Use of low density polyethylene membranes for assessment of genotoxicity of PAHs in the Seine River.

The genotoxicity of river water dissolved contaminants is usually estimated after grab sampling of river water. Water contamination can now be obtained with passive samplers that allow a time-integrated sampling of contaminants. Since it was verified that low density polyethylene membranes (LDPE) accumulate labile hydrophobic compounds, their use was proposed as a passive sampler. This study was designed to test the applicability of passive sampling for combined chemical and genotoxicity measurements. The LDPE extracts were tested with the umu test (TA1535/pSK1002 ± S9) and the Ames assay (TA98, TA100 and YG1041 ± S9). We describe here this new protocol and its application in two field studies on four sites of the Seine River. Field LDPE extracts were negative with the YG1041 and TA100 and weakly positive with the TA98 + S9 and Umu test. Concentrations of labile mutagenic PAHs were higher upstream of Paris than downstream of Paris. Improvement of the method is needed to determine the genotoxicity of low concentrations of labile dissolved organic contaminants.

Genotoxicity and activation of cellular defenses in transplanted zebra mussels Dreissena polymorpha along the Seine river.

The aim of the present study was to confirm the relevance of studying DNA adduct formation in a field study. In that context, freshwater mussels Dreissena polymorpha, collected in a reference station, were transplanted in different sites with a pollution gradient. After one and two months, mussels were collected and DNA adduct formation was analyzed using the (32)P post labelling technique on both gills and digestive glands. In addition, the expression of genes involved in the detoxification system (catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), HSP70, aryl hydrocarbon receptor (AHR), P glycoprotein (PgP), metallothionein (MT)) was assessed by RT-PCR. DNA adducts were observed at amount comparable to data from literature. Increase of DNA adducts after two months of transplantation could be correlated with strong modulation of gene expression implicated in detoxification processes. Indeed, PgP and HSP70 gene expressions were similarly induced in gills and digestive glands while SOD and CAT expressions were down regulated in both tissues. AHR, GST and MT genes were differently regulated depending upon the tissue studied and the level of contamination in the different sites. We demonstrated that mussels transplanted in the different stations with pollution gradient were able to biotransform PAHs, assessed by DNA adduct formation and the high decrease of detoxification genes. Specific DNA adducts pattern obtained after one and two month mussel transplantations demonstrated the relevance of DNA adduct as biomarker of environmental pollution.

DNA adduct formation and induction of detoxification mechanisms in Dreissena polymorpha exposed to nitro-PAHs.

Derived polycyclic aromatic hydrocarbons (PAHs) such as nitro-PAHs are present in the environment and are known to be much more toxic than PAHs compounds. However, very few studies have analysed their effects on the aquatic environment and none have investigated the freshwater environment. In the present study, we determined whether 1-nitropyrene (1-NP), a model of nitro-PAHs, can induce DNA adducts in gills and digestive glands of the freshwater mussel Dreissena polymorpha. Two concentrations of 1-NP (50 and 500 µM) were tested. In addition, in order to understand the metabolic pathways involved in 1-NP genotoxicity, mRNA expression of genes implicated in biotransformation mechanisms was assessed by quantitative reverse transcription-PCR. Results showed the presence of DNA adducts in both gills and digestive glands, with highest levels obtained after 5 days of exposure to 500 µM. Metallothionein mRNA levels were enhanced in digestive glands exposed to 50 µM. Surprisingly, at the higher concentration (500 µM), aryl hydrocarbon receptor and HSP70 genes were only up-regulated in digestive glands while PgP mRNA levels were increased in both tissues. Results suggested a cytotoxic and genotoxic effect of 1-NP. Mussels seemed to be able to partially detoxify this compound, in view of the low amount of DNA adducts observed after 5 days exposure to 50 µM. For the first time, 1-NP biotransformation and detoxification systems have been characterised in D. polymorpha.

Quantifying diet-borne metal uptake in Gammarus pulex using stable isotope tracers.

