F Thibon
email: fanny.thibon@obs-vlfr.fr

L Weppe

N Vigier

C Churlaud

T Lacoue-Labarthe

M Metian

Y Cherel

P Bustamante

Large-scale survey of lithium concentrations in marine organisms

Keywords: ecotoxicology, bioaccumulation, trophic webs, bio-reduction, biogeography, multiple correspondence analyses

Trace metals such as Cu, Hg, and Zn have been widely investigated in marine ecotoxicological studies considering their bioaccumulation, transfer along trophic webs, and the risks they pose to ecosystems and human health. Comparatively, Li has received little attention, although this element is increasingly used in the high-tech, ceramics/glass, and medication industries. Here, we report Li concentrations in more than 400 samples, including whole organisms and different organs of bivalves, cephalopods, crustaceans, and fish. We investigated species from three contrasting biogeographic areas, i.e. temperate (Bay of Biscay, northeast Atlantic Ocean), tropical (New Caledonia, Pacific Ocean) and subpolar climates (Kerguelen Islands, southern Indian Ocean), among diverse trophic groups (filter-feeders to meso-predators) and habitats (benthic, demersal, and pelagic). Although Li is homogeneously distributed in the ocean (at 0.18 µg/mL), Li concentrations in soft tissues vary greatly, from 0.01 to 1.20 µg/g dry weight. Multiple correspondence analyses reveal two clusters of high and low Li concentrations. Li distributions in marine organisms appear to be mostly geographically independent, though our results highlight a temperature dependency in fish muscles. Li is consistently bio-reduced through the trophic webs, with filter-feeders showing the highest concentrations and predatory fish the lowest.

Strong variations are observed among organs, consistent with the biochemical similarity between Na and Li during transport in the brain and in osmoregulatory organs. Fish gills and kidneys show relatively high Li concentrations (0.26 and 0.15 µg/g, respectively) and fish brains show a large range of Li contents (up to 0.34 µg/g), whereas fish liver and muscles are Li depleted (0.07 ± 0.03 and 0.06 ± 0.08 µg/g, respectively). Altogether, these results provide the first exhaustive baseline for future Li ecotoxicology studies in marine environments.

Introduction

Numerous trace metals are released to rivers, coastal areas, and ultimately the oceans by human activities, and their behaviors in many marine taxa such as bivalves, cephalopods, crustaceans, and fish have been thoroughly investigated in past decades (e.g. [START_REF] Bustamante | Distribution of trace elements in the tissues of benthic and pelagic fish from the Kerguelen Islands[END_REF][START_REF] Eisler | Compendium of trace metals and marine biota Volume 2 vertebrates[END_REF][START_REF] Metian | Trace element bioaccumulation in reef fish from New Caledonia: Influence of trophic groups and risk assessment for consumers[END_REF]. However, lithium (Li) concentrations in marine organisms have received little attention, despite the exponentially increasing use of this element in hightech industries due to its unique physicochemical properties. Li in the oceans is dominantly derived from two natural sources, i.e. high-temperature hydrothermal fluxes at mid-ocean ridges and river inputs. It exits the ocean mainly via the formation of marine authigenic aluminosilicate clays on the seafloor [START_REF] Chan | Lithium isotopic composition of marine sediments[END_REF]. Due to its long oceanic residence time (~1.2 million years)

and its weak capacity to adsorb onto marine particles [START_REF] Decarreau | Partitioning of lithium between smectite and solution: An experimental approach[END_REF], Li is homogeneously distributed throughout the water column [START_REF] Misra | Lithium isotope history of Cenozoic seawater: Changes in silicate weathering and reverse weathering[END_REF]. Thus, the oceanic concentration of dissolved Li is constant at 0.183 ± 0.003 µg/mL, irrespective of latitude and depth [START_REF] Aral | Lithium: environmental pollution and health effects[END_REF][START_REF] Misra | Lithium isotope history of Cenozoic seawater: Changes in silicate weathering and reverse weathering[END_REF]Riley and Tengudai, 1964).

Worldwide anthropogenic Li production has strongly increased in the past decade (from 28 to 43 thousand tons between 2010 and 2017), with its growing use in several industries [START_REF] Labbé | Panorama 2011 du marché du lithium[END_REF]USGS, 2017). Thus, Li has the highest predicted annual growth rate among metals during 2017-2025 (+18%), far surpassing cobalt (+8.8%; Léguérinel et al., 2018). Indeed, Li-ion batteries are expected to account for more than 80% of the total Li market in 2025, although Li in batteries has the worst recycling recovery rate compared to Al, Co, Cu, Mn, and Ni [START_REF] Harper | Recycling lithium-ion batteries from electric vehicles[END_REF]. Li from waste-disposal practices in the USA contaminates groundwater at levels toxic to some freshwater organisms (50-170 µg/L) and far above Li concentrations in most rivers in the USA and worldwide (~1.5 µg/L) (e.g. [START_REF] Huh | Lithium and its isotopes in major world rivers: Implications for weathering and the oceanic budget[END_REF][START_REF] Kszos | Review of lithium in the aquatic environment: Distribution in the United States, toxicity and case example of groundwater contamination[END_REF]. Li contamination also results from the use of agricultural amendments [START_REF] Millot | Multi-isotopic composition (δ7Liδ11B-δD-δ18O) of rainwaters in France: Origin and spatio-temporal characterization[END_REF][START_REF] Négrel | Lithium isotopes as tracers of groundwater circulation in a peat land[END_REF].

Recently, a positive correlation was reported between population density and Li content in the Han River and tap waters of the megalopolis of Seoul (Korea), highlighting the first known largescale Li pollution in an urban area [START_REF] Choi | The impact of anthropogenic inputs on lithium content in river and tap water[END_REF]. Consequently, considerable further anthropogenic contributions of Li to the environment are expected in the coming decades, raising the necessity to understand the role and impact of Li in coastal and marine ecosystems.

It remains unclear how Li affects ecosystems. Li may be somewhat beneficial to organism health, as is the case for humans [START_REF] Giotakos | Lithium in the public water supply and suicide mortality in Greece[END_REF][START_REF] Liaugaudaite | Lithium levels in the public drinking water supply and risk of suicide: A pilot study[END_REF][START_REF] Ohgami | Lithium levels in drinking water and risk of suicide[END_REF][START_REF] Schrauzer | Lithium in drinking water and the incidences of crimes, suicides, and arrests related to drug addictions[END_REF]: some authors consider Li to be an essential trace element, recommending a daily Li intake of 14.3 µg/kg [START_REF] Schrauzer | Lithium: Occurrence, dietary intakes, nutritional essentiality[END_REF]. Indeed, since 1949, various forms of Li (mostly Li2CO3) have been used as the major treatment for bipolar disorder because Li presents a neuroprotective effect [START_REF] Machado-Vieira | Lithium, stress, and resilience in bipolar disorder: Deciphering this key homeostatic synaptic plasticity regulator[END_REF][START_REF] Szklarska | Is lithium a micronutrient? From biological activity and epidemiological observation to food fortification[END_REF][START_REF] Won | An oldie but goodie: Lithium in the treatment of bipolar disorder through neuroprotective and neurotrophic mechanisms[END_REF]. However, Li2CO3 is poisonous beyond the maximum therapeutic dose (10.4 mg/L in blood; [START_REF] Timmer | Lithium intoxication[END_REF] and may lead to death at concentrations above 20 mg/L [START_REF] Aral | Toxicity of lithium to humans and the environment-A literature review[END_REF][START_REF] Schou | Lithium Poisoning[END_REF]. Above a given critical concentration, Li is also toxic to aquatic organisms, affecting some of their metabolic functions. For instance, Li-enriched seawater disrupts embryogenesis in urchins, zebrafish, and amphibians [START_REF] Hall | The mode of action of lithium salts in amphibian development[END_REF][START_REF] Kao | Lithium-induced respecification of pattern in Xenopus laevis embryos[END_REF][START_REF] Kiyomoto | Distinct embryotoxic effects of lithium appeared in a new assessment model of the sea urchin: the whole embryo assay and the blastomere culture assay[END_REF][START_REF] Stachel | Lithium perturbation and goosecoid expression identify a dorsal[END_REF].

The bioconcentration of trace elements is a well-known process [START_REF] Bryan | Bioaccumulation of marine pollutants[END_REF]) that depends on their bioavailability in a given media and their bio-accessibility in trophic sources. Thus, the chemical form of an element (dissolved, particulate, adsorbed, or complexed) influences its capacity for absorption and assimilation by living organisms. Furthermore, the concentrations of some contaminants increase along trophic levels [START_REF] Atwell | Biomagnification and bioaccumulation of mercury in an arctic marine food web: insights from stable nitrogen isotope analysis[END_REF]. Such biomagnification is well documented in various food webs around the world for Hg and organic contaminants [START_REF] Kelly | Food Web-Specific Biomagnification of Persistent Organic Pollutants[END_REF]. In the ocean and coastal areas, abiotic (temperature, salinity, pH) and biotic factors (physiological characteristics, habitats, trophic ecology) can also affect organism metalaccumulation capacities. To date, Li concentrations in marine organisms and their controlling factors have not been intensively investigated [START_REF] Ansari | Heavy metals in marine pollution perspective-a mini review[END_REF][START_REF] Luoma | Bioavailability of trace metals to aquatic organisms -A review[END_REF]. Although significant differences in Li concentrations have been observed between selected marine species and organs [START_REF] English | Trace element and stable isotope analysis of fourteen species of marine invertebrates from the Bay of Fundy, Canada[END_REF][START_REF] Guérin | Determination of 20 trace elements in fish and other seafood from the French market[END_REF][START_REF] Noël | Se and Mo levels in foodstuffs from the Second French TDS[END_REF], changes of Li concentrations along the trophic webs remains unknown.

