Metallothioneins in aquatic invertebrates: Their role in metal detoxification and their use as biomarkers

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1 Aquatic Toxicology 76 (2006) Review Metallothioneins in aquatic invertebrates: Their role in metal detoxification and their use as biomarkers J.-C. Amiard a, C. Amiard-Triquet a, S. Barka b, J. Pellerin c, P.S. Rainbow d, a Université de Nantes, Pôle Mer et Littoval, SMAB (EA 2160), Service d écotoxicologie, 2 Rue de la Houssinière, BP 92208, Nantes Cedex 3, France b Laboratoire de Physiologie des êtres Marins, Institut Océanographique, 195 Rue Saint-Jacques, Paris, France c Université duquébec à Rimouski, ISMER, 310, allée des Ursulines, Rimouski, Qué., Canada G5L 3A1 d Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK Received 25 February 2005; received in revised form 5 August 2005; accepted 5 August 2005 Abstract The literature on metallothioneins (MT) and metallothionein-like proteins (MTLP) in aquatic invertebrates is large and increasing, and yet inconsistencies and confusion remain, not least over the physiological role of MT and their use as biomarkers in environmental monitoring programmes. We have collated published information on MT in three important groups of aquatic invertebrates the molluscs, crustaceans and annelid worms, and attempted to seek explanations for some of the apparent inconsistencies present in the dataset. MTs can be induced by the essential metals Cu and Zn and the non-essential metals Cd, Ag and Hg in both vertebrates and invertebrates, but their induction is variable. Such variation is intraspecific and interspecific, and is down to a variety of reasons environmental and physiological explored here. Against this background of variability MTs do appear to play roles both in the routine metabolic handling of essential Cu and Zn, but also in the detoxification of excess amounts intracellularly of these metals and of non-essential Cd, Ag and Hg. Different isoforms of MT probably play different physiological roles, and the dependence on MT in detoxification processes varies environmentally and between zoological groups. MTs can be used as biomarkers if used wisely in well-designed environmental monitoring programmes Elsevier B.V. All rights reserved. Keywords: Metallothionein; Review; Aquatic invertebrates; Biomarker; Detoxification Corresponding author. Tel.: ; fax: address: p.rainbow@nhm.ac.uk (P.S. Rainbow) X/$ see front matter 2005 Elsevier B.V. All rights reserved. doi: /j.aquatox

2 J.-C. Amiard et al. / Aquatic Toxicology 76 (2006) Contents 1. Introduction Principal characteristics of MTs and their biological roles MT concentrations in different species and distribution between organs MT induction in response to metal exposure: evidence and inconsistencies Is MT induction dependent on the metal tested? Is MT induction dependent on biological characteristics? Is MT induction dependent on the period and dose of exposure? Tentative explanations Relative importance of MT in metal detoxification Increased MT turnover rates versus increased MT concentrations MT induction and toxicity MT induction and physiological variability The use of MT as a biomarker of metal exposure Comparisons of different species Comparisons of different organs Confounding factors MT as a biomarker Conclusions References Introduction Metallothioneins (MTs) are non-enzymatic proteins with a low molecular weight, high cysteine content, no aromatic amino acids and heat stability. The thiol groups ( SH) of cysteine residues enable MTs to bind particular heavy metals. The first MT was found in equine renal cortex (Margoshes and Vallee, 1957) and true MT or MT-like proteins have since been reported in many vertebrates including many species of fish (Olsson et al., 1998; Roeva et al., 1999) and aquatic invertebrates (Roesijadi and Fowler, 1991), mainly molluscs (Langston et al., 1998; Isani et al., 2000) and crustaceans (Roesijadi, 1992; Engel and Brouwer, 1993; Barka, 2000). As noted by Templeton and Cherian (1991), the behaviour of MT is dominated by the chemistry of the thiol group, such that any metal sharing stoichiometric characteristics with copper or zinc, may also be bound to MT. The proteins are usually not saturated by a single metal but contain several atoms of Cu, Zn, Cd, or Hg and Ag when present (Amiard and Cosson, 1997). Multiple isoforms have been identified and polymorphism appears to be particularly important in invertebrates compared to mammals. Variations in molecular mass have also been observed, suggesting the presence of monomeric and dimeric forms (Langston et al., 1998). The biological functions of metallothioneins are still a subject of controversy. Given the metal-binding capacity of MTs, it is generally considered that these proteins play a role in the homeostatic control of essential metals (Cu, Zn) as they can act as essential metal stores ready to fulfill enzymatic and other metabolic demands (Brouwer et al., 1989; Viarengo and Nott, 1993; Roesijadi, 1996). Non-essential metals such as Cd and Hg are, however, able to displace normally MT-associated essential metals (Amiard and Cosson, 1997). Furthermore, because enhanced metal tolerance has been associated with MT induction (Roesijadi et al., 1982a; George and Olsson, 1994; Pavicic et al., 1994a; Roesijadi, 1996; Wallace and Lopez, 1997; Ritterhoff and Zauke, 1998), MTs are thought also to be involved in the detoxification of excess amounts of both essential and non-essential trace metals. Recent progress in molecular biology will allow the more precise characterization of these different forms (Lemoine et al., 2000; Syring et al., 2000; Tanguy et al., 2001; Tanguy and Moraga, 2001), and clarify any double role for MTs in homeostasis and detoxification by detecting specific MT gene expression devoted to either role (Palmiter, 1999; Davis and Cousins, 2000; Butler and Roesijadi,

3 162 J.-C. Amiard et al. / Aquatic Toxicology 76 (2006) ). Molecular biological techniques should also allow the sensitive detection of small incremental changes over basal MT expression which are undetectable using other methods (Butler and Roesijadi, 2001). In many species (annelids, molluscs, crustaceans, fish,...) induction of metallothionein synthesis by metal contaminants (Ag, Cd, Cu, Hg,...) has been demonstrated, suggesting the potential use of MT concentrations in organisms as biomarkers of metal exposure. MT is now part of a core suite of biomarkers recognized at European level and examined in the framework of biological effect quality assurance in monitoring programmes (BEQUALM) (Mathiessen, 2000). Nevertheless, literature data present many contradictions and inconsistencies in MT induction. At sites where metals are present and bioavailable at high concentrations, some species do not show increased MT concentrations, at least in some organs (Pedersen and Lundebye, 1996; Géret and Cosson, 2000; Geffard et al., 2001; Berthet et al., 2003). Some laboratory experiments have shown induction over very short time periods (Viarengo et al., 1985; Bodar et al., 1988; Del Ramo et al., 1995; Martinez et al., 1996; Barka et al., 2001), whereas in the field long exposures of many months have been necessary to show any significant rise in MT concentrations in bivalves transplanted from a clean to a contaminated site (e.g. Couillard et al., 1995a; Geffard et al., 2001). The processes that generate these inconsistencies need to be understood in order to validate the use of MTs as biomarkers. In vertebrates, and particularly in fish, trace metal detoxification processes depend mainly on metal binding to metallothioneins. As a consequence, any MTmetal exposure relationship is easier to demonstrate in fish than in invertebrates, as there is less or no interference from biomineralization processes, which are common in invertebrate trace metal detoxification (Mason and Jenkins, 1995). However, if MTs are to be considered potential biomarkers in monitoring programmes, it needs to be remembered that fish, even those considered to be sedentary, are less strictly representative than relatively less mobile invertebrates of the site at which they have been caught. Furthermore the sampling of fish often seems to be less controllable than that of (usually more numerous) invertebrates, particularly as regards presence/absence, selection of size, age or weight categories, and the influence of these factors on the inducibility of fish MT is well-recognized (George and Olsson, 1994; Hamza-Chaffai et al., 1997; Tom and Auslander, 2005). The literature on metallothionein synthesis is vast, but many references are either dated (Fischer, 1980; Suzuki, 1987) or are limited to a historical aspect (Nordberg, 1998), spectroscopic properties (Stillman, 1995), structure (Romero-Isart and Vasak, 2002) or a single zoological group (mammals Miles et al., 2000; molluscs Cosson, 2000; Isani et al., 2000). In this review we emphasize three major invertebrate taxa the molluscs, the crustaceans and the annelids, although some data exist for other invertebrate phyla (e.g. echinoderms: Riek et al., 1999; sponges: Philp, 1999; Schröder et al., 2000; Berthet et al., 2005; coelenterates: Andersen et al., 1988). These three taxa are among most ecologically important of the aquatic invertebrates. In addition to the usefulness of MT as a biomarker in these three groups, MT plays a potentially key role in the adaptation to metal exposure of these three important groups of invertebrates, which contribute greatly to estuarine and coastal ecosystems, in terms of biomass, and ecosystem structure and functioning. 2. Principal characteristics of MTs and their biological roles The most important and original MT characteristic is their high cysteine content. Cysteines account for 33% of the 61 constitutive amino acids of mammalian MTs while crustacean MTs contain 18 cysteines in amino acids (Binz and Kägi, 1999). The alignment of Cys-Cys, Cys-X-Cys and Cys-X-Y- Cys sequences where X and Y are amino acids other than cysteine, is the criterion that allows the distinction between different structural MT classes and that leads to many isoforms of the same protein. MTs have been divided into three classes (Fowler et al., 1987). Class I comprises all proteinaceous MTs with locations of cysteine closely related to those in mammals. Some molluscan and crustacean MTs belong to this class, such as those characterized in mussels (Mackay et al., 1990), oysters (Roesijadi et al., 1989), crabs (Lerch et al., 1982) and lobsters (Brouwer et al., 1989). Class II includes proteinaceous MTs which lack this close similarity to mammalian MTs,

