Enantiomeric fractionation as a tool for quantitative assessment of biodegradation: The case of metoprolol.

An efficient chiral liquid chromatography high resolution mass spectrometry method has been developed for the determination of metoprolol (MTP) and three of its major metabolites, namely O-desmethylmetoprolol (O-DMTP), α-hydroxymetoprolol (α-HMTP) and metoprolol acid (MTPA) in wastewater treatment plant (WWTP) influents and effluents. The optimized analytical method has been validated with good quality parameters including resolution >1.3 and method quantification limits down to the ng/L range except for MTPA. On the basis of this newly developed analytical method, the stereochemistry of MTP and its metabolites was studied over time in effluent/sediment biotic and sterile microcosms under dark and light conditions and in influents and effluents of 5 different WWTPs. MTP stereoselective degradation was exclusively observed under biotic conditions, confirming the specificity of enantiomeric fraction variations to biodegradation processes. MTP was always biotransformed into MTPA with a (S)-enantiomer enrichment. The results of enantiomeric enrichment pointed the way for a quantitative assessment of in situ biodegradation processes due to a good fit (R(2) > 0.98) of the aerobic MTP biodegradation to the Rayleigh dependency in all the biotic microcosms and in WWTPs because both MTP enantiomers followed the same biodegradation kinetic profiles. These results demonstrate that enantiomeric fractionation constitutes a very interesting quantitative indicator of MTP biodegradation in WWTPs and probably in the environment.

Insights into reductive dechlorination of triclocarban in river sediments: Field measurements and in vitro mechanism investigations.

Triclocarban (TCC) reductive dechlorination was investigated using a combination of field and laboratory experiments. Field monitoring revealed that TCC reductive dechlorination in river sediments leads to formation of two isomers of its lesser chlorinated congener namely 3,4'-dichlorocarbanilide and 4,4'-dichlorocarbanilide. Monochlorocarbanilide was not detected in sediments supporting that transformation of dichlorocarbanilide into monochlorocarbanilide is the rate limiting step of TCC dechlorination. In vitro experiments were conducted to study TCC potential reductive dechlorination mechanisms. These experiments demonstrated that 4,4'-dichlorocarbanilide was specifically formed upon a nucleophilic addition reaction under anaerobic conditions. The 3,4'-diclorocarbanilide was formed upon a two steps radical mechanism under aerobic conditions which includes TCC activation by one electron oxidation followed by a nucleophilic substitution reaction with glutathione. In vitro results suggested that strict anaerobic conditions might not be required for TCC reductive dechlorination in the environment. Moreover, in vitro reactions were performed using biomimetic or enzymatic systems supposing that TCC dechlorination might occur through microbial action in situ. Measured dichlorocarbanilide isomers/triclocarban ratios were used to evaluate the relative significance of both dechlorination pathways and the pathway leading to 3,4'-dichlorocarbanilide was found significant in all investigated river sediment samples.

Screening triclocarban and its transformation products in river sediment using liquid chromatography and high resolution mass spectrometry.

An analytical method was developed and validated for the target screening of triclosan (TCS), triclocarban (TCC) and its lesser and higher chlorinated congeners namely, 4,4'-dichlorocarbanilide (DCC) 3,3',4,4'-tetrachlorocarbanilide (3-Cl-TCC) and 2,3',4,4'-tetrachlorocarbanilide (2-Cl-TCC) in river sediment. Sediment samples were extracted by pressurized liquid extraction and quantification and identification of target compounds were carried by liquid chromatography high resolution mass spectrometry (LC-HRMS). The overall method recoveries were 89% with relative standard deviations below 6%. Method detection limits ranged from 0.01 to 0.12 ng/g. The usefulness of the method was demonstrated on sediment samples collected downstream of three wastewater treatment plants in an attempt to provide with a set of occurrence data of these biocides in France and for a better understanding of their fate in river. Major results are the following: TCC, DCC and 3-Cl-TCC were ubiquitously detected demonstrating that these emerging contaminants have been probably overlooked in France. Reductive dechlorination of TCC into DCC was also ubiquitous but predominated in anoxic sediment. 3-Cl-TCC is probably more persistent than TCC and LC-HRMS enabled the detection and identification of a suite of other chlorinated biocides in river sediment.

