Magalie Baudrimont

Impacts of gold nanoparticle exposure on two freshwater species: a phytoplanktonic alga (Scenedesmus subspicatus) and a benthic bivalve (Corbicula fluminea)

For years, nanotechnologies have developed the use of common materials, such as iron or silica, at an extremely small scale because of their new properties (reactivity, conductivity, optical sensitivity). More precisely, gold nanoparticles are used in numerous technologies such as electronics, new paints or research on cancer. But, despite their promising future and expansive utilization, only a few studies deal with their behaviors or impacts on the environment. Thus, we decided to explore the impacts of amine-coated 10nm gold nanoparticle (AuNp) contaminations on two freshwater aquatic models. The green algaScenedesmus subspicatus was submitted to 24 h-direct exposures at four AuNp concentrations (1.6×102, 1.6×103, 1.6×104 and 1.6×105 AuNp/cell) along with a control condition. The process used for the freshwater bivalvesCorbicula fluminea was a trophic exposure during 7 days to three AuNp concentrations (1.6×103, 1.6×104 and 1.6×105 AuNp/cell). These conditions were tested in triplicate with controls. For these experiments, OD measurements (γ= 520nm) were performed to verify AuNp concentrations in the water (stability). Cell numerations of algae were used to determine the growth/mortality effects on this species. Cellular impacts and AuNp distributions in the two species were revealed by transmission electron microscopy (TEM). The bioaccumulation rates were assessed by gold dosagesvia MS-ICP procedures. Molecular impacts were analyzed by quantifications of metallothionein concentrations (metal detoxification protein) and genetic expressions via real-time RT-PCR. Our study focused on the expression of six genes encoding proteins involved in: metal detoxification (metallothionein), the response to oxidative stress (catalase and superoxide-dismutase), the mitochondrial respiratory chain (subunit 1 of the cytochrome-C-oxidase), the concentration of mitochondria (RNA12s) and the response to xenobiotics (glutathione S transferase); using the β-actin as reference of the basal rates of gene expressions.The results showed a marked impact on the algae after a 24h-exposure to amine-coated 10nm gold nanoparticles, leading to 20% of mortality for the lowest contamination condition, while the highest one reached 50%. TEM examinations showed that AuNp were strongly adsorbed by the cell wall of algae, leading to progressive intracellular and wall disturbances. The bivalve contaminations revealed the ability of these particles to be bioaccumulated and to penetrate gills and digestive epithelia. Their lysosomial localization leads to the loss of their coating, which brought on an oxidative stress.

Seasonal variations of metallothionein concentrations in the asiatic clam (Corbicula fluminea)

Natural variations of metallothionein (MT) concentrations in the Asiatic clam Corbicula fluminea were analyzed over a 1-year period in specimens collected from an unpolluted site (Cazaux-Sanguinet lake, southwest France). Sampling was carried out from November 1994 to December 1995, one to three times per month, according to the season. At each sampling time, lake temperature was measured and concentrations of MTs, Cd, Hg, Zn and Cu were determined in the whole soft body and in four organs or tissue samples. A histological study was conducted simultaneously to follow the development of the gonads in relation to the reproductive cycle of this bivalve. Results showed very high fluctuations in MT concentrations over the whole year, with a maximum value measured in the middle of May and ratios of around 4 at the whole organism level between extreme MT values. The tissue compartment presenting the most important variations was the visceral mass, which contains the gonads. Metal accumulation in the organisms did not seem to be involved among the factors likely to account for these variations in MT concentrations; metal concentrations remained at low and relatively constant levels throughout the whole year. The histological study revealed one spawning period from late May to the middle of June, appearing just after the MT “peak,” with maximum incubation of the embryos in the gills in late June. Because MT biosynthesis can be induced by hormonal secretions implicated during reproductive phenomena, the variations in MT concentrations appeared to be directly related to the biological cycle of this freshwater mollusc rather than to the direct or indirect effects of metal bioaccumulation.

RAD sequencing reveals within‐generation polygenic selection in response to anthropogenic organic and metal contamination in North Atlantic Eels

Measuring the effects of selection on the genome imposed by human‐altered environment is currently a major goal in ecological genomics. Given the polygenic basis of most phenotypic traits, quantitative genetic theory predicts that selection is expected to cause subtle allelic changes among covarying loci rather than pronounced changes at few loci of large effects. The goal of this study was to test for the occurrence of polygenic selection in both North Atlantic eels (European Eel, Anguilla anguilla and American Eel, A. rostrata), using a method that searches for covariation among loci that would discriminate eels from ‘control’ vs. ‘polluted’ environments and be associated with specific contaminants acting as putative selective agents. RAD‐seq libraries resulted in 23 659 and 14 755 filtered loci for the European and American Eels, respectively. A total of 142 and 141 covarying markers discriminating European and American Eels from ‘control’ vs. ‘polluted’ sampling localities were obtained using the Random Forest algorithm. Distance‐based redundancy analyses (db‐RDAs) were used to assess the relationships between these covarying markers and concentration of 34 contaminants measured for each individual eel. PCB153, 4′4′DDE and selenium were associated with covarying markers for both species, thus pointing to these contaminants as major selective agents in contaminated sites. Gene enrichment analyses suggested that sterol regulation plays an important role in the differential survival of eels in ‘polluted’ environment. This study illustrates the power of combining methods for detecting signals of polygenic selection and for associating variation of markers with putative selective agents in studies aiming at documenting the dynamics of selection at the genomic level and particularly so in human‐altered environments.

