Alice Carravieri
email: alice.carravieri@cebc.cnrs.fr

Yves Cherel

Pierre Blévin

Maud Brault-Favrou

Olivier Chastel

Paco Bustamante

Mercury exposure in a large subantarctic avian community

Keywords: Feeding ecology, Kerguelen, Procellariiformes, seabirds, Southern Ocean

contamination poses potential threats to ecosystems worldwide.

In order to study Hg bioavailability in the poorly documented southern Indian Ocean, Hg exposure was investigated in the large avian community of Kerguelen Islands.

Adults of 27 species (480 individuals) showed a wide range of feather Hg concentrations, from 0.4 ± 0.1 to 16.6 ± 3.8 µg g -1 dry weight in Wilson's storm petrels and wandering albatrosses, respectively. Hg concentrations increased roughly in the order crustacean-< fish-≤ squid-≤ carrion-consumers, confirming that diet, rather than taxonomy, is an important driver of avian Hg exposure. Adults presented higher Hg concentrations than chicks, due to a longer duration of exposure, with the only exception being the subantarctic skua, likely because of feeding habits' differences of the two age-classes in this species. High Hg concentrations were reported for three species of the poorly known gadfly petrels, which merit further investigation.

Introduction

Mercury (Hg) is a pervasive non-essential metal affecting ecosystem health. Despite its natural origin, Hg has been mobilized by human activities such as mining and fossilfuel combustion (UNEP 2013), thus resulting in a significant increase in Hg available for cycling among land, air and the ocean since pre-industrial times [START_REF] Selin | Global biogeochemical cycling of mercury: A review[END_REF]. Hg emissions are transported through the atmosphere on a hemispheric-to-global scale, allowing for transport to remote locations such as sub-polar and polar regions [START_REF] Fitzgerald | The case for atmospheric mercury contamination in remote areas[END_REF]. After atmospheric deposition and through biotic and abiotic mechanisms, Hg is readily transformed in methyl-Hg, the highly toxic form that bioaccumulates in the tissues of living organisms and biomagnifies up food webs, especially in aquatic environments [START_REF] Fitzgerald | Marine biogeochemical cycling of mercury[END_REF]). Top predators are thus exposed to significant quantities of Hg via their diet, providing information on Hg bioavailability within their food webs [START_REF] Morel | The chemical cycle and bioaccumulation of mercury[END_REF]). Among consumers, birds have varied levels of ecological, spatial and temporal integration of contaminants depending on species, and they have been identified as effective indicators of Hg bioavailability in both terrestrial and marine environments (Burger andGochfeld 2004, Solonen andLodenius 1990).

The Kerguelen Islands are a remote subantarctic archipelago in the southern Indian Ocean, where the level of Hg bioavailability is poorly documented [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], Bustamante et al. 2003[START_REF] Cipro | Trace elements in the white-chinned-petrel (Procellaria aequinoctialis) from the Kerguelen Islands, Southern Indian Ocean[END_REF][START_REF] Cossa | Mercury in the Southern Ocean[END_REF]. This archipelago hosts a large and highly diverse avian assemblage (35 different breeding species). The community includes a few terrestrial species and many seabirds, with Sphenisciformes (penguins) and Procellariiformes (albatrosses and petrels) dominating by mass and numbers, respectively [START_REF] Guinet | Consumption of marine resources by seabirds and seals in Crozet and Kerguelen waters: changes in relation to consumer biomass 1962-85[END_REF][START_REF] Weimerskirch | The avifauna of the Kerguelen Islands[END_REF]. Overall Kerguelen seabirds feed on a few key species of marine organisms, including some crustaceans (euphausiids, hyperiids), fish (myctophids, notothenioids) and cephalopods (oceanic squids) [START_REF] Bocher | Amphipod-based food web: Themisto gaudichaudii caught in nets and by seabirds in Kerguelen waters, southern Indian Ocean[END_REF], Cherel et al. 2010[START_REF] Cherel | Stable isotopes, beaks and predators: a new tool to study the trophic ecology of cephalopods, including giant and colossal squids[END_REF][START_REF] Guinet | Consumption of marine resources by seabirds and seals in Crozet and Kerguelen waters: changes in relation to consumer biomass 1962-85[END_REF], with some seabirds relying extensively on carrion. This biological richness can be related to the large and productive shelf surrounding the archipelago [START_REF] Blain | A biogeochemical study of the island mass effect in the context of the iron hypothesis: Kerguelen Islands, Southern Ocean[END_REF]. Kerguelen seabirds show a wide range of contrasted feeding strategies, with species foraging in the benthic and pelagic environments and ranging from neritic to oceanic waters. Noticeably, the oceanic species forage over a large latitudinal gradient, from subtropical to Antarctic waters (Supplementary Table S1). Kerguelen seabirds therefore offer a unique opportunity to study Hg bioavailability over diverse water masses of the Southern Ocean.

The present study aims to assess Hg bioavailability in the southern Indian Ocean by using birds from the Kerguelen Islands as bioindicators. Hg exposure was evaluated by using body feathers, because feathers are the main route of Hg excretion in birds [START_REF] Braune | Mercury levels in Bonaparte's gulls , Larus philadelphia. during autumn molt in the Quoddy region, New Brunswick, Canada[END_REF]. Importantly, this work complements a recent investigation on Hg in chicks [START_REF] Blévin | Wide range of mercury contamination in chicks of Southern Ocean seabirds[END_REF]) by focusing on breeding adults and by including more species. While Hg concentrations in chick feathers are representative of a welldefined, relatively short period of exposure (the chick-rearing period), adult feathers provide a wider perspective on Hg exposure of the species over their whole life cycles [START_REF] Evers | Patterns and interpretation of mercury exposure in freshwater avian communities in northeastern North America[END_REF]. Thus adult feather Hg concentrations were determined in 27 representative species, including the only two terrestrial birds of the assemblage, in order to: (i) describe Hg exposure in a large number of sympatric bird species from the poorly documented southern Indian Ocean; (ii) compare the exposure pattern to that of avian communities from other subantarctic and oceanic remote locations worldwide; (iii) test the effect of age-class on feather Hg concentrations by using the recently published Hg data on chicks [START_REF] Blévin | Wide range of mercury contamination in chicks of Southern Ocean seabirds[END_REF], and (iv) investigate the influence of various factors (taxonomy, diet, feeding habitats, moulting patterns) on Hg exposure. Taxonomy, which was not tested in [START_REF] Blévin | Wide range of mercury contamination in chicks of Southern Ocean seabirds[END_REF], was expected to play a minor role in explaining feather Hg concentrations when compared to feeding strategies, since diet is considered to be the main factor driving Hg variation in birds [START_REF] Becker | Feather mercury levels in seabirds at South Georgia: influence of trophic position, sex and age[END_REF][START_REF] Blévin | Wide range of mercury contamination in chicks of Southern Ocean seabirds[END_REF], Bocher et al. 2003[START_REF] Monteiro | Relationship between mercury levels and diet in Azores seabirds[END_REF][START_REF] Stewart | Influence of phylogeny, diet, moult schedule and sex on heavy metal concentrations in New Zealand Procellariiformes[END_REF]). In addition, adult birds were expected to show higher feather Hg concentrations than chicks, as they are exposed over a longer period to Hg via their diet and moulting patterns [START_REF] Catry | The role of stable isotopes and mercury concentrations to describe seabird foraging ecology in tropical environments[END_REF][START_REF] Stewart | Influence of species, age and diet on mercury concentrations in Shetland seabirds[END_REF].

Materials and methods

Fieldwork was carried out from 2003 to 2011 on the Kerguelen Islands (49°21'S, 70°18'E, Fig. 1), which are located in the southern part of the Polar Frontal Zone, in the immediate vicinity of the Polar Front (Orsi et al. 1995, Park andGambéroni, 1997).

Breeding adults from 27 bird species belonging to 5 orders and 10 families were sampled (n = 5 to 33 individuals per species, Supplementary Table S2). Sampling was conducted at different locations of the archipelago, depending on the species breeding sites. Resident neritic seabird species (Kerguelen shag and gentoo penguin) were sampled at colonies close to the open sea, while terrestrial species (lesser sheathbill and Kerguelen pintail) were sampled on islands of the large Morbihan Bay (closed sea).

