Marine J Briand
email: marine.briand@univ-nc.nc

Yves Letourneur

Xavier Bonnet

Emmanuel Wafo

Thomas Fauvel

François Brischoux

Gaël Guillou

Paco Bustamante

Spatial variability of metallic and organic contamination of anguilliform fish in New Caledonia

Keywords: trace elements, PCBs, pesticides, POPs, carnivorous fish, coral reefs, SW Pacific Ocean

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Coral reefs are threatened by a wide range of natural and anthropic disturbances, including chemical pollution [START_REF] Richmond | Coral reefs: present problems and future concerns resulting from anthropogenic disturbance[END_REF]. Three main contaminant classes are particularly involved: polychlorinated biphenyls (PCBs), pesticides (both classes are part of persistent organic pollutants or POPs), and metallic trace elements. These ubiquitous contaminants can be toxic, even at low concentrations; they are resistant to degradation, transported over long distances and can bioaccumulate in marine organisms (e.g. [START_REF] Phillips | The chemistries and environmental fates of trace metals and organochlorines in aquatic ecosystems[END_REF][START_REF] Robertson | PCBs: Recent advances in environmental toxicology and health effects[END_REF]. The half-life of contaminants in the marine environment is estimated to be at least a decade for the most persistent PCBs [START_REF] Sinkkonen | Degradation half-life times of PCDDs, PCDFs and PCBs for environmental fate modeling[END_REF][START_REF] Robertson | PCBs: Recent advances in environmental toxicology and health effects[END_REF], several years to a decade for trace elements such as Hg [START_REF] Lodenius | Mercury concentrations in an aquatic ecosystem during twenty years following abatement the pollution source[END_REF], and several months to years for pesticides [START_REF] Hellawell | Toxic substances in rivers and streams[END_REF]. After their release into the marine environment, some contaminants can be strongly accumulated by organisms of different trophic levels [START_REF] Van Ael | Persistent organic pollutants in the Scheldt estuary: environmental distribution and bioaccumulation[END_REF][START_REF] Dummee | Bioaccumulation of heavy metals in water, sediments, aquatic plant and histopathological effects on the golden apple snail in Beung Boraphet reservoir, Thailand[END_REF].

Southwest Pacific coral reefs are generally in good health. However, for a few decades, toxic wastes released by human activities threaten some of them [START_REF] Richmond | Coral reefs: present problems and future concerns resulting from anthropogenic disturbance[END_REF]. New Caledonia's lagoon, the largest in the world, is no exception. This hot spot of biodiversity is subjected to increasing contamination pressure resulting from industrialisation and urbanisation. New Caledonia is the third producer of nickel ore in the world [START_REF] Dalvi | The past and the future of nickel laterites[END_REF]). Since the end of the 19 th century, many open-cast mines and three metallurgical factories have been opened. Mining activities and natural soil erosion due to rainfall (Ambatsian et al. 1997) generate massive sediment deposits and discharge of associated trace elements (Co, Cr, Fe, Mn and Ni,) into the lagoon. Furthermore, with the expansion of industrial factories and urban development, the lack of efficient treatment of wastewater and the use of pesticides for agriculture, the contamination by POPs is also an important issue.

Nearly 250 pesticides are officially authorized in New Caledonia (DAVAR, 2001). Twentysix of these are banned but in Europe are still extensively used in NC.

Several studies focused on trace metal contaminants in coastal waters, in particular around Nouméa (the main urban region) [START_REF] Fernandez | A combined modelling and geochemical study of the fate of terrigenous inputs from mixed natural and mining sources in a coral reef lagoon (New Caledonia)[END_REF]Metian et al. 2008a, b;[START_REF] Hédouin | The tropical brown alga Lobophora variegata as a bioindicator of mining contamination in the New Caledonia lagoon: A field transplantation study[END_REF][START_REF] Hédouin | Trends in concentrations of selected metalloid and metals in two bivalves from the coral reefs in the SW lagoon of New Caledonia[END_REF][START_REF] Hédouin | Validation of two tropical marine bivalves as bioindicators of mining contamination in the New Caledonia lagoon: Field transplantation experiments[END_REF]. Bioaccumulation of mining (Co, Cr, Fe, Mn and Ni) and urban trace elements (Ag, As, Cd, Cu, Hg, Pb, Se, V and Zn) have been investigated in various taxa (crustaceans, molluscs, ascidians) [START_REF] Bustamante | Bioaccumulation of 12 trace elements in the tissues of the Nautilus Nautilus macromphalus from New Caledonia[END_REF][START_REF] Hédouin | Allometric relationship in the bioconcentration of heavy metals by the edible tropical clam Gafrarium tumidum[END_REF][START_REF] Hédouin | Nickel bioaccumulation in bivalves from the New Caledonia lagoon: Seawater and food exposure[END_REF][START_REF] Hédouin | Trends in concentrations of selected metalloid and metals in two bivalves from the coral reefs in the SW lagoon of New Caledonia[END_REF][START_REF] Hédouin | Metal and metalloid bioconcentration capacity of two tropical bivalves for monitoring the impact of land-based mining activities in the New Caledonia lagoon[END_REF][START_REF] Hédouin | Validation of two tropical marine bivalves as bioindicators of mining contamination in the New Caledonia lagoon: Field transplantation experiments[END_REF][START_REF] Pernice | Comparative bioaccumulation of trace elements between Nautilus pompilius and Nautilus macromphalus (Cephalopoda: Nautiloidea) from Vanuatu and New Caledonia[END_REF]Metian et al. 2008a[START_REF] Metian | Metal and metalloid bioaccumulation in the Pacific blue shrimp Litopenaeus stylirostris (Stimpson) from New Caledonia: Laboratory and field studies[END_REF]. One study recently revealed high contamination levels by trace elements in fish from the south-western lagoon, extending from the coast to the barrier reef [START_REF] Bonnet | Anguilliform fish reveal large scale contamination by trace elements in the coral reefs of New Caledonia[END_REF]. However, there is still little information available about trace element contamination of fish across the whole lagoon in New Caledonia (Chouvelon et al. 2009, Fernandez and[START_REF] Fernandez | Impact potentiel de l'effluent de Vale NC sur le milieu marin (2005---2011[END_REF][START_REF] Metian | Trace element bioaccumulation in reef fish from New Caledonia: influence of trophic groups and risk assessment for consumers[END_REF].

Furthermore, to our knowledge there is no information available to date on PCB and pesticide concentrations in marine organisms in New Caledonia.

Analyses of spatial patterns of contamination allow the identification of their environmental sources. They also lead to a better understanding of the physical, chemical and biological processes involved in contaminant accumulation [START_REF] Robertson | PCBs: Recent advances in environmental toxicology and health effects[END_REF][START_REF] Johnson | Polychlorinated Biphenyls[END_REF]). However, with the exception of the coastal region (especially in Nouméa), contamination by trace elements is insufficiently documented and its impact on marine ecosystems remains unclear. Considering the immense surface of the south-western lagoon of New Caledonia, it is necessary to extend investigations to a large spatial scale and to other contaminants such as POPs.

Marine top predators, mainly long-living species, bioaccumulate high levels of metallic elements (e.g. Cd and Hg) or organochlorine pollutants in their tissues (e.g. [START_REF] Adams | Mercury levels in four species of sharks from the Atlantic coast of Florida[END_REF][START_REF] Wafo | PCBs and DDTs in Stenella coeruleoalba dolphins from the French Mediterranean coastal environment (2007-2009): Current state of contamination[END_REF]. Fish are particularly useful to assess contamination status and distribution of pollutants; especially if they are site-attached or even territorial, living in benthic habitats and have a high trophic level (carnivorous) (e.g. [START_REF] Kojadinovic | Bioaccumulation of trace elements in pelagic fish from the Western Indian Ocean[END_REF][START_REF] Dierking | Spatial patterns in PCBs, pesticides, mercury and cadmium in the common sole in the NW Mediterranean Sea, and a novel use of contaminants as biomarkers[END_REF]). The sedentary nature of fish is a crucial aspect in spatial variability studies of contamination. Anguilliform fish (morays, congers and snake eels), which are ubiquitous organisms highly represented in tropical marine waters, fulfil the conditions of good bioindicators in contamination monitoring [START_REF] Bonnet | Anguilliform fish reveal large scale contamination by trace elements in the coral reefs of New Caledonia[END_REF]. These sedentary benthic predators, which mainly live in coral reef matrices of shallow waters, are good candidates for studying the impact and spatial variability of contamination in marine environments. Surprisingly, to date they have received little interest, which could be due to their cryptic lifestyle and the difficulty to collect them [START_REF] Ineich | Anguilliform fishes and sea kraits: neglected predators in coral-reef ecosystems[END_REF].

In this general context, the main purposes of this study were 1) to determine the level of contamination of two anguilliform fish species by trace elements and POPs, 2) to study the spatial variability of these contaminants along two coast-barrier reef gradients which are differently exposed to human activities. As the influence of ecological and biological factors on the concentration of contaminants is important, three biological parameters (body size, trophic position, and lipid content in tissue) and their link to the bioaccumulation processes were also investigated.

Materials and methods

Fish sampling and study areas

Two anguilliform species were studied, Gymnothorax chilospilus (Muraenidae) and Conger sp. (Congridae). Capture of specimens was carried out using sea kraits (Laticauda spp.) because the prey of these predators is almost exclusively anguilliform fish. This method has been successfully used in previous studies (e.g. [START_REF] Reed | Sea kraits (Squamata: Laticauda spp.) as a useful bioassay for assessing local diversity of eels (Muraenidae, Congridae) in the Western Pacific Ocean[END_REF][START_REF] Ineich | Anguilliform fishes and sea kraits: neglected predators in coral-reef ecosystems[END_REF][START_REF] Brischoux | Isotopic signatures, foraging habitats and trophic relationships between fish and seasnakes on the coral reefs of New Caledonia[END_REF]. The prey items were obtained by a gentle massage of the sea krait abdomen. Because the eels consumed are non-spiny, the snake readily regurgitates their prey (Brischoux and Bonnet 2009) without any effect on their survival [START_REF] Fauvel | Do researchers impact their study populations? Assessing the effect of field procedures in a long term population monitoring of sea kraits[END_REF]. The spatial accuracy of sea snake sampling and their use as sentinels was described in previous studies (Brischoux et al. 2007a(Brischoux et al. , 2009;;[START_REF] Brischoux | Estimating the impact of sea kraits on the anguilliform fish community (Muraenidae, Congridae, Ophichthidae) of New Caledonia[END_REF][START_REF] Bonnet | Long-term field study of sea kraits in New Caledonia: fundamental issues and conservation[END_REF]. Sea kraits probe the surrounding seafloors within a radius of 10-15km and anguilliform fish are extremely sedentary. Considering the large spatial scale of the current study, this accuracy was sufficient to examine possible local influences along a coast-barrier reef gradient.

