Bioaccumulation and detoxification processes of Hg in the king scallop Pecten maximus: field and laboratory investigations.

Hg bioaccumulation was investigated in the king scallop Pecten maximus in the laboratory and in the field. In controlled conditions, scallops were exposed to (203)Hg through seawater, sediment and food in order to determine its uptake and depuration kinetics. In the field, Hg and metallothionein (MT) concentrations and the metal subcellular distribution were determined in scallops from two sites of the Bay of Seine (France) differently subjected to the Seine river inputs. While Hg concentrations in the whole soft parts and kidneys (viz. the highest accumulator organ) did not differ between scallops from both sites (74-156 ng g(-1)dry wt), they did for the digestive gland and the gills. According to the experimental results, a higher exposure to dissolved Hg might occur in the site close to the estuary whereas Hg would be mainly incorporated via the dietary pathway in the site away from the estuary. Within the cells of wild scallops, Hg was mainly associated to the cytosolic fraction in the digestive gland and gills (60-100%). However, the lack of relationship between Hg and MT levels suggests that Hg detoxification in P. maximus involves other, non-MT, soluble compounds. In kidneys, insoluble compounds played an important role in Hg sequestration. No effect of scallop age was observed neither on Hg and MT concentrations nor on the subcellular distribution of the metal. Finally, according to FAO/WHO recommendations (maximum weekly Hg intake), our results clearly indicate that the low Hg contents in the edible part of the king scallops from the Bay of Seine prevent any risk for human consumers.

Effects of subchronic digestive exposure to organic or inorganic cadmium on biomarkers in rat tissues.

In an experimental food chain, Wistar rats were fed cadmium (Cd) in an inorganic (CdCl(2)) or organic (mainly associated with metallothionein from Helix aspersa snail viscera) form. After 1 month of exposure to 100 microg inorganic Cd g(-1) in food, an induction of metallothionein was observed in all target tissues. In liver, glutathione peroxidase (GSH-Px) activity decreased and alanine aminotransferase (ALAT) activity increased, suggesting that Cd causes hepatotoxicity. However, lipid peroxidation as well as catalase and caspase 3 (a marker of apoptosis) activities were not modified. At a rather low exposure (2.5 microg Cd g(-1)), metallothionein level in the kidney was found to be the most sensitive biomarker of exposure for both Cd forms. In the small intestine of rats ingesting inorganic Cd, metallothionein expression was significantly higher than that observed for rats fed organic Cd. Present results allowed proposing a simple design to assess the effect of a chemical in a trophic transfer approach.

Comparison of transfer and effects of Cd on rats exposed in a short experimental snail-rat food chain or to CdCl2 dosed food.

Transfer and toxic effects of two cadmium (Cd) forms, inorganic (CdCl2 dosed rat food) or organic (contaminated snail-based rat food) were studied in Wistar rat. Cd concentrations in rat food were 0 and 2.5 microg Cd g(-1) for both inorganic and organic forms and a high concentration of 100 microg Cd g(-1) was also tested for the inorganic form. Rats were exposed for four weeks to contaminated food. Both forms of Cd were bioavailable to rats, with a percentage of transfer from food to rats of around 1% for all contaminated groups. Cd concentrations in rat tissues increased with increasing Cd concentrations in the food. Rats fed with organic form of Cd accumulated significantly more Cd in the main organ for Cd toxicity, the kidney, than those eating the inorganic form. Survival was not affected for any rat group but a decrease in growth and food consumption was observed for the inorganic form. As a defence system against Cd toxicity, rats increased their metallothionein (MT) synthesis at the highest Cd concentration in the target organs (kidney, liver and small intestine) and even did the same at low Cd concentrations (2.5 microg Cd g(-1)) in the kidney. At this low Cd concentration, MT induction was lower in the small intestine of rats ingesting organic Cd than those ingesting inorganic Cd. Bioavailability of organic and inorganic forms of Cd was similar, but subsequent Cd distribution within organs was different. This quantification of the trophic transfer of both inorganic and organic forms of a toxicant is a basis for a better assessment of the fate and effects of chemicals in food webs.

Trace elements in three marine birds breeding on Reunion Island (Western Indian ocean): part 2-factors influencing their detoxification.