Birds were non-destructively captured by mist net or by hand, depending on species, and released immediately after sampling. A few whole body feathers (6-10) were pulled out from the lower back of the birds and then stored dry in sealed plastic bags until analysis at the University of La Rochelle, France.

Depending on bird and hence feather size, 1 to 5 whole feathers per individual were cleaned, oven-dried to a constant mass and homogenised as described in [START_REF] Blévin | Wide range of mercury contamination in chicks of Southern Ocean seabirds[END_REF]. An Advanced Mercury Analyzer spectrophotometer (Alted AMA 254) was used to measure total Hg, which approximates the amount of methyl-Hg in feathers [START_REF] Bond | Relationships between stable isotopes and metal contaminants in feathers are spurious and biologically uninformative[END_REF]Diamond 2009, Thompson andFurness 1989a). For each individual, analyses were run in duplicate-triplicate by taking sub-samples of the homogenised feathers (relative standard deviation < 10% for each individual). Accuracy was checked using certified reference material (Tort-2 Lobster Hepatopancreas, NRC, Canada; certified Hg concentration: 0.27 ± 0.06 µg g -1 dry weight). Our measured values were 0.24 ± 0.01 µg g -1 dry weight, n = 22. Blanks were analysed at the beginning of each set of samples and the detection limit of the method was 0.005 µg g -1 dry weight. Hg concentrations are presented µg g -1 dry weight (dw).

Statistical analyses were performed using R 2.15.1 (R Core Team 2012). Data exploration was performed mainly following [START_REF] Zuur | A protocol for data exploration to avoid common statistical problems[END_REF]. The influence of taxa (species, genus, family and order) on adult feather Hg concentrations was tested by using generalized linear models (GLM) with a gamma distribution and an inverse link function. Model selection was based on Akaïke's Information Criteria adjusted for small sample sizes (AICc) [START_REF] Burnham | Model selection and multi-model inference: a practical information-theoretic approach[END_REF]. The sampling year was not included in the models because most species were sampled in only one year (Supplementary Table S2) and thus the year effect would be confounded by the species effect.

Nonetheless, no inter-annual differences in feather Hg concentrations were found on the six species that were sampled in two different years (light-mantled sooty albatross, softplumaged and Kerguelen petrels, black-bellied storm petrel, South Georgia diving petrel and lesser sheathbill, data not shown). As biometric measurements were not performed on individual birds during the sampling procedure, the effect of size and mass on feather Hg concentrations could not be incorporated in the models. However, mean values of size and mass were obtained for each species from the literature (Supplementary Table S1) and their correlations with mean feather Hg concentrations were tested. Finally, the effect of age-class on feather Hg concentrations was investigated on 21 out of the 27 species by comparing adult data from the present study with chick data from the same Kerguelen locations [START_REF] Blévin | Wide range of mercury contamination in chicks of Southern Ocean seabirds[END_REF]. A significance level of α < 0.05 was used for all statistical tests. Results are means ± SD.

General information on the feeding ecology of Kerguelen birds was based on published and unpublished data obtained using various methods (stomach content and stable isotope analyses and tracking devices), and is summarized in Supplementary Table S1. Importantly, dietary information was restricted to the chick food, because parent birds carry significant amounts of food in their stomach at that time only. By contrast, adult diet is poorly known both during and outside the breeding period. The relationship between Hg contamination and trophic ecology was not studied here using stable isotopes, because of the uncoupled temporal integration of Hg and stable isotopes in feathers of adult birds [START_REF] Bond | Relationships between stable isotopes and metal contaminants in feathers are spurious and biologically uninformative[END_REF][START_REF] Thompson | Feathers as a means of monitoring mercury in seabirds: insights from stable isotope analysis[END_REF].

Results

Feather Hg concentrations were measured in a total of 480 adult birds from the Kerguelen Islands (details in Supplementary Table S2). Feather Hg concentrations varied widely within the avian community, with means ranging from 0.42 ± 0.13 to 16.6 ± 3.8 µg g -1 dw in Wilson's storm petrels and wandering albatrosses, respectively (Fig. 2). The lowest feather Hg concentration occurred in a South-Georgian diving petrel and the highest in a northern giant petrel (0.10 and 32.1 µg g -1 , respectively). Model selection showed that species was the most important factor explaining feather Hg concentrations when compared to other taxonomic levels (Table 1). Coefficients of variations (CV) also varied considerably between species, ranging from 13 to 109% (Supplementary Table S2). Mean feather Hg concentration was significantly related to species size (Pearson correlation, r = 0.50, p = 0.008, n = 27), but not to species mass (r = 0.23, p = 0.256, n = 27).

By combining feather Hg data of adults from this study with those of chicks from [START_REF] Blévin | Wide range of mercury contamination in chicks of Southern Ocean seabirds[END_REF], a total of 654 individuals from 21 seabird species were analysed. The model including species, age-class and their interaction as explaining factors of feather Hg concentrations showed the best fit to the data (Table 1). Feather Hg concentrations were significantly higher in adults than in chicks in all but two species: the blue petrel (no statistical difference) and subantarctic skua (higher chick level) (Fig. 3). The ratio of adult to chick feather Hg concentrations varied between species, ranging from 0.5 to 20.7 in the subantarctic skua and South-Georgian diving petrel, respectively (Supplementary Table S2).

Discussion

To the best of our knowledge, this study is the first to report feather Hg concentrations for such a large number of adult sympatric birds, including representative species of all the four families of the order Procellariiformes, namely albatrosses, petrels, storm petrels and diving petrels. Hg exposure varied widely within the community, showing a remarkable 40-fold difference between the species with the lowest and highest Hg concentrations. The community exposure pattern agrees with results on chicks [START_REF] Blévin | Wide range of mercury contamination in chicks of Southern Ocean seabirds[END_REF]): small petrels, penguins and terrestrial species generally showed low levels of exposure (< 2.5 µg g -1 dw), coastal seabirds, Procellaria petrels and small albatrosses had intermediate concentrations (< 10 µg g -1 dw), while Pterodroma petrels, northern giant petrels and wandering albatrosses were the species having the highest concentrations (> 10 µg g -1 dw).

Comparisons with other avian communities

The pattern of Hg concentrations among members of the Kerguelen avian community fits well with that of other subantarctic sites, such as South Georgia, southern Atlantic Ocean (Fig. 4). Feather Hg concentrations were comparable in seabirds from the two localities, with the exception of the northern giant petrel and wandering albatross [START_REF] Anderson | Influence of trophic position and foraging range on mercury levels within a seabird community[END_REF][START_REF] Becker | Feather mercury levels in seabirds at South Georgia: influence of trophic position, sex and age[END_REF]. This could be related to inter-site dietary differences of these two top predators. Moreover, similar feather Hg concentrations were reported for some species of albatrosses and petrels from the southern Pacific Ocean [START_REF] Thompson | Using seabirds to monitor mercury in marine environments: The validity of conversion ratios for tissue comparisons[END_REF][START_REF] Thompson | Temporal and spatial variation in mercury concentrations in some albatrosses and petrels from the sub-Antarctic[END_REF]. Hg bioavailability thus seems to be similar within the three sectors of the Southern Ocean, which agrees well with its circumpolar annular oceanographic structure [START_REF] Sokolov | On the relationship between fronts of the Antarctic Circumpolar Current and surface chlorophyll concentrations in the Southern Ocean[END_REF]. At lower latitudes of the Southern Hemisphere, only the tropical seabird communities from the western Indian Ocean have been investigated, revealing much lower feather Hg concentrations (~ 0.05-1.5 µg g -1 ; [START_REF] Catry | The role of stable isotopes and mercury concentrations to describe seabird foraging ecology in tropical environments[END_REF][START_REF] Kojadinovic | Mercury in seabird feathers: Insight on dietary habits and evidence for exposure levels in the western Indian Ocean[END_REF]) than in Kerguelen birds. This trend could result from differences in the physical and biological factors driving methyl-Hg production and food web transfer in the two regions (i.e., the atmospheric deposition of inorganic Hg, the rate of primary productivity, the abundance of sinking organic matter and the structure of the microbial community, [START_REF] Mason | Mercury biogeochemical cycling in the ocean and policy implications[END_REF]. The wide range of Hg exposure of Kerguelen birds compares well with avian communities of remote archipelagos of the Northern Hemisphere, namely the tropical Midway Atoll, North Pacific Ocean and the subtropical Azores Islands, North Atlantic Ocean (both ~ 1-20 µg g -1 ; Burger and Gochfeld 2000a[START_REF] Gochfeld | Metals in feathers of bonin petrel, Christmas shearwater, wedge-tailed shearwater, and red-tailed tropicbird in the Hawaian Islands, northern Pacific[END_REF][START_REF] Monteiro | Relationship between mercury levels and diet in Azores seabirds[END_REF]). These wide ranges of contamination are related to the presence of highly contaminated species of albatrosses and gadfly petrels within the communities (see below). Indeed, the avian assemblage of the temperate Machias Seal Island, North Atlantic Ocean, which includes neither albatrosses nor gadfly petrels, had lower feather Hg concentrations (0.7-7 µg g -1 ;

