Gilles Rivière
email: gilles.riviere@anses.fr

Julien Jean

Sébastien Gorecki

Marion Hulin

Martine Kolf-Clauw

Cyril Feidt

Nicole Picard-Hagen

Paule Vasseur

Bruno Le Bizec

Véronique Sirot

Nicole Picard- Hagen

Dietary exposure to perfluoroalkyl acids, brominated flame retardants and health risk assessment in the French infant total diet study

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

INTRODUCTION

Early stages of life, i.e. the fetal and early postnatal periods, correspond to periods of increased susceptibility [START_REF] Makri | Children's susceptibility to chemicals: a review by developmental stage[END_REF][START_REF] Sly | Susceptibility of children to environmental pollutants[END_REF][START_REF] Kandarakis | Endocrine-disrupting chemicals: an endocrine society scientific statement[END_REF]. Several pieces of evidence suggest that fetuses and infants may be more sensitive to pollutants [START_REF] Landrigan | Children's health and the environment: public health issues and challenges for risk assessment[END_REF]. The French Agency for Food, Environmental and Occupational Health & Safety (ANSES) launched a specific TDS on infants to complete its overall chemical food safety program for the general population [START_REF] Hulin | Assessment of infant exposure to food chemicals: the French Total Diet Study design[END_REF]. More than 500 chemical substances were analyzed in food products consumed by children under 3 years old, including persistent organic pollutants. community to cooperate in limiting the production and use of PFAAs and in developing safer non fluorinated alternatives (scientists, governments, chemical and product manufacturers [START_REF] Blum | The Madrid statement on poly-and perfluoroalkyl substances (PFASs)[END_REF].

PFAAs contaminate several compartments of the environment (water, soil, air) and accumulate in the food chain [START_REF] Kowalczyk | Transfer of Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS) From Contaminated Feed Into Milk and Meat of Sheep: Pilot Study[END_REF]. Food, particularly seafood products, is a significant source of exposure to PFAAs in humans [START_REF] Cornelis | First assessment of population exposure to perfluorinated compounds in Flanders, Belgium[END_REF][START_REF] Efsa | Scientific Opinion of the Panel on Contaminants in the Food chain. Perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA) and their salts (Question No EFSA-Q-2004-163)[END_REF][START_REF] Trudel | Estimating Consumer Exposure to PFOS and PFOA[END_REF][START_REF] Rivière | Food risk assessment for perfluoroalkyl acids and brominated flame retardants in the French population: results from the second French total diet study[END_REF]. PFOS and PFOA persist in the environment and can accumulate in animals and humans. High concentrations are generally observed in the liver, blood and kidneys. Toxicity studies on PFAAs generally only investigate PFOS and PFOA [START_REF] Olsen | Half-Life of Serum Elimination of Perfluorooctanesulfonate, Perfluorohexanesulfonate, and Perfluorooctanoate in Retired Fluorochemical Production Workers[END_REF]. Their apparent elimination half-lives in humans are approximately 4 to 5 years [START_REF] Olsen | Half-Life of Serum Elimination of Perfluorooctanesulfonate, Perfluorohexanesulfonate, and Perfluorooctanoate in Retired Fluorochemical Production Workers[END_REF][START_REF] Bartell | Rate of decline in serum PFOA concentrations after granular activated carbon filtration at two public water systems in Ohio and West Virginia[END_REF]. The main toxic effects reported in animals have been observed in the liver [START_REF] Seacat | Sub-chronic dietary toxicity of potassium perfluorooctanesulfonate in rats[END_REF], on reproductive and developmental functions and immune and hormonal systems [START_REF] Seacat | Subchronic toxicity studies on perfluoroctanesulfonate potassium salt in cynomolgus monkeys[END_REF][START_REF] Kang | Transcriptional changes in steroidogenesis by perfluoroalkyl acids (PFOA and PFOS) regulate the synthesis of sex hormones in H295R cells[END_REF][START_REF] Grandjean | Serum vaccine antibody concentrations in children exposed to perfluorinated compounds[END_REF], neurobehavior (Johansson et al., 2007) as well as on lipid metabolism [START_REF] Efsa | Scientific Opinion of the Panel on Contaminants in the Food chain. Perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA) and their salts (Question No EFSA-Q-2004-163)[END_REF]. PFOS and PFOA have neoplastic effects but have not been shown to be genotoxic [START_REF] Efsa | Scientific Opinion of the Panel on Contaminants in the Food chain. Perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA) and their salts (Question No EFSA-Q-2004-163)[END_REF].

Brominated flame retardants (BFRs) are mixtures of chemical substances produced and incorporated in many products due to their fire-retardant properties. They are incorporated in plastics, textiles as well as in electronic equipments. This class encompasses different compounds, including hexabromocyclododecanes (HBCDDs), polybrominated biphenyls (PBBs), polybrominated diphenyl ethers (PBDEs) and tetrabromobisphenol A (TBBPA). Food appears to be the main route of exposure for some BFRs such as PBDEs [START_REF] Frederiksen | Human internal and external exposure to PBDEs-a review of levels and sources[END_REF][START_REF] Domingo | Polybrominated diphenyl ethers in food and human dietary exposure: a review of the recent scientific literature[END_REF]. The characterisation of chronic human toxicity of BFRs is difficult since they are often experimentally studied as mixtures. In experimental studies, these compounds have been shown to trigger neurodevelopmental effects (EFSA, 2011a).

