C Munschy
email: cmunschy@ifremer.fr

N Bely

K Héas-Moisan

N Olivier

C Pollono

S Hollanda

N Bodin

Tissue-specific bioaccumulation of a wide range of legacy and emerging persistent organic contaminants in swordfish (Xiphias gladius) from Seychelles, Western Indian Ocean

Keywords: Persistent Organic Pollutants (POPs), Brominated Flame Retardants, Perfluoroalkyl Substances, Synthetic Musks, Organotropism, Large Pelagic Fish

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

Introduction

Apical marine fish predators represent a major marine resource and play an essential role in marine ecosystems. In addition, they are of high economic value to industrial and artisanal fisheries and provide important nutrients to local populations [START_REF] Sioen | Comparison of the nutritional-toxicological conflict related to seafood consumption in different regions worldwide[END_REF]. However, due to their position at the top of trophic webs, coupled with a long lifespan, they are prone to accumulate high levels of various contaminants, including toxic compounds such as Persistent Organic Pollutants (POPs) and various other persistent hydrophobic contaminants. POPs are defined by their persistent, bioaccumulative, toxic properties and propensity to travel far from their emission sources, hence leading to global distribution [START_REF] Wania | Tracking the distribution of persistent organic pollutants[END_REF]Lohmann et al., 2007). Various classes of organic contaminants, although not listed under the United Nations Stockholm Convention for POPs, share similar properties and are therefore raising concerns with regards to ocean health. The contaminants reported in this study were organochlorines (OCs), including polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), polychlorinated biphenyls (PCBs) and organochlorinated pesticides (OCPs) (namely, dichlorodiphenyl-trichloroethane -DDT and its isomers, hexachlorobenzene -HCB, hexachlorocyclohexanes -HCHs), brominated flame retardants (BFRs) including polybrominated diphenyl ethers (PBDEs), hexabromocyclododecanes (HBCDDs) and various other non-PBDE BFRs, perfluoroalkyl substances (PFASs) including perfluorosulfonates (PFSAs) and perfluorocarboxylic acids (PFCAs), and synthetic musks (SMs). These diverse contaminant families feature various physico-chemical properties and anthropogenic pressures, i.e. industrial, domestic and agricultural usages. Most of them have been phased-out, especially in the Northern

Hemisphere, but their global distribution, in addition to potential contemporary use and secondary emissions, emphasize the need to characterize their occurrence in the marine environment.

Swordfish (Xiphias gladius) is a highly-migratory large pelagic species inhabiting tropical and temperate waters in world oceans [START_REF] Varghese | Biological aspects of swordfish, Xiphias gladius Linnaeus, 1758, caught during tuna longline survey in the Indian Seas[END_REF]. This top predator species is kwown to undertake large diel vertical migrations from the surface to more than 1000 m depth, foraging in deep waters during the day and staying in the mixed layer at night [START_REF] Abascal | Horizontal and vertical movements of swordfish in the Southeast Pacific[END_REF]. Although swordfish are opportunistic predators, their diet in the western Indian Ocean is dominated by mesopelagic fishes and cephalopods [START_REF] Potier | Forage fauna in the diet of three large pelagic fishes (lancetfish, swordfish and yellowfin tuna) in the western equatorial Indian Ocean[END_REF].

The main objectives of this study were i) to assess the state of contamination of the Seychelles marine ecosystem using swordfish as a high bioaccumulating species at the top end of the pelagic food chain, ii) to characterize the bioaccumulation of a wide range of persistent hydrophobic organic contaminants, both legacy and of emerging concern, iii) to determine contaminant sources through levels and profiles iv) to explore the distribution of the studied contaminants in various fish tissues. To the best of our knowledge, the data presented here are among the first obtained for such a high diversity of organic contaminants in swordfish from this part of the world. In particular, some contaminants, such as non-PBDE BFRs and synthetic musks are reported for the first time. As such, the results could serve as a benchmark for future evaluation of the contamination of Indian Ocean ecosystems.

Material and Methods

Sample collection

Ethical approval was not required for this study, as all fish were collected as part of routine professional fishing procedures. A total of 17 fish (9 males and 8 females) were collected between November 2013 and November 2014 offshore of the Seychelles islands (Figure 1), on board a pelagic semi-industrial longline vessel fishing.

All fish were measured (lower jaw fork length -LJFL-in cm), sexed and further processed at sea: whole liver, whole gonads and 5 cm 3 squares of front dorsal (FD) muscle were collected from each specimen, stored in aluminium foil taking all necessary precautions to avoid external contamination and kept frozen on board until landing (for a maximum of 10 days).

On selected individuals (n = 5 males and 3 females), additional muscle samples were collected from back dorsal (BD), middle dorsal (MD) and middle ventral (MV) parts of the fish and analysed for contaminants (Table 3). Upon unloading at Victoria Fishing Port (Mahe, Seychelles), vessel logbook information (sampling dates and coordinates) were retrieved and the samples were rapidly transferred to the Seychelles Fishing Authority Research Laboratory for further processing: a sub-sample was collected from the central part of each liver (L), gonad (G) and muscle (FD, BD, MD, MV) sample and stored frozen in amber glassware prior to freeze-drying. The freeze-dried samples were transported to the IFREMER Laboratory of Biogeochemistry of Organic Contaminants (Nantes, France), homogenized using a blender and finely ground with a ball mill MM400 (Retsch) prior to organic contaminant and total lipid content analysis.

Total lipid content (TLC) analysis

An aliquot of each freeze-dried sample (0.5 g dw) used for organic contaminant analysis was extracted with a mixture of hexane / acetone (80:20) at 100 °C under 100 bars using pressurized liquid extraction (PLE) with a Dionex accelerated solvent extractor (ASE 200,Dionex Corp.,USA). The extracts were evaporated to dryness at 105°C for 12 hr to determine total lipid content (TLC) expressed in % of wet weight (ww).

Contaminant analysis

Detailed analytical procedures for PCDD/Fs, PCBs-OCPs, BFRs and PFASs can be found in [START_REF] Munschy | Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) in marine mussels from French coasts: Levels, patterns and temporal trends from 1981 to 2005[END_REF], [START_REF] Munschy | Persistent organic pollutants in albacore tuna (Thunnus alalunga) from Reunion Island (Southwest Indian Ocean) and South Africa in relation to biological and trophic characteristics[END_REF], [START_REF] Munschy | Classic and novel brominated flame retardants (BFRs) in common sole (Solea solea L.) from main nursery zones along the French coasts[END_REF]2015) and Munschy et al. (2019), respectively. The analyses were processed using gas chromatography coupled to high resolution mass spectrometry (GC-HRMS), ultra performance liquid chromatograph (UPLC)-MSMS.

Synthetic musk analysis was described in [START_REF] Tixier | Occurrence of synthetic musks in marine shellfish along French coasts[END_REF] for musk xylene (2,4,6-trinitro-1,3-dimethyl-5-tert-butylbenzene -MX) and musk ketone (4'-tert-butyl-2',6'-dimethyl-3',5'dinitroacetopheneone -MK). HHCB (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-(γ)-2-benzopyran -Galaxolide) and 1,3,4,4,hexamethyl-1,2,3,4-tetrahydronaphthalene -Tonalide) were analysed using an Agilent 7890 Table 1 summarizes the general analytical procedures used for the various contaminant classes. PCDD/Fs, PCBs, OCPs and BFRs were analysed in all samples (n = 74, see Table 3), while HBCDDs and SMs were analysed in a limited number of samples (n = 6 male and 4 female muscle, n = 5 male and 3 female gonads and n = 5 male and 3 female liver for HBCDDs and n = 5 male and 5 female muscles for SMs). Contaminant concentrations were calculated in pg g -1 wet weight (ww), with a mean humidity percentage of 75% (mean value calculated on all samples). Concentrations were converted into ng g -1 lipid weight (lw) using the total lipid content determined in each sample.

QA/QC

In order to minimize external and cross-contaminations, all samples were processed in the laboratory in a clean, low-dust atmosphere and under positive pressure, and windows were UV-protected. All chemicals were carefully selected to satisfy trace analysis requirements.

Glassware was oven-baked at 450°C for 8 hr.

QA / QC procedures were followed during each sequence analysis; they included the analysis of in-house quality control samples, procedural blanks, quantifications using external calibration (5 to 6 calibration levels), addition of labelled compounds before extraction, used to calculate recoveries, and participation in interlaboratory comparison tests for the marine environment. Detailed information on QA/QC performances can be found in the supplementary material.

Statistical analysis

Statistical analyses were performed using StatSoft Statistica software v 7.1. Correlations (e.g. between total lipid content, fish size / age and POP concentrations) were tested using simple linear regression coefficients. In view of the small sample size per group, data comparisons (biological parameters, POP concentrations and ratios) across fish groups were performed using non-parametric tests (Mann-Whitney test for comparison of two independent samples, or one-way ANOVA Kruskal-Wallis's test), with a significance level (α) of 0.05. Results were considered to be significant only when both tests gave significant results.

Results and discussion

Biometric parameters

The analysed samples were chosen to cover a broad range of swordfish sizes: female and male fish analysed for organic contaminants were 124-203 and 110-204 cm LJFL, respectively (Table 2). Swordfish age was estimated using growth curves from [START_REF] Farley | Determination of swordfish growth and maturity relevant to the southwest Pacific stock[END_REF]. Calculations gave estimated ages ranging from 2 to 20 years in males and between 2 and 9 years in females.

Fish sizes and ages showed no significant differences between males and females, although female swordfish are reported to grow faster and to longer lengths than males [START_REF] Varghese | Biological aspects of swordfish, Xiphias gladius Linnaeus, 1758, caught during tuna longline survey in the Indian Seas[END_REF]. The biometric characteristics were in the range of those collected in 1998-2001 in the western Indian Ocean (around Reunion Island) [START_REF] Poisson | Reproductive dynamics of swordfish (Xiphias gladius) in the southwestern Indian Ocean (Reunion Island). Part 1: oocyte development, sexual maturity and spawning[END_REF]. Most swordfish were collected during the reproduction season identified in the western Indian Ocean, i.e. from October to April [START_REF] Poisson | Reproductive dynamics of swordfish (Xiphias gladius) in the southwestern Indian Ocean (Reunion Island). Part 1: oocyte development, sexual maturity and spawning[END_REF]; only two females were collected during the resting season.