The study was realized in the South Western lagoon of New Caledonia. Both fish species were caught in two main areas: Grand Nouméa (GN) and Grand Lagon Sud (GS) (Fig. 1). Grand Nouméa, which is close to the main city of Nouméa, is subject to a variety of anthropic pollution such as industrial activities, farming industries and wastewater. In particular a nickel factory has functioned since 1880 (SLN, Société Le Nickel, Fig. 1). Grand Lagon Sud is less influenced by industrial and urban pollution. Nevertheless, since 1950 mining activity has developed near the Bay of Prony (Goro-Nickel, Vale Inco, Fig. 1), increasing the erosion of lateritic soil in this region. For each area, three stations located on a coast-barrier reef gradient inside the lagoon were sampled: Kuendu beach (coast; CO1), Signal islet (intermediate-reef; IR1) and Amédée islet (barrier-reef; BR1) inside GN and Ouen island (coast; CO2), Mato islet (intermediate-reef; IR2) and N'da islet (barrier-reef; BR2) inside GS (Fig. 1). Sampling was carried out from January to April 2011 and from August to September 2011, corresponding respectively to the hot and wet versus cool and dry seasons in New Caledonia.

Each fish was identified, measured and weighed prior to dissection (Table 1). As some fish collected were partially digested, their total length (TL) was estimated applying allometric equations using either the snout vent length, or the tail length (Brischoux et al. 2007b). For each individual of both species, a piece of white muscle was sampled on the nondigested part and immediately frozen at -30°C for subsequent analyses. Muscle tissues were freeze-dried and ground to powder with a porcelain mortar and pestle.

Trace element analyses

The total Hg concentration in the powder obtained from the tissues was determined by analysing the Hg, with an Advanced Mercury Analyzer (ALTEC AMA 254), directly on aliquots ranging from 5 to 50 mg of dry sample weighed to the nearest 0.01 mg [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]. From 150 to 300 mg of each sample were digested using a 3:1 v:v nitrichydrochloric acid mixture with 65% ultrapure HNO 3 and ultrapure 37% HCl. The acidic digestion was performed overnight under ambient temperature and then heated in a microwave for 30 min, increasing the temperature up to 105 °C, and 15 min at 105 °C (1200 W). After the mineralization process, each sample was diluted to 30 or 50 ml with milli-Q quality water, according to the volume of acid added to the mineralization (3 and 4.5 ml, respectively).

The analysis of Ag, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Se, V and Zn required an extra step in the preparation protocol. These elements were analysed by Inductively Coupled Plasma Atomic Emission Spectrometry (Varian Vista-Pro ICP-OES) and Mass Spectrometry (ICP-MS II Series Thermo Fisher Scientific). Reference tissues -dogfish liver DOLT-4 (NRCC), lobster hepatopancreas TORT-2 (NRCC) -were treated and analysed in the same way as the samples. Results were in line with the certified values, and the standard deviations were low, proving good repeatability of the method. The results for standard reference materials displayed recovery of the elements ranging from 73% to 116%. For each set of analyses, blanks were included in each analytical batch. The detection limits (µg.g -1 dw) were 0.005 (Hg), 0.015 (Cd), 0.017 (Ag), 0.02 (Cr, Co, Pb), 0.03 (Ni), 0.08 (Mn), 0.1 (Cu, Se), 0.2 (As), 0.3 (V) and 3.3 (Fe and Zn). All trace element concentrations are given on a dry weight basis (µg.g -1 dw).

POP analyses 2.3.1 Choice of PCB congeners and pesticides

The concentrations of 15 individual congeners (IUPAC Nos. 18, 20, 28, 31, 44, 52, 101, 105, 118, 138, 149, 153, 170, 180, 194;[START_REF] Ballschmiter | Analysis of polychlorinated biphenyls (PCB) by glass capillary gas chromatography[END_REF] were determined in each sample. This list contains the seven target congeners (PCBs 28,52,101,118,138,153,180) proposed by the International Council for the Exploration of the Sea (ICES) as indicators of PCB contamination [START_REF] Valoppi | Use of PCB congener and homologue analysis in ecological risk assessment[END_REF].

The samples and blank were analyzed for the following pesticides: Aldrin, Diazinon, Dieldrin, Endosulfan I, Endosulfan II, Endrin, Heptachlor, Heptachlor-epoxide A, Heptachlorepoxide B, Lindane and, pp'-DDD, pp'-DDE, pp'-DDT.

Sample extraction and quantification

Compounds were extracted and quantified following the procedures described by [START_REF] Dierking | Spatial patterns in PCBs, pesticides, mercury and cadmium in the common sole in the NW Mediterranean Sea, and a novel use of contaminants as biomarkers[END_REF]. About 1g of lyophilized sample was extracted and concentrated to 2 ml. About 100 µL of this extract was reserved to determine the content of lipid (Lp, %) of each sample (see next paragraph). The remaining fraction was purified with concentrated sulphuric acid, followed by additional purification by liquid chromatography on a silicaalumina column [START_REF] Murphy | Sulfuric acid for the cleanup of animal tissues for analysis of acid stable chlorinated hydrocarbons residues[END_REF][START_REF] Wells | Separation of organochlorine residue by absorption chromatography prior to capillary gas chromatography[END_REF]. Four fractions were eluted: fraction Ia (Aldrin, pp'-DDT, Heptachlor (50%)), fraction Ib (Heptachlor (50%), pp'-DDT), fraction II (Endosulfan I, Endosulfan II, Lindane, pp'-DDD) and fraction III (Dieldrin, Endrin, Heptachlor-epox A and B).

PCBs and pesticides were analysed by gas chromatography (GC) coupled to mass spectrometry (MS). Calibration was done using fifteen individual standard solutions for PCBs and a standard mixture containing all pesticides was used for pesticides. All organic pollutant concentrations are given on a dry weight basis (ng.g -1 dw). Detection limits were 0.01 ng.g -1 for PBC congeners, 0.1 ng.g -1 for DDT and its metabolites, 0.01 ng.g -1 for Heptachlor-epoxide A and B, 0.1 ng.g -1 for Lindane, Diazinon, Heptachlor, Aldrin, Endosulfan II, Endrin and 0.2 ng.g -1 for Endosulfan I and Dieldrin.

 15 N and lipid quantification

The nitrogen stable-isotope ratio ( 15 N) was used to give an approximate trophic position. Analyses were carried out on dorsal white muscle, as this tissue gives the most reliable values [START_REF] Pinnegar | Differential fractionation of δ 13 C and δ 15 N among fish tissues: implications for the study of trophic interactions[END_REF]. Then 1 ± 0.1 mg of powdered freeze-dried samples was weighed out and sealed in tin capsules for analyses. 15 N/ 14 N ratios were determined by continuous-flow isotope-ratio mass spectrometry with a Thermo Scientific Delta V Advantage mass spectrometer coupled to a Thermo Scientific Flash EA1112 elemental analyser [START_REF] Chouvelon | Species and ontogenic-related differences in  13 C and  15 N values and Hg and Cd concentrations of cephalopods[END_REF]. Results are expressed as isotope ratios δ 15 N (‰) relative to international standard (atmospheric N 2 for nitrogen), according to the formula:

δ 15 N = [( R sample / R standard ) -1] * 10 3
where R = 15 N/ 14 N [START_REF] Peterson | Stable isotopes in ecosystem studies[END_REF]. Replicate measurements of internal laboratory standards (acetanilide) indicated a precision of approximately 0.2‰ for δ 15 N values.

To evaluate the lipid content (Lp, %) in each fish, 100 µL of the evaporated extraction residue was introduced in an initially tarred (tablet) container; then the whole was deposited into a desiccator. After drying to constant mass, the lipid content is determined by gravimetry.

Statistical analysis

Several indices were used to characterize the POP contamination. Our PCB profiles revealed that PCBs detected in the environment mainly came from capacitors (pyralene: DP3, DP4 or Arochlor1230, 1242). Consequently it was decided to express the global footprint of PCB contamination (PCBTot) using the estimation proposed by [START_REF] Annema | Evaluation of PCB fluxes in the environment[END_REF]: PCBTot =  (28 + 52 + 101 + 138 + 153 + 180)*5. ICES (sum of concentrations of the 7 ICES congeners), and the proportional contribution of each PCB class (tri-to octachlorinated) was also calculated using all of the PCBs analysed. The total DDT concentration, DDT, was calculated as the sum of pp'-DDD, pp'-DDE and pp'-DDT. An indication of the timing of PCB inputs was estimated through the ratio of the two congeners CB153/CB138 [START_REF] Monod | PCB congeners in the marine Biota of Saint Paul and Amsterdam Islands, Southern Indian Ocean[END_REF][START_REF] Wafo | Accumulation and distribution of organochlorines (PCBs and DDTs) in various organs of Stenella coeruleoalba and a Tursiops truncatus from Mediterranean littoral environment[END_REF]: a high ratio (> 1) indicates continuous inputs or persistence of PCBs in the environment, whereas a low ratio (< 1) reflects a decrease or sporadicity of PCB inputs or non-persistence. Finally, the ratio of the total concentration of pesticides to total concentration of PCBs analysed (Pest/PCB) was used as an approximate indication of the relative importance of agricultural versus industrial sources of organic pollutants in the environment (de [START_REF] De Mora | Organochlorinated compounds in Caspian Sea sediments[END_REF]); a ratio higher than 1 indicates prevalence of agricultural sources, whereas a ratio less than 1 indicates prevalence of industrial sources.

Data were log transformed to satisfy conditions of normality (Shapiro-Wilk test) and homogeneity of variance (Bartlett test). However for some compounds (Aldrin, Diazinon, Dieldrin, Endrin, pp'-DDD and pp'-DDT), transformation was not sufficient. When conditions were satisfied, parametric tests were used. In other cases non-parametric analogues were applied.

Principal component analysis (PCA) was used to obtain an overview of the contribution of each contaminant according to sites. Trace elements and POP concentrations were analysed in separate PCAs and as no clear patterns were revealed from POP analysis, only trace element results were presented. Spatial variability of contaminant concentrations in each species was also tested using 2-way ANOVA and Tukey's post hoc multiple comparisons. For all contaminants, Pearson or Spearman correlations were used to explore the relationship between both contaminant concentration and fish size (TL) and contaminant concentration and trophic level ( 15 N). For organic pollutants the relationship between contaminant concentration and the percentage of lipids in muscle (Lp) was also examined.

When a correlation existed, ANCOVA, instead of ANOVA was applied, using TL,  15 N or Lp as a covariate. R project (R version 2.13.2) was the statistical program used for all analyses.

The levels of significance for statistical analyses was always set at α = 0.05.

Results

Accumulation levels in anguilliform fish

All trace elements measured were detected in anguilliform fish, except Ag and V which were below the detection limit. Mean concentrations and ranges of detected values of contaminants (trace elements and POPs) in anguilliform fish muscle are presented in Table 2.

Several elements showed a high inter-individual variability (i.e. Cd, Cr, Ni, Pb; coefficient of variation CV > 100%). However it was most often the same individuals that accumulated high concentrations of several trace elements linked to their origin (e.g. a G. chilospilus specimen from the coastal site of Grand Nouméa; 35.7 µg.g -1 dw of Cr and 17.4 µg.g -1 dw of Ni).

Overall, few differences in mean contaminant concentrations were found between species. However, significantly higher concentrations of As, Hg and Se were measured in Conger sp., whereas G. chilospilus exhibited higher concentrations of Cd and Zn (p ANOVA < 0.001). PCBs had been similarly accumulated by both species, albeit weak interspecific differences were detected for pesticides: Conger sp. presented significantly higher concentrations of Heptachlor, Heptachlor-epoxy B (p ANOVA < 0.01), while slightly higher concentrations of Endosulfan I and Aldrin were measured in G. chilospilus (p ANOVA < 0.05).