For all these compounds, dietary intakes appear to be a major contributor to the overall exposure. On the other hand, some of these compounds trigger effects that can be of particular importance for the youngest population (behavioral, neurodevelopmental or endocrine disruptor effects). It is consequently deemed of importance to estimate the dietary intake of the youngest population to these compounds and to assess the risk related to these exposures. The objective of the present study is to assess the risk to children aged from 0 to 3 years of age related to the presence of PFAA and BFR compounds in food. Exposures were calculated using concentrations of 16 PFAAs, 8 PBDEs, 3 PBBs, 3 HBCDDs and TBBPA measured in food samples collected in the context of the infant total diet study (TDS) performed in France [START_REF] Hulin | Assessment of infant exposure to food chemicals: the French Total Diet Study design[END_REF].

MATERIALS & METHOD

Consumption data for children under 3

For children under 3 years of age, consumption data came from a national consumption survey conducted specifically on this population [START_REF] Fantino | Apports nutritionnels en France en 2005 chez les enfants non allaités âgés de moins de 36 mois[END_REF]. Briefly, this cross-sectional study was conducted from January to March 2005 by the Syndicat Français des Aliments de l'Enfance et de la Nutrition Clinique, © « Etude SOFRES 2005 / Université de Bourgogne -Pr M. Fantino pour le Syndicat Français des Aliments de l'Enfance » [START_REF] Fantino | Etude SFAE sur la consommation alimentaire des nourrissons et enfants en bas âge français de 1 mois à 36 mois -Analyse des données nutritionnelles[END_REF][START_REF] Fantino | Apports nutritionnels en France en 2005 chez les enfants non allaités âgés de moins de 36 mois[END_REF]. Individual, consecutive 3-day weight food records were collected from a sample of 705 children selected through proportionate quota sampling based on the living area, the age of the children, the occupation of the mother and the family socio-economic category. Totally or partially breastfed infants (during the survey period) were excluded from the study.

Food sampling

Food samples were collected as previously described [START_REF] Hulin | Assessment of infant exposure to food chemicals: the French Total Diet Study design[END_REF]. To obtain a representative and general view of infant food consumption, food items were selected based on the results of the consumption survey [START_REF] Fantino | Apports nutritionnels en France en 2005 chez les enfants non allaités âgés de moins de 36 mois[END_REF] based on two main criteria: -The most consumed food in terms of quantity and/or percentage of consumers -Food items that are known or supposed to be the main contributors to the exposure to one or more substances of interest Food items were sampled in the same region and prepared as consumed by the same contractor (i.e. peeled, cooked, etc.) based on a result of a national study on parents' food preparation/cooking processes carried out by Anses [START_REF] Hulin | Assessment of infant exposure to food chemicals: the French Total Diet Study design[END_REF].

Infant products were updated before selecting food items due to the changing market. Food items included common foods such as vegetables, fruits or cakes as well as specific infant foods such as infant formula or jarred baby food. The sampling plan covered over 95% of the total diet of 0 to 3 years old children. To take into account seasonal variability, possible different flavours, and different ways of consuming or preparing foods, all the analysed samples, referred to as "composites" or "pools", were formed of 12 sub-samples of the same food and of equal weight. The products purchased were prepared in a way that reflected as closely as possible what is done in the home: preparation (removal of the non-edible part, washing of foods, etc.) and cooking (duration and power, addition or not of salt, fat, etc.). Specificities in infant food consumption and habits were considered. The infant formulas were diluted and heated most of the time, prepared baby dishes were reheated, vegetables (excluding raw vegetables), meat and fish were cooked according to the practices reported in the survey on practices [START_REF] Hulin | Assessment of infant exposure to food chemicals: the French Total Diet Study design[END_REF]. The sampling phase took place between July 2011 and July 2012 in the center region of France.

Analytical procedure

Perfluoroalkyl acids

The analyses were performed in food items known or supposed to be contaminated by PFAAs (EFSA, 2012), corresponding to 199 samples out of 457.

The analytical method was used to determine the concentration of 5 perfluoroalkyl sulfonates: PFOS, perfluorobutane sulfonate (PFBS), perfluorohexane sulfonate (PFHxS), perfluoroheptane sulfonate (PFHpS) and perfluoro[-1-]decanesulfonate (PFDS), 11 perfluorocarboxylic acids: PFOA, perfluorobutanoic acid (PFBA), perfluoropentanoic acid (PFPA) perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnA), perfluorododecanoic acid (PFDoA), perfluorotridecanoic acid (PFTrDA) and perfluorotetradecanoic acid (PFTeDA) and one perfluoroalkyl sulfonate (PFOSi) [START_REF] Kadar | Development of an analytical strategy based on liquid chromatography-high resolution mass spectrometry for measuring perfluorinated compounds in human breast milk: Application to the generation of preliminary data regarding perinatal exposure in France[END_REF]. Solid food samples were freeze-dried and extracted with methanol. After evaporation, food extracts were purified onto two consecutive solid-phase extraction (SPE) columns (copolymeric reversed phase and charcoal). Final purified extracts were analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS); electrospray ionisation in the negative ion mode was preferred and at least two transitions were monitored per analyte. Water samples (100 mL), were directly loaded on a reversed-phase SPE column. For milk and dairy products, samples were first extracted using acetone. For fish samples, a dispersive SPE based on charcoal particles was used.

Quantification was performed according to isotopic dilution principles. Each sample was spiked by two 13 Clabelled quantification standards ( 13 C4-PFOA and 13 C4-PFOS) for quantification. A recovery standard (fluorometholone) was added at the end of the analytical process. Depending on the matrix, these recoveries ranged from 30 to 80%. Limits of detection (LOD) and quantification (LOQ), were determined based on the signal-to-noise ratio and depended on the matrix and analyte, overall, they ranged from 0.2 pg.g -1 fresh weight (fw) to 3.7 ng.g -1 fw. Repeatability of the signal was also dependent on the matrix, but was always better than 18.6 % for all analytes.

Brominated flame retardants

The analyses were performed in food items known or supposed to be contaminated by BFRs [START_REF] Efsa | Scientific Opinion of the EFSA Panel on Contaminants in the Food Chain on Polybrominated Biphenyls (PBBs) in Food[END_REF]EFSA, 2011a, b, c) , corresponding to 206 samples out of 457.