Reducing PEC uncertainty in coastal zones: a case study on carbamazepine, oxcarbazepine and their metabolites.

Concentrations of the antiepileptic drugs carbamazepine (Cbz), oxcarbazepine (OxCz) and their main metabolites were predicted in a wastewater treatment plant (WTP) and in the vicinity of its submarine outfall located in a Mediterranean coastal zone. Refined predicted environmental concentrations (PECs) were calculated in effluents based on consumption data and human excretion rates. PECs were estimated in the sea using the hydrodynamic MARS 3D model integrating meteorological data, oceanic conditions (wind, tide, atmospheric pressure), freshwater and sewage inputs. Measured environmental concentrations (MECs) were compared to PECs to assess the estimation relevance. In the coastal zone, PEC and MEC were in the same magnitude range. Modeling of Cbz diffusion and advection just above the submarine outfall showed the influence of the thermocline during summer, with low diffusion of Cbz from the bottom to the surface. This work allowed understanding the dispersion of target compounds and deserved further development for a better acknowledgement of vulnerability at local scales.

Ecological risk assessment of the presence of pharmaceutical residues in a French national water survey.

In this study, we focused on the list of 33 chemicals that was established through a French national prioritisation strategy. Assessing the potential risks to the environment was a step-wise procedure: (i) we determined the Predicted Environmental Concentration (PEC) of all molecules measured in the national survey based on the highest recommended dose used, (ii) we used the Measured Environmental Concentration (MEC) and the Predicted No-Effect Concentration (PNEC) to establish the Risk Quotient (RQ) based on either a PEC/PNEC (estimated risk) or MEC/PNEC (real risk) ratio. The risk assessment was performed using a binary ecological classification suggesting that appreciable risk is likely (RQ⩾1). Of the 15 molecules quantified in the survey, 12 had a PEC higher than the action limit value of 0.01µg/L. According to the EU Guideline, environmental risk was estimated as likely for the following five compounds: acetaminophen (RQ=1.6), ibuprofen (RQ=600), diclofenac (RQ=15), oxazepam (RQ=2.1) and carbamazepine (RQ=3.2). Only ibuprofen was identified as posing real environmental risk based on its MEC (RQ=1.9).

Occurrence, removal and accumulation in sludge of protozoan cysts and helminth eggs in a full-scale anaerobic pond in Burkina Faso.

The present paper investigates the occurrence, removal, and accumulation of protozoan cysts and helminth eggs in a large anaerobic pond treating municipal wastewater of Ouagadougou (Burkina Faso). With a hydraulic retention time of 6.5 days, the anaerobic pond achieved 100% removal of helminth eggs and protozoan cysts most of the time, except during the hot period. The average residual concentrations of helminth eggs and protozoan cysts in the effluent were respectively 0.45 eggs/L (minimum 0 and maximum 3), and 5.4 cysts/L (minimum 0 and maximum 26). Protozoan cysts accumulation in sludge averaged 1,613 cysts/g total solids. Ancylostoma duodenale, Ascaris lumbricoides and Hymenolepis nana were the main helminth species found in the sludge. After 7 years of operation, the sludge in the pond still contained a high level of viable helminth eggs evaluated at 42%.

UV filters, ethylhexyl methoxycinnamate, octocrylene and ethylhexyl dimethyl PABA from untreated wastewater in sediment from eastern Mediterranean river transition and coastal zones.

