Immunocompetence analysis of the aquatic snail Lymnaea stagnalis exposed to urban wastewaters.

Wastewater treatment plant effluents from urban area are a well-known source of chronic multiple micropollution to the downstream living organisms. In this study, ecologically relevant laboratory-bred freshwater gastropods, Lymnaea stagnalis, were exposed for 29 days to raw effluents of a wastewater treatment plant in Lyon area (France). A time-course analysis of individual markers of immunocompetence (hemocyte density and viability, hemocyte NADPH activity, phenol oxidase activity, and capacity of phagocytosis) has shown slight trends of inflammatory-like responses induced by the 100% effluents. So far, no short-term hazard for L. stagnalis can be revealed. However, over the long term, such environmental stress-stimulating immune responses could provoke deleterious life history trade-offs because the immune system is known to be highly energy-consuming.

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.

Influence of temperature on age-related lipid peroxidation in a short-lived vertebrate (Nothobranchius furzeri).

In ectotherms, temperature modulates oxidative stress, a key driver of aging. However, the effects of temperature on oxidative stress have not been investigated at several life stages of an ectotherm. In order to improve understanding of aging processes, we conducted a cross-sectional study in short-lived ectotherm vertebrates, the killifish Nothobranchius furzeri. Malondialdehyde contents were assessed at three stages of life, in the liver and muscles of fish acclimatized to optimal or sub-optimal temperatures during all their life cycle. In accordance with the "free-radical theory," our results highlighted an increase in lipid peroxidation in senescent organisms. In the liver, this lipid peroxidation increase was more intense in senescent fish acclimatized to sub-optimal temperature than in fish acclimatized to their optimal temperature.

Integrative biomarker assessment of the effects of chemically and mechanically dispersed crude oil in Pacific oysters, Crassostrea gigas.

The impact of dispersed crude oil and dispersant on adult Pacific oysters, Crassostrea gigas, was evaluated through an integrative biomarker approach including (1) biochemical (plasma catecholase- and laccase-type phenoloxidase and superoxide dismutase), (2) histological (digestive cell lysosomal responses, digestive gland histopathology) and (3) physiological (flesh condition index) endpoints in the haemolymph and digestive gland. Adult oysters were exposed to non-contaminated water (control), chemically-dispersed oil (Brut Arabian Light), mechanically-dispersed oil and dispersant (FINASOL®) alone for 2days, and further depurated in non-contaminated water for 4weeks. After exposure to chemically and mechanically dispersed oil oysters exhibited induction of plasma laccase-type phenoloxidase and superoxide dismutase activities, enlargement of digestive cell lysosomes, lipofuscin accumulation, reduced neutral lipid content and atrophy of digestive gland diverticula; more markedly on exposure to chemically dispersed oil. From the studied biomarkers, only lysosomal biomarkers were significantly affected after exposure to the dispersant alone. This included lysosomal enlargement, neutral lipid depletion and lipofuscin accumulation in the digestive gland epithelium. A recovery of plasma enzyme activities was observed after 4weeks of depuration. The integrative biological response index indicated that chemically dispersed oil caused significantly higher stress to C. gigas than the mechanically-dispersed one or the dispersant alone; nevertheless, the response seems to be reversible after depuration.

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.

First evidence of marine diesel effects on biomarker responses in the Icelandic scallops, Chlamys islandica.

The decrease of ice cover in polar areas is expected to lead to an increase in ship traffic. In this context, the risk associated with exposure of the aquatic environment to oil-related chemicals from spills and/or accidental fuel discharges from ships will increase in the near future, potentially causing negative impacts on sensitive Arctic species. This study investigated for the first time the biomarker responses of the Icelandic scallops (Chlamys islandica) to marine diesel exposure. Antioxidant response, neurotoxic effect, gonad maturation, and lipid content were assessed in male and female scallops, Chlamys islandica after a 7-day exposure to marine diesel. At the end of exposure, results showed an inhibition of acetylcholinesterase activity in Icelandic scallops exposed to high concentration of diesel. At the end of exposure, results showed an increase in naphthalene residues in hemolymph, together with an inhibition of AChE activity in Icelandic scallops exposed to high concentration of diesel. In addition, alteration of the escape capacity was observed suggesting effects on behavior responses. Overall, this study contributes to the better knowledge of physiological effects of marine diesel in Arctic marine organisms.

