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.

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.

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.

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.

Dissolved organic matter from treated effluent of a major wastewater treatment plant: characterization and influence on copper toxicity.

A combination of reverse osmosis (RO) concentration and DAX-8/XAD-4 resin adsorption techniques is used to isolate the various constituents of urban dissolved organic matter (DOM) from inorganic salts. Three fractions: hydrophobic (HPO), transphilic (TPI) and hydrophilic (HPI) accounting respectively for 35%, 20% and 45% of extracted carbon, are isolated from effluents of a major French wastewater treatment plant. This atypical DOC distribution, in comparison with natural water where the HPO fraction dominates, shows the significance of HPI fraction which often gets neglected because of extraction difficulties. A number of analytical techniques (elemental, spectroscopic: UV, FTIR) allow highlighting the weak aromaticity of wastewater effluent DOM (EfOM) due to fewer degradation and condensation processes and the strong presence of proteinaceous structures indicative of intense microbial activity. Copper toxicity in the presence of DOM is estimated using an acute toxicity test on Daphnia Magna (Strauss). Results reveal the similar protective role of each EfOM fraction compared to reference Suwannee river fulvic acid despite lower EfOM aromaticity (i.e. specific UV absorbance). The environmental implications of these results are discussed with respect to the development of site-specific water quality criteria.

More than inorganic copper is bioavailable to aquatic mosses at environmentally relevant concentrations.

The present study investigates how dissolved organic matter (DOM) alters copper bioavailability at environmentally relevant concentrations (1-5 microg/L of dissolved copper, 1-4 mg/L of dissolved organic copper). A methodology combining two biological endpoints (short-term and steady-state bioaccumulation of copper by the aquatic moss Fontinalis antipyretica) and a sampling of labile copper with diffusion gradient in thin films (DGT) is proposed for batch experiments conducted with mineral water and various DOM, ethylenediaminetetra-acetic acid (EDTA), humic acid, and natural Seine River (France) extracts (hydrophobic and transphilic fractions). All types of DOM reduce the bioavailability of copper to aquatic mosses, and this reduction was more pronounced for the short-term biological endpoint, which was taken as being representative for environmental exposure. Labile copper sampled with DGT made it possible to estimate short-term bioaccumulation in the case of EDTA and natural Seine River extracts. With humic acid solutions, however, labile copper was lower than bioavailable copper. This result suggests that at realistic metal concentrations and with certain types of natural DOM, bioavailable copper might comprise not only inorganic copper but also some weak organic complexes. Hence, labile copper, in situ sampled with DGT, might not systematically overestimate bioavailable copper, as suggested previously on the basis of in vitro toxicity studies.

Effects of chronic dietary and waterborne cadmium exposures on the contamination level and reproduction of Daphnia magna.

Regulatory assessments of metal toxicity on freshwater organisms assume that toxic effects are caused by dissolved metals. In aquatic systems, organisms are exposed to both dissolved and particulate-bound metals. In this study, the chronic toxicity of dietary cadmium (Cd) on the reproduction and Cd body burden of Daphnia magna was investigated. Daphnids (<24 h) were successively exposed to dissolved Cd (8 h) and then to uncontaminated or contaminated algae (16 h) for 21 d. The results show a higher Cd burden in daphnids because of the addition of contaminated food and reveal that Cd uptake by D. magna from water and food was additive for the lowest Cd concentrations tested. Similar Cd distributions (cytosolic and insoluble fractions) were observed in the two groups of organisms, showing similar potential toxicity of Cd accumulated from the two exposure routes. Dietary Cd induces deleterious effects on D. magna reproduction. On the basis of Cd body burden of daphnids, the results support the claim that waterborne and dietary Cd exposures were additive in causing toxicity for Cd concentrations lower than 25 microg/L. At the highest Cd concentrations, the importance of dietary Cd on the daphnid contamination level decreases and confounding factors such as feeding rate reduction seem to appear, which induce an effect on neonate reproduction. In this study, we illustrate the need to take the dietary pathway into account in regulatory assessments and to establish effective concentrations with particulate-bound metals.

Primary production in headwater streams of the Seine basin: the Grand Morin river case study.

Periphytic biomass has an important influence on the water quality of many shallow streams. The purpose of this paper is to synthesize the knowledge obtained on periphyton during the PIREN Seine research program. Periphyton was sampled using chl a measurements by acetone extraction and oxygen measurements with microelectrodes. The experiments reveal the presence of an important fixed biomass ranging between 123 and 850 mgchl a m(-2) and the mean gross production (photosynthesis) is shown to range between 180 and 315 mgC m(-2) h(-1). An independent approach was performed using the ProSe model, which simulates transport and biogeochemical processes in 22 km of the Grand Morin stream. A strong agreement between in situ measurements and the model results was obtained. The gross production obtained using ProSe is 220 mgC m(-2) h(-1) for the periphyton, which matches the experimental data. Although the net photosynthetic activity of the phytoplankton (0.84 gC gC(-1) d(-1)) is higher than the periphytic one (0.33 gC gC(-1) d(-1)), the absolute periphytic activity is greater since the mean biomass (3.4 gC m(-)(2)) is 10 times higher than the phytoplanktonic one (0.3 gC m(-2)), due to the short residence time of the water body (1.5d).

