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.

Biochemical and behavioural responses of the endobenthic bivalve Scrobicularia plana to silver nanoparticles in seawater and microalgal food.

Because of their bactericidal effects, Ag nanoparticles (Ag NPs) have promising industrial development but could lead to potential ecological risks. The aim of this study was to examine the uptake and effect of silver (soluble or as lactate Ag NPs of 40 nm) at low concentrations (10 µg L(-1)) in the endobenthic bivalve Scrobicularia plana exposed, for 14 days, directly (water) or via the diet (microalgae). The stability of Ag NPs in seawater was examined using dynamic light scattering. Release of soluble Ag from Ag NPs in the experimental media was quantified by using diffusive gradient in thin film. Bioaccumulation of Ag in bivalves was measured by electrothermal atomic absorption spectrometry. Behavioural and biochemical biomarkers were determined in bivalves. Aggregation of Ag NPs and the release of soluble Ag from Ag NPs were observed in the experimental media. For both forms of Ag, bioaccumulation was much more important for waterborne than for dietary exposure. The response of oxidative stress biomarkers (catalase, glutathion S-transferase, superoxide dismutase) was more important after dietary than waterborne exposure to Ag (soluble and NPs). These defences were relatively efficient since they led to a lack of response of damage biomarkers. Burrowing was not affected for bivalves exposed directly or through the diet to both Ag forms but feeding behaviour was impaired after 10 days of dietary exposure. Since no differences of responses to Ag either soluble or nanoparticulate were observed, it seems that labile Ag released from Ag NPs was mainly responsible for toxicity.

Fate of isotopically labeled zinc oxide nanoparticles in sediment and effects on two endobenthic species, the clam Scrobicularia plana and the ragworm Hediste diversicolor.

Although it is reported that metal and metal oxide nanoparticles, which are among the most rapidly commercialized materials, can cause toxicity to organisms, their fate in the environment and toxicity to marine organisms are not well understood. In this study, we used a stable isotope labelling approach to trace the fate of nanoparticles (NPs) in sediments and also investigated bio-uptake in two estuarine intra-sedimentary invertebrates Scrobicularia plana and Nereis diversicolor. We selected exposure to 3 mg kg(-1) sediment ZnO NPs since this level is a realistic prediction of the environmental concentration in sediments. 67ZnO NPs (DLS: 21-34 nm, positively charged: 31.3 mV) suspensions were synthesised in diethylene glycol (DEG). We explored the fate of 67ZnO NPs in sediment, 67Zn bioaccumulation and the biochemical (biomarkers of defence and damage) and behavioural (burrowing kinetics and feeding rates) biomarkers in both species to 67ZnO NPs and DEG on its own during a 16 d laboratory exposure. After exposure, 67Zn concentrations in sediment showed higher levels in the upper section (1cm: 2.59 mg kg(-1)) decreasing progressively (2 cm: 1.63 mg kg(-1), 3 cm: 0.90 mg kg(-1), 4 cm: 0.67 mg kg(-1)) to a minimum value at the bottom (5 cm: 0.31 mg kg(-1)). 67Zn bioaccumulation was observed in both organisms exposed to 67ZnO NPs in DEG but no major inter-species differences were found. At the biochemical level, 67ZnO NPs exposure significantly induced increased glutathione-S-transferase activity in worms and catalase activity in clams whereas superoxide dismutase activity and thiobarbituric acid reactive substance levels were not affected in any species. Exposure to DEG on its own leads to a significant increase of metallothionein-like protein levels in clams compared with those exposed to 67ZnO NPs or controls. Burrowing behaviour as well as feeding rate were significantly impaired in both species exposed to 67ZnO NPs. Concerning exposure to DEG on its own, burrowing behaviour impairments were also shown in both species and feeding rate was impaired in bivalves. At environmentally realistic concentration of 67ZnO NPs in sediment, there is no strong evidence for a severe nanoparticle effect since most effects were also observed in the presence of DEG alone.