The extraction of fat was adapted to the physical characteristics of the samples (Cariou et al., 2006;Debrauwer et al., 2005). For all matrices, quantification standards were added before extraction (eight 13 C12-labelled PBDE congeners, one 13 C12-labelled PBB congener and three 13 C12-labelled HBCD congeners). The solid samples were freeze-dried, ground, and extracted by Accelerated Solvent Extraction (ASE) using a Dionex TM ASE 300 TM . Pressure and temperature were set to 100 bars and 120°C respectively. Liquid samples underwent protein precipitation through the addition of potassium oxalate. Two additional successive extractions using a solvent mixture of ethanol, ether and hexane were performed to extract the fat.

Purification involved three stages involving silica, Florisil® and charcoal-celite® columns. A recovery standard was added to each vial for each class of compounds ( 13 C12-PBDE 138 for PBDEs and PBBs and fluorometholone for HBCDDs) just before injection.

Detection and identification of PBDEs and PBBs were carried out using a Hewlett-Packard 6890 gas chromatograph, equipped with a DB-5MS capillary column (30 m x 0.25 mm ID, 0.25 µm film thickness) coupled to a Jeol JMS-800D (Jeol, Tokyo, Japan), operating at a resolving power of 10,000 (10% valley). Concentrations of the following PBDE congeners were measured: . Concentrations of PBB-52, PBB-101 and PBB-153 were also measured. For PBDEs, analytical limits in infant specific food items ranged from 0.01 to 1.4 pg.g -1 fw and from 0.1 to 15 pg.g -1 fw for common food items. For PBBs, analytical limits ranged from 0.007 to 5.4 pg.g -1 fw depending on the matrix. Detection rates were 8, 4 and 12% for PBB-52, -101 and -153, respectively.

HBCDD were analysed by LC-MS/MS. HBCDD isomers were separated by reverse-phase chromatography using a Hypersil Gold column (100 mm × 2.1 mm; 1.9µm, Thermo), with a mobile phase containing acetonitrile/methanol (1:1) (A) and 20 mM ammonium acetate (B). The detection and quantification MS instrument was a triple quadrupole 6410 (Agilent Technologies, Santa Clara, CA, USA), fitted with an electrospray ion source, operating in the negative ion mode. Analytical limits for infant specific food items ranged from 0.007 to 4 pg.g -1 fw. For common food items, the median limit of detection ranged from 1.7 to 5 pg.g -1 fw.

TBBPA was analyzed on a reversed-phase chromatography column (Gemini, 50 mm × 2 mm; 3µm, Phenomenex) with a mobile phase containing acetonitrile+0.1% acetic acid (A) and water+0.1% acetic acid (B). The detection and quantification was achieved on an Orbitrap system (Exactive, Thermo) operating in the negative electrospray mode and full scan acquisition. Analytical limits were highly dependent on the matrix and ranged from 0.1 to 3 ng.kg -1 fw.

Internal quality controls

LABERCA operates an ISO/IEC 17025:2005-certified Quality Assurance system requiring strict controls with regard to personnel, instrument conditions, and experimental situations.

Blank samples and quality control were analyzed and monitored in each series of ten to twenty samples: the signal of each compound in the blanks was checked to avoid contamination throughout the analytical procedure, and concentrations of analytes in quality control were monitored to ensure repeatability and followed in charts. In addition, every year the laboratory takes part in an interlaboratory comparison tests and achieves satisfactory results for both of these methods.

Exposure calculation

Censored data (results below the limits of detection (LOD) and quantification (LOQ)) were processed according to a substitution method that involved framing the actual level using the lowest (low assumption or lowerbound (LB)) and highest (high assumption or upper-bound (UB)) values possible. The low assumption was calculated by assuming that all values below the LOD were equal to zero and those between the LOD and the LOQ were equal to the LOD. The high assumption was calculated by assuming that all values below the LOD were equal to the LOD and those between the LOD and the LOQ were equal to the LOQ.

Using data on individual consumption and levels of PFAAs and BFRs measured in food, exposure was calculated with the following equation:

= , ×
where:

-Ei is the total daily exposure of an individual i (µg.kg body weight -1 .day -1 ), -Ci,k is the daily consumption of the food k by an individual i (g.day -1 ), -Lk is the estimated level for the studied contaminant in the food k (µg.g -1 fresh weight), -BWi is the body weight of the individual i (kg), -n is the total number of foods consumed by the individual i.

For common food items, concentrations data were completed with data from the second French TDS focusing on the general population [START_REF] Rivière | Food risk assessment for perfluoroalkyl acids and brominated flame retardants in the French population: results from the second French total diet study[END_REF].

According to WHO recommendations (GEMS/Food-EURO. 2013), exposure data were estimated according to both the upper bound hypothesis (LB and UB, respectively). In the present article health risk was assessed considering the worst case scenario, i.e., considering the UB hypothesis.

Mean and 90 th percentile (P90) of exposure were calculated for the population divided into four age groups: 1-4 months, 5-6 months, 7-12 months and 13-36 months. The mean exposure of the 10% of the most exposed children (over the P90) was also calculated. The percentage (and corresponding confidence interval at 95%, CI95%) of children exceeding the respective health based guidance value was calculated for each age group. Margins of safety were calculated (when necessary) as the ration between BMDLs and exposure (mean or P90).

The ratio of the exposure through individual food items over the total exposure was calculated to determine the contribution (in %) of each food item to the total exposure. Food items contributing to more than 10% of the total exposure were identified as main contributors to the exposure.

Collective appraisal

The collective assessment of the risk linked to PFAA and BFR exposure has been conducted with the the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) Expert Committee on food contaminants.