Total lipid content

TLC values in all tissues of fish of both sexes showed high variability, with mean values ranging from 4.0 ± 2.6 (gonads) and 7.9 ± 5.4 (muscles) % ww in females and from 3.7 ± 2.1 (gonads) to 7.7 ± 5.4 (muscles) % ww in males (Table 3), i.e. similar to the values reported in swordfish specimens worldwide (Kannan et al., 2002a;[START_REF] Stefanelli | Organochlorine compounds in tissues of swordfish (Xiphias gladius) from Mediterranean Sea and Azores islands[END_REF][START_REF] Corsolini | Polybrominated diphenyl ethers, perfluorinated compounds and chlorinated pesticides in swordfish (Xiphias gladius) from the Mediterranean Sea[END_REF]. No significant differences in TLC were found in males and females in any tissue. TLC was not significantly different in the various muscle parts; gonads showed significantly lower TLC than liver and muscle.

Major contaminant levels and biological parameters

Concentrations of the major studied classes of contaminants are presented in Table 3.

Globally, in all swordfish, contaminants ranked in the order OCPs > PCBs > PFASs > PBDEs in muscle (Figure 2), which reflects a higher exposure of swordfish to OCPs in this oceanic region. Organochlorinated and organobrominated contaminants covariated strongly with each other, but not with PFASs, highlighting the different sources and bioaccumulation behaviours of these contaminant families. Different contaminant distributions were observed in gonads and liver, where PFASs and OCPs showed the highest levels (Figure 2). Lipophilic contaminants such as PCBs and OCPs showed similar distribution to TLC, to which they were significantly correlated (p < 0.001 for PCBs and OCPs). As a result, when normalized to TLC, the differences in PCB and OCP concentrations across tissues were not significant.

PBDE concentrations expressed in ww did not show any significant differences across tissues, although TLC and PBDE concentrations were positively correlated (p < 0.01).

Conversely, PFOS and PFCAs were significantly higher in liver and gonads than muscle.

PFASs have been previously shown to partition preferentially to phospholipids and proteinrich tissues such as gonads and liver in various fish species [START_REF] Jürgens | Bioconcentration and tissue distribution of perfluorinated acids in rainbow trout (Oncorhynchus mykiss)[END_REF][START_REF] Zafeiraki | Occurrence and tissue distribution of perfluoroalkyl substances (PFASs) in sharks and rays from the eastern Mediterranean Sea[END_REF]. Liver to muscle and gonads to muscle concentration ratios (on a ww basis) were 21 ± 8 and 12 ± 6, respectively, for PFASs, 1.4 ± 0.6 and 0.6 ± 0.1 for PCBs and 2.1 ± 2.0 and 0.5 ± 0.1 for OCPs. These ratios calculated with lipid-normalized concentrations were 1.0 ± 0.3 and 0.9 ± 0.3, respectively, for PCBs and 1.0 ± 0.3 and 0.8 ± 0.3 for OCPs.

The various muscle parts (FD, BD, MD, MV) did not show major differences in contaminant concentrations (in ww or in lw), hence highlighting the homogeneity in contaminant distribution in the studied muscle parts and suggesting that contaminant transfer in swordfish muscle is homogeneous. This could have a practical outcome for the design of future tissue sampling strategies, enabling sampling of any muscle part without affecting organic contaminant bioaccumulation results.

Although globally (all contaminant families considered together), a tendency towards higher contamination levels in male tissues was observed, due to high inter-individual variability in concentrations, the difference was significant for DDTs in the liver of males only (in ww and lw). As male and female individuals have similar feeding habits, supported by similar carbon and nitrogen stable isotope signatures in both sexes (δ 13 C: -16.3 ± 0.4 ‰ in males versus -16.5 ± 0.3 ‰ in females; δ 15 N: 14.2 ± 0.6 ‰ in males versus 14.0 ± 0.4 ‰ in females, as determined in the muscle of the same individuals; [START_REF] Munschy | Legacy and emerging organic contaminants: Levels and profiles in top predator fish from the western Indian Ocean in relation to their trophic ecology[END_REF], the lack of differences in contaminant burden between sexes suggests that female swordfish spawning did not lead to significant decontamination over time in the studied individuals. Similarly, some authors have suggested that spawning does not lead to a significant drop in contaminant concentrations in female fish in response to growth rate and other life history characteristics (Madenjian et al., 2010;[START_REF] Ng | Tracking bioaccumulation in aquatic organisms: a dynamic model integrating life history characteristics and environmental change[END_REF]. In fact, the importance of contaminant loss through spawning depends upon lipid utilisation and spawning strategies (Larsson et al., 1993;[START_REF] Rypel | Variations in PCB concentrations between genders of six warmwater fish species in Lake Logan Martin, Alabama, USA[END_REF]. In tropical areas such as the south Indian Ocean, swordfish has multiple spawnings with extended spawning periods [START_REF] Poisson | Reproductive dynamics of swordfish (Xiphias gladius) in the southwestern Indian Ocean (Reunion Island). Part 1: oocyte development, sexual maturity and spawning[END_REF]. However, their high and continuous feeding rate may counteract contaminant loss through spawning.

Organochlorinated pollutants levels and profiles

PCDD/Fs

PCDD/Fs were seldom detected in swordfish samples. 2,3,7,8-TCDF and 2,3,4,7,8-PeCDF were the two most-detected congeners (24% of the samples) above the limits of quantification, at levels ranging from 0.02 to 0.14 pg g -1 ww and from 0.03 to 0.19 pg g -1 ww, respectively. 1,2,3,7,8-PeCDF was the second most-detected congener (7% of the samples) at 0.02-0.09 pg g -1 ww. The concentrations of ∑PCDD/Fs expressed in TEQ were, on average, 0.0038 ± 0.020 pg TEQ g -1 ww in muscle (lower bound values, < LOQ = 0), i.e. 93 times below EU regulations for food consumption (EU, 2011). These levels are among the lowest found in marine biota worldwide, including in long-lived predatory fish. Indeed, in swordfish collected from Spanish markets, PCDD/F concentrations were 0.050 pg WHO-TEQ g -1 ww (lower bound) in 2005 [START_REF] Bocio | Concentrations of PCDD/PCDFs and PCBs in fish and seafood from the Catalan (Spain) market: Estimated human intake[END_REF] and 0.079 pg WHO-TEQ g -1 ww (middle bound, < limit of detection -LOD = LOD / 2) in 2012 [START_REF] Perello | Human exposure to PCDD/Fs and PCBs through consumption of fish and seafood in Catalonia (Spain): Temporal trend[END_REF].

PCBs

Among the 6 i-PCBs, CB-180, CB-153 and CB-138 were detected in the majority of samples (in 91%, 88% and 87% of samples respectively). The frequency of detection of CB-101, CB-52 and CB-28 was 81%, 69% and 51% respectively. Regarding dl-PCBs, CB-114 was below the limit of detection in all samples, CB-77 and CB-81 were detected in 47% and 43% of samples respectively, while the other dl-PCBs were detected in 71% (CB-123) to 93% (CB-167) of samples. ∑i-PCB concentrations in male swordfish were between 59 and 1514 pg g -1 ww in muscle (FD, MD, BD and MV muscle samples mixed), between 149 and 592 pg g -1 ww in liver and between 196 and 726 pg g -1 ww in gonads. In females, i-PCB concentrations ranged from 169 to 1007 pg g -1 ww, from 133 to 453 pg g -1 ww and from 38 to 382 pg g -1 ww in muscle, liver and gonads respectively. ∑dl-PCB concentrations were found to be about 5 times lower and strongly correlated to ∑i-PCB concentrations (r = 0.99, all tissues). Mean concentrations of quantified PCBs in muscle, liver and gonads of swordfish analysed in this study are summarized in Table 3. The levels are far lower than those reported in swordfish from the Mediterranean Sea (13-399 ng g -1 ww) (Kannan et al., 2002a;[START_REF] Stefanelli | Organochlorine compounds in tissues of swordfish (Xiphias gladius) from Mediterranean Sea and Azores islands[END_REF][START_REF] Corsolini | Persistent organic pollutants in edible fish: a human and environmental health problem[END_REF][START_REF] Bocio | Concentrations of PCDD/PCDFs and PCBs in fish and seafood from the Catalan (Spain) market: Estimated human intake[END_REF][START_REF] Perello | Human exposure to PCDD/Fs and PCBs through consumption of fish and seafood in Catalonia (Spain): Temporal trend[END_REF] and from the Brazilian coast (6.5 ng g-1 ww) (de Azevedo e [START_REF] De Azevedo E Silva | Polychlorinated biphenyls and DDT in swordfish (Xiphias gladius) and blue shark (Prionace glauca) from Brazilian coast[END_REF]. ∑dl-PCB levels reported in swordfish from the North Atlantic Ocean were 0.28 ng g -1 ww [START_REF] Mezzetta | Concentration of DL-PCBs in fish from market of Parma city (north Italy): Estimated human intake[END_REF], i.e. in the range of those found in our study.

On average, i-PCB profiles were dominated by CB-153 > CB-180 > CB-138 in all tissues and in both sexes. These three highly bioaccumulative and non-metabolizable congeners accounted for 85% of ∑i-PCBs. Among dl-PCBs, CB-118 was predominant (49 ± 3% of ∑dl-PCBs), followed by CB-105, CB-156 and CB-167 (each between 12% and 16%). Non-ortho PCBs (CB-77, -81, -126 and -169) represented 6% of ∑dl-PCBs, with CB-126 and CB-169 being the most prevalent. Similar profiles have already been reported worldwide in marine fish including top predators [START_REF] Ueno | Global pollution monitoring of polychlorinated dibenzo-p-dioxins (PCDDs), furans (PCDFs) and coplanar polychlorinated biphenyls (coplanar PCBs) using skipjack tuna as bioindicator[END_REF][START_REF] Bhavsar | Composition of dioxin-like PCBs in fish: an application for risk assessment[END_REF][START_REF] Corsolini | HCB, p,p '-DDE and PCB ontogenetic transfer and magnification in bluefin tuna (Thunnus thynnus) from the Mediterranean Sea[END_REF][START_REF] Munschy | Persistent organic pollutants in albacore tuna (Thunnus alalunga) from Reunion Island (Southwest Indian Ocean) and South Africa in relation to biological and trophic characteristics[END_REF]. Globally, hexachlorinated congeners were predominant in all tissues, followed by heptachlorinated and pentachlorinated congeners (Figure 3).