UVF may occur in the aquatic environment through two principal sources: direct inputs from recreational activities and indirect wastewater- and river-borne inputs. The aim of this study was to obtain a first overview of levels of three UVF (EHMC, OC and OD-PABA) in coastal areas subjected to river inputs, untreated wastewater discharges and dumpsite leachates. We selected three eastern Mediterranean rivers that have been impacted for decades by untreated wastewater release and collected sediment in the coastal zone during the hot and humid seasons. Western Mediterranean sites receiving treated wastewaters were analyzed for comparison. The results gave an overview of sediment contamination under these two contrasted situations representative of Mediterranean coastal areas without bathing activities. The analysis of the three UVF revealed the ubiquity and high point source contamination by EHMC and OC in transition and coastal zones, with levels as high as 128 ng g(-1)d.w. OD-PABA was also frequently detected, but at lower concentrations (

Carbamazepine, carbamazepine epoxide and dihydroxycarbamazepine sorption to soil and occurrence in a wastewater reuse site in Tunisia.

Treated wastewater is being increasingly used for irrigation and aquifer replenishment through artificial recharge. However, wastewater reuse can result in contamination of exposed soil and groundwater by chemicals such as some pharmaceuticals and their metabolites. The fate of these molecules depends largely on their capacity to sorb onto soil and aquifer materials during infiltration. In this study, the sorption isotherm of carbamazepine (CBZ), an anti-seizure medication, and two of its metabolites, i.e. carbamazepine-10,11-epoxide (CBZ-EP) and 10,11-dihydro-10,11-dihydroxycarbamazepine (DiOH-CBZ), were determined in two soils in laboratory assays. In the field, the presence of CBZ and its metabolites were investigated in soil and in groundwater underlying an irrigated area with treated wastewater. The results showed that CBZ had the highest carbon normalised sorption coefficients in the two tested soils (irrigated soil and a Lufa SP2.4 reference soil) followed by CBZ-EP and DiOH-CBZ, indicating the relatively higher mobility of CBZ metabolites compared to CBZ. The chromatographic analysis revealed that CBZ and its two metabolites were present in treated wastewater used for irrigation and in groundwater. In soil samples, CBZ concentrations showed a build-up taking place with irrigation. The mobility of metabolites in soil and their potential biodegradation require further investigation.

Organic UV filter concentrations in marine mussels from French coastal regions.

The accumulation of EHMC, OCT and OD-PABA, three common UV filter compounds, was investigated in marine mussels. Wild Mytilus edulis and Mytilus galloprovincialis were sampled in ten sites along the French Atlantic and Mediterranean coasts from June to November. In mussel tissues, 100% of the samples had quantifiable EHMC concentrations ranging from 3 to 256ngg(-1) dry weight, while 55% of the samples had detectable OCT concentrations ranging from under 2 to 7 112ngg(-1) dry weight. These concentrations significantly increased with the rising air temperature in summer, the recreational pressure and the geomorphological structure of the sampling sites (its lack of openness to the wide). This is the first study to report bioaccumulation of UV filters in marine mussels, thus highlighting the need for further monitoring and assessment.

In vitro biomonitoring of contamination by estrogenic compounds in coastal environments: comments on the use of M. galloprovincialis.

The use of mussel extracts in in vitro bioassays which express the estrogen receptor could provide valuable information on the bioavailability of endocrine disruptors in coastal environments. The aim of this study was to assess the temporal variability of the estrogenic responses in bioassays in Mytilus galloprovincialis. A 6-month in situ experiment was conducted in order to follow the estrogenic activity on MELN cell line during the reproduction stages of mussels. Estradiol equivalents (EEQ) determined in mussels using the MELN cell lines ranged from 0.79 to 3.72 ng/g dry weight (d.w.) in males, from 0.42 to 2.33 ng/g d.w. in females and from 3.41 to 4.2 d.w. in undifferentiated bivalves. We observed an increase in EEQ values during the spawning stage for males, not for female. The maximal EEQ values were observed at the indifferent stage. We discuss these results in regards to the actual knowledge on mussels' reproductive cycle and to the possible impact of xeno-estrogens. Variations of E2 levels in mussels must be taken into account for further studies on xeno-estrogens monitoring using hER reporter cell-lines bioassays.

