Seasonal variations of low pesticides contamination and biomarker responses in marine bivalves from French estuaries.

Biomarkers involved in detoxification process (GST), oxidative stress (SOD and MDA), immune response (Laccase) and neurotoxic disorders (AChE) were analysed in Pacific oysters and blue mussels collected from 4 locations within the Pertuis sea (France). Seasonal variations of total pesticide mean concentrations were found in seawater with metolachlor being the main pesticide measured (up to 32 ng/L). The majority of pesticide concentrations in sediment were below the LOD. Seasonal contamination differences were evidenced for chlortoluron, especially in mussels where concentrations reached 16 ng/g (wet weight) during the winter, in the Charente estuary, but no relationships with any of the biomarkers selected arisen. Actually, low concentrations of alpha-, beta-BHC and alachlor were correlated to GST activity, and low levels of hexachlorobenzene were linked to the AChE activity and MDA content in oysters. In mussels, low concentrations of methylparathion, parathion and beta-BHC were correlated to laccase.

Preliminary inter-port study of the quality of environments using physiological responses of invertebrates exposed to chronic trace element and organic contamination in Corsica (Mediterranean Sea).

Port areas are socio-ecosystems impacted by chronic mixture pollution. Some marine species benefit from living there and may be studied to define the ecological state of such environments. In this study, the risks of chronic chemical contamination and its consequences on three marine molluscs were evaluated in North Corsica (France) port areas. Mediterranean mussel Mytilus galloprovincialis, tubular sea cucumber Holothuria tubulosa and Mediterranean limpet Patella sp. were sampled in three port areas and a reference location. A set of biomarkers was analysed to evaluate oxidative stress, detoxification, energetic metabolism, neurotoxicity, immunity and bioaccumulation (metallic trace elements and organic pollutants). The objectives were to assess pollution-induced effects in organisms, to determine the best bioindicator species for the selected locations and to validate a "pool" sampling technique (when the analysis is done on a single pool of samples and not on individual samples). The results validate the sampling techniques as "pool" for management purposes. St-Florent was demonstrated as the most contaminated location. All the other locations present a low contamination, below the recommended threshold values (for metallic trace elements and organic pollutants). Finally, the limpet appears to be the best bioindicator for the selected locations. Mussel and sea cucumber are inappropriate due to their absence in this oligotrophic region and the lack of responses observed, respectively.

First characterization of seasonal variations in biomarkers baseline in Patella sp. from Mediterranean ports (North Corsica, France).

In port areas the identification of contamination sources is necessary for an efficient management. Biomonitoring provides information on the environmental impact of the pollutants. It is often difficult to differentiate the natural variations of biomarkers from those induced by pollution. The present study aims to define a baseline level for biochemical biomarkers in limpet (Patella sp.) collected in four North-Corsica port areas. Reference data for five biomarkers (superoxide dismutase, glutathione S-transferase, laccase, pyruvate kinase and acetylcholinesterase) were described in a model, using length of the limpet shell, temperature and salinity. The measured biomarkers responses on potentially polluted sites usually fell within the range of the expected values for an unaffected site, suggesting that a main part of the variations is explained by environmental conditions. Not included in the model, biological factors (sex, development stage, etc.), annual variation or other physico-chemical parameter could explain the variations in the model.

First quantitative biomonitoring study of two ports (marina, commerce) in French littoral area: Evaluation of metals released into the marine environment and resulting from galvanic anodes.

Coastal areas are important interface environments between marine and terrestrial ecosystems and are also areas of high economic activity related, among others, to trade, fishing and tourism. The port areas of La Rochelle (France) are part of these areas with strong anthropic activities and are therefore subject to potential contamination, particularly with trace metals. Among the various sources of discharges, galvanic (or sacrificial) cathodic protection can be questioned. Indeed, few studies have been carried out on the potential impact of the degradation of sacrificial anodes in ports on the health of bivalves. A study was therefore carried out on the black scallop Mimachlamys varia in the commercial port and the marina of La Rochelle using the caging method. Several biomarkers of the health status of individuals were observed in the laboratory (SOD, GST, MDA, AChE, and LAC). Among the different results obtained over two exposure periods, it appears that the potential effects of sacrificial anodes, whether new or present on site for several years, are masked by meteorological conditions, as well as by harbor activities.

