Moncef Rjeibi

Marc Metian

Tarek Hajji

Thierry Guyot

Rafika Ben Chaouacha- Chekir

Paco Bustamante

Prof Paco Bustamante

INTERSPECIFIC AND GEOGRAPHICAL VARIATIONS OF TRACE METAL CONCENTRATIONS IN CEPHALOPODS FROM TUNISIAN WATERS

Keywords: Octopus, Cuttlefish, Squid, Trace elements, Bioaccumulation, Mediterranean sea

The concentrations of 6 metals (Ag, Cd, Cu, Hg, Pb, and Zn) were investigated and compared in three tissues (arms, digestive gland and mantle) of three cephalopod species from the Tunisian waters: the common octopus (Octopus vulgaris), the common cuttlefish (Sepia officinalis) and the European squid (Loligo vulgaris). Whatever the species or the sites, the digestive gland displayed the highest concentrations of Ag, Cd, Cu, Pb and Zn highlighting its major role in their bioaccumulation and detoxification. This is also true for Hg but only for the digestive gland of O.vulgaris. Muscle from the arms and the mantle contained thus relatively low trace metal concentrations except for Hg in L.vulgaris and S.officinalis.

Geographic comparison of metal concentrations in Tunisian cephalopods from 3 locations indicates that higher concentrations of Ag, Pb and Hg were observed in cephalopods from northern and eastern coasts, whereas the highest Cd levels was detected in the southeastern reflecting different conditions of exposure. Comparing the trace element concentrations between species, Ag, Cd, Cu, Hg and Zn concentrations were the highest in the digestive gland of octopuses. This may be related to the differences in ecological features and swimming behaviour among different cephalopod species. Effects of length and sex on metal levels were also considered, indicating a limited influence of sex on metal concentration.

INTRODUCTION

Metals in cephalopods have received increasing attention in last decades as these molluscs play a major role both as predator and prey in marine ecosystems [START_REF] Boyle | Cephalopods: Ecology and Fisheries[END_REF]. As cephalopods bioaccumulate large quantities of various metals, they represent an important source of contaminants for their predators (e.g. Bustamante et al., 1998a[START_REF] Dorneles | Cephalopods and cetaceans as indicators of offshore bioavailability of cadmium off Central South Brazil Bight[END_REF]. Cephalopods also represent an increasing resource for humans [START_REF] Piatkowski | Impact of cephalopods in the food chain and their interaction with the environment and fisheries: an overview[END_REF] and consequently cephalopod catches raised dramatically these last decades [START_REF] Boyle | Cephalopods: Ecology and Fisheries[END_REF]FAO, 2007).

Cephalopods are generally short-lived species, most of them reproducing between 1 to 2 years old and then die. Because of their short-life span and their capacity to bioaccumulate very high metal concentrations in their tissues, cephalopods were proposed as reliable biomonitoring species to follow the variation of pollutants in the marine environment over time [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]Bustamante et al. 2006;[START_REF] Miramand | Variation of heavy metal concentrations (Ag, Cd, Co, Cu, Fe, Pb, V, and Zn) during the life cycle of the common cuttlefish Sepia officinalis[END_REF][START_REF] Pierce | Geographic, seasonal and ontogenetic variation in cadmium and mercury concentrations in squid (Cephalopoda: Teuthoidea) from UK waters[END_REF][START_REF] Kojadinovic | Multi-elemental concentrations in the tissues of the oceanic squid Todarodes filippovae from Tasmania and the southern Indian Ocean[END_REF]).

In the Mediterranean Sea, trace metal concentrations have been reported in different cephalopod species (i.e., Sepia officinalis, Octopus vulgaris and Eledone cirrhosa) collected from the Northern and Northeastern region [START_REF] Renzoni | Mercury concentration in the water, sediments and fauna of an area of the Tyrrhenian coast[END_REF][START_REF] Miramand | High concentrations of some heavy metals in tissues of the Mediterranean octopus[END_REF][START_REF] Ayas | The Effects of Season and Sex in the Metal Levels of Mature Common Cuttlefish (Sepia officinalis) in Mersin Bay, Northeastern Mediterranean[END_REF]. On the contrary, there is still limited information available on cephalopods from African coasts of the Mediterranean Sea [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]. To the best of our knowledge, no data on metal concentrations in cephalopod species from Tunisia have been reported while the fisheries of these molluscs have a very high economic importance in this country.

In this context, this study was undertaken (a) to investigate the geographical variations of trace metals in the different tissues of the European squid Loligo vulgaris, the common octopus Octopus vulgaris and the common cuttlefish Sepia officinalis in three locations of the Tunisian coast and (b) to establish comparison between species in every sampling sites (c) to examine the influence of size and the sex on metal concentration in cephalopods. For this purpose, Ag, Cd, Cu, Hg, Pb and Zn were determined in the digestive gland and the muscles (both mantle and arms) of Loligo vulgaris, O. vulgaris and S. officinalis.

