Michaela Klotz

Idris Amirouche

Christian Guizard

Céline Viazzi

Sylvain Deville
email: sylvain.deville@saint-gobain.com

Ice templating -an alternative technology to produce micro-monoliths

Hierarchical ceramics with porosities defined at multiple length scales are of particular interest as catalyst support materials. In fixed-bed reactors, catalytically active particles or porous pellets are packed in the reactor. A combination of high specific surface area, accessibility to the active sites and mechanical strength is therefore required and yet difficult to achieve. This problem is tackled using the ice templating process. Two approaches are investigated to obtain micromonoliths combining macroand mesoporosities. Ice templating of boehmite suspensions provides materials with macropores ranging from 10 to 26 µm and mesopores from 3.6 to 6.2 nm throughout the entire volume of the sample. Total porosity of the samples ranges from 70 to 98%, and specific surface areas from 170 to 300 m 2 g -1 can be reached. The mesopore characteristics can be adapted by modifying the particle size and the freezing rate. However, the mechanical properties are not sufficient for application as catalyst carriers. An alternative method based on wash-coating a mechanically resistant ice templated monolith is described. These monoliths have 64% porosity and straight, continuous, 4.5 µm diameter channels. Uniform 100 to 200 nm thick coatings, with a 360 m g -1 specific surface are deposited without altering the initial mechanical strength of the monolith: 134 ± 30 MPa. We expect such architectures, combining high specific surface area, low pressure drop, and high mechanical strength, to be of special interest as catalyst carriers.

Ceramics with porosities defined and controlled at multiple length scales are currently investigated for various energy-related applications. In fixed bed reactors, for example, catalytically active particles or porous pellets are packed in the reactor volume and only the easily accessible part of the catalytic sites are chemically active. Simply decreasing the particle size in order to increase the available catalytically active surface necessarily decreases the pore size, which corresponds to the inter-particles space in this case. This results in an excessive increase of the pressure drop across the catalytic bed making it unsuitable for some applications. Currently, the solution to this problem has been using packed beds of porous pellets in which the catalytic phase is dispersed on the internal surface of the pellets. However most conventional porous pellets used in packed bed reactors have a monomodal porous structure. With such microstructures, it is thus difficult to combine at the same time a high dispersion of the catalyst and a low diffusion path for the reactant. It is therefore of interest to use a hierarchical pore system comprising macro-, meso-, and/or micropores. Meso-and micropores will provide high specific surface area, required for a good dispersion of the catalytic sites. Macropores are necessary for rapid fluid distribution and pressure drop limitation. Their size, shape, and interconnectivity must be controlled in order to facilitate both mass transport through the macrochannels and local diffusion to the catalytic sites. Monoliths with monodispersed macroporous channels on the order of a few to tens micrometers, oriented along the flux of reactants and products, are thus of particular interest if they can be connected to high surface area mesopores. This configuration should allow rapid access to the catalytic sites, advantageous diffusion path, and limited pressure drop.

Ice templating is a simple way of making such macroporous channels in a pellet. In this process, first reported for ceramics by Fukasawa et al., 1 unidirectional freezing of an aqueous suspension or gel triggers ice crystal growth along a thermal gradient. Rejection of the particles by the growing crystals concentrates the particles between the ice crystals. Subsequent sublimation of the ice leads to a porous body where the porosity is the replica of the ice crystals. The morphologies and characteristics of the ice crystals depend on the process and formulation parameters. As ice templating is mostly based on physical interactions and mechanisms, materials can be templated independently of their nature. To date, metals, polymers, and ceramics starting either from slurries, suspensions or gels have been ice-templated. Several reviews of this process give a detailed description of the process and its potential applications. 2,3 Hierarchically porous silica obtained by ice templating has been investigated by Nishihara and Mukai. [4][5][6] Starting either from hydrocolloids or hydrogels, they obtained silica monoliths with 10-15 µm sized macropores possessing micro-and mesoporosity resulting from the packing of the colloidal silica particles between the ice crystals. Icetemplated systems of mixed nanoparticles with specific properties were also investigated, adding zeolites, graphite, or photo-catalyticly active TiO2 to the colloidal silica, which in this case acted as a binder.7 Mixed alumina-silica 8 and titania-silica monoliths 9 were also reported. Ohshima and coworkers 10 introduced monodispersed poly[styrene-(co-2-hydroxyethyl methacrylate)] latex particles into a silica sol and obtained a beautiful reverse opal structure in the walls between the macroporosity. However, there is only limited work on hierarchical ice-templated alumina or boehmites. Winnubst and coworkers 11 reported meso-macroporous alumina monoliths starting with a mixture of sub-micrometer sized α-alumina and nanometer sized γalumina. The nanosized γ-alumina is used as a binder; the samples consequently do not require freeze-drying and can be dried under room pressure and temperature conditions.

In the present work, we investigate the ice templating of nanosized boehmite particles aqueous suspensions, chosen for their wide use as catalyst carriers. [START_REF] Misra | Industrial Alumina Chemicals[END_REF] More specifically, the impact of the boehmite particle size and the freezing speed on the porous structure is investigated. An alternative method is described, to produce monoliths with hierarchic porous structure resulting from the deposition of a mesoporous boehmite washcoat in sintered ice-templated monoliths.

Experimental

Boehmite Micromonoliths with Mesoporous Walls

Boehmite micromonoliths with mesoporous walls are manufactured by directional freezing of boehmite suspensions and gentle thermal treatment to avoid densification by sintering. Three types of boehmite powders were tested: Disperal, Catapal B from Sasol, and boehmite synthesized following the procedure described by Yoldas. 13 Suspensions of Disperal and Catapal B are prepared by dispersing 35 wt% of the commercial powder in deionized water (DI). The suspension pH is adjusted to 4 using nitric acid (69 wt%, Sigma-Aldrich). Dispersion is promoted by ultrasound application.

Colloidal boehmite is synthesized following the Yoldas method. 3.7 × 10 -2 mol of aluminum tri-sec butoxyde (Sigma-Aldrich, 97%) is first refluxed with 34 ml of DI water. After 15 min refluxing at 90 °C, 2.6 × 10 -3 mol of nitric acid (Aldrich, 69%) is added for peptization. A transparent sol is obtained and refluxed for 45 min.

The suspension is poured into PTFE moulds and cooled from the bottom using a copper rod cooled by liquid nitrogen. The cooling rates are adjusted through a thermocouple and a ring heater placed around the copper rod. After complete freezing, ice crystals are removed by sublimation using a commercial freeze dryer (FREE Zone 2.5Plus, Labconco, Kansas City, Missouri, USA). The samples are maintained at low temperature (-82 °C) and reduced pressure (0.42 mbar) for at least 48 h in order to ensure complete removal of the ice crystals. The boehmite samples were then thermally treated at 550 °C for 2 h. Throughout the paper, the samples are referred to using the starting boehmite powder and the freezing speed.

Zirconia Micromonolith with Mesoporous Boehmite Layer

In a first step, yttria stabilized zirconia (YSZ) monoliths are prepared following the experimental procedure described in ref. 14 . The as prepared zirconia suspensions are poured into a 21.5 mm diameter and 25 mm height PTFE mould. Freezing and sublimation are performed as described previously. Samples are thermally treated at 1350 °C for binder removal and sintering. The sintered monoliths are machined to a 5 mm thickness. To remove cutting debris, they are extensively cleaned ultrasonically with large quantities of DI water.

These sintered zirconia monoliths are washcoated with a boehmite suspension, prepared by dispersing commercial boehmite powder (Disperal, Sasol) in DI water. Dispersion is promoted by ultrasound and pH adjustment to 3.5 with nitric acid (69 wt%, Sigma-Aldrich). A binder solution containing polyethylene glycol (PEG2M, Sigma-Aldrich) and isopropanol is then added. Final mass percentages are: 2.29 Disperal/90.64 DI water/0.55 PEG2M/6.52 Isopropanol. Washcoating is performed by immersing the YSZ monoliths into the boehmite suspension under vacuum. This serves to outgas the air from the channels of the monolith and promote suspension infiltration. After this washcoating step, excess solution is eliminated from the monolith surfaces and the sample is dried in a controlled atmosphere oven at 40 °C and 80% relative humidity. The samples are thermally treated at 550 °C for 2 h.