Seabird tissues collected between 2002 and 2004 from Barau's Petrel (Pterodroma baraui), Audubon's Shearwater (Puffinus lherminieri bailloni), and White-Tailed Trop icbird (Phaethon lepturus) colonies on Reunion Island were analyzed for metallothioneins (MTs) and trace element content. The subcellular distribution between soluble and insoluble fractions of Cd, Cu, Fe, Mn, Se, and Zn was determined in liver and kidney. In both, the soluble fraction of the cell concentrated most of the Cd and Se, whereas Fe, Mn, and Zn were preferentially accumulated in the insoluble fraction. The distribution of these elements varied with the tissue, age of the bird, and species. Furthermore, the distributions of Fe and Mn were somewhat influenced by the bird's physical condition. MT levels were measured in the soluble fraction after heat denaturation. The levels of these proteins varied from 5.5 +/- 2.7 mg x g(-1) dry weight (dw) to 11.4 +/- 6.2 mg x g(-1) dw depending on the species and the tissue considered. MT levels were significantly different between liver and kidney only in the White-Tailed Tropicbird. In the three species, MT levels in kidney were significantly higher in adult than juvenile birds. The bird's weight also had an influence on hepatic and renal MT levels, but not the sex nor the reproductive status. The implication of MTs in Cu and Zn homeostasis and Cd and Hg detoxification are discussed. In addition, clues on Hg regulation by Se were found, especially in Barau's Petrel, where the levels of these two elements were significantly correlated.

Metals in the shell of Bathymodiolus azoricus from a hydrothermal vent site on the Mid-Atlantic Ridge.

Specimens of the mussel Bathymodiolus azoricus were collected from Menez Gwen, a relatively shallow (850 m) hydrothermal vent field on the Mid-Atlantic Ridge. Each bivalve shell (n = 21) was individually cleaned by selective chemical. The residual crystal matrix of each shell was individually analysed for the concentrations of the minor elements magnesium and strontium and the trace elements iron, manganese, copper and zinc. The chemical composition of the crystal matrix is unusual. B. azoricus is identified as a species having one of the most strontium impoverished shells amongst the marine molluscs. For a bimineral species the magnesium concentration is also extraordinary low. Despite originating from a trace metal rich environment; the metal concentrations in the shells were exceptionally low. Mean concentrations of iron, manganese, copper and zinc were 20.6, 3.7, 0.6 and 9.4 microg g(-1) respectively. Minor and trace element concentrations exhibited a marked intra-population variability. Copper concentrations increased and iron and zinc concentrations decreased with increasing shell weight. Due to its insensitivity to the high environmental levels of trace elements and the variability in intra-population concentrations induced by shell weight the crystal matrix of the shell of B. azoricus has little potential for use in environmental trace metal monitoring in areas contiguous to deep-sea hydrothermal vents.

Trace elements in three marine birds breeding on Reunion Island (Western Indian ocean): part 1-factors influencing their bioaccumulation.

This work aimed to use seabirds as bioindicators of trace element levels in the tropical waters and food webs of the Western Indian Ocean. The accumulation patterns of selected toxic (Cd and Hg) and essential (Cu, Fe, Mn, Se, and Zn) elements were determined in liver, kidney, and pectoral muscle of 162 marine birds belonging to 3 species collected in Reunion Island between 2002 and 2004. These pelagic seabirds belong to the following species: Barau's Petrel (Pterodroma baraui), Audubon's Shearwater (Puffinus lherminieri bailloni), and White-Tailed Tropicbird (Phaethon lepturus). Hg levels were also measured in breast feathers. Highest mean kidney Cd and liver Hg levels (respectively, 27.79 +/- 13.78 microg x g (-1) dry weight (dw) and 24.31 +/- 14.13 microg x g (-1) dw) were found in the squid-eating Barau's Petrel. Barau's Petrel feather Hg levels fell in the range of 0.6 to 2.7 microg x g(-1) dw previously reported for other petrels and shearwaters. The values of the other elements were also in the same range as those previously reported in the published literature concerning related seabirds, although Se and Zn burdens in the Reunion birds were among the highest values. Levels of Zn, Fe, and, to a lesser extent, Cu appeared to be regulated in seabird tissues. Uptake and pathways of metabolism and storage seemed to be similar for the five essential elements. The reproductive status of the bird did not seem to affect element levels, which, moreover, were not significantly different between male and female birds. However, trace elements in sampled birds varied according to the tissue considered, the age of the animal, and its species. Diet was seemingly a major influencing factor. Health status also appeared to have an impact on element levels.

Antioxidant systems and lipid peroxidation in Bathymodiolus azoricus from Mid-Atlantic Ridge hydrothermal vent fields.