MATERIALS AND METHODS

Biological material and sample preparation

Seventy one individuals belonging to three cephalopod species, the european squid Loligo vulgaris (n=24), the common octopus Octopus vulgaris (n=20) and the common cuttlefish Sepia officinalis (n=27) (see Table 1 for details), were sampled in April 2010 in three locations, all situated in the authorized fishing area of the Tunisian coast (Fig. 1).

The first location, Bizerte, lies in the north of Tunisia and characterized with many industrial activities especially plastic and iron industries [START_REF] El Ati-Hellal | Lead and cadmium concentrations in seawater and algae of the tunisian coast[END_REF]. The second site, Monastir, is located in the middle of east coast of Tunisia; the coastal area of this city can be characterized as unpolluted, given the non apparent sources of contamination (Hamza-Chaffai et al., 1997;[START_REF] Banni | Assessment of heavy metal contamination using real-time PCR analysis of mussel metallothionein mt10 and mt20 expression: a validation along the Tunisian coast[END_REF][START_REF] Jebali | Oxidative DNA damage levels and catalase activity in the clam Ruditapes decussatus as pollution biomarkers of Tunisian marine environment[END_REF]. The last sampling site, Sfax, is located in the Gulf of Gabés (southeastern coast of Tunisia) and is considered as a major hub for Tunisian aquatic resources, contributing to approx. 65% of the national production [START_REF] Cgp | Annuaire des statistiques des pêches en Tunisie[END_REF]. Important industrial activities, mainly crude phosphate treatments, chemical industries, tannery and plastic plants, are being developed along this region, possibly affecting the local marine ecosystems [START_REF] Hamza-Chaffai | Physico-chemical forms of storage of metals (Cd, Cu and Zn) and metallothionein-like proteins in gills and liver of marine fish from the Tunisian coast: ecotoxicological consequences[END_REF]; [START_REF] Boujelben | Dosage de métaux lourds (Plomb, Cadmium, Cuivre, Zinc) dans les poissons péchés dans la région de Sfax[END_REF]; [START_REF] Banni | Monitoring pollution in Tunisian coasts: application of a classification scale based on biochemical markers[END_REF].

Cephalopods were collected by nets or trawling by professional fishermen. From the moment organisms were caught, they were placed on ice for the time they were brought to the laboratory and then rapidly frozen at -25° C in individual plastic bags until dissections. Each individual was then defrosted, weighed (total body weight) and measured (dorsal mantle length -DML-). The determination of the sex and maturity stage were also realized. The maturity stage was evaluated by direct observation of the reproductive structures colors. The origin, number of individuals and biological details of individuals for each species of cephalopods are given in Table 1. Subsequently, samples of arm muscle, mantle muscle and digestive gland were removed for trace metal analysis.

Metal analysis

After dissection, tissue samples were frozen at -20°C, freeze-dried during 3 days and then finely ground in a porcelain mortar with a pestle. One aliquot of approximately 300 mg of each homogenized dry sample was digested with 5 ml of 65% ultrapure HNO 3 (Merck ®) at 80°C until the solution was clear. Then acid was evaporated and the residue was dissolved in 0.3N ultrapure nitric acid. Concentrations of Ag, Cd, Cu, Pb and Zn were determined by flame and graphite furnace atomic absorption spectrophotometry with a Hitachi Z5000 with Zeeman correction. The total Hg concentrations in the powder obtained from the tissues were determined by analyzing Hg directly with an Advanced Mercury Analyzer (ALTEC AMA 254) on aliquots ranging from approx. 10 mg of dry sample weighed to the nearest 0.01 mg (Bustamante et al. 2006a). Certified reference materials, Dogfish Liver DOLT-4 (NRCC), Lobster Hepatopancreas Tort-2 (NRCC) and Dogfish Muscle DORM-2 (NRCC), were treated and analysed in the same way as the samples. The results for standard reference materials were in good agreement with certified values with recoveries ranging from 91% for Ag to 98% for Hg (Table 2). The detection limits (μg g -1 dw) were 0.03 for Ag, 0.034 for Cd, 0.5 for Cu, 0.005 for Hg, 3.2 for Pb and 3 for Zn.

Statistical analysis

All data were tested for normality by Shapiro-test and homogeneity of variances by Bartlett's test. Since data did not respect the former assumptions of parametric analysis, non-parametric tests were applied. Kruskal-Wallis test (K-W test) followed by a multiple comparison test with Holm adjustment method was performed to detect differences between geographical areas, and between tissues in species. The level of significance for statistical analyses was always set at α=0.05.

Principal Component Analysis (PCA) was used to investigate the distribution of metal concentrations in the selected tissues within the species from the 3 locations (for details on PCA used, see [START_REF] Metian | Trace element bioaccumulation in reef fish from New Caledonia: Influence of trophic groups and risk assessment for consumers[END_REF].