Sample Characterization

Dispersed particle size is measured by quasi elastic light spectroscopy (Zetasizer, Malvern), by dilution of the slurry used for ice templating at constant pH. Specific surface area and pore size distributions are determined using nitrogen adsorption/desorption at 77 K (Micromeritics-ASAP 2010) after outgassing at 280 °C for at least 2 h. Hg intrusion is measured using an AutoPore IV from Micromeritics, the pressure range varied between 0.44 and 30 000 psi. SEM observations are performed using either a TM1000 from Hitachi or a Nova NanoSEM 230 from FEI. Boehmite samples made following the first method are prepared by cutting the frozen monoliths at half height before lyophilization and thermal treatment. Samples obtained following the second method were either machined (views perpendicular to freezing direction) or fractured (views along freezing direction) after thermal treatment. Transmission electron microscopy (TEM) observations are performed on crushed samples on a FEI Tecnai G2 20. Compressive strength (test equipment from Lloyd) was measured on five samples (10 mm diameter for a 15 mm height), with a 150 kN sensor and a crosshead speed of 0.5 mm s -1 .

Results and Discussion

In a first approach, hierarchical micromonoliths with mesoporous walls are synthesized by directional freezing of boehmite suspensions. The impact of the powder granulometry on the mesoporosity is studied using powders with increasing size in the nanometer-range: commercial powders from Sasol (referred to hereafter by "Catapal" and "Disperal") as well as a boehmite suspension synthesized following Yoldas method (referred to hereafter by "Yoldas"). The mean particle sizes of dispersed Catapal and Disperal, as indicated in the technical data sheet from Sasol, are, respectively, 100 and 80 nm. A 28 nm particle size value is measured for the Yoldas powder, using quasielastic light scattering.

Using aqueous suspensions of these powders, monoliths are frozen at a constant freezing rate of 10 µm s -1 . A gentle heat treatment at 550 °C is chosen to avoid the sintering and transformation of the boehmite into α-alumina. The final porosity characteristics are thus directly related to particle packing during the freezing process. The ice crystallization seems to occur similarly for the three powders, yielding elongated and highly dendritic macropores, as shown on the SEM images in Figure 1. A lamellar macroporous structure is obtained at the micrometer scale with a mean pore width of approximately 10 µm. These values are confirmed by Hg intrusion measurements: the measured mean macropore diameters for Catapal and Disperal samples are, respectively, 10 and 10.3 µm. The use of commercial powders with larger particle size leads to denser walls than Yoldas samples. In this case, the primary macropores are separated by bundles of sheets generating a second level of macropores. Due to lower mechanical resistance towards the Hg pressure, the macropore size was not assessed with Hg intrusion. It can be noticed that for the monolith produced from the Catapal powder, dendrites are only situated on one side of the sheets, whereas in the case of Yoldas, the sheets appear smooth. Mesoporosity is assessed by N2 adsorption-desorption measurements (Figure 2). The resulting textural properties together with particle size and freezing conditions are summarized in Table 1. As expected, increasing the particle size leads to a decrease of the specific surface area of the monoliths from 292 to 174 m 2 g -1 . High values of specific surface area are measured for all starting boehmite particles, showing that loose contact occurs between particles in the monolith walls. The mesopore diameter calculated from N2 adsorption-desorption measurements is, respectively, 4.8, 5.4, and 6.2 nm for Yoldas, Disperal, and Catapal monoliths, but otherwise pore volume of the walls is equivalent for the three types of boehmite powders. When Yoldas boehmite particles are used as the starting material, a typical type IV isotherm is obtained (Figure 2). The initial part of the isotherm indicates the presence of microporosity, unlike Catapal and Disperal samples. In Figure 2b, one can see that mesopore size distribution is narrow for Yoldas and much larger for Catapal and Disperal monoliths. This can explain why the pore volume is almost constant due to the contribution of the largest pore size. A TEM image (Figure 3) of a Yoldas monolith obtained at a freezing speed of 10 µm s -1 shows the mesoporous structure of the walls formed by the nanosized Yoldas particles. The pore size is in agreement with the value obtained from N2 adsorption-desorption whereas typical platelet morphology of boehmite particles can be seen on this image. The freezing speed, or correspondingly the ice crystal growth velocity, impacts the redistribution and packing behavior of the particles. In the case of Yoldas boehmite suspensions, decreasing the freezing speed from 10 to 2 µm s -1 decreases the pore diameter from 4.8 to 3.6 nm. The corresponding pore volume of the walls also decreases from 0.37 to 0.20 cm 3 g -1 showing that lower freezing speed allows particles to arrange in a closer manner. As reported in Table 1, the specific surface area is not affected by this reorganization of the porous structure. One explanation could be the contribution of the microporosity, almost identical for both samples. These ice-templated monoliths exhibit a high specific surface area and a hierarchic porosity, which are of potential interest for catalyst carriers. However the one step process used for their preparation leads to very brittle pellets with insufficient mechanical strength for applications in a catalytic bed. We therefore propose an alternative method to combine the unique macroporous structure of the ice templated ceramic monoliths and the requirement of high surface area for the active catalytic sites. High mechanical strength monoliths are produced by wash coating mesoporous boehmite layer on a sintered ice templated monolith. This strategy yields pellets combining large continuous macropores, providing access to the catalyst without the usual large pressure drop and lowering the diffusion path, while the boehmite washcoat provides high specific surface area needed for a good dispersion of the catalytic sites. Ice templated YSZ monoliths with a particular macroporosity are used as substrates for the bohemite coating. As recently described by Deville et al., 14 the use of specific ice-shaping compounds in the ice-templating process leads to a honeycomb-like structure with hexagonally shaped pores running continuously throughout the monoliths (Figure 4a andb). The macropores exhibit a smooth and dendrites-free surface (Figure 4d), optimal to minimize the pressure drop. The mean macropore diameter as determined by Hg intrusion is 4.5 µm, with a narrow pore size distribution (Figure 4c). The measured compressive strength for such monoliths with 64% macroporosity is 134 ± 30 MPa. In addition, high temperature sintered zirconia are known for their high abrasion resistance. All these characteristics make this substrate well adapted as catalyst carrier pellets for fixed-bed reactors. These substrates are washcoated under reduced pressure using a boehmite suspension to obtain a mesoporous structure. The Disperal powder was chosen to produce mesoporous coatings with high specific surface area. As shown by SEM images in Figure 4, a 100-200 nm thick homogeneous coating is obtained at the surface of the macroporous icetemplated structure. The thin coating only marginally affects the macropore size. The SEM images in Figure 4e-f show close up views of a fractured wall. The YSZ ice-templated monolith appears as light grey, while the porous bohemite is darker. The coating thickness is indicated by arrows. Specific surface area of the monoliths before and after washcoating increases from 0.2 to 3.8 m 2 g -1 . The boehmite coating represents 1% of the total monolith mass; the SSA of the coating thus can be estimated to be 360 m 2 g -1 . Low transport resistance of the reactants and short diffusion paths to the catalytic sites are expected from pellets based on this architecture. Work is currently under progress for precious metal deposition and catalytic performance evaluation.

Conclusions

Combining high surface area, accessibility to catalytically active sites, and mechanical strength is a tough requirement for many catalytic carriers. We demonstrate that approaches based on ice-templating can address this requirement, in particular for the fabrication of pellets used in fixed-bed reactors. Boehmite micromonoliths with a hierarchic porosity can be obtained using a one-step process although the mechanical properties do not meet the application requirements. We demonstrate a two-step process in which an ice-templated support with microchannels is prepared, followed by the deposition of a thin mesoporous layer inside the channels. High mechanical resistance, hierarchic porosity, and high specific surface area for catalyst dispersion can thus be advantageously combined. Although the functional catalytic properties remain to be assessed, the combination of continuous and calibrated macropores channels, high strength, and large amount of meso-and micropores is appealing for such applications.

Figure 1 :

 1 Figure 1 : SEM images of ice templated monoliths manufactured using (a) Catapal B, (b) Disperal, and (c) Yoldas. Scale bars 250 µm.

Figure 2 :

 2 Figure 2: N2 adsorption/desorption isotherms and corresponding pore size distribution. (a, b) Impact of boehmite powder: Yoldas, Dispersal, and Catapal powders. (c, d) Impact of freezing kinetics, crystal growth velocity of 2 and 10 µm s -1 .

Figure 3 :

 3 Figure 3 : TEM image of the wall mesoporous structure in a Yoldas monolith obtained at a freezing speed of 10 µm s -1 . Scale bar 10 nm.