RESULTS AND DISCUSSION

Contamination data

PFAAs

Analyses covered 96% of the diet theoretically contributing to the total exposure. Due to the limited available toxicological data for PFAAs other than PFOS, PFOA, PFBS and PFHxA, only the results from these compounds are reported in the present article, concentrations of all other PFAAs are available as supplementary materials.

Excluding water, overall detection rates were very low (between 0 for PFDS and PFHpS) to 5.9% for PFTrDA.

PFAAs were not detected in any of the infant specific food items, so results are only presented for common food items (Table 1). Highest concentrations of PFOA and PFOS were determined in crustaceans and mollusks: respectively, 0.014 ng.g -1 fw and 0.312 ng.g -1 fw [START_REF] Rivière | Food risk assessment for perfluoroalkyl acids and brominated flame retardants in the French population: results from the second French total diet study[END_REF]. The contamination data of common food items were those already reported [START_REF] Rivière | Food risk assessment for perfluoroalkyl acids and brominated flame retardants in the French population: results from the second French total diet study[END_REF].

In 2012, the European Food Safety Authority (EFSA) reported PFAAs concentration in fish ranging from 0.0064 to 18.2 ng.g -1 fw for PFOA and from 0.04 to 211 ng.g -1 fw for PFOS [START_REF] Efsa | Scientific report of EFSA on Perfluoroalkylated substances in food: occurence and dietary exposure[END_REF]. A Spanish study published in 2010 reported concentrations of PFOA and PFOS ranging from 0.17 to 0.72 ng.g -1 fw and from 0.16 to 1.1 ng.g -1 fw, respectively [START_REF] Llorca | Infant exposure of perfluorinated compounds: levels in breast milk and commercial baby food[END_REF]. In both studies, detection rates were low (8% in the study reported by EFSA in 2012 for instance). The concentrations reported in our study are slightly lower than those previously reported.

BFRs

For PBDEs, overall analyses covered 96% of the contributing diet. Detection rates for the 7 PBDEs (BDE-28+BDE-47+BDE-99+BDE-100+BDE-153+BDE-154+BDE-183) ranged from 20% to 93% for BDE-183 and BDE-99, respectively. Highest mean concentrations were measured in dairy desserts and infant cereals for BDE-209 (respectively 69.57 and 66.94 pg.g -1 fw considering the LB hypothesis) and in vegetables/meat or vegetable/fish ready to eat jars, infant cereals and dairy desserts for the sum of the 7 PBDEs (4.35 pg.g -1 fw, 3.67 pg.g -1 fw and 3.22 pg.g -1 fw, respectively considering the LB hypothesis, Table 2). For common food items, highest BDE-209 mean concentrations were measured in sweet and savoury biscuits and bars (244 pg.g -1 fw) and in margarine (105 pg.g -1 fw). For the sum of the 7 PBDEs, highest mean concentrations were measured in fish (578 ng.g -1 fw). The contamination data of common food items were those already reported [START_REF] Rivière | Food risk assessment for perfluoroalkyl acids and brominated flame retardants in the French population: results from the second French total diet study[END_REF]. In 2011, EFSA reported concentrations of PBDE in fish and seafood ten-fold higher than those reported in the present study (around 400 versus 40 pg.g -1 fw; EFSA, 2011a). Similarly in infant food items, EFSA reported concentrations of BDE-209 of 130 pg.g -1 fw versus 8 to 70 pg.g -1 fw in the present study.

For HBCDDs, overall analyses covered 96% of the contributing diet. HBCDDs were detected in 82, 36 and 38% of the samples for isomers α, β and γ, respectively. Detection rates were very variable depending on the matrix analyzed (from 0 to 100%, in, respectively, cereals and dairy desserts and milk-based beverages). Measured concentrations in infant specific food items ranged from 2.46 to 42.85 pg.g -1 fw (considering the sum of the three isomers and the UB hypothesis, Table 3). In one sample of infant formula, a concentration of 307 pg. g -1 fw was measured whereas all other infant formula samples had concentrations ranging from 0.22 to 8.15 pg. g - 1 fw. A similar situation has been observed in the past where high concentrations of HBCDD were measured in different matrices (especially turkey and eggs, Anses 2012). However, except an occasional contamination, no relevant hypothesis could be drawn to explain this observation. For common food items, highest mean concentrations were measured in fish (between 177 pg.g -1 fw and 185 pg.g -1 fw depending on the hypothesis) and in delicatessen meat (between 140 pg.g -1 fw and 150 pg.g -1 fw, depending on the hypothesis). The contamination data of common food items were those already reported [START_REF] Rivière | Food risk assessment for perfluoroalkyl acids and brominated flame retardants in the French population: results from the second French total diet study[END_REF]. EFSA, in 2011, reported concentrations of HBCDD in infant food in the same order of magnitude (between 10 and 30 pg. g -1 fw, EFSA 2011c) than those reported in the present study.

For PBBs, overall, analyses covered 96% of the contributing diet. The three congeners analyzed were detected in 4 to 12% of samples tested. In infant specific food items, measured concentrations were close to the limits of detection, with values ranging from 0.26 to 4.88 pg.g -1 (considering the UB hypothesis, Table 4) for the sum of the three congeners. Highest PBB-52 concentrations were measured in an infant specific dairy based dessert containing fruits (3.28 pg.g -1 fw) and in a ready to eat jar containing salmon, rice and vegetables (1.23 pg.g -1 fw). For common food items, concentrations were slightly higher than those for infant specific food items with mean concentrations ranging from 0.19 to 20.4 pg.g -1 fw (considering the UB hypothesis). The contamination data of common food items were those already reported (Riviere et al., 2014). These results are consistent with the poor use of these compounds in Europe since their ban in 2000 (EFSA 2010). In 2010, EFSA reported concentrations of PBB in fish in the same order of magnitude than those reported here (between 1.86 and 18.9 ng.kg -1 fw depending on the congener, EFSA 2010).