Bond and Diamond 2009). Therefore, the specific composition of the avian communities rather than the proximity to highly industrialized countries seems to be a key factor driving the level of Hg exposure within avian assemblages from open sea regions. In this context, the range of Hg exposure within the Kerguelen avian community is remarkable, as it encompasses the concentrations reported worldwide in remote oceanic locations [START_REF] Blévin | Wide range of mercury contamination in chicks of Southern Ocean seabirds[END_REF].

Influence of taxonomy

The best taxonomic explanatory variable of feather Hg concentrations in the Kerguelen community was species, as it integrates a large range of ecological, behavioural, physiological and life-history traits that are susceptible to drive variation in feather Hg concentrations (Anderson et al. 2009, Bond and[START_REF] Bond | Mercury concentrations in seabird tissues from Machias Seal Island, New Brunswick, Canada[END_REF]. Although interspecific differences in avian Hg exposure have often been investigated, taxonomicrelated variations were rarely tested in a large number of species [START_REF] Anderson | Influence of trophic position and foraging range on mercury levels within a seabird community[END_REF], Ochoa-Acuña et al. 2002). Here, statistical models including genus, family or order as explanatory variables had a poor fit to feather Hg data. Indeed, closely-related species at Kerguelen often showed very different levels of exposure. For example, black-bellied storm petrels displayed higher Hg concentrations than the other two Hydrobatidae species, despite similar size and life-history traits. The same pattern was highlighted for Sphenisciformes, with the gentoo penguins having higher feather Hg concentrations than the other three penguin species [START_REF] Carravieri | Penguins as bioindicators of mercury contamination in the Southern Ocean: birds from the Kerguelen Islands as a case study[END_REF]. Therefore, the effect of taxonomy seems to play a minor role in avian Hg exposure when compared to other ecological factors [START_REF] Becker | Feather mercury levels in seabirds at South Georgia: influence of trophic position, sex and age[END_REF][START_REF] Lock | Metal concentrations in seabirds of the New Zealand region[END_REF][START_REF] Stewart | Influence of phylogeny, diet, moult schedule and sex on heavy metal concentrations in New Zealand Procellariiformes[END_REF].

Nevertheless, the present study provides new and interesting results regarding a particular taxonomic group: the gadfly petrels (genus Pterodoma, [START_REF] Warham | The Petrels: Their Ecology & Breeding Systems[END_REF]), which were amongst the species with the highest Hg concentrations (Fig. 2). Our data together with a review of the scientific literature (Table 2) point out the high level of Hg contamination of almost all the Pterodroma petrels so far investigated, including species living in different marine ecosystems. Nevertheless, the Barau's Petrel P. baraui from La Réunion Island, western Indian Ocean, showed surprisingly low feather Hg concentrations (1.0 µg g -1 ), suggesting again a regional trend of low Hg bioavailability at tropical latitudes of the Indian Ocean (see subsection 4.1).

Influence of diet and feeding habitat

Inter-specific variability in Hg contamination is typically attributed to diet [START_REF] Arcos | Mercury levels in seabirds and their fish prey at the Ebro Delta, NW Mediterranean: the role of trawler discards as a source of contamination[END_REF][START_REF] Monteiro | Relationship between mercury levels and diet in Azores seabirds[END_REF][START_REF] Stewart | Influence of phylogeny, diet, moult schedule and sex on heavy metal concentrations in New Zealand Procellariiformes[END_REF]). Since Hg is efficiently biomagnified up food webs [START_REF] Atwell | Biomagnification and bioaccumulation of mercury in an arctic marine food web: insights from stable nitrogen isotope analysis[END_REF][START_REF] Campbell | Mercury and other trace elements in a pelagic Arctic marine food web , Northwater Polynya, Baffin Bay[END_REF][START_REF] Jarman | Influence of trophic position and feeding location on contaminant levels in the Gulf of the Farallones food web revealed by stable isotope analysis[END_REF], high trophic level prey, such as fish and cephalopods show higher Hg concentrations than crustaceans and other planktonic organisms [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][START_REF] Kojadinovic | Mercury content in commercial pelagic fish and its risk assessment in the Western Indian Ocean[END_REF][START_REF] Stewart | Influence of species, age and diet on mercury concentrations in Shetland seabirds[END_REF]. This is consistent with feather Hg concentrations of Kerguelen birds increasing roughly in the order crustacean-< fish-≤ squid-≤ carrionconsumers (Fig. 2b), as previously shown in chicks [START_REF] Blévin | Wide range of mercury contamination in chicks of Southern Ocean seabirds[END_REF]. This confirms that Hg is efficiently biomagnified and that diet plays a key role in explaining Hg exposure. Accordingly, the positive correlation highlighted between bird size and feather Hg concentrations is likely explained by a trophic effect, as larger avian species tend to occupy higher trophic positions and/or to consume larger prey items (Burger and Gochfeld 2000a).

Differences in dietary exposure over diverse habitats within and outside the breeding period can also account for important variability in feather Hg concentrations [START_REF] Anderson | Influence of trophic position and foraging range on mercury levels within a seabird community[END_REF][START_REF] Blévin | Wide range of mercury contamination in chicks of Southern Ocean seabirds[END_REF]. Here, inshore non-migratory species (Kerguelen shag, kelp gull, gentoo penguin), which feed on benthic organisms, showed intermediate to high Hg concentrations. This agrees with high Hg bioavailability in benthic environments due to methyl-Hg production in coastal marine sediments [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][START_REF] Fitzgerald | Marine biogeochemical cycling of mercury[END_REF]). In the oceanic domain, methyl-Hg concentration reaches a maximum in mesopelagic waters [START_REF] Driscoll | Mercury as a Global Pollutant: Sources, Pathways, and Effects[END_REF][START_REF] Fitzgerald | Marine biogeochemical cycling of mercury[END_REF], resulting in enhanced contamination of mesopelagic prey (Chouvelon et al. 2013[START_REF] Choy | The influence of depth on mercury levels in pelagic fishes and their prey[END_REF]). This could explain the high feather Hg concentrations of oceanic species relying extensively on mesopelagic fish and cephalopods (e.g. gadfly petrels, Ochoa-Acuña et al. 2002[START_REF] Ridoux | The diets and dietary segregation of seabirds at the subantarctic Crozet Islands[END_REF]). On the other hand, migratory seabirds can travel thousands of kilometres away from their breeding sites after reproduction (e.g., [START_REF] Warham | The Petrels: Their Ecology & Breeding Systems[END_REF], being potentially exposed to different quantities of Hg. Here, species visiting northern subtropical and neritic waters during the non-breeding period (e.g. the wandering albatross) tended to have higher feather Hg concentrations than those that forage predominantly within the limits of the Southern Ocean year-round (e.g. the light-mantled sooty albatross) [START_REF] Cherel | A comprehensive isotopic investigation of habitat preferences in nonbreeding albatrosses from the Southern Ocean[END_REF]. However, further investigations on i) the poorly known feeding strategies outside the breeding season and ii) Hg distribution and speciation in the Southern Ocean could elucidate the rationale of this latitudinal trend (see [START_REF] Blévin | Wide range of mercury contamination in chicks of Southern Ocean seabirds[END_REF].

