Minet Antoine

Alain Manceau

Valada-Mennuni Anaïs

Brault-Favrou Maud

Churlaud Carine

Jérôme Fort

Nguyen Thành

Spitz Jérôme

Paco Bustamante

Lacoue-Labarthe Thomas

Mercury in the tissues of five cephalopods species: first data on the nervous system

Keywords: ] muscles Muscles: 100% MeHg Optic lobes: 100% MeHg Digestive gland: 67% MeHg 33% iHg Organotropism, mantle muscle, digestive gland, optic lobes, Hg, speciation, HR-XANES

Highlights

 Hg levels were measured in digestive gland, muscle and brain in 5 cephalopod species  Hg concentrations in the optic lobes can be predicted from muscle ones  Highest Hg concentrations were found in the optic lobes of the octopus E. cirrhosa  XANES spectrometry showed 33% of iHg in octopus digestive gland suggesting demethylation  100% of MeHg in brain and muscle implies dietary MeHg storage in these tissues Graphical

Introduction

Mercury (Hg) is a globally distributed pollutant that poses considerable risk to the health of humans and wildlife [START_REF] Scheuhammer | Effects of environmental methylmercury on the health of wild birds, mammals, and fish[END_REF]. Special attention to this contaminant within the marine environment, a final receptor for a large proportion of atmospheric, freshwater and terrestrial Hg contamination is needed [START_REF] Driscoll | Mercury as a Global Pollutant: Sources, Pathways, and Effects[END_REF]. Mercury is mostly derived from anthropogenic sources and is principally found in inorganic (iHg) free forms and complexed ions, and as organic forms, which include methylmercury (MeHg) [START_REF] Mason | Organomercury compounds in the environment[END_REF]. In fact, under anaerobic conditions in sediments or deep waters, microorganisms such as iron and sulphate reducing bacteria convert iHg to organic MeHg [START_REF] Benoit | Geochemical and Biological Controls over Methylmercury Production and Degradation in Aquatic Ecosystems[END_REF][START_REF] Hammerschmidt | Geochemical Controls on the Production and Distribution of Methylmercury in Near-Shore Marine Sediments[END_REF].

Metagenome and metatranscriptome analyses have revealed that aerobic microorganisms are also potentially involved in MeHg production in subsurface oxic seawater (Villar et al., 2020). Hg mobility and toxicity depend on its molecular speciation [START_REF] Weiner | Basis for ecotoxicological concern in aquatic ecosystems contaminated by historical mercury mining[END_REF], which confers strong neurotoxic properties to MeHg against biota including humans [START_REF] Farina | Mechanisms of methylmercury-induced neurotoxicity: Evidence from experimental studies[END_REF][START_REF] Eagles-Smith | Modulators of mercury risk to wildlife and humans in the context of rapid global change[END_REF]. Furthermore, MeHg accumulates in marine organisms throughout their lifespans and biomagnifies along trophic chains to reach concentrations well above its toxicity threshold in top predators [START_REF] Bisi | Trophic relationships and mercury biomagnification in Brazilian tropical coastal food webs[END_REF][START_REF] Van Der Velden | Basal mercury concentrations and biomagnification rates in freshwater and marine food webs: Effects on Arctic charr (Salvelinus alpinus) from eastern Canada[END_REF][START_REF] Chouvelon | Oligotrophy as a major driver of mercury bioaccumulation in medium-to high-trophic level consumers: A marine ecosystem-comparative study[END_REF][START_REF] López-Berenguer | A critical review about neurotoxic effects in marine mammals of mercury and other trace elements[END_REF].

Among the oceanic trophic compartments, cephalopods are a central link in food webs and play a role as vector for many contaminants such as cadmium (Cd) and Hg [START_REF] Clark | Cephalopods as prey. 3. Cetaceans[END_REF][START_REF] Bustamante | Cephalopods as a vector for the transfer of cadmium to top marine predators in the north-east Atlantic Ocean[END_REF][START_REF] Maz-Courrau | Bioaccumulation and Biomagnification of Total Mercury in Four Exploited Shark Species in the Baja California Peninsula, Mexico[END_REF]. Our knowledge of Hg in cephalopods is essentially limited to the species of interest to fisheries (e.g. [START_REF] Penicaud | Metal bioaccumulation and detoxification processes in cephalopods: A review[END_REF] and levels are generally measured in edible portions of organisms (i.e. muscles of the mantle and arms of squids and octopods, respectively) that store more than 70% whole body Hg burden [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][START_REF] Bustamante | Total and organic Hg concentrations in cephalopods from the North East Atlantic waters: influence of geographical origin and feeding ecology[END_REF][START_REF] Lacoue-Labarthe | Bioaccumulation of inorganic Hg by the juvenile cuttlefish Sepia officinalis exposed to 203 Hg radiolabelled seawater and food[END_REF]. Most of the Hg found in cephalopod muscles is in its organic form [START_REF] Bustamante | Total and organic Hg concentrations in cephalopods from the North East Atlantic waters: influence of geographical origin and feeding ecology[END_REF][START_REF] Seco | Mercury levels in Southern Ocean Squid: variability over the last decade[END_REF], and the speciation of Hg in tissues other than muscles has not been well documented to date (see below).

Hg concentrations reported in cephalopod muscles vary by two orders of magnitude [START_REF] Bustamante | Total and organic Hg concentrations in cephalopods from the North East Atlantic waters: influence of geographical origin and feeding ecology[END_REF] among species. Overall, median Hg concentrations in cuttlefish, octopods and squids range from 0.2 and 0.5 µg.g -1 dry weight (dw) [START_REF] Penicaud | Metal bioaccumulation and detoxification processes in cephalopods: A review[END_REF]. These levels are moderately high with respect to their central trophic position and their short life span (an average of two years) that limits the Hg bioaccumulation within their tissues [START_REF] Chouvelon | Species and ontogenic differences in δ 13 C and δ 15 N values and Hg and Cd concentrations of cephalopods[END_REF]. Higher concentrations were reported in some species or individuals, up to 1.76 µg.g -1 dw in the curled octopus, Eledone cirrhosa, from Mediterranean Sea (i.e. 0.44 µg.g -1 wet weight (ww) reported by Storelli et al., 2010 assuming 75% water content as reported in [START_REF] Spitz | Proximate composition and energy content of forage species from the Bay of Biscay: high or low quality food?[END_REF]. Hg concentrations in mantle muscle and the digestive gland have been used to infer Hg environmental contamination, since Hg mostly accumulates in its organic form in muscles and is highly concentrated in the digestive gland, where Hg detoxification has been reported to occur [START_REF] Bustamante | Total and organic Hg concentrations in cephalopods from the North East Atlantic waters: influence of geographical origin and feeding ecology[END_REF][START_REF] Lacoue-Labarthe | Bioaccumulation of inorganic Hg by the juvenile cuttlefish Sepia officinalis exposed to 203 Hg radiolabelled seawater and food[END_REF].