Generalized Additive Models (GAMs) were used to identify size-related, spatial and sex trends in explaining variability in metals concentrations [START_REF] Zuur | Analysing Ecological Data[END_REF]. GAMs were fitted to average log-transformed on metal concentrations in digestive gland, arms, and mantle for each species and the dorsal mantle (DML) was considered as a continuous explanatory variable, while the sex and the location were treated as categorical explanatory variables in the model. The assumption of Gaussian error distributions was finally checked through the residuals of the model (homogeneity, normality, and no obvious pattern in residuals in general). All statistical analyses were performed using the free software R (R Development Core Team, 2010).

RESULTS

Metal concentrations and tissue distribution

Ag, Cd, Cu, Hg, Pb and Zn concentrations in the 3 selected tissues (digestive gland, arms, and mantle) of L. vulgaris, O. vulgaris, and S. officinalis are illustrated in Figure 2. Compared with the other tissues examined and regardless of the geographic location, the digestive gland contained the highest levels of Ag, Cd, Cu, Pb, and Zn (K-W test, p < 0.001) in all species.

Concentration of Hg was also significantly higher (K-W, p < 0.001) in the digestive gland of O. vulgaris whereas its concentrations did not show significant differences among tissues of L. vulgaris and S. officinalis (K-W test, p > 0.05). In all species and with the exception of Hg, metal concentrations in the muscle were relatively low and were in the same range of concentration among the species.

Geographical variation

Metal concentrations in the tissues of cephalopods greatly differ both with species and sampling sites (Fig. 2). Hg concentrations were significantly higher (K-W, p<0.05) in L. vulgaris from Bizerte and Monastir compared to Sfax for all tissues. The same pattern was observed for Cd, Cu and Zn concentrations with significantly higher Cd and Zn concentrations in the arms and in the digestive gland in squids from Monastir (K-W, p < 0.05) and higher Cu concentrations in the mantle in squids from Bizerte (K-W, p < 0.01) compared to those from Sfax.

In O. vulgaris, specimens from Monastir and Bizerte showed also significantly higher Cu and Hg concentrations (K-W, p < 0.05) in the arms than those of Sfax. On the contrary, Cd concentrations in the digestive gland and the muscle tissues were higher (K-W, p<0.05) in Sfax than in the other sites. Similarly, the southeastern coast exhibited higher Zn concentrations (K-W, p<0.05) in the muscle tissues of the common octopus (Fig. 2).

Metal levels in the tissues of S. officinalis showed significant spatial variation as well.

Concentrations were significantly higher in Bizerte and Monastir for Ag in the digestive gland (K-W, p<0.05) and for Cu and Zn in both digestive gland and arms (K-W, p<0.05). In contrast, cuttlefish from Sfax showed higher concentrations for Cd in both digestive gland and mantle (K-W, p<0.05), and equal or higher for Hg according to the tissue (Fig. 2).

Interspecies comparison

Considering all sites together, Kruskal-Wallis one-way analysis of variance indicated that trace element concentrations between species are most of the time significantly different (p<0.05; comparison by tissues and trace elements). However, no difference was observed for Cd concentrations comparing mantles of the 3 species (p=0.45). Multiple comparisons were then performed and highlighted some specific differences between species. Considering the digestive gland for instance, O. vulgaris showed the highest concentrations of Ag, Cd and Hg among species (K-W, p<0.05); Cu and Zn concentrations were similar to S. officinalis and higher (K-W, p<0.05) than those of L. vulgaris. According to the multiple comparisons, Cu and Zn concentrations in the mantle were the highest in O. vulgaris compared to the other species (K-W, p<0.05) whereas Hg concentrations were significantly higher in squids than octopus (K-W, p<0.05). Finally, the common squid displayed the highest (K-W, p<0.05) levels for Cd and Hg in their arms compared to other investigated species whereas it presented the lowest Cu and Zn levels (K-W, p<0.05).

The Principal component analyses (PCA; integrating all the trace elements) were realized for each tissues (Figs. 3, 4, and5). They showed a clear pattern of trace element concentrations for interspecies variation. Indeed, this analysis tore more or less species apart, especially for the digestive glands and arms (Figs. 3C and4C). The first 2 principal components accounted for 74.44% of the variability in the metal concentrations in the digestive gland, with 52.54% on axis 1 and 21.90% on axis 2 (Fig. 3). Species were separated according to the axis 1 which clearly discriminated O. vulgaris and L. vulgaris relatively to the levels of Ag, Cd, Cu and Zn.

On the second axis, O. vulgaris and L. vulgaris were mainly distributed on the PCA according the relative levels of Hg (Fig. 3C).