Biochemical, physiological and behavioural markers in the endobenthic bivalve Scrobicularia plana as tools for the assessment of estuarine sediment quality.

The aim of this study was to link the responses at different levels of biological organisation of the endobenthic bivalve Scrobicularia plana differentially exposed to anthropogenic pressure. Clams were collected in April 2008 from three estuaries along a pollution gradient (Goyen < Loire < Seine). Biomarkers of defence (metallothionein concentration and glutathione-S-transferase activity) were activated in the Loire and the Seine. Biomarkers of damage revealed neurotoxicity (decreased AChE activity) and impairment of digestive enzyme activities (cellulase or amylase) in these estuaries. The highest lactate dehydrogenase activity was registered in the Loire estuary, in parallel with enhanced levels of vanadium (a metal present in petroleum), likely as a consequence of a small oil spill that occurred one month before the sampling collection. Physiological biomarkers (energy reserves as glycogen, lipids and proteins, condition and gonado-somatic indices) showed a few intersite differences. However, the median size was significantly lower in clams exposed to direct (chemicals) or indirect (available food) effects in the most contaminated site. Burrowing behaviour was disturbed in clams from both of the Loire and Seine estuaries, a response probably due to physiological impairment rather than to avoidance of contaminated sediment. The activation of defence mechanisms towards metals (metallothionein) and other classes of contaminants (the biotransformation enzyme glutathione-S-transferase) do not ensure a total protection since a number of impairments were observed at the infra-organismal (AChE and digestive enzyme activities) and individual (burrowing behaviour) levels in relation to the degree of anthropogenic pressure. However, even in the most contaminated estuary (Seine), historical records do not show a consistent decrease of S. plana populations.

Metal distributions in Tigriopus brevicornis (Crustacea, Copepoda) exposed to copper, zinc, nickel, cadmium, silver, and mercury, and implication for subsequent transfer in the food web.

The marine copepod Tigriopus brevicornis is a benthic microcrustacean inhabiting splashed rock pools in the intertidal zone. The genus has a worldwide distribution, and some species play an important role as a link in trophic webs. It is well established that many crustaceans are able to survive in contaminated environments by storing metallic pollutants in detoxified form, and thus, they may represent a source of contaminants for their predators. The present study was designed to determine the distribution of bioaccumulated metals, both essential and nonessential, with a view to assessing their availability to the next trophic level. Groups of 1000-1500 adult copepods were exposed for 1-14 days to Ag, Cd, Cu, Hg, Ni, and Zn in water. Three concentrations, chosen to be realistic in comparison with those encountered in polluted environments, were tested for each metal. Copepods were homogenized and metals were analyzed in supernatants (i.e., metals stored in soluble form in soft tissues or easily remobilized from the exoskeleton) and pellets (including metal-rich detoxificatory granules or with cellular debris) recovered after centrifugation. Another experiment, consisting of desorption tests, was designed to evaluate the fraction of metals loosely bound onto the exoskeleton. The bioaccumulation ability is highly variable, as shown by the ratios between the concentrations reached in T. brevicornis experimentally exposed to different metals (at higher dose and at day 14) and those in controls (7, 12, 18, 26, 53, and 99, respectively, for Zn, Cu, Ni, Cd, Ag, and Hg). Cu, Zn, and Ni concentrations in copepods increased linearly with time over the whole range of exposure concentrations, whereas a plateau body metal concentration was reached with time particularly at the highest exposure concentrations for Cd, Ag, and Hg. Metals bound onto the exoskeleton were remobilized to different extents, the percentages of desorption being 11, 14, 16, 19, 31, and 32, respectively, for Zn, Cd, Cu, Ni, Ag, and Hg. Metals mainly present in the supernatant (Cd, Ni, Zn) are probably more able to be transferred along a food chain than those which were equally distributed between soluble and insoluble fractions (Ag, Cu, and Hg).

Bioaccessibility of essential and non-essential metals in commercial shellfish from Western Europe and Asia.