Influence of moulting patterns

Feather Hg concentrations reflect blood Hg levels at the time of moult [START_REF] Bearhop | Dynamics of mercury in blood and feathers of great skuas[END_REF][START_REF] Evers | Adverse effects from environmental mercury loads on breeding common loons[END_REF]. This means dietary Hg but also Hg accumulated over the intermoult period and remobilized during feather synthesis [START_REF] Furness | Using bird feathers to measure mercury in the environment: relationships between mercury content and moult[END_REF][START_REF] Thompson | Feathers as a means of monitoring mercury in seabirds: insights from stable isotope analysis[END_REF]. Another important intrinsic driver of Hg concentrations in feathers is therefore the timing, duration and frequency of moult. For instance, the irregular and infrequent moulting patterns of large albatrosses are believed to contribute significantly to high Hg concentrations in their feathers [START_REF] Anderson | Influence of trophic position and foraging range on mercury levels within a seabird community[END_REF][START_REF] Becker | Feather mercury levels in seabirds at South Georgia: influence of trophic position, sex and age[END_REF].

However, enhanced feather Hg concentrations were also reported in Kerguelen species with annual moult cycles, like great-winged and white-chinned petrels. This indicates that moulting patterns alone cannot explain all the inter-specific variation in Hg contamination. In Procellariiformes, demethylation of Hg in the liver appears to be a significant detoxification strategy (Thompson andFurness 1989b, Thompson et al. 1993). The efficiency of demethylation mechanisms is species-dependent and could contribute to explain the inter-specific differences in feather Hg concentrations among members of the Kerguelen avian community.

Adults and chicks

As previously shown by several other studies (e.g., Burger andGochfeld 2000b, Catry et al. 2008), feather Hg concentrations were higher in adults than in chicks in almost all Kerguelen species (Fig. 3). Indeed, adults have more time to bioaccumulate Hg in their tissues during the long inter-moult period (≥ one year) before excreting it in feathers [START_REF] Monteiro | Mercury levels in seabirds from the Azores, midnorth Atlantic Ocean[END_REF][START_REF] Thompson | Mercury accumulation in great skuas Catharacta skua of known age and sex, and its effects upon breeding and survival[END_REF]. By contrast, chick feather Hg concentrations represent the dietary exposure over the chick-rearing period [START_REF] Ackerman | Bird mercury concentrations change rapidly as chicks age: Toxicological risk is highest at hatching and fledging[END_REF][START_REF] Becker | The value of chick feathers to assess spatial and interspecific variation in the mercury contamination of seabirds[END_REF], which ranges from several weeks to several months in Kerguelen species (Supplementary Table S2). Assuming a similar rate of Hg intake of adults and chicks, species with short and long chick-rearing periods should show high and low adult-to-chick ratios in feather Hg concentrations, respectively. The hypothesis was verified in the two diving petrels (high ratios, ≥ 9) and in the wandering albatross and northern giant petrel (low ratios, ~ 2-4), respectively. Exceptions to this pattern are likely indicative of differential relative Hg exposures in the two age-classes, as observed in blue petrels and subantarctic skuas (ratios: 1.3 and 0.5, respectively). Adult blue petrels from the Kerguelen Islands feed at the same low trophic level in cold Antarctic waters both during the breeding and moulting periods (Cherel et al. 2002a[START_REF] Cherel | Stable isotope evidence of diverse speciesspecific and individual wintering strategies in seabirds[END_REF]. By contrast, chick food includes a significant proportion of mesopelagic fish (Cherel et al. 2002a[START_REF] Connan | Interannual dietary changes and demographic consequences in breeding blue petrels from Kerguelen Islands[END_REF], which is consistent with their enhanced Hg exposure over adults. At Kerguelen, subantarctic skua chicks are mainly fed with blue petrels [START_REF] Mougeot | Predation on burrowing petrels by the brown skua , Catharacta skua lönnbergi. at Mayes Island, Kerguelen[END_REF], thus explaining their high feather Hg concentrations. The chick data are in accordance with previous results on chicks of the closely-related great skua that feeds on bird meat [START_REF] Stewart | Influence of species, age and diet on mercury concentrations in Shetland seabirds[END_REF]. The low Hg contamination of adult subantarctic skuas is puzzling and it strongly suggests that moulting adults do not rely on small petrels for feeding. Indeed, previous findings on subantarctic skuas from South Georgia suggest that they have a mixed diet of zooplankton and low trophic-level prey over the wintering grounds [START_REF] Phillips | Movements, winter distribution and activity patterns of Falkland and brown skuas: insights from loggers and isotopes[END_REF]. Therefore, both the duration of Hg exposure and feeding habits are key factors explaining differences in feather Hg concentrations between seabird chicks and adults.

Conclusions

Results from this study reinforce previous findings showing that taxonomy plays a minor role in determining avian Hg exposure when compared to feeding strategies [START_REF] Stewart | Influence of phylogeny, diet, moult schedule and sex on heavy metal concentrations in New Zealand Procellariiformes[END_REF]. Our results confirm that Hg concentrations are very high in some subantarctic birds, with many species showing levels of potential concern. The most common used toxic threshold of feather Hg concentration in birds is 5 µg g -1 (e.g., [START_REF] Evers | Adverse effects from environmental mercury loads on breeding common loons[END_REF]. Here, this level was exceeded by some individuals of 11 seabird species and by all individuals of wandering albatross, northern giant petrel, and whiteheaded and great-winged petrels. Although subantarctic species may have evolved to cope with high Hg exposure in their environment [START_REF] Blévin | Wide range of mercury contamination in chicks of Southern Ocean seabirds[END_REF][START_REF] Thompson | Temporal and spatial variation in mercury concentrations in some albatrosses and petrels from the sub-Antarctic[END_REF], there is an urgent need to investigate the inter-and intra-specific physiological differences of Hg metabolism, excretion and toxicity, in order to establish whether some species, or some particular individuals, could be at risk. Evidence of Hg consequences on breeding, hatchling and fledging success has been reported in polar birds (Goutte et al. in press, Tartu et al. 2013). Such investigations on risk related to Hg exposure are even more relevant in the context of global warming that would favour the methylation rate of Hg in the Ocean [START_REF] Cossa | Marine biogeochemistry: Methylmercury manufacture[END_REF]. Finally, the present study enables selecting the white-headed petrel as a good bioindicator species of Hg bioavailability in the Southern Ocean, considering its high level of exposure and low intra-specific variation. The white-headed petrel adds to the list of Kerguelen bioindicator species recently identified according to their foraging ecology and exposure patterns, i.e. the gentoo and king penguins, the black-browed, light-mantled sooty and wandering albatrosses [START_REF] Blévin | Wide range of mercury contamination in chicks of Southern Ocean seabirds[END_REF], Carravieri et al. 2013). The periodic examination of feather Hg concentrations in species from these remote regions over the long-term will make it possible to monitor The authors declare that there are no conflicts of interest. Values are means ± SD. Species abbreviations: WSP, Wilson's storm petrel; GBSP, grey-backed storm petrel; TBP, thin-billed prion; BP, blue petrel; CDP, common diving petrel; SGDP, South Georgian diving petrel; LS, lesser sheathbill; KPi, Kerguelen pintail; AP, Antarctic prion; SRP, southern rockhopper penguin; KP, king penguin; MP, macaroni penguin; KeP, Kerguelen petrel; SS, subantarctic skua; BBA, black-browed albatross; BBSP, black-bellied storm petrel; KS, Kerguelen shag; WCP, white-chinned petrel; GP, gentoo penguin; KG, kelp gull; GrP, grey petrel; LMSA, light-mantled sooty albatross; SPP, soft-plumaged petrel; WHP, white-headed petrel; GWP, great-winged-petrel; NGP, northern giant petrel; WA, wandering albatross. WCP, white-chinned petrel; GP, gentoo penguin; KG, kelp gull; GrP, grey petrel; LMSA, light-mantled sooty albatross; WHP, white-headed petrel; GWP, great-winged-petrel; NGP, northern giant petrel; WA, wandering albatross.