Gammarids are aquatic amphipods widely used for water quality monitoring. To investigate the copper and cadmium diet-borne metal uptake in Gammarus pulex, we adapted the pulse-chase stable isotopes-based approach to determine the food ingestion rate (IR), the gut retention time (GRT) and the metal assimilation efficiencies (AE). G. pulex were fed with (65)Cu-, (106)Cd-, and (53)Cr-labeled alder leaves for 7.5h and then with unlabeled leaves for 5d. The metal stable isotope contents in the gammarids, leaves, filtered water and periodically collected feces were determined. Chromium was poorly assimilated by the gammarids; thus, Cr was used as an unassimilated tracer. The first tracer defecation occurred before the first feces harvest, indicating a gut passage time of less than 9h. A 24-h GRT and a 0.69gg(-1)d(-1) IR were estimated. The Cd AE value was estimated as 5-47%, depending on the assimilation determination method applied. The Cu AE value could not be evaluated regardless of the determination method used, most likely because of the rapid Cu regulation in gammarids in addition to analytical uncertainties when determining the Cu content in leaves. Application of the Cd AE value in the framework of the biodynamic bioaccumulation model shows that the diet-borne uptake of Cd significantly contributes (66-95%) to the metal bioaccumulation in G. pulex fed with alder leaves.

Seasonal and PAH impact on DNA strand-break levels in gills of transplanted zebra mussels.

Genotoxicity endpoints are useful tools to biomonitor the physicochemical and biological quality of aquatic ecosystems. A caging study on the freshwater bivalve Dreissena polymorpha was planned to run over four seasons in the Seine River basin in order to assess whether DNA damage measured in transplanted mussels to polluted area vary according to seasonal changes. Three sites were chosen along the Seine River, one upstream from Paris and two downstream, corresponding to a chemical gradient of water contamination. The DNA strand break (comet assay) and chromosomal damage (micronucleus test) were measured in caged mussels at each site and in winter, spring and summer, along with PAH water contamination, PAH bioaccumulation, the mussel condition index (CI), the gonado-somatic index (GSI) and the filtration rate (FR). The level of DNA strand break measured in winter was low and increased in spring, concomitantly with FR and GSI. Over the same period, micronucleus (MN) frequency and PAH bioaccumulation decreased significantly in caged mussels, with both parameters positively correlated to each other. DNA strand-break levels and MN frequencies showed inter-site variations corresponding to the chemical contamination gradient. These two genotoxicity endpoints usefully complement each other in field studies. These results show that the MN test and comet assay, when applied to gill cells of caged zebra mussels, are sensitive tools for freshwater genotoxicity monitoring.

Impact of biofouling on diffusive gradient in thin film measurements in water.

The technique of diffusive gradient in thin film (DGT) is commonly used to assess metal contamination in natural waters. In this paper, we assess the effect of biofouling on DGT measured labile concentrations in water and investigate whether an additional nuclepore polycarbonate membrane on the surface of DGT devices can limit biofilm growth. Simultaneous field deployments of DGT equipped with and without the additional membrane in a canal receiving wastewater were compared. The effect of the biofilm was also assessed in controlled laboratory experiments, completed by the experimental determination of several metals diffusion coefficients in the hydrogel and membrane systems. The biofilms effect was problematic only from the 10th day of accumulation. Accumulation of some elements is highly biased by the presence of a thick biofilm (Zn, Ni, Cd). The polycarbonate membrane improved the quantification of Cd and Ni but adversely affects the quantification of Cr and Co. A kinetic model is proposed to explain the biofilm role on the DGT measurement. Depending on the metals of interest, it is possible to limit bias due to biofilms by using an additional polycarbonate membrane.

Genotoxicity assessment and detoxification induction in Dreissena polymorpha exposed to benzo[a]pyrene.