Dissolved and bioavailable contaminants in the Seine river basin.

Diffusive Gradient in Thin Films (DGT) and Semi-Permeable Membrane Devices (SPMDs) were deployed in the Seine river basin in order to assess labile metals and truly dissolved Polycyclic Aromatic Hydrocarbons. We show that the tools are reliable in aquatic environments to assess the speciation of dissolved contaminants and hence provide a good insight into the potential bioavailability of contaminants. The deployment of the DGT and SPMDs in contrasting environments in the Seine river basin allowed distinction to be made of availability of contaminants between headwater streams and much more impacted river reaches and an assessment of bioavailability. At the stations under urban influence, the impact of dissolved organic matter on both copper and PAHs bioavailability is less pronounced than at upstream stations, where humic substances dominate.

Evaluation of DGT as a metal speciation tool in wastewater.

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

Short-term variability of dissolved trace element concentrations in the Marne and Seine rivers near Paris.

The concentrations of dissolved trace elements (Li, B, Mn, Cu, As, Rb, Sr, Mo, Cd, Ba, Pb) in the Marne and Seine rivers in the Paris urban area were monitored over a 2-year period. The resulting data indicated moderate contamination of waters by the most toxic elements (Cu, As, Cd and Pb). The River Marne upstream and the River Seine downstream of the city of Paris displayed similar concentrations. However higher fluxes of trace elements were observed in the Seine than in the Marne due to their different discharges. Li, B, Rb, Sr and Ba concentrations were correlated with river discharge and concentrations were higher during high river flow. This was interpreted as a dilution by discharge from a major natural or anthropogenic source. Mn, Cu, Mo, Cd and Pb concentrations were not correlated with discharge. Dissolved Mn, Cu and Cd increased rapidly in summer, whereas the concentration of Mo decreased. These variations were attributed to redox processes. During summer when the dissolved oxygen concentrations decrease, Mn, Cu, Cd and Pb are released into solution whereas Mo is immobilised. Like metals, variations in arsenic contents were not linked with discharge. Its similarity with phosphate distribution suggests similar controls involving phytoplankton uptake and release from sediments through organic matter mineralization.

Trace metal speciation and fluxes within a major French wastewater treatment plant: impact of the successive treatments stages.

Seven metals (Cd, Co, Cr, Cu, Fe, Ni and Pb) were monitored at the Seine-Aval wastewater treatment plant during 6 sampling campaigns in April 2004. Particulate and dissolved metals have been measured in 24h composite samples at each treatment stage (primary settling, secondary activated sludge and tertiary flocculation by FeCl(3)). In addition, the diffusive gradient in thin film technique (DGT) was used to determine the dissolved inert and labile metal fraction. Although all treatment stages were able to decrease particulate metals concentrations in wastewater, most dissolved metals concentrations were mainly affected during primary settling. This unexpected result was attributed to tertiary sludge filtrate recirculation. Metals added via the FeCl(3) reagent at the tertiary treatment were shown to lower the overall Cr removal from wastewater and to enrich Ni in effluents. The plant operating conditions (recirculation and reagent addition) appear therefore as important as treatment processes for the metals removal. Total metal fluxes were highly decreased by the whole treatment plant for Cd, Cr, Cu and Pb and to a lesser extend for Co and Ni. However, the labile metal fluxes were poorly decreased for Cu (18%), not significantly decreased for Ni and increased for Fe. The labile fraction of Cd, Co and Cr was not detectable at any stage of the plant. Discharged labile fluxes, at least for Ni, were potentially significant compared to the labile metal fluxes in the river measured downstream the plant. Treated urban wastewater discharges should be carefully considered as a possible source of bioavailable trace metals.

Variability estimation of urban wastewater biodegradable fractions by respirometry.

This paper presents a methodology for assessing the variability of biodegradable chemical oxygen demand (COD) fractions in urban wastewaters. Thirteen raw wastewater samples from combined and separate sewers feeding the same plant were characterised, and two optimisation procedures were applied in order to evaluate the variability in biodegradable fractions and related kinetic parameters. Through an overall optimisation on all the samples, a unique kinetic parameter set was obtained with a three-substrate model including an adsorption stage. This method required powerful numerical treatment, but improved the identifiability problem compared to the usual sample-to-sample optimisation. The results showed that the fractionation of samples collected in the combined sewer was much more variable (standard deviation of 70% of the mean values) than the fractionation of the separate sewer samples, and the slowly biodegradable COD fraction was the most significant fraction (45% of the total COD on average). Because these samples were collected under various rain conditions, the standard deviations obtained here on the combined sewer biodegradable fractions could be used as a first estimation of the variability of this type of sewer system.