The aims of the present study are (1) to establish a biogeochemical baseline for future ecotoxicological studies in marine and coastal environments, and (2) to provide a preliminary assessment of the behavior of Li in food webs. Thus, we provide an extensive dataset of Li concentrations across different marine groups (bivalves, cephalopods, crustaceans, and fish) and assess their variations in terms of ecological and geographical factors. We analyzed organs (brain, digestive gland, gills, gonads, kidneys, liver, and muscles) and whole organisms of 33 species from three climatically contrasted zones (tropical: New Caledonia; temperate: Bay of Biscay; subantarctic: Kerguelen Islands).

Materials and methods

Sampling

For our analyses, we selected 33 species from three distinct biogeographic areas: the Bay of Biscay (northeast Atlantic Ocean), New Caledonia (western tropical Pacific Ocean), and the Kerguelen Islands (southern Indian Ocean; see Supplementary Fig. S1). These areas were chosen to explore the effect of climate on Li bioaccumulation in marine organisms: the Bay of Biscay represents a temperate system, New Caledonia a tropical system, and the Kerguelen Islands a subpolar environment. Species were carefully selected to be comparable across the biogeographic areas in terms of taxonomy, position in the water column (habitat), and trophic ecology.

Samples from the Bay of Biscay were bought from fishermen at the local seafood market in La Rochelle (France) between October 2018 and March 2019. Pelagic organisms from the Kerguelen Islands were collected on cruises of the RV "La Curieuse" in February 1998 (austral summer) with a IYGPT trawl (International Young Gadoid Pelagic Trawl, opening 12 x 7 m 2 ) with 10 mm mesh size in the cone, while benthic fish were collected from net fishing at the same period. Samples from New Caledonia were collected in March and October 2007. The samples from Kerguelen and New Caledonia were gathered from a former laboratory collection and have been the subject of several previous studies focusing on other trace elements [START_REF] Bustamante | Distribution of trace elements in the tissues of benthic and pelagic fish from the Kerguelen Islands[END_REF][START_REF] Chouvelon | Hg concentrations and related risk assessment in coral reef crustaceans, molluscs and fish from New Caledonia[END_REF][START_REF] Cipro | Trace elements in invertebrates and fish from Kerguelen waters, southern Indian Ocean[END_REF][START_REF] Metian | Trace element bioaccumulation in reef fish from New Caledonia: Influence of trophic groups and risk assessment for consumers[END_REF]. The analyzed organisms included molluscs (bivalves and cephalopods), crustaceans (Malacostraca), and fish (Chondrichthyes and Actinopterygii). The organisms were divided into trophic groups to assess the influence of food and feeding ecology characteristics on Li bioaccumulation [START_REF] Metian | Trace element bioaccumulation in reef fish from New Caledonia: Influence of trophic groups and risk assessment for consumers[END_REF][START_REF] Cipro | Trace elements in invertebrates and fish from Kerguelen waters, southern Indian Ocean[END_REF]. We defined the trophic groups according to feeding mode (filter-feeders, grazers/scavengers, predators) and preferred predator diet (i.e., invertebrates, small fish, or both). In addition, the trophic level of each fish species [START_REF] Froese | FishBase[END_REF] was used to better define any relationship between an organism's Li content and its position in the food webs.

These trophic levels are based on the organism diet and the trophic level of their food items. They are estimated using nitrogen isotopes (δ 15 N) to quantitatively position species in their respective food webs [START_REF] Kline | Cross-validation of trophic level estimates from a mass-balance model of Prince William Sound using 15N /14N data[END_REF].

Immediately after sampling, all organisms were weighed (wet weight, ww) and measured (total length, i.e., from the head to the base of the caudal fork for fish, mantle length for squids, shell width for bivalves, and carapace length for some crustaceans). The collected species and their weights (wet and dry) and lengths are reported in Supplementary Table S1

Sample dissolution and Li concentration analyses

To minimize procedural blanks, the following procedures were performed in a pressurized clean laboratory, under a fume hood, and using distilled reagents and pre-cleaned vessels.

Between 200-and 250-mg aliquots of each freeze-dried tissue sample were weighed and poured into a Corning® 50 mL centrifuge tube. Sample dissolution was performed overnight at room temperature in a mixture of 6 mL of 65% ultrapure HNO3 and 2 mL of 37% ultrapure HCl (reverse aqua regia). For mineralization, the dissolved samples were transferred to a microwave and heated to 120°C over 30 min, then held at 120°C for 15 min. After mineralization, all samples were diluted to 50 mL with Milli-Q water. Procedural blanks and the reference material IAEA 436 (tuna fish flesh; International Atomic Energy Agency, Austria) were prepared via the same protocol to evaluate contamination and data precision and accuracy.

Li concentrations were analyzed using a Thermo Fisher X-Series II Inductively Coupled Plasma Mass Spectrometer. Li concentrations are reported in µg/g dw. Calibration and spike solutions were prepared using a 1,000 mg/L Li monoelemental commercial solution. The biological reference material IAEA 436 was analyzed several times during each analytical session to monitor the accuracy and reproducibility of the measured Li concentrations; the Li concentration of this biological reference material is not certified, but was estimated by Azemard et al., 2006 (0.040 ± 0.009 µg/g dw, confidence interval from 0.018 to 0.062 µg/g dw). Due to the uncertainties on the Li concentration of this standard, we developed a spike-addition method to verify (1) the accuracy of our Li measurements and (2) the completeness of the mineralization process. For each of the twelve mineralization sessions, four aliquots of ~250 mg of IAEA 436 were digested. In three of these tubes, spikes of different Li concentrations (0.5, 1.0, or 5.0 µg/L) were added. We achieved good recovery rates for the spike concentrations (98.2 ± 3.4%, n = 12), demonstrating the near-100% yield of our entire chemical procedure. The calibration solutions were run several times during each analytical session to monitor instrumental drift and correct for the memory effect. Blank contributions were about 5%, and internal errors on measurements were 2%. Non-spiked IAEA 436 reference material provided good reproducibility (4%) between the twelve analytical sessions, and a mean Li concentration of 0.020 ± 0.001 µg/g dw (n = 12), within the confidence interval published by [START_REF] Azemard | Report on the world-wide intercomparison exercise for the determination of trace elements and methylmercury in fish homogenate IAEA-436[END_REF] During our analytical sessions, the detection limit was 0.0125 µg/g dw for 250 mg of sample taken up in 50 mL of diluted reverse aqua regia; only two samples had concentrations below this value and were sidelined: the liver of Scyliorhinus canicula and the brain of Spondyliosoma cantharus.

Statistical data processing

As the Li concentrations in our samples are not normally distributed, even transformed, differences among data groups (i.e., between biogeographic areas, taxonomic groups, trophic groups and levels, habitat, and organs) were tested statistically using the R software package (R Core Team, 2017) following two non-parametric tests: the Mann-Whitney-Wilcoxon test for comparisons between two data series, and the Kruskal-Wallis test for comparisons between three or more series. The level of significance for statistical analyses was set at α = 0.05. The detailed statistical treatment is presented in the Supplementary Material.

The relationships between Li concentrations and biological parameters were investigated using multiple correspondence analyses (MCA, see section 3.1), a statistical tool that reduces the number of original qualitative variables to a smaller number of dimensions that explain the majority of the observed variability. In this study, MCA was performed in R considering the following variables: habitat, location, trophic group, taxonomical group, and organ type (predictor categorical variables); Li concentration was then added as a single dependent quantitative variable. We used the "ACM" function of "FactoMineR" R package (quanti.sup subfunction was filled to take Li into account).

Results and Discussion

Li concentrations ranged from the detection limit (0.01 µg/g dw) to 1.20 µg/g dw (digestive gland of Pecten maximus) (Supplementary Table S2). As the oceanic Li concentration is homogeneous at 0.18 µg/mL [START_REF] Aral | Lithium: environmental pollution and health effects[END_REF][START_REF] Misra | Lithium isotope history of Cenozoic seawater: Changes in silicate weathering and reverse weathering[END_REF]Riley and Tengudai, 1964), the large range of Li concentrations observed in marine organisms must result from different bioaccumulation dynamics related to biotic and abiotic processes. To investigate this, we first examine the entire dataset to identify which factors are related to high -or low -Li concentrations, and then investigate these factors in more details.