Interactive effects of metal contamination and pathogenic organisms on the marine bivalve Cerastoderma edule.

The present study evaluated the interactive effects of cadmium contamination and pathogenic organisms (trematodes Himasthla elongata and bacteria Vibrio tapetis) singularly and in combination during 7 days on the bivalve Cerastoderma edule. Some defense-related activities were analyzed such as genetic expression, metallothionein and immune responses. Trematode metacercarial infection, similar whatever the treatment, induced the strongest responses of immune parameters. Particularly, the interaction between cadmium and parasite exposures induced unusual responses on gene expression and immune responses. No effect of bacterial challenge appeared on bivalve responses, nevertheless a strong mortality of V. tapetis infected cockles occurred between 7 and 14 days. Cadmium bioaccumulation was significantly modulated by both pathogenic organisms. Furthermore, an antagonistic effect of trematodes and bacteria was shown on metal bioaccumulation of co-infected cockles. These results highlighted the importance of considering the multiplicity of perturbation sources in coastal ecosystems to assess the health status of organisms.

Metallothionein gene expression and protein levels in triploid and diploid oysters Crassostrea gigas after exposure to cadmium and zinc.

Quantitative real-time polymerase chain reaction (PCR) was used to compare for the first time the differential expression of metallothionein (MT) isoform genes, together with biosynthesis of the total MT proteins, in the gills of triploid and diploid juvenile Pacific oyster Crassostrea gigas in response to cadmium (Cd) and zinc (Zn) exposure. Oysters were exposed to Cd (0.133 microM), Zn (15.3 microM), and Cd+Zn for 14 d. Results showed similar response capacities to metal exposures in the two populations. No significant difference was revealed in terms of MT gene expression, MT protein synthesis, and Cd accumulation. However, triploid oysters bioaccumulated Zn 30% less efficiently than diploid oysters. Among the three MT isoform genes, CgMT2 appeared to be more expressed than CgMT1, whereas CgMT3 appeared to be anecdotal (10(6) times lower than CgMT2). CgMT2 and CgMT1 gene expression levels were increased sevenfold in the presence of Cd, whereas Zn appeared to have no effect. A twofold increase in MT protein levels occurred in response to Cd exposure. Discrepancies between mRNA and protein levels suggest that in C. gigas MT are regulated at the transcriptional level, as well as at the translational level.

Effects of copper and cadmium spiked-sediments on embryonic development of Japanese medaka (Oryzias latipes)

Because of their high capacity to accumulate contaminants such as persistent organic pollutants and heavy metals, aquatic sediments are considered as a long-term source of contamination for aquatic organisms. In compliance with the increasing interest both for sediment quality evaluation and the use of fish early life stage (ELS) toxicity assays, we proposed an embryo-larval test to evaluate embryotoxicity and genotoxicity of sediment-bound contaminants. Pre-blastula stage medaka (Oryzias latipes) embryos were exposed by static sediment contact to two model heavy metals (cadmium and copper) at environmental concentrations during the whole 10-day embryonic development. Lethal and sub-lethal effects were recorded in both embryos and larvae for 20 days post fertilisation (dpf) using several global toxicity and phenotypic endpoints. The comet assay was also performed on medaka prolarvae to evaluate genotoxic effects of the tested chemicals. Environmental concentrations of cadmium (Cd) and copper (Cu) did not affect embryo and larval survival. However, both heavy metals significantly induced morphological abnormalities, particularly spinal and cardiovascular deformities. Cd but not Cu induced tachycardia. Both heavy metals induced a significant increase in DNA damage at all tested concentrations. Resulting LOEC values for Cd and Cu corresponded to 1.9 and 8.5 μg/g d.w. sediment, respectively. Although metal bioavailability is probably lower for naturally contaminated sediments, the relatively low toxicity thresholds for both Cd and Cu raise the question of possible risk for fish embryos developing in direct contact to sediments. This study demonstrates the applicability, sensitivity and relevance of the Japanese medaka embryo-larval assay (MELA) to evaluate sediment hazardous potency at environmental concentrations of heavy metals.