OCPs

All DDT isomers were identified at concentrations above the limits of quantification in 100% of the samples. HCB was detected in 91% of the samples, while β-HCH and α-HCH were detected in 49% and 24% of the samples, respectively. γ-HCH was below the limit of quantification in all samples. Concentrations of the main detected OCPs are shown in Table 3. In males, ∑DDT concentration ranges (min-max) were between 196 and 16567 pg g -1 ww in muscle (FD, MD, BD and MV muscle samples mixed), between 1804 and 9146 pg g -1 ww in liver and between 210 and 8852 pg g -1 ww in gonads. In females, ∑DDT concentrations ranged from 313 to 13790 pg g -1 ww in muscle, from 1184 to 4294 pg g -1 ww in liver and from 186 to 4020 pg g -1 ww in gonads. These concentrations are lower than those reported in swordfish from the Mediterranean Sea (38-57 ng g -1 ww range) (Kannan et al., 2002a;[START_REF] Stefanelli | Organochlorine compounds in tissues of swordfish (Xiphias gladius) from Mediterranean Sea and Azores islands[END_REF][START_REF] Corsolini | Polybrominated diphenyl ethers, perfluorinated compounds and chlorinated pesticides in swordfish (Xiphias gladius) from the Mediterranean Sea[END_REF] and in a similar range to those from specimens collected from the Brazilian coast (2.47 ng g -1 ww) (de Azevedo e [START_REF] De Azevedo E Silva | Polychlorinated biphenyls and DDT in swordfish (Xiphias gladius) and blue shark (Prionace glauca) from Brazilian coast[END_REF]. HCB concentrations in males were in the 33-638 pg g -1 ww range, and in the 34-472 pg g -1 ww range in females, i.e. much lower than those reported in specimens collected in 2005 in the Southern Tyrrhenian Sea [START_REF] Corsolini | Polybrominated diphenyl ethers, perfluorinated compounds and chlorinated pesticides in swordfish (Xiphias gladius) from the Mediterranean Sea[END_REF]. β-HCH concentrations were in the 2-90 pg g -1 ww range in males and in the 1-83 pg g -1 ww range in females. All OCP concentrations were cross-correlated, indicating similar sources and behaviour in the analysed fish. Overall, the organochlorine concentrations determined in our study were low in comparison to those reported in other oceans, in particular in the Northern Hemisphere. These results are coherent with previous studies on various top predator fish worldwide [START_REF] Ueno | Global pollution monitoring of PCBs and organochlorine pesticides using skipjack tuna as a bioindicator[END_REF]Nicklish et al., 2017) and reflect higher contaminant inputs in the Northern Hemisphere from industrialized and urban areas [START_REF] Gioia | Factors affecting the occurrence and transport of atmospheric organochlorines in the China Sea and the northern Indian and south east Atlantic Oceans[END_REF][START_REF] Tanabe | Monitoring temporal and spatial trends of legacy and emerging contaminants in marine environment: Results from the environmental specimen bank (es-BANK) of Ehime University, Japan[END_REF].

DDT profiles in swordfish muscle were dominated by p,p'-DDE (71% ± 6% of DDTs), followed by p,p'-DDT (19% ± 5%), p,p'-DDD (5% ± 1%), o,p'-DDT (4% ± 0.5%) and o,p'-DDD (0.9% ± 0.2%). Due to the higher stability of the p,p' metabolites DDE and DDD versus the parent compound p,p'-DDT, DDT isomeric profiles in fish are mainly dominated by p,p'-DDE followed by p,p'-DDD (Binelli and Provini, 2003a;[START_REF] Jürgens | Bioconcentration and tissue distribution of perfluorinated acids in rainbow trout (Oncorhynchus mykiss)[END_REF]. Contamination profiles similar to the one found in our study (i.e. p,p'-DDE > p,p'-DDT > p,p'-DDD) have been reported in fish from Southern China coasts where DDT originates from antifouling paints and dicofol impurities [START_REF] Guo | Levels and bioaccumulation of organochlorine pesticides (OCPs) and polybrominated diphenyl ethers (PBDEs) in fishes from the Pearl River estuary and Daya Bay, South China[END_REF] et al., 2013). The fact that p,p'-DDT, which half-life in fish is 8 months (Binelli and Provini, 2003a), ranked second, suggests continuous inputs of DDT , which is consistent with various publications reporting DDT use in Southern Asia, South-Eastern Africa and India [START_REF] Bogdal | Worldwide distribution of persistent organic pollutants in air, including results of air monitoring by passive air sampling in five continents[END_REF][START_REF] Ali | Organochlorine pesticides (OCPs) in South Asian region: a review[END_REF][START_REF] Bouwman | Chlorinated, brominated and fluorinated organic pollutants in African Penguin eggs: 30 years since the previous assessment[END_REF][START_REF] Van Den Berg | Global trends in the production and use of DDT for control of malaria and other vector-borne diseases[END_REF]. The past and potential contemporary use of DDT in various Southern countries is also reflected by the ∑DDT / ∑PCB concentration ratio of 13.7 ± 3.8 (average value in muscle), which reveals a higher DDT exposure of swordfish from the Seychelles islands versus PCBs. Interestingly, this ratio was far higher than that determined in albacore tuna from another location in the southwestern Indian Ocean (i.e. 3.33 ± 1.39 at Reunion Island, [START_REF] Munschy | Persistent organic pollutants in albacore tuna (Thunnus alalunga) from Reunion Island (Southwest Indian Ocean) and South Africa in relation to biological and trophic characteristics[END_REF], which suggests higher inputs of DDTs in the northern and central western Indian Ocean versus southwestern areas. In addition, higher emissions due to increased volatilization of DDT are more likely to occur in tropical regions than temperate regions. Together, higher usage and higher emissions would explain the higher ∑DDT / ∑PCB concentration ratio found in swordfish inhabiting tropical and subtropical regions of the western Indian Ocean versus albacore tuna inhabiting more temperate waters.

No significant differences in p,p'-DDE contributions were observed across tissues in either sex. Conversely, higher p,p'-DDD and o,p'-DDD contributions and lower p,p'-DDT and o,p'-DDT contributions were found in liver versus gonads and muscle (Figure 3). The ratio of o,p'-DDD + p,p'-DDD / ∑ DDTs was significantly higher in liver followed by gonads and muscle (both genders). It has previously been reported that fish are able to metabolize DDT and that the bioaccumulation of DDT metabolites and biotransformation are tissue-specific (Kwong et al., 2008). The higher contribution of the DDT metabolites p,p'-DDD and o,p'-DDD in liver suggests that DDT degradation occurred preferentially in liver. -28, -47, -49, -66, -100, -154 and -155 were quantified in over 50% of the samples. The detection frequency of the remaining congeners decreased in the order BDE-208 (31%) > BDE-153 (29%) > BDE-99 (22%). ∑ 8 PBDE 153, concentrations in male swordfish ranged from 12 to 242 pg g -1 ww in muscle (FD, MD, BD and MV muscle samples mixed), 24 to 55 pg g -1 ww in liver and 13 to 36 pg g -1 ww in gonads. In females, ∑PBDE concentrations ranged from 14 to 86 pg g -1 ww in muscle, 13 to 124 pg g -1 ww in liver and 14 to 72 pg g -1 ww in gonads. Mean PBDE concentrations are presented in Table 3 and are similar to those reported in swordfish from markets in Valencia, Spain in 2007-2012 [START_REF] Pardo | Probabilistic risk assessment of the exposure to polybrominated diphenyl ethers via fish and seafood consumption in the Region of Valencia (Spain)[END_REF], but lower than those reported in the muscle and liver of male and female individuals collected in 2005 in the Southern Tyrrhenian Sea (Italy) [START_REF] Corsolini | Polybrominated diphenyl ethers, perfluorinated compounds and chlorinated pesticides in swordfish (Xiphias gladius) from the Mediterranean Sea[END_REF], or in swordfish from fish markets in Catalonia, Spain in 2005 [START_REF] Domingo | Exposure to PBDEs and PCDEs associated with the consumption of edible marine species[END_REF]. Concerns have been raised over a possible future increase in BFR inputs in Southern Hemisphere countries such as South Africa due to the absence of restrictions or regulations on imports of BFRs and BFR-containing products [START_REF] Quinn | Brominated flame retardants in wild bird eggs from the industrialised heartland of South Africa[END_REF].

BFR concentrations and profiles

BDE

PBDE profiles in muscle were dominated by BDE-47 (49% ± 14% of the ∑PBDE), followed by BDE-154 (22% ± 7%) and BDE-49 (16% ± 3%). PBDE profiles were significantly different between male and female fish muscles, with higher contributions of BDE-28 and BDE-47 and a lower contribution of BDE-100 in females versus males. BDE-47 contributed to 43% ± 15% and 45% ± 12% of the ∑PBDE in gonads and liver, respectively, i.e. similar to contribution in muscle.

PBDEs undergo metabolism in marine biota, including fish, which metabolise PBDEs via debromination processes in a species-specific way [START_REF] Roberts | Species-specific differences and structure-activity relationships in the debromination of PBDE congeners in three fish species[END_REF]. Although BDE-47 was -as often in marine biota-found to be the predominant PBDE congener in all tissues, BDE-154 was also shown to be a fairly large contributor in swordfish. This congener may originate from the degradation of higher brominated congeners such as BDE-183, as shown in other fish species [START_REF] Roberts | Species-specific differences and structure-activity relationships in the debromination of PBDE congeners in three fish species[END_REF]. BDE-183 was indeed seldom detected (i.e. in only 7 samples in total) and its contribution was low (2% ± 2%), hence supporting the degradation hypothesis. Similarly, BDE-49, which can result from BDE-99 debromination, was found in relatively high proportions in swordfish (17 ± 4%). The BDE-99 / BDE-100 ratio, used as an indicator of fish capacity to metabolize PBDEs [START_REF] Voorspoels | Polybrominated Diphenyl Ethers in marine species from the Belgian North Sea and the Western Scheldt estuary: levels, profiles, and distribution[END_REF], was 0.4 ± 0.1 on average in muscle. This ratio has been shown to decrease with increasing trophic levels [START_REF] Wan | Trophodynamics of polybrominated diphenyl ethers in the marine food web of Bohai Bay, North China[END_REF] and would suggest swordfish are capable of metabolizing PBDEs.

However, determining the PBDE profiles of main swordfish preys would be essential to determine if these profiles originated from metabolism in swordfish or from its prey.

Non-PBDE BFRs were detected in less than 50% of the analysed samples and ranked in the order BB-153 (46%) > HBB (26%) > BTBPE (22%) > DBDPE (18%). Their concentrations ranged from 0.1 to 1.7 pg g -1 ww, 0.2 to 0.8 pg g -1 ww, to 11 pg g -1 ww and 4 to 26 pg g -1 ww, respectively. Average concentrations ranked in the order DBDPE > BTBPE ~ HBB ~ BB-153.

This low occurrence is consistent with low biomagnification capacities, although reported results remain inconclusive for DBDPE and BTBPE [START_REF] Tao | Biomagnification of PBDEs and alternative brominated flame retardants in a predatory fish: Using fatty acid signature as a primer[END_REF]. To our knowledge, these results are the first to be reported for top predator fish from the Indian Ocean.