Figure 4 :

 4 Figure 4 : Macroporous YSZ ice templated monoliths (a) macroscopic view (b) SEM micrograph perpendicular to ice solidification direction, showing the macroporous channels (c) mercury porosimetry results (d) SEM micrograph along ice solidification direction (e) SEM micrograph of the monolith washcoated with boehmite (f) close-up view of the washcoat, the arrows indicates the thickness of the coating. Scale bars (b) 50 µm, (d) 20 µm, (e) 2 µm, and (f) 250 nm.

  ]

	Yoldas	28	2	293	3.6	0.20
	Yoldas	28	10	292	4.8	0.37
	Disperal	80	10	224	5.4	0.39
	Catapal	100	10	174	6.2	0.38

Table 1 :

 1 Particle size, freezing conditions and samples characteristics

	Boehmite powder	Dispersed particle diameter (nm) Ice velocity front (µm.s -1 )	SSA (m².g -1 )	Mesopore diameter (nm)	In-wall volume (cm 3 .g -1 ) pore
	Yoldas	28	2	293	3.6	0.20
	Yoldas	28	10	292	4.8	0.37
	Disperal	80	10	224	5.4	0.39
	Catapal	100	10	174	6.2	0.38


Dr Paco Bustamante

V Lahaye

C Durnez

C Churlaud

F Caurant

Total and organic Hg concentrations in cephalopods from the North Eastern Atlantic waters: influence of geographical origin and feeding ecology

Keywords: Bioaccumulation, Speciation, Body distribution, Squid, Octopus, Cuttlefish

Total (T-Hg) and organic (O-Hg) mercury concentrations and tissue distribution were examined in 20 species of cephalopods (n = 278) from the north eastern Atlantic waters, i.e. from the Bay of Biscay to the Faroe Islands. Concentrations of T-Hg in whole cephalopods showed elevated variations among species, i.e. from 40 to 3560 ng.g -1 dwt, but a low variability within each species (mean CV% = 39%). With the exception of oceanic squids, the digestive gland globally displayed higher T-Hg concentrations than the remaining tissues. In contrast, O-Hg concentrations determined in selected species were generally higher in the remaining tissues. Despite higher T-Hg concentrations, the digestive gland weakly contributed to the total body burden of both T-Hg and O-Hg (< 25% and <15%, respectively). In fact, from 75 to 95% of the T-Hg and O-Hg were contained in the muscular remaining tissues. Therefore, O-Hg may have a strong affinity to proteins in cephalopods. Sex and size only significantly influenced the bioaccumulation of Hg for the Loliginidae family.

T-Hg and O-Hg concentrations were also influenced by geographical origin: Celtic Sea > Bay of Biscay > Faroe Islands, corresponding to the seawater Hg concentrations in these areas. In the Faroe Islands and the Celtic Sea, benthic cephalopods contained significant higher Hg concentrations compared to pelagic ones. This suggests that diet is not the main pathway of Hg uptake in cephalopods as pelagic species were expected to be more exposed to O-Hg through fish consumption than benthic ones.

INTRODUCTION

Transfer of metals in marine food webs appears to be mainly directed by their physicochemical forms, which therefore defines their bioavailability to the upper level (Wallace & Lopez 1997). Among metals of environmental concern, mercury (Hg) is readily methylated by micro-organisms, bioaccumulates in marine biota and consistently biomagnifies through the food chain, predators showing higher tissue concentrations than in their prey.

Among organic Hg compounds, methyl Hg is the most stable form in the marine environment but also the most toxic one to organisms [START_REF] Cossa | Le mercure en milieu marin. Biogéochimie et écotoxicologie[END_REF]). Hg methylation generally increases its toxicity as a result of its enhanced penetration through lipid membranes (Boudou et al. 1983). In top predators such as Humans, the nervous system is the critical organ for chronic Hg exposure and methyl Hg can react directly with important receptors (WHO, 1990).

Overall, Hg is considered as the most toxic metal since the accident of the Minnamata Bay in Japan and seafood consumption constitutes the main ingestion source of Hg in Man [START_REF] Gesamp | Scientific aspects of Marine Pollution review of Potentially Harmfull Substances: Arsenic, Mercury and Selenium[END_REF].

A wealth of scientific information on Hg in fish is available due tot the fact that (1) they are important components of marine, estuarine and freshwater ecosystems and (2) they constitute a major food source for Man. Even if Hg concentrations in fish may vary according to their trophic level and age [START_REF] Honda | Organ and tissue distribution of heavy metals, and their growth-related changes in Antarctic fish, Pagothenia borchgrevinki[END_REF][START_REF] Clarkson | Mercury[END_REF][START_REF] Riisgård | Biomagnification of mercury in a marine grazing food-chain: algal cells Phaeodactylum tricornutum, mussels Mytilus edulis and flounders Platichthys flesus studied by means of a stepwise-reduction-CVAA method[END_REF], virtually all the Hg (i.e. > 95%) in fish is methylated [START_REF] Bloom | On the chemical form of mercury in edible fish and marine invertebrate tissue[END_REF]) and is therefore bioavailable for upper trophic levels. Hence, fish consumption is an important source of dietary Hg [START_REF] Svensson | Fish as a source of exposure to mercury and selenium[END_REF]) and is of special health concern [START_REF] Clarkson | Human health risks from methylmercury in fish[END_REF].

In contrast to fish, information on Hg in cephalopod tissues is scarce. Most of the studies about total Hg concentrations has mainly addressed on edible tissues (e.g. [START_REF] Nagakura | Mercury content of whales[END_REF][START_REF] Stoepler | Comparative studies on trace metal levels in marine biota. I. Mercury in marine organisms from Western Italian coast, the Strait of Gibraltar and the North Sea[END_REF], Cappon & Smith 1982[START_REF] Buzina | Effect of industrial pollution on seafood content and dietary intake of total and methylmercury[END_REF][START_REF] Cappon | Speciation of selected trace elements in edible seafood[END_REF], Sapunar et al. 1989[START_REF] Plessi | Mercury and selenium content in selected seafood[END_REF] and very few studies have discussed the ratio between organic and inorganic Hg in cephalopods (i.e. [START_REF] Buzina | Effect of industrial pollution on seafood content and dietary intake of total and methylmercury[END_REF], Cappon & Smith 1982).

Contradictory results have been published in the current literature, as for instance [START_REF] Nagakura | Mercury content of whales[END_REF] showed a higher percentage of O-Hg in cephalopod tissues whereas [START_REF] Cappon | Speciation of selected trace elements in edible seafood[END_REF] found higher inorganic Hg in octopus and squid flesh. Therefore, the assessment of the chemical state of Hg is of ground importance since this strongly determines the bioavailability of this metal to the upper trophic level [START_REF] Cossa | Le mercure en milieu marin. Biogéochimie et écotoxicologie[END_REF]).

Several studies concerning other heavy metals in cephalopods tissues have demonstrated the major role of the digestive gland in metal metabolism, as this organ is deeply involved in assimilation processes, detoxification and storage of both essential and non-essential metals (e.g. [START_REF] Martin | High copper concentrations in squid livers in association with elevated levels of silver, cadmium, and zinc[END_REF][START_REF] Smith | Concentrations of the elements Ag, Al, Ca, Cd, Cu, Fe, Mg, Pb and Zn, and the radionuclides 210 Pb and 210 Po in the digestive gland of the squid Nototodarus gouldi[END_REF][START_REF] Miramand | Concentration and distribution of heavy metals in tissues of two cephalopods, Eledone cirrhosa and Sepia officinalis, from the French coast of the English Channel[END_REF][START_REF] Bustamante | Bioaccumulation of 12 trace elements in the tissues of the nautilus Nautilus macromphalus from New Caledonia[END_REF], 2002ab, 2004). Moreover, cephalopods also accumulate organic contaminants such as organotin, organochlorine pesticide or PCBs in the digestive gland [START_REF] Tanabe | Polychlorobiphenyls, DDT, and hexachlorocyclohexane isomers in the western North Pacific ecosystem[END_REF][START_REF] Kawano | Biological accumulation of chlordane compounds in marine organisms from the northern North Pacific and Bering Sea[END_REF][START_REF] Yamada | Organotin compounds and polychlorinated biphenyls of livers in squid collected from coastal waters and open oceans[END_REF][START_REF] Weisbrod | Organochlorine bioaccumulation and risk for whales in a Northwest Atlantic food web[END_REF], 2001[START_REF] Ueno | Specific accumulation of polychlorinated biphenyls and organochlorine pesticides in Japanese common squid as a bioindicator[END_REF][START_REF] Danis | Bioaccumulation of PCBs in the cuttlefish Sepia officinalis from seawater, sediment and food pathways[END_REF]. Conversely, very little information is available concerning Hg in the digestive gland of cephalopods and mainly focuses on common octopus (e.g. [START_REF] Renzoni | Mercury concentration in the water, sediments and fauna of an area of the Tyrrhenian coast[END_REF][START_REF] Raimundo | Geographical variation and partition of metals in tissues of Octopus vulgaris along the Portuguese coast[END_REF][START_REF] Seixas | Accumulation of mercury in the tissues of the common octopus Octopus vulgaris (L.) in two localities on the Portuguese coast[END_REF].

