[Micropollutants in aquatic environments and residues of human health care products]. / Micropolluants des milieux aquatiques et résidus des produits de soins humains.

The contamination of water and natural environments by micropollutants linked to residues from health care activities (drugs, detergents, parapharmaceuticals, etc.) generates invisible, silent and alarming levels of pollution for the living world. The management of these phenomena is potentially a public health issue. It is necessary to raise awareness among caregivers in order to develop strategies for reducing pollution at the site, compatible with the multiple constraints of their care activities.

Biomarkers as tools for monitoring within the Water Framework Directive context: concept, opinions and advancement of expertise.

The Water Framework Directive (WFD) currently supports chemical and ecological monitoring programmes in order to achieve the good water surface status. Although chemical and ecological assessments are necessary, they have some limitations. Chemical approaches focus on certain substances identified as priorities, but they do not take into account other potentially harmful substances and also ignore the hazards related to contaminant cocktails. On the other hand, while ecological approaches provide holistic information on the impairment of biological communities in ecosystems, they do not distinguish the role of contaminants in these alterations, and consequently do not allow the establishment of contaminant impact reduction plans. Consequently, ecotoxicologists suggest the use of effect-based tools such as biomarkers. Biomarkers highlight the effect of potentially harmful substances (or a cocktail), and their specificity towards the chemicals makes it possible to properly discriminate the role of toxicants within biological community impairments. Thus, the integration of such tools (besides existing chemical and ecological tools) in the WFD could considerably improve its biomonitoring strategy. The B n' B project (Biomarkers and Biodiversity) exposes key objectives that will allow to (i) establish an inventory of the biomarkers developed by French laboratories; (ii) determine their methodological advancement and limits and, on this basis, formulate recommendations for biomonitoring use and future research needs; (iii) discuss the biomarkers' ecological significance, specificity to contaminants and interpretation capacity; (iv) establish, in fine, a selection of valuable biomarkers to enter the WFD; and (iv) propose integrative tools to facilitate the decision-taking by stakeholders.

Multisubstance Indicators Based on Caged Gammarus Bioaccumulation Reveal the Influence of Chemical Contamination on Stream Macroinvertebrate Abundances across France.

Most anthropogenic stressors affecting freshwater systems are qualitatively known. However, the quantitative assessment of contaminant exposure and effects to aquatic communities is still difficult, limiting the understanding of consequences on aquatic ecosystem functioning and the implementation of effective management plans. Here, multisubstance indicators based on caged gammarid bioaccumulated contamination data are proposed (for metals and persistent organic pollutants, POPs) to map the bioavailable contamination level of freshwater ecosystems at a large spatial scale. We assessed the ability of these indicators to highlight the relationships between chemical exposure gradients and alteration in the abundance of macroinvertebrate populations on a data set of 218 watercourses distributed throughout France. We identified spatial regional heterogeneities in the levels of bioavailable contamination of metals (18 compounds) and POPs (43 compounds). Besides this, a degradation of Gammaridae, Ephemeridae, and Hydrobiidae densities with increasing levels of metal contamination are identified relative to Baetidae, Chironomidae, and Hydropsychidae. We show here that active biomonitoring allows the establishment of multisubstance indicators of bioavailable contamination, which reliably quantify chemical exposure gradients in freshwater ecosystems. Our ability to identify species-specific responses to chemical exposure gradients demonstrates the promising possibility to further decipher the effects of chemical contamination on macroinvertebrate assemblages through this type of indicator.

Evidence for the widespread occurrence of short- and medium-chain chlorinated paraffins in fish collected from the Rhône River basin (France).

Chlorinated paraffins (CPs) are high-volume chemicals used in numerous industrial applications. Their quantitative analysis is extremely challenging and this work presents the optimization of an analytical method based on gas chromatography hyphenated with electron capture negative ionization time-of-flight high-resolution mass spectrometry (GC-ECNI-TOF HRMS) for the simultaneous determination of short-chain and medium-chain CPs (SCCPs and MCCPs, respectively) in fish tissues (i.e. dorsal muscle). The resolution of the TOF-MS analyzer reduced or eliminated isobaric interferences and the CP response was optimized through Design of Experiment. A simple clean-up procedure based on adsorption chromatography further removed some potentially interfering organochlorines. Good selectivity, linearity and accuracy were achieved; method detection limits or limits of reporting were compatible with expected levels in wild fish (0.03-0.35 ng g-1 wet weight, ww, depending on the congener). This method was proven suitable for the analysis of CPs in tissues of common barbel Barbus, a fish species frequently used for water quality monitoring purposes in Europe. SCCPs and MCCPs were found to be widespread within the Rhône river basin (France). At all locations, MCCP concentrations (1.3-72.7 ng g-1 ww) were higher than those of SCCPs (0.3-10.6 ng g-1 ww) and levels were systematically lower than the proposed Predicted No Effect Concentrations (PNECs). Spatial variations of SCCP composition profiles largely surpassed those of MCCPs, suggesting the influence of local sources.

