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.

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.

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.

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.

Some methodological aspects of metallothionein evaluation.

The specificity of the methods used for metallothioneins (MTs) determination in both field and laboratory studies is crucial to a relevant interpretation of the results. The technique applied in the present study is commonly used, but several potential problems may limit its validity: (i). the stability of the metal-SH links during heating and the possibility of metal exchanges between MTs and others compounds; (ii). the presence of heat-stable thiol compounds with high molecular weight or very low molecular weight, which may subsist in the supernatant and interfere during global analysis of MTs; (iii). the subsistence of metals not bound to MT in the supernatant after heating; and (iv). the impact of reducing agents such as mercaptoethanol on the metal-binding ligands. Our investigations were conducted mainly on oysters (Crassostrea gigas) and polychaetes (Hediste/Nereis diversicolor) sampled, respectively, in two French metal-rich sites, the Gironde estuary and Boulogne harbour. This study, which allowed us to clarify most of the potential problems noted above, indicates the importance of dealing with methodological difficulties.