Overall, analyses of TBBPA covered 94% of the contributing diet. TBBPA was detected in 30% of the samples. Highest mean concentrations were measured in croissant-like pastries (914 ng.kg -1 fw, Table 5) and infant and follow-on formulae (between 45 and 60 ng.kg -1 fw depending on the hypothesis), poultry (between 42 ng.kg -1 fw and 54 ng.kg -1 fw) and chocolate (between 32 and 62 ng.kg -1 fw).

Exposure data

PFAAs

Considering the UB hypothesis with regards to censored data, mean daily exposures to PFOA ranged from 1.49 ng.kg bw -1 .d -1 (90 th percentile 1.77 ng.kg bw -1 .d -1 ) in the 1 to 4 months of age children to 2.95 ng.kg bw -1 .d -1 (90 th percentile 5.16 ng.kg bw -1 .d -1 ) in the 1 to 3-year old children (Table 6). For PFOS, the mean exposures ranged from 1.61 ng.kg bw -1 .d -1 (90 th percentile 3.56 ng.kg bw -1 .d -1 ) in the 5-6 months of age children to 2.66 ng.kg bw -1 .d -1 (90 th percentile 4.63 ng.kg bw -1 .d -1 ) in the 1 to 3 years of age children. The mean exposure of the most exposed children (whose exposure were higher than the 90 th percentile), ranged from 6.25 to 7.45 ng.kg bw -1 .d -1 for PFOA and from 5.54 to 6.37 for PFOS ng.kg bw -1 .d -1 . In 2012, EFSA reported exposures higher than those reported in the present study [START_REF] Efsa | Scientific report of EFSA on Perfluoroalkylated substances in food: occurence and dietary exposure[END_REF]. In newborns, reported exposures to PFOS and PFOA were 0.16 and 0.29 ng.kg bw -1 .d -1 , respectively, whereas in young children these exposures ranged from 0.20 to 17 ng.kg bw -1 .d -1 for PFOA depending of the hypothesis with regards to censored data and from 0.58 to 14 ng.kg bw -1 .d -1 for PFOS. In France, in the last total diet study, exposures to PFOA and PFOS of the 3 to 6 years of age children were estimated at 2.45 ng.kg bw -1 .d -1 and 2.18 ng.kg bw -1 .d -1 , respectively [START_REF] Anses | Avis de l'Anses et rapport d'expertise relatifs à l'Etude de[END_REF], which is the same range as for 1-3 years-old children of the present study. Due to the very limited number of food where PFBS and PFHxA were detected, no conclusions can be drawn with regard to the exposures to these compounds.

BFRs

Considering the UB hypothesis, mean daily exposure to BDE-209 ranged from 1.12 ng.kg bw -1 .d -1 to 2.62 ng.kg bw -1 .d -1 , for the 1 to 3 years of age children and the 1 to 4 months of age children, respectively (Table 7). For the sum of the 7 PBDEs, mean exposures ranged from 0.448 to 0.926 ng.kg bw -1 .d -1 (Table 8). The 90 th percentiles ranged from 1.88 to 3.91 ng.kg bw -1 .d -1 for BDE-209, whereas they ranged from 0.694 to 1.56 ng.kg bw -1 .d -1 for the sum of the 7 PBDEs depending on the age groups (4.30 ng.kg bw -1 .d -1 ). Two children had exposures of the 7 PBDEs much higher than the mean of their respective age groups. In both cases, these children consumed during the survey high quantities of salmon. Exposures of the 3 to 6 years of age children in France previously reported were in the same order of magnitude than those reported in the present study (ranging from 0.481 to 0.510 ng.kg bw -1 .d -1 ) whereas exposure to BDE-209 reported by EFSA were higher (ranging from 2.61 ng.kg bw -1 .d -1 to 6.02 ng.kg bw -1 .d -1 depending on the hypothesis for censored data, EFSA 2011a). It is of interest to note that the ratio of exposures to BDE-209 to exposures to the sum of PBDEs is higher in the present study (around 3-fold) than in some previously reported studies (around 2-fold, Anses 2011, EFSA 2011a). This being possibly due to the increasing use of BDE-209 compared to that of the other congeners.

For the sum of HBCDDs and following the UB hypothesis, mean daily exposure ranged from 0.505 ng.kg bw -1 .d -1 for the 1 to 3 years of age children to 8.27 ng.kg bw -1 .d -1 for the 1 to 4 months of age children (Table 9). The 90 th percentile ranged from 0.880 to 43.2 ng.kg bw -1 .d -1 for the same age groups, the last value being due to the atypical contamination value measured in one sample of infant formula. For this reason the median values have also been reported in Table 9. Mean exposures of the 3 to 6 years of age children previously reported in the previous total diet study ranged from 0.35 to 0.49 ng.kg bw -1 .d -1 (Anses 2011). EFSA, in 2011, reported similar values (between 0.36 and 1.27 ng.kg bw -1 .d -1 for the sum of the three HBCDDs, EFSA 2011c).

Mean exposure to the sum of the three PBBs ranged from 0.049 ng.kg bw -1 .d -1 in the 1 to 3 years of age children (90 th percentile: 0.074 ng.kg bw -1 .d -1 ) to 0.097 ng.kg bw -1 .d -1 in the 1 to 4 months of age children (90 th percentile: 0.204 ng.kg bw -1 .d -1 , Table 10). These exposure values were in the same order of magnitude than those reported for the 3 to 6 years of age children in the previously conducted total diet study [START_REF] Anses | Avis de l'Anses et rapport d'expertise relatifs à l'Etude de[END_REF].