Enzymatic defenses involved in protection from oxygen radical damage were determined in gills and mantle of Bathymodiolus azoricus collected from three contrasting Mid-Atlantic Ridge (MAR) hydrothermal vent fields (Menez-Gwen, Lucky Strike and Rainbow). The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx) (total and Se-dependent), and levels of total oxyradical scavenging capacity (TOSC), metallothioneins (MT) and lipid peroxidation (LPO) were determined in B. azoricus tissues and the impact of metal concentrations on these antioxidant systems and lipid peroxidation assessed. SOD, CAT, TOSC, MTs and LPO levels were higher in B. azoricus gills while glutathione peroxidases (total and Se-dependent) were higher in the mantle, and with the exception of CAT, were of the same order of magnitude as in other molluscs. TOSC levels from Menez-Gwen indicate that the vent environment at this site is less stressful and the formation of ROS in mussels is effectively counteracted by the antioxidant defense system. TOSC depletion indicates an elevated ROS production in molluscs at the other two vent sites. Cytosolic SOD, GPx and LPO were more relevant at Lucky Strike (Bairro Alto) where levels of essential (Cu and Zn) and toxic metals (Cd and Ag) were highest in the organisms. CAT activity and LPO were predominant at the Rainbow vent site, where an excess of Fe in mussel tissues and in vent fluids (the highest of all three vent sites) may have contributed to increased LPO. Therefore, three distinct pathways for antioxidant enzyme systems and LPO based on environmental metal speciation of MAR vent fields are proposed for Bathymodiolus gills. At Menez-Gwen, TOSC towards peroxyl and hydroxyl radicals and peroxynitrite are predominant, while at Lucky Strike cytosolic SOD activity and GPx are the main antioxidant mechanisms. Finally at Rainbow, catalase and lipid peroxidation are dominant, suggesting that resistance of mussels to metal toxicity at these vent fields decreases in the sequence Menez-Gwen > Lucky Strike and Rainbow.

Cadmium detoxification processes in the digestive gland of cephalopods in relation to accumulated cadmium concentrations.

The high concentrations of cadmium recorded in the digestive gland of cephalopods from various temperate and subpolar waters suggest that these molluscs have developed efficient cadmium detoxification mechanisms. The subcellular distribution of cadmium in the digestive gland cells was investigated in seven cephalopod species from the Bay of Biscay (France) and the Faroe Islands. In most species, cadmium was mainly found in the cytosolic fraction of the digestive gland cells, reaching up to 86% of the total cadmium for the squid Loligo vulgaris from the Bay of Biscay. But species with the highest total level of cadmium showed a higher percentage of cadmium associated to insoluble compounds. The quantification of metallothioneins (MTs) by the polarographic method was performed in order to evaluate the involvement of these proteins in the detoxification of the high amounts of bioaccumulated cadmium. Metallothionein levels in cephalopods ranged form 742 +/- 270 to 3478 +/- 1572 microg/g wet weight. No relationship could be established between total cadmium, cytosolic cadmium and MT levels suggesting the occurrence of other Cd-binding ligands. Although these proteins have not been characterised, as cadmium in the digestive gland of cephalopods is mainly associated with soluble ligands, a high potential transfer to predators can be predicted.

Induction of specific isoforms of metallothionein in mussel tissues after exposure to cadmium or mercury.

The synthesis of metallothioneins has been established for Mytilus edulis exposed to cadmium and mercury. We checked if this induction resulted in the synthesis of tissue- or metal-specific isoforms in the gills, the mantle, and the digestive gland that could be used as tool for the characterization of undefined metallic contamination of aquatic ecosystems. An accumulation of metals was observed in the selected organs after 21 days of exposure. The levels of metallothioneins measured by using the polarographic method were significantly increased by cadmium and mercury in the gills (21 days). Size exclusion chromatography showed the presence of a monomer and a dimer of metallothionein of respective apparent molecular weight about 12 kDa and 20 kDa in all samples. They were resolved into five components by anion exchange chromatography in the gills of control or Hg-treated mussels, whereas a sixth isoform was isolated in the gills of cadmium-exposed mussels. In the mantle of mussels exposed or not, five isoforms were separated, and in the digestive gland of mussels exposed or not, six isoforms were separated. The occurrence of a specific cadmium-binding isoform in the gills has to be confirmed in cadmium-contaminated specimens collected in situ before its detection may be used as biomarker of cadmium contamination.

Bivalve metallothionein as a biomarker of aquatic ecosystem pollution by trace metals: limits and perspectives.