In the arms, the PCA showed that the first two axes accounted 68.66% of variability, with 40.02% explained by axis 1 and 28.64% by axis 2 (Fig. 4). The first axis takes into account the relative levels of Cd and Zn, while the second axis is correlated with the Cu and Hg levels.

Squids were clearly discriminated according to their high Hg and Cd levels whereas octopus and cuttlefish, thight on this PCA, were differentiated from squids according to their Cu and Zn levels (Fig. 4C).

Figure 5 shows the PCA realized for the mantle. Axis 1 and 2 of the PCA here accounted for 65.08% of the variability in metal levels in the mantle between individuals, with 36.69% on axis 1 and 28.38% on axis 2 (Fig. 5). Cu and Zn strongly influenced variability between species along axis 1 whereas Cd and Hg concentrations contributed to axis 2. Species were not clearly separated in comparison to the 2 previous PCA perfomed on digestive gland and arms (Fig. 5C vs. Figs. 3C and4C). Nevertheless, L. vulgaris seems to be related to elevetated concentrations of Hg and Cd as in arms.

Although the aim of these PCAs was to illustrate the interspecies comparison, the regroupment of samples through sampling location (Figs. 3D, 4D and 5D) complement our first approach showing, when metals are integrated through 2 components, the superposition of Sfax and Monastir profile and therefore the different profile of Bizerte. For instance, Hg seems the main driver of Bizerte profile (Figs. 3D, 4D and5D).

Effects of sex and size on metals accumulation

Results of Generalized additive models (GAMs) fitted to the average log-transformed metal concentrations in the three selected tissues of L. vulgaris did not show any significant differences for Ag, Cd and Cu concentrations considering sex and size. However, the GAMs established for Hg concentrations showed significant size effects, being negatively correlated with the body size (DML) in the digestive gland (P=0.015), mantle (P< 0.01) and arms (P=0.018) (Figs. 6A, 6B and 6C). In contrast, a strong positive linear effect of DML (P<0.01) on Hg concentrations was detected in the mantle (Fig. 6D).

In the case of O. vulgaris, the effect of size was detected for Cu, Hg and Pb concentrations: negative correlation was found between size and the digestive gland (P<0.01) and the mantle (P<0.001) for Hg (Figs. 7A and7B), the digestive gland (P=0.049) for Pb (Fig. 7C) and in the arms (P=0.027) for Cu (Fig. 7D). An effect of sex on which males showed significantly higher concentrations than females, was also found for Hg in the digestive gland (p=0.020), for Hg and Cu in the mantle (P=0.033 and P=0.023, respectively, and for Zn in the arms (P<0.01).

For S. officinalis, significant differences for size were included in the GAMs fitted for Hg and Cu concentrations. A linear positive correlation was detected between Hg levels and the body size in the digestive gland (P=0.021), the mantle (P<0.001) and the arms (P<0.01) (Figs. 8A, 8B and 8C) whereas Cu concentration decreased with size (P=0.011) (Fig. 8D). Considering the sex effect, males showed significantly lower Hg levels in the mantle (P=0.034) and the arms (P=0.035) than females.

DISCUSSION

Cephalopods are characterized by a strong ability to highly bioaccumulate trace elements in their tissues [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., 1998b;[START_REF] Manso | Characterization of essential and toxic elements in cephalopod tissues by EDXRF and AAS[END_REF]; the digestive gland appears to be the key organ, being deeply involved in the storage and/or detoxification of various essential and non essential metals of cephalopods (Bustamante et al., 2002a;[START_REF] Miramand | Variation of heavy metal concentrations (Ag, Cd, Co, Cu, Fe, Pb, V, and Zn) during the life cycle of the common cuttlefish Sepia officinalis[END_REF][START_REF] Raimundo | Metallothioneins and trace elements in digestive gland, gills, kidney and gonads of Octopus vulgaris[END_REF]. Since they are short-lived organisms, cephalopods are also interesting in reflecting the variation of metal concentrations in the environment where they live [START_REF] Pierce | Geographic, seasonal and ontogenetic variation in cadmium and mercury concentrations in squid (Cephalopoda: Teuthoidea) from UK waters[END_REF]. In the present study, the analysis of 6 metals (Ag, Cd, Cu, Hg, Pb and Zn) in edible tissues (muscles of both arms and mantle) and in the digestive gland of 3 major cephalopod species from Tunisia. It provides new dataset on an important seafood resources in the south Mediterranean Sea and it informs on the variation of contamination status along the entire tunisian coast.

