High denitrification potential but low nitrous oxide emission in a constructed wetland treating nitrate-polluted agricultural run-off.

Constructed wetlands (CW) can efficiently remove nitrogen from polluted agricultural run-off, however, a potential caveat is nitrous oxide (N2O), a harmful greenhouse gas and stratospheric ozone depleter. During five sampling campaigns, we measured N2O fluxes from a 0.53 ha off-stream CW treating nitrate-rich water from the intensively fertilized watershed in Rampillon, France, using automated chambers with a quantum cascade laser system, and manual chambers. Sediment samples were analysed for potential N2 flux using the HeO2 incubation method. Both inlet nitrate (NO3-) concentrations and N2O emission varied significantly between the seasons. In the Autumn and Winter inlet concentrations were about 11 mg NO3--N L-1, and < 6.5 mg NO3--N L-1 in the Spring and Summer. N2O emission was highest in the Autumn (mean ± standard error: 9.7 ± 0.2 µg N m-2 h-1) and lowest in the Summer (wet period: 0.2 ± 0.3 µg N m-2 h-1). The CW was a very weak source of N2O emitting 0.32 kg N2O-N ha-1 yr-1 and removing around 938 kg NO3--N ha-1 yr-1, the ratio of N2O-N emitted to NO3--N removed was 0.033%. The automated and manual chambers gave similar results. From the potential N2O formation in the sediment, only 9% was emitted to the atmosphere, the average N2 N 2O ratio was high: 89:1 for N2-Npotential: N2O-Npotential and 1353:1 for N2-Npotential: N2O-Nemitted. These results indicate complete denitrification. The focused principal component analysis showed strong positive correlation between the gaseous N2O fluxes and the following environmental factors: NO3--N concentrations in inlet water, streamflow, and nitrate reduction rate. Water temperature, TOC and DOC in the water and hydraulic residence time showed negative correlations with N2O emissions. Shallow off-stream CWs such as Rampillon may have good nitrate removal capacity with low N2O emissions.

Role of Biofilms in Contaminant Bioaccumulation and Trophic Transfer in Aquatic Ecosystems: Current State of Knowledge and Future Challenges.

In freshwater environments, microbial assemblages attached to submerged substrates play an essential role in ecosystem processes such as primary production, supported by periphyton, or organic matter decomposition, supported by microbial communities attached to leaf litter or sediments. These microbial assemblages, also called biofilms, are not only involved in nutrients fluxes but also in contaminants dynamics. Biofilms can accumulate metals and organic contaminants transported by the water flow and/or adsorbed onto substrates. Furthermore, due to their high metabolic activity and their role in aquatic food webs, microbial biofilms are also likely to influence contaminant fate in aquatic ecosystems. In this review, we provide (1) a critical overview of the analytical methods currently in use for detecting and quantifying metals and organic micropollutants in microbial biofilms attached to benthic substrata (rocks, sediments, leaf litter); (2) a review of the distribution of those contaminants within aquatic biofilms and the role of these benthic microbial communities in contaminant fate; (3) a set of future challenges concerning the role of biofilms in contaminant accumulation and trophic transfers in the aquatic food web. This literature review highlighted that most knowledge on the interaction between biofilm and contaminants is focused on contaminants dynamics in periphyton while technical limitations are still preventing a thorough estimation of contaminants accumulation in biofilms attached to leaf litter or sediments. In addition, microbial biofilms represent an important food resource in freshwater ecosystems, yet their role in dietary contaminant exposure has been neglected for a long time, and the importance of biofilms in trophic transfer of contaminants is still understudied.

Application of a multidisciplinary and integrative weight-of-evidence approach to a 1-year monitoring survey of the Seine River.

Quality assessment of environments under high anthropogenic pressures such as the Seine Basin, subjected to complex and chronic inputs, can only be based on combined chemical and biological analyses. The present study integrates and summarizes a multidisciplinary dataset acquired throughout a 1-year monitoring survey conducted at three workshop sites along the Seine River (PIREN-Seine program), upstream and downstream of the Paris conurbation, during four seasonal campaigns using a weight-of-evidence approach. Sediment and water column chemical analyses, bioaccumulation levels and biomarker responses in caged gammarids, and laboratory (eco)toxicity bioassays were integrated into four lines of evidence (LOEs). Results from each LOE clearly reflected an anthropogenic gradient, with contamination levels and biological effects increasing from upstream to downstream of Paris, in good agreement with the variations in the structure and composition of bacterial communities from the water column. Based on annual average data, the global hazard was summarized as "moderate" at the upstream station and as "major" at the two downstream ones. Seasonal variability was also highlighted; the winter campaign was least impacted. The model was notably improved using previously established reference and threshold values from national-scale studies. It undoubtedly represents a powerful practical tool to facilitate the decision-making processes of environment managers within the framework of an environmental risk assessment strategy.

Behavioural and biochemical responses to metals tested alone or in mixture (Cd-Cu-Ni-Pb-Zn) in Gammarus fossarum: From a multi-biomarker approach to modelling metal mixture toxicity.