Bioconcentration of two pharmaceuticals (benzodiazepines) and two personal care products (UV filters) in marine mussels (Mytilus galloprovincialis) under controlled laboratory conditions.

Bioaccumulation is essential for gaining insight into the impact of exposure to organic micropollutants in aquatic fauna. Data are currently available on the bioaccumulation of persistent organic pollutants, but there is very little documentation on the bioaccumulation of pharmaceuticals and personal care products (PPCPs). The bioconcentration of selected PPCPs was studied in marine mussels (Mytilus galloprovincialis). The selected PPCPs were two organic UV filters, i.e., 2-ethylhexyl-4-trimethoxycinnamate (EHMC) and octocrylene (OC), and two benzodiazepines (BZP), i.e., diazepam (DZP) and tetrazepam (TZP). Laboratory experiments were performed in which M. galloprovincialis was exposed to these compounds either directly from water, for the less lipophilic substances (BZP) or via spiked food for lipophilic UV filters. M. galloprovincialis uptook and eliminated BZP following first-order kinetics. The biological half-life (t (1/2)) of TZP was 1.4 days, resulting in a bioconcentration factor of 64 and 99 mL g(-1) dry weight (dw), respectively, for 2.3 and 14.5 µg L(-1) of exposure, while the biological half-life (t (1/2)) of DZP was 0.4 days, resulting in a bioconcentration factor of 51 mL g(-1) dw for 13.2 µg L(-1) of exposure. The uptake of UV filter was rapid in mussels, followed by elimination within 24 h. EHMC increased from 15 to 138 ng g(-1) dw in 1 h and decreased to 25 ng g(-1) after 24 h for 11.9 µg L(-1) exposure. OC reached 839 ng g(-1) dw after 1 h and decreased to 33 ng g(-1) after 24 h for 11.6 µg L(-1) exposure. However, EHMC and OC were slightly accumulated in 48 h, i.e., 38 and 60 ng g(-1) dw, respectively.

Monitoring organic contaminants in small French coastal lagoons: comparison of levels in mussel, passive sampler and sediment.

In this study, the distribution of organic contaminants was investigated in the particular context of three Mediterranean coastal lagoons, where pollution input was hypothesised to come mainly from sediments resuspension. Mussels and semi-permeable membrane devices (SPMDs) were exposed to the water column for one month and then their content in estrogen-, benzo[a]pyrene- and dioxin-like substances as well as polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls and alkylphenols was determined with biological and chemical analyses. PAH concentration was high in sediments (up to 1028 ng g(-1) dry weight), however the aqueous PAH concentrations estimated from SPMD data could be considered below the levels inducing adverse effects according to the environmental quality standards proposed by the Water Framework Directive. Dioxin-like activity was observed in sediments but not in mussels and SPMDs. In the two sewage-impacted lagoons, nonylphenols could be quantified in sediments, SPMDs and mussels. Nonylphenol concentrations in mussels were among the highest found in the literature. However, since nonylphenols contributed only to a small part of the estrogenic activities observed, natural or synthetic steroids originating from wastewater discharges could be also implicated in these responses in sediments.

Estrogenic and AhR activities in dissolved phase and suspended solids from wastewater treatment plants.

The distribution of estrogen receptor (ERalpha) and Aryl Hydrocarbon Receptor (AhR) activities between the dissolved phase and suspended solids were investigated during wastewater treatment. Three wastewater treatment plants with different treatment technologies (waste stabilization ponds (WSPs), trickling filters (TFs) and activated sludge supplemented with a biofilter system (ASB)) were sampled. Estrogenic and AhR activities were detected in both phases in influents and effluents. Estrogenic and AhR activities in wastewater influents ranged from 41.8 to 79 ng/L E(2) Eq. and from 37.9 to 115.5 ng/L TCDD Eq. in the dissolved phase and from 5.5 to 88.6 ng/g E(2) Eq. and from 15 to 700 ng/g TCDD Eq. in the suspended solids. For both activities, WSP showed greater or similar removal efficiency than ASB and both were much more efficient than TF which had the lowest removal efficiency. Moreover, our data indicate that the efficiency of removal of ER and AhR activities from the suspended solid phase was mainly due to removal of suspended solids. Indeed, ER and AhR activities were detected in the effluent suspended solid phase indicating that suspended solids, which are usually not considered in these types of studies, contribute to environmental contamination by endocrine disrupting compounds and should therefore be routinely assessed for a better estimation of the ER and AhR activities released in the environment.