4 J.-C. Amiard et al. / Aquatic Toxicology 76 (2006) while Class III MTs consists of non-proteinaceous MTs also known as phytochelatins. Binz and Kägi (1999) have recently proposed a new classification of MTs taking into account phylogenetic features as an additional classification criterion. Class I and Class II MTs known so far are distinguishable into 15 families. According to this recent MT classification of Binz and Kägi (1999), molluscan MTs belong to family 2 whereas crustacean MTs belong to family 3. Binz and Kägi (1999) did not allocate annelid MTs to a family. The highly conservative character of MTs, together with their ubiquity, leads to the conclusion that these proteins play an essential role in vital processes. Mason and Jenkins (1995) proposed two roles for MTs. Firstly, these proteins comprise a non-toxic zinc and copper reservoir available for the synthesis of metalloenzymes, allowing the homeostasis of many cellular processes (Brouwer et al., 1989; Viarengo and Nott, 1993; Roesijadi, 1996). Secondly, MTs can reduce the nonspecific binding of non-essential metals within cells, and so restrict their toxic potential (Roesijadi, 1992, 1996; Zaroogian and Jackim, 2000). Many authors now accept that metallothioneins may control the homeostasis of essential metals such as zinc and copper, as they represent stocks able to fulfill enzymatic and other metabolic demands. When non-essential metals (Cd, Hg or Ag) enter a cell, there is inevitably competition between them and existing essential metals (Cu, Zn) for intracellular ligands like metalloproteins, and a detoxificatory role for MT here would be of advantage. Metallothioneins also seem to have other roles including protection against ionizing radiation (Cai et al., 1999) and a more general antioxidant defense (Viarengo et al., 2000; Cavaletto et al., 2002; Correia et al., 2002b; Rodriguez-Ortega et al., 2002). Thus organisms pre-exposed to a metal such as cadmium resist oxidising stress better, as a result of the induction of MT which seems to limit the effects of hydroxyl (OH) and superoxide (O 2 ) radicals by scavenging them. Regulation of MT genes by heavy metals appears to be mediated by a zinc-sensitive inhibitor that interacts with a constitutively active transcription factor (Palmiter, 1994). The in vitro affinity of the protein decreases in the hierarchical sequence Hg 2+ >Cu +, Ag +,Bi 3+ Cd 2+ >Pb 2+ >Zn 2+ >Co 2+ (Vasak, 1991) showing that Zn is likely to be displaced by a number of other metals including those generally considered to be the most toxic. Furthermore, Ni ions have a very high affinity for cysteine (Costa et al., 1994). Thus Roesijadi (1996) has proposed a model for the coupled MT induction and rescue of target ligands compromised by inappropriate metal binding (Fig. 1). As MTs, like other proteins, have limited lives, it is reasonable to consider that they will be integrated into the classical cytophysiological circuit in order to be expelled (or stored) as cellular wastes from the cytoplasm via lysosomes where protein degradation occurs (Isani et al., 2000). The mechanisms of incorporation Fig. 1. Model for coupled MT induction and rescue of target ligands comprised by inappropriate metal binding, by Cd in this example. (MT, metallothionein; MRE, metal regulatory element; MTF, metal transcription factor; MTI, metal transcription inhibitor) (after Roesijadi, 1996).

5 164 J.-C. Amiard et al. / Aquatic Toxicology 76 (2006) of metals into lysosomes have been relatively ignored. Nevertheless, several authors have observed the concomitant presence of S and metals in lysosomes, an observation which probably results from the incorporation of MTs into these cellular organelles (Brown, 1982 and literature therein; Martoja et al., 1988; Vogt and Quinito, 1994; Barka, 2000; Nassiri et al., 2000; Marigomez et al., 2002 and literature therein). In fact the origins of lysosomal organic or inorganic elements, in particular of sulphur, have never been proved (Mason and Jenkins, 1995), although Viarengo and Nott (1993) put forward a synthesis of the mechanisms implied in the metabolism of metals. However it has been shown that, in vitro, rat lysosomal extracts degraded MTs associated with zinc and cadmium, whereas those MTs associated with copper resisted degradation because of the stability of the disulphide links in their molecular conformation (Mehra and Bremner, 1985, cited in Bremner, 1991). This last result may explain the fact that copper in particular is the metal found associated with sulphur in lysosomes, Cu-MT being the most resistant to lysosomal degradation. The involvement of MTs (and potentially also of lysosomes) in metal accumulation has led many authors to associate metal tolerance with MT induction and to conclude that these proteins are very likely to be involved in detoxification processes (Roesijadi et al., 1982a; Klaverkamp and Duncan, 1987; Roesijadi and Fellingham, 1987; Bodar et al., 1990). Moreover, it has been established that a rise in MT concentration can be linked to a decrease of the sensitivity of an organism to metals in excess (Pavicic et al., 1994a,b; Roesijadi and Fellingham, 1987; Stuhlbacher et al., 1992; Stuhlbacher and Maltby, 1992; Pavicic et al., 1994a,b). Some authors have noted that in a single species, the populations that live in a medium polluted by metals have higher concentrations of MT (e.g. Stuhlbacher et al., 1992; Ross et al., 2002). Several authors have carried out pre-exposures of animals to a given metal (Cd, Zn,...) and subsequently noted a strong induction of MT (Bodar et al., 1990; Unger and Roesijadi, 1996; Roesijadi et al., 1997a). During the embryogenesis of the mussel Mytilus galloprovincialis, stages with different sensitivities to cadmium and zinc exposure (recognized by abnormality during development) were noted and the less sensitive stages corresponded with those with the highest MT concentrations (Pavicic et al., 1994b). In the freshwater bivalve Pyganodon grandis exposed in the field along a Cd gradient, total gill MT concentrations varied from 92 to 271 nmol g 1 whereas total gill Cd concentrations varied over a wider range ( nmol g 1 ) (Wang et al., 1999). The authors underlined the fact that bivalves exposed to concentrations of dissolved free Cd higher than 1 nm in the external medium exhibited a marked increase of Cd in the low relative molecular mass ligand pool. Symptoms of toxic effects at different levels of biological organization were associated with these biochemical changes, re-inforcing a contrario the hypothesis of a detoxification role for MT (even if it appeared to be efficient only over a restricted range of concentrations). At the moment, most published works only provide total MT concentrations, whereas many isoforms coexist within organisms. Dallinger et al. (1997) for example reported evidence for the existence of distinct MT isoforms in terrestrial snails, one dedicated to cadmium detoxification and the other to copper regulation. The same conclusions were reached in the case of Drosophila melanogaster (Lauverjat et al., 1989). These results are also in agreement with the findings of Brouwer et al. (1992) for the crab Callinectes sapidus in which different MT isoforms have been found to play different physiological roles. DNA characterization and quantification of expression in different organs of mussels Mytilus edulis have shown that Zn exposure was responsible for high levels of a MT-10 messenger RNA whereas Cd exposure induced two MT messenger RNAs, MT-10 but especially an MT-20 (Lemoine et al., 2000). Molecular biological advances using MT gene amplification or duplication have confirmed that functions of different isoforms are different, some of them being involved in metal homeostasis and others in nonessential metal detoxification. MT encoding genes have now been identified in many crustaceans and molluscs: Scylla serrata (Olafson et al., 1979b; Lerch et al., 1982; Otvos et al., 1982), C. sapidus (Narula et al., 1995; Syring et al., 2000; Brouwer et al., 2002), Crassostrea virginica (Roesijadi et al., 1989; Butler and Roesijadi, 2001), Homarus americanus (Brouwer et al., 1989), M. edulis (Mackay et al., 1993), Astacus astacus and Potamon potamios (Pedersen et al., 1996), Portunus pelagicus (Ang and Chong, 1998), Macoma balthica (Rigaa et al., 1998), Dreissena polymorpha (Englelken and Hildebrandt, 1999), Jasus edwardsii

