Keywords: mercury, seabirds, Southern Antarctic Territories, speciation, stable isotopes, ecology, trophic transfer, biogeochemistry

Despite their distance from industrial pressure, marine southern and Antarctic environments are contaminated by worldwide distributed pollutants, such as mercury (Hg), through atmospheric transport and oceanic currents. So far, Hg contamination pathways in the Southern Ocean remains poorly understood, particularly in the Indian sector, and new studies are required to elucidate its fate and impact in these regions. Seabirds, as top predators of marine food webs, are exposed to elevated concentrations of biomagnified methylmercury (MeHg) via dietary intake and moreover, they forage in the different marine compartments both in spatial and depth terms. Therefore, they are considered as effective bioindicators of Hg environmental contamination and the good knowledge of their ecological characteristics permits their application for tracing Hg in such remote environments otherwise of difficult access. The main objective of this doctoral work is the characterization of the exposure pathways of the MeHg accumulated in model seabirds and the identification of the processes involved in the Hg biogeochemical cycle in the Southern Ocean (from Antarctic to subtropical waters). The proposed methodological approach consisted on the combination of Hg isotopic composition and Hg speciation in tissues of a precise selection of seabirds of the Southern Ocean. In a first step, the evaluation of tissue-specific Hg isotopic signatures was accomplished notably in blood and feathers, as they can be non-lethally sampled. In chicks, both tissues can be effectively and indifferently used for biomonitoring of local contamination using Hg isotopes, whereas in adults each tissue provides access to different temporal exposure: blood at recent scale (i.e. exposure during the breeding period) and feathers at annual scale, thus providing complementary isotopic information at the different stages of seabird annual cycle. A second part was focused on the exploration of MeHg sources in four penguin species within a same subantarctic location, the Crozet Islands. Hg isotopes effectively discriminated the four populations and species-specific foraging habitats and latitudinal movements were found the main factors determining their exposure to distinct environmental MeHg sources. In a third part, Hg isotopes were investigated in two ubiquitous seabird models (skua chicks and penguins) over a large a latitudinal scale from Antarctica to the subtropics. Latitudinal variations of Hg isotopic values (δ 202 Hg, Δ 199 Hg) appeared to be influenced by different extent of photochemical processes and other biogeochemical pathways such as Hg reduction, and methylation/demethylation processes, as well as trophic or metabolic processes.

Résumé

Malgré leur distance de la pression industrielle, les milieux marins austraux et antarctiques sont contaminés par des polluants globaux, tels que le mercure (Hg), par le transport atmosphérique et les courants océaniques. Jusqu'à présent, les voies de contamination du Hg dans l'Océan Austral restent peu connues, en particulier dans le secteur Indien, et de nouvelles études sont nécessaires pour élucider son devenir et son impact dans ces régions. Les oiseaux marins, en tant que prédateurs supérieurs des chaînes alimentaires marines, sont exposés à des concentrations élevées de méthylmercure (MeHg) via leur régime alimentaire. De plus, ils présentent des habitats de prédation contrastés dans les différents compartiments marins (en termes spatiaux et de profondeur). Par conséquent, ils sont considérés comme des bioindicateurs efficaces de la contamination par Hg et la bonne connaissance de leurs caractéristiques écologiques permet leur application comme traceurs du Hg dans des environnements éloignés, qui sont autrement difficiles à échantillonner. L'objectif principal de ce travail de doctorat est la caractérisation des voies d'exposition des MeHg accumulé par les oiseaux marins et l'identification des processus impliqués dans le cycle biogéochimique du Hg dans l'Océan Austral (des eaux antarctiques aux eaux subtropicales). L'approche méthodologique proposée consiste à combiner la composition isotopique du Hg et la spéciation du Hg dans les tissus d'espèces d'oiseaux marins de l'Océan Austral choisis précisément. Dans une première étape, une évaluation des signatures isotopiques de Hg spécifique de chaque tissu a été réalisée, notamment dans le sang et les plumes, car ceux-ci peuvent être échantillonnés de manière non létale. Chez les poussins, les deux tissus peuvent être utilisés efficacement et indifféremment comme bioindicateurs de la contamination locale en utilisant des isotopes du Hg, alors que chez les adultes, chaque tissu donne accès à une exposition temporelle différente: le sang à l'échelle récente (c'est-à-dire l'exposition pendant la période de reproduction) et les plumes à l'échelle annuelle, fournissant ainsi des informations isotopiques complémentaires aux différentes étapes du cycle annuel des oiseaux. Une deuxième partie a été axée sur l'exploration des sources de MeHg dans quatre espèces de manchots d'une même zone subantarctique, les îles de Crozet. Les populations ont été discriminées par leurs signatures isotopiques du Hg en fonction de leurs habitats de prédation et leurs mouvements latitudinaux, qui se trouvent être les facteurs principaux déterminant leur exposition à différentes sources de MeHg. Dans une troisième partie, les isotopes du Hg ont été étudiés dans deux modèles d'oiseaux (poussins de labbes et manchots adultes) sur une grande échelle latitudinale de l'Antarctique à la zone subtropicale. Les variations latitudinales des valeurs isotopiques du Hg (δ 202 Hg, Δ 199 Hg) semblent être influencées par une différente amplitude des processus photochimiques et d'autres voies biogéochimiques telles que la réduction du Hg, et les processus de méthylation / déméthylation, ainsi que les processus trophiques ou métaboliques.

Mots clés: mercure, oiseaux marins, Terres Australes et Antarctiques Françaises, spéciation, isotopes stables, écologie, transfert trophique, biogéochimie Preface iii

Remerciements/Acknowledgements

Tout d'abord, je voudrais remercier mon directeur et co-directeur de thèse, Paco Bustamante et David Amouroux, d'avoir fait confiance à moi dans un premier temps et m'avoir soutenue au long de cette étape, pour vos conseils, vos lectures et relectures, votre patience… j'ai énormément appris de vous pendant ces trois années. Paco, merci beaucoup pour ton encadrement, ta disponibilité en tout moment, tes réponses et corrections super rapides et efficaces malgré la distance tu as été toujours là et tu as énormément facilité l'organisation et le bon déroulement de la thèse. David, mille merci pour tous tes conseils, réflexions et discutions enrichissantes, pour m'avoir communiqué tout ce savoir sur le mercure et les isotopes… et science appart, merci pour ton aide, ta compréhension et même pour m'avoir permis de faire des voyages incroyables pendant cette thèse ! Un énorme merci à Zoyne Pedrero pour la confiance que tu m'as faite du début, c'est avec toi que j'ai fait mes premiers pas dans le monde de la recherche et je suis très reconnaissante de m'avoir encouragé à faire cette thèse. Pour ton aide, ton amitié et tes conseils… mil gracias! A Yves Cherel, merci beaucoup pour tes conseils, corrections et pour tout ce que tu m'as appris sur l'écologie et ce monde merveilleux des oiseaux. Merci beaucoup à toutes les personnes qui ont fait partie de ce projet, à tous les fieldworkers qui ont contribué à la collection des échantillons dans le cadre du programme no. 109 (H. Weimerskirch) soutenu par l'Institut Polaire Français Paul Emile Victor (IPEV) et Terres Australes et Antarctiques Françaises (TAAF) et par l'Agence Nationale de la Recherche (program POLARTOP, O. Chastel). Merci également à Alice Carravieri, pour l'organisation des échantillons, les fichiers de données et les réponses super rapides.

Merci aussi à Aurélien

Un grand merci à Emmanuel Tessier et Sylvain Bérail pour vos conseils techniques, votre attention en tout moment, les coups de main dans les grosses sessions d'analyses et surtout votre patience ! J'ai beaucoup appris à votre côté et ça a été un vrai plaisir de travailler avec vous.

Merci beaucoup à Julien Barre qui m'a supporté au labo mais aussi au bureau pendant ces presque quatre ans, pour m'aider et solutionner toutes les doutes et m'accompagner pendant les longues semaines d'analyse. A Sylvain Boucher, pour les coups de main avec la machine et les conseils. A Bastien, pour ton aide lors de la deuxième année, grâce à ta bonne humeur les grosses étapes de travail ont été un plaisir avec les rockeurs, les punks et les métalleurs. Thanks to Joana and Caiyan for being my guides and for all your help and encouragement inside and outside the lab.

Merci aussi à toutes les autres personnes du LCABIE pour la bonne ambiance de travail, à mes collègues de bureau, les anciens et les nouveaux: Nagore, Ariane, Alice, Marine A., Tiago, Alyssa, Wahid. Un super merci à tous mes collègues et amis, qui ont été un grand soutien pour moi, à Sara, Javi, Maxime, Marine, Manue, Mathieu, Andy, Jota … pour tous les repas, pauses cafés, rires, sorties et soirées ensemble.

Preface iv

Un millón de gracias a los colegas de la Universidad de Valparaíso, a Gaby Lobos y su equipo, Paty, Dani, Felipe por su acogida, su buen humor y toda su ayuda durante mi estancia y mis viajes por Chile, fue una experiencia inolvidable en gran parte gracias a vosotros. Y por último muchas gracias a todas esas personas increíbles que tengo la buenísima suerte de tener a mi lado. A mi pequeña familia paloise, Iris, Izar, Amaia, Jesús, Clément, Aralar, Jérôme, Leire, Iñigo, Dani…porque os adoro a pesar de las infinitas anchoas y traquenards. A Sara, mi compi de aventuras, por estos cuatro años (y todos los que quedan) siendo también protagonista de mi vida. A Diego, por todos los ánimos y también por cuidarme y alimentarme estas últimas semanas. A Sofi y Quike, mi inspiración y referentes allá donde vayan, gracias por ser tan geniales. A Krikri, mein Wunderprofessor, thank you so much for your inspiring words, your humour and all the laughs together. A Christian, Luci y Ana por ser capaces de hacerme soltar una carcajada y apoyarme desde cualquier parte del mundo. A Claupi, Moni y a mi gente de Somo y Loredo por ayudarme a desconectar y por todos los ánimos sobre todo este último verano. Y un millón de gracias a mi familia, por todo el cariño, apoyo y todos los empujoncitos hacia arriba… ¡gracias! . Mercury is a chemical element represented by the symbol Hg from the Greek "hydrargyros", meaning silver water. It is a transition metal which appears under its liquid form at normal conditions of temperature and pressure. Hg atomic number is 80, its atomic mass 200.59 g•mol -1 .

Preface v

Table of content

Regarding its electronic configuration, Hg is classified as a transition metal IIB presenting an unsaturated "d" layer, hence it is easily polarizable. Hg presents three oxidation states: Hg 0 (metallic), Hg2 2+ and Hg 2+ . It tends to form stable complexes with similar ligands such as thiols (-SH) and cyanides (-CN).

Due to its physicochemical properties, Hg naturally occurs in all types of environmental compartments, is easily transported over long distances and accumulated in the environment. One important characteristic of Hg is its volatility (due to its relatively high vapour pressure for being a metal) [START_REF] Stein | Environmental Distribution and Transformation of Mercury Compounds[END_REF], which facilitates its release in the air and, consequently, the direct discharges into the sea via rainfall and river runoff. The global Hg cycle is therefore dominated by exchanges between the atmosphere and the aquatic environment. Hg can appear under different chemical forms: elemental mercury (Hg 0 ), divalent inorganic form (Hg 2+ or iHg), and organic forms including monomethylmercury (CH3Hg or MeHg), dimethylmercury ((CH3)2Hg o DMHg) and ethylmercury (C2H5Hg).

Toxicity and effects of Hg in health

Hg is nowadays considered as a priority and global pollutant especially due to its accumulation and biomagnification in aquatic food webs. Hg causes severe toxic effects on animal and human health and its degree of toxicity strongly depends on its speciation [START_REF] Tan | The endocrine effects of mercury in humans and wildlife[END_REF]. Its organometallic form, MeHg, is recognized by its capacity to bioaccumulate in the tissues of the organisms and biomagnificate during trophic transfer. Indeed, seafood consumption remains the major contributor to Hg contamination in humans and is generally used as a reliable criteria for the risk assessment associated to Hg exposition. Moreover, MeHg is a potent neurotoxin and causes irreversible damages to the nervous system and brain. This problematic has positioned Hg (and especially MeHg) as pollutants of major concern and have been widely investigated in the last decades for better understanding of the mechanisms of MeHg generation, transformations and occurrence in the environment and led to the agreement of the Minamata Convention with the objective of reducing the utilisation and emission of Hg. Exposure of Hg in humans Despite its utilisation in the past as a cure of syphilis and various infections, it has been established that Hg can produce serious and irreversible damages on reproduction, behaviour, endocrinology and development [START_REF] Grandjean | Adverse effects of methylmercury: Environmental health research implications[END_REF]. Although the main route of human exposure to Hg compounds is related to fish consumption (mainly in form of MeHg), it can also occur by inhalation or skin contact. Indeed, Hg poisoning by inhalation of Hg 0 is the most fatal intoxication since around 80% penetrates easily into the blood where it is retained by oxidation.

This can produce harmful effects on the nervous, digestive, and immune systems and the lungs and kidneys [START_REF] Who | Exposure tonMercury: A major public health concern[END_REF]. The negative effect of Hg is aggravated by its capacity of binding proteins and biomolecules. Indeed, the intoxication by Hg in humans results in the inhibition of the biological functions of certain enzymes that contain thiol groups (-SH).

As a consequence of Hg neurotoxicity, brain is considered a primary target organ and, at sufficient concentrations, Hg may disrupt neurological processes due to this high affinity for protein thiols [START_REF] Clarkson | The toxicology of mercury and its chemical compounds[END_REF]. In the 19 th century, the use of liquid Hg by English hat manufacturers caused them severe neurological symptoms (depression, tremors and slurred speech), a grave disease illustrated by the widely known character of the "mad hatter" in Lewis Carroll writing "Alice's adventures in wonderland".

Intoxications by Hg in the History

The first known large scale Hg poisoning occurred in Minamata Bay, Japan, in 1956 by the release of Hg in the wastewater of a factory where Hg was used as a catalyst for the production of acetaldehyde. Following the subsequent methylation of the released Hg by microorganisms in the sediment, MeHg enters the food chain and contaminated the local fishermen and their families. This disaster caused more than 1000 deaths during 36 years and nearly 2 million people suffered from a neurological syndrome, so called Minamata disease, due to the consumption of intensely contaminated fish and seafood. Hg concentrations in fish ranged between 5.61 and 37.5 μg.g -1 [START_REF] Harada | Minamata disease: methylmercury poisoning in Japan caused by environmental pollution[END_REF]. Analyses of hair samples from contaminated people displayed concentrations that could reach 705 μg.g -1 (threshold considered "at risk" 30 μg.g -1 [START_REF] Legrand | Methylmercury Blood Guidance Values for Canada[END_REF]). Between 1964 and 1965, a similar same tragedy took place in Niigata, Japan [START_REF] Tsubaki | Minamata disease. Methylmercury poisoning in Minamata and[END_REF], known as "Second Minamata", where victims experienced the first symptoms up to 5 years after MeHg intake. The agent was identified earlier than in Minamata and several studies demonstrated the neurotoxic effects of Hg, which were particularly severe among children exposed during their prenatal period [START_REF] Harada | Minamata disease: methylmercury poisoning in Japan caused by environmental pollution[END_REF]. Several sequences of poisonings from seed flours coated with fungicide containing MeHg occurred in Iraq in 1956, 1960[START_REF] Snyder | Congenital Mercury Poisoning[END_REF][START_REF] Bakir | Methylmercury Poisoning in Iraq[END_REF]; as well as in New Mexico in 1969 [START_REF] Snyder | Congenital Mercury Poisoning[END_REF].

Nowadays, Hg is already largely used for gold extraction by the artisanal and small-scale gold mining (ASGM) community. Gold is extracted with Hg by the formation of an amalgam which is later heated so that Hg is evaporated from the mixture leaving gold. This method is the cheapest alternative for gold extraction and it is quick, easy and can be performed by one single person (UNEP 2013;AMAP 2011). Hence, ASGM industry has rapidly expanded over the last two decades in developing countries [START_REF] Hagan | Mercury hair levels and factors that influence exposure for residents of Huancavelica, Peru[END_REF]; principally in South America, Africa, and Asia; but also in North America and Australia (UNEP 2013). It has been estimated that approximately 15 million people participate in the ASGM industry in 70 countries (UNEP 2012).

Although most uses of Hg are diminishing, ASGM represents the most significant source of global Hg emissions (UNEP 2013) and is expected to increase even more. Indeed, between 2005 and 2010, Hg emissions produced by ASGM doubled (UNEP 2013).

Figure 1.1 Artisanal and small-scale gold mining in San Juanito mine, Bolivia. Photo from [START_REF] Laffont | Anomalous mercury isotopic compositions of fish and human hair in the Bolivian amazon[END_REF]

Regulation/ implications

In 2009, the Governing Council of the United Nations Environment Programme (UNEP) initiated the development of a legally necessary global implementation to protect the human health and the environment from anthropogenic emissions and releases of Hg. In 2013, governments from 127 countries signed for this agreement which was called the Minamata Convention on Mercury. This convention was principally addressed to the development of national plans for public health, application of controlled measurements on Hg releases to the environmental compartments and regulation of ASGM and exposure to its communities, including strategies for compilation of health data, training for health-care workers and educating for consciousness.

Many governments control legal limits on maximum levels of MeHg in fish for consumption.

World Health Organization (WHO) actually give their recommendations in terms of daily oral intake of MeHg and estimated that the normal concentration for a non-contaminated population should be comprised between 5 et 10 μg L -1 in blood [START_REF] Who | Mercury Assessing the environmental burden of disease at national and local levels[END_REF]. Consumption of long-lived predatory species such as shark and swordfish have been especially disapproved. Hg 0 air concentrations in areas surrounding ASGM burning sites have been found to exceed the WHO limit for public exposure of 1.0 μg m -3 [START_REF] Who | Mercury Assessing the environmental burden of disease at national and local levels[END_REF]. It was reported that workers exposed to 30 μg m -3 Hg experienced tremble, but renal diseases and changes in plasma enzymes are expected at 15 μg m -3 [START_REF] Who | Health Systems: Improving Perfomance-The World Health Report 2000[END_REF]. Furthermore, Hg exposure by ASGM affects not only the health of workers but also the people from adjacent communities (UNEP, 2012). Given this problematic situation, the continuous application of studies on Hg contamination is necessary to assemble health data and levels of exposure in order to favour the implementation of control policy to regulate and prevent Hg pollution.

Main sources of Hg in the environment

Although Hg natural sources such as soils, rocks, volcanoes or biomass can be involved in the remobilization of Hg at a global scale, the main source of this metal comes from the anthropogenic activities. Its high electrical conductivity and its ability to form amalgams with other metals like gold and silver, have potentiated the heavy exploitation of Hg in mining, in metallurgical field and in the process of fossil fuels combustion.

Natural sources

Natural sources represent all the direct emissions of Hg coming from processes such as volcanic eruptions, geothermal activities or soil erosion or minerals that contain Hg naturally, mainly as cinnabar (HgS). Globally, it is estimated that about 30% of Hg emissions derive from natural sources [START_REF] Amos | Legacy impacts of all-time anthropogenic emissions on the global mercury cycle[END_REF]. Volcanoes, geothermal and terrestrial sources are considered primary natural sources of Hg, whereas the re-emission of previously deposited Hg on surface waters, vegetation or land are related to biomass burning, meteorological conditions and exchange of Hg 0 at air-water, air-land or snow-ice interfaces [START_REF] Mason | Mercury Fate and Transport in the Global Atmosphere[END_REF]. Oceans (which represent 70% of the planetary surface) are considered a substantial natural input of Hg into the atmosphere [START_REF] Morel | The Chemical Cycle and Bioaccumulation of Mercury[END_REF] estimated as 52% of Hg natural sources [START_REF] Pirrone | Global mercury emissions to the atmosphere from anthropogenic and natural sources[END_REF].

Table 1.1. Global Hg emissions by natural sources estimated for 2008 [START_REF] Pirrone | Global mercury emissions to the atmosphere from anthropogenic and natural sources[END_REF].

Natural source

Hg (Mg

Anthropogenic sources

The anthropogenic activity is considered the main source of Hg in the environment and is the result of its exploitation in mining, electrolytic industries using Hg electrodes, the fumes of fossil fuels, pesticide based on Hg compounds and alkaline batteries [START_REF] Spalding | Methylmercury accumulation in tissues and its effects on growth and appetite in captive great egrets[END_REF]. Current anthropogenic emissions correspond approximately to 2000 Mg•year -1 [START_REF] Streets | All-time releases of mercury to the atmosphere from human activities[END_REF].

Mineral exploitation and fuel fossil combustion are nowadays the major contributors of human Hg emissions (UNEP 2013). Actually, coal combustion critically enhanced the natural cycle of Hg in environmental compartments during the Industrial Revolution and, at the present time, it continues to be the most expanded source of anthropogenic Hg as a result of its widespread use for electricity and heat production around the world. Emissions from fuel fossil combustion have been estimated around 35-45 % of the total Hg input [START_REF] Pacyna | Global anthropogenic mercury emission References 205 inventory for 2000[END_REF]. In South America, ASGM is today responsible for the largest proportion of the emissions (about 60%) (UNEP 2013).

Since the Ancient times, the exploitation of Hg natural reservoirs has been extensively accomplished for precious metal recovery in South America, predominantly at the time of Spanish colonization (the release of nearly 2•10 5 Mg of Hg was estimated from 1550 to 1850). Hg mineral exploitation achieved its apogee from 1860 to 1920, during which emissions achieved 2500 Mg•year -1 [START_REF] Streets | All-time releases of mercury to the atmosphere from human activities[END_REF]. Indeed, it has been suggested that the remarkable Hg concentrations exiting nowadays could be likely the heritage of the significant exploitation of Hg for gold extraction at this time. Today, the main Hg extraction mines all around the world are located in the periphery of the Pacific Ocean (principally in Peru and Mexico). [START_REF] Streets | All-time releases of mercury to the atmosphere from human activities[END_REF] Emission inventories showed that Asian countries accounted for more than 50% of the global anthropogenic Hg, especially China which is now responsible of 25% of worldwide Hg emissions [START_REF] Tang | Mercury speciation and emissions from coal combustion in Guiyang, southwest China[END_REF]. Coal burning and power plants are responsible of the total combustion emissions of Hg in China. However the global situation is nowadays more encouraging since emission control policies have been restructured in order to reduce the environmental impact of air pollutants from the Chinese sector in recent years (AMAP 2011).

In the marine environment, anthropogenic emissions have produced an extreme impact, involving the substantial increase of Hg levels in the ocean (concentrations have duplicated in the first 100 meters of the world's oceans in the last 100 years) (UNEP 2013). As a consequence, Hg content in several species of marine top predators have amplified by 12 times, implying that over 90% of their accumulated Hg comes from anthropogenic sources.

Hg atmospheric concentration is determined by the combination of primary direct sources (natural and anthropogenic) and the continuous re-emission from environmental compartments.

Actually, it is believed that, before the industrialisation, Hg levels in the environment were in equilibrium, with a balance between mobilization from long-term geological reservoirs and removal from the different reservoir compartments (atmosphere, soils, and oceans) (AMAP 2011). The extreme amplification of anthropogenic emissions since the beginning of the industrial period has perturbed this natural balance so that Hg became a contaminant of major concern through global atmospheric, oceanic circulation and biological turnover. 1.4 Biogeochemical cycle of Hg in the marine environment 1. [START_REF] Choi | Metabolic Pathways Leading to Mercury Methylation in Desulfovibrio desulfuricans LS[END_REF].1 Which are the potential sources of Hg in remote marine areas such as the Southern Ocean?

In the Ocean, the primary source of Hg is direct atmospheric input, however, it is known that mobilisation from sediments and submarine hydrothermal inputs also contribute to Hg amounts in the marine compartment [START_REF] Mason | Mercury biogeochemical cycling in the ocean and policy implications[END_REF]. Despite the distance from industrial focus, the Southern Ocean is affected by the anthropogenic stress mainly via atmospheric circulation of contaminants and ocean currents [START_REF] Pacyna | Global emission of mercury to the atmosphere from anthropogenic sources in 2005 and projections to 2020[END_REF]. The relatively long residence time of Hg in the atmosphere favours its redistribution at a global scale [START_REF] Schroeder | Atmospheric mercury -An overview[END_REF] and therefore, significant amounts of Hg arrive to remote zones mainly due to long-range transport from human sources at lower latitudes.

Other potential source of Hg in the Ocean is sediment erosion and mobilization [START_REF] Mason | Mercury biogeochemical cycling in the ocean and policy implications[END_REF][START_REF] Lamborg | A global ocean inventory of anthropogenic mercury based on water column measurements[END_REF]. Due to its affinity to organic matter, Hg can be released from the water column and deposited on sediments. Different iHg species are distributed among the dissolved, colloidal and particulate phases, bound to different ligands that form a variety of complexes. In anoxic environments, sulphurs (S 2-) are the most abundant species but when they are absent, Hg forms complexes preferentially with the organic matter. Chlorinated complexes (HgClx2-x) and hydroxides (Hg(OH)x2-x) are predominant in oxygenated and slightly eutrophic environments. This fact involves a different Hg speciation distribution between marine and freshwater systems. Coastal areas are thus under considerably higher Hg pressure [START_REF] Lamborg | A global ocean inventory of anthropogenic mercury based on water column measurements[END_REF] so that Hg export to the open ocean areas is not negligible [START_REF] Amos | Gas-particle partitioning of atmospheric Hg(II) and its effect on global mercury deposition[END_REF]. In the Southern Ocean, Hg release to the opened areas may be thus more significant in water masses surrounding Antarctic continent or coastal zones in the vicinity of subantarctic islands whereas open waters are mainly between 4000 and 5000 meters deep.

An important Hg input into polar oceanic zones is melting ice, snow and permafrost (AMAP 2011;[START_REF] Sheppard | Heavy metal content of meltwaters from the Ross Dependency , Antarctica. New Zeal[END_REF]. Additionally, oxidation of Hg in the atmosphere has been found to be an important source into polar oceans with meaningful inputs occurring during springtime as a result of atmospheric Hg depletion events (AMDEs), when halogen radicals oxidize Hg 0 of the air [START_REF] Ebinghaus | Antarctic springtime depletion of atmospheric mercury[END_REF]. Thus, sea ice represents an important Hg reservoir derived from freeze rejection, scavenging of atmospheric Hg and discharge from sea ice cover [START_REF] Chaulk | Mercury distribution and transport across the ocean-sea-ice-atmosphere interface in the arctic ocean[END_REF]. Because Hg is deposited under inorganic forms, its rate of methylation (and demethylation) in the physical environment, prior its transfer within food webs, is crucial for Hg accumulation in Antarctic biota, in which high levels of MeHg have been detected [START_REF] Bargagli | Biomagnification of mercury in an Antarctic marine coastal food web[END_REF].

Main processes involved in Hg biogeochemical cycle in the marine system

Marine systems constitute an important part of the Hg biogeochemical cycle. Actually, the presence of Hg in oceanic compartments can represent an important source of contamination for the atmosphere due to volatilization of the gaseous form Hg 0 , for sediments as iHg and for trophic webs through the MeHg accumulation. This last process is of major concern since it is the main way of Hg exposure for humans and animals by the consumption of seafood. Since Hg is highly reactive in aqueous medium, it undergoes different transformations (oxidation, reduction, methylation and demethylation) which are carried out by biological and abiotic processes. Abiotic reactions are mainly induced by photochemistry so that they take place in the photic zone of the water column [START_REF] Hammerschmidt | Aqueous phase methylation as a potential source of methylmercury in wet deposition[END_REF][START_REF] Monperrus | Mercury methylation, demethylation and reduction rates in coastal and marine surface waters of the Mediterranean Sea[END_REF][START_REF] Lehnherr | Importance of ultraviolet radiation in the photodemethylation of methylmercury in freshwater ecosystems[END_REF]. The bio-physicochemical characteristics of a given ecosystem will define Hg speciation, and consequently, its mobility and toxicity. Hg is emitted into the atmosphere in the form of elemental vapour (Hg 0 ) which passes into its Hg divalent form (Hg 2+ ). This oxidized form of Hg is very soluble, then it can react rapidly with rainwater or snow, or can even be adsorbed by small particles, and subsequently deposited in the environment. Therefore, it is widely recognized that Hg 0 constitutes the largest component of the total gaseous Hg concentration in the troposphere and it is transported on a hemispheric scale [START_REF] Slemr | Distribution, speciation, and budget of atmospheric mercury[END_REF]. The residence time of the Hg 0 in the troposphere is around 0.8 to 1.6 years [START_REF] Selin | Chemical cycling and deposition of atmospheric mercury: Global constraints from observations[END_REF], while the soluble forms remain only few weeks. Divalent Hg species can be introduced on the soil (in the form of dry or wet deposit) where they can be methylated into more toxic organic forms by biological processes.

In marine systems, two main processes control the atmospheric-water compartment interactions: iHg reduction and Hg 0 oxidation. Since iHg is the substrate for methylation, its reduction into Hg 0 and sea to air exchanges are also important processes regulating MeHg levels in surface waters. The reduction of iHg will produce volatile Hg 0 reducing the Hg amount in the water system, whereas the opposite Hg 0 oxidation reaction will decrease the volatile Hg 0 pool and the water-air Hg transfer contributing to the increase of iHg concentrations in the aquatic compartment and its later methylation.

Biotic reduction mechanisms can be mediated by Hg-resistant bacteria found in oceans and estuarine areas [START_REF] Mason | Alkylmercury species in the equatorial Pacific[END_REF][START_REF] Mason | Bioaccumulation of mercury and methylmercury[END_REF], including in the Arctic [START_REF] Barkay | Bacterial mercury resistance from atoms to ecosystems[END_REF][START_REF] Møller | Mercuric reductase genes (merA) and mercury resistance plasmids in High Arctic snow, freshwater and sea-ice brine[END_REF] and Antarctic sea ice [START_REF] Gionfriddo | Microbial mercury methylation in Antarctic sea ice[END_REF]. These microorganisms are able to reduce Hg 2+ into Hg 0 by the mercuric reductase (merA), which is a part of the mer operon that encodes a group of proteins involved in the detection, transport and reduction of Hg. Phototrophic organisms, such as phytoplankton has also been found to reduce iHg [START_REF] Poulain | Redox transformations of mercury in an Arctic snowpack at springtime[END_REF].

Although biological Hg reduction occurs in the Ocean, Hg photochemical reduction is the main mechanism occurring in surface waters and is responsible of the re-emission of Hg 0 to the atmosphere. The production of dissolved gaseous Hg is mostly photomediated and involves the reduction of iHg species by solar irradiation in the presence of dissolved organic carbon (DOC), which is assumed to be crucial in the production of dissolved Hg 0 [START_REF] Amyot | Production and loss of dissolved gaseous mercury in coastal seawater[END_REF]. DOC plays a complex role in the Hg aquatic chemistry since it creates strong bounds with Hg controlling its distribution and availability, but also provides highly redox active radicals that may serve as reductants or oxidants during photochemical reactions [START_REF] Zhang | Sunlight and iron(III)-induced photochemical production of dissolved gaseous mercury in freshwater[END_REF].

Different factors can also potentiate Hg 0 photo-oxidation, such as the presence of -OH, -O2 -and chloride ions [START_REF] Lalonde | The role of mercury redox reactions in snow on snow-to-air mercury transfer[END_REF]. Direct photolysis of NO3 -, NO2 -and H2O2 can potentiate the release of active halogens and OH radicals in seawater [START_REF] Mopper | Hydroxyl Radical Photoproduction in the Sea as Its Potential Impact on Marine Processes[END_REF] and in snow [START_REF] Poulain | Redox transformations of mercury in an Arctic snowpack at springtime[END_REF]. The -O2 -radical also presents the capability to oxidize Hg 0 and can be produced by photochemistry in natural surface waters. Both processes have been found to occur in snow packs and are induced by photochemistry [START_REF] Lalonde | The role of mercury redox reactions in snow on snow-to-air mercury transfer[END_REF][START_REF] Poulain | Redox transformations of mercury in an Arctic snowpack at springtime[END_REF] after the formation of initial radicals.

Polar zones present a unique Hg cycle, mainly characterized by the occurrence of atmospheric phenomena (AMDEs) and the presence of sea ice cover, which both contribute to particularly different processes at the air-ocean interface. AMDEs consist in the rapid oxidation of atmospheric Hg 0 into gaseous Hg 2+ (also known as reactive gaseous Hg) during the spring season through reactions with halogens such as bromine [START_REF] Lindberg | Dynamic oxidation of gaseous mercury in the arctic troposphere at polar sunrise[END_REF][START_REF] Ariya | Reactions of gaseous mercury with atomic and molecular halogens: Kinetics, product studies, and atmospheric implications[END_REF], which is released from sea-salt to the atmosphere during sea ice formation [START_REF] Schroeder | Atmospheric mercury -An overview[END_REF][START_REF] Lindberg | Dynamic oxidation of gaseous mercury in the arctic troposphere at polar sunrise[END_REF]. These events involve the transfer of oxidized iHg into particles which are deposited on the snow pack, involving Hg depletion from atmosphere [START_REF] Temme | Measurements of atmospheric mercury species at a coastal site in the antarctic and over the south atlantic ocean during polar summer[END_REF] and its potential transfer to the ocean during melting [START_REF] Lindberg | Dynamic oxidation of gaseous mercury in the arctic troposphere at polar sunrise[END_REF]. AMDEs have been shown

to concur with an abrupt simultaneous decrease in atmospheric ozone concentrations (Lindberg et al., 2002, Schroeder and[START_REF] Schroeder | Atmospheric mercury -An overview[END_REF]. Although the exact mechanism is still undefined, previous studies have proposed the occurrence of reactions involving ozone and certain halogenated compounds (including BrOx, Br, BrO) that lead to ozone depletion events [START_REF] Simpson | Halogens and their role in polar boundary-layer ozone depletion[END_REF]. The existence of this phenomenon was first discovered in the Arctic [START_REF] Schroeder | Atmospheric mercury -An overview[END_REF] and later observed in coastal Antarctica [START_REF] Ebinghaus | Antarctic springtime depletion of atmospheric mercury[END_REF][START_REF] Bargagli | Enhanced deposition and bioaccumulation of mercury in Antarctic terrestrial ecosystems facing a coastal polynya[END_REF]. Indeed, AMDEs have been detected all along the fringes of Antarctic sea ice, in polynyas and between pack ice and fast ice [START_REF] Dommergue | Overview of mercury measurements in the Antarctic troposphere[END_REF]. These events are assumed to have been occurring in coastal Antarctica over long periods, becoming especially important during the last glacial period, when sea ice recorded much higher concentrations of sea salt and dust [START_REF] Jitaru | Atmospheric depletion of mercury over Antarctica during glacial periods[END_REF]. Hg 0 in coastal Antarctic marine boundary layer is known to be present in lower concentrations than further north ecosystems [START_REF] Soerensen | Global concentrations of gaseous elemental mercury and reactive gaseous mercury in the marine boundary layer[END_REF]. Nevertheless, much higher concentrations of Hg 2+ have been documented comparing to northern latitudes [START_REF] Soerensen | Global concentrations of gaseous elemental mercury and reactive gaseous mercury in the marine boundary layer[END_REF], which could lead to greater Hg deposition on coastal snow packs enhancing Hg concentrations in the surface snow and, consequently, into the ocean during sea ice melting [START_REF] Brooks | Springtime atmospheric mercury speciation in the McMurdo, Antarctica coastal region[END_REF]. The observation of a constant increase of oxidized Hg in the Antarctic environment during the sunlight period could suggest the existence of more elevated Hg 0 oxidation rates comparing to the Arctic sea ice [START_REF] Dommergue | Overview of mercury measurements in the Antarctic troposphere[END_REF]. Moreover, the fast reactivity of Hg 0 and the presence of Hg 2+ from late winter to summer are proposed as substantial contributors of net input of atmospheric Hg on Antarctic surfaces [START_REF] Dommergue | Overview of mercury measurements in the Antarctic troposphere[END_REF]. After the deposition of Hg 2+ on snow, the most part of this Hg 2+ (50-80%, [START_REF] Dommergue | The fate of mercury species in a subarctic snowpack during snowmelt[END_REF] is believed to be photoreduced and re-emitted to the atmosphere as Hg 0 in a few days [START_REF] Steffen | Atmospheric mercury in the Canadian Arctic. Part I: A review of recent field measurements[END_REF][START_REF] Poulain | Redox transformations of mercury in an Arctic snowpack at springtime[END_REF][START_REF] Lalonde | The role of mercury redox reactions in snow on snow-to-air mercury transfer[END_REF].

The presence of sea ice cover in Polar Regions has been demonstrated to influence Hg cycling in marine systems, due to inhibition of aquatic photochemistry. Antarctica is an ice-covered continent isolated from the other continents by marine currents that favour the accumulation of ice in the continent. However, the decline of sea ice in the Antarctic ecosystems is accelerated by currents of warmer waters from lower latitudes and the action of large ocean waves that also involve ice fragmentations [START_REF] Comiso | Large-scale Characteristics and Variability of the Global Sea Ice Cover. Sea Ice An Introd. to its Physics[END_REF]. Sea ice is composed of frozen seawater, brine, gases, minerals, particles, and biota [START_REF] Beattie | Total and methylated mercury in arctic multiyear sea ice[END_REF]. Annually, sea ice around East Antarctica melts releasing nutrients [START_REF] Arrigo | Large scale importance of sea ice biology in the Southern Ocean[END_REF]) and also Hg [START_REF] Cossa | Mercury in the Southern Ocean[END_REF]. The sequestration of atmospheric Hg in sea ice compartment is indeed responsible of an additional input of Hg in the ocean. It is believed that Antarctic sea ice melting contributes over 9.6 Mg of Hg to seawater each year [START_REF] Gionfriddo | Microbial mercury methylation in Antarctic sea ice[END_REF], which is more considerable than some Arctic Ocean estimations [START_REF] Beattie | Total and methylated mercury in arctic multiyear sea ice[END_REF]. Recent measurements in the east coast of Antarctica indicated elevated concentrations of total Hg in surface snow samples and evidenced that Hg atmospheric deposition processes are enhanced comparing to the Arctic ecosystem (Angot et al., 2016a). Furthermore, coastal Antarctic ecosystems are affected by intense katabatic winds that transport Hg from Antarctic inland atmospheric reservoir towards the coast (Angot et al., 2016a), which can strongly influence the cycle of atmospheric Hg at a continental scale [START_REF] Temme | Measurements of atmospheric mercury species at a coastal site in the antarctic and over the south atlantic ocean during polar summer[END_REF]Angot et al., 2016a;Angot et al., 2016b;[START_REF] Sprovieri | Five-year records of Total Mercury Deposition flux at GMOS sites in the Northern and Southern Hemispheres[END_REF]. Angot et al., 2016a 1.4.2.2 Methylation and demethylation of Hg in marine and polar systems Hg methylation and demethylation are two key processes regulating the Hg cycle in marine environments because they determine the fate of MeHg in the water column, contributing in a major way to its bioaccumulation in food webs. Methylation and demethylation in the marine environment can be driven by both biological activities and abiotic mechanisms [START_REF] Cossa | The origin of methylmercury in open Mediterranean waters[END_REF]. Biological reactions pathways have been proposed to be of major importance for Hg species transformations in seawater [START_REF] Barkay | Bacterial mercury resistance from atoms to ecosystems[END_REF] and anoxic lake waters [START_REF] Eckley | Determination of mercury methylation potentials in the water column of lakes across Canada[END_REF]. Nevertheless, most studies highlighted the requirement of anaerobic metabolism or reducing conditions to achieve a substantial production of MeHg, taking into account the reversible demethylation pathways (Rodrı ǵuez [START_REF] Rodrı Ǵuez Martı Ń-Doimeadios | Mercury methylation/demethylation and volatilization pathways in estuarine sediment slurries using species-specific enriched stable isotopes[END_REF].

DMHg is considered the main product of biotic methylation [START_REF] Mason | The distribution and speciation of mercury in the South and equatorial Atlantic[END_REF]) and its production is closely linked to primary productivity in marine polar regions [START_REF] Pongratz | Production of methylated mercury, lead, and cadmium by marine bacteria as a significant natural source for atmospheric heavy metals in polar regions[END_REF][START_REF] Lehnherr | Methylation of inorganic mercury in polar marine waters[END_REF] Figure 1.7. Conceptual model of Hg methylation/demethylation in marine waters. Figure from Lehnherr et al., 2011 1.4.2.2

.1 Abiotic/biotic methylation

Natural abiotic methylation requires the availability of methyl-donor compounds that have been observed both in sediments and in the water column [START_REF] Krishnamurthy | Biomethylation and environmental transport of metals[END_REF][START_REF] Cossa | The origin of methylmercury in open Mediterranean waters[END_REF]. This process is strongly affected by numerous environmental factors such as pH, temperature, salinity, organic matter, redox conditions, among others [START_REF] Ullrich | Mercury in the aquatic environment: a review of factors affecting methylation[END_REF]. These methyl-donor compounds can be small organic molecules such as methylcobalamin (MeCo), methyl-iodide or methyltin; or larger molecules belonging to dissolved organic matter (DOM), such as fulvic and/or humic acids [START_REF] Thayer | Methylation: Its role in the environmental mobility of heavy elements[END_REF][START_REF] Celo | Abiotic methylation of mercury in the aquatic environment[END_REF]. MeCo is a naturally occurring coenzyme of vitamin B12 considered the main potential environmental agent of Hg abiotic methylation in aquatic environments [START_REF] Thayer | Methylation: Its role in the environmental mobility of heavy elements[END_REF][START_REF] Chen | Methylation of inorganic mercury by methylcobalamin in aquatic systems[END_REF]. This reaction is carried out by transfer of methyl groups in the form of methylcarbanion (CH3 -), radical (-CH3) or carbonium ion (CH3 + ) [START_REF] Ridley | Biomethylation of toxic elements in the environment[END_REF][START_REF] Krishnamurthy | Biomethylation and environmental transport of metals[END_REF]. In the environment, abiotic methylation may occur in the atmosphere [START_REF] Gårdfeldt | A kinetic study on the abiotic methylation of divalent mercury in the aqueous phase[END_REF] resulting from the presence of acetate as methyl-donor and Hg 2+ in the atmospheric aqueous phase. Biotic methylation is considered to be significantly faster than abiotic methylation [START_REF] Choi | Metabolic Pathways Leading to Mercury Methylation in Desulfovibrio desulfuricans LS[END_REF] and includes the transfer of methyl groups from MeCo to Hg 2+ mediated by bacteria. In marine systems, it is mainly carried out by sulphate and iron reducing bacteria (SRB and IRB, respectively) [START_REF] Gilmour | Sulfate Stimulation of Mercury Methylation in Freshwater Sediments[END_REF][START_REF] Fleming | Mercury Methylation from Unexpected Sources : Molybdate-Inhibited Freshwater Sediments and an Iron-Reducing Bacterium[END_REF]. SRB (representative of the genus Desulfovibrio) are recognized as the principal Hg organisms methylating iHg in aquatic anoxic environments [START_REF] Compeau | Sulfate-Reducing Bacteria: Principal Methylators of Mercury in Anoxic Estuarine Sedimentt[END_REF]. Experiments in microcosms have demonstrated that

Methylcarbanion (CH 3 + )

Methylation

CH 3 Hg + the absence of SRB involves a significant decrease of the methylation rates [START_REF] Avramescu | Biogeochemical factors influencing net mercury methylation in contaminated freshwater sediments from the St. Lawrence River in Cornwall, Ontario, Canada[END_REF]. Numerous studies have focused in the investigation of Desulfovibrio desulfuricans ND132 as a model organism [START_REF] Gilmour | Sulfate-reducing bacterium Desulfovibrio desulfuricans ND132 as a model for understanding bacterial mercury methylation[END_REF]. One recent study [START_REF] Parks | The Genetic Basis for Bacterial Mercury Methylation[END_REF] identified the two gene clusters associated to bacterial methylation (hgcA and hgcB), which has supposed an important advance for the application of genomics to investigate microbial Hg methylation pathways in the environment. Hg metabolic methylation processes are not completely understood

and it has been suggested as a protective mechanism against Hg toxicity in some microorganisms (Pedrero et al., 2012a). However, not all SRB present Hg methylation capacities and the methylation yields are variable between strains [START_REF] Bridou | Simultaneous determination of mercury methylation and demethylation capacities of various sulfate-reducing bacteria using speciesspecific isotopic tracers[END_REF]. In the case of complete oxidizers, Hg methylation seems to require the activation of acetyl coenzyme A synthetase in the cytoplasm [START_REF] Choi | Metabolic Pathways Leading to Mercury Methylation in Desulfovibrio desulfuricans LS[END_REF], whereas incomplete oxidizers can methylate independently of the acetyl coenzyme A [START_REF] Ekstrom | Mercury Methylation Independent of the Acetyl-Coenzyme A Pathway in Sulfate-Reducing Bacteria Mercury Methylation Independent of the Acetyl-Coenzyme A Pathway in Sulfate-Reducing Bacteria[END_REF].

SRB are anaerobic bacteria that oxidize organic substances using sulphate as the terminal electron acceptor [START_REF] Harmon | Using sulfate-amended sediment slurry batch reactors to evaluate mercury methylation[END_REF], then total sulphur is believed to play an important role in Hg 2+ availability and is also an indicator of the presence of SRB [START_REF] Schartup | Sediment-Porewater Partitioning, Total Sulfur, and Methylmercury Production in Estuaries[END_REF]. The rate of Hg methylation is therefore dependent on many environmental parameters, including pH, temperature, sulphate and DOM concentrations and redox potential in sediments [START_REF] Choi | Metabolic Pathways Leading to Mercury Methylation in Desulfovibrio desulfuricans LS[END_REF]. All these conditions will influence the speciation and availability of mobile Hg 2+ and the abundance and activity of microorganisms. MeHg can also apparently be produced during detrital remineralization in oxic marine waters, associated with nutrient maxima and oxygen utilization [START_REF] Sunderland | Mercury sources, distribution, and bioavailability in the North Pacific Ocean: Insights from data and models[END_REF][START_REF] Cossa | The origin of methylmercury in open Mediterranean waters[END_REF].

Figure 1.9. Hypothetical explanatory model at the molecular scale of the Hg methylation process by encoded protein by genes hgcA and hgcB. Figure from [START_REF] Parks | The Genetic Basis for Bacterial Mercury Methylation[END_REF] Some studies in the Arctic region have proposed the presence of occurring bacterial Hg methylation in the lower parts of sea ice [START_REF] Beattie | Total and methylated mercury in arctic multiyear sea ice[END_REF], sea ice brine [START_REF] Douglas | Influence of Snow and Ice Crystal Formation and Accumulation on Mercury Deposition to the Arctic[END_REF], snow packs [START_REF] Larose | Bioavailable mercury cycling in polar snowpacks[END_REF] and frost flowers [START_REF] Bowman | The genetic potential for key biogeochemical processes in Arctic frost flowers and young sea ice revealed by metagenomic analysis[END_REF], although this has not been proved by the identification of Hg-methylating microbes (i.e, containing functional hgc genes) in sea ice or polar surface waters. However, in a recent study in Antarctic sea ice [START_REF] Gionfriddo | Microbial mercury methylation in Antarctic sea ice[END_REF], potential Hg methylation genes from a marine nitrite-oxidizing bacterium (Nitrospina) and mer operons from Proteobacteria have been identified, providing a considerable advance for understanding Hg methylation in polar regions. Furthermore, Nitrospina-like microorganisms have been found to be present across the sea ice-brine-sea water interface [START_REF] Ngugi | Diversification and niche adaptations of Nitrospinalike bacteria in the polyextreme interfaces of Red Sea brines[END_REF][START_REF] Delmont | Phaeocystis antarctica blooms strongly influence bacterial community structures in the Amundsen Sea polynya[END_REF].

Figure 1.10. Proposed mechanism by [START_REF] Gionfriddo | Microbial mercury methylation in Antarctic sea ice[END_REF] for methylation of iHg mediated by a microaerophilic nitrite-oxidizing bacterium, Nitrospina, in Southern Ocean sea ice containing an hgcA-like protein. Figure from [START_REF] Sunderland | Biogeochemistry: Mercury methylation on ice[END_REF] Despite the large amount of anaerobic Hg-methylating bacteria [START_REF] Gilmour | Sulfate-reducing bacterium Desulfovibrio desulfuricans ND132 as a model for understanding bacterial mercury methylation[END_REF], methylation has also been observed in well-oxygenated polar surface waters [START_REF] Heimbürger | Shallow methylmercury production in the marginal sea ice zone of the central Arctic Ocean[END_REF][START_REF] Lehnherr | Methylation of inorganic mercury in polar marine waters[END_REF][START_REF] Schartup | Freshwater discharges drive high levels of methylmercury in Arctic marine biota[END_REF]. Hg methylation by anaerobic bacteria is known to be partially dependent on the activity of phytoplankton, as it has been confirmed in freshwater biofilms [START_REF] Desrosiers | Mercury methylation in the epilithon of boreal shield aquatic ecosystems[END_REF][START_REF] Hamelin | Methanogens: Principal methylators of mercury in lake periphyton[END_REF] and in sea ice interface [START_REF] Gionfriddo | Microbial mercury methylation in Antarctic sea ice[END_REF]. This has been reasoned from the observation of higher MeHg concentrations observed in Antarctic sea ice enriched in phytoplankton comparing to the water column. During this process, the release of metabolites (such as extracellular thiols) could influence iHg bioavailability [START_REF] Leclerc | Relationship between Extracellular Low-Molecular-Weight Thiols and Mercury Species in Natural Lake Periphytic Biofilms[END_REF] or the interaction of iHg or Hg 0 with methylating ligands such as haloalkanes [START_REF] Yin | Fumigant methyl iodide can methylate inorganic mercury species in natural waters[END_REF][START_REF] Celo | Abiotic methylation of mercury in the aquatic environment[END_REF][START_REF] Larose | Springtime changes in snow chemistry lead to new insights into mercury methylation in the Arctic[END_REF]. However, the exact role of phytoplankton with respect to the marine production of MeHg remains unknown [START_REF] Grégoire | A little bit of light goes a long way: the role of phototrophs on mercury cycling[END_REF].

Another study in the Arctic [START_REF] Pućko | Transformation of mercury at the bottom of the arctic food web: An overlooked puzzle in the mercury exposure narrative[END_REF] proposed that iHg could be methylated by intestinal microbial communities in a zooplankton species (Calanus hyperboreus), however further studies are needed to confirm this hypothesis.

Abiotic/biotic demethylation

Photoinduced MeHg demethylation is the main abiotic process of MeHg degradation in aquatic environments [START_REF] Barkay | Bacterial mercury resistance from atoms to ecosystems[END_REF][START_REF] Lehnherr | Importance of ultraviolet radiation in the photodemethylation of methylmercury in freshwater ecosystems[END_REF]. The extent of MeHg photodegradation is strongly dependent on the type of MeHg-binding ligands [START_REF] Zhang | Photolytic degradation of methylmercury enhanced by binding to natural organic ligands[END_REF]. Greater and faster photodemethylation rates have been observed in freshwater comparing to the oceans, where this process is much slower particularly when compared to MeHg degradation processes mediated by microorganisms [START_REF] Whalin | Factors influencing the oxidation, reduction, methylation and demethylation of mercury species in coastal waters[END_REF]. These differences are the result of different MeHg complexes in both ecosystems, in freshwater MeHg is mainly bound to sulphur-containing ligands whereas in seawater it is mainly associated with DOM and Hg-chloride complexes [START_REF] Zhang | Photolytic degradation of methylmercury enhanced by binding to natural organic ligands[END_REF]. The high stability of chloride compounds such as methyl-chloride (CH3HgCl) makes MeHg degradation much difficult [START_REF] Whalin | Factors influencing the oxidation, reduction, methylation and demethylation of mercury species in coastal waters[END_REF]. Different radicals have been evidenced as reactive intermediates responsible for photodegradation: hydroxyl radicals (-OH) [START_REF] Chen | Degradation of monomethylmercury chloride by hydroxyl radicals in simulated natural waters[END_REF], singlet oxygen (O2) from DOM [START_REF] Chen | Degradation of monomethylmercury chloride by hydroxyl radicals in simulated natural waters[END_REF][START_REF] Zhang | Photolytic degradation of methylmercury enhanced by binding to natural organic ligands[END_REF].

Regarding biotic demethylation, two principal types of processes have been observed: reductive demethylation and oxidative demethylation. Reductive demethylation is carried out by bacteria possessing the mer operon, occurring mostly in the aerobic zone [START_REF] Barkay | Bacterial mercury resistance from atoms to ecosystems[END_REF].

Briefly, this process consists on a first demethylation of MeHg by an enzyme (merB, organomercurial lyase) resulting in iHg that is then degraded inside the bacterial cell by another enzyme (merA) and then evacuated from the cell as Hg 0 [START_REF] Barkay | Bacterial mercury resistance from atoms to ecosystems[END_REF][START_REF] Kritee | Mass dependent stable isotope fractionation of mercury during mer mediated microbial degradation of monomethylmercury[END_REF].

Oxidative demethylation by anaeorobic bacteria is still poorly understood but it possibly occurs as a by-product of metabolism by SRB [START_REF] Marvin-Dipasquale | Methyl-Mercury Degradation Pathways: A Comparison among Three Mercury-Impacted Ecosystems[END_REF]. MeHg is thus degraded to CO2 and a small amount of CH4, involving the release of iHg that would be available again for methylation [START_REF] Marvin-Dipasquale | Methyl-Mercury Degradation Pathways: A Comparison among Three Mercury-Impacted Ecosystems[END_REF][START_REF] Barkay | Bacterial mercury resistance from atoms to ecosystems[END_REF]. It has been suggested by several environmental studies that oxidative demethylation mechanisms are predominant at lower Hg concentrations (<100 ng•g -1 ) in anaerobic conditions, whereas reductive mer-operon mediated demethylation seems to dominate at high Hg concentrations in more aerobic medium [START_REF] Marvin-Dipasquale | Methyl-Mercury Degradation Pathways: A Comparison among Three Mercury-Impacted Ecosystems[END_REF][START_REF] Oremland | Methylmercury decomposition in sediments and bacterial cultures: Involvement of methanogens and sulfate reducers in oxidative demethylation[END_REF][START_REF] Hines | Mercury biogeochemistry in the Idrija River, Slovenia, from above the mine into the Gulf of Trieste[END_REF]. In contrast to reductive demethylation, catalyzed by the enzymatic system encoded by the operon mer, oxidative demethylation does not appear to be a process for MeHg detoxification [START_REF] Marvin-Dipasquale | Methyl-Mercury Degradation Pathways: A Comparison among Three Mercury-Impacted Ecosystems[END_REF]. Experimental studies in laboratory have indicated that most part of methylating SRB are also capable of demethylating MeHg, however not all the bacteria that demethylate do necessarily methylate [START_REF] Bridou | Simultaneous determination of mercury methylation and demethylation capacities of various sulfate-reducing bacteria using speciesspecific isotopic tracers[END_REF]. As well as for methylation, demethylation rates appear to be largely influenced by environmental parameters such as pH, redox conditions and salinity [START_REF] Compeau | Sulfate-Reducing Bacteria: Principal Methylators of Mercury in Anoxic Estuarine Sedimentt[END_REF]. However, biological methylation has been supposed to be slower and controlled by the assimilation of iHg, whereas demethylation is faster and does not depend on the MeHg concentration [START_REF] Bridou | Simultaneous determination of mercury methylation and demethylation capacities of various sulfate-reducing bacteria using speciesspecific isotopic tracers[END_REF]. Biotic demethylation has been suggested to constitute a regulator process of the MeHg accumulation in contaminated environments [START_REF] Barkay | Bacterial mercury resistance from atoms to ecosystems[END_REF].

1.4.3 Bioaccumulation and biomagnification in marine food webs 1.4.3.1 Incorporation of Hg in the food web Microorganisms such as bacteria, phytoplankton and protozoan are considered the primary entrance access of Hg in marine food webs [START_REF] Pickhardt | Algal blooms reduce the uptake of toxic methylmercury in freshwater food webs[END_REF][START_REF] Chen | Mercury and arsenic bioaccumulation and eutrophication in Baiyangdian Lake, China[END_REF].

Phytoplankton is known to concentrate 10 4 -10 5 higher Hg levels than ambient water concentrations and this accumulation represents the largest relative increase in MeHg levels at any point in a food web [START_REF] Mason | Bioaccumulation of mercury and methylmercury[END_REF]. The specific bioaccumulation of the different Hg compounds is controlled by biological processes, being MeHg the preferentially bioaccumulated due to a better efficiency in the trophic transfer [START_REF] Morel | The Chemical Cycle and Bioaccumulation of Mercury[END_REF]. Hg 0 and DMHg are not reactive and diffuse both inside and outside unicellular microorganisms [START_REF] Mason | Uptake, toxicity and trophic transfer of Hg in a coastal diatom[END_REF]. The difference in bioaccumulation between MeHg and iHg is more delicate and depends on the concentrations of MeHg and iHg in the studied compartment, the proportion of each species under its lipophilic form (HgCl2, CH3HgCl or organic complexes in the water) and their ability to be assimilated by organisms [START_REF] Mason | Uptake, toxicity and trophic transfer of Hg in a coastal diatom[END_REF]. In an aquatic system, and more specifically in an oceanic environment, iHg concentration is ~20 times higher than MeHg, however, the ability to form lipophilic complexes with chloride ions is greater for MeHg (100%) than for iHg (45%) [START_REF] Morel | The Chemical Cycle and Bioaccumulation of Mercury[END_REF]. Although the two lipophilic compounds are assimilated equally, MeHg is concentrated in the cytoplasm and associated with the soluble fraction of phytoplankton whereas iHg mainly remains adsorbed on the cell membrane [START_REF] Morel | The Chemical Cycle and Bioaccumulation of Mercury[END_REF]. This different distribution in the unicellular organisms involves that the efficiency of MeHg assimilation is four times greater than iHg, even if HgCl2 complexes are more lipophilic (Kow = 3.3) than CH3HgCl (Kow = 1.7) [START_REF] Morel | The Chemical Cycle and Bioaccumulation of Mercury[END_REF].