Practical advice for selecting or determining trophic magnification factors for application under the European Union Water Framework Directive.

European Union Directive 2013/39/EU, which amended and updated the Water Framework Directive (WFD; 2000/60/EC) and its daughter directive (2008/105/EC), sets Environmental Quality Standards for biota (EQSbiota ) for a number of bioaccumulative chemicals. These chemicals pose a threat to both aquatic wildlife and human health via the consumption of contaminated prey or the intake of contaminated food originating from the aquatic environment. EU member states will need to establish programs to monitor the concentration of 11 priority substances in biota and assess compliance against these new standards for the classification of surface water bodies. An EU-wide guidance effectively addresses the implementation of EQSbiota . Flexibility is allowed in the choice of target species used for monitoring to account for both diversity of habitats and aquatic community composition across Europe. According to that guidance, the consistency and comparability of monitoring data across member states should be enhanced by adjusting the data on biota contaminant concentrations to a standard trophic level by use of the appropriate trophic magnification factor (TMF), a metric of contaminant biomagnification through the food web. In this context, the selection of a TMF value for a given substance is a critical issue, because this field-derived measure of trophic magnification can show variability related to the characteristics of ecosystems, the biology and ecology of organisms, the experimental design, and the statistical methods used for TMF calculation. This paper provides general practical advice and guidance for the selection or determination of TMFs for reliable application within the context of the WFD (i.e., adjustment of monitoring data and EQS derivation). Based on a series of quality attributes for TMFs, a decision tree is presented to help end users select a reasonable and relevant TMF. Integr Environ Assess Manag 2019;15:266-277. © 2018 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Looking at biological community level to improve ecotoxicological assessment of freshwater sediments: report on a first French-Swiss workshop.

The first French-Swiss workshop on ecotoxicology of freshwater sediment communities was co-organized by the French Research Institute of Science and Technology for Environment and Agriculture (Irstea) and the Swiss Centre for Applied Ecotoxicology (Ecotox Centre EAWAG-EPFL) in Villié-Morgon (Beaujolais Region, France) on April 27-28, 2017. The workshop brought together scientists working in different fields of expertise (ecotoxicologists, ecologists, environmental chemists…), environmental stakeholder groups and managers, as well as economic players (start-ups and consultancies) to better connect research needs of potential end-users with research outputs. The objectives of this workshop were (i) to establish the state of the art of research in the characterization of sediment contamination and in the evaluation of the effects on sediment-associated biological communities and ecosystem functioning and (ii) to give an overview of the French and Swiss regulations dealing with the assessment of contaminated sediments in freshwater ecosystems. The ultimate goal was to collectively identify research needs and knowledge gaps, as well as to highlight ways to improve the ecotoxicological assessment of sediments in freshwater environments by further considering the structure and functions of associated microbial and invertebrate communities.

Responses of two sentinel species (Hexagenia limbata--mayfly; Pyganodon grandis--bivalve) along spatial cadmium gradients in lakes and rivers in northwestern Québec.

Specimens of the mayfly larva Hexagenia limbata and of the floater mussel Pyganodon grandis were sampled in rivers and lakes contaminated by trace metals in the Abitibi-James Bay region in northwestern Québec. Water samples were collected at each sampling site with in situ diffusion samplers and analyzed for major cations, anions and trace metals (Cd, Cu, Mn, Zn). Surficial sediment samples were also collected at each site and analyzed for Cd, Cu and Zn. In response to Cd contamination at river and lake sites, both sentinel organisms accumulated the metal and synthesized metallothionein (MT), a metal-binding protein synthesized by organisms as a defence mechanism against excess metals in the surrounding media. At the river sites, H. limbata unexpectedly maintained much higher concentrations of MT per unit of accumulated Cd than at the lake sites; this difference between lentic and lotic environments may reflect the response of the species to the more stressful hydrodynamic conditions that prevail in a river. The accumulation of Cd in the mayflies at lake and river sites decreased as a function of the ambient manganese concentration. We hypothesize that dissolved Mn protects against Cd bioaccumulation in H. limbata. The present results support the contention that one cannot extrapolate conclusions drawn from the use of a single sentinel species to a larger set of freshwater invertebrates--both the mayfly and the bivalve are promising biomonitors.