However, reports of Hg concentrations in tissues that are likely sensitive to Hg, such as the nervous system, are scarce.

Although Hg neurotoxicity has been recognised, information regarding Hg brain tropism in marine organisms are scattered [START_REF] Berntssen | Maximum limits of organic and inorganic mercury in fish feed[END_REF]. Previous studies have focused on fish provided nervous system Hg based on concentrations present in the environment. Alternatively, they assessed classically targeted organs such as muscle and liver. Hg concentrations in the tissues of bluefish, Pomatomus saltatrix from New Jersey showed Hg levels one order of magnitude lower in the brain than muscles and kidneys [START_REF] Burger | Mercury and selenium levels, and selenium:mercury molar ratios of brain, muscle and other tissues in bluefish (Pomatomus saltatrix) from New Jersey, USA[END_REF]. In more contaminated fish such as the spotted seatrout, Cynoscion nebulosus and European bass, Dicentrarchus labrax that were isolated from polluted areas, brain Hg concentrations were less than 3-fold lower than those reported in muscles and liver [START_REF] Adams | Mercury contamination in spotted seatrout, Cynoscion nebulosus: an assessment of liver, kidney, blood, and nervous system health[END_REF][START_REF] Mieiro | Evaluation of Species-Specific Dissimilarities in Two Marine Fish Species: Mercury Accumulation as a Function of Metal Levels in Consumed Prey[END_REF]. In addition, Hg in brain of the golden grey mullet, Liza aurata, from different areas of the Portuguese coastline faithfully reflected local water and sediment contamination variability [START_REF] Pereira | Fish eyes and brain as primary targets for mercury accumulation -A new insight on environmental risk assessment[END_REF]. Through an experimental approach, autoradiography revealed a specific affinity of 203 iHg in the brain of brown trout Salmo trutta and rainbow trout Oncorhynchus mykiss following waterborne exposure, suggesting that the transport of this metal occurs from sensory or motor systems to the brain [START_REF] Rouleau | Accumulation of waterborne mercury(II) in specific areas of fish brain[END_REF]. Hg exposure and subsequent Hg accumulation in the nervous system can then lead to oxidative stress, metallothionein production, brain lesions, morphometric alterations and consequently affected swimming behaviour in Atlantic salmon Salmo salar, European bass Dicentrarchus labrax, zebrafish Danio rerio, and white seabream Diplodus sargus [START_REF] Berntssen | Chronic dietary mercury exposure causes oxidative stress, brain lesions, and altered behaviour in Atlantic salmon (Salmo salar) parr[END_REF][START_REF] Mieiro | Brain as a critical target of mercury in environmentally exposed fish (Dicentrarchus labrax)-Bioaccumulation and oxidative stress profiles[END_REF][START_REF] Gentès | Specific Effects of Dietary Methylmercury and Inorganic Mercury in Zebrafish (Danio rerio) Determined by Genetic, Histological, and Metallothionein Responses[END_REF][START_REF] Pereira | Inorganic mercury accumulation in brain following waterborne exposure elicits a deficit on the number of brain cells and impairs swimming behavior in fish (white seabream-Diplodus sargus)[END_REF].

Cephalopods are known as an alternative model to vertebrates for the evolution of complex brains and high intelligence, and they are renowned for their advanced cognitive performances [START_REF] O'brien | he Current State of Cepha opod Science and Perspectives on the Most Critical Challenges Ahead From Three Early-Career Researchers[END_REF] and complex behaviour [START_REF] Hanlon | Cephalopod Behaviour[END_REF]. Such abilities require a much more evolved and developed brain than is needed in mollusks like bivalves and gastropods [START_REF] Hanlon | Cephalopod Behaviour[END_REF]. The cephalopod nervous system is composed of the central brain (around 40 interconnected ganglia), two optic lobes and the peripheral nervous system. Their large-sized brain, compared to other invertebrates, is involved in behavioural plasticity, cognitive capacity as well as skin control [START_REF] Shigeno | Cephalopod Brains: An Overview of Current Knowledge to Facilitate Comparison With Vertebrates[END_REF] and is of increased concern when considering impacts of neurotoxicants (e.g. [START_REF] Chabenat | Hidden in the sand: Alteration of burying behaviour in shore crabs and cuttlefish by antidepressant exposure[END_REF]. Very few studies have examined the accumulation of Hg in tissues of the nervous system, and Hg levels in the muscular mantle, branchial hearts, kidneys, gills, ink sack and the digestive gland [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][START_REF] Pereira | Metal concentrations in digestive gland and mantle of Sepia officinalis from two coastal lagoons of Portugal[END_REF]. In fact, to the best of our knowledge, only one study that examined a giant squid, Architeuthis dux, individual (1.25 μg.g -1 dw; [START_REF] Bustamante | Metal and metalloid concentrations in the giant squid Architeuthis dux from Iberian waters[END_REF] in which its brain Hg concentration was in the same order of magnitude as that of other tissues. The extent to which Hg bioaccumulation occurs and speciation in the brains of cephalopods requires further examination.

In this context, the current study aims to investigate levels of Hg contamination in five different species of cephalopods from the Bay of Biscay including Eledone cirrhosa, Sepia officinalis, Loligo vulgaris, Todaropsis eblanae and Illex coindetii. Hg concentrations were measured in the tissues with major roles in Hg bioaccumulation and detoxification including the mantle muscles, which are responsible for storing most the majority of the Hg burden, and the digestive gland, which can demethylate and depurate Hg from the body. Hg levels were also determined in the optic lobes of the brain, since it is a tissue that is potentially sensitive to Hg toxicity. In addition, the molecular form of Hg in the optic lobes, digestive gland and mantle muscles of some curled octopus, E. cirrhosa, individuals were investigated via high energy-resolution XANES spectroscopy.