Seabird species

Feather Hg (µg g (South Georgia, Anderson et al. 2009[START_REF] Becker | Feather mercury levels in seabirds at South Georgia: influence of trophic position, sex and age[END_REF] a Species average for size (length from the tip of the bill to the end of the tail) and mass (body weight) from [START_REF] Shirihai | The complete guide to Antarctic wildlife: birds and marine mammals of the Antarctic continent and the Southern Ocean[END_REF].

b [START_REF] Stahl | Le régime alimentaire du goéland dominicain Larus dominicanus de l'île de la Possession, archipel Crozet , 46° 25'S, 51° 45[END_REF] and [START_REF] Ridoux | The diets and dietary segregation of seabirds at the subantarctic Crozet Islands[END_REF] refer to the related Crozet Islands.

  temporal trends of Hg bioavailability to predators in the open ocean in relation to global trends of Hg emissions.

Fig. 1 .

 1 Fig. 1. Map and location of the Kerguelen Islands and of the main oceanic fronts and zones within the southern Indian Ocean. Abbreviations: STF, Subtropical Front; PF, Polar Front; STZ, Subtropical Zone; SAZ, Subantarctic Zone; AZ, Antarctic Zone.

Fig. 2 .

 2 Fig.2. Inter-specific differences in adult feather Hg concentrations within the Kerguelen avian community. Species are presented according to a) taxonomic groups: penguins (grey), procellariiform seabirds (white) and other species (black); and b) dietary groups: terrestrial species and crustacean-(dark grey), fish-(grey), squid-(black) and carrion-(white) consumers. Species sharing the same letter are not statistically different (Tukey HSD, p < 0.05). Values are means ± SD. Species abbreviations: WSP, Wilson's storm petrel; GBSP, grey-backed storm petrel; TBP, thin-billed prion; BP, blue petrel; CDP, common diving petrel; SGDP, South Georgian diving petrel; LS, lesser sheathbill; KPi, Kerguelen pintail; AP, Antarctic prion; SRP, southern rockhopper penguin; KP, king penguin; MP, macaroni penguin; KeP, Kerguelen petrel; SS, subantarctic skua; BBA, black-browed albatross; BBSP, black-bellied storm petrel; KS, Kerguelen shag; WCP, white-chinned petrel; GP, gentoo penguin; KG, kelp gull; GrP, grey petrel; LMSA, light-mantled sooty albatross; SPP, soft-plumaged petrel; WHP, white-headed petrel; GWP, great-winged-petrel; NGP, northern giant petrel; WA, wandering albatross.

Fig. 3 .

 3 Fig. 3. Age-class differences in feather Hg concentrations within the Kerguelen seabird community. * Statistically different (Wilcoxon, p < 0.05); NS: not significant. Values are means ± SD. Species abbreviations: TBP, thin-billed prion; BP, blue petrel; CDP, common diving petrel; SGDP, South Georgian diving petrel; AP, Antarctic prion; SRP, southern rockhopper penguin; KP, king penguin; MP, macaroni penguin; KeP, Kerguelen petrel; SS, subantarctic skua; BBA, black-browed albatross; KS, Kerguelen shag;

Fig. 4 .

 4 Fig. 4. Comparison of feather Hg concentrations between the same seabird species breeding in the southern Indian Ocean (Kerguelen Islands, present study) and in the southern Atlantic Ocean

Table 1

 1 AIC c model ranking for adult feather Hg concentrations within the Kerguelen avian community. Models are GLM with a gamma distribution and an inverse link function.Abbreviations: AIC c , Akaike's Information Criteria adjusted for small sample-sizes values; w i , AIC c weights. Scaled ΔAIC c ; ΔAIC c = 0.00 is interpreted as the best fit to the data among the models b Weight of evidence interpreted as a proportion. Weights across all models sum to 1.00

	Models	N° parameters	AIC c	ΔAIC c	a	w i b
	Adults (N = 480)
	Species	28	1698	0		1
	Genus	22	1719	20		0
	Family	11	2226	528		0
	Order	6	2422	723		0
	Null	2	2459	760		0
	Adults and chicks (N = 654)
	Species * Age-class	43	1684	0		1
	Species + Age-class	23	2276	592		0
	Species	22	2471	787		0
	Age-class	3	2915	1231		0
	Null	2	3037	1353		0

a

Acknowledgements

The authors thank the numerous fieldworkers who helped with collecting feathers, F. Capoulun and A. Jaeger for preparing some of the samples and L. Thiers for helpful suggestions on R coding. The present work was supported financially and logistically by the Poitou-Charentes Region through a PhD grant to A. Carravieri, the Agence Nationale de la Recherche (program POLARTOP, O. Chastel), the Institut Polaire Français Paul Emile Victor (IPEV, program no. 109, H. Weimerskirch) and the Terres Australes et Antarctiques Françaises (TAAF). The Contrat de Projet Etat Région (CPER 13) is also acknowledged for funding the AMA.

Conflict of interest

Appendix A -Supplementary material Supplementary Table S1. Species, biometric data, foraging habitats and dietary habits of marine and terrestrial birds breeding at the Kerguelen Islands.


Alice Carravieri
email: alice.carravieri@cebc.cnrs.fr

Paco Bustamante

Carine Churlaud

Aymeric Fromant

Yves Cherel

Moulting patterns drive within-individual variations of stable isotopes and mercury in seabird body feathers: implications for monitoring of the marine environment

Keywords: biomonitoring, birds, moult, Southern Ocean, trophic ecology

come

Moulting patterns drive within-individual variations of stable isotopes and mercury in seabird body feathers: implications for monitoring of the marine environment

Introduction

Feathers are used extensively in avian biology, chemoecology and toxicology because they can be easily collected and are metabolically inert after synthesis, thus preserving their chemical composition almost indefinitely. For instance, stable isotope analyses of feathers have provided insights into many aspects of avian ecology [START_REF] Inger | Applications of stable isotope analyses to avian ecology[END_REF][START_REF] Hobson | Isotopic ornithology: a perspective[END_REF]) and feathers are considered as excellent non-destructive tools for monitoring contaminants, including heavy metals and persistent organic pollutants [START_REF] Burger | Metals in avian feathers: bioindicators of environmental pollution[END_REF][START_REF] García-Fernández | Feathers as a biomonitoring tool of polyhalogenated compounds: a review[END_REF]. More recently, the application of the stable isotope method to ecotoxicological studies has granted insight into patterns of contamination among sites, species and individuals (e.g. [START_REF] Ofukany | Connecting breeding and wintering habitats of migratory piscivorous birds: implications for tracking contaminants (Hg) using multiple stable isotopes[END_REF][START_REF] Ramos | Trophodynamics of inorganic pollutants in a wide-range feeder: the relevance of dietary inputs and biomagnification in the yellow-legged gull (Larus michahellis)[END_REF]. Feathers from museum collections have been further used to depict historical changes in birds' foraging ecology and contaminant exposure (Vo et al. 2011;[START_REF] Jaeger | Isotopic investigation of contemporary and historic changes in penguin trophic niches and carrying capacity of the Southern Indian Ocean[END_REF].