Once in the organism, the complexation of MeHg with aminoacids containing thiol groups (e.g. cysteine and methionine) or non-sulphured aminoacids (e.g., histidine), will determined the bioavailability for the microorganisms predators and this complexation appears therefore essential to regulate Hg solubility in the gastrointestinal tract and its subsequent assimilation. Figure from Morel et al., 1998. 1.4.

Bioaccumulation and bioamplification of MeHg

Since MeHg accumulates mainly in the cytoplasm of the cell and iHg is mainly sequestered by membrane thiols [START_REF] Mason | Uptake, toxicity and trophic transfer of Hg in a coastal diatom[END_REF], the transfer to the upper trophic level is enhanced for MeHg while iHg is mainly excreted, especially when digestive process are fast. Moreover, the assimilation efficiency is much high for MeHg compare to iHg [START_REF] Wang | Bioaccumulation kinetics and exposure pathways of inorganic mercury and methylmercury in a marine fish, the sweetlips Plectorhinchus gibbosus[END_REF]. This specific selectivity of Hg compounds explains the exponential increase of MeHg fractions (and decrease of iHg) with trophic levels observed along food webs [START_REF] Watras | Bioaccumulation of mercury in pelagic freshwater food webs[END_REF], with 20 and 50% of MeHg accumulated in the phytoplankton and more than 75% in the second trophic level (zooplankton), reaching values close to 90-100% in muscles of predators such as fish.

Physiological factors also determine Hg accumulation since concentrations are known to increase with size, weight and age of the species considered [START_REF] Hammerschmidt | Bioaccumulation and trophic transfer of methylmercury in Long Island Sound[END_REF]. In general, an increase in Hg concentrations is observed in an individual's tissues during its life span. However Hg exposure strongly depends on foraging strategies (i.e. benthic or pelagic) within a same trophic level. Hg bioaccumulation in aquatic food webs is thus controlled by many parameters, some of them still not verified or completely understood, involving some incertitude about this phenomenon particularly in cases where predators belonging to lowcontaminated ecosystems accumulate elevated Hg concentrations (e.g., [START_REF] Carravieri | From Antarctica to the subtropics : Contrasted geographical concentrations of selenium , mercury , and persistent organic pollutants in skua chicks ( Catharacta spp .)[END_REF].

The processes of Hg accumulation in the top predators of marine food webs (i.e. large fish, seabirds or marine mammals) appears to be relatively complex due to the developed metabolic strategies face to Hg contamination. Diet represent the main pathway of exposure for these predators, it is absorbed in the intestine and transported by the blood stream towards the main storage tissues (muscle, liver) and target organs (kidney and brain). Specific Hg complexes (with ligands containing thiols, e.g. cysteine, or with Se) appear to regulate the accumulation and elimination in the different organs [START_REF] Wagemann | Methylmercury and total mercury in tissues of arctic marine mammals[END_REF][START_REF] Bridges | Transport of inorganic mercury and methylmercury in target tissues and organs[END_REF]Kim et al., 1996). Subsequently, Hg can be stored definitely or temporarily, metabolized or remobilized to be eliminated through excreta, skin appendages (nails, claws, feathers, hairs, etc.) and to the offspring (fetuses or eggs) (Thompson et al., 1998b).

MeHg is mainly accumulated in muscles (storage tissue) and also in brain tissue (target organ), associated to the potent neurotoxic effects of MeHg (AMAP 2011). Marine mammals and seabirds can remobilise MeHg stored in the muscles and excrete it through replacement of hair and feathers, which constitutes an effective excretory pathway [START_REF] Hoefs | Seasonal changes in mercury accumulation in the black-eared kite, Milvus migrans lineatus[END_REF][START_REF] Braune | Comparison of Total Mercury Levels in Relation to Diet and Molt for Nine Species of Marine Birds[END_REF].

During feather moult in birds, Hg is transferred from the internal tissues and organs into the growing feathers, leading to a decrease of Hg concentrations in storage tissues [START_REF] Furness | Using bird feathers to measure mercury in the environment: Relationships between mercury content and moult[END_REF]. Avian Hg dynamics are thus highly influenced by this sequential moult and the rates of excretion, whereas in mammals Hg is excreted by hair or fur continuously with the notable exception of cetaceans. Indeed, baleen and toothed whales, have no fur and are thus deprived of this route of Hg excretion [START_REF] Dietz | What are the toxicological effects of mercury in Arctic biota?[END_REF]. A key detoxification mechanism of Hg is its coprecipitation with Se to form HgSe granules or tiemanite, process that appears to be particularly developed in the case of hairless mammals or in top predators strongly exposed to Hg such as albatrosses [START_REF] Palmisano | Speciation of mercury in dolphin liver: A two-stage mechanism for the demethylation accumulation process and role of selenium[END_REF][START_REF] Nakazawa | The presence of mercury selenide in various tissues of the striped dolphin: evidence from l-XRF-XRD and XAFS analyseswz[END_REF][START_REF] Arai | Chemical forms of mercury and cadmium accumulated in marine mammals and seabirds as determined by XAFS analysis[END_REF][START_REF] Ikemoto | Environmental Contamination and Detoxification Mechanism of Heavy Metals in Marine Mammals and Seabirds : Interaction of Selenium with Mercury , Silver , Copper , Zinc , and Cadmium in Liver[END_REF][START_REF] Wagemann | Methylmercury and total mercury in tissues of arctic marine mammals[END_REF]. This efficient detoxification process allows to reduce Hg bioavailability and thus its toxicity [START_REF] Cuvin-Aralar | Mercury and selenium interaction: A review[END_REF].

Figure 1.12. Hypothetical demethylation mechanism with consequent formation of inert HgSe granules (tiemanite) in the liver. Figure from [START_REF] Wagemann | Methylmercury and total mercury in tissues of arctic marine mammals[END_REF] As a consequence of the complexity of Hg dynamics in the organism, MeHg demethylation mechanisms are still relatively poorly understood and it is still not clear if Hg demethylation exclusively takes place in liver or also in other organs and tissues. Due to the high fractions of iHg in livers of marine mammals and seabirds [START_REF] Kehrig | Trace-elements, methylmercury and metallothionein levels in Magellanic penguin (Spheniscus magellanicus) found stranded on the Southern Brazilian coast[END_REF][START_REF] Wagemann | Methylmercury and total mercury in tissues of arctic marine mammals[END_REF], liver is considered the main organ responsible of MeHg demethylation. However, it is still not completely understood whether the fraction of iHg found in different organs depends on the specific capacity of each organ to demethylate MeHg, or if it is the result of Hg species-specific storage after transport from a key organ. Demethylation of MeHg have been observed in kidney and brain [START_REF] Dock | Mercury in the Pregnant Hamster[END_REF][START_REF] Vahter | Demethylation of methylmercury in different brain sites of Macaca fascicularis Monkeys during long-term subclinical methylmercury exposure[END_REF], by anaerobic microbes of the intestinal tract [START_REF] Clarkson | Mechanisms of mercury disposition in the body[END_REF][START_REF] Dock | Mercury in the Pregnant Hamster[END_REF][START_REF] Rowland | Interactions of the Gut Microflora and the Host in Toxicology *[END_REF], red and white blood cells [START_REF] Berglund | Inter-individual variations of human mercury exposure biomarkers: a cross-sectional assessment[END_REF][START_REF] Clarkson | The Three Modern Faces of Mercury Methyl Mercury in Fish History of Human Exposure[END_REF] and hair follicles [START_REF] Berglund | Inter-individual variations of human mercury exposure biomarkers: a cross-sectional assessment[END_REF]. After in vivo demethylation, the produced iHg can be eliminated from the body in faeces or is transported to the kidney and excreted by urine [START_REF] Smith | The kinetics of intravenously administered methyl mercury in man[END_REF]). Blood, which is responsible of transporting Hg by thiolconjugate ligands [START_REF] Bridges | Transport of inorganic mercury and methylmercury in target tissues and organs[END_REF][START_REF] Clarkson | Mechanisms of mercury disposition in the body[END_REF], is an equilibrium vector between Hg uptake, tissue storage and remobilisation due to the continuous inter-organ circulation of Hg.

Part 2. Mercury stable isotopes: tracing sources and transformations in the environment

Introduction to stable isotopic fractionation

An isotope of a given element is an atom whose nucleus has the same number of protons (Z) but a different number of neutrons (N), and thus occupy the same position in the periodic table.

Therefore, two isotopes of a same element have a similar atomic number (number of protons in the nucleus), but different mass number (sum of the number of neutrons and the number of protons in the nucleus). Each isotope is then identified by its mass number. Most elements have several natural isotopes which can be stable or unstable (also called radioactive because they can decay into more stable isotopes emitting radiation).

The interest of using stable isotopes started at the mid-20 th century [START_REF] Urey | The Thermodynamic Properties of Isotopic Substances[END_REF], where differences in physicochemical behaviour between isotopes of the same element were explained by their mass difference. The first studies on the isotopic fractionation of the elements was focused on light isotopes (C, H, O, N, S) because their relative mass differences (Δm / m) are higher compared to heavier elements, and consequently the expected fractionation between their isotopes is greater (and easily measured). The isotopic fractionation of these elements has made it possible to investigate numerous geochemical processes and to study paleoclimates. At the end of the 20 th century, the development of high-performance analytical devices (MC-ICPMS) allowed to measure with high precision and accuracy the slight variations of isotopic ratios and thus to extend the application of isotopic fractionation studies also to heavier stable isotopes, such as iron (Fe), calcium (Ca), copper (Cu), zinc (Zn), selenium (Se), thallium (Th), lead (Pb) or mercury (Hg).

Hg has seven stable isotopes with the following masses and relative abundances: 196 Hg (0.15%); 198 Hg (9.97%); 199 Hg (16.87%); 200 Hg (23.10%); 201 Hg (13.18%); 202 Hg (29.86%) and 204 Hg (6.87%). During the last decades, In the last decades, the measurement of Hg isotopic fractionation has become an essential tool in identifying sources of Hg and biogeochemical processes within the different compartments of the environment [START_REF] Blum | Mercury Isotopes in Earth and Environmental Sciences[END_REF].

Hg isotopic fractionation: theories and mechanisms

Isotopic fractionation is defined as the enrichment of one isotope relative to another due to physical or chemical processes. In a chemical reaction, molecules containing lighter isotopes will react more easily than those containing heavy isotopes due to the less dissociation energy of light isotopes [START_REF] Hoefs | Seasonal changes in mercury accumulation in the black-eared kite, Milvus migrans lineatus[END_REF]. Thus, the isotopic fractionation of an element is the distribution of its isotopes between two substances or between two phases of the same substance and can be defined by the alpha fractionation factor (α) which is given by the ratio of two isotopes in one compound A (RA) divided by the ratio of these same two isotopes in a compound B (RB), as represented in this equation:

𝛼 𝐴-𝐵 = 𝑅 𝐴 𝑅 𝐵 = 𝐴 𝑖𝑠𝑜𝑡𝑜𝑝𝑒1 𝑖𝑠𝑜𝑡𝑜𝑝𝑒2 𝐵 𝑖𝑠𝑜𝑡𝑜𝑝𝑒1 𝑖𝑠𝑜𝑡𝑜𝑝𝑒2
In the case of Hg this equation is defined as:

𝛼 𝑥𝑥𝑥 = ( 𝐻𝑔 𝑥𝑥𝑥 / 𝐻𝑔) 𝐴 198 ( 𝐻𝑔 𝑥𝑥𝑥 / 𝐻𝑔) 𝐵 198
Hg isotopes show mass dependent fractionation and mass independent fractionation depending on the chemical reaction or environmental compartment studied.

Mass dependent fractionation (MDF)

MDF involves an enrichment of products in lighter isotopes whereas heavier isotopes will remain in the reactant during a given process. Hg isotopic signatures are normalized with a reference standard iHg solution (NIST 3133) that is used by the international community to facilitate inter-laboratory comparisons. Isotope composition is reported using delta notation (δ) which is the per mil (‰) deviation with respect to this standard [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF].

𝛿 𝑥𝑥𝑥 = ( 𝐻𝑔 𝑥𝑥𝑥 198 𝑠𝑎𝑚𝑝𝑙𝑒 𝐻𝑔 𝑥𝑥𝑥 198 𝑁𝐼𝑆𝑇 3133 -1) × 1000 (‰)
where xxx is the mass of each Hg isotope between (196, 199, 200, 201, 202, or 204), the isotope 198 is used as the reference isotope since it is the lightest and most abundant isotope (isotope 196 represents only 0.15%). Generally, Hg isotopic studies focus on the 5 most abundant isotopes 198, 199, 200, 201 and 202. Two main processes induce isotopic fractionation of an element: equilibrium fractionation and kinetic fractionation.

-Equilibrium fractionation

The equilibrium fractionation corresponds to the isotope exchange reactions between two equilibrium substances. This fractionation is induced by the difference of vibrational energy between two isotopes of one element bound to another atom. Heavy isotopes require a greater vibrational energy than the light ones to be dissociated from a chemical bond, and consequently, they are more stable in a molecule with the strongest chemical bonds.

-Kinetic fractionation

The kinetic isotopic fractionation is involved by the difference in reaction rates between two isotopically distinct molecules. This fractionation is associated with unidirectional and incomplete processes such as evaporation, dissociation, diffusion, oxidation or reduction reactions, as well as during biological transformations [START_REF] Hoefs | Seasonal changes in mercury accumulation in the black-eared kite, Milvus migrans lineatus[END_REF]. Since light isotopes have higher translation rates and higher diffusion coefficients, and form weaker bonds that are more easily breakable, they will react faster involving an enrichment of heavy isotopes in the residue of the reaction.

Mass independent fractionation (MIF)

Hg MIF is reported using Δ which is the deviation of the measured isotope ratio from the theoretical ratio predicted by MDF.

∆ 𝑥𝑥𝑥 𝐻𝑔 = 𝛿 𝑥𝑥𝑥 𝐻𝑔 -(𝛽 𝑥𝑥𝑥 • 𝛿 202 𝐻𝑔)
where xxx is the mass of the Hg isotope and βxxx is the kinetic or equilibrium fractionation factor appropriate for each isotope relative to the more abundant isotope (202) and is defined by the following equation, slightly different if considering equilibrium or kinetical fractionation [START_REF] Young | Kinetic and equilibrium mass-dependant isotope fractionation laws in nature and their geochemical and cosmochemical significance[END_REF]:

𝛽 𝑥𝑥𝑥 𝑒𝑞𝑢𝑖𝑙𝑖𝑏𝑟𝑖𝑢𝑚 = ln ( 1 198 ) -ln ( 1 𝑥𝑥𝑥 ) ln ( 1 198 ) -ln ( 1 202 ) 𝛽 𝑥𝑥𝑥 𝑘𝑖𝑛𝑒𝑡𝑖𝑐 = ln ( 198 𝑥𝑥𝑥 ) ln ( 198 202 )
The differentiation of these two processes (equilibrium vs kinetic) in Hg isotopes would require accurate measurements (in the order of 0.01 ‰) that is rarely observed, and therefore, will not have impact in the measurement of the observed isotopic ratios. Values for factor β were recommended by [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF] for each Hg isotope: Two mass-independent mechanisms are responsible of isotope fractionation: the nuclear field shift effect (NFS) and the magnetic isotope effect (MIE). A third minor mechanism has been recently observed: self-shielding effect (SSE).

∆ 204 𝐻𝑔 = 𝛿 204 𝐻𝑔 -(1.
-Nuclear field shift effect (NFS)

NFS depends on the nuclear volume and nuclear charge radius [START_REF] Schauble | Role of nuclear volume in driving equilibrium stable isotope fractionation of mercury, thallium, and other very heavy elements[END_REF]. Since the nucleus of isotopes differ on the number of neutrons, they will have different masses and size, however, nuclear volume does not increase linearly. Odd isotopes are often smaller than expected based on linear relationship relative to even isotopes (Figure 1.14). This relative difference in size leads to a higher nuclear charge density so that the electrons will be more strongly linked to the nucleus and consequently, it will influence the ground-state energies of atoms and their efficiency and participation in chemical reactions. If all the isotopes are in the form of elemental Hg, the odd isotopes will form weak covalent bonds and will be more volatile [START_REF] Bigeleisen | Nuclear size and shape effects in chemical reactions. Isotope chemistry of the heavy elements[END_REF][START_REF] Schauble | Role of nuclear volume in driving equilibrium stable isotope fractionation of mercury, thallium, and other very heavy elements[END_REF]. -Magnetic isotope effect (MIE) MIE only occurs during kinetic reactions in which reactants absorb the photonic or thermal energy to produce a pair of R radicals, involving spin conversion. Nuclides with an odd number of protons or neutrons are characterized by a non-zero nuclear spin that induces a magnetic moment in the nuclei (isotopes 199 Hg and 201 Hg present a nuclear +1/2 and +3/2 and a magnetic moment of +0.5029 and -0.5602 µB, respectively). Since radical pairs with magnetic nuclei can undergo spin conversion more rapidly than radical pairs without magnetic nuclei, odd isotopes are more likely to recombine whereas even isotopes are more likely to become products [START_REF] Buchachenko | Magnetic isotope effect for mercury nuclei in photolysis of bis(ptrifluoromethylbenzyl)mercury[END_REF]. MIE is an effect of light exposure and is considered the more probable responsible of the large MIF observed in photochemical reactions [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF]. -Self-shielding effect (SSE) SSE has been observed in one experimental study compact fluorescent lamps during which they found significant unusual fractionation of even Hg isotopes (Δ 200 Hg =-10.69‰) [START_REF] Mead | Unique Hg stable isotope signatures of compact fluorescent lamp-sourced Hg[END_REF]. In these lamps, the light is generated by excitation of vapour Hg 0 . Due to different nuclear spin and mass of the seven Hg isotopes, Hg absorption line at 254 nm is divided into distinct lines due to thermal and collisional increase and the specific transfers at each line is not identical and will be related to the isotopic abundance [START_REF] Mead | Unique Hg stable isotope signatures of compact fluorescent lamp-sourced Hg[END_REF]. Therefore, this mechanism induces an unusual isotopic fractionation that does not change linearly with mass but it favours the photoexcitation of Hg isotopes with low abundance (e.g., 196 Hg) rather than those with high abundance (e.g., 202 Hg). Since only odd isotopes 199 Hg and 201 Hg have non-zero nuclear spins and magnetic moments, and the NFS is assumed to generate negligible MIF of the even Hg isotopes [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF], SSE is the most possible mechanism creating even-MIF.

MIF of even Hg isotopes had been determined in natural samples mainly related to the atmosphere [START_REF] Gratz | Isotopic composition and fractionation of mercury in Great Lakes precipitation and ambient air[END_REF]Sherman et al., 2012b;[START_REF] Chen | Unusual fractionation of both odd and even mercury isotopes in precipitation from Peterborough, ON, Canada[END_REF][START_REF] Demers | Mercury isotopes in a forested ecosystem: Implications for airsurface exchange dynamics and the global mercury cycle[END_REF][START_REF] Rolison | Isotopic composition of speciesspecific atmospheric Hg in a coastal environment[END_REF][START_REF] Štrok | The mercury isotope composition of Arctic coastal seawater[END_REF][START_REF] Wang | Massdependent and mass-independent fractionation of mercury isotopes in precipitation from Guiyang, SW China[END_REF][START_REF] Yuan | Large Variation of Mercury Isotope Composition During a Single Precipitation Event at Lhasa City, Tibetan Plateau, China[END_REF]. Nevertheless, the different conditions existing in nature (low concentration of Hg 0 in the atmosphere and the different spectrum of sunlight) indicate that SSE mechanisms must not be representative in nature and therefore, more information is needed to confirm the appropriate cause of even-MIF in the environment.

Experimental determination of Hg isotopic fractionation

Processes inducing MDF and MIF of Hg

In the last decades, the measurement of Hg isotopic MDF and MIF has become an essential tool in identifying sources of Hg and biogeochemical processes within the different compartments of the environment. The area of research on Hg isotopes in the environment is continuously growing and is permitting to better elucidate fractionation mechanisms and to apply Hg isotopes as useful tracers of Hg sources and pathways in the environment. Also, numerous experimental studies have been carried out to provide a basic outline for better understanding of Hg isotopic fractionation mechanisms during biotic and abiotic processes.

Briefly, all chemical reactions have been documented to induce Hg MDF, which is known to affect all Hg isotopes during equilibrium or chemical reactions. MDF reactions follow the conventional kinetic fractionation law and favour the reduction of light isotopes leaving a residual pool of Hg enriched in heavy isotopes. Biotic and dark abiotic reactions do not produce significant MIF. Only photochemical reactions have been established to involve the production of large MIF [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF]Estrade et al., 2009) and are consequently of primary importance in the field of Hg isotopes.

Since Hg isotopic fractionation (especially MDF) can be caused by such numerous processes, the discrimination of Hg processes involved in the fate of Hg from its emission to its accumulation in the environment remains a great challenge. In order to better determine these processes, several studies focused on the characterization of the magnitude of isotopic fractionation in kinetic or equilibrium reactions during physical, chemical and biological processes. The measurement of fractionation factors is based in a Rayleigh-distillation system in which the product of a kinetic or equilibrium process will be enriched in lighter isotopes compared to the remaining fraction. In the case of Hg, the fractionation factors are usually reported as α 198/202 corresponding to the variation in the isotope ratio 202/198 ( 202 ∆Hg product-reactant). For the anomalous fractionation of the isotope ratio 199/198 the fractionation is reported as (∆199Hg) ∆Hg product-reactant.

The first experimental study about Hg isotopic fractionation demonstrated that iHg photoreduction and MeHg photodemethylation induced at the same time Hg MDF and MIF in aquatic systems [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF]. These authors confirmed that only Hg odd isotopes (199 and 201) showed a significant MIF signature (∆ 199 Hg and ∆ 201 Hg), whereas even isotopes followed a MDF law. A latter study about iHg photoreduction in natural waters confirmed both types of fractionation between Hg 0 product and the remaining iHg (Zheng and Hintelmann, 2009a). Since only odd isotopes present a non-zero nuclear spin and a nuclear magnetic moment that can interact with the magnetic moments of electrons, some authors proposed that MIF on odd isotopes was caused by the isotopic MIE [START_REF] Malinovsky | Experimental study of mass-independence of Hg isotope fractionation during photodecomposition of dissolved methylmercury[END_REF]. However, the NFS appears to be more pronounced for the isotope 199 than for the isotope 201 and thus occurs mainly during equilibrium processes [START_REF] Schauble | Role of nuclear volume in driving equilibrium stable isotope fractionation of mercury, thallium, and other very heavy elements[END_REF].

Significant MDF of Hg isotopes has been shown to be produced during microbial reduction of iHg and microbial demethylation of MeHg [START_REF] Kritee | Mercury Stable Isotope Fractionation during Reduction of Hg(II) to Hg(0) by Mercury Resistant Microorganisms[END_REF][START_REF] Kritee | Mass dependent stable isotope fractionation of mercury during mer mediated microbial degradation of monomethylmercury[END_REF] as well as methylation of iHg by SRB (Rodriguez [START_REF] Gonzalez | Species-Specific Stable Isotope Fractionation of Mercury during Hg ( II ) Methylation by an Anaerobic Bacteria ( Desulfobulbus propionicus ) under Dark Conditions[END_REF]. Hg isotopic fractionation induced by microorganisms was first investigated during biological reduction in Hg-resistant bacteria containing the enzyme merA, which involved a α 198/202 ranging from 1.0013 to 1.0020, meaning an enrichment of δ 202 Hg between -1.3 and -2.0‰ [START_REF] Kritee | Mercury Stable Isotope Fractionation during Reduction of Hg(II) to Hg(0) by Mercury Resistant Microorganisms[END_REF]. One year later, [START_REF] Kritee | Mercury Stable Isotope Fractionation during Reduction of Hg(II) by Different Microbial Pathways[END_REF] measured isotopic fractionation during reduction by two Hg-resistant strains and a Hg-sensitive strain without the enzyme merA and obtained in both cases similar results as their previous study (α 202/198 =1.0016±0.0004). [START_REF] Kritee | Mass dependent stable isotope fractionation of mercury during mer mediated microbial degradation of monomethylmercury[END_REF] measured a lower MDF for degradation of MeHg to Hg 0 (α 202/198 =1.0004±0.0002). On the contrary, no MIF is induced during these processes, which is coherent with the absence of MIE in biological transformations. MDF in reactions mediated by bacteria was demonstrated to be dependent of both cell-specific physiological factors (e.g., enzyme catalysis rate, gene expression, cell transport) and external environmental factors determining Hg bioavailability [START_REF] Kritee | Mass dependent stable isotope fractionation of mercury during mer mediated microbial degradation of monomethylmercury[END_REF]. Hg isotopic fractionation during bacterial methylation under dark conditions and in anoxia indicated a higher extent of the MDF was than biotic or abiotic reduction reactions (α 202/198 = 1.0026 ± 0.0004) [START_REF] Gonzalez | Species-Specific Stable Isotope Fractionation of Mercury during Hg ( II ) Methylation by an Anaerobic Bacteria ( Desulfobulbus propionicus ) under Dark Conditions[END_REF]. Under these conditions, since some methylator bacterial strains are also able to demethylate [START_REF] Bridou | Simultaneous determination of mercury methylation and demethylation capacities of various sulfate-reducing bacteria using speciesspecific isotopic tracers[END_REF], the simultaneous bacterial demethylation may affect the Hg kinetic fractionation effect of the methylation, since the iHg in the medium would constitute a mixed pool of residual isotopically heavier iHg from methylation and isotopically lighter iHg from demethylation (Rodriguez [START_REF] Gonzalez | Species-Specific Stable Isotope Fractionation of Mercury during Hg ( II ) Methylation by an Anaerobic Bacteria ( Desulfobulbus propionicus ) under Dark Conditions[END_REF]. Indeed, the impact in Hg isotopic compositions of the bacterial demethylation process was demonstrated in SRB cultures [START_REF] Perrot | Identical Hg Isotope Mass Dependent Fractionation Signature during Methylation by Sulfate-Reducing Bacteria in Sulfate and Sulfate-Free Environment[END_REF], in which a steady-state between iHg and MeHg isotopic composition was observed after 24h of experiment. During microbial demethylation, the lighter Hg isotopes of the MeHg molecules are preferentially transformed and have been found to influence the Hg species isotope composition when significant amounts of MeHg are accumulated (Pedrero et al., 2012a). It has also been demonstrated that a lower MeHg bacterial production yield is obtained under sulphate respiration comparing to fumarate conditions because of the formation of insoluble Hg sulphide complexes which reduce the bioavailability of iHg for the cells [START_REF] Benoit | Aspects of bioavailability of mercury for methylation in pure cultures of Desulfobulbus propionicus (1pr3)[END_REF][START_REF] Gilmour | Sulfate-reducing bacterium Desulfovibrio desulfuricans ND132 as a model for understanding bacterial mercury methylation[END_REF]. [START_REF] Perrot | Identical Hg Isotope Mass Dependent Fractionation Signature during Methylation by Sulfate-Reducing Bacteria in Sulfate and Sulfate-Free Environment[END_REF] investigated Hg isotopic fractionation during bacterial methylation under sulphate and fumarate respiration conditions, reporting similar "apparent" kinetic fractionation factors. The authors referred to the term "apparent" fractionation because methylation and demethylation were occurring simultaneously so that the product of the reaction (MeHg) would be converted back to the product of the reaction, leading to uncertainty in the modelled fractionation factor.

Figure 1.16. Variation of amplitude of the isotopic fractionation factor in the isotope ratio 202/198 ( 202 ∆Hg product-reactant) measured in experimental studies for kinetic and equilibrium reactions. References correspond to (1) Bergquist and Blum, 2007, (2) Yang and Sturgeon, 2009 (3) Zheng et al., 2007, (4) Zheng and Hintelmann, 2010a, (5) Zheng andHintelmann, 2010b, (6) Sherman et al., 2010, (7) Kritee et al., 2007, (8) Kritee et al., 2009 (9) Rodriguez Gonzalez et al., 2009, (10) Perrot et al., 2015, (11) Janssen et al., 2016, (12) Perrot et al., 2013, (13) Jiménez-Moreno et al., 2013, (14) Estrade et al., 2009, (15) [START_REF] Sun | Mass-dependent and -independent fractionation of mercury isotope during gas-phase oxidation of elemental mercury vapor by atomic Cl and Br[END_REF] In the case where MIF has been demonstrated, the MIE or NFS type is reported. Updated from [START_REF] Bergquist | The odds and evens of mercury isotopes: Applications of mass-dependent and mass-independent isotope fractionation[END_REF] MDF has also been identified during experimental abiotic methylation of iHg by MeCo with slightly lower amplitude (α 202/198 ranging from 1.0005 to 1.0015) [START_REF] Malinovsky | Mercury isotope fractionation during abiotic transmethylation reactions[END_REF]. During kinetic experiments with MeCo with the presence of chloride, a decrease of the methylation extent was observed especially significant under visible light conditions due to the enhancement of MeHg photodemethylation [START_REF] Jiménez-Moreno | Chemical kinetic isotope fractionation of mercury during abiotic methylation of Hg(II) by methylcobalamin in aqueous chloride media[END_REF].

However, these experiments did not produce MIF, probably due to the complexation by chloride and/or the lack of higher energy UV radiation. ∆ 199 Hg/∆ 201 Hg ratios [START_REF] Blum | Mercury Isotopes in Earth and Environmental Sciences[END_REF] because Hg pathways have the particularity to lead to a different ∆ 199 Hg/∆ 201 Hg ratio, that is therefore used as indicator of MIF mechanisms.

Experimental studies on aqueous solutions with DOC demonstrated that photochemical reduction of iHg into Hg 0 resulted in a ∆ 199 Hg/∆ 201 Hg ratio of 1.02±0.02 [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF]. A similar ratio was later obtained in Arctic snow samples during iHg photoreduction (Sherman et al., 2010). Photochemical demethylation of MeHg displayed a ∆ 199 Hg/∆ 201 Hg ratio of 1.36±0.03 [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF]. [START_REF] Malinovsky | Experimental study of mass-independence of Hg isotope fractionation during photodecomposition of dissolved methylmercury[END_REF] reported a lower ratio (1.28±0.03) during experimental MeHg photodemethylation under UV-C radiation. Recently, other studies have focused in the influence of the type of solar radiation and DOC ligands in MeHg photodemethylation processes in aquatic systems. [START_REF] Chandan | Mercury isotope fractionation during aqueous photoreduction of monomethylmercury in the presence of dissolved organic matter[END_REF] documented variable Δ 199 Hg/Δ 201 Hg ratios for MeHg photodemethylation under different types and concentrations of DOC (with different content of reduced sulphur). Under low MeHg/S-red-DOC ratios Δ 199 Hg/Δ 201 Hg slopes were consistent and less variable (1.38±0.02); whereas high MeHg/S-red-DOC ratios presented lower slopes varying from 1.17±0.04 to 1.30±0.02. [START_REF] Rose | Effects of ultraviolet radiation on mercury isotope fractionation during photo-reduction for inorganic and organic mercury species[END_REF] investigated the effect of solar radiation (different intensities and frequencies) on Hg MIF during MeHg aquatic photodemethylation and obtained a ratio similar to [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF].

MIF slopes have also been determined during equilibrium and dark processes thus corresponding to NFS. Dark iHg reduction exhibited a Δ 199 Hg/Δ 201 Hg ratio near to 1.6 in presence of DOC and SnCl2 (Zheng and Hintelmann 2010b), similar to equilibrium fractionation between dissolved iHg and thiol-binding Hg [START_REF] Wiederhold | Equilibrium mercury isotope fractionation between dissolved Hg(II) species and thiol-bound Hg[END_REF] and during liquid Hg 0 evaporation (Estrade et al., 2009;[START_REF] Ghosh | Estimation of nuclear volume dependent fractionation of mercury isotopes in equilibrium liquid-vapor evaporation experiments[END_REF].

Determination of ∆ 199 Hg/δ 202 Hg ratio in experimental processes

A useful parameter used for determining sources and processes in environmental samples is the ∆ 199 Hg/δ 202 Hg ratio. Photochemical reduction of iHg from an aqueous solution in the presence of DOC has been observed to produce a ∆ 199 Hg /δ 202 Hg slope of 1.15 ± 0.07 [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF] whereas photochemical reduction of MeHg into Hg 0 induced a ∆ 199 Hg / δ 202 Hg ratio of 2.43 ± 0.10 [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF]. Other photochemical reactions such as iHg reduction from snow crystals [START_REF] Sherman | Mass-independent fractionation of mercury isotopes in Arctic snow driven by sunlight[END_REF] and artificial light-induced iHg reduction via thiol ligands (Zheng and Hintelmann 2010b) have also reported significant MIF, which is mainly attributed to the MIE. Contrastingly, equilibrium evaporation with liquid Hg 0 is fractionated to a small extent (MIF due to the NFS) and presented a ∆ 199 Hg/δ 202 Hg slope of ∼0.1 (Estrade et al., 2009;[START_REF] Ghosh | Estimation of nuclear volume dependent fractionation of mercury isotopes in equilibrium liquid-vapor evaporation experiments[END_REF]. Non-photochemical abiotic reductions can be easily distinguished from photochemical reductions because without MIE they generate a significantly lower MIF (Zheng and Hintelmann 2010b). Also, photochemical reduction of Hg is often accompanied by a concurrent non-photochemical reduction (Zheng and Hintelmann 2010b). Therefore, all photochemical reactions that have been studied produce changes in both MDF and MIF and their ratio of ∆ 199 Hg to δ 202 Hg appears to be indicative of the type of reaction. An overview of the some of the fractionation slopes (∆ 199 Hg vs δ 202 Hg) that have been observed experimentally is presented in Figure 1.17.

Figure 1.17. Summary of ∆ 199 Hg vs δ 202 Hg slopes of Hg isotopic fractionation observed in experimental studies. References: [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF][START_REF] Zheng | Mercury isotope fractionation during volatilization of Hg(0) from solution into the gas phase[END_REF], Zheng and Hintelmann, 2010b, Sherman et al., 2010[START_REF] Kritee | Mercury Stable Isotope Fractionation during Reduction of Hg(II) to Hg(0) by Mercury Resistant Microorganisms[END_REF][START_REF] Kritee | Mass dependent stable isotope fractionation of mercury during mer mediated microbial degradation of monomethylmercury[END_REF][START_REF] Gonzalez | Species-Specific Stable Isotope Fractionation of Mercury during Hg ( II ) Methylation by an Anaerobic Bacteria ( Desulfobulbus propionicus ) under Dark Conditions[END_REF], Estrade et al., 2009[START_REF] Jiskra | Solution speciation controls mercury isotope fractionation of Hg(II) sorption to goethite[END_REF][START_REF] Wiederhold | Equilibrium mercury isotope fractionation between dissolved Hg(II) species and thiol-bound Hg[END_REF][START_REF] Ghosh | Estimation of nuclear volume dependent fractionation of mercury isotopes in equilibrium liquid-vapor evaporation experiments[END_REF] In the case where MIF has been demonstrated, the MIE or NFS type is reported. Figure from the review of [START_REF] Blum | Mercury Isotopes in Earth and Environmental Sciences[END_REF] The approach of experimental studies is very helpful to estimate the different reactions originating Hg isotopic fractionation in nature. The degree of fractionation (δ 202 Hg, Δ 199 Hg) as well as certain parameters such as the Δ 199 Hg/Δ 201 Hg and ∆ 199 Hg/δ 202 Hg ratios observed in the different environmental samples are diagnostic of the sources and undergone reactions since the initial Hg isotopic signature is later modified by intermediate reactions inducing Hg isotopic fractionation.

Hg isotopic composition in environmental studies

Variation of Hg isotopic composition in environmental samples

Hg isotopic fractionation enables the characterisation of different processes but also the determination of Hg sources and mechanisms in the environment. However, Hg isotopic variations occurring in natural samples result from multiple transformations and strongly depend on environmental conditions, thus it is very probable that they are slightly modified from modelled experimental studies. A total δ 202 Hg variation of 10 ‰ was observed in nature so far, varying from -4.5 ‰ to +3.4 ‰ [START_REF] Blum | Mercury Isotopes in Earth and Environmental Sciences[END_REF]. The variationsin ∆ 199 Hg values was greater and ranged from -5 ‰ to +6.5‰. (∆ 199 Hg) values reported in several natural and environmental samples in literature. Figure from [START_REF] Blum | Mercury Isotopes in Earth and Environmental Sciences[END_REF] Environmental samples such as soils, snow, lichens and peat bogs have been found to be good Hg tracers of natural and anthropogenic inputs since they incorporate atmospheric Hg deposition [START_REF] Estrade | Tracing and quantifying anthropogenic mercury sources in soils of northern France using isotopic signatures[END_REF][START_REF] Sherman | Mass-independent fractionation of mercury isotopes in Arctic snow driven by sunlight[END_REF][START_REF] Estrade | Isotope tracing of atmospheric mercury sources in an urban area of northeastern France[END_REF][START_REF] Enrico | Atmospheric Mercury Transfer to Peat Bogs Dominated by Gaseous Elemental Mercury Dry Deposition[END_REF]. One characteristic of these samples is their generally negative MIF values that seem to represent the signature of Hg 0 evaporated to the atmosphere after photoreduction in aquatic systems [START_REF] Bergquist | The odds and evens of mercury isotopes: Applications of mass-dependent and mass-independent isotope fractionation[END_REF]Estrade et al., 2009;[START_REF] Sherman | Mass-independent fractionation of mercury isotopes in Arctic snow driven by sunlight[END_REF]. A preferential sequestration of isotopically light iHg (lower δ 202 Hg) has been demonstrated when binding to mineral phases and organic matter, leading to a residual pool of heavier isotopes in soils and sediments [START_REF] Jiskra | Solution speciation controls mercury isotope fractionation of Hg(II) sorption to goethite[END_REF][START_REF] Wiederhold | Equilibrium mercury isotope fractionation between dissolved Hg(II) species and thiol-bound Hg[END_REF]. In natural organic matter, Hg dominantly bounds to reduced sulphur groups (thiols, -SH) [START_REF] Skyllberg | Complexation of mercury(II) in soil organic matter: EXAFS evidence for linear two-coordination with reduced sulfur groups[END_REF] as a result of their exceptionally high affinity for Hg.

Hg isotopic compositions in precipitation and gaseous Hg over North America great lakes first reported an enrichment in heavier isotopes the vapour phase compared to precipitation, while ∆ 199 Hg values were positive in precipitation and negative in the vapour phase [START_REF] Gratz | Isotopic composition and fractionation of mercury in Great Lakes precipitation and ambient air[END_REF].

In addition, these authors first documented even isotope anomalies (reported as Δ 200 Hg) of up to 0.25‰ in atmospheric precipitation samples over North America. Later, [START_REF] Chen | Unusual fractionation of both odd and even mercury isotopes in precipitation from Peterborough, ON, Canada[END_REF] confirmed the occurrence of significant even-MIF by determination of Δ 200 Hg up to 1.2‰ in rain and snowfall samples in Canada, also observing seasonal variation of Δ 200 Hg, with greater values in winter than in summer. Since then, numerous studies have observed even-MIF in atmospheric samples and therefore, it is thought to be associated to mechanisms occurring in the atmosphere, such as photo-oxidation in the tropopause and/or neutron capture in space [START_REF] Gratz | Isotopic composition and fractionation of mercury in Great Lakes precipitation and ambient air[END_REF]Sherman et al., 2012b;[START_REF] Chen | Unusual fractionation of both odd and even mercury isotopes in precipitation from Peterborough, ON, Canada[END_REF][START_REF] Demers | Mercury isotopes in a forested ecosystem: Implications for airsurface exchange dynamics and the global mercury cycle[END_REF][START_REF] Rolison | Isotopic composition of speciesspecific atmospheric Hg in a coastal environment[END_REF][START_REF] Štrok | The mercury isotope composition of Arctic coastal seawater[END_REF][START_REF] Wang | Massdependent and mass-independent fractionation of mercury isotopes in precipitation from Guiyang, SW China[END_REF][START_REF] Yuan | Large Variation of Mercury Isotope Composition During a Single Precipitation Event at Lhasa City, Tibetan Plateau, China[END_REF]. However, the exact process inducing significant even Hg isotopes-MIF remains still unclear and the understanding of even-MIF could provide useful information about the biogeochemical cycle of Hg, mainly about atmospheric processes, such as air-surface exchanges, and the influence of climate changes [START_REF] Cai | Mass-independent fractionation of even mercury isotopes[END_REF].

Due to the complex Hg cycle in aquatic systems, characterized by numerous biogeochemical transformations, numerous studies have focused on Hg isotopic composition of aquatic organisms to help elucidating Hg pathways in these ecosystems (e.g. [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF][START_REF] Laffont | Anomalous mercury isotopic compositions of fish and human hair in the Bolivian amazon[END_REF][START_REF] Senn | Stable isotope (N, C, Hg) study of methylmercury sources and trophic transfer in the northern gulf of Mexico[END_REF][START_REF] Perrot | Higher massindependent isotope fractionation of methylmercury in the pelagic food web of Lake Baikal (Russia)[END_REF][START_REF] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF][START_REF] Das | Tracing mercury seawater vs. atmospheric inputs in a pristine SE USA salt marsh system: Mercury isotope evidence[END_REF]. Most of these studies have reported significantly positive ∆ 199 Hg values in organisms, while δ 202 Hg values exhibited a wide range of negative and positive values. Since Hg MIF isotopic signatures are preserved during in vivo processes, aquatic organisms (mainly fish) have been used to provide valuable information about MeHg bioaccumulation and its degree of degradation before it is incorporated into food webs [START_REF] Bergquist | The odds and evens of mercury isotopes: Applications of mass-dependent and mass-independent isotope fractionation[END_REF].

Tracking Hg sources and processes in freshwater and marine ecosystems

Hg isotopic signatures have been widely applied for studying biogeochemical processes in different aquatic compartments; such as lakes [START_REF] Gantner | Variations in stable isotope fractionation of Hg in food webs of Arctic lakes[END_REF][START_REF] Perrot | Higher massindependent isotope fractionation of methylmercury in the pelagic food web of Lake Baikal (Russia)[END_REF]Sherman et al., 2013), rivers [START_REF] Tsui | Sources and transfers of methylmercury in adjacent river and forest food webs[END_REF] or oceans [START_REF] Senn | Stable isotope (N, C, Hg) study of methylmercury sources and trophic transfer in the northern gulf of Mexico[END_REF][START_REF] Point | Methylmercury photodegradation influenced by sea-ice cover in Arctic marine ecosystems[END_REF][START_REF] Gehrke | Sources of mercury to San Francisco Bay surface sediment as revealed by mercury stable isotopes[END_REF][START_REF] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF]. Fish have been usually applied for investigation of Hg sources and exposure pathways to the food web. Variations in Hg isotopic signatures between oceanic and coastal compartments have been attributed to both different sources and Hg cycles in the two reservoirs [START_REF] Gantner | Variations in stable isotope fractionation of Hg in food webs of Arctic lakes[END_REF][START_REF] Senn | Stable isotope (N, C, Hg) study of methylmercury sources and trophic transfer in the northern gulf of Mexico[END_REF][START_REF] Point | Methylmercury photodegradation influenced by sea-ice cover in Arctic marine ecosystems[END_REF][START_REF] Gehrke | Sources of mercury to San Francisco Bay surface sediment as revealed by mercury stable isotopes[END_REF][START_REF] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF]. Although marine organisms characteristically exhibit positive δ 202 Hg values [START_REF] Senn | Stable isotope (N, C, Hg) study of methylmercury sources and trophic transfer in the northern gulf of Mexico[END_REF][START_REF] Point | Methylmercury photodegradation influenced by sea-ice cover in Arctic marine ecosystems[END_REF], geologic samples such as sediments or rocks typically report negative δ 202 Hg and close-to-zero ∆ 199 Hg values [START_REF] Smith | Isotope geochemistry of mercury in source rocks, mineral deposits and spring deposits of the California Coast Ranges, USA[END_REF][START_REF] Feng | Tracing mercury contamination sources in sediments using mercury isotope compositions[END_REF][START_REF] Gehrke | Sources of mercury to San Francisco Bay surface sediment as revealed by mercury stable isotopes[END_REF][START_REF] Donovan | An isotopic record of mercury in San Francisco Bay sediment[END_REF][START_REF] Mil-Homens | Tracing anthropogenic Hg and Pb input using stable Hg and Pb isotope ratios in sediments of the central Portuguese Margin[END_REF]Sherman et al, 2013;[START_REF] Yin | Identifying the sources and processes of mercury in subtropical estuarine and ocean sediments using hg isotopic composition[END_REF]. Some studies also revealed that coastal organisms are influenced by continental inputs and thus reflect Hg coming from a recent geological source (δ 202 Hg values~0‰) [START_REF] Senn | Stable isotope (N, C, Hg) study of methylmercury sources and trophic transfer in the northern gulf of Mexico[END_REF][START_REF] Day | Mercury stable isotopes in seabird eggs reflect a gradient from terrestrial geogenic to oceanic mercury reservoirs[END_REF][START_REF] Perrot | Tracing sources and bioaccumulation of mercury in fish of Lake Baikal--Angara River using Hg isotopic composition[END_REF]. A compilation of Hg isotopic composition in pelagic and coastal fish, as well as other organisms (whales, birds) of other aquatic compartments (large lakes, estuaries), is illustrated in Figure 1. 19. Figure 1.19. Compilation of Hg isotopic composition (δ 202 Hg, Δ 199 Hg) in marine and freshwater organisms (fish, whales and bird blood) in literature. Oceanic fish (Gulf of Mexico), [START_REF] Senn | Stable isotope (N, C, Hg) study of methylmercury sources and trophic transfer in the northern gulf of Mexico[END_REF] Coastal fish (Gulf of Mexico), [START_REF] Senn | Stable isotope (N, C, Hg) study of methylmercury sources and trophic transfer in the northern gulf of Mexico[END_REF] Fish (San Francisco Bay, Mississipi river), [START_REF] Gehrke | Sources of mercury to San Francisco Bay surface sediment as revealed by mercury stable isotopes[END_REF] Fish (San Francisco Bay, Topsmelt), [START_REF] Gehrke | Sources of mercury to San Francisco Bay surface sediment as revealed by mercury stable isotopes[END_REF] Pelagic fish (Baikal Lake), [START_REF] Perrot | Higher massindependent isotope fractionation of methylmercury in the pelagic food web of Lake Baikal (Russia)[END_REF] Coastal fish (Baikal Lake), [START_REF] Perrot | Higher massindependent isotope fractionation of methylmercury in the pelagic food web of Lake Baikal (Russia)[END_REF] Fish (estuarines, NE USA), [START_REF] Kwon | Mercury Isotope Study of Sources and Exposure Pathways of Methylmercury in Estuarine Food Webs in the Northeastern U.S[END_REF] Bird blood (estuarines NE USA), [START_REF] Kwon | Mercury Isotope Study of Sources and Exposure Pathways of Methylmercury in Estuarine Food Webs in the Northeastern U.S[END_REF] Fish (Seine estuary), (Cransveld et al., in prep) Fish (Black Sea), (Cransveld et al., in prep) Pilot whales (Faroe Islands) [START_REF] Li | Assessing sources of human methylmercury exposure using stable mercury isotopes[END_REF] Mesopelagic fish (>200 m) (Pacific Ocean) [START_REF] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF] Epipelagic fish (<200 m) (Pacific Ocean) [START_REF] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF]) [START_REF] Cransveld | Mercury stable isotopes discriminate different populations of European seabass and trace potential Hg sources around Europe[END_REF]) [START_REF] Cransveld | Mercury stable isotopes discriminate different populations of European seabass and trace potential Hg sources around Europe[END_REF] By way of example, one study in fish from the Gulf of Mexico depicted clear Hg isotopic differences between coastal and oceanic species, which was associated to distinct sources of MeHg since coastal species were highly influenced by Hg input from the Mississippi river [START_REF] Senn | Stable isotope (N, C, Hg) study of methylmercury sources and trophic transfer in the northern gulf of Mexico[END_REF]. Differences in Hg isotopic signatures between coastal and oceanic fish were in the order of ~0.8‰ for δ 202 Hg and ~1‰ for ∆ 201 Hg values visibly indicating different sources of MeHg and different photodemethylation extent between the two reservoirs. Another study in Lake Baikal (Russia) revealed much higher Δ 199 Hg values in the pelagic food web (3.15-6.65‰) in comparison to coastal fish (0.26-1.65‰), allowing then to discriminate between the coastal and pelagic sources and cycling of MeHg (Perrot et al., 2012, Figure 1.20) Figure 1.20. Schematic design of Hg isotopic results in the food web (amphipods, phytoplankton, zooplankton, fish and seals) of Lake Baikal (Russia) revealing the existence of different Hg cycling between coastal and pelagic food webs [START_REF] Perrot | Higher massindependent isotope fractionation of methylmercury in the pelagic food web of Lake Baikal (Russia)[END_REF].

Likewise, studies on seabirds have also demonstrated the efficiency of Hg isotopic analyses in avian eggs for investigation and identification of different Hg sources between coastal and oceanic environments in the Arctic [START_REF] Day | Mercury stable isotopes in seabird eggs reflect a gradient from terrestrial geogenic to oceanic mercury reservoirs[END_REF]. It has been assumed that the oceanic MeHg is submitted to a substantial degree of photochemical processes, hence oceanic organisms present more positive MIF (∆ 199 Hg and ∆ 201 Hg). In the contrary, coastal organisms reflect an Hg coming from contribution of benthic sources and have undergone more limited extent of photochemical reactions, then display lower MIF values.

Other studies have focused in the gradient of δ 202 Hg, Δ 199 Hg and Δ 201 Hg values for depicting MeHg sources and pathways in the open ocean. One study demonstrated the relationship between Hg MIF values (Δ 199 Hg and Δ 201 Hg) and foraging depth of fish foraging in the Pacific Ocean [START_REF] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF], due to a higher extent of photochemical reactions in the surface layer. These authors evidenced the occurrence of microbial methylation in the pycnocline that produces additional MeHg without MIF, and thus dilutes the MIF of MeHg exported from the surface mixed layer [START_REF] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF]. [START_REF] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF].

In polar marine ecosystems, two studies have revealed the influence of ice cover on Hg marine photochemistry by the investigation of Hg isotopic signatures of seabird eggs [START_REF] Point | Methylmercury photodegradation influenced by sea-ice cover in Arctic marine ecosystems[END_REF] and seal livers [START_REF] Masbou | Hg Stable Isotope Time Trend in Ringed Seals Registers Decreasing Sea Ice Cover in the Alaskan Arctic[END_REF] over a latitudinal gradient in different regions of the Gulf of Alaska. These studies observed a decrease of MIF signatures with the effect of the sea ice cover, which prevents light penetration and thus inhibit photochemical reactions. These authors estimated that the disappearance of ice cover in the Arctic Ocean as a result of global warming would therefore intensify the evasion of MeHg to the atmosphere via photodemethylation.

The biogeochemical cycle of Hg in polar ecosystems is mainly controlled by interactions between the atmospheric and oceanic compartments. AMDEs have been assumed to constitute a substantial pathway of Hg input in the polar marine environment and several studies have explored the mechanisms associated to the oxidation of Hg 0 during these processes (e.g, [START_REF] Ariya | Reactions of gaseous mercury with atomic and molecular halogens: Kinetics, product studies, and atmospheric implications[END_REF][START_REF] Lindberg | Dynamic oxidation of gaseous mercury in the arctic troposphere at polar sunrise[END_REF]. Substantial negative MIF has been observed in Arctic snow after deposition of iHg by AMDEs (Δ 199 Hg < -1.2‰), meaning that Hg is depleted in odd Hg isotopes during halogen oxidation [START_REF] Sherman | Mass-independent fractionation of mercury isotopes in Arctic snow driven by sunlight[END_REF]. Δ 199 Hg/Δ 201 Hg ratios observed for air-snow and snow-frost flowers were, respectively, 1.07± 0.04‰ [START_REF] Sherman | Mass-independent fractionation of mercury isotopes in Arctic snow driven by sunlight[END_REF] and 1.26±0.10‰ (Sherman et al., 2012b). Since there is well-supported evidence that AMDEs are related to bromine and chlorine radicals [START_REF] Barrie | Ozone destruction and photochemical reactions at polar sunrise in the lower Arctic atmosphere[END_REF][START_REF] Mao | Arctic mercury depletion and its quantitative link with halogens[END_REF], being Br and BrO the dominant oxidizers [START_REF] Stephens | The relative importance of chlorine and bromine radicals in the oxidation of atmospheric mercury at Barrow, Alaska[END_REF], Hg isotope fractionation during gas-phase oxidation of Hg 0 by Cl and Br atoms were studied to better recognise the initiation of these processes occurring in polar regions [START_REF] Sun | Mass-dependent and -independent fractionation of mercury isotope during gas-phase oxidation of elemental mercury vapor by atomic Cl and Br[END_REF]. Nevertheless, a recent study demonstrated that direct Hg 0 deposition was the dominant source to the Arctic tundra (70%), which is enhanced during summertime, meanwhile AMDEs represent a minor contribution to Hg input [START_REF] Obrist | Tundra uptake of atmospheric elemental mercury drives Arctic mercury pollution[END_REF]. As observed in Figure 1.22, Hg isotopic studies in the Antarctic Polar Regions are still scarce comparing to Arctic environments. To date, Hg isotopic signatures have only been measured measured in ornithogenic sediments in the coastal Antarctica (Ross Sea) and in fresh seal and penguin feces [START_REF] Zheng | Mercury Stable Isotopes in Ornithogenic Deposits As Tracers of Historical Cycling of Mercury in Ross Sea, Antarctica[END_REF]. This study explored Hg isotopic composition in historical and modern biological deposits in order to trace temporal trends in Hg sources and cycling in the Antarctic Ocean and to discern different bioaccumulation pathways in Antarctic marine biota. Sediment cores were collected from the top to 38 cm depth permitting to assess Hg contamination sources between the present time and around the year 1280 AD. Regarding the trends observed for the more ancient deep layers, seal and penguin excrements were considered the major contributor of Hg inputs into the sediment respectively for the first period (1280-1400 AD) and for the second period (1400-1650 AD), coherent with the increase of penguin population during from that period. During 1650-1990 AD, penguin guano continued to be considered the major source of Hg inputs into the sediment (36-75%), but a lower ∆ 199 Hg/ ∆ 201 Hg slope observed in this period (1.20±0.04) relative to the precedent period (1.34±0.05) was interpreted as substantial inputs deriving also from bedrock sediment and atmospheric Hg deposition that would explain the dilution in ∆ 199 Hg signatures. The most top layer, corresponding to 1990 to the present time, recorded higher Hg concentrations and lower ẟ 202 Hg and ∆ 199 Hg signatures, which was attributed to enhanced atmospheric Hg input due to increasing anthropogenic emissions, also sequestered by terrestrial vegetation or algae in the surface.

∆ 199 Hg/∆ 201 Hg ratios in aquatic systems

As previously said, it is important to consider that experimental values often represent an approximation of more complex processes in nature. Moreover, the ligands associated with Hg in aqueous solutions (such as organic matter, dissolved cations and halogens in seawater) lead to a slight differences in ∆ 199 Hg/∆ 201 Hg ratios and therefore, the theoretical slopes obtained in laboratory medium are only approximate for interpretation of environmental samples of natural freshwater and marine ecosystems. [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF].

As observed in Figure 1.23, fish from Lake Michigan presented a Δ 199 Hg/Δ 201 Hg ratio of 1.28±0.03, corresponding to the accumulation of residual MeHg that has previously undergone photochemical demethylation [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF]. This is coherent with the predominance of MeHg in fish (~95%) and makes possible to estimate the rate of photodemethylation in the water column of a given ecosystem [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF]. Latter studies in freshwater fish also reported similar Δ 199 Hg/Δ 201 Hg ratios close to 1.3 [START_REF] Laffont | Anomalous mercury isotopic compositions of fish and human hair in the Bolivian amazon[END_REF][START_REF] Gantner | Variations in stable isotope fractionation of Hg in food webs of Arctic lakes[END_REF]Sherman et al., 2013a). However, Hg isotopic studies in marine fish (and seabirds that feed on marine fish) have documented slightly lower Δ 199 Hg/Δ 201 Hg ratios. For example, fish from the Gulf of Mexico and from the Pacific Ocean exhibited respective ∆ 199 Hg/∆ 201 Hg slopes of 1.20±0.07 [START_REF] Senn | Stable isotope (N, C, Hg) study of methylmercury sources and trophic transfer in the northern gulf of Mexico[END_REF] and 1.20±0.01 [START_REF] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF]. Hg isotopic studies in seabird eggs from Alaska reported ∆ 199 Hg/∆ 201 Hg slopes of 1.26±0.06 [START_REF] Point | Methylmercury photodegradation influenced by sea-ice cover in Arctic marine ecosystems[END_REF] and 1.09±0.04 [START_REF] Day | Mercury stable isotopes in seabird eggs reflect a gradient from terrestrial geogenic to oceanic mercury reservoirs[END_REF].

Although all the MIF slopes compiled in fish derive from MeHg photochemical degradation in both freshwater and marine systems, lower ∆ 199 Hg/∆ 201 Hg can be the consequence of differences in organic matter or the influence of dissolved cations and halogens present in seawater. Moreover, freshwater ecosystems can be impacted by iHg inputs from riverine inputs whereas in opened marine areas Hg atmospheric deposition is considered the main source [START_REF] Mason | Mercury biogeochemical cycling in the ocean and policy implications[END_REF]

Biogeochemical characteristics

Carbon cycle and stable carbon isotopes

There is evidence of the important role of organic carbon remineralization on the production of Hg methylated compounds in the open Ocean [START_REF] Sunderland | Mercury sources, distribution, and bioavailability in the North Pacific Ocean: Insights from data and models[END_REF][START_REF] Cossa | The origin of methylmercury in open Mediterranean waters[END_REF].