Comparison between species

The analysis of metals in the three species (L. vulgaris, O. vulgaris and S. officinalis) with different lifestyle highlighted differences in their capacity of metal accumulation, each species consistently exhibiting different metal concentrations along the Tunisian coasts. For instance, the highest concentrations of Ag, Cd, Cu and Zn were found in octopuses and the lowest in squids from each locality. This result is in accordance with previous observations for the two same species in the Portuguese waters [START_REF] Lourenço | Elemental composition of cephalopods from Portuguese continental waters[END_REF]. Furthermore, Bustamante et al. 2002) showed that benthic cephalopods displayed significantly higher Cd, Cu and Zn concentrations than pelagic ones, which could be explained by differences in their feeding behavior and their physiology. On the contrary, L. vulgaris showed the highest levels of Hg for all tissues in the northern coast, in both arms and mantle in the eastern coast and only in the arms in the southeastern coast. Food represents the main accumulation pathway for Hg in cephalopods (Lacoue-Labarthe et al., 2009b) but benthic cephalopods exhibit more complex diets than pelagic ones. Indeed, they feed on polychets, crustaceans, molluscs, echinoderms and fish [START_REF] Mcquaid | Feeding behaviour and selection of bivalve prey by Octopus vulgaris Cuvier[END_REF] whereas squids mainly feed on fish and other cephalopod species [START_REF] Pierce | Diets of squid Loligo forbesi and Loligo vulgaris in the northeast Atlantic[END_REF]. In contrast to benthic prey, pelagic fish and cephalopods contain higher proportion of organic Hg [START_REF] Bloom | On the chemical form of mercury in edible fish and marine invertebrate tissue[END_REF]Bustamante et al., 2006b) which is more bioavailable than inorganic Hg. Such a pelagic diet would lead to higher Hg exposure and therefore to higher Hg bioaccumulation in pelagic cephalopods since their prey contain higher organic Hg loads than those of benthic ones [START_REF] Cossa | Le mercure en milieu marin. Biogéochimie et écotoxicologie[END_REF].

Metal distribution among tissues

Highest concentrations of Ag, Cd, Cu, Pb and Zn were found in the digestive gland of all species similarly to the same species from other locations [START_REF] Miramand | High concentrations of some heavy metals in tissues of the Mediterranean octopus[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., 2002a;Bustamante et al., 2002b ;[START_REF] Nessim | Bioaccumulation of heavy metals in Octopus vulgaris from coastal waters of Alexandria (Eastern Mediterranean)[END_REF][START_REF] Raimundo | Geographical variation and partition of metals in tissues of Octopus vulgaris along the Portuguese coast[END_REF][START_REF] Napoleão | Elemental characterization of tissues of Octopus vulgaris along the Portuguese coast[END_REF][START_REF] Raimundo | Fe, Zn, Cu and Cd concentrations in the digestive gland and muscle tissues of Octopus vulgaris and Sepia officinalis from two coastal areas in Portugal[END_REF]Bustamante et al., 2008;[START_REF] Pereira | Metal concentrations in digestive gland and mantle of Sepia officinalis from two coastal lagoons of Portugal[END_REF]. The intrinsic capacity of this organ to store these elements is likely related to its major role in the assimilation of trace metals and nutrients [START_REF] Martin | High copper concentrations in squid livers in association with elevated levels of silver, cadmium, and zinc[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., 2002a;Bustamante et al., 2006b;Lacoue-Labarthe et al., 2009a). Cephalopods are known for their high feeding rates and their elevated assimilation efficiencies to satisfy their fast growth rates. As a consequence of strong assimilation of essential and non essential metals, cephalopods evolved efficient detoxification processes in order to cope with their toxicity (Bustamante et al., 2002a). In this context, Hg generally appears to be an exception in the present study. Hg levels in the digestive gland of L. vulgaris and S. officinalis were generally similar than those measured in the muscle (both from arms and mantle), this result being consistent with previous works on squids from the Northern atlantic [START_REF] Pierce | Geographic, seasonal and ontogenetic variation in cadmium and mercury concentrations in squid (Cephalopoda: Teuthoidea) from UK waters[END_REF] and from the Indian and Pacific oceans [START_REF] Kojadinovic | Multi-elemental concentrations in the tissues of the oceanic squid Todarodes filippovae from Tasmania and the southern Indian Ocean[END_REF]. In addition, Lacoue-Labarthe and coauthors (Lacoue-Labarthe et al., 2009b) reported that juvenile cuttlefish exposed to inorganic Hg via seawater accumulated mainly Hg in muscular parts (>70%). However, the proportion of the whole body Hg content associated with the digestive gland increased during exposure and depuration phases, suggesting that the metal was detoxified from the muscles towards this organ.

Our results confirmed that Hg organotropism in cephalopods differs according to the phylogeny (Sepiida and Teuthida vs Octopoda). Indeed, the digestive gland of the common octopus O. vulgaris displayed the highest Hg concentrations among the three tissues, which contrasts with L. vulgaris and S. officinalis. It is also consistent with previous works on different octopus species along the European coasts [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]Bustamante et al., 2006a) that reported elevated Hg concentrations in ocopus. The preferential concentration of Hg in digestive gland of O. vulgaris might be related to different Hg uptake pathway in O. vulgaris compared to the other species and most probably a higher contribution of food pathway to global upake or a higher retention of Hg from octopuses than the others (Bustamante et al. 2006).