Ideally, a monitoring tool must show little within-and between-individual variations in the levels of targeted compounds in order to facilitate the statistical description of spatiotemporal trends within a population. Indeed, a main limitation in the use of feathers is withinindividual heterogeneity both between and within feather types. For example, changes in foraging habitat or diet during moult lead to large variations in feather δ 13 C and δ 15 N values, respectively [START_REF] Jaeger | Use of stable isotopes to quantify seasonal changes of trophic niche and levels of population and individual specialisation in seabirds[END_REF], and feathers that grow at different times present different mercury (Hg) concentrations, as the Hg body pool is progressively depleted during the moult [START_REF] Furness | Using bird feathers to measure mercury in the environment: relationships between mercury content and moult[END_REF][START_REF] Braune | Mercury levels in Bonaparte's gulls (Larus philadelphia) during autumn molt in the Quoddy Region, New Brunswick, Canada[END_REF]. For both scientific and ethical reasons, body feathers are generally considered as the best feather type to sample [START_REF] Furness | Using bird feathers to measure mercury in the environment: relationships between mercury content and moult[END_REF][START_REF] Jaeger | Using carbon and nitrogen isotopic values of body feathers to infer inter-and intra-individual variations of seabird feeding ecology during moult[END_REF]), but few investigations looked at their stable isotope values and contaminant levels of within-individual heterogeneity (Thompson et al. 1993;[START_REF] Bond | High within-individual variation in total mercury concentration in seabird feathers[END_REF][START_REF] Jaeger | Using carbon and nitrogen isotopic values of body feathers to infer inter-and intra-individual variations of seabird feeding ecology during moult[END_REF][START_REF] Jaeger | Use of stable isotopes to quantify seasonal changes of trophic niche and levels of population and individual specialisation in seabirds[END_REF][START_REF] Brasso | Unique pattern of molt leads to low intra-individual variation in feather mercury concentrations in penguins[END_REF]. Since body feathers grow asynchronously during the whole moulting period, a main recurrent problem is that the precise timing of synthesis of a given body feather remains unknown. However, the temporal drawback can be eluded in the case of synchronous or almost synchronous moult of body feathers. Indeed, assuming a constant growth rate for all body feathers, a synchronous growth theoretically means that all body feathers should have the same chemical composition and thus should show identical stable isotope values and contaminant levels.

Here, we tested this hypothesis by measuring δ 13 C and δ 15 N values and Hg concentrations in four body feathers from the same individuals of three groups of marine birds showing different moulting patterns. Seabirds are useful organisms to biomonitor marine ecosystems, because they are long-lived animals that prey at the top of the marine food webs [START_REF] Furness | Seabirds as monitors of the marine environment[END_REF][START_REF] Burger | Marine birds as sentinels of environmental pollution[END_REF]. Selected models were:

1. Chicks of white-chinned petrels Procellaria aequinoctialis. Chicks of this species were selected as representative of bird chicks, which present a complete moult with body feathers growing almost synchronously towards the end of the chick-rearing period. Whitechinned petrel chicks have the advantage of showing significant between-individual variations in their feather stable isotope values [START_REF] Jaeger | Use of stable isotopes to quantify seasonal changes of trophic niche and levels of population and individual specialisation in seabirds[END_REF], being thus a potentially good model to compare feather heterogeneity between and within individuals.

2. Adults of Antarctic prions Pachyptila desolata. Adults of this species were selected as representative of adults of flying seabirds that disperse after breeding and moult far away from their breeding grounds during the inter-nesting period. Moult is sequential and generally protracted over several weeks to months [START_REF] Bridge | Influences of morphology and behavior on wing-molt strategies in seabirds[END_REF]. Antarctic prions breed within the Southern Ocean and their feather δ 13 C values indicate moulting primarily in northern warmer waters [START_REF] Cherel | Stable isotope evidence of diverse species-specific and individual wintering strategies in seabirds[END_REF].

3. Adults of king penguins Aptenodytes patagonicus. Adults of this species were selected as representative of penguins, which present a unique moulting pattern among birds.

Penguins renew their whole plumage while fasting ashore, because transient reduction in thermal insulation during moult prevents them from going at sea. Consequently, all their body feathers grow simultaneously at the expense of energy reserves that are built up during the pre-moulting foraging period of hyperphagia at sea [START_REF] Groscolas | How to molt while fasting in the cold: the metabolic and hormonal adaptations of emperor and king penguins[END_REF][START_REF] Cherel | Energy and protein requirements for molt in the king penguin Aptenodytes patagonicus[END_REF].

As body feathers grow simultaneously in white-chinned petrel chicks and king penguin adults, but not in Antarctic prion adults, our driving hypothesis was that feather δ 13 C and δ 15 N values and Hg concentrations would present lower within-than between-individual variations in the two former groups and would show an opposite pattern in the latter species.

The main consequence of these moulting strategies is that chicks and penguins should be more suitable avian models than adults of flying species for long-term monitoring of the marine environment.

Materials and methods

Body feathers from white-chinned petrel chicks and king penguin adults were collected on Possession Island (46° 30' S, 51° 45' E), Crozet Archipelago in 2007 and 2005, respectively. King penguin adults were sampled during their annual moult, while well-feathered whitechinned petrel chicks were sampled at the end of the chick-rearing period. AP adults were collected on the Kerguelen Islands (49°21'S, 70°18'E) during the 2011-2012 austral summer.

Birds were found dead or dying after being trapped in the vegetation (Acaena adscendens); they were stored at -20°C until dissection at the Centre d'Etudes Biologiques de Chizé, France. For all species, several whole body feathers per individual were pulled out from the lower back (dorsal tract) and stored dry in sealed plastic bags until analysis at the University of La Rochelle, France. Prior to chemical analysis, whole feathers were cleaned as described in [START_REF] Blévin | Wide range of mercury contamination in chicks of Southern Ocean seabirds[END_REF] and then oven dried for 48 hr at 50°C. Every whole feather was homogenized by cutting it with scissors into small fragments and a subsample of ~0.3 mg was packed into tin containers for stable isotope analysis. The relative abundance of carbon and nitrogen isotopes were determined with a continuous flow mass spectrometer (Thermo Scientific Delta V Advantage) coupled to an elemental analyser (Thermo Scientific Flash EA 1112). Results are presented in the usual  notation relative to Vienna PeeDee Belemnite and atmospheric N 2 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.

The remaining homogenized subsample of every feather was then analysed for total Hg in an Advanced Mercury Analyzer spectrophotometer (Altec AMA 254) following [START_REF] Blévin | Wide range of mercury contamination in chicks of Southern Ocean seabirds[END_REF]. Whenever possible, Hg analysis was repeated twice and the relative standard deviation between runs calculated (< 10% for all samples). Accuracy was checked using a certified reference material (Tort-2 Lobster Hepatopancreas, NRC, Canada; certified Hg concentration: 0.27 ± 0.06 µg g -1 dry weight). Our measured values were 0.268 ± 0.022 µg g -1 dry weight (n = 14). Blanks were analysed at the beginning of each set of samples and the detection limit of the method was 0.005 µg g -1 dry weight. Data of Hg concentrations are presented relative to the dry weight (dw).

Statistical analyses were performed using R 2.15. (R Core Team 2012). Linear mixed effect models were used in order to test the repeatability of feather stable isotope values and Hg concentrations within individuals. Random effect models with the individual as a random intercept were constructed for the three groups of birds. The variance explained by the model (d), i.e. the between-individual variance, and the residual variance (σ) were used to calculate the intra-class correlation coefficient (ICC) as d 2 /(d 2 +σ 2 ), which is a measure of repeatability [START_REF] Nakagawa | Repeatability for Gaussian and non-Gaussian data: a practical guide for biologists[END_REF]. ICC ranges from 0 to 1, with values close to 1 meaning that most of the variance is explained by between-individual differences. Stable isotope values and Hg concentrations are means ± standard deviation (SD).

Results

Feather stable isotope values and Hg concentrations were measured in four different body feathers from 10 white-chinned petrel chicks, 10 Antarctic prion adults and 7 king penguin adults (Table 1;Fig 1). Individual SD were very low and ICC very high for feather δ 13 C and δ 15 N values and Hg concentrations of white-chinned petrel chicks and king penguin adults (Tables 1 and2). By contrast, Antarctic prion adults showed large within-individual variations, i.e. high SD and low ICC in feather δ 13 C and δ 15 N values and Hg concentrations.