Multifactorial control of Li bioaccumulation by marine species

The relationship between Li and biological parameters (modalities of the predicted categorical variables) was investigated using MCA, as described in section 2.3. In Figure 1, the calculated coordinates of all variable modalities are plotted in a two-dimensions space. Li concentration was added as a quantitative variable, and is represented by the grey arrow. The proximity between different modalities in Figure 1 indicates a connection between them, such as for "bivalves" and "filter-feeders", or "fish" and "predators of invertebrates".

It is notable that the modalities "fish", "predators of invertebrates", "predators of small fish", and "demersal" are associated (group 1, Fig. 1), which was expected considering that most fish are demersal. "Bivalves", "benthic", and "filter-feeders" form a second group (group 2), which is consistent with the fact that collected filter-feeders are benthic bivalves. "Cephalopods" are associated with "pelagic" and "predators of invertebrates and small fish" in group 3, as this group is represented by four pelagic squid vs. only two species of benthic cuttlefish. "Crustaceans" and "grazers/scavengers" constitute group 4. The association of these two last modalities is also related to sampling: the only collected grazer is a crustacean. Each group includes several parameters (biogeographic area, trophic group, taxonomy, habitat, or tissue), indicating that most of the modalities are confounded. There is clearly an imbalance across potential predictors that is related to the sampled organisms. However, sampling location is reasonably well crossed with other predictors and it is possible to evaluate independent effects of this variable (see section 3.2).

The Li concentration trend (grey arrow) points almost directly towards group 2 and away from group 1, indicating that samples whose modalities belong to those groups 2 and 1 correspond to high and low Li concentrations, respectively. Whether feeding mode (filter-feeding)

or taxonomy (bivalve) is related to high Li values cannot be discerned with this data set, however, the MCA shows that filter feeding bivalves display higher Li values. The impact of each variable on Li concentration is further discussed in the following sections.

Impact of biogeographic area

To determine whether Li concentration differs according to biogeographic areas, we chose three locations characterized by contrasted climates and environments, here represented by the annual mean sea surface temperature (SST): New Caledonia (SST = 21 °C), the Bay of Biscay (SST = 16 °C), and the Kerguelen Islands (SST = 1 °C) (https://www.watertemperature.org/).

For comparing Li concentrations of similar tissues, we choose muscle, the only ubiquitous organ among the various studied organisms (note that gonads from Pectinidae are also considered, since gonads and muscle were pooled together in New Caledonian individuals). At first glance, the observed differences in Li concentrations between biogeographic areas are not drastic, as shown in Figure 2 and Supplementary Table S3. The largest difference (of 0.17 µg/g dw, p < 0.01) is observed between Li concentrations in crustacean muscles from the Bay of Biscay and New Caledonia. For fish and cephalopod muscles, differences between biogeographic areas are less than 0.06 µg/g dw (on average). Li concentrations in muscles and gonads of Pectinidae from the Bay of Biscay are similar to or slightly lower than those of Pectinidae from New Caledonia. In the MCA, the predicted variable "Location" is not highly confounded with other variables (cf. Fig. 1 section 3.1), allowing discussion on its direct effect on Li concentrations. The modalities "Bay of Biscay", "Kerguelen Islands", and "New Caledonia" are not aligned with observed variations in Li concentrations (Fig. 1), indicating that, considering all species, there is no clear and simple relationship between tissue Li concentrations and a biogeographic / climatic gradient. The similar Li concentrations in Pectinidae are also consistent with the results of [START_REF] Chassard-Bouchaud | Bioaccumulation of lithium by marine organisms in European, American, and Asian coastal zones: microanalytic study using secondary ion emission[END_REF], which showed that Li retention by Mytilus edulis (common mussel) is geographically independent.

Nevertheless, in fish, Li concentrations in muscles (n > 20 per location) increase significantly (p < 0.0001) from 0.03 ± 0.01 µg/g dw (New Caledonia) to 0.14 ± 0.17 µg/g dw (Kerguelen Islands)

with decreasing SST, similar to crustaceans (although we have fewer data on crustaceans) (Fig. 2).

The fish, as poikilotherms, are metabolically influenced by seawater temperature: typically, lower temperatures mean slower metabolism [START_REF] Mehta | Impact of temperature on contaminants toxicity in fish fauna: a review[END_REF]. Therefore, the higher Li concentrations in muscles of fish from the Kerguelen Islands are consistent with a slower Li turnover (and therefore a higher Li retention time), compared to fish from the Bay of Biscay and New Caledonia. This trend is not observed for cephalopods, but we only had a few samples from New Caledonia (n=8). It is consistent with the temperature effect on Hg and Pb retention in in the black-lip oyster Saccostrea echinata [START_REF] Denton | Influence of temperature and salinity on the uptake, distribution and depuration of mercury, cadmium and lead by the black-lip oyster Saccostrea echinata[END_REF], with increasing metal half-life in visceral mass when temperature decreases. Accumulation and depuration experiments of Cu and Pb conducted on the girdled horn sea snail Cerithidea cingulata and the Starry flounder Platichthys stellatus, respectively, also reported higher tissue concentrations after depuration at low temperature compared to higher temperatures (Prabhakara [START_REF] Rao | Influence of temperature on copper accumulation and depuration in a tropical cerithiid Cerithidea (cerithideopsilla) cingulata (Geelin 1790)[END_REF][START_REF] Varanasi | Biological fate of metals in fish. Marine biological effects of OCS petroleum development[END_REF]. A more detailed investigation of Li bioaccumulation processes is however necessary to explore the relationship between Li concentrations and metabolic rate among different species.

Impact of trophic group

Collected organisms allow exploring Li accumulation in different trophic groups (detailed in section 2.1). Considering all tissues, Li concentration among all filter-feeders is 0.42 ± 0.25 µg/g dw, a value significantly higher than for any other trophic groups (p < 0.0001; Fig. 3). In contrast,

Li concentrations in grazers/scavengers and in predators of invertebrates, of invertebrates and small fish, and of small fish are low (0.10 ± 0.02, 0.10 ± 0.11, 0.09 ± 0.07, and 0.07 ± 0.00 µg/g dw, respectively) and not statistically different (p > 0.05). These differences are in agreement with our MCA results (section 3.1) indicating that Li concentrations align from the modality "predators of small fish" towards the modality "filter-feeders".

Considering muscle tissues alone avoids potential bias due to the incorporation of Lienriched sedimentary particles in the gut, for instance in the digestive gland of filter-feeders (see section 3.4). Muscle tissues display the same trend between filter-feeders and other trophic groups (Fig. 3B), although not significant due to the paucity of muscles sampled among filterfeeders (n=10).

The same Li decrease along trophic groups and among taxonomic groups is observed in the three biogeographic areas (Fig. S3). All areas combined, Li concentrations decrease from bivalves to fish (p < 0.05) as: bivalves (0.42 ± 0.50 µg/g dw) > crustaceans (0.19 ± 0.19 µg/g dw) > cephalopods (0.12 ± 0.10 µg/g dw) > fish (0.08 ± 0.17 µg/g dw; Fig. 4). The variability among Li concentrations was limited, except for fish (0.01-0.77 µg/g dw) and, to a lesser extent, cephalopods (0.02-0.23 µg/g dw), as previously reported for other trace elements (e.g., Rainbow

2002). This variability may partly be explained by the various habitats (benthic, demersal, and pelagic) occupied by species within these two groups. However, sampled organisms did not show a significant effect of habitats on Li concentrations. In contrast, the MCA (Fig. 1) indicates that the benthic habitat is close to the high Li concentration group (group 2). This is strongly influenced by the dominance of Li-rich filter-feeders among benthic species, whose Li enrichment in the digestive gland is discussed in more details in the following section. The Li concentration variability within groups can also be related to the presence of various tissues (see section 3.4), and result from species differences in ecological need, swimming behavior, or metabolic activities [START_REF] Canli | The relationships between heavy metal (Cd, Cr, Cu, Fe, Pb, Zn) levels and the size of six Mediterranean fish species[END_REF].

Since filters-feeders are generally at lower trophic levels than predatory fish, our results strongly suggest that trophic ecology influences Li concentrations in marine organisms, but a bioreduction, not biomagnification, of Li is observed along the trophic webs (Fig. 3A). Both comparisons including all organs (Figs. 3A,4) and muscles only (Fig. 3B) demonstrate the consistent bio-reduction of Li along marine food webs, similar to that previously described for the two essential trace metals, Cu and Zn. In contrast, Hg is known to significantly bioamplify towards the top of the food webs [START_REF] Atwell | Biomagnification and bioaccumulation of mercury in an arctic marine food web: insights from stable nitrogen isotope analysis[END_REF]. Consensus has not been reached on other elements such as As, Cd, and Se, although several studies argue for their bioamplification in specific cases [START_REF] Barwick | Biotransference and biomagnification of selenium copper, cadmium, zinc, arsenic and lead in a temperate seagrass ecosystem from Lake Macquarie Estuary, NSW, Australia[END_REF][START_REF] Rahman | Bioaccumulation, biotransformation and trophic transfer of arsenicin the aquatic food chain[END_REF]; see review in [START_REF] Suedel | Trophic Transfer and Biomagnification Potential of Contaminants in Aquatic Ecosystems[END_REF]. These

observations suggest that the risk assessment on human health is limited to Li intake from shellfish consumption and not from Li-poor mesopredators (e.g. fish and cephalopods), which is in marked contrast to Hg [START_REF] Atwell | Biomagnification and bioaccumulation of mercury in an arctic marine food web: insights from stable nitrogen isotope analysis[END_REF][START_REF] Chouvelon | Enhanced bioaccumulation of mercury in deep-sea fauna from the Bay of Biscay (north-east Atlantic) in relation to trophic positions identified by analysis of carbon and nitrogen stable isotopes[END_REF][START_REF] Lavoie | Biomagnification of Mercury in Aquatic Food Webs: A Worldwide Meta-Analysis[END_REF].