Oceanic carbon cycle can be impacted by physical mechanisms such as upwelling [START_REF] Marshall | Closure of the meridional overturning circulation through Southern Ocean upwelling[END_REF] or the biological pumps [START_REF] Ducklow | Upper ocean carbon export and the biological pump[END_REF]. Stable carbon signatures, reported as δ 13 C, are slightly affected with trophic level and are widely used to determine primary carbon sources in a food web. The Southern Ocean is defined by a well-recognised latitudinal δ 13 C gradient which effectively characterise different water masses at the basal food web [START_REF] Trull | Insights into Southern Ocean carbon export from the δ13c of particles and dissolved inorganic carbon during the SOIREE iron release experiment[END_REF] and which is preserved in consumers at higher trophic levels (Cherel and Hobson, 2007;[START_REF] Jaeger | Seabird satellite tracking validates the use of latitudinal isoscapes to depict predators' foraging areas in the Southern Ocean[END_REF]. In the marine environment, δ 13 C values enables the determination of inshore-offshore and pelagic-benthic gradients as well as large latitudinal scales. Indeed, this isotopic method has been successfully applied in areas surrounding Southern Ocean islands to discriminate pelagic from neritic feeders (Cherel and Hobson, 2007) permitting the utilisation of δ 13 C signatures to assess the main foraging zones of seabirds [START_REF] Cherel | A comprehensive isotopic investigation of habitat preferences in nonbreeding albatrosses from the Southern Ocean[END_REF]Cherel and Hobson, 2007). Furthermore, phytoplankton δ 13 C values are positively correlated to higher primary productivity, which is known to be extremely elevated in the vicinity of subantarctic islands during austral summer [START_REF] Blain | A biogeochemical study of the island mass effect in the context of the iron hypothesis: Kerguelen Islands, Southern Ocean[END_REF][START_REF] Pollard | Physical controls on biogeochemical zonation in the Southern Ocean[END_REF]. Importantly, although δ 13 C signatures provide valuable latitudinal trends of the foraging zones, they do not offer indication about longitude variations.

Primary production

The Southern Ocean is also characterised by spatial and seasonal heterogeneity in primary production. Generally, these southern marine regions present high nutrient concentrations such as nitrate, phosphate or manganese; whereas phytoplankton production is contrastingly low [START_REF] Boyd | A mesoscale phytoplankton bloom in the polar Southern Ocean stimulated by iron fertilization[END_REF][START_REF] Blain | A biogeochemical study of the island mass effect in the context of the iron hypothesis: Kerguelen Islands, Southern Ocean[END_REF][START_REF] Sanial | Study of the phytoplankton plume dynamics off the Crozet Islands (Southern Ocean): A geochemical-physical coupled approach[END_REF], considered then as "high nutrient-low chlorophyll" waters. Factors such as sunlight incidence, water temperature and complex hydrodynamics have been proposed as influential factors on biological activity rates [START_REF] Boyd | The role of iron in the biogeochemistry of the Southern Ocean and equatorial Pacific: A comparison of in situ iron enrichments[END_REF]. Low Fe concentrations in these waters have been shown to limit phytoplankton growth [START_REF] Boyd | A mesoscale phytoplankton bloom in the polar Southern Ocean stimulated by iron fertilization[END_REF]. Nevertheless, the Southern Ocean areas experience seasonal supply of Fe and other macro and micro-nutrients that arrive to the surface, resulting in high primary production and phytoplankton bloom during the austral summer [START_REF] Mongin | Winter advection of iron can explain the summer phytoplankton bloom that extends 1000 km downstream of the Kerguelen Plateau in the Southern Ocean[END_REF][START_REF] Blain | Effect of natural iron fertilization on carbon sequestration in the Southern Ocean[END_REF], especially in the Antarctic zone by the transit of the circumpolar deep waters [START_REF] Cossa | Mercury in the Southern Ocean[END_REF].

In Antarctic coastal zones, this phytoplankton bloom phenomenon is known to be enhanced by sea ice melting during spring and summer seasons [START_REF] Sokolov | Chlorophyll blooms in the Antarctic Zone south of Australia and New Zealand in reference to the Antarctic Circumpolar Current fronts and sea ice forcing[END_REF][START_REF] Riaux-Gobin | Environmental conditions, particle flux and sympagic microalgal succession in spring before the sea-ice break-up in Adélie Land, East Antarctica[END_REF], demonstrating that sea ice from Adélie Land contains high biomass concentrations and primary production [START_REF] Riauxgobin | A pigment analysis of feeding modes of Thelepus extensus (Polychaeta, Terebellidae) in relation to wave exposure at the Iles Kerguelen[END_REF], especially fast ice, which is associated to high microalgal biomass and nutrient regeneration. Determined areas of the Subantarctic Zone are highly productive due to seasonally release of nutrients in regions of the Kerguelen Plateau, a phenomenon known as "island fertilisation effect" because nutrients are deposited on the borders of the Crozet, Kerguelen and South Georgia archipelagos [START_REF] Blain | A biogeochemical study of the island mass effect in the context of the iron hypothesis: Kerguelen Islands, Southern Ocean[END_REF][START_REF] Korb | Contrasting primary production regimes around South Georgia, Southern Ocean: Large blooms versus high nutrient, low chlorophyll waters[END_REF][START_REF] Sanial | Study of the phytoplankton plume dynamics off the Crozet Islands (Southern Ocean): A geochemical-physical coupled approach[END_REF]. Upwelling waters are also characteristic of these zones and drive the supply of nutrients to surface waters [START_REF] Plancke | Phytoplankton biomass and productivity in the Subtropical Convergence area and shelves of the western Indian subantarctic islands[END_REF].

3.2 Bioindicators models of the study: seabirds of the Southern Ocean

Seabirds as bioindicators of Hg contamination

A bioindicator of chemical contamination is considered an organism able to reflect the contaminant exposure in a given ecosystem by integration of local and global sources of contamination over different spatial and temporal scales. Wildlife bioindicators present several advantages because they reflect the contamination of the food webs on which they depend and thus allow assessing and understanding the contamination in the ecosystems. The choice of an appropriate bioindicator must be based on its capacity to bioaccumulate the concerned contaminants and, ideally, the environmental concentrations and measurements in the tissues of the bioindicator should be proportionally related. Such direct proportionality is verified for mammals and seabirds for biomagnifiable contaminants such as Hg. The effect of physiologic factors (metabolism and detoxification processes) and ecological aspects (specific life-history and foraging behaviour) must also be taken into consideration when marine mammals and seabirds are used for a correct interpretation of the measured concentrations or interspecific variations.

Birds have demonstrated to be satisfactory bioindicators of ecosystems health in general and of chemical contamination in particular [START_REF] Furness | Seabirds as monitors of the marine environment[END_REF]. Thus they have been widely used for contamination biomonitoring, particularly in marine ecosystems [START_REF] Burger | Marine Birds as Sentinels of Environmental Pollution[END_REF]. In some cases they have contributed to unchain regulation or prohibition of pollutant chemicals. This is the case of peregrine falcon whose population became endangered due to the extensive use of some pesticides, mainly DTT, before its ban in 1970 [START_REF] Ratcliffe | British Birds Changes Attributable to Pesticides in Egg Breakage Frequency and Egshell Thickness in some British Birds[END_REF][START_REF] Walker | Neurotoxic Pesticides and Behavioural Effects Upon Birds[END_REF].

Since they are top predators within marine food webs, seabirds are exposed to high levels of Hg by dietary uptake, especially under the form of MeHg, leading to Hg-related health effects.

Severe consequences of Hg toxicity have been documented on seabirds at the individual level.

Since Hg is an endocrine disrupter, it has led to reproductive disturbance and decreased hatching rates [START_REF] Tartu | To breed or not to breed: endocrine response to mercury contamination by an Arctic seabird[END_REF], resulting in substantial demographic decline (Goutte et al., 2014ab). Therefore, it is hypothesized that an increase in the incorporation of MeHg in the trophic webs, as a consequence of the progressive warming of the ocean [START_REF] Cossa | Marine biogeochemistry: Methylmercury manufacture[END_REF], could provoke the decrease in seabird populations due to the reprotoxic effects of Hg (Goutte et al., 2014b).

The use of seabirds to study the sources of Hg and the processes associated to its geochemistry in the ocean is particularly interesting since they forage in diverse positions at sea, in the horizontal and vertical scales, according to the species. Seabirds present some ecological characteristics that make them ideal models for evaluation of Hg fate and bioavailability in marine Although some of seabird characteristics could present disadvantages for environmental monitoring studies (such as their high mobility and exploitation of large areas), a careful choice of species whose ecological characteristics are well-known may ensure the procurement of relevant information about of Hg contamination and its correct interpretation (Thompson et al., 1993ab;[START_REF] Blévin | Wide range of mercury contamination in chicks of southern ocean seabirds[END_REF]Carravieri et al., 2014ab). During this doctoral dissertation, the principles of selection of the most appropriate seabird models were detailed and justified according to the specific objective of each study (Chapters 3 and 4). Combining information from previous studies on satellite-tracking, carbon isotopic measurements and direct field observations [START_REF] Jaeger | Seabird satellite tracking validates the use of latitudinal isoscapes to depict predators' foraging areas in the Southern Ocean[END_REF]Cherel and Hobson, 2007;[START_REF] Lescroël | Foraging under contrasting oceanographic conditions: The gentoo penguin at Kerguelen Archipelago[END_REF] enabled to create a primary database of the specific foraging strategies of each seabird species. Furthermore, a previous doctoral work [START_REF] Carravieri | Seabirds as bioindicators of Southern Ocean ecosystems: concentrations of inorganic and organic contaminants, ecological explanation and critical evaluation[END_REF] already provided key insights for the identification of the most relevant bioindicator species for long-term monitoring of Hg contamination, by measurements of Hg concentrations in more than 30 seabird species with contrasting feeding ecology of the Southern Indian Ocean. Among others indications, Carravieri (2014) highlighted the importance of food intake as the main pathway of exposure of seabirds to contaminants, diet being the first factor explaining Hg concentrations across a single seabird community [START_REF] Carravieri | Penguins as bioindicators of mercury contamination in the southern Indian Ocean: geographical and temporal trends[END_REF]. Nevertheless, numerous other factors such as phylogeny, moulting patterns, sex, life span and diet composition are likely to influence Hg bioaccumulation in seabird tissues.

Biodiversity of seabirds in the Southern Ocean and ecological characteristics

French Southern and Antarctic territories represent the ideal region for scientific ecological research because they encompass a great biodiversity characterized by massive colonies of birds and marine mammals. A large number of seabirds of the Southern Indian Ocean (about 380 millions of individuals) return to these localities to breed each year [START_REF] Van Franeker | Carbon fluxes to Antarctic top predators[END_REF], of which penguins correspond to the largest biomass (90%) (Woehler, 1995). Another abundant seabird community corresponds to the family Procellariiformes (albatrosses and petrels),

including emblematic species such as the wandering albatross, for which the highest feather Hg concentration has been reported (40 µg g -1 on average, and up to 95 µg g -1 recorded for a single individual, [START_REF] Bustamante | High feather mercury concentrations in the wandering albatross are related to sex, breeding status and trophic ecology with no demographic consequences[END_REF]. These species are extremely long-lived seabirds, in some cases achieving 80 years old, so are ideal models for monitoring temporal evolution of seabird populations investigating their life history, reproduction, mortality, survival and physical conditions.

Breeding period takes place during the austral summer for the most part of seabird species although there are also some exceptions of winter breeders such as the emperor penguin. While breeding, seabird individuals are constrained to return frequently to their breeding colonies in order to incubate or provision their offspring, thus they are spatially restricted to surrounding areas when foraging at sea. Therefore, seabird diet at this time is mainly composed of local prey.

One exception are king penguins that are known to forage in the waters of the Antarctic Polar

Front irrespectively of their breeding location [START_REF] Pütz | Spatial and temporal variability in the foraging areas of breeding king penguins[END_REF]. During the breeding season, sampling collection is thus more accessible and feather and blood can be sampled on live adult individuals and chicks.

Outside the breeding period, seabirds are not linked to a central place and have developed specific and contrasting feeding strategies to optimise food resources and elude inter and intraspecific competition. Migratory seabirds can cover long distances during their non-breeding period, in some cases arriving up to North Atlantic and Pacific Ocean zones. The use of migratory seabirds as a bioindicator could thus be perceived as problematic, but their mobility also has some advantages. Indeed, compared to resident species that reflect contamination of a specific exposure zone, migratory seabirds integrate Hg into different areas: breeding and wintering areas.

Nevertheless, this characteristic requires the ability to discriminate Hg accumulated in each of these zones, as previously mentioned by combination of Hg measurements and spatial ecology studies, in order to monitor the movement and determine the distribution of organisms during their life cycle [START_REF] Fort | Spatial Ecotoxicology: Migratory Arctic Seabirds Are Exposed to Mercury Contamination While Overwintering in the Northwest Atlantic[END_REF]. Penguins, and other diving seabirds, disperse over wide marine areas exploiting different depths of the water column, foraging at epipelagic, mesopelagic or benthic zones depending on species. Moulting is a necessary process in seabirds for preservation of their thermic isolation and flying capacities. Most flying birds moult annually during the less productive austral winter [START_REF] Jaeger | Isotopic investigation of contemporary and historic changes in penguin trophic niches and carrying capacity of the Southern Indian Ocean[END_REF]. Their feather replacements is sequential and prolonged generally over 2-3 months to one year [START_REF] Bridge | Influences of morphology and behavior on wing-molt strategies in seabirds[END_REF], between two breeding periods. Some seabird species present more complex moulting patterns such as the wandering albatross that moults intermittently over several inter-breeding periods [START_REF] Weimerskirch | Sex-specific differences in molt strategy in relation to breeding in the wandering albatross[END_REF]. Unlike flying seabirds, adult penguins renew their whole plumage in the course of austral summer immediately before or after breeding during 2-5 weeks. This process is crucial in the annual cycle of penguins since they experience a progressive thermal reduction that prevents them from going at sea. Therefore, they evolve a previous foraging period of hyperphagia at sea to build up the energy reserves required during moult (e.g, [START_REF] Thiebot | Adjustment of pre-moult foraging strategies in Macaroni Penguins Eudyptes chrysolophus according to locality, sex and breeding status[END_REF]. Penguins exhibit a synchronous moult as a consequence of their entire feather renewal, therefore their feathers present a quite homogenous chemical composition [START_REF] Brasso | Unique pattern of molt leads to low intraindividual variation in feather mercury concentrations in penguins[END_REF]Carravieri et al. 2014a). Moreover, since penguins stay on land during extended periods while fasting, they are easily accessible in particular at the beginning of the breeding cycle and during moult.

As previously mentioned, the synthesis of feathers represents a major pathway for excretion of Hg that is remobilized from storage tissues at the time of moulting. Approximately 80% of the Hg contained in the internal tissues is excreted in feathers [START_REF] Hoefs | Seasonal changes in mercury accumulation in the black-eared kite, Milvus migrans lineatus[END_REF][START_REF] Braune | Comparison of Total Mercury Levels in Relation to Diet and Molt for Nine Species of Marine Birds[END_REF].

Once feathers are synthesized, Hg concentrations do not change and therefore are assumed to reflect the Hg accumulated over two moulting periods. Consequently, feathers analyses provides information on the integrated Hg contamination over different periods of several months. In migratory seabirds such as Alcids, analysis on feathers thus make possible to discriminate the Hg exposure at their breeding and wintering sites [START_REF] Fort | Spatial Ecotoxicology: Migratory Arctic Seabirds Are Exposed to Mercury Contamination While Overwintering in the Northwest Atlantic[END_REF]. In chicks, feathers are synthesized continuously throughout their rearing period, providing access to local Hg contamination over that time since their parents nourish them with prey in the vicinity of the colony. Thus, the use of chick feathers is very relevant for monitoring of Hg contamination of the environment because the integration time and spatial resolution of Hg exposure are clearly defined [START_REF] Carravieri | Seabirds as bioindicators of Southern Ocean ecosystems: concentrations of inorganic and organic contaminants, ecological explanation and critical evaluation[END_REF][START_REF] Blévin | Wide range of mercury contamination in chicks of southern ocean seabirds[END_REF]Carravieri et al., 2014a). Meanwhile, blood presents a different Hg turnover as it a metabolically active tissue representative of recent Hg exposure that is accumulated dynamically in storage tissues. Therefore, blood reflects a shorter-term Hg intake than feathers, in the order of few weeks/months before sampling (Bearhop et al., 2000b) and, since it is collected during the breeding period, both adult and chick blood samples are assumed to provide information about Hg contamination near the colonies.

Seabirds from the Southern Ocean are considered competent samplers to investigate Hg contamination since they provide access to different marine environmental compartments exploiting interspecific foraging strategies and feeding on large diversity of prey horizontally (from coastal to oceanic) and vertically (epipelagic to mesopelagic zones). Besides, investigating ubiquitous species breeding at colonies of the different locations of the Southern Indian Ocean enables to explore a vast latitudinal gradient from Antarctica (Adélie Land) to the subtropics (Amsterdam Island). In a last instance, exploration of diverse avian tissues (feathers, blood or internal tissues) can give information of different integration times of Hg exposure and/or metabolic response face to Hg contamination.

Main objectives of this doctoral work

Despite its distance from anthropogenic pressure, the Southern Ocean and Antarctic zones are affected by inputs of contaminants, such as Hg, mainly by oceanic currents and atmospheric deposition [START_REF] Pacyna | Global emission of mercury to the atmosphere from anthropogenic sources in 2005 and projections to 2020[END_REF]. As a globally distributed contaminant, Hg can be easily transported over long distances and is present in substantial levels in these considered pristine ecosystems [START_REF] Cossa | Mercury in the Southern Ocean[END_REF]. Several studies have documented meaningful levels of Hg bioaccumulation in tissues of biota and in particular of top predators (e.g., [START_REF] Bocher | Influence of the diet on the bioaccumulation of heavy metals in zooplankton-eating petrels at Kerguelen archipelago, Southern Indian Ocean[END_REF][START_REF] Anderson | Influence of trophic position and foraging range on mercury levels within a seabird community[END_REF]Carravieri et al., 2014a;[START_REF] Bustamante | High feather mercury concentrations in the wandering albatross are related to sex, breeding status and trophic ecology with no demographic consequences[END_REF]. Nevertheless, Hg contamination in the Southern Ocean, especially in the Indian sector, remains largely unknown and determining the origin and fate of Hg in such remote areas involves a major challenge. Indeed, previous results of Hg latitudinal variations reported in marine birds and in the water column have exhibited a reverse gradient. While MeHg concentrations seemed to be higher in Antarctic than in the subantarctic and subtropical waters [START_REF] Cossa | Mercury in the Southern Ocean[END_REF], analyses on seabird tissues have documented increasing MeHg concentrations from Antarctic through subantarctic to subtropical colonies [START_REF] Blévin | Wide range of mercury contamination in chicks of southern ocean seabirds[END_REF]Carravieri et al., 2014b;Goutte et al., 2014;[START_REF] Carravieri | From Antarctica to the subtropics : Contrasted geographical concentrations of selenium , mercury , and persistent organic pollutants in skua chicks ( Catharacta spp .)[END_REF].

Under these circumstances, a better understanding of the formation sources of MeHg in these This dissertation has been focused in different aspects and methodological actions that have been previously assessed in order to ensure the procurement of valuable information by Hg isotopic measurements in different avian tissues and to guarantee its correct interpretation and application for elucidating Hg biogeochemical processes. In a first step, an optimisation and validation of analytical methods and techniques (Chapter 2) was required for some avian matrixes that were analysed by Hg speciation and Hg isotopic composition studies (principally feathers and blood). Secondly, a methodological approach (Chapter 3) was addressed with the triple objective of i) inquire the detoxification mechanisms and metabolic response of seabirds face to Hg by analyses of different avian tissues (feathers, blood and diverse internal tissues and organs), ii) evaluate the level of information provided by tissue-specific isotopic signatures and iii) determine the most effective tissue for biomonitoring depending on the scientific purpose.

Once the methodological features were assessed and the choice of the most pertinent bioindicator tissue was justified, we focused on the exploration of the ecological and environmental aspects of this doctoral work (Chapter 4). In this chapter, a first part was focused on the investigation of MeHg exposure pathways as a function of specific ecological characteristics of seabirds (i.e., trophic ecology and foraging strategies) in order to determine distinct environmental MeHg sources at variable marine compartments in a same ecosystem (i.e., the Crozet Islands). The *Corresponding authors: marina.renedoelizalde@univ-pau.fr; david.amouroux@univ-pau.fr

Introduction

Mercury (Hg) is a globally distributed pollutant of major concern for humans and wildlife, whose toxicity is known to be dependent on its molecular speciation. Methylmercury (MeHg) is considered the most toxic Hg species and, once acquired by dietary uptake, it accumulates in organisms and biomagnifies within the food webs [START_REF] Bargagli | Biomagnification of mercury in an Antarctic marine coastal food web[END_REF][START_REF] Bargagli | Enhanced deposition and bioaccumulation of mercury in Antarctic terrestrial ecosystems facing a coastal polynya[END_REF]. In aquatic systems, anaerobic microorganisms such as sulphate and iron reducing bacteria (SRB and IRB) transform inorganic Hg (iHg) in 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][START_REF] Fleming | Mercury Methylation from Unexpected Sources : Molybdate-Inhibited Freshwater Sediments and an Iron-Reducing Bacterium[END_REF], resulting in its incorporation into the food chain. Hence top predators, particularly those linked to aquatic ecosystems, are at highest risk for increased dietary Hg exposure, especially MeHg, leading to potential Hg-related health effects [START_REF] Wiener | Ecotoxicology of mercury. Handbook of Ecotoxicology[END_REF] and to consequences at the population level (Goutte et al. 2014a).

Birds have been extensively used as effective bioindicators of Hg contamination in the environment, particularly of marine ecosystems [START_REF] Burger | Marine Birds as Sentinels of Environmental Pollution[END_REF]. Due to their high position on the aquatic food webs and their long life span, seabirds accumulate significant levels of Hg in their tissues. Feathers are interesting samples to analyse because they represent the main route of Hg elimination in seabirds, so contain most of their Hg body burden [START_REF] Braune | Comparison of Total Mercury Levels in Relation to Diet and Molt for Nine Species of Marine Birds[END_REF][START_REF] Monteiro | Mercury concentrations in prey fish indicate enhanced bioaccumulation in mesopelagic environments[END_REF][START_REF] Hoefs | Seasonal changes in mercury accumulation in the black-eared kite, Milvus migrans lineatus[END_REF][START_REF] Furness | Using bird feathers to measure mercury in the environment: Relationships between mercury content and moult[END_REF]. Moreover, feathers can be easily and non-destructively sampled on live individuals. During moult, most of the Hg stored within internal tissues (70-90%) is remobilised into growing feathers [START_REF] Hoefs | Seasonal changes in mercury accumulation in the black-eared kite, Milvus migrans lineatus[END_REF], where it is sequestered in the sulphydryl groups of the keratin molecules and cannot be reincorporated into internal tissues. Once bound to keratin, Hg is physically and chemically stable [START_REF] Appelquist | Mercury monitoring: Mercury stability in bird feathers[END_REF] and resistant to a variety of rigorous treatments (Thompson et al., 1998a). Although other metals such as lead or cadmium are supposed to be incorporated in feathers by atmospheric input, the homogenous distribution pattern of Hg previously observed in feathers has led to hypothesize that Hg contamination is more likely due to endogenous causes (food and physiology) and is not affected by atmospheric exposure [START_REF] Hahn | Bird feathers as bioindicators in areas of the German Environmental Specimen Bank -bioaccumulation of mercury in food chains and exogenous deposition of atmospheric pollution with lead and cadmium[END_REF]. Nevertheless, gaseous Hg adsorption has been recently demonstrated in human hair under Hg vapour exposure (Queipo [START_REF] Abad | Evidence of the direct adsorption of mercury in human hair during occupational exposure to mercury vapour[END_REF][START_REF] Laffont | Hg speciation and stable isotope signatures in human hair as a tracer for dietary and occupational exposure to mercury[END_REF], suggesting the possibility of direct Hg deposition also in feathers under high gaseous Hg ambient concentrations which could potentially have a repercussion on the use of bird feathers from museum collections for retrospective investigation on Hg temporal trends.

However, this process is unlikely to occur in feathers of seabirds inhabiting non-contaminated areas. The first research study of Hg speciation in feathers was carried out by Thompson and Furness (Thompson and Furness 1989a) in various seabird species, in which they found that Hg incorporated into this tissue was mainly composed of MeHg (77-118%). A dominance of MeHg in feathers was also observed in following studies (Kim et al., 1996;Thompson et al., 1998b;[START_REF] Bond | Total and Methyl Mercury Concentrations in Seabird Feathers and Eggs[END_REF]. Hg has been therefore assumed to be present almost exclusively under its organic form in feathers while no accurate determination of iHg has been achieved so far. Total Hg concentrations (THg) are often measured for MeHg quantification in feathers as an economical alternative to speciation analyses. Actually, direct analyses of MeHg in feathers are usually considered on studies focused on temporal variations on Hg concentrations using historical feather collections from museums, e.g [START_REF] Thompson | Historical changes in mercury concentrations in the marine ecosystem of the north and north-east Atlantic ocean as indicated by seabird feathers[END_REF]Thompson et al., 1993b;[START_REF] Furness | Spatial and temporal variation in mercury contamination of seabirds in the North Sea[END_REF][START_REF] Bond | Rapidly increasing methyl mercury in endangered ivory gull (Pagophila eburnea) feathers over a 130 year record[END_REF]; where a potential contamination of iHg has been found to be produced by the successive application of preservatives containing HgCl2 and methylbromide [START_REF] Vo | Temporal increase in organic mercury in an endangered pelagic seabird assessed by century-old museum specimens[END_REF]. Analyses of Hg speciation in feathers are therefore essential to better evaluate Hg exposure and metabolic processes in birds.

Different analytical methods for Hg speciation in feather samples have been reported in previous studies. Thompson and Furness (Thompson and Furness 1989b) proposed the first method to determine concentrations of THg and MeHg in feathers by MeHg selective extraction (adapted from Uthe et al. [START_REF] Uthe | Longitudinal study of methylmercury and inorganic mercury in blood and urine of pregnant and lactating women, as well as in umbilical cord blood[END_REF]) for subsequent analyses by cold-vapour atomic fluorescence spectroscopy (CV-AFS). This procedure has been widely used by studies of Hg speciation in feathers [START_REF] Thompson | Historical changes in mercury concentrations in the marine ecosystem of the north and north-east Atlantic ocean as indicated by seabird feathers[END_REF]Thompson et al., 1993ab;Kim et al., 1996;[START_REF] Monteiro | Accelerated increase in mercury contamination in North Atlantic mesopelagic food chains as indicated by time series of seabird feathers[END_REF]Thompson et al., 1998b;[START_REF] Spalding | Methylmercury accumulation in tissues and its effects on growth and appetite in captive great egrets[END_REF]. More developed analytical techniques were later performed by using gas chromatography in order to separate Hg species: gas chromatography coupled to atomic fluorescence spectroscopy (GC-AFS) [START_REF] Bond | Total and Methyl Mercury Concentrations in Seabird Feathers and Eggs[END_REF][START_REF] Mallory | Mercury concentrations in feathers of marine birds in Arctic Canada[END_REF]; gas chromatography coupled to electron-capture detector (GC-ECD) [START_REF] Kehrig | Trace-elements, methylmercury and metallothionein levels in Magellanic penguin (Spheniscus magellanicus) found stranded on the Southern Brazilian coast[END_REF], and single isotope dilution analysis by gas chromatography coupled to ICPMS (S-IDA-GC-ICPMS) with single isotope spike (only isotopically labelled MeHg was added) [START_REF] Vo | Temporal increase in organic mercury in an endangered pelagic seabird assessed by century-old museum specimens[END_REF]. However, these analytical techniques do not allow correcting possible losses or transformations between Hg species and provide uniquely the quantification of MeHg and THg concentrations, so the determination of iHg concentrations needs to be calculated as the difference between both compounds' concentrations. A synthesis of the results published in previous studies have been compiled in Table 2.1. It can be observed that in some cases MeHg proportion values of feathers exceed 100% of THg, indicating a lack of accuracy or precision in MeHg quantification by classical techniques. This observation enhances the interest of our developed method for accurate and precise Hg species analyses.

The quantification of both Hg species involves an analytical challenge due to potential losses or species transformation reactions (i.e., MeHg demethylation or iHg methylation processes) which could occur during the whole analytical procedure, leading to erroneous results in the quantification of Hg species concentrations [START_REF] Clémens | Mercury speciation analysis in seafood by species-specific isotope dilution : method validation and occurrence data[END_REF]. GC-ICPMS using a double isotopic dilution method provides the simultaneous measurement of both MeHg and iHg, and subsequently THg as THg = MeHg + iHg, with high precision [START_REF] Rodríguez-González | Isotope dilution analysis for elemental speciation: a tutorial review[END_REF][START_REF] Clémens | Mercury speciation in seafood using isotope dilution analysis: A review[END_REF]. Obtained data can also be processed by Isotope Pattern Deconvolution (IPD), a general model for isotope dilution that takes into consideration both spikes (isotopically enriched solutions) and natural species, and enables the determination of potential interconversion reactions and the consequent correction of Hg species concentrations [START_REF] Clémens | Mercury speciation analysis in seafood by species-specific isotope dilution : method validation and occurrence data[END_REF].

Accordingly, isotope dilution methods guarantee a better precision and accuracy than conventional quantification by external calibration [START_REF] Clémens | Mercury speciation in seafood using isotope dilution analysis: A review[END_REF]. Previous studies published method developments (extraction and derivatisation) for Hg speciation by ID-GC-ICPMS in biological samples, such as seafood [START_REF] Clémens | Mercury speciation in seafood using isotope dilution analysis: A review[END_REF] or human hair [START_REF] Laffont | Mercury speciation analysis in human hair by species-specific isotope-dilution using GC-ICP-MS[END_REF]. Preliminary work was carried out on human hair thus allowing an initial approach of Hg species analyses on keratin samples by evaluating different extraction methods [START_REF] Laffont | Mercury speciation analysis in human hair by species-specific isotope-dilution using GC-ICP-MS[END_REF].

Our References: [1] Thompson and Furness 1989a, [2] Kim et al. 1996, [3] Spalding et al. 2000, [4] Bond et al., 2009, [5] Vo et al. 2011, [6] Bond et al., 2015, [7] Kehrig et al. 2015, [8] [START_REF] Mallory | Mercury concentrations in feathers of marine birds in Arctic Canada[END_REF].

Experimental

Feather samples and reference materials Kerguelen Islands (49°21′S, 70°18′E) and Amsterdam Island (37°50'S, 77°31'E). Feather sampling dates of each seabird species are indicated in Table 2.4

Sample preparation and extraction procedures

Feathers were cleaned in a 2:1 chloroform:methanol solution for 5 min in an ultrasonic bath, followed by two methanol rinses to remove surface impurities, and then oven dried at 50ºC during 48h [START_REF] Carravieri | Penguins as bioindicators of mercury contamination in the Southern Ocean: birds from the Kerguelen Islands as a case study[END_REF]. They were afterwards well homogenised in order to acquire accurate analytical results avoiding within-feather variation in Hg sequestration, which could produce fluctuations in observed Hg measurement [START_REF] Hahn | Bird feathers as bioindicators in areas of the German Environmental Specimen Bank -bioaccumulation of mercury in food chains and exogenous deposition of atmospheric pollution with lead and cadmium[END_REF]. Feathers were finely cut with scissors to obtain a homogenous sample. In the particular case of king penguin (F-KP), whitechinned petrel and Antarctic prion (used for the extraction method assessment since more quantity of sample was available), feathers were cut with scissors and additionally grinded in a planetary ball mill (Retsch PM400) at 400 rpm. We noted that during homogenization with planetary ball mill, a potential contamination of iHg could occur and therefore, this homogenisation method was later discarded. The extraction was carried out in CEM Pyrex vessels by 1 min of warming up to 75ºC and 3 min at 75ºC with magnetic agitation to homogenise the samples. HB extractions were performed in closed PFA vessels (Savillex) of 50 mL at 85ºC during 2 hours in a SC100-36 Hotblock (Environmental Express, South Carolina, USA). The addition of isotopic enriched standard solutions was tested before and after the extraction process. In the case of spike addition before extraction, standard solutions were added directly to the solid sample whereas for spike addition after extraction solutions were added to the extract. All samples were extracted in triplicate.

Prior to Hg species analyses, samples were derivatized at pH 4 by ethylation using sodium tetraethylborate (NaBEt4, 5%), in order to produce volatile ethylated forms of Hg that could be separated by gas chromatography, and then extracted in isooctane by mechanical shaking using an orbital shaker during 20 min. Hg species analyses were carried out by GC-ICPMS Trace Ultra GC equipped with a Triplus RSH autosampler coupled to an ICP-MS XSeries II (Thermo Scientific, USA) as detailed in previous works [START_REF] Clémens | Mercury speciation analysis in seafood by species-specific isotope dilution : method validation and occurrence data[END_REF].

Total Hg analyses

Total Hg concentrations were quantified by using an advanced Hg analyser spectrophotometer (AMA-254, Altec). Homogenised samples (aliquots between 10 and 15 mg) were analysed after thermal destruction and gold amalgamation (drying time 60 s, decomposition time 180 s, waiting time for quantitative trapping of released mercury on the gold amalgamator 45 s). A matrix dependent calibration was performed with human hair reference material (NIES-13) by addition of different masses of sample, following EPA method 7473 (US EPA 2007). This calibration was validated by quantification of a second human hair reference material (IAEA-086), providing an accuracy of 92±5% (n=5) relative to recommended reference value. Feather THg concentrations were calculated by this calibration in order to correct matrix effects associated to keratinised samples. Several blanks were analysed at the beginning of each analytical session.

Limit of detection (LOD), calculated for blank average values (15 blanks) plus three times the standard deviation (SD) of these blanks (IUPAC), was 0.15 ng g -1 .

Quantification methods for isotopic dilution calibration

Hg species concentrations were determined by different quantification approaches in order to deeply assess analytical performances on keratin samples. Two quantification methods for isotope dilution technique were used: single-IDA and IPD. The concentrations were calculated by both methods and the transformation factors were calculated using IPD, allowing to evaluate interconversion reactions (M% and D%) that occur during both extraction and derivatisation of each analytical procedure tested. For double isotope dilution technique, the sample is spiked with known amounts of two isotope tracers (in this case 199 iHg and 201 MeHg) to alter the natural isotopic abundance of the studied endogenous species ( 202 iHg and 202 MeHg). Quantification is then based on the measurement of the mixed isotope ratios, as explained elsewhere [START_REF] Clémens | Mercury speciation in seafood using isotope dilution analysis: A review[END_REF][START_REF] Rodríguez-González | Isotope dilution analysis for elemental speciation: a tutorial review[END_REF]. Single-IDA model consists on the specific measurement of Hg species separately. Only two isotopes are considered for the quantification of each Hg species (R 202/201 for MeHg and R 202/199 for iHg). IPD takes into account all the different isotopic patterns of both spikes and endogenous species, providing the determination of possible inter-species transformations (M% and D%) and the consequent correction of concentrations [START_REF] Clémens | Mercury speciation in seafood using isotope dilution analysis: A review[END_REF]. The reported results of [THg] were calculated as the sum of [MeHg] and

Adaptation of MeHg selective extraction method (MSE)

A specific extraction technique of MeHg was applied in feather samples for the analysis of Hg speciation by ID-GC-ICPMS. The method, adapted from Uthe et al. [START_REF] Uthe | Longitudinal study of methylmercury and inorganic mercury in blood and urine of pregnant and lactating women, as well as in umbilical cord blood[END_REF], consists in a first extraction of MeHg in an organic phase (toluene) followed by a reverse extraction in aqueous phase. F-KP and feather samples from different marine bird species (Antarctic prion and white-chinned petrel) and one terrestrial species (pheasant) were used to assess the selectivity of this method, as they exhibit a wide range of Hg concentrations and different Hg species distribution. NIES-13 was also extracted in triplicate and analysed. Firstly, 0.15-0.20 g of feather samples were extracted by alkaline extraction in a HB system in Savillex vessels with sodium hydroxyde (NaOH [reagent grade], 10M, 4 mL) during 2 h at 60ºC, following Thompson et al. (Thompson and Furness 1989b). Then, 0.5 mL of extract was diluted with 4.5 mL of milliQ water in 50 mL tubes and neutralized afterwards with 0.1 mL of sulfuric acid (H2SO4, ACS Grade 95-98%). 5 mL acidic NaBr (30% w/w NaBr [Ultragrade, 99.5%] in H2SO4 4 M), 10 mL of aqueous CuSO4 (2.5% w/w [reagent grade, 99%]) and 10 mL of toluene (anhydrous, 99.8%) were added to the extracts. Samples were agitated at 420 rpm for 1 hour in an orbital shaking table. Secondly, an amount of 4 mL of toluene (MeHg passes into the organic phase) was transferred in a Falcon tube with 4 mL of sodium thiosulphate (Na2S2O3, 0.005 M [ACS grade, 98%]). Samples were vortexed during 1 min. An aliquot of sodium thiosulphate (MeHg-thiosulphate) was collected in 5 mL tubes and kept at 4ºC until analyses. The addition of isotopically enriched solutions ( 201 MeHg and 199 iHg) was tested in two different steps: 1) before NaOH extraction and 2) before specific MeHg extraction (after NaOH extraction) (Figure 2.2).

Prior to derivatisation, the addition of a solution of HCl to the MeHg-thiosulphate extract is required to reduce competition with sulphur groups during derivatisation, then different concentrations of HCl solution were tested. The concentrations of the NaBEt4 solution for ethylation were as well optimised. These tests were performed and validated for NIES-13. The best results were achieved when adding 2 mL of HCl solution (5% v/v) and 200 µL of NaBEt4 (5% v/v) to 200 µL of thiosulphate extract. The limit volume of thiosulphate extract for derivatisation is 500 µL, since a higher volume involved matrix effects.

Statistical tests for environmental feather samples

Statistical analyses were performed using XLSTAT 2016. Normality and homoscedasticity were firstly checked for the whole dataset by using Shapiro-Wilk and Breusch-Pagan tests, respectively. Since not all the results all the samples presented a normal distribution and homoscedasticity, non-parametric test was used (Kruskal-Wallis coupled to Conover-Iman procedure with Bonferroni correction). The significance level was fixed of α=0.05 for all tests.

Values are means ± SD. This result indicates that spike addition before extraction is highly recommended for hair and feather samples for correcting possible losses and/or species interconversion processes occurring also during the acid extraction step.

TMAH extracts exhibited lower sensitivity for both keratinised matrixes, which consequently induced a higher standard deviation (SD) in determination and a source of error in quantification, even if no substantial alteration of the obtained isotopic ratio was observed. This response demonstrates the occurrence of undesirable competing reactions during derivatisation when using TMAH as extraction reagent. In an acid medium, MeHg from the spike solution is less complexed with the thiol ligands of keratin. Thus, MeHg is more available for ethylation so the derivatization step will be more effective. In the opposite case, in an alkaline medium there is a stabilization of the MeHg by the complexes formed with the thiols, a minor degradation exists but, at the same time, MeHg will be less reactive and there could be interferences by other derivatized molecules (competition) [START_REF] Tseng | Rapid determination of inorganic mercury and methylmercury in biological reference materials by hydride generation, cryofocusing, atomic absorption spectrometry after open focused microwave-assisted alkaline digestion[END_REF]. Thus, when extracting keratinised samples in alkaline medium the risk of error by integration of attenuated peaks must be taken into account.

Generally, no significant differences in concentration values were observed between single-IDA and IPD quantification methods for our two reference materials. was chosen in this study to avoid the long data treatment required by IPD. Nevertheless, IPD permits the calculation of conversion reactions among Hg species and the consequent correction of concentrations related to these transformations and therefore, it is considered a more powerful approach. Indeed, IPD generally provides more accurate results than a single-IDA except under particular circumstances in which Hg species concentrations are substantially different within the same sample [START_REF] Monperrus | Evaluating the potential and limitations of double-spiking species-specific isotope dilution analysis for the accurate quantification of mercury species in different environmental matrices[END_REF]. In the rest of cases, the level of accuracy of IPD is higher, although it is less precise because the correction of species interconversion is carried out at the expense of the precision of the obtained amount of interconverting analytes [START_REF] Meija | Isotope scrambling and error magnification in multiple-spiking isotope dilution[END_REF].

In conclusion, since satisfactory results were obtained either using single-IDA or IPD in hair and feather samples, IPD remains a reference accurate method for metrology and analytical development while single-IDA can be easily used for routine monitoring analyses.

Intercomparison methods for THg concentrations quantification

Results of THg concentrations were compared for direct quantification by AMA-254 and for the sum of Hg compounds by speciation analyses (SSE) (Table 2.S1). F-KP feather sample was analysed several times in order to obtain a representative THg concentration value appeared to be much higher for SSE method for reference materials (1.26% for NIES-13 and 1.53% for F-KP) and feather samples (mean value 1.17% (0.27-2.32%)). This could mean that MSE is less precise, maybe due to its higher complexity and elevated number of analytical steps.

([THg]=3816±275 ng•g -1 , n=12
As a general trend, results for both extraction methods agree with the expected values for reference materials and matched for the rest of feathers samples (R 2 = 0.997).

MSE can be thus considered as an efficient and valid method to quantitatively extract MeHg in hair and feather samples covering a great range of Hg concentrations. However, it is important to highlight that, in this study, MeHg extracted by MSE has been measured by isotope dilution and not by external calibration, allowing to obtain more precise measurements and to correct possible losses or transformations during the whole analytical procedure and providing a more precise quantification of MeHg concentrations. Determination of Hg concentrations by external calibration, as it was performed by previously published studies on Hg speciation using MSE (Thompson and Furness, 1989a;[START_REF] Thompson | Mercury Accumulation in Great Skuas Catharacta Skua of Known Age and Sex, and Its Effects Upon Breeding and Survival[END_REF]Kim et al., 1996;Thompson et al., 1998b), could induce a lack of accuracy and precision and therefore errors in MeHg quantification.

Figure 2.3. Comparison of MeHg concentrations (ng•g-1, logarithmic representation) obtained by simultaneous species extraction (SSE) and MeHg selective extraction (MSE) methods by isotope dilution analyses with spike addition before extraction in certified reference material (NIES-13

) and feather samples for the key species studied. Trend line y = 1.0399x -0.0798 (Pearson's correlation,r=0.998,p<0.001,n=5) (Kim et al., 1996;Thompson et al., 1998b;[START_REF] Bond | Total and Methyl Mercury Concentrations in Seabird Feathers and Eggs[END_REF]. It indicates that Hg speciation in feathers is not influenced by the levels of Hg concentrations, with MeHg being the most abundant compound since it is preferentially excreted via feathers for detoxification purposes (e.g., Thompson et al., 1998b;Bearhop et al., 2000)).

0 1 2 3 4 0 1 2 3 4 ln([MeHg] SSE) (ng•g -1 ) ln([MeHg] MSE) (ng•g -1 )
Despite the predominance of MeHg in seabird feathers, the amounts of iHg appeared to be nonnegligible (reaching almost 20% of total Hg in some individuals). This result highlights the necessity of measuring both Hg compounds in feathers to better investigate Hg exposure and metabolic response of birds. [START_REF] Martin-Doimeadios | Application of Isotopically Labeled Methylmercury for Isotope Dilution Analysis of Biological Samples Using Gas Chromatography/ICMS[END_REF][START_REF] Point | Simultaneous determination of inorganic mercury, methylmercury, and total mercury concentrations in cryogenic fresh-frozen and freeze-dried biological reference materials[END_REF][START_REF] Monperrus | Evaluating the potential and limitations of double-spiking species-specific isotope dilution analysis for the accurate quantification of mercury species in different environmental matrices[END_REF][START_REF] Clémens | Mercury speciation analysis in seafood by species-specific isotope dilution : method validation and occurrence data[END_REF][START_REF] Navarro | Simultaneous determination of mercury and butyltin species using a multiple species-specific isotope dilution methodology on the European, Anguilla anguilla glass eel and yellow eel[END_REF]. Accurate and precise quantification of Hg speciation requires exigent parameters such as quite low LOQ in the order of ng L -1 , important specificity to avoid matrix effects and satisfactory repeatability and reproducibility of the analytical method. The objective of this assessment was to find the most adequate extraction method for Hg species extraction in seabird internal tissues by testing different systems and reagents. The procedures were validated for MeHg and THg concentrations with a CRM (liver and muscle) and IRM (blood).

Experimental

Samples and reference materials

Total Hg analyses

THg concentrations were quantified by using an advanced Hg analyser spectrophotometer (AMA-254, Altec). Homogenised samples (aliquots between 10 and 15 mg) were analysed in triplicate. Several blanks were analysed at the beginning of each analytical session. Limit of detection (LOD), calculated for blank average values (15 blanks) plus three times the standard deviation (SD) of these blanks (IUPAC), was 0.15 ng g -1 .

Results and discussion

Liver samples

All the extraction methods tested in liver samples provided satisfactory results for Hg concentrations except MW acid extraction, which resulted in an insufficient recovery for iHg species relative to CRM values (DOLT-4). The absence of substantial methylation factor observed by IPD results suggests that transformations between Hg compounds during the derivatisation step could not explain the uncomplete extraction for iHg by acid MW method. Two explanations are therefore possible: 1) an important methylation occurred during acid MW extraction (and could not be corrected since spike solutions were added after extraction); or 2) MW conditions were not adjusted to this kind of extraction. By contrast, acid extraction using HB provided good recoveries for both MeHg (87.8±5.5%) and THg (94.5±2.5%) so it appeared to be a valid reagent for liver sample extraction. Nevertheless, better recoveries were obtained for alkaline extraction in TMAH with both HB (MeHg: 93.4±2.8%, THg: 96.0±2.9%) and MW (MeHg: 95.4±3.4%, THg: 97.5±3.2%) extraction systems, therefore indicating the best recoveries for MW TMAH extraction. Generally, no significant differences in concentration values were observed between single-IDA and IPD calculation methods for DOLT-4 reference material. A slightly higher D% factor was observed for DOLT-4 alkaline extracts by MW (1.4±0.7%) and HB (0.9±0.2%) but did not involve a substantial difference in final Hg species concentrations (Table 2.5). As for livers, alkaline extraction appeared the most adequate method for muscle digestion.

Both HB and MW extraction techniques were suitable and valid for the complete extraction of MeHg and iHg species, however MW is a more robust and easily controlled technique so it was selected for digestion of muscles. Both single-IDA and IPD quantification methods seemed to be valid analytical approaches for data treatment and, as observed for feathers, were proposed and preferred for routine analysis (single-IDA is suggested) or analytical development (IPD). For this work, once the assessment and development of the analytical approaches was accomplished, single-IDA was applied due to practical aspects and the numerous samples to be analysed.

Blood samples

For the blood sample (RBC-KP), satisfactory recoveries were obtained for all the extraction methods and reagents relative to reference concentration value (AMA-254), indicating their suitability for blood samples digestion and also suggesting that the organic matrix of the blood cells is much more easier to digest compare to the other tissues. Slight methylation (M% ~1%)

and demethylation reactions (D%~2%) were observed during derivatization of blood samples irrespectively of the method used but no differences were found among the two quantification methods (single-IDA and IPD). Alkaline extraction was preferred because it enables that the matrix is completely dissolved so that extract recuperation is much easier than for acid extracts.

Moreover, TMAH MW extraction exhibited the most accurate results (100.4±2.3%) and was thus preferred among the rest of methods. Single-IDA was chosen since it is the most practical quantification option (Table 2.9).

Analytical performances and long-term internal reproducibility of the Hg speciation method (SSE)

After the evaluation of different extraction techniques and reagents, we concluded that:

-MW assisted extraction was the most robust and easily controlled technique so it was selected for Hg species extraction in all the studied matrixes.

-Acid extraction appeared the most suitable technique for keratinised samples (human hair and feathers) whereas alkaline extraction provided the most satisfactory results for internal tissues (livers and muscles) and blood samples.

-Both single-IDA and IPD quantification methods were considered valid analytical approaches for data treatment. Single-IDA is suggested for routine analysis or when the number of samples is substantial (due to its practical advantages), whereas IPD is recommended for analytical development.

Sensitivity, precision and accuracy of the optimised extraction methods were evaluated for the different matrixes (Table 2.10). The sensitivity was assessed by the measurement of calibration slopes calculated as the relation of the peak area obtained for 202 MeHg (counts per second, cps) divided by the concentration of MeHg injected (ng•L -1 ) for the injection in 2 µL of isooctane. Sensitivity is reported as the mean value for the 9 injections. The precision was calculated as the mean RSD of 3 injections of each extract. Accuracy was calculated by recoveries of MeHg and THg relative to certified values for CRM (NIES-13, DOLT-4 and ERM-CE-464). LOD obtained is 3.24 ng g -1 and 11.62 ng g -1 and LOQ is 9.04 ng g -1 and 30.37 ng g -1 , for MeHg and iHg, respectively. In the case of alkaline extraction for internal tissues and blood (extraction of 0.10 g of blood or internal tissue sample in 5 mL of TMAH), LOD obtained is 4.05 ng g -1 and 12.45 ng g -1 and LOQ is 11.04 ng g -1 and 28.49 ng g -1 , for MeHg and iHg, respectively (20 blanks of extraction). analyses of low concentrated samples when optimal conditions of sensitivity were achieved.

Sessions

Experimental

Optimisation of the extraction method Sample preparation of feathers, internal tissues and blood was carried out as detailed in Part 2.1. Amounts of approximately 0.2 g of feathers and 0.1 g of muscle, liver and blood samples were extracted in 5 mL of HNO3 (65%, INSTRA quality) after a predigestion step overnight at room temperature. Two different mineralization systems were tested: High Pressure Asher (HPA, Anton Paar, Austria) and HB. HPA mineralization was performed at high conditions of pressure (130 bar) and temperature (temperature ramp: 80ºC-120 ºC (2ºC/min) -300 ºC (2.5 h) -80 ºC (1 h), total time with cooling = 4.5 h). HB mineralization was effectuated in Savillex Teflon vessels at 75ºC during 8 h (6 h in HNO3 and 2 h more after the addition of 1/3 of the volume (1.66 mL) of H2O2 (30%, ULTREX quality). Tl is used because its fractionation in the instrument is quite similar to Hg and it is located in the same mass range of the analyte. The solution is introduced continuously and the 205 Tl/ 203 Tl ratio is monitored. Then the instrumental mass-bias is calculated using an exponential law as described below:

The external mass bias correction used is simply a direct consequence of the use of the delta notation against the NIST 3113 using a standard-sample bracketing sequence. This correction supposes the instrumental mass bias is the same for the sample and the NIST 3133 standard. Another secondary standard UM-Almadén (now distributed as NIST 8610) is introduced during the analysis to validate the measurements and is prepared from cinnabar ore from Almadén (Spain).

Results and discussion

Secondary standard UM-Almadén was analyzed several times during the session focused on method optimization and displayed a mean δ 202 Hg value of -0.54±0.17‰ (Mean±2SD, n = 45), coherent with certified δ 202 Hg value, permitting to validate the analytical session for extraction method optimization. Regarding the results obtained for Hg extraction in the three reference materials evaluated, both HPA and HB provided accurate results for human hair IAEA-086 and dogfish liver DOLT-4, according to previous isotopic values from the literature (Table 2.11). Therefore, both HPA and HB extractions appeared appropriate extraction methods for human hair and fish liver samples. However, in the case of fish muscle tissue (i.e., tuna fish muscle ERM-CE-464), HB extracts exhibited satisfactory ∆ 199 Hg values whereas δ 202 Hg values were lower than reference values, indicating that HB digestion did not provide a full Hg extraction, even after addition of H2O2 and supplementary heating (2h). Therefore, HPA appeared the most suitable extraction method for muscle. It is also important to highlight that the HPA extract of ERM-CE-464 required an additional heating cycle in HB (2h) with H2O2 (1.66 mL) to ensure a complete extraction.

A clear limitation of this method for Hg isotopic analyses is that HPA extractions in reference materials were not carried out in triplicate as it was performed for avian samples.

Therefore, the precision of HPA method could not be assessed. Precision of Hg isotopic values for HB triplicate extractions (evaluated as 2SD‰ for δ 202 Hg and ∆ 199 Hg, respectively) were satisfactory for IAEA-086 (0.10 and 0.02 ‰) and DOLT-4 (0.18 and 0.08 ‰) but less precise results were obtained for ERM-CE-464 (0.13 and 0.15 ‰). satisfactory for HB extraction, as it was previously observed for the CRM of tuna fish muscle.

Meanwhile, HPA provided satisfactory results for the three extractions and seemed to be the best choice for muscle digestion at this range of THg concentration (4.11-5.24 µg g -1 ) and for a mass sample digested (0.20-0.25 mg), although it also required an additional heating step with H2O2

(during 2h). In the case of southern giant petrel liver, HPA and HB extractions presented similar isotopic values, as obtained for dogfish liver CRM and both were considered suitable extraction methods for a wide range of THg concentrations (2 to 208 µg g -1 ) and 0.18-0.20 mg of digested sample. The availability of both extraction methods was also observed for king penguin blood sample (RBC-KP) for which HB and HPA provided similar mean delta values for THg concentration of 1.9 µg g -1 and 0.15-0.17 mg of digested sample.

In terms of precision (evaluated as 2SD‰), Hg extraction of king penguin feathers showed slightly more precise δ 202 Hg values for HPA (0.06 ‰) than HB (0.12 ‰), although ∆ 199 Hg values were less precise in HPA (0.12 ‰) than HB (0.05 ‰). In the case of giant petrel liver, HPA also provided more precise δ 202 Hg values (0.05 ‰) than HB (0.10 ‰), whereas ∆ 199 Hg values were less precise for HPA extraction (0.05 ‰) than HB (0.02 ‰). Precision of extraction in muscles was satisfactory for both delta values (respectively for δ 202 Hg and ∆ 199 Hg) for HPA (0.09 and 0.01 ‰) whereas quite high 2SD were obtained for HB extraction (0.23 and 0.13 ‰).Slightly more precise delta values were however observed for blood HB extraction (0.10 and 0.02%) than for HPA (0.14 and 0.11 ‰). In conclusion on the preparation before stable isotope Hg analyses:

-Both HPA and HB extractions can be used as efficient extraction methods for human hair and feathers, avian blood samples and also for the both types of liver (dogfish CRM and avian liver).

-Both methods also showed similar precision for the triplicate of extraction for feathers and blood. Thus HB was chosen due to practical reasons because it permits a higher number of samples per cycle (36 samples for HB, 5 samples for HPA) and easier sample preparation.

-In the case of avian liver, although both extraction methods are suitable, HPA provided more precise δ 202 Hg values (0.05 ‰) than HB (0.10 ‰), as 2SD‰. Therefore, although HPA implies a tedious preparation comparing to HB, it may deliver more satisfactory delta values for this type of matrix.

-In the case of fish and avian muscles, HB did not provide a complete mineralisation therefore HPA is the unique recommended method.

-Therefore, in this doctoral work, HB extraction was applied for hair, feathers and blood samples whereas HPA was preferred for livers and muscles.

Medium-term and long-term internal reproducibility

Internal reproducibility of the analytical method was performed on the medium and longterm measurements of CRM and IRM for avian samples (F-KP and RBC-KP) prepared following identical protocols and by the same operator and equipment during each analytical session. This permits the evaluation of both precision of the global method (extraction and MC-ICP-MS measurement) and instrumental precision (MC-ICP-MS) for the same solution analysed.

Extractions were performed following the specific developed method for each matrix and one extract of each reference material was also analysed several times during the whole analytical session. Long-term internal reproducibility was also determined with 1-6 months for the different analytical sessions during this doctoral work (Table 2.13).

Since there are no certified reference isotopic values for the used reference materials

Abstract

Seabirds bioaccumulate important Hg concentrations in their tissues as a direct consequence of their medium to high trophic position within marine food webs. Blood and feathers are the most targeted avian tissues for environmental biomonitoring studies, as they respectively reflect Hg short and long-term exposure and can be easily and non-destructively sampled on live individuals. Studying Hg isotopic ratios in seabird tissues can provide valuable information for avian ecology studies but it also represents a promising approach for tracking sources and pathways of Hg in marine ecosystems. Nevertheless, a good knowledge of the Hg isotopic differences between blood and feathers is required for the interpretation of the information provided by each tissue. This study presents the first comparison of Hg isotopic composition of blood and feathers, which were sampled on seabird chicks and adults from diverse breeding colonies of the Southern Ocean. Chicks of Antarctic and subantarctic skuas (n=40) and adults from six species of penguins (n=62) were chosen for this purpose. Our results indicated a strong correlation between blood and feathers Hg isotopic ratios in the case of skua chicks, with almost identical values for both tissues in all the individuals (mean difference of -0.01±0.25 ‰ (p=0.741) and -0.05±0.12 ‰ (p=0.008) for δ 202 Hg and Δ 199 Hg, respectively). Conversely, penguin adults displayed a clear divergence between tissue-specific Hg isotopic values (mean differences 0.28±0.19 ‰ (p<0.0001) and 0.25±0.13 ‰ (p<0.0001) for δ 202 Hg and Δ 199 Hg, respectively).

Different Hg integration times between tissues and dietary or foraging habitat variations were hypothesized as the main explanatory factors of such Hg isotopic mismatch between blood and feathers in adults. We concluded that both blood and feathers were valid bioindicator tissues for Hg isotopic studies and, in the case of adults, they should be chosen depending on the scientific question: feathers are useful for the investigation of long-term (i.e. annual) monitoring, whereas blood samples are more suitable for short-term (i.e. seasonal) and physiological monitoring.

Keywords: skua, penguins, bioaccumulation, marine ecosystems, isotopes

Introduction

Mercury (Hg) and more specifically methylmercury (MeHg) have been reported as highly toxic environmental pollutants with severe risks for animal and human health. The amount of Hg released into the environment has worthily increased since the pre-industrial times as a consequence of its large utilisation in human activities. Due to its persistence and biomagnification in marine food webs, high levels of Hg have been reported in high trophic level predators. Seabirds, as top predators, present elevated Hg amounts in their tissues and have been reported as efficient bioindicators of the environmental pollution (e.g. [START_REF] Burger | Marine Birds as Sentinels of Environmental Pollution[END_REF]. Since they display contrasted foraging strategies and feed at different trophic levels, they are considered appropriate models to assess Hg contamination of the marine environment.

