Hélène Thomas-Guyon

A mesocosm study of fate and effects of CuO nanoparticles on endobenthic species (Scrobicularia plana, Hediste diversicolor).

The fate and effects of CuO nanoparticles (CuO NPs) were examined in endobenthic species (Scrobicularia plana , Hediste diversicolor), under environmentally realistic conditions in outdoor mesocosms (exposure to Cu at 10 μg L(-1) in particulate (CuO NPs) or soluble salt (CuNO(3)) forms) for 21 days. Labile Cu was determined in water and sediment by using diffusive gradient in thin films. No labile Cu being detected from CuO NPs; the observed effects in invertebrates exposed to CuO NPs were mainly attributed to the toxicity of nanoparticulate rather than dissolved Cu toxicity. Bioaccumulation of CuO NPs was observed in both species. Biomarkers were examined at different levels of biological organization: biochemical markers of defense and damage, biomarkers of genotoxicity (comet assay), and behavioral biomarkers (feeding and burrowing). Behavioral biomarkers, antioxidant defenses (catalase, glutathion S-transferase, metallothionein), and genotoxicity are the most sensitive tools to highlight the effect of soluble or nanoparticulate metal forms. Concerning other biomarkers of defense (superoxide dismutase, lactate dehydrogenase, laccase) and damage (thiobarbituric acid reactive substances, acetylcholinesterase, acid phosphatase), no significant effects were detected. This experiment shows the suitability of mesocosms for studying the environmental effects of nanoparticles.

A marine mesocosm study on the environmental fate of silver nanoparticles and toxicity effects on two endobenthic species: The ragworm Hediste diversicolor and the bivalve mollusc Scrobicularia plana

Silver nanoparticles are widely used in a range of products and processes for their antibacterial properties, electrical and thermal conductivity. The fate and effects of Ag nanoparticles were examined in two endobenthic species (Scrobicularia plana, Hediste diversicolor), under environmentally realistic conditions in outdoor mesocosms exposed to Ag at 10 μg L(-1) in nanoparticulate (Ag NPs) or soluble salt (AgNO3) forms for 21 days. Labile Ag was determined in water and sediment by using diffusive gradient in thin films. Ag levels were equivalent in contaminated Ag NPs mesocosms to those contaminated with the soluble form. Bioaccumulation of Ag was observed for both species exposed to either Ag in the nanoparticulate or ionic forms. Concerning biomarker responses, both soluble and nanoparticulate Ag forms, induced defenses against oxidative stress, detoxification, apoptosis, genotoxicity and immunomodulation. Nevertheless, DNA damages measured by the comet assay in the digestive gland of S. plana, and Phenoloxidase and lysozyme activities in S. plana and H. diversicolor, respectively, were higher in the presence of Ag NPs compared to soluble Ag suggesting a specific nano effect.

Liver antioxidant and plasma immune responses in juvenile golden grey mullet (Liza aurata) exposed to dispersed crude oil

Dispersants are often used after oil spills. To evaluate the environmental cost of this operation in nearshore habitats, the experimental approach conducted in this study exposed juvenile golden grey mullets (Liza aurata) for 48 h to chemically dispersed oil (simulating, in vivo, dispersant application), to dispersant alone in seawater (as an internal control of chemically dispersed oil), to mechanically dispersed oil (simulating, in vivo, natural dispersion), to the water-soluble fraction of oil (simulating, in vivo, an oil slick confinement response technique) and to seawater alone (control condition). Biomarkers such as fluorescence of biliary polycyclic aromatic hydrocarbon (PAH) metabolites, total glutathione liver content, EROD (7-ethoxy-resorufin-O-deethylase) activity, liver antioxidant enzyme activities, liver lipid peroxidation and an innate immune parameter (haemolytic activity of the alternative complement pathway) were measured to assess the toxicity of dispersant application. Significant responses of PAH metabolites and total glutathione content of liver to chemically dispersed oil were found, when compared to water-soluble fraction of oil. As was suggested in other studies, these results highlight that priority must be given to oil slick confinement instead of dispersant application. However, since the same patterns of biomarker responses were observed for both chemically and mechanically dispersed oil, the results also suggest that dispersant application is no more toxic than the natural dispersion occurring in nearshore areas (due to, e.g. waves). The results of this study must, nevertheless, be interpreted cautiously since other components of nearshore habitats must be considered to establish a framework for dispersant use in nearshore areas.