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.

Effect of crude oil exposure and dispersant application on meiofauna: an intertidal mesocosm experiment.

Dispersant application is used as a response technique to minimize the environmental risk of an oil spill. In nearshore areas, dispersant application is a controversial countermeasure: environmental benefits are counteracted by the toxicity of dispersant use. The effects of the use of chemical dispersants on meiobenthic organisms and nematodes were investigated in a mesocosm experiment. A 20 day experiment was performed in four experimental sets of mesocosms. In three of them, sediments were contaminated, respectively by oil (500 mg kg(-1)), dispersed oil (oil + 5% dispersant), and dispersant alone, whereas in the last set sediments were kept undisturbed and used as a reference (Re). Our results showed that the meiobenthic response to oil contamination was rapid, for copepods and nematodes. One-way ANOVA showed a significant decrease of the abundance of copepods. In the case of nematodes, univariate and multivariate analyses indicated a clear decrease of the abundance of the species after only 20 days of pollutant exposure and thus reducing Shannon-Wiener diversity and Pielou's evenness. In contrast, Sphaerolaimus gracilis and Sabateria sp. became more frequent within disturbed assemblages and appeared to be resistant and/or opportunistic species in the presence of these kinds of toxicants. Moreover, responses of copepods and nematodes to the treatment seemed to be the same irrespective of whether only oil or oil + dispersant was performed. The main toxicities of dispersed oil come not from the "composition of a newly formed oil and oil spill dispersant mixture" but from the "quantities of increased dispersed oil droplets".

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.

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.

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.

Dispersant use as a response to oil spills: toxicological effects on fish cardiac performance.

Dispersant use is a controversial technique used to respond to oil spills in nearshore areas. In order to assess the toxicity of this technique, this study evaluated the cardiac toxicological effects on juvenile golden grey mullets Liza aurata exposed for 48 h to either dispersant alone, chemically dispersed oil, mechanically dispersed oil, the water-soluble fraction of oil or a control condition. Following exposure, the positive inotropic effects of adrenaline were assessed in order to evaluate a potential impairment on the cardiac performance. The results revealed an impairment of the positive inotropic effects of adrenaline for all the contaminants (single dispersant, dispersed and undispersed oil, water-soluble fraction of oil). This suggests that: (1) cardiac performance is a valuable parameter to study the physiopathological effects of dispersed oil; (2) dispersant application is likely to impair cardiac performance.

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.

Toxicological effects of crude oil and oil dispersant: biomarkers in the heart of the juvenile golden grey mullet (Liza aurata).

Dispersant use is a controversial oil spill response technique in coastal areas. Using an experimental approach, this study evaluated the toxicity of dispersant use upon juveniles of golden grey mullet (Liza aurata). Fish were exposed for 48 h to either dispersant only, chemically dispersed oil, mechanically dispersed oil, the water soluble fraction of oil or to control conditions. Following exposure and a depuration period, biomarkers were assessed in fish hearts, namely the total glutathione content and the activity of four enzymes (glutathione S-transferase, superoxide dismutase, catalase and glutathione peroxides). Comparing biomarker responses between the different treatments, this study revealed that 48 h exposure to dispersed oil (whether mechanically or chemically dispersed) resulted in a toxicity that was still detectable after a 14 days depuration period. Comparing biomarkers responses after an exposure to chemically and mechanically dispersed oil, this study suggests that chemical dispersion of the oil slick would not be more toxic than its natural dispersion under certain turbulent meteorological conditions (e.g. waves). Furthermore, the results indicated that the heart could be a target organ of interest in further studies investigating the toxicity of hydrocarbons. This study, which has been integrated into the DISCOBIOL project (Dispersant et techniques de lutte en milieu côtier: effets biologiques et apport à la réglementation), presents information of interest when attempting to provide a framework for dispersant applications in coastal areas.