HBCDDs were analysed in a selection of samples (n = 10 and 16 female and male tissues, respectively). Among the three HBCDD analysed isomers, α-HBCDD was the only one detected, at a frequency of 38%. In female individuals, α-HBCDD was detected at concentrations ranging from 4 to 29 pg g -1 ww in muscle, of 9 pg g -1 ww in liver and below the limits of quantification in gonads. In males, α-HBCDD was detected at 7-17 pg g -1 ww in muscle, 6-13 pg g -1 ww in liver and below the limits of quantification in gonads. In swordfish collected from Italian markets but originating from western Indian Ocean (i.e. FAO zone 51),

HBCDDs were detected in one sample out of two at 16 pg g -1 ww [START_REF] Tavoloni | Single sample preparation for brominated flame retardants in fish and shellfish with dual detection: GC-MS/MS (PBDEs) and LC-MS/MS (HBCDs)[END_REF], i.e.

similar to the levels found in this study.

PFAS concentrations and profiles

Among the analysed PFASs, PFOS, PFDA and PFUnDA were detected in 100% of samples.

PFDoDA and PFTrDA were detected in 91% of samples and PFNA and PFTeDA in 77% and 59% of samples, respectively. The presence of both PFOS and PFCAs in swordfish indicates biomagnification of these compounds at the top of food webs and is consistent with their global distribution in tropical and subtropical oceans [START_REF] Gonzalez-Gaya | Perfluoroalkylated substances in the global tropical and subtropical surface oceans[END_REF]. Table 3 shows the mean concentrations of PFOS and ∑PFCAs in the various tissues of male and female swordfish. PFOS concentrations in all tissues were in the 23-1713 pg g -1 ww range, while remaining PFASs, i.e. long chain PFCAs, were in the 9-4777 pg g -1 ww range. PFOS was identified at < 1 to 8 ng g -1 ww in the liver of swordfish from the Southern Tyrrhenian Sea in 1999 (Kannan et al., 2002b) and was below 1.5 ng g -1 ww in both muscle and liver of male and female individuals collected from the same area in 2005 [START_REF] Corsolini | Polybrominated diphenyl ethers, perfluorinated compounds and chlorinated pesticides in swordfish (Xiphias gladius) from the Mediterranean Sea[END_REF]. In swordfish consumed in France (no reported geographical origin), ∑ PFASs were in the 0.28-1.11 ng g -1 ww range [START_REF] Yamada | Perfluoroalkyl acid contamination and polyunsaturated fatty acid composition of French freshwater and marine fishes[END_REF].

PFCA concentrations were higher than PFOS concentrations in all samples. PFCA / PFOS ratio was significantly higher in gonads (9.0 ± 3.2) than in liver (6.3 ± 2.3) or muscle (6.1 ± 2.5), which highlights a tissue-specific distribution of PFASs. Only long-chain PFASs (C ≥ 8)

were detected in swordfish tissue samples, corresponding to the higher bioaccumulative abilities and biomagnification propensities of PFASs of higher carbon-chain length in trophic webs versus their short-chain counterparts [START_REF] Jürgens | Bioconcentration and tissue distribution of perfluorinated acids in rainbow trout (Oncorhynchus mykiss)[END_REF][START_REF] Conder | Are PFCAs bioaccumulative? A critical review and comparison with regulatory lipophilic compounds[END_REF]). More specifically, PFCA profiles showed a predominance of odd-chain length compounds (PFUnDA and PFTrDA) versus even-chain ones (PFDA and PFDoDA). This has previously been observed in fish and partially explained by the degradation of neutral precursors such as 10:2 perfluorodecanol (fluorotelomer alcohol -FTOH) [START_REF] Ellis | Degradation of fluorotelomer alcohols: A likely atmospheric source of perfluorinated carboxylic acids[END_REF][START_REF] Hart | Skipjack tuna as a bioindicator of contamination by perfluorinated compounds in the oceans[END_REF].

The origin of PFUnDA has also been attributed to the industrial production of fluoropolymers or impurities from the oxidation of fluorotelomer olefins [START_REF] Prevedouros | Sources, fate and transport of perfluorocarboxylates[END_REF][START_REF] Harada | Odd-numbered perfluorocarboxylates predominate over perfluorooctanoic acid in serum samples from Japan, Korea and Vietnam[END_REF].

No differences in PFAS profiles were observed between sexes. PFOS and some PFCAs showed different relative contributions according to tissue: PFOS was more abundant in liver than in gonads, while the opposite was observed for PFTrDA; PFDA and PFUnDA were more abundant in muscle than in gonads (Figure 3). These results demonstrate tissuespecific and compound-specific PFAS distribution, probably reflecting complex interrelated effects according to tissue biochemical composition and PFAS binding affinities [START_REF] Verreault | Perfluorinated alkyl substances in plasma, liver, brain, and eggs of glaucous gulls (Larus hyperboreus) from the Norwegian Arctic[END_REF].

Synthetic musk concentrations and profiles

The contamination of swordfish by synthetic musks (SMs) was investigated in a limited number of muscle samples (n = 5 males and 5 females). Among the four investigated SMs, the two polycyclic musks, HHCB and AHTN were found in the highest concentrations, at 428 ± 221 and 274 ± 238 pg g -1 ww respectively. HHCB was detected in all samples, while AHTN was only detected in four samples. MX and MK were detected in four samples only, at 10 ± 3 and 69 ± 54 pg g -1 ww respectively. The differences observed in nitromusk and aromatic musk contamination levels is consistent with current usages and regulations [START_REF] Rimkus | Polycyclic musk fragrances in the aquatic environment[END_REF]. High variations were observed across individuals and globally, no differences in HHCB or AHTN were observed according to sex. Levels in swordfish from the western Indian Ocean were in the lower ranges of those measured in other marine fish species worldwide [START_REF] Rainieri | Occurrence and toxicity of musks and UV filters in the marine environment[END_REF]. For instance, in hammerhead shark livers collected in 2004 in the Ariake Sea in Japan, only HHCB was detected at 16-48 ng g -1 ww, i.e. 1 or 3 orders of magnitude below those in fish from Canada, the US and Europe [START_REF] Nakata | Occurrence of synthetic musk fragrances in marine mammals and sharks from Japanese coastal waters[END_REF].

Contaminant levels versus human health regulations

Due to POP toxicity and accumulation in seafood, which is one of the major routes of human exposure to these pollutants [START_REF] Törnkvist | PCDD/F, PCB, PBDE, HBCD and chlorinated pesticides in a Swedish market basket from 2005 -Levels and dietary intake estimations[END_REF][START_REF] Shin | Progressive risk assessment of polychlorinated biphenyls through a Total Diet Study in the Korean population[END_REF], maximum POP residues in foodstuffs have been established in various countries worldwide. Globally, contamination levels were several orders of magnitude below regulations. i-PCBs were 26 to 1200 times lower in muscle, and 200 to 1400 times lower in liver, than the maximum levels set by the European Commission for non dl-PCBs in foodstuffs (EU, 2011), i.e. 75 ng g -1 ww and 200 ng g -1 ww in muscle and liver respectively, and below the maximum values set by the governments of Japan (500 ng g -1 ww), Australia (500 ng g -1 ww) and the US (2000 ng g -1 ww) for total PCBs [START_REF] Vizzini | Comparison of stable isotope composition and inorganic and organic contaminant levels in wild and farmed bluefin tuna, Thunnus thynnus, in the Mediterranean Sea[END_REF]. dl-PCB concentrations calculated in toxic equivalents (TEQs) using the toxic equivalent factors (TEFs) set by the World Health Organization in 2005 (Van den [START_REF] Van Den Berg | The 2005 World Health Organization reevaluation of human and mammalian toxic equivalency factors for dioxins and dioxin-like compounds[END_REF] showed average levels of 0.35 ± 0.24 pg TEQ g -1 ww, i.e. below the maximum level set by the European Commission for dl-PCBs in foodstuffs of 3 pg TEQ g -1 ww in muscle (EU, 2011). Non-ortho PCBs and, in particular, CB-126 (74% of dl-PCB TEQs) and CB-169 (24%), contributed the most to TEQ values. They were also below the guideline value of 0.79 pg TEQ g -1 ww set by Canada to protect wildlife consumers of aquatic biota (CCME, 2001). p,p'-DDE maximum levels were below those set by Japan (3000 ng g -1 ww) and Australia (1000 ng g -1 ww) [START_REF] Vizzini | Comparison of stable isotope composition and inorganic and organic contaminant levels in wild and farmed bluefin tuna, Thunnus thynnus, in the Mediterranean Sea[END_REF]. DDT concentrations were below the maximum admissible concentrations set by the EU (Binelli and Provini, 2003b) but slightly exceeded the limit of 14 ng g -1 ww set by Canada (CCME, 1999) in two male muscle samples (at 17.7 and 16.6 ng g -1 ww). Other OCPs were below EU and China maximum limits for lindane and HCH and below the environmental quality standards (EQS) of 10 µg kg -1 set by the European Union (EU, 2013) for HCB to protect human health. PBDEs (sum of 6 congeners = BDE-28, 47, 99, 100, 153 and 154) were above the EQS of 0.0085 µg kg -1 in muscle set to protect human health (7 times higher in muscle on average) in 78% of muscle samples. However, the EQS set by the EU for PBDEs is recognized as being extremely low, leading to environmental concentrations exceeding EQS in most cases [START_REF] Fliedner | Current levels and trends of selected EU Water Framework Directive priority substances in freshwater fish from the German environmental specimen bank[END_REF]. The HBCDD EQS of 167 µg kg -1 ww, although established on whole fish to protect wildlife (secondary poisoning of fish-eating predators), was never exceeded, nor was the PFOS NQS of 9.1 µg kg -1 ww in muscle (human health purpose).

Conclusions

This study provides the first data on the contamination of the top predator swordfish by a wide range of persistent organic contaminants in the Seychelles archipelago, western Indian

Ocean. The results highlight the widespread contamination of southern oceans by persistent organic contaminants, albeit at low levels. Swordfish from the study area were more impacted by DDTs than PCBs, which is consistent with current knowledge on global environmental inputs of these POPs in the Southern Hemisphere. PFASs and, in particular, long-chain PFCAs, were found in relative abundance, especially in gonads and liver.