Spatio-temporal contamination of microplastics in shellfish farming regions: A case study.

The seasonal pattern of microplastics (MPs) contamination of the French littoral area of the Pertuis Charentais, one of the main French shellfish production regions, was assessed for the first time, between May 2019 and May 2020 at four different sites. The reference site was located at "Ile de Ré" and the other sites were located in the estuaries of the Sèvre Niortaise, Charente and Seudre rivers. Both blue mussels (Mytilus edulis) and Pacific oysters (Magallana gigas), that are considered sentinel species for the quality of the marine environment were analysed, along with sediment and seawater samples. MPs were extracted from each sample, counted, measured and sorted by colour and type. Micro-Raman spectroscopy was used to determine the proportion of confirmed MPs and the polymer types. The results showed that the contamination of mussels by fibres and fragments (1.9 ± 2.1 MPs/g ww) was significantly higher than for oysters (0.4 ± 0.4 MPs/g ww). Specifically, the contamination by fibres in both species was significantly greater than the contamination by fragments. Significant variations of MPs contamination were observed across the seasons and sites in bivalves, and depended on the species and the type of MPs (fibres or fragments). Mean concentrations of MPs measured in water and sediment were 0.007 MPs/L and 210 MPs/kg dw, respectively. Finally, blue was the dominant colour for fibres (79 %) and fragments (81 %). Blue fragments were mainly made of PS (70 %) followed by PC (18 %) and PP, PA or PLA (3 %) whereas blue fibres were mainly made of PA (80 %) followed by PET (13 %) or PP (7 %). This rare environmental case study of long-term chronic exposure of farming areas to MPs provides new knowledge on in situ variations of plastic fibres and fragments contamination throughout the seasons.

Intergenerational effects of environmentally-aged microplastics on the Crassostrea gigas.

This study focused on the impacts of aged aquaculture microplastics (MPs) on oysters (Crassostrea gigas). Adult oysters were exposed for two months to a cocktail of MPs representative of the contamination of the Pertuis Charentais area (Bay of Biscay, France) and issuing from oyster framing material. The MPs mixture included 28% of polyethylene, 40% of polypropylene and 32% of PVC (polyvinyl chloride). During the exposure, tissues were sampled for various analyzes (MP quantification, toxicity biomarkers). Although no effect on the growth of adult oysters was noted, the mortality rate of bivalves exposed to MPs (0.1 and 10 mg. L-1 MP) increased significantly (respectively 13.3 and 23.3% of mortalities cumulative). On the one hand, the responses of biomarkers revealed impacts on oxidative stress, lipid peroxidation and environmental stress. At 56 days of exposure, significant increases were noted for Glutathione S-Transferase (GST, 10 mg. L-1 MP), Malondialdehyde (MDA, 10 mg. L-1 MP) and Laccase (LAC, 0.1 and 10 mg. L-1 MP). No variations were observed for Superoxyde Dismutase (SOD). Besides, ingestion of MPs in oyster tissues and the presence in biodeposits was highlighted. In addition, in vitro fertilisations were performed to characterize MPs effects on the offspring. Swimming behavior, development and growth of D-larvae were analysed at 24-, 48- and 72-h after fertilisation. D-larvae, from exposed parents, demonstrated reduced locomotor activity. Developmental abnormalities and arrest as well as growth retardation were also noted. This study highlighted direct and intergenerational effects of MPs from aged plastic materials on Pacific oysters.

Biomonitoring of Mimachlamys varia transplanted to areas impacted by human activities (La Rochelle Marina, France).