Long-term trends in accumulated metals (Cd, Cu and Zn) and metallothionein in bivalves from lakes within a smelter-impacted region.

Temporal monitoring studies are needed to detect long-term trends in ecosystem health. In the present study, we tested metallothionein (MT) as a potential biomarker for long-term variations in trace metal levels in lakes subject to atmospheric metal inputs from a nearby copper smelter. Over a 13-year period, we estimated on several occasions ambient free Cd2+, Cu2+ and Zn2+ concentrations at the sediment-water interface in six lakes with contrasting metal levels, and measured metal and metallothionein concentrations in gills of bivalves (Pyganodon grandis) living in these lakes. All but one of the study lakes had comparable drainage ratios, so inter-lake differences in hydrological export of metals from contaminated watersheds to receiving waters were likely minimal. Declines in the metal emissions from the smelter (especially for Cd and Zn) during the 1980s led to appreciable decreases in both calculated free Cd2+ ion concentrations in the study lakes (-59+/-21% between 1989 and 1998) and accumulated Cd levels in their clam populations (-46+/-12% between 1989 and 2002). Taking all lakes into account, MT concentrations in bivalves have comparatively dropped by 44% (+/-10%) since 1989. In contrast to what we found for Cd, there were no significant reductions in the calculated free Cu2+ and Zn2+ concentrations in the various lakes during our study period (-2 and -10%, respectively, with 95% confidence intervals spanning zero). Overall, observed decreases in MT in bivalves over time were best correlated with similar decreases in both ambient and accumulated Cd levels (r = 0.77, P = 0.0003 and r = 0.79, P < 0.0001, respectively, both P-values corrected for temporal autocorrelation), suggesting that long-term trends in gill MT concentrations in P. grandis may be useful as predictors of temporal changes in environmental Cd contamination in lakes.

Linking changes in subcellular cadmium distribution to growth and mortality rates in transplanted freshwater bivalves (Pyganodon grandis).

Relationships between Cd accumulation and subcellular distribution, and growth and mortality rates were examined in the freshwater bivalve Pyganodon grandis in a transplant experiment. Organisms were transferred from a clean lacustrine site to four lakes situated along a Cd concentration gradient in the mining region of Rouyn-Noranda. The bivalves were maintained in open enclosures placed in the bottom sediments of the littoral zone of all five lakes for 400 days. At the end of the experiment, metallothionein (MT) was measured in the bivalve gills with a Hg-saturation assay and Cd partitioning among the various cytosolic protein pools was determined by size-exclusion chromatography. Marked differences were observed among the five sites: the range in calculated free-cadmium ion concentrations in water overlying the sediments was 35-fold whereas Cd concentrations in the gill cytosol of the transplanted bivalves varied three-fold. In the transplanted bivalves, the distribution of gill Cd among the various cytosolic complexes also varied significantly among sites. For bivalves transplanted to the three most contaminated sites, Cd concentrations in the high molecular weight pool (HMW>25 kDa) were significantly higher than the baseline levels determined from bivalves caged at the reference site; a similar trend was seen for Cd concentrations in the metallothionein pool (Cd-MT). For bivalves transferred to two of the high contamination sites, proportionately less of the gill cytosolic Cd was sequestered (i.e. detoxified) by MT-like proteins. Reductions in survival were also observed at these two sites, and these elevated mortalities, in turn, were consistent with the absence of indigenous bivalve populations at these sites. This result is compatible with our recent work on P. grandis populations living in lakes of the Rouyn-Noranda area, in which we demonstrated that excessive accumulation of Cd in the HMW pool of the gill cytosol of the individual mollusks could be related to the impairment of population health status.

Sub-cellular partitioning of Cd, Cu and Zn in tissues of indigenous unionid bivalves living along a metal exposure gradient and links to metal-induced effects.