Further studies on the contamination of top predators and their prey in the Indian Ocean would be required on a larger geographical scale to gain more insight into global ocean contamination in this part of the world and better characterize contaminant biomagnification in tropical food webs.

organochlorine pesticide burden in eels in the lower Thames River (UK -15, -28, -47, -77, -99, -100, -126, -153, -154, -169, -183, -197, -205, -207, -209

  gas chromatograph coupled to a Waters Xevo® TQ-S micro (Waters Corp.) mass spectrometer (GC-MS/MS) using multiple reaction monitoring (MRM). Quantification and confirmation were performed in the multiple reaction monitoring using atmospheric pressure chemical ionization (APCI) mode (HHCB, MRM = 258.1 > 243.1, 258.1 > 213.1; AHTN, MRM = 273.4 > 255.2, 258.1 > 243.1).

Figure 1 :

 1 Figure 1: Map of swordfish fishing positions in the Seychelles, western Indian Ocean in 2013-2014.

Figure 3 .

 3 Figure 3. Contributions (%) of (A) PCBs (grouped per number of chlorine atom), (B) DDTs (5 isomers) and (C) PFASs (PFOS and long-chain PFCAs) in the muscle, gonads and liver of swordfish collected in Seychelles in 2013-2014.PCBsDDTs PFASs

  . Based on the knowledge of technical DDT isomeric

	composition (i.e. 75% of p,p'-DDT, 15% of o,p'-DDT, 5% of p,p'-DDE and 5% of other
	isomers, Zhou et al., 2014), several ratios using DDT isomers are commonly-used to
	investigate DDT origin and ageing. The o,p'-DDT / p,p'-DDT ratio, used to determine the
	origins of DDT, was similar in all samples (0.25 ± 0.14 on average, regardless tissue or sex),
	indicating similar exposure sources for all fish and indicative of technical DDT (Suarez et al.,
	2013). The ratio of (p,p'-DDE + p,p'-DDD) / ∑ DDTs, commonly-used to distinguish new and
	old DDT sources, showed an average value of 0.77 ± 0.06 in muscle, characteristic of
	relatively old DDT inputs (recent DDT inputs would be characterized by a ratio < 0.5, Suarez

  Madenjian, C.P., Schrank, C.S., Begnoche, L.J., Elliott, R.F., Quintal, R.T., 2010. Sexual difference in PCB concentrations of coho salmon (Oncorhynchus kisutch). Science of the TotalEnvironment 408, 1719Environment 408, -1724Environment 408, . doi: 10.1016Environment 408, /j.scitotenv.2009.12.023. .12.023.

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Acknowledgements

The authors would like to thank the Seychelles Fishermen Boat Owners Association (FBOA), especially Mr. Elvis Hoareau and his crew from the MV Albacore for supplying us with swordfish samples throughout, as well as Mr. Beatty Hoareau and Ms. Virginie Lagarde for their support and help in project and sampling design. A special thanks also to the SFA Research technicians, especially Ms. Maria Rose, Ms. Clara Belmont and Ms. Kettyna Gabriel, for their invaluable help in processing the samples. We acknowledge Laura Valentine from "English Assistance for Industry" for the English corrections made to the manuscript.

Funding

This study is part of the CONSWO research project ("Balancing risks with benefits associated with consumption of swordfish") led by the Seychelles Fishing Authority (SFA) in collaboration with the French Research Institute for Sustainable Development (IRD), with the financial support of the European Fisheries Partnership Agreement (EU-FPA).

Contaminants

Freeze-dried sample mass (g dw)


Caroline Fritz
email: caroline.fritz@aphp.fr

Nicolas Viault

; Baptiste Fohlen

Maximilian Edlinger- Stanger

Hanna Mcgregor

Bernard Cholley

Neal Fleming

Percutaneous cannulation for extracorporeal membrane oxygenation (ECMO): a method for pig experimental models Authors

Keywords: Extracorporeal membrane oxygenation, Percutaneous cannulation, Ultrasound-guided cannulation

Pigs are often used for experimental models of cardiogenic shock, cardiac arrest or acute lung injury with veno-arterial (VA) or veno-venous (VV) extracorporeal membrane oxygenation (ECMO) implementation. Percutaneous (as opposed to surgical) cannulation in experimental models has potential advantages, including, less surgical trauma or stressful stimuli and less bleeding complications when compared to open chest cannulation. However, pig anatomy can also be a challenge because of the deep location and angled anatomy of the femoral artery. The Seldinger technique and the use of a percutaneous cannulation kit is feasible in pigs.

Summarized here we present (Graphical Abstract):

• Percutaneous ECMO cannulation method for non-cardiac surgeon.

• Establishment of this simple and rapid methods for pig experimental models • Predictable complications of this method

*Methods details

Principles of ECMO. Extracorporeal membrane oxygenation is widely used in human intensive care to support lung injury (veno-venous (VV) ECMO) or circulatory failure (veno-arterial (VA) ECMO) (1). The global concept is to remove blood from the venous compartment, pass it through an oxygenator membrane and then return the oxygenated blood in to the circulatory system at a defined flow rate. In cases of lung failure without cardiac failure, the oxygenated blood is returned to the venous circulation. In case of heart failure, the oxygenated blood is returned to the arterial circulation. This technique was developed in the early 60's, but its use is now growing, opening new perspectives and new interrogation options for researchers.

Principles of percutaneous cannulation. Percutaneous access using the Seldinger technique is well known in clinical practice for catheter insertion (central venous or arterial) [START_REF] Frykholm | Clinical guidelines on central venous catheterisation. Swedish Society of Anaesthesiology and Intensive Care Medicine[END_REF]. For establishing ECMO, due to the size of the cannula, the Seldinger technique is rarely used (3) and surgical access is still the preferred method (4). The reported complications of this technique are mainly related to the insertion process, and include insertion failure due to a mismatch between vessel size and diameter of the cannula, as well as potential bleeding complications [START_REF] Chhor | Risk factors of percutaneous cannulation failure by intensivists for veno-arterial extracorporeal life support for refractory cardiac arrest[END_REF]. Long term complications include infection, limb ischemia and subsequent sensory-motor complications in humans [START_REF] Pilarczyk | Comparison of a New Miniaturized Extracorporeal Membrane Oxygenation System With Integrated Rotary Blood Pump to a Standard System in a Porcine Model of Acute Lung Injury[END_REF][START_REF] Ostadal | Direct comparison of percutaneous circulatory support systems in specific hemodynamic conditions in a porcine model[END_REF].

Pig specificity. Pigs are often used for experimental models of cardiogenic shock, cardiac arrest or acute lung injury with VA or VV ECMO implementation [START_REF] Fritz | High Versus Low Blood-Pressure Target in Experimental Ischemic Prolonged Cardiac Arrest Treated with Extra Corporeal Life Support[END_REF][START_REF] Luo | Low versus standard-blood-flow reperfusion strategy in a pig model of refractory cardiac arrest resuscitated with Extra Corporeal Membrane Oxygenation[END_REF] . Percutaneous cannulation in experimental models has potential advantages beyond the need for advanced surgical expertise: less stressful surgical stimuli are applied to the animal and less bleeding complications arise. But pig anatomy can also be a challenge because of the deep location and angled anatomy of the femoral artery.

In this protocol we will present the material and the procedure to perform percutaneous femoral cannulation for ECMO in Landras pigs. We will present only VA-ECMO cannulation, but VV-ECMO cannulation follows the same procedure with one jugular and one femoral venous access.

Material and methods.

Ethics. All experiments pictured and described in this article were reviewed and approved by the Paris University Ethics Committee for Animal Experimentation (APAFIS #23056). The procedure for the care and sacrifice of study animals was in accordance with the European Community Standards on the Care and Use of Laboratory Animals.

Animal model and anesthesia. The procedure must be performed under general anesthesia to avoid pain and leg movement. Pigs must be in supine position. Ultrasound. Vascular ultrasound should be used to: 1) guide the percutaneous needle puncture, and 2) control the guide wire position in the vessel.

Transthoracic ultrasound. Cardiac ultrasound can be used to localize the distal tip of the venous cannula in the right atrium or in the inferior vena cava.

Fluoroscopy. Fluoroscopy can also be used to localize the cannulas, mainly for VV ECMO to make sure the distance between cannula tips is great enough to avoid recirculation.

Additional information.

Major complications. Bleeding is the main complication to be concerned about. Because of the angled anatomy of the femoral artery, the vessel dilatation can create an arterial lesion that cannot be fixed without open surgery. To avoid this complication, vessel dilatation and cannulation must also be smooth and careful. Another major and unpredictable complication can be the creation of an intra-abdominal false path. If the cannula (venous or arterial) perforates the vessel wall, it will rapidly induce hemorrhagic shock when the ECMO flow begins, with no chance to save the animal.

Cannula size. The cannula size depends on the animal weight (femoral artery diameter) and on the ECMO flow target.

The larger the cannulae, the lower the resistance to flow.

ECMO anticoagulation. Even if the circuit and cannulae are coated with bioline, a bolus of unfractioned heparin (5000 UI) should be added in the pump priming solution, followed by repeated intravenous heparin injections of 100 UI.kg -1 every hour.

ECMO weaning. The percutaneous cannulation approach is only feasible for non-weaning ECMO protocols. If the animals are to be maintained alive after ECMO removal, a surgical repair of the artery is required.

Training: percutaneous ECMO implantation as described in this method article does not require specific surgical competencies and can be achieved by investigators trained in ultrasound and Seldinger's technique.

Acknowledgements :

We thank Pr David Smadja, Julie Piquet and Alexis Anaes from the Biosurgical Research Lab (Carpentier Foundation) in Paris Cardio Vascular Research Center (PARCC), France for allowing us to use their facility and for their expert technical help. We thank Dr Marine Di Méglio for her ultrasound expertise and teaching.

Declaration of interests:

☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

X The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Additional information:

---- 2 )-

 2 Ultrasound (US) with a 8-12 MHz linear transducer probe for the vessel puncture guidance -Local anesthetic (Lidocaine) -Venous cannula (21 Fr, Biomedicus cannulae, Medtronic, USA) and percutaneous insertion kit (Maquet, Germany) including guide wire, vessel dilator, puncture needle, scalpel, syringe -Arterial cannula (15 Fr or 16 Fr, Biomedicus cannulae, Medtronic, USA) percutaneous insertion kit (Maquet, Germany), including guide wire, vessel dilator, puncture needle, scalpel, syringe -Centrifugal pump (Rotaflow Centrifugal Pump System, Maquet, Germany) Procedure for VA-ECMO femoral cannulation (Figure 1) -Assemble the ECMO -Administer a bolus of intravenous unfractionated heparin (100 UI.kg -1 ) to the animal -Localize the vessels (artery and vein) using US -Inject local anesthesia (Lidocaine) to limit pain in case of femoral nerve stimulation and to provide a vasodilatory effect Puncture the femoral vein with the needle, insert the guide wire (diameter and size adapted to the cannula, minimum length 150 cm) Puncture the ipsi-or contralateral femoral artery with the needle, insert the guide wire (diameter and size adapted to the cannula, minimum length 100cm) Make a 1 cm skin incision around each wire -Start with the venous cannulation first -Dilate the vessel progressively with increasing size dilators -Confirm repeatedly that the wire is not bent by checking the smooth and free motion of the wire into the dilatator -Insert the cannula after surface lubrication with saline -Remove the wire and the introducer/dilator -Clamp immediately clamp to avoid blood loss -Repeat the same procedure of progressive dilation and immediate clamping for the arterial cannula Procedure for cannulae connection to ECMO circuit and for pump start (Figure Connect both cannulae to the ECMO circuit while dripping saline over tubing junction to avoid air bubbles in the circuit. -Start the centrifugal pump while both cannulae are still clamped -Remove the venous clamp first -Then remove the arterial line clamp Methods validation.