Owing to their induction by metals metallothioneins (MTs) have been proposed as biomarkers of the metallic contamination of the environment. On the other hand, bivalves are regarded as very convenient bioindicators of the aquatic ecosystems and an extensive literature has been dedicated to their response to metals. Among studies supporting the involvement of MTs in metal detoxification some discrepancy appears due to inter- and intra-specific variations, or to heterogeneous exposure conditions. A lesser number of papers are dealing with the use of metallothionein levels as biomarkers, and sometimes they evidence that natural factors influencing metallothionein synthesis have to be taken into account before final conclusions can been drawn. Moreover, there is still a large number of non-intercalibrated protocols used to quantify amounts of metallothioneins in organisms. As comparisons are necessary to assess the relative abundance of metallothioneins in a studied species, more work has to be completed before such comparisons could be validated. In the present paper we wish to establish the limits of the use of mollusc metallothioneins as a biomarker of aquatic ecosystem contamination by trace metals, using published and recent data as support for our conclusions and perspectives.

Relationship between metallothioneins and metals in a natural population of the clam Ruditapes decussatus from Sfax coast: a non-linear model using Box-Cox transformation.

Cadmium, copper and zinc were determined concomitantly with metallothionein-like proteins (MTLPs) in the subcellular fractions of Ruditapes decussatus digestive gland. This study covered 4 months and aimed to evaluate the effect of metal pollution and other factors such as sex, size and reproductive state on MTLP levels. Copper concentrations did not vary with month, however Cd and Zn concentrations showed high levels during August. Organisms showing low cadmium concentrations presented the highest cadmium percentages in the soluble fraction (SF) containing MTLPs. However for high cadmium concentrations, the insoluble fraction (IF) was implicated in cadmium association. MTLP levels varied according to the month, the sex and the size of the organisms. A non-linear model based on the Box-Cox transformation, was proposed to describe a positive and a significant relationship between MTLPs and the studied metals. A model including sex and size showed that these two factors affected MTLP levels, but were less important than metals. Males of R. decussatus showed higher significant correlations between MTLP levels and cadmium than females. Moreover, the effect of size and reproductive state on MTLP levels was less perceptible in males than in females. As a result, MTLPs in males of R. decussatus could be proposed as suitable biomarker for detecting metal contamination.

Heavy metal intracellular balance and relationship with metallothionein induction in the gills of carp. After contamination by Ag, Cd, and Hg following pretreatment with Zn or not.

Determination of metal levels (Cu, Zn, Cd, Ag, Hg) in soluble and insoluble fractions of gill homogenates has been performed after 7 d exposure of carp (Cyprinus carpio) to moderate concentrations of Cd, Ag, and Hg in water. Metallothionein levels have been quantified by polarographic method before and after contamination and a subsequent decontamination phase (7 d). The influence of pretreatment by zinc (7 d) has also been evaluated. Metallothionein level variations have been interpreted as having regard to interrelated flows of metal between subcellular fractions. Special interest has been focused on heat-stable compound (HSC)-bound heavy metal flows within the cytosol, taking in account that MT is the major component of these ligands. Our data showed differences between the ability of metals to bind cytosolic ligands and HSCs, and their respective potency for MT induction in gill. Regardless of pretreatment, mercury gave the highest increase of gill MT, and after the decontamination MT level remained high compared to control. Cadmium and silver gave similar increases, but a significant difference with control appeared only after the decontamination step with silver, whereas 1 week of contamination was enough for cadmium. Our experimental conditions gave the following order of potency for MT induction in gill: Hg > > Cd > Ag > Zn.

Trace elements in little egrets and flamingos of Camargue, France.

Trace elements (Cd, Cu, Hg, Pb, Se, Zn) were measured in nine organs (liver, kidney, breast muscle, lungs, breastbone, stomach, gizzard, spleen, feathers) of several specimens of Greater Flamingos (Phaenicopterus ruber (Pallas] and Little Egrets (Egretta garzetta (L.] from the Camargue, in the Rhône river delta. In both species, individual fluctuations of Cd, Hg, Pb, and Se levels were important in all organs, whereas Zn and Cu, essential metals, exhibited only moderate variations. The organotropism of trace elements supports the hypothesis that major routes of contamination would be both nutritional and aerial for flamingos, and prevailingly alimentary for egrets. In both species we found the highest levels in liver and kidney, with the exception of lead, the highest levels of which were found in breastbone and feathers. Our study indicates that trace element concentrations in flamingos and egrets are higher than or equivalent to those reported in the literature for birds living in polluted areas. Because the Camargue is a unique area in western Europe it would be of interest to develop more studies to evaluate the impact of surrounding human activities on this Biosphere Reserve.