The use of DNA adduct analysis has previously focused on the use of marine organisms for biomonitoring, whereas similar investigations in freshwater organisms are sparse. In that context, we have investigated the relevance of DNA adducts as biomarkers of genotoxicity in the freshwater mussels Dreissena polymorpha. The objective of the present study is to determine the stability of DNA adducts induced by benzo[a]pyrene (B[a]P) in zebra mussels. Mussels were exposed to dissolved B[a]P (10-100 µg/l) for 4 days. Afterwards, mussels were kept in clean water for 28 days and DNA adduct levels were subsequently measured in two different organs, the digestive glands and the gills, using the (32)P-postlabelling technique. In parallel, the expression of genes involved in the detoxification system was assessed by qPCR (catalase, superoxide dismutase, glutathione S transferase, HSP70, aryl hydrocarbon receptor, P glycoprotein). We observed a higher level of DNA adducts in the digestive glands compared to the gills. Moreover, in gills, the level of DNA adduct was dependent on the B[a]P concentration. The levels of adducts tended to decrease in both organs after 28 days in clean water. In addition, an early induction of HSP70, PgP, AHR and SOD mRNA levels was noticed in the gills compared to the digestive glands. CAT and GST gene expression increased from 12h exposure in both organs. A higher gene expression level of those genes was observed in the gills, except for AHR and CAT genes. Data converge towards the fact that DNA adducts hence represent a very promising biomarker of B[a]P contamination and potentially of exposure to polycyclic aromatic hydrocarbons. In addition, for the first time in this study, B[a]P detoxification system was characterised in D. polymorpha.

Pollution-induced community tolerance of freshwater biofilms: measuring heterotrophic tolerance to Pb using an enzymatic toxicity test.

This study aims at investigating the impacts of Pb on freshwater biofilms with a pollution-induced community tolerance (PICT) approach using a recently developed short-term toxicity test based on ß-glucosidase activity to measure biofilms' tolerance to Pb. We first investigated more closely the influence of the total suspended solids (TSS) concentrations of biofilm suspensions used for short-term toxicity tests performed to assess Pb tolerance. The Pb EC(50) values of four dilutions of the same biofilm suspension increased with their TSS concentrations. TSS-normalization allowed to obtain a unique measure of Pb tolerance, thus confirming that TSS-normalization of EC(50) values is a good means to estimate biofilm tolerance to Pb. The experiment was repeated with three different biofilm samples collected at different sites and dates. Second, biofilms were exposed to Pb (0, 1, 10 and 100 µg/L) for 3 weeks in microcosms to assess the impacts of Pb exposure on the communities. An increase in Pb tolerance was observed for the biofilm exposed to 100 µg/L. Automated Ribosomal Intergenic Spacer Analysis revealed modifications of bacterial and eukaryotic community structure with Pb exposure. Moreover, exposure to 100 µg/L Pb also led to an increase in Zn tolerance but not Cu tolerance. This study shows that tolerance acquisition to Pb can be detected after exposure to environmental concentrations of Pb using a PICT methodology and normalized EC(50) values as measures of Pb tolerance.

Modelling copper bioaccumulation in Gammarus pulex and alterations of digestive metabolism.

Bioaccumulation enables to integrate the ability of aquatic organisms to regulate metals and effects of water chemistry on metal bioavailability. Linking this process to biological responses offers thus promising lines of enquiry for protecting aquatic ecosystems. This study aims at characterizing the mechanisms involved in waterborne Cu bioaccumulation and assessing metal impact on digestive metabolism in an ecosystem engineer widely distributed in Europe, Gammarus pulex. The organism was exposed to several Cu concentrations (from 0.5 to 100 µg/L) in aquatic microcosms to establish kinetic parameters for the construction and comparison of two bioaccumulation models, i.e. the biodynamic and saturation models. Cu uptake was recorded in waters exhibiting various concentrations of Na, Mg and Ca at environmental levels to assess the influence of cationic composition on bioaccumulation. Then, the effect of increasing Cu in exposure media on the digestive metabolism of G. pulex was investigated by measuring enzymatic activities (ß-glucosidase, N-acetyl-ß-glucosaminidase, ß-galactosidase). We showed that the saturation model is more suitable than the biodynamic model to describe Cu bioaccumulation in gammarids due to a maximal capacity of animals to accumulate the metal. Cationic composition of water affected insignificantly Cu uptake. All activities of tested enzymes decreased with increasing Cu in exposure media but with different degrees. High correlations were established between the inhibition of enzymatic activities and amounts of Cu bioaccumulated by gammarids. These biological responses could thus provide early-warming of Cu impact on aquatic biota.