Material and methods

Sample collection and sample preparation

In this study, 80 cephalopods were sampled along the continental shelf of the Bay of Biscay throughout EVHOE surveys conducted by the ns tut ran ais de echerche pour ' p oita on de a Mer (IFREMER) in autumn of 2016and 2017(Leaute et al., 2016;;[START_REF] Duhamel | EVHOE 2017 cruise, RV Thalassa[END_REF]. Collected organisms belonged to five cephalopod species from four different families (see Table 1), as follows: curled octopus, E. cirrhosa (n = 20); common cuttlefish, S. officinalis (n = 21); common squid, L. vulgaris (n = 20); the lesser flying squid, T. eblanae (n = 9); and southern shortfin squid, I. coindetii (n = 11). Specimens were frozen at -20°C on board and then stored in the laboratory prior to dissection and sample preparation.

In the laboratory, individuals were thawed and weighed (g) before measurements obtained. Dorsal mantle length (DML; mm) was measured as a reference for measuring the size of the cephalopod. Whenever possible, sex was determined by visual observation of gonads and hectocotylised arms (Table 1). During dissection, the digestive gland, mantle and the optic lobes were collected and wet weighed (g ww). The sampled tissues were frozen and freeze-dried for 48 hours. Samples were dry weighed (g dw), ground manually with a porcelain pestle and mortar into a fine powder, and hermetically stored in 40 mL plastic vials.

Mercury analyses

Total Hg concentrations within samples were analysed at the LIttoral ENvironnement et Sociétés laboratory (LIENSs, La Rochelle, France) using an Advanced Mercury Analyser spectrophotometer (Altec AMA 254) as described in [START_REF] Chouvelon | Hg concentrations and related risk assessment in coral reef crustaceans, molluscs and fish from New Caledonia[END_REF]. All analyses were carried out in duplicate or triplicate, and had relative standard deviation values < 10%. Hg analyses were run according to a thorough quality control programme that included an analysis of procedural blanks at the beginning of each analytical sessions and a certified reference material (CRM) was assessed every 10 samples.

Thus, the accuracy of the analytical procedure was verified via assessing CRM TORT-2 (lobster hepatopancreas, NRC, Canada; Hg concentration of 0.27 ± 0.06 μg•g -1 dw). Results indicated an average Hg concentration of 0.257 ± 0.009 μg.g -1 dw, with an average recovery of 95 ± 3%, and were in good agreement with certified values. The detection limit of the method was 0.005 μg.g -1 dw. Mercury concentrations will be expressed as µg.g -1 dw in subsequent portions of the text.

HR-XANES measurements

Freeze-dried powdered digestive gland, mantle muscle and optic lobe of E. cirrhosa were pressed into pellets, and their Hg L 3 -edge XANES (for X-ray Absorption Near Edge Structure) spectra were measured using high energy-resolution (HR-XANES) with high-luminosity analyser crystals on beamline ID26 at the European Synchrotron Radiation Facility (ESRF, [START_REF] Rovezzi | High energy-resolution x-ray spectroscopy at ultra-high dilution with spherically bent crystal analyzers of 0.5 m radius[END_REF]. Mantle muscles and the digestive gland of the most contaminated individual were selected for further examination, whereas the optic lobes of the six individuals that were most contaminated were pooled because each lobe had low mass (~50-80 mg). Spectra were collected at a temperature of 10-15 K and a scan time of 15 s to reduce exposure, and repeated at using pristine positions of the sample to increase the signal-to-noise ratio. Scans were monitored carefully for any evidence of radiation damage. Incident energy was scanned from 12260 to 12360 eV in increment of 0.2 eV and the spectra were normalised to unity at E = 12,360 eV. The identities and relative proportions of Hg species were determined using a spectral database [START_REF] Bourdineaud | Divalent mercury from dissolved organic matter is bioavailable to fish and accumulates as dithiolate and tetrathiolate complex[END_REF]. The precision of the fit of components was estimated to be equal to the variation of their best-fit values when the fit residual (NSS) was increased by 20%. NSS was defined as the normalised sum-squared difference between data and fit, was expressed as follows: Σ[(y data -y fit ) 2 ]/Σ(y data 2). Details regarding data acquisition and analysis were performed as previously described [START_REF] Manceau | Mercury(II) binding to metallothionein in Mytilus edulis revealed by high energy-resolution XANES spectroscopy[END_REF].

Statistical and data analyses

Statistics were computed using R version 3.6.1 (R Development Core Team, 2011). The influence of the tissue on Hg concentration was tested by using generalised linear models (GLM) with a gaussian distribution and an identity in func on. ode se ec on as ased on a e's Information Criteria adjusted for small sample sizes (AICc) (Burnham and Anderson, 2002).

Concentrations of Hg analysed in the mantle, digestive gland and optic lobes of the five species assessed were compared using a one-way ANOVA followed by a Tukey test. Normality and heteroscedasticity of values were tested using a Barlett and Shapiro test, respectively, and each of the cephalopods relative to the others were considered separately. When the conditions of the statistical tests were not met, non-parametric tests of Wilcoxon followed the post-hoc test of Nemenyi were applied. A linear model was applied to describe relationships between Hg concentrations in the optic lobe and mantle muscle and an analysis of covariance (ANCOVA) was used to test differences in the slopes of regression lines for the five species considered.

Hg concentrations reported from the literature were converted when necessary from ww to dw using measured moisture values from the different tissues, as follows: dw represents 20% of the ww for muscle and brain tissues and 35% of the ww of the digestive gland [START_REF] Suppes | Seasonal Variations of Fat, Protein, and Moisture in Channel Catfish[END_REF]Arai et al., 2016 and personal data).

Results

The mantle size and weight of animals varied from 4.2 to 19.0 cm and 19 to 764 g ww, respectively. Measured Hg concentrations were 0.02-1.48 µg.g -1 dw, from 0.09-1.35 µg.g -1 dw, and 0.03-1.89 µg.g -1 dw in mantle muscles, the digestive gland and optic lobes, respectively (Table 1).

GLMs revealed that Hg concentrations differed according to species and tissue assessed (Fig. 1; Table 2). Overall, although concentrations among the three analysed tissues were of the same order of magnitude, Hg concentrations were significantly higher in mantle muscles and the digestive gland than optic lobes in all species assessed, except in E. cirrhosa. Relative Hg concentrations in mantle muscles of species assessed were as follows: E. cirrhosa ≈ S. officinalis ≈ T. eblanae > L. vulgaris > I. coindetii (values measured were 0.52 ± 0.35, 0.46 ± 0.24, 0.29 ± 0.06, 0.16 ± 0.03, and 0.08 ± 0.01 μg.g -1 dw, respectively).