Figure 1 :

 1 Figure 1: Femoral percutaneous cannulation. A: ultrasound allows localization of the femoral artery and vein. B: Needle puncture under ultrasound guidance. C: arterial and venous guidewires in place. D: percutaneous insertion kit ready with two long wires. E: Arterial dilatation, venous canula already in place. F: arterial and venous cannulae in place and clamped before ECMO circuit connection.

Figure 2 :

 2 Figure 2: Procedure for cannulae connection to ECMO circuit and pump start. A: cannulae in place. B. Connection of the cannula to the ECMO circuit while dripping saline over the junction to avoid air bubbles in the circuit. C. Cannulae and circuit are connected. D. Starting the centrifugal pump prior to remove the venous clamp. E. Remove the arterial line clamp after the venous clamp. F. Ensure that the two cannulae have different color


Christophe J Dubois
email: christophe.dubois@u-bordeaux.fr

Olivier Pierrefiche

Perinatal exposure to ethanol in rats induces permanent disturbances of breathing and chemosensitivity during adulthood

Keywords: (6 max) Ethanol, perinatal, adult, breathing, hypoventilation, hypoxia. 1 1 SIDS: Sudden Infant Death Syndrome, CPEE: Chronic Perinatal Ethanol Exposure, Rf: Respiratory frequency, VT: Tidal Volume, VE: Minute ventilation

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

Introduction

Consumption of drugs of abuse during pregnancy is associated with a greater incidence of unexplained Sudden Infant Death Syndrome (SIDS) [START_REF] Ward | Sudden infant death syndrome in infants of substance-abusing mothers[END_REF]. In particular, maternal alcohol (ethanol) consumption during pregnancy has been correlated with a higher risk of SIDS [START_REF] Klug | A comparison of the effects of parental risk markers on pre-and perinatal variables in multiple patient cohorts with fetal alcohol syndrome, autism, Tourette syndrome, and sudden infant death syndrome: an enviromic analysis[END_REF][START_REF] O'leary | Maternal alcohol use and sudden infant death syndrome and infant mortality excluding SIDS[END_REF]. However, because of the association of ethanol consumption with consumption of other addictive drugs including nicotine through cigarette smoking, the correlation between ethanol exposure during pregnancy and the occurrence of SIDS remains difficult to establish [START_REF] Kandall | Maternal substance use and subsequent sudden infant death syndrome (SIDS) in offspring[END_REF][START_REF] Lester | The maternal lifestyle study: effects of substance exposure during pregnancy on neurodevelopmental outcome in 1-month-old infants[END_REF].

In order to investigate whether exposure to ethanol during brain development could impinge the respiratory function, our lab developed an animal model of chronic perinatal ethanol exposure (CPEE) that consisted in ethanol exposure of rats throughout the gestation and lactation periods [START_REF] Dubois | Early chronic ethanol exposure in rats disturbs respiratory network activity and increases sensitivity to ethanol[END_REF][START_REF] Naassila | Effect of prenatal and postnatal ethanol exposure on the developmental profile of mRNAs encoding NMDA receptor subunits in rat hippocampus[END_REF]. With this model we have shown that, in vivo CPEE rats exhibited a strong slowdown of breathing activity from early postnatal days [START_REF] Kervern | Perinatal alcohol exposure in rat induces long-term depression of respiration after episodic hypoxia[END_REF] to the juvenile state, just before weaning and thus before ethanol withdrawal [START_REF] Dubois | Early chronic ethanol exposure in rats disturbs respiratory network activity and increases sensitivity to ethanol[END_REF][START_REF] Dubois | Blunted response to low oxygen of rat respiratory network after perinatal ethanol exposure: involvement of inhibitory control[END_REF]. Analysis of the respiratory activity in situ [START_REF] Dubois | Early chronic ethanol exposure in rats disturbs respiratory network activity and increases sensitivity to ethanol[END_REF][START_REF] Dubois | Blunted response to low oxygen of rat respiratory network after perinatal ethanol exposure: involvement of inhibitory control[END_REF] also revealed that the respiratory rhythm was slower after CPEE. This hypoventilation was associated to a polycythemia and an increase in both arterial blood oxygen content and hematocrit [START_REF] Dubois | Blunted response to low oxygen of rat respiratory network after perinatal ethanol exposure: involvement of inhibitory control[END_REF] suggesting a physiological adaptation. In addition, in both in situ and in vivo approaches, the response to hypoxia was blunted in juvenile CPEE rats [START_REF] Dubois | Blunted response to low oxygen of rat respiratory network after perinatal ethanol exposure: involvement of inhibitory control[END_REF]. Similarly to our model, chronic ethanol exposure during the tadpole development induced alterations in both the response to hypoxia and hypercapnia tested both in vivo and in vitro [START_REF] Taylor | Chronic nicotine and ethanol exposure both disrupt central ventilatory responses to hypoxia in bullfrog tadpoles[END_REF][START_REF] Taylor | Developmental exposure to ethanol or nicotine inhibits the hypercapnic ventilatory response in tadpoles[END_REF][START_REF] Taylor | Chronic, but not acute, ethanol exposure impairs central hypercapnic ventilatory drive in bullfrog tadpoles[END_REF].

Other features of importance regarding early exposure to ethanol is the duration and the time window of exposure. In rats, prenatal ethanol exposure during the last 4 days of gestation did not modify ventilation at rest one week after birth [START_REF] Cullere | Neonatal sensitization to ethanol-induced breathing disruptions as a function of late prenatal exposure to the drug in the rat: modulatory effects of ethanol's chemosensory cues[END_REF]. In the same vein, an acute exposure to ethanol in tadpoles during development failed to reproduce the respiratory effects observed after a chronic exposure during the same period [START_REF] Taylor | Chronic, but not acute, ethanol exposure impairs central hypercapnic ventilatory drive in bullfrog tadpoles[END_REF]. Interestingly, when a chronic ethanol exposure is performed during adulthood, rats presented a slower respiratory rhythm and a transient alteration of the response to hypoxia without change in the response to hypercapnia at the end of ethanol exposure [START_REF] Sabino | Effect of chronic ethanol exposure on rat ventilatory responses to hypoxia and hypercapnia[END_REF]. Altogether this suggests that a chronic exposure to ethanol produce dramatic effects on the respiratory system regardless of the time period of exposure. Nonetheless to date, the respiratory effects of ethanol exposure during brain development have only been evaluated at the end of the exposure that is at a time when the subjects might be considered still under the influence of ethanol. Therefore, an important question that had not been investigated so far was whether the reported effects would persist during adulthood, long after ethanol withdrawal. Additionally, whether the response to hypercapnia in rats would be preserved after CPEE remained to be explored. Therefore, we investigated the activity and chemosensitivity of the respiratory system in CPEE rats one month after ethanol withdrawal using whole-body plethysmography performed during normoxia, acute hypoxia and acute hypercapnia. In addition, we analyzed arterial blood samples in normoxia during resting conditions.

Material and methods

Animals

Experimental procedures were in accordance with guidelines for care and use of laboratory animals (European Community, EU Directive 2010/63/EU). Sprague-Dawley rats were purchased from Janvier labs (France) and bred in our animal facility, with food and water provided ad libitum. Control rats were offspring from dams never exposed to ethanol, while CPEE rats were the progeny of rats that received a 10% (v/v) ethanol solution as their only drinking fluid one month before mating and until weaning of the offspring [START_REF] Dubois | Early chronic ethanol exposure in rats disturbs respiratory network activity and increases sensitivity to ethanol[END_REF][START_REF] Naassila | Effect of prenatal and postnatal ethanol exposure on the developmental profile of mRNAs encoding NMDA receptor subunits in rat hippocampus[END_REF][START_REF] Kervern | Perinatal alcohol exposure in rat induces long-term depression of respiration after episodic hypoxia[END_REF][START_REF] Dubois | Blunted response to low oxygen of rat respiratory network after perinatal ethanol exposure: involvement of inhibitory control[END_REF]. Ethanol consumption by the dams progressively increased to reach a peak daily average of 13.95 ± 2.02 g of ethanol/kg body weight before weaning of the pups. Typically, similar ethanol exposure with equivalent daily average ethanol consumption leads to blood ethanol concentrations in the dams of about 110 mg/ dL at the end of exposure [START_REF] Naassila | Effect of prenatal and postnatal ethanol exposure on the developmental profile of mRNAs encoding NMDA receptor subunits in rat hippocampus[END_REF][START_REF] Barbier | Long-term alterations in vulnerability to addiction to drugs of abuse and in brain gene expression after early life ethanol exposure[END_REF]. Twenty-one days after birth, offspring from both groups were weaned, and therefore ethanol was withdrawn from the CPEE group. One month later rats from both groups were exposed either to a hypoxic or to a hypercapnic acute challenge. Only male rats were used to avoid any sex difference in the response to hypoxia [START_REF] Mortola | Ventilatory response to hypoxia in rats: gender differences[END_REF], which have also been observed after other prenatal chronic experimental procedures such as maternal separation [START_REF] Kinkead | Neonatal maternal separation and early life programming of the hypoxic ventilatory response in rats[END_REF].