First evidence of a potential antibacterial activity involving a laccase-type enzyme of the phenoloxidase system in Pacific oyster Crassostrea gigas haemocytes

Phenoloxidases (POs) are a group of copper proteins including tyrosinase, catecholase and laccase. In several insects and crustaceans, antibacterial substances are produced through the PO cascade, participating in the direct killing of invading microorganisms. However, although POs are widely recognised as an integral part of the invertebrate immune defence system, experimental evidence is lacking that these properties are conserved in molluscs, and more particularly in the Pacific oyster Crassostrea gigas. In the present study, Vibrio splendidus LGP32 and Vibrio aestuarianus 02/041 growths were affected, after being treated with C. gigas haemocyte lysate supernatant (HLS), and either a common substrate of POs, l-3,4-dihydroxyphenylalanine (L-DOPA), to detect catecholase-type PO activity, or a specific substrate of laccase, p-phenylenediamine (PPD), to detect laccase-type PO activity. Interestingly, a higher bacterial growth inhibition was observed in the presence of PPD than in the presence of L-DOPA. These effects were suppressed when the specific PO inhibitor, phenylthiourea (PTU), was added to the medium. Results of the present study suggest, for the first time in a mollusc species, that antibacterial activities of HLS from C. gigas potentially involve POs, and more particularly laccase catalysed reactions.

First evidence of laccase activity in the Pacific oyster Crassostrea gigas

Phenoloxidases (POs) are a family of enzymes including tyrosinases, catecholases and laccases, which play an important role in immune defence mechanisms in various invertebrates. The aim of this study was to thoroughly identify the PO-like activity present in the hemolymph of the Pacific oyster Crassostrea gigas, by using different substrates (i.e. dopamine and p-phenylenediamine, PPD) and different PO inhibitors. In order to go deeper in this analysis, we considered separately plasma and hemocyte lysate supernatant (HLS). In crude plasma, oxygraphic assays confirmed the presence of true oxidase activities. Moreover, the involvement of peroxidase(s) was excluded. In contrast to other molluscs, no tyrosinase-like activity was detected. With dopamine as substrate, PO-like activity was inhibited by the PO inhibitors tropolone, phenylthiourea (PTU), salicylhydroxamic acid and diethyldithio-carbamic acid, by a specific inhibitor of tyrosinases and catecholases, i.e. 4-hexylresorcinol (4-HR), and by a specific inhibitor of laccases, i.e. cetyltrimethylammonium bromide (CTAB). With PPD as substrate, PO-like activity was inhibited by PTU and CTAB. In precipitated protein fractions from plasma, and with dopamine and PPD as substrates, PTU and 4-HR, and PTU and CTAB inhibited PO-like activity, respectively. In precipitated protein fractions from hemocyte lysate supernatant, PTU and CTAB inhibited PO-like activity, independently of the substrate. Taken together, these results suggest the presence of both catecholase- and laccase-like activities in plasma, and the presence of a laccase-like activity in HLS. To the best of our knowledge, this is the first time that a laccase-like activity is identified in a mollusc by using specific substrates and inhibitors for laccase, opening new perspectives for studying the implication of this enzyme in immune defence mechanisms of molluscs of high economic value such as C. gigas.

Effects of 16 pure hydrocarbons and two oils on haemocyte and haemolymphatic parameters in the Pacific oyster, Crassostrea gigas (Thunberg).

The in vitro effects of polycyclic aromatic hydrocarbons (PAHs) on haemocyte and haemolymphatic parameters of the Pacific oyster, Crassostrea gigas, were tested using field concentrations (10(-7) and 10(-9) mg mL(-1)) observed in the Marennes-Oleron Basin (Charente-Maritime, France) and high concentrations (10(-3) and 10(-5) mg mL(-1)) observed during oil spills. As a first step, the effects of pollutants, after a 24 h contamination period, were monitored on individual and pooled haemolymph samples and similar results were observed for both sample types. In a second step, haemolymphs from 45 oysters were withdrawn and pooled. Eighteen pollutants were tested in vitro after a 4 and 24h contamination period and 10 of them showed significant modulations of the five haemocyte parameters tested. Seven pollutants (fluorene, pyrene, anthracene, phenanthrene, chrysene, indeno[1,2,3-c,d]pyrene and heavy fuel oil (HFO)) induced a decrease in haemocyte mortality. Fluorene, pyrene and HFO significantly decreased phagocytosis activity. Percentage of non-specific esterase positive cells, phenoloxidase activity and lysosome presence were increased by naphthalene, benzo[b]fluoranthene and dibenz[a,h]anthracene, respectively. Modulation of immune parameters in the Pacific oyster by PAHs suggested that PAH pollution may be related to enhanced susceptibility to diseases.