Hg exposure in birds is essentially attributed to dietary uptake (especially MeHg) which appears to be easily uptaken and distributed by the blood stream to internal organs and tissues (Thompson and Furness, 1989b;[START_REF] Braune | Comparison of Total Mercury Levels in Relation to Diet and Molt for Nine Species of Marine Birds[END_REF][START_REF] Hoefs | Seasonal changes in mercury accumulation in the black-eared kite, Milvus migrans lineatus[END_REF]. Seabirds present a considerable opportunity of detoxification by their capacity to efficiently excrete environmental contaminants through moulting feathers (annually in the case of most species). Thus, moulting represents the main detoxification route for Hg in most seabird species [START_REF] Furness | Using bird feathers to measure mercury in the environment: Relationships between mercury content and moult[END_REF].

Indeed, Hg is assumed to be sequestered in feathers binding to the keratin molecules [START_REF] Appelquist | Mercury monitoring: Mercury stability in bird feathers[END_REF], which impedes its reincorporation into internal tissues. Between 70 and 90% of the whole Hg body burden is assumed to be remobilised from internal tissues and excreted into the growing feathers (Honda, Nasu, and Tatsukawa 1986), where the main part of Hg is known to be present under its organic form (more than 90% of THg as MeHg) (Thompson and Furness 1989b;[START_REF] Thompson | Using seabirds to monitor mercury in marine environments. The validity of conversion ratios for tissue comparisons[END_REF][START_REF] Renedo | Assessment of mercury speciation in feathers using species-specific isotope dilution analysis[END_REF].

In seabirds, blood and feathers are the most frequently used tissues for biomonitoring studies because they do not involve lethal-sampling. However, each tissue presents a specific Hg turnover and they potentially provide access to different temporal scales of Hg exposure. Feathers are representative of Hg incorporation during the inter-moult periods and are metabolically inert after their synthesis. In contrast, blood is a metabolically active tissue representative of a shorter-term Hg intake than feathers, meaning a few weeks/months before sampling (Bearhop et al., 2000b).

Therefore, tissue-specific integration times must be considered for the selection of the most appropriate avian tissue depending on the scientific purposes (Carravieri al., 2014a). Since chick moult occurs at the end of the chick-rearing period, both blood and simultaneously-growing feathers reflect recent Hg intake over a similar period (Carravieri al., 2014a). Consequently, sampling chick tissues could allow reducing the temporal mismatch of Hg integration between feathers and blood.

In the last decades, the measurement of Hg isotopic mass-dependent (MDF) and massindependent fractionation (MIF) has become a documented tool for identifying sources of Hg and biogeochemical processes within the different compartments of the environment [START_REF] Blum | Mercury Isotopes in Earth and Environmental Sciences[END_REF]. MDF can occur during all Hg specific transformation processes such as volatilization [START_REF] Zheng | Mercury isotope fractionation during volatilization of Hg(0) from solution into the gas phase[END_REF], reduction [START_REF] Kritee | Mercury Stable Isotope Fractionation during Reduction of Hg(II) to Hg(0) by Mercury Resistant Microorganisms[END_REF], bacterial methylation or demethylation reactions (Rodriguez [START_REF] Gonzalez | Species-Specific Stable Isotope Fractionation of Mercury during Hg ( II ) Methylation by an Anaerobic Bacteria ( Desulfobulbus propionicus ) under Dark Conditions[END_REF][START_REF] Kritee | Mass dependent stable isotope fractionation of mercury during mer mediated microbial degradation of monomethylmercury[END_REF][START_REF] Perrot | Successive methylation and demethylation of methylated mercury species (MeHg and DMeHg) induce mass dependent fractionation of mercury isotopes[END_REF][START_REF] Perrot | Identical Hg Isotope Mass Dependent Fractionation Signature during Methylation by Sulfate-Reducing Bacteria in Sulfate and Sulfate-Free Environment[END_REF] and metabolic processes [START_REF] Feng | Specific Pathways of Dietary Methylmercury and Inorganic Mercury Determined by Mercury Speciation and Isotopic Composition in Zebrafish ( Danio rerio )[END_REF][START_REF] Perrot | Natural Hg isotopic composition of different Hg compounds in mammal tissues as a proxy for in vivo breakdown of toxic methylmercury[END_REF]. Significant Hg MIF is known to be induced exclusively during photochemical reactions and mainly concerns the two odd isotopes (199 and 201) [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF]Zheng and Hintelmann 2010a). Unlike MDF, MIF is thus not affected by biological processes [START_REF] Kritee | Mercury Stable Isotope Fractionation during Reduction of Hg(II) to Hg(0) by Mercury Resistant Microorganisms[END_REF][START_REF] Kritee | Mass dependent stable isotope fractionation of mercury during mer mediated microbial degradation of monomethylmercury[END_REF][START_REF] Perrot | Identical Hg Isotope Mass Dependent Fractionation Signature during Methylation by Sulfate-Reducing Bacteria in Sulfate and Sulfate-Free Environment[END_REF] and its signature is thus assumed to be conserved throughout the food web (Kwon et al. 2012b;[START_REF] Feng | Specific Pathways of Dietary Methylmercury and Inorganic Mercury Determined by Mercury Speciation and Isotopic Composition in Zebrafish ( Danio rerio )[END_REF]. Therefore, both MDF and MIF provide complementary information and are used as a double tracers for both Hg potential sources and transformation pathways.

Numerous studies have exploited and demonstrated the usefulness of Hg isotopes for tracing Hg exposure and sources in aquatic organisms [START_REF] Li | Environmental Origins of Methylmercury Accumulated in Subarctic Estuarine Fish Indicated by Mercury Stable Isotopes[END_REF][START_REF] Perrot | Higher massindependent isotope fractionation of methylmercury in the pelagic food web of Lake Baikal (Russia)[END_REF][START_REF] Jackson | Evidence for mass-independent and massdependent fractionation of the stable isotopes of mercury by natural processes in aquatic ecosystems[END_REF][START_REF] Gehrke | Sources of mercury to San Francisco Bay surface sediment as revealed by mercury stable isotopes[END_REF][START_REF] Senn | Stable isotope (N, C, Hg) study of methylmercury sources and trophic transfer in the northern gulf of Mexico[END_REF]. Hg isotopic signatures have also been successfully used in ecotoxicology studies for a better understanding of detoxification and excretion processes and Hg metabolic responses, such as hepatic demethylation in marine mammals [START_REF] Masbou | Hg Stable Isotope Time Trend in Ringed Seals Registers Decreasing Sea Ice Cover in the Alaskan Arctic[END_REF][START_REF] Perrot | Natural Hg isotopic composition of different Hg compounds in mammal tissues as a proxy for in vivo breakdown of toxic methylmercury[END_REF] or human exposure by excretion biomarkers such as urine (Sherman et al., 2013) and hairs [START_REF] Laffont | Anomalous mercury isotopic compositions of fish and human hair in the Bolivian amazon[END_REF]. Concerning avian samples, two studies have revealed the efficiency of Hg isotopic analyses in seabird eggs for investigating factors controlling Hg cycling in aquatic ecosystems [START_REF] Point | Methylmercury photodegradation influenced by sea-ice cover in Arctic marine ecosystems[END_REF][START_REF] Day | Mercury stable isotopes in seabird eggs reflect a gradient from terrestrial geogenic to oceanic mercury reservoirs[END_REF].

In this work, we present the first data on Hg isotopic composition of blood and feathers of the same individual seabirds with the double objective of investigating Hg isotopic association between both tissues and of evaluating their suitability as tracers of Hg fate in marine ecosystems.

This study was performed on different seabird species exhibiting contrasted ecological characteristics and breeding in distant sites in order to cover a wide range of Hg concentrations.

Assuming that Hg integrated in chick blood and feathers correspond to a similar time frame, we first focused on chicks for exploring inter-tissue isotopic relationships and the allocation of Hg following transport and fractioning from blood to feathers. Potential biologic processes or transport among the two tissues were hypothesized to induce Hg MDF, leading to differences in Hg MDF values between blood and feathers. Because the temporal mismatch between chick tissues is supposed to be reduced and Hg MIF is not induced by in vivo processes, Hg MIF values in blood and feathers of chicks were predicted to be similar. Chicks of two very close species of skua were chosen to test these hypotheses. Complementary to this, we investigated feathers and blood samples of adult seabirds to verify the potential influence of different tissue-specific temporal windows of Hg exposure on Hg isotopic signatures. We expected that shifts on foraging habits and zones during adult annual cycle could produce significant Hg isotopic variations among tissues. Penguins from six different species, all belonging to different populations, were selected to confirm this hypothesis.

Material and methods

Sites, field collection and ecological characteristics of sampled seabirds Reference materials, sample preparation and total Hg analysis Due to the absence of feather and bird blood certified reference materials (CRM), two internal reference samples were prepared with pooled samples collected from different individuals of king penguin (KP) from Crozet Islands: F-KP (feathers) and RBC-KP (red blood cells). The two reference samples were analysed at each session. For the validation of the results, human hair CRM (IAEA-086) was additionally analysed due to its similar chemical composition to feathers (keratin). Feathers samples were cleaned, oven dried and homogenised as detailed in [START_REF] Carravieri | Penguins as bioindicators of mercury contamination in the Southern Ocean: birds from the Kerguelen Islands as a case study[END_REF]). Blood samples were collected from a wing vein, centrifuged, and red blood cells were kept frozen at -20°C until analysis. Total Hg concentration was also quantified by using an advanced Hg analyser spectrophotometer (AMA-254, Altec) thus allowing intercomparing with Hg total concentrations obtained by Hg speciation analyses, meaning the sum of inorganic and organic Hg. For feather analyses, a matrix dependent calibration was performed with human hair reference material (NIES-13) as described in [START_REF] Renedo | Assessment of mercury speciation in feathers using species-specific isotope dilution analysis[END_REF]. Blood samples analyses were performed as described in [START_REF] Carravieri | From Antarctica to the subtropics : Contrasted geographical concentrations of selenium , mercury , and persistent organic pollutants in skua chicks ( Catharacta spp .)[END_REF].

Hg species concentrations analysis

Feathers were prepared following a previously developed method [START_REF] Renedo | Assessment of mercury speciation in feathers using species-specific isotope dilution analysis[END_REF]. Hg from blood samples (0.10-0.15 g) was extracted by alkaline microwave digestion with 5 mL of tetramethylammonium hydroxide (25% TMAH in H2O, Sigma Aldrich) [START_REF] Rodrigues | Mercury speciation in whole blood by gas chromatography coupled to ICP-MS with a fast microwave-assisted sample preparation procedure[END_REF].

Details of the extraction method, analysis and quantification of Hg species are included in [START_REF] Renedo | Assessment of mercury speciation in feathers using species-specific isotope dilution analysis[END_REF].

Total Hg isotopic composition analysis Samples (0.05-0.10 g) were digested with 3 or 5 mL of HNO3 acid (65%, INSTRA quality) after a predigestion step overnight at room temperature. Two different mineralisation systems were successfully tested and used: High Pressure Asher (HPA) and Hotblock. HPA mineralisation was performed at high conditions of pressure (130 bar) and temperature (temperature ramp: 80ºC-120 ºC (2ºC/min) -300 ºC (2.5 h) -80 ºC (1 h)). Hotblock mineralisation was effectuated in Savillex vessels at 75ºC during 8 h (6 h in HNO3 and 2 h more after the addition of 1/3 of the total volume of H2O2 (30%, ULTREX quality)). Hg isotopic composition was determined using coldvapour generator (CVG)-MC-ICPMS (Nu Instruments), as detailed in [START_REF] Perrot | Higher massindependent isotope fractionation of methylmercury in the pelagic food web of Lake Baikal (Russia)[END_REF]. Hg isotopic values were reported as delta notation, calculated relative to the bracketing standard NIST SRM-3133 reference material to allow inter-laboratory comparisons, as described in SI. NIST SRM-997 thallium standard solution was used for the instrumental mass-bias correction using the exponential law.

Recoveries of extraction were verified for all samples by checking the signal intensity obtained on the MC-ICPMS for diluted extracts relative to NIST 3133 standard (with an approximate uncertainty of +/-15%). Total Hg concentrations in the extract solution were compared to the concentrations found by AMA-254 analyses. Average recoveries obtained were 98 ±14% for feathers (n=104) and 111 ±11% for blood samples (n=102). Accuracy was evaluated by analyses of human hair IAEA-086 and NIES-13 reference materials for keratin matrixes and tuna fish ERM-CE-464 for soft-tissues. Internal reference samples of feathers (F-KP) and avian blood (RBC-KP) were also measured. Repeatability was estimated by analysing the same extract of each reference material on the long term (during 2 weeks of analysis). Uncertainty for delta values was calculated using 2SD typical errors for each internal reference material (Table 3.S2). Internal reproducibility was also assessed for numerous measurements of the two internal reference samples of feathers (F-KP) and avian blood (RBC-KP) within 2-10 months of interval (Table 3.S3).

Results

Skua chicks

Mean skua chicks' δ 202 Hg values of feathers (blood) ranged widely between populations, varying from 0.00±0.36 ‰ (0.23±0.13 ‰) in Adélie Land to 1.56±0.08 ‰ (1.51±0.20 ‰) in Amsterdam Island. Lower variability was found in Δ 199 Hg between the four sites, with mean values of feathers (blood) ranging from 1.46±0.07 ‰ (1.51±0.08 ‰) in Adélie Land to 1.76±0.05 ‰ (1.70±0.05 ‰) in Amsterdam Island (Table 3.1).

Individual paired differences of δ 202 Hg and Δ 199 Hg (feathers minus blood) were respectively -0.01±0.25‰ and -0.05±0.12‰, when pooling all the individuals. In general, insignificant differences in tissue-specific Hg isotopic ratios were found, with the highest δ 202 Hg difference obtained for Adélie Land (-0.16±0.33‰) and Kerguelen (0.19±0.18‰) and the highest Δ 199 Hg difference for the Crozet population (-0.06±0.17‰). No statistically differences between feathers and blood δ 202 Hg values were observed for the 40 individuals of skua. However, when evaluating each skua population separately, only Kerguelen individuals presented significant differences between feather and blood δ 202 Hg values, probably associated to high SD of tissues-paired differences (δ 202 Hg difference = 0.19±0.18‰). Δ 199 Hg mean paired differences observed in skua chicks were statistically significant for the overall data set. Paired differences in Amsterdam individuals were marginally significant (Table 3.1).

Linear regressions between blood and feather Hg isotopic values were determined for all the individuals (Figure 3 Significantly different Hg isotopic signatures (feathers minus blood) were observed among penguin tissues. Feathers displayed heavier δ 202 Hg and Δ 199 Hg values than blood in almost all the individuals (except some individuals of gentoo penguins). Respective mean paired differences for δ 202 Hg and Δ 199 Hg were 0.28±0.19 ‰ and 0.25±0.13 ‰, when considering the 62 individuals.

Gentoo penguins showed much lower mean paired differences -0.02±0.12 ‰ (for δ 202 Hg) and 0.10±0.12 ‰ (for Δ 199 Hg) comparing to other penguin species. King penguins displayed the highest δ 202 Hg difference (0.45±0.19‰) and northern rockhopper penguins the highest Δ 199 Hg difference (0.33±0.08‰). Paired differences were statistically significant for both δ 202 Hg values and Δ 199 Hg values considering the six populations. Regarding each population separately, feather and blood δ 202 Hg values were statistically similar only for Adélie and gentoo penguins. However, differences of Δ 199 Hg values between the two tissues were substantial for every penguin population.

A high linear correlation among tissues was obtained for both δ 202 Hg (Pearson correlation, R 2 =0.93 p<0.0001, n=62) and ∆ 199 Hg values (Pearson correlation, R 2 =0.87, p<0.0001, n=62) in all the penguin individuals (Figure 3.2). Regressions slopes were not statistically different from slope 1:1 neither for δ 202 Hg (t=-22.01, p<0.0001) nor for ∆ 199 Hg values (t=-14.41, p<0.0001).

Discussion

The information about Hg speciation in each tissue is essential for the interpretation of total Hg isotopic signatures. Feather and blood samples analysed in this study presented a vast majority of MeHg content (data already published [START_REF] Renedo | Assessment of mercury speciation in feathers using species-specific isotope dilution analysis[END_REF]). This observation is consistent with previous reported results indicating that MeHg accumulates in seabirds' feathers as a detoxification mechanism, where it typically comprises the most abundant proportion of total Hg (Thompson and Furness 1989a;[START_REF] Renedo | Assessment of mercury speciation in feathers using species-specific isotope dilution analysis[END_REF] and reporting blood as a Hg transport tissue within the organism (Bearhop, Ruxton, and Furness 2000b).

Chicks: identical Hg isotopic composition in feathers and blood Skua chicks exhibited identical Hg MIF (∆ 199 Hg) and MDF (δ 202 Hg) values in blood and feathers, suggesting that both tissues reflect equally Hg acquisition during a similar temporal window. This finding represents substantial practical advantages for avian ecology studies, firstly because during fieldwork the sampling can be done indifferently on feathers or blood of chicks;

and secondly, and more importantly, because blood and feathers of all the individuals can be compared in interpretative terms directly without applying corrections, as it must be done for carbon and nitrogen stable isotopes [START_REF] Cherel | Differences in δ13C and δ15N values between feathers and blood of seabird chicks: Implications for non-invasive isotopic investigations[END_REF]).

Figure 3.1. Feather versus blood (red blood cells) δ 202 Hg and Δ 199 Hg values in skua chicks (individual values and mean population values). Regression equations for δ 202

Hg blood-feather and Δ 199 Hg blood-feather values are y=1.12x-0.17 (R 2 =0.81, p<0.0001), and y=0.63x-0.56 (R 2 =0.33, p<0.0001), respectively. Abbreviations for the different populations are AL (Adélie Land), Ker (Kerguelen), Cro (Crozet) and Ams (Amsterdam).

Differences in Hg isotopic differences between feathers and blood of adult penguins

Compared to skua chicks, penguin adults generally displayed a higher degree of variations between tissue-specific Hg isotopic values, with feathers showing slightly heavier δ 202 Hg and Δ 199 Hg values. Potential explanatory factors of those inter-tissues isotopic differences are discussed in the following sections.

Differences between feathers and blood: metabolic origin Metabolic processes are complex and constituted by several steps that potentially induce MDF between internal tissues. Intrinsic processes such as inter-tissue transport or Hg complexation to proteins could therefore induce significant MDF between blood and feathers. The similar Hg isotopic signatures observed between blood and feathers of skua chicks could be interpreted as an absence of Hg isotopic fractionation during internal mechanisms in chick tissues. However, we cannot ensure an absence of Hg isotopic fractionation between blood and feathers in chicks because this effect could be linked to specific conditions of their young age such as a low Hg bioaccumulation in which no limiting-step is responsible of significant MDF during the different metabolic steps. However, penguins exhibited heterogeneous blood and feather paired differences between the six penguin populations, with some species exhibiting similar values for both samples (primarily gentoo penguins) while other species displayed high discordance between tissuespecific isotopic signatures. Since blood and feather paired isotopic differences do not follow a similar pattern within all the penguins' populations, intrinsic metabolic processes do not seem the major cause of MDF. Therefore, the influence of extrinsic factors related to the specific ecological characteristics for each penguin species (such as geographical and temporal mismatch among tissues) seems to be a more plausible explanatory factor of Hg isotopic variations within adults' blood and feathers. Furthermore, this is consistent with MIF differences between blood and feathers, which are not likely linked to metabolic processes.

Geographical and ecological influence

The accumulation of a greater extent of heavier (δ 202 Hg) and odd (Δ 199 Hg) Hg isotopes in adult' feathers relative to blood could be likely the consequence of significant shifts in feeding ecology (foraging habitat and prey) that take place during the different stages of their annual cycle. Since feathers of adult penguins are assumed to reflect Hg acquisition during approximatively one complete year, we expected that significant changes in foraging habitats during this time would alter feather Hg isotopic signatures as a result of integration of Hg from diverse sources. This integrative effect could explain the dissimilarities obtained here between gentoo penguins, considered to be resident all year long [START_REF] Lescroël | Spatial and temporal variation in the diet of the gentoo penguin (Pygoscelis papua) at Kerguelen Islands[END_REF], and the other five penguin populations, known to spread out during winter. Gentoo penguins displayed limited isotopic differences among tissues (-0.02±0.12 ‰ for δ 202 Hg and 0.10±0.12 ‰ for Δ 199 Hg), whereas the other penguins provided statistically different tissue-specific isotopic values. Based on our hypothesis, feathers of gentoo penguins would hypothetically reflect Hg exposure from more restricted areas diminishing the Hg isotopic differences between blood and feathers.

Owing to their flightlessness, adult penguins are known to be restricted to the Southern Ocean during their wintery dispersion. In contrast, flying seabirds reproducing in this area are able to cover long distances during their non-breeding period at the time of feather growth, and therefore, Hg accumulated into their feathers represents the acquisition of remote and extended areas [START_REF] Fort | Spatial Ecotoxicology: Migratory Arctic Seabirds Are Exposed to Mercury Contamination While Overwintering in the Northwest Atlantic[END_REF]. Therefore, the higher shifts in latitudinal zones or depths that are produced during the different stages of the annual cycle (inter-moult and breeding periods) could potentially enhance Hg isotopic differences between blood and feathers tissues (Chapter 3.

. Feather versus blood (red blood cells) δ 202 Hg and Δ 199 Hg values in adult penguins (individual values and mean population values). Regression equations for δ 202

Hg blood-feather and Δ 199 Hg blood-feather values are y=1.08x+0.13 (R 2 =0.86, p<0.0001) and y=1.18x-0.04 (R 2 =0.75, p<0.0001), respectively. Abbreviations for the different populations are AP (Adélie penguin), GP (gentoo penguin), KP (king penguin), MP (macaroni penguin), RP (eastern rockhopper penguin) and NRP (northern rockhopper penguin).

Influence of different integration times

The mismatch of Hg isotopic signatures between blood and feathers of adults could also be the result of the different Hg integration times among both tissues. Adults' feathers sequestrate the Hg acquired between two moults (approximatively 12 months in our seabird species), presenting a much longer Hg accumulation interval compared to blood. In contrast, chicks start synthetizing their first feathers at the second part of their rearing-period (which is about 2 months of total duration in the case of skuas) and therefore the two tissues should represent similar periods of Hg dietary intake reducing this mismatch.

Contrary to other adult flying seabirds, penguins present a synchronous moult so they renew their whole plumage entirely in a single venue, resulting in high homogeneity in feathers in terms of chemical composition, including THg [START_REF] Brasso | Unique pattern of molt leads to low intraindividual variation in feather mercury concentrations in penguins[END_REF]Carravieri, et al., 2014a). The discordance in Hg isotopic composition between feathers and blood observed in adult penguins at the individual level supports the expected influence of longer Hg integration times in feathers.

Our results suggest that Hg isotopic studies on recent contamination or physiological monitoring should be preferentially applied on adult blood samples instead of feathers. Working on adult seabirds' feathers is however suitable for tracking whole annual Hg contamination.

Choice of the most adequate monitoring sample

For the selection of the most pertinent avian sample for Hg isotopic studies, it is important to consider the advantages and limitations of each tissue. According to practical issues, feathers permit an easy collection, storage and long-term conservation which is clearly advantageous for retrospective studies on environmental pollution and make them a relevant tissue for comparison between bird biomonitoring studies. Otherwise, blood requires a more invasive sample collection compared to feathers and it is more problematic to collect, preserve and store in the field. Apart practical considerations, the use of adult feathers present several constraints such as the potentially higher degree of heterogeneity within individuals (except penguins, see above), the asynchronous growth occurring in some species (Carravieri et al., 2014a) and the reflect of year round exposure for adults (especially in flying adults that migrate over large distances and change their diet). Hence, chick feathers should be preferred for local and recent contamination whereas adult feathers could be interesting for investigating Hg contamination over their whole annual cycle. However blood is more appropriate if we want information about recent assimilation or local contamination in adult individuals sampled at the colonies during their breeding period.

Conclusion

This work presents the first measurements of Hg isotopic composition on seabirds' blood and feathers of the same individuals and validates the efficient utilisation of both as effective key target tissues for tracing local Hg isotopic signatures. Briefly, feathers are interesting tissues for the investigation of historical contamination trends and annual monitoring whereas blood samples represent ecosystem-specific tissues more suitable for seasonal and physiological monitoring. Our study also highlights the great potential of combining Hg isotopes studies and seabirds for depicting Hg routes in marine ecosystems and a better understanding of the Hg cycle.

Main findings of this work -Blood and feather Hg stable isotopes were measured in skua and penguin populations from distant sites in the Southern Ocean, from the fringing subtropics to Antarctica.

-In adult birds, Hg isotopic composition differed between blood and feathers, most likely as a consequence of different tissue-specific temporal windows of Hg exposure.

-In chicks, blood and feathers had identical Hg isotopic values meaning that the two tissues carry the same amount of information and can be easily compared with each other without any correction factor.

-Hg isotopic studies in blood and feathers open a new perspective for exploring Hg cycle and exposure pathways within marine ecosystems.

Introduction

Hg presents several adverse effects which involves serious consequences for the health of avian species, affecting particularly their behaviour, physiology and development [START_REF] Evers | Adverse effects from environmental mercury loads on breeding common loons[END_REF][START_REF] Tan | The endocrine effects of mercury in humans and wildlife[END_REF]) with consequences at the population level (Goutte, et al., 2014ab). Hg intake in seabirds occurs mainly by ingestion of prey containing MeHg, which is readily absorbed into the blood and transported to internal tissues, especially liver, kidney, brain and muscles by the blood stream [START_REF] Wolfe | Effects of mercury on wildlife: A comprehensive review[END_REF]. Dietary MeHg is also efficiently transferred to avian eggs and therefore, reproduction is consequently one of the most affected aspects of Hg toxicity in seabirds [START_REF] Wolfe | Effects of mercury on wildlife: A comprehensive review[END_REF]. The main route for Hg elimination in seabirds is excretion by moulting feathers (Thompson and Furness 1989b;[START_REF] Thompson | Using seabirds to monitor mercury in marine environments. The validity of conversion ratios for tissue comparisons[END_REF], where Hg (predominantly under its MeHg form, [START_REF] Renedo | Assessment of mercury speciation in feathers using species-specific isotope dilution analysis[END_REF] is sequestered in relatively high concentrations. These feather Hg concentrations are correlated with blood Hg levels at the time of feather growth (Bearhop et al., 2000). Feathers are thus considered as archives of Hg exposure since the previous feather synthesis and they reflect both site-specific dietary uptake of MeHg and the stored then remobilised body burden Hg [START_REF] Monteiro | Mercury concentrations in prey fish indicate enhanced bioaccumulation in mesopelagic environments[END_REF]. During seasonal moult, Hg accumulated in muscles is remobilised into growing feathers, and the Hg accumulated in the organism is gradually reduced [START_REF] Braune | Comparison of Total Mercury Levels in Relation to Diet and Molt for Nine Species of Marine Birds[END_REF][START_REF] Furness | Using bird feathers to measure mercury in the environment: Relationships between mercury content and moult[END_REF]. Hg dynamic in seabirds are consequently complex and highly variable between species, since detoxification rates and strategies are potentially influenced by their degree of exposure to Hg, their moult frequency and the efficiency of Hg excretion by moulting (Bearhop et al. 2000).

Metallothioneins are cysteine-rich proteins with high affinity to Hg (and other trace elements, such as Cd or Zn) that have been suggested to play an important role in Hg detoxification. These proteins have been detected in the tissues of seabirds [START_REF] Elliott | Heavy metal and metallothionein concentrations in seabirds from the Pacific coast of Canada[END_REF][START_REF] Elliott | Heavy metal and metallothionein concentrations in Atlantic Canadian seabirds[END_REF][START_REF] Andrews | Avian Metallothioneins : Structure , regulation and evolution[END_REF] and their amount is proportional to iHg concentrations in the liver [START_REF] Kehrig | Trace-elements, methylmercury and metallothionein levels in Magellanic penguin (Spheniscus magellanicus) found stranded on the Southern Brazilian coast[END_REF]. To date, the binding Hg-metallothioneins has only been identified in dolphin livers (Pedrero et al., 2012b). Additionally, Hg has also been found to accumulate significantly as an inert Hg-Se complex called tiemannite in the liver of marine mammals [START_REF] Palmisano | Speciation of mercury in dolphin liver: A two-stage mechanism for the demethylation accumulation process and role of selenium[END_REF][START_REF] Nakazawa | The presence of mercury selenide in various tissues of the striped dolphin: evidence from l-XRF-XRD and XAFS analyseswz[END_REF] and seabirds [START_REF] Arai | Chemical forms of mercury and cadmium accumulated in marine mammals and seabirds as determined by XAFS analysis[END_REF][START_REF] Ikemoto | Environmental Contamination and Detoxification Mechanism of Heavy Metals in Marine Mammals and Seabirds : Interaction of Selenium with Mercury , Silver , Copper , Zinc , and Cadmium in Liver[END_REF], thus reducing its bioavailability and impeding its remobilisation (Cuvin-Aralar and Furness, 1991).

Demethylation of MeHg into less toxic iHg in the liver appears to be a significant detoxification mechanism in seabirds (Thompson and Furness 1989b;Thompson et al., 1993b) and is supposed to be a complementary process to Hg excretion by feathers (Muirhead and Furness, 1988;[START_REF] Nigro | Intracellular storage of mercury and selenium in different marine vertebrates[END_REF]. Consequently, seabird species that present both a Hg-rich diet and a complex moulting pattern associate excretion of MeHg into feathers with a high capacity of hepatic demethylation (Kim et al., 1996). This overall process appears to be especially significant in Procellariiforms, such as albatrosses and petrels [START_REF] Wolfe | Effects of mercury on wildlife: A comprehensive review[END_REF]. Understanding Hg bioaccumulation, inter-tissue distribution and detoxification or excretion processes in seabirds represents a great challenge. Numerous studies have investigated Hg levels and speciation in internal tissues in the last decades focusing on the toxicokinetics [START_REF] Monteiro | Kinetics, dose-response, and excretion of methylmercury in freeliving adult Cory's shearwaters[END_REF][START_REF] Spalding | Methylmercury accumulation in tissues and its effects on growth and appetite in captive great egrets[END_REF] or on their use as bioindicators of Hg contamination [START_REF] Norheim | The Degree of Methylation and Organ Distribution of Mercury in Some Birds of Prey in Norway[END_REF][START_REF] Dietz | Organic mercury in Greenland birds and mammals[END_REF]Kim et al., 1996;[START_REF] Kehrig | Trace-elements, methylmercury and metallothionein levels in Magellanic penguin (Spheniscus magellanicus) found stranded on the Southern Brazilian coast[END_REF]. Here, we measured for the first time Hg isotopic composition in several avian tissues from the same individuals to investigate Hg biotransformation processes within the whole body. Since each tissue presents a different metabolic turnover, they are supposed to provide variable Hg speciation and Hg isotopic signatures, which both combined, can provide some information about the occurring biological processes and Hg fate in seabird tissues.

Hg isotopes can undergo mass-dependent and mass-independent isotope fractionation (MDF and MIF, respectively). MDF is known to affect all Hg isotopes and is induced during transformations such as methylation and demethylation (Rodriguez [START_REF] Gonzalez | Species-Specific Stable Isotope Fractionation of Mercury during Hg ( II ) Methylation by an Anaerobic Bacteria ( Desulfobulbus propionicus ) under Dark Conditions[END_REF][START_REF] Kritee | Mass dependent stable isotope fractionation of mercury during mer mediated microbial degradation of monomethylmercury[END_REF][START_REF] Perrot | Successive methylation and demethylation of methylated mercury species (MeHg and DMeHg) induce mass dependent fractionation of mercury isotopes[END_REF][START_REF] Perrot | Identical Hg Isotope Mass Dependent Fractionation Signature during Methylation by Sulfate-Reducing Bacteria in Sulfate and Sulfate-Free Environment[END_REF], reduction [START_REF] Kritee | Mercury Stable Isotope Fractionation during Reduction of Hg(II) to Hg(0) by Mercury Resistant Microorganisms[END_REF]) and volatilization [START_REF] Zheng | Mercury isotope fractionation during volatilization of Hg(0) from solution into the gas phase[END_REF]. Numerous studies have evidenced Hg MDF during trophic transfer leading to accumulation of isotopically heavier MeHg in predators relative to their prey [START_REF] Perrot | Tracing sources and bioaccumulation of mercury in fish of Lake Baikal--Angara River using Hg isotopic composition[END_REF][START_REF] Senn | Stable isotope (N, C, Hg) study of methylmercury sources and trophic transfer in the northern gulf of Mexico[END_REF][START_REF] Li | Assessing sources of human methylmercury exposure using stable mercury isotopes[END_REF]. Therefore, in vivo processes such as Hg species-specific accumulation in target organs, inter-tissue transport or Hg detoxification mechanisms (such as demethylation in the liver) involve Hg MDF, which has been widely used as a proxy of these processes in aquatic organisms such as fish [START_REF] Feng | Specific Pathways of Dietary Methylmercury and Inorganic Mercury Determined by Mercury Speciation and Isotopic Composition in Zebrafish ( Danio rerio )[END_REF]Kwon et al., 2012a;[START_REF] Zhu | Methylmercury in fish from the South China Sea: geographical distribution and biomagnification[END_REF] and marine mammals [START_REF] Masbou | Hg Stable Isotope Time Trend in Ringed Seals Registers Decreasing Sea Ice Cover in the Alaskan Arctic[END_REF][START_REF] Perrot | Natural Hg isotopic composition of different Hg compounds in mammal tissues as a proxy for in vivo breakdown of toxic methylmercury[END_REF]. In contrast, Hg MIF seems not to be induced by biological processes [START_REF] Kritee | Mercury Stable Isotope Fractionation during Reduction of Hg(II) to Hg(0) by Mercury Resistant Microorganisms[END_REF][START_REF] Kritee | Mass dependent stable isotope fractionation of mercury during mer mediated microbial degradation of monomethylmercury[END_REF][START_REF] Feng | Specific Pathways of Dietary Methylmercury and Inorganic Mercury Determined by Mercury Speciation and Isotopic Composition in Zebrafish ( Danio rerio )[END_REF]; hence, Hg MIF is assumed to not vary within food webs. Significant Hg MIF variations are driven by photochemical reactions, the only mechanism known to date [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF]Zheng and Hintelmann 2010a). The interest of this study is the combination of Hg speciation and Hg isotopic composition analyses of avian internal tissues (pectoral muscle, liver, brain, kidney and blood) and feather samples to i) contribute to the elucidation of Hg transformations and metabolic fate of Hg among seabird tissues and ii) evaluate the level of information on Hg contamination provided by the most targeted non-lethal samples: feathers and blood. We hypothesize that hepatic demethylation of ingested MeHg may induce a Hg MDF between tissues, resulting in the accumulation of lighter isotopes in the produced iHg in liver and heavier isotopic composition in the remaining MeHg with its potential remobilisation into feathers during moult.

Due to the assumed absence of Hg MIF during in vivo processes, no anomalous Hg fractionation was expected between internal tissues. However, the different integration times and turnover of Hg between feathers and internal tissues was presumed to potentially influence Hg MIF variations between internal tissues (muscle and liver) and feathers, as previously observed between feathers and blood in adult individuals (Chapter 3.1).

Material and methods

Description of ecological characteristics of the studied seabirds

The Antarctic prion (Pachyptila desolata) is a small petrel that moves far from the colonies between breeding periods (mainly to subtropical waters) and returns to breed in the Southern Ocean [START_REF] Cherel | Combination of At-Sea Activity , Geolocation and Feather Stable Isotopes Documents Where and When Seabirds Molt[END_REF]. Antarctic prions obtain most of their food near the surface. They are opportunistic epipelagic foragers, feeding on the most available swarming crustaceans, mainly the hyperiid amphipod Themisto gaudichaudii and the subantarctic krill Euphausia vallentini [START_REF] Cherel | Food and feeding ecology of the sympatric thinbilled Pachyptila belcheri and Antarctic P . desolata prions at Iles Kerguelen[END_REF]. In Kerguelen Islands, they forage from subantarctic to Antarctic waters during the chick-rearing period [START_REF] Cherel | Food and feeding ecology of the sympatric thinbilled Pachyptila belcheri and Antarctic P . desolata prions at Iles Kerguelen[END_REF].

White-chinned petrel (Procellaria aequinoctialis) is a large petrel and one of the most abundant seabirds in the Southern Ocean. This species is known to be effective competitors and excellent divers [START_REF] Péron | Seasonal variation in oceanographic habitat and behaviour of white-chinned petrels Procellaria aequinoctialis from[END_REF]. In Kerguelen, their diet is mostly composed of fish, but it also includes crustaceans, cephalopods, and offal and discards from fishing vessels [START_REF] Delord | At-sea distribution and diet of an endangered top predator : relationship between white-chinned petrels and commercial longline fisheries[END_REF]. During the chick rearing period, seabirds from the Kerguelen Islands forage from subantarctic to Antarctic waters [START_REF] Delord | At-sea distribution and diet of an endangered top predator : relationship between white-chinned petrels and commercial longline fisheries[END_REF], and they winter in the Benguela Current where they feed primarily on fish [START_REF] Péron | Seasonal variation in oceanographic habitat and behaviour of white-chinned petrels Procellaria aequinoctialis from[END_REF][START_REF]Diets of the white-chinned petrel and sooty shearwater in the Southern Benguela Region, South Africa[END_REF].

Together with the closely-related northern giant petrel (Macronectes halli), the southern giant petrel (M. giganteus) is the largest petrel and the dominant scavenger in the Southern Ocean (González-Solís et al., 2002). During the breeding period, the species presents sex-related foraging strategies, with males mainly scavenging on land and females primarily feeding at sea [START_REF] Thiers | Foraging zones of the two sibling species of giant petrels in the Indian Ocean throughout the annual cycle : implication for their conservation[END_REF]. During the inter-breeding periods, both males and females are pelagic within the southern Indian Ocean [START_REF] Thiers | Foraging zones of the two sibling species of giant petrels in the Indian Ocean throughout the annual cycle : implication for their conservation[END_REF]. As white-chinned petrels, giant petrels are shipfollowers, being thus at risk to be killed by fishing gears.

Sample collection, preparation and reference material used

Dead individuals of Antarctic prions (n=10) and white-chinned petrels (n=10) were collected at the Kerguelen archipelago (49°21′S, 70°18′E) in January 2012 and October 2003, respectively.

Loss of labels precludes precise knowledge of both origins and sampling dates of southern giant petrels (n=3); however seabirds were collected dead either at Crozet Islands (subantarctic waters) and/or in Adélie Land (high-Antarctica). Internal tissues analysed in this study (pectoral muscle, liver, kidney and brain) were sampled, weighed and stored individually in plastic bags. Blood samples were obtained by collecting clotted blood from heart atria and stored in Eppendorf microtubes. After dissection, all the samples were stored at -20°C. Body feathers were removed from breast and stored dry. Flying feathers from white-chinned petrels were also collected. Prior to the analyses, tissue samples were cryogenically pulverised and freeze-dried. Feathers were cleaned, oven-dried and homogenised as detailed in previous work [START_REF] Carravieri | Penguins as bioindicators of mercury contamination in the Southern Ocean: birds from the Kerguelen Islands as a case study[END_REF].

Feathers, muscles and livers from the 10 individuals of both Antarctic prions and white-chinned petrels were analysed, as were kidneys, brains and blood samples of the three southern giant petrels.

Three certified reference materials (CRM), with certified concentration values of both THg and MeHg, were used in this study for validation of our speciation and isotopic results: NIES-13 (human hair), DOLT-4 (dogfish liver) and ERM-CE-464 (tuna fish). Human hair CRM IAEA-086 was also used for validation of Hg isotopic analyses. King penguin feather and red blood cells samples (F-KP and RBC-KP, respectively) were used as internal reference materials.

Hg species concentration analysis

Samples were digested by microwave-assisted extraction using a CEM microwave system (Discover SP-D, CEM Corporation) coupled to an autosampler Explorer 4872 96 (USA). The extraction was carried out in CEM Pyrex vessels by 1 min of warming up to 75ºC and 3 min at 75ºC with magnetic agitation to homogenise the samples. Feathers (~ 0.10 g) were extracted using nitric acid (HNO3•6N, INSTRA quality) with addition of isotopic enriched standard solutions before the extraction process as detailed in previous work [START_REF] Renedo | Assessment of mercury speciation in feathers using species-specific isotope dilution analysis[END_REF]. Blood and internal tissues (0.10-0.15 g) were extracted by alkaline digestion with 5 mL of tetramethylammonium hydroxide (25% TMAH in H2O, Sigma Aldrich). Quantification of Hg species was carried out by isotope dilution analysis, using a GC-ICP-MS Trace Ultra GC equipped with a Triplus RSH autosampler coupled to an ICP-MS XSeries II (Thermo Scientific, USA) as detailed elsewhere [START_REF] Clémens | Mercury speciation analysis in seafood by species-specific isotope dilution : method validation and occurrence data[END_REF]. THg concentrations were also quantified by using an advanced Hg analyser spectrophotometer (AMA-254, Altec) for method intercomparison and corroboration of Hg total concentrations obtained by Hg speciation analyses. Hg speciation results were validated for each CRM (Table 3.S4).

Hg isotopic composition analysis

Amounts of 0.10-0.15 g of sample were digested with 5 mL of HNO3 acid (65%, INSTRA quality) after a predigestion step overnight at room temperature. Feathers and blood samples were extracted by Hotblock effectuated in Savillex vessels at 75ºC during 8 h (6 h in HNO3 and 2 h more after the addition of 1.66 mL of H2O2, 30% ULTREX quality). Internal tissues (muscle, liver, kidney and brain) were extracted by High Pressure Asher (HPA) mineralisation performed at high conditions of pressure (130 bar) and temperature (temperature ramp: 80ºC-120ºC (2ºC/min) -300ºC (2.5 h) -80ºC (1 h)). Hg isotopic composition was determined for the six most abundant stable Hg isotopes ( 198 Hg, 199 Hg, 200 Hg, 201 Hg, 202 Hg and 204 Hg) using cold-vapour generator (CVG)-MC-ICPMS (Nu Instruments), as detailed in [START_REF] Perrot | Higher massindependent isotope fractionation of methylmercury in the pelagic food web of Lake Baikal (Russia)[END_REF]. Hg isotopic values were reported as delta notation, calculated relative to the bracketing standard NIST SRM-3133 CRM to allow inter-laboratory comparisons. NIST SRM-997 thallium standard solution was used for the instrumental mass-bias correction using the exponential law. Uncertainty for delta values was calculated using 2 SD typical errors for each reference material (Table 3.S5).

Statistical analyses

Statistical tests were performed using RStudio. Before analyses, data were checked for normality of distribution and homogeneity of variances using Shapiro-Wilk and Breusch-Pagan tests, respectively. According to this, parametrical (One-way ANOVA) or non-parametrical tests (Kruskal-Wallis with Conover-Iman test) were performed. Statistically significant results were set at α= 0.05.

Results

Antarctic prions

THg concentrations, Hg species distribution and Hg isotopic composition in Antarctic prions samples varied significantly depending on the type of tissue analysed (Table 3.3, Figure 3.3). All the 10 individuals exhibited highest mean THg levels in feathers and livers, with lower concentrations in muscle (2.6±0.9, 2.1±0.5, 0.3±0.1 µg•g -1 , respectively; Kruskal-Wallis, H= 19.86, p<0.0001). Feather and liver Hg concentrations were highly variable between individuals, although muscle exhibited quite homogenous THg concentrations. Kidney and blood from five Antarctic prions were also analysed, providing intermediate mean THg concentrations (0.9±0.2 and 0.7±0.1 µg•g -1 ; respectively).

Hg species distribution was also variable among the different tissues (Kruskal-Wallis, H= 24.28, p<0.001). As expected, MeHg was the most abundant Hg species in both feathers and muscle of Antarctic prions (94±3 and 84±5%, respectively). MeHg also predominates in the liver (mean 66±6%, up to 74%) (Table 3.3).

Hg isotopic composition showed large MDF variations among internal tissues and feathers (Kruskal-Wallis, H= 18.10, p<0.0001), with individual δ 202 Hg values ranging from -0.28 to 1.55 ‰. Liver and muscle exhibited the lowest mean δ 202 Hg signatures (0.01±0.25 and 0.37±0.41 ‰, respectively) whereas feathers presented significantly heavier values (1.32±0.13 ‰). MIF signatures were also significantly different between internal tissues and feathers (Kruskal-Wallis, H= 17.36, p<0.0001), ranging from 1.77 to 2.52 ‰. Δ 199 Hg values were similar in liver and muscle, whereas they were 0.95 ‰ higher in feathers relative to muscles. Hg and Δ 199 Hg, respectively) D) Hg MIF signatures (Δ 199 Hg versus Δ 201 Hg). The red and blue dashed lines represent the theoretical Δ 199 Hg/Δ 201 Hg slope for MeHg photodemethylation and for iHg photoreduction in the water column [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF]. Δ 199 Hg/Δ 201 Hg slope for Antarctic prions was 1.11±0.08 (R 2 =0.86, p<0.0001). White-chinned petrels All the 10 individuals of white-chinned petrels presented significantly different THg concentrations in their tissues (Table 3 Hg and Δ 199 Hg, respectively) D) Hg MIF signatures (Δ 199 Hg versus Δ 201 Hg). The red and blue dashed line represents the theoretical Δ 199 Hg/Δ 201 Hg slope for MeHg photodemethylation and for photoreduction of iHg in the water column [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF]. White-chinned petrel samples Δ 199 Hg/Δ 201 Hg line regression is 1.19±0.12 (R 2 =0.85, p<0.0001).

Southern giant petrels

As previously observed in the other seabird species, Hg concentrations, speciation and isotopic signatures were highly dependent on the type of tissue analysed. However, in this case, high interindividual variability was also observed between the three southern giant petrels (Table 3.5). In general, similar trends of Hg concentrations were observed for the three southern giant petrels, with the significantly highest mean THg concentrations in livers and the lowest in brains (309.7±135.1 and 5.9±6.4 µg•g -1 , respectively). Kidneys were also highly concentrated (39.4±11.7 µg•g -1 ). Feathers and muscles also showed similar THg concentrations (13.3±6.9 and 11.6±15.2 µg•g -1 , respectively) comparing to other MeHg accumulative tissues, such as blood (9.9±12.1 µg•g -1 ) (H= 10.70, p=0.058).

Despite inter-individual variability, Hg species distributions in liver and kidney tissues were characterized by a great predominance of iHg comparing to other tissues (H= 14.36, p=0.012), with livers presenting a slightly higher mean iHg percentage (96±2%) than kidneys (84±16%).

The proportion of MeHg was homogenous for the three individuals in both feathers (mean 97.4±0.1%) and blood samples (mean 90±5%). However, Hg speciation in the storage organs differed widely between individuals, with muscles displaying a mean proportion of MeHg of 49% (37-58%), similar to brain 49% (34-74%) (Table 3.S8).

Concerning Hg isotopic composition, southern giant petrels displayed a large range of δ 202 Hg values (from -0.93 to 2.35 ‰), which were statistically different among tissues (H= 12.47, p=0.029). No significant variations in Δ 199 Hg values were neither obtained among different tissues (H= 5.54, p=0.353) that ranged between 1.23 and 1.57 ‰.

Figure 3.5 A) THg concentrations and Hg MDF (as δ 202 Hg) in liver (blue), kidneys (dark blue), muscles (yellow), feathers (red), brains (green) and blood (grey) from southern giant petrels. B)

Hg MDF relative to the fraction of MeHg (regression line: y=2.75x-0.48, Pearson correlation coefficients were R 2 =0.92, p<0.0001). C) Hg MDF versus Hg MIF (expressed as δ 202 Hg and Δ 199 Hg, respectively) D) Hg MIF signatures (Δ 199 Hg versus Δ 201 Hg). The red and blue dashed line represents the theoretical Δ 199 Hg/Δ 201 Hg slope for MeHg photodemethylation and for photoreduction of iHg in the water column [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF]. For the southern giant petrel samples Δ 199 Hg/Δ 201 Hg line regression is 1.17±0.09 (R 2 =0.85, p<0.0001). from Lake Baikal ranging from 0.64 ± 0.26‰ in fish (sculpins) to 1.83 ± 0.29‰ in seal muscles.

High differences in MDF values were observed in feathers (a tissue that accumulates nondemethylated Hg) among organisms corresponding to different trophic levels, with giant petrels exhibiting higher feather mean δ 202 Hg values (2.33±0.02 ‰) than Antarctic prions (1.32±0.13‰) and white-chinned petrels (1.10±0.14‰). However, this trend is not coherent with the assumption of heavier δ 202 Hg values with increasing trophic levels. Since Antarctic prions mainly feed on zooplankton, they are exposed to lower Hg levels by diet than the other two species [START_REF] Bocher | Influence of the diet on the bioaccumulation of heavy metals in zooplankton-eating petrels at Kerguelen archipelago, Southern Indian Ocean[END_REF][START_REF] Fromant | Wide range of metallic and organic contaminants in various tissues of the Antarctic prion, a planktonophagous seabird from the Southern Ocean[END_REF]. Similarly, white-chinned petrels, which mostly feed on pelagic fish, presented significantly lower Hg concentrations and MDF values than southern giant petrels that scavenge on seal and other seabird carcasses [START_REF] Cipro | Trace elements in tissues of white-chinned petrels (Procellaria aequinoctialis) from Kerguelen waters, Southern Indian Ocean[END_REF]. Therefore, an influence of the diet could be proposed as the most plausible explanation of such variations in Hg concentrations and isotopic values among the three species.

Differences on MIF extent were also noticed between the three seabird populations. The highest Δ 199 Hg values were measured in Antarctic prions' tissues (ranging from 1.77 to 2.52‰) that were significantly greater than those registered in white-chinned petrels (from 1.05 to 1.71‰) and southern giant petrels (from 1.23 to 1.57‰) H=49.117,p<0.0001,n=72).

Previous biomonitoring studies have revealed variably positive Hg MIF (Δ 199 Hg or Δ 201 Hg) in aquatic organisms that have acquired MeHg that was photochemically degraded in different extents [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF][START_REF] Tsui | Sources and transfers of methylmercury in adjacent river and forest food webs[END_REF][START_REF] Perrot | Tracing sources and bioaccumulation of mercury in fish of Lake Baikal--Angara River using Hg isotopic composition[END_REF][START_REF] Point | Methylmercury photodegradation influenced by sea-ice cover in Arctic marine ecosystems[END_REF][START_REF] Senn | Stable isotope (N, C, Hg) study of methylmercury sources and trophic transfer in the northern gulf of Mexico[END_REF]Sherman et al., 2013;[START_REF] Sherman | Assessment of mercury exposure among small-scale gold miners using mercury stable isotopes[END_REF]. Large variations in Δ 199 Hg among the two petrel species and Antarctic prions could be due to specific feeding behaviour or distinct foraging zones. Antarctic prions are known to migrate to northern latitudes during inter-breeding periods [START_REF] Cherel | Combination of At-Sea Activity , Geolocation and Feather Stable Isotopes Documents Where and When Seabirds Molt[END_REF], where photochemical reactions are potentially more substantial.

Therefore, the higher MIF in their tissues could be attributed to the exposure to MeHg having undergone greater photodemethylation extent before incorporation to the food web.

Hg species distribution and MDF variations: proxies of metabolic processes Hg species distribution among tissues followed a general pattern for the three seabird models, with liver and kidneys being predominantly enriched in iHg, relative to muscles and feathers that tend to accumulate mainly MeHg, which is in good agreement with previous studies on seabirds (Thompson and Furness 1989b;[START_REF] Kim | Mercury levels and its chemical form in tissues and organs of seabirds[END_REF][START_REF] Uthe | Longitudinal study of methylmercury and inorganic mercury in blood and urine of pregnant and lactating women, as well as in umbilical cord blood[END_REF][START_REF] Kehrig | Trace-elements, methylmercury and metallothionein levels in Magellanic penguin (Spheniscus magellanicus) found stranded on the Southern Brazilian coast[END_REF]. Tissue-specific δ 202 Hg values were also observed on the three seabird species, since tissues that contained high amounts of iHg exhibited much lighter (in some cases negative) δ 202 Hg values.

In contrast, tissues presenting similar proportions of both Hg compounds (i.e. iHg and MeHg)

showed intermediate δ 202 Hg signatures (muscles and brains). Finally, feathers and blood, which had a majority of MeHg, were characterized by much positive δ 202 Hg values and higher interindividual homogeneity. The correlation between MeHg fractions and MDF values can be attributed to intrinsic metabolic processes (such as demethylation and/or inter-organ transport)

that would induce changes in Hg isotopic composition before the posterior accumulation of Hg species among different tissues.

As previously mentioned, liver is assumed to play a detoxification role in Hg metabolism by demethylation of MeHg into a less toxic species iHg, as it has been reported extensively in studies on seabirds [START_REF] Thompson | Using seabirds to monitor mercury in marine environments. The validity of conversion ratios for tissue comparisons[END_REF]Kim et al., 1996;Thompson et al., 1998ab;[START_REF] Cipro | Trace elements in tissues of white-chinned petrels (Procellaria aequinoctialis) from Kerguelen waters, Southern Indian Ocean[END_REF].

Evidences of Hg MDF during MeHg demethylation processes have been reported in experimental studies [START_REF] Perrot | Successive methylation and demethylation of methylated mercury species (MeHg and DMeHg) induce mass dependent fractionation of mercury isotopes[END_REF][START_REF] Gonzalez | Species-Specific Stable Isotope Fractionation of Mercury during Hg ( II ) Methylation by an Anaerobic Bacteria ( Desulfobulbus propionicus ) under Dark Conditions[END_REF] and during in vivo processes in organisms [START_REF] Feng | Specific Pathways of Dietary Methylmercury and Inorganic Mercury Determined by Mercury Speciation and Isotopic Composition in Zebrafish ( Danio rerio )[END_REF][START_REF] Kwon | Application of mercury isotopes for tracing trophic transfer and internal distribution of mercury in marine fish feeding experiments[END_REF][START_REF] Perrot | Natural Hg isotopic composition of different Hg compounds in mammal tissues as a proxy for in vivo breakdown of toxic methylmercury[END_REF]. This is explained by the preferential demethylation of isotopically lighter MeHg (lower δ 202 Hg values) which produced light iHg and lead to the deposition of residual MeHg fraction enriched in heavier Hg isotopes (greater δ 202 Hg values). Hg demethylation process seemed to occur in significant degree in livers of white-chinned petrels and southern giant petrels, since both presented almost exclusively iHg and an important enrichment in lighter isotopes from muscles to livers, especially for whitechinned petrels (~ 0.94 ‰). On the contrary, livers from Antarctic prions displayed a minor presence of iHg (from 26 to 45%) and a lower difference in MDF was observed between muscles and livers (~ 0.36 ‰) comparing to the two other seabird species, possibly indicating less efficient MeHg hepatic demethylation.. It is widely recognized that in vivo MeHg demethylation is quite dependent on individual maturity and exposition to Hg levels [START_REF] Wagemann | Methylmercury and total mercury in tissues of arctic marine mammals[END_REF]Wang et al., 2013). Therefore, another non-exclusive explanation is that the apparent slight demethylation extent in livers from Antarctic prions could be attributed to their exposure to low levels of Hg by dietary intake, since they feed at lower pelagic trophic levels comparing to petrels, and therefore, their necessity of Hg detoxification processes such as hepatic demethylation may be less significant. Furthermore, Antarctic prions presented much higher Hg levels excreted in feathers relative to their concentrations registered in liver (comparing to the two petrel species), suggesting that they possibly present higher capacities of Hg elimination by moulting feathers. Contrastingly, the predominance of iHg in liver and kidney tissues from the two petrel species, together with their very elevated concentrations accumulated in such internal tissues, may be indicative of significant MeHg hepatic (and possibly renal) demethylation (Kim et al., 1996;[START_REF] Cipro | Trace elements in tissues of white-chinned petrels (Procellaria aequinoctialis) from Kerguelen waters, Southern Indian Ocean[END_REF].

After in vivo demethylation, the remaining non-metabolised and heavier MeHg fraction would be potentially redistributed and accumulated in other organs (muscle, brain) and, in the case of seabirds, excreted by feathers. This effect could explain the MDF variations obtained between storage and target organs in seabirds, which matches with previous observations in fish (Feng et al. 2015). Several toxicokinetic studies on MeHg metabolism and elimination in other organisms, such as fish and humans, have evidenced that faeces and urine excretion are the predominant elimination pathway of iHg resulting from MeHg in vivo demethylation and/or from direct ingestion [START_REF] Van Walleghen | Elimination of mercury by yellowperch in the wild[END_REF][START_REF] Li | Assessing sources of human methylmercury exposure using stable mercury isotopes[END_REF]Sherman et al., 2013;[START_REF] Feng | Specific Pathways of Dietary Methylmercury and Inorganic Mercury Determined by Mercury Speciation and Isotopic Composition in Zebrafish ( Danio rerio )[END_REF].

The isotopic characterization of urine/faeces [START_REF] Li | Assessing sources of human methylmercury exposure using stable mercury isotopes[END_REF][START_REF] Feng | Specific Pathways of Dietary Methylmercury and Inorganic Mercury Determined by Mercury Speciation and Isotopic Composition in Zebrafish ( Danio rerio )[END_REF]Sherman et al., 2013) evidenced that the excreted iHg is isotopically lighter than the remaining Hg in the body, where it is principally present as MeHg.

Variations in MIF and MDF values between feathers and internal tissues

A common trend observed in the three seabird species was the enrichment in higher δ 202 Hg values from muscles to feathers, which was particularly noteworthy in giant petrels (~ 1.88 ‰), followed by Antarctic prions (~0.95 ‰) and white-chinned petrels (~0.81 ‰). Significant anomalous Hg fractionation (MIF) was also observed between muscles and feathers in whitechinned petrels (enrichment of Δ 199 Hg values +0.20‰) and particularly in Antarctic prions (+0.44 ‰). However, no substantial Hg MIF was obtained from muscles to feathers in the case of giant petrels.