Influence of algal and bacterial particulate organic matter on benzo[a]pyrene bioaccumulation in Daphnia magna.

In order to better asses the influence of organic matter on the bioavailability of hydrophobic organic contaminants, the effect of algae and POM of bacterial origin on the bioaccumulation of benzo[a]pyrene in Daphnia magna was evaluated. The bioaccumulation was monitored with increasing concentrations of particulate organic matter (POM) and dissolved organic matter (DOM). In all experiments, the presence of POM greatly reduced the bioaccumulation of benzo[a]pyrene. The reduction was more pronounced in the presence of algae, for which we observed a 99%-reduction effect in the presence of 6 x10 (5) cell/mL (equivalent to 5.3 mg C/L). The bioaccumulation of benzo[a]pyrene was decreased by 49% by organic matter of bacterial origin at 4.7 mg C/L. Assuming that benzo[a]pyrene was partitioned between water, DOM and POM and supposing that D. magna accumulated free benzo[a]pyrene via respiration and POM-bond benzo[a]pyrene via ingestion, bioaccumulation data allowed to estimate the dietary uptake rate of benzo[a]pyrene as well as partitioning coefficients K(POC) and K(DOC). Despite the ingestion of contaminated particles, we could not observe any dietary uptake of benzo[a]pyrene in daphnids. We verified, as usually supposed, that the bioaccumulation of benzo[a]pyrene to D. magna occurs mainly via direct contact. Very high partitioning coefficients (log K(POC) between 5.2 and 6.2) were estimated. This study pointed out the great influence of biogenic organic matter on the fate and the bioavailability of benzo[a]pyrene in aquatic ecosystems.

Biodegradable fraction of organic carbon estimated under oxic and anoxic conditions.

The biodegradability of water samples is usually estimated with bioassays under oxic conditions. In order to overcome some of the drawbacks linked to the incubation of the samples in aerobic batches, a new protocol is proposed and tested, which is based on an organic carbon (OC) balance after a 45 days incubation under anoxic conditions with excess nitrate. The biodegradable fractions of organic matter obtained with the anoxic protocol are slightly lower than those obtained under oxic conditions. Several possible reasons for a systematic underestimation of the biodegradable organic matter under anoxic conditions are evaluated and discussed: a reduced microbial metabolic potential, significantly reduced degradation rates for the slowly biodegradable organic matter, an additional production of refractory organic compounds during the incubation, or the inhibition of the recycling of the organic matter stored in bacterial biomass. Nevertheless, the 7% difference observed on the biodegradable total OC estimations keeps low enough so that the anoxic protocol can be proposed as a convenient alternative to the oxic one.

Effect of dissolved organic matter of various origins and biodegradabilities on the bioaccumulation of polycyclic aromatic hydrocarbons in Daphnia magna.

As a preliminary study of the influence of urban organic matter on the bioavailability of polycyclic aromatic hydrocarbons (PAHs), the effect of different types of dissolved organic matter (DOM) on the bioaccumulation of fluoranthene, pyrene, or benzo[a]pyrene in Daphnia magna was studied. Commercial humic substances, DOM from the aeration basin of a wastewater treatment plant, and highly biodegradable DOM (algae or animal extracts) were tested. The bioaccumulation of benzo[a]pyrene was reduced by each DOM (up to 80% reduction with humic substances). Pyrene bioaccumulation was also decreased by each DOM to a lesser extent. Fluoranthene bioaccumulation was affected by the presence of humic acids only. In each experiment, the solution containing humic DOM led to the lowest bioaccumulation. Supposing that only dissolved PAHs were bioavailable, the reduction of bioaccumulation allowed a biological estimate of the partition coefficients of DOM and PAH, K(DOC). The estimated coefficients were positively related to the aromaticity of DOM and negatively related to its biodegradability.

Performance of diffusion gradient in thin films to evaluate the toxic fraction of copper to Daphnia magna.

This study investigates the relevance of the diffusion gradient in thin films technique (DGT) to measure copper's induced lethality on Daphnia magna in natural water spiked with various organic ligands. Ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), and glycine were used as artificial organic ligands in controlled solutions of mineral water. With EDTA, DGT measurement makes it possible to predict the toxicity of the mixture because Cu-EDTA complexes are inert, whereas DGT is of no help in the case of NTA, because Cu-NTA complexes are fully labile. The Cu-glycine complexes appear as partly labile and toxic. Humic acids as well as fresh and aged algae extracts also were used as models for natural dissolved organic matters. All three of them form copper complexes that are not toxic to Daphnia magna and appear as partly labile with open-pored DGT. However, the use of restricted gels in DGT greatly reduces the contribution of labile complexes, at least for humic acids and aged algae copper complexes. The DGT with restrictive gels, therefore, appears to be a powerful tool for measuring bioavailable copper in natural water bodies, especially when the dissolved organic matter mostly is of humic origin. The DGTs potential ability to mimic the lability induced by the biological uptake also is discussed.

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.