Andrea Luna-Acosta

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.

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.

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.

Enhanced immunological and detoxification responses in Pacific oysters, Crassostrea gigas, exposed to chemically dispersed oil.

The aim of this study was to evaluate the effects of chemically dispersed oil on an economically and ecologically important species inhabiting coasts and estuaries, the Pacific oyster Crassostrea gigas. Studies were carried out with juveniles, known to generally be more sensitive to environmental stress than adults. A set of enzyme activities involved in immune defence mechanisms and detoxification processes, i.e. superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), catecholase-type phenoloxidase (PO), laccase-type PO and lysozyme were analysed in different oyster tissues, i.e. the gills, digestive gland and mantle, and in the plasma and the haemoycte lysate supernatant (HLS) of the haemolymph. Results indicated that total PAH body burdens were 2.7 times higher in the presence than in the absence of the chemical dispersant. After 2 days of exposure to chemically dispersed oil, alkylated naphthalenes accounted for 55% of the total PAH body burden, whereas alkylated fluorenes and alkylated dibenzothiophenes accounted for 80% when the chemical dispersant was absent. Importantly, a higher number of enzyme activities were modified when oil was chemically dispersed, especially in the plasma and gills. Moreover, independently of the presence or absence of chemical dispersant, oil exposure generally inhibited enzyme activities in the gills and plasma, while they were generally activated in the mantle and haemocytes. These results suggest that the gills and plasma constitute sensitive compartments in C. gigas, and that the mantle and haemocytes may play an important role in protection against xenobiotics. Among the six enzyme activities that were analysed in these body compartments, five were modulated in the chemical dispersion (CD) treatment while only half of the enzyme activities were modulated in the mechanical dispersion treatment. Furthermore, CD treatment effects were often observed following exposure, but also during depuration periods. These results suggest that immune and/or detoxification responses are likely to be affected when dispersants are used to treat oil spills in shallow waters.

Differential tissue distribution and specificity of phenoloxidases from the Pacific oyster Crassostrea gigas

Phenoloxidases (POs) play a key role in melanin production, are involved in invertebrate immune mechanisms, and have been detected in different bivalves. Recently, we identified catecholase- and laccase-like PO activities in plasma and haemocyte lysate supernatant (HLS) of the Pacific oyster Crassostrea gigas. To go further in our investigations, the aims of this study were (i) to determine the tissue distribution of PO activities in C. gigas, and (ii) to identify and characterise the different sub-classes of POs (i.e. tyrosinase, catecholase and/or laccase) involved in these oxido-reductase activities. With dopamine and p-phenylenediamine (PPD) but not with l-tyrosine used as substrates, PO-activities were detected by spectrophotometry in the gills, digestive gland, mantle, and muscle. These results suggest the presence of catecholase and laccase but not of tyrosinase activities in oyster tissues. The highest activity was recovered in the digestive gland. PO-like activities were all inhibited by 1-phenyl-2-thiourea (PTU) and by the specific laccase inhibitor, cethyltrimethylammonium bromide (CTAB). With dopamine as substrate, the catecholase inhibitor 4-hexylresorcinol (4-HR) only inhibited PO in the muscle. SDS-PAGE zymographic assays with dopamine and PPD elicited a unique ~40kDa protein band in the muscle. In the other tissues, laccase-like activities could be related to ~10kDa and/or ~200kDa protein bands. The ~10kDa protein band was also detected in plasma and HLS, confirming the presence of a laccase in the later compartments, and probably in most of the tissues of C. gigas. This is the first time to our knowledge that a ~10kDa protein band is associated to a laccase-like activity in a mollusc species, contributing to the characterisation of phenoloxidase activities in marine bivalves.

Persistent Organic Pollutants in a marine bivalve on the Marennes-Oléron Bay and the Gironde Estuary (French Atlantic coast) -Part 2: Potential biological effects