Plethysmography

A built-in whole-body plethysmograph based on Drorbaugh and Fenn's principle was used [START_REF] Drorbaugh | A barometric method for measuring ventilation in newborn infants[END_REF]. Awake and unrestrained rats were positioned in a 3.6 liters cylindrical chamber where a 1.6 l min -1 flow of dry air (Bronkhorst Hi-Tec airflow stabilizer 10 l min-1 air, Ruurlo, Holland) was flushed to avoid CO2 and water accumulation. The chamber temperature was maintained between 21 ± 1 °C and 25.6 ± 1.0 °C. Pressure changes in the measurement chamber caused by external pressure changes or by airflow fluctuations were equalized between the measurement and the 3.6 liters reference chambers. Measurement of pressure changes due to the animal ventilation were measured with a DRUCK-EFFA transducer (Asnières, France pressure transducer, range: ± 0.1 cm H2O). The differential pressure signal was filtered (bandwidth: 0.05 to 15 Hz) and converted into a digital signal (MacAdios analog-to-digital 14-bit converter; GW-Instruments, Somerville, MA) at a sampling rate of 100 Hz. Calibration of the plethysmograph was done by injecting 100 µl of air into the measurement chamber via a Hamilton syringe. A homemade software (derived from Superscope II software; GW-Instruments, Somerville, MA) detected the onset and amplitude of each breath and calculated the breath duration (and thus the respiratory frequency, Rf) and the tidal volume (VT) was calculated according to the Drorbaugh and Fenn's equation [START_REF] Drorbaugh | A barometric method for measuring ventilation in newborn infants[END_REF][START_REF] Epstein | A theoretical analysis of the barometric method for measurement of tidal volume[END_REF]. Minute ventilation (VE) was calculated as the product of Rf by VT. Estimations of the concentrations of O2 and CO2 inside the animal chamber were obtained from the outflow concentrations using an infrared CO2 analyzer and a paramagnetic O2 analyzer (Arelco, Fontenay-sous-Bois, France), calibrated with known gas mixtures. Hypoxic conditions were obtained by flushing during 5 minutes an isocapnic gas mixture containing 11% O2 while hypercapnic gas mixture contained 5% CO2 and 21% O2. To avoid restraining the rats, body temperature was measured before the plethysmography session and assumed to be stable [START_REF] Gordon | Thermal biology of the laboratory rat[END_REF].

Arterial blood sample analysis

Blood sample analysis was performed as previously described [START_REF] Dubois | Blunted response to low oxygen of rat respiratory network after perinatal ethanol exposure: involvement of inhibitory control[END_REF]. Briefly, after deep anesthesia, arterial blood was rapidly collected from the common carotid artery and immediately analyzed using an ABL725 analyzer (Radiometer, Copenhagen, Denmark).

After blood collection, euthanasia of the animal was immediately performed. Parameters for the analysis were relevant to the acid-base equilibrium (pCO2, pH), oxygen uptake (pO2, pCO2), transport (Total O2 content, hematocrit, pO2, O2 saturation) and release (p50, total O2 content) and tissue oxygenation (lactate).

Data analysis and representation

Statistical analyses were performed on raw data using the estimation statistics webpage (https://www.estimationstats.com). To compare each respiratory parameter at rest between control and CPEE rats, 2-sided Mann-Whitney tests were performed. Within each group, the response to hypoxia or hypercapnia was assessed using the 2-sided Wilcoxon test. Finally in order to test whether the responses to hypoxia or hypercapnia was affected by CPEE, 2 ways ANOVAs with repeated measurements were performed. A p<0.05 was considered as significant.

For graphical representations, the mean difference between normoxia recordings and chemorespiratory responses are shown in Cumming estimation plots. Individual values are plotted on the left while the mean differences are plotted on the right as a bootstrap sampling distribution (5000 bootstraps samples). On the later, the population means are represented as a dot and the 95% confidence interval (bias-corrected and accelerated) are indicated by the ends of the vertical error bar [START_REF] Ho | Moving beyond P values: data analysis with estimation graphics[END_REF].

Results

Persistent hypoventilation after ethanol withdrawal in adult CPEE rats

We had previously shown that CPEE induced a hypoventilation in non-weaned juvenile rats that remained under ethanol influence [START_REF] Dubois | Early chronic ethanol exposure in rats disturbs respiratory network activity and increases sensitivity to ethanol[END_REF][START_REF] Dubois | Blunted response to low oxygen of rat respiratory network after perinatal ethanol exposure: involvement of inhibitory control[END_REF] that was probably compensated by a polycythemia allowing for a higher arterial blood content [START_REF] Dubois | Blunted response to low oxygen of rat respiratory network after perinatal ethanol exposure: involvement of inhibitory control[END_REF]. Nonetheless it was not known whether those alterations of the respiratory physiology would persist after a long period of ethanol withdrawal.

After CPEE and one month of ethanol withdrawal, rat respiratory frequency was significantly reduced by 12% when compared to control rats (p<0.05, table 1), while the tidal volume remained unchanged (p>0.05, table 1). As a consequence, the minute ventilation was significantly decreased after CPEE and ethanol withdrawal (p<0.05, table 1). Body weight and temperature were unaffected by CPEE and ethanol withdrawal (p>0.05 for both parameters, table 1).

Lower blood oxygen content after ethanol withdrawal in adult CPEE rats

We next investigated whether this persistent hypoventilation was associated with a change in arterial blood oxygen parameters. One month after ethanol withdrawal, the acidbase equilibrium and the hematocrit in CPEE rats were similar to that of rats never exposed to ethanol (p>0.05, Table 2). Nonetheless, the total blood oxygen content was significantly lower when compared to control rats (p<0.05, table 2), suggesting a long-term alteration of the blood oxygenation status. This was not associated to changes in oxygen saturation (Pearson product moment correlation, P>0.05). A subset of animals was tested for both plethysmography and blood sample analysis (n=8, including 3 CPEE withdrawn rats). For these rats, there was a significant positive correlation between resting minute ventilation and total oxygen content (data not shown, Pearson product moment correlation, p<0.05), suggesting that anesthesia unlikely interfered with the analysis of arterial blood oxygen parameters.

Persistent blunted response to hypoxia after ethanol withdrawal in CPEE rats

We have shown that the response to an acute hypoxia was greatly dampened in CPEE juvenile rats before ethanol withdrawal [START_REF] Dubois | Blunted response to low oxygen of rat respiratory network after perinatal ethanol exposure: involvement of inhibitory control[END_REF]. Therefore, we examined whether these perturbations would persist after ethanol withdrawal.

The response to 5 minutes of hypoxia (11% O2) in 9 control rats consisted in increases in both respiratory frequency (+37 ± 5 %, p<0.05 compared to before hypoxia, fig 1A-B) and tidal volume (+27 ± 5%, p<0.05 compared to before hypoxia, fig1A, C). Overall, the minute ventilation was increased by 71 ± 10 % (p<0.05, fig 1A ,D).

After one month of ethanol withdrawal, the response to hypoxia in CPEE rats consisted of an increase in tidal volume (+28 ± 11% p<0.05, fig 1A ,C), without any significant increase in respiratory frequency (+18 ± 7%, p>0.05, fig 1A -B). The increase in minute ventilation was significant (+43 ± 7%, p<0.05, Fig 1A ,D), but this response remained significantly lower than in control rats (2W-ANOVA RM, p<0.05 for both respiratory frequency and minute ventilation).

Normal response to hypercapnia after ethanol withdrawal in CPEE rats

Since the response to hypoxia was persistently altered after ethanol withdrawal, we investigated whether this could be due to a general alteration of the chemosensory adaptative responses of the respiratory system. Therefore control (n=8) and CPEE rats withdrawn from ethanol (n=7) were submitted to a 5-minute hypercapnia challenge (5% CO2, fig 2).

During hypercapnia, both groups of rats presented a similar increase in respiratory frequency (control +44 ± 6% and CPEE rats +41 ± 5% compared to normocapnia, p < 0.05 for both) associated to an increase in tidal volume (control rats +56 ± 9%, CPEE rats +71 ± 11%, p<0.05 for both). Minute ventilation was increased by 125 ± 15% and 137 ± 15% in control and CPEE rats, respectively. Furthermore, 2-way ANOVAs revealed no differences between groups for these different parameters (p>0.05 for all). Therefore, the response to hypercapnia was unaltered after ethanol withdrawal in CPEE rats.

Discussion

We found that one month after ethanol withdrawal, adult animals that were exposed to ethanol during gestation and lactation periods exhibited a persistent hypoventilation at rest during normoxia that was accompanied with a lower blood oxygen content. In addition, the response to hypoxia was significantly attenuated in these rats whereas the response to hypercapnia remained unaltered.

We and others have shown that at the end of a chronic ethanol exposure in rats, performed either during perinatal period [START_REF] Dubois | Early chronic ethanol exposure in rats disturbs respiratory network activity and increases sensitivity to ethanol[END_REF][START_REF] Dubois | Blunted response to low oxygen of rat respiratory network after perinatal ethanol exposure: involvement of inhibitory control[END_REF] or adulthood [START_REF] Sabino | Effect of chronic ethanol exposure on rat ventilatory responses to hypoxia and hypercapnia[END_REF], the animals, still under the influence of ethanol, presented a marked reduction in respiratory frequency. However, the question whether this basal slower respiratory rhythm was persistent in absence of ethanol remained unanswered. Here, we report that one month after ethanol withdrawal from a perinatal exposure, the respiratory rhythm remained slower than in control rats. This reveals a long-lasting effect on rhythmic respiratory activity of an ethanol exposure during brain development. Our data stands out from other studies carried out after perinatal exposure to other drugs of abuse such as cocaine or nicotine where the breathing abnormalities found at birth disappear within the first 2 weeks of life and thus are only transient [START_REF] Bamford | Effect of nicotine exposure on postnatal ventilatory responses to hypoxia and hypercapnia[END_REF][START_REF] Bamford | Dynamic ventilatory responses in rats: normal development and effects of prenatal nicotine exposure[END_REF][START_REF] Olsen | In utero cocaine exposure: effect on neonatal breathing in guinea pigs[END_REF][START_REF] Simakajornboon | Effect of prenatal nicotine exposure on biphasic hypoxic ventilatory response and protein kinase C expression in caudal brain stem of developing rats[END_REF]. In this context and to our knowledge, the present report is the first to show a persistent reduction in respiratory frequency in young adulthood after an early-life exposure to a drug of abuse, suggesting that ethanol (i.e. alcohol) may be the most significant drug of abuse not to be consumed during pregnancy in regard to breathing physiology.