Maximum acceptable concentrations of metals in food - based on total concentrations - have been established in many countries. To improve risk assessment, it would be better to take into account bioaccessible concentrations. A total of seven species of molluscs from France, UK and Hong Kong was examined in this study including clams, mussels, oysters, scallops and gastropods. The species which have total metal concentrations higher than the most severe food security limits are mainly oysters (all of the three studied species), the gastropod Buccinum undatum for cadmium and zinc, and scallops for cadmium. The lowest bioaccessibility (in % extractability with gut juices) was observed for silver (median for all of the species: 14%), it was moderate for lead (median: 33%) and higher for cadmium, zinc and copper (medians were respectively 54%, 65%, and 70%). In most cases, bioaccessible concentrations remained higher than the safety limits, except for cadmium in scallops and zinc in B. undatum. The influence of feeding habit (masticated or swallowed, addition of vinegar or lemon) on metal bioaccessibility in oysters is limited. On the contrary, cooking the gastropods decreased the bioaccessibility of metals, except silver.

Environmental quality assessment in estuarine ecosystems: use of biometric measurements and fecundity of the ragworm Nereis diversicolor (Polychaeta, Nereididae).

The ability to cope with environmental stress may be expensive in terms of energy and this cost of tolerance is suspected to have negative counterparts such as reduced growth and fecundity. To date, condition indices based on biometric measurements are currently used in bivalves or fish but do not exist in endobenthic worms, despite their interest as bioindicators for the sedimentary compartment in which the major part of pollutants is stored in aquatic environments. In the present work, several biometric variables (jaw and total body length, number of segments, the length of the first three segments L3, wet or dry weight) were measured in the ragworm Nereis diversicolor originating from clean (Authie) and polluted (Seine) estuaries (France) to study size-weight relationships. The production of oocytes by females (which represent 80-90% of the population) was quantified in the same specimens. In females from the polluted site, the condition was shown to be altered and, in parallel, the production of oocytes was lower than in females from the comparatively clean site. From an operational point of view, we recommend the use of the length L3 and the wet weight. Because fecundity is mainly dependent on size and weight in N. diversicolor, biometric measurements would be useful tools for assessing the biological quality of estuarine sediments.

Decoupling of cadmium biokinetics and metallothionein turnover in a marine polychaete after metal exposure.

This study investigated the kinetics of Cd bioaccumulation, detoxification, subcellular distribution, and efflux in the nereid polychaete Perinereis aibuhitensis after Cd pre-exposure. Cd pre-exposure increased the Cd body burden in the worms, but did not affect the overall Cd uptake and efflux rates and metallothionein-like protein (MTLP) concentrations. During short-term exposure to dissolved Cd, Cd in the cytosolic fraction increased after Cd pre-exposure, and this fraction also increased during the Cd efflux period, indicating that the insoluble fraction of Cd was presumably lost at a faster rate than the loss of cytosolic Cd. Even though the MTLP concentration remained comparable after Cd pre-exposure, both the MTLP synthesis rate and the degradation rate increased, thus leading to a high MTLP turnover in the Cd-exposed worms. However, Cd uptake and efflux into different protein size fractions did not follow the patterns of MTLP synthesis and degradation, strongly suggesting that Cd kinetics is decoupled from the MTLP kinetics in the worms. Our study adds to an increasing body of evidence on the complicated relationship between metal biokinetics and MTLP kinetics in different groups of marine invertebrates which have strong contrasts in their metal handling strategies.

Biomonitoring in a clean and a multi-contaminated estuary based on biomarkers and chemical analyses in the endobenthic worm Nereis diversicolor.