Li distribution in the tissues of marine organisms

Our dataset allows us to further explore the internal distribution and accumulation of Li among various organs. Comparison of the different tissues within each taxonomic group (Fig. 5, Table 1; excluding crustaceans, for which only muscles were analysed) shows that cephalopods are the only group for which there is no significant difference (p > 0.05) between Li concentrations in muscles and digestive gland. For the other groups, Li is unequally distributed among organs (Fig. 5, Table 1). In fish, Li is most concentrated in the gills, moderately concentrated in the kidneys and brain, and least concentrated in the liver and muscles. In bivalves (P. maximus), both gills and digestive gland display high Li concentrations compared to muscles and gonads. These observations are supported by the MCA results (section 3.1): the Li trend extends from the modalities "liver", "brain", "muscles", and "kidneys" towards "gills" and "gonads" in the high Li concentration group (group 2; Fig. 1).

In fish, the liver is usually reported to be a storage organ for many trace elements (Cd, Cu, Fe, Hg, Pb, and Zn;[START_REF] Metian | Trace element bioaccumulation in reef fish from New Caledonia: Influence of trophic groups and risk assessment for consumers[END_REF]. However, our data show that the liver is depleted in Li compared to the gills and kidneys (Figs. 5,6), regardless of geographic origin (Table S2).

Nonetheless, liver Li concentrations are higher than those in muscles, except for S. canicula and C. gunnari (Fig. 6, Table S2). The relatively low Li concentrations in fish liver (average 0.07 ± 0.03 µg/g dw) demonstrate that Li is not stored in this organ; our results instead show that the brain, gills, and kidneys play a key role in Li bioaccumulation. The kidneys and gills are involved in ionoregulation, and brain functions mostly depend on Na channels that areable to transport Li as well. Gills are in direct contact with seawater and, together with the kidneys, play a critical role in osmoregulation of marine organisms and the maintenance of their internal Na balance [START_REF] Greenwell | Osmoregulation in fish: Mechanisms and clinical implications[END_REF]. Li uptake in fish most likely occurs through Na channels [START_REF] Tkatcheva | Physiological and biochemical effects of lithium in rainbow trout[END_REF] and ubiquitous Na-H Exchangers (NHEs) located in cell membranes [START_REF] Counillon | Na+/H+ antiporter (NHE1) and lactate/H+ symporters (MCTs) in pH homeostasis and cancer metabolism[END_REF].

Li + ions can penetrate gill cells via NHEs by following Na + pathways [START_REF] Milosavljevic | The intracellular Na+/H+ exchanger NHE7 effects a Na+ coupled, but not K+ coupled proton-loading mechanism in endocytosis[END_REF][START_REF] Tkatcheva | Physiological and biochemical effects of lithium in rainbow trout[END_REF]. The high Li concentrations measured in gills and kidneys is thus consistent with Li accumulation primarily controlled by NHEs during water filtration, explaining why these two organs appear crucial to Li distribution in marine organisms. This implies that seawater is likely a major pathway of Li incorporation, although additional particulate pathways, such as predation, might participate to Li incorporation. Future studies should now further investigate the relative importance of different Li incorporation pathways in fish.

Gills functions may also partly explain the observed Li bio-reduction between filterfeeders and other trophic groups (cf. section 3.3). Indeed, Li becomes more concentrated in the muscles of organisms that use gills both for respiration and feeding (e.g., filter-feeders) compared to organisms that use gills for respiration only (crustaceans, cephalopods, and fish). Thus, organ function may play a key role in how Li is distributed and internally regulated. Unfortunately, gills from filter-feeders could not be further analyzed to investigate this relationship. Among fish, aside from muscles, we observe no trend according to trophic groups for Li concentrations in liver, kidneys, or gills (Fig. S4). However, we note that Li concentrations in fish brain increase with trophic level (from 3 to 5, p < 0.01, Fig. 7), a trend deserving further investigation.

In the king scallop Pecten maximus, the highest Li concentration was observed in the digestive gland (0.80 ± 0.27 µg/g dw, Fig. 6), apparently consistent with its major role in detoxification and the storage of several trace elements, such as Ag, As, Cd, Cu, and Zn, which reach high concentrations in this tissue [START_REF] Bustamante | Subcellular and body distributions of 17 trace elements in the variegated scallop Chlamys varia from the French coast of the Bay of Biscay[END_REF]Metian et al., 2008a, b;[START_REF] Saavedra | Anatomical distribution of heavy metals in the scallop Pecten maximus[END_REF]. Nevertheless, the considered Pectinidae live in benthic sediments and are likely to ingest fine particles from the muddy sand, such as silts and clays [START_REF] Mikulich | Food of the scallop[END_REF][START_REF] Shumway | Food resources related to habitat in the scallop, Placopecten magellanicus (Gmelin 1791): A qualitative study[END_REF], which can contain Li at concentrations >100 µg/g [START_REF] Tardy | Le lithium dans les minéraux argileux des sédiments et des sols[END_REF], several orders of magnitude above that dissolved in seawater. Therefore, the high digestive gland Li concentrations may be due to the presence of sediment particles that remained in the gut portions sampled with the digestive gland tissue, as the Pecten were not depurated before dissection. This interpretation is consistent with the presence of Li-rich particles observed by [START_REF] Chassard-Bouchaud | Bioaccumulation of lithium by marine organisms in European, American, and Asian coastal zones: microanalytic study using secondary ion emission[END_REF] in the intestine lumen of the pearl oyster Pinctada fucata martensi. Similarly, the Li concentrations in gonads may be overestimated as this organ is crossed by the intestine, which serves also for gamete emission (Fig. 6). Although not evidenced yet, Li + ions from the ingested food and sediment particles may pass through the intestine to be stored in the digestive gland and gonads, as suggested for the blue mussel (Mytilus edulis) and the transparent razor shell (Cultellus pellucidus) [START_REF] Chassard-Bouchaud | Bioaccumulation of lithium by marine organisms in European, American, and Asian coastal zones: microanalytic study using secondary ion emission[END_REF].

Conclusions

This work provides the first extensive dataset of Li concentrations and distributions among marine organisms and their tissues in contrasted biogeographic areas (the Bay of Biscay, New Caledonia, and the Kerguelen Islands). Within trophic webs, Li is consistently bio-reduced in the three regions. Li heterogeneities among organs are consistent with the biological incorporation of Li by Na transporters. Although further study is required at high trophic levels, the observed Li bio-reduction towards predators suggests a weak impact of their consumption on human health.

However, this investigation also highlights the need to assess Li bio-accumulation in bivalves and shellfish and their potential impact on human health in case of Li contamination.

Table 1. Average Li concentrations (μg/g dw ± SD) in various organs of bivalves, cephalopods, crustaceans, and fish from the entire dataset (Supplementary Tables S1 andS2). n: number of individuals. S2). Modality coordinates are reported in the space of dimensions 1 and 2 (Dim1 and Dim2), the first two dimensions resulting from MCA, which are ordered according to the percentage of the data variability that they explain. Qualitative variables considered here are trophic group, taxonomic group, habitat, organ type, and location. The grey arrow shows Li concentration as a quantitative variable. Although this model cannot discriminate which factor is the major control on Li distribution, it highlights that some modalities of different variables (e.g. filter feeders, bivalves, benthic, and gonads) are grouped and have higher Li concentrations than others (demersal, liver, fish, and predator of small fish, in particular). Abbreviations: FF, filter feeder; GS, grazer/scavenger; PI, predator of invertebrates; PISM, predator of invertebrates and small fish; PSM, predator of small fish; BB, Bay of Biscay; NC, New Caledonia; Kerg., Kerguelen Islands; Dig. Gl., digestive gland. All statistical analyses were performed using the R software. For every test, the level of significance for statistical analyses was set at α = 0.05. With continuous qualitative variables such as lithium concentration, statistical tests were employed to determine if any differences observed between groups or species were significant or not. This is given by the p-value (p-val, Wasserstein and Lazar, 2016), calculated using R software functions of adapted statistical tests (cf. table below): when the calculated p-value is < 0.05, the observed difference is considered as significant.

When p-value is > 0.05, the difference is not considered as significant.