In Antarctic prion adults, individual ranges of stable isotopes values were as large as 4.9 ‰ and 5.6 ‰ in feather δ 13 C and δ 15 N values, respectively. Noticeably, while feather Hg concentrations were within the same order of magnitude for the three groups of birds (overall individual range: 1.1 to 3.9 μg g -1 dw), CV were much lower in individual white-chinned petrel chicks (3 to 9%) and king penguin adults (<1 to 8%) than in Antarctic prion adults (15 to 60%).

Discussion

To the best of our knowledge, this study is the first to compare the levels of heterogeneity in stable isotope values and contaminant concentrations of body feathers both within and between individuals in relation to different moulting patterns. The results verified the initial hypothesis stating that a synchronous moult leads to lower within-than between-individual variations in feather δ 13 C and δ 15 N values and Hg concentrations, while asynchronous feather growth induces higher levels of feather heterogeneity within individual birds. Adult moult is potentially of variable duration according to sex, age, breeding status, species, phylogeny and environmental constraints. Hence, the synchronous/protracted moult hypothesis merits further investigations focusing on slow-moulting birds (e.g. albatrosses, Bridge 2006), species undergoing two moults per year (e.g. terns, [START_REF] Burger | Metal levels in regrown feathers: Assessment of contamination on the wintering and breeding grounds in the same individuals[END_REF]), and species with complex moulting patterns (e.g. alcids, [START_REF] Bridge | The effects of intense wing molt on diving in alcids and potential influences on the evolution of molt patterns[END_REF]).

Feather δ 13 C and δ 15 N values and Hg concentrations showed low variances and high ICC, thus indicating low levels of within-individual variations in white-chinned petrel chicks and king penguin adults. Noticeably, SD of δ 13 C and δ 15 N values were close to the accuracy of the isotopic method. These findings have three important implications.

1. They suggest that all body feathers that grow simultaneously have the same δ 13 C and δ 15 N values and Hg concentrations.

2. When considering the overall feather chemical composition, they predict negligible within-individual variations in other useful stable isotope values of keratin, like δ 2 H and δ 34 S [START_REF] Hobson | Isotopic ornithology: a perspective[END_REF][START_REF] Ramos | Trophodynamics of inorganic pollutants in a wide-range feeder: the relevance of dietary inputs and biomagnification in the yellow-legged gull (Larus michahellis)[END_REF] and in the concentrations of other compounds that are deposited in the keratin structure, like trace metals and metalloids, persistent organic pollutants and hormones [START_REF] Burger | Metals in avian feathers: bioindicators of environmental pollution[END_REF][START_REF] Bortolotti | Tracking stress: localisation, deposition and stability of corticosterone in feathers[END_REF][START_REF] García-Fernández | Feathers as a biomonitoring tool of polyhalogenated compounds: a review[END_REF]). This prediction is not verified for at least one group of molecules, the pigments, which can vary qualitatively and quantitatively from one body feather to the other (Stettenheim 2000). It also merits further investigations for those compounds that show a time-dependent deposition in feathers, like steroid hormones [START_REF] Bortolotti | Flaws and pitfalls in the chemical analysis of feathers: bad news-good news for avian chemoecology and toxicology[END_REF].

3. Analysis of one to several pooled body feathers will provide identical stable isotope values and concentrations in various chemical compounds, meaning that any quantity of body feathers that grow simultaneously is equally representative of the individual. This is particularly relevant for studies including several elements and molecules, because some measurements necessitate only a single body feather (e.g. Hg, δ 13 C and δ 15 N), but others require much more material, and hence several feathers (e.g. organic pollutants; García-

Fernández et al. 2013).

In contrast to white-chinned petrel chicks and king penguin adults, the amount of variability in feather δ 13 C and δ 15 N values and Hg concentrations within individuals was high in AP adults. Such high levels of heterogeneity were previously found in the feather isotopic values of adults of light-mantled (Phoebetria palpebrata) and wandering (Diomedea exulans) albatrosses [START_REF] Jaeger | Using carbon and nitrogen isotopic values of body feathers to infer inter-and intra-individual variations of seabird feeding ecology during moult[END_REF][START_REF] Jaeger | Use of stable isotopes to quantify seasonal changes of trophic niche and levels of population and individual specialisation in seabirds[END_REF], and in body feather Hg concentrations of adults of Arctic (Sterna paradisaea) and common terns (S. hirundo), and of Leach's storm petrels (Oceanodroma leucorhoa) [START_REF] Bond | High within-individual variation in total mercury concentration in seabird feathers[END_REF]. The within-individual variation of adult flying birds likely results from body feathers being synthesized and replaced at different times during the moulting period (e.g. [START_REF] Battam | Chemical composition and tissue energy density of the cuttlefish (Sepia apama) and its assimilation efficiency by Diomedea albatrosses[END_REF]. This heterogeneity reflects the birds' movements within different water masses and associated dietary shifts [START_REF] Jaeger | Use of stable isotopes to quantify seasonal changes of trophic niche and levels of population and individual specialisation in seabirds[END_REF]), together with changes in body burdens of Hg [START_REF] Furness | Using bird feathers to measure mercury in the environment: relationships between mercury content and moult[END_REF][START_REF] Braune | Mercury levels in Bonaparte's gulls (Larus philadelphia) during autumn molt in the Quoddy Region, New Brunswick, Canada[END_REF] during the protracted moult. A positive aspect of feather variability is that measuring the isotopic values of several body feathers per individual can provide valuable information on the foraging strategies of adult birds during the moulting period [START_REF] Jaeger | Using carbon and nitrogen isotopic values of body feathers to infer inter-and intra-individual variations of seabird feeding ecology during moult[END_REF][START_REF] Jaeger | Use of stable isotopes to quantify seasonal changes of trophic niche and levels of population and individual specialisation in seabirds[END_REF].

A negative aspect is that it complicates the use of body feathers as an effective monitoring tool because increasing variability can blur temporal and spatial trends. Hence, in a first step, within-individual heterogeneity must be accurately quantified, as well as the minimum number of body feathers to sample, as described for example by [START_REF] Jaeger | Using carbon and nitrogen isotopic values of body feathers to infer inter-and intra-individual variations of seabird feeding ecology during moult[END_REF] and [START_REF] Brasso | Unique pattern of molt leads to low intra-individual variation in feather mercury concentrations in penguins[END_REF]. In a second step, body feathers of a given individual can be pooled in order to perform a unique measurement per bird. However, uncertainties in the biological interpretation of the data will remain, since, in most cases, practical reasons preclude determining the precise timing of synthesis of the sampled body feathers in relation to the food and feeding ecology of the individuals. Minimizing heterogeneity related to analytical procedure also requires thorough feather homogenisation.

We therefore recommend that long-term routine monitoring investigations on the trophic structure and contamination levels of ecosystems focus on birds presenting synchronous rather than asynchronous moult of body feathers. This means targeting chicks rather than adults [START_REF] Blévin | Wide range of mercury contamination in chicks of Southern Ocean seabirds[END_REF]; this study), and, in the Southern Hemisphere, adult penguins rather than adult flying seabirds [START_REF] Carravieri | Penguins as bioindicators of mercury contamination in the Southern Ocean: birds from the Kerguelen Islands as a case study[END_REF]this study). Chicks may represent the disadvantage of presenting low magnitude variation in feather δ 15 N (not δ 13 C) in relation to varying chick growth rates (~0. 6‰, Sears et al. 2009), and museum specimens include only a few well-feathered chicks, thus precluding determining historical changes.

Nevertheless, chicks present several advantages over other age-classes.

1. Most seabird chicks can be easily handled since they remain on land, while being fed exclusively by their parents. In addition, chick feathers can be sampled before fledging with minimum disturbance (for example during the annual ringing session in a long-term study colony).

2. The food of chicks and the foraging ecology of parent seabirds can be investigated by collecting stomach samples and using bio-logging. Hence, feather stable isotope values and contaminant levels can be related to the feeding ecology of the animals. Since adult seabirds are central place foragers when breeding, stable isotopes and contaminants in chick feathers likely represent primarily the local environment.