In the digestive gland, the same trend was observed, and significantly higher levels of Hg were measured in E. cirrhosa and S. officinalis than in other squid species. Relative values were as follows:

E. cirrhosa ≈ S. officinalis > L. vulgaris ≈ T. eblanae > I. coindetii (0.47 ± 0.34, 0.43 ± 0.12, 0.29 ± 0.06, 0.24 ± 0.06, and 0.12 ± 0.02 μg.g -1 dw, respectively). In the optic lobes, relative Hg concentrations were similar, however, a greater difference between E. cirrhosa and other species was observed, as follows: E. cirrhosa > S. officinalis ≈ T. eblanae > L. vulgaris ≈ I. coindetii (0.60 ± 0.47, 0.23 ± 0.09, 0.18 ± 0.07, 0.09 ± 0.02, and 0.04 ± 0.01, μg.g -1 dw, respectively). Interestingly, two E. cirrhosa individuals (53 and 90 mm mantle size) had elevated Hg concentrations that reached more than 1 µg.g -1 in the mantle and the digestive gland and more than 1.5 µg.g -1 in the optic lobes, whereas values rarely exceeded 0.5 μg.g -1 dw in other species, in any tissue assessed.

The concentrations of Hg in the optic lobes were linearly and strongly associated with those of mantle muscles (Fig. 2, Table 3). ANCOVA revealed that slopes of the regressions were significantly different among when E. cirrhosa (R 2 = 0.978), S. officinalis and T. eblanae (R 2 = 0.829), and I. coindetii and L. vulgaris (R 2 = 0.663) were compared. This implies that the ratio of brain and muscle Hg concentrations differed among the three groups. Interestingly, E. cirrhosa had an optic lobe Hg concentration that was slightly higher (1.15-fold greater) than that of mantle muscles, whereas S. officinalis/T. eblanae and I. coindetii/L. vulgaris had optic lobe concentrations that were 0.35 and 0.50-fold lower, respectively, than those of mantle muscles.

HR-XANES spectra of the optic lobes and mantle muscles were statistically indistinguishable, indicating similar or identical chemical forms of Hg were present in each tissue (Fig. 3a). The best spectral match with our database of model compounds was obtained with MeHg bound to cysteine residues in fish (Fig. 3b). In this complex, Hg is linearly binds a methyl group and the aliphatic thiol from a cysteinyl group (forming a MeHgCys complex). This geometry gives a characteristic absorption peak at 12,279.8 eV, which is not observed when Hg is coordinated to three or four thiols [START_REF] Bourdineaud | Divalent mercury from dissolved organic matter is bioavailable to fish and accumulates as dithiolate and tetrathiolate complex[END_REF]. This peak occurs at 12,279.6 eV in the digestive gland and its intensity was lower (Fig. 3c). These two features are diagnostic of the linear coordination to two thiol groups, as it is observed in an inorganic Hg(Cys) 2 complex [START_REF] Manceau | Chemical forms of mercury in human hair reveal sources of exposure[END_REF]. Proportions of methylated and inorganic Hg, as determined by a linear combination fit of the optic lobe spectrum and the Hg(Cys) 2 standard, were 68 ± 11% for MeHgCys and 33 ± 11% for Hg(Cys) 2 (Fig. 3d).

Discussion

Among marine organisms, most available information regarding Hg contamination has been determined in bivalves, fish and top predators (mammals and birds), mainly due to their importance as a food source for humans and their roles as bioindicators of Hg contamination of the ocean [START_REF] Renzoni | Mercury Levels along the Food Chain and Risk for Exposed Populations[END_REF]. In contrast, the information regarding Hg contamination in cephalopod tissues remains scarce, despite the fact that molluscs are known to efficiently accumulate chemical contaminants, including non-essential metals that may be concentrated at very high levels in their tissues [START_REF] Dorneles | Cephalopods and cetaceans as indicators of offshore bioavailability of cadmium off Central South Brazil Bight[END_REF][START_REF] Lischka | High cadmium and mercury concentrations in the tissues of the orange-back flying squid, Sthenoteuthis pteropus, from the tropical Eastern Atlantic[END_REF]. Although Hg concentrations are moderate in cephalopods, significant differences between taxons have been observed, and values have been shown to vary by two orders of magnitude among cephalopod families [START_REF] Penicaud | Metal bioaccumulation and detoxification processes in cephalopods: A review[END_REF]. The cephalopods have a pivotal place in trophic webs, and have a key role in the Hg intake of top predators including humans [START_REF] Jackson | Applying new tools to cephalopod trophic dynamics and ecology: perspectives from the Southern Ocean Cephalopod Workshop, February 2-3, 2006[END_REF], therefore a better understanding of the Hg bioaccumulation processes in cephalopods is needed. Finally, despite their invertebrate status, cephalopods have an exceptionally developed nervous system and display complex cognitive abilities that confer great adaptive capacity, despite the short lifespan of the animals [START_REF] Hanlon | Cephalopod Behaviour[END_REF]. However, the effects of Hg in the brains of cephalopods remains unknown, despite the fact that Hg is known for its high degree of toxicity on the nervous system of vertebrates [START_REF] Syversen | The toxicology of mercury and its compounds[END_REF].

Mercury concentrations varied among species collected in the same geographical location (i.e. the Bay of Biscay) at the same time, suggested that individuals were exposed to Hg to variable extents.

More precisely, E. cirrhosa and S. officinalis had the highest levels of Hg in their tissues, relative to levels in other species assessed. This is congruent with the previous observation that benthic octopus and nektobenthic cuttlefish (i.e. Octopodidae and Sepiidae) generally display significantly higher Hg concentrations in their tissues than pelagic squids, including Ommastrephidae and Loliginidae, from the same area (Pénicaud et al., 2017). For example, among seven cephalopods from the Mediterranean Sea, Octopodidae (E. moschata, E. cirrhosa, and Octopus salutii) had the highest Hg concentrations, followed by Sepiidae (Sepia elegans and Sepia orbignyana). The lowest concentrations were found in Ommastrepidae (I. coindetii) and Loliginidae (L. vulgaris) [START_REF] Storelli | Intake of essential and nonessential elements from consumption of octopus, cuttlefish and squid[END_REF]. To date, these inter-specific differences have mainly been attributed to ecological factors, such as the proximity of benthic species to sediment where Hg methylation is located [START_REF] Storelli | Mercury speciation in the muscle of two commercially important fish, hake (Merluccius merluccius) and striped mullet (Mullus barbatus) from the Mediterranean Sea: estimated weekly intake[END_REF]. Nevertheless, squids generally displayed elevated trophic levels than octopus and cuttlefish, indicating that they likely feed on MeHg-enriched prey such as fish and/or cephalopods [START_REF] Pierce | Diets of squid Loligo forbesi and Loligo vulgaris in the northeast Atlantic[END_REF][START_REF] Coelho | A comparative study of the diet of Loligo vulgaris (Lamarck, 1799) (Mollusca: Cephalopoda) from the south coast of Portugal and the Saharan Bank (Central-East Atlantic)[END_REF]) compared to benthic cephalopods, which prey on invertebrates including crustaceans and bivalves with lower concentrations of Hg and lower proportions of MeHg. This incongruency indicates that species-dependent physiological processes, such as demethylation and elimination capacities, could explain the interspecific Hg variation.