6 J.-C. Amiard et al. / Aquatic Toxicology 76 (2006) (Khoo and Sin, 1999), Crassostrea gigas (Tanguy and Moraga, 2001; Tanguy et al., 2001), M. galloprovincialis (Ceratto et al., 2002), Perna viridis (Khoo and Patel, 1999), Littorina brevicula (Park et al., 2002), Ruditapes decussatus (Simes et al., 2003), Megathura crenulata (Lieb, 2003). Expression of MT genes shows interspecific diversity. MT expression in the foot of the gastropod mollusc Littorina littorea represents a good example (English and Storey, 2003). The UTRs (untranslated regions) were found to be longer than the corresponding regions of mammalian and D. melanogaster MT sequences but comparable to those of the sea urchin (Strongylocentrotus purpuratus) and lobster (H. americanus). With a predictable molecular mass of 10 kda, the protein encoded by LL Met with 100 amino acids in length was significantly larger than most other MTs. However no significant matches in the cdna sequence were found with other species. LL Met transcripts were detected in low amounts in control gastropods but transcript levels increased significantly after exposure to anoxia or freezing conditions. This L1-MT protein is thought to be similar to the Cd binding protein found in the digestive gland of L. littorea during exposure to high cadmium availability (Langston and Zhou, 1986). This protein was different from a metal binding protein of approximately 20 kda found in L. littorea from unpolluted sites, thus suggesting a role in detoxification for the former protein and in homeostasis of metals for the latter one. In the blue mussel M. edulis, a different pattern is observed. Cadmium was found to induce the transcription of both the MT-20 gene and the MT-10 gene (Lemoine and Laulier, 2003), a result consistent with those reported in crustaceans (Pedersen et al., 1998). MT gene organization and regulation sequences (MREs) need to be explored more deeply in each species to understand more fully the roles of the MTs. Exploring the role of untranslated regions as well as the MRE sequences is also needed to understand regulatory elements of MT synthesis. Even though evidence has been provided that mrna increased after Cd exposure and could be used as a biomarker of metal contamination in the gills of M. edulis, correlation between MT mrna and MT concentrations has yet to be established (Lemoine and Laulier, 2003). Novel genes coding for MTs are still being characterized and we cannot preclude that discrepancies between results will not be explained by the discovery of other genes present. In M. galloprovincialis, observation of a putative MT protein compared to other sequences showed a 100% identity with the MT20-III isoform from M. edulis with amino acids at positions 25 (glutamine) and 46 (arginine) and a relatively high (68%) AT content, a feature only found in these species. This high AT content may be a sign of an unknown means of regulation or adaptation to different habitats (Ceratto et al., 2002). Surprisingly, a MT clone in the bivalve Anadara granosa (Chan et al., 2002) also encoded a putative MT with the highest homology to M. edulis (Chan et al., 2002). Only one isoform of MT was present in A. granosa challenged with cadmium, similar to the MT-20 in the blue mussel. The deduced amino acid sequence exhibited conserved sequences present in mollusc MTs similar to the MT gene that encodes Class I MTs, another sign of the conserved nature of MT and its similar function in a variety of organisms. In the giant keyhole limpet M. crenulata, a clone containing the complete cdna for a novel molluscan MT and the corresponding gene were recently isolated. It appeared to be almost part of the Class I MT despite the presence of indels (Lieb, 2003) and shares with M. galloprovincialis a high amount of glycine residues (Ceratto et al., 2002). Evolution however, seems to have promoted exon shuffling in M. crenulata, adding new functional sites in the protein as reflected by parts of the keyhole limpet sequence being similar to other published sequences: the first third to the sequence in the snail Helix pomatia linked to Cu-binding, the second third related to the H. pomatia Cd-binding MT, and the last third resembling mussel MTs. Two genes coding for two different MTs have been characterized in the oyster C. gigas and an ELISA developed to further their use as biomarkers of exposure to metals (Boutet et al., 2002). It appears that the MT gene controls the synthesis of a novel MT which could be specifically dedicated to detoxification in C. gigas (Tanguy and Moraga, 2001). On the other hand, regulation of MT gene transcription is due to the presence of metal responsive elements (MRE), the core region of which is well conserved across species (Imbert et al., 1990). Multiple copies of MRE have been shown to activate MT gene transcription in parallel. Apart from studies of the expression of MT genes in the presence of heavy metals, new nuclear genetic markers have been developed, and extensive studies have now been carried out on the polymorphism of

7 166 J.-C. Amiard et al. / Aquatic Toxicology 76 (2006) selected genes in relation to environmental stress factors such as pollution (Tanguy et al., 2002). A low level of polymorphism in the coding sequences of MT genes was observed in C. gigas. The low level of polymorphism in CgMT2 could be explained by its recent appearance in the genome of this oyster. As for M. crenulata, the C. gigas MT gene may have been submitted to mutational events transforming the CgMT1 gene into the CgMT2 gene, conserving most of the 5 and 3 untranslated regions (Tanguy and Moraga, 2001). Increases in frequency of types C in exon 2 and B in exon 3 in CgMT1 were observed in Cd-resistant populations. CgMT1 and CgMT2 genes can therefore be envisaged as MTs essentially involved in detoxification and CgMT2 would be activated by increased metal exposure (Tanguy and Moraga, 2001). These two MT types in C. gigas are now considered as appropriate genetic indicators for monitoring metal polluted sites (Tanguy et al., 2002). However, in another oyster Ostrea edulis, Tanguy et al. (2003) observed no significant induction of MTs, either in the gills or the digestive gland, nor MT-RNA expression. These results led to the conclusion that MTs are not involved in metal detoxification in this oyster, consistent with the lack of genetic diversity in field populations (Tanguy et al., 2003). Transgenic gain-of-function or loss-of-function experiments have been carried out to examine the potential functions of MTs. In yeast, deletion of the MT gene resulted in loss of ability to tolerate the toxicity of Cu (Hamer et al., 1985). In mammals the disruption of the MT-I and MT-II genes and abolishment of MT synthesis resulted in a loss of tolerance to Cd (Masters et al., 1994), whereas MTs were shown not to be required for the biosynthesis of metalloproteins, for homeostasis of essential metals (Zn and Cu), nor for protection against oxidative stress, at least under normal conditions (Palmiter, 1999). These latter findings contrast with many data acquired previously in field and laboratory experiments. 3. MT concentrations in different species and distribution between organs Tissues directly involved in metal uptake, storage and excretion have a high capacity to synthesise MTs. In aquatic organisms, these proteins have been identified in the digestive gland (also termed the midgut gland or hepatopancreas) (Viarengo et al., 1984; Olafson et al., 1979a,b) and gills (Roesijadi and Klerks, 1989; Viarengo et al., 1980; Nolan and Duke, 1983; Engel et al., 1985; Roesijadi and Klerks, 1989; Mouneyrac et al., 1998) of molluscs and crustaceans. Basic levels of metallothionein (or MTLP) concentrations in bivalves and crustaceans sampled from sites considered to be uncontaminated are shown in Tables 1 and 2. The quantification of the MT does not provide an absolute value, and several methods of MT quantification have been proposed (Cosson and Amiard, 2000; Dabrio et al., 2002), with additional later improvements (El Hourch et al., 2003, 2004). Comparative assays of different techniques have been carried out, showing a good correlation between results obtained for instance using differential pulse polarography (DPP) and a metal saturation assay (Onosaka and Cherian, 1982), and between DPP and spectrophotometric determination (Roméo et al., 1997). However, the various techniques produce concentration values that do differ and the results are often expressed in different units. Most of the work carried out on annelids has been focussed on characterization and does not include quantitative analyses. In the estuarine and coastal polychaete Hediste diversicolor (often still referred to as Nereis diversicolor), we have determined the concentrations of compounds which share many characteristics of MT (cytosolic, heatstable, thiol-rich compounds CHSTC) in specimens originating from an uncontaminated site (Blackwater estuary, Essex, UK) (Mouneyrac et al., 2003). The measured concentration was near 10 mg g 1 (dw), a high value compared to those obtained in other zoological groups (Tables 1 and 2) with the same technique of determination (DPP). However, it has been shown that these CHSTC include not only molecules with molecular masses corresponding to MT but also lower molecular mass compounds (about 2 kda), and thus the concentration mentioned above is probably an overvaluation of the true MT concentration. Data can be compared strictly only when they have been obtained with the same analytical technique (Table 1). However, it may be noted that the amphipod crustacean Echinogammarus echinosetosus showed MT concentrations orders of magnitude lower than all the other species (Table 1). This cannot be attributed to its phylogenetic status since another amphipod Orchestia gammarellus showed values considerably higher

8 J.-C. Amiard et al. / Aquatic Toxicology 76 (2006) Table 1 Basal concentrations of metallothionein in crustaceans from sites considered to be uncontaminated Species Tissue Concentration (mg g 1 dw or ww) Reference Artemia salina Whole animal 0.45 ww a Del Ramo et al. (1995) Daphnia magna 1.8 ± 0.7 dw b Bodar et al. (1990) Echinogammarus echinosetosus ± ww a Martinez et al. (1996) Orchestia gammarellus Whole animal dw c Mouneyrac et al. (2002) Penaeus vannamei Hepatopancreas 2.5 ± 0.4 ww c Moknes et al. (1995) Nephrops norvegicus Hepatopancreas ww c Canli et al. (1997) Gills ww c Canli et al. (1997) Procambarus clarkii Hepatopancreas 0.1 ww a Del Ramo et al. (1995) ww a Martinez et al. (1993) Carcinus maenas Midgut gland ± dw d Pedersen and Lundebye (1996) ± dw d Lundebye and Depledge (1998) 67 ± 18 nmol g 1 dw d Pedersen et al. (1997) ww d Wedderburn et al. (1998) ww d Pedersen et al. (1994) Gills 0.7 ± 0.25 nmol g 1 ww d Pedersen et al. (1997) a Silver saturation. b 109 Cd/haemoglobin assay. c DPP. d Purification in acetone, SH spectrophotometer and calculation after Cd + Cu + Zn concentrations. (taking into account the fact that the concentrations are expressed in relation to wet weight in the former and dry weight in the latter). Low values of MT were determined in the hepatopancreas of the crab Carcinus maenas (Pedersen et al., 1997) but heterogeneous values were obtained although the same technique was used (purification of MT with acetone before spectrometric analysis). Taking into account the same set of data (Pedersen et al., 1997), MT concentrations were considerably higher in the hepatopancreas of C. maenas than in the gills. In Nephrops norvegicus, MT concentrations in the hepatopancreas were also higher than in the gills, but the difference was not so marked (Canli et al., 1997). In the case of bivalves (Table 2), we have only selected results acquired using the most commonly used quantification technique, namely DPP, to compare strictly concentrations in different species and organs. If one takes into account the fact that the concentrations are expressed either as wet weight or dry weight, the concentrations in the different species are relatively homogeneous, except for a very low value in the kidney of Tridacna crocea. However, this has been the only species in which this organ was isolated (due to the large size of these bivalves) and the comparison is of limited significance since inter-organ differences are generally found. In all the studied species, MT concentrations were higher in the digestive gland of bivalves than in the gills. Inter-organ differences have been systematically investigated in mussels and oysters. Based on the assumption that MT is induced by metals, it would be expected that tissues with the highest accumulated metal concentrations should have the highest MT concentrations. The ability of oysters to store metals to a considerably higher degree than can mussels is welldocumented in biomonitoring programmes (Beliaeff et al., 1998). The results of a translocation study carried out between a clean and a metal-rich site with mussels M. edulis and oysters C. gigas corroborate these findings (Figs. 2 and 3). However, despite differences reaching one to several orders of magnitude depending on various metals, MT concentrations were not so very different, reaching a few hundreds of mg kg 1 (ww) in the gills of both species and a few thousands in their digestive glands (Figs. 2 and 3). These remarkable inter-organ differences in MT concentrations cannot be explained by inter-organ comparisons in accumulated metal concentrations. The accumulated concentrations of each of the three studied metals were of the same order of magnitude in both organs in mussels (Fig. 2), and the same situation is depicted in Fig. 3 for oysters.