Biological analysis of endocrine-disrupting compounds in Tunisian sewage treatment plants.

Endocrin-disrupting compounds (EDCs) are frequently found in wastewater treatment plants (WWTPs). So far, research has been mainly focused on the detection of estrogenic compounds and very little work has been carried out on other receptors activators. In this study, we used reporter cell lines, which allow detecting the activity of estrogen (ERalpha), androgen (AR), pregnane X (PXR), glucocorticoid (GR), progesterone (PR), mineralocorticoid (MR), and aryl hydrocarbon (AhR) receptors, to characterise the endocrine-disrupting profile of the aqueous, suspended particulate matter, and sludge fractions from three Tunisian WWTPs. The aqueous fraction exhibited estrogenic and androgenic activities. Suspended particulate matter and sludge extracts showed estrogenic, aryl hydrocarbon and pregnane X receptor activities. No GR, MR, or PR (ant) agonistic activity was detected in the samples, suggesting that environmental compounds present in sewage might have a limited spectrum of activity. By performing competition experiments with recombinant ERalpha, we demonstrated that the estrogenic activity detected in the aqueous fraction was due to EDCs with a strong affinity for ERalpha. Conversely, in the sludge fraction, it was linked to the presence of EDCs with weak affinity. Moreover, by using different incubation times, we determined that the EDCs present in suspended particulate matter and sludge, which can activate AhR, are metabolically labile compounds. Finally, we showed in this study that environmental compounds are mainly ER, AR, PXR, and AhR activators. Concerning AR and PXR ligands, we do not to know the nature of the molecules. Concerning ER and AhR compounds, competition experiments with recombinant receptor and analysis at different times of exposure of the AhR activation gave some indications of the compound's nature that need to be confirmed by chemical analysis.

Presence and fate of carbamazepine, oxcarbazepine, and seven of their metabolites at wastewater treatment plants.

Many pharmaceuticals are excreted in wastewater as parent substances or metabolites subsequent to therapeutic or diagnostic application in medical care. This includes the antiepileptic carbamazepine, which is not removed during conventional wastewater treatment and was found to be ubiquitous in the aquatic environment. Some carbamazepine metabolites have also been found in treated wastewater, but only five of them have been studied to date. However, at least 30 carbamazepine metabolites have been identified in humans, including some pharmacologically active or genotoxic compounds. Oxcarbazepine, an antiepileptic which is increasingly used, generates metabolites common to those of carbamazepine. The present work focuses on the presence of carbamazepine, oxcarbazepine, and seven of their metabolites (carbamazepine-10,11-epoxide, 10-hydroxy-10,11-dihydrocarbamazepine, 10,11-dihydro-10,11-trans-dihydroxycarbamazepine, 2-hydroxy-carbamazepine, iminostilbene, acridine, and acridone) at three different treatment plants (conventional activated sludge, trickling filter, and stabilization ponds) selected in France. The main aim of this work was to identify selected compounds in wastewater after therapeutic use and to measure concentrations in influents and effluents at the three wastewater treatment plants. Except for iminostilbene, all of these compounds were detected in wastewater. The metabolite common to carbamazepine and oxcarbazepine, i.e., 10,11-dihydro-10,11-trans-dihydroxycarbamazepine, was detected at a higher concentration than the parent substances in wastewater. The presence of parent molecules was noted in inlet and outlet water samples. Carbamazepine, as expected, was not removed by conventional activated sludge treatment. Nevertheless, in a wastewater treatment plant with a 78-day hydraulic retention time, a 73% decrease in carbamazepine concentration was observed. For the first time, oxcarbazepine was found in environmental samples. A decrease in oxcarbazepine concentrations was observed at the three sewage treatment plants, with removal ranging from 24 to 73%. No metabolite removal was observed after activated sludge treatment. In the two other sewage treatments plants, the fate of the metabolites differed. The concentration of some metabolites, e.g., 10,11-dihydro-10,11-trans-dihydroxycarbamazepine and acridine, increased, possibly via different processes such as cleavage of glucuronide conjugates or biotic and abiotic degradation of parent compounds. The behavior of the studied substances is discussed in terms of the treatment process and hydraulic retention time.

