acute toxicity to marine bacteria vibrio fischeri was measured before and after ultrasonic irradiation. At the conditions employed in this study, irradiated TPPO samples were always more toxic than TPPO itself with toxicity levels being a function of treatment conditions.     

An interlaboratory comparison of nanosilver characterisation and hazard identification: Harmonising techniques for high quality data

Within the FP7 EU project NanoValid a consortium of six partners jointly investigated the hazard of silver nanoparticles (AgNPs) paying special attention to methodical aspects that are important for providing high-quality ecotoxicity data. Laboratories were supplied with the same original stock dispersion of AgNPs. All partners applied a harmonised procedure for storage and preparation of toxicity test suspensions. Altogether ten different toxicity assays with a range of environmentally relevant test species from different trophic levels were conducted in parallel to AgNP characterisation in the respective test media. The paper presents a comprehensive dataset of toxicity values and AgNP characteristics like hydrodynamic sizes of AgNP agglomerates and the share (%) of Ag⁺-species (the concentration of Ag⁺-species in relation to the total measured concentration of Ag). The studied AgNP preparation (20.4 ± 6.8 nm primary size, mean total Ag concentration 41.14 mg/L, 46–68% of soluble Ag⁺-species in stock, 123.8 ± 12.2 nm mean z-average value in dH₂O) showed extreme toxicity to crustaceans Daphnia magna, algae Pseudokirchneriella subcapitata and zebrafish Danio rerio embryos (EC50 < 0.01 mg total Ag/L), was very toxic in the in vitro assay with rainbow trout Oncorhynchus mykiss gut cells (EC50: 0.01–1 mg total Ag/L); toxic to bacteria Vibrio fischeri, protozoa Tetrahymena thermophila (EC50: 1–10 mg total Ag/L) and harmful to marine crustaceans Artemia franciscana (EC50: 10–100 mg total Ag/L). Along with AgNPs, also the toxicity of AgNO₃ was analyzed. The toxicity data revealed the same hazard ranking for AgNPs and AgNO₃ (i.e. the EC50 values were in the same order of magnitude) proving the importance of soluble Ag⁺-species analysis for predicting the hazard of AgNPs. The study clearly points to the need for harmonised procedures for the characterisation of NMs. Harmonised procedures should consider: (i) measuring the AgNP properties like hydrodynamic size and metal ions species in each toxicity test medium at a range of concentrations, and (ii) including soluble metal salt control both in toxicity testing as well as in Ag⁺-species measurements. The present study is among the first nanomaterial interlaboratory comparison studies with the aim to improve the hazard identification testing protocols.     

Use of millet root elongation for toxicity tests of phenolic compounds

Millet root elongation was used for toxicity tests of phenol and seven chlorophenols. Each compound was tested twice, all with a control and six concentration levels in quadruplicates. Each test solution contained 15 seeds. The results showed that the order of decreasing toxicity is trichlorophenol, dichlorophenol, chlorophenol, and phenol. The root elongation method is more sensitive than the biomass method; however, it is time consuming. Phenolic compounds at low concentration stimulated the growth of root, but when the concentration is further increased, the stimulation effect is reversed and inhibition takes place.     

Predictive abilities of numerical sediment quality guidelines in Sydney Harbour, Australia, and vicinity

Matching chemical and toxicological data of surficial sediments from Sydney Harbour, Australia, and vicinity, were collected to evaluate predictive abilities of Effects Range-Low (ERL), Effects Range-Median (ERM) and other sediment quality guidelines (SQGs). Samples (n=103) containing a wide range of chemicals and concentrations were subjected to a battery of 4-6 toxicity tests. ERLs and functionally equivalent low-range SQGs were highly predictive of non-toxicity when not exceeded, as incidences of toxicity were 0-8%. ERMs and other mid-range SQGs were predictive of toxicity in combined test data with > or = 80% of toxic samples with one or more SQG exceeded. Predictive abilities of mid-range SQGs were low for amphipod survival tests (generally < 20% toxic samples), but the incidence of toxicity increased with increasing numbers of SQGs exceeded and increasing mean SQG quotients. Predictive abilities of SQGs generally matched their narrative intent when outcomes of multiple toxicity tests were considered, and were consistent with North American data. Functionally equivalent SQGs gave comparable results (except where developed for single chemical classes), indicating that predictive abilities were primarily influenced by sensitivities of test species and/or the bioavailability of contaminants.     

Assessment of ecotoxicological risks related to depositing dredged materials from canals in northern France on soil

The implementation of an ecological risk assessment framework is presented for dredged material deposits on soil close to a canal and groundwater, and tested with sediment samples from canals in northern France. This framework includes two steps: a simplified risk assessment based on contaminant concentrations and a detailed risk assessment based on toxicity bioassays and column leaching tests. The tested framework includes three related assumptions: (a) effects on plants (Lolium perenne L.), (b) effects on aquatic organisms (Escherichia coli, Pseudokirchneriella subcapitata, Ceriodaphnia dubia, and Xenopus laevis) and (c) effects on groundwater contamination. Several exposure conditions were tested using standardised bioassays. According to the specific dredged material tested, the three assumptions were more or less discriminatory, soil and groundwater pollution being the most sensitive. Several aspects of the assessment procedure must now be improved, in particular assessment endpoint design for risks to ecosystems (e.g., integration of pollutant bioaccumulation), bioassay protocols and column leaching test design.     

Evaluation of the sensitivity of marine microalgal strains to the heavy metals, Cu, As, Sb, Pb and Cd

The sensitivity of nine marine microalgal species (consisting of five divisions and seven genera) to the five heavy metals, Cu(II), As(V), Sb(III), Pb(II) and Cd(II) was studied by using a fluorometric growth-inhibition assay with 96-well microplates. The algal strains studied were Cylindrotheca sp. and the LPP group that respectively characterize aggregating and filamentous types, and Chlorococcum littorale, Chlorococcum sp., Isochrysis galbana, Tetraselmis tetrathele, Heterocapsa sp., Synechococcus sp. and Prasinococcus sp. for types that occur as single cells. A good linear relationship was observed between the chlorophyll a concentration and intensity of chlorophyll fluorescence (485-nm excitation filter and 645-nm emission filter) when the chlorophyll a concentration was within the range of 0.10-5.0 microg ml(-1). A starting cell concentration of 0.10 or 0.25 microg Chl a ml(-1) was therefore selected. In accordance with OECD 201 standard procedures, the IC(50) value (concentration of a metal producing 50% growth inhibition relative to the control) was determined 72 h after adding a heavy metal by using the biomass integral. The microplate toxicity test used in this study is considered to be applicable to diverse algae, not only enumerating species but also hardly enumerating ones.