The development of human activities on French Atlantic coastlines (La Rochelle) lead to chronic pollution of the environment by organic (pesticides, hydrocarbons, agrochemicals) and inorganic (heavy metals) contaminants. These past years, several regulations have been implemented to preserve coastal environments. The purpose of this study was to perform biomonitoring of bivalve species using an outdoor caging technique. The goal of our work was to assess the impact of harbour's trace elements on the state of health of the marine bivalve Mimachlamys varia. First, various molecular defence biomarkers were measured: SOD (oxidative stress), GST (detoxification process), MDA (lipid peroxidation), and Laccase (immune reaction). Thus, in April 2016, scallops were collected at three caging sites, which differ by their levels of pollution, after transplantation into port areas (fairing, rainwater) and a control site (marsh). Bivalve samples were taken at three sampling dates (D0, D07, D21). Biomarker assays were performed in the digestive glands due to their bioaccumulation properties. The second aim was to explore the impacts of inorganic pollutants placed in environmental harbour's sites. After 21 days, the biomarker response of transplanted bivalves revealed a SOD decrease, Laccase and GST stimulations, higher concentrations in Cu, Fe, As, Co, Mn, Zn, Sn and no significant variation of MDA concentration. Our ecological relevance of biomarker approaches opens interesting perspectives to identify M. varia such as a pertinent marine sentinel species. The several selected biomarkers determined could confirm their ability to appraise the water quality of hydro-systems located in French coastlines, such as port areas.

Ocean warming combined with lower omega-3 nutritional availability impairs the cardio-respiratory function of a marine fish.

Highly unsaturated fatty acids of the omega-3 series (HUFA) are major constituents of cell membranes, yet are poorly synthesised de novo by consumers. Their production, mainly supported by aquatic microalgae, has been decreasing with global change. The consequences of such reductions may be profound for ectotherm consumers, as temperature tightly regulates the HUFA content in cell membranes, maintaining their functionality. Integrating individual, tissue and molecular approaches, we examined the consequences of the combined effects of temperature and HUFA depletion on the key cardio-respiratory functions of the golden grey mullet, an ectotherm grazer of high ecological importance. For 4 months, fish were exposed to two contrasting HUFA diets [4.8% eicosapentaenoic acid (EPA)+docosahexaenoic acid (DHA) on dry matter (DM) versus 0.2% EPA+DHA on DM] at 12 and 20°C. Ventricular force development coupled with gene expression profiles measured on cardiac muscle suggest that combining HUFA depletion with warmer temperatures leads to: (1) a proliferation of sarcolemmal and sarcoplasmic reticulum Ca2+ channels and (2) a higher force-generating ability by increasing extracellular Ca2+ influx via sarcolemmal channels when the heart has to sustain excessive effort due to stress and/or exercise. At the individual scale, these responses were associated with a greater aerobic scope, maximum metabolic rate and net cost of locomotion, suggesting the higher energy cost of this strategy. This impaired cardiac performance could have wider consequences for other physiological performance such as growth, reproduction or migration, all of which greatly depend on heart function.

Assessment of the biological quality of port areas: A case study on the three harbours of La Rochelle: The marina, the fishing harbour and the seaport.

This work was designed to investigate biological impacts at 3 dates (day 0, day 7 and day 21) on black scallops (Mimachlamys varia) in the three ports areas of La Rochelle town in winter 2017. In order to assess the biological effects on the wild population of black scallops, bivalves were place in four different locations: in the three ports (semi-closed areas), and in a marshland uncontaminated site (closed area). Biomarkers of effects (heavy metals) and exposure (oxidative stress and immunological effects) were assessed in the digestive glands of specimens in order to compare two techniques of sampling: "pool" technique and "inter-subject" technique. Our findings reported in the both techniques show significant modulation of GST (detoxification), SOD (antioxidant response) and MDA (lipid peroxidation) in bivalves exposed to a specific contamination in each port. Laccase-type enzyme also highlighted an important aspect in terms of biomarker response of the immune function at the 7th day of exposition. Overall, our study demonstrated that the "pool" technique using the same quality indicator M. varia could be used to obtain reliable results at lower costs. In contrast, in fundamental context, the "inter-subject" technique could bring more precise results to light. However, it requires burdensome and costly handling.

What is the relationship between the bioaccumulation of chemical contaminants in the variegated scallop Mimachlamys varia and its health status? A study carried out on the French Atlantic coast using the Path ComDim model.