9 168 J.-C. Amiard et al. / Aquatic Toxicology 76 (2006) Table 2 Basal metallothionein concentrations determined by Differential Pulse Polarography in bivalves from sites considered to be uncontaminated Species Tissue Concentration (mg g 1 dw or ww) Reference Cerastoderma edule Whole animal 4.55 dw Bebianno and Langston (1989) Chlamys opercularis Digestive gland 3.6 ww Bustamante (1998) Chlamys varia Digestive gland 3.5 ww Bustamante (1998) Corbicula fluminea Whole animal ww Rainglet (1998) Crassostrea gigas Whole animal 0.1 ww Amiard-Triquet et al. (1998a) Digestive gland 5 7 dw Imber et al. (1987) ww Geffard et al. (2001) Gills ww Geffard et al. (2002a) Donax vittatus Whole animal 6.37 dw Bebianno and Langston (1989) Macoma balthica Whole animal 0.98 ± 0.11 dw Bordin et al. (1994) 3.40 ± 1.58 dw Bordin et al. (1997) 2.3 dw Amiard-Triquet et al. (1998b) 3 5 dw Hummel et al. (2001) Mytilus edulis Whole animal 2.43 dw Bebianno and Langston (1989) 2.75 ± 0.99 dw Bebianno and Langston (1991) ww Amiard-Triquet et al. (1998a) Digestive gland ww Amiard et al. (1998) 8.04 dw Bebianno and Langston (1989) 8dw Bebianno and Langston (1991) 8.8 dw Amiard-Triquet et al. (1998b) Gills 0.3 ww Amiard et al. (1998) 2.2 dw Bebianno and Langston (1991) dw Amiard-Triquet et al. (1998b) Mytilus galloprovincialis Whole animal dw Bebianno and Machado (1997) ww Raspor et al. (1999a) 2.81 ± 0.66 dw Bebianno and Langston (1992) 5.73 ± 0.99 ww Mourgaud et al. (2002) Digestive gland ww Raspor et al. (1999a) 2.1 ww Pavicic et al. (1993) Gills ww Raspor et al. (1999a) Larvae 48 h 1.8 dw Geffard et al. (2002b) Larvae 96 h 0.5 dw Geffard et al. (2002b) Ostrea edulis Gills 1.25 dw Langston et al. (1998) Remaining in soft tissue 1.48 dw Langston et al. (1998) Ruditapes decussatus Whole animal dw Bebianno and Langston (1989) Whole animal 2.05 ± 0.41 dw Bebianno et al. (1993) Digestive gland 3.40 ww (2 6) Hamza-Chaffai et al. (1999) 4.7 dw Bebianno et al. (1993) Gills 1.97 dw Bebianno et al. (1993) Tridacna crocea Kidney ± ww Duquesne and Coll (1995) 4. MT induction in response to metal exposure: evidence and inconsistencies In polluted environments, animals are generally exposed to a mixture of different metals and when MT induction is shown, it is generally impossible to attribute this additional synthesis to one element or another. In the laboratory, several authors have exposed aquatic organisms to mixtures of metals with the same consequence for any interpretation of the role of each element. In many works, metallothionein concentrations were not quantified and the authors focussed their attention on the cytoplasmic distribution of metals. Many authors consider that increased accumulation of

10 J.-C. Amiard et al. / Aquatic Toxicology 76 (2006) Fig. 2. Metal and MT concentrations (mg kg 1 ww) in gills and digestive glands of mussels Mytilus edulis. BB: specimens originating from a clean site (Bay of Bourgneuf, France); BG: specimens from the clean site translocated to the metal-rich Gironde estuary, France for 2 months in spring and autumn. Concentrations with the same superscript in capitals do not differ according to the site of collection. Concentrations with the same superscript in lower case do not differ according to the date of collection. (After Mousseau, unpublished and Geffard, 2001). one or several metals in the metallothionein-like ligand pool of an aquatic organism exposed to increased concentrations in their medium reflects induction of the protein (e.g. Mason and Jenkins, 1991; Klerks and Bartholomew, 1991). Pedersen and Lundebye (1996) have compared such an indirect method in which the metal content (Cu and Zn) of partially purified MT was measured and a direct method in which MT was quantified by spectrophotometric measurements of SH. The two techniques yielded similar results. Thus in the tables discussed below, we have accepted as a proof of induction the fact that the concentration of MT-bound metals increased in exposed organisms. However, it must be kept in mind that at least in some cases, metals added in the medium could have replaced constitutive metals in existing MT, according to their different affinities for the SH groups. In annelids (Table 3), the quoted authors examined MT induction in whole individuals whereas in molluscs (Table 4) and crustaceans (Table 5), many results were given for individual organs (digestive gland, gills). Contrary to the general hypothesis that metals induce MT synthesis, a number of papers also showed the absence of any induction in invertebrates exposed to metals, or even a decrease of MT concentrations upon metal exposure. Taking into account this and other inconsistencies, we will try to answer the following questions:

11 170 J.-C. Amiard et al. / Aquatic Toxicology 76 (2006) Fig. 3. Metal and MT concentrations (mg kg 1 ww) in gills and digestive glands of oysters Crassostrea gigas. BB: specimens originating from a clean site (Bay of Bourgneuf, France); BG: specimens from the clean site translocated to the metal-rich Gironde estuary, France for 2 months in spring and autumn; GG: specimens originating from the metal-rich Gironde estuary. Concentrations with the same superscript in capitals do not differ according to the sampling group. Concentrations with the same superscript in lower case do not differ according to the date of collection. (After Mousseau, unpublished and Geffard, 2001). Is MT induction dependent on the metal tested? Is MT induction dependent on biological characteristics (species, population, organ)? Is MT induction dependent on the period and dose of exposure? 4.1. Is MT induction dependent on the metal tested? Since MT was first described by Margoshes and Vallee (1957) in their attempt to isolate a cadmiumbinding protein, many authors have favoured the study of Cd as inducer for MT synthesis in different species. Particularly in the case of laboratory experiments, the results for responses to Cd are over-represented (Tables 3 5). However, Cu and Zn also appear to be efficient inducers, and other studies devoted to Ag and Hg (in molluscs and crustaceans, Tables 4 and 5) and Ni and Tc (in crustaceans, Table 5) have shown MT induction. Occasional studies have also demonstrated the binding of Mn to MTLP in the kidneys of bivalves Mercenaria mercenaria and Placopecten magellanicus exposed to known MT inducers (Cd alone, or Mn in a mixture with Cd and Zn) (Carmichael et al., 1980; Fowler and Gould, 1988). In the field, Lafontaine et al. (2000) reported MT induction in zebra mussels D. polymorpha in the presence of excess Se but, since the site of collection was also contaminated with Cu, it is difficult to decide the individual role of each element. In mussels (M. edulis) impacted following the Erika oil spill in December 1999, vanadium, which was present at high concentration in the oil, was accumulated and

12 J.-C. Amiard et al. / Aquatic Toxicology 76 (2006) Table 3 Annelid worm species in which MT or MTLP has been shown to be induced by metal exposure in the field (Field) or in the laboratory (Lab) Zoological group Species Condition Inducer Reference Cd Cu Zn Others Oligochaetes Limnodrilus hoffmeisteri Field + Klerks and Levinton (1989) Lab + Klerks and Bartholomew (1991) Field + Wallace et al. (1998) Lab + Wallace et al. (1998) Limnodrilus udekemianus Lab + Deeds and Klerks (1999) Monopylephorus cuticulatus Lab + Thompson et al. (1982) Polychaetes Alvinella pompejana Field + (1) Cosson-Mannevy et al. (1986) Chaetozone setosa Field + + Eriksen et al. (1990) Eudystylia vancouveri Lab + Young and Roesijadi (1983) Eurythoe complanata Lab (+) + Marcano et al. (1996) Glycera dibranchiata Lab + Rice and Chien (1979) Goniada maculata Field + + Eriksen et al. (1990) Field + Eriksen et al. (1989) Laeonereis acuta Lab + Geracitano et al. (2004) Lumbrineris fragilis Field + +? (+)? Eriksen et al. (1988) Melinna cristata Field No No Eriksen et al. (1989) Neanthes arenaceodentata Lab + Jenkins and Sanders (1986) Lab + Jenkins and Mason (1988) Lab + Mason and Jenkins (1991) Nereis (Hediste) diversicolor Field No No No Berthet et al. (2003) Orbinia norvegica Field No No Eriksen et al. (1989) Pectinaria belgica Field No No Eriksen et al. (1989) MT concentrations were correlated positively and significantly with those of vanadium (Amiard et al., 2004). A few studies do allow a comparison of the relative importance of different metals as MT inducers. In the crustacean S. serrata, Olafson et al. (1979a), using gel chromatography (Sephadex G75), have shown that MT was induced by Cd and Zn whereas no induction was observed in specimens contaminated with Hg and Cu. Entering into the detail of the experimental procedure, a noticeable point is that experimental doses, delivered by injection, were higher for the essential elements Cu and Zn (5 mg kg 1 body weight) than for non-essential Cd and Hg ( mg kg 1 ) for successive injections carried out every 3 over 10 days. This procedure is in agreement with the general observation that Cu and Zn are not as toxic as Cd and Hg. A study by Narayanan et al. (1999) has shown that the 96 h LC50s for S. serrata were 32 gznl 1,8 gcdl 1 and only 0.09 ghgl 1 (not determined for Cu). Even if it is not possible to establish a direct link between these results and the doses tested by Olafson et al. (1979b), it must be underlined that Hg is nearly 100 times more toxic than Cd to S. serrata and thus, identical injected doses do not have the same biological significance at all. Barka et al. (2001) have tried to get over this difficulty, testing a range of metal concentrations, the highest of which was equal to 1/10 of the LC50 determined for copepods Tigriopus brevicornis (Barka et al., 1997). In Fig. 4, MTLP concentrations, determined by using differential pulse polarography, are shown in controls and in different groups of copepods exposed to six different metals at the doses mentioned in Table 6 for 1 14 days. All metals have induced MTLP synthesis, but Cu and Zn clearly appeared as the most potent whereas Ni and Ag had much less influence. Induction due to Cd and Hg is highly variable according to the conditions of exposure, a point which will be discussed below (see Section 4.3) Is MT induction dependent on biological characteristics? The easiest way to answer this question is to compare the responses of different species or populations