There are two statistical approaches: parametric, and non-parametric. The parametric approach is used when data distribution follows the dispersion of a normal probability low. Nonparametric approach does not involve any hypothesis about data distribution and uses a rank order method. Here is a summary This mode is classically used to represent groups using boxplots. The basic description of a

Boxplot is summarized below for information (the central horizontal bar being for the median value)

Figures

PI

Crust. for crustacean. FF, filter-feeders; GS, grazers/scavengers; PI, predator of invertebrates; PISM, predator of invertebrates and small fish; PSM, predator of small fish. Trophic group were defined according to fishbase.org for fish and sealifebase.se for bivalves, cephalopods, and crustaceans.

Table S2. Average Li concentration (µg/g dw) measured in the various tissues of the marine organisms from the Bay of Biscay, the Kerguelen Islands and New Caledonia.

Figure 1 .

 1 Figure 1. Results of the multiple correspondence analyses (MCA) performed over the entire dataset (Supplementary TableS2). Modality coordinates are reported in the space of dimensions 1 and 2 (Dim1 and Dim2), the first two dimensions resulting from MCA, which are ordered according to the percentage of the data variability that they explain. Qualitative variables considered here are trophic group, taxonomic group, habitat, organ type, and location. The grey arrow shows Li concentration as a quantitative variable. Although this model cannot discriminate which factor is the major control on Li distribution, it highlights that some modalities of different variables (e.g. filter feeders, bivalves, benthic, and gonads) are grouped and have higher Li concentrations than others (demersal, liver, fish, and predator of small fish, in particular). Abbreviations: FF, filter feeder; GS, grazer/scavenger; PI, predator of invertebrates; PISM, predator of invertebrates and small fish; PSM, predator of small fish; BB, Bay of Biscay; NC, New Caledonia; Kerg., Kerguelen Islands; Dig. Gl., digestive gland.

Figure 2 .

 2 Figure 2. Li concentrations (μg/g dw) in different marine organisms grouped by biogeographic area and indicating mean annual sea surface temperature (SST): NC, New Caledonia, SST = 21 °C (red); BB, Bay of Biscay, SST = 16 °C (orange); Kerg., Kerguelen Islands, SST = 1 °C (blue). Abbreviations: Biv., bivalves; Crust., crustaceans; Ceph., cephalopods. Numbers at the right of each plot indicate the number of analyses (n) for each location. We note that n = 5 for NC and BB crustaceans and NC Pectinidae, n = 8 for NC cephalopods, and n > 20 for other groups. Asterisks indicate p-values from the Mann-Whitney-Wilcoxon test (see Supplementary Material for details) and highlight the significance of the observed differences between pairs of groups: ns (not significant) for p > 0.05, * for p < 0.05, ** for p < 0.01, *** for p < 0.001, **** for p < 0.0001 (highly significant difference). Outliers are plotted as grey circles.

Figure 3 .

 3 Figure 3. Box plots showing Li concentrations (μg/g dw) of (A) all organs/tissues and (B) muscles vs. trophic group. The numbers at the top of each plot indicate the number of analyses for each trophic group (n). Trophic groups are: FF, filter-feeders; GS, grazers/scavengers; PI, predator of invertebrates; PISM, predator of invertebrates and small fish; PSM, predator of small fish. Asterisks and 'ns' indicate statistical significance as in Figure 2. Outliers are plotted as gray circles. The high variability of Li concentrations observed at the PI level is mainly related to the fish G. piabilis and G. nicholsi, which are mostly planktivores.

Figure 4 .

 4 Figure 4. The median and range of Li concentrations (μg/g dw -all tissue considered) among different taxonomic groups. The numbers at the top of the plot indicate the number of analyses (n) per taxonomic group. The dashed blue line indicates the seawater Li concentration (0.18 μg/mL). Subdivisions are made by the habitat: benthic species (brown) live on the seafloor, demersal species (or benthopelagic, orange) move in the water column just above the seafloor, and pelagic species (yellow) live in the water column. Abbreviations: Biv., bivalves; Crust., crustaceans; Ceph., cephalopods. Asterisks and 'ns' indicate statistical significance as in Figure 2. Outliers are plotted as gray circles.

Figure 5 .

 5 Figure 5. Li concentrations (μg/g dw) measured in the whole body ('whole') and different tissues of the analyzed marine organisms. The dashed blue line indicates the seawater Li concentration (0.18 μg/mL). Fish show the greatest variability in Li concentrations, both within each organ type and among different organs. Abbreviations: Biv., bivalves; Crust., crustaceans; Ceph., cephalopods.

Figure 6 :

 6 Figure 6: Average Li concentrations (μg/g dw) in the organs of selected fish and bivalve species from the Bay of Biscay. Colors indicate organ types, small gray dots indicate individual analyses, and vertical lines indicate the range of observed values. Abbreviations: PI, predator of invertebrates; PISM, predator of small fish and invertebrates; FF, filter-feeders. In most studied species, gills display higher Li concentrations than other organs, whereas muscles and liver show lower Li concentrations.

Figure 7 .

 7 Figure 7. Average Li concentrations (μg/g dw) in fish brain vs. (a) trophic groups and (b) trophic levels. Fish trophic levels are from Froese and Pauly (2020). Small grey circles are individual measurements. Vertical lines reflect the range of values per trophic group or level. Numbers at the top indicate the number of analyses (n) per trophic group or level. Asterisks and 'ns' indicate statistical significance as in Figure 2.

  Figure S1. Locations of sampling sites.

Figure S2 .

 S2 Figure S2. Soft tissue Li concentration ([Li] in µg/g dw) vs. length of individuals. Color code for the group to whom studied individuals belong.

Figure S3 .

 S3 Figure S3. Box plots showing Li concentrations (µg/g dw) separated by geographical area of (1) all organs/tissues and (2) muscles vs. trophic group. Numbers at the top indicate the number of analyses (n) per trophic group. Trophic groups are: FF, filter-feeders; GS, grazers/scavengers; PI, predator of invertebrates; PISM, predator of invertebrates and small fish; PSM, predator of small fish.

Acknowledgments

We acknowledge financial support from the ANR ISO2MET (//anr.fr/Projet-ANR-18-CE34-0002, www.iso2met-project.fr/) and we are grateful for access to facilities provided by the laboratory LIttoral ENvironnement et Sociétés (LIENSs). This work benefited from the support of the PNEC Programme (Chantier Nouvelle-Calédonie), the IRD (Institut de Recherche pour le Développement), the IPEV (Institut Paul Emile Victor, program 309, C. Barbraud), and the TAAF (Terres Australes et Antarctiques Françaises). The authors are grateful to O. Pringault (IRD-Nouméa) for his help in organizing the fieldwork in New Caledonia, and to Captain M. Clarque (IRD-Nouméa) and the IRD Diver Team for providing access to their diving equipment. We thank G. Duhamel and the crew of the RV 'La Curieuse' for their help with collecting organisms in the Kerguelen Islands. We thank Michel Warnau for his support during fieldwork in New Caledonia, and Maud Brault-Favrou and Elise Noger-Huet for their help during sample preparation. We are also extremely grateful to Jean-Olivier Irisson (LOV) for his help with statistical computation of the data (MCA). We thank the CPER (Contrat de Projet Etat-Région) and the FEDER (Fonds Européen de Développement Régional) for funding ICP-MS analyses at the LIENSs laboratory. The IUF (Institut Universitaire de France) is acknowledged for its support to PB as a Senior Member.

Author contributions

PB and NV led the project. TLL, PB, LW, and YC participated in sampling. CC and LW performed sample preparation and Li concentration measurements. FT and LW modeled the data and wrote the manuscript. All authors contributed to interpretation and manuscript correction.

Supplementary Material

Large scale survey of lithium concentrations in marine organisms

Data tables


Alexandre Cugerone
email: alexandre.cugerone@umontpellier.fr

Bénédicte Cenki-Tok

Emilien Oliot

Manuel Munoz

Alain Chauvet

Kalin Kouzmanov

Vincent Motto-Ros

Elisabeth Le Goff

Rare metal concentration associated to recrystallization: example from the Pyrenean Axial Zone

Germanium is often found in trace concentration in non-deformed sphalerite (ZnS). However, locally in sphalerite from the Variscan Pyrenean Axial Zone, the presence of Ge-minerals (oxides, sulphides or chloritoids with up to 70% wt Ge) is remarkable. The rise question is thus what are the chemical and/or mechanical processes that control rare element contents of sulphides which have undergone deformation and metamorphism? In order to answer this question, we document microstructural and chemical heterogeneities in sphalerite, performed with EBSD (electron backscatter diffraction) coupled to LA-ICPMS in situ analysis. Deformation could induce recrystallization of primary sphalerite. Recrystallized domains contain low Ge contents (1-50 ppm Ge) whereas porphyroclastic sphalerite grains present higher Ge concentrations (up to 550 ppm Ge). Geminerals (up to 70 wt% Ge) are exclusively hosted in the Ge-poor recrystallized domains. We propose that Germanium was removed from the primary sphalerite crystal lattice during sulphide recrystallization and subsequently concentrated in Ge-minerals, leaving behind a Ge-depleted fine-grained recrystallized sphalerite matrix. Numerous sulphide ores enriched in rare elements similar to Pyrenean deposits may present recrystallization features like deformed Mississippi-Valley-Type, Kipushi-type or Volcanic-Hosted-Massive-Sulphide deposits. Consequently, the potential of this type of deposits shall be evaluated and we suggest that exploration methods must integrate precise textural and microstructural information coupled with trace element in situ and mapping techniques.