Spatial patterns in contaminants

Trace elements

A significant spatial variability of several trace elements, in particular those linked with mining activity (Co, Cr, Fe, Mn and Ni), was detected along the coast-barrier reef gradient (Fig. 2 and Table 3). The PCAs in G. chilospilus indicated that the coastal sites were characterized by Co, Mn and Ni elements, in contrast to the barrier reef sites which were characterised by As and Cd concentrations (the plot of the first two PCA dimensions explained respectively 51.1% and 59.0% of data variability in G. chilospilus and Conger sp., Fig. 2). In Conger sp., the difference between sites was even stronger, with most trace elements (As, Co, Cr, Fe, Ni, Se, and Zn) characterising coastal sites. These general trends were confirmed by the significantly higher concentrations of mine elements at coastal sites for both species (p ANOVA or p ANCOVA < 0.05, Table 3).

The same spatial pattern (coast vs. barrier reef) was revealed in the two studied areas (GN and GS, Table 3 andS1). The coastal site of Grand Nouméa showed significantly higher concentrations of Ni in G. chilospilus (p ANOVA = 0.02) and Cr and Fe in Conger sp. (p ANOVA < 0.05, Table 3 andS1), with the same trends followed by several other trace elements (Cr, Fe in G. chilospilus, As, Co, Cu, Ni, Se, and Zn in Conger sp., results of PCA not shown).

However in Grand Lagon Sud the spatial gradient was less pronounced. The coastal site was characterized by higher Co and Mn concentrations in both species (results of PCA not shown), but spatial analyses only suggested significantly lower concentrations of As and Cd in G. chilospilus (p ANCOVA = 0.001, Tables 3 andS1).

The coastal site of Grand Nouméa was characterized by significantly higher levels of several trace elements compared to Grand Lagon Sud (e.g. As, Cd and Ni in G. chilospilus, and Cr, Fe in Conger sp.; p ANCOVA < 0.05, Tables 3 andS1).

Organic pollutants

The PCAs did not reveal any clear spatial pattern for POPs in either species (results not shown). Analyses suggested a low spatial variability along the coast-barrier reef gradient (Table 4).

The PCB contamination in G. chilospilus did not vary significantly between sites (see PCBTot index; p ANOVA > 0.05), in spite of a slight increase from the coast to the barrier reef.

However in Conger sp. the intermediate sites were characterized by high concentrations (p ANCOVA < 0.05, Table 4). The CB153/CB138 ratio, which was generally below 1, showed few spatial variations and only G. chilospilus from coastal sites showed a significantly higher ratio (p ANCOVA < 0.05, Tables 4 andS2). The PCB chlorination classes differed slightly along the coast-barrier reef gradient and between studied areas, with a low variability detected in each case (< 10%, p ANCOVA or p ANOVA < 0.05, Table 4). However the results highlighted a remarkable increase in the proportion of the most toxic chlorinated classes in coastal sites (i.e. 8Cl in GN for G. chilospilus and 7Cl in GS for Conger sp., p ANOVA < 0.05).

The results for pesticides indicated few spatial variations (Tables 4 andS2). The coastal sites highlighted significantly higher concentrations of some pesticides in G. chilospilus (Aldrin and Heptachlor, p ANCOVA or p KW < 0.05), whereas the intermediate reef sites revealed significantly higher rates of several other pesticides in Conger sp. (e.g. Diazinon, Heptachlor epoxide A and pp'-DDT; p ANCOVA or p KW < 0.05).

Finally, spatial analyses of POP contamination according to sources of pollution (agriculture vs. industrial) reinforce previous results (see Pest/PCBs; Table 4). Along the gradient, G. chilospilus from the coastal sites were significantly different from other sites, with quite homogeneous sources of contamination (ratio ≥ 1). In comparison industrial sources dominated in other sites (ratio 0.50, p ANOVA < 0.05, Table 4). A strong influence of industrial pollution in intermediate sites (ratio 0.50, p ANCOVA < 0.05) was also highlighted in Conger sp.

Influence of size, trophic position and lipid content on contamination level

Several trace elements (Co, Cr, Ni, Pb, Se, and Zn in G. chilospilus, Cd and Mn in Conger sp.) were not correlated with either the size or the trophic position of fish (p Pearson > 0.05), but some trace elements were significantly correlated with fish size (Table 5). Only As and Hg highlighted a positive relationship with the trophic position of fish ( 15 N) in Conger sp., whereas no correlation was found for G. chilospilus.

Organic pollutant concentrations were linked to size, trophic position or lipid content, depending on the contaminant. Most of them were mainly significantly correlated with the lipid contents in fish (Table 5). For example, PCBs were only correlated with lipids in both species. Pesticides were also correlated with Lp, and to a lesser extent with  15 N and TL (Table 5).

Discussion

This study confirms, reinforces and extends the high trace element contamination status of anguilliform fish in New Caledonia [START_REF] Bonnet | Anguilliform fish reveal large scale contamination by trace elements in the coral reefs of New Caledonia[END_REF]. It also constitutes the first substantial baseline on organic pollutant contamination of fish in New Caledonia.

Importantly, our results show that large scale contamination reaches remote parts of the lagoon, more than 30km offshore (SW lagoon), and thus well beyond the urbanized Nouméa region.

Contaminant levels and spatial variability

Trace element patterns

Anguilliform fish concentrate both significant levels of trace elements associated with mining exploitation (Cr, Fe, Mn, and Ni for both species), and also some elements linked to urban activities (e.g. Cd, Cu and Zn, especially in G. chilospilus) (Table 2). This elevated typical mining signature detected in sedentary benthic species contrasts with other studies which highlight low contaminant levels in some more mobile fish in both the lagoons of New Caledonia and other tropical regions (Eisler 2010; [START_REF] Bonnet | Anguilliform fish reveal large scale contamination by trace elements in the coral reefs of New Caledonia[END_REF]Table 6).

Although coastal sites in New Caledonia are highly contaminated by trace elements, directly or indirectly linked to mining activity (Co, Cr, Fe, Mn and Ni), the contamination has spread throughout the entire lagoon as far as the remote barrier reef of the extremely vast South-Western lagoon. This is probably due to dispersal by local currents [START_REF] Fichez | Origine, transport et devenir des apports naturels et anthropiques dans le lagon sud-ouest de Nouvelle-Calédonie[END_REF]).

However, it should be noted that concentrations decrease significantly from the coast to the barrier reef.

Unsurprisingly, fish from the coastal site in Grand Nouméa present important concentrations in trace elements, especially Cr, Fe and Ni (Table 3). This urbanized and industrialized region is subjected to clear impact from the metallurgic industry [START_REF] Hédouin | Trends in concentrations of selected metalloid and metals in two bivalves from the coral reefs in the SW lagoon of New Caledonia[END_REF][START_REF] Hédouin | Validation of two tropical marine bivalves as bioindicators of mining contamination in the New Caledonia lagoon: Field transplantation experiments[END_REF]Metian et al. 2008[START_REF] Metian | Trace element bioaccumulation in reef fish from New Caledonia: influence of trophic groups and risk assessment for consumers[END_REF]. In contrast, the Grand Lagon Sud is less impacted by trace element inputs. Even if coastal sites in this area seem to be less threatened, trends still reveal important concentrations of Co, Mn and Ni (Table 3). This strong contamination level likely results from severe soil erosion, due to deforestation and mining exploitation in this region over several decades. Given the numerous mines in New Caledonia and the intensive development of mining activities, assessing to what extent other areas of the New Caledonian lagoon have been affected is timely.

We expected a strong impact of urban pollution at least in Grand Nouméa sites, given the intense activity in the coastal industrial area of Ducos. However, while spatial patterns are obvious for trace elements associated with mining activities, those associated with urban pollution (As, Cd, Cu, Pb and Zn) are less clear. Whereas Se and Zn are mostly concentrated in coastal sites for Conger sp., Cu and Pb are homogeneously distributed along the coastbarrier reef gradient. Furthermore, the cases of As and Cd are more complex with sometimes higher concentrations (e.g. As in Conger sp.) and sometimes lower concentrations in coastal sites (e.g. As and Cd in G. chilospilus). Finally elements issued from urban pollution concern the entire lagoon, maybe due to their wide dispersal by local currents [START_REF] Fichez | Origine, transport et devenir des apports naturels et anthropiques dans le lagon sud-ouest de Nouvelle-Calédonie[END_REF].

Another possible explanation is that in the past some islets were used as rubbish tips for decades (e.g. Amédée islet in Nouméa region, [START_REF] Bonnet | Anguilliform fish reveal large scale contamination by trace elements in the coral reefs of New Caledonia[END_REF]. Further information on these issues is thus needed.

Organic pollutant patterns

Contamination by POPs detected in anguilliform fish was rather low. However, considering the high toxicity of some POPs, even at very low doses, this contamination should be considered as important. Levels of PCB concentrations recorded in New Caledonia are generally lower than in fish from other tropical or temperate regions (e.g. ICES index in New Caledonia: 14.0 ± 12.2 ng.g -1 dw (G. chilospilus) and 16.5 ± 14.3 ng.g -1 dw (Conger sp.); in Wallis Central Pacific: 31.3 ± 9.6 ng.g -1 dw (Cephalopholis argus) and 49.7 ± 45.3 ng.g -1 dw (Epinephelus merra) (Letourneur et al., unpublished data); in the Mediterranean Sea: 100.6 ng.g -1 dw (Solea solea) [START_REF] Dierking | Spatial patterns in PCBs, pesticides, mercury and cadmium in the common sole in the NW Mediterranean Sea, and a novel use of contaminants as biomarkers[END_REF]). Exposure profiles of PCBs showed a contribution of chlorination classes in both species, with the prevalence of moderately chlorinated classes (5Cl and 6Cl) and the scarcity of highly chlorinated classes (7Cl and 8Cl).

These results emphasize a recent or even current use of PCBs in New Caledonia, otherwise only the more persistent classes (highly chlorinated) should be detected. Further studies are needed to confirm this hypothesis.

In New Caledonian fish, pesticide concentrations are equivalent or lower than in other regions (e.g. Lindane in New Caledonia: 1.3 ± 0.6 ng.g -1 dw (G. chilospilus) and 1.7 ± 1.6 (Conger sp.); in Wallis Central Pacific: 3.5 ± 1.03 ng.g -1 dw (C. argus) and 3.6 ± 2.07 ng.g -1 dw (E. merra) (Letourneur et al., unpublished data); in French Polynesia: 73.3 ± 34.5 ng.g -1 dw (Epinephelus hexagonatus) [START_REF] Salvat | Recherches sur la contamination par les pesticides d'organismes marins des réseaux trophiques récifaux de Polynésie Française[END_REF]; in the Mediterranean Sea: 35.4 ng.g -1 dw (Solea solea) [START_REF] Dierking | Spatial patterns in PCBs, pesticides, mercury and cadmium in the common sole in the NW Mediterranean Sea, and a novel use of contaminants as biomarkers[END_REF]). Despite the quantities and diversity of pesticides imported into New Caledonia, these low levels detected reflect their relatively moderate use, which probably comes from local and non-professional activities rather than from the agricultural industry. In addition, Nouméa is located far from the most important agricultural areas. This type of non-professional use could also explain a non-negligible detection of pesticides such as DDT and its metabolites (i.e. ΣDDT: 1.55 ± 1.50 ng.g -1 dw in G. chilospilus and 1.57 ± 1.48 in Conger sp.), which are supposed to be prohibited by international regulations and not homologated in New Caledonia (DAVAR, 2001).