Accumulation differences between sites

As mentioned previously, cephalopods are short-lived species and thus they are supposed to reflect short-term ambient concentrations of metals in the surrounding environment [START_REF] Bustamante | Metal and metalloid concentrations in the giant squid Architeuthis dux from Iberian waters[END_REF][START_REF] Pierce | Geographic, seasonal and ontogenetic variation in cadmium and mercury concentrations in squid (Cephalopoda: Teuthoidea) from UK waters[END_REF]. Due to the low metal concentrations found in the muscle tissues and their low variability of all species, the digestive gland was used to compare the Ag, Cu, Cd, Pb and Zn concentrations between sampling sites. Geographical variation of metal concentrations was not similar for all the species: concentrations of Ag and Pb found in cuttelfish and octopus were significatly higher in the northern (Bizerte) and eastern (Monastir) coasts than in the southeastern (Sfax). Interestingly, Ag and Pb concentrations did not vary in squids, which can be related the difference in diet but aslo the different habitats. Squids are neritic and/or oceanic cephalopods eaten mainly on fish whereas octopuses and cuttlefishes are more benthic cephalopods (closer to the coast) feeding most of the time on bottom invertebrates such crustaceans, bivalves and polychetes [START_REF] Boyle | Cephalopod biology in the fisheries context[END_REF][START_REF] Mcquaid | Feeding behaviour and selection of bivalve prey by Octopus vulgaris Cuvier[END_REF]. A previous study conducted along the Tunisian coast on algae showed that the highest concentration of Pb was observed in Bizerte; important industrial releases, the sewage wastes, harbors and the ferry traffic in Bizerte could be the source [START_REF] El Ati-Hellal | Lead and cadmium concentrations in seawater and algae of the tunisian coast[END_REF]. A similar geographical trend was observerd for the Cu concentrations in cuttlefish and Zn concentrations in both squids and cuttlefish. This variation of Cu and Zn in the digestive gland is difficult to confront with the environmental data due to the lack of reliable measurements. However, the uniform concentrations of Zn in octopuses and Cu in squids and octopuses captured along the Tunisian coast may be explained by metabolic functions such as homeostatic mechanisms that regulate these levels in the organism [START_REF] Canli | Metal (Cd, Pb, Cu, Zn, Fe, Cr, Ni) Concentrations in Tissues of a Fish Sardina pilchardus and a Prawn Peaenus japonicus from Three Stations on the Mediterranean Sea[END_REF].

Cd behavior was distincted from the other metals, especially in the case Cd concentrations in octopus and cuttlefishes from southeastern coast: they were higher than the Cd concentrations from the northern and eastern coasts. This could be related to the higher level of Cd in the coastal waters of Sfax subjected to the continuous discharge of metals from local industrial activities and from the phosphogypsum stock (Hamza-Chaffai et al., 1997). Several reports indicated the contaminated status of the coast, especially by Cd [START_REF] Hamza-Chaffai | Health assessment of a marine bivalve Ruditapes decussatus from the Gulf of Gabès (Tunisia)[END_REF][START_REF] Smaoui-Damak | Preliminary study of the clam Ruditapes decussatus exposed in situ to metal contamination and originating from the Gulf of Gabès, Tunisia[END_REF][START_REF] Banni | Monitoring pollution in Tunisian coasts: application of a classification scale based on biochemical markers[END_REF][START_REF] Banni | Assessment of heavy metal contamination using real-time PCR analysis of mussel metallothionein mt10 and mt20 expression: a validation along the Tunisian coast[END_REF]. Thus, high Cd concentrations have been previously measured in other marine organisms collected in this area such as the clam Ruditapes decussates [START_REF] Hamza-Chaffai | Health assessment of a marine bivalve Ruditapes decussatus from the Gulf of Gabès (Tunisia)[END_REF][START_REF] Smaoui-Damak | Preliminary study of the clam Ruditapes decussatus exposed in situ to metal contamination and originating from the Gulf of Gabès, Tunisia[END_REF] and the scorpionfish Scorpaena porcus [START_REF] Hamza-Chaffai | Physico-chemical forms of storage of metals (Cd, Cu and Zn) and metallothionein-like proteins in gills and liver of marine fish from the Tunisian coast: ecotoxicological consequences[END_REF]. In addition, the higher Cd concentrations in the muscle from arms and mantle in Sfax indicate that the sequestration capacity of the digestive gland was exceeded and resulted in a transfer of the metal toward muscle tissues. This Cd transfer from digestive glands to edible tissues and the impact of increased Cd contamination coul lead to a food safety problem of the human consumer in this highly Cd contaminated area. Geographical variations were also observed for Hg concentrations in the digestive gland and the muscle. Clearly, specimens captured in the northern and the eastern coasts exhibited higher levels of Hg in all tissue than those from the south. A similar pattern has been shown in small pelagic fish in this areas [START_REF] Joiris | Total and Methylmercury in Sardines Sardinella aurita and Sardina pilchardus from Tunisia[END_REF]. Eastern sites may be subjected to potential natural inputs from nearby Hg ferous belts and previous mining areas [START_REF] Joiris | Total and Methylmercury in Sardines Sardinella aurita and Sardina pilchardus from Tunisia[END_REF].