Early genotoxic effects in gill cells and haemocytes of Dreissena polymorpha exposed to cadmium, B[a]P and a combination of B[a]P and Cd.

The aim of this study was to assess the genotoxic potential of environmentally relevant concentrations of Cd on the zebra mussel, an important freshwater sentinel organism, and to determine the stability of DNA damage in gill cells and haemocytes. The oxidative DNA damage and the co-genotoxicity of Cd in combination with B[a]P were investigated. We measured DNA damage in haemocytes and gill cells of zebra mussels exposed for 11 days to a constant concentration of Cd (10µg/L), B[a]P (10µg/L) or the two combined chemicals (10µg/L+1µg/L). Enzymatic dissociation of gills with dispase gave the lower percentage DNA in tail, compared with collagenase/dispase or collagenase. Bioaccumulation of cadmium in the soft tissues of mussels exposed to CdCl(2) or CdCl(2)+B[a]P increased in a time-dependent manner indicating that both exposures were effective. Cd (10µg/L) is genotoxic only during the first 3 days of exposure in gill cells, while in haemocytes the genotoxicity of Cd was observed later. B[a]P (10µg/L) induced an early increase of DNA damage in gill cells (after 10h and 1 day), while in both gill cells and haemocytes, B[a]P caused a marked increase of DNA damage after 3 days of exposure. The Cd+B[a]P mixture decreased the DNA-damaging effect of Cd and B[a]P in both cell types. Cd induced an increase of DNA damage in Fpg-treated slides, indicating that Cd contributed to oxidative DNA damage. Cadmium induced a cytogenetic effect in gill cells, assessed by the number of micronuclei, throughout the duration of the exposure, while B[a]P did not induce any cytogenetic effect. B[a]P, Cd and Cd+B[a]P induced a transient increase in the number of bi-nucleated cells. Our data clearly show that gills are more sensitive to Cd and B[a]P, which makes them more suitable for future bio-monitoring studies.

Low exposure levels of urban metals induce heterotrophic community tolerance: a microcosm validation.

The biological response of periphyton chronically exposed to metals of urban origin (Cd, Ni and Zn) was investigated with a Pollution-Induced Community Tolerance (PICT) approach using a previously developed short-term toxicity test based on ß-glucosidase (heterotrophic) activity. Periphyton was grown on plastic membranes immersed in indoor aquaria contaminated with metals at realistic contamination levels (0.3, 3 µg/l for Cd, 5, 50 µg/l for Ni, 20, 200 µg/l for Zn). After 3 weeks of exposure, biofilms' parameters (dry-weight, chlorophyll a concentration, heterotrophic activity) were analyzed and tolerance acquisition of the heterotrophic communities was assessed using the toxicity test. Modifications of bacterial and eukaryotic community structure were assessed with Automated Ribosomal Intergenic Spacer Analysis (ARISA). Effects of metal exposure were observed on biofilms parameters in the Cd and Zn experiments. Tolerance levels increased for both Cd-exposed biofilms, and for the high metal treatment biofilms in the Ni and Zn experiments. Analysis of the ARISA profiles showed that metal exposure affected the structure of both bacterial and eukaryotic communities. Moreover, Cd tolerance of the Zn-exposed heterotrophic communities was evaluated, which showed that the Zn-tolerant community (high metal treatment in the Zn experiment) also became tolerant to Cd (co-tolerance). The study shows that tolerance acquisition can be detected after exposure to environmental metal concentrations using ß-glucosidase activity as an endpoint in short-term toxicity tests.