13 172 J.-C. Amiard et al. / Aquatic Toxicology 76 (2006) Table 4 Mollusc species in which MT or MTLP has been shown to be induced by metal exposure in the field (Field) or in the laboratory (Lab) Zoological group Species Condition Inducer Reference Bivalves Cd Cu Zn Others Adamussium colbecki Gills Lab + Viarengo et al. (1997) Anadara granosa Blood Lab + Chan et al. (2002) Anodonta anatina Kidney Lab + Streit and Winter (1993) Anodonta cygnea Lab No No Tallandini et al. (1986) Anodonta grandis Gills Field + Couillard et al. (1993), Giguère et al. (2003) Digestive gland Field + Couillard et al. (1993) Remaining soft tissue Field + Couillard et al. (1993) (Anodonta) Pyganodon grandis Whole soft tissue Field + Couillard et al. (1995a) Gills Field + Wang et al. (1999) Calyptogena magnifica Kidneys Field + Roesijadi et al. (1985) Chamaelea gallina Digestive gland Field + + Rodriguez-Ortega et al. (2002) Chloromytilus meridionalis Whole soft tissue Lab + Hennig (1986) Corbicula fluminea Gill, mantle and adductor Lab + Doherty et al. (1988) muscle Visceral mass Lab + Doherty et al. (1988) Whole soft tissue Field + + Baudrimont et al. (1999) Crassostrea gigas Embryos, Larvae Field Geffard et al. (2003) Whole soft tissue Lab + + Frazier and George (1983) Whole soft tissue Lab + Fowler et al. (1986) Whole soft tissue Lab + Roesijadi et al. (1989) Whole soft tissue Field Mouneyrac et al. (1998) Digestive gland Field (+) ++ Imber et al. (1987) Gills Field Geffard et al. (2001) Crassostrea glomerata Whole soft tissue Field No No Nordberg et al. (1986) Crassostrea virginica Embryos Lab + + Ringwood and Brouwer (1993, 1995) Whole soft tissue Lab + Ridlington and Fowler (1977) Whole soft tissue Lab + Ridlington and Fowler (1979) Gills Lab + Roesijadi and Klerks (1989) Gills Lab + Unger et al. (1991) Gills Field + + Roesijadi (1992) Gills Lab + Roesijadi (1994a) Gills Field + + No Roesijadi (1994a) Dreissena polymorpha Whole soft tissue Field + Se Lafontaine et al. (2000) Whole soft tissue Lab + No Lecoeur et al. (2004) Macoma balthica Whole soft tissue Lab + Johansson et al. (1986) Whole soft tissue Field + + Ag Johansson et al. (1986) Whole soft tissue Lab + a + a + a Bordin et al. (1994, 1997)

14 J.-C. Amiard et al. / Aquatic Toxicology 76 (2006) Table 4 (Continued ) Zoological group Species Condition Inducer Reference Cd Cu Zn Others Whole soft tissue Field Bordin et al. (1997) Whole soft tissue Lab + Ag, Hg Mouneyrac et al. (2000) Whole soft tissue Field + Ag Bray et al. (1983) Mercenaria mercenaria Kidneys Lab + Robinson et al. (1985) Mizuhopecten yessoensis Gills Lab + Evtushenko et al. (1986) Hepatopancreas Lab + Evtushenko et al. (1986) Mytilus edulis Whole soft tissue Lab + Noël-Lambot (1976) Whole soft tissue Lab + George et al. (1979) Whole soft tissue Field + Frankenne et al. (1980) Whole soft tissue Lab + Nolan and Duke (1983) Whole soft tissue (add Lab + Frazier (1986) muscle and exclude foot) Whole soft tissue Lab + Langston et al. (1989) Whole soft tissue Lab + Bebianno and Langston (1991) Whole soft tissue Lab + Mackay et al. (1993) Whole soft tissue Lab + + Bebianno and Langston (1999) Whole soft tissue Field V Amiard et al. (2004) Mantle Lab + Carpenè et al. (1980) Gills Lab + Carpenè and George (1981) Gills Lab Hg Roesijadi et al. (1982a) Gills Lab Hg Roesijadi et al. (1982b) Gills Lab Hg Roesijadi (1986) Gills Lab Roesijadi and Fellingham (1987) Gills Field Talbot and Magee (1978) Gills Lab + + No Hg Roesijadi et al. (1988) Digestive gland Lab Ag George et al. (1986) Digestive gland Lab + Harrison et al. (1983) Digestive gland Lab + Barsyte et al. (1999) Digestive gland Field Geffard et al. (2001) Viscera Field Talbot and Magee (1978) Kidney Lab + George and Pirie (1979) Mytilus galloprovincialis Whole soft tissue Lab + Viarengo et al. (1984) Whole soft tissue Lab + Bebianno and Langston (1992) Whole soft tissue Field + + (+) Bebianno and Machado (1997) Whole soft tissue Field Ni Mourgaud et al. (2002) Gills Lab + Viarengo et al. (1980) Gills Lab + Viarengo et al. (1981a) Gills Lab + Isani et al. (1997) Gills Lab + Viarengo et al. (1997) Gills Lab + Bebianno and Serafim (1998) Gills Lab + Serra et al. (1999) Mantle Lab + Viarengo et al. (1981a) Digestive gland Lab + Viarengo et al. (1981a) Digestive gland Lab + Viarengo et al. (1985) Digestive gland Field + Viarengo et al. (1997) Digestive gland Lab + + Pb Pavicic et al. (1993) Digestive gland Lab + Langston et al. (1989) Adductor mucle Lab + Carpenè et al. (1983) Adductor mucle Lab + Isani et al. (1997)

15 174 J.-C. Amiard et al. / Aquatic Toxicology 76 (2006) Table 4 (Continued ) Zoological group Species Condition Inducer Reference Gastropods Cd Cu Zn Others Foot Lab + Carpenè et al. (1983) Gonad Lab + Carpenè et al. (1979) Viscera Lab + Isani et al. (1997) Embryo-larval Lab + + Pavicic et al. (1994a,b) Embryo-larval Lab + a + a + a Geffard et al. (2002b) Ostrea edulis Whole soft tissue Lab + + Frazier and George (1983) Whole soft tissue Field + Fayi and George (1984) Gonads, mantle, digestive Lab + + Alonso and Martin-Mateo (1996) tract Ostrea lutaria Whole soft tissue Lab + Sharma (1983) Whole soft tissue Field + No Nordberg et al., 1986 Gonads Lab + Alonso and Martin-Mateo (1996) Mantle, Muscle Lab + Alonso and Martin-Mateo (1996) Gut, Plasma + Alonso and Martin-Mateo (1996) Pecten maximus Digestive gland Field + Stone et al. (1986) Perumytilus purpuratus Gonads, Gills, remaining Field + Riveros et al. (2003) Placopecten magellanicus Kidney Lab + Fowler and Megginson (1986) Protothaca straminea Viscera and Kidney Lab (+) (+) (+) Roesijadi (1980) Rangia cuneata Whole soft tissue Field + Ag Bray et al. (1983) Ruditapes decussatus Whole soft tissue Lab + Bebianno et al. (1993) Digestive gland Lab + Bebianno et al. (1993) Gills Lab + Bebianno et al. (1993) Digestive gland Lab + Hamza-Chaffai et al. (1998) Gills Lab + Roméo and Gnassia-Barelli (1995) Gills Lab + Bebianno and Serafim (1998) (=Ruditapes) Tapes philippinarum Gills Field + Irato et al. (2003) Digestive gland Lab + No Ag Ng and Wang (2004) Whole soft tissue Lab + Ishiguro et al. (1982) Scapharca inoequivalis Lab + Serra et al. (1995) Tridacna crosea Gills, Mantle Lab + Duquesne et al. (1995) Kidneys Field + + Duquesne and Coll (1995) Unio elongates Whole soft tissue Lab No + Tallandini et al. (1986) Batillus cornutus Hepatopancreas Field + Dohi et al. (1986) Buccinum tenuissimum Hepatopancreas Field + Dohi et al. (1986) Bulla digitalis Whole soft tissue Field Hennig (1986)