Temporal study of estrogenic responses of mussel (Mytilus galloprovinciallis) extracts applied to reporter cell lines.

In vitro bioassays would facilitate monitoring of estrogen-like compounds in mussels (Mytilus galloprovincialis) since they tend to accumulate lipophilic compounds in their fat and muscle tissue. However, estradiol (E2) steroid (already identified in mussels) could induce false positive responses. This study focuses on temporal variability in estrogenic responses of mussel extracts and the possible relation of this response with E2 levels. The reproductive cycle and growth were thus monitored for six months in sexed mussels. E2 levels were similar between genders and these levels varied with reproductive stages. human estrogen receptor (hER) activities were similar at all stages except in February. E2 present in male and female extracts accounted for part of the hER activation observed, but no correlation was found between E2 levels and hER activities.

Modulation of aromatase activity and mRNA by various selected pesticides in the human choriocarcinoma JEG-3 cell line.

Aromatase enzyme plays a central role in steroidogenesis by converting androgens to estrogens and has been proposed as an important molecular target for many environmental endocrine disrupters chemicals. In this study, we have screened 30 selected pesticides with known, unknown or supposed effects on aromatase activity, for their ability to modulate aromatase activity in the human choriocarcinoma JEG-3 cell line after both short (2 h) and long exposure (24 h). All pesticides were tested at concentrations up to 10 microM that did not cause cytotoxicity after 24h of exposure, as verified by the MTT viability assay. Four pesticides inhibited aromatase activity after 2 h of exposure: prochloraz (IC(50)<1 microM), fenbuconazole (IC(50)=1.1 microM), propiconazole (IC(50)=1.5 microM) and fenarimol (IC(50)=3.3 microM). Among them, prochloraz and fenbuconazole also exerted inhibitory effects after 24h. Toxaphen (10 microM) and heptachlor (10 microM) inhibited aromatase activity after 24h exposure only. Nine pesticides induced aromatase activity: aldrin, chlordane, cypermethrin, parathion-methyl, endosulfan, methoxychlor, oxadiazon, metolachlor and atrazine after 24 h of exposure, while tributyltin induced aromatase activity at 1 nM and 3 nM after both 2 h and 24 h of exposure, respectively. To further investigate the mechanisms of aromatase induction we measured CYP19 mRNA expression and showed that methoxychlor, aldrin, chlordane and tributyltin induced the transcription of the cyp19 gene. In addition, none of the aromatase inducers transactivated the retinoic acid receptor (RAR) in JEG-3 stably transfected with a RARE-luciferase plasmid while the RAR agonist TTNPB induced both aromatase and luciferase expression in these cells. Our results, which provide new data for fenbuconazole, as an inhibitor of human aromatase, and for eight pesticides as aromatase inducers, are discussed with regards to the regulation of aromatase expression in the JEG-3 cellular context.

Identification of new human pregnane X receptor ligands among pesticides using a stable reporter cell system.