Spatio-temporal variability of solid, total dissolved and labile metal: passive vs. discrete sampling evaluation in river metal monitoring.

In order to obtain representative dissolved and solid samples from the aquatic environment, a spectrum of sampling methods are available, each one with different advantages and drawbacks. This article evaluates the use of discrete sampling and time-integrated sampling in illustrating medium-term spatial and temporal variation. Discrete concentration index (CI) calculated as the ratio between dissolved and solid metal concentrations in grab samples are compared with time-integrated concentration index (CI) calculated from suspended particulate matter (SPM) collected in sediment traps and labile metals measured by the diffusive gel in thin films (DGT) method, collected once a month during one year at the Seine River, upstream and downstream of the Greater Paris Region. Discrete CI at Bougival was found to be significantly higher than at Triel for Co, Cu, Mn, Ni and Zn, while discrete metal partitioning at Marnay was found to be similar to Bougival and Triel. However, when using time-integrated CI, not only was Bougival CI significantly higher than Triel CI, CI at Marnay was also found to be significantly higher than CI at Triel which was not observed for discrete CI values. Since values are time-averaged, dramatic fluctuations were smoothed out and significant medium-term trends were enhanced. As a result, time-integrated concentration index (CI) was able to better illustrate urbanization impact between sites when compared to discrete CI. The impact of significant seasonal phenomenon such as winter flood, low flow and redox cycles was also, to a certain extent, visible in time-integrated CI values at the upstream site. The use of time-integrated concentration index may be useful for medium- to long-term metal studies in the aquatic environment.

Adapting an enzymatic toxicity test to allow comparative evaluation of natural freshwater biofilms' tolerance to metals.

A simple, low-cost and non-radioactive short-term toxicity test was developed to study the effects of urban metals on natural freshwater periphytic communities. ß-glucosidase activity of natural freshwater biofilms collected in situ was chosen as an endpoint. Metals (Cd, Cu, Ni, Pb, and Zn) successfully inhibited bacterial enzymatic activity after a 1-h exposure enabling the calculation of EC(50). The EC(50) value of a biofilm sample varied with the Total Suspended Solids concentration (TSS) of the biofilm suspension, showing that EC(50) values (expressed as total added metal concentrations) are not representative of the bioavailable metal concentration during the toxicity test. For Cu, Cd, Ni, Zn and Pb, the EC(50) values increased linearly with the TSS concentration leading us to define a normalized EC(50): the value of the EC(50) divided by the corresponding TSS concentration. Normalized EC(50) proved to be a robust, reliable way to assess metal tolerance of a biofilm for Cd, Cu, Ni, Zn and Pb. Normalized EC(50) obtained, expressed as kg(metal)/g(TSS), varied between 0.2 to 7.6 for Cu, 1 to 8 for Cd, 1.8 to 92.3 for Ni, 1.8 to 76.6 for Zn and 25 to 189 for Pb.

ISTA 14-in-situ accumulation of PAHs in low-density polyethylene membranes in sediment.

The use of passive samplers for the assessment of organic contaminants has been extended to solid matrixes for the past decade. Passive sampling is usually applied to sediment in laboratory experiments involving significant upheaval, whereas in-situ experiments remain rare. In this study, low-density polyethylene (LDPE) strips were deployed within the sediments of a small river contaminated with polycyclic aromatic hydrocarbons (PAHs). LDPE strips were deployed in the 3-cm depth sediment layer. Over a period of 36 days, LDPE strips were regularly retrieved and accumulated PAHs in LDPE were extracted and analyzed. Accumulations of hydrophobic contaminants in LDPE directly exposed in the sediment were observed. Accumulations in LDPE were observed for moderately hydrophobic PAHs with the highest concentrations in the sediment. Low accumulations were observed for more hydrophobic compounds, despite their presence in high concentrations in the sediment. This was explained by very low exchange rates and competitive interactions with particles in the sediment.