Mercury concentrations in mantle muscles ranged from 0.06 to 1.48 μg.g -1 dw and were similar to those reported by Rjeibi and collaborators (2015) in cephalopods from Tunisia (0.01 to 0.95 μg.g -1 dw). They were also comparable to concentrations reported for cephalopods from the Adriatic Sea (L. vulgaris from 0.20 to 1.50 μg.g -1 dw, O. vulgaris from 0.12 to 1.30 μg.g -1 dw, S. officinalis from 0.25 to 4.78 μg.g -1 dw) and the Atlantic Ocean (S. officinalis > 1 μg.g -1 dw) [START_REF] Brambilla | Mercury occurrence in Italian seafood from the Mediterranean Sea and possible intake scenarios of the Italian coastal population[END_REF][START_REF] Pereira | Metal concentrations in digestive gland and mantle of Sepia officinalis from two coastal lagoons of Portugal[END_REF], which showed that a homogeneous level of contamination occurs between locations.

Heterogeneity in Hg concentrations among tissues [START_REF] Lischka | High cadmium and mercury concentrations in the tissues of the orange-back flying squid, Sthenoteuthis pteropus, from the tropical Eastern Atlantic[END_REF][START_REF] Manso | Characterization of essential and toxic elements in cephalopod tissues by EDXRF and AAS[END_REF] is driven by uptake, detoxification, storage and/or excretion (including MeHg) differences [START_REF] Penicaud | Metal bioaccumulation and detoxification processes in cephalopods: A review[END_REF]. Hg concentrations were highest in the digestive gland and in mantle muscles in all species considered, except for the curled octopus, E. cirrhosa, for which higher Hg concentrations were found in optic lobes (see discussion thereafter). High Hg concentrations in the digestive gland of cephalopods have previously been documented in several species [START_REF] Bustamante | Total and organic Hg concentrations in cephalopods from the North East Atlantic waters: influence of geographical origin and feeding ecology[END_REF][START_REF] Manso | Characterization of essential and toxic elements in cephalopod tissues by EDXRF and AAS[END_REF][START_REF] Rjeibi | Interspecific and geographical variations of trace metal concentrations in cephalopods from Tunisian waters[END_REF]. Mercury, and notably the MeHg within prey, mainly accumulates via food consumption [START_REF] Lacoue-Labarthe | Bioaccumulation of inorganic Hg by the juvenile cuttlefish Sepia officinalis exposed to 203 Hg radiolabelled seawater and food[END_REF][START_REF] Chouvelon | Species and ontogenic differences in δ 13 C and δ 15 N values and Hg and Cd concentrations of cephalopods[END_REF], which implies that the physiological functions of digestion and absorption of the digestive gland contribute to the high Hg levels found in the tissue [START_REF] Boucaud-Camou | Fine structure and function of the digestive cell of Sepia officinalis (Mollusca: Cephalopoda)[END_REF][START_REF] Boucher-Rodoni | Feeding and digestion[END_REF].

The digestive gland is also known as the main organ of metal detoxification and storage in cephalopods [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 two cephalopods Eledone cirrhosa and Sepia officinalis, from the French coast of the English Channel[END_REF][START_REF] Lacoue-Labarthe | Ecotoxicology of early-life stages in the common cuttlefish Sepia officinalis: review and perspectives[END_REF][START_REF] Bustamante | Biokinetics of zinc and cadmium accumulation and depuration at different stages in the life cycle of the cuttlefish Sepia officinalis[END_REF]. Although the digestive gland represents only 6 to 10% of the mass of an animal, the organ may contain more than 80% of the body burden of some metals such as Ag, Cd, and Co [START_REF] Penicaud | Metal bioaccumulation and detoxification processes in cephalopods: A review[END_REF]. However, Hg in the digestive gland contributes to far less of the total body burden than muscles [START_REF] Bustamante | Total and organic Hg concentrations in cephalopods from the North East Atlantic waters: influence of geographical origin and feeding ecology[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]. Concentrations of Hg were in the same order of magnitude in both tissues, but as muscles accounted for more than 70% of the total body weight, and contained 70 to 90% of the total Hg body burden [START_REF] Bustamante | Total and organic Hg concentrations in cephalopods from the North East Atlantic waters: influence of geographical origin and feeding ecology[END_REF][START_REF] Seco | Mercury levels in Southern Ocean Squid: variability over the last decade[END_REF]. This tissue distribution is likely due to two main mechanisms: 1) translocation and binding of MeHg to muscle proteins because they are rich in sulfhydryl groups with a high affinity for Hg, as shown in fish [START_REF] Bloom | On the chemical form of mercury in edible fish and marine invertebrate tissue[END_REF]. Consequently, the muscles constitute a good bioindicator tissue of MeHg exposure in cephalopods as like in many organisms, most of the Hg found in the muscular parts is under organic form [START_REF] Bustamante | Total and organic Hg concentrations in cephalopods from the North East Atlantic waters: influence of geographical origin and feeding ecology[END_REF][START_REF] Seco | Mercury levels in Southern Ocean Squid: variability over the last decade[END_REF][START_REF] Chételat | Methylmercury exposure in wildlife: A review of the ecological and physiological processes affecting contaminant concentrations and their interpretation[END_REF]. Relatively similar Hg concentrations observed in the digestive gland compared and mantle muscles may also result from 2) the demethylation of MeHg, which facilitates iHg elimination by the digestive gland. Indeed, more than 70% of the Hg within the muscles of cephalopods is methylated, but more than a half of the Hg found in their digestive glands is inorganic [START_REF] Bustamante | Total and organic Hg concentrations in cephalopods from the North East Atlantic waters: influence of geographical origin and feeding ecology[END_REF][START_REF] Seco | Mercury levels in Southern Ocean Squid: variability over the last decade[END_REF]). Here, HR-XANES spectrometry speciation analyses revealed that one third of the total Hg within the digestive gland of E. cirrhosa was made up of an inorganic form bound to peptides (e.g. glutathione, GSH) or proteins as a dicysteinate complex, which was modeled using the Hg(Cys) 2 reference. This suggests that 1) iHg may come from the environment [START_REF] Sun | Methylmercury produced in upper oceans accumulates in deep Mariana Trench fauna[END_REF] through both dissolved and trophic pathways. Indeed, it has been demonstrated that cuttlefish efficiently bioconcentrate dissolved i 203 Hg, which is then distributed throughout the digestive gland [START_REF] Lacoue-Labarthe | Bioaccumulation of inorganic Hg by the juvenile cuttlefish Sepia officinalis exposed to 203 Hg radiolabelled seawater and food[END_REF]. Thus, this organ contained 20% of accumulated i 203 Hg after 10 days of exposure. In addition, ~90% of the i 203 Hg from prey is assimilated and then mostly (~70%) found in the digestive gland. Alternatively, 2) iHg may result from in vivo MeHg demethylation [START_REF] Sun | Methylmercury produced in upper oceans accumulates in deep Mariana Trench fauna[END_REF], which likely occurs in the digestive gland, facilitating the rapid elimination of produced iHg, based on experimental findings in which a short biological half-life (i.e. 4 days) of dietary 203 iHg was reported [START_REF] Lacoue-Labarthe | Bioaccumulation of inorganic Hg by the juvenile cuttlefish Sepia officinalis exposed to 203 Hg radiolabelled seawater and food[END_REF]. The biochemical mechanism of demethylation that occurs in the digestive gland has not been determined.