Overall, POP concentrations are homogeneous throughout the lagoon, without any clear spatial pattern along the coast-barrier reef gradient, or between studied areas.

Nevertheless, some high pesticide concentrations in G. chilospilus were observed in several coastal sites and in intermediate reefs, with high PCB and some high pesticide levels in Conger sp. (Tables 4 andS2). Storage organs (i.e. liver, kidney or digestive gland) usually accumulate higher concentrations than muscle (e.g. [START_REF] Bustamante | Distribution of trace elements in the tissues of benthic and pelagic fish from the Kerguelen Islands[END_REF][START_REF] Chouvelon | Hg concentrations and related risk assessment in coral reef crustaceans, molluscs and fish from New Caledonia[END_REF][START_REF] Metian | Trace element bioaccumulation in reef fish from New Caledonia: influence of trophic groups and risk assessment for consumers[END_REF]Eisler 2010). Further research is required to test this notion in Anguilliforms and thus to better assess spatial contamination gradients, especially for POPs.

The results underlined the complexity in identifying the agricultural and/or industrial origin of organic contamination for each site (see Pest/PCBs ratio, Table 4). Trends seem to emphasize a slight prevalence of agricultural contamination sources at coastal sites, in comparison with other sites dominated by industrial pollution. Furthermore, the Nouméa area generally seems to be more exposed to industrial contamination, whereas Grand Lagon Sud has revealed strong local agricultural pollution (except at intermediate reefs, Table S2). In Grand Nouméa, anguilliform fish have not been exposed to PCB inputs in recent times in view of their low CB153/CB138 ratio (i.e. ratio < 0.8,  0.75 in G. chilospilus and  0.78 in Conger sp., Table S2). On the other hand, the PCB inputs in Grand Lagon Sud seem to be more recent mainly in coastal sites, as detected in both species (ratio > 1, 1.64 in G. chilospilus and 1.04 in Conger sp., Table S2). Industrial activities in this area, including the recent construction of a mining factory, could explain in part the continuing inputs of PCBs in this area.

Influence of biological parameters on contamination level

As mentioned in several studies, concentrations of contaminants can also vary according to ecological and biological factors such as fish size (or age), trophic position and feeding habits or lipid content [START_REF] Monteiro | Mercury content in relation to sex, size, age and growth in two scorpionfish (Helicolenus dactylopterus and Pontinus kuhlii) from Azorean waters[END_REF][START_REF] Geyer | Bioaccumulation and Occurrence of Endocrine-Disrupting Chemicals (EDCs), Persistent Organic Pollutants (POPs), and Other Organic Compounds in Fish and Other Organisms Including Humans. Bioaccumulation, New Aspects and Developments[END_REF][START_REF] De Pinho | Total mercury in muscle tissues of five shark species grom Brazilian offshore waters: effects of feeding habit, sex, and length[END_REF]. These complex relationships can induce interspecific differences in bioaccumulation processes and so emphasize the relevance in detecting the contamination rate at a specific level.

Our results indicated the influence of the three factors tested (body size, trophic position and lipid content) on several contaminants in both species. The link between body size and bioaccumulation is complex, depending on both the contaminants and the fish species considered. In some cases, trace elements accumulated with increasing fish size (e.g.

As, Cd and Hg in G. chilospilus, Table 5), which is consistent with continuous bioaccumulation in muscle during life span [START_REF] Braune | Mercury accumulation in relation to size and age of Atlantic herring (Clupea harengus harengus) from the southwestern Bay of Fundy, Canada[END_REF][START_REF] Burger | Mercury levels and potential risk from subsistence foods from the Aleutians[END_REF][START_REF] Bloom | On the chemical form of mercury in edible fish and marine invertebrate tissue[END_REF]. In some other cases, trace element concentration is negatively correlated with size (e.g. Cu and Fe in both species, or Co, Cr, Ni, Pb, Se and Zn in Conger sp.). This trend could be explained by a decrease in assimilation or by more efficient elimination processes with increasing fish size [START_REF] Braune | Mercury accumulation in relation to size and age of Atlantic herring (Clupea harengus harengus) from the southwestern Bay of Fundy, Canada[END_REF][START_REF] Swaileh | Effect of body size and season on the concentrations of Cu, Cd, Pb and Zn in Diastylis rathkei (kröyer) (Crustacea: Cumacea) from Kiel Bay, Western Baltic[END_REF][START_REF] Warnau | Allometry of heavy metal bioconcentration in the echinoid Paracentrotus lividus[END_REF]. Such a relationship can also suggest that an ontogenetic diet shift might occur, implying a variation in the exposure to contaminants through the food pathway, as found in other species [START_REF] Chouvelon | Species and ontogenic-related differences in  13 C and  15 N values and Hg and Cd concentrations of cephalopods[END_REF][START_REF] Chouvelon | Sizerelated patterns in stable isotopes and mercury concentrations in fish help refining marine ecosystem indicators and evidence distinct management units for hake in the north-eastern Atlantic[END_REF]). However, with the exception of Hg, and to a lesser extent Se, biomagnification of trace elements in trophic webs is still unclear and difficult to identify [START_REF] Wang | Interactions of trace metal and different marine food chains[END_REF]. For anguilliform fish from New Caledonia, the trophic position influenced As and Hg concentrations only in Conger sp. The POP results are in accordance with their hydrophobic nature, which allows their sequestration in fat tissues during the process of organochlorine accumulation [START_REF] Robertson | PCBs: Recent advances in environmental toxicology and health effects[END_REF]. Most of them were indeed positively linked to the lipid content of fish, in particular in Conger sp., where lipid variation ranged between 0.2 and 5.5% (Table 1).

Measures of  15 N in fish confirm the high trophic level of anguilliform fish [START_REF] Brischoux | Isotopic signatures, foraging habitats and trophic relationships between fish and seasnakes on the coral reefs of New Caledonia[END_REF]. When this is associated with other life history traits (size, age, longevity, philopatry, etc.), the detection of high trace element levels appears consistent. These benthic predators are also probably good candidates for monitoring organic pollution because of the strong relationship of POPs with lipid content (even if the flesh of these fish is not very fatty).

The specific biological characteristics of each species may well explain interspecific differences in bioaccumulation of contaminants.

Moreover, contamination of anguilliform fish (high trophic level predators) suggests that trophic networks in general are contaminated [START_REF] Monniot | Cellular sites of iron and nickel accumulation in ascidians related to the naturally and anthropic enriched New Caledonian environment[END_REF][START_REF] Hédouin | Allometric relationship in the bioconcentration of heavy metals by the edible tropical clam Gafrarium tumidum[END_REF][START_REF] Hédouin | Nickel bioaccumulation in bivalves from the New Caledonia lagoon: Seawater and food exposure[END_REF]Metian et al. 2008b). As a consequence, it is important to carry out contaminant analysis in prey, top predators (e.g. sea snakes, Bonnet 2012) and in the global trophic web. This will provide a more complete assessment of contamination of the environment and lead to better understanding of the mechanisms and processes involved (e.g. bioaccumulation, biomagnification, [START_REF] Bryan | Pollution due to heavy metals and their compounds[END_REF][START_REF] Rainbow | Cosmopolitan biomonitors of trace metals[END_REF]. A good understanding of both the organisms (i.e. biology, ecology and metabolic capacities) and the trophic network structures, plus environmental conditions, is necessary to understand the dynamics of bioaccumulation and biomagnification throughout the trophic webs [START_REF] Harmelin-Vivien | Difference of mercury bioaccumulation in red mullets from the north-western Mediterranean and Black seas[END_REF].

Conclusion

This study strongly confirms a clear accumulation of trace metal contaminants in the muscles of anguilliform fish and the obvious coastal contamination by trace elements linked to mining activities. It is also shown that contamination by POPs occurs in the whole lagoon.

These results raise an alert about complex contaminations throughout the lagoon on a large spatial scale. Various contaminants may show different behaviour in abiotic and biotic environments. Due to their lipophilic nature, POPs have different targets compared to trace elements. This characteristic can explain in part their divergences in mobility in comparison to trace elements. Thus, the more widespread repartition of POPs within the lagoon is probably due to their great capacity of dispersion, through atmospheric transport and currentdriven dilution during tropical rain events. This highlights the importance of considering multiple sources of contamination. Moreover, the possibility of a cocktail effect of these various contaminants is not known and could increase the threat. Therefore, in the future, long term monitoring studies and experiments on toxicity are necessary.

A) B)

Table 1. Number (N) of fish analyzed from the coastal (CO), intermediate (IR) and barrier reef (BR) sites in Grand Nouméa (GN) and Grand Lagon Sud (GS). Mean (± SD) fish total length (TL, cm), trophic position (express with  15 N, ‰) and lipid content (Lp, %) are given, with minimal and maximal values (min-max) indicated in brackets. No variation of TL,  15 N and Lp between sites was observed (p ANOVA > 0.05).

Species

Site N N (GN) N (GS) TL (cm) (min-max) δ 15 N (‰) (min-max) Lp (%) (min-max)

Gymnothorax

Figure 1 .

 1 Figure 1. Location of the sampled sites distributed on two coast-barrier reef gradients in New Caledonia: Kuendu (CO1), Signal (IR1) and Amédée (BR1) in Grand Nouméa (GN) and Ouen (CO2), Grand Mato (IR2) and N'Da (BR2) in Grand Lagon Sud. CO = coastal sites; IR = intermediate reef sites; BR = barrier reef sites. Stars indicate areas of human activities, which are the undustrial area of Ducos (1), the Nikel factory « SLN » (2) and the Nikel factory « Goro-Nikel » (3). The emerged land is indicated in black, grey areas represent coral reefs; the dark grey line represents the slope of the barrier reef.

Figure 2 .

 2 Figure 2. Plot of the principal component analysis (PCA) assessing spatial patterns of anguilliform fish contamination by trace elements on the coast-barrier reef gradient (left panel) and the contribution of the contaminants analyzed to site differentiation (right panel) in G. chilospilus (A) and Conger sp. (B). CO = coastal sites; IR = intermediate reef sites; BR = barrier reef sites. The most contributing variables in both species were Cr, Cu, Fe, Ni concentrations for the first axis and Cd and Mn concentrations for the second axis. The significant differences between sites are illustrated by confidence ellipses of centroids at 95%.

Table 3 .

 3 Trace element concentrations (mean (SD); µg.g -1 dw) in G. chilospilus and Conger sp. along the coastal-barrier reef gradient. ANOVA (ANV) or ANCOVA (ACV) with Tukey's pairwise comparisons were used to analyze spatial patterns.

	Gradient

Note: GN: Grand Nouméa; GS: Grand Lagon Sud; CO: coastal sites; IR: intermediate reef sites; BR: barrier reef sites ns = not significant (p > 0.05); * p < 0.05, ** p < 0.01, *** p < 0.001.

Table 4 .

 4 PCBs and pesticide concentrations (mean (SD); ng.g -1 dw, except % of classes) in G. chilospilus (A) and Conger sp. (B) along the coastbarrier reef gradient. ANOVA or ANCOVA with Tukey's pairwise comparisons were used to analyze spatial patterns A)

	Gradient

a only one value.

Table 5 .

 5 Summary of correlations performed on trace elements and organic pollutants (PCBs and pesticides) concentrations versus total length (TL), trophic position (δ 15 N) and lipid content (Lp) in G. chilospilus and Conger sp.