A model predicting waterborne cadmium bioaccumulation in Gammarus pulex: the effects of dissolved organic ligands, calcium, and temperature.

Metal bioavailability depends on the presence of organic ligands in the water and on the concentrations of competitive cations. The present study aims at testing whether the diffusive gradient in thin films technique (DGT) could be used to take into account Cd speciation and its consequences on bioavailability in a bioaccumulation model and whether the influences of the Ca concentration and temperature also should be considered. Four kinetic experiments were conducted on Gammarus pulex: a calibration of Cd turnover rates and of the DGT lability in mineral water, a study of the influence f ethylenediaminetetraacetic acid (EDTA) and humic acids (HA) on uptake rates, and two experiments testing the influence of the Ca concentrations and temperature on Cd uptake clearance rates (ku). In mineral water, where Cd was considered fully labile, the ku was 0.46 L g⁻¹ d⁻¹, and the depuration rate was 0.032 d⁻¹. The initial Cd influxes were lowered significantly by additions of 10 µg L⁻¹ of EDTA or 10 mg L⁻¹ of HA in the water but not at 5 mg L⁻¹HA, even if DGT measurements proved that Cd formed Cd-HA complexes in that treatment. Increasing Ca concentrations lowered ku values, and a competitive inhibition model between Ca and Cd fitted the data. A 30% enhancement of k, values was observed when the temperature was increased by 8°C, which appeared comparatively as a weak effect. Thus, taking into account the metal speciation and the influence of the Ca concentration should improve Cd bioaccumulation modeling in amphipods. In freshwater, where metal bioavailability is reduced by the presence of dissolved organic matter, forecasting Cd waterborne uptake using the labile concentrations should allow robust comparisons between laboratory and field studies.

In situ measurement with diffusive gradients in thin films: effect of biofouling in freshwater.

Concerning in situ passive sampler deployment, several technical priorities must be considered. In particular, deployment time must be sufficiently long not only to allow a significant quantity to be accumulated to facilitate analysis but also to ensure that the signal is above the quantification limit and out of the blank influence. Moreover, regarding the diffusive gradient in thin films (DGT) technique, deployment time must also be sufficiently long (at least 5 days) to avoid the interactions of the solutes with the material diffusion layer of the DGT and for the steady state to be reached in the gel. However, biofouling occurs in situ and modifies the surface of the samplers. In this article, we propose a kinetic model which highlights the biofouling effect. This model was able to describe the mitigation of the flux towards the DGT resin observed on Cd, Co, Mn, Ni and Zn during a 22-day deployment in the Seine River. Over a period of 22 days, biofouling had a significant impact on the DGT concentrations measured, which were decreased twofold to threefold when compared to concentrations measured in unaffected DGTs.

Assessing the relation between anthropogenic pressure and PAH concentrations in surface water in the Seine River basin using multivariate analysis.

Understanding the relation between polycyclic aromatic hydrocarbons (PAHs) in freshwater and anthropogenic pressure is fundamental to finding a solution to reduce the presence of PAHs in water, and thus their potential impact on aquatic life. In this paper we propose to gain greater insight into the variability, sources and partitioning of PAHs in labile (or freely dissolved=not associated to the organic matter), dissolved and particulate phases in freshwater. This study was conducted using land use data as a marker of anthropogenic pressure and coupling it with chemical measurements. This study was conducted on 30 sites in the Seine River basin, which is subjected to a strong human impact and exhibits a wide range of land uses. Half of the sites were studied twice. Labile PAHs were measured by semi-permeable membrane devices (SPMDs), and dissolved and particulate phases by grab samples. Partial least squares regressions were performed between chemical measurements and data of anthropogenic pressure. The results indicate different sources for the dissolved phase and particles. Dissolved and labile phases were more related to the population density of the watershed, while particles were more related to a local pressure. Season and land use data are necessary information to correctly interpret and compare PAH concentrations from different sites. Furthermore, the whole data set of the 45 field deployments comprising labile, dissolved, total and particulate PAH concentrations as well as the physico-chemical parameters is available in the supplementary information.