For TBBPA, mean daily exposures, following the UB hypothesis, ranged from 0.968 to 9.94 ng.kg bw -1 .d -1 for the 1 to 3 years of age group and the 1 to 4 months of age group, respectively (Table 11). One of the children from the 5 to 6 months of age group had exposure estimated at 77.1 ng.kg bw -1 .d -1 , i.e. 14 times higher than the mean exposure of that group of age. The reason for this lying in the fact that this child consumed a follow-on formula highly contaminated compared to other formulae. In 2006, the United-Kingdom committee on toxicity of chemicals in food reported exposures similar to those reported in the present study in 1.5 to 4.5 years of age children (between 7.0 and 4.6 ng.kg bw -1 .d -1 , COT 2006).

Risk assessment

PFAAs

Both PFOA and PFOS bind to plasma protein and are non-genotoxic, therefore allowing the setting of a health based guidance value based on a threshold effect (US-EPA, 2009; EFSA 2018). US-EPA in 2009 set a Reference Dose (RfD) at 0.08 µg.kg bw -1 .d -1 for PFOS based on the effects observed on thyroid in a 182-day study in monkeys [START_REF] Seacat | Subchronic toxicity studies on perfluoroctanesulfonate potassium salt in cynomolgus monkeys[END_REF]. For PFOA, the American environmental protection agency (US-EPA) in 2009 also established a reference dose for PFOA set at 0.2 µg.kg bw -1 .d -1 based on a developmental study performed in mice (Lau et al., 2006). On the other hand, the EFSA set provisional tolerable daily intakes for PFOA and PFOS at, respectively, 0.8 and 1.8 ng.kg bw -1 .d -1 (EFSA, 2018). These health based guidance values were based on, the increase in serum total cholesterol in adults and a decrease in antibody response at vaccination in children for PFOS and on the increase in serum total cholesterol for PFOA.

Anses recently reported some health based guidance values for Perfluorobutanesulfonic acid (PFBS) and Perfluorohexanoic acid (PFHxA) (0.08 and 0.32 mg.kg bw -1 .d -1 , both based on renal toxicity; Anses, 2017a and2017b).

With regard to PFOA and PFOS, based on the RfD set by the US EPA in 2009 for PFOA (0.2 µg.kg bw -1 .d -1 ) and PFOS (0.08 µg.kg bw -1 .d -1 ) the situation appears to not be of health concern (Table 6). However, in 2016, the US EPA reported lower RfD values, based on the result of pharmacokinetic modelling of serum values in animals in conjunction with the criteria of selected effects and their extrapolation to humans (respectively 20 and 30 µg.kg bw -1 .d -1 for PFOA and PFOS, respectively, US-EPA, 2016a and 2016b). We observed that for PFOS, no children included in the present study exceeded this RfD whereas for PFOA, only one child exceeded it. Considering the provisional health based guidance values set by EFSA in 2018 for PFOA and PFOS, high percentages of the 4 populations showed exceedances of the respective health based guidance values when considering the upper bound hypothesis (from 20% to almost 100% of the populations, Table 6).

For PFBS and PFHxA, mean and 90 th percentile exposures were much lower than the respective health based guidance values set by Anses (Anses, 2017a and2017b). For the other PFAAs, given the absence of data that would enable a toxicological point of departure to be established, it was not possible to reach any conclusions as to the health risk associated with dietary exposure to these substances.

BFRs

None of the compounds analyzed in the present study are genotoxic, therefore allowing the setting of health based guidance values based on threshold effects (EFSA 2011). These compounds are lipophilic and accumulate in the adipose tissues.

Regarding the 209 congeners of PBDEs, their chemical structure being similar to that of non-dioxin-like polychlorinated biphenyls (NDL-PCBs) and due to their common mechanism of action [START_REF] Pellacani | Synergistic interactions between PBDEs and PCBs in human neuroblastoma cells[END_REF][START_REF] Miller | Developmental coexposure to polychlorinated biphenyls and polybrominated diphenyl ethers has additive effects on circulating thyroxine levels in rats[END_REF][START_REF] Kodavanti | Polybrominated diphenyl ether (PBDE) effects in rat neuronal cultures: 14C-PBDE accumulation, biological effects, and structure-activity relationships[END_REF] and pending the definition of a health-based guidance value for PBDEs, the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) Expert Committee on food contaminants compared exposure to the following congeners: BDE-28,-47, -99, -100, -153, -154, -183 with the threshold of 10 ng/kg bw/day set for the six NDL-PCBs that are the most frequently found in food (AFSSA, 2007[START_REF] Rivière | Food risk assessment for perfluoroalkyl acids and brominated flame retardants in the French population: results from the second French total diet study[END_REF]. The European Food Safety Authority (EFSA) also set benchmark dose lower confidence limits (BMDLs) for a benchmark response of 10% (BMDL10) for the four most "of concern" PBDEs based on their neurodevelopmental effects: 172 ng.kg bw -1 for BDE-47, 4.2 ng.kg bw -1 for BDE-99, 9.6 ng.kg bw -1 for BDE-153 and 1,700 μg.kg bw -1 for BDE-209 (EFSA, 2011a). Regarding HBCDDs, EFSA identified neurodevelopmental effects on behavior as the critical endpoint, and derived a BMDL10 of 0.79 mg.kg bw -1 . Due to the limitations and uncertainties in current knowledge, EFSA concluded that it was inappropriate to use this BMDL to establish a health-based guidance value, and instead recommended using the margin of safety (MOS) approach for the risk characterisation of HBCDDs by comparing dietary intake with the estimated dietary human intake associated with the body burden at the BMDL10, set at 0.003 ng.kg bw -1 .d -1 . In its opinion on PBBs, EFSA could not define a health-based guidance value, but suggested comparing data on exposure to PBBs with a no observed adverse effect level (NOAEL) of 0.15 mg/kg bw/day based on the induction of hepatic carcinomas in rats (EFSA, 2010a). With regards to TBBPA, the toxicology point of departure (BMDL10) was established in rat reprotoxicity study and based on the effects on the homeostasis of thyroid hormones (decrease of the circulating levels of hormone T4 in the first generation). It was set at 16 mg.kg bw -1 .d -1 (EFSA 2011b). Due to its specific characteristics in terms of pharmacokinetics and toxicity, BDE-209 was considered separately from the other seven congeners, whose chemical structure and mechanism of action are similar to those of NDL-PCBs.