Additionally, there is a high industrial activities along the Italian coast, Italy facing Tunisia.

Influence of biological parameters

The potential impact of length (dorsal mantle length; DML) and sex have been preliminary considered here, the "preliminary" character need to be emphasized given the limited number of samples to draw a conclusion but at least it gave us a hint of potential biological parameters in these cephalopods caught in North Africa. Considering all tissues in all species, metal concentrations did not show significant differences between males and females except for few cases. In O. vulgaris, males showed always significant higher values than females for Hg concentrations in the digestive gland and mantle, Cu concentrations in the mantle and Zn concentrations in the arms. On contrary, females presented the highest Hg concentrations in the muscle (mantle and arms) of S. officinalis. This is probably due to the existence of physiological and/ or ecological inter-sex differences [START_REF] Monteiro | Inter-and intra-specific variation of mercury levels in muscle of cephalopods from the Azores[END_REF] such as different behaviour [START_REF] Jozet-Alves | Sex differences in spatial cognition in an invertebrate: The cuttlefish[END_REF], sexual dimorphism for some species [START_REF] Jackson | Temporal life history plasticity of the Southern Ocean squid Todarodes filippovae from waters off Tasmania, Australia[END_REF] or maternal transfer/elimination of metals through eggs (showed in cuttlefish; [START_REF] Lacoue-Labarthe | First experiments on the maternal transfer of metals in the cuttlefish Sepia officinalis[END_REF]. A previous study on cephalopods provides conflicting results in terms of metal concentrations and sex: thus O. vulgaris from the East Mediterranean coast showed higher Cd and Cu concentrations in mantle tissue in female than male [START_REF] Nessim | Bioaccumulation of heavy metals in Octopus vulgaris from coastal waters of Alexandria (Eastern Mediterranean)[END_REF].

Additionally, [START_REF] Ayas | The Effects of Season and Sex in the Metal Levels of Mature Common Cuttlefish (Sepia officinalis) in Mersin Bay, Northeastern Mediterranean[END_REF] described such influence of gender for Cd, Cu, Pb and Zn levels in the mantle of S. officinalis from the Turkish waters. However, other works on cephalopods [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] Barghigiani | Size Related Mercury Accumulations in Edible Marine Species from an Area of the Northern Tyrrhenian Sea. Water, Air[END_REF][START_REF] Kojadinovic | Multi-elemental concentrations in the tissues of the oceanic squid Todarodes filippovae from Tasmania and the southern Indian Ocean[END_REF] reported the lack of relation between heavy metals concentrations and sex.

Results from different correlations estimated between heavy metal concentrations in tissues of different species and size (viz. mantle length) showed significant negative relationships for Hg in all tissues of L. vulgaris, for Hg and Pb in the digestive gland, Hg in the mantle and Cu in the arms in O. vulgaris and for Cu in the mantle in S. officinalis. The negative relationships between heavy metal concntrations in the tissues and cephalopods sizes are consistent with previous studies: Cd concentrations decreased with length in the squid L. forbesi from the waters [START_REF] Pierce | Geographic, seasonal and ontogenetic variation in cadmium and mercury concentrations in squid (Cephalopoda: Teuthoidea) from UK waters[END_REF] as well as for Octopus salutii and Eledone cirrhosa from the Mediterranean Sea [START_REF] Barghigiani | Interaction between selenium and cadmium in Eledone cirrhosa of the northern Tyrrhenian Sea[END_REF][START_REF] Storelli | Cadmium and total mercury in some cephalopods from the South Adriatic Sea (Italy)[END_REF]. A plausible explanation for the negative correlations between these metals and cephalopods size could be attributed to the existence of a «dilution» effect linked to the rapid increase of tissue mass due to growth (that is very rapid in cephalopods) with regards to proportionally lower intake of trace elements [START_REF] Pierce | Geographic, seasonal and ontogenetic variation in cadmium and mercury concentrations in squid (Cephalopoda: Teuthoidea) from UK waters[END_REF]. On the contrary to the above results, Hg concentrations were positively correlated with size in all tissues of S. officinalis. This result is an agreement with observations described for the same metal in Eledone cirrhosa (Rossi etal. 1993), in loliginid squid (Bustamante et al. 2006;[START_REF] Pierce | Geographic, seasonal and ontogenetic variation in cadmium and mercury concentrations in squid (Cephalopoda: Teuthoidea) from UK waters[END_REF] and in cuttlefish [START_REF] Chouvelon | Inter-specific and ontogenic differences in δ 13 C and δ 15 N values and Hg and Cd concentrations in cephalopods[END_REF]. The discrepancy of these observations resulted probably from the more prominent effect of different factors, such as food availability (i.e., quality and quantity of food) and growth rates (which may be affected by temperature) [START_REF] Villanueva | Growth and proteolytic activity of Octopus vulgaris paralarvae with different food rations during first feeding, using Artemia nauplii and compound diets[END_REF], on the metal accumulation in cephalopods.