Introduction

Critical metals like Germanium (Ge), Gallium (Ga) or Indium (In) are often hosted in sphalerite lattice. These elements are presently exploited as by product in base metal deposits in low concentrations (up to several thousand of ppm ; [START_REF] Höll | Metallogenesis of germanium-A review[END_REF]). These concentrations are observed in non-deformed sulphides (Cugerone et al., 2018a). But the largest concentrations of zinc sulphide are hosted in deformed/metamorphosed environments [START_REF] Wilkinson | Sediment-Hosted Zinc-Lead Mineralization: Processes and Perspectives: Processes and Perspectives, Treatise on Geochemistry[END_REF]) and the presence of critical metals in these deposit-type has been poorly explored.

The impact of recrystallization on sulphide composition has been studied especially in pyrite or arsenopyrite [START_REF] Cook | Arsenopyrite-Pyrite Association in an Orogenic Gold Ore : Tracing Mineralization History from Textures and Trace Elements[END_REF][START_REF] Dubosq | Pyrite deformation and connections to gold mobility : Insight from micro-structural analysis and trace element mapping[END_REF]) but only few studies are available for sphalerite. [START_REF] Lockington | Trace and minor elements in sphalerite from metamorphosed sulphide deposits[END_REF] compare diverse sphalerites which naturally endured different metamorphic imprints. A loss of trace elements like Pb, Bi, Ag is observed associated to a reincorporation of Fe, Cd, Mn, In. Re-homogenization of Cu in the sphalerite lattice is observed but data for Ge in sphalerite are absent. The studied sphalerites are located in the Pyrenean Axial Zone (PAZ) Pb-Zn district deposits. These Pb-Zn mineralizations were sampled from deposits exploited in the past and represent an interesting natural material for studying the impact of recrystallization on sphalerite texture and chemistry. The occurence of Ge-minerals hosted in sphalerite is reported in the Pyrenean Axial Zone [START_REF] Johan | Analogues germanifères et gallifères des silicates et oxydes dans les gisements de zinc des Pyrénées centrales, France; argutite et carboirite, deux nouvelles espèces minérales[END_REF]Cugerone et al. 2018a).like brunogeierite (GeFe 2 O 4 ), briartite (GeCu 2 (Fe, Zn) S 4 ) or carboirite (GeFeAl 2 O 5 (OH) 2 ).

Our study aims at investigating the role of recrystallization on trace elements composition of sphalerite, as well as the mode of formation of these hyper concentrated Ge-minerals. In this study, we have combined textural (EBSD) and chemical (EPMA and LA-ICP-MS) analyses.

Methods

Electron-BackScattered Diffraction (EBSD) maps were performed with a Camscan Crystal Probe X500FE SEM-EBSD at Geosciences Montpellier (CNRS-University of Montpellier, France). Condition of acquisition were 20 keV for the voltage, 5 nA for the probe current with a working distance of 25 mm under 2 Pa low vacuum. Samples were positioned horizontally and at the standard 70° angle of the EBSD detector. EDS maps were acquired simultaneously with electron backscatter pattern data. The measurement step was systematically below 5 µm. Oxford Instruments Aztec and Channel 5 softwares were used to generate EDS and EBSD grain size maps.

Microprobe analysis (EMPA) were carried out using a Cameca SX100 electron microprobe analyzer at the Service inter-regional Microsonde-Sud, Montpellier. Major, minor and trace elements were measured with a beam current of 100nA and accelerating voltage of 20 kV. 14 elements were analyzed: standards, spectral lines, and spectrometers were as follows: Zn (Zn, Lα, TAP); S (FeS 2 ,Kα, PET); Fe (Fe 2 O 3 ,Kα, LLif); Cd (CdS, Lα, LPET); Ge (Ge, Kα, LLif); Sb (GaSb, Lα, LPET); Cu (CuS, Kα, LLif); Ga (GaSb, Kα, LLif); Ag (Ag, Lα, LPET); Mn (Mn, Kα, LPET); Sn (Sn, Lα, PET); Pb (Pb, Mα, PET); As (GaAs, Lα, TAP); Si (CaSiO 3 ,Kα, TAP). Peak counting times ranged from 30 to 240 s (240 s for Ge). Germanium is analyzed on 2 LLif monochromators. The limit of de-tection for Ge and Cu, calculated by internal Cameca procedures, is reduced to 84 ppm and 110 ppm respectively. Gallium, As, Ag, Pb, Sn, Sb, and Mn were below detection limit. Zinc contents in sphalerite measured with EMPA were used as internal calibration for LA-ICP-MS analysis.

Laser ablation inductively coupled plasma-mass spectrometry (LA-ICP-MS) was used to determine trace elements concentrations in sphalerite. Analyses were carried out using an Excimer CompEx 102 coupled with a ThermoFinnigan Element XR available at the OSU-OREME AETE platform at the University of Montpellier. Laser ablation was performed using a constant 5 Hz pulse rate at 140 mJ laser energy. Each LA-ICP-MS analysis comprises 180 s of background measurement and 60 s of sample ablation (signal measurement), followed by a 60 s retention time to ensure a proper cell washout. Data were processed using the Glitter 4.0 software package. The following isotopes were measured: 29 and 208 Pb. MASS-1 reference was used as external standard (Dr. Stephen Wilson, personal communication) with a corrected 57 ± 1.75 ppm value for Ge. NIST SRM 610 was used as secondary external standard to identify possible instrumental drift. The elements Ni, As, Mo, Se, Pd and Si are in all analyses below the limits of detection. Only Ge and Cu contents measured in sphalerite will be discussed in this paper.

Samples

Pb-Zn deposits in Pyrenean Axial Zone

Pyrenean Pb-Zn deposits are hosted in the Variscan deformed (~ 325-290 Ma) Pyrenean Axial Zone exhumed during the collision between the Iberia and Eurasian plates since the Lower Cretaceous. Foliation trajectories exhibit a monotonous N080-N110°E trend with variable dip angles. Two main Variscan deformation events are recorded in the host-rock. A poorly expressed S 1 cleavage is often parallel to S 0 and is associated to regional M 1 Medium Pressure and Low-Temperature (MP-LT) metamorphic conditions. A wellexpressed S 2 cleavage is sub-vertical and superimposed to the previous structures. S 2 axial planar cleavage is associated to M 2 contact (HT-LP) metamorphic conditions, marked in the studied area with the appearance of cordierite.

Three Pb-Zn mineralizations types are present in the PAZ and fully described in Cugerone et al. 2018b. In Figure 1, schematic 3D sketch is shown and describe the major mineralization types. Type 1 corresponds to minor disseminated mineralization, probably syngenetic and from an exhalative source. (II) Type 2a is a stratabound mineralization, epigenetic and synchronous to the Variscan D 1 regional deformation event and (III) Type 2b is a vein mineralization, epigenetic and supposed synchronous to the late Variscan D 2 regional deformation event.

Herein, we will only focus on the Type 2b Ge-rich mineralization and more precisely on Arre deposit (Cugerone et al. 2018b). Type 2b is an epigenetic subvertical vein-type mineralization (Fig. 2A) which has endured at least one deformation event, probably Variscan in age (D 2 in Cugerone et al. 2018b). These veins are composed of sphalerite and galena, with a small amount of pyrite, chalcopyrite, arsenopyrite and gangue of quartz-carbonate (Fig. 2B). Numerous Type 2b deposits are present in the Axial Zone (Cugerone et al. 2018b) and in this paper, we will only focus on the Arre deposit. This Pb-Zn(-Ge) mineralization is hosted in Devonian calc-schist and marble rocks. Veins are oriented N070 E with sub-vertical dip (Fig. 2A).

Macrotextures and microtextures

EBSD has been performed on several Type 2b mineralizations and especially in Arre sphalerite (Fig. 3A and3B) to obtain textural and microstructural information. Sphalerite appear with diverse grain size (from 10 μm to > 1 mm). Coarse grained are considered as parent grains are internally deformed Smaller grains (< 100µm) are located around the parent grains locally Cugerone et al., 2018b) in twin boundaries. Three different texture in sphalerite are distinguished: dark domains and light domains in coarse grains and recrystallized small grains.

On the same area, EDS chemical mapping is performed during EBSD acquisition, and location of Geminerals is noted in Figure 3A. Ge-minerals are essentially composed of brunogeierite (GeFe 2 O 4 ) and carboirite (GeFeAl 2 O 5 (OH) 2 ). Ge-minerals are preferentially hosted in the small recrystallized sphalerite fraction or close to twin boundaries.