			G. chilospilus
		Trace elements	PCBs	Pesticides
	TL	As*, Cd**, Cu*, Fe*, Hg*, Mn*		Ald.**, Dia.**
	 15 N			Endo.*
	Lp		CB153/138**, 4 Cl**, 8 Cl* Hept.**
			Conger sp.
		Trace elements	PCBs	Pesticides
	TL	Co**, Cr*, Cu**, Fe*, Ni*, Pb*, Se*, Zn*		Hept.**
	 15 N As***, Hg***		Ald.*
	Lp		CTotal PCBs**, SICES *, CB153/138*, 3 Cl*, 4 Cl***, 5 Cl**, 6 Cl*, 8 Cl**	Dia.*, Diel.*, Endo.I**, Endo.II**, Endr.**, Hept.A*, Hep.B*, Lind.**, DDT**
	Note: 3 Cl to 8 Cl : classes of chlorination; Lind.: Lindane; Dia.: Diazinon; Hept.: Heptachlor; Ald.:
	Aldrin; Hept.B: Heptachlor-epox.B; Hept.A: Heptachlor-epox.A; Endo.I: Endosulfan I; Diel: Dieldrin; Endr.; Endrin; DDT : pp'-DDT.
	Significativity: * p < 0.05, ** p < 0.01, *** p < 0.001.
		correlations are underlined.

Table 6 .

 6 Comparison of several trace element concentrations (mean ± SD and range of values; µg.g -1 dw) measured in muscle of G. chilospilus, Conger sp. and other lagoon fish species in New Caledonia.

	Trace element Concentration (µg.g -1 dw)	species	reference
	As	11.0 ± 7.4 (0.9-31.9)	G. chilospilus	This study
	As	25.7 ± 25.6 (3.0-117.9)	Conger sp.	This study
	As	13.7 ± 7.57 (<7.87-19.1)	Lethrinus laticaudis	Metian et al. (2013)
	As	9.47 ± 1.47 (<7.85-10.7)	Priacanthus hamrur	Metian et al. (2013)
	As	< 6.01-7.77	Cymbacephalus beauforti	Metian et al. (2013)
	As	1.2 -52.5	mix	Fernandez and Breau (2011)
	Cd	0.41 ± 0.47 (0.01-2.91)	G. chilospilus	This study
	Cd	0.04 ± 0.04 (0.01-0.16)	Conger sp.	This study
	Cd	<0.06	mix	Fernandez and Breau (2011)
	Co	0.11 ± 0.08 (0.02-0.45)	G. chilospilus	This study
	Co	0.13 ± 0.15 (0.02-0.68)	Conger sp.	This study
	Co	< 0.03	Lethrinus laticaudis	Metian et al. (2013)
	Co	< 0.03	Priacanthus hamrur	Metian et al. (2013)
	Co	< 0.02	Cymbacephalus beauforti	Metian et al. (2013)
	Co	<0.1 -1.1	mix	Fernandez and Breau (2011)
	Cr	4.53 ± 7.15 (0.86-35.71)	G. chilospilus	This study
	Cr	4.03 ± 5.36 (0.45-23.60)	Conger sp.	This study
	Cr	< 0.79	Lethrinus laticaudis	Metian et al. (2013)
	Cr	1.39 (<0.70-1.39)	Priacanthus hamrur	Metian et al. (2013)
	Cr	< 0.60	Cymbacephalus beauforti	Metian et al. (2013)
	Cr	<0.1 -5.7	mix	Fernandez and Breau (2011)
	Cu	1.6 ± 0.7 (0.7-3.4)	G. chilospilus	This study
	Cu	1.9 ± 0.9 (0.7-4.5)	Conger sp.	This study
	Cu	< 0.79	Lethrinus laticaudis	Metian et al. (in press)
	Cu	< 0.83	Priacanthus hamrur	Metian et al. (in press)
	Cu	< 0.53-0.64	Cymbacephalus beauforti	Metian et al. (in press)
	Cu	0.25 -3.0	mix	Fernandez and Breau (2011)
	Fe	40.2

± 36.9 (12.2-196.8) G. chilospilus This study Fe 34.3 ± 31.1 (7.9-134.4) Conger sp.

				This study
	Fe	9.19 ± 1.42 (7.56-10.1)	Lethrinus laticaudis	Metian et al. (2013)
	Fe	10.4 ± 3.56 (7.82-16.9)	Priacanthus hamrur	Metian et al. (2013)
	Fe	7.01-7.60	Cymbacephalus beauforti	Metian et al. (2013)
	Fe	4.0 -211	mix	Fernandez and Breau (2011)
	Mn	2.3 ± 1.4 (0.6-7.2)	G. chilospilus	This study
	Mn	2.7 ± 2.3 (0.4-10.1)	Conger sp.	This study
	Mn	< 0.79	Lethrinus laticaudis	Metian et al. (2013)
	Mn	< 0.83	Priacanthus hamrur	Metian et al. (2013)
	Mn	< 0.60	Cymbacephalus beauforti	Metian et al. (2013)
	Mn	<0.03 -3.3	mix	Fernandez and Breau (2011)
	Ni	1.95 ± 3.19 (0.30-17.38)	G. chilospilus	This study
	Ni	1.44 ± 1.77 (0.10-7.74)	Conger sp.	This study
	Ni	<0.06 -1.35	mix	Fernandez and Breau (2011)

Note: mix = 144 fish from 27 species and 10 families (Acanthuridae, Carangidae, Haemulidae, Labridae, Lethrinidae, Lutjanidae, Mugilidae, Scaridae, Serranidae and Siganidae).

Table S1 .

 S1 Trace element concentrations (mean (SD); µg.g -1 dw) in G. chilospilus and Conger sp. along the gradient in Grand Nouméa and Grand Lagon Sud.

			Grand Nouméa (GN)			Grand Lagon Sud (GS)
			CO1	IR1	BR1	CO2	IR2	BR2
		As	16.27 (13.65) 8.57 (7.52) 16.75 (4.87)	2.42 (1.26)	7.90 (3.62)	13.34 (6.49)
		Cd	0.36 (0.31)	0.36 (0.30) 0.36 (0.18)	0.06 (0.07)	0.56 (0.85)	0.54 (0.34)
		Co	0.21 (0.10)	0.10 (0.05) 0.09 (0.06)	0.22 (0.14)	0.11 (0.06)	0.05 (0.03)
	G. chilospilus	Cr Cu Fe Hg Mn	16.83 (16.87) 3.21 (2.04) 4.28 (3.30) 2.35 (0.85) 1.75 (0.89) 1.38 (0.30) 95.2 (64.4) 33.8 (19.0) 35.4 (15.7) 0.07 (0.03) 0.05 (0.02) 0.04 (0.02) 2.92 (0.89) 2.13 (1.18) 1.70 (0.43)	1.76 (0.74) 1.51 (0.57) 26.5 (13.0) 0.05 (0.04) 5.04 (2.41)	4.28 (8.41) 1.74 (0.69) 46.4 (54.0) 0.07 (0.04) 1.72 (0.51)	4.05 (7.77) 1.49 (0.50) 35.5 (36.6) 0.04 (0.02) 2.00 (0.98)
		Ni	9.21 (7.09)	1.33 (0.79) 1.09 (0.81)	1.17 (0.76)	1.84 (3.27)	1.73 (3.23)
		Pb	0.07 (0.03)	0.06 (0.07) 0.05 (0.04)	0.06 (0.03)	0.03 (0.03)	0.04 (0.03)
		Se	1.17 (0.28)	1.21 (0.47) 1.00 (0.19)	1.07 (0.28)	1.31 (0.28)	1.25 (0.54)
		Zn	50.2 (6.5)	59.9 (23.3) 52.7 (11.4)	47.0 (12.1)	57.3 (9.3)	63.0 (20.7)
			Grand Nouméa (GN)			Grand Lagon Sud (GS)
			CO1		BR1	CO2	IR2	BR2
		As	42.51 (33.77)	20.68 (18.76)	29.77 (28.29) 20.57 (21.08) 9.67 (10.84)
		Cd	0.05 (0.04)	0.04 (0.04)	0.02 (0.01)	0.03 (0.04)	0.08 (0.07)
		Co	0.23 (0.20)	0.07 (0.04)	0.23 (0.21)	0.10 (0.06)	0.03 (0.01)
		Cr	11.69 (6.00)	3.13 (1.61)	1.02 (0.45)	0.95 (0.31)	0.86 (0.14)
	Conger sp.	Cu Fe Hg Mn	2.59 (1.21) 78.7 (34.2) 0.08 (0.04) 3.06 (1.73)	2.20 (0.81) 25.7 (8.2) 0.07 (0.03) 2.61 (3.34)	1.98 (0.61) 19.1 (7.60) 0.18 (0.09) 4.86 (2.93)	1.32 (0.43) 15.3 (4.0) 0.07 (0.03) 1.76 (0.95)	1.42 (0.55) 21.6 (12.3) 0.28 (0.22) 1.74 (1.06)
		Ni	3.90 (2.05)	1.15 (0.67)	0.77 (0.25)	0.35 (0.14)	0.32 (0.20)
		Pb	0.08 (0.06)	0.12 (0.17)	0.06 (0.02)	0.03 (0.01)	0.03 (0.01)
		Se	2.77 (1.06)	1.65 (0.43)	1.50 (0.28)	1.37 (0.21)	1.28 (0.34)
		Zn	58.7 (27.6)	40.8 (29.6)	60.9 (26.5)	33.8 (6.3)	36.2 (6.9)

Note: CO: coastal sites; IR: intermediate reef sites; BR: barrier reef sites.

Table S2 .

 S2 Trace element concentrations (mean (SD); µg.g -1 dw) in G. chilospilus (A) and Conger sp. (B) along the gradient in Grand Nouméa and Grand Lagon Sud.