Effect of dispersed crude oil exposure upon the aerobic metabolic scope in juvenile golden grey mullet (Liza aurata).

This study evaluated the toxicity of dispersant application which is, in nearshore area, a controversial response technique to oil spill. Through an experimental approach with juveniles of Liza aurata, the toxicity of five exposure conditions was evaluated: (i) a chemically dispersed oil simulating dispersant application; (ii) a single dispersant as an internal control of chemically dispersed oil; (iii) a mechanically dispersed oil simulating natural dispersion of oil; (iv) a water soluble fraction of oil simulating an undispersed and untreated oil slick and (v) uncontaminated seawater as a control exposure condition. The relative concentration of PAHs (polycyclic aromatic hydrocarbons) biliary metabolites showed that the incorporation of these toxic compounds was increased if the oil was dispersed, whether mechanically or chemically. However, toxicity was not observed at the organism level since the aerobic metabolic scope and the critical swimming speed of exposed fish were not impaired.

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.

Effect of an experimental oil spill on vertebral bone tissue quality in European sea bass (Dicentrarchus labrax L.).

In order to identify biomarkers of oil pollution in fish we tested the effects of an experimental Light Cycle Oil (LCO) exposure on vertebral bone of sea bass, Dicentrarchus labrax L. A total of 60 adult fish were acclimated for fifteen days, then twenty were collected as controls (Day 0) while 40 were exposed to a soluble fraction of LCO (1136 ng L(-1) of ten Polycyclic Aromatic Hydrocarbons, PAHs) for seven days. Twenty of them were sampled at the end of the exposure period and the twenty last after a recovery period of fourteen days in clean seawater. Vertebral abnormalities were counted and bone mineralization, total bone area and bone density profiles were established for several post-cranial and caudal vertebrae. In sea bass, seven days of LCO exposure did not affect the frequency and severity of the vertebral abnormalities. No significant differences were observed in bone density and bone repartition (parameters of bone area profiles) between unexposed (Day 0), exposed (D7) and decontaminated (D21) fish. In contrast, bone mineralization of the vertebrae decreased in contaminated sea bass, but in a reversible way, which confirms a previous study in trout showing that this parameter is an early stress indicator. Our results suggest that vertebral bone mineralization could be used as a biomarker of PAH pollution in sea bass. It would be interesting to check this new biomarker in other teleost species exposed to various xenobiotics.

In vivo effects of the soluble fraction of light cycle oil on immune functions in the European sea bass, Dicentrarchus labrax (Linné).

Hydrocarbons are major contaminants that may affect biota at various trophic levels in estuaries and coastal ecosystems. The effects of accidental pollution by light cycle oil (LCO), a refined product of heavy fuel oil, on bioaccumulation, depuration processes and immune-related parameters in the European sea bass, Dicentrarchus labrax, were investigated in the laboratory after 7 days of exposure and a 2-week recovery period. Exposure of fish to the soluble fraction of LCO (1600ngL(-1)) for 7 days led to the bioaccumulation of some polycyclic aromatic hydrocarbons (PAHs) in muscles: naphthalene, acenaphthene, fluorene, phenanthrene and anthracene. After 7 days of recovery period, half-elimination of naphthalene was reported in fish muscles due to facilitated diffusive loss by the epithelium and a faster elimination rate proven by the presence of a high level of naphthalene biliary metabolites. The other bioaccumulated molecules displayed a slower depuration rate due to their elimination by the formation of hydrophobic metabolites excreted through bile or urine. Three days after the beginning of the recovery period, each contaminated fish showed severe external lesions (tissue necrosis, suppurative exudates, haemorrhagic area). The hypothesis of a possible link with inflammatory phenomenon was supported by (i) an inversion of the leucocyte sub-population percentage, (ii) a significant up-expression in the spleen of the tumour necrosis factor alpha gene, (iii) a significant increase in ACH(50). Moreover, the lack of C3 gene regulation in the spleen suggested a non-renewal of this component. The reduction of phagocytic activity and lysozyme concentration reflected immune suppression. Finally, LCO toxicity in this fish was clearly demonstrated to be related to inflammatory reaction and immune depletion.