Metallothionein mRNA induction is correlated with the decrease of DNA strand breaks in cadmium exposed zebra mussels.

We have previously shown that cadmium (Cd) and benzo[a]pyrene (BaP) induced early DNA damages in zebra mussels, and that the level of DNA strand breaks (SB) returned to a basal level after 3 days of exposure to Cd. The aim of the present study was to go further in the mechanisms of Cd and BaP detoxification. For that purpose, expression of genes encoding for metallothionein (MT), aryl hydrocarbon receptor (AHR), P-gp, catalase, glutathione S-transferase and heat shock protein 70 (HSP70) proteins have been measured using RT-qPCR. Data reported here show that Cd is a strong inducer of MT and HSP70 genes, and that BaP is a strong inducer of P-gp and AHR genes. Exposure to Cd and BaP resulted in moderate changes in antioxidant enzymes mRNA. Since the increase of MT mRNA occurred when the DNA SB level returned to its basal level, we can suggest that MT is implicated in cadmium detoxification.

Linking community tolerance and structure with low metallic contamination: a field study on 13 biofilms sampled across the Seine river basin.

It is difficult to assess the biological consequences of diffuse water contamination by micropollutants which are present in rivers at low, even sublethal levels. River biofilms, which respond quickly to changes of environmental parameters, are good candidates to acquire knowledge on the response of aquatic organisms to diffuse chemical contamination in the field. The study was designed as an attempt to link biofilm metal tolerance and metallic contamination in a field survey covering 13 different sampling sites in the Seine river basin (north of France) with low contamination levels. Cd and Zn tolerance of heterotrophic communities was assessed using a short-term toxicity test based on ß-glucosidase activity. Metal tolerance levels varied between sites but there was no obvious correlation between tolerance and corresponding water contamination levels for Cd and Zn. Indeed, metallic contamination at the sampling sites remained subtle when compared to water quality standards (only two sampling sites had either Zn or both Cu and Zn concentrations exceeding the Environmental Quality Standards set by the EU Water Framework Directive). Yet, multivariate analysis of the data using Partial Least Squares Regression revealed that both metallic and environmental parameters were important variables explaining the variability of metal tolerance levels. Automated Ribosomal Intergenic Spacer Analysis (ARISA) was also performed on both bacterial and eukaryotic biofilm communities from the 13 sampling sites. Multivariate analysis of ARISA fingerprints revealed that biofilms with similar tolerance levels have similar ARISA profiles. Those results confirm that river biofilms are potential indicators of low, diffuse contamination levels of aquatic systems.

Essential metal contents in indigenous gammarids related to exposure levels at the river basin scale: metal-dependent models of bioaccumulation and geochemical correlations.

Biomonitoring, assumed to be an integrative measurement of the chemical exposure of aquatic organisms, is not straightforward for essential metals because they can be actively regulated by animals. Although increasing bioaccumulation with exposure levels is a crucial endpoint for the development of biomonitors, it is rarely verified in real environments, where the metal concentrations are rather low and vary little. This study was designed at the scale of a river basin to assess the ability of Gammarus pulex indigenous populations to accumulate Cu, Zn and Mn in realistic exposure conditions. During two annual campaigns, water and gammarids were collected at various sites contrasted in terms of physicochemistry and contamination. The results show significant relationships between metal concentrations in animals and in freshwaters established by conceptual models of bioaccumulation, but with patterns specific to each metal (base level, internal regulation and maximal accumulation). In particular, a saturation process of Cu accumulation occurs at environmental exposure levels, unlike Mn and Zn. Statistical analyses performed from field data show that Cu and Zn bioaccumulations may be influenced by a complex combination of geochemical variables, unlike Mn. We conclude that G. pulex is a useful candidate to monitor metal bioavailability in freshwaters due to its responsiveness to low exposures of surrounding environments. Nevertheless, a reliable quantification of bioavailability of essential metals requires characterizing some geochemical effects on metal bioaccumulation.