Increasing activity along the French Atlantic coast has led to chronic pollution with, in particular, mixtures of contaminants such as hydrocarbons, phytosanitary products, PCBs and heavy metals. Based on previous research, pollution biomarkers were used in this study as they can indicate health status when monitoring the impact of pollutants on coastal species such as the marine bivalve Mimachlamys varia. Mollusc bivalves were sampled in March 2016, in open and semi-open areas (a harbour zone), from thirteen sites which differed in terms of their level of pollution, and were located along the Atlantic coast from Brittany down to the Nouvelle-Aquitaine region. First, analyses of heavy metals and organic contaminants (e.g. pesticides, polycyclic aromatic hydrocarbons, polychlorobiphenyl) in the digestive gland of bivalves were performed. Second, biochemical assays were used to study defence biomarkers: oxidative stress with Superoxide Dismutase (SOD), detoxification of organic compounds with Glutathione-S Transferase (GST), lipid peroxidation with Malondialdehyde (MDA), and immune processes with Laccase. In addition to the biochemical assays, a genetic approach was used to measure genetic diversity (haplotype and nucleotide diversity) at each site. Biomarker assays and genetic diversity were correlated with the chemical contaminants in bivalves using the Path-ComDim statistical model. Our results showed specific correlations between biochemical assays in the digestive glands with heavy metal contaminants, and between genetic diversity and organic pollution. Blocks of responses were analysed for correlations in order to develop standardized tools and guidelines that could improve our understanding of the short-term and long-term impact of contaminants on physiological parameters.

Spatial and temporal impacts of the Skjervøy harbour diesel spill on native population of blue mussels: A sub-Arctic case study.

This work was designed to investigate biological impacts on blue mussels (Mytilus edulis spp) after being exposed to diesel spill. On December 2013, an 180,000-litre accidental acute diesel spill was reported in a small harbour of northern Norway (Skjervøy). In order to assess the biological effects on the wild population of blue mussels, bivalves were collected at three different locations: at the oil-spill spot, at the other side of the harbour (opposite the oil-spill area), and in an uncontaminated site. Body burden and seawater samples were collected from a few days up to five months after the diesel spill. Biomarkers of oxidative stress and immunological effects were assessed in the blue mussels digestive glands. Our findings reported significant modulation of GST (detoxification), SOD (antioxidant response) and MDA (lipid peroxidation) in bivalves exposed to diesel with a similar response at two and five months after the spill. Laccase-type enzyme also highlighted an important aspect in terms of biomarker response of the immune function. Overall, our study demonstrated that some biomarkers returned to basal levels a few months after the diesel spill. Consequently, it highlighted the usefulness of normalised tools and guidelines for biomonitoring strategies after a diesel spill.

Alterations of tissue metallothionein and vitellogenin concentrations in tropical cup oysters (Saccostrea sp.) following short-term (96h) exposure to cadmium.

Metallothioneins and vitellogenins are low molecular weight proteins that have been used widely in environmental monitoring as biomarkers of exposure and damage to metals and estrogenic compounds, respectively. In the present study, the responses of metallothionein and vitellogenin tissue concentrations were measured following acute (96h) aqueous exposures to cadmium in Saccostrea sp., a tropical cup oyster native to the Western Pacific Ocean that has recently established itself in the Caribbean Sea. Adult oysters (1.5-5.0cm shell length) collected from the municipal marina of Santa Marta, Colombia (Caribbean Sea) and acclimated for 5days in the laboratory, were exposed to Cd at five concentrations (0, 1, 10, 100 and 1000µg/L) and their tissues (gills, digestive gland and adductor muscle) were analyzed in pools of 5 individuals (3 replicates per concentration). Metallothioneins in digestive glands of oysters exposed to Cd concentrations≥100µg/L showed a significant increase, from 8.0 to 14.8µg MT/mg total protein, whereas metallothionein concentrations in gills increased to lesser extent, and no differences were observed in adductor muscle. Metallothionein concentrations in digestive gland and gills correlated directly with whole soft tissue Cd concentrations (ranging from 2 to 297µg/g dw Cd). Vitellogenin in homogenates of oyster gonad tissue, after 96h of exposure to 1000µg/L Cd, were significantly lower (0.04mg P/g gonad) compared to control oysters (0.68mg P/g gonad), suggestive of an anti-estrogenic effect of Cd at high concentrations, whereas no significant changes in vitellogenin concentrations were observed at intermediate Cd exposure concentrations. This study confirms acute responses of metallothionein and vitellogenin concentrations in tissues of Saccostrea sp. exposed to high concentrations of cadmium (Cd≥100µg/L, 96h). The present results are first step towards validating the use of these two proteins as biomarkers of metal exposure in this species.