Contaminant effects on defence responses of ecologically and economically important organisms, such as the Pacific oyster Crassostrea gigas, are likely to influence their ability to resist infectious diseases, particularly at the young stages. The aim of this study was to explore the potential relationships between organic contaminants accumulated in the soft tissues of juvenile oysters, defence responses and physiological condition. Oysters were transplanted during summer and winter periods in different sites in the Marennes-Oléron Bay, the first area of oyster production in France, and in the Gironde Estuary, the biggest estuary in Occidental Europe. Amongst the battery of biochemical and physiological biomarkers applied in the present work [superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), malondyaldehyde (MDA), catecholase, laccase and lysozyme in gills, digestive glands, mantle and haemolymph, glycogen, proteins and lipids in the digestive gland and the condition index at the whole-organism level], MDA and lysozyme in the digestive gland and SOD, GPx and laccase in plasma contributed in order to significantly discriminate the sites in which oysters bioaccumulated different levels of heavy polycyclic aromatic hydrocarbons (HPAHs), polychlorobiphenyls (PCBs), polybromodiphenylethers (PBDEs), dichlorodiphenyltrichloroethanes (DDTs) and lindane. These results strengthen the hypothesis that it is possible to differentiate sites depending on their contamination levels and biological effects by carrying out studies with transplanted juvenile oysters. In addition, correlations were found between antioxidant and immune-defence responses, and PAH and DDT body burdens in the first area of oyster production in France (the Marennes-Oléron Bay) and where considerable oyster mortalities have been reported. This result suggests that the presence of organic chemical contaminants in the Marennes-Oléron Bay may influence defence responses in juveniles of C. gigas, and, therefore, could influence their ability to resist infectious diseases.

Bioaccumulation and metabolisation of 14C-pyrene by the Pacific oyster Crassostrea gigas exposed via seawater

The first objective of this study was to determine the bioaccumulation kinetics of pyrene in the soft tissues of Crassostrea gigas (mantle, muscle, gills, digestive gland, and the remaining soft tissues). As bivalves can biotransform hydrocarbons in more polar compounds (metabolites) that are more easily excreted, the second objective was to investigate the oyster capacity to metabolize pyrene into its metabolite, the 1-hydroxypyrene. To these ends, oysters were exposed 24 h to waterborne (14)C-pyrene then placed in depuration conditions for 15 d. Oysters efficiently bioaccumulated pyrene in their soft tissues and equilibrium was reached within the exposure time. The metabolite1-hydroxypyrene was also detected in oyster tissues but represented only 4-14% of the parent pyrene. At the end of the exposure period, the gills and the mantle showed the highest pyrene proportion of total soft tissue content, i.e. 47% and 26%, respectively. After 15 d of depuration, the mantle contained 32% and 30% of the remaining pyrene and 1-hydroxypyrene, respectively. As C. gigas did not display a high capacity for metabolizing pyrene, it can be considered as a good bioindicator species to survey and monitor pyrene contamination in the coastal marine environment.

Persistent Organic Pollutants in a marine bivalve on the Marennes-Oléron Bay and the Gironde Estuary (French Atlantic coast) -Part 1: Bioaccumulation

The aim of this study was to determine 1) the relevance of using the Pacific oyster Crassostrea gigas as a sentinel organism, at a juvenile stage, for polycyclic aromatic hydrocarbon (PAH) and persistent organic pollutant (polychlorobiphenyl, PCB, polybromodimethylether, PBDE, and organochlorine pesticide, OCP) contamination, 2) the potential levels of chemical organic contamination in the Marennes-Oléron Bay, and their potential sources and 3) the potential influence of physiological or environmental factors on contaminant body burdens in oysters. To this end, juvenile oysters purchased from an oyster hatchery were transplanted to a reference site, in Bouin, and to different transplantation sites in the Marennes-Oléron Bay, the first oyster production area in France, and in the Gironde Estuary, the biggest estuary in Occidental Europe. Transplantations were done during summer and winter. Whole oyster soft tissues from each site were analysed for PAHs, PCBs, PBDEs and OCPs. Results obtained with a transplantation period of 3months suggest that C. gigas, at the juvenile stage, is a relevant sentinel organism for short-term assessment of contamination for these contaminants. In addition, no significant effects of physiological factors on contaminant body burdens were observed. Principal component analysis revealed two distinct groups of contaminants (PAHs and OPCs, and PCBs and PBDEs) and three groups of sites: 1) the reference site, 2) Les Palles (LP) and Boyard (BOY) in winter and 3) all the other sites. The group of LP and BOY was clearly defined by the levels of PAHs and OCPs, suggesting higher levels of contamination of these chemical compounds on these sites, potentially due to local contamination sources. In addition, no relevant effects of physiological or environmental factors on contaminant body burdens were observed. Results suggest also a predominance of contaminants related to agricultural activities along the Marennes-Oléron Bay, and therefore, further studies on the presence of pesticides in this region should be considered.