Ge and Cu in sphalerite

LA-ICP-MS analyses were all performed associated to EBSD grain size map to link in situ chemistry to the type of texture ablated, on the surface at least. Figure 3A shows EBSD grain size map on sphalerite ore. LA-ICP-MS spots are visible in Figure 3B associated to their Gecontents with a sphalerite observed in transmitted light. Germanium appear highly zoned. In dark and light domains, median content reach 433 ± 21 ppm and 46 ± 2 ppm respectively. In recrystallized domains, Ge content is typically low with median Ge concentrations of 4 ± 2 ppm. Heterogeneous Ge in sphalerite is positively correlated to Cu, especially in the dark and light domains in Arre deposit with Cu median content of 1012 ± 75 ppm and 102 ± 7 ppm respectively. Cu is lower in recrystallized grains with median values of 29 ± 9 ppm.

Calculation of Ge content in the sphalerite compared to Ge-minerals shows that Ge concentration in Geminerals is equivalent to a supposed primary nondeformed sphalerite with mean content of 700 ppm Ge (Cugerone et al., in prep).

Discussion and conclusions

The studied sphalerites from Arre deposit show remarkable heterogeneous sphalerite texture and related chemistry. Formation of Ge-minerals is intimely associated to the deformation (supposed Variscan D 2 ) and recristallization due to their preferentially occurence in recrystallized fraction (Fig. 3a). Germanium was probably removed from the primary sphalerite crystal lattice during recrystallization and subsequently concentrated in Ge-minerals, leaving behind a Gedepleted fine grained recrystallized sphalerite matrix (Cugerone et al., in prep).

Several textural and chemical analogues to these sphalerites may be present in deformed-MVT deposits [START_REF] Ye | Trace and minor elements in sphalerite from base metal deposits in South China: A LA-ICPMS study[END_REF], in Kipushi deposits [START_REF] Belissont | Distribution and oxidation state of Ge, Cu and Fe in sphalerite by u-XRF and K-edge u-XANES: Insights into Ge incorporation, partitioning and isotopic fractionation[END_REF][START_REF] Horn | Textural characteristics and trace element distribution in carbonatehosted Zn-Pb-Ag ores at the Paleoproterozoic Black Angel deposit, central West Greenland[END_REF]) but also in other sulphides like chalcopyrite from VHMS deposit [START_REF] Reiser | Mineralogy and geochemistry of tin-and germanium-bearing copper ore, Barrigao re-mobilized vein deposit, Iberian Pyrite Belt, Portugal[END_REF]. These studies present recrystallized textures and, in some cases reported Ge-minerals. Ores affected by recrystallization are likely to contain accessory minerals that can be potentially enriched in rare metals.

This study will integrate Laser induced breakdown spectroscopy (LIBS) mapping on these ore samples to better understand the trace element heterogeneities in sphalerite. It is an excellent technique to map Ge because there is no interference with another element in sphalerite, contrary to WDS or µ-XRF mapping.

Figure 1 :

 1 Figure 1: Schematic 3D sketch presenting the three main mineralization types which are typically observed in the studied area and related to each studied deposit. Type 2b vein mineralizations appear in intensely S2 deformed domains. Location of Arre deposit is noted (modified from Cugerone et al., 2018b)

Figure 2 :

 2 Figure 2: A: Pb-Zn Type 2b Vein mineralization in Arre deposit. B: Typical Pb-Zn Type 2b ore mineralization with color zonation in sphalerite and presence of superimposed S2 cleavage (modified from Cugerone et al., 2018b, S0-S1: S0-S1 foliation; S2: S2 foliation; Qtz: quartz; Sph: sphalerite.

Figure 3 .

 3 Figure 3. A: EBSD grain size map in sphalerite (Arre sample) with location of Ge and Cu rich phases. These minerals are preferentially preferentially located in the recrystallized sphalerite domains. B: Corresponding area in transmitted light with LA-ICP-MS spot analyses associated to Ge-contents (in ppm). Note the color zonation which is corresponding to variations in Ge content. (S2: S2 foliation; Qtz: quartz; Sph: sphalerite). This figure is from Cugerone et al., in prep.

  Si; 34 S; 55 Mn; 57 Fe; 59 Co; 61 Ni; 63 Cu; 65 Cu; 64 Zn; 69 Ga; 71 Ga; 74 Ge; 75 As; 77 Se; 95 Mo; 105 Pd; 107 Ag; 109 Ag, 110 Cd; 111 Cd, 115 In; 118 Sn; 119 Sn; 120 Sn; 121 Sb; 123 Sb

Acknowledgements

The authors thank the French Geological Survey (Bureau de Recherches Géologiques et Minières; BRGM) for funding through the national program "Référentiel Géologique de France" (RGF-Pyrénées). The authors gratefully acknowledge Christophe Nevado and Doriane Delmas for thin section preparation, Fabrice Barou for EBSD analysis. Bernard Boyer and Olivier Bruguier for respectively, their involvement in EMPA (Service inter-régional Microsonde-Sud, Montpellier) and LA-ICP-MS analysis (Plateforme AETE, OSU-OREME, Montpellier)


Enora Tourneur

Alain Chauvet

Kalin Kouzmanov

Johann Tuduri

Stanislas Sizaret

Textural and mineralogical constraints on the mode of formation of the Bou Azzer Co-Ni arsenide mineralization (Anti-Atlas, Morocco): Tectonic implications

The Bou Azzer inlier, supposed to be the suture zone of the Neoproterozoic orogeny, is composed of an assumed ancient oceanic crust of serpentinized ultramafic rocks, gabbro and basaltic pillow rocks. This inlier hosts the Co-Ni ore deposit of Bou Azzer with two types of mineralization: i) a massive, elongated ore bodies, known as "contact" type mineralization, along the contact between serpentinite, quartz diorite and Precambrian volcanic rocks; and ii) a "cross-cutting" type represented by faults that cut all units, only mineralized at the vicinity with the "contact" type. A detailed mineralogical and textural study brings new arguments on the ore-formation processes at Bou Azzer. First, it appears that both types are developed within a progressive and continuous tectonohydrothermal stage as shown by the mineralogical evolution. Second, the "contact" type exhibits two various aspects: i.e., Laminated Contact Mineralization (LCM) and Breccia-Related Contact Mineralization (BRCM) that are related to different initial textures of the pre-mineralization host rocks. Third, Ni-Co mineralization seems developing by replacement of spinel/magnetite grains of the serpentinite. All these observations are consistent with ore formation along a tectonic contact thus explaining the existence of serpentinite fragments slope breccia.

Introduction

Various cobalt mineralization styles on Earth can not be attributed to a single ore-forming model. At least, six major mineralization styles are known: 1) sedimenthosted stratiform Cu-Co deposits (Fay and Barton 2012); 2) ortho-magmatic sulfide deposits [START_REF] Naldrett | A model for the formation of the Ni-Cu-PGE deposits of the Noril'sk Region: In International Platinum[END_REF]; 3) Co from lateritic profiles mainly exploited for Ni (Orloff 1968); 4) Co-rich crusts associated with Mnnodules within seawater hydrothermal oxidizing venting environment [START_REF] Hein | Cobalt rich ferromanganese crusts in the pacific: In Marine mineral deposits, 1st edition[END_REF]; 5) hydrothermal/volcanogenic deposits (the Bou Azzer district) [START_REF] Leblanc | Ophiolites précambriennes et gîtes arseniés de cobalt (Bou Azzer, Maroc)[END_REF][START_REF]Contribution à l'étude du district à Co, As[END_REF]; 6) Five elements (Ni-Co-As-Ag-Bi) vein-type deposits [START_REF] Kissin | Nickel-cobalt-native silver (five elemnt) veins: A rift-related ore type[END_REF][START_REF] Markl | Natural fracking and the genesis of five-element veins[END_REF]Burish et al. 2017). At Bou Azzer, two types of ore bodies occur -"contact" and "cross-cutting" ones, both with unclear mode of formation, especially regarding the relationships with larger scale geodynamic processes. In this contribution, we present and discuss: i) mineralogical and paragenetic features that favor coeval processes of formation for both mineralization types at Bou Azzer; ii) new textural observations helping to discuss the premineralization tectonic context and its implication on the presently admitted classical model of Precambrian ophiolite for the Bou Azzer inlier [START_REF] Leblanc | Proterozoic oceanic crust at Bou Azzer[END_REF][START_REF] Gasquet | Contribution to a geodynamic reconstruction of the Anti-Atlas (Morocco) during Pan-African times with the emphasis on inversion tectonics and metallogenic activity at the Precambrian-Cambrian transition[END_REF].

The Bou Azzer -El Graara inlier and deposits

The Bou Azzer -El Graara inlier, oriented NW-SE, is located in the central part of the Moroccan Anti-Atlas, south of Ouarzazate. The inlier is composed of a dismembered but complete sequence of an assumed ancient oceanic crust composed by serpentinized ultramafic rocks overlaid by gabbro and basaltic pillow rocks [START_REF] Leblanc | Ophiolites précambriennes et gîtes arseniés de cobalt (Bou Azzer, Maroc)[END_REF][START_REF] Leblanc | Proterozoic oceanic crust at Bou Azzer[END_REF][START_REF] Leblanc | The Late Proterozoic ophiolites of Bou Azzer (Morocco): evidence for Pan-African plate tectonics[END_REF][START_REF] Bodinier | Geochemistry of Precambrian ophiolites from Bou Azzer, Morocco[END_REF].