Relationships between biochemical and physiological biomarkers (acetylcholinesterase [AChE], catalase, and glutathione S-transferase [GST] activities, thiobarbituric acid reactive substances, glycogen, lipids and proteins) and accumulated concentrations of contaminants (polychlorinated biphenyls [PCBs], polycyclic aromatic hydrocarbons and metals) were examined in the keystone species Nereis diversicolor. The chemical analyses of worms and sediments allowed the designation of the Seine estuary and the Authie estuary as a polluted and relatively clean site respectively. Worms from the Seine estuary exhibited higher GST and lower AChE activities. Generally, larger worms had higher concentrations of energy reserves. Principal component analyses clearly highlighted intersite differences: in the first plan, GST activities and chemical concentrations were inversely related to concentrations of energy reserves; in the second one, PCB concentrations and AChE activity were inversely related. Depleted levels of energy reserves could be a consequence of combating toxicants and might predict effects at higher levels of biological organization. The use of GST and AChE activities and energy reserve concentrations as biomarkers is validated in the field in this keystone species.

From biomarkers to population responses in Nereis diversicolor: assessment of stress in estuarine ecosystems.

Suborganismal responses to toxicants can be sensitive tools to assess marine pollution, but their ecological significance is a matter of debate. Among these biomarkers, those linked to reproduction are most probably related to populational effects. To test this hypothesis, Nereis diversicolor were collected in the multipolluted Seine estuary and the comparatively clean Authie estuary (France). Energy reserves were higher in Authie worms, suggesting a better physiological status. The number of oocytes per female was higher for the polychaetes from the Authie, but it was related to the size of animals, which was higher at this site. Densities of worms were depleted in the Seine compared to those in Authie. Demographic structure of the Seine population was also altered. The concomitant changes in energy reserves, egg production, and population structure and density suggest that the effects on biomarkers and at the population level are related.

Peptaibols: stable markers of fungal development in the marine environment.

Different peptaibols were observed in both fresh and frozen marine sediments collected from a marine area devoted to bivalve culture (Fier d'Ars, Atlantic coast, France). The identification of the peptaibols was based on a three-step mass-spectrometric analysis: observation of doubly charged ions with a characteristic isotopic profile, cleavage and observation of C- and N-terminal fragments, and partial sequencing of the N-terminal segments. The MS characteristics indicated numerous similarities between the peptaibols detected and those produced by different strains of Trichoderma species isolated from fresh sediments. Peptaibols were also detected in mussel samples collected at the same site. This constitutes the first observation of contamination of the marine human-food chain by fungal metabolites. Since peptaibols were readily observed both in fresh sediments and in samples kept frozen for several years, these compounds can be considered as stable markers of the development of Trichoderma in the marine environment.

Influence of biological and ecological factors on the bioaccumulation of polybrominated diphenyl ethers in aquatic food webs from French estuaries.

Previous studies have shown the worldwide presence of six congeners of polybrominated diphenyl ethers (PBDEs) in marine biota (BDE-28, -47, -99, -100, -153 and -154). The objective of the present study was to document their presence, their level and their transfer in the food web of two major estuaries in France, the Loire and the Seine. PBDEs were quantified in eight principal species from the Loire, representing primary consumers (the bivalve Scrobicularia plana), omnivores (the worm Nereis diversicolor, the shrimps Crangon crangon, Palaemon elegans and Palaemon serratus, the flatfish Platichthys flesus and Solea solea) and supercarnivores (the eel Anguilla anguilla). In the Seine, only worms, bivalves, sole and eels have been studied. Parameters, which can interfere with the interpretation of contamination data (organ distribution, influence of weight or size of specimens, lipid richness, intrinsic variability), have been examined. BDE-47 was the predominant congener in all biota. Higher contamination was observed in most of the species collected from the Seine, in agreement with the higher human presence and economic activity in the Seine than in the Loire basin. PBDEs have been shown to biomagnify in both of the studied estuarine food webs. However, assessment of PBDE transfer from seafood products exposed to contaminants in the Seine estuary showed that human daily intake is far below the no observed adverse effect levels.

Size dependent bioaccumulation and ecotoxicity of gold nanoparticles in an endobenthic invertebrate: the Tellinid clam Scrobicularia plana.