Cephalopods are widespread in a great variety of marine habitats from coastal waters to very deep ocean environments. They are benthic (e.g. octopus), nectobenthic (cuttlefish, bottlesquid), neritic and oceanic (mainly squids), and constitute a primary food source for many marine predators such as fish, marine mammals or seabirds [START_REF] Clarke | Cephalopods as prey. III. Cetaceans[END_REF][START_REF] Croxall | Cephalopods as prey. I. Seabirds[END_REF][START_REF] Klages | Cephalopods as prey. II. Seals[END_REF][START_REF] Smale | Cephalopods as prey. IV. Fishes[END_REF]. It follows that they could represent an important vector of contaminant to the consumers as shown for Cd [START_REF] Bustamante | Cephalopods as a vector for the transfer of cadmium to top marine predators in the north-east Atlantic Ocean[END_REF]. However, estimations of top predator exposures to Hg are limited because of a poor knowledge about Hg levels and bioavailability in this prey. Cephalopods are also extensively fished and consumed by humans [START_REF] Amaratunga | Advances in assessment of world cephalopod resources[END_REF]) and thus, are also of direct concern to public health.

In this framework, the present study aims at providing baseline data on Hg concentrations in cephalopods for a large range of species from the North-eastern Atlantic waters and at investigating their variations according to biological and geographical factors. To this end, total and organic Hg concentrations have been determined in the digestive gland and remaining muscular tissues of several squid, cuttlefish, and octopus species caught at various latitudes, i.e. from the Bay of Biscay to the Faroe Islands. We then discuss the observed variations of the Hg concentration and its body distribution and finally show the correlation between organic and inorganic Hg.

MATERIAL & METHODS

Sampling and sample preparation

The sampling of cephalopods includes 278 individuals belonging to 20 species (see Table 1), which were caught by trawl between 1996 and 2003 in the waters from the Bay of Biscay, the Celtic Sea, the Scottish waters and the Faroe Islands waters.

Cephalopods were fished either by scientific or commercial vessels. Collected specimens were immediately frozen on board in individual plastic bags. In the laboratory, each individual was weighed and measured (mantle length, total length), and the sex determined.

The origin, number of individuals and body weight of each species of cephalopods are given in Table 1. In most of the individuals, the digestive gland was dissected to be treated separately. Therefore, Hg concentrations in the whole animals were calculated according to the concentrations in the digestive gland and in the remaining tissues. The latter are in fact mainly composed by muscle tissues (arms, fins and mantle accounting for 70 to 80% of the body weight).

Analytical procedure

Separated tissue samples were either dried to a constant weight for several days at 60°C or freeze-dried and then homogenised in a motar and porcelain pestle. For T-Hg, two aliquots ranging from 10 to 50 mg of dried material were directly analysed in an Advanced Mercury Analyser spectrophotometer (Altec AMA 254). Hg determination involved evaporation of the metal by progressive heating until 800°C was reached and then held under oxygen atmosphere for 3 min, and subsequent amalgamation on a gold-net. Afterwards, the net was heated to liberate the collected Hg, which was then measured by atomic absorption spectrophotometry. The same procedure was performed for the analysis of O-Hg after extraction adapted from [START_REF] Uthe | Rapid semimicro method for the determination of methyl mercury in fish tissue[END_REF]. O-Hg was extracted from 2 aliquots of approx. 500 mg of each homogenised dry sample using 2 ml of acidic sodium bromide (30% NaBr in 4N H 2 SO 4 ), 4 ml of cupric sulfate (2.5% CuSO 4 in milli-Q quality water) and 10 ml of toluene under agitation for 10 min in glass flasks. The organic phase was then separated and centrifuged 10 min at 1500 G to eliminate residues of the tissue material. The supernatants were used for the analysis in the AMA.

Quality control

Mercury analyses were ran with respect to a thorough quality control program including analyses of reference materials dogfish liver DOLT-2 and lobster hepatopancreas TORT-2 purchased from the National Research Council, Canada. These standards were treated and analysed under the same conditions as the samples. The results were in good agreement with the certified values (Table 2). Detection limits (ng.g -1 dry wt) were 5 for total Hg and 20 for organic Hg. Metal concentrations in tissues are also reported in ng.g -1 dry wt.

Data analyses

Prior to statistical analyses, Hg concentrations and mantle length were tested for normality and equality of variances. Comparisons of Hg concentrations between tissues within each species were tested by t-test. Gender variations of Hg concentrations with size were tested by ANCOVA. Comparisons of Hg concentrations among several species were performed using one-way ANOVA followed by Tukey multiple comparison test. Statistical analyses were performed using XLStat 7.0. The significance for statistical analyses was always set at α = 0.05.

RESULTS

Levels of concentrations and tissue distribution

T-Hg has been determined in both the digestive glands and the remaining tissues of 278 cephalopods sampled in the north-eastern Atlantic waters and its concentrations in the whole individuals have been calculated accordingly (Table 3). T-Hg in the whole cephalopods varies greatly among species, from 40 to 3560 ng.g -1 dwt but generally displays a low variability in each species. Indeed, the coefficients of variation (CV) of T-Hg concentrations in the whole individuals, calculated as the standard deviation divided by the mean multiplied by 100, vary from 14 to 98% (mean CV% = 39%).

With a few exceptions concerning oceanic squids (Cranchidae, Histioteuthidae and Ommastrephidae), T-Hg concentrations are higher in the digestive gland of cephalopods than in the remaining tissues (Table 3). This difference is significant (P < 0.05) for various species from the Bay of Biscay (the squid Loligo vulgaris, the bobtail squid Rossia Macrosoma, the cuttlefishes Sepia elegans, S. officinalis and S. orbignyana, and the octopuses Bathypolypus baierii, B. sponsalis, Eledone cirrhosa, Octopus vulgaris and O. salutii), for Bathypolypus baierii from West Scotland, and for Loligo forbesi from the Faroe Islands. Between the two compartments analysed, the remaining tissues contain most of the T-Hg burdens whichever the species, varying from 60 to 95% of the total body burden (Fig. 1).

Organic Hg (O-Hg) has been determined in the digestive gland and in the remaining tissues of selected species (Table 4). As L. forbesi, Todarodes sagittatus, and E. cirrhosa were found in three sampling areas (i.e. Bay of Biscay, Celtic Sea and Faroe Islands), these species have been retained to carry out further geographical comparisons. In addition, O-Hg has been determined in the remaining tissues of the other cephalopod species. Regardless of the origin and independently of the levels of the concentrations, O-Hg is strongly correlated with T-Hg in the remaining tissues of the species analysed (Fig. 2). The remaining tissues contain a high percentage of O-Hg representing 84 to 98% of the total O-Hg in the organism. Therefore, as muscular remaining tissues constitute the main proportion of cephalopod body mass (i.e. around 90%), they contain the greatest fraction of the whole body burden, of which 67 to 83% of the T-Hg is under organic form in this compartment (Table 4).

In the digestive gland, the percentage of O-Hg given relatively to T-Hg is lower and varies widely among species, i.e. from 22 to 65 %. However, when considering the total body burden of O-Hg, the digestive gland always contains less than 16% of O-Hg for all the considered species (Table 4).

Influence of size and sex

The variations of T-Hg concentrations with size in whole-body cephalopods according to the sex have been tested by ANCOVA only for the largest samples (i.e. more than 10 individuals). Hg concentrations significantly vary with size only for a few species, all belonging to the Loliginidae family (Table 5). Results generally do not show any significant differences due to sex but Loligo forbesi from the Faroe Islands and from the Celtic Sea exhibit a significant interaction between size and sex, meaning that the bioaccumulation rate of Hg is different between sexes, females showing the highest values. dry wt for E. cirrhosa, from 100 to 458 ng.g -1 dry wt for L. forbesi and from 40 to 879 ng.g -1 dry wt for T. sagittatus. Significant differences (P<0.05) of T-Hg concentrations in whole individuals are hence found among such areas for each species. Indeed, the cephalopods from the Faroe Islands exhibit the lowest values whereas the highest are found in those from the Irish waters (Fig. 3). A similar pattern is observed for O-Hg in the remaining tissues because of its correlation with T-Hg (Fig. 2).