We studied organ and sub-cellular distributions of several trace metals in a freshwater bivalve that has been proposed for use as a metal biomonitor. Specimens of Pyganodon grandis were collected from nine lakes located along a Cd, Cu and Zn concentration gradient (Rouyn-Noranda area, Quebec). Gills and digestive gland were isolated, homogenized and six sub-cellular fractions were separated by differential centrifugation and analysed for their Cd, Cu and Zn content. Metallothionein was quantified independently. Gill tissues contained abundant calcium concretions that accounted for over 60% of the total gill burden of each metal. Cadmium and Zn concentrations in this granule fraction reflected ambient metal concentrations. Metal concentrations in the digestive gland also responded to the metal contamination gradient, but to a lesser extent than the gills, reflecting the lower abundance of granules in the digestive gland. Metals (Cd, Cu) in this organ were present largely in the "heat-stable proteins" fraction, and metal concentrations in this fraction were strongly correlated with those of both metallothionein and, to a lesser extent, the "lysosomes+microsomes" and "mitochondria" fractions. In both organs, Cd concentrations in the "heat-denaturable protein" fraction remained low and constant, suggesting reasonably effective metal detoxification. Some evidence for oxidative stress was noted in the gills but not in the digestive gland. Overall, we conclude that in nature metals in P. grandis are bound differently in the gills and in the digestive gland and that metal detoxification in the former organ may be less effective than in the latter.

Sub-cellular partitioning of metals (Cd, Cu, Zn) in the gills of a freshwater bivalve, Pyganodon grandis: role of calcium concretions in metal sequestration.

Indigenous unionid molluscs, Pyganodon grandis, were collected from nine lakes in the Rouyn-Noranda area (Quebec, Canada) along a polymetallic concentration gradient (Cd, Cu, Zn). After excision, the gills were gently homogenised and the cellular compartments were separated by a differential centrifugation procedure that yielded the following particulate fractions: "nuclei + cellular debris", "mitochondria", "lysosomes + microsomes" and "granules". The supernatant remaining after the final ultracentrifugation step, i.e., the operationally-defined cytosol, was separated into a "heat-denaturable proteins" (HDP) fraction and a "heat-stable proteins" (HSP) fraction containing metallothionein (MT). The Cd, Cu and Zn content of each particulate and cytosolic fraction was determined and gill metallothionein was quantified independently by a mercury saturation assay. Cytosolic Cd concentrations were significantly related to the dissolved Cd concentrations at each site, but cytosolic Cu and Zn (essential metals) were not related to their respective ambient dissolved metal concentrations. Metallothionein concentrations increased along the metal contamination gradient and were related to cytosolic Cd (and Zn) in a concentration-dependent manner. However mass balance calculations showed that binding to metallothionein could only account for a small proportion of total gill metal ( approximately 10% Cd; approximately 3% Cu; approximately 1% Zn). Under these chronic exposure conditions, the three metals (Cd, Cu and Zn) were mainly located in calcium concretions present in the gills (respectively 58 +/- 13% of the total gill Cd, 64 +/- 6% of the total gill Cu and 73 +/- 6% of the total gill Zn). The overall contribution of granules to the total gill dry weight remained relatively constant among the different lakes, suggesting that lake-to-lake variations in granule synthesis were independent of the metal contamination gradient, i.e., these constituent elements of unionid gills act as non-inducible metal sinks at the cellular level. Metal concentrations increased proportionally in both the granules and the MT pool along the polymetallic gradient, suggesting a constant partitioning between these two compartments. Overall, despite an increase in Cd in the "mitochondria" fraction, metal sequestration mechanisms seem to be reasonably effective in detoxifying cadmium: in the cytosol, Cd concentrations in the potentially metal-sensitive HDP fraction remained relatively low and constant, even in specimens collected from the most contaminated lakes.

Metal-induced stress in bivalves living along a gradient of Cd contamination: relating sub-cellular metal distribution to population-level responses.