First data on three bivalve species exposed to an intra-harbour polymetallic contamination (La Rochelle, France).

Evaluating diffuse sediment contamination in the environment is a major concern with the aim of reaching a good chemical and ecological state of the littoral zone. In this study the risks of chronic chemical contamination and consequences in the bivalves Crassostrea gigas, Mytilus sp. and Mimachlamys varia were evaluated in coastal environments. The objective here was to understand the anthropological phenomena that affect the functioning of the marina of La Rochelle (semi-closed environment). Harbours seeking ecomanagement accreditations (such as the international reference ISO 14001) constitute zones of interest to implement biomonitoring studies. The biological effects of chemical pollution in the Marina of La Rochelle were studied to develop a multi-biomarker biomonitoring approach on specific marine species of this site. Moreover, a genetic (DNA barcoding) approach was applied to validate the species identity of collected bivalves. Of the three species tested the scallop, M. varia, was the most sensitive to metal exposure.

Short-Term and Long-Term Biological Effects of Chronic Chemical Contamination on Natural Populations of a Marine Bivalve.

Understanding the effects of chronic chemical contamination on natural populations of marine organisms is complex due to the combined effects of different types of pollutants and environmental parameters that can modulate the physiological responses to stress. Here, we present the effects of a chronic contamination in a marine bivalve by combining multiple approaches that provide information on individual and population health. We sampled variegated scallops (Mimachlamys varia) at sites characterized by different contaminants and contamination levels to study the short and long-term (intergenerational) responses of this species to physiological stress. We used biomarkers (SOD, MDA, GST, laccase, citrate synthase and phosphatases) as indicators of oxidative stress, immune system alteration, mitochondrial respiration and general metabolism, and measured population genetic diversity at each site. In parallel, concentration of 14 trace metals and 45 organic contaminants (PAHs, PCBs, pesticides) in tissues were measured. Scallops were collected outside and during their reproductive season to investigate temporal variability in contaminant and biomarker levels. Our analyses revealed that the levels of two biomarkers (Laccase-type phenoloxidase and malondialdehyde) were significantly correlated with Cd concentration. Additionally, we observed significant seasonal differences for four of the five biomarkers, which is likely due to the scallop reproductive status at time of sampling. As a source of concern, a location that was identified as a reference site on the basis of inorganic contaminant levels presented the same level of some persistent organic pollutants (DDT and its metabolites) than more impacted sites. Finally, potential long-term effects of heavy metal contamination were observed for variegated scallops as genetic diversity was depressed in the most polluted sites.

Biomarker modulation associated with marine diesel contamination in the Iceland scallop (Chlamys islandica).

The decrease of ice cover in the Arctic will lead to an increase of ship traffic in the upcoming decades. Consequently, oil pollution is expected. In this context, the goals of this study were to evaluate the biological impact of marine diesel contamination and, on this basis, to determine analytical tools of interest (biomarkers) for future biomonitoring of diesel spills. Using a 7-day contamination protocol, this study investigated biochemical modulations in the digestive gland of the Iceland scallop (Chlamys islandica). Incorporation of contaminants was verified assessing haemolymph metabolites. Results showed a response of glutathione-S-transferase to contamination suggesting detoxification processes and the suitability of such a tool for diesel spill biomonitoring. The lack of modulation of superoxide dismutase activity and lipid peroxidation suggests no oxidative stress and the unsuitability of these molecular tools for biomonitoring.