Taking into account in vivo processes, it is well established that MeHg presents a high affinity for thiol-containing compounds such as cysteine, the principal constituent of keratin which forms the growing feather. MeHg-cysteine complexes are known to be transferred across the bloodbrain barrier [START_REF] Clarkson | The toxicology of mercury and its chemical compounds[END_REF] and across membranes to liver and kidneys [START_REF] Bridges | Transport of inorganic mercury and methylmercury in target tissues and organs[END_REF]. Therefore, as a first explanation, we could associate increasing δ 202 Hg values from muscles to feathers to an isotopic fractionation induced by intrinsic process, such as formation and transport of MeHg-cysteine complexes; or even a combination of other reactions occurring during inter-tissue transport or inside the feather itself. A priori, differences in Δ 199 Hg signatures could be also explained by internal processes related to photochemistry, so inducing significant MIF, such as a potential photochemical demethylation in the feather. However, this hypothesis appears unlikely because Hg is known to be sequestered in the sulphydryl groups of the keratin molecules where it is supposed to be physically and chemically stable [START_REF] Appelquist | Mercury monitoring: Mercury stability in bird feathers[END_REF]. Therefore, Δ 199 Hg variations could be mostly explained by the influence of extrinsic factors, such as the combination of different temporal and geographic integration windows between feathers and internal tissues samples. Feathers are known to reflect Hg exposure between two moults, integrating Hg acquired both at the breeding site and during their wintery migration far from their colonies. However, significant shifts in feeding ecology (foraging habitat and prey)

that are produced during the different stages of their annual cycle (inter-moult and breeding periods) could potentially enhance Hg isotopic differences (both MDF and MIF) between feathers and internal organs. Changing latitudinal zones or depths during their migration period would also influence the uptake of isotopically different MeHg due to distinct aquatic photochemistry.

Indeed, MIF variations between tissues in adult seabirds demonstrated to be an effective proxy of changes in foraging behaviour (horizontal, vertical and latitudinal dimensions) along their annual cycle (Chapter 3.1). Thus, migrating to southern latitudes with higher photochemistry extent would involve integration of MeHg with greater Δ 199 Hg values into growing feathers. For instance, Antarctic prions exhibited here the highest differences in MIF among organs and feathers, which is consistent with their pre-breeding moult in warmer subtropical waters [START_REF] Cherel | Combination of At-Sea Activity , Geolocation and Feather Stable Isotopes Documents Where and When Seabirds Molt[END_REF]) and supported by the results obtained for δ 13 C isotopic analyses, for which feathers exhibited a subtropical signature whereas internal tissues showed Antarctic signatures (Figure 3.S6). However, although this hypothesis could explain the variations in MIF between internal organs and feathers observed in Antarctic prions and white-chinned petrels, it is not coherent with the absence of MIF for this tissues on giant petrels. A first suggestion that could explain this effect is the extended moulting time interval of southern giant petrels. Their moult initiates at the end of their breeding period so, possibly, a fraction of Hg integrated in feathers could correspond to their diet at the beginning of moult in the colonies and, consequently, reduce the fluctuations observed in MIF values among internal tissues and feathers. Another possible cause agent of this absence of MIF between feathers and internal organs could be due to the elevated Hg concentrations in the tissues of these apex predators that would induce a "reservoir effect", leading to the attenuation of Hg isotopic composition of recently acquired Hg during inter-organ equilibration producing more homogeneity in MIF values among the different tissues.

White-chinned petrels exhibited also significantly different MIF values among all the three analysed tissues. According to the other seabird species, the difference between internal organs and feathers seems to be clearly due to different Hg sources (as a result of different integration periods). Furthermore, this idea is supported by the observed different Δ 199 Hg/Δ 201 Hg ratios between internal tissues and feathers of white-chinned petrels, thus suggesting different magnitudes of MeHg photodemethylation. As a first hypothesis, MIF differences between muscle and liver of this species could be associated to a difference in the ingested MeHg and iHg. Based on previous studies, dietary iHg is principally accumulated in liver and later redistributed to other organs [START_REF] Feng | Specific Pathways of Dietary Methylmercury and Inorganic Mercury Determined by Mercury Speciation and Isotopic Composition in Zebrafish ( Danio rerio )[END_REF]. However, the diet of white-chinned petrels is mainly composed on mesopelagic fish and cephalopods, which are known to be almost exclusively constituted by MeHg (more than 90%), thus the potential ingestion of some others organisms contributing to a significant higher iHg fraction is improbable [START_REF] Monteiro | Mercury concentrations in prey fish indicate enhanced bioaccumulation in mesopelagic environments[END_REF][START_REF] Bustamante | Total and organic Hg concentrations in cephalopods from the North Eastern Atlantic waters: Influence of geographical origin and feeding ecology[END_REF]).The similar Δ 199 Hg/Δ 201 Hg ratios displayed by muscles and livers also supports the idea of a similar source of MeHg between tissues. An alternative explanation could be an influence of different Hg residence time between tissues. Muscles are assumed to reflect Hg acquisition since the last moult, after remobilisation into growing feathers, therefore representing Hg accumulated during one year (considering species with annual moulting). However, Hg in liver (mainly as metabolite iHg) is considered to represent long-term accumulation, although it is known that a part of the produced iHg from hepatic demethylation is excreted by urine or faeces. This different Hg turnover and residence time between muscles and liver could involve distinct sources of Hg accumulated specifically in each tissue.

Conclusion

In conclusion, the exploration of Hg isotopes in tissues from three seabird species with contrasting ecological behaviour (foraging habits, trophic level, type of prey, etc. Hg integration times between feathers and internal organs permits the access to different temporal and geographical windows. Although internal organs can provide valuable information about long-term Hg accumulation and physiological aspects, their longer Hg turnover relative to other "metabolically recent" tissues that give access to more precise Hg temporal integration windows, such as blood (weeks to few months) or feathers (~1 year), makes them less sensitive to trace Hg spatio-temporal exposure.

Main findings of this work:

-Hg species distribution among tissues followed a general pattern for the three seabird models, with livers being predominantly enriched in iHg, certainly as a consequence of in vivo demethylation, whereas muscles and feathers tend to accumulate mainly MeHg.

- Chapter 4.

Ecological and biogeochemical aspects

Introduction

As a result of its severe toxicity, mercury (Hg) is considered as a worldwide pollutant of major concern for humans and wildlife (e.g. [START_REF] Tan | The endocrine effects of mercury in humans and wildlife[END_REF]. It is present in all compartments, transported over long distances and accumulated in the environment, where once methylated, it biomagnifies (e.g. [START_REF] Atwell | Biomagnification and bioaccumulation of mercury in an arctic marine food web: insights from stable nitrogen isotope analysis[END_REF]. In aquatic systems, Hg inorganic forms (iHg) can be methylated by microbiological [START_REF] Compeau | Sulfate-Reducing Bacteria: Principal Methylators of Mercury in Anoxic Estuarine Sedimentt[END_REF][START_REF] Jensen | Biological methylation of mercury in aquatic organisms[END_REF] or abiotic processes [START_REF] Celo | Abiotic methylation of mercury in the aquatic environment[END_REF], leading to the incorporation and posterior biomagnification of methylmercury (MeHg) in marine food webs. Vertical profiles of MeHg concentrations have been globally found in oceans [START_REF] Cossa | The origin of methylmercury in open Mediterranean waters[END_REF][START_REF] Mason | Alkylmercury species in the equatorial Pacific[END_REF][START_REF] Hammerschmidt | Vertical methylmercury distribution in the subtropical North Pacific Ocean[END_REF], including the Southern Ocean [START_REF] Cossa | Mercury in the Southern Ocean[END_REF], presenting lower concentrations in surface waters due to the degradation of MeHg into iHg by photodemethylation processes [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF] and increasing concentrations with depth peaking at lowoxygen intermediate waters, where microbial methylation is assumed to occur [START_REF] Mason | Alkylmercury species in the equatorial Pacific[END_REF][START_REF] Heimbürger | Methyl mercury distributions in relation to the presence of nano-and picophytoplankton in an oceanic water column (Ligurian Sea, North-western Mediterranean)[END_REF][START_REF] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF]. Consequently, an increasing gradient of Hg concentrations was found in fish caught from the surface (epipelagic zone) to deeper waters (mesopelagic zone) [START_REF] Monteiro | Mercury concentrations in prey fish indicate enhanced bioaccumulation in mesopelagic environments[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] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF].

Seabirds are positioned at medium to high trophic levels, being thus exposed to elevated concentrations of MeHg via dietary uptake. They are recognized as efficient bio-indicators of marine Hg contamination at different spatial scales according to their life cycle [START_REF] Burger | Marine Birds as Sentinels of Environmental Pollution[END_REF][START_REF] Fort | Spatial Ecotoxicology: Migratory Arctic Seabirds Are Exposed to Mercury Contamination While Overwintering in the Northwest Atlantic[END_REF]. In contrast to flying birds, penguins exploit relatively spatially restricted foraging zones and are thus representative of Hg contamination in more limited areas, which make them interesting models for local biomonitoring studies [START_REF] Carravieri | Penguins as bioindicators of mercury contamination in the Southern Ocean: birds from the Kerguelen Islands as a case study[END_REF][START_REF] Carravieri | Penguins as bioindicators of mercury contamination in the southern Indian Ocean: geographical and temporal trends[END_REF]. Investigating diverse penguin species provide access to different marine environmental compartments since they have species-specific foraging ecologies. They feed on a large diversity of prey in different oceanographic ecosystems, both horizontally (from the neritic to the oceanic domains) and vertically, as they forage at different depths of the water column (from the epipelagic to the mesopelagic zone). Taking advantage of these specificities, we have measured here the stable isotope ratios of Hg in blood samples of four sympatric penguin species that breed at the subantarctic Crozet Islands (southern Indian Ocean) in order to characterize a priori different MeHg exposure pathways.

Mercury has seven stable isotopes that undergo mass dependent fractionation (MDF) as a consequence of many physical, chemical or biological processes [START_REF] Gonzalez | Species-Specific Stable Isotope Fractionation of Mercury during Hg ( II ) Methylation by an Anaerobic Bacteria ( Desulfobulbus propionicus ) under Dark Conditions[END_REF][START_REF] Kritee | Mercury Stable Isotope Fractionation during Reduction of Hg(II) to Hg(0) by Mercury Resistant Microorganisms[END_REF][START_REF] Kritee | Mass dependent stable isotope fractionation of mercury during mer mediated microbial degradation of monomethylmercury[END_REF]Estrade et al. 2009;Zheng and Hintelmann 2010a).

Moreover, photochemical reactions induce significant Hg mass independent fractionation (MIF), wherein the odd isotopes are enriched or depleted in reaction products relative to the even isotopes (Zheng and Hintelmann 2009a;[START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF][START_REF] Sherman | Mass-independent fractionation of mercury isotopes in Arctic snow driven by sunlight[END_REF][START_REF] Perrot | Higher massindependent isotope fractionation of methylmercury in the pelagic food web of Lake Baikal (Russia)[END_REF]. However, no substantial MIF has been observed during trophic processes (Kwon et al. 2012b;[START_REF] Perrot | Higher massindependent isotope fractionation of methylmercury in the pelagic food web of Lake Baikal (Russia)[END_REF], meaning that MIF values can be used as conservative tracer of MeHg sources in predators.

Increasing number of studies have successfully applied Hg isotope analysis for elucidating sources and pathways of MeHg in the environment (e.g. [START_REF] Senn | Stable isotope (N, C, Hg) study of methylmercury sources and trophic transfer in the northern gulf of Mexico[END_REF][START_REF] Gehrke | Sources of mercury to San Francisco Bay surface sediment as revealed by mercury stable isotopes[END_REF][START_REF] Day | Mercury stable isotopes in seabird eggs reflect a gradient from terrestrial geogenic to oceanic mercury reservoirs[END_REF][START_REF] Chen | Isotopic evidence for distinct sources of mercury in lake waters and sediments[END_REF]. In aquatic ecosystems, MIF is highly sensitive to photochemical reactions and varies as a function of light penetration extent at different locations or depths. In a previous study, [START_REF] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF] evidenced a relationship between fish foraging depth and Hg isotopic values as a consequence of different extent of photochemical degradation of MeHg in the water column. These authors showed that MeHg produced at the subsurface of the open ocean is effectively uptake by fish with higher MIF values in epipelagic fish than in deep ones. Higher magnitudes of MIF have also been observed in pelagic organisms relative to coastal organisms [START_REF] Senn | Stable isotope (N, C, Hg) study of methylmercury sources and trophic transfer in the northern gulf of Mexico[END_REF][START_REF] Day | Mercury stable isotopes in seabird eggs reflect a gradient from terrestrial geogenic to oceanic mercury reservoirs[END_REF][START_REF] Perrot | Higher massindependent isotope fractionation of methylmercury in the pelagic food web of Lake Baikal (Russia)[END_REF]. This gradient was mainly attributed to enhanced light penetration in oceanic waters due to minor water turbidity and lower dissolved organic carbon (DOC), thus leading to higher rate and extent of MeHg demethylation in the pelagic water column.

Stable isotopes of carbon (δ 13 C) and nitrogen (δ 15 N) serve as powerful indicators of foraging habitat and trophic position, respectively. Isotopic measurements have already been validated in the Southern Ocean, with seabird δ 13 C values indicating their latitudinal foraging grounds and depicting offshore versus inshore consumers and benthic vs pelagic ones, while δ 15 N values increased with trophic level (e.g., Cherel et al., 2007;[START_REF] Jaeger | Seabird satellite tracking validates the use of latitudinal isoscapes to depict predators' foraging areas in the Southern Ocean[END_REF]. Thus, we used δ13C and δ15N values to explain the potential variations in Hg stable isotopes in seabirds and to trace the origin, trophic transfer and bioaccumulation processes of Hg in marine food webs, as it was previously demonstrated in fish [START_REF] Senn | Stable isotope (N, C, Hg) study of methylmercury sources and trophic transfer in the northern gulf of Mexico[END_REF][START_REF] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF][START_REF] Li | Environmental Origins of Methylmercury Accumulated in Subarctic Estuarine Fish Indicated by Mercury Stable Isotopes[END_REF][START_REF] Cransveld | Mercury stable isotopes discriminate different populations of European seabass and trace potential Hg sources around Europe[END_REF].

The main objective of this work was to investigate the effectiveness of Hg stable isotope ratios in seabird tissues to discern and quantify MeHg sources and exposure pathways in the different marine compartments in which they forage. Because blood samples are known to reflect recent Hg exposure (month scale) and penguins are generally restricted to areas near their colonies at the time of sample collection (end of the breeding period), Hg isotopic composition of penguin blood samples were considered as indicative of local Hg values. Thus, we hypothesized that contrasting ecological strategies among penguins (four species) would determine the uptake of distinct environmental MeHg sources and would lead to interspecies differences in blood Hg isotopic composition. According to MIF dynamics in aquatic systems, we did the following hypothesis that more positive Δ 199 Hg values are expected for penguins foraging in shallower waters (rockhopper and macaroni penguins) than mesopelagic (king penguins) feeders; while lower Δ 199 Hg values are predictable for benthic predators (gentoo penguins) relative to the other three pelagic species. The main question addressed in this study is: what is the effectiveness of Hg isotopes to elucidate the environmental origin and exposure pathways of MeHg accumulated in seabirds depending on their ecological characteristics?

Material and methods

Study site and sampling procedure Sample collection was conducted during the austral summer 2011-2012 (from October to February; Table 4.S1) at Ile de la Possession, Crozet Islands (Subantarctic Zone, 46°26'S, 51°45'E). Ten to 11 randomly-chosen breeding adults were blood sampled at the end of the chickrearing period by venepuncture of a flipper vein using heparinized syringes. Whole blood was centrifuged to separate plasma from blood cells (hereafter blood). Blood samples were kept frozen at -20°C until Hg and isotopic analyses in France.

Diet and foraging habits of penguin populations

All the four sympatric species of penguins that breed at the Crozet Islands were investigated, including the king Aptenodytes patagonicus, gentoo Pygoscelis papua, macaroni

Eudyptes chrysolophus and eastern rockhopper E. chrysocome filholi penguins. The king penguins (KP) is a large oceanic species that feed on mesopelagic fish (myctophids) at deep depths (100-300 m) in distant southern foraging grounds located in the vicinity of the Polar Front [START_REF] Kooyman | Diving Behavior and Energetics During Foraging Cycles in King Penguins[END_REF]Cherel et al., 2007;[START_REF] Bost | Large-scale climatic anomalies affect marine predator foraging behaviour and demography[END_REF]. In contrast, the medium-sized gentoo penguin (GP) is a coastal neritic species that dive both pelagically and benthically to feed opportunistically on a large diversity of prey, including swarming crustaceans and benthic fish [START_REF] Bost | Maximum diving depth and diving patterns of the gentoo penguin Pygoscelis papua at the Crozet Islands[END_REF][START_REF] Ridoux | Marine Ornithology: The diets and dietary segregation of seabirds at the Subantarctic Crozet Islands[END_REF]. The smaller and closely-related macaroni (MP) and rockhopper (RP) penguins forage in offshore waters where they primarily target swarming crustaceans (euphausiids and hyperiids) in the top 70 m of the water column [START_REF] Ridoux | Marine Ornithology: The diets and dietary segregation of seabirds at the Subantarctic Crozet Islands[END_REF][START_REF] Tremblay | Geographic variation in the foraging behaviour, diet and chick growth of rockhopper penguins[END_REF]Cherel et al. 2007;[START_REF] Bon | Influence of oceanographic structures on foraging strategies: Macaroni penguins at Crozet Islands[END_REF].

Sample preparation and analytical methods

Hg speciation and Hg isotopic analyses

For Hg speciation analyses, Hg was extracted from blood samples (0.10-0.15 g) by alkaline microwave digestion with 5 mL of tetramethylammonium hydroxide (25% TMAH in H2O, Sigma Aldrich) [START_REF] Rodrigues | Mercury speciation in whole blood by gas chromatography coupled to ICP-MS with a fast microwave-assisted sample preparation procedure[END_REF]. Details of the extraction method, analysis and quantification of Hg species are included in (Chapter 3.1).

Prior to Hg isotopic analyses, blood samples (0.05-0.10 g) were digested with 3 or 5 mL of HNO3 acid (65%, INSTRA quality) after a predigestion step overnight at room temperature and later extraction in Hotblock at 75ºC during 8 h (6 h in HNO3 and 2 h more after the addition of 1/3 of the total volume of H2O2 (30%, ULTREX quality)). Details of Hg isotopic composition analyses are included in (Chapter 3.1).

For the validation of the analytical results, two certified reference material were analysed:

human hair IAEA-086 and tuna fish ERM-CE-464. An internal reference sample was prepared with pool samples collected from different individuals of king penguin from Crozet Islands: RBC-KP (red blood cells) and was analysed at each analytical session. Uncertainty for delta values was calculated using 2SD typical errors for reference material (Table 4.S2).

Carbon and nitrogen stable isotopes analyses

Carbon (δ 13 C) and nitrogen (δ 15 N) stable isotope ratios were determined in red blood cells with a continuous flow mass spectrometer (Thermo Scientific Delta V Advantage) coupled to an elemental analyser (Thermo Scientific Flash EA 1112) (aliquots mass: ~0.3 mg). Results are in delta notation relative to Vienna PeeDee Belemnite and atmospheric N2 for δ 13 C and δ 15 N, respectively. Replicate measurements of internal laboratory standards (acetanilide) indicated measurement errors < 0.15‰ for both δ 13 C and δ 15 N values.

Statistical analyses

Statistical tests were performed using RStudio. Before analyses, data were checked for normality of distribution and homogeneity of variances using Shapiro-Wilk and Breusch-Pagan tests, respectively. According to this, parametrical (One-way ANOVA) or non-parametrical tests (Kruskal-Wallis with Conover-Iman test) were performed. Statistically significant results were set at α= 0.05.

Results

Blood Hg concentrations and Hg isotopic composition

Blood THg concentrations differed amongst penguin species (Kruskal Wallis, H=30.09, p<0.0001), with KP and GP being more contaminated than MP and RP (Table 4.1). Accordingly, blood MeHg was overall different (H=28.87, p<0.0001), with KP and GP showing higher concentrations than MP and RP. All individual penguins presented a large predominance of MeHg in their blood (94±2%, range: 91-98%, n = 42).

Blood samples showed large ranges of individual δ 202 Hg (MDF) and ∆ 199 Hg (MIF) values

(1.28-2.12 ‰ and 1.31-1.95 ‰, respectively, n = 42). Both blood δ 202 Hg and ∆ 199 Hg values differed amongst penguins (H=26.94 and 32.27, respectively, both p<0.0001), except KP and GP that showed identical δ 202 Hg values. Blood δ 202 Hg and ∆ 199 Hg values increased in the order GP = KP < MP < RP and GP < MP < KP < RP, respectively. RP notably showed high inter-individual variability in their blood ∆ 199 Hg values (from 1.55 to 1.93 ‰).

Measured blood δ 202 Hg values followed the predicted theoretical MDF line. In contrast, measured blood δ 199 Hg values diverged from the δ 199 Hg theoretical MDF line (Figure 4.S1),

indicating that all blood samples showed MIF of the 199 Hg and 201 Hg odd isotopes. Overall penguin blood samples displayed a ∆ 199 Hg/∆ 201 Hg slope of 1.16±0.05 (R 2 =0.98, p<0.0001) (Figure 4.1). Individual ∆ 199 Hg/∆ 201 Hg ratios for each penguin species are included in Table 4.1.

Blood δ 13 C and δ 15 N values

Penguins were segregated by their δ 13 C and δ 15 N values (Figure 4.S2), with both variables being significantly and negatively correlated (Pearson correlation R 2 = -0.74, p<0.0001). The four species presented distinct δ 13 C values (Kruskal Wallis, H=35.30, p<0.0001), with a progressive 13 C enrichment from KP to GP. Blood δ 15 N values were also different (H=25.24, p<0.0001). They allow splitting species into two groups, with MP, RP and GP differing from KP by their 1.5-1.8

‰ lower δ 15 N values (Table 4.1). Δ 199 Hg/Δ 201 Hg slope expected for photoreduction of iHg in the water column [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF].

Relationship between blood Hg isotopic composition and δ 13 C and δ 15 N values Without considering KP population, highly negative correlations were found for δ 13 C values with δ 202 Hg values (R 2 =-0.73, p<0.0001) and with ∆ 199 Hg values (R 2 =-0.71, p<0.0001) ().

An absence of correlation was observed between blood ∆ 199 Hg and δ 15 N values (R 2 =0. 03, p=0.30) and between blood δ 202 Hg and δ 15 N values for the overall individuals (R 2 =-0.10, p=0.52) (Figure

Discussion

Because MeHg is the predominant Hg species in all the blood samples, irrespective of THg concentration or penguin species, the measured Hg isotopic composition of this tissue corresponds essentially to MeHg values. This predominance of MeHg in blood allows directly comparing the four penguin species, and the exploration of MDF and MIF values to trace MeHg pathways in each marine compartment used by penguins in Crozet waters. In the following sections, variations of Hg isotopic composition among penguins are discussed and interpreted in function of their specific ecological characteristics in order to estimate sources and processes involving MeHg.

Variations in foraging habitats (δ 13 C) between penguin populations

In areas surrounding subantarctic islands, δ 13 C values decrease from neritic to oceanic waters (inshore-offshore δ 13 C gradient), and from warm to cold waters (latitudinal δ 13 C gradient), thus allowing using δ 13 C values to assess the main foraging grounds of seabirds [START_REF] Cherel | A comprehensive isotopic investigation of habitat preferences in nonbreeding albatrosses from the Southern Ocean[END_REF]Cherel and Hobson, 2007). A large range of δ 13 C values was observed across penguin populations with decreasing values from GP to KP (a 3.6 ‰ difference). KP exhibited much lower δ 13 C values than the other two oceanic penguins (MP and RP), which is in agreement with their well-known southern foraging grounds down to the Polar Front (Cherel et al., 2007;[START_REF] Bost | Large-scale climatic anomalies affect marine predator foraging behaviour and demography[END_REF]. RP showed significantly lower δ 13 C values than MP, indicating that they forage at more southern latitudes than MP during the breeding period (Cherel and Hobson, 2007). Finally, the more positive δ 13 C values of GP compared to the other species are in agreement with the inshore feeding habits of the species (Cherel and Hobson, 2007). Species-specific δ 13 C values clearly demonstrate that the four penguins breeding at the Crozet Islands document each a different marine environment, thus allowing studying processes affecting Hg behaviour and its fate in the Southern Ocean.

Relation between Hg isotopes and trophic ecology (δ 15 N) Independently of the species, the lack of correlation between blood ∆ 199 Hg and δ 15 N values is consistent with the absence of MIF during trophic transfer [START_REF] Laffont | Anomalous mercury isotopic compositions of fish and human hair in the Bolivian amazon[END_REF][START_REF] Perrot | Tracing sources and bioaccumulation of mercury in fish of Lake Baikal--Angara River using Hg isotopic composition[END_REF]Sherman et al., 2013;[START_REF] Li | Assessing sources of human methylmercury exposure using stable mercury isotopes[END_REF]. While MDF could be produced during trophic transfer [START_REF] Laffont | Anomalous mercury isotopic compositions of fish and human hair in the Bolivian amazon[END_REF][START_REF] Perrot | Tracing sources and bioaccumulation of mercury in fish of Lake Baikal--Angara River using Hg isotopic composition[END_REF], no correlation was observed between δ 202 Hg and δ 15 N values. Differences in dietary composition amongst pelagic penguins is well illustrated by δ 15 N values being higher in the fish-consumer species (KP) than in crustacean feeders (MP and RP).

KP preferentially rely on mesopelagic fish so they have a higher trophic position than MP and RP that rely mainly on swarming euphausiids and hyperiids (Cherel et al. 2007). Moreover, mesopelagic fish are known to accumulate elevated MeHg concentrations as a result of enhanced Hg methylation in deep waters [START_REF] Monteiro | Mercury concentrations in prey fish indicate enhanced bioaccumulation in mesopelagic environments[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]. Both the higher trophic position and the mesopelagic foraging habitat of KP explain why their THg and MeHg concentrations were twice higher compared to the epipelagic crustacean-eaters MP and RP. Based on the existing knowledge of Hg isotopic fractionation dynamics, we hypothesized that the observed MIF variations between penguin species are linked to distinct MeHg sources having undergone different degrees of photochemical reactions. Since no effect of trophic level was found for δ 202 Hg and ∆ 199 Hg values, the most plausible explanation of Hg isotopic variations is the consequence of species-specific foraging habitats within the marine environmental compartments. Hg isotopic values and penguin species-specific foraging habitats MIF ratios and estimation of the extent of photochemical processes Photochemical reactions of MeHg and iHg in the water column are recognised as the main driver of Hg MIF values and each process is characterized by a different ∆ 199 Hg/∆ 201 Hg ratio.

Since this ∆ 199 Hg/∆ 201 Hg ratio is assumed to be preserved in the food chain after MeHg assimilation by primary producers and during bio-magnification up the food web, it is used to identify mechanisms involving MIF variations [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF]. Theoretical slopes have been experimentally designed in aquatic systems [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF], corresponding to 1.36±0.02 for MeHg photodemethylation and 1.00±0.02 for iHg photoreduction in freshwater with natural DOC. Although studies in freshwater fish also reported Δ 199 Hg/Δ 201 Hg ratios close to 1.3 [START_REF] Laffont | Anomalous mercury isotopic compositions of fish and human hair in the Bolivian amazon[END_REF][START_REF] Gantner | Variations in stable isotope fractionation of Hg in food webs of Arctic lakes[END_REF]Sherman et al., 2013), slightly lower Δ 199 Hg/Δ 201 Hg ratios have been observed in marine organisms [START_REF] Senn | Stable isotope (N, C, Hg) study of methylmercury sources and trophic transfer in the northern gulf of Mexico[END_REF][START_REF] Point | Methylmercury photodegradation influenced by sea-ice cover in Arctic marine ecosystems[END_REF][START_REF] Gehrke | Sources of mercury to San Francisco Bay surface sediment as revealed by mercury stable isotopes[END_REF][START_REF] Day | Mercury stable isotopes in seabird eggs reflect a gradient from terrestrial geogenic to oceanic mercury reservoirs[END_REF][START_REF] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF][START_REF] Kwon | Mercury Isotope Study of Sources and Exposure Pathways of Methylmercury in Estuarine Food Webs in the Northeastern U.S[END_REF]. This effect may be the result of different ligands associated with Hg in aqueous solutions (such as organic matter, dissolved cations and the presence of halogens in seawater). Indeed, variable Δ 199 Hg/Δ 201 Hg ratios have been recently documented for MeHg photodemethylation under different types and concentrations of DOC [START_REF] Chandan | Mercury isotope fractionation during aqueous photoreduction of monomethylmercury in the presence of dissolved organic matter[END_REF], indicating that low concentrations of DOC relative to MeHg could involve lower ∆ 199 Hg/∆ 201 Hg ratios. The overall ∆ 199 Hg/∆ 201 Hg ratio for penguin blood samples (1.16±0.05) is consistent with those previously reported in marine fish and seabirds [START_REF] Senn | Stable isotope (N, C, Hg) study of methylmercury sources and trophic transfer in the northern gulf of Mexico[END_REF][START_REF] Day | Mercury stable isotopes in seabird eggs reflect a gradient from terrestrial geogenic to oceanic mercury reservoirs[END_REF][START_REF] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF]. Due to the predominance of MeHg in penguin blood, and assuming a potential diminution in MIF slope due to low DOC concentrations in subantarctic waters, the obtained ratio seems to indicate an accumulation of residual MeHg that has principally undergone photochemical demethylation.

Hg MIF in marine fish has been used to estimate the relative proportion of MeHg formed in the open ocean that is photochemically degraded prior its entry into the food web [START_REF] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF]). Thus, it has been shown to be an effective proxy of fish foraging depths [START_REF] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF].

Assuming MeHg photodemethylation as the major photochemical process, we estimated the percentage of presumed photodemetylated MeHg before entering the food web based on experimental studies [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF]) (detailed in Annexes). The overall difference between penguins (~3%) was surprisingly low when taking into account the range of habitats used by the penguins (from coastal benthic/pelagic feeders to mesopelagic feeders). However, considering the existence of higher amounts of DOC in benthic waters relative to the pelagic domains, more substantial differences should be found between sites, even for equivalent MeHg concentrations among the compartments. Previous studies in fish from the Gulf of Mexico estimated that MeHg in coastal fish was ∼10-20% degraded in contrast to oceanic fish whose percentage of photodemethylated MeHg was ∼40-65% [START_REF] Senn | Stable isotope (N, C, Hg) study of methylmercury sources and trophic transfer in the northern gulf of Mexico[END_REF]. Even higher in surface waters, photodegradation appears overall limited in the southern Indian Ocean subpolar waters compared to subtropical waters as a consequence of lower sunlight extent and slighter angle of incidence. However, further investigations are needed to evaluate if photodemethylation really varies across a larger latitudinal range (Chapter 4.2).

MIF variations with penguin foraging depths

The decrease of light penetration with depth leads to the reduction of the rate of MeHg photodemethylation with the subsequent less extent of Hg MIF [START_REF] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF]. Although Hg MIF is much more sensitive to photochemical reactions, photodemethylation also induces MDF by production of isotopically lighter MeHg and so that the remaining iHg is enriched in heavier isotopes [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF]). In the North Pacific Ocean, [START_REF] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF] documented a ∆ 199 Hg offset of ~5 ‰ between fish feeding at the surface mixed layer and at 600 m depth. Here, we aimed to assess the correlation between Hg MIF and penguin foraging depths in the Southern Ocean. Although MIF values of RP, MP and GP were in good agreement with the predicted relationship between ∆ 199 Hg values and foraging depth, this trend did not hold for KP since they 4.3 Blood Hg MIF versus MDF values (Δ 199 Hg versus δ 202 Hg) of subantarctic penguins from Possession Island, Crozet archipelago. Regression equation is y=-0.39x-0.93, R 2 =0.60, p<0.0001. Abbreviations: PF, Polar Front; GP, gentoo penguin; KP, king penguin; MP, macaroni penguin; RP, rockhopper penguin.

Tracing distinct MeHg sources over inshore-offshore and pelagic-benthic gradients Considering only Hg MIF values is not enough for the complete understanding of the exposure pathways to MeHg relative to specific foraging habitats in penguins. Therefore, we further explored and compared Hg isotopic values between penguins, both in a horizontal (inshore-offshore) and in a vertical (benthic-pelagic) dimension. Previous studies have documented Hg isotopic differences between coastal and oceanic marine organisms [START_REF] Senn | Stable isotope (N, C, Hg) study of methylmercury sources and trophic transfer in the northern gulf of Mexico[END_REF][START_REF] Day | Mercury stable isotopes in seabird eggs reflect a gradient from terrestrial geogenic to oceanic mercury reservoirs[END_REF][START_REF] Perrot | Higher massindependent isotope fractionation of methylmercury in the pelagic food web of Lake Baikal (Russia)[END_REF], indicating the existence of contrasted environmental MeHg sources with distinct Hg isotopic baseline and different extent of aquatic photochemistry. ‰) [START_REF] Gleason | Sources and cycling of mercury in the paleo Arctic Ocean from Hg stable isotope variations in Eocene and Quaternary sediments[END_REF]) and the Antarctic coasts (δ 202 Hg:-0.39±0.49 ‰; ∆ 199 Hg: 0.71±0.43 ‰) [START_REF] Zheng | Mercury Stable Isotopes in Ornithogenic Deposits As Tracers of Historical Cycling of Mercury in Ross Sea, Antarctica[END_REF]. Indeed, [START_REF] Zheng | Mercury Stable Isotopes in Ornithogenic Deposits As Tracers of Historical Cycling of Mercury in Ross Sea, Antarctica[END_REF] observed similar MIF values between historical sediment profiles and penguin and seal fresh faeces, suggesting that faeces were the dominant sources of Hg to the sediments at different time periods. Due to the representative penguin community in Crozet Islands, a significant fraction of Hg accumulated in coastal sediments of could be of ornithogenic origin and therefore display similar values as Antarctic sediments. GP presented the lowest δ 202 Hg and ∆ 199 Hg values, indicative of their higher exposure to benthic MeHg. Greater methylation in benthic waters may also implicate the production of isotopically lighter MeHg at depth and its accumulation in the benthic food web, contributing to the lower δ 202 Hg values and the high levels of MeHg observed in GP. The significant correlations between δ 13 C and Hg isotopic values seem to indicate a progressive transition from terrestrial to marine values both in a horizontal foraging trend (inshore-offshore) and vertical gradient (benthicpelagic). This suggests that the lower Hg isotopic values observed in benthic ecosystems may derive from in situ Hg methylation in sediment and reduced exposure to sunlight (due to depth) while a gradual increase in ∆ 199 Hg (and δ 202 Hg) was observed through offshore shallower areas as a consequence of a higher magnitude of photochemistry. Nevertheless, the low variations in Hg isotopes values between benthic and pelagic penguins compared to previous inshore-offshore values measured in other marine ecosystems [START_REF] Senn | Stable isotope (N, C, Hg) study of methylmercury sources and trophic transfer in the northern gulf of Mexico[END_REF][START_REF] Day | Mercury stable isotopes in seabird eggs reflect a gradient from terrestrial geogenic to oceanic mercury reservoirs[END_REF] seem to indicate a higher degree of mixture between benthic and pelagic MeHg sources. The remote location of the Crozet Islands, which are surrounded by deep oceanic waters (4000-5000 m), could explain a lower impact of the sediment-derived MeHg inputs compared to continental coastal zones.

Moreover, these islands have a plateau of around 150 km wide that interacts with different water masses derived from the Antarctic Circumpolar Current [START_REF] Pollard | Large-scale circulation around the Crozet Plateau controls an annual phytoplankton bloom in the Crozet Basin[END_REF]

Conclusions

The combination of blood isotopic values (Hg, C and N) of penguins together with the documented information about their feeding ecology allows characterizing the major factors explaining their levels of MeHg exposure in relationship with their contrasted ecological habits.

The observed Hg isotopic variations from benthic to pelagic penguins suggest the existence of mixing sources deriving from MeHg production at depth and increased photochemical processes when going to shallower offshore waters. Due to the scarce data on Hg isotopic composition in the Southern Ocean, further research is needed to investigate biogeochemical sources of MeHg formation and the processes leading to accumulation of Hg in biota from these remote ecosystems.

Main findings of this work:

-Penguins with contrasted feeding ecology were used to test the variability of Hg isotopic values according to their known feeding habitats and diets.

- Keywords: mercury, isotopes, Southern Ocean, seabirds, sources, biogeochemistry

Introduction

Although Antarctic and subantarctic environments have been usually perceived as remote areas untouched by anthropogenic pressure, they are reached by contaminants through ocean circulation and atmospheric transport [START_REF] Fitzgerald | Marine biogeochemical cycling of mercury[END_REF]. Mercury (Hg) is considered a globally distributed pollutant of major concern for humans and wildlife especially under its form of methylmercury (MeHg), a potent neurotoxin which is capable to accumulate in tissues of living organisms and to biomagnify within the food webs [START_REF] Mason | Uptake, toxicity and trophic transfer of Hg in a coastal diatom[END_REF][START_REF] Watras | Bioaccumulation of mercury in pelagic freshwater food webs[END_REF]. Hg biogeochemical cycle is highly complex and comprises several chemical processes and biological transformations, such as methylation/demethylation and oxidation/reduction reactions that control its abundance and speciation in the water column. In the aquatic compartment, Hg methylation is mainly produced by anaerobic micro-organisms [START_REF] Compeau | Sulfate-Reducing Bacteria: Principal Methylators of Mercury in Anoxic Estuarine Sedimentt[END_REF]. Due to its intense biomagnification along marine food webs, top predators are exposed to high levels of MeHg through the food pathway. Such high exposure poses severe health risks and has impact on the populations (e.g. [START_REF] Tartu | To breed or not to breed: endocrine response to mercury contamination by an Arctic seabird[END_REF]Goutte et al. 2014a).

In the Southern Ocean, particularly in the Indian Ocean sector, various studies have reported worryingly increasing levels of Hg bioaccumulation in biota, especially in top predators (e.g., [START_REF] Bocher | Influence of the diet on the bioaccumulation of heavy metals in zooplankton-eating petrels at Kerguelen archipelago, Southern Indian Ocean[END_REF]Carravieri et al., 2014bc). Nevertheless, scarce information is available on Hg concentrations, spatial distribution and speciation in the water column from this remote area of the world ocean [START_REF] Cossa | Mercury in the Southern Ocean[END_REF][START_REF] Lamborg | A global ocean inventory of anthropogenic mercury based on water column measurements[END_REF][START_REF] Canario | Mercury and methylmercury in the Atlantic sector of the Southern Ocean[END_REF]. Despite its distance from industrial sources of Hg contamination, the most important pathways for Hg transport to the Southern Ocean and Antarctic zones involve a combination of atmospheric input, oceanic currents and sea ice processes that can explain the consequential concentrations of MeHg observed in these remote areas [START_REF] Cossa | Mercury in the Southern Ocean[END_REF]. Observations on Hg species distribution in the Southern Ocean suggested distinct features of Hg cycle such as net atmospheric Hg inputs on surface water near the ice edge, Hg enrichment in brine during sea ice formation and net Hg methylation [START_REF] Cossa | Mercury in the Southern Ocean[END_REF]. Geographical trends on MeHg in southern oceanic waters suggested more elevated concentrations in Antarctic waters (including the sea ice zone) whereas progressively decreasing MeHg amounts were observed in subantarctic and subtropical seawaters [START_REF] Cossa | Mercury in the Southern Ocean[END_REF]. Surprisingly, this north to south increasing gradient is opposed to the observed trend in tissues of seabirds that increase from Antarctic, through subantarctic to subtropical populations [START_REF] Blévin | Wide range of mercury contamination in chicks of southern ocean seabirds[END_REF]Carravieri et al , 2014b;Goutte et al., 2014, Carraveri et al., 2017).

Understanding the Hg cycle in the Southern Ocean and tracing the processes that transform Hg on its various chemical forms are key in determining how it is transported to these remote areas and what is its fate and impact once it gets there. The use of Hg stable isotopes offers a new and powerful perspective for exploring the cycle of this element and have demonstrated to be useful to depict Hg sources or quantify its reactivity within different environmental compartments [START_REF] Blum | Mercury Isotopes in Earth and Environmental Sciences[END_REF]). Hg has seven isotopes which experience mass-dependent and massindependent isotopic fractionation (MDF and MIF, respectively). Most biogeochemical processes such as volatilisation, reduction, demethylation and methylation induce MDF (Rodriguez [START_REF] Gonzalez | Species-Specific Stable Isotope Fractionation of Mercury during Hg ( II ) Methylation by an Anaerobic Bacteria ( Desulfobulbus propionicus ) under Dark Conditions[END_REF][START_REF] Kritee | Mercury Stable Isotope Fractionation during Reduction of Hg(II) to Hg(0) by Mercury Resistant Microorganisms[END_REF][START_REF] Kritee | Mass dependent stable isotope fractionation of mercury during mer mediated microbial degradation of monomethylmercury[END_REF]Zheng and Hintelmann 2010a). Only photochemical demethylation and reduction processes have been identified as producing MIF on odd Hg isotopes (199,201) at significant levels (Zheng and Hintelmann 2009a;[START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF][START_REF] Sherman | Mass-independent fractionation of mercury isotopes in Arctic snow driven by sunlight[END_REF][START_REF] Perrot | Higher massindependent isotope fractionation of methylmercury in the pelagic food web of Lake Baikal (Russia)[END_REF][START_REF] Gantner | Variations in stable isotope fractionation of Hg in food webs of Arctic lakes[END_REF]. Consequently, the combination of Hg MDF and MIF signatures provide interesting and complementary information for Hg potential sources and transformation pathways.

Seabirds are exposed to large quantities of Hg via the marine food webs and have been identified as effective biomonitors of Hg contamination, particularly due to ecological and practical reasons [START_REF] Burger | Marine Birds as Sentinels of Environmental Pollution[END_REF][START_REF] Furness | Seabirds as monitors of the marine environment[END_REF]. Indeed, marine birds are generally colonial and philopatric, enabling to take samples during their reproduction period and to follow numerous individuals simultaneously and repeatedly over time. Moreover, they are particularly powerful samplers of ocean environments, exploiting a wide variety of prey and a great diversity of foraging habitats and thus providing access to different marine compartments otherwise difficult to sample. Numerous studies have focused on the biomonitoring of Hg using seabirds' tissues, preferentially by analyses of blood and feathers which can be nonlethally collected. Due to their ecological characteristics, chicks and penguins are the most pertinent seabird models for tracing Hg contamination. Indeed, they reflect local contamination since adult penguins are restricted to more precise areas than migratory flying seabirds, and chicks are fed with prey from waters around the colony [START_REF] Blévin | Wide range of mercury contamination in chicks of southern ocean seabirds[END_REF]Carravieri et al., 2014c).

In this research work, the objective was to investigate Hg dynamics over a wide latitudinal gradient across the Southern Indian Ocean, from Antarctica to the subtropics. Analyses of Hg isotopic composition were performed in blood tissues of two seabird models from the French Southern and Antarctic Territories: skua chicks (2 species) and adult penguins (6 species).

Although both feathers and blood have been validated as effective tracers for Hg isotopic studies (Chapter 3.1), blood samples were chosen for this study since they provide more-specific Hg isotopic signatures and hence, a better discrimination between populations. As a consequence of different geographical and climatic parameters, we expected to observe significant variations in Hg isotopic signatures between distant southern regions that could provide new insights for tracking Hg cycle in the Southern Ocean.

Material and methods

Sampling sites

Sample collection was conducted during the austral summer 2011-2012 (from October to February) in four sites of the French Southern and Antarctic Territories, depending on the seabird species breeding colonies: Adélie Land (66°40'S, 140°10'E), Crozet Islands (46°26'S, 51°45'E), Kerguelen Islands (49°21′S, 70°18′E) and Amsterdam Island (37°50'S, 77°31'E). Chicks from two species of skua (Antarctic and subantarctic skua) and adults from five species of penguins (Adélie, king, macaroni, eastern rockhopper and northern rockhopper) were investigated. Exact dates of sample collection of each seabird species are specified in Annexes (Table 4.S9). Blood samples were collected and centrifuged as detailed elsewhere [START_REF] Carravieri | From Antarctica to the subtropics : Contrasted geographical concentrations of selenium , mercury , and persistent organic pollutants in skua chicks ( Catharacta spp .)[END_REF]. Details of ecological characteristics of each seabird species at the different locations are specified in Annexes.

Reference materials, sample preparation and analytical description

For the validation of the analytical results, two certified reference material were analysed: human hair IAEA-086 and tuna fish ERM-CE-464. An internal reference sample was prepared with pool samples collected from different individuals of king penguin from Crozet Islands: RBC-KP (red blood cells) and was analysed at each analytical session.

For Hg speciation analyses, Hg was extracted from blood samples (0.10-0.15 g) by alkaline microwave digestion with 5 mL of tetramethylammonium hydroxide (25% TMAH in H2O, Sigma Aldrich) [START_REF] Rodrigues | Mercury speciation in whole blood by gas chromatography coupled to ICP-MS with a fast microwave-assisted sample preparation procedure[END_REF]. Details of the extraction method, analysis and quantification of Hg species are included in (Chapter 3.1). For Hg isotopic analyses, blood samples (0.05-0.10 g) were digested with 3 or 5 mL of HNO3 acid (65%, INSTRA quality) after a predigestion step overnight at room temperature and later extraction in Hotblock at 75ºC during 8 h (6 h in HNO3 and 2 h more after the addition of 1/3 of the total volume of H2O2 (30%, ULTREX quality)).

Details of Hg isotopic analyses are included in (Chapter 3.1).

Statistical tests

Statistical tests were performed using RStudio. Before analyses, data were checked for normality of distribution and homogeneity of variances using Shapiro-Wilk and Breusch-Pagan tests, respectively. According to this, parametrical (One-way ANOVA) or non-parametrical tests (Kruskal-Wallis with Conover-Iman test) were performed. Statistically significant results were set at α= 0.05.

Results

Hg concentrations and Hg speciation trends

Hg accumulation in seabird tissues showed a latitudinal gradient with THg concentrations increasing from Antarctic through subantarctic to subtropical populations of both seabird models.

MeHg was the dominant Hg form accumulated in blood tissues for all the individuals (92±7%), meaning that blood Hg isotopic composition corresponds essentially to MeHg isotopic values.

Skua blood and penguin blood exhibited significantly different MeHg concentrations between sites (Kruskal-Wallis, H=31.09 and 43.05, respectively, both p<0.0001). Skua blood MeHg concentrations increased from 0.51 to 3.78 µg•g -1 (n=40), respectively for Antarctic and subtropical populations. Penguin blood MeHg also increased from Antarctic to subtropical populations (0.43 to 1.95 µg•g -1 , respectively, n=51), in the order AP < RP < MP < KP < NRP (Table 4.2). Measured blood δ 202 Hg values followed the predicted theoretical MDF line whereas significant MIF of odd Hg isotopes (∆ 199 Hg and ∆ 201 Hg) was observed for all the individuals (Figure 4.S5), indicating that MeHg accumulated in seabird tissues had undergone photochemical processes before its incorporation into the food web.

Correlations between Hg isotopic signatures and latitudinal trends

A strong correlation was found between Δ 199 Hg and δ 202 Hg values in both skuas and penguins (R 2 =0.76 and 0.61, respectively, both p<0.0001;

Discussion

The solid relationship of Δ 199 Hg and δ 202 Hg values with latitude (and between Δ 199 Hg and δ 202 Hg themselves) could be explained by two possible factors: i) different sources of Hg with distinct isotopic signatures (MDF and MIF) or ii) different processes before Hg incorporation and/or during uptake and bioaccumulation in the food web that produce both Hg MDF and MIF across the different sites. We expected that the most plausible explanation would be a combination of both factors, meaning that there is a different Hg isotopic baseline (due to distinct MeHg sources) and an influence of processes associated to geographically changing parameters which would induce Hg isotopic fractionation across the study sites. In the following sections, potential sources and processes explaining the observed Hg isotopic latitudinal trends were conceptually proposed, tested and discussed.

Potential Hg sources affecting MDF and MIF variations over latitude

The Southern Ocean ecosystems present a Hg cycle principally characterised by inputs from oceanic currents, net Hg atmospheric deposition to the ocean surface and substantial net Hg methylation [START_REF] Pacyna | Global emission of mercury to the atmosphere from anthropogenic sources in 2005 and projections to 2020[END_REF][START_REF] Cossa | Mercury in the Southern Ocean[END_REF].

Different water masses of the studied area of the Southern Ocean derive from distinct origins, such as the Antarctic circumpolar current, Polar Front and subtropical waters, then present contrasted characteristics (temperature, biological productivity, etc.) and are exposed to different sunlight conditions that could affect the magnitude of Hg photochemical processes. Furthermore, assuming greater Hg reduction and volatilisation extents as a result of higher temperature, subtropical waters could exhibit heavier MeHg isotopic signatures than Antarctic waters.

Nevertheless, seawater Hg isotopic information is still scarce with only one study in the Arctic Canadian coastal area that exhibited negative δ 202 Hg values (-2.85 to -1.10 ‰) and mostly slightly positive Δ 199 Hg values (< 0.37 ‰).

In Antarctic zones, the sequestration of atmospheric Hg in the sea ice compartment is supposed to be responsible of an additional input of Hg to the ocean [START_REF] Gionfriddo | Microbial mercury methylation in Antarctic sea ice[END_REF][START_REF] Cossa | Mercury in the Southern Ocean[END_REF]. Polar Regions are characterized by unique phenomena called atmospheric mercury depletion events (AMDEs) which consist in rapid oxidation of gaseous Hg 0 in air through reactions with halogens such as bromine [START_REF] Lindberg | Dynamic oxidation of gaseous mercury in the arctic troposphere at polar sunrise[END_REF][START_REF] Ariya | Reactions of gaseous mercury with atomic and molecular halogens: Kinetics, product studies, and atmospheric implications[END_REF]. Supposing that we could extrapolate Arctic AMDEs for interpreting the dynamics in Antarctic ecosystem, substantial deposition of oxidized Hg in Antarctic snowpack could have an influence in Hg MIF signatures of Antarctic seabirds. In the Arctic, snow samples impacted by AMDEs in Alaska displayed near to zero δ 202 Hg values (from -0.5 to 0.7 ‰) and highly negative ∆ 199 Hg values (down to -5 ‰) [START_REF] Sherman | Mass-independent fractionation of mercury isotopes in Arctic snow driven by sunlight[END_REF][START_REF] Obrist | Tundra uptake of atmospheric elemental mercury drives Arctic mercury pollution[END_REF]. Arctic ice cores reported negative δ 202 Hg values (down to -2.48‰) and mostly near-to zero or positive Δ 199 Hg (up to 2.44‰), opposite to Arctic snow during AMDEs but overlapping with Hg 0 samples [START_REF] Sherman | Mass-independent fractionation of mercury isotopes in Arctic snow driven by sunlight[END_REF] and surface Arctic seawater (Štrok et al., 2015). Moreover, there are additional processes that could bring oxidized Hg to Antarctic coasts by katabatic winds (Angot et al., 2016b). Direct measurements of wet deposition (iHg) have documented slightly negative δ 202 Hg (-0.44 to -4.27 ‰) and positive Δ 199 Hg values (0.19 to 1.16 ‰). Significant even-MIF of 200 Hg has also been observed in precipitation samples, leading to slightly negative Δ 200 Hg in the vapour Hg 0 and consistently positive Δ 200 Hg in precipitation (Sherman et al. 2012b;[START_REF] Chen | Unusual fractionation of both odd and even mercury isotopes in precipitation from Peterborough, ON, Canada[END_REF][START_REF] Štrok | The mercury isotope composition of Arctic coastal seawater[END_REF][START_REF] Demers | Mercury isotopes in a forested ecosystem: Implications for airsurface exchange dynamics and the global mercury cycle[END_REF][START_REF] Gratz | Isotopic composition and fractionation of mercury in Great Lakes precipitation and ambient air[END_REF][START_REF] Rolison | Isotopic composition of speciesspecific atmospheric Hg in a coastal environment[END_REF][START_REF] Wang | Massdependent and mass-independent fractionation of mercury isotopes in precipitation from Guiyang, SW China[END_REF][START_REF] Yuan | Large Variation of Mercury Isotope Composition During a Single Precipitation Event at Lhasa City, Tibetan Plateau, China[END_REF], hypothetically induced by oxidation processes of atmospheric Hg 0 [START_REF] Chen | Unusual fractionation of both odd and even mercury isotopes in precipitation from Peterborough, ON, Canada[END_REF]). An influence of wet deposition could thus affect iHg isotopic baseline in a given ecosystem.

However, the slight differences in Δ 199 Hg values between Antarctic and the subantarctic seabird communities seem to indicate an absence of a high influence of Hg inputs from snow or sea ice melting in Antarctic food web. These results seems to indicate that atmospheric deposition is not substantial enough or cannot be detected in the Hg accumulated in the blood of skua and penguins and sea ice melting and wet deposition seems unlikely an important factor of Hg latitudinal trends.

Sediment erosion and its mobilization to opened areas could also contribute to Hg release into the aquatic compartment in water masses surrounding the Antarctic continent or the coastal zones in the vicinity of the subantarctic islands. Soils, sediments and vegetation from remote areas samples typically report negative δ 202 Hg and close-to-zero ∆ 199 Hg values [START_REF] Obrist | Tundra uptake of atmospheric elemental mercury drives Arctic mercury pollution[END_REF][START_REF] Gleason | Sources and cycling of mercury in the paleo Arctic Ocean from Hg stable isotope variations in Eocene and Quaternary sediments[END_REF]. Antarctic ornithogenic sediments showed negative δ 202 Hg values (around -0.5 ‰) and slightly positive ∆ 199 Hg (~0.71 ‰) [START_REF] Zheng | Mercury Stable Isotopes in Ornithogenic Deposits As Tracers of Historical Cycling of Mercury in Ross Sea, Antarctica[END_REF]. On the assumption that a higher interaction between coastal sediment and biota is produced in the Antarctic zone near the continent comparing to subantarctic and subtropical islands, Antarctic seabirds may exhibit lower δ 202 Hg and ∆ 199 Hg values relative to subantarctic and subtropical communities. However, Hg sediment inputs in coastal waters of the subantarctic and subtropical islands are not negligible, since benthic penguin populations have been shown to be also influenced by sediment-derived Hg at the local scale of Crozet archipelago (Chapter 4.1). Therefore, continental influence has to be considered likely to ice melting under climate warming conditions. It is however unlikely a major factor driving the observed latitudinal trend in Hg isotopes.

MIF: effect of different photochemistry over latitude Spatial variability in MeHg photodemethylation at a large scale Because significantly positive Hg MIF is assumed to be the consequence of photochemical processes before incorporation into the food webs [START_REF] Bergquist | The odds and evens of mercury isotopes: Applications of mass-dependent and mass-independent isotope fractionation[END_REF][START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF], the variability of ∆ 199 Hg values among the three geographically distant areas (Antarctic, subantarctic and subtropical waters) was considered to be mainly the consequence of distinct surface ocean photochemistry conditions. In this study, we covered a southern to northern gradient that extends from Polar Regions off the Antarctic continent to subtropical oceanic environments, through which different climatic factors may contribute to the observed latitudinal Hg MIF pattern.

Aquatic photochemical MeHg demethylation is known to induce an increase in MIF (Δ 199 Hg and Δ 201 Hg) and MDF values (δ 202 Hg) in the residual MeHg pool, resulting in a Δ 199 Hg/Δ 201 Hg ratio of 1.36±0.04, as reported by experimental studies in freshwater with natural content of dissolved organic carbon (DOC) [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF]. Meanwhile, photochemical reduction of iHg originated a Δ 199 Hg/Δ 201 Hg ratio of 1.00±0.01 [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF].∆ 199 Hg/∆ 201 Hg slopes obtained for blood samples of penguins (1.21±0.04) and skuas

(1.14±0.04) are in good agreement with those previously obtained in marine fish and birds [START_REF] Senn | Stable isotope (N, C, Hg) study of methylmercury sources and trophic transfer in the northern gulf of Mexico[END_REF][START_REF] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF][START_REF] Point | Methylmercury photodegradation influenced by sea-ice cover in Arctic marine ecosystems[END_REF][START_REF] Day | Mercury stable isotopes in seabird eggs reflect a gradient from terrestrial geogenic to oceanic mercury reservoirs[END_REF]. 4.6. Hg MIF signatures (Δ 199 Hg versus Δ 201 Hg) for blood samples of skua chicks (left) and penguins (right) from the Southern Ocean. See Figure 4.4 for legend. The solid line represents the Δ 199 Hg/Δ 201 Hg slope of the samples. The red dashed line represents the theoretical Δ 199 Hg/Δ 201 Hg slope for MeHg photodemethylation in the water column and the blue dashed line represents the theoretical Δ 199 Hg/Δ 201 Hg slope expected for photoreduction of iHg in the water column [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF]. Assuming that MeHg photodegradation is the dominant photochemical process before MeHg assimilation in seabird tissues, we estimated the fraction of photodemethylated MeHg of each area based on three different experimental models [START_REF] Chandan | Mercury isotope fractionation during aqueous photoreduction of monomethylmercury in the presence of dissolved organic matter[END_REF][START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF] (detailed in Annexes) and considering both different types and concentrations of DOC (MeHg:DOC ratios) and also the amount of reduced sulphur content in organic matter (MeHg:Sred-DOC ratios). Basing our estimations on the most similar conditions to our study areas, we obtained that the difference on the extent of photodemethylated MeHg from Antarctic and subantarctic to subtropical zones was in the order of 2% (based on Chandan et al., 2015 models) and 5% (based on [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF]) (Table 4.S5). No substantial changes in latitudinal variations were observed in the calculated results when changing these parameters.