Interspecific and geographical variations

Figure 4 compares the T-Hg concentrations between benthic and pelagic cephalopods from the Bay of Biscay, the Celtic Sea and the Faroe Islands. Benthic cephalopods display higher T-Hg concentrations in the Faroe Islands and the Celtic Sea (F=5.39 P=0.024 and F=23.39 P<0.001, respectively) compared to pelagic ones (Fig. 4). However, such is not the case for cephalopods from the Bay of Biscay (F=2.1 P=0.150).

DISCUSSION

The distribution of Hg was considered between two separated body compartments, the digestive gland, and the remaining tissues of cephalopods (Figure 2). The digestive gland was first selected as this organ plays a major role in the energetic metabolism of cephalopods [START_REF] Boucaud-Camou | Feeding and digestion in cephalopods[END_REF] and is known to accumulate metal in large amounts (e.g. [START_REF] Martin | High copper concentrations in squid livers in association with elevated levels of silver, cadmium, and zinc[END_REF][START_REF] Finger | Molecular association of Cu, Zn, Cd and 210 Po in the digestive gland of the squid Nototodarus gouldi[END_REF][START_REF] Miramand | Concentration and distribution of heavy metals in tissues of two cephalopods, Eledone cirrhosa and Sepia officinalis, from the French coast of the English Channel[END_REF][START_REF] Bustamante | Bioaccumulation of 12 trace elements in the tissues of the nautilus Nautilus macromphalus from New Caledonia[END_REF]. The second compartment included the rest of the animal (around 90% of the total body weight). This compartment is mainly constituted by muscles, which could also store Hg in significant amounts (e.g. [START_REF] Renzoni | Mercury concentration in the water, sediments and fauna of an area of the Tyrrhenian coast[END_REF], Cappon & Smith 1982[START_REF] Cappon | Speciation of selected trace elements in edible seafood[END_REF][START_REF] Monteiro | Inter-and intra-specific variation of mercury levels in muscle of cephalopods from the Azores[END_REF][START_REF] Seixas | Accumulation of mercury in the tissues of the common octopus Octopus vulgaris (L.) in two localities on the Portuguese coast[END_REF].

Although cephalopods have been demonstrated to have special capabilities to concentrate heavy metals at various levels in their tissues, dissimilar information on Hg concentrations in cephalopods and on their variations has been reported (see Table 6 for review). Overall, Hg concentrations have been mainly studied in a few cephalopod species for human health purposes but most of these works generally remained limited to edible tissues, i.e. mantle muscle, arms and fins. Very little data consider various species at the same time or over a large area [START_REF] Stoepler | Comparative studies on trace metal levels in marine biota. I. Mercury in marine organisms from Western Italian coast, the Strait of Gibraltar and the North Sea[END_REF][START_REF] Monteiro | Inter-and intra-specific variation of mercury levels in muscle of cephalopods from the Azores[END_REF]). To the best of our knowledge, this study is the first one to investigate Hg concentrations and tissue distribution in a wide range of cephalopod species including benthic and pelagic species living in neritic and oceanic habitats over a large geographical scale.

T-Hg concentrations in cephalopods from the north-eastern Atlantic waters were globally within the same range of concentrations as other related species from the south West Atlantic and the Japanese waters (Table 6). In contrast, higher T-Hg concentrations have been reported

for cephalopods from the Tyrrhenian and Adriatic Seas (e.g. [START_REF] Renzoni | Mercury concentration in the water, sediments and fauna of an area of the Tyrrhenian coast[END_REF][START_REF] Storelli | Cadmium and total mercury in some cephalopods from the South Adriatic Sea (Italy)[END_REF]. Higher Hg concentrations in Mediterranean organisms are typically explained by high temperature and absence of solar radiation in the deep environment that favors a high methylation rate. Moreover, natural sources of Hg in the Mediterranean Sea may contribute to Hg enrichment through the benthic food webs, as it constitutes the richest natural reserve of this element [START_REF] Bacci | Mercury in the Mediterranean[END_REF].

Regardless of the species and the origin, it is striking to note that the coefficients of variation of T-Hg concentrations in the whole individuals are relatively low for a toxic element (i.e. always < 98%). Similar coefficients of variation have been previously reported for essential Cu and Zn which might be under homeostatic control [START_REF] Bustamante | Etude des processus de bioaccumulation et de détoxication d'éléments traces (métaux lourds et terres rares) chez les mollusques céphalopodes et bivalves pectinidés. Implication de leur biodisponibilité pour le transfert vers les prédateurs[END_REF], suggesting also a relative ability of cephalopods to regulate Hg. However, T-and O-Hg concentrations varied between the digestive gland and the remaining tissues, the former generally showing higher T-Hg but lower O-Hg concentrations (Tables 3 &4). The digestive gland plays a major role in the digestive processes and also in the detoxification of xenobiotics in cephalopods: on the one hand, the digestive gland synthesizes the digestive enzymes and is a place of absorption and assimilation of nutrients [START_REF] Boucaud-Camou | Feeding and digestion in cephalopods[END_REF][START_REF] Boucher-Rodoni | Feeding and digestion[END_REF]; on the other hand, this organ is involved in the storage and detoxification of several trace elements such as Ag, Cd, Cu, or Zn (e.g. [START_REF] Miramand | High concentrations of some heavy metals in tissues of the Mediterranean octopus[END_REF][START_REF] Finger | Molecular association of Cu, Zn, Cd and 210 Po in the digestive gland of the squid Nototodarus gouldi[END_REF][START_REF] Miramand | Concentration and distribution of heavy metals in tissues of two cephalopods, Eledone cirrhosa and Sepia officinalis, from the French coast of the English Channel[END_REF], Bustamante et al. 2002ab, 2004) and organic compounds such as PCBs or TBT [START_REF] Yamada | Organotin compounds and polychlorinated biphenyls of livers in squid collected from coastal waters and open oceans[END_REF][START_REF] Ueno | Specific accumulation of polychlorinated biphenyls and organochlorine pesticides in Japanese common squid as a bioindicator[END_REF][START_REF] Danis | Bioaccumulation of PCBs in the cuttlefish Sepia officinalis from seawater, sediment and food pathways[END_REF]. Sources of metals to cephalopods are 1) seawater, as it passes through the skin and through the gills during respiration, and 2) food, which probably represents the main pathway for most of transition and heavy metals [START_REF] Koyama | Bioaccumulation of waterborne and dietary cadmium by oval squid Sepioteuthis lessoniana, and its distribution among organs[END_REF], Bustamante et al. 2002a[START_REF] Bustamante | Uptake, transfer and distribution of silver and cobalt in tissues of the common cuttlefish Sepia officinalis at different stages of its life cycle[END_REF]. However, it is difficult to ascertain the relative importance of each pathways of uptake for Hg, which can be influenced by the speciation of this metal in both seawater and prey. Moreover, to the best of our knowledge, neither inorganic nor organic Hg uptake has been studied in cephalopods and would be particularly interesting to be investigated in the future.

In the digestive gland, the fraction of O-Hg varied widely (i.e. from 20 to 60 %) but remained always lower to that determined in the remaining tissues (i.e. from 67 to 83%) (Table 4).

Lower O-Hg contents in the digestive gland suggests that demethylation of O-Hg could occur in this organ like in fish liver [START_REF] Lockhart | Methylmercury in northern pike and some biochemical characteristics of contaminated fish[END_REF][START_REF] Boudou | Trophic chains and experimental ecosystems: study of bioaccumulation and transfer processes[END_REF]. However, it is largely admitted that the biomagnification of Hg resembles that of hydrophobic organic pollutants rather than that of ionic metals, and it is generally thought to result from the lipid solubility of methyl Hg [START_REF] Monteiro | Mercury concentrations in prey fish indicate enhanced bioaccumulation in mesopelagic environments[END_REF]. High lipid contents in the digestive gland of cephalopods would therefore lead to high O-Hg concentrations. However, such prediction needs to consider Hg chemical properties. In fact, Hg has a low octanol-water distribution coefficient (K ow ) compared to other fat soluble compounds such as DDT or PCBs [START_REF] Bienvenue | Transport of mercury compounds across biomolecular lipid membranes: effect of lipid composition, pH, chloride concentration[END_REF][START_REF] Veith | An evaluation of using coefficients and water solubility to estimate BCF for organic chemicals in fish[END_REF]. Unlike other hydrophobic compounds, O-Hg seems to have a strong affinity for the sulphydryl groups of certain proteins and resides in muscular proteins rather than in fat tissue in fish [START_REF] Bloom | On the chemical form of mercury in edible fish and marine invertebrate tissue[END_REF]). Thus, a second reason for the low O-Hg contents in the digestive gland of cephalopods could be a preferential redistribution to muscular tissues. It is therefore not surprising that most of the Hg contained in the muscular remaining tissues was under organic form (Figure 2).