Gold nanoparticles (AuNPs) have important technological applications resulting in an increased potential for release to the environment, and a greater possibility of toxicological effects. The marine bivalve Scrobicularia plana was exposed to AuNPs of size 5, 15 and 40 nm during a 16 d laboratory exposure at 100 µg Au L(-1). After exposure to AuNPs forming aggregates (>700 nm), the clams accumulated Au in their soft tissues. Biochemical (biomarkers) and behavioral (burrowing and feeding) responses were investigated. Au NPs were responsible of metallothionein induction (5, 40 nm), increased activities of catalase (15, 40 nm) and superoxide dismutase (40 nm) and of glutathione S-transferase by the three sizes of AuNPs indicating defense against oxidative stress. Exposure to AuNPs impaired burrowing behavior. However, it must be underlined that these effects were observed at a dose much higher than expected in the environment.

Potential influence of confounding factors (size, salinity) on biomarkers in the sentinel species Scrobicularia plana used in programmes monitoring estuarine quality.

INTRODUCTION: To use biomarkers in monitoring programmes, potential confounding factors must be considered. In the clam Scrobicularia plana, the influence of size and salinity on biomarkers at different levels of biological organisation has been examined. MATERIAL AND METHODS: Biochemical (glutathione-S-transferase, lactate dehydrogenase, acetylcholinesterase, digestive enzymes, metallothionein), physiological (energy reserves) and behavioural (burrowing) responses were compared (a) in specimens of different sizes from the Loire estuary; (b) in specimens from the Belon estuary at two sites with salinities of 30.1 or 11.5. RESULTS: Amongst the biomarkers able to reveal pollution effects, several are influenced by the size of the clams (Ag, Cu, Ni and glycogen concentrations, GST and AChE activities, condition indices). Salinity differences induced variations of the same order of magnitude (GST, AChE) or even higher (lactate dehydrogenase, digestive enzymes in the crystalline style) than contamination-induced variations. In burrowing tests, the number of burrowed specimens was similar at both salinities after an experiment time <3 h. CONCLUSIONS: Size is a factor necessarily but easily controlled. Because the weight may be different in clams of identical size, correction factors may be used to minimise the influence of weight changes on biomarkers. A correction factor taking into account salinity levels can also be used. The protein concentrations in the clams did not differ with salinity, a very favourable outcome since all enzyme activities are classically expressed by reference to total protein concentrations. For burrowing tests, the number of burrowed specimens at a particular time is an endpoint that is preferable to measures of burrowing speed.

Behavioural and biochemical responses of two marine invertebrates Scrobicularia plana and Hediste diversicolor to copper oxide nanoparticles.

Engineered nano-sized Cu oxide particles are extensively used in diverse applications. Because aquatic environments are the ultimate "sink" for all contaminants, it is expected that nanoparticles (NP) will follow the same fate. In this study, two marine invertebrates Scrobicularia plana and Hediste diversicolor were chosen as ecotoxicological models. The aim was to evaluate behavioural (burrowing kinetics, feeding rate) and biochemical (biomarkers) responses of S. plana and H. diversicolor exposed in the laboratory to Cu (10 µg L(-1)) added in natural seawater either in the form of engineered nanoparticles (NPs) of CuO or as dissolved Cu in 2% HNO(3). Exposure was characterized by considering (i) the physico-chemical fate of NP (ii) the fraction of labile Cu in experimental media and (iii) Cu bioaccumulation. Results showed high aggregation of CuO NPs in seawater and no additional bioavailable Cu concentrations. Behavioural impairments were observed in S. plana exposed to CuO NPs or soluble Cu whereas in H. diversicolor, only the exposure to soluble Cu led to a burrowing decrease. No obvious neurotoxicity effects were revealed since in both species, no changes in cholinesterasic activity occurred in response to both forms of Cu exposure. Biomarkers of oxidative-stress catalase and glutathione-S-transferase were enhanced in both species whereas superoxide dismutase was increased only in S. plana exposed to CuO NPs. Metallothionein-like protein was increased in bivalves exposed to both forms of Cu. Since, no detectable release of soluble Cu from CuO NPs occurred during the time of experiment, ecotoxicity effects seem to be related to CuO NPs themselves.