16 J.-C. Amiard et al. / Aquatic Toxicology 76 (2006) Table 4 (Continued ) Zoological group Species Condition Inducer Reference Chitons Cephalopods Cd Cu Zn Others Crepidula fornicata Whole soft tissue Lab Hg Harrison et al. (1987) Littorina brevicula Whole soft tissue Lab + Park et al. (2002) Littorina littorea Whole soft tissue Lab + Noël-Lambot et al. (1978) Whole soft tissue Lab + Langston and Zhou (1986) Whole soft tissue Field (+) Langston and Zhou (1986) Whole soft tissue Field + Leung and Furness (1999) Digestive gland Lab + Langston et al. (1989) Digestive gland Lab + Bebianno et al. (1992) Gills, kidney Lab + Bebianno and Langston (1995) Remaining tissues Lab + Bebianno et al. (1992) Murex trunculus Whole soft tissue Lab + Bouquegneau et al. (1983) Hepatopancreas + kidney Lab + Dallinger et al. (1989) Nassarius reticulates Different tissues Lab No Hylland et al. (1994) Nucella lapillus Leiblein gland Lab + Leung and Furness (2001a,b) (=Nucella) Purpura lapillus Whole soft tissue Field + Noël-Lambot et al. (1978) Whole soft tissue Lab + Noël-Lambot et al. (1978) Patella aspersa Field No No Bebianno et al. (2003) Patella granularis Whole soft tissue Lab + Hennig (1986) Patella intermedia Whole soft tissue Field + Howard and Nickless (1977) Patella vulgata Whole soft tissue Field + + Howard and Nickless (1977) Whole soft tissue Field + Noël-Lambot et al. (1978) Whole soft tissue Lab + Noël-Lambot et al. (1978) Whole soft tissue Field + Noël-Lambot et al. (1980) Whole soft tissue Lab + Noël-Lambot et al. (1980) Cryptochiton stelleri Whole soft tissue Field + Olafson et al. (1979b) Loligo forbesi Field No No Craig and Overnell (2003) Todarodes pacificus Hepatopancreas Field + Dohi et al. (1986) a Mixture. exposed to metals under identical conditions, to eliminate any risk of interference with other variable factors. Some examples are given below for the three taxa of interest in this review for both field and laboratory exposures. Since MTs are probably involved in detoxification processes, it is relevant to compare populations chronically exposed to metal contaminants in their environment and populations living in sites considered to be uncontaminated. Among annelids, Cd-resistant oligochaetes Limnodrilus hoffmeisteri inhabiting the metal (Cd, Cr, Ni)-contaminated Foundry Cove on the Hudson River (New York, USA) produced metallothionein-like proteins as well as metal-rich

17 176 J.-C. Amiard et al. / Aquatic Toxicology 76 (2006) Table 5 Crustacean species in which MT or MTLP has been shown to be induced by metal exposure in the field (Field) or in the laboratory (Lab) Zoological group Species Condition Cd Cu Zn Others Reference Phyllopoda Cladocera Copepoda Amphipoda Decapoda Artemia sp. Whole organism Lab + Del Ramo et al. (1995) Daphnia magna Whole organism Lab + Bodar et al. (1988) Whole organism (Daphnids) Lab + Bodar et al. (1990) Moina macrocopa Whole organism Lab + Yamanura et al. (1983) Tigriopus brevicornis Whole organism Lab Ni; Hg; Ag Barka et al. (2001) Echinogammarus echinosetosus Whole organism Lab + Martinez et al. (1996) Gammarus locusta Whole organism Lab + Correia et al. (2002a) Gammarus pulex Hepatopancreas Lab + Stuhlbacher and Maltby (1992) Orchestia gammarellus Whole organism Field + + Mouneyrac et al. (2002) Themisto abyssorum Whole organism Field + Ritterhoff and Zauke (1998) Themisto libellula Whole organism Field + Ritterhoff and Zauke (1998) Acetes sibogae Whole organism Lab + Olafson et al. (1979a,b) Astacus astacus Hepatopancreas Lab + Pedersen et al. (1996) Austropotamobius pallipes Hepatopancreas Lab (+) (+) Lyon et al. (1983) Callinectes sapidus Gills Lab + Wiedow et al. (1982) Gills Lab + Brouwer et al. (1984) Gills Field + Engel and Brouwer (1984) Thoracic muscle Lab + Wiedow et al. (1982) Hepatopancreas Lab + Wiedow et al. (1982) Hepatopancreas Field + + Schlenk and Brouwer (1991) Hepatopancreas Lab + Schlenk and Brouwer (1991) Hepatopancreas Lab + Brouwer et al. (1984) Hepatopancreas Field + + Engel and Brouwer (1984) Cancer magister Hepatopancreas Field + Olafson et al. (1979b) Cancer pagurus Hepatopancreas Field + Overnell and Trewhella (1979) Hepatopancreas Lab + + Overnell (1982) Hepatopancreas Lab + Overnell and Trewhella (1979) Carcinus maenas Gills Field No + + Pedersen et al. (1997) Gills Field + Legras et al. (2000) Hepatopancreas Lab + Pedersen et al. (1994) Hepatopancreas Lab Pedersen et al. (1998) Hepatopancreas Field + Pedersen and Lundebye (1996) Hepatopancreas Field No (+) No Pedersen et al. (1997) Hepatopancreas Lab Wong and Rainbow (1986a,b)

18 J.-C. Amiard et al. / Aquatic Toxicology 76 (2006) Table 5 (Continued ) Zoological group Species Condition Cd Cu Zn Others Reference a Mixture Cu + Zn. b Mixture Cd + Cu + Zn. Hepatopancreas Field Legras et al., 2000 Muscle Lab + Jennings et al. (1979) Hepatopancreas Field Rainbow and Scott (1979) Crangon crangon Abdominal muscle Lab + Napierska and Radlowska (1998) Diogenes brevirostris -Soft tissues Lab + + Hennig (1986) Homarus americanus Hepatopancreas Lab + Engel and Brouwer (1986) Homarus gammarus Hepatopancreas Lab Tc Goudard et al. (1991) Jasus lalandii Hepatopancreas Lab + a + a Hennig (1986) Tail meat Lab + a + a Hennig (1986) Leander intermedius Field Ross et al. (2002) Metapenaeopsis crassissima Abdominal muscle (female) Field + Francesconi et al. (1998) Nephrops norvegicus Gills (male/female) Lab + No No + b Canli et al. (1997) Hepatopancreas (male) Lab + + No + b Canli et al. (1997) Hepatopancreas (female) Lab + No No + b Canli et al. (1997) Hepatopancreas Lab + Canli (1995) Pachygrapsus marmoratus Gills Field No Legras et al., 2000 Hepatopancreas ++ Legras et al. (2000) Palaemon elegans Hepatopancreas Field + White and Rainbow, 1986 Hepatopancreas Lab + + White and Rainbow (1986) Palaemon pacificus Soft tissues Lab + a + a Hennig (1986) Palaemonetes pugio Whole organism Lab Hg Kraus et al. (1988) Whole organism Lab + Howard and Hacker (1990) Whole organism Lab + Wallace et al., 2000 Penaeus vannamei Hepatopancreas Lab + Moknes et al. (1995) Potamon potamios Hepatopancreas Lab + Pedersen et al. (1996) Procambarus clarkii Hepatopancreas Lab + Martinez et al. (1993) Hepatopancreas Lab + Del Ramo et al. (1989, 1995) Rhithropanopeus harrissii Crab zoeae Lab + Sanders et al. (1983) Scylla serrata Hepatopancreas Lab + No + Hg: no Olafson et al. (1979a) Hepatopancreas Field + Olafson et al. (1979b) Hepatopancreas Lab + Olafson et al. (1979b)

19 Table 6 Examples of apparent inconsistencies in the induction of metallothionein Species Studied organ Exposure conditions Studied contaminant Exposure doses Length of exposure MT or MTLP induction a Bivalves Corbicula fluminea Soft tissues Laboratory Pb 160, 640 gl 1 15 days No induction Rainglet (1998) Cu 60, 240 gl 1 No induction Zn 250, 1000 gl 1 No induction Cd 25, 100 gl 1 No induction Soft tissues Field (River Lot, Cd, Zn Decreasing gradient 21 days Site 1: no induction Baudrimont et al. (1999) France) From sites days Site 2 (21d): 2.5 Site 3 (150d): 9 + or no r with Cd and Zn Crassostrea gigas Gills Laboratory Water borne Géret et al. (2000) Cd 200 gl 1 4 or 21 days + Cu 40 gl 1 No induction Zn 1000 gl 1 No induction Laboratory Contaminated food Cd 1000 gl 1 4 or 21 days + Cu 50 gl 1 No induction Zn 2000 gl 1 No induction Field Cd, Cu, Zn Gironde estuary vs. + Geffard et al. (2002a,b) Bourgneuf Crassostrea gigas Digestive gland Laboratory Water borne Géret et al. (2000) Cd 200 gl 1 4 or 21 days + Cu 40 gl 1 No induction Zn 1000 gl 1 No induction Laboratory Contaminated food Cd 1000 gl 1 4 or 21 days + Cu 50 gl 1 No induction Zn 2000 gl 1 No induction Field Cd, Cu Gironde vs. Bourgneuf + Geffard et al., 2001 Zn No induction Macoma balthica Soft tissues Laboratory Cd, Cu, Zn Mixture: Cd (100 gl 1 ) 3 days 9.2 Bordin et al. (1997) and Cu (100 gl 1 ) and Zn (600 gl 1 ) Soft tissues Lab (Somme, Fr.) Cu Cu (30 and 40 gl 1 ) 13 days No induction Ballan-Dufrançais et al. (2001) Lab (Loire, Fr.) Cu (30 and 40 gl 1 ) 13 days + Mytilus edulis Gills Laboratory Cd 50 gl 1 28 days Cd (514) Roesijadi et al. (1988) Cu 5 gl 1 Cu (7.4) Hg 5 gl 1 Hg (3560) Zn 10, 50, 250 gl 1 Zn (no induction) Reference Mytilus edulis Digestive gland Laboratory Water borne Geffard et al. (2000) Cd 200 gl 1 4 or 21 days No induction Cu 40 gl 1 No induction 178 J.-C. Amiard et al. / Aquatic Toxicology 76 (2006)