Effects of dispersed oil exposure on the bioaccumulation of polycyclic aromatic hydrocarbons and the mortality of juvenile Liza ramada

Dispersing an oil slick is considered to be an effective response to offshore oil spills. However, in nearshore areas, dispersant application is a controversial countermeasure: environmental benefits are counteracted by the toxicity of dispersant use. In our study, the actual toxicity of the dispersant response technique in the nearshore areas was evaluated through an experimental approach using juvenile Liza ramada. Fish were contaminated via the water column (i) by chemically dispersed oil, simulating dispersant application, (ii) by dispersant, as an internal control of chemical dispersion, (iii) by mechanically dispersed oil, simulating only the effect of natural mixing processes, without dispersant application, and (iv) by the water soluble fraction of oil, simulating the toxicity of an oil slick before recovery. Bioconcentrations of polycyclic aromatic hydrocarbons (PAH) and mortality were evaluated, and related to both total petroleum hydrocarbon (TPH) and polycyclic aromatic hydrocarbon (PAH) concentrations in seawater. Fish exposed to chemically dispersed oil showed both a higher bioconcentration of PAH and a higher mortality than fish exposed to either the water soluble fraction of oil or the mechanically dispersed oil. These results suggest that (i) dispersion is a more toxic response technique than containment and recovery of the oil slick; (ii) in turbulent mixing areas, dispersant application increases the environmental risk for aquatic organisms living in the water column. Even if the experimental aspects of this study compel us to be cautious with our conclusions, responders could consider these results to establish a framework for dispersant use in nearshore areas.

Fate and effects of metal-based nanoparticles in two marine invertebrates, the bivalve mollusc Scrobicularia plana and the annelid polychaete Hediste diversicolor

The objective of this paper is to synthesize results from seven published research papers employing different experimental approaches to evaluate the fate of metal-based nanoparticles (Ag NPs, Au NPs, CuO NPs, CdS NPs, ZnO NPs) in the marine environment and their effects on two marine endobenthic species, the bivalve Scrobicularia plana and the ragworm Hediste diversicolor. The experiments were carried out under laboratory (microcosms) conditions or under environmentally realistic conditions in outdoor mesocosms. Based on results from these seven papers, we addressed the following research questions: (1) How did the environment into which nanoparticles were released affect their physicochemical properties?, (2) How did the route of exposure (seawater, food, sediment) influence bioaccumulation and effects?, (3) Which biomarkers were the most responsive? and (4) Which tools were the most efficient to evaluate the fate and effects of NPs in the marine environment? The obtained results showed that metal‐based NPs in general were highly agglomerated/aggregated in seawater. DGT tools could be used to estimate the bioavailability of metals released from NPs under soluble form in the aquatic environment. Both metal forms (nanoparticulate, soluble) were generally bioaccumulated in both species. Among biochemical tools, GST and CAT were the most sensitive revealing the enhancement of anti-oxidant defenses in both species exposed to sub-lethal concentrations of metal-based NPs. Apoptosis and genotoxicity were frequently observed.

Detection of early effects of a single herbicide (diuron) and a mix of herbicides and pharmaceuticals (diuron, isoproturon, ibuprofen) on immunological parameters of Pacific oyster (Crassostrea gigas) spat

In the context of massive summer mortality events of the Pacific oyster Crassostrea gigas, the aim of this study was to investigate the early effects on genes, enzymes and haemocyte parameters implicated in immune defence mechanisms in C. gigas oysters exposed to a potentially hostile environment, i.e. to an herbicide alone or within a mixture. Following 2 h of exposure to the herbicide diuron at 1 μg L(-1), the repression of different genes implicated in immune defence mechanisms in the haemocytes and the inhibition of enzyme activities, such as laccase-type phenoloxidase (PO) in the plasma, were observed. The inhibition of superoxide dismutase (SOD) activity in the plasma was also observed after 6 and 24 h of exposure. In the mixture with the herbicides diuron and isoproturon, and the pharmaceutical ibuprofen, catecholase-type PO activity in the plasma and the percentage of phagocytosis in the haemocytes were reduced after 6 h of exposure. Our results showed that early effects on molecular, biochemical and cellular parameters can be detected in the presence of diuron alone or within a mixture, giving an insight of its potential effect in situations that can be found in natural environments, i.e. relatively high concentrations for short periods of time.

Study of Atrazine Effects on Pacific Oyster, Crassostrea gigas, Haemocytes

Shellfish farming is an important economic activity around the world. This activity often takes place in areas subjected to various recurring pollutions. The recrudescent use of herbicides in agriculture including atrazine implies pollutant transfer towards aquatic environment in estuarine areas. Harmful effects of such substances on animals in marine environment, particularly on cultured bivalves, are poorly documented. Bivalve molluscs such as mussels and oysters have been postulated as ideal indicator organisms because of their way of life. They filter large volumes of seawater and may therefore accumulate and concentrate contaminants within their tissues. Moreover, development of techniques allowing effect analysis of such compounds on bivalve biology may lead to the development of diagnosis tools adapted to analyze pollutant transfer towards estuarine areas. In this context, influence of atrazine on defence mechanisms was analyzed in Pacific oysters, Crassostrea gigas. Atrazine was tested in vitro and in vivo on oyster haemocytes, and its effects were analyzed by flow cytometry. Haemocyte viability, cell cycle and cellular activities were monitored. Atrazine induced no significant effect in oyster under tested conditions except for peroxidase activity.