		B)	Grand Nouméa (GN)	Grand Lagon Sud (GS)
			CO1	IR1	BR1	CO2	IR2	BR2
		PCBs (ng.g -1 dw)
		A) PCBTot SICES	Grand Nouméa (GN) 75.59 (56.91) 40.16 (13.36) 31.95 (27.93) 99.47 (67.68) 15.43 (4.98) Grand Lagon Sud (GS) 19.12 (13.14) 10.93 (3.53) 8.38 (7.86) 26.32 (18.94) 3.89 (1.70)
		153/138	CO1 0.77 (0.44)	IR1	BR1 0.78 (0.15)	CO2 1.04 (0.47)	IR2 0.88 (0.36) 1.43 (0.61) BR2
		PCBs index (ng.g -1 dw) PCB chlorination classes (% of CTotal PCBs)
		PCBTot 3 Cl	25.79 (8.98) 70.14 (78.71) 59.07 (56.06) 28.13 (11.28) 66.95 (57.15) 62.18 (29.27) 15.28 (7.70) 11.24 (2.15) 17.86 (5.84) 19.94 (6.08) 25.78 (3.73)
		ICES 4 Cl	5.80 (1.67) 17.37 (3.83)	17.59 (18.5) 13.65 (11.80) 6.25 (2.90) 22.43 (4.85) 19.26 (19.14) 11.07 (4.59) 20.35 (13.76) 16.33 (13.90) 13.83 (5.80)
		153/138 5 Cl	0.71 (0.10) 33.38 (7.02)	0.75 (0.15)	0.78 (0.08) 36.39 (4.06)	1.62 (0.20) 25.86 (13.80) 32.74 (7.86) 31.72 (6.57) 0.77 (0.13) 0.82 (0.11)
		6 Cl	27.21 (2.42)		24.05 (2.66)	23.33 (13.93) 32.37 (7.73) 18.73 (5.74)
		PCB chlorination classes (% of CTotal PCBs) 7 Cl 5.04 (2.92) 4.24 (2.29)	13.04 (13.56) 3.62 (1.38)	2.63 (1.42)
		3 Cl 8 Cl	14.34 (4.50) 14.09 (4.40) 14.51 (4.11) 1.72 (1.43) 1.66 (1.07)	26.44 (4.10) 19.51 (4.90) 13.74 (2.16) 0.66 (0.87) 0.26 (0.24) 0.78 (0.74)
		4 Cl	24.48 (12.55) 16.06 (10.80) 18.52 (7.00)	15.71 (5.60) 13.47 (2.70) 17.71 (3.19)
	Conger sp.	5 Cl 6 Cl 7 Cl 8 Cl Pesticides (ng.g -1 dw) 24.42 (5.30) 33.18 (7.50) 30.82 (4.90) 28.72 (10.30) 30.73 (11.20) 30.32 (7.60) 3.64 (1.58) 4.67 (2.30) 5.02 (3.50) 4.40 (3.25) 1.27 (1.50) 0.81 (0.98) Ald. 0.13 (0.07) 0.09 (0.02) Dia. 0.10 (0,01) 0.69 (1.15) Diel. 0.88 (0.66) 0.41 (0.18)	26.87 (5.20) 35.60 (5.40) 30.14 (6.92) 27.42 (3.40) 28.24 (5.20) 34.59 (7.04) 3.46 (0.89) 2.87 (0.88) 3.64 (1.44) 0.11 (0.05) 0.31 (0.36) 0.18 (0.14) 0.10 a 0.12 (0.04) 0.12 (0.04) 10.64 (6.58) 3.99 (2.31) 3.26 0.29 (0.13) 0.25 (0.09) 0.24 (0.04)
	G. chilospilus	Endo.I Pesticides (ng.g -1 dw) 1.20 (0.93) Ald. 0.11 (0.03) Endo.II 4.24 (2.49) Dia. 5.18 (4.63) Endr. 0.10 (0.003) Diel. 0.30 (0.17) Hept. 0.59 (0.41) Endo.I 0.83 (0.58) Hept.A 2.74 (2.72) Endo.II 1.30 (0.09) Hept.B 0.89 (0.84) Endr. 0.18 (0.13) Lind. 2.82 (1.44) Hept. 0.47 (0.17) DDE 0.52 (0.65) Hept.A 0.59 (0.15) DDD 0.33 (0.32) Hept.B 0.20 (0.16) DDT 0.62 (0.71) Lind. 1.49 (0.51) ΣDDT 1.47 (1.03)	0.11 (0.03) 1.72 (2.30) 0.43 (0.28) 0.95 (0.87) 2.73 (2.10) 0.43 (0.85) 0.59 (0.57) 1.66 (1.40) 0.40 (0.39) 1.72 (0.91)	0.57 (0.59) 0.07 (0.04) 2.18 (0.80) 1.08 (1.00) 0.10 (0.01) 0.44 (0.50) 0.47 (0.23) 1.17 (1.80) 1.14 (0.49) 1.87 (1.60) 0.43 (0.14) 0.13 (0.06) 1.53 (0.50) 0.23 (0.17) 0.43 (0.31) 1.46 (1.20) 0.17 (0.11) 0.41 (0.78) 1.58 (2.27) 1.28 (0.58) 2.18 (2.49)	0.44 (0.33) 0.21 (0.15) 1.71 (0.48) 2.64 (1.70) 0.86 (0.16) 0.51 (0.33) 0.55 (0.49) 1.35 (1.50) 0.69 (0.66) 1.77 (1.30) 0.30 (0.41) 0.13 (0.05) 1.37 (1.61) 1.18 (0.49) 0.12 (0.02) 0.73 (0.29) 0.10 0.40 (0.27) 1.73 (2.30) 1.15 (0.46) 1.94 (2.32)	0.61 (0.73) 0.12 (0.04) 2.61 (2.60) 1.20 (0.67) 1.69 (3.19) 0.28 (0.16) 0.51 (0.55) 1.64 (1.10) 2.47 (1.77) 1.99 (2.30) 0.30 (0.48) 0.10 (0.01) 1.25 (0.93) 0.25 (0.28) 0.37 (0.21) 1.73 (1.70) 0.65 (0.22) 0.04 (0.03) 0.40 (0.61) 1.26 (0.77) 1.42 (0.71)	2.77 (4.64) 2.44 (3.26) 0.06 (0.04) 1.47 (2.67) 1.63 (0.78) 0.60 (0.46) 0.29 (0.23) 0.35 (0.21) 1.56 (1.60) 0.12 (0.06) 2.12 (1.60) 0.62 (0.27) 0.55 (1.40) 0.23 (0.24) 0.18 (0.18) 0.10 1.71 (1.00) 0.41 (0.27) 0.13 (0.32) 1.16 (0.32) 0.74 (0.39)
		DDE SPest/	0.35 (0.29)	0.45 (0.45)	0.68 (1.00)	0.11 (0.01)	0.22 (0.20)	0.33 (0.35)
		DDD SPCB	0.29 (0.18) 0.50 (0.09)	0.28 (0.29)	0.50 (0.72) 0.52 (0.17)	0.29 (0.21) 2.82 (2.79)	0.19 (0.05) 0.51 (0.21)	0.31 (0.19) 1.78 (1.54)
		DDT	3.10 (1.21)	0.94 (1.50)	0.52 (0.74)	0.65 (0.50)	0.77 (0.73)	0.42 (0.54)
		ΣDDT	3.74 (1.31)	1.67 (2.00)	1.71 (1.80)	1.05 (0.31)	1.18 (0.90)	1.06 (0.55)
		Pest/ PCB	1.27 (0.28)	0.53 (0.12)	0.52 (0.14)	0.99 (0.31)	0.47 (0.11)	0.51 (0.10)

Note: CO: coastal sites; IR: intermediate reef sites; BR: barrier reef sites. a only one value.

Acknowledgments

This work was funded by the Fondation Total and the Ecole Doctorale du Pacifique (ED 469) through a PhD grant to MB. We thank the DENV (Province Sud) and E. Potut (Scaphca) for logistic support during the sampling. We are grateful to the many volunteers who participated in the fieldwork. We also thank C. Pigot and C. Goiran (Université de la Nouvelle-Calédonie) for their contribution in field and laboratory work. The study was carried out under permit 930-2011/ARR/DENV (28 March 2001) issued by the DENV, Province Sud, New Caledonia.

2.1 ± 1.7 (<0.1 -7.3) 2.9 ± 2.3 (<0.1 -8.9) Endrin 0.3 ± 0.8 (<0.1 -4.5) 0.8 ± 2.0 (0.07 -9.9) Heptachlor 0.4 ± 0.4 (<0.1 -1.9) 0.5 ± 0.4 (<0.1 -1.9) Heptachlor-epox.A 1.5 ± 1.2 (0.03 -5.6)

1.8 ± 1.9 (0.1 -9.1) Heptachlor-epox.B 0.3 ± 0.5 (<0.01 -2.6) 0.5 ± 0.6 (<0.01 -2.7) Lindane 1.3 ± 0.6 (0.3 -3.1) 1.7 ± 1.2 (0.2 -5.1)

DDT

1.6 ± 1.5 (0.3 -6.9) 1.6 ± 1.5 (0.3 -7.2)


Fabiola Camargo

Gaëlle Lucas-Leclin

Paul Dumont

Patrick Georges

Jean-Marie Danet

David Holleville

Stéphane Guerandel

Grégoire Pillet

Ghaya Baili

Loïc Morvan

Daniel Dolfi

Iryna Gozhyk

Grégoire Beaudoin

Isabelle Sagnes

LASE'14 -VECSELs IV [8966-19]

Keywords: Vertical External Cavity Surface-Emitting Lasers, Atomic Clocks, Metrology, Microwave Photonics

Coherent population trapping (CPT) is an interesting technique for the development of compact atomic frequency references. We describe an innovating laser source for the production of the two cross-polarized coherent laser fields which are necessary in CPT-based atomic clocks. It relies on the dual-frequency and dual-polarization operation of an optically-pumped vertical external-cavity semiconductor laser. This particular laser emission is induced by intracavity birefringent components which produce a controllable phase anisotropy within the laser cavity and force emission on two cross-polarized longitudinal modes. The laser emission is tuned at the Cs D 2 line (λ = 852.14 nm), and the frequency difference ∆ν between the two laser modes is tunable in the microwave range. The laser line wavelength is stabilized onto an atomic hyperfine transition, and concurrently the frequency difference is locked to an ultra-low noise RF oscillator at 9.2 GHz. The high spectral purity of the optically-carried microwave signal resulting from the beatnote of the two cross-polarized laser lines is assessed through its narrow spectral linewidth (<30 Hz) as well as its low phase noise (≤ -100 dBrad 2 /Hz). The performance of this laser source is already adequate for the interrogation of atoms in a CPT atomic clock, and should result in an estimated relative stability of 3.10 -13 τ -1/2 -one order of magnitude better than commercial atomic clocks.

INTRODUCTION

Microwave photonics is an emerging field which studies the generation, distribution, processing and analysis of microwave signals carried on optical frequency carriers, using the advantages of photonics technologies [START_REF] Yao | A Tutorial on Microwave Photonics[END_REF][START_REF] Marpaung | Integrated microwave photonics[END_REF]. It enables access to high-frequency signals generation and processing, without the state-of-the-art electronics and also to long range transmission of microwave signals through optical fiber links. Firstly developed for defense applications, microwave photonics spreads to a large number of civil applications such as cellular, wireless, and satellite communications and medical imaging. It is also used in metrology, in the development of frequency standards and inertial captors.

Here we are interested in the realization of compact and stable atomic clocks using the coherent population trapping (CPT) phenomenon in which the radiofrequency (RF) probe frequency is optically carried [START_REF] Vanier | Atomic clocks based on coherent population trapping: a review[END_REF][START_REF] Knappe | A microfabricated atomic clock[END_REF]. More precisely, in a CPT-based atomic clock, the microwave interrogation that delivers the atomic reference signal is induced by two phasecoherent laser fields at resonance with a common excited state. When the frequency difference between the two laser fields corresponds exactly to the ground-state hyperfine splitting (equal to 9.192 631 770 GHz with Cs atoms), the atoms are trapped in a dark state and stop interacting with the laser light. The resulting narrow transmission line in the atomic absorption spectrum is used as a frequency reference. In order to guarantee the stability of the clock frequency, CPT atomic clocks require a high purity, optically carried, microwave signal as well as low-noise properties for the laser source (intensity and frequency fluctuations). Among the different optical configurations which influence the CPT signal, those combining cross-polarized excitation scheme and temporal Ramsey-like pulsed interrogation have demonstrated highly contrasted and narrow-linewidth CPT resonance [START_REF] Zanon | High contrast Ramsey fringes with coherent-population-trapping pulses in a double lambda atomic system[END_REF]. Such configuration was recently proven to reach short-term frequency stability of 3.2 × 10-13 !!/! [START_REF] Danet | Dick effect in a pulsed atomic clock using coherent population trapping[END_REF].