For the six NDL-PCBs the threshold value (10 ng/kg bw/day) was not observed to have been exceeded (Table 8).

The margins of safety calculated for BDE-209 (with regard to the BMDL10 of 1 700 µg.kg bw -1 .d -1 ) ranging from 430,000 to 1,5 million, are much larger than the 2.5 margin deeming tolerable by EFSA (Table 7). For PBDEs, the situation is therefore deemed to be tolerable considering these health-based guidance values.

Regardless of the age group, the margins of safety calculated with regard to the BMDL10 established by EFSA for the sum of the three HBCDDs (3 000 ng.kg bw -1 .d -1 ) ranged 70 for the 1-4 months using the 90th percentile of exposure to 5940 using the median of exposure of the 13-36 months (Table 9). They were higher than the critical margin of safety value set at 25 to consider the exposure as being of no health concern.

The margins of safety calculated using the no adverse effect level observed by EFSA for the sum of the three PBBs (0.15 mg.kg bw -1 .d -1 ) ranged 740,000 to more the 3,000,000, depending on the age group and the exposure value (mean or P90) (Table 10). They are then considered as high enough to conclude that the situation is of no health concern.

For TBBPA, the margins of safety calculated using the BMDL10 established by EFSA (16 mg.kg bw -1 .d -1 ), ranging 511,000 to more than 16 million (Table 11), are also large enough (higher than 1 000 considered by EFSA as the critical margin of exposure) to conclude that the situation is of no health concern.

Overall, the risk related to the dietary exposures to these compounds appeared not to be of concern, for infants, similarly to the general population older than 3 years old [START_REF] Rivière | Food risk assessment for perfluoroalkyl acids and brominated flame retardants in the French population: results from the second French total diet study[END_REF]. However, it seems of importance to note that the present analysis focused on general infant population and then does not take into account some specific situations such as accidental contaminations. Moreover, the present study does not take into account breastfed infants where lipophilic compounds are known to accumulate [START_REF] Colles | Fourth WHO-coordinated survey of human milk for persistent organic pollutants (POPs): Belgian results[END_REF].

Uncertainties

Some uncertainties are important to mention in parallel to the presented results. Indeed, risk analysis processes suppose some hypotheses that can lead to over or underestimation of the risk. The main identified uncertainties are the following. First, the available data to set health based guidance values. For many perfluoroalkyl acids and brominated flame retardants, there is, as of today, not enough robust toxicological data to establish robust health based guidance values. Additionally, considering the population of interest of this study, one has to mention that some toxicological endpoints specific to this population could exist due to the development of the organisms. Efforts have been made to consider values that should be protective to the infant population, however, experimental data to come could lead to lower health based guidance values than the one considered in the present study, as it was the case for PFOS and PFOA with decreasing health-based guidance values with time. Moreover, synergistic or antagonistic effects could occur due to the exposure of substances from the same chemical family. Some uncertainties are due to the consumption data used to perform exposure calculations. Since only main practices are considered in the present study, specific dietary habits that could lead to different exposure patterns are not observed. Finally, analytical methodology could lead to uncertainties due to the precision of the method but also due to the limits of detection and quantification that can be achieved. This, indeed, has some impact on the exposure calculation as seen above for PFOS and PFOA. In the case of a limit of detection far above the concentrations measured in the food items, the upper bound hypothesis would lead to a significant overestimation of the exposure.

Conclusion

The results presented above allow to assess the risk related to the dietary exposure of some PFAAs and of some BFRs. For the compounds analyzed, dietary exposures appear to be of no health concern in the 0 to 3 years of age children with regard to the selected health based guidance values. However, considering the newly established RfD for PFOS and PFOA by US-EPA, for some children with specific diets the risk can not be excluded. This analysis also reveals that form some congeners (especially for some PFAAS), no health based guidance values are as of today available due to the paucity of robust toxicological data, preventing to perform a risk assessment. It is also of importance to consider that new toxicological data becoming available, health based guidance values could be lowered, especially for compounds for which some endocrine disruptor effects have been reported but no characterized enough as of today to set a health based guidance value.