Pregnane X receptor (PXR, NR1I2) is activated by various chemically unrelated compounds, including environmental pollutants and drugs. We proceeded here to in vitro screening of 28 pesticides with a new reporter system that detects human pregnane X receptor (hPXR) activators. The cell line was obtained by a two-step stable transfection of cervical cancer HeLa cells. The first transfected cell line, HG5LN, contained an integrated luciferase reporter gene under the control of a GAL4 yeast transcription factor-binding site. The second cell line HGPXR was derived from HG5LN and stably expressed hPXR ligand-binding domain fused to GAL4 DNA-binding domain (DBD). The HG5LN cells were used as a control to detect nonspecific activities. Pesticides from various chemical classes were demonstrated, for the first time, to be hPXR activators: (1) herbicides: pretilachlor, metolachlor, and alachlor chloracetanilides, oxadiazon oxiconazole, and isoproturon urea; (2) fungicides: bupirimate and fenarimol pyrimidines, propiconazole, fenbuconazole, prochloraz conazoles, and imazalil triazole; and (3) insecticides: toxaphene organochlorine, permethrin pyrethroid, fipronil pyrazole, and diflubenzuron urea. Pretilachlor, metolachlor, bupirimate, and oxadiazon had an affinity for hPXR equal to or greater than the positive control rifampicin. Some of the newly identified hPXR activators were also checked for their ability to induce cytochrome P450 3A4 expression in a primary culture of human hepatocytes. HGPXR, with HG5LN as a reference, was grafted onto nude mice to assess compound bioavailability through in vivo quantification of hPXR activation. Altogether, our data indicate that HGPXR cells are an efficient tool for identifying hPXR ligands and establishing pesticides as hPXR activators.

Assessment of copper bioavailability and toxicity in vineyard runoff waters by DPASV and algal bioassay.

The aim of this study was to assess the toxicity of runoff waters in an agricultural multipollution context through an in-depth assessment of copper bioavailability and toxicity. Runoff waters were screened for major ions, metals and diuron. The potential environmental impact of these runoff waters was evaluated using the conventional 72-h growth inhibition test with the green alga Pseudokirchneriella subcapitata. The results suggested that the toxicity detected in the calcareous vineyard field was due to the presence of diuron, whereas the non-calcareous runoff waters were non-toxic. Chemical speciation modelling by MINEQL revealed that most of the copper present in the non-toxic natural runoff waters was complexed by organic matter. These samples were spiked with copper, and then the toxicity and the electrochemically bioavailable copper fraction were measured. Differential pulse anodic stripping voltammetry (DPASV) was used to detect labile complexes and free copper. This combined approach highlighted the presence of some labile copper complexes in samples reaching the EC10-these could have contributed to the copper toxicity.

Binding of estrogenic compounds to recombinant estrogen receptor-alpha: application to environmental analysis.

Estrogenic activity in environmental samples could be mediated through a wide variety of compounds and by various mechanisms. High-affinity compounds for estrogen receptors (ERs), such as natural or synthetic estrogens, as well as low-affinity compounds such as alkylphenols, phthalates, and polychlorinated biphenyls are present in water and sediment samples. Furthermore, compounds such as polycyclic aromatic hydrocarbons, which do not bind ERs, modulate estrogen activity by means of the aryl hydrocarbon receptor (AhR). In order to characterize compounds that mediate estrogenic activity in river water and sediment samples, we developed a tool based on the ER-alphaligand-binding domain, which permitted us to estimate contaminating estrogenic compound affinities. We designed a simple transactivation assay in which compounds of high affinity were captured by limited amounts of recombinant ER-alpha and whose capture led to a selective inhibition of transactivation. This approach allowed us to bring to light that water samples contain estrogenic compounds that display a high affinity for ERs but are present at low concentrations. In sediment samples, on the contrary, we showed that estrogenic compounds possess a low affinity and are present at high concentration. Finally, we used immobilized recombinant ER-alpha to separate ligands for ER and AhR that are present in river sediments. Immobilized ER-alpha, which does not retain dioxin-like compounds, enabled us to isolate and concentrate ER ligands to facilitate their further analysis.