20 Table 6 (Continued ) Species Studied organ Exposure conditions Studied contaminant Exposure doses Length of exposure MT or MTLP induction a Zn 1000 gl 1 No induction Laboratory Contaminated food Cd 1000 gl 1 4 or 21 days No induction Cu 50 gl 1 No induction Zn 2000 gl 1 No induction Field Cd, Cu, Zn Gironde vs. Bourgneuf + Mytilus edulis Gills Laboratory Water borne Geffard et al. (2000) Cd 200 gl 1 4 or 21 days + Cd Cu 40 gl 1 No induction Zn Zn 1000 gl 1 No induction Laboratory Contamination food Cd 1000 gl 1 4 or 21 days No induction Cu 50 gl 1 No induction Zn 2000 gl 1 No induction Field Cd, Cu, Zn Gironde vs. Bourgneuf No induction Ruditapes decussates Gills Laboratory Cd 400 gl 1 30 days + (1.8) Bebianno et al. (1993) Digestive gland + (1.2) Remainder + (0.3) Digestive gland Field 30 days + Cd, Cu, Zn Hamza-Chaffai et al. (1999, 2000) Crustaceans Carcinus maenas Midgut gland Laboratory Cu mg l 1 36d + 30d recovery No induction Lundebye and Depledge (1998) Midgut gland Laboratory Cu gl 1 7 days Induction at 68.1 gl 1 Brown et al. (2004) Midgut gland Field (Cu) + or no induction Pedersen et al. (1997) Gills (Cu, Zn) + Midgut gland Field (Cu) Decreasing gradient from Site 1: slight induction, Pedersen and Lundebye (1996) site 1 to site 5 no sign. Midgut gland Field Decreasing gradient from No induction Wedderburn et al. (1998) site 1 to site 5 Gammarus locusta Whole body Laboratory (water) Cu 3, 5 and 10 gl 1 4 days + Correia et al. (2002a) Whole body Laboratory (sediment) Cu 1, 3 and 6 mg kg 1 28 days No induction Correia et al. (2002a) Orchestia gammarellus Whole body Field Cu, Zn + (2) Mouneyrac et al. (2002) Whole body Laboratory Cu, Zn Cu and/or Zn (1-100 M) 5 25 days No induction Cd M 17 days No induction Tigriopus brevicornis Whole body Lab Cu Cu (1.5, 9, 15 gl 1 ) 1, 2, 4, 7, 14 days + Barka et al. (2001) Zn Zn (2, 18, 35 gl 1 ) + Ni Ni (1, 5, 20 gl 1 ) (+) Ag Ag (1.5, 15, 30 gl 1 ) (+) Cd Cd (0.47, 2.35, 4.7 gl 1 ) Day 1:+; days 2 14: No induction Hg Hg (0.75, 3, 7.5 gl 1 ) No induction at highest dose a Values between brackets = ratios between MT levels in exposed specimens and controls when available. Reference J.-C. Amiard et al. / Aquatic Toxicology 76 (2006)

21 180 J.-C. Amiard et al. / Aquatic Toxicology 76 (2006) Fig. 4. Comparative MT concentrations in control and metalexposed Tigriopus brevicornis ( gg 1 ww) after short and longterm contamination to different nominal metal concentration ranges (details of conditions of exposure shown in Table 6) (after Barka et al., 2001). granules for Cd storage and detoxification (lysosomal degradation products of Cd-MT?), whereas nonresistant worms only produced MT (Wallace et al., 1998). In Baltic clams M. balthica exposed to Cu (30 or 40 ng ml 1 ), metal and metallothionein-like protein concentrations were correlated in bivalves from an industrialized site but not in those originating from a comparatively clean site (Table 6) (Ballan-Dufrançais et al., 2001). Several comparisons between species exist. In oysters, C. gigas accumulates more Cd than O. edulis and the proteins binding accumulated metals are different (Frazier and George, 1983). A large translocation experiment has been carried out by Geffard (2001). For 7 months (March to October 1999), oysters and mussels from a clean site (Bay of Bourgneuf, France) were translocated to the metal-rich Gironde estuary. Specimens were collected monthly at the site of origin and from among the translocated individuals. Cd, Cu, Zn and MT concentrations were determined in different tissues. In oysters C. gigas, the study demonstrated that MT concentrations in the digestive gland were only occasionally correlated with accumulated metal concentrations (Geffard et al., 2001) whereas in the gills, such correlations were observed over the major part of the year (Geffard et al., 2002a). However, even in the gills, seasonal variations of MT concentrations were high enough to conceal intersite differences when annual means were calculated for all the individuals analysed monthly. The same situation of high Fig. 5. Grand means of MT and metal concentrations (mg kg 1 ww) in gills (A) and digestive glands (B) of mussels Mytilus edulis based on all the individual samples collected from March to October at two sites: control: bb (n = 54) and transplanted to from the control site to the metal-contaminated site: bg (n = 50). * Difference significant (after Geffard et al., 2005). variability concealing intersite differences was seen in the gills of the mussels (M. edulis) (Fig. 5A). In contrast, annual means of MT concentrations differed significantly between the digestive glands of mussels originating from the clean site or translocated to the metal-rich site (Fig. 5B). The physiology of trace metal accumulation differs greatly between mussels and oysters (Rainbow, 1992), and thus it is not surprising that the relative importance of natural versus contamination factors in affecting MT concentrations is not the same in each species. Three species of bivalves (M. edulis, M. galloprovincialis, R. decussatus) were exposed to the same Cd concentration for the same period of time (Bebianno and Langston, 1991, 1992; Bebianno et al., 1993). A linear relationship was observed between

22 J.-C. Amiard et al. / Aquatic Toxicology 76 (2006) Cd concentrations (whole soft tissues) and metallothionein concentrations, this increase being higher in both species of mussels than in R. decussatus. Similarly Bebianno and Serafim (1998) showed that the mussel M. galloprovincialis responds more strongly to contamination by Cd than the clam R. decussatus (MT induction in the gills was two-fold higher). To turn to crustaceans, the crabs C. maenas and Pachygrapsus marmoratus were abundant in the metalrich Gironde estuary (France). Both species were sampled at three locations along a salinity gradient (Legras et al., 2000). MT and metal (Cd, Cu, Zn) concentrations were analysed in hepatopancreas and gills. Regression analyses were performed to determine which parameters were the most important in controlling MT concentrations among natural (salinity, sex, season, concentrations of total proteins) and contamination factors. In agreement with the results of Pedersen et al. (1997), accumulated metal concentrations in C. maenas were among the major factors statistically related to MT concentrations. On the other hand, MT concentrations were mainly influenced by natural factors in P. marmoratus, MT concentrations being linked more closely to changes in general protein metabolism associated with different salinities than to accumulated concentrations of metals. It is possible that P. marmoratus might have more marked physiological adaptations to low salinity than C. maenas, with increased effects on protein metabolism. It is not unreasonable that an intertidal crab with a more southerly distribution and warmer water temperature (like P. marmoratus) might experience a higher range of physical variables including salinity and temperature than a temperate crab like C. maenas and be adapted accordingly (Legras et al., 2000). Following exposure to Cu, the crab C. maenas shows MT induction, which is absent, however, in either the limpet Patella vulgata or the mussel M. edulis (Brown et al., 2004) Is MT induction dependent on the period and dose of exposure? In several species, MT or MTLP induction has been shown to be rapid in specimens exposed to different metals. For instance, MT induction was observed after 4 days in the gills of oysters C. virginica exposed to 50 gcdl 1 (Roesijadi and Klerks, 1989), and after 1 3 days of exposure in different organs of mussels M. galloprovincialis exposed to gl 1 Cu (Viarengo et al., 1980, 1981a,b, 1985). Bordin et al. (1994) observed increased concentrations of MTLP in the Baltic clam M. balthica after 2 days of exposure to a mixture of Cd, Cu and Zn (100, 100 and 600 gl 1 respectively). After 1 day of exposure to a range of doses of Ag, Cd, Cu, Hg, Ni or Zn, significant increases in MTLP concentrations were present in the copepod crustacean T. brevicornis (Barka et al., 2001). In response to Cd exposure (100 gl 1 to 10 mg l 1 ) induction has also been observed after 2 24 h in freshwater and marine crustaceans belonging to different taxa (the amphipod E. echinosetosus, Martinez et al., 1996; the cladoceran Daphnia magna, Bodar et al., 1988; the phyllopodan Artemia sp., and the crayfish Procambarus clarkii, Martinez et al., 1993; Del Ramo et al., 1995). On the other hand, in the bivalve Mizuhopecten yessoensis exposed to Cd (0.5 mg l 1 ) Cd-HMW proteins were enhanced throughout the experiment (days 1 60) whereas Cd-LMW proteins similar to MT increased in concentration only after 60 days (Evtushenko et al., 1986). In oysters C. gigas translocated from a reference site to a Cd-contaminated estuary, the percentage of Cd linked to MTLP increased from 5% at day 0 to 21% after 1 month and 67% after 3 months, the latter being equal to the percentage of MTLP-bound Cd in resident oysters from the contaminated site (Mouneyrac et al., 1999). In the clam R. decussatus exposed to Cd (400 gl 1 ) for 30 days, a two-fold increase in MT concentrations was detected in the gills whereas only a slight increase was observed in the digestive gland and no significant increase was shown in the remainder of the soft tissues (Bebianno et al., 1993; Table 6). MT induction in the digestive gland of this clam has been confirmed by translocation studies in the Mediterranean Sea (Hamza-Chaffai et al., 1999, 2000; Table 6). In many cases, strong differences have been shown in comparisons of MT induction in specimens chronically exposed in the field and those submitted to more or less acute exposures in the laboratory with the argument that high doses are justified to produce clearer results (Table 6). Experimental (Géret, 2000) and field (Geffard, 2001) results have been compared in mussels M. edulis and oysters C. gigas analysed using the same laboratory procedures. Mussels from a clean site (Bay of