The common solution for the production of the coherent pair of laser beams in such a polarization configuration is the optical phase-locking of two independent lasers. However, as miniaturization is a challenge for such clocks, a simplified optical bench using a single laser source would be a serious improvement for CPT clocks. Recently, the dual-frequency emission of a solid-state laser has been demonstrated in the GHz range with a Er:Yb:Glass laser [START_REF] Pillet | Dual-Frequency Laser at 1.5 µm for Optical Distribution and Generation of High-Purity Microwave Signals[END_REF] and a Nd:YAG laser [START_REF] Le Gouët | Dual-frequency single-axis laser using a lead lanthanum zirconate tantalate (PLZT) birefringent etalon for millimeter wave generation: beyond the standard limit of tunability[END_REF], both based on the simultaneous emission of two cross-polarized longitudinal modes inside the same laser cavity. However, the main limitation of these lasers is their strong intensity noise inherent to their class-B dynamical behavior. In order to overcome this limitation, dual-frequency emission has been demonstrated in a class A optically pumped vertical-external-cavity semiconductor laser (OP-VECSEL) which does not suffer from relaxation oscillation since the photon lifetime inside the cavity is much longer than the excited carriers lifetime. This configuration has been demonstrated at 1 µm [START_REF] Baili | Experimental demonstration of a tunable dual-frequency semiconductor laser free of relaxation oscillations[END_REF] and 852 nm [START_REF] Camargo | Coherent Dual-Frequency Emission of a Vertical External-Cavity Semiconductor Laser at the Cesium D2 Line[END_REF].

DESIGN AND OPERATION OF THE DUAL-FREQUENCY AND DUAL-POLARIZATION OP-VECSEL

The optically-pumped VECSEL design enables the introduction of free-space intracavity elements to force the dualfrequency and dual-polarization emission of the cavity, while using specially designed semiconductor structure emitting at the targeted wavelength [START_REF] Okhotnikov | Semiconductor Disk Lasers: Physics and Technology[END_REF].

2.1.

Semiconductor structure The semiconductor chip is a conventional multilayer active structure, grown with metal-organic chemical-vapor deposition on a 350 µm-thick GaAs substrate. A distributed Bragg reflector (DBR), composed of 32.5 pairs of λ/4-thick AlAs/Al 0.225 Ga 0.775 As layers, provides a 80-nm large high reflectivity centered at 850 nm, with > 99.94% at 852 nm. The 30 λ/4-thick active structure is composed of seven 8 nm-thick GaAs quantum wells (QW) embedded in pumpabsorbing Al 0.225 Ga 0.775 As barriers; the QW are positioned at the antinodes of the optical standing wave following a 11110101001000 sequence (starting at the air surface, see Figure 1) resulting in almost identical pumping for each QW. The pump absorption is 90 % of the internal pump power in a single pass. Two 30-nm thick Al 0.39 Ga 0.61 As layers form potential barriers on each side of the active cavity, for carrier confinement. Finally, a 50 nm-thick InGaP capping layer protects the cavity from Al-oxidation. The design of this structure, without anti-reflection coating, results in an enhancement of the modal gain by a factor of 3 thanks to constructive interferences inside the semiconductor sub-cavity.

2.2.

Laser cavity The laser cavity is composed of the semiconductor chip and a = 15 mm concave output coupler, transmitting 0.5 % at 850 nm. The pump source is a broad-area laser diode emitting at 670 nm, coupled into a 100 µm diameter, = 0.22, multimode fiber. This source delivers up to 1 W at the fiber end. The pump beam is focused on the semiconductor chip with two doublets ( ! = 25 mm, ! = 19 mm) under a 50° angle, yielding a 70 µm by 110 µm-elliptical spot on the semiconductor structure. About 30% of the incident pump power is reflected onto the surface. The dual-polarization emission is obtained by introducing a birefringent element in the laser cavity, which raises the degeneracy between polarizations and thus results in the stable oscillation of two cross-polarized modes. Here we use a 500 µm-thick antireflection coated YVO 4 plate cut at 45° to its optical axis, which induces a spatial separation of 50 µm between the extraordinary and ordinary beams in the structure along the longer axis of the pump ellipse (Figure 2). This spatial separation reduces the coupling between the two polarizations which ensures the stable dualpolarization, dual-frequency emission [START_REF] Pal | Measurement of the coupling constant in a two-frequency VECSEL[END_REF]; nevertheless the spatial separation is small enough to maintain a good overlap between the pump beam and the two laser spots. Finally, the single-frequency operation at each polarization, as well as the wavelength coarse tunability, are obtained using a 50 µm-thick uncoated silica etalon with a free spectral range !"#$%& = 2 THz. Fine tuning of the laser wavelength is obtained using a piezo-transducer glued on the output coupler. To control the laser frequency difference, a 5 % MgO-doped stoichiometric lithium tantalate (SLT) crystal is placed within the cavity. Its high transparency at 852 nm minimizes additional intra-cavity losses. The crystal axes are oriented to limit the ordinary-polarized mode frequency dependency with the SLT applied voltage. With all intra-cavity elements, the FSR of the laser cavity is !"# = 12 GHz, higher than the targeted frequency difference ∆ !" = 9.192 GHz, and corresponding to an optical cavity length !"# = 12.5 mm. The laser cavity design has focused on compactness as well as high mechanical and thermal stability. The pump optics, the semiconductor chip and the intra-cavity elements are integrated in a compact 90 mm × 90 mm × 40 mm casing (see Figure 2). This limits mechanical and acoustic vibrations as well as air temperature fluctuations inside the external cavity. The temperature of the whole set-up is stabilized to 24°C. The semiconductor chip temperature is maintained at 16°C using a Peltier element, and the SLT crystal and YVO 4 plate are fixed on the same temperature-controlled copper mount.

2.3.

Frequency difference tunability The birefringence introduced inside the cavity generates two combs of cross-polarized longitudinal modes. For two adjacent cross-polarized oscillating modes (i.e. with a frequency difference below !"# ), the laser beatnote frequency, defined as the frequency difference ∆ = | !"# -!"# |, is given by the formula:

∆ν = FSR !"# !∆! ! (1)
where ∆ = !"# !"# -!"# !"# corresponds to the optical path difference due to the birefringence inside the cavity, and is defined modulo /2. Thus ∆ is tunable over one FSR of the cavity. The tunability of ∆ is calculated from the contributions of both YVO 4 and SLT crystals, taking into account three main effects: thermal expansion, thermo-optic and electro-optic effects. The birefringence variation of SLT with the applied transverse voltage follows the relationship:

∆n !"# !" V = ! ! ! ! ! ! r !! - ! ! ! ! r !" (2)
where = 2 mm is the distance between the two electrodes used to apply a voltage on the crystal, ! and ! are the refraction indexes for the extraordinary and ordinary polarizations, !! and !" are the electro-optic coefficients along the axes x and z, respectively (x is the axis on which the voltage is applied and z is along the beam propagation). The contribution of thermal changes on birefringence can be estimated with the following relationship:

!(∆!) !! = !! !! n ! -n ! + L !! ! !! - !! ! !! (3)

MgO-doped SLT material parameters

Refractive index at 852 nm n o = 2.1441 n e = 2.1418

Thermo-optic coefficients (K -1 ) a 0 = 5.586 × 10 -5 a 1 = 0.92168 × 10 -8 a 0 = 1.8017 ×10 -5 a 1 = 6.1500 ×10 -8 Thermal expansion coefficient (K -1 ) ! = 1.5 x 10 -5 unknown Electro-optic coefficient (pm/V) r 13 = 6.96 r 33 = 29.6

From material parameters values in Tables 1 &2, we compute the sensitivity of the frequency difference to temperature and to EO applied voltage of, respectively, 1.41 GHz/K at 25°C and 1.56 MHz/V. Thus the crystal temperature allows a coarse tunability of the frequency difference to the targeted value of 9.192 GHz.

Laser characterization

Without intra-cavity elements, the laser threshold is obtained at 0.25 W of incident pump power. The laser output power reaches 83 mW for the maximal pump power limited by thermal effects in the semiconductor structure; the laser emission exhibits then a multimode spectrum centered at 855 nm. With the Fabry-Perot etalon, the laser operates in single mode emission with a wavelength tunability of almost 5 nm.

With all intra-cavity elements, the laser threshold increases to 0.35 W and the laser output power decreases to 26 mW at 852.1 nm -corresponding to 13 mW per polarization. The fine tunability of the cross-polarized laser lines is achieved with the rotation of the Fabry-Perot etalon and the adjustment of the cavity length. Proper adjustments of the cavity and pump alignments allows to force the laser emission on two adjacent longitudinal modes on orthogonal polarizations, with a frequency difference ∆ below the free-spectral range of the cavity. The beatnote frequency is evidenced by mixing the cross-polarized modes with a polarizer oriented at 45° of their axes, and coupling the beam into a fiber-coupled fast photodiode followed by two low-noise RF amplifiers (Gain = 22 dB each) and a RF amplifier. The generated beatnote frequency is around 9.2 GHz with a spectral linewidth at -3 dB below 600 kHz on a time scale of 300 ms, measured with a resolution bandwidth of 100 kHz (Figure . 3). The tunability of the frequency difference is 1.4 GHz/K with the temperature of the SLT and YVO 4 crystals, and 1.3 MHz/V with the voltage applied to the SLT crystal -in good agreement with our numerical estimation in §2.3. 3. LASER STABILIZATION Under free-running conditions, the laser performance is not compatible with the interrogation of Cs atoms in a CPTbased atomic clock, which requires a high-purity optically-carried microwave signal. Thus the laser emission has to be further stabilized. In this section we detail the stabilization of the ordinary-polarized laser line onto a hyperfine Cs atomic transition, and the concurrent locking of the frequency difference onto a RF oscillator.

At the output of the laser, the two cross-polarized modes are separated by a polarization beam splitter (see Figure 4). The laser is protected from any optical feedback with two -30 dB optical Faraday isolators (one per polarization). A fraction (0.5 mW) of the ordinary-polarized power is injected into a saturated absorption set-up for its stabilization on a Cs atomic transition at 852.1 nm. Finally, both beams are recombined with the same polarization axis in order to measure the beatnote frequency and lock it on the RF frequency reference.

Optical frequency stabilization at Cs transition

The laser frequency of the ordinary polarized beam is locked onto a Doppler-free transition (F = 4 towards F' = 4'/5') of the Cs D 2 line using a pump-probe saturated absorption technique, as shown in Figure 4. An acousto-optic crystal is used to modulate the so-called pump beam at 100 kHz to generate an error signal ! . This method avoids the direct modulation of the pump diode current, which would simultaneously introduce a modulation of the laser power. The error signal is null at the top of each atomic transition, and its slope is 12 MHz/V (Figure 5). It is integrated and the correction is made by a low-frequency two-integration stages servo loop on the piezo-electric transducer glued to the output coupler.

Frequency difference stabilization

The frequency difference between the two laser modes (the so-called beatnote frequency) is locked onto a RF synthesizer, which generates a stable RF reference at the frequency !" , using an optical phase-lock loop described in this section. After recombining the beams and superimposed their polarization axes, the two laser frequencies are mixed on a fast GaAs photodiode (Figure 4). The subsequent beatnote signal is :

s !"#$%&$" t = A sin 2π ν ! -ν ! t + ϕ ! + ϕ !" t (4)
with ϕ 0 a constant phase term and ϕ !" t the residual phase-noise fluctuations.