Metals are usually present as mixtures at low concentrations in aquatic ecosystems. However, the toxicity and sub-lethal effects of metal mixtures on organisms are still poorly addressed in environmental risk assessment. Here we investigated the biochemical and behavioural responses of Gammarus fossarum to Cu, Cd, Ni, Pb and Zn tested individually or in mixture (M2X) at concentrations twice the levels of environmental quality standards (EQSs) from the European Water Framework Directive. The same metal mixture was also tested with concentrations equivalent to EQSs (M1X), thus in a regulatory context, as EQSs are proposed to protect aquatic biota. For each exposure condition, mortality, locomotion, respiration and enzymatic activities involved in digestive metabolism and moult were monitored over a 120h exposure period. Multi-metric variations were summarized by the integrated biomarker response index (IBR). Mono-metallic exposures shed light on biological alterations occurring at environmental exposure levels in gammarids and depending on the considered metal and gender. As regards mixtures, biomarkers were altered for both M2X and M1X. However, no additive or synergistic effect of metals was observed comparing to mono-metallic exposures. Indeed, bioaccumulation data highlighted competitive interactions between metals in M2X, decreasing subsequently their internalisation and toxicity. IBR values indicated that the health of gammarids was more impacted by M1X than M2X, because of reduced competitions and enhanced uptakes of metals for the mixture at lower, EQS-like concentrations. Models using bioconcentration data obtained from mono-metallic exposures generated successful predictions of global toxicity both for M1X and M2X. We conclude that sub-lethal effects of mixtures identified by the multi-biomarker approach can lead to disturbances in population dynamics of gammarids. Although IBR-based models offer promising lines of enquiry to predict metal mixture toxicity, further studies are needed to confirm their predictive quality on larger ranges of metallic combinations before their use in field conditions.

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.

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.

Contribution of aqueous and dietary uptakes to lead (Pb) bioaccumulation in Gammarus pulex: From multipathway modeling to in situ validation.

Although dynamic approaches are nowadays used increasingly to describe metal bioaccumulation in aquatic organisms, the validation of such laboratory-derived modeling is rarely assessed under environmental conditions. Furthermore, information on bioaccumulation kinetics of Pb and the significance of its uptake by dietary route is scarce in freshwater species. This study aims at modeling aqueous and dietary uptakes of Pb in the litter-degrader Gammarus pulex and assessing the predictive quality of multipathway modeling from in situ bioaccumulation data. In microcosms, G. pulex were exposed to environmentally realistic concentrations of Pb (from 0.1 to 10µg/L) in the presence of Pb-contaminated poplar leaves, which were enclosed or not in a net to distinguish aqueous and dietary uptakes. Results show that water and food both constitute contamination sources for gammarids. Establishing biodynamic parameters involved in Pb aqueous and dietary uptake and elimination rates enabled to construct a multipathway model to describe Pb bioaccumulation in gammarids. This laboratory-derived model successfully predicted bioaccumulation measured in native populations of G. pulex collected in situ when local litter was used as dietary exposure source. This study demonstrates not only the suitable applicability of biodynamic parameters for predicting Pb bioaccumulation but also the necessity of taking dietary uptake into account for a better interpretation of the gammarids' contamination in natural conditions.

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.

Impact of an urban multi-metal contamination gradient: metal bioaccumulation and tolerance of river biofilms collected in different seasons.

The aim of this study was to investigate the repeatability and seasonal variability of the biological response of river biofilms chronically exposed to a multi-metal pressure in an urban contamination gradient. Biofilms were grown on immersed plastic membranes at three sites on the Seine river upstream (site 1) and downstream (sites 2 and 3) from Paris (France). They were collected in four different seasons (autumn, spring, summer and winter). Biofilm tolerance to Cu, Ni, Pb and Zn was measured using a PICT (Pollution-Induced Community Tolerance) approach with a previously developed short-term toxicity test based on ß-glucosidase (heterotrophic) activity. Metal concentrations in the river and also in the biofilm samples (total and non-exchangeable bioaccumulated metals) were also monitored. Biofilm-accumulated metal concentrations reflected the increase of the multi-metal exposure along the urban gradient. These concentrations were strongly correlated with dissolved and particulate organic carbon and with the total metal fraction in the river water, which recalls the significant influence of the environmental parameters on metal uptake processes in river biofilms. Overall, natural biofilms allow monitoring water quality by integrating the variations of a diffuse metal contamination overtime. Tolerance levels globally increased from site 1 to site 3 reflecting the metal pollution gradient measured in the river water collected at the three sites. Cu tolerance tended to increase during warm seasons but no clear seasonal tendency could be found for Ni, Pb and Zn. Furthermore, principal component analysis clearly discriminated samples collected upstream (site 1) from samples collected downstream (sites 2 and 3) along the first principal component which was correlated to the metal gradient. Samples collected in winter were also separated from the others along the second principal component correlated to parameters like water temperature and Total Suspended Solids concentration. This study shows that chronic in situ exposure to environmental metal concentrations has a significant impact on natural biofilms. Biofilm tolerance to metals and biofilm metal bioaccumulation both reflect metal exposure levels although they remain low when compared to Environmental Quality Standards from the European Water Framework Directive. Yet temperature appears as an important environmental variable shaping community structure and response to toxic exposure which shows that the sampling date is an important parameter to consider when using natural river biofilms to assess the impacts of urban pressure.