Calculated photodemethylation extents in Antarctic coastal ecosystem by the same experimental approximation was similar to our findings [START_REF] Zheng | Mercury Stable Isotopes in Ornithogenic Deposits As Tracers of Historical Cycling of Mercury in Ross Sea, Antarctica[END_REF]. It is noteworthy to highlight that the applied fractionation models represent more realistic environmental conditions (low MeHg:

Sred-DOC ratios) than previous experimental studies so that they are more representative of the processes occurring under natural conditions in the environment. Nevertheless, these estimations are also based on laboratory experimental models so that the degree of extrapolation must be taken into account. The insignificant variations in MeHg photodemethylation extent over the studied latitudinal gradient (~ 2%) suggests that MeHg photodemethylation is, for instance, not influencing the observed increasing MeHg concentrations from Antarctic to subtropical seabird communities.

The slight MIF latitudinal variations between such distant sites of the Southern Ocean strongly suggest that the extent of photodemethylation is closely related to the distinct conditions for the photochemical processes to occur. Even if the annual mean day photoperiod is similar in the different sites (~12h, Jasmine et al., 2009), Hg isotopic signatures of penguin blood samples reflect only the summer conditions when the daily photoperiod is longer in Antarctic zones.

However, the different angles of solar incidence between the different latitudes affect the UV/visible light and the transmission in surface waters that control directly the extent of the various photochemical processes [START_REF] Rose | Effects of ultraviolet radiation on mercury isotope fractionation during photo-reduction for inorganic and organic mercury species[END_REF]. As it was commented above, the DOC quality and quantity is a controlling factor of the photochemical demethylation of MeHg and the resulting MIF [START_REF] Chandan | Mercury isotope fractionation during aqueous photoreduction of monomethylmercury in the presence of dissolved organic matter[END_REF]. For instance, we have shown that DOC concentrations would rather be homogenous over the different latitudes [START_REF] Tremblay | Origin and fate of particulate and dissolved organic matter in a naturally iron-fertilized region of the Southern Ocean[END_REF][START_REF] Canario | Mercury and methylmercury in the Atlantic sector of the Southern Ocean[END_REF][START_REF] Ogawa | Vertical distributions of dissolved organic carbon and nitrogen in the Southern Ocean[END_REF] but the higher biological productivity usually observed in Antarctic and the Polar Front zones compared to subtropical waters may lead to different DOM quality and photochemical reactivity. For example, we can expect higher photodemethylation efficiency in the southern latitudes which may also compensate the lack of efficient solar radiation (UV/visible light). Furthermore, it is important to consider that specific foraging habitats between seabird species inhabiting the same latitudes could also induce variations in Hg MIF, especially when using penguins as models (Chapter 4.1). extent in winter as a result of lower solar radiation and a potentially higher influence of sea ice cover, as already demonstrated in previous studies in the Arctic [START_REF] Point | Methylmercury photodegradation influenced by sea-ice cover in Arctic marine ecosystems[END_REF][START_REF] Masbou | Hg Stable Isotope Time Trend in Ringed Seals Registers Decreasing Sea Ice Cover in the Alaskan Arctic[END_REF]. However, greater foraging depths of emperor penguins could also affect Δ 199 Hg variations between summer and winter seabird populations [START_REF] Wienecke | Extreme dives by free-ranging emperor penguins[END_REF]). experimentally obtained for photochemical demethylation [START_REF] Chandan | Mercury isotope fractionation during aqueous photoreduction of monomethylmercury in the presence of dissolved organic matter[END_REF], we estimated that around 0.3‰ of the total δ 202 Hg enrichment may also be induced by this photochemical process (details in Annexes). This means that the remaining 1.0-1.3 ‰ of latitudinal δ 202 Hg variation may be the consequence of different Hg trophic sources and/or biogeochemical processes inducing an enrichment in heavier isotopes when decreasing latitude.

Temporal variability in MIF values

Apart the potential sources mentioned above, kinetically process-driven fractionation have also been found to alter Hg isotopic composition. For instance, solar radiation can directly affect water surface temperatures, and the presence of ice cover can also create a thermal barrier in surface water masses on the vicinity of the Antarctic continent. There are at least three potential biogeochemical pathways that could produce an enrichment in heavier Hg isotopes (δ 202 Hg) as a consequence of progressively increasing water temperature with latitude: i) reduction and volatilisation of inorganic Hg 0 from the aquatic compartment to the atmosphere leading to a pool of dissolved Hg 0 enriched in heavier isotopes, ii) increasing microbial methylation/demethylation ratios leading to the enrichment of the remaining MeHg in heavier Hg isotopes and/or iii) MeHg assimilation efficiency, trophic processes or metabolic responses inducing Hg MDF at different degrees.

Reduction and volatilisation

We postulate that higher temperature in subtropical water comparing to Antarctic waters would result in a higher degree of biotic reduction and greater volatilisation extent. This would thus produce a residual pool of Hg 2+ enriched in heavier isotopes in the aqueous phase, which would be coherent with the MDF enrichment presently found with decreasing latitude. However, this fact would be contradictory with the higher MeHg concentrations at higher latitudes reported for the water column [START_REF] Cossa | Mercury in the Southern Ocean[END_REF]. [START_REF] Kritee | Mercury Stable Isotope Fractionation during Reduction of Hg(II) to Hg(0) by Mercury Resistant Microorganisms[END_REF] demonstrated that Hg bacterial reduction result in Hg MDF leaving the remaining inorganic fraction enriched in heavy isotopes and defining a Rayleigh system for which the fractionation factor (expressed as difference of δ 202 Hg) appeared to be between -1.3 and -2.0 ‰. A substantially higher biological reduction in subtropical ecosystems relative to Antarctic zones could therefore explain a part of latitudinal MDF variations. Hg volatilisation process of aqueous Hg 0 into gaseous Hg 0 has been found to induce MDF with a low fractionation factor of the order of 0.47 ‰ [START_REF] Zheng | Mercury isotope fractionation during volatilization of Hg(0) from solution into the gas phase[END_REF]). Based on water temperature recorded on the approximate latitudinal sites [START_REF] Cossa | Mercury in the Southern Ocean[END_REF][START_REF] Canario | Mercury and methylmercury in the Atlantic sector of the Southern Ocean[END_REF], we estimated the fraction of volatilised Hg by calculation of Henry's law constant (KH) reported by experimental studies on saline water [START_REF] Andersson | Determination of Henry's law constant for elemental mercury[END_REF]) (detailed in Annexes).

We estimated that volatilisation processes could only contribute to 0.03 ‰ of MDF variation, indicating that this conceptual hypothesis is not sufficient to explain total Hg MDF differences across the investigated latitudinal gradient.

Biotic Hg methylation and demethylation

Water temperature and concentration of organic matter are two factors assumed to influence methylation activity [START_REF] Fitzgerald | Marine biogeochemical cycling of mercury[END_REF]. The binding of iHg to sinking organic particles facilitates its transport through depth, where it can be methylated by microorganisms with rates that will depend on the type of microbial communities associated with phytoplankton degradation [START_REF] Heimbürger | Methyl mercury distributions in relation to the presence of nano-and picophytoplankton in an oceanic water column (Ligurian Sea, North-western Mediterranean)[END_REF]. Based on our findings, we hypothesized that higher MeHg concentrations in subtropical seabirds were possibly the result of higher net biotic methylation occurring there in the mesopelagic zone. Experimental measurements of isotopic fractionation during net MeHg methylation (i.e simultaneous methylation and demethylation) by sulphatereducing bacteria suggested a fractionation factor in the order of -2.5 ‰ [START_REF] Perrot | Identical Hg Isotope Mass Dependent Fractionation Signature during Methylation by Sulfate-Reducing Bacteria in Sulfate and Sulfate-Free Environment[END_REF], so that the theoretical magnitude of isotopic enrichment of this process could largely explain the Ocean. An increasing methylation/demethylation ratio from Antarctic to subtropical zones would produce an enrichment in heavier isotopes of the residual pool of MeHg + (i.e. assuming steady state) with decreasing latitude. This supposition could be thus in good agreement with our isotopic latitudinal trends findings and with the higher MeHg levels found in subtropical seabirds compared to those from Antarctic zones. Hg bioavailability for primary producers. For instance, colder and low productivity systems are assumed to be more susceptible to Hg bioaccumulation due to the inhibition of growth biodilution, slow MeHg excretion rates and simpler food webs with less diversity of species [START_REF] Lavoie | Biomagnification of Mercury in Aquatic Food Webs: A Worldwide Meta-Analysis[END_REF]). In our context, the difference in water temperature between sites are not an explanatory factor since lower MeHg concentrations were observed in Antarctic seabirds comparing to the other communities. However, this could be due to an influence of higher productivity in Antarctic and Polar Front zones as a result of seasonal release of nutrients and algal blooms during austral summer [START_REF] Sullivan | Dsributions of Phytoplankton Blooms in the Southern Ocean[END_REF][START_REF] Sokolov | Chlorophyll blooms in the Antarctic Zone south of Australia and New Zealand in reference to the Antarctic Circumpolar Current fronts and sea ice forcing[END_REF], which is enhanced by sea ice melting in Antarctic coastal zones [START_REF] Sokolov | Chlorophyll blooms in the Antarctic Zone south of Australia and New Zealand in reference to the Antarctic Circumpolar Current fronts and sea ice forcing[END_REF][START_REF] Riaux-Gobin | Environmental conditions, particle flux and sympagic microalgal succession in spring before the sea-ice break-up in Adélie Land, East Antarctica[END_REF]. This phenomenon could thus reduce the uptake of MeHg at the basal food web [START_REF] Pickhardt | Algal blooms reduce the uptake of toxic methylmercury in freshwater food webs[END_REF] inducing a biomass dilution of Hg concentrations [START_REF] Karimi | Stoichiometric controls of mercury dilution by growth[END_REF][START_REF] Chen | Patterns of Hg bioaccumulation and transfer in aquatic food webs across multi-lake studies in the northeast US[END_REF] and lower Hg concentrations in final predators. Moreover, this effect could potentially explain the increase of non-assimilated MeHg pool in the aquatic compartment, coherent with [START_REF] Cossa | Mercury in the Southern Ocean[END_REF]. Additionally, a higher complexity of the food web could explain Hg increasing levels from Antarctic to subtropical ecosystems, as already hypothesized by [START_REF] Carravieri | Seabirds as bioindicators of Southern Ocean ecosystems: concentrations of inorganic and organic contaminants, ecological explanation and critical evaluation[END_REF], and also induce a higher enrichment in MDF as a consequence of higher number of trophic levels. Antarctic ecosystem is characterised by a less complex food web in which predators feed on key available species, such as the Antarctic krill Euphausia superba [START_REF] Brasso | A comprehensive assessment of mercury exposure in penguin populations throughout the Southern Hemisphere: Using trophic calculations to identify sources of population-level variation[END_REF][START_REF] Cherel | Isotopic niches of emperor and Adélie penguins in Adélie Land, Antarctica[END_REF]. By contrast, subantarctic and subtropical ecosystems dispose of a larger diversity of prey species including four species of Euphausiids (Cherel et al. 2007;[START_REF] Bost | Where do penguins go during the interbreeding period? Using geolocation to track the winter dispersion of the macaroni penguin[END_REF], constituting a more complex food web.

The lower latitudinal MDF enrichment observed for skua chicks in comparison with penguins could potentially be linked to different level of Hg exposure or contrasted metabolic response.

Although higher blood Hg concentrations were obtained for skua chicks relative to penguins at the different latitudes, lower δ 202 Hg values were exhibited by skuas despite their higher trophic level. These observations could be explained by potential less efficient metabolic processes in chicks (such as hepatic demethylation) as a consequence of age, which could induce lower in vivo

Conclusion

The Chapter 5.

Conclusions and perspectives

Chapter Conclusions and perspectives

The present doctoral work includes new and valuable information about the exposure pathways of MeHg accumulated in seabirds that help identifying the processes involved in the Hg geochemical cycle in the Southern Ocean. This dissertation has been focused in the investigation, for the first time, on the origin of Hg, the production processes of MeHg and its transfer to marine -For absolutely all the seabird individuals analysed (adults and chicks), MeHg appeared to be the major species independently of total Hg concentrations.

-All the single feathers displayed more than 80% of Hg as MeHg and represents on average 92±2%. This finding was in good agreement with previous observations and with the

A) B)
evidence that seabirds excrete MeHg in moulting feathers which therefore represents a very efficient Hg detoxification strategy.

-However, this study also permitted to highlight that non-negligible amounts of iHg were present in feathers from some individuals, meaning that it cannot be considered the analysed THg in feathers equals MeHg.

-The precise quantification of Hg species is essential when Hg isotopic analyses are performed afterwards because Hg speciation strongly influences the measured Hg isotopic signatures.

-Moreover, the application of methods measuring both Hg compounds' concentrations in feathers is recommended in cases of accidental iHg contamination, as already detected in feather museum collections.

5.2 Blood vs feathers: which level of information is provided by Hg isotopes of each bioindicator tissue?

The choice of the best bioindicator tissue for Hg contamination is fundamental for interpreting the level of Hg environmental contamination. Since blood and feathers can be nonlethally collected, a deep investigation of Hg isotopic signatures in blood and feathers was performed in chicks using skuas and in adults using penguins (Chapter 3.1), permitting to conclude that:

-In chicks, blood and feathers had identical Hg isotopic values, meaning that the two tissues can be easily compared in interpretative terms without any correction factor. Since chicks present a simultaneous moult of all the feathers before fledging, blood and feathers are both representative of a well-defined period of Hg uptake during the breeding period when they are fed by their parents with local prey. Therefore both tissues provide the same information by Hg isotopic analyses and can be sampled indifferently in chicks during fieldwork.

-In adult penguins, Hg isotopic composition differed between blood and feathers, most likely as a consequence of different tissue-specific temporal windows of Hg exposure.

Feathers of adult penguins are supposed to be representative of local conditions around the colonies year round. However, the difference between feather and blood Hg isotopic signatures reflects changes in foraging habitats and or prey in the inter-breeding period, as a result of integration of Hg from diverse sources during the whole year (between two moults). Although this pattern was not accomplished in the case of giant petrel, mostly due to a "reservoir effect", it was concluded that extrinsic factors in terms of temporal and geographical aspects of Hg exposure highly affects Hg isotopic information between feathers and internal tissues, as previously observed between feathers and blood in adult individuals (Chapter 3.1).

- Although some metabolic aspects were identified according to Hg speciation and isotopic variations among tissues, the redistribution and excretion pathways of Hg in seabirds need to be further investigated in order to clarify their metabolic responses. For example, it is still not clear if the isotopically heavier fraction of non-demethylated MeHg in the liver is later redistributed into muscle, thus enriching δ 202 Hg values in this storage organ, or if it is directly excreted in feathers (composed of the isotopically heaviest fraction). In the first case, MeHg stored in muscles would be assumed to be a combination of non-metabolised MeHg uptaken from diet and the non-demethylated MeHg fraction redistributed from liver, indicating that muscle is an intermediate storage organ influenced by Hg demethylation in liver and MeHg excretion by feathers. In the second case, the preferential excretion of isotopically heavier MeHg through feathers would be easily explained as the excretion of MeHg remaining fraction from demethylation. Another observation that needs to be clarified in future studies is the presence of high fractions of iHg in muscles observed in giant petrels, especially in old individuals. This could suggest a redistribution of iHg from livers to muscles as a result of a high MeHg demethylation rate (and subsequent iHg concentrations accumulated in liver during their life) or due to high amounts of iHg by dietary uptake since they feed on seabird or mammal carrion (livers).

Figure 5.3. Schematic design of the metabolic processes inducing MDF in the organisms o birds and the reflect of distinct Hg sources as a result of different times of integration between internal tissues (long-term accumulation) and feathers (seasonal acquisition during interbreeding periods).

Perspectives. MDF signatures (δ 202 Hg) were significantly different among tissues of seabirds and mainly dependent on the fraction of MeHg and were thus assumed as a valuable tool for explore metabolic processes in response to Hg contamination (demethylation in liver, excretion by feathers, transfer among tissues, etc). As a consequence, an interesting perspective would be the performance of Hg-Compound Specific Isotopic Analysis (CSIA) that consists in the simultaneous determination of Hg isotopic signatures in both MeHg and iHg by the coupling GC-MC-ICPMS (objective of TIMOTAAF project, EC2CO, CNRS, 2 nd year). This approach, satisfactory applied in internal tissues of marine mammals [START_REF] Perrot | Natural Hg isotopic composition of different Hg compounds in mammal tissues as a proxy for in vivo breakdown of toxic methylmercury[END_REF], would provide information on the influence of metabolism responses on in vivo distribution of Hg. Indeedn this is the objective For example, similar Δ 199 Hg isotopic signatures of MeHg and iHg accumulated in internal tissues would indicate an unique source of Hg assimilated through food, essentially in the form of MeHg, with iHg being the metabolised Hg fraction (i.e., the result of hepatic demethylation). According to this, we could also clarify if the total amount of iHg in livers derives uniquely from in vivo demethylation or if a fraction of this iHg could be directly come from food intake. Significant ingestion of iHg could occur in the case of white-chinned and giant petrels, as they respectively feed on offal from fish fisheries and on seabird or mammals carrion [START_REF] Cipro | Trace elements in tissues of white-chinned petrels (Procellaria aequinoctialis) from Kerguelen waters, Southern Indian Ocean[END_REF]González-Solís et al., 2002), possibly influencing MIF signatures of iHg in their livers. ∆ 199 Hg) values of penguin blood samples (diamonds) with Arctic seabird eggs from oceanic zones Bering Sea (55-65ªN, 170ºW) and Gulf of and coastal zones (Norton Sound,64ºN,164ºW) represented with dark blue circles (oceanic) and yellow circles (coastal) [START_REF] Day | Mercury stable isotopes in seabird eggs reflect a gradient from terrestrial geogenic to oceanic mercury reservoirs[END_REF] 0.0 ). This could possibly be a result of more efficient remobilisation from muscle to moulting feathers for excretion of Hg in mature adults and feathers tended to accumulate mainly MeHg, Moreover, the two younger petrels presented an almost equal composition of both Hg compounds (52 and 58% of MeHg, respectively) however, the old one displayed a higher percentage of iHg (63%) which was surprising since muscles are generally considered MeHg storage organs. Blood Hg concentrations of the two younger petrels were very similar (2.97±0.01 and 3.03±0.05 µg•g -1 ), whereas the oldest presented 8 times higher blood Hg concentrations (23.9±0.1 µg•g -1 ), Much lower Hg amounts were also found in brain for the two youngest (2.8±0.2 and 1.6±0.1 µg•g -1 ) compared to the oldest individual (13.2±0.2 µg•g -1 ), however brain Hg speciation differed widely between individuals, present a similar trend for the youngest and oldest birds (around 40% of MeHg), whereas medium age individual presented much higher extent of MeHg (73%). Skua chicks' diet is usually composed of other birds and marine mammals scavenge or seabirds eggs, and also of fish when feeding at sea. Depending on the location, skuas present different feeding ecology. Antarctic skuas from Adélie Land feed their chicks with eggs and carcasses of Adélie penguins Pygoscelis adeliae [START_REF] Carravieri | From Antarctica to the subtropics : Contrasted geographical concentrations of selenium , mercury , and persistent organic pollutants in skua chicks ( Catharacta spp .)[END_REF]). In the Kerguelen Islands, their diet is particularly composed of blue petrels Halobaena caerulea and thin-billed prions, Pachyptila belcheri [START_REF] Mougeot | Predation on burrowing petrels by the brown skua (Catharacta skua lo È nnbergi) at Mayes Island, Kerguelen[END_REF]. Crozet skuas rely mainly on eggs and carcasses of crested penguins (Eudyptes chrysolophus and E. chrysocome filholi), but also on black rats Rattus rattus introduced by humans. In Amsterdam Island, skua diet is not completely recognised but, according to field observations, chicks are fed with other seabirds' eggs and carcasses and with subantarctic fur seals Arctocephalus tropicalis scavenge, but also with introduced brown rats Rattus norvegicus [START_REF] Carravieri | From Antarctica to the subtropics : Contrasted geographical concentrations of selenium , mercury , and persistent organic pollutants in skua chicks ( Catharacta spp .)[END_REF].

Hg isotopic composition of key tissues documents Hg metabolic processes in seabirds

Adults from five species of penguins were studied: king penguin Aptenodytes patagonicus, Adélie Pygoscelis adeliae, macaroni Eudyptes chrysolophus, eastern rockhopper E. chrysocome filholi and northern rockhopper E. chrysocome moseleyi penguins. King penguins mainly feed on pelagic fish, especially myctophids such as Krefftichthys anderssoni [START_REF] Bost | Feeding of diving predators and diel vertical migration of prey: King penguins' diet versus trawl sampling at Kerguelen Islands[END_REF]Cherel et al., 2010). Adélie penguins also consume fish but their diet is mostly composed of krill (especially, Euphausia superba and E. crystallorophias) [START_REF] Rodary | Diving behaviour of Adelie penguins ( Pygoscelis adeliae ) at Dumont D'Urville, Antarctica: nocturnal patterns of diving and rapid adaptations to changes in sea-ice condition[END_REF]. Macaroni penguins primarily feed on crustaceans, such as hyperiids (e.g. Themisto gaudichaudii, and Primno macropa) and euphausiids (e.g. E. vallentini, Thysanoessa spp.), and on fish such as the myctophid Krefftichthys anderssoni (Cherel and Hobson 2007;[START_REF] Cherel | Isotopic niches and trophic levels of myctophid fishes and their predators in the Southern Ocean[END_REF][START_REF] Thiebot | Adjustment of pre-moult foraging strategies in Macaroni Penguins Eudyptes chrysolophus according to locality, sex and breeding status[END_REF]. Eastern rockhopper penguins from Crozet Islands are known to rely upon the subantarctic krill E. vallentini, while northern rockhopper penguins from Amsterdam Island mainly feed on the euphasiid Thysanoessa gregaria and squid [START_REF] Tremblay | Geographic variation in the foraging behaviour, diet and chick growth of rockhopper penguins[END_REF]. Chicks of emperor penguins (Aptenodytes forsteri) are winter breeders and were studied in order to evaluate seasonal variations (summer vs winter) in the Antarctic continent. In winter, emperor penguins mainly feed over the Antarctic continental shelf and they present excellent diving capacities foraging mainly on fish (and squids) of benthic areas between 200 and 400 m depth [START_REF] Wienecke | Extreme dives by free-ranging emperor penguins[END_REF]). Due to their seasonal mismatch relative to the other studied seabirds, they were not considered for the exploration of latitudinal trends. [START_REF] Chandan | Mercury isotope fractionation during aqueous photoreduction of monomethylmercury in the presence of dissolved organic matter[END_REF] for midrange MeHg:Sred-DOC, SRFA organic matter conditions, 35% S-red).

Reference data for this study (1) Other published data (2) Reference data (3) Reference data (4) Zone [START_REF] Cossa | Mercury in the Southern Ocean[END_REF] (2) AZ: Nerentorp Mastromonaco et al. 2017; SAZ: Canario et al. 2017; STZ: Bratkic et al. 2016 (3) Tremblay et al. 2015; Canario et al. 2017; Ogawa et al., 1999 (4) [START_REF] Ksionzek | Dissolved organic sulfur in the ocean: Biogeochemistry of a petagram inventory[END_REF] Table 4.S3. Calculation of MeHg:Sred-DOC ratios for the estimation of MeHg photodemethylation based on experimental fractionation factors [START_REF] Chandan | Mercury isotope fractionation during aqueous photoreduction of monomethylmercury in the presence of dissolved organic matter[END_REF] for low-range MeHg:Sred-DOC, NL NOM organic matter conditions, 50% Sred).

Reference data for this study (1) Other published data (2) Reference data (3) Reference data (4) Zone [START_REF] Cossa | Mercury in the Southern Ocean[END_REF] (2) AZ: Nerentorp Mastromonaco et al. 2017; SAZ: Canario et al. 2017; STZ: Bratkic et al. 2016 (3) Tremblay et al. 2015; Canario et al. 2017; Ogawa et al., 1999 (4) [START_REF] Ksionzek | Dissolved organic sulfur in the ocean: Biogeochemistry of a petagram inventory[END_REF] Estimation of the MDF enrichment factor associated to MeHg photodemethylation

In order to estimate the enrichment of MDF values associated to the calculated photodemethylation extent at each site, we performed a recalculation of the fractionation factor of δ 202 Hg values (α (δ 202 Hg)) for our environmental conditions. We based this calculation on the enrichment factors attributed to mid-range MeHg;S red-DOC and low MeHg;S red-DOC, which seem to be the closest model to our conditions in the Southern Ocean (Tables 4.S6 and 4.S7). We

Calculation of the fraction of volatilised Hg and fractionation factor

Calculation of Henry's law constant (KH)

The calculation of Henry's law constant permits to estimate the evasion (or volatilisation) extent of Hg from oceanic waters in function of the water surface temperature. This constant represents the fraction of gaseous Hg 0 in function of Hg 0 remaining in the aqueous phase.

An equation for artificial sea water (concentration conditions of 1.5 M of NaCl) was proposed by [START_REF] Andersson | Determination of Henry's law constant for elemental mercury[END_REF].

Data of water temperature were taken from previous studies in austral autumn [START_REF] Cossa | Mercury in the Southern Ocean[END_REF]) and summer [START_REF] Canario | Mercury and methylmercury in the Atlantic sector of the Southern Ocean[END_REF]. Based on approximate values of temperature water, we estimated yearly volatilisation extents taking place at each site. Obtained values were KH=0.21 (T=273 K), 0.25 (T=281 K) and 0.32 (T=291 K) respectively for Antarctic, subantarctic and subtropical zones. Hg isotopic fractionation during volatilisation is independent of initial solution volumes and volatilization rates [START_REF] Zheng | Mercury isotope fractionation during volatilization of Hg(0) from solution into the gas phase[END_REF]. According to this volatilization experiment, we calculated α( 198 Hg/ 202 Hg) from the slope when plotting log(1000-δf/1000-δi) vs log(fR) following this equation:

where δ is the isotopic composition of the remaining reactant at time t, δi is the initial isotopic composition of the reactant, fR is the fraction of the remaining reactant at time t, and α is the kinetic fractionation factor. Based on experimental calculations, we determined the Rayleigh-type fractionation slope for our model, assuming that initial δ 202 Hg (δi) is 0 and taking α( 198 Hg/ 202 Hg) of 1.00047 [START_REF] Zheng | Mercury isotope fractionation during volatilization of Hg(0) from solution into the gas phase[END_REF]

  Dommergue et Krishna Das d'avoir accepté d'être rapporteurs de cette thèse et merci à David Point, Lars-Eric Heimbürger et Jérôme Fort d'être les examinateurs.

  Remerciements/Acknowledgements ............................................................................... iii Table of content ................................................................................................................ v List of abbreviations and acronyms ................................................................................. ix

Figure 1 . 2 .

 12 Figure 1.2. Temporal trends in Hg anthropogenic emissions by (a) source type and (b) geographical region. Figure from[START_REF] Streets | All-time releases of mercury to the atmosphere from human activities[END_REF]

  Figure 1.3 Global Hg budgets in the main environmental compartments and pathways that are of importance in the global Hg cycle. Estimations of natural and anthropogenic releases to air land and water are based in models. Figure from UNEP 2013.

Figure 1 . 4

 14 Figure 1.4 Schematic diagram of the Hg cycle in the Arctic Ocean. Figure from AMAP 2011.

Figure 1 . 5 .

 15 Figure 1.5. Schematic diagram of proposed reactions of bromine, Hg species and ozone involved in depletion events during light and dark conditions.Figure from Nerentorp[START_REF] Mastromonaco | Antarctic winter mercury and ozone depletion events over sea ice[END_REF]

  Figure 1.5. Schematic diagram of proposed reactions of bromine, Hg species and ozone involved in depletion events during light and dark conditions.Figure from Nerentorp[START_REF] Mastromonaco | Antarctic winter mercury and ozone depletion events over sea ice[END_REF]

Figure 1

 1 Figure 1.6. Illustration of the processes of transport and transformations of Hg occurring in coastal Antarctica (Dumont d'Urville, Adélie Land) during summer. Figure fromAngot et al., 2016a

Figure 1

 1 Figure 1.8. Transfer of a methyl group via the MeCo (or CH3CoB12) in form of methylcarbanion for methylation of iHg.

Figure 1 .

 1 Figure 1.11. Specific assimilation of Hg species at the first trophic levels of the food web.Figure from[START_REF] Morel | The Chemical Cycle and Bioaccumulation of Mercury[END_REF]

Figure 1 .

 1 Figure 1.13 Schematic diagram of kinetic and equilibrium stable isotope fractionation. Figure from Wiederhold, 2015.

Figure 1 .

 1 Figure 1.14. Root mean squared charge radii of Hg stable isotopes showing that odd isotopes( 199 Hg and 201 Hg) tend to be smaller than a trend line through even isotopes. Figure from[START_REF] Schauble | Role of nuclear volume in driving equilibrium stable isotope fractionation of mercury, thallium, and other very heavy elements[END_REF]

Figure 1 .

 1 Figure 1.15. Schematic illustration of fractionation mechanisms for Hg isotope system. NVE represents fractionation due to NFS. MDE means mass difference effect. Figure from Wiederhold, 2015.

Figure 1 .

 1 Figure 1.18. Compilation of MDF (δ 202 Hg) and MIF(∆ 199 Hg) values reported in several natural and environmental samples in literature. Figure from[START_REF] Blum | Mercury Isotopes in Earth and Environmental Sciences[END_REF]

Figure 1 .

 1 Figure 1.21. Schematic representation of a simplified model for Hg cycling between atmosphere, surface mixed layer, pycnocline and marine food web (figure from[START_REF] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF].

Figure 1 .

 1 Figure 1.22. Hg isotopic composition in environmental samples from Polar Regions (Arctic and Antarctic) from literature.

Figure 1 .

 1 Figure1.23. Δ 201 Hg versus ∆ 199 Hg for the photochemical reduction experiments and fish tissue samples from the Lake Michigan[START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF].

  foraging strategies among species, involving different trophic and different compartments of the food web; ii) they have a long life span, leading to high Hg concentrations in their tissues; iii) they are generally colonial and philopatric, allowing to easily sample several individuals simultaneously during the reproduction and follow these individuals repeatedly over time; iv) they reflect Hg contamination from vast and remote areas, otherwise difficult to sample and monitor with conventional sampling methods.

Figure 1 .

 1 Figure 1.25. Northern giant petrel at Crozet Islands. Photo: Yves Cherel.

Figure 1 .

 1 Figure 1.26. King penguins breeding at colonies of Crozet Islands. Photo: Paco Bustamante.

  ecosystems remains necessary. Therefore, the main objective of the present doctoral work is the characterisation of the exposure pathways of MeHg accumulated in seabirds to identify the sources and processes involved in the Hg biogeochemical cycle in these remote marine environments. With this intention, the proposed methodological approach consists on the determination of Hg isotopic signatures (δ 202 Hg, Δ 199 Hg) and Hg species distribution in tissues of an accurate selection of seabirds of the Southern Ocean (French Southern and Antarctic territories). The combination of Hg speciation and isotopic composition, together with the information of trophic and habitat isotopic tracers (δ 13 C, δ 15 N), is expected to provide useful insights for the elucidation the sources of MeHg in these few explored marine areas. This doctoral work is based in important insights documented in the doctoral work of A.Carravieri[START_REF] Carravieri | Seabirds as bioindicators of Southern Ocean ecosystems: concentrations of inorganic and organic contaminants, ecological explanation and critical evaluation[END_REF] that enabled a better understanding of the intrinsic and extrinsic factors influencing Hg exposure in seabird as a function of their ecological characteristics, and therefore, provided valuable information concerning the selection of the most preferable bioindicators species. The present work continues the evaluation of the main factors influencing Hg exposure pathways and aims to achieve further innovative information such the determination of the origin (or formation sources) of MeHg accumulating in marine birds according to the latitude, trophic level and ecology of these organisms. This work comprises totally innovative results of Hg isotopic composition of seabird tissues, representing the first data of Hg isotopic composition in the Southern Ocean, including Antarctic Polar Regions, thus providing valuable information about distinct and variable marine ecosystems of these remote zones.

(

  second part was focused at a larger scale (i.e., latitudinal gradient) with the objective of exploring potential MeHg sources and major processes involved in the Hg biogeochemical cycle across the Southern Ocean, from Antarctica to the subtropics, by interpretation of Hg isotopic variations in marine birds. The main conclusions and perspectives of the doctoral work, including the descriptive aspects, are presented in Chapter 5. Published in Talanta journal in 2017, N°174, pp. 100-110) Marina Renedo 1,2* , Paco Bustamante 1 , Emmanuel Tessier 2 , Zoyne Pedrero 2 , Yves Cherel 3 , David Amouroux 2* 1 Littoral Environnement et Sociétés (LIENSs), UMR 7266 CNRS-Université de la Rochelle, 2 rue Olympe de Gouges, 17000 La Rochelle, France 2 CNRS/ UNIV PAU & PAYS ADOUR, Institut des Sciences Analytiques et de Physico-chimie pour l'Environnement et les Materiaux , UMR5254, 64000, Pau, France 3 Centre d'Etudes Biologiques de Chizé (CEBC), UMR 7372 CNRS-Université de La Rochelle, 79360 Villiers-en-Bois, France

  research work includes the assessment of two keratin-based materials (feathers and hair) and considers further analytical strategies, such as the isotopically enriched spiking technique in all the extraction methods tested or the additional evaluation of classic MeHg selective extraction.This study evaluates in depth analytical performances on keratin samples and provides information about non-desirable reactions occurring during both extraction and derivatisation of each analytical procedure tested. Hg speciation in feathers of a great number of seabirds from the Southern Ocean have been successfully determined by applying the developed method.

  Due to the non-existence of commercialised feather reference material, we prepared a pool sample of feathers collected from different individuals of king penguin (Aptenodytes patagonicus) from Crozet Islands which was used as internal reference standard (IRM) for our laboratory feather analyses and named F-KP. For the validation of the results, all the analyses were performed on a human hair certified reference material (NIES-13). Human hair has been chosen as the most appropriate matrix for validation of feather analysis since they have a similar composition, almost completely composed of keratin. NIES-13 presents high Hg concentrations and contains ~90% of MeHg ([THg]= 4420 ± 200 ng•g -1 and [MeHg]= 3800 ± 400 ng•g -1 , certified values).The evaluation of MSE and SSE methods was accomplished with feather samples of two marine bird species: the white-chinned petrel (Procellaria aequinoctialis) and Antarctic prion (Pachyptila desolata). A pool of feathers from a raised pheasant (Phasianus colchicus) was used as a control since terrestrial birds are known to accumulate lower amounts of Hg in their tissues than aquatic birds[START_REF] Burger | Marine Birds as Sentinels of Environmental Pollution[END_REF].The developed SSE method was applied to feathers from several seabird species exhibiting a large range of Hg concentrations. The selection of marine birds comprises seven species of penguins: emperor (Aptenodytes forsteri), king (A. patagonicus), Adélie (Pygoscelis adeliae), gentoo (P. papua), macaroni (Eudyptes chrysolophus), southern rockhopper (E. chrysocome filholi) and northern rockhopper (E. chrysocome moseleyi) penguins, and the wandering albatross (Diomedea exulans), northern (Macronectes halli) and southern (M. giganteus) giant petrels, Antarctic prion (Pachyptila desolata) and Antarctic (Catharacta maccormicki) and subantarctic (C. lönnbergi) skuas. Feather sampling was conducted in four sites of the French Southern and Antarctic Territories: Adélie Land (66°40'S, 140°10'E), Crozet Islands (46°26'S, 51°45'E),

  Different reagents were tested for the optimisation of the extraction method: acid digestion using nitric acid (HNO3•6N, INSTRA quality) and alkaline digestion by tetramethylammonium hydroxide (25% TMAH in H2O, Sigma Aldrich). Sample amounts between 0.20-0.25 g were digested in 5 mL of reagent. Two different extraction systems were also tested: microwave (MW) and Hotblock (HB) (Figure 2.1). MW assisted extraction was performed using a CEM MW system (Discover SP-D, CEM Corporation) coupled to an autosampler Explorer 4872 96 (USA).

Figure 2

 2 Figure 2.1. Optimisation procedure for Hg speciation analyses in feathers: scheme of the extraction methods tested.

  [iHg] determined by ID-GC-ICPMS, and were compared to [THg] determined by AMA-254 in order to evaluate their similarity and verify the recovery of the extraction.

Figure 2

 2 Figure 2.2. Sample preparation flow chart of the two methods compared: speciation extraction and MeHg specific extraction in human hair and feather samples.

1. 2

 2 Method optimisation for Hg speciation analyses in avian blood and internal tissues Introduction The performance of an analytical technique for Hg speciation by GC-ICPMS requires the optimisation of different procedures and parameters such as sample preparation, extraction, derivatisation, chromatographic separation and selective detection. Moreover, when applying the isotope dilution technique, the optimisation of the spike procedure (addition before or after extraction) and the choice of the quantification method are crucial for the assessment and correction of possible Hg species interconversions during the whole analytical procedure. Most of the studies focusing on Hg species extraction, especially in organs and other biological tissues, used MW assisted extraction with a preference for alkaline extraction in TMAH since it enables the total hydrolysis of biological tissues conserving intact C-Hg bounds (Rodriguez Martin-

4 (

 4 Internal tissues (pectoral muscle and liver) from a dead individual of southern giant petrel (Macronectes giganteus) were sampled, weighed and stored individually in plastic bags. This species was used because it is both a large animal when adults and it display high Hg concentrations in its tissues (González-Solís, Sanpera, and Ruiz 2002) so large quantities of tissues were available for the technique development and their assessment. In order to optimise and validate the developed methods for Hg speciation analyses, two CRM were used: DOLT-Dogfish liver, NRCC) and ERM-CE-464 (Tuna fish, IRMM) for liver and muscle extractions, respectively. Both CRM are certified in THg and MeHg concentrations. Since no CRM of bird blood was obtainable, a pool sample of blood collected from different individuals of king penguin was prepared and used as IRM.Sample preparation and extraction proceduresAfter dissection, bird tissues were stored at -80°C, and prior to the extraction they were cryogenically pulverized and freeze-dried. Two reagents were tested for extraction of Hg species on the studied avian samples: the classically used alkaline extraction in TMAH and a nitric acid extraction (HNO3•6N). As for feathers, MW and HB extraction systems were also evaluated and compared, but in this case spike addition procedure was uniquely accomplished after extraction of blood and tissues because the conversion between Hg compounds has not been documented for this kind of samples. Blanks of extraction were performed in triplicate for each reagent and extraction system and then each extract was analysed in triplicate injection. Sample amounts of seabird tissues and CRM (between 0.10-0.15 g) were digested in 5 mL of reagent (Figure2.4).

Figure 2 . 4 .

 24 Figure 2.4. Optimisation procedure for Hg speciation analyses in tissues: scheme of the extraction methods tested

  long-term internal reproducibility and repeatability of the optimised method was performed on CRM (NIES-13, DOLT-4 and ERM-CE-464) and on our IRM for feather samples (F-KP) and avian blood samples (RBC-KP). Internal reproducibility was assessed for triplicate injection of the three extracts of each reference material (n=9), prepared following identical protocols and by the same operator and equipment in each analytical session, with 1-6 months of interval. Since the term reproducibility implies the involvement of measurement by different operators and laboratories, we use the term internal reproducibility. Repeatability was estimated by analysing in triplicate the same extract the same day, under identical conditions (n=3). For keratinised matrixes by acid extraction, limits of detection (LOD) and quantification (LOQ) were calculated as the sum of spiked HNO3•6N blank average values analysed in triplicate (15 blanks of extraction) plus three times the standard deviation (SD) of these blanks for LOD or ten times for LOQ (IUPAC). For extraction of 0.25 g of hair or feathers in 5 mL of HNO3•6N,

Figure 2 . 5 .

 25 Figure 2.5. Control charts of measured MeHg and THg concentrations for keratinised reference materials NIES-13 and F-KP for each analytical session.

  analyses in biological tissues require a rigorous sample preparation with previous homogenisation and a full extraction of Hg from the matrix. Acid digestion is the most common strategy for Hg extraction in biological samples and is followed by a dilution of the extract in acidic matrix. A complete THg extraction is essential in order to avoid potential Hg isotopic fractionation during sample preparation and to reduce possible matrix effects associated to the presence of lipids or other organic ligands in such complex biological tissues. Numerous tests of sample digestion were performed to assure a complete extraction of Hg in the samples with addition of H2O2 in some cases to guarantee a full oxidation of Hg. The final solution of the sample extract in an acidic matrix is 10% HNO3 and 2% HCl for analyses. Final concentration in these diluted extracts was generally 1 ng g -1 and could decrease to a minimum of 0.5 ng.g -1 for

Figure 2

 2 Figure 2.7. Optimisation procedure for Hg isotopic analyses in feathers, blood and internal tissues: scheme of the extraction methods tested

Figure 2

 2 Figure 2.8. Schematic design of sample introduction into MC-ICPMS instrument. Range of [Hg] (0.5-1 ppb) and 10% HNO3 / 2% HCl for the sample. Double entry plasma torch.

  except for UM-Almadén (NIST-8160), the accuracy of the method was approximate and based on published data in previous studies as reference. Precision (2SD‰) at medium-term reproducibility was evaluated for diary measurements of UM-Almadén during a whole week of analyses showing a mean precision (δ 202 Hg and ∆ 199 Hg values) of 0.15 ‰ and 0.08 ‰ for the six performed analytical sessions (n=30-45 analyses/session). Unlike other CRM, avian reference samples were analysed a representative number (see below) of times during each analytical session permitting an evaluation of medium-term reproducibility in this kind of samples. F-KP presented a mean precision at medium term of 0.18 ‰ and 0.10 ‰ (n=5-10 analyses/session) and RBC-KP a mean precision of 0.07 ‰ and 0.07 ‰, (n=3-5 analyses/session), both corresponding to mean precision values for three analytical sessions.Mean isotopic values for the overall analytical sessions were correct for reference material UM-Almadén according to previously reported values in other studies with a δ 202 Hg value of -0.55±0.08 ‰ (Mean±2SD, n=173). The precision (2SD) at long-term for Hg isotopic values was satisfactory for the most analysed reference samples: UM-Almadén (0.15 ‰ and 0.05 ‰, n=173), IAEA-086 (0.07 ‰ and 0.07 ‰, n=6), NIES-13 (0.09 ‰ and 0.07 ‰, n=8) and for the avian internal standards F-KP (0.12 ‰ and 0.07 ‰, n=22) and RBC-KP (0.05 ‰ and 0.06 ‰, n=17) for δ 202 Hg and ∆ 199 Hg values, respectively.

  2). Alternatively, measurements of Hg isotopic composition in feathers of flying migratory seabirds open a new perspective for exploring Hg cycle and fate since they denote a powerful tool to trace Hg exposure pathways in large zones of the marine environment.

Figure

  Figure 3.2. Feather versus blood (red blood cells) δ 202 Hg and Δ 199 Hg values in adult penguins (individual values and mean population values). Regression equations for δ 202 Hg blood-feather and Δ 199 Hg blood-feather values are y=1.08x+0.13 (R 2 =0.86, p<0.0001) and y=1.18x-0.04 (R 2 =0.75, p<0.0001), respectively. Abbreviations for the different populations are AP (Adélie penguin), GP (gentoo penguin), KP (king penguin), MP (macaroni penguin), RP (eastern rockhopper penguin) and NRP (northern rockhopper penguin).

  isotopic variations within the trophic levels and feeding strategies Wide ranges of Hg concentrations and isotopic values were obtained among tissues from the three pelagic seabird species investigated in this study. Increasing MDF signatures with trophic level have already been found in aquatic food webs by Hg isotopic analyses in fish or marine mammal muscle. For example,[START_REF] Perrot | Higher massindependent isotope fractionation of methylmercury in the pelagic food web of Lake Baikal (Russia)[END_REF] measured δ 202 Hg values in pelagic food web

  ) and different biological characteristics (metabolic response, detoxification strategies and capacities) enables to evaluate the influence of these factors in Hg exposure pathways and bioaccumulation. The observed MDF variations among internal organs and feathers provided information about detoxification processes occurring in seabirds (Hg hepatic demethylation, inter-organ redistribution or Hg excretion by moulting), indicating different metabolic mechanisms depending on species, physiology or the extent of Hg dietary exposure. The existence of different

  Differences in δ 202 Hg values between livers and muscles indicates Hg fractionation during MeHg demethylation in livers. -Different temporal windows of Hg exposure and different Hg turnover between internal tissues (life span accumulation) and feathers (annual incorporation) are reflected in distinct Hg MIF values, with feathers showing significantly greater Δ 199 Hg values potentially as a consequence of the exposure to distinct Hg sources during wintery migration. -Heavier δ 202 Hg values in feathers relative to muscles could be due to a combination of Hg fractionation during metabolic pathways (preferential excretion of isotopically heavier MeHg from muscles to growing feathers during moult) and extrinsic factors linked to different temporal windows of Hg exposure in feathers (wintery acquisition).

Figure 4 .

 4 Figure 4.1 Blood Hg MIF values (Δ 199 Hg versus Δ 201 Hg) of subantarctic penguins from Possession Island, Crozet archipelago. The solid line represents the Δ 199 Hg/Δ 201 Hg slope of the samples. The red dashed line represents the theoretical Δ 199 Hg/Δ 201 Hg slope for MeHg photodemethylation in the water column, and the blue dashed line represents the theoretical Δ 199 Hg/Δ 201 Hg slope expected for photoreduction of iHg in the water column[START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF].

Figure 4 .

 4 Figure 4.2. A) Blood δ 202 Hg versus δ 13 C values of subantarctic penguins from Possession Island, Crozet archipelago. Regression equation (excluding KP) is y=--0.17x-1.64, R 2 =-0.73, p<0.0001. B) Blood Δ 199 Hg versus δ 13 C values. Regression equation is y=-0.12x-0.74, R 2 =-0.71, p<0.0001. Abbreviations: GP, gentoo penguin; KP, king penguin; MP, macaroni penguin; RP, rockhopper penguin.

  feed at deeper depths and should show lower blood ∆ 199 Hg values than MP. Since KP adults are representative of Polar Front water masses during this time, this result is likely associated to a latitudinal effect in Hg isotopic values (Chapter 4.2). Anyway, Hg isotopic values allow visualising penguin foraging depths, which are generally complex to determine accurately due to penguin seasonal foraging changes or vertical movements of prey among other factors. Indeed, by interpretation of δ 13 C values we can only determine different foraging habitats in a spatial scale (inshore to offshore or latitudinal movements), as well as satellite tracking that enables only horizontal monitoring. Since Hg MIF values offers efficient tracking of foraging depths, measuring Hg isotopes represents a valuable tool for complementary interpretation of avian ecological habits.

Figure

  Figure 4.3 Blood Hg MIF versus MDF values (Δ 199 Hg versus δ 202 Hg) of subantarctic penguins from Possession Island, Crozet archipelago. Regression equation is y=-0.39x-0.93, R 2 =0.60, p<0.0001. Abbreviations: PF, Polar Front; GP, gentoo penguin; KP, king penguin; MP, macaroni penguin; RP, rockhopper penguin.

  Marine sediments are characterized by negative to slightly positive MDF values and low or nearto-zero MIF values and therefore lighter Hg isotopic values are typically found in coastal biota relative to oceanic organisms. Moreover, reduced light penetration as a consequence of higher turbidity in coastal ecosystems limits Hg photochemistry. Hence, it contributes to the lower Hg MIF values of MeHg accumulated in coastal relative to open ocean food webs, excepting Apart from different extent of photochemical processes, biotic methylation is known to occur in benthic sediments and in the open water column below the surface mixed layer[START_REF] Blum | Methylmercury production below the mixed layer in the North Pacific Ocean[END_REF]. Since microbiological methylation produces MeHg enriched in lighter isotopes compared to the residual iHg pool (Rodriguez[START_REF] Gonzalez | Species-Specific Stable Isotope Fractionation of Mercury during Hg ( II ) Methylation by an Anaerobic Bacteria ( Desulfobulbus propionicus ) under Dark Conditions[END_REF], greater methylation yields than demethylation promote the accumulation of isotopically lighter MeHg (lower δ 202 Hg values)[START_REF] Perrot | Identical Hg Isotope Mass Dependent Fractionation Signature during Methylation by Sulfate-Reducing Bacteria in Sulfate and Sulfate-Free Environment[END_REF].For easily tracking MeHg sources between Crozet marine compartments, KP were not considered in the discussion since they are representative of Polar Front waters during the studied period. Assuming different MeHg sources between inshore-offshore Crozet areas, benthic GP were expected to accumulate Hg with a sediment origin, i.e. lower δ 202 Hg and ∆ 199 Hg values, relative to oceanic foragers. Due to the lack of Hg isotopic data on sediments from Crozet Islands, we estimated negative δ 202 Hg values and close to zero ∆ 199 Hg values, as commonly observed in sediments from other sites such as in the Arctic Ocean (δ 202 Hg: -1.37±0.38 ‰; ∆ 199 Hg: -0.02±0.07

  , potentially favouring the recirculation and mixing of MeHg from different sources. The relationship between Hg isotopes (δ 202 Hg and ∆ 199 Hg) and MeHg concentration (Figure 4.S4) indicates that each penguin species reflects an exposure to different MeHg sources. A potentially higher productivity in the Polar Front compared to subantarctic waters would lead to greater bacterial methylation, which could then explain the lower δ 202 Hg values and higher MeHg concentrations obtained in KP compared to the other pelagic penguins (Chapter 4.2). This indicates that KP feed on a specific food web which is potentially enriched in MeHg compared to other subantarctic penguins. Therefore, Hg isotopes in sympatric penguins of the Crozet Islands represent an ecosystem characterised by three distinct food webs exposed to different levels and sources of MeHg, clearly dominated by a pelagic-benthic gradient and a latitudinal effect (potentially linked to productivity).

  photodemethylation was not the main explanatory factor of the heavier isotopic signatures (δ 202 Hg) accumulated in seabirds when decreasing latitude. Although MDF latitudinal variations can be partially explained by photochemical processes, the existence of biogeochemical pathways (methylation/demethylation/reduction) or even biological processes (trophic effects and/or metabolic mechanisms) could induce additional MDF between Antarctic and subtropical marine environments. Overall, this work demonstrates that Hg isotopic variations recorded in seabird blood samples provide new information on the major biogeochemical processes involved in the Hg cycle in the water column of the Southern Ocean ecosystems.

Figure 4 . 4

 44 Figure 4.4 Field sampling legend of skua chick and penguin samples at colony locations over a latitudinal gradient from Antarctic (Adélie Land), subantarctic (Crozet and Kerguelen Islands) and subtropical zones (Amsterdam Island).

Figure 4 .

 4 5). ∆ 199 Hg/∆ 201 Hg slopes obtained for blood samples of penguins (1.21±0.04) and skuas (1.14±0.04)(Figure 4.6). A significant and positive relationship was observed between latitude and blood δ 202 Hg values for both skua and penguins (R 2 =0.91 and 0.85, respectively, both p<0.0001). Blood ∆ 199 Hg values were also correlated with latitude in skuas (R 2 =0.56, p<0.0001) and penguins (R 2 =0.19, p=0.001) (Figure4.8).

Figure 4 .

 4 Figure 4.5 Hg MDF versus Hg MIF (expressed as δ 202 Hg and Δ 199 Hg, respectively) for blood samples of A) skua chicks and B) penguin adults (right) from the Southern Ocean. See Figure 4.4 for legend.

Figure

  Figure 4.6. Hg MIF signatures (Δ 199 Hg versus Δ 201 Hg) for blood samples of skua chicks (left) and penguins (right) from the Southern Ocean. See Figure4.4 for legend. The solid line represents the Δ 199 Hg/Δ 201 Hg slope of the samples. The red dashed line represents the theoretical Δ 199 Hg/Δ 201 Hg slope for MeHg photodemethylation in the water column and the blue dashed line represents the theoretical Δ 199 Hg/Δ 201 Hg slope expected for photoreduction of iHg in the water column[START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF].

  : winter and summer periods in Antarctic zone Antarctic ecosystems are characterised by contrasted annual insolation conditions between the dark period of the austral winter, when the sun does not rise above the horizon, and summertime during which day length accounts ~16h. As previously said, the lower angle of solar incidence probably inhibits the photodegradation of dissolved MeHg in surface water. Therefore, lower MeHg photodemethylation efficiency could be expected in Antarctic waters comparing to northern latitudes. Despite the extreme sunlight conditions in Antarctic ecosystems, Antarctic seabird populations (Adélie penguins and Antarctic skua chicks) presented similar ∆ 199 Hg values compared to subantartic communities. Hg isotopic composition of seabird blood in chicks and adult penguins is representative of the recent Hg exposure before sampling, i.e., the breeding period (Chapter 3.1), in the case of Adélie penguins and Antarctic skuas during summertime. We compared blood Hg isotopic values of these two summer breeding species with a winter breeder penguins inhabiting Antarctic ecosystems, the emperor penguin that are then representative of the Antarctic winter period (Figure4.7). Significantly lower Δ 199 Hg values of emperor penguin relative to the other two summer breeders (~0.5 ‰) could suggest lower photodemethylation

Figure 4 .

 4 Figure 4.7. Comparison of Hg isotopic composition (δ202Hg, Δ199Hg) of Antarctic seabirds representative of the summer period (AP: Adélie penguins, AS: Antarctic skua) and winter period (EP: emperor penguins) of Adélie Land.

  obtained MDF latitudinal variation. Experimental abiotic methylation/demethylation by MeCo exhibited a fractionation factor of -1.6 ‰[START_REF] Jiménez-Moreno | Chemical kinetic isotope fractionation of mercury during abiotic methylation of Hg(II) by methylcobalamin in aqueous chloride media[END_REF]. However, specific enrichment factors must be extrapolated to our environmental conditions in order to estimate if the obtained latitudinal variations in δ 202 Hg values in seabirds could be explained by different extent of methylation/demethylation pathways across the three studied areas in the Southern

Figure 4 .

 4 Figure 4.8. Mean Hg isotopic composition versus latitude for blood samples of skua chicks (left) and penguin adults (right) from the Southern Ocean. A) Regression equations for skua δ 202 Hg blood-latitude (y=-0.05+3.43, R 2 =0.91, p<0.0001) and for penguin δ 202 Hg blood-latitude(y=-0.05x-4.21, R 2 =0.85, p<0.0001). B) Regression equations for skua Δ 199 Hg blood-latitude (y=-0.01+2.07, R 2 =0.56, p<0.0001) and for penguin Δ 199 Hg blood-latitude (y=-0.01x+2.07, R 2 =0.19, p=0.001).

  Hg isotopic fractionation leading to lower blood δ 202 Hg values compared to adult penguins (Chapter 3.2). Apart different levels of Hg exposure or metabolic response, variations in δ 202 Hg values between seabird species have been shown to be strongly dependent on Hg sources associated to different integration times between tissues (Chapter 3.1) or specific foraging ecologies (Chapter 4.1).

-

  photodemethylation extent despite most favourable solar conditions at lower latitude. The larger degree of MDF (δ 202 Hg) with latitude suggest the existence of additional processes between sites, such as biological reduction, methylation and/or demethylation processes. Also, trophic or metabolic processes could also contribute to the observed trend although this hypothesis seems less probable. This work includes the first Hg isotopic data of the Southern Ocean and Antarctic ecosystems over a unique latitudinal gradient, which is representative of the MeHg cycle in the different study areas. In consequence, the present exploration of MDF and MIF values in seabirds allows the elucidation and quantification of potential biogeochemical pathways of Hg in the Southern Ocean and contributes to better understanding the oceanic and global Hg cycling.

  ecosystems in the Southern Ocean, more precisely in the Indian Ocean sector. Based on previous investigations achieved during the doctoral work of A. Carravieri on the determination of Hg baseline in the southern Indian Ocean seabirds and on the main factors driving its accumulation in these species, this work directly focused in a precise selection of seabird species with specific ecological habits that enabled to provide key information of the main factors influencing seabird MeHg exposure and bioaccumulation pathways. By combination of total Hg isotopic composition (δ 202 Hg, Δ 199 Hg) and Hg speciation in this unique collection of avian samples, an important database of Hg isotopic information has been created in these poorly understood ecosystems, facilitating a better comprehension of the Hg cycle in remote areas of the Southern Ocean at different spatial . The main findings concluded from this doctoral work are that: -The investigation of Hg isotopes in seabirds permits to establish relationships between Hg accumulation in the food web, Hg biogeochemistry in marine coastal and oceanic environments and internal metabolic mechanisms in response to Hg contamination in seabirds; -Due to the different Hg turnover, residence time and integration of Hg exposure between seabird tissues, tissue-specific Hg isotopes give access to distinct of sources Hg acquired in individuals at different temporal and geographical windows, providing information of Hg exposure in vast spatial zones and at different stages of their annual cycle; -Blood and feathers in chicks can be effectively and indifferently used for biomonitoring of local contamination using Hg isotopes, whereas in adults each tissue provides access to different temporal exposure: blood at recent scale (i.e. exposure during the breeding period) and feathers at seasonal scale (i.e., exposure during wintery period), thus providing complementary isotopic information at different stages of seabird annual cycle; -Seabirds effectively represent the specific Hg environmental exposure in each marine compartments in function of their by their foraging habitats and latitudinal movements and Hg isotopes in their tissues can be useful to trace sources and exposure pathways of MeHg of these areas of difficult to access.5.1 Hg speciation in feathers of a large seabird community of the Southern OceanA first important contribution of this thesis is the development and adaptation of an analytical technique that allows an accurate, precise and simultaneous quantification of Hg compounds (MeHg and iHg) in feather samples (Chapter 2). Even it was already admitted that most of the Hg present in seabird's feathers is MeHg, only organic Hg had been analysed leading to estimation of MeHg content up to 130% (e.g.,Bond et al., 2009a). It was therefore necessary to better evaluate the fraction of THg which is under MeHg and iHg forms. Our work on this issue was published in Talanta and comprises a detailed compilation of Hg speciation methods in feathers exposing the principal limitations of classical techniques. The optimised extraction method, the analytical technique (by GC-ICPMS) together with species-specific isotope dilution analysis allowed the achievement of very good analytical performances. The developed method was efficaciously applied to feather samples from 13 marine bird species from the Southern Ocean (penguins, albatrosses, petrels and skuas) to investigate the variability of Hg speciation across a large range of Hg exposure conditions and concentrations, including adult individuals and chicks (Figure5.1).

Figure 5 . 1 .