The use of biomarkers to assess the impacts of contaminants on aquatic ecosystems has noticeably increased over the past few years. Few of these studies, however, have contributed to the prediction of ecologically significant effects (i.e., at the population or community levels). The present field study was designed to evaluate the potential of metallothionein (MT) and sub-cellular metal partitioning measurements for predicting toxic effects at higher levels of the biological organization in freshwater bivalves (Pyganodon grandis) chronically exposed to Cd. For that purpose, we quantitatively sampled P. grandis populations in the littoral zone of nine lakes on the Precambrian Canadian Shield during two consecutive summers (1998 and 1999); lakes were characterized by contrasting Cd levels but similar trophic status. We tested relationships between the population status of P. grandis (i.e., growth parameters, density, biomass, secondary production, turnover ratio and cumulative fecundity) and (i) ambient Cd concentrations, (ii) sub-organismal responses (MT concentrations in the gill cytosol of individuals and Cd concentrations in three metal-ligand pools identified as M-HMW, the high molecular weight pool, M-MT, the metallothionein-like pool and M-LMW, the low molecular weight pool) and (iii) ecological confounding factors (food resources, presence of host fishes for the obligatory parasitic larval stage of P. grandis). Our results show that littoral density, live weight, dry viscera biomass, production and cumulative fecundity decreased with increasing concentrations of the free-cadmium ion in the environment (Pearson's r ranging from -0.63 to -0.78). On the other hand, theoretical maximum shell lengths (L( infinity )) in our populations were related to both the dissolved Ca concentration and food quality (sestonic C and N concentrations). Overall, Cd concentrations in the gill cytosolic HMW pool of the individual molluscs were the biomarker response that was most frequently and most strongly correlated with the population variables (Pearson's r ranging from -0.58 to -0.80). Our findings demonstrate, however, the difficulty of currently assigning to sub-cellular metal partitioning measurements (mainly Cd bound to the HMW fraction) any predictive role for population health, notably because of the influence of ecological confounding variables (e.g., the cumulative number of degree-days in the littoral zone, as is the case here). Metal contamination of our lakes has decreased markedly in the past 10 years and consequently we believe that the toxic effects of metals may have been replaced by some natural factors as the main agent for structuring the clam populations in these lakes.

Steady-state distribution of metals among metallothionein and other cytosolic ligands and links to cytotoxicity in bivalves living along a polymetallic gradient.

The present study was designed to assess the environmental effects of metals in a field setting. We explored exposure-->bioaccumulation-->effects relationships in freshwater molluscs exposed to metals in their natural habitat. Indigenous floater mussels (Pyganodon grandis) were collected from ten limnologically similar lakes located along a Cd, Cu and Zn gradient. Ambient free-metal ion concentrations were estimated as a measure of metal exposure. Metallothionein (MT) was measured in mussel gills and metal partitioning among the various cytosolic protein pools was determined by size exclusion chromatography. Various biomarkers were also measured, including malondialdehyde (MDA) concentrations in the gills and in the digestive gland, glutathione-peroxidase and glutathione-reductase activities in the digestive gland, and lipid concentrations in the gonad. Cadmium and MT concentrations in the gill cytosol increased along the contamination gradient, but Cu and Zn levels were independent of the ambient free-metal ion concentrations. The distribution of Cd among the various cytosolic complexes remained quite constant: 80% in the MT-like pool, 7% in the low molecular weight pool (LMW<1.8 kDa) and 13% in the high molecular weight pool (HMW>18 kDa). For these chronically exposed molluscs there was thus no threshold exposure concentration above which spillover of Cd occurred from the MT pool to other cytosolic ligands. However, the presence of Cd in the LMW and HMW fractions suggests that metal detoxification was imperfect, i.e. that P. grandis was subject to some Cd-related stress at low chronic exposure concentrations. Consistent with this suggestion, MDA concentrations, an indicator of oxidative stress, increased with gill cytosolic Cd. In the digestive gland, MDA concentrations were unrelated to any of the measured metals, but glutathione-peroxidase and glutathione-reductase activities increased with gill cytosolic copper. We speculate that cytosolic Cu catalyses the production of reactive oxygen species, to which the organism reacts by increasing activities of the two enzymes, thus preventing the accumulation of reactive oxygen species. Lipid concentrations in the gonad did not decrease with any of the measured toxicological parameters, suggesting that energy reserves for reproduction were not compromised in the metal-contaminated mussels. The results of the present study, where chronically exposed bivalves were collected from their natural habitat along a metal contamination gradient, contrast markedly with what would have been predicted on the basis of experimental metal exposures, and clearly demonstrate the need to study metal exposure-->bioaccumulation-->effects relationships in natural populations.