Assuming that cephalopods are exposed to the MeHg form of Hg by prey, remaining MeHg from the pool of ingested Hg entering the bloodstream may become distributed throughout other tissues. Some studies have investigated the binding and transport of Hg(II) to hemocyanin [START_REF] Brouwer | Heavy Metal Ion Interactions with Callinectes sapidus Hemocyanin: Structural and Functional Changes Induced by a Variety of Heavy Metal Ions[END_REF][START_REF] Brouwer | Metal Ion Interactions with Limulus polyphemus and Callinectes sapidus Hemocyanins: Stoichiometry and Structural and Functional Consequences of Calcium(II), Cadmium(II), Zinc(II), and Mercury(II) Binding[END_REF]), but little is known regarding MeHg binding to hemocyanin (blood oxygen transport proteins) in cephalopods. Nevertheless, it can be assumed its transport is facilitated by sulphydryl or tryptophanyl binding sites of the molecule, which allows MeHg transfer to other tissues [START_REF] Brouwer | Heavy Metal Ion Interactions with Callinectes sapidus Hemocyanin: Structural and Functional Changes Induced by a Variety of Heavy Metal Ions[END_REF].

As mentioned previously, only mercury concentration (i.e. 1.25 μg.g -1 dw) has been reported for the brain of the giant squid, Architeuthis dux, to our knowledge [START_REF] Bustamante | Metal and metalloid concentrations in the giant squid Architeuthis dux from Iberian waters[END_REF]. In the present study, the Hg concentrations were determined in the optic lobes, which is part of the nervous central system of cephalopods and are responsible for the highest level of coordinated function such as association, learning and visual analysis [START_REF] Young | The anatomy of the nervous system of Octopus vulgaris[END_REF]. In this tissue, Hg concentrations ranged from 0.03 to 1.89 μg.g -1 dw among analysed individuals, which demonstrated that Hg accumulates in this area of the central nervous system. Values were comparable to, even slightly higher than, Hg concentrations reported in fish brains including 0.02 to 0.80 μg.g -1 dw in the golden grey mullet, Liza aurata [START_REF] Pereira | Fish eyes and brain as primary targets for mercury accumulation -A new insight on environmental risk assessment[END_REF]; 0.10 to 1.45 μg.g -1 dw in the bluefish, Pomatomus saltatrix (i.e. 0.02 to 0.29 μg.g -1 ww; [START_REF] Burger | Mercury and selenium levels, and selenium:mercury molar ratios of brain, muscle and other tissues in bluefish (Pomatomus saltatrix) from New Jersey, USA[END_REF]; and 0.10 to 1.60 μg.g -1 dw in the seabass, Dicentrarchus labrax (i.e. 0.02 to 0.32 μg.g -1 ww; [START_REF] Mieiro | Brain as a critical target of mercury in environmentally exposed fish (Dicentrarchus labrax)-Bioaccumulation and oxidative stress profiles[END_REF]. In addition to the lack of information regarding contamination of the central nervous system of cephalopods, such as Hg concentrations in the brain, knowledge regarding Hg neurotoxicity in this Class is also incomplete.

Interestingly, Hg concentrations in the brain are strongly correlated with that of the muscles in different species investigated here, which indicates that Hg concentrations of the optic lobes are not regulated independently from Hg bioaccumulation throughout the organism. In addition, R 2 values ranged from 0.66 to 0.98, depending on the group of species considered (see Fig. 2; Table 3), which indicated that Hg accumulation in the brain may be driven by the Hg concentrations in muscles in accordance with a defined brain/muscle ratio that is determined for each species. L. vulgaris and I. coindetii had a brain/muscle ratio of 0.35, S. officinalis and T. eblanae had a brain/muscle ratio of 0.50, and E. cirrhosa was the only species that displayed a ratio greater than 1 (about 1.3) as a result of elevated Hg concentrations in the brain than mantle (Fig. 2). A positive correlation between Hg concentrations in the brain and muscles has been reported previously in eight species of catfish (Brachyplatystoma filamentosum; B. platynemum; B. rousseauxii; B. vaillantii; Phractocephalus hemiliopterus; Pseudoplatystoma punctifer; P. tigrinum; Zungaro zungaro) (R 2 = 0.917; p < 0.0001; [START_REF] Bastos | Mercury in muscle and brain of catfish from the Madeira river, Amazon, Brazil[END_REF]. More importantly, the brain/muscle ratio never exceeded 0.6 when 21 fish species whose Hg concentrations in muscles ranged from 0.25 to 20.50 µg.g -1 dw were examined (i.e. 0.05 to 4.10 µg.g -1 ww; [START_REF] Bastos | Mercury in muscle and brain of catfish from the Madeira river, Amazon, Brazil[END_REF]. Similarly, the brain/muscle Hg ratio (0.054/0.274 µg.g -1 ww) did not exceeded 0.2 in yellow perch, Perca flavescens [START_REF] Khadra | Mercury and selenium distribution in key tissues and early life stages of Yellow Perch (Perca flavescens)[END_REF]. Furthermore, ratios lower than 1 are also common in birds. In the following species, ratios less than one were determined, as follows: 0.43 for the semipalmated sandpiper, Calidris pusilla [START_REF] Burger | Metals in tissues of migrant semipalmated sandpipers (Calidris pusilla) from Delaware Bay, New Jersey[END_REF]; 0.60 for the Atlantic puffin, Fratercula arctica; 0.68 for the common guillemot, Uria aalge; and 0.55 for the black-legged kittiwake, Rissa tridactyla (Fort et al., 2015). Thus, ratios observed for the squids and cuttlefish in our study are in accordance with previously reported data and suggest that Hg accumulation in the brain is limited compared to muscle tissue in cephalopods.