 51 Figure 5.1. Overall results of total Hg concentrations (µg•g -1 , dw) and MeHg proportion (%) in feathers from adult (A) and chick (B) individuals of 13 seabird species from the Southern Ocean.

Figure 5 . 2 .

 52 Figure 5.2. Schematic explanation of the integration windows of Hg reflected by blood and feathers in chick and adult individuals providing their consequent tissue-specific Hg isotopic signatures.

  The different Hg turnover and residence time between muscles (annual scale) and liver (long-term/ life-accumulation) could affect not only δ 202 Hg values but also Δ 199 Hg values, potentially providing insights of distinct Hg sources during the life of an individual. This is the case of white-chinned petrels, which exhibited significantly different Δ 199 Hg values between liver and muscles. Indeed, muscles are assumed to reflect Hg acquisition since the last moult, where a high fraction of MeHg (~80%) has been remobilised into growing feathers. Consequently, Hg accumulated in muscles corresponds to Hg acquired the following year relative to Hg present in feathers (in case of annual moulting). However, Hg in liver is considered to represent long-term accumulation, probably during all their life time. Distinct sources of Hg accumulated specifically in each tissue maybe associated with variability of foraging ecology during their annual cycle or even with age could potentially affect isotopic variability between muscles and liver. However, this hypothesis needs to be further clarified.

5. 4 -

 4 Can the use Hg isotopic analyses in seabirds provide a better understanding of the Hg cycle in southern marine ecosystems?As demonstrated in Chapter 4, Hg isotopic composition in seabirds constitutes a valuable tool for the definition of MeHg sources and a potential variability of processes among the different marine compartments although, as extensively discussed, this requires a precise selection of bioindicator tissues and seabird species of well-known trophic ecology. Hg isotopic signatures in blood samples of chicks (skuas) and of adults (penguins) permitted to reveal different MeHg sources and levels of exposure depending on specific foraging behaviour variability within a same ecosystem. It also showed a latitudinal effect in Hg isotopic signatures suggesting an influence of the processes involved in the Hg biogeochemical cycle between the studied sites of the Southern Ocean. The main results of this part permitted some general conclusions:-A progressive increase in Hg isotopic signatures from benthic to pelagic penguins (δ 202 Hg:1.45-1.93 ‰ and ∆ 199 Hg: 1.41-1.77 ‰) suggested an influence of different MeHg biogeochemical characteristics across inshore-offshore marine compartments. Specific foraging habitats and latitudinal movements of penguins highly determine their exposure to distinct environmental MeHg sources in the variable marine ecosystems of the Southern Ocean.-Variations of Hg MIF signatures in function of seabird foraging depths permit an estimation of the extent of MeHg photodemethylation at different depths of the water column with a total difference of ~3% of MeH photodemethylated between pelagic and benthic ecosystems.-Latitudinal variations in Hg MIF signatures in function of seabird breeding sites allow to estimate the extent of MeHg photodemethylation over a latitudinal gradient of the Southern Ocean from Antarctica to the subtropics (~2%), suggesting that photodemethylation is not the main explanatory factor of the latitudinal enrichment in heavier isotopic values (δ 202 Hg) while they have no impact on the fact that higher MeHg concentrations are bioaccumulated in subtropical seabirds relative to Antarctic communities.-High latitudinal variations in Hg MDF over the studied latitudinal gradient could partly explained by photochemistry, but also by different extent of Hg volatilisation, biotic reduction, bacterial methylation/demethylation and/or trophic and metabolic processes associated to different climatic conditions (water temperature, presence of organic matter, pH, etc.).In conclusion, this doctoral work demonstrates the relevance of the use of seabirds as bioindicators to understand changing dynamics and processes involved in the Hg cycle in these marine compartments and includes the first screening of Hg isotopic results in the Southern Ocean. The study and comparison of different seabird species with contrasted ecological characteristics from a same region allows to reflect the Hg contamination of different habitats and thus to obtain information about the contamination of a given ecosystem. As an example, Figure5.4 includes mean Hg isotopic composition (δ 202 Hg, Δ 199 Hg) in feathers from all the seabird species analysed in the subantarctic zone (Kerguelen and Crozet Islands). This compilation permits a global view of Hg isotopic variation between seabirds in function of their foraging habitats (in terms of space and depth), trophic ecology, age or influence of latitudinal movements between migratory and resident species.

Figure 5 . 4 .

 54 Figure 5.4. Compilation of mean MDF and MIF values of feathers of seabirds from subantarctic waters of the Southern Ocean including Antarctic prions, white-chinned petrels and southern giant petrels, subantarctic skuas and the four subantarctic penguins. PF means Polar Front.

Figure 5 . 5 .

 55 Figure 5.5. Comparison of MDF (ẟ 202 Hg) and MIF(∆ 199 Hg) values of penguin blood samples (diamonds) with Arctic seabird eggs from oceanic zones Bering Sea(55-65ªN, 170ºW) and Gulf of and coastal zones(Norton Sound, 64ºN, 164ºW) represented with dark blue circles (oceanic) and yellow circles (coastal)[START_REF] Day | Mercury stable isotopes in seabird eggs reflect a gradient from terrestrial geogenic to oceanic mercury reservoirs[END_REF]

Figure 2 .

 2 Figure 2.S1. Comparison of THg concentrations (ng•g -1 , logarithmic representation) obtained by simultaneous species extraction SSE (MeHg+iHg) and by AMA-254 for all feather samples of the 13 seabird species studied (n=175 individuals).

Figure 3 .Figure 3 .

 33 Figure 3.S3. Blood and feathers Δ 199 Hg values for the four populations of skua chicks. Diamonds indicate mean values. Black lines indicate median values. Red asterisk indicates outlier.

Figure 3 .

 3 Figure 3.S5. Blood and feathers Δ 199 Hg values for the six populations of penguin adults. Diamonds indicate mean values. Black lines indicate median values. Red asterisk indicates outliers.

Figure 3 .

 3 Figure 3.S6. Hg MIF relative to the fraction of MeHg A) Livers (blue), muscles (yellow) and feathers (red) of Antarctic prions (triangles). B) Livers (blue), muscles (yellow) and feathers (red) of white-chinned petrels (diamonds). C) Livers (light blue), kidneys (dark blue), muscles (yellow), feathers (red), brains (green) and blood (grey) of southern giant petrels (circles)

Figure 4 .Figure 4 .Figure 4 .Figure 4 .

 4444 Figure 4.S1. Blood Hg MDF and MIF values of subantarctic penguins from Possession Island, Crozet Archipelago A) blood ẟ 200 Hg versus ẟ 202 Hg values, B) blood ẟ 199 Hg versus ẟ 202 Hg values. Dotted lines represent the theoretically predicted MDF based on ẟ 202 Hg. Abbreviations: GP, gentoo penguin; KP, king penguin; MP, macaroni penguin; RP, rockhopper penguin.

  could estimate a α (δ 202 Hg)/α (∆ 199 Hg) near 1 in both cases (and both seabird models), meaning that during photodemethylation of MeHg the same degree of fractionation is experienced by δ 202 Hg and ∆ 199 Hg. Consequently, considering that the overall latitudinal MIF variations are attributed to MeHg photodemethylation and are in the order of 0.3 ‰ (~0.25 ‰ and ~0.32 ‰, respectively for skuas and penguins), we could explain ~0.3 ‰ of latitudinal MDF variations due to MeHg photodemethylation.

  Calculation of kinetical fractionation factor α( 198 Hg/ 202 Hg) in function of Hg remaining fraction (fR) at each Hg remaining fraction (fR) was calculated from volatilisation constant KH in order to estimate Hg fractionation magnitude during volatilisation at each location. Experimental volatilisation of aqueous Hg 0 into gaseous Hg 0 indicated MDF with a low kinetic fractionation factor α( 198 Hg/ 202 Hg) obtained during experimental volatilisation process was similar for accumulative fractionation (1.00044) and instantaneous fractionation (1.00047), indicating that

Figure 4 .

 4 Figure 4.S5. Hg MDF and MIF signatures for blood of skua chicks (left side) and of penguins (right side) from the Southern Ocean. A) ẟ 202 Hg versus ẟ 200 Hg, B) ẟ 202 Hg versus ẟ 199 Hg. Blue (Adélie Land), green (Crozet and Kerguelen) and red (Amsterdam). The dotted lines represent the theoretically predicted MDF based on ẟ 202 Hg.

  Determination of ∆ 199 Hg/∆ 201 Hg ratio for identifying photochemical processes MIF signatures can provide supplementary information on Hg sources and biogeochemical cycle. For samples that display ∆ 199 Hg values greater than ±0.30‰, it is useful to consider

	2.3.2
							28
			-0.6 ‰ + MIE -1.5 ‰ + MIE -1.7 ‰			Hg 2+ (aq) natural sunlight, fulvic acid (1) MeHg + (aq) natural sunlight, fulvic acid (1) Hg 2+ (aq) dark organically mediated reduction (1)
			-0.5 ‰					Hg 2+ (aq) chemical reduction (1)
			-0.6 ‰				23	Hg 2+ (aq) ethylation (2)
		-0.5 ‰ + MIE -1.3 ‰ -1.7 ‰ + reverse MIE +			Hg 2+ (aq) UV photolysis with formic acid (2) Hg 2+ (aq) natural sunlight, natural DOM (3) Hg 2+ (aq) natural sunlight, LMWOC (4) Hg 2+ (aq) dark natural DOM reduction (4)
			-1.9 ‰					Hg 2+ dark chemical reduction (5)
			-1.7 ‰				18	Hg 2+ snow, natural sunlight (6)
		-0.8 ‰ + reverse MIE			Hg 0 (aq) photochemical oxidation with Cl-(15)
			-0.7‰					Hg 0 (aq) photochemical oxidation with Br* + NFS (15)
			+0.6‰
			-2.6 ‰ -4.4 ‰	-1.6 ‰ -0.4 ‰		13	Hg 2+ (aq) bacterial reduction (7) MeHg + (aq) bacterial reduction (8) Hg 2+ (aq) bacterial methylation (9) Hg 2+ (aq) "apparent" bacterial methylation fumarate respiration (10) Hg 2+ (aq) "apparent" bacterial methylation sulphate respiration (10)
			-2.5 ‰			Hg 2+ (aq) bacterial methylation (IRB) (11)
							8	Hg 2+ (aq) bacterial methylation (SRB) (11)
					-1.1 ‰
		-9 ‰	-4 ‰					Hg 2+ (aq) dark abiotic methylation by MeCo chloride medium (12) Hg 2+ (aq) visible abiotic methylation by MeCo chloride medium (12)
				-1.6 ‰		3	MeHg + (aq) equilibrium abiotic dimethylation by MeCo (13)
			-6.7‰		-0.5‰		Hg 0 (aq) volatilisation (3)
								Hg 0 (liq) evaporation (14)
							-2
	-10	-8	-6	-4	-2	0	2
					δ 202 Hg (‰)

Table 1 .

 1 2. . ∆ 199 Hg/∆ 201 Hg slopes calculated during experimental or environmental processes.

Reference Process Conditions Slope ∆ 199 Hg/∆ 201 Hg

	Bergquist and Blum, 2007	Aqueous MeHg photodemethylation	Natural sunlight, high Hg/DOC ratio	1.36±0.03
	Malivnosky et al., 2010	Aqueous MeHg photodemethylation	UVC radiation, alkaline and acid solutions	1.28±0.03
	Chandan et al., 2015	Aqueous MeHg photodemethylation	Artificial light, low MeHg/DOC ratio	1.38±0.02
	Chandan et al., 2015	Aqueous MeHg photodemethylation	Artificial light, high MeHg/DOC ratio	1.17±0.04 to 1.30±0.02
	Rose et al., 2015	Aqueous MeHg photodemethylation	UVA and UVB radiation	1.35±0.16
	Bergquist and Blum, 2007	Aqueous iHg photoreduction	Natural sunlight, high Hg/DOC ratio	1.02±0.02
	Zheng and Hintelmann, 2009	Aqueous iHg photoreduction	Increasing Hg/DOC ratios	1.19±0.01 to 1.31±0.07
	Sherman et al., 2010	iHg photoreduction from snow	Natural sunlight, snow	1.07±0.04
	Sun et al., 2016	Hg 0 photoxidation (NFS)	Presence of Br-	1.64±0.30
	Sun et al., 2017	Hg 0 photoxidation	Presence of Cl-	1.89±0.18
	Zheng and Hintelmann, 2009	Dark iHg reduction (NFS)	Presence of DOC	1.53±0.17/ 1.54±0.06
	Zheng and Hintelmann, 2010b	Dark iHg reduction (NFS)	Presence of SnCl2	1.59±0.11 / 1.62±0.08
	Wiederhold et al., 2010	Equilibrium fractionation (NFS)	Dissolved iHg-thiol binding	1.54±0.22
	Estrade et al., 2009	Evaporation liquid Hg 0		2.00±0.60
	Ghosh et al., 2013	Evaporation liquid Hg 0		1.59±0.05

Blum, et al. 2013

Table 1 .

 1 3. Compilation ofHg isotopic composition (δ 202 Hg, ∆ 199 Hg, ∆ 201 Hg) and ∆ 199 Hg/∆ 201 Hg slopes reported in aquatic/marine biota from the literature.

	Study	Sample	Location	n	δ

202 Hg (‰) ∆ 201 Hg (‰) ∆ 199 Hg (‰) Slope ∆ 199 Hg/∆ 201 Hg (‰)

				(min,max) (-0.25,+0.60) (+0.38,+1.25) (+0.46,+1.55)
	Blum et al, 2013	Fish muscle	North Pacific Ocean	28 Mean±SD	0.63±0.41	1.95±1.08	2.50±1.19	1.20±0.01
				(min,max) (+0.00,+1.84) (+0.16,+4.59) (+1.00,+5.50)
	Freshwater ecosystem Kwon et al, 2014	Fish muscle	Northeastern coast USA 9 Mean±SD -0.03±0.20	0.58±0.17	0.71±0.20
	Bergquist and Blum, 2007	Fish muscle	Michigan Lake (USA) 16 Mean±SD (min,max) (-0.34,+0.23) (+0.29,+0.77) (+0.37,+0.98) 0.60±0.38 2.79±0.51 3.60±0.65	1.28±0.03
		Mussel	(min,max) (-0.29,+1.17) (+2.16,+3.64) (+2.73,+4.64) Northeastern coast USA 7 Mean±SD 0.06±0.36 0.12±0.09 0.38±0.10
	Gantner et al, 2009	Fish muscle	Arctic Lakes (Canada) 64 Mean±SD -0.34±0.78 (min,max) (-0.31,+0.77) (+0.02,+0.27) (+0.21,+0.52) 1.39±0.87 1.83±1.07	1.21±0.13
		Crab	(min,max) (-1.68,+1.28) (-0.11,+3.89) (0.0,+4.90) Northeastern coast USA 6 Mean±SD -0.11±0.23 0.29±0.27 0.59±0.23	1.22±0.07
	Laffont et al., 2009	Fish muscle	Amazon lakes (Bolivia) 20 Mean±SD (min,max) (-0.45,+0.15) (+0.03,+0.72) (+0.23,+0.87)	1.28±0.12 (overall data)
		Bird blood (eider)	(min,max) (-0.92,-0.40) (-0.14,+0.38) (-0.09, +0.55) Northeastern coast USA 5 Mean±SD 0.85±0.28 0.53±0.07 0.70±0.07
		Hair fish consumers Amazon lakes (Bolivia) 13 Mean±SD (min,max) (+0.59,+1.39) (+0.46,+0.63) (+0.62,+0.82) 1.15±0.16 0.12±0.08 0.19±0.06	1.16±0.04
	Point et, al 2011	Seabird eggs (murre)	Alaska	(min,max) (+1.04,+1.42) (+0.25,+0.81) (+0.15, +0.66) 43 Mean±SD 0.51±0.55 0.91±0.19 1.05±0.26	1.26±0.06
	Perrot et al, 2010	Fish muscle (perch)	Baikal Lake (Russia)	12 Mean±SD -0.48±0.14 (min,max) (-0.84,+1.45) (+0.40,+1.12) (+0.35,+1.63) 0.89±0.33 1.14±0.46	1.28±0.05
	Day et al, 2012	Seabird eggs (murre)	Alaska	(min,max) (-0.57,-0.33) (-0.05,+0.08) (+0.87,+1.39) 45 Mean±SD 0.69±0.25 0.72±0.25 0.88±0.28	1.09±0.04
		Fish muscle (roach)	Baikal Lake (Russia)	12 Mean±SD -0.61±0.14 (min,max) (+0.26,+1.18) (+0.33,+1.21) (+0.50,+1.41) 0.44±0.21 0.58±0.30	1.30±0.05
	Masbou et al. 2015	Ringed seal liver	Alaska	(min,max) (-0.73,-0.47) (+0.20,+0.59) (+0.26,+0.79) 53 Mean±SD -0.17±0.33 0.41±0.17 0.51±0.17	0.97±0.03
		Fish muscle (perch) Bratsk Reservoir (Russia) 12 Mean±SD -0.55±0.45 (min,max) (-0.87,+0.72) (-0.04,+0.91) (+0.07,+1.04) 0.39±0.41 0.52±0.50	1.37±0.10
				(min,max) (-0.90,-0.16) (+0.13,+0.79) (+0.20,+1.04)
		Fish muscle (roach) Bratsk Reservoir (Russia) 12 Mean±SD -0.37±0.30	1.08±0.29	1.28±0.38	1.34±0.04
				(min,max) (-0.80,-0.22)	(0.30,1.39) (+0.41,+1.87)
	Sherman et al., 2013	Freshwater fish	Florida Lake (USA)	22 Mean±SD	0.09±0.38		1.35±1.22	1.30±0.01
				(min,max) (-0.60, 0.68)		(+0.18, +4.43)
				Mean±SD				1.25±0.03
	Li et al., 2016	Freshwater fish	Lake Melville (Canada) 28 (min,max) (-0.76, +0.15)		(+0.72, 3.14)
	Marine ecosystem
	Senn et al, 2010	Fish muscle	Gulf of Mexico	32 Mean±SD	0.03±0.48	0.93±0.52	1.18±0.62	1.20±0.07
				(min,max) (-1.10,+0.70) (+0.24,+2.30) (+0.35:+2.82)
	Gehrke et al, 2011	Fish muscle	San Francisco Bay	34 Mean±SD	0.15±0.23	0.61±0.20	0.78±0.26	1.26±0.01

Part 3. Presentation of the doctoral work: scientific context, bioindicators and main objectives

	Figure 1.24. Map of the
	studied zones of the Southern
	Ocean divided into the three zones
	AZ (Antarctic Zone), SAZ 3.1 Scientific context: The Southern Ocean (Indian sector) (subantarctic zone) and STZ
	(subtropical zone) corresponding 3.1.1 Oceanographic characteristics to different water masses
	determined by the oceanic fronts: The Southern Ocean corresponds to all the water masses at the south of the Subtropical Front Polar Front, Subantarctic Front
	(40 º S). The hydrological features of the Southern Ocean play an important role in the worldwide and Subtropical Front.
	oceanic circulation since it is the nexus of the water masses belonging to the three main oceans
	of the globe: the Pacific, Indian and Atlantic Oceans. The Southern Ocean helps to drive the global
	ocean circulation, and stores and recirculates heat, oxygen or carbon. It is characterized by a
	predominance of strong westerly winds (The Roaring Forties) which are not stopped by any land
	and then involve a hydrological annular structure around the Antarctic continent (called Antarctic
	circumpolar current). This results in the presence of different water masses defined by oceanic
	fronts that separate physically the Southern, Atlantic, Indian and Pacific Ocean basins (Sokolov
	and Rintoul, 2007).
	The climatic and environmental zones strongly influence the environmental conditions of the
	different ecosystems surrounding the Antarctic continent, distributed here as the Antarctic zone
	(situated between the South Pole and the Polar Front), the Subantarctic zone (between the Polar
	Front and the Subtropical Front) and the Subtropical zone (north the Subtropical Front). The
	Subtropical Zone does not strictly make part of the Southern Ocean since it comprises the area
	further north of the Subtropical Zone, but in this doctoral work it is included as a Southern Ocean
	locality for simplification. These regions correspond to the French Southern and Antarctic
	Territories and cover a long north-south gradient. In this doctoral work, avian samples were
	collected from 4 territories:
	-Adélie Land, on the Antarctic continent (66°39′S, Antarctic Zone)
	-Kerguelen (49°21′S, 70°18′E) and Crozet Islands (46°25′S, 52°E) (Subantarctic Zone)
	-Amsterdam Island, (37°47'S, Subtropical Zone)

Table 2 .

 2 1. Synthesis of main previous studies on Hg speciation in feathers in order of publication. Result values are represented as mean±SD.

	Hg speciation	Analytical				THg	iHg	MeHg	MeHg
	approach	method	Species	Region	N	(µg•g -1 )	(µg•g -1 )	(µg•g -1 )	(%)	Ref
			Wandering albatross complex	Gough Island (South Atlantic Ocean) Ocean) Marion Island (Indian	26	30.7±11.7	n/a	29.2±11.6	95
			Sooty albatross	Gough Island (South Atlantic Ocean)	7	9.4±3.9	n/a	9.1±4.0	97
	Hg species measured:		Northern fulmar	Foula (Shetland)	15	1.8±0.8	n/a	2.0±0.7	111
	MeHg, THg	CV-AFS	European shag Great skua	Foula (Shetland) Foula (Shetland)	14 14	1.7±0.7 6.8±4.4	n/a n/a	2.0±0.8 7.3±5.5	118 107	[1]
	MeHg specific extraction		Arctic skua	Foula (Shetland)	9	2.2±1.7	n/a	1.7±1.8	77
			Kittiwake	Foula (Shetland)	14	2.4±0.6	n/a	2.2±0.7	92
			Razorbill	Foula (Shetland)	16	2.1±0.3	n/a	2.1±0.6	100
			Common guillemot	Foula (Shetland)	17	1.5±0.4	n/a	1.7±0.5	113
			Puffin	Foula (Shetland)	10	5.2±2.7	n/a	5.1±2.1	98
	Hg species measured:		Oldsquaw	Chaun (Northeast Siberia)	5	0.7±0.2	n/a	0.9±0.2	128
	MeHg, THg MeHg specific extraction	CV-AFS	Herring gull Arctic tern	Chaun (Northeast Siberia) Chaun (Northeast Siberia)	5 5	6.1±4.6 0.9±0.1	n/a n/a	6.5±4.5 1.1±0.1	106 122	[2]
	Hg species measured:
	MeHg, THg	CV-AFS	Great egret	Everglades (Florida)	6	2.0±0.2	n/a	n/a	120	[3]
	MeHg specific extraction
	Hg species measured: MeHg, THg		Arctic tern Common murre Common tern	New Brunswick (Canada) New Brunswick (Canada) New Brunswick (Canada)	5 5 5	0.9±0.5 1.0±0.4 1.4±0.6	n/a n/a n/a	0.8±0.3 1.2±0.3 1.6±1.8	95±15 133±33 114±65
	MeHg specific extraction	GC-AFS	Razorbill	New Brunswick (Canada)	5	1.8±0.7	n/a	1.1±0.4	82±35	[4]
			Atlantic puffin	New Brunswick (Canada)	5	4.9±2.8	n/a	1.6±0.7	100±50
			Leach's storm petrel	New Brunswick (Canada)	5	4.9±2.8	n/a	5.3±3.4	99±40
	Hg species measured:
	MeHg, iHg,THg Chromatographic	ID-GC-ICPMS	Black-footed albatross (post-1990)	Pacific Ocean	10	47.9±35.7	0.5±1.2	42.1±30.9	89±14	[5]
	separation
	Hg species measured: MeHg, THg MeHg specific extraction	CV-AFS	Ivory gulls (post-1975)	Canada	5	3.5±2.1	n/a	2.7±2.2	68±37	[6]

MeHg Selective Extraction (adapted from Uthe et al. 1972)

	Ethylation with sodium tetraethylborate
	(NaBEt4, 5%)
	Extraction in isooctane

SS-IDA-GC-ICP-MS

	Human hair CRM (NIES-13) Results and discussion	Feathers (4 seabird species: Antarctic prion, white-chinned petrel, king penguin, giant petrel / 1 terretrial bird species : pheasant)
	0.1-0.2 g sample Comparison of speciation extraction procedures: optimisation 0.1-0.2 g sample * before extraction
	5 mL HNO3•6N INSTRA	4 mL NaOH 10M in HotBlock
	(60ºC/2h) Results of [MeHg], [iHg] and [THg] (as [MeHg]+[iHg]) determined by single-IDA and IPD Microwave assisted extraction Ambiance Temp (1 min) -->75ºC (3 min) Addition of isotopically enriched standard
	solutions ( 199 IHg 2+ and 201 MeHg) and transformation factors (M% and D%) calculated by IPD for each selected extraction Aliquot 500 uL + 4.5 mL MilliQ water + Aliquot 500 uL 100 uL H2SO4 after extraction procedure are presented for both reference materials NIES-13 (Table 2.2) and F-KP (Table 2.3).
	* All the extraction methods tested provided satisfactory results for Hg concentrations for the
	two reference materials, except acid extractions with spike addition after extraction, which
	resulted in an insufficient recovery for both Hg species. In the case of NIES-13, recoveries for
	Addition of: 5 mL acidic NaBr (30%w/w) MW acid extractions with spike addition after extraction were 80.6±1% and 76.5±4.6% for MeHg
		10 mL CuSO4 (2.5% w/w)
		10 mL toluene
		Shaking 1h/420 rpm (horizontal)
		Aliquot 4 mL of toluene
		+ 4 mL Na2S2O3 (0.005 M)
		Vortex 1 min + Centrifugation 4500 rpm
		Aliquot Na2S2O3
		+ HCl (diluted x 10)

and THg, respectively, while recoveries of 96.0±1.2% and 95.9±0.2% were achieved for spike addition before extraction. For F-KP, differences between both spiking procedures were not as remarkable as for NIES-13, but better results of THg were obtained when spike addition was added before ([THg]=3899±62 ng•g -1 ) than after MW acid extraction ([THg]=3249±118 ng•g -1 ).

  before extraction, lower but also important D% factors was obtained: MW acid (5.5±1.0% and 4.5±0.2%), HB acid (8.4±0.1% and 3.3±0.8%) and HB alkaline (7.7±0.3% and 6.5±1.5%) for NIES-13 and F-KP samples, respectively. Indeed, for the same extraction conditions but spike addition after extraction, no significant D% were observed on hair and feathers. This result means that significant demethylation reactions mainly occurred during extraction, particularly in the case of MW extraction with TMAH. This could be explained by an influence of different behaviour or transformation rates between endogenous Hg and isotopically enriched Hg from spike solutions[START_REF] Point | Consideration and influence of complexed forms of mercury species on the reactivity patterns determined by speciated isotope dilution model approaches: A case for natural biological reference materials[END_REF]. Differences of complexation and lability patterns between Hg from matrix and enriched Hg potentially occur during the extraction procedure, affecting the accuracy of Hg analysis by species-specific isotope dilution. In the previous assessment on human hair samples[START_REF] Laffont | Mercury speciation analysis in human hair by species-specific isotope-dilution using GC-ICP-MS[END_REF], demethylation during extraction was uniquely observed for HNO3 oven extraction at 80 °C (4.6±2.5%). Contrary to our results, no demethylation reactions occurred during TMAH extraction.Table 2.2. Results of Hg species concentrations and recoveries obtained for the different extraction methods tested in CRM and calculated by the two different quantification methods(Single-IDA and IPD). Species interconversion factors were calculated by IPD. N is referred to number of extractions.Table 2.3. Results of Hg species concentrations and recoveries obtained for the different extraction methods tested in feather IRM sample (F-KP) and calculated by the two different quantification methods(Single-IDA and IPD). Species interconversion factors were calculated by IPD. N is referred to number of extractions.Since no significant differences were observed between MW and HB extraction systems, MW was chosen as the most suitable extraction system since it provides a better-quality control of the extraction, permitting a homogenisation of the sample by electromagnetic stirring and automatic and individually controlled temperature and pressure conditions. Results of MW extraction with spike addition before extraction were evaluated in order to choose the most appropriate reagent.The sensitivity was assessed by the measurement of calibration slopes calculated as the relation of the peak area obtained for 202 MeHg (counts per second, cps) divided by the concentration of MeHg injected (ng•L -1 ) for the injection in 2 µL of isooctane. Much higher sensitivity was obtained for HNO3 than for TMAH extracts obtained for both hair and feathers. Better precision (RSD) was achieved by HNO3 extraction than by TMAH for the two reference samples. Such important difference is also a consequence of lower sensitivity achieved after alkaline extraction.

	Comparison of analytical performances
	NIES-13	Human hair CRM				Concentrations (ng•g -1 )	Recoveries (%)	Interconversion factors (%)
	Calculation method F-KP		System King penguin feathers (IRM) Reagent Spike addition n	MeHg		iHg	THg Concentrations (ng•g -1 ) MeHg (%)	THg(%)	M (%) Interconversion factors (%) D (%)
			System		Certified values Reagent Spike addition 3800 ± 400	n	MeHg	4420 ± 200 iHg	THg	M (%)	D(%)
	Microwave Calculation method	HNO3•6N after extraction 3 3064 ± 55		319 ± 148	3383 ± 158	81 ± 1	77 ± 5
						before extraction 3 3647 ± 46 THg AMA-254 (n=12)		591 ± 53	4238 ± 52	96.0 ± 1.2 3816 ± 275 95.9 ± 0.2
			Microwave Microwave	TMAH HNO3•6N after extraction 3 3467 ± 236 after extraction	594 ± 8 3 2219 ± 50	4202 ± 104 1029 ± 106 96 ± 3 3249 ± 118 95 ± 2
	Single-IDA					before extraction	3 2539 ± 39	1360 ± 49	3899 ± 62
			Microwave	before extraction 3 3651 ± 417 TMAH after extraction	1463 ± 398 3 2461 ± 159	5114 ± 589 928 ± 83	96 ± 11 3389 ± 179 116 ± 13
	Single-IDA	HotBlock	HNO3•6N after extraction 3 3022 ± 157 before extraction	661 ± 16 3 2581 ± 256	3683 ± 149 1592 ± 311 80 ± 4 4173 ± 237 83 ± 3
			HotBlock	before extraction 3 3403 ± 28 HNO3•6N after extraction	621 ± 64 3 2238 ± 40	4025 ± 48 1267 ± 77	90 ± 1 3506 ± 87 91 ± 1
			HotBlock		TMAH	after extraction 3 3364 ± 150 before extraction	845 ± 99 3 2376 ± 22	4210 ± 192 1315 ± 13	89 ± 4 3691 ± 28 95 ± 4
			HotBlock	before extraction 3 3549 ± 263 TMAH after extraction	964 ± 257 3 2733 ± 70	4514 ± 499 1003 ± 50	93 ± 7 3737 ± 86 102 ± 11
			Microwave	HNO3•6N after extraction 3 3022 ± 18 before extraction	218 ± 53 3 2384 ± 192	3240 ± 62 1438 ± 137 79.5 ± 0.5 3822 ± 225 73 ± 1	2.8 ± 1.2	0.3 ± 0.1
			Microwave	before extraction 3 3578 ± 53 HNO3•6N after extraction	565 ± 82 3 2175 ± 43	4143 ± 103 1162 ± 91	94 ± 1 3337 ± 101 94 ± 2	1.5 ± 1.2 1.3 ± 1.1	5.5 ± 1.0 0.5 ± 0.7
			Microwave		TMAH	after extraction 3 3574 ± 104 before extraction	616 ± 56 3 2585 ± 22	4191 ± 92 1405 ± 46	96 ± 1 3990 ± 51 96 ± 2	3.0 ± 1.5 0.3 ± 0.3	1.3 ± 0.6 4.5 ± 0.2
	IPD		Microwave	before extraction 3 3803 ± 112 TMAH after extraction	574 ± 68 3 2161 ± 23	4377 ± 44 964 ± 67 100 ± 3 3125 ± 71 99 ± 1	1.0 ± 0.3 8.8 ± 1.9	26.6 ± 1.0 0.2 ± 0.3
	IPD		HotBlock	HNO3•6N after extraction 3 2826 ± 23 before extraction	704 ± 9 3 2505 ± 284	3529 ± 31 1217 ± 114 74 ± 1 3722 ± 323 80 ± 1	0.7 ± 0.2 0.1 ± 0.8	-1.1 ± 0.1 11.3 ± 10.4
			HotBlock	before extraction 3 3421 ± 45 HNO3•6N after extraction	479 ± 22 3 2165 ± 106	3900 ± 23 1240 ± 77	90 ± 1 3404 ± 131 88 ± 1	1.0 ± 0.3 2.8 ± 1.5	8.4 ± 0.1 0.2 ± 0.2
			HotBlock		TMAH	after extraction 3 3146 ± 186 before extraction	654 ± 37 3 2374 ± 24	3800 ± 148 1194 ± 24.4 83 ± 5 3568 ± 40 86 ± 3	1.3 ± 1.0 0.5 ± 0.2	2.9 ± 0.4 3.3 ± 0.8
			HotBlock	before extraction 3 3346 ± 32 TMAH after extraction	494 ± 55 3 2118 ± 138	3840 ± 87 1110 ± 47	88 ± 1 3228 ± 145 87 ± 2	2.7 ± 1.4 11.9 ± 8.6	7.7 ± 0.3 1.0 ± 0.9
						before extraction	3 2452 ± 156	1266 ± 90.9	3718 ± 184	1.4 ± 1.3	6.5 ± 1.5
									The only exception was
	found in the case of NIES-13 MW TMAH with spike addition before extraction. The correction
	of a substantial D% factor by IPD (26.6±1.0%) involved a significant difference on iHg

concentrations calculated by single-IDA (1463±398 ng•g -1 ) and IPD (574±68 ng•g -1 ). This phenomenon also occurred under the same extraction conditions for F-KP, which exhibited a D% of 11.3±10.4%. According to the results obtained for the rest of extraction methods when spiked solutions were added Concerning the results obtained for procedures with spike addition after extraction, a significant M% was observed for alkaline extractions of F-KP by both systems: MW (8.8±1.9%) and HB (11.9±8.6%). Much lower M% factors were obtained in the case of hair TMAH extracts by MW (3.0±1.5%) and HB (1.3±1.0%). Therefore, important methylation reactions occurred in feather TMAH extracts when spike was added after but not when it was added before. It should also be considered that an additional source of error could exist in quantification of TMAH extraction due to low-sensitivity. No significant D% was obtained for extractions with spike addition after extraction, which means that demethylation artefacts barely occurred during derivatisation step whatever the reagent used. Laffont et al.

[START_REF] Laffont | Mercury speciation analysis in human hair by species-specific isotope-dilution using GC-ICP-MS[END_REF]

did not observe methylation but considerable demethylation (4.2±0.8%) during derivatisation of hair TMAH extracts. Notable demethylation reactions occurred during extraction whatever the reagent and extraction system used, and particularly when using TMAH. Due to the existence of important inter-species conversion reactions during extraction and derivatization of keratin-based material samples, the addition of the enriched solutions before acid or alkaline extraction is highly recommended independently of the method used.

Accuracy was calculated by recoveries of MeHg and THg relative to NIES-13 certified values.

Although the most satisfactory recoveries (for both MeHg and THg of NIES-13) were obtained for TMAH MW extraction with spike addition before the extraction by IPD quantification, this kind of extraction was refused as an pertinent method due to the low sensitivity obtained when using TMAH reagent. Nitric acid MW extraction with spike addition before extraction provided much higher sensitivity and precision, consequently it was chosen for simultaneous species extraction (SSE) on hair and feather samples. For this selected SSE method, mean recoveries for MeHg and THg in NIES-13 quantified by single-IDA (96.0±1.2% and 95.9±0.2) and IPD (94.2±1.4% and 93.7±2.3%) were satisfactory and very similar, suggesting the possibility of using both quantification approaches as valid analytical solutions. Due to practical reasons, single-IDA

Table 2 .

 2 4. Results of MeHg, iHg and THg concentrations obtained for feathers of 13 seabird species of the Southern Ocean. Values are expressed as mean±SD. N means number of individuals analysed. Groups with different letter presented statistically different values (Kruskal-Wallis).ConclusionsA method for the simultaneous determination of Hg speciation in feathers was optimised and validated. The evaluation of different extraction procedures, spiking strategies and quantification methods was performed, concluding that nitric acid microwave assisted extraction with spike addition before the extraction was found the most adequate for feathers (and hair) samples. Both single-IDA and IPD quantification methods are proposed as valid analytical approaches for either routine analysis or monitoring issues (single-IDA) or metrology and analytical development purposes (IPD). In our case, due to the high number of feather samples, single-IDA was favoured because it is a more practical option. The developed method demonstrates the capability of the GC-ICPMS by using species-specific isotope dilution for the precise and accurate measurement of MeHg, iHg and thus THg as MeHg+iHg concentrations and the correction of potential

	Species	Locality	Sampling	Status	Chick diet	n	THg		iHg			MeHg
			dates
							(ng g -1 )	(ng g -1 )	(ng g -1 )	Statistic	%		Statistic
	Spheniscidae
	Emperor penguin	Terre Adélie	Nov 2011	Chicks	pelagic fish	10	812±100	(662-1047)	48±19	764±96	A,B	94±2	(88-97%)	B,C.D.E
	King penguin	Crozet	Oct 2011	Adults	pelagic fish	11	2291±704	(1658-4141)	178±82	2113±633	D,E,F	92±2	(89-95%)	A,B,C,D
	Adélie penguin	Terre Adélie	Feb 2012	Adults	pelagic	10	375±87	(289-532)	28±16	347±76	A	93±3	(88-98%)	A,B,C,D,E
					crustaceans &
					fish
	Gentoo penguin	Crozet	Oct 2011	Adults crustaceans &	11	4330±1853	(1548-7590)	430±181	3899±1686	E,F	90±2	(87-92%)	A
					fish
	Macaroni penguin	Crozet	Jan 2012	Adults	pelagic	10	2274±244	(1852-2575)	222±49	2053±231	F,G	90±2	(87-94%)	A
					crustaceans &
					fish
	Southern rockhopper	Crozet	Feb 2012	Adults	pelagic	10	1388±243	(1055-1685)	110±36	1279±214	A,B,C	92±2	(90-95%)	A,B,C
	penguin				crustaceans &
					fish
	Northern rockhopper	Amsterdam	Nov 2011	Adults	pelagic	10	1692±241	(1250-2033)	122±43	1571±243	B,C,D	93±3	(88-95%)	A,B,C,D,E
	penguin				crustaceans,
					fish & squid

transformations between MeHg and iHg compounds during the different analytical steps. It was successfully applied in environmental feather samples where MeHg appeared to be the major species for all the feathers analysed independently of THg concentrations. This finding fits well with the evidence that seabirds excrete MeHg in moulting feathers as a Hg detoxification strategy.

However, non-negligible amounts of iHg were present in feathers from some individuals. This finding, together with the existence of accidental iHg contamination in feather museum collections, supports the recommended application of methods measuring both Hg compounds' concentrations in feathers.

Table 2 .

 2 5. Results of Hg species concentrations and recoveries obtained for the different extraction methods tested in CRM and calculated by Single-IDA and IPD. Species interconversion factors were calculated by IPD. n is referred to number of extractions. Table 2.6. Results of Hg species concentrations and recoveries obtained for avian liver sample (giant petrel, F-PGA01) and calculated by the two different calculation methods(Single-IDA and IPD). Species interconversion factors were calculated by IPD. n is referred to number of extractions. This result could be due to interconversion reactions during extraction or maybe a loss of matter during the extraction step that cannot be corrected by isotope dilution because spike solutions were added later. Although acid HB extraction provided a better recovery for MeHg (86.2±0.9%), the extraction was not complete for iHg species, which was due to the high M% occurring during derivatisation, as indicated by IPD quantification. Regarding the alkaline extraction procedures, good THg recoveries for ERM-CE-464 were exhibited by MW (96.8±1.3%) and HB (96.2±0.3%), indicating the validity of both extraction systems when using TMAH reagent. Nevertheless, it can be observed that for both alkaline systems there was an overestimation of iHg species and underestimation of MeHg concentrations, which could be attributed to interconversion reactions during extraction. Substantial differences in concentration values were observed between single-IDA and IPD quantification methods for HB extractions in both alkaline and acid medium, meaning the occurrence of interconversion reactions during derivatisation. The calculation of important M% factors (46.5±4.3% for TMAH HB and 41.1±15.2% for acid HB extracts) involves the substantial differences of Hg species concentrations between both quantification methods and indicates the presence of strong methylation reactions occurring during derivatisation of HB extracts. Lower but also important M% factors were found for MW TMAH (7.2±14.7%) and MW acid (16.0±7.9%). Demethylation reactions were however insignificant whatever the extractant used (Table2.7). Table 2.7. Table. Results of Hg species concentrations and recoveries obtained for tuna fish CRM (ERM-CE-464) and calculated by the two different quantification methods (Single-IDA and IPD). Species interconversion factors were calculated by IPD. n is referred to number of extractions. Table 2.8. Results of Hg species concentrations and recoveries obtained for avian muscle (giant petrel, M-PGA01) and calculated by the two different quantification methods (Single-IDA and IPD). Species interconversion factors were calculated by IPD. n is referred to number of extractions.

	DOLT-4	Dogfish liver CRM
					Concentrations (ng•g -1 )	Recoveries (%)
	Quant.			Spike
	Method System	Extr	addition n MeHg	iHg	THg	MeHg	THg	M %	D%
				Certified
				values	1330±120	~1250	2580±220
				after
		MW	TMAH	extraction 3 1202±13	1154±42	2516±81 95.4±3.4 97.5±3.2
			HNO3•	after
	Single-	MW	6N	extraction 3 1192±23	735±21	1927±12 89.6±1.7 74.7±0.5
	IDA			after
		HB	TMAH	extraction 3 1242±37	1235±38	2478±75 93.4±2.8 96.0±2.9
			HNO3•	after
		HB	6N	extraction 3 1134±16	1271±18	2439±64 87.8±5.5 94.5±2.5
				after
		MW	TMAH	extraction 3 1236±4	1226±41	2462±44 92.9±0.3 95.4±1.7	0.3±0.2	1.4±0.7
			HNO3•	after
	IPD	MW	6N	extraction 3 1119±25 after	737±18	1855±11 84.1±1.9 71.9±0.4	0.4±0.1	0.5±0.2
		HB	TMAH	extraction 3 1241±32	1174±28	2416±59 93.3±2.4 93.6±2.3	0.5±1.0	0.9±0.2
			HNO3•	after
		HB	6N	extraction 3 1168±7	1344±30	2511±37 87.8±0.5 97.3±1.4	0.7±0.3	0.1±0.6
	As for the reference liver material, the digestion of giant petrel liver (F-GPA01) also
	resulted in an insufficient recovery for iHg by MW acid extraction relative to the reference THg
	concentration value obtained by AMA-254. Calculated recoveries for HB extraction procedures
	tested in F-PGA01 were satisfactory for acid HB (103.7±6.2%), TMAH HB (94.9±4.0%), and
	TMAH MW extraction (114.1±9.2%). Although no substantial differences in concentration
	values were observed between single-IDA and IPD calculation methods for giant petrel liver F-
	PGA01, important D% factors were obtained for acid HB extract (5.5±3.6%) and for alkaline
	MW extract (4.9±9.0%), suggesting that this seabird liver sample can potentially undergo

demethylation during the derivatisation step independently of the extraction reagent. Despite these relatively high M%, MeHg concentrations calculated by single-IDA and IPD are not significantly different (Table 2.6). In conclusion, TMAH MW extraction provided the most satisfactory results for Hg species extraction in DOLT-4 CRM relative to reference values (MeHg and THg). For the avian liver, TMAH MW was also chosen since it provided good recoveries relative to AMA-254 obtained concentration. Both single-IDA and IPD quantification methods seemed to be valid analytical approaches for data treatment.

Muscle samples

As observed in livers, acid extractions in MW did not provide sufficient recoveries for MeHg concentrations in tuna fish ERM-CE-464 reference material (45.7±0.5%), indicating an uncomplete extraction.

Table 2 .

 2 9. Results of Hg species concentrations and recoveries obtained for avian blood samples (red blood cells, RBC-KP) and calculated by the two different quantification methods(Single-IDA and IPD). Species interconversion factors were calculated by IPD. n is referred to number of extractions.

	RBC-KP				Concentrations (ng•g -1 )	Recovery (%)
	Quant. Method	System	Extr.	Spike addition n	MeHg	iHg	THg	THg	M %	D%
				AMA-254 value			1980±76
	Single-IDA	MW	TMAH after extraction 3 1956±38	62±13	1988±39	100.4±2.3
		MW	HNO3•6 N	after extraction 3 1896±32	54±21	1951±42	98.5±2.1
		HB	TMAH after extraction 3 1917±43	85±7	2003±45	101.1±2.3
		HB	HNO3•6 N	after extraction 3 1979±78	70±16	2079±93	105.0±4.7
	IPD	MW	TMAH after extraction 3 1989±42	72±9	2061±41	104.1±1.3	0.9±0.5	1.3±0.3
		MW	HNO3•6 N	after extraction 3 2028±54	64±7	2092±59	105.6±3.0	1.1±0.5	1.9±0.3
		HB	TMAH after extraction 3 1954±36	77±9	2031±32	102.6±1.6	1.0±0.4	1.7±0.1
		HB	HNO3•6 N	after extraction 3 1980±78	68±4	2048±76	103.4±3.9	1.1±0.4	1.9±0.2

Table 2 .

 2 10. Analytical performances of the optimised MW assisted extraction (3 extractions) for the studied matrixes in acid (keratinised samples) and in TMAH (internal tissues and blood samples)

	Matrix	Sample		Sensitivity	Precision (%)	Accuracy (%)
				(cps/ng L -1 )
					MeHg	iHg	MeHg	THg
	Hair	CRM human hair	NIES-13	8.14E+05	0.38±0.05 1.59±0.31 95.9±1.2 95.9±0.2
	Feathers IRM King penguin	F-KP	1.05E+06	1.30±0.34 1.58±0.23	-	102±1.5
	Liver	CRM Dogfish liver	DOLT-4	7.07E+07	1.04±1.01 3.64±1.28 95.4±3.4 97.5±3.2
		Giant petrel liver	F-PGA01	8.12E+04	2.87±2.42 1.22±1.19	-	114.1±9.2
	Muscle	CRM Tuna fish	ERM-CE-464	6.19E+07	1.30±0.85 4.16±1.20 93.9±1.2 96.8±1.3
		Giant petrel muscle	M-PGA01	4.83E+05	1.61±1.08 2.15±1.34	-	98.9±3.3
	Blood	IRM King penguin	RBC-KP	1.32E+06	1.36±1.

  Figure 2.6. Control charts of measured MeHg and THg concentrations for reference materials ERM-CE-464 and DOLT-4 and for IRM RBC-KP for each analytical session.

	(ng•g -1 ) Concentration MeHg	6000 (ng•g -1 ) 5500 Concentration MeHg 4000 4500 5000		5000 4000 0 1000 2000 3000 420 90.00 421 10.00	421 00	421 30.00	421 50.00	422 00	421 70.00		423 00 421 90.00	422 10.00	424 00	422 30.00	425 00 422 50.00	422 70.00	426 00	422 90.00	427 00	-1 ) (ng•g Concentration THg	0 1000 2000 3000 4000 6000 5000 Concentration THg -1 ) 5500 (ng•g 420 90.00 4000 4500 5000	421 00	421 10.00	421 30.00	422 00	421 50.00	421 70.00	423 00	421 90.00	422 10.00	424 00	422 30.00	422 50.00	425 00	422 70.00	422 90.00	426 00	427 00 ERM-CE-464 NIES-13
											Sessions															Sessions Sessions
	Concentration MeHg (ng•g -1 )	1000 1500 Concentration MeHg 3000 -1 ) (ng•g 2000 2500	0 1000 2000 3000 4000 5000 420 90.00	421 10.00	421 00	421 30.00	422 00 421 50.00		421 70.00	423 00	421 90.00	422 10.00	424 00	422 30.00	425 00 422 50.00	422 70.00	426 00	422 90.00	427 00	Concentration THg (ng•g -1 )	0 1000 2000 3000 4000 5000 420 90.00 1000 Concentration THg (ng•g -1 ) 1500 3000 2000 2500		421 00	421 10.00	421 30.00	422 00	421 50.00	421 70.00	423 00	421 90.00	422 10.00	424 00	422 30.00	422 50.00	425 00	422 70.00	422 90.00	426 00	427 00	F-KP
											Sessions Sessions																	Sessions
		2500																			2500
	Concentration (ng•g -1 ) MeHg	0 500 1000 1500 2000	424 00.00			424 20.00		424 40.00			424 60.00		424 80.00		425 00.00		425 20.00			-1 ) (ng•g Concentration THg	0 2000 500 1000 1500	424 00.00			424 20.00		424 40.00			424 60.00		424 80.00		425 00.00		425 20.00		RBC-KP
											Sessions																	Sessions

Table 2 .

 2 11. Results Hg isotopic composition obtained for reference materials extracted by the two different mineralisation methods: HPA and Hotblock. Results are compared to values in literature. n means number of extractions.Concerning avian samples, Hg isotopic results were similar for the two extraction methods in all the matrixes (Table2.12). King penguin feathers (F-KP) presented identical isotopic values for the two types of extraction methods, as observed for IAEA-086, indicating that both HPA and HB are appropriate for Hg extraction in keratinized matrixes whether hairs or feathers, for a range of THg concentrations in dry material of 0.57-3.25 µg g -1 and for a mass a sample digested ranging from 0.22 mg to 0.25 mg. Regarding giant petrel muscle, results for HB extraction presented much lower δ 202 Hg values in comparison with HPA extracts possibly indicating an incomplete mineralization of the matrix (due to presence of fat, organic matter, etc.) that can create matrix effects in the CVG. Nevertheless, MIF values(∆ 199 Hg and ∆ 201 Hg) were

	Certified
	reference	[Hg] (µg	Digested	δ 204	δ 202	δ 201	δ 200	δ 199	∆ 204	∆ 201	∆ 200	∆ 199
	material	g -1 )	mass (mg)	Hg	Hg	Hg	Hg	Hg	Hg	Hg	Hg	Hg n
	Human hair
	(IAEA-086)	0.57
	HPA extraction		0.21
	Average (‰)			0.74 0.63 0.68 0.29 0.35 -0.20 0.21 -0.03 0.19
	2SD (‰)			-	-	-	-	-	-	-	-	-
	HB extraction		0.15-0.17
	Average (‰)			0.95 0.69 0.75 0.33 0.37 -0.07 0.23 -0.02 0.20
	2SD (‰)			0.38 0.10 0.12 0.07 0.06 0.25 0.04 0.04 0.05
	Yamawaka et
	al., 2016
	Average (‰)			0.87 0.58 0.64 0.31 0.41 0.00 0.20 0.02 0.26
	2SD (‰)			0.12 0.09 0.09 0.04 0.02 0.04 0.03 0.04 0.02
	Dogfish liver
	(DOLT-4)	2.58±0.22
	HPA extraction		0.20
	Average (‰)			-0.49 -0.35 0.73 -0.09 1.08 0.03 0.99 0.08 1.17
	2SD (‰)			-	-	-	-	-	-	-	-	-
	HB extraction		0.18-0.19
	Average (‰)			-0.50 -0.35 0.65 -0.16 1.00 0.02 0..92 0.01 1.09
	2SD (‰)			0.08 0.18 0.09 0.05 0.09 0.02 0.18 0.04 0.08
	Masbou et al.,
	2014
	Average (‰)			-	-0.35 0.71 -0.14 1.03	-	0.98 0.00 1.12
	2SD (‰)			-	0.2	0.06 0.06 0.07	-	0.19 0.03 0.11
	Tuna fish
	(ERM-CE-464) 5.24±0.10
	HPA extraction		0.17	0.87 0.65 2.36 0.40 2.50 -0.10 1.87 0.07 2.33
	Average (‰)			-	-	-	-	-	-	-	-	-
	2SD (‰)
	HB extraction		0.16-0.18	0.69 0.55 2.31 0.33 2.51 -0.06 1.90 0.05 2.36
	Average (‰)			0.11 0.13 0.08 0.03 0.10 0.00 0.01 0.03 0.15
	Li et al., 2014
	Average (‰)			0.94 0.70 2.49 0.43 2.55 -0.10 1.96	0	2.38
	2SD (‰)			0.06 0.04 0.06 0.05 0.08 0.05 0.06 0.04 0.07

Table 2 .

 2 12. Results Hg isotopic composition obtained for the studied avian samples (feathers, muscle, liver and blood) extracted by the two different mineralisation methods: HPA and Hotblock. n means number of extractions.

			Digested
		[Hg]	mass	δ 204	δ 202	δ 201	δ 200	δ 199	∆ 204	∆ 201	∆ 200	∆ 199
		(µg g -1 )	(mg)	Hg	Hg	Hg	Hg	Hg	Hg	Hg	Hg	Hg n
	King penguin
	feathers (F-
	KP)	3.25±1.18
			0.23-
	HPA extraction		0.24
	Average (‰)			2.36	1.58	2.70	0.80 2.10 0.00 1.52 0.01 1.70 3
	2SD (‰)			0.17	0.06	0.07	0.06 0.12 0.14 0.03 0.07 0.12
			0.23-
	HB extraction		0.25
	Average (‰)			2.35	1.52	2.65	0.76 2.12 0.09 1.51 0.00 1.74 3
	2SD (‰)			0.15	0.12	0.06	0.07 0.04 0.16 0.07 0.07 0.05
	Giant petrel
	muscle (M-
	PGA01)	4.11±0.15
			0.23-
	HPA extraction		0.24
	Average (‰)			0.49	0.38	1.54	0.17 1.50 -0.08 1.26 -0.02 1.41 3
	2SD (‰)			0.08	0.09	0.04	0.08 0.04 0.17 0.04 0.04 0.01
			0.23-
	HB extraction		0.25
	Average (‰)			0.04	0.06	1.23	0.02 1.41 -0.05 1.18 -0.01 1.40 3
	2SD (‰)			0.35	0.23	0.17	0.12 0.17 0.01 0.03 0.01 0.13
	Giant petrel
	liver
	(F-PGA01)	208.7±6.8
			0.19-
	HPA extraction		0.21
	Average (‰)			-0.44 -0.28 0.98 -0.14 1.27 -0.02 1.19 0.00 1.34 3
	2SD (‰)			0.30	0.05	0.11	0.08 0.05 0.34 0.12 0.06 0.05
			0.18-
	HB extraction		0.19
	Average (‰)			-0.29 -0.22 1.04 -0.05 1.33 0.03 1.21 0.05 1.38 3
	2SD (‰)			0.19	0.10	0.09	0.11 0.02 0.16 0.03 0.07 0.02
	King penguin
	blood (RBC-
	KP)	1.98±0.76
			0.15-
	HPA extraction		0.17
	Average (‰)			1.86	1.27	2.22	0.63 1.79 -0.04 1.27 -0.01 1.47 3
	2SD (‰)			0.26	0.14	0.09	0.09 0.15 0.04 0.02 0.01 0.11
			0.15-
	HB extraction		0.17
	Average (‰)			1.95	1.32	2.28	0.65 1.80 -0.01 1.28 -0.01 1.47 3
	2SD (‰)			0.10	0.10	0.04	0.03 0.04 0.15 0.11 0.03 0.02

Table 2 .

 2 13. Results Hg isotopic composition obtained for reference materials (CRM UM-Almadén, human hair, liver, muscle and avian feathers and blood) for the overall analytical sessions performed during this doctoral work. N means number of analyses.