3. Chick moult is easy to study and the time window integrated by chick feathers is well-defined, because growth of body feathers takes place on land in the mid to the second half of the chick-rearing period [START_REF] Bost | The breeding biology of the gentoo penguin Pygoscelis papua on the Crozet Islands[END_REF][START_REF] Phillips | Postnatal development of northern fulmar chicks, Fulmarus glacialis[END_REF].

4. Working on chicks minimizes the temporal mismatch resulting from different integration times between feather stable isotopes and some contaminants, like Hg [START_REF] Bond | Relationships between stable isotopes and metal contaminants in feathers are spurious and biologically uninformative[END_REF]. Feather δ 13 C and δ 15 N values reflect the feeding ecology of the birds at the time of feather synthesis [START_REF] Hobson | Assessing avian diets using stable isotopes. I. Turnover of 13 C in tissues[END_REF][START_REF] Bearhop | Factors that influence assimilation rates and fractionation of nitrogen and carbon stable isotopes in avian blood and feathers[END_REF], while feathers integrate Hg accumulated in internal tissues during two successive moults [START_REF] Furness | Using bird feathers to measure mercury in the environment: relationships between mercury content and moult[END_REF]. In chicks, this means the second part of the chick-rearing period (see above) versus a slightly longer period corresponding to the end of down growth (initial chick moult) to the beginning of body feather growth (Stewart et al. 1997), respectively.

In agreement with Stewart et al. (1997) and [START_REF] Burger | Marine birds as sentinels of environmental pollution[END_REF], the present work provides new evidence showing that chick feathers are a useful avian tool for routine biomonitoring of the trophic structure and contaminant bioavailability in the marine environment. By targeting species with different breeding locations and chick-rearing periods (for example summer versus winter breeders), different, but well-defined spatio-temporal scales of the marine environment can be investigated. The present work focuses on seabirds, but it can be generalized to water birds and terrestrial species, since a synchronous or almost synchronous moult of body feathers in chicks before fledging is a general pattern amongst avian species.

Table 1

 1 Feather δ 13 C and δ 15 N values and Hg concentrations in white-chinned petrel chicks, Antarctic prion adults and king penguin adults. Abbreviations: CV, coefficient of variation; Id: individual. Values are means ± SD of four feathers per individual bird

			δ 13 C (‰)		δ 15 N (‰)		Hg (µg g -1 dw)
	Id	Min	Max	Mean ± SD	Min	Max	Mean ± SD	Min	Max Mean ± SD CV (%)
	White-chinned petrel chicks
	1	-21.7	-21.0	-21.3 ± 0.3	11.4	12.2	11.9 ± 0.3	1.22	1.51	1.35 ± 0.12	8.86
	2	-20.7	-20.6	-20.6 ± 0.1	12.5	12.9	12.7 ± 0.2	2.06	2.20	2.11 ± 0.06	2.95
	3	-23.0	-22.7	-22.8 ± 0.1	10.8	11.1	11.0 ± 0.1	1.01	1.22	1.09 ± 0.09	8.35
	4	-20.6	-20.5	-20.6 ± 0.1	12.5	13.2	12.8 ± 0.3	1.93	2.33	2.18 ± 0.17	7.91
	5	-22.3	-21.8	-22.0 ± 0.2	11.7	11.9	11.8 ± 0.1	2.47	2.93	2.72 ± 0.19	6.99
	6	-21.5	-21.0	-21.2 ± 0.2	12.0	12.3	12.2 ± 0.1	1.47	1.74	1.59 ± 0.12	7.70
	7	-20.3	-20.1	-20.2 ± 0.1	12.7	13.1	12.9 ± 0.2	2.85	3.42	3.11 ± 0.25	8.12
	8	-21.6	-21.4	-21.5 ± 0.1	12.2	12.3	12.2 ± 0.1	1.61	1.81	1.74 ± 0.09	5.08
	9	-22.1	-22.0	-22.1 ± 0.1	11.3	11.4	11.4 ± 0.1	1.28	1.40	1.34 ± 0.06	4.13
	10 -21.1	-20.7	-20.9 ± 0.2	12.7	13.0	12.8 ± 0.1	2.40	2.73	2.59 ± 0.14	5.32
	Antarctic prion adults
	1	-18.7	-18.4	-18.6 ± 0.2	8.8	9.4	9.1 ± 0.2	1.73	3.79	2.87 ± 1.07	37.3
	2	-18.4	-16.9	-18.0 ± 0.7	9.7	15.2	11.7 ± 2.4	1.35	2.89	2.39 ± 0.71	29.7
	3	-21.6	-16.7	-18.6 ± 2.1	9.0	13.0	10.9 ± 1.7	1.13	3.45	2.40 ± 0.96	39.9
	4	-19.0	-17.9	-18.4 ± 0.5	8.5	10.8	9.2 ± 1.1	1.14	2.86	1.86 ± 0.78	42.0
	5	-19.1	-18.6	-18.8 ± 0.2	8.2	9.4	8.6 ± 0.6	1.00	1.77	1.35 ± 0.40	29.8
	6	-21.3	-18.3	-19.4 ± 1.3	8.3	9.9	9.3 ± 0.7	1.68	2.42	2.13 ± 0.36	15.3
	7	-18.6	-16.4	-17.9 ± 1.0	8.4	14.0	10.0 ± 2.7	1.53	3.11	2.26 ± 0.84	37.3
	8	-18.3	-17.8	-18.0 ± 0.2	9.1	10.1	9.7 ± 0.4	0.97	1.59	1.30 ± 0.25	19.6
	9	-18.7	-16.3	-17.4 ± 1.2	9.1	11.3	10.4 ± 1.0	1.47	4.89	2.70 ± 1.61	59.8
	10 -19.3	-17.8	-18.5 ± 0.6	8.6	10.8	9.4 ± 1.0	2.40	4.77	3.95 ± 1.08	27.3
	King penguin adults
	1	-21.4	-21.0	-21.2 ± 0.2	10.7	11.1	11.0 ± 0.1	2.18	2.36	2.27 ± 0.08	3.52
	2	-20.1	-19.8	-19.9 ± 0.1	11.3	11.7	11.5 ± 0.2	3.78	3.82	3.80 ± 0.02	0.54
	3	-21.2	-21.0	-21.1 ± 0.1	11.3	11.5	11.4 ± 0.1	2.07	2.36	2.22 ± 0.14	6.38
	4	-21.5	-21.2	-21.4 ± 0.1	10.7	11.1	10.8 ± 0.2	2.78	2.97	2.88 ± 0.08	2.79
	5	-20.6	-20.3	-20.5 ± 0.1	11.0	11.1	11.1 ± 0.1	2.48	2.74	2.57 ± 0.12	4.69
	6	-20.0	-19.8	-19.9 ± 0.1	12.1	12.3	12.2 ± 0.1	3.15	3.77	3.41 ± 0.28	8.20
	7	-22.9	-22.1	-22.3 ± 0.4	11.1	11.6	11.4 ± 0.2	2.33	2.41	2.37 ± 0.04	1.84

Table 2

 2 Variance parameters and intra-class correlation coefficients (ICC) of linear mixed effects models with the individual as a random intercept (random effects models) for whitechinned petrel chicks, Antarctic prion adults and king penguin adults. Abbreviations: d, variance explained by the model, here the between-individual variance; σ, residual variance

	δ 13 C	δ 15 N	Hg

Figure 1. Simultaneously-moulting white-chinned petrel chicks (WCP; white circles) and king penguin adults (KP; black diamonds) show smaller SD in both feather δ13C and Hg values than sequentially-moulting Antarctic prion adults (AP; grey squares). Values are means ± SD of four feathers per individual bird.

Acknowledgements

The authors thank fieldworkers who helped with collecting bird feathers, A. Jaeger for the preparation of some samples, G. Guillou for running stable isotope analysis, and S. Patrick for helpful advice in statistical analyses. The present work was supported financially and logistically by the Région Poitou-Charentes through a PhD grant to AC, and by the Agence Nationale de la Recherche (program POLARTOP, O. Chastel), the Institut Polaire Français Paul Emile Victor (IPEV, program no. 109, H. Weimerskirch) and the Terres Australes et Antarctiques Françaises (TAAF).