The hypoxic ventilatory response in adults rats is reduced after a chronic ethanol exposure performed during adulthood [START_REF] Sabino | Effect of chronic ethanol exposure on rat ventilatory responses to hypoxia and hypercapnia[END_REF]. After chronic ethanol exposure during the tadpole development, it has been observed an impairment of the central hypoxic ventilatory response in vitro [START_REF] Taylor | Chronic nicotine and ethanol exposure both disrupt central ventilatory responses to hypoxia in bullfrog tadpoles[END_REF] and in rats, hypoxic ventilatory response was also blunted in vivo in juvenile animals at the end of CPEE [START_REF] Dubois | Blunted response to low oxygen of rat respiratory network after perinatal ethanol exposure: involvement of inhibitory control[END_REF]. At that developmental stage, the failure to increase minute ventilation during hypoxia in CPEE rats was associated to the lack of increase in tidal volume [START_REF] Dubois | Blunted response to low oxygen of rat respiratory network after perinatal ethanol exposure: involvement of inhibitory control[END_REF]. In the present study we have shown that after ethanol withdrawal the increase in minute ventilation during hypoxia is however associated to a lower increase in respiratory frequency and to a significant increase in tidal volume. This suggests that the physiological mechanisms responsible for the response to hypoxia could be differentially affected at the end of ethanol exposure and after one month of ethanol withdrawal. However, more experiments are required to clearly define the mechanisms involved. Interestingly, the consequences of perinatal ethanol exposure on the response to hypercapnia differ from the effects observed on the response to hypoxia. We found that perinatal exposure to ethanol does not alter in the long term the response to hypercapnia. This result is different from experiments performed during tadpole development, in which the hypercapnia-induced increase in lung burst frequency was abolished, regardless the stage of exposure [START_REF] Taylor | Developmental exposure to ethanol or nicotine inhibits the hypercapnic ventilatory response in tadpoles[END_REF][START_REF] Taylor | Chronic, but not acute, ethanol exposure impairs central hypercapnic ventilatory drive in bullfrog tadpoles[END_REF]. However, our result is consistent with exposure to other drugs of abuse (i.e. cocaine and nicotine) performed in mammals [START_REF] Bamford | Effect of nicotine exposure on postnatal ventilatory responses to hypoxia and hypercapnia[END_REF][START_REF] Bamford | Dynamic ventilatory responses in rats: normal development and effects of prenatal nicotine exposure[END_REF][START_REF] Olsen | In utero cocaine exposure: effect on neonatal breathing in guinea pigs[END_REF][START_REF] Simakajornboon | Effect of prenatal nicotine exposure on biphasic hypoxic ventilatory response and protein kinase C expression in caudal brain stem of developing rats[END_REF][START_REF] Autret | Ventilatory control in newborn mice prenatally exposed to cocaine[END_REF]. Altogether this result suggests that early life exposure to drugs of abuse have only limited impact on the response to hypercapnia in mammals and that the effects of early life ethanol exposure on hypercapnia depends upon the specie studied. Nonetheless, further work is needed to determine whether this difference could also be due to the type of chronic ethanol exposure used or to the duration of withdrawal per se, since the withdrawal consequences have been investigated only in the present study. Importantly, the fact that the alteration of chemosensitivity is specific to the response to hypoxia suggests that CPEE does not alter chemosensitivity integration in a global manner but rather affects the neuronal network involved in the response to hypoxia.

Previously, the dampened response to hypoxia in juvenile animals was related to a stronger bulbar glycinergic neurotransmission [START_REF] Dubois | Blunted response to low oxygen of rat respiratory network after perinatal ethanol exposure: involvement of inhibitory control[END_REF]. It would be then interesting to know whether glycinergic dysfunction persists after ethanol withdrawal within the respiratory network. One limitation of the present study lies in the absence of metabolic information during plethysmography. Such information could unmask possible metabolic disruption or an associated pro-inflammatory state induced by the CPEE that could possibly bias the respiratory measurements. Therefore future studies will require a more extensive exploration of the metabolic status of the animals.

We found that the hematocrit of young adult CPEE rats withdrawn from ethanol is similar to that of control rats. This result is different from what we obtained in juvenile rats at the end of ethanol exposure [START_REF] Dubois | Blunted response to low oxygen of rat respiratory network after perinatal ethanol exposure: involvement of inhibitory control[END_REF]. Therefore, the hematocrit normalizes during ethanol withdrawal suggesting that it is the presence of ethanol early in life (possibly during the fetal period) that triggered such adaptive response. However such normalization of the hematocrit could also be explained by the physiological decay of hematopoietic activity that occurs after the first three weeks of postnatal life [START_REF] Kouno | Development of haemoglobin subtypes and extramedullary haematopoiesis in young rats. Effects of hypercapnic and hypoxic environment[END_REF]. In our experiments, we also found that the total oxygen blood content was reduced after withdrawal from CPEE. Importantly for breathing physiology, such situation could lead to a chronic state of hypoxia. Since this lower oxygen blood content was detected after ethanol withdrawal but not before [START_REF] Dubois | Blunted response to low oxygen of rat respiratory network after perinatal ethanol exposure: involvement of inhibitory control[END_REF], this suggest that such putative chronic state of hypoxia would only develop after ethanol withdrawal. Chronic hypoxia on its own may induce long lasting changes to the respiratory activity that depends on the severity, period and duration of hypoxia. Interestingly, it has been reported that the basal respiratory activity and the response to an acute hypoxia were increased following a sustained hypoxia protocol performed during adulthood [START_REF] Reeves | Effect of long-term intermittent and sustained hypoxia on hypoxic ventilatory and metabolic responses in the adult rat[END_REF]. Therefore, a chronic hypoxic state after ethanol withdrawal in CPEE rats is unlikely to be the origin of the hypoventilation and reduced response to hypoxia that we report here. Rather, the normalization of the hematocrit (and therefore the lower oxygen transport capacity) associated to the hypoventilation after ethanol withdrawal in CPEE rat is more likely to be responsible for the lower arterial blood oxygen content. Despite our efforts to minimize the time to collect blood samples, anesthesia may have biased our analysis through a differential depression of respiratory activity between groups. However, the correlation we found between resting minute ventilation and total oxygen content in a subset of animals suggests that if this bias exists, it remains of minimal impact.

An alteration of the ventilatory function and in particular of chemosensitivity has been proposed as a likely origin of the sudden infant death syndrome [START_REF] Kahn | Sudden infant deaths: from epidemiology to physiology[END_REF][START_REF] Leiter | Mechanisms of pathogenesis in the Sudden Infant Death Syndrome[END_REF][START_REF] Ribas-Salgueiro | Central chemoreceptors and sudden infant death syndrome[END_REF]. The model of ethanol exposure we used typically induce blood ethanol concentration of about 100 mg/dL in dams [START_REF] Naassila | Effect of prenatal and postnatal ethanol exposure on the developmental profile of mRNAs encoding NMDA receptor subunits in rat hippocampus[END_REF][START_REF] Barbier | Long-term alterations in vulnerability to addiction to drugs of abuse and in brain gene expression after early life ethanol exposure[END_REF], a level comparable to that observed in pregnant women moderately dependent to alcohol [START_REF] Eckardt | Effects of Moderate Alcohol Consumption on the Central Nervous System[END_REF][START_REF] Halmesmäki | Alcohol counselling of 85 pregnant problem drinkers: effect on drinking and fetal outcome[END_REF]. Children that were exposed to ethanol in utero exhibit typical physical features that include an elongated face, or a flattened jaw which are features shared with children with breathing-related sleep disorders [START_REF] Burd | Diagnosis of FAS: a comparison of the Fetal Alcohol Syndrome Diagnostic Checklist and the Institute of Medicine Criteria for Fetal Alcohol Syndrome[END_REF], a population more prone to the sudden infant death syndrome [START_REF] Kahn | Sudden infant deaths: from epidemiology to physiology[END_REF][START_REF] Leiter | Mechanisms of pathogenesis in the Sudden Infant Death Syndrome[END_REF][START_REF] Morris | Invited review: Neural network plasticity in respiratory control[END_REF][START_REF] Thach | The role of respiratory control disorders in SIDS[END_REF]. In this context, altered breathing and impaired chemosensitivity appear as an aggravating factor in the occurrence of unexplained sudden death, although the exact origin of these impairments remains to be determined. Therefore, there remains a gap in our knowledge on the long-term effects of perinatal exposure to drugs of abuse, including ethanol, and more studies, both at preclinical and clinical levels, are required. In this report we show that a perinatal exposure to ethanol in rats dramatically alters the respiratory function even after one month of ethanol withdrawal. This highlights the long-term deleterious effects of early ethanol exposure after withdrawal that could possibly translate in Human as a wide range of comorbidities occurring not only at birth, but potentially several months or years later. Furthermore, and besides the respiratory-related consequences of CPEE, the present alterations of the respiratory function could also be associated to a broad panel of comorbidities including sleep disorders, memorization or cardiovascular pathologies.

Figure 1 .

 1 Figure 1. Persistent weakening of the response to hypoxia after ethanol withdrawal in CPEE rats. A. Typical recordings of breathing activity in control (n=9) and CPEE withdrawn rats (n=7) under normoxic conditions (top) and at the end of a 5 min hypoxia (bottom). Inspiration is upwards. Analysis of respiratory frequency (B.), tidal volume (C.) and minute ventilation (D.) before and at the end of a 5 min hypoxia. For each, the left panel indicates individual variations of the parameter and on the right panel the mean difference between before and after hypoxia is shown in Cumming estimation plots. *, p<0.05 hypoxia compared to baseline values (Wilcoxon test); #, p<0.05 CPEE withdrawn rats compared to control rats (2 way repeated measurement ANOVA).

Figure 2 .

 2 Figure 2. Normal response to hypercapnia after ethanol withdrawal in CPEE rats. A. Examples of plethysmographic recordings in control (n=8) and CPEE withdrawn rats (n=7) under normoxic conditions (top) and at the end of a 5 min hypercapnia (bottom). Inspiration is upwards. Analysis of respiratory frequency (B.), tidal volume (C.) and minute ventilation (D.) before and at the end of a 5 min hypoxia. For each, the left panel indicates individual variations of the parameter and on the right panel the mean difference between before and after hypercapnia is shown in Cumming estimation plots. *, p<0.05 hypercapnia compared to baseline values (Wilcoxon test).

Table 1 . Alteration of ventilatory parameters in normoxia after ethanol withdrawal in CPEE rats.

 1

	Control group	CPEE withdrawn
		group
	(n=17)
		(n=14)

Table 2 . Arterial blood status in normoxia after ethanol withdrawal in CPEE rats.

 2

	Control group	CPEE withdrawn
		group
	(n=7)
		(n=8)

Acknowledgements

The authors would like to thank Guy Vardon for providing the plethysmographic equipment and useful experimental advices.

Funding

This work was supported by the Council of Région de Picardie.

CRediT author statement

Christophe Dubois: Conceptualization, Methodology, Investigation, Formal analysis, Data Curation, Writing-Original draft preparation, Visualization, Writing-Reviewing and Editing. Olivier Pierrefiche: Conceptualization, Writing-Reviewing and Editing, Funding acquisition, Supervision.

Declaration of competing interest

The authors declare no competing interest.