Seasonal variability and inter-species comparison of metal bioaccumulation in caged gammarids under urban diffuse contamination gradient: implications for biomonitoring investigations.

Although caging of Gammarus species offers promising lines of inquiry to monitor metal bioavailability in freshwaters, the interspecies responsiveness to metal exposures is still unclear. In addition, abiotic factors inherent to transplantation can hamper the interpretation of field bioaccumulation data. To assess the relevance of using gammarids as biomonitors, we investigated the seasonal influence on metal bioaccumulation in two common species, Gammarus pulex and Gammarus fossarum. During four seasons, caged gammarids were deployed on three sites along the Seine River exhibiting a diffuse gradient of multi-metal contamination: a site upstream and two sites downstream from the Paris megacity. For each seasonal deployment, metal concentrations in animals were determined after 7d-exposure in situ (Ag, Cd, Co, Cu, Mn, Ni, Pb and Zn). Results show that the seasonal patterns of metal contaminations are similar between both Gammarus species, and closely related to the river axis' contamination gradient. Statistical analyses indicate that bioaccumulation of essential metals in both species is influenced by season, especially by water temperature. This highlights the necessity to consider this climatic factor inherent to the deployment period for a reliable interpretation of bioaccumulation data in the field. The comparison of accumulation factors suggests that these two species coming from different geochemical origins display similar abilities to internalize metals. This generic responsiveness of caged gammarids supports their use as sentinel organisms to quantify low spatiotemporal variations in metal bioavailabilities.

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.

DGT measurement in low flow conditions: diffusive boundary layer and lability considerations.

Recent papers have alerted the scientific community that a diffusive boundary layer (DBL) forming in front of diffusive gradients in thin film (DGT) devices when they are immersed in water might have a significant impact on the results and have suggested a method to assess the DBL. This paper aims at evaluating to what extent the DBL impacts the results of metal measurement in water by DGT and providing new information on the dissociation kinetics of metal complexes in wastewater by using DBL calculation. A careful study of the influence of the water velocity on the measurement with DGTs equipped with restricted gels is presented. Deployments took place in the laboratory with a range of stirring speeds (0-400 rpm) and in a canal receiving treated wastewater with increasing controlled water velocity (0.07-3 cm s(-1)). Even under extreme low flow conditions, the error made in using the equation that does not take into account that the DBL was lower than the analytical error. Nevertheless, the DBL is the seat of dissociation of complexes and increases the lability window beyond the steric constraints of the hydrogel. The capacity of restricted gels to only sample inorganic species under these conditions is questioned. This study also is an opportunity to provide information on metal-ligand interactions in wastewater by creating the kinetic signature of the wastewater. Unlike previous studies which used different types of water, Pb was the more limited metal and interacted strongly with the ligands.

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.

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.

Waterborne nickel bioaccumulation in Gammarus pulex: comparison of mechanistic models and influence of water cationic composition.

The biodynamic and saturation models offer promising lines of enquiry to predict the bioaccumulation of metals by aquatic organisms. However, in order to construct these models, the accumulation strategies have to be defined for each metal/organism couple in controlled conditions. This study aims at modelling the waterborne bioaccumulation of Ni and the influence of the water's geochemical properties on this process in a crustacean that is widely distributed in Europe, Gammarus pulex. In the laboratory, G. pulex was exposed to several Ni concentrations (from 0.001 to 100 mg L(-1)) in aquatic microcosms. Our results show that G. pulex is very tolerant to Ni (LC50(48 h)=477 mg L(-1) Ni). Time course experiments enabled the construction of a biodynamic model by determining the uptake (k(u)) and elimination (k(e)) rate constants. When the exposure concentration exceeded 1 mg L(-1) Ni, the metal uptake reached a maximum due to a limited number of binding sites for Ni. Therefore, the organism's maximal capacity to accumulate the metal (B(max)) and the half-saturation constant (K) were determined to establish the saturation model. We showed that the two models are comparable for the lowest exposure concentrations (<1 mg L(-1) Ni), with k(u)/k(e)=B(max)/K. Then, the bioaccumulation of Ni was recorded in waters exhibiting various concentrations of three major ions (Na(+), Mg(2+) and Ca(2+)). Only Ca had an inhibitory effect on the Ni uptake. This study reports for the first time the bioaccumulation of Ni in G. pulex. Because of its high tolerance to Ni and its high capacity to accumulate this metal, this crustacean could be used as an indicator of Ni bioavailability in freshwaters.

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.