As for other metals, Hg concentrations in cephalopod may vary with biological and environmental factors such as age (size), sex, lifestyle and geographical origin (e.g. [START_REF] Monteiro | Inter-and intra-specific variation of mercury levels in muscle of cephalopods from the Azores[END_REF], Bustamante et al. 1998[START_REF] Raimundo | Geographical variation and partition of metals in tissues of Octopus vulgaris along the Portuguese coast[END_REF][START_REF] Seixas | Accumulation of mercury in the tissues of the common octopus Octopus vulgaris (L.) in two localities on the Portuguese coast[END_REF]. It is striking that only the squids belonging to the Loliginidae family exhibited a significant increase of Hg concentrations with size and displayed different metal accumulation rates between sexes (Table 5). Despite lack of data on the influence of such intra-specific factors on Hg bioaccumulation, it is generally admitted that Hg concentrations in cephalopod tissues are positively correlated with size [START_REF] Monteiro | Inter-and intra-specific variation of mercury levels in muscle of cephalopods from the Azores[END_REF][START_REF] Rossi | Mercury in Eledone cirrhosa from the Northern Tyrrhenian Sea: contents and relations with life cycle[END_REF][START_REF] Storelli | Cadmium and total mercury in some cephalopods from the South Adriatic Sea (Italy)[END_REF]. The higher accumulation rate of Hg in Loligo forbesi females, probably due to the existence of physiological and/ or ecological inter-sex differences, has been previously cited by [START_REF] Monteiro | Inter-and intra-specific variation of mercury levels in muscle of cephalopods from the Azores[END_REF]. However, this paper does not focus on this issue and further research should be carried out on this aspect.

When considering the different species, Hg concentrations seem to be influenced by the lifestyle. Indeed, benthic cephalopods generally displayed significantly higher T-Hg and O-Hg concentrations than pelagic ones (Fig. 4). The only exception was the Bay of Biscay because of the different cuttlefish species showing lower Hg concentrations than octopus. The lower Hg concentrations in cuttlefish are likely due to the very low Hg concentrations found in the cuttlebone (Bustamante, unpublished data) leading to the dilution of the whole body Hg concentrations. Overall, benthic cephalopods exhibit more complex diets than pelagic ones and feed on polychets, crustaceans, molluscs, echinoderms and fish [START_REF] Boyle | Prey handling and salivary secretions in octopuses[END_REF][START_REF] Mcquaid | Feeding behaviour and selection of bivalve prey by Octopus vulgaris Cuvier[END_REF]. In contrast, squid mainly feed on fish and other cephalopod species [START_REF] Nixon | Cephalopod diets[END_REF][START_REF] Rocha | The diet of Loligo vulgaris and Loligo forbesi (Cephalopoda: Loliginidae) in the Galician waters (NW Spain)[END_REF][START_REF] Pierce | Diets of squid Loligo forbesi and Loligo vulgaris in the Northeast Atlantic[END_REF]. Such a pelagic diet would lead to higher O-Hg concentrations in pelagic cephalopods as their prey contain higher O-Hg concentrations than those of benthic ones [START_REF] Cossa | Le mercure en milieu marin. Biogéochimie et écotoxicologie[END_REF]). Moreover, squids have higher feeding rates than octopus [START_REF] Nixon | Cephalopod diets[END_REF]. Surprisingly, both oceanic and neritic squids displayed significant lower Hg concentrations compared to benthic octopus. It is therefore difficult to explain these results when only considering Hg exposure through the diet and it derives that trophic ecology is not the only factor controlling the intake of Hg in cephalopods. Because they live close to the sea floor, benthic cephalopods would be exposed to higher dissolved O-Hg concentrations resulting from the methylation by micro-organisms living in the sediment. Experiments to determine the affinity of dissolved O-Hg for cephalopods appear therefore necessary to assess this hypothesis.

The influence of the geographical area was considered using the three species of cephalopods (i.e. E. cirrhosa, L. forbesi and T. sagittatus) living between the Bay of Biscay (southern part) and the Faroe Islands (northern part) (Fig. 3). Our results indicated that geographical origin is an important factor contributing to both intra-specific and inter-specific variations of Hg concentrations. In fact, cephalopods from the Celtic Sea always displayed the highest T-and O-Hg concentrations and those from the Faroe Islands the lowest (Fig. 3). Around Ireland, waters showed 10 to 100 times higher Hg concentrations compared to those from the Bay of Biscay (Garner 1978[START_REF] Cossa | Concentrations of mercury in nearshore surface waters of the Bay of Biscay and in the Gironde estuary[END_REF]. Although Faroe Islands are poorly documented regarding Hg levels in seawater, it is likely that metal concentrations in this area would be very low according to the absence of heavy industry in this region. Assuming that cephalopods are short-lived species, they would be useful to provide information on the variation of Hg concentrations in seawater.

CONCLUSION

This study shows that Hg is mainly present under organic form in cephalopods, in which the metal is likely to be bound to proteins in muscles. Because O-Hg is highly bioavailable, cephalopods should therefore be considered as a significant source of Hg for consumers and predators. Among tissues, the digestive gland does not appear as a storage organ of Hg but may allow the partial demethylation of the organic form. In addition, our results suggest that the dissolved pathway could be an important route of uptake for Hg in cephalopods. On this basis, cephalopods would reflect the environmental concentrations of Hg. As they are shortlived and abundant, cephalopods would provide information on short-term variations of Hg concentrations in seawater. Therefore, cephalopods could be an interesting tool to monitor Hg contamination in the marine environment. their participation to the sampling. We are also very grateful to Drs A. Guerra and A.F. González for their help in the determination of the species of octopuses and oceanic squids.

Part of this work was supported financially by the European Union through the BIOCET program (EVK3 -2000 -00027).

Fig. 3

 3 Fig. 3 compares the T-Hg concentrations in the whole individuals of E. cirrhosa, L. forbesi

Fig. 2 .Fig. 3 .

 23 Fig. 2. Relationship between O-Hg and T-Hg (ng.g -1 dwt) in the remaining tissues of cephalopods from the North Eastern Atlantic waters.

Fig. 4 .

 4 Fig. 4. Comparison of the T-Hg concentrations (Mean ± SD; ng.g -1 dwt) in benthic (black) and pelagic (white) cephalopods (whole body). * indicates a significant difference (P ANOVA < 0.05) between the concentrations.

Table 1 .