	Reference material	Date		δ 204 Hg	δ 202 Hg	δ 201 Hg	δ 200 Hg	δ 199 Hg	∆ 204 Hg	∆ 201 Hg	∆ 200 Hg	∆ 199 Hg	n
	UM-Almadén	May 2015	Average (‰)	-0.81	-0.54	-0.44	-0.26	-0.17	0.03	-0.04	0.01	-0.03	45
	(CRM cinnabar)		2SD (‰)	0.25	0.17	0.14	0.13	0.10	0.17	0.07	0.08	0.09
		June 2015	Average (‰)	-0.81	-0.54	-0.44	-0.26	-0.17	-0.01	-0.04	0.01	-0.03	45
			2SD (‰)	0.25	0.17	0.14	0.13	0.10	0.15	0.07	0.08	0.09
		December
		2015	Average (‰)	-1.01	-0.66	-0.50	-0.30	-0.18	-0.03	-0.01	0.03	-0.01	4
			2SD (‰)	0.11	0.10	0.01	0.01	0.05	0.19	0.08	0.04	0.04
		March
		2016	Average (‰)	-0.85	-0.57	-0.46	-0.28	-0.16	0.01	-0.03	0.01	-0.01	29
			2SD (‰)	0.23	0.18	0.13	0.13	0.10	0.18	0.12	0.09	0.11
		July 2016	Average (‰)	-0.85	-0.56	-0.46	-0.28	-0.18	-0.02	-0.04	0.00	-0.04	30
			2SD (‰)	0.19	0.09	0.10	0.14	0.11	0.14	0.07	0.13	0.10
		May 2017	Average (‰)	-0.74	-0.49	-0.41	-0.24	-0.16	-0.01	-0.04	0.00	-0.04	7
			2SD (‰)	0.13	0.07	0.08	0.10	0.17	0.09	0.07	0.09	0.16
		Reference
		values	Average (‰)	-0.82	-0.56	-0.46	-0.27	-0.17	-	-0.04	0.00	-0.03
		(NIST
		8610)	2SD (‰)	0.07	0.03	0.02	0.01	0.01	-	0.01	0.01	0.02
	IAEA-086	June 2015	Value (‰)	1.23	0.81	0.76	0.45	0.50	0.02	0.15	0.04	0.29	1
		March
		2016	Average (‰)	0.94	0.73	0.77	0.37	0.44	-0.14	0.22	0.00	0.26	5
	(RM humain hair)		2SD (‰)	0.25	0.13	0.09	0.11	0.16	0.26	0.08	0.09	0.15
		Yamakawa
		et al. 2016	Average (‰)	0.87	0.58	0.64	0.31	0.41	0.00	0.20	0.02	0.26	3
			2SD (‰)	0.12	0.09	0.09	0.04	0.02	0.04	0.03	0.04	0.02
	NIES-13	June 2015	Value (‰)	2.98	2.07	3.07	1.10	2.29	-0.11	1.51	0.06	1.76	1
		March
		2016	Average (‰)	2.99	2.11	3.11	1.08	2.34	-0.14	1.52	0.05	1.80	5
	(CRM humain hair)		2SD (‰)	0.28	0.13	0.16	0.01	0.14	0.24	0.05	0.07	0.08
		July 2016	Value (‰)	3.08	2.21	3.18	1.18	2.42	-0.22	1.52	0.07	1.87	1
		May 2017	Value (‰)	2.96	2.10	3.05	1.05	2.35	-0.01	1.52	0.06	1.85	1

. Methodological approach: biological aspects Part 3.1: Blood and feathers from seabirds as efficient biomonitoring tissues for Hg isotopic studies: implications of using chicks and adults

  Table 3.1. Blood (red blood cells) and feather δ 202 Hg and Δ 199 Hg values, associated statistics, and isotopic differences between feathers and blood of skua chicks. Both tissues were sampled simultaneously in large chicks. Statistically significant results are marked in bold. Values are means ± SD

	Penguins
	Penguins' feathers (blood) mean δ 202 Hg values increased from Adélie penguins (0.56±0.20 ‰
	(0.67±0.13 ‰)) to northern rockhopper penguins (2.16±0.14 ‰ (2.42±0.13 ‰)). In the case of	Paired t-test	Paired δ 202 Hg differences
	Species Δ 199 Hg values, gentoo penguins displayed the lowest values (1.41±0.06 ‰ (1.51±0.12 ‰)) and Location n Blood δ 202 Hg (‰) Feathers δ 202 Hg (‰)	(δ 202 Hg values)	δ 202 Hg (‰)
	Mean ± SD northern rockhopper penguins the highest ones (1.89±0.12 ‰ (2.22±0.10 ‰)) (Table 3.2). Mean ± SD	t	p	Mean ± SD
	Antarctic skua	Adélie Land	9	0.23±0.13	0.00±0.36	1.455 0.184	-0.16±0.33
	Subantarctic skua	Kerguelen	10	1.04±0.08	1.22±0.19	-0.680 0.009	0.19±0.18
		Crozet	11	1.39±0.18	1.35±0.14	-3.209 0.512	-0.04±0.18
		Amsterdam	10	1.56±0.08	1.51±0.20	0.884 0.400	-0.07±0.20
		All populations 40			-0.333 0.741	-0.01±0.25
						Paired t-test	Paired Δ 199 Hg differences
	Species	Location	n Blood Δ 199 Hg (‰)	Feathers Δ 199 Hg (‰)	(Δ 199 Hg values)	(‰)
				Mean ± SD	Mean ± SD	t	p	Mean ± SD
	Antarctic skua	Adélie Land	9	1.51±0.08	1.46±0.07	-1.096 0.305	-0.04±0.11
	Subantarctic skua	Kerguelen	10	1.61±0.06	1.55±0.12	-1.166 0.271	-0.05±0.12
		Crozet	11	1.69±0.11	1.64±0.12	-1.454 0.180	-0.06±0.17
		Amsterdam	10	1.76±0.05	1.70±0.05	-2.440 0.037	-0.05±0.08
		All populations 40			-2.745 0.008	-0.05±0.12
	.1) showing high and linear correlation for δ 202 Hg values (Pearson
	correlation, r=0.91, p<0.0001, n=40). ∆ 199 Hg values were also significantly correlated but to a
	lesser extent (Pearson correlation, r=0.53, p<0.0001, n=40). Regressions slopes were not
	statistically different from slope 1:1 neither for δ 202 Hg (t=-12.68, p<0.0001) nor for ∆ 199 Hg values
	(t=-6.54, p<0.0001).

Table 3 .

 3 2. Blood (red blood cells) and feather δ 202Hg and Δ 199 Hg values, associated statistics, and isotopic differences between feathers and blood. Both tissues were sampled simultaneously in adults. Statistically significant results are marked in bold. Values are means ± SD

			Paired t-test	Paired δ 202 Hg differences
	Blood δ 202 Hg (‰)	Feathers δ 202 Hg (‰)	(δ 202 Hg values)	(‰)

Table 3 .

 3 3. THg, Hg speciation (iHg, MeHg), and Hg isotopic composition (δ 202 Hg, Δ 199 Hg) in Antarctic prions from Kerguelen Islands. Values not sharing the same superscript letter are significantly different. Values are means ± SD

	Tissue	n	THg	iHg	MeHg		δ 202 Hg	∆ 199 Hg	∆ 199 Hg/∆ 201 Hg
			µg g -1	µg g -1	µg g -1	%	‰	‰	‰
	Liver	5 2.1±0.5 A 0.71±0.20 1.4±0.4 66±6 A 0.01±0.25 A 1.90±0.05 A	1.15±0.05 A
	Muscle 10 0.3±0.1 B 0.05±0.02 0.3±0.1 84±5 B 0.37±0.41 A 1.93±0.14 A	1.16±0.03 A
	Feathers 10 2.6±0.9 A 0.15±0.07 2.4±0.9 94±3 C 1.32±0.13 B 2.37±0.10 B	1.15±0.01 A
	According to theoretical Δ 199 Hg/Δ 201 Hg slopes obtained experimentally for MeHg
	photodemethylation (1.3) and iHg photoreduction (1.0) in aquatic compartments (Bergquist and
	R 2 =0.89, p<0.0001) and displayed a MIF slope of 1.11±0.08.

Blum, 2007)

, Hg accumulated in Antarctic prions' tissues should have undergone both processes as it represents a Δ 199 Hg/Δ 201 Hg slope between both theoretical lines (1.16, for the overall samples). Δ 199 Hg/Δ 201 Hg ratios were calculated for each tissue separately, indicating no statistically differences between samples (ANOVA, F=0.005, p=0.995, n=25). Δ 199 Hg and Δ 201 Hg values obtained for Antarctic prions' feathers were highly correlated (Pearson correlation,

Table 3 .

 3

		4. THg, Hg speciation (iHg, MeHg) and Hg isotopic composition (δ 202 Hg, Δ 199 Hg) in
	white-chinned petrels from Kerguelen Islands. Values not sharing the same superscript letter are
	significantly different. Values are means ± SD
	Tissue	n	THg	iHg	MeHg	δ

202 Hg ∆ 199 Hg ∆ 199 Hg/∆ 201 Hg

	Figure 3.4. A) THg concentrations and Hg MDF (as δ 202 Hg) in liver (blue), muscle (yellow)
	and feathers (red) from white-chinned petrels. B) Hg MDF relative to the fraction of MeHg.
	(regression line: y=1.87x-0.89, Pearson correlation coefficients were R 2 =0.93, p<0.0001. C) Hg
	µg g -1 MDF versus Hg MIF (expressed as δ 202 µg g -1 µg g -1	%	‰	‰
	Liver	10	54.3±18.7 A 49.4±18.8 4.9±2.1 10±5 A -0.64±0.27 A 1.17±0.05 A 1.14±0.04 A
	Muscle	10	4.7±1.8 B	1.0±0.3 3.7±1.8 77±9 B 0.30±0.28 B 1.29±0.07 B 1.15±0.04 A
	Feathers	10	5.4±2.2 B	0.2±0.1 5.2±2.1 96±2 C 1.10±0.14 C 1.49±0.21 C 1.26±0.08 B
	Δ 199 Hg/Δ 201 Hg line exhibited by the overall samples of white-chinned petrel presented a slope
	of 1.46. However, Δ 199 Hg/Δ 201 Hg ratios calculated individually for each tissue were significantly
	different between livers and muscles (1.14±0.04 and 1.16±0.05) and feathers (1.26±0.08) (H=
	13.80, p=0.001).

Table 3 .

 3 5. THg, Hg speciation (iHg, MeHg), and Hg isotopic composition (δ 202 Hg, Δ 199 Hg) in southern giant petrels. Values not sharing the same superscript letter are significantly different. Values are means ± SD.MIF slopes obtained for the overall samples of southern giant petrels displayed a Δ 199 Hg/Δ 201 Hg slope of 1.17±0.09. Δ 199 Hg/Δ 201 Hg ratios calculated individually for each tissue were statistically similar between tissues (H= 8.03, p=0.154).

	Tissue n	THg	iHg	MeHg	δ 202 Hg	∆ 199 Hg	∆ 199 Hg/∆ 201 Hg
		µg g -1	µg g -1	µg g -1	%	‰	‰
	Liver 2 309.7±135.1 A 295.4±122.0 14.3±13.0	4±2 A	0.20±0.68 AB 1.29±0.08 A 1.11±0.03 A
	Kidney 3 39.4±11.7 AB	32.2±6.8	7.2±8.7 16±16 AB -0.22±0.77 AB 1.37±0.08 A 1.21±0.05 A
	Blood 3 9.9±12.1 AB	0.6±0.4	9.4±11.7 90±5 CD 2.01±0.36 BC 1.48±0.12 A 1.20±0.01 A
	Muscle 3 11.7±15.2 AB	7.1±9.9	4.6±5.3 49±11 BC 0.45±0.09 AB 1.37±0.07 A 1.15±0.03 A
	Feathers 3 13.3±6.9 AB	0.4±0.2	13.0±6.7 97.4±0.1 D 2.33±0.02 C 1.30±0.05 A 1.16±0.01 A
	Brain 3 5.88±6.39 B	3.64±4.51 2.25±1.90 49±21 ABC 0.94±0.84 ABC 1.39±0.04 A 1.17±0.06 A

Table 4 .

 4 1. Food and feeding ecology, including blood δ 13 C (foraging habitat) and δ 15 N (diet) values, together with blood Hg characteristics of subantarctic penguins from Possession Island, Crozet archipelago (n, number of individuals). Values not sharing the same superscript letter are statistically different. Values are means ± SD.

	Species	Diet	Diving	N	δ 13 C	δ 15 N	THg	MeHg	MeHg	δ 202 Hg	∆ 199 Hg	∆ 199 Hg/∆ 201 Hg
			behaviour		(‰)	(‰)	(µg g -1 )	(µg g -1 )	(%)	(‰)	(‰)	ratio
	King penguin	fish	mesopelagic	11	-21.8±0.4 A 10.1±0.2 A 2.01±0.29 A	1.88±0.28 A	93±1 1.49±0.11 A 1.60±0.04 A 1.16±0.04 A
	Gentoo penguin	crustaceans, fish	epipelagic, benthic	11	-18.6±0.3 B	8.2±0.6 B	2.04±1.00 A 1.89±0.93 A	93±1 1.45±0.12 A 1.41±0.06 B 1.18±0.04 A
	Macaroni penguin	crustaceans (fish)	epipelagic	10	-20.0±0.7 C	8.6±0.4 B	1.06±0.16 B 1.01±0.15 B	96±2 1.66±0.11 B 1.54±0.06 C	1.14±0.02 A
	Eastern rockhopper penguin	crustaceans (fish)	epipelagic	10	-20.8±0.3 D	8.6±0.4 B	0.97±0.20 B 0.93±0.19 B	95±1 1.93±0.18 C 1.77±0.13 D	1.15±0.04 A

Table 4 .

 4 2. Total and MeHg concentrations (Mean ±1SD, µg.g -1 dw),Hg isotopic composition (δ 202 Hg and ∆ 199 Hg, Mean ± SD, ‰) of the skua and penguin species breeding at the different studied sites. n means the number of individuals analysed. Values not sharing the same superscript letter are statistically different.

	Species	Code	Location	Sampling date	n	THg	MeHg	δ 202 Hg	∆ 199 Hg
						µg g -1	µg g -1	‰	‰
	Antarctic skua	AL	Adélie Land	Dec 2011-Jan 2012	9	0.53±0.08 A	0.51±0.09 A	0.23±0.13 A	1.51±0.08 A
	Subantarctic skua	Ker	Kerguelen	Dec 2011	10	2.32±0.34 B	2.16±0.33 B	1.04±0.08 B	1.61±0.06 A
	Subantarctic skua	Cro	Crozet	Jan-Feb 2012	11	1.81±1.19 B	1.70±1.16 B	1.39±0.18 C	1.69±0.11 B
	Subantarctic skua	Ams	Amsterdam	Dec 2011	10	4.00±0.89 C	3.78±0.79 C	1.56±0.08 D	1.76±0.05 B
	Adélie penguin	AP	Adélie Land	Feb 2012	10	0.47±0.12 A	0.43±0.11 A	0.56±0.20 A	1.54±0.11 A
	King penguin	KP	Crozet	Oct 2011	11	2.01±0.29 B	1.88±0.28 B	1.49±0.11 B	1.60±0.04 A
	Macaroni penguin	MP	Crozet	Jan 2012	10	1.06±0.16 C	1.01±0.15 C	1.66±0.11 C	1.54±0.06 A
	Eastern rockhopper penguin	RP	Crozet	Feb 2012	10	0.97±0.20 C	0.93±0.19 C	1.93±0.18 D	1.77±0.13 B
	Northern rockhopper penguin	NRP	Amsterdam	Nov 2011	10	2.40±0.56 B	1.95±0.43 B	2.16±0.14 E	1.89±0.12 B

  Moreover, the investigation of Hg species-specific δ 202 Hg values in key organs would provide insights about in vivo demethylation of MeHg, which is widely assumed to occur mainly in livers, but could be observed in other tissues such as kidneys, brain or even muscles.

		Feathers
								70-90% MeHg mobilized
								during feather moult
		MeHg				MeHg storage	Muscle	MeHg iHg?
						organs
								MeHg
		MeHg		MeHg		MeHg target	Brain	iHg?
	Diet	iHg ?	Blood	iHg?	Organs	organs	Kidneys?	iHg	?
						MeHg
						detoxification organs	Liver	MeHg	iHg
									Excreta/Urine

Table 3 .

 3 S2: Mean values of Hg isotopic composition obtained for reference materials: UM-Almadén, NIES-13 (CRM human hair), IAEA-086 (RM human hair), ERM-CE-464 (CRM tuna fish), F-KP (IRM penguin feathers) and RBC-KP (IRM penguin red blood cells)

	Sample	Reference	n δ

204 Hg 2SD δ 202 Hg 2SD δ 201 Hg 2SD δ 200 Hg 2SD δ 199 Hg 2SD ∆ 204 Hg 2SD ∆ 201 Hg 2SD ∆ 200 Hg 2SD ∆ 199 Hg

																			2SD
				‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰
	UM Almadén	This study	77 -0.84 0.22 -0.55 0.16 -0.45 0.13 -0.27 0.15 -0.17 0.12 -0.01 0.16 -0.04 0.09 0.00 0.11 -0.03 0.11
		(Sherman, Blum,
		Keeler, et al.
		2012)	61	-	-	-0.57 0.05 -0.46 0.05 -0.28 0.03 -0.16 0.04	-	-	-0.03 0.02 0.01 0.02 -0.02 0.03
	NIES-13	This study	8 2.99 0.22 2.08 0.15 3.07 0.16 1.09 0.10 2.34 0.10 -0.12 0.28 1.51 0.06 0.04 0.04 1.81 0.09
		(Yamakawa et al.
		2016)	11 2.76 0.16 1.89 0.10 2.77 0.10 0.98 0.08 2.13 0.07 -0.04 0.11 1.36 0.07 0.04 0.04 1.65 0.06
	IAEA-086	This study	6 0.94 0.25 0.73 0.13 0.77 0.09 0.37 0.11 0.44 0.16 -0.14 0.26 0.22 0.08 0.00 0.09 0.26 0.15
		(Yamakawa et al.
		2016)	3 0.87 0.12 0.58 0.09 0.64 0.09 0.31 0.04 0.41 0.02 0.00 0.04 0.20 0.03 0.02 0.04 0.26 0.02
	ERM-CE-464	This study	2 0.91 0.13 0.64 0.04 2.37 0.03 0.40	0 2.49 0.01 -0.04 0.19 1.89 0.05 0.08 0.01 2.33 0.01
		(Li et al. 2014)	9 0.94 0.06 0.70 0.04 2.49 0.06 0.43 0.05 2.55 0.08	-	-	1.96 0.06 0.07 0.04 2.38 0.07
	F-KP	This study	17 2.40 0.25 1.58 0.22 2.67 0.21 0.80 0.16 2.11 0.18 0.04 0.24 1.48 0.13 0.01 0.09 1.71 0.15
	RBC-KP	This study	7 1.98 0.18 1.30 0.13 2.26 0.14 0.64 0.13 1.78 0.17 0.04 0.10 1.28 0.09 -0.01 0.09 1.46 0.14

Table 3 .

 3 S3: Internal reproducibility of Hg isotopic composition obtained for internal reference materials: F-KP (IRM penguin feathers) and RBC-KP (IRM penguin red blood cells) during the performed analytical sessions. N means number of analysis for the same sample extracts, which were prepared under similar procedures for each session

	Sample	Analytical session	δ

204 Hg δ 202 Hg δ 201 Hg δ 200 Hg δ 199 Hg ∆ 204 Hg ∆ 201 Hg ∆ 200 Hg ∆ 199 Hg n

  Figure 3.S2. Blood and feathers δ 202 Hg values for the four populations of skua chicks. Diamonds indicate mean values. Black lines indicate median values. Red asterisks indicate outliers.

	King penguin feathers	F-KP May 2015	Average (‰) 2.30	1.52	2.64	0.77	2.11	0.09	1.50	0.00	1.73	8
			2SD (‰)	0.32	0.22	0.20	0.11	0.08	0.16	0.06	0.06	0.08
		March 2016	Average (‰) 2.40	1.58	2.68	0.80	2.11	0.04	1.49	0.00	1.71 10
			2SD (‰)	0.24	0.26	0.17	0.15	0.18	0.31	0.12	0.06	0.15
		July 2016	Average (‰) 2.44	1.60	2.70	0.85	2.15	0.05	1.50	0.04	1.74	5
			2SD (‰)	0.26	0.15	0.16	0.18	0.15	0.11	0.07	0.12	0.12
	King penguin blood RBC-KP March 2016	Average (‰) 1.95	1.32	2.28	0.65	1.80	-0.01	1.28	-0.01	1.47	3
			2SD (‰)	0.10	0.10	0.04	0.03	0.04	0.15	0.11	0.03	0.02
		April 2016	Average (‰) 1.60	0.99	2.08	0.52	1.84	0.12	1.34	0.02	1.59	3
			2SD (‰)	0.37	0.20	0.29	0.36	0.08	0.15	0.14	0.25	0.05
		July 2016	Average (‰) 1.99	1.30	2.26	0.62	1.77	0.05	1.28	-0.03	1.44	5
			2SD (‰)	0.15	0.10	0.12	0.11	0.16	0.10	0.10	0.07	0.15

  Hg concentrations and speciation of the three individuals of giant petrels at the individual level: influence of ageIncreasing Hg concentrations were observed with age (Table3.S8), with the oldest specimen displaying much higher Hg amounts compared to medium age adult and to the youngest one for both kidneys (50.8±0.7, 39.9±0.4 and 27.5±0.4 µg•g -1 ; respectively) and livers (405.2±6.6 and 214.2±34.2 µg•g -1 ; respectively for the oldest and youngest individual). Indeed, Hg concentrations obtained in these two organs from the oldest bird were almost twice higher comparing to the youngest one. Hg species distributions were characterized by a great predominance of iHg with livers presenting percentages of 98% (young) and 95% (old). Although kidneys also present a higher extent of iHg, its proportion was more variable between individuals with the younger birds displaying much higher fraction of iHg (more than 90%) than the old individual, whose iHg composition only achieved 66 %. Feathers also displayed increasing Hg levels with age, ranging from 8.5±0.1 through 10.3±0.1 and 21.2±0.1 µg•g -1 , respectively for the youngest, medium age and oldest individuals. Hg concentrations in muscle were not correlated with age, since the youngest individual exhibited higher Hg content (4.1±0.1 µg•g -1 ) than the medium age giant petrel (1.7±0.1 µg•g -1

Table 3 .

 3 S5. Mean values of Hg isotopic composition obtained for reference materials: UM-Almadén (CRM cinnabar), DOLT-4 (CRM dogfish liver), ERM-CE-464 (CRM tunafish), NIES-13 (CRM human hair), IAEA-086 (RM human hair), P-KP (IRM penguin feathers) and RBC-KP (IRM penguin red blood cells).

	Sample	Reference	n δ

204 Hg 2SD δ 202 Hg 2SD δ 201 Hg 2SD δ 200 Hg 2SD δ 199 Hg 2SD ∆ 204 Hg 2SD ∆ 201 Hg 2SD ∆ 200 Hg 2SD ∆ 199 Hg

																					2SD
				‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰
	UM Almadén This study 45 -0.81 0.25 -0.54 0.17 -0.44 0.14 -0.26 0.13 -0.17 0.10 -0.01 0.15 -0.04	0.07	0.01	0.08	-0.03	0.09
		(Sherman,
		Blum,
		Keeler, et al.
		2012)	61	-	-	-0.57 0.05 -0.46 0.05 -0.28 0.03 -0.16 0.04	-	-	-0.03	0.02	0.01	0.02	-0.02	0.03
	DOLT-4	This study		-0.50 0.08 -0.33 0.07 0.66 0.03 -0.13 0.10 1.01 0.04 -0.01 0.02	0.91	0.02	0.04	0.06	1.09	0.02
		(Masbou
		2014)		-		-0.35 0.20 0.71 0.06 -0.14 0.06 1.03 0.07	-	-	0.98	0.19	0.02	0.03	1.12	0.11
	ERM-CE-464 This study		0.91 0.13 0.64 0.04 2.37 0.03 0.40 0.00 2.49 0.01 -0.04 0.19	1.89	0.05	0.08	0.01	2.33	0.01
		(Li et al.
		2014)		0.94 0.06 0.70 0.04 2.49 0.06 0.43 0.05 2.55 0.08	-	-	1.96	0.06	0.07	0.04	2.38	0.07
	NIES-13	This study		2.98	-	2.07	-	3.07	-	1.10	-	2.29	-	-0.11	-	1.51	-	0.06	-	1.76	-
		(Yamakawa
		et al. 2016) 11	2.76 0.16 1.89 0.10 2.77 0.10 0.98 0.08 2.13 0.07 -0.04 0.11	1.36	0.07	0.04	0.04	1.65	0.06
	IAEA-086	This study		1.23	-	0.81	-	0.76	-	0.45	-	0.50	-	0.02	-	0.15	-	0.04	-	0.29	-
		(Yamakawa
		et al. 2016)		0.87 0.12 0.58 0.09 0.64 0.09 0.31 0.04 0.41 0.02 0.00 0.04	0.20	0.03	0.02	0.04	0.26	0.02
	F-KP	This study		2.35 0.15 1.52 0.12 2.65 0.06 0.76 0.07 2.12 0.04 0.09 0.16	1.51	0.07	0.00	0.07	1.74	0.05
	RBC-KP	This study		1.95 0.10 1.32 0.10 2.28 0.04 0.65 0.03 1.80 0.04 -0.01 0.15	1.28	0.11	-0.01	0.03	1.47	0.02

Table 3 .

 3 S6. Individual and mean values of MeHg, iHg and THg concentrations (Mean ±1SD, µg g -1 dw) and Hg isotopic composition (δ 202Hg and Δ 199 Hg, Mean ± 2SD, ‰) of tissues Antarctic prions (n=10) from Kerguelen Islands. 57±0.92 2.00±0.17 1.32±0.13 3.04±0.15 0..69±0.06 2.70±0.12 0.02±0.07 2.05±0.09 0.02±0.02 2.37±0.10

	Sample	Tissue	MeHg	iHg	THg	δ 204 Hg	δ 202 Hg	δ 201 Hg	δ 200 Hg	δ 199 Hg	∆ 204 Hg	∆ 201 Hg	∆ 200 Hg	∆ 199 Hg
			µg g -1	µg g -1	µg g -1	‰	‰	‰	‰	‰	‰	‰	‰	‰
	FA01-LivA	Liver 1.94±0.22	1.00±0.09	2.94±0.29	NA	NA	NA	NA	NA	NA	NA	NA	NA
	FA02-LivA	Liver 1.32±0.05	0.59±0.01	1.89±0.02	NA	NA	NA	NA	NA	NA	NA	NA	NA
	FA03-LivA	Liver 1.22±0.04	0.66±0.01	1.87±0.05	NA	NA	NA	NA	NA	NA	NA	NA	NA
	FA04-LivA	Liver 0.95±0.04	0.56±0.01	1.50±0.05	NA	NA	NA	NA	NA	NA	NA	NA	NA
	FA05-LivA	Liver 1.95±0.14	0.73±0.11	2.68±0.25	NA	NA	NA	NA	NA	NA	NA	NA	NA
	FA06-LivA	Liver 0.89±0.16	0.60±0.04	1.49±0.14	-0.45	-0.28	1.55	-0.20	-0.03	1.83	1.76	-0.06	1.90
	FA07-LivA	Liver 1.13±0.02	0.47±0.14	1.60±0.11	0.51	0.30	1.90	0.21	0.06	1.99	1.68	0.06	1.91
	FA08-LivA	Liver 1.25±0.12	1.04±0.14	2.30±0.26	-0.19	-0.10	1.56	-0.03	-0.05	1.95	1.63	0.02	1.98
	FA09-LivA	Liver 1.65±0.23	0.87±0.11	2.52±0.24	-0.15	-0.09	1.50	-0.11	-0.02	1.82	1.57	-0.06	1.84
	FA10-LivA	Liver 1.55±0.17	0.55±0.11	2.10±0.20	0.35	0.24	1.82	0.04	0.00	1.95	1.64	-0.08	1.89
	Average	Liver 1.38±0.38	0.71±0.20	2.09±0.51 0.02±0.40 0.01±0.25 1.66±0.18 -0.02±0.16 -0.00±0.04 1.91±0.08 1.65±0.07 -0.02±0.06 1.90±0.05
	FA01-MusA Muscle 0.24±0.02 0.027±0.0003 0.28±0.02	0.47	0.15	1.92	0.13	0.26	2.13	1.81	0.06	2.09
	FA02-MusA Muscle 0.25±0.01 0.028±0.002	0.29±0.01	0.07	-0.10	1.65	-0.03	0.21	1.94	1.72	0.02	1.97
	FA03-MusA Muscle 0.18±0.00 0.077±0.004	0.26±0.01	-0.02	0.00	1.77	0.00	-0.01	2.16	1.78	0.00	2.16
	FA04-MusA Muscle 0.20±0.01 0.031±0.001	0.23±0.01	0.99	0.57	2.02	0.22	0.15	2.04	1.60	-0.07	1.90
	FA05-MusA Muscle 0.35±0.01	0.08±0.01	0.43±0.01	1.13	0.70	2.10	0.34	0.08	2.00	1.58	-0.01	1.82
	FA06-MusA Muscle 0.20±0.01 0.033±0.002	0.24±0.01	1.09	0.79	2.38	0.43	-0.08	2.30	1.79	0.04	2.10
	FA07-MusA Muscle 0.22±0.01 0.046±0.008	0.27±0.01	1.12	0.77	2.16	0.37	-0.02	1.98	1.58	-0.01	1.79
	FA08-MusA Muscle 0.27±0.01 0.042±0.003	0.31±0.01	1.34	0.89	2.32	0.44	0.02	2.06	1.65	0.00	1.83
	FA09-MusA Muscle 0.29±0.01 0.041±0.001	0.33±0.01	-0.18	-0.19	1.46	-0.13	0.10	1.73	1.60	-0.04	1.77
	FA10-MusA Muscle 0.32±0.01 0.068±0.003 0.386±0.004	0.25	0.12	1.73	0.01	0.06	1.92	1.63	-0.05	1.89
	Average	Muscle 0.25±0.05	0.05±0.02	0.30±0.06 0.63±0.57 0.37±0.41 1.95±0.30 0.18±0.21 0.08±0.11 2.03±0.16 1.67±0.09 -0.01±0.04 1.93±0.14
	FA01-pool-B1 Feather 1.98±0.01 0.287±0.002	2.27±0.02	1.81	1.19	2.97	0.61	2.71	0.03	2.08	0.02	2.41

Table 3 .

 3 S7. Individual and mean values of MeHg, iHg and THg concentrations (Mean ±1SD, µg g -1 dw) and Hg isotopic composition (δ 202Hg and Δ 199 Hg, Mean ± 2SD, ‰) of tissues white-chinned petrels (n=10) from Kerguelen Islands.

	Species	Tissue	MeHg	iHg	THg	δ 204 Hg	δ 202 Hg	δ 201 Hg	δ 200 Hg	δ 199 Hg	∆ 204 Hg	∆ 201 Hg	∆ 200 Hg	∆ 199 Hg
			µg g -1	µg g -1	µg g -1	‰	‰	‰	‰	‰	‰	‰	‰	‰
	WCP01-LivA	Liver	1.92±0.05 57.90±0.71 59.82±0.71	-1.34	-0.91	0.36	-0.45	1.00	0.03	1.05	0.01	1.23
	WCP02-LivA	Liver	1.73±0.06 79.27±0.64 81.00±0.64	-1.13	-0.80	0.32	-0.43	0.92	0.07	0.92	-0.02	1.13
	WCP03-LivA	Liver	5.54±0.04 69.36±0.68 74.90±0.68	-1.21	-0.80	0.42	-0.40	0.96	-0.01	1.02	0.00	1.16
	WCP04-LivA	Liver	4.36±0.09 33.96±0.70 38.32±0.71	-0.20	-0.11	1.03	-0.10	1.16	-0.03	1.11	-0.04	1.19
	WCP05-LivA	Liver	4.60±0.06 41.34±0.45 45.95±0.46	-0.65	-0.45	0.71	-0.27	1.05	0.02	1.05	-0.04	1.16
	WCP06-LivA	Liver	7.95±0.20 69.30±0.27 77.26±0.33	-1.50	-0.99	0.17	-0.54	0.80	-0.02	0.91	-0.04	1.05
	WCP07-LivA	Liver	4.97±0.05 27.98±0.54 32.94±0.54	-1.08	-0.76	0.51	-0.38	1.03	0.05	1.08	0.00	1.22
	WCP08-LivA	Liver	8.15±0.06 49.46±0.24 57.62±0.25	-0.99	-0.68	0.47	-0.35	0.98	0.02	0.98	-0.01	1.15
	WCP09-LivA	Liver	4.55±0.10 40.29±0.29 44.83±0.31	-0.83	-0.54	0.68	-0.26	1.09	-0.02	1.08	0.01	1.23
	WCP10-LivA	Liver	5.01±0.03 25.64±0.18 30.65±0.18	-0.57	-0.35	0.79	-0.25	1.08	-0.05	1.05	-0.07	1.17
	Average	Liver	4.88±2.10 49.45±18.76 54.33±18.67 -0.95±0.39 -0.64±0.27 0.54±0.26 -0.34±0.13 1.01±0.10	0.01±0.04	1.03±0.07	-0.02±0.03 1.17±0.05
	WCP01-MusA	Muscle 1.67±0.13 0.60±0.02	2.27±0.13	-0.19	-0.17	0.90	-0.12	1.06	0.07	1.03	-0.04	1.10
	WCP02-MusA	Muscle 1.33±0.01 0.90±0.04	2.23±0.04	-0.08	-0.14	1.08	-0.09	1.30	0.13	1.18	-0.02	1.33
	WCP03-MusA	Muscle 4.27±0.02 1.07±0.03	5.34±0.03	0.83	0.60	1.60	0.33	1.52	-0.07	1.15	0.03	1.37
	WCP04-MusA	Muscle 3.37±0.13 0.79±0.06	4.17±0.14	0.55	0.33	1.42	0.19	1.45	0.05	1.17	0.02	1.37
	WCP05-MusA	Muscle 4.15±0.06 1.06±0.02	5.21±0.06	0.59	0.41	1.39	0.29	1.42	-0.02	1.08	0.09	1.32
	WCP06-MusA	Muscle 4.75±0.11 0.82±0.04	5.57±0.12	0.58	0.40	1.37	0.17	1.29	-0.01	1.07	-0.03	1.19
	WCP07-MusA	Muscle 4.07±0.09 1.01±0.03	5.08±0.10	0.69	0.48	1.54	0.20	1.46	-0.03	1.18	-0.04	1.34
	WCP08-MusA	Muscle 6.15±0.06 1.66±0.04	7.81±0.07	0.38	0.21	1.22	0.10	1.31	0.06	1.06	-0.01	1.26
	WCP09-MusA	Muscle 3.60±0.02 0.87±0.06	4.46±0.06	0.64	0.44	1.47	0.20	1.46	-0.02	1.13	-0.02	1.35
	WCP10-MusA	Muscle 3.70±0.11 1.11±0.07	4.81±0.13	0.56	0.41	1.42	0.20	1.39	-0.05	1.11	-0.01	1.29
	Average	Muscle 3.71±1.82 0.99±0.34	4.69±1.78 0.45±0.37 0.30±0.28 1.34±0.21 0.15±0.16 1.37±0.12	0.01±0.06	1.12±0.05	0.00±0.04	1.29±0.07
	WCP01-pool-B1 Feathers 8.23±0.06 0.28±0.01	8.51±0.04	1.41	0.95	1.86	0.52	1.69	-0.01	1.14	0.05	1.45

Table 3 .

 3 S8. Individual and mean values of MeHg, iHg and THg concentrations (Mean ±1SD, µg g -1 dw), MeHg proportion (Mean ±1SD, %) and Hg isotopic composition(δ 202 Hg and Δ 199 Hg, Mean ± 2SD, ‰) of tissues of southern giant petrels (n=3) from Kerguelen Islands. PGA01 is the young specimen, PGA02 a medium age individual and PGA03 the oldest individual.

	Sample	Tissue	MeHg	iHg	THg	δ 204 Hg	δ 202 Hg	δ 201 Hg	δ 200 Hg	δ 199 Hg	∆ 204 Hg	∆ 201 Hg	∆ 200 Hg	∆ 199 Hg
			µg g -1	%	µg g -1	µg g -1	‰	‰	‰	‰	‰	‰	‰	‰	‰
	PGA01-
	LivA	Liver	5.09±0.80 2.21±0.21	209.14±34.22 214.22±34.23	-0.44	-0.28	0.98	-0.14	1.27	-0.02	1.19	0.00	1.34
	PGA03-
	LivA	Liver	23.50±0.10 5.80±0.11	381.69±6.56	405.19±6.57	1.06	0.68	1.64	0.32	1.40	0.05	1.13	-0.02	1.23
														-
			14.29±13.0			309.71±135.0	0.31±1.0	0.20±0.6		0.09±0.3	1.34±0.0	0.01±0.0	1.16±0.0	0.01±0.0	1.29±0.0
	Average	Liver	2	3.89±2.15 295.41±122.01	3	6	8	1.31±0.47	3	9	5	4	1	8
	PGA01-
	KidA	Kidney 2.73±0.12 9.93±0.45	24.80±0.41	27.53±0.43	-0.48	-0.35	0.87	-0.21	1.21	0.04	1.13	-0.03	1.30
	PGA02-
	KidA	Kidney 1.64±0.11 4.13±0.30	38.23±0.43	39.88±0.44	-1.63	-0.93	0.40	-0.44	1.13	-0.24	1.10	0.03	1.36
	PGA03-
	KidA	Kidney 17.24±0.53 33.90±0.50	33.60±0.40	50.84±0.67	0.77	0.60	1.63	0.28	1.61	-0.12	1.18	-0.03	1.46
							-	-		-				-
				15.99±15.7			0.44±1.2	0.22±0.7		0.12±0.3	1.14±0.0	0.11±0.1	1.14±0.0	0.01±0.0	1.37±0.0
	Average	Kidney 7.21±8.71	9	32.21±6.83	39.42±11.66	0	7	0.97±0.62	6	4	4	4	3	8
	PGA01-
	PGAt	Blood	2.66±0.01 89.01±0.31	0.31±0.01	2.97±0.01	3.25	2.13	2.86	1.06	2.06	0.08	1.26	0.00	1.52
	PGA02-
	PGAt	Blood	2.59±0.05 84.72±0.24	0.431±0.003	3.03±0.05	2.41	1.61	2.33	0.79	1.74	0.00	1.12	-0.02	1.34
	PGA03-
	PGAt	Blood 22.89±0.12 95.52±0.12	1.04±0.03	23.93±0.13	3.43	2.30	3.05	1.14	2.15	-0.01	1.32	-0.01	1.57
														-
							3.03±0.5	2.01±0.3		1.00±0.1	1.98±0.2	0.03±0.0	1.23±0.1	0.01±0.0	1.48±0.1
	Average	Blood 9.38±11.70 90.34±5.00	0.59±0.39	9.98±12.08	5	6	2.74±0.37	8	1	5	0	1	2
	PGA01-
	MusA	Muscle 2.09±0.01 53.15±2.12	2.03±0.14	4.11±0.14	0.49	0.38	1.54	0.17	1.50	-0.08	1.26	-0.02	1.41

Table 4 .

 4 S1. Blood Hg isotopic composition of subantarctic penguins from Possession Island, Crozet archipelago (n, number of individuals).Values are means ± SD.

	Species	Date	n	δ 204 Hg	δ 202 Hg	δ 201 Hg	δ 200 Hg	δ 199 Hg	∆ 204 Hg	∆ 201 Hg	∆ 200 Hg	∆ 199 Hg
				‰	‰	‰	‰	‰	‰	‰	‰	‰
	King penguin	October 2011 11 2.18±0.17	1.49±0.11	2.50±0.08	0.73±0.06	1.98±0.05	-0.05±0.09	1.38±0.03	-0.02±0.03	1.60±0.04
	Gentoo penguin	October 2011 11 2.20±0.18	1.45±0.12	2.29±0.12	0.72±0.07	1.77±0.08	0.03±0.03	1.20±0.04	-0.01±0.02	1.41±0.06
	Macaroni penguin	January 2012 10 2.49±0.16	1.66±0.11	2.60±0.12	0.83±0.08	1.96±0.07	0.01±0.05	1.35±0.05	0.00±0.04	1.54±0.06
	Eastern rockhopper penguin February 2012 10 2.89±0.25	1.93±0.18	2.99±0.04	0.98±0.07	2.25±0.12	0.01±0.06	1.54±0.08	0.07±0.07	1.77±0.13

Table 4 .

 4 S2. Mean values of Hg isotopic composition obtained for reference materials: UM-Almadén, NIES-13 (CRM human hair), hair), ERM-CE-464 (CRM tuna fish) and RBC-KP (IRM penguin red blood cells).

	Sample	Reference	n δ

204 Hg 2SD δ 202 Hg 2SD δ 201 Hg 2SD δ 200 Hg 2SD δ 199 Hg 2SD ∆ 204 Hg 2SD ∆ 201 Hg 2SD ∆ 200 Hg 2SD ∆ 199 Hg 2SD

				‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰	‰
	UM Almadén	This study	38	-0.84 0.23 -0.54 0.14 -0.45 0.14 -0.27 0.18 -0.17 0.14	-0.03 0.13	-0.04 0.08	0.00 0.14	-0.03 0.12
		Sherman et al., 2012a 61 -	-		-0.57 0.05 -0.46 0.05 -0.28 0.03 -0.16 0.04 -	-		-0.03 0.02	0.01 0.02	-0.02 0.03
	NIES-13	This study	5	2.99 0.28	2.11 0.13	3.11 0.16	1.08 0.01	2.34 0.14	-0.14 0.24	1.52 0.05	0.05 0.07	1.80 0.08
		Yamakawa et al. 2016 11	2.76 0.16	1.89 0.10	2.77 0.10	0.98 0.08	2.13 0.07	-0.04 0.11	1.36 0.07	0.04 0.04	1.65 0.06
	ERM-CE-464	This study	2	0.91 0.13	0.64 0.04	2.37 0.03	0.40 0.00	2.49 0.01	-0.04 0.19	1.89 0.05	0.08 0.01	2.33 0.01
		Li et al. 2014	9	0.94 0.06	0.70 0.04	2.49 0.06	0.43 0.05	2.55 0.08 -	-		1.96 0.06	0.07 0.04	2.38 0.07
	RBC-KP	This study	5	2.00 0.15	1.32 0.12	2.28 0.14	0.64 0.14	1.79 0.18	0.04 0.10	1.29 0.09	-0.02 0.09	1.46 0.16

Table 4 .

 4 S2. Calculation of MeHg:Sred-DOC ratios for the estimation of MeHg photodemethylation based on experimental fractionation factors

Table 4 .

 4 S6. Calculation of fractionation factors of MDF (α (δ 202 Hg)) corresponding to MeHg photodemethylation at each study area based on experimental studies under mid-rangeMeHg:Sred-DOC conditions (SRFA organic matter conditions, 35% S-red)[START_REF] Chandan | Mercury isotope fractionation during aqueous photoreduction of monomethylmercury in the presence of dissolved organic matter[END_REF].

		Conditions: mid-range MeHg;S red-DOC (35% S-red)
	Population/
	Sites	ε ∆ 199 Hg (‰) α (∆ 199 Hg) ε δ 202 Hg (‰)	α (δ 202 Hg)	Ratio α (δ 202 Hg)/α (∆ 199 Hg)
	Skuas
	AZ	-5.23895	0.99476	-4.73312	0.99527
	SAZ	-5.24037	0.99476	-4.73440	0.99527
	STZ	-5.20641	0.99479	-4.70372	0.99530	1.00051
	Penguins
	AZ	-5.30277	0.99470	-4.79078	0.99521
	SAZ	-5.26504	0.99473	-4.75669	0.99524
	STZ	-5.163113	0.99484	-4.66461	0.99534	1.00050

Table 4 .

 4 S7. Calculation of fractionation factors of MDF (α (δ 202 Hg)) corresponding to MeHg photodemethylation at each study area based on experimental studies under lowMeHg:Sred-DOC conditions (NL NOM organic matter conditions, 50% S-red)[START_REF] Chandan | Mercury isotope fractionation during aqueous photoreduction of monomethylmercury in the presence of dissolved organic matter[END_REF].

			Conditions: low MeHg;S red-DOC (50% S-red)
	Population/
	Sites	ε ∆ 199 Hg (‰) α (∆ 199 Hg) ε δ 202 Hg (‰)	α (δ 202 Hg)	Ratio α (δ 202 Hg)/α (∆ 199 Hg)
	Skuas
	AZ	-1.501006	0.998499	-1.520006	0.998480
	SAZ	-1.500575	0.998499	-1.519569	0.998480
	STZ	-1.495628	0.998504	-1.514560	0.998485	0.99998
	Penguins
	AZ	-1.438243	0.998562	-1.456449	0.998544
	SAZ	-1.504152	0.998496	-1.523191	0.998477
	STZ	-1.489287	0.998511	-1.508138	0.998492	0.99998

Table 4 .

 4 (Table 4.S8). The obtained slope represents a α( 198 Hg/ 202 Hg) factor of 1.0005, meaning a total mass-dependent fractionation of 0.03 ‰ due to volatilization. S8. Calculation of the fraction of volatilised Hg and enrichment factor between the studied sites

	Site	Water temperature (K)	Henry's constant (KH)	Hg 0 (air) Hg 0 (aq)	fR (Hg 0 aq)	log (fR)	ε δ 202 Hg (‰)
	AZ	273	0.21	0.8	3.87	0.829	-0.188	0.088
	SAZ	281	0.25	0.9	3.58	0.799	-0.224	0.105
	STZ	291	0.31	1.0	3.23	0.763	-0.270	0.127

Acknowledgements

The present work was supported financially and logistically by the Région Poitou-Charentes through a PhD grant to MR, and by the Institut Polaire Français Paul Emile Victor (IPEV, program no. 109, H. Weimerskirch) and the Terres Australes et Antarctiques Françaises (TAAF) and by the Agence Nationale de la Recherche (program POLARTOP, O. Chastel). The IUF (Institut Universitaire de France) is acknowledged for its support to PB as a Senior Member. The authors thank all the fieldworkers that contributed to the collection of samples. Field procedures were authorised by the Ethics Committee of IPEV and by the Comité de l'Environnement Polaire. Acknowledgements The present work was supported financially and logistically by the Région Poitou-Charentes through a PhD grant to MR, and by the Institut Polaire Français Paul Emile Victor (IPEV, program no. 109, H. Weimerskirch) and the Terres Australes et Antarctiques Françaises (TAAF) and by the Agence Nationale de la Recherche (program POLARTOP, O. Chastel). The IUF (Institut Universitaire de France) is acknowledged for its support to PB as a Senior Member. The authors thank all the fieldworkers that contributed to the collection of samples. Field procedures were authorised by the Ethics Committee of IPEV and by the Comité de l'Environnement Polaire.

Acknowledgments

The authors thank M. Loubon and F. Théron who helped collecting blood samples in the field, A. Carravieri for managing the sample database, E. Tessier, S. Bérail and J. Barre (IPREM) for technical assistance during Hg speciation and Hg isotopic analyses, and M. Brault-Favrou and G.

Guillou for preparing and running stable isotope samples, respectively. The present work was supported financially and logistically by the Institut Polaire Français Paul Emile Victor (IPEV, programme no. 109, H. Weimerskirch), and the Terres Australes et Antarctiques Françaises (TAAF) and by the Agence Nationale de la Recherche (program POLARTOP, O. Chastel). It was also supported financially by the Région Poitou-Charentes (now Région Nouvelle Aquitaine) through a PhD grant to MR, and by the French national program EC2CO Biohefect/Ecodyn//Dril/MicrobiEen (TIMOTAAF project, to DA). The IUF (Institut Universitaire de France) is acknowledged for its support to PB.

Acknowledgments

The authors thank all the fieldworkers that contributed to the collection of seabird blood and feather samples in the frame of the program no. 109 (H. Weimerskirch) supported by the Institut Polaire Français Paul Emile Victor (IPEV) and the Terres Australes et Antarctiques Françaises (TAAF) and by the Agence Nationale de la Recherche (program POLARTOP, O. Chastel). Special thanks are due to A. Carravieri who managed the sample database. The present work was supported financially by the Région Poitou-Charentes (now Région Nouvelle Aquitaine) through a Ph.D. grant to MR, and by the French national program EC2CO Biohefect/Ecodyn//Dril/MicrobiEen (TIMOTAAF project, P.I. D. Amouroux). The IUF (Institut Universitaire de France) is acknowledged for its support to PB.

Abstract

Seabirds are considered as effective sentinels of environmental marine contamination and their feathers are extensively used as non-lethal samples for contaminant biomonitoring. This tissue represents the main route for mercury (Hg) elimination in seabirds and contains predominantly methylmercury (MeHg). In this work, we developed a robust analytical technique for precise and accurate simultaneous quantification of MeHg, inorganic Hg (iHg) and consequently total Hg (THg), in feathers by gas-chromatography (GC)-ICPMS analyses using species-specific isotope dilution technique. An optimisation of the extraction method was carried out by testing different extraction systems, reagents and spiking procedures using an feather IRM.

The procedure was validated for MeHg and THg concentrations with a human hair certified reference material. Microwave nitric acid extraction with spike addition before the extraction provided the best recovery and was chosen as the most appropriate species simultaneous extraction method (SSE). An additional assessment was performed by comparison of our developed extraction method and a MeHg specific extraction technique (MSE) classically used for Hg speciation studies on feathers. The developed method was applied to feather samples from a large number of seabirds from the Southern Ocean (penguins, albatrosses, petrels and skuas) to investigate the variability of Hg speciation across a large range of Hg exposure conditions and concentrations. In all cases, MeHg accounted for > 90% of THg, thus verifying the predominance of organic Hg over iHg in feathers.

Keywords: seabirds, Southern Ocean, GC-ICPMS, methylmercury, inorganic mercury, interspecies transformations, keratin

Part 3.2: Hg isotopic composition of key tissues documents Hg metabolic processes in seabirds

Abstract

Seabirds bioaccumulate significant Hg amounts in their tissues as a consequence of their medium to high position in marine food webs and of their long-life span. Since they display contrasted foraging strategies, feeding on different trophic levels, seabirds are appropriate models to assess Hg bioaccumulation processes as well redistribution and detoxification mechanisms in their organism. The aim of this work is thus the evaluation of Hg metabolic responses by analyses of different tissues of seabirds from the Southern Ocean. Internal tissues (pectoral muscle, liver, brain, kidneys and blood) and feather samples were studied by the combination of Hg speciation and Hg isotopic composition analyses. Three seabird species with contrasting feeding ecology were chosen for this assessment: Antarctic prion (zooplankton eaters), white-chinned petrel (mesopelagic fish consumers) and southern giant petrel (other-seabird meat and carrion eaters).

High variability in Hg speciation between tissues evidenced their different roles in metabolic processes such as demethylation in liver, recognized as a Hg detoxification organ. Hg isotopic signatures exhibited the same trend in the three seabird species, with increasing mass-dependent fractionation (MDF, δ 202 Hg) values from livers through muscles to feathers, attributed to preferential demethylation of isotopically lighter Hg in liver and preferential sequestration of isotopically heavier Hg in feathers (preferential complexation of heavier isotopes and/or integration of distinct Hg sources). Mass-independent fractionation (MIF, Δ 199 Hg) values differed widely between internal tissues and feathers, suggesting the existence of different Hg sources due to distinct Hg turnover between tissues, consistent with organs as long-term accumulative tissues and feathers as representative of annual Hg contamination between inter-moult periods. Annexes: Chapter 3. Methodological approach: Biological aspects

Keywords

Feathers and blood from seabirds as efficient biomonitoring tissues for Hg isotopic studies: implications of using chicks and adults

Experimental

Hg isotopic composition analyses

Hg isotopic compositions of samples are reported using delta notation according to the following equations:

MIF signatures are expressed using capital delta notation, as suggested elsewhere [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF], according to the following equations: Hg±2SD δ 202 Hg±2SD δ 201 Hg±2SD δ 200 Hg±2SD δ 199 Hg±2SD ∆ 204 Hg±2SD ∆ 201 Hg±2SD ∆ 200 Hg±2SD ∆ 199 Hg±2SD (µg g -1 ) (µg g -1 ) (µg g -1 ) Total Hg and Hg species concentrations Pronounced differences in total Hg concentrations were found between sites for both skua and penguin populations. However, no variation in Hg species distribution was observed between tissues, individuals, species or sites. Both feathers and blood presented a majority of MeHg for all the individuals (skuas and penguins) independently of Hg concentration. Percentages of MeHg mean values for feathers and blood of the four skua populations were 94±2% and 94±3%, respectively.

Overall penguins' individuals presented a mean percentage of MeHg of 92±2% and 92±7%

respectively for feathers and blood samples. Skua chick feathers displayed higher MeHg concentrations (µg/g dry mass) compared to blood samples for all the analysed individuals (3 times higher, on average for the four populations). In the case of penguins, similar concentrations were obtained in both tissues for some penguin species (Adélie, king and both eastern and northern rockhopper penguins), whereas feathers presented higher MeHg levels than blood in all the individuals of gentoo and macaroni penguins (2 times higher on average for the two populations). As a general trend, blood and feathers MeHg concentrations were highly correlated for skuas (Pearson correlation, R 2 =0.89, p<0.0001, n=40) and penguin individuals (Pearson correlation, R 2 =0.74, p<0.0001, n=72), as shown in Figure 3.S1.

Figure 3.S1. A) Feather versus blood (red blood cells) MeHg concentrations in skua chicks. B) Feather versus blood (red blood cells) MeHg concentrations in adult penguins (individual values and mean population values). Abbreviations for the different populations are AP (Adélie penguin), GP (gentoo penguin), KP (king penguin), MP (macaroni penguin), RP (eastern rockhopper penguin) and NRP (northern rockhopper penguin).

AL A B

Estimation of the fraction of photodemethylated MeHg for each penguin population

The extent of photodemethylation undergone by MeHg before its incorporation into the aquatic food web was estimated by following Bergquist and Blum reference model [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF]. MIF fractionation factors (α∆199Hg and α∆201Hg) were determined experimentally as a function of MeHg/DOC ratio by Rayleigh-fractionation equations in waters with the presence of 2 mg/L and 20 mg/L DOM. This same model was later extrapolated to environmental conditions in marine medium taking into account different conditions of MeHg/DOC in the Arctic Ocean [START_REF] Point | Methylmercury photodegradation influenced by sea-ice cover in Arctic marine ecosystems[END_REF]. In a more recent study by [START_REF] Chandan | Mercury isotope fractionation during aqueous photoreduction of monomethylmercury in the presence of dissolved organic matter[END_REF] photodemethylation extent was evaluated at different ratios between MeHg and reduced sulfur content in DOC (MeHg: Sred-DOC). In our study, we calculated MeHg:DOC according to previous data on the subantarctic zone of the Southern Ocean [START_REF] Canario | Mercury and methylmercury in the Atlantic sector of the Southern Ocean[END_REF][START_REF] Tremblay | Origin and fate of particulate and dissolved organic matter in a naturally iron-fertilized region of the Southern Ocean[END_REF], MeHg amount in the pelagic zone (100-200 m depth) which is approximatively 0.48 ng per mg of dissolved organic carbon. Concentrations of DOC in surface waters were not considered since photochemical reactions are known to photodegrade organic matter and that MeHg is produced under the photic zone, where phytoplankton is degraded by bacteria. We calculated by extrapolation the MIF fractionation factors for the Crozet Islands to obtain fitting equations based on Chandan calculations (low concentrations of DOC) [START_REF] Chandan | Mercury isotope fractionation during aqueous photoreduction of monomethylmercury in the presence of dissolved organic matter[END_REF]. MeHg accumulated in GP was 13.1±0.6% photodegraded, whereas slightly greater extent of MeHg photodegradation was observed in pelagic penguins: 14.7±0.3% (KP) and 14.3±0.5% (MP) and 16.2±1.1% (RP).

Estimation of the fraction of photodemethylated MeHg in surface waters over latitude

Variations in surface ocean MeHg photodemethylation can be estimated from the MIF recorded in seabird blood based on a Rayleigh model:

where Δ 199 Hgi corresponds to the initial MIF of marine MeHg before undergoing photodemethylation, which was assumed as 0 due to the negligible total Hg Δ 199 Hg values in seawater (Štrok et al., 2015) and because MeHg is assumed to have a similar MIF as the total Hg before photodemethylation since it likely converted from inorganic Hg in the surface ocean. The ε(Δ 199 Hg) is the enrichment factor, reported as ε = 1000*( α∆199Hg -1) in function of the kinetic fractionation factor (α∆199H) determined in laboratory experiments. f is the remaining fraction of non-photodemethylated MeHg.

Experimental estimations of enrichment factors of MeHg aquatic photodemethylation was first performed as a function of MeHg/DOC ratio by [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF]. The relation between MIF values and the fraction of remaining MeHg was used in fish from the Lake Michigan to estimate the photodegradation extent. This reference fractionation model was later applied for estimation of the MeHg loss due to photodemethylation in marine environments [START_REF] Gehrke | Sources of mercury to San Francisco Bay surface sediment as revealed by mercury stable isotopes[END_REF][START_REF] Senn | Stable isotope (N, C, Hg) study of methylmercury sources and trophic transfer in the northern gulf of Mexico[END_REF][START_REF] Point | Methylmercury photodegradation influenced by sea-ice cover in Arctic marine ecosystems[END_REF]. [START_REF] Point | Methylmercury photodegradation influenced by sea-ice cover in Arctic marine ecosystems[END_REF] extrapolated the fractionation model to the presence of DOC in Arctic marine natural waters (lower DOC content) [START_REF] Point | Methylmercury photodegradation influenced by sea-ice cover in Arctic marine ecosystems[END_REF]. In a recent study, [START_REF] Chandan | Mercury isotope fractionation during aqueous photoreduction of monomethylmercury in the presence of dissolved organic matter[END_REF] investigated Hg isotopic fractionation factors considering changing DOC binding sites and concluded that MIF extent is strongly dependent on the amount of reduced sulphur content in organic matter (Sred-DOC). Enrichment factors were evaluated under different types and concentrations of organic matter standards:

Suwanee River fulvic acid (SRFA), Pony Lake fulvic acid (PL FAR) and Nordic Lake natural organic matter (NL NOM). These two reference organic matter models present contrasted proportions of reduced sulphur organic matter (35% for SRFA, 59% for PL FAR and 50% for NL NOM). Furthermore, these models represent more realistic environmental conditions (low MeHg:

Sred-DOC ratios) than previous experimental studies and can be more representative of the processes occurring under natural conditions in the environment.

In order to estimate the extent of photochemical demethylation of MeHg over the studied latitudinal gradient in the Southern Ocean we calculated the corresponding enrichment factors by application of experimental models in our own environmental conditions of MeHg:DOC and MeHg: S red-DOC. Recent published data on MeHg concentrations [START_REF] Cossa | Mercury in the Southern Ocean[END_REF][START_REF] Mastromonaco | Speciation of mercury in the waters of the Weddell, Amundsen and Ross Seas (Southern Ocean)[END_REF][START_REF] Canario | Mercury and methylmercury in the Atlantic sector of the Southern Ocean[END_REF], DOC concentrations [START_REF] Tremblay | Origin and fate of particulate and dissolved organic matter in a naturally iron-fertilized region of the Southern Ocean[END_REF]Table 4.S4. Calculation of MeHg: DOC ratios for the estimation of MeHg photodemethylation based on experimental fractionation factors [START_REF] Bergquist | Mass-dependent and -independent fractionation of Hg isotopes by photoreduction in aquatic systems[END_REF] for high MeHg:DOC conditions Reference data for this study (1) Other published data

(2)

Reference data

(3) Zone Depth MeHg (pmol L -1 ) MeHg (pmol L -1 ) DOC (mg L -1 ) MeHg/DOC [START_REF] Cossa | Mercury in the Southern Ocean[END_REF] (2) AZ: Nerentorp Mastromonaco et al. 2017; SAZ: Canario et al. 2017; STZ: Bratkic et al. 2016 (3) [START_REF] Tremblay | Origin and fate of particulate and dissolved organic matter in a naturally iron-fertilized region of the Southern Ocean[END_REF][START_REF] Canario | Mercury and methylmercury in the Atlantic sector of the Southern Ocean[END_REF][START_REF] Ogawa | Vertical distributions of dissolved organic carbon and nitrogen in the Southern Ocean[END_REF]