For this reason, this area is interpreted as being a suture zone related to the Neoproterozoic orogeny (e.g. [START_REF] Choubert | Histoire géologique du Précambrien de l'Anti-Atlas de l'Archéen à l'aurore des temps primaires[END_REF][START_REF] Leblanc | Interprétation géodynamique du domaine panafrican (Précambrian terminal) de l'Anti-Atlas (Maroc) à partir de données geologique et géochronologique[END_REF][START_REF] Saquaque | Un exemple de suture-arc: Le Precambrien de l'Anti-Atlas centre oriental (Maroc)[END_REF]. The tectono-magmatic evolution of this area [START_REF] Tuduri | Structural control, magmatic-hydrothermal evolution and formation of hornfels-hosted, intrusion-related gold deposits: Insight from the Thaghassa deposit in Eastern Anti-Atlas, Morocco[END_REF]) involves a first tectono-magmaticevent, associated with massive high-K calc-alkaline magmatism emplaced in deformed meta-sediments under transpressive tectonic control (the Lower Complex); and a second transtensive deformational event, associated with intense magmatism, volcanism and volcano-clastic sediments deposition (the Upper Complex). The Precambrian group of Ouarzazate, characterizing the Upper Complex, is composed of dacitic to rhyolitic ignimbrites and andesitic tuffs [START_REF] Mifdal | Datations U/Pb et Rb/Sr du volcanisme acide de l'Anti-Atlas marocain et du socle sous-jacent dans la région de Ouarzazate. Apport au problème de la limite Précambrien-Cambrien[END_REF]; all intruded by a calc-alkaline to highly potassic plutonic rocks. All these Precambrian units are covered by the Cambrian sedimentary cover [START_REF] Pouit | Paléogéographie et répartition des minéralisations stratifomes de cuivre dans l'Anti-Atlas occidental (Maroc)[END_REF][START_REF] Bouchta | L'aire cuprifère de l'Anti-Atlas (Maroc) : permanence et arêtes riches[END_REF]Gasquet et al. 2001).

3 Textural and paragenetic constraints on the mineralization

The Co-Ni ore deposits of Bou Azzer

Two types of ore bodies occur in the Bou Azzer district, as defined by [START_REF] Leblanc | Ophiolites précambriennes et gîtes arseniés de cobalt (Bou Azzer, Maroc)[END_REF] and En-naciri (1995) (Fig. 1):

i) The massive "contact" type is composed by Co-Ni-Fe arsenides hosted within a siliceous or carbonaceous gangue. This mineralization is developed systematically at the contact between serpentinites and quartz-diorite and/or Upper complex volcanic rocks south and north of the serpentinite occurrences, respectively.

ii) The "cross-cutting" type is composed of Co-Fe arsenide mineralization, associated with K-feldspar, quartz and carbonate gangue in veins. These veins intersect all units, and are only mineralized at the vicinity of the "contact" type ore bodies. Six paragenetic stages are recognized at Bou Azzer: 1) a Ni-arsenide stage with nickeline (NiAs) and rammelsbergite (NiAs 2 ); 2) a Co-arsenide stage with skutterudite (CoAs 3 ) and safflorite (CoAs 2 ); 3) a Co-sulfo arsenide stage with cobaltite (CoAsS); 4) a Fe-arsenide stage with loellingite (FeAs 2 ); 5) a Fe sulfo-arsenide stage with arsenopyrite (FeAsS); and 6) a sulfide stage with chalcopyrite, sphalerite, galena, and fahlores. The six stages are observed within the contact type mineralization, all included in a carbonaceous and a siliceous gangue whereas only the last fourth stages occur in the cross-cutting type.

A petro-structural model is proposed in order to integrate the six mineralization stages within three main events which lead to the formation of the Bou Azzer deposits (Fig. 1): 1) a Ni-Co-rich arsenide stage composed of nickeline, rammelsbergite and subsequent cobaltite and skutterudite; 2) the formation of the siliceous or carbonaceous gangue associated with the late Co-rich arsenide and the earlier Fe-rich sulfoarsenide minerals; 3) the final formation of the crosscutting structures that mainly host the final stage of the Co-rich arsenide mineralization event and the Fe-Cubearing sulfide minerals, both occurring in veins mainly composed of K-feldspar (minor), quartz and carbonate.

Textural observations on the massive type

Detailed textural analysis of the "contact" type mineralization led to the following observations: 1. Massive arsenide-rich parts of the mineralization show two distinct textural features:

-Massive lenses of lamellar nickeline surrounded by skutterudite sometimes alternating with carbonate forming a banded texture (Fig. 2a).

-Massive lenses of arsenides with isolated nuclei of elongated rammelsbergite surrounded by cobaltite, carbonate, skutterudite and chalcopyrite, all included within large euhedral skutterudite and with late carbonate (Fig. 2b); 2. The gangue of the contact mineralization is mainly carbonaceous and may exhibit several inherited fragments of serpentinite (Fig. 3a), indicating the nature of the host rock (serpentinite) prior to the formation of the mineralized structure; 3. Skutterudite is commonly fractured, with neoserpentine infilling (Fig. 3b), thus demonstrating that serpentinization was still active at least during the earliest mineralization stages. 4. Spinels and rammelsbergite which entirely replace spinel are observed within the serpentinite close to the contact (Fig. 3b). Spinels are systematically bordered by magnetite (Fig. 3c) or rammelsbergite (Fig. 3b). In addition, cathodoluminescence imaging shows relics of spinel also frequently occurring in the core of the carbonate gangue (Fig. 3c), thus demonstrating again that serpentinite was the precursor of the mineralized gangue. 5. Cathodoluminescence imaging of the gangue carbonate shows that two textures co-exist; one dominated by large euhedral crystals evolving toward fragmented grains with a lot of space between them, similar to textures related to dilatation breccia formed within a stress-free open space (Fig. 3d) [START_REF] Jébrak | Hydrothennal breccias in vein-type ore deposits: A review of mechanisms, morphology and size distribution[END_REF][START_REF] Chauvet | Structural Control of Ore Deposits: The Role of Pre-Existing Structures on the Formation of Mineralised Vein Systems[END_REF]. Two main conclusions could be drown from the textural and mineralogical analysis of the Bou Azzer mineralization:

-The mineralogical evolution of both contact and cross-cutting mineralization types results from a progressive and continuous process that begins with Ni-Co ore formation followed by Co-Fe-Cu-rich stages. Ni-Co stages are encountered exclusively within the contact type while the Co/Fe/Cu stages are developed within both structures. This suggests that both mineralizations, i.e. the contact and the cross-cutting ones, are formed during the same tectono-mineralogical event even if the contact type ore bodies were the ones that formed first. -Textural features within the "contact" type demonstrate that these domains result from replacement and hydrothermal processes that affect a serpentinite rock. Textural constraints indicate that most of the ore bodies derived from serpentinite breccia could form Breccia-related Contact Mineralization (BRCM); although some can form, by the transformation of banded serpentinite levels, Laminated Contact Mineralization (LCM) (Fig. 4). It is suggested that such brecciated areas, characterized by high permeability, are favorable domains for fluid circulation and subsequent ore formation.

-The occurrence of relictic spinel bordered by magnetite and rammelsbergite confirms the hypothesis of replacement of serpentinite levels. It is suggested that spinel and magnetite fragments are at the origin of the Ni/Co-rich arsenides whereas serpentinite ones formed the gangues.

A first hypothesis regarding the tectonic context of ore formation at Bou Azzer is illustrated in figure 3: the brecciated levels as precursors for the formation of the contact orebodies can result from the activity of normal faults, as expected by the geometrical constraints. Such normal faults are not consistent with the classical obduction scenario and can help to the exhumation of the serpentinized mantle in this area.

Figure 1 .

 1 Figure 1. Model of formation of the Bou Azzer Co-Ni-rich arsenide ore deposit showing the distribution of the mineralization stages within the "contact" and the "cross-cutting" type mineralization.

Figure 2

 2 Figure 2. a. The "contact" type composed of laminated contact mineralization (LCM) of Ni-Co rich arsenide then surrounding by carbonate (image in reflected light). b. Nucleus of Rammelsbergite surrounding by skutterudite, carbonate, chalcopyrite, in a massive gangue of skutterudite and carbonates, indicates a brecciatedrelated environment of the contact mineralization (BRCM) (image in reflected light). Carb: carbonate, Co: Cobalt, Cp: Chalcopyrite, Ni: Nickel, Ram: Rammelsbergite, Sk: skutterudite

Figure 4 .

 4 Figure 4. Example of hypothetic tectonic context consistent with the formation of serpentinite breccia that serves as receptacle for mineralization. In that case, fault motion need to be normal. (BRCM: Brecciated-related contact mineralization; LCM: Laminated contact mineralization)

Acknowledgements

Managem and CTT mining companies are acknowledged for their support during field work and access to the mine sites. Part of the laboratory work was supported by the TelluS Program of CNRS/INSU. We also acknowledge Christophe Nevado and Doriane Delmas for their meticulous works on thin sections.