 1 Characteristics of sampled cephalopods. ND: Not determined.

	Family	Sampling area	Sample Mantle length Fresh weight	Sex		Water content (%)
	Species		Size	(mm)	(g)		Digestive gland	Remaining tissues	Whole individual
	Loliginidae
	Alloteuthis subulata	Bay of Biscay	15	152 ± 32	123 ± 57	7 ♂, 8 ♀	70 ± 3	78 ± 3	78 ± 2
	Loligo forbesi	Faroe Islands	21	152 ± 36	115 ± 59 11 ♂, 10 ♀	73 ± 5	92 ± 2	78 ± 1
	"	Celtic Sea	18	139 ± 26	88 ± 36	13 ♂, 5 ♀	69 ± 3	78 ± 1	78 ± 1
	"	Bay of Biscay	12	119 ± 48	81 ± 81	8 ♂, 4 ♀	69 ± 5	78 ± 1	77 ± 1
	L. vulgaris	Bay of Biscay	21	151 ± 47	117 ± 71 7 ♂, 14 ♀	71 ± 3	78 ± 3	78 ± 3
	Ommastrephidae
	Illex coindetii	of Biscay	22	130 ± 54	158 ± 191 11 ♂, 11 ♀	55 ± 7	80 ± 2	79 ± 2
	Todarodes sagittatus Faroe Islands	37	213 ± 21	203 ± 89 13 ♂, 24 ♀	46 ± 5	83 ± 4	75 ± 2
	"	West Scotland	6	345 ± 50	1244 ± 906 2 ♂, 4 ♀	49 ± 6	78 ± 0	74 ± 2
	"	Celtic Sea	5	449 ± 16	2682 ± 417	5 ♀	32 ± 13	80 ± 2	76 ± 3
	"	Bay of Biscay	5	98 ± 34	93 ± 112	4 ♂, 1 ♀	62 ± 12	84 ± 4	83 ± 4
	Todaropsis eblanae	Bay of Biscay	9	101 ± 43	106 ± 112 5 ♂, 4 ♀	58 ± 7	80 ± 2	78 ± 3
	Histioteuthidae
	Histioteuthis reversa Bay of Biscay	6	38 ± 22	25 ± 47	2 ♀, 4 ND	60 ± 8	87 ± 3	85 ± 3
	Cranchidae
	Teuthowenia megalops Bay of Biscay	1	180	40	ND	46	85	83
	Sepiolidae
	Rossia macrosoma	Bay of Biscay	4	31 ± 4	16 ± 9	3 ♂, 1 ♀	71 ± 5	83 ± 1	81 ± 1
	Sepiidae
	Sepia elegans	Bay of Biscay	14	60 ± 8	28 ± 11	4 ♂, 10 ♀	63 ± 6	76 ± 2	75 ± 2
	S. officinalis	Celtic Sea	1	85	77	1 ♀	70	78	78
	"	Bay of Biscay	23	82 ± 26	96 ± 80	14 ♂, 8 ♀	64 ± 4	78 ± 1	78 ± 1
	S. orbignyana	"	15	74 ± 10	57 ± 22	7 ♂, 8 ♀	60 ± 6	77 ± 2	76 ± 2
	Octopodidae
	Bathypolypus baierii West Scotland	2	44 ± 21	50 ± 51	1 ♂, 1 ♀	71 ± 2	85 ± 0	84 ± 0
	"	Bay of Biscay	2	76 ± 0	203 ± 18	2 ♂	73 ± 10	85 ± 0	85 ± 0
	B. sponsalis	Bay of Biscay	5	41 ± 13	28 ± 15	1 ♂, 4 ♀	72 ± 5	85 ± 1	84 ± 1
	Benthoctopus sp.	Celtic Sea	1	153	1500	1 ♀	56	88	87
	Eledone cirrhosa	Faroe Islands	4	100 ± 8	423 ± 105	4 ♂	66 6	93 ± 4	81 ± 1
	"	Celtic Sea	2	51 ± 4	53 ± 23	1 ♂, 1 ♀	56 ± 14	82 ± 1	81 ± 1
	"	Bay of Biscay	6	85 ± 16	201 ± 142 4 ♂, 2 ♀	53 ± 4	83 ± 1	81 ± 1
	Octopus vulgaris	of Biscay	13	115 ± 19	1388 ± 540 7 ♂, 6 ♀	58 ± 6	83 ± 1	82 ± 1
	O. salutii	Bay of Biscay	6	58 ± 15	94 ± 75	3 ♂, 3 ♀	66 ± 8	82 ± 3	81 ± 4
	Scaergus unicirrhus	Bay of Biscay	1	62	89	1 ♂	60	80	79
	Opisthoteuthidae
	Opisthoteuthis sp.	Celtic Sea	1	-	1111	1 ♂	79	94	94

Table 2 .

 2 Comparison of total and O-Hg concentrations (µg.g -1 dry wt) of certified standards from the NRCC determined in the present study with certified values.

	Metal		DOLT-2 (n = 15)			TORT-2 (n = 15)
		Certified values Present study	Recovery	Certified values Present study	Recovery
	Total Hg	2.14 ± 0.28	2.13 ± 0.03	100%	0.270 ± 0.060	0.269 ± 0.006	100%
	Organic Hg 0.693 ± 0.053	0.763 ± 0.021	109%	0.152 ± 0.060	0.156 ± 0.011	97%

Table 3 .

 3 T-Hg concentrations (Mean ± SD; ng.g -1 dwt) in cephalopods from the North Eastern

	Atlantic. N: sample size
	Family	Sampling area	N	Digestive gland	Remaining tissues	Whole individual
	Species			Mean ± SD	Range	Mean ± SD	Range	Mean ± SD	Range
	Loliginidae
	Alloteuthis subulata	Bay of Biscay	15	ND	-	196 ± 40	121-262	ND	ND
	Loligo forbesi	Faroe Islands	21	196 ± 40	121-262	136 ± 27	86-208	139 ± 27	87-211
	"	Celtic Sea	18	382 ± 232	202-1030	260 ± 68	188-445	265 ± 74	188-458
	"	Bay of Biscay	12	235 ± 104	165-512	179 ± 53	91-645	181 ± 151	95-640
	L. vulgaris	Bay of Biscay	21	406 ± 171	113-681	264 ± 86	113-398	269 ± 88	115-404
	Ommastrephidae
	Illex coindetii	Bay of Biscay	22	192 ± 76	81-357	193 ± 78	61-331	191 ± 75	63-333
	Todarodes sagittatus	Faroe Islands	37	54 ± 16	25-91	71 ± 22	39-160	67 ± 17	35-116
	"	West Scotland	6	318 ± 154	198-613	489 ± 245	251-945	440 ± 220	245-855
	"	Celtic Sea	5	511 ± 552	186-1485	740 ± 208	424-991	660 ± 207	372-879
	"	Bay of Biscay	5	168 ± 52	112-231	188 ± 89	73-289	178 ± 67	81-236
	Todaropsis eblanae	Bay of Biscay	9	217 ± 108	120-463	281 ± 129	130-500	284 ± 120	140-497
	Histioteuthidae
	Histioteuthis reversa	Bay of Biscay	6	88 ± 44	31-137	102 ± 31	65-147	99 ± 31	57-144
	Cranchidae
	Teuthowenia megalops Bay of Biscay	1	-	172	-	205	-	199
	Sepiolidae
	Rossia macrosoma	Bay of Biscay	4	916 ± 83	310-2279	313 ± 190	52-743	394 ± 388	88-913
	Sepiidae
	Sepia elegans	Bay of Biscay	14	614 ± 160	332-937	237 ± 92	90-406	265 ± 92	111-443
	S. officinalis	Celtic Sea	1	-	650	-	980	-	960
	"	Bay of Biscay	23	531 ± 274	196-1269	235 ± 105	98-596	262 ± 119	132-677
	S. orbignyana	"	15	467 ± 161	241-757	118 ± 35	63-176	144 ± 34	90-195
	Octopodidae
	Bathypolypus baierii	West Scotland	2	791± 239	622-961	415 ± 92	350-480	453 ± 104	379-526
	"	Bay of Biscay	2	1650 ± 453 1330-1970	337 ± 71	287-387	424 ± 110	346-501
	B. sponsalis	Bay of Biscay	5	1003 ± 233	796-1370	216 ± 32	185-269	304 ± 47	274-387
	Benthoctopus sp.	Celtic Sea	1	-	1090	-	921	-	942
	Eledone cirrhosa	Faroe Islands	4	184 ± 65	91-241	144 ± 71	79-234	146 ± 70	80-233
	"	Celtic Sea	2	243 ± 168	124-362	504 ± 418	208-800	477 ± 390	201-753
	"	Bay of Biscay	6	457 ± 36	422-524	298 ± 52	265-402	325 ± 47	295-419
	Octopus vulgaris	Bay of Biscay	13	1141 ± 242	844-1690	381 ± 99	236-640	445 ± 92	297-662
	O. salutii	Bay of Biscay	6	1950 ± 923 1001-3560	462 ± 161	302-767	617 ± 297 419-1207
	Scaergus unicirrhus	Bay of Biscay	1	-	979	-	422	-	496
	Opisthoteuthidae
	Opisthoteuthis sp.	Celtic Sea	1	-	610	-	993	-	971
	ND: Not determined.

Table 4 .

 4 O-Hg concentrations (Mean ± SD; ng.g -1 dwt) and their relative contributions (% of the T-Hg) to tissue content and to whole body content in selected cephalopod species.