Interestingly, the most contaminated species among the cephalopod species panel investigated was in E. cirrhosa, which had a brain/muscle Hg ratio of 1.3. This finding suggested that Hg brain-tropism is most significant in E. cirrhosa. To the best of our knowledge, a similar ratio not been described in fish, except blackchin tilapia, Sarotherodon melanotheron, which had a brain/muscle Hg ratio of 1.5 (Hg concentrations were of 1.25 μg.g -1 dw (i.e. 0.25 μg.g -1 ww) in the brain and 0.85 μg.g -1 dw (i.e. 0.17 μg.g -1 ww) in muscle [START_REF] Coulibaly | Seasonal Accumulations of Some Heavy Metal in Water, Sediment and Tissues of Black-Chinned Tilapia Sarotherodon melanotheron from Biétri Bay in Ebrié Lagoon, Ivory Coast[END_REF]. Similar to seabirds, the razorbill, Alca torda, had a Hg brain/muscle ratio close to 1 (i.e. 2.05 and 2.04 μg.g -1 dw, respectively). Finally, the highest ratios have been determined humans, and have reached 3.6 (i.e. 0.090 and 0.025 μg.g -1 dw in the occipital cortex and abdominal muscles, respectively; [START_REF] Björkman | Mercury in human brain, blood, muscle and toenails in relation to exposure: an autopsy study[END_REF]. Despite these observations, no clear explanation has been provided to enhance our understanding of how Hg accumulation preferentially occurs in the brain versus muscles in these species.

In addition, our results revealed that Hg accumulation in the brain with respect to muscles Hg does not depend on contamination level. In other words, the brain/muscle ratio did not increase as the range of Hg muscle concentrations measured for each species increased. Indeed, with the exception of two individuals that were highly contaminated (> 1.35 µg.g -1 dw), mantle muscle Hg concentrations in E. cirrhosa specimens ranged from 0.24 to 0.57 µg.g -1 dw, which was similar to the range recorded in S. officinalis specimens (0.18-0.75 µg.g -1 dw). In fish, species reported to contaminated throughout a large range of Hg concentrations (e.g. from 0.25 to 20.50 µg.g -1 dw i.e. 0.05 to 4.10 µg.g -1 ww from [START_REF] Bastos | Mercury in muscle and brain of catfish from the Madeira river, Amazon, Brazil[END_REF] had low brain/muscle ratios that were similar to values determined for cephalopods, with the exception of E. cirrhosa. Consequently, Hg accumulation in brain of the octopus may have a species-specific explanation. First, Hg concentrations in the musculature were measured in mantle muscles to facilitate comparison between squids and cuttlefish. However, the majority of muscle mass in octopus species is found in their arms. Not surprisingly, Hg concentrations do not differ between both muscle types [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]. This finding indicates the brain/muscle ratio was likely not overestimated as a result of the analysis of mantle muscles in this study. Further, fostered Hg accumulation in the octopus brain may result from a more permeable tissue to metal intake. Cephalopods have linker junctions between glial cells, which form a blood-brain barrier (BBB) that surrounds the central nervous system [START_REF] Abbott | A fibre matrix model for the restricting junction of the blood-brain barrier in a cephalopod mollusc: implications for capillary and epithelial permeability[END_REF]. Once in the bloodstream, transported MeHg is transferred across the BBB and is capable of reaching the brain thanks to its lipophilicity [START_REF] Boening | Ecological effects, transport, and fate of mercury: a general review[END_REF][START_REF] Holmes | Is low-level environmental mercury exposure of concern to human health?[END_REF]. The BBB structure and function are known to vary among different taxa [START_REF] O'brown | Bridging barriers: a comparative look at the blood-brain barrier across organisms[END_REF]) and may have permeabilities with respect to Hg and/or MeHg [START_REF] Aschner | Mercury Neurotoxicity: Mechanisms of Blood-Brain Barrier Transport[END_REF]). However, no findings have indicated that BBB structures differ among coleoid species that would explain the elevated Hg concentrations observed in the optic lobes of E. cirrhosa.

Alternatively, elevated levels of Hg accumulation in the brain that result from increased Hg exposure likely are a result of the tight binding between the metal and chemical components of optic lobes.

Despite of a lack of comparative data and knowledge, HR-XANES spectrometry analyses performed using optic lobes of E. cirrhosa revealed (1) that Hg was entirely methylated, as in mantle muscle in E. cirrhosa [START_REF] Mieiro | Brain as a critical target of mercury in environmentally exposed fish (Dicentrarchus labrax)-Bioaccumulation and oxidative stress profiles[END_REF], and (2) that MeHg is complexed to thiol groups. Importantly, Hg detoxification via with selenium (Se) association can be excluded because no Hg bond signature to Se was observed via HR-XANES spectrometry in the tissues of E. cirrhosa. Thus, cysteine residues from peptides (e.g. glutathione) or proteins (e.g. metallothionein) are the most likely candidate ligands, since they are by far the most predominant biological thiols [START_REF] Manceau | Mercury(II) binding to metallothionein in Mytilus edulis revealed by high energy-resolution XANES spectroscopy[END_REF]. Nevertheless, it should be noted that it was not possible to determine whether another form of MeHg association occurred in other cephalopod species, since brain concentrations in other species assessed were too low to examine via XANES spectrometry.

Conclusions and perspectives

The present study provided information regarding the bioaccumulation and tissue distribution of Hg in five cephalopod species (E. cirrhosa, S. officinalis, L. vulgaris, T. eblanae and I. coindetii) from the Bay of Biscay and has provided new information regarding Hg concentrations and speciation of the digestive gland, muscles and central nervous system. Results showed that efficient Hg accumulation occurred in the optic lobes of cephalopods, which was strongly correlated with Hg concentrations measured of the mantle. The present study also pointed out interspecific differences in Hg concentrations among the five studied species and the unique pattern of Hg bioaccumulation in the brain of E. cirrhosa.