CONCLUSION

The present work provides new information on bioaccumulation and tissue distribution of 6 heavy metals in three species (O. vulgaris, L. vulgaris and S. officinalis) from the Tunisian waters. Among the major information provided in the present work, which by the way represents a first investigation in North Africa, we confirmed the important role of digestive gland and the variation of Hg organotropisms among species. It also keep the debate ongoing concerning the effect of sex and lengths on metal concentrations in cephalopods. In addition, metal concentrations measured in the edible tissues indicated that for elevated consumption of flesh, toxic risk may be reached for Cd in all the species collected in contaminated areas and for Hg in squid and cuttlefish. Evaluation of this risk deserves further investigations.

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: Sampling locations (Bizerte, Monastir and Sfax) along the Tunisian coast.

Figure 4 :

 4 Figure 4: Principal component analysis (PCA) of cephalopods based on metal levels in the arms. A) Correlation bi-plot showing the distribution of the variables. B) Projection of individuals on the correlation bi-plot with eigenvalue of the first two components. C) Grouping individuals by species. D) Grouping individuals by sampling sites.

Figure 5 :Figure 6 :Figure 7 :Figure 8 :

 5678 Figure 5: Principal component analysis (PCA) of cephalopods based on metal levels in the mantle. A) Correlation bi-plot showing the distribution of the variables. B) Projection of individuals on the correlation bi-plot with eigenvalue of the first two components. C) Grouping individuals by species. D) Grouping individuals by sampling sites.

Table 1 :

 1 Biological measurements obtained for the cephalopod samples. N: number of individuals; DML: Dorsal mantle length.

	Species	Site	N Sex	Maturity DML (mm)		Weight (g)
				stage	Mean ± SD Range	Mean ± SD Range
	L. vulgaris	Bizerte	4 4♀	III	196 ± 29	167 -227 220 ± 81	145-318
		Monastir 10 7♂ ; 3♀ III, II	188 ± 21	162 -225 130 ± 23	97-157
		Sfax	10 4♂ ; 6♀ III, II	213 ± 20	190 -256 220 ± 36	184-297
	O. vulgaris	Bizerte	7 2♂ ; 5 ♀ III, II	116 ± 18	98 -150 623 ± 348	343-1267
		Monastir 5 3♂ ; 2♀ III, II	126 ± 20	112 -160 746 ± 143	624-983
		Sfax	8 2♂ ; 6♀ III, II	133 ± 16	113 -155 851 ± 157	607-1033
	S. officinalis	Bizerte	7 1♂; 6♀ III	119 ± 16	97 -140 208± 79	105-328
		Monastir 10 8♂ ; 2♀ III	119 ± 11	103 -135 186 ± 47	117 -258
		Sfax	10 4♂ ; 6♀ III, II, I	99 ± 20	65 -130 136 ± 66	41-248

Table 2 :

 2 Comparison of metal concentrations (µg.g -1 d.w) of certified reference materials from the NRCC determined in the present study with certified values.

	Metal	TORT-2 (n=6)		DOLT-4 (n=3)		DORM-2 (n=4)
		Certified values	Present study	Certified values	Present study	Certified values	Present study
	Zn	180 ± 6	170 ± 12	-		25.3 ± 2.3	23. 5 ± 0.7
	Cu	106 ± 10	98 ± 10	-	-	2.34 ± 0.16	2.29 ± 0.18
	Cd	26.7 ± 0.6	25.62 ± 1.82	-	-	0.043 ± 0.008	0.042 ± 0.016
	Pb	0.35 ± 0.13	0.43 ± 0.27	-	-	-	-
	Ag	-	-	0.93 ± 0.07	0.85 ± 0.01	-	-
	Hg	0.27 ± 0.06	0.26 ± 0.03	2.58 ± 0.22	2.54 ± 0.18	-	-

Acknowledgments: MR benefits from a PhD fellowship provided by the Tunisian Ministery of Higher Education and Scientific Resarch. MM is Nippon Foundation Senior Nereus Fellow at the Stockholm Resilience Centre. We are grateful to C. Churlaud for her help in the Hg analyses. This study has been financially supported by the laboratory LIENSs and the CPER (Contrat de Projet Etat Région).