	Species	Sampling area	N		O-Hg concentrations		% of O-Hg / T-Hg concentration	% of whole body O-Hg
					(ng.g -1 dwt)
				Digestive gland Remaining tissues Whole individual	Digestive gland Remaining tissues Whole individual	Digestive gland Remaining tissue
	Loligo forbesi	Faroe Islands	5	83 ± 17	110 ± 21	106 ± 19	40 ± 8	79 ± 8	72 ± 9	15 ± 1	85 ± 1
		Celtic Sea	5	181 ± 50	233 ± 69	272 ± 82	44 ± 18	86 ± 4	84 ± 4	3 ± 1	98 ± 1
		Bay of Biscay	5	153 ± 40	115 ± 38	136 ± 50	53 ± 2	77 ± 13	75 ± 12	5 ± 2	95 ± 2
	Todarodes sagittatus	Faroe Islands	10	38 ± 11	82 ± 31	70 ± 18	60 ± 8	80 ± 12	74 ± 11	12 ± 4	88 ± 4
		Celtic Sea	5	171 ± 175	637 ± 234	531 ± 207	37 ± 12	84 ± 9	79 ± 7	6 ± 3	94 ± 3
		Bay of Biscay	3	93 ± 31	137 ± 78	126 ± 62	56 ± 8	68 ± 17	67 ± 15	16 ± 13	84 ± 13
	Histioteuthis reversa	Bay of Biscay	1	63	143	119	46	98	83	7	93
	Teuthowenia megalops	Bay of Biscay	1	63	192	166	37	94	84	5	95
	Benthoctopus sp.	Celtic Sea	1	706	818	804	65	89	85	10	90
	Eledone cirrhosa	Faroe Islands	4	102 ± 38	124 ± 55	123 ± 54	55 ± 7	88 ± 9	85 ± 8	14 ± 4	86 ± 4
		Celtic Sea	2	153 ± 138	469 ± 377	437 ± 349	57 ± 17	95 ± 3	93 ± 3	6 ± 1	94 ± 1
		Bay of Biscay	6	100 ± 38	267 ± 66	239 ± 39	22 ± 9	89 ± 7	73 ± 7	7 ± 3	93 ± 3
	Scaergus unicirrhus	Bay of Biscay	1	352	413	405	36	98	82	16	84

Table 5 .

 5 Sex and size (mantle length) variations of T-Hg concentrations in cephalopods from the North Eastern Atlantic waters (whole body): results of ANCOVA tests give the F-value and the associated probability (p) in brackets. Statistical significance is shown in bold face.

	Location Species	N	Size	Sex	Size * Sex
	Faroe Islands
	Loligo forbesi	21	29.737	4.255	7.972
			(<0.0001)	(0.055)	(0.012)
	Todarodes sagittatus	37	2.461	0.547	0.828
			(0.126)	(0.465)	(0.370)
	Celtic Sea
	Loligo forbesi	18	20.811	0.010	10.571
			(0.0004)	(0.922)	(0.006)
	Bay of Biscay
	Loligo forbesi	12	2.114	0.001	0.037
			(0.184)	(0.978)	(0.852)
	L. vulgaris	21	13.048	3.853	4.155
			(0.002)	(0.066)	(0.057)
	Illex coindetii	22	3.025	0.275	0.949
			(0.099)	(0.607)	(0.343)
	Sepia elegans	14	0.925	1.900	1.904
			(0.359)	(0.198)	(0.198)
	S. officinalis	23	0.002	1.222	0.070
			(0.964)	(0.283)	(0.794)
	S. orbignyana	15	2.530	0.038	0.001
			(0.140)	(0.849)	(0.980)
	O. vulgaris	13	2.336	3.208	2.480
			(0.161)	(0.107)	(0.150)

Table 6 .

 6 Mean ± SD (ng.g -1 dwt) of T-Hg and O-Hg concentrations and percentage of the O-Hg in the tissues of cephalopods from the literature. Percentage of the whole body burden of T-Hg (% dwt) contained in the digestive gland (black) and the remaining tissues (white) of cephalopods from the North Eastern Atlantic waters.

	Family	Sampling area	Tissue	N	T-Hg	% O-Hg	Reference
	Species				Mean ± SD
	Cephalopod	Adriatic Sea	Edible parts	37	528 ± 282*	-	Sapunar et al (1989)
	Squid	Japan	Edible parts	6	120-1320*	39-64	Nagakura et al (1974)
	Squid	United Sates	Edible parts	1	486*	35	Cappon & Smith (1982)
	Loliginidae
	Loligo forbesi	Azores	Muscle	72	648 ± 396*	-	Monteiro et al. (1992)
	L. patagonica	Argentina	Mantle	3	72*	-	Falandysz (1989)
	Loligo vulgaris	Adriatic Sea	Edible parts	NA	1530*	63	Buzina et al. (1989)
	Loligo vulgaris	North Morocco	Edible parts	10	1920*	-	Stoepler et al. (1979)
	Loligo vulgaris	North Sea	Edible parts	10	600*	-	"
	Loligo vulgaris	Adriatic Sea	Edible parts	NA	534 ± 42 *	-	Plessi et al. (2001)
	Ommastrephidae
	Illex coindetii	Adriatic Sea	Muscle	15	17980 ± 906*	-	Storelli & Marcotrigiano
	Illex coindetii	Adriatic Sea	Digestive gland	15	396 ± 165*	-	(1999)
	Todarodes pacificus	Japan	Muscle	NA	510* a	-	Ichihaschi et al. (2001b)
	Todarodes pacificus	Japan	Digestive gland	NA	149* a	-	"
	Todarodes sagittatus	Azores	Muscle	2	300 ± 72*	-	Monteiro et al. (1992)
	Ommastrephes bartrami	Azores	Muscle	14	282 ± 186*	-	"
	Sthenoteuthis oualaniensis	Japan	Digestive gland	8	231*	-	Ichihashi et al. (2001a)
	Sepiidae
	Sepia officinalis	Adriatic Sea	Edible parts	NA	1416*	83	Buzina et al. (1989)
	Sepia officinalis	Sardinia	Edible parts	8	1200*	-	Stoepler et al. (1979)
	Sepia officinalis	Adriatic Sea	Edible parts	34	930*	-	"
	Sepia officinalis	North Morocco	Edible parts	15	750*	-	"
	Sepia officinalis	North Sea	Edible parts	13	480*	-	"
	Sepia officinalis	Adriatic Sea	Edible parts	NA	444 ± s *	-	Plessi et al. (2001)
	Octopodidae
	Octopus	United States	Edible parts	1	1356	47	Cappon & Smith (1982)
	Eledone moschata	Adriatic Sea	Edible parts	NA	138*	-	Plessi et al. (2001)
	Eledone sp.	Mediterranean	Edible parts	3	2400*	-	Stoepler et al. (1979)
	Octopus salutii	Adriatic Sea	Muscle	17	1620 ± 480*	-	Storelli & Marcotrigiano
	Octopus salutii	Adriatic Sea	Digestive gland	17	2772 ± 1518*		(1999)
	Octopus vulgaris	Tyrrhenian Sea	Muscle	5	9900 ± 3800*	-	Renzoni et al. (1973)
	Octopus vulgaris	Tyrrhenian Sea	Digestive gland	5	367000 ± 274000*	-	"
	Octopus vulgaris	Tyrrhenian Sea	Kidney	4	31100 ± 9400*	-	"
	Octopus vulgaris	Tyrrhenian Sea	Gills	5	7000 ± 3300*	-	"
	Octopus vulgaris	Tyrrhenian Sea	Gonad	5	5600 ± 1900*	-	"
	Octopus vulgaris	Adriatic Sea	Edible parts	NA	3120	95	Buzina et al. (1989)
	Octopus vulgaris	Azores	Muscle	96	384 ± 348*	-	Monteiro et al. (1992)
	Octopus vulgaris	Sardinia	Edible parts	5	1728*	-	Stoepler et al. (1979)
	Octopus vulgaris	North Morocco	Edible parts	6	690*	-	"
	Octopus vulgaris	Adriatic Sea	Edible parts	4	690*	-	"
	* calculated from fresh weight; NA: not available.

BBA : Bathypolypus baierii ; BER: Benthoctopus sp.; BSP: Bathypolypus sponsalis ; ECI: Eledone cirrhosa; HRE : Histioteuthis reversa; ICO: Illex coindetii; LFO : Loligo forbesi; LVU : L. vulgaris; OPI: Opisthoteuthis sp.; OSA : Octopus salutii ; OVU: Octopus vulgaris; RMA: Rossia macrosoma ; SEL: Sepia elegans; SOF: S. officinalis; SOR: S. orbignyana; SUN : Scaergus unicirrhus; TEB: Todaropsis eblanae; TME : Teuthowenia megalops; TSA: Todarodes sagittatus.

Acknowledgements

We thank the 'Fiskirannsoknarstovan' (Torshavn, Faroe Islands) and IFREMER (Direction des Ressources Halieutiques, Nantes) for facilitating collections of the cephalopods on the RVs Magnus Heinasson and Gwen-Drez, and E. Gironnet, R. Mouritsen and J. Labastie for