The collection of data regarding Hg concentrations and speciation in this specific tissue is essential for evaluating Hg exposure risk in cephalopods, which are characterised by the presence of centralised nervous systems, their high cognitive capabilities, and their sophisticated behaviour.

While many studies have demonstrated that MeHg impairs motor function, senses, communication (e.g. [START_REF] Basu | An interspecies comparison of mercury inhibition on muscarinic acetylcholine receptor binding in the cerebral cortex and cerebellum[END_REF][START_REF] Zahir | Low dose mercury toxicity and human health[END_REF][START_REF] Puga | Unveiling the neurotoxicity of methylmercury in fish (Diplodus sargus) through a regional morphometric analysis of brain and swimming behavior assessment[END_REF], and induces oxidative stress responses [START_REF] Mieiro | Brain as a critical target of mercury in environmentally exposed fish (Dicentrarchus labrax)-Bioaccumulation and oxidative stress profiles[END_REF], no information has been reported regarding Hg neurotoxicity in cephalopods to date. Since Hg concentrations of some E. cirrhosa specimens (i.e. 1.9 µg.g -1 dw) were 3 times above the neurotoxic threshold found for the white seabream, D. sargus [START_REF] Pereira | Inorganic mercury accumulation in brain following waterborne exposure elicits a deficit on the number of brain cells and impairs swimming behavior in fish (white seabream-Diplodus sargus)[END_REF], further research will be needed to investigate how unique features of species may explain the high Hg levels recorded in octopodidae, and more generally, how Hg contamination in cephalopods may affect their functional brain plasticity, behaviour and fitness [START_REF] Liscovitch-Brauer | Trade-off between Transcriptome Plasticity and Genome Evolution in Cephalopods[END_REF][START_REF] Cosmo | Octopus vulgaris: An Alternative in Evolution[END_REF].

Table 1. Mantle size range and mean (± sd in cm), mantle weight range and mean (± sd in g) and total Hg concentrations (mean ± sd in µg.g -1 dw) in mantle muscles, digestive gland and optic lobes of Eledone cirrhosa (n=20), Illex coindetii (n=11), Todaropsis eblanae (n=9), Loligo vulgaris (n=20), Sepia officinalis (n=21). a Scaled AICc; AICc = 0.00 is interpreted as the best fit to the data among the models.

FigureFigure 1

 1 Figure

Figure 3 .

 3 Figure 3. Chemical forms of Hg determined by Hg L 3 -edge HR-XANES spectroscopy and ball-and-stick representation of the MeHgCys and Hg(Cys) 2 complexes. a-c) Comparison of the spectrum for the optic lobes with the spectra (a) for the mantle muscle, (b) for MeHgCys in fish muscle, and (c) for the digestive gland. d) Linear combination fit of the spectrum for the digestive gland with 68 ± 11% of the optic lobes spectrum and 33 ± 11% of the dicysteinate complex Hg(Cys) 2 . NSS = 1.50 × 10 -4 .

Table 2 .

 2 AICc model ranking for log Hg concentrations within the five studied cephalopod species from Bay of Biscay. Models are GLMs with a gaussian distribution and an identity link function. reviations: Cc, ai e's nformation Criteria adjusted for sma samp e-sizes values; w i , AICc

	Tissues	Family	Mantle	Mantle size	Weight	Weight	[Hg] mantle	[Hg] digestive	[Hg] optic lobes
			size range	mean ± sd	range (g)	mean ± sd	muscles	gland
			(cm)	(cm)		(g)
	Species
	Eledone cirrhosa	Octopodidae	4.2 -14.9	8.3 ± 2.4	19 -764	197 ± 177	0.52 ± 0.35	0.47 ± 0.34	0.60 ± 0.47
	Illex coindetii	Ommastrephidae	14.3 -18.0	15.5 ± 1.4	62 -131	85 ± 19	0.08 ± 0.01	0.12 ± 0.02	0.04 ± 0.01
	Todaropsis eblanae	Ommastrephidae	7.8 -13.0	10.6 ± 1.8	43 -167	105 ± 46	0.29 ± 0.15	0.24 ± 0.06	0.18 ± 0.07
	Loligo vulgaris	Loliginidae	13.2 -19.0	16.6 ± 1.8	83 -198	139 ± 34	0.16 ± 0.03	0.29 ± 0.06	0.09 ± 0.02
	Sepia officinalis	Sepiidae	12.5 -17.0	14.6 ± 1.2	240 -544	397 ± 105	0.46 ± 0.24	0.43 ± 0.12	0.23 ± 0.09

Table 3 .

 3 Parameters of the linear relationships between Hg concentrations in the optic lobes[Hg] ol and the mantle muscles [Hg] m . When the slopes between species were not significantly different, only one model was fitted within pooled data. See Figure2

	Species	Model Equation	P value	R 2
	E. cirrhosa	[Hg] ol = 1.32 x [Hg] m -0.09	p < 0.001	0.978
	S. officinalis -T. eblanae	[Hg] ol = 0.35 x [Hg] m + 0.08	p < 0.001	0.829
	I. coindetii -L. vulgaris	[Hg] ol = 0.50 x [Hg] m + 0.01	p < 0.001	0.663

Acknowledgments

This work is a contribution to the MERCy project funded by the Ministère de la Transition Ecologique et Solidaire et de la Fondation pour la Recherche sur la Biodiversité. The Région Nouvelle Aquitaine is acknowledged for its support to the PhD grant of AM through the EXPO project. We are grateful for the use of the facilities of the Plateforme Analyses Elémentaires of the LIENSs laboratory and to IFREMER for allowing the sampling during the EVHOE cruises (2016)(2017). Thanks are due to the CPER (Contrat de Projet Etat-Région) and the FEDER (Fonds Européen de Développement Régional) for funding the AMA of the LIENSs laboratory. The Institut Universitaire de France (IUF) is acknowledged for its support to PB as a Senior Member. This work benefitted from the French GDR "Aquatic Ecotoxicology" framework which aims at fostering stimulating scientific discussions and collaborations for more integrative approaches. The spectroscopic work was supported by the EcoX project funded by the Equipex initiative of the Agence Nationale pour la Recherche (ANR) under grant ANR-10-EQPX-27-01.