23 182 J.-C. Amiard et al. / Aquatic Toxicology 76 (2006) Fig. 6. Ratios between controls and contaminated animals (experimentally or in situ) of MT and metal concentrations (mg kg 1 ww) in gills and digestive glands of mussels Mytilus edulis in two contamination experiments (via water and via food) and in the field (after Geffard and Géret, unpublished). Bourgneuf, France) were exposed separately to Cd, Cu or Zn added to water (200, 40 and 1000 gl 1 respectively) or food (1000, 50 and 2000 gl 1 respectively in the culture medium of microalgae), while other specimens were translocated for 6 months to a metalrich estuary (Gironde, France). Results are shown in Fig. 6. In the laboratory, Cd was accumulated strongly from food and from water in both gills and digestive glands of the mussels. A strong accumulation of Cu was also observed but only in the gills after direct exposure in solution, a fact that may be linked to differential Cu bioavailability from food and water. In the field, metal accumulation was comparatively low but always significant for all three metals, the highest ratio being reached for Cd in the digestive gland. Surprisingly, despite the strong experimental enhancement of bioaccumulated Cu and especially Cd, MT induction was limited, the most striking response being observed in gills of Cd-exposed mussels (but the ratio of MT concentrations between contaminated specimens and controls was about 4, compared to 342 for Cd concentrations). In contrast, MT concentrations were not enhanced in the gills of mussels translocated to the metal-rich estuary, whereas a highly significant induction was observed in the digestive gland (Table 6). Similarly, in the oysters, despite much more important enhancement of metal bioaccumulation under laboratory conditions, MT concentrations showed no greater changes than those observed in the oysters translocated into the metal-rich Gironde estuary (Amiard-Triquet et al., 1999; Géret et al., 2000). In M. balthica, the induction of MT was clear after 3 days of exposure to a mixture of contaminants (Bordin et al., 1997). However, MT induction in the same species following Cu exposure varied to the point of absence according to the origin of the population tested (Somme or Loire) (Ballan-Dufrançais et al., 2001). In Corbicula fluminea no induction was obtained in the laboratory by Rainglet (1998), while Baudrimont et al. (1999) observed clear induction in situ. Mouneyrac et al. (2002) showed similar inconsistencies in MT induction between field and laboratory exposures for the amphipod crustacean O. gammarellus. In another amphipod Gammarus locusta, dissolved metal exposure induced MT, while metal contamination via sediment did not (Correia et al., 2002a). In the crab C. maenas (Table 6), MT induction was clearly shown in the gills in relation to the presence of Cu and Zn in the field (Pedersen et al., 1997). The situation was less clear in the hepatopancreas (Table 6), perhaps as a result of the various methods used to quantify the MT (Pedersen et al., 1997). No induction of MT in the hepatopancreas was observed in the laboratory by Cu exposure at mg l 1 (Lundebye and

24 J.-C. Amiard et al. / Aquatic Toxicology 76 (2006) Depledge, 1998), whereas induction was observed at 68.1 gl 1 (Brown et al., 2004). The mode of contamination thus has great importance in the induction of MT. The time period of contamination also affects the induction of MT. In the copepod T. brevicornis, Cd induces MT on the first day of exposure, but not on the following days (Table 6) (Barka et al., 2001). The concentration dose of the contamination is also important for this species. Thus in T. brevicornis mercury does not induce MT at the highest concentration tested in the laboratory (7.5 gl 1 ), although induction was observed at lower dissolved exposures, perhaps as a general toxic effect of the raised mercury availability affecting protein synthesis (Barka et al., 2001). Changes in MTLP concentrations have been examined in the amphipod O. gammarellus exposed for between 5 and 25 days to Cu or Zn or to a mixture of these metals (1 100 M) (Mouneyrac et al., 2002). The MTLP concentrations hardly varied according to the time of exposure, but in specimens exposed to the highest dose of Cu a significant decrease in MTLP concentration was observed after 17 and 25 days of exposure. The MTLP concentrations were also generally constant with increasing exposure doses. However, at 100 M of the mixture at days 5, 10 and 17; at 10 M Cu + Zn at day 25 as well as at 100 M Cu alone at days 17 and 25, lower MTLP concentrations were observed (Mouneyrac et al., 2002), perhaps again in reflection of a general toxic effect. In another amphipod E. echinosetosus exposed to Cd for 24 h, MT was significantly induced in a dose-dependent way over the range of exposure concentrations from 100 to 1000 gl 1.For the highest dose tested (2000 gl 1 ), MT concentration in the amphipod was higher than in controls specimens but not so high as in specimens exposed to 1000 gl 1 only (Martinez et al., 1996). In copepods T. brevicornis (Barka et al., 2001) exposed to Ag, Cu, Ni or Zn, after a noticeable induction during the first 24 h of exposure, a MTLP induction plateau was typically reached (example given for Ag in Fig. 7A). No Cd-MTLP induction threshold was reported at day 1 but a longer exposure decreased MTLP concentrations throughout (Fig. 7B). For the highest mercury exposure (7.5 gl 1 ), at any considered time exposure, MTLP levels in copepods dropped to values close to those of controls (Fig. 7C) (Barka et al., 2001) Tentative explanations Relative importance of MT in metal detoxification In invertebrates, two major mechanisms of detoxification involving intracellular ligands have been welldocumented: metal-binding to cytosolic compounds including metallothioneins (or metallothionein-like proteins) and biomineralisation (Mason and Jenkins, 1995; Marigomez et al., 2002). Depending on the species, the relative importance of these two detoxification mechanisms varies considerably. Any distinction between the detoxification mechanisms, however, is not complete, given the possibility for the lysosomal products of MT (particularly Cu-MT) breakdown remaining as insoluble residual bodies. Extreme examples have been observed for particular metals in particular species, such as the insoluble storage of more than 90% of Cu body burden in worms (H. diversicolor) exposed to high availabilities of this element in the field (Berthet et al., 2003) or of Ag in oysters (C. gigas) exposed in the laboratory (Martoja et al., 1988). Only a fraction of cytosolic metals is bound to metallothionein in these two cases. In the worm H. diversicolor, the percentage of cytosolic metals bound to MTLPs varies for each element and according to sites of origin of the specimens (9 47% for Cd, 25 40% for Cu, 14 21% for Zn) (Berthet et al., 2003). Limited or negligible involvement of MT in metal binding has been reported for several bivalves such as Scrobicularia plana, Anondonta cygnea and Unio elongatulus (Langston et al., 1998 and literature cited therein). In scallops Pecten maximus, MTLP sequesters only 15% of cytosolic Cd. In the Baltic clam M. balthica, a detectable portion of cytosolic Ag, Cu and Zn is associated with MTLP, most significantly at impacted sites in San Francisco Bay (Johansson et al., 1986). In the oyster O. edulis, MT has only a minor role in sequestering Cd, Cu and Zn at highly contaminated sites (Restronguet Creek, UK) since most of these cytosolic metals are associated with very low molecular mass ligands (Langston et al., 1998). Under polluted conditions in the field (Patuxent River, USA), MT binds up to 20% of gill Cd in C. virginica (Roesijadi, 1994a). Amounts of Cu (1%) and Zn (0.3%) bound to MT are negligible in both oyster species. However, MT in C. virginica embryos binds more Cu and Zn than any other cytosolic pool (Ringwood and Brouwer, 1995). In larvae of M. gal-

25 184 J.-C. Amiard et al. / Aquatic Toxicology 76 (2006) Fig. 7. Metallothionein concentrations ( gg 1 ww) in Tigriopus brevicornis exposed to increasing nominal dissolved concentrations of nonessential metals for between 1 and 14 days (after Barka et al., 2001). loprovincialis after 48 and 96 h exposure respectively, 50 and 75% of Cd, 50 and 50% of Cu, and 60 and 80% of Zn are present in insoluble form (Geffard et al., 2002b). The competition between sequestration mechanisms (non-mt cytosolic ligands, biomineralised granules, tertiary lysosomes,...) can interfere with the response of MT to metal exposure (George and Olsson, 1994). The complexities of such inter-relationships are particularly high in those species (malacostracan crustaceans, gastropod molluscs) which contain the copper-bearing respiratory pigment haemocyanin. Furthermore, in crustaceans, the moult cycle introduces very pronounced cellular and tissue effects on haemocyanin and copper metabolism (Engel and Brouwer, 1993).