In situ evaluation of oxidative stress and immunological parameters as ecotoxicological biomarkers in a novel sentinel species (Mimachlamys varia).

Although the variegated scallop Mimachlamys varia seems to be a suitable sentinel species for contaminant monitoring, no study has identified biomarkers in this species. In order to fill this gap, this study conducted an in situ biomarker approach. M. varia were collected in contaminated and uncontaminated areas and responsiveness of oxidative stress and immunological biomarkers was evaluated in the digestive gland. In parallel, 14 trace element concentrations were evaluated in the same organ. Superoxide dismutase activity and malondialdehyde content responded efficiently to in situ contamination when a certain degree of contamination was reached. Laccase-type phenoloxidase showed a high sensitivity but saturation of the response was highlighted for the highest contaminations. Additionally, correlations were found between biomarkers and trace element concentrations. Taken together, results showed that biomarker approach conducted in the digestive gland of M. varia represents a sensitive analytical tool to highlight ecotoxicological issues in coastal marine ecosystems.

First description of French V. tubiashii strains pathogenic to mollusk: II. Characterization of properties of the proteolytic fraction of extracellular products.

Extracellular products (ECPs) of the French Vibrio tubiashii strain 07/118 T2 were previously reported to be toxic for the Pacific oyster Crassostrea gigas. In this study we now assessed host cellular immune responses and bacterial potential effectors by which these ECPs can be associated with host damages. The adhesion capacity (28% inhibition) and phagocytosis ability (56% inhibition) of oyster hemocytes were the main functions affected following in vitro contact between hemocytes and V. tubiashii ECPs. This may be linked to the demonstration of the capability of ECPs to cleave various cellular substrates as oyster collagen. Moreover, a strong metalloproteolytic activity was recorded with general (azocasein) and specific (ADAM) substrates and characterized by the use of standard inhibitors and metal ions. The addition of 1,10-phenanthroline and Zn2+ decreased proteolytic activity by about 80% and 50% respectively, confirming the presence of zinc metalloproteolytic activity in the ECPs. Mass spectrometry analyses of crude ECPs identified an extracellular zinc metalloprotease encoded by a gene with an open reading frame of 1821 bp (606 aa). Consensus zinc-binding motifs specific to thermolysin family and some glycosylation and phosphorylation sites were located on the deduced protein sequence. Taken together, our results suggest that this (these) zinc metalloprotease(s) might contribute to the impairment of hemocyte immunological functions; however, their direct involvement in ECPs toxicity remains to be demonstrated.

Pelagic food web patterns: do they modulate virus and nanoflagellate effects on picoplankton during the phytoplankton spring bloom?

As agents of mortality, viruses and nanoflagellates impact on picoplankton populations. We examined the differences in interactions between these compartments in two French Atlantic bays. Microbes, considered here as central actors of the planktonic food web, were first monitored seasonally in Arcachon (2005) and Marennes-Oléron (2006) bays. Their dynamics were evaluated to categorize trophic periods using the models of Legendre and Rassoulzadegan as a reference framework. Microbial interactions were then compared through 48 h batch culture experiments performed during the phytoplankton spring bloom, identified as herbivorous in Marennes and multivorous in Arcachon. Marennes was spatially homogeneous compared with Arcachon. The former was potentially more productive, featuring a large number of heterotrophic pathways, while autotrophic mechanisms dominated in Arcachon. A link was found between viruses and phytoplankton in Marennes, suggesting a role of virus in the regulation of autotroph biomass. Moreover, the virus-bacteria relation was weaker in Marennes, with a bacterial lysis potential of 2.6% compared with 39% in Arcachon. The batch experiments (based on size-fractionation and viral enrichment) revealed different microbial interactions that corresponded to the spring-bloom trophic interactions in each bay. In Arcachon, where there is a multivorous web, flagellate predation and viral lysis acted in an opposite way on picophytoplankton. When together they both reduced viral production. Conversely, in Marennes (herbivorous web), flagellates and viruses together increased viral production. Differences in the composition of the bacterial community composition explained the combined flagellate-virus effects on viral production in the two bays.