Long-term records of cadmium and silver contamination in sediments and oysters from the Gironde fluvial-estuarine continuum - Evidence of changing silver sources

The Gironde fluvial estuarine system is impacted by historic metal pollution (e.g. Cd, Zn, Hg) and oysters (Crassostrea gigas) from the estuary mouth have shown extremely high Cd concentrations for decades. Based on recent work (Chiffoleau et al., 2005) revealing anomalously high Ag concentrations (up to 65 mg kg(-1); dry weight) in Gironde oysters, we compared long-term (~1955-2001) records of Ag and Cd concentrations in reservoir sediment with the respective concentrations in oysters collected between 1979 and 2010 to identify the origin and historical trend of the recently discovered Ag anomaly. Sediment cores from two reservoirs upstream and downstream from the main metal pollution source provided information on (i) geochemical background (upstream; Ag: ~0.3 mg kg(-1); Cd: ~0.8 mg kg(-1)) and (ii) historical trends in Ag and Cd pollution. The results showed parallel concentration-depth profiles of Ag and Cd supporting a common source and transport. Decreasing concentrations since 1986 (Cd: from 300 to 11 mg kg(-1); Ag: from 6.7 to 0.43 mg kg(-1)) reflected the termination of Zn ore treatment in the Decazeville basin followed by remediation actions. Accordingly, Cd concentrations in oysters decreased after 1988 (from 109 to 26 mg kg(-1), dry weight (dw)), while Ag bioaccumulation increased from 38 up to 116 mg kg(-1), dw after 1993. Based on the Cd/Ag ratio (Cd/Ag~2) in oysters sampled before the termination of zinc ore treatment (1981-1985) and assuming that nearly all Cd in oysters originated from the metal point source, we estimated the respective contribution of Ag from this source to Ag concentrations in oysters. The evolution over the past 30 years clearly suggested that the recent, unexplained Ag concentrations in oysters are due to increasing contributions (>70% after 1999) by other sources, such as photography, electronics and emerging Ag applications/materials.

Evidence of an altered protective effect of metallothioneins after cadmium exposure in the digenean parasite-infected cockle (Cerastoderma edule).

The aim of the present study was to analyse the relation between parasitism and subsequent metallothioneins (MT) in the case of metal contamination. Experimental exposure of parasitized and unparasitized cockles (Cerastoderma edule) to cadmium (Cd) was performed, with the cockle as first or second intermediate host of 2 digenean species. After 7 days of Cd exposure in microcosms, cockles infected as first intermediate host by Labratrema minimus exhibited metal concentrations in tissues double that in uninfected cockles. Jointly, MT concentrations of parasitized cockles were not modified in comparison with uninfected individuals in which concentrations were increased 4.3-fold compared with controls. In cockles experimentally infected as the second intermediate host by Himasthla elongata, cadmium concentrations significantly increased again in parasitized cockles compared with uninfected individuals in contaminated conditions. Simultaneously, MT concentrations in healthy cockles increased, whereas they significantly decreased in parasitized individuals. Therefore, the presence of digenean parasites in Cd-exposed cockles leads to a maintenance or a decrease in MT concentrations compared with healthy individuals, whereas Cd accumulation in tissues is significantly increased. These experiments indicate a significant alteration of the protective effect of metallothioneins towards metals which could consequently enhance cockle vulnerability. Moreover, these results highlight the limit of the use of MT as a biomarker of metal pollution in field monitoring if parasitism is not taken into account.

Bivalve population health: Multistress to identify hot spots

This study investigated some stress (metals, parasites) and response (immunity, metallothionein) factors in two cockle and two Manila clam populations. Data from eight seasons were averaged to obtain global baseline values. Stress/response characteristics of each population were compared to population health status that was determined through population dynamics parameters. Four different scenarios were discussed: (1) a lightly stressed cockle population with correct population health but with a risk of deterioration (hot spot); (2) a lightly stressed introduced cockle population threatened of extinction. In this case ecological factors were suspected; (3) a moderately stressed clam population with moderate adaptative response. The population was sustainable but the level of stress should not increase (hotspot); and (4) a stressed clam population and unfavourable ecological conditions preventing clam settlement. This monitoring highlighted that the discrepancy between population health and stress levels could be due to insufficient response by bivalves and/or by unfavourable ecological factors.

Cadmium uptake by the European eel: Trophic transfer in field and experimental investigations

Due to its status of threatened species and being heavily contaminated by metals, the European eel (Anguilla anguilla) was selected to investigate cadmium contamination levels of fish settled along a historically cadmium-contaminated hydrosystem, the Garonne-Gironde continuum (France), according to its various location sites and fish length. Results have shown an important site effect on cadmium concentrations in liver but not in gills, highlighting the possible predominance of the trophic exposure route. Subsequently, uncontaminated eels were experimentally exposed to cadmium by water uptake and/or trophic route(s). Eels were fed with different preys: white shrimps collected in an unpolluted area in the Gironde estuary, and cadmium-enriched shrimps. Data obtained tend to show that the use of cadmium-enriched food during experimental investigations triggers an underestimation of the metal trophic transfer rate. These two complementary approaches provide some elements to suggest that the trophic route plays an important role in cadmium contamination of wild eels.