The beatnote signal is then amplified and mixed with the RF reference to generate an error signal ! proportional to (Δ -!" ). The error signal is then amplified by a high-bandwidth proportional-integrator corrector and applied to the intracavity electro-optic crystal to compensate for the frequency-difference fluctuations. Figure 3 compares the beatnote spectra of the free-running laser and of the laser when the frequency difference Δ = ν ! -ν ! is locked to the RF reference at 9.2 GHz. The linewidth of the beatnote spectrum is dramatically narrowed, below the RF analyzer resolution of 30 kHz, which evidences correction of the uncorrelated frequency fluctuations. The servo loop bandwidth is about 1 MHz, which depends on the proportional gain as well as on the electro-optic crystal response. The signal-to-noise ratio, measured between maximum of the peak and the minimum close the frequency carrier, is 55 dB.

The two servo-loops -of the absolute frequency difference on a Cs atomic transition and of the frequency difference on the RF local oscillator -operate together for 30 minutes under laboratory conditions.

LASER NOISE AND DYNAMICS

The noise properties of the stabilized laser emission are investigated in order to evaluate the contribution of the dual frequency OP-VECSEL to the performance of a CPT-Ramsey atomic clock. We study three main noise sources: the laser frequency noise, the beatnote phase noise and the laser intensity noise.

Laser frequency noise

The laser frequency noise reveals the fluctuations of the absolute optical frequency as compared to the Cs transition. It affects the atomic clock by reducing the interaction between Cs atoms and the laser beams. To quantify this frequency noise, we measure the power spectral density of the error signal ! with a Fast Fourier Transform analyzer, and normalized it by the sensitivity coefficient of the set-up (12 MHz/V). When the optical frequency locking is activated, we observed a (in-loop) noise level below the detection noise floor, meaning we reach a 60 dB frequency noise reduction at 1 Hz. The low-frequency technical noise is suppressed on a bandwidth of 300 Hz, which is limited by the piezo transducer resonance (Figure 6).

Beatnote phase noise

The local oscillator (LO) which provides the RF interrogation signal is a key element an atomic frequency reference.

In CPT atomic clocks, the LO frequency is optically carried for the atom interrogation process. Conversion from electrical to optical domain is obtained by locking the laser frequency difference ∆ to the LO frequency. This conversion may add some noise, originating from the RF synthesizer (LO), the phase-lock loop and the laser itself. To characterize the additive phase noise ϕ !" t induced on the beatnote signal by the laser and the phase-lock loop, we measure the beatnote signal in an independent setup (photodiode + gain) and compare it to the RF reference (Figure 7): then the LO noise is rejected as a common contribution in the phase noise measurement. Figure 8 shows the additive phase noise under free running operation and locked operation (for a frequency difference ∆ locked at the reference frequency of 9.6 GHz). Under free-running operation, we believe that the phase noise results mainly from the partially uncorrelated pump-induced thermal fluctuations of the semiconductor chip on each laser beam. With the phase-lock loop, the phase noise level is -105 dBrad²/Hz, below the LO standard phase noise, leading to a 100 dB noise reduction of the beatnote phase noise at 100 Hz, compared to the free running operation. At frequencies above 5 kHz, the noise level reaches -90 dBrad²/Hz up to 1 MHz which corresponds to the optical phase-lock loop bandwidth.

Laser intensity noise

The relative intensity noise (RIN), which is converted into a frequency noise in the phase-lock loop, has been the object of a specific study. A white noise floor of -115 dB/Hz for each polarization has been measured directly at the laser output. Polarization resolved measurements show no difference of the RIN behaviour between the two modes. Moreover, the two frequency-stabilization servo-loops do not disturb the RIN level (Figure 9). The laser intensity noise is limited by the pump intensity noise with a gain factor of +5 dB consistent with theoretical estimation under our operating conditions [START_REF] Baili | Experimental Investigation and Analytical Modeling of Excess Intensity Noise in Semiconductor Class-A Lasers[END_REF]. We have verified that the RIN of the pump diode is not limited by its injected current noise, and we suspect the mode partition noise resulting from the competition between laser modes to be the main limiting factor of the pump source RIN.

EVALUATION OF CLOCK FREQUENCY STABILITY

In this section, we evaluate the laser noise contribution to the atomic clock performance and therefore the ultimate clock frequency stability reachable with a CPT-Ramsey atomic clock using our dual frequency laser source. The atomic clock set-up is described in Figure 10. The two cross-polarized laser modes, stabilized on Cs transition lines (852 nm and with a frequency difference of ∆ = 9.2 GHz), illuminate a cell full of Cs atoms. An acousto-optic modulator is used for temporal pulse shaping. The clock configuration is detailed in [START_REF] Danet | Dick effect in a pulsed atomic clock using coherent population trapping[END_REF]. The clock sequence of duration ! = 6 ms is composed of a first pumping pulse ! = 2 ms, a free evolution time ! = 4 ms and finally a very short detection pulse ! = 25 µs, during which the transmitted power through the cell is measured with a photodiode. The clock frequency () is defined as the following:

ν t = ν !" 1 + ε + y t (5)
where !" is the ground-state splitting, the inaccuracy caused by deterministic effects, and () the frequency stability which corresponds to the relative frequency fluctuations of the clock signal measured on a duration time . To characterize the frequency stability of the signal (), Allan variance is commonly used and is defined as:

σ ! ! τ = (7)
The major contribution of the laser power fluctuations (IN) is on the amplitude noise of the detected signal () which is converted into an additional clock frequency noise through the LO lock loop. Since the signal () is measured during a time ! = 25 µs, the atomic clock is sensitive to the laser intensity noise on a bandwidth of 1/ ! = 40 kHz. The clock frequency is sensitive to the laser frequency fluctuations (FN) during the pumping time ! of the Cs atoms. A laser frequency variation from the Cs optical transition reduces the interaction between light and atoms. Thus it induces a fluctuation of the number of atoms trapped into the dark-state, converted into amplitude noise on signal (). The calculated sensitivity bandwidth is about 700 Hz under usual operating conditions. Finally, the Dick effect arises from the lack of information on the probe frequency during the pumping of the Cs atoms, equivalent to a dead time during which the LO noise is not probed and then not corrected [START_REF] Dick | Local oscillator induced instabilities in trapped ion frequency standards[END_REF]. It can be described by the down-conversion of the LO frequency noise at Fourier frequencies higher than the interrogation frequency. The LO frequency noise originates from its intrinsic noise and the laser beatnote frequency noise, i.e. the beatnote phase noise evaluated previously. The LO intrinsic noise will not be considered here, as we are only interested here in the noise added by the bifrequency laser source. The contributions to the Allan variance of the clock signal are grouped in Table 3. White noise assumption is made to simplify calculations, at the maximum measured levels. From these calculations, we estimate a clock stability of ! = 10 -12 !!/! , limited by the laser intensity noise. In order to improve the short-term stability of the clock frequency, it would thus be necessary to normalize the CPT signal with the instantaneous laser power, by monitoring the laser beam power in front of the atomic cell. This technique has already been applied to CPT atomic clocks and a RIN contribution reduced at the level of 2×10 -13 !!/! seems realistic [START_REF] Danet | Dick effect in a pulsed atomic clock using coherent population trapping[END_REF]. With a further improvement of the phase-lock loop to reduce the phase-noise level on the whole servo-loop bandwidth, a short-term stability of the clock frequency in the range of 3 × 10 - 13 !!/! is a reasonable target.

CONCLUSION

The dual-frequency emission of an optically-pumped VECSEL has been demonstrated at 852 nm with a frequency difference of 9.2 GHz. Stabilized operation of the laser with both servo-loops activated has been demonstrated. Under this working operation, the laser noise properties have been carefully investigated. Numerical evaluations based on measurements of the frequency noise, the intensity noise and the beatnote phase noise, demonstrate that a clock frequency stability of 2.7 x 10 -12 on 1 s may be achieved, limited by the intensity noise contribution. Assuming that we reduce the laser intensity noise contribution by changing either the clock or the laser set-up, the laser contribution to clock frequency stability would be limited by the Dick effect and could be reduced below 3 x 10 -13 , paving the way for future compact atomic clocks with 1.10 -13 frequency stability level.

Figure 1 .

 1 Figure 1. Description of the semiconductor active chip; (black line, left axis) intensity of the resonant field at λ = 850 nm; (blue line, right axis) refractive index.

Figure 2 .

 2 Figure 2. (left) scheme of the laser setup; (right) photograph of the dual-frequency laser prototype.

!=

  and using the thermal expansion coefficients = and the thermo-optic coefficients ! , ! = ∑ ! ! for the ordinary and extraordinary polarizations.

Figure 3 .

 3 Figure 3. Beatnote frequency spectrum in free-running operation (RBW = 100 kHz) and in lock operation (RBW = 10 kHz), at 9.2 GHz. Noise is rejected 1 MHz away from the carrier frequency.

Figure 4 .

 4 Figure 4. Stabilization set-up; EO: electro-optic crystal, PZT: piezo-electric transducer, PBS: polarization beam splitter, AOM: acousto-optic modulator, OSA: optical spectrum analyser, ESA: electrical spectrum analyzer, FFT : Fast Fourier Transform spectrum analyzer.

Figure 5 .

 5 Figure 5. Saturated absorption spectrum (top) and resulting error signal (bottom)

Figure 6 .

 6 Figure 6. Power spectrum density of the frequency noise of the ordinary polarized mode, under free-running operation and under locked operation.

Figure 7 .

 7 Figure 7. Additive phase noise measurements set-up. LO

Figure 8 .

 8 Figure 8. Power spectrum density of the beatnote phase noise due to the laser contribution under free-running operation and under locked operation (LO frequency: 9.6 GHz) compared to the standard phase noise of the LO.

Figure 9 .

 9 Figure 9. Spectral density of the laser relative intensity noise for each polarization, and of the pump relative intensity noise.

Figure 10 .

 10 Figure 10. Scheme of the CPT atomic clock based on the dual-frequency laser source, with the three servo-loops (of the optical frequency, the frequency difference and the RF reference).

Table 1 .

 1 Material parameters of YVO 4 crystal at 25°C by Ter-Gabrielyan & al.[START_REF] Ter-Gabrielyan | Linear thermal expansion and thermo-optic coefficients of YVO4 crystals the 80-320 K temperature range[END_REF]

YVO 4 material parameters at 25°C

	Refractive index at 852 nm	n o = 1.9702	n e = 2.1811
	Thermo-optic coefficients (K -1 )	a 0 = -3.67 x 10 -6 a 1 = 9.79 x 10 -8 a 2 = -1.13 x 10 -10	a 0 = 8.07 x 10 -7 a 1 = 2,89 x 10 -8
	Thermal expansion coefficient (K -1 )	! = 2.06 x 10 -6	∥ = 8.44 x 10 -6

Table 2 .

 2 Material parameters of SLT crystal doped with MgO at 25°C byDolev & al. [14]

Table 3 :

 3 Calculations of Allan variance for each laser noise contributions × 10 -12 !!/! Beatnote phase noise -90 dBrad²/Hz 2.7 × 10 -13 !!/! Laser frequency noise 10 10 Hz²/Hz 7.7 × 10 -14 !!/!

	Parameter	Maximum Noise Level Allan Variance
	Laser intensity noise	-110 dB/Hz	1.5

ACKNOWLEDGMENTS

Part of this work was funded by the French Agence Nationale de la Recherche [ANR-07-BLAN-0320-03]. The authors gratefully aknowledge support by Triangle de la Physique [2010-089T]; the Délégation Générale de l'Armement; the Labex First-TF.