Significance of physicochemical forms of storage in microalgae in predicting copper transfer to filter-feeding oysters (Crassostrea gigas).

Copper distribution has been examined in two microalgae (Haslea ostrearia, Diatom; Tetraselmis suecica, Prasinophyceae) exposed to Cu at 30 microg/L(-1). Exchangeable copper linked at the cell surface was desorbed using 8-hydroxyquinoline-5-sulfonate as complexing agent. Then, incorporated copper was separated between soluble and insoluble fractions. In addition, algae were resuspended in acid solutions, the pHs of which covered the range existing in the digestive tract of bivalves. Considering that the soluble fraction is the most easily transferred in the food chain and that exchangeable Cu is easily desorbed, the percentages of Cu potentially available in microalgae have been assessed. These percentages have been compared with those retained in oysters Crassostrea gigas fed with contaminated microalgae in previous studies. In H. ostrearia, the potentially available fraction of Cu (90%) was very similar to the percentage retained by oysters (93%) when the bivalves were acclimated to this food for 3 weeks. Only half (21%) of the potentially available Cu of T. suecica (42%) was readily assimilated in oysters after 3 weeks. This is in agreement with the results of the desorption tests at physiological pHs which showed that only 15-25% of Cu was lost, despite solubilization of other constituents of T. suecica as demonstrated by the decrease in their dry weight. Bioavailability determined from metal speciation in food allows a relevant prediction of the trophic transfer in the case of H. ostrearia, but caution is recommended in generalizing this mode of assessment as shown in the case of T. suecica.

Metallothionein concentration in sponges (Spongia officinalis) as a biomarker of metal contamination.

The synthesis of metallothioneins (MTs) is often induced when organisms are exposed to heavy metals in the field. They are among the major "specific" biomarkers identified to date. With a view to include MTs in biomonitoring programs, the organisms most commonly studied are bivalves. Sponges present most of the characteristics researched in bioindicators of pollution and consequently have been proposed to constitute a "Sponge Watch Program". The detection of large quantities of metals in sponges suggests the existence of detoxification systems and indeed, the presence of metallothionein-like proteins (MTLPs) has been reported in two different species of sponges. In Spongia officinalis, the present study has demonstrated the presence of compounds exhibiting most of the characteristics of MTs: cytosolic, heat-stable, with apparent molecular mass of 4 to 15 kDa and binding (at least) Ag, Cu and Zn. Specimens have been collected along the French Mediterranean coast from three sites differing by their degree of contamination. Relationships between MTLP and metal concentrations have been established. For copper, mercury and zinc, the correlations were significantly positive.

Do seasonal changes affect metallothionein induction by metals in mussels, Mytilus edulis?

Mussels have been proposed as biomonitors of metal pollution based on the determination of metallothionein (MT) concentrations as a biomarker, but a comprehensive study taking into account both intersite and long-term temporal variations in MT and metal concentrations in different organs is lacking. Thus, the present study was designed to examine the concentrations of cytosolic and insoluble Cd, Cu, Zn, and MT in gills and digestive gland of mussels (Mytilus edulis) of homogeneous size and age obtained from aquaculture and kept on a reference site or translocated to a metal-rich site throughout their reproductive season (March-October 1997). Relatively significant binding of metals to the insoluble fraction was observed in both tissues. In the digestive gland, monthly MT concentrations were strongly correlated to cytosolic metal levels. Moreover, despite significant temporal variations, the grand mean MT concentration based on all individual determinations in the digestive gland (reference, n=54; transplants, n=50) was significantly higher in mussels from the metal-rich site. On the other hand, gill MT concentrations did not reflect metal contamination as reliably.