Table 1

 1 Mean concentrations (ng.kg -1 fw) of PFOA and PFOS in common and infant specific food items

						PFBS				PFHxA				PFOA					PFOS
				LB		UB			LB		UB				LB	UB				LB		UB
	Food	N a					a Mean	(Min-	Mean	(Min-	a	Mean	(Min-Max)	Mean	(Min-	a	Mean	(Min-	Mean	(Min-
	category								±SD	Max)	±SD	Max)		±SD		±SD	Max)		±SD	Max)	±SD	Max)
												Common food
	Non-alcoholic beverages	1	0	0±0	(0-0)	9.19±0	(9.19-9.19)	0	0±0	(0-0)	9.19 ±0	(9.19-9.19)	0	0±0	(0-0)	13.78 ±0	(13.78-13.78)	0	0±0	(0-0)	9.18 ±0	(9.18-9.18)
	Water	12 0	0±0	(0-0)	0.462±0.006	(0.454-0.474)	0	0±0	(0-0)	0.462 ±0.006	(0.454-0.474)	0	0±0	(0-0)	0.462 ±0.0061	(0.454-0.474)	0	0±0	(0-0)	1.08 ±0.53	(0.91-2.77)
	Dairy-based desserts	1	0	0±0	(0-0)	27.55±0	(27.55-27.55)	0	0±0	(0-0)	27.55 ±0	(27.55-27.55)	0	0±0	(0-0)	41.33 ±0	(41.33-41.33)	0	0±0	(0-0)	27.55 ±0	(27.55-27.55)
	Milk	1	0	0±0	(0-0)	4.54±0	(4.54-4.54)	0	0±0	(0-0)	4.54 ±0	(4.54-4.54)	0	0±0	(0-0)	4.54 ±0	(4.54-4.54)	0	0±0	(0-0)	45.40 ±0	(4.54-4.54)
	Vegetables (excluding potatoes)	1	0	0±0	(0-0)	12.35±0	(12.35-12.35)	0	0±0	(0-0)	12.35 ±0	(12.35-12.35)	0	0±0	(0-0)	18.53 ±0	(18.53-18.53)	0	0±0	(0-0)	12.35 ±0	(12.35-12.35)
	Mixed dishes	1	0	0±0	(0-0)	42.66±0	(42.66-42.66)	0	0±0	(0-0)	42.66 ±0	(42.66-42.66)	0	0±0	(0-0)	63.99 ±0	(63.99-63.99)	0	0±0	(0-0)	42.66 ±0	(42.66-42.66)
	Fish	1	0	0±0	(0-0)	25.91±0	(25.91-25.91)	0	0±0	(0-0)	25.91 ±0	(25.91-25.91)	100	30.14 ±0	(0.030139-0.030139)	30.14 ±0	(30.14-30.14)	100	277.4 ±0	(277.4-277.4)	277.4 ±0	(277.4-277.4)
	Ultra-fresh dairy products	1	0	0±0	(0-0)	21.76±0	(21.76-21.76)	0	0±0	(0-0)	21.76 ±0	(21.76-21.76)	0	0±0	(0-0)	32.64 ±0	(32.64-32.64)	0	0±0	(0-0)	21.76 ±0	(21.76-21.76)
	Meat	1	0	0±0	(0-0)	37.97±0	(37.97-37.97)	0	0±0	(0-0)	37.97 ±0	(37.97-37.97)	0	0±0	(0-0)	56.95 ±0	(56.95-56.95)	0	0±0	(0-0)	37.97 ±0	(37.97-37.97)
	Poultry and game	2	0	0±0	(0-0)	37.22±1.59	(36.09-38.34)	0	0±0	(0-0)	37.22 ±1.59	(36.09-38.34)	0	0±0	(0-0)	55.82 ±2.38	(54.14-57.51)	0	0±0	(0-0)	37.22 ±1.59	(36.09-38.34)
												Infant food
	Milk-based beverage	8	0	0±0	(0-0)	16.22±2.92	(12.53-19.34)	0	0±0	(0-0)	16.22 ±2.92	(12.53-19.34)	0	0±0	(0-0)	32.24 ±5.84	(25.05-38.69)	0	0±0	(0-0)	16.22 ±2.92	(12.53-19.34)
	Cereals-based food	5	0	0±0	(0-0)	36.93±0.17	(36.66-37.11)	0	0±0	(0-0)	36.93 ±0.17	(36.66-37.11)	0	0±0	(0-0)	36.93 ±0.17	(36.66-37.11)	0	0±0	(0-0)	18.46 ±0.084	(18.33-18.56)

N : number of composite samples analysed LB : Lower bound hypothesis ; UB :Upper bound hypothesis a: %age of detection

Table 2

 2 Mean concentrations (pg.g -1 fw) of PBDEs in food

	Food item

Table 3

 3 Mean concentrations (pg.g -1 fw) of HBCDDs in food

	Food item	N	HBCDD α % detection LB	UB	HBCDD β % detection LB	UB	HBCDD γ % detection LB	UB	Sum of the HBCDDs % detection LB	UB
					Infant specific food
	Milk-based beverage	8	100	14.27 14.27	88	3.36	3.39	88	5.14	5.31	92	22.77 22.97
	Cereals	5	40	11.04 12.31	0	0.00	0.44	0	0.00	1.85	13	11.04 14.59
	Milk-based desserts	6	100	26.81 26.81	83	6.94	7.03	83	8.87	9.02	89	42.61 42.85
	Growing-up milk	9	89	3.56	3.63	67	0.80	0.91	67	0.96	1.21	74	5.32	5.74
	Soups and puree	11	18	0.96	1.48	9	0.25	0.38	9	0.27	0.61	12	1.49	2.46
	Fruit puree	4	75	4.28	4.55	25	0.48	1.01	50	1.24	1.84	50	5.99	7.40
	Vegetable-based ready-to-eat meal	20	70	5.16	5.67	50	1.04	1.27	45	1.35	1.71	55	7.54	8.65
	Meat/fish-based ready-to-eat meal	45	76	8.90	9.16	49	1.75	1.87	44	2.25	2.54	56	12.89 13.57
	Infant formula	28	61	8.41	8.74	18	1.95	2.20	18	2.46	2.89	32	12.81 13.83
	Follow-on formula	33	33	2.45	3.13	12	0.48	0.72	12	0.67	1.03	19	3.60	4.88

Table 4

 4 Mean concentrations (pg.g -1 fw) of PBBs in food

	Food item	N	BB 52	BB 101	BB 153	Somme des PBB

AKNOWLEDGMENTS

The authors are grateful to the Anses expert committee panel in charge of assessing the chemical risk in food and the experts from the infant TDS scientific committee, namely: Jean-Pierre Cravédi, Claude Atgié, Pierre-Marie Badot, Jacques Bélégaud, Catherine Bennetau-Pelissero, Valérie Camel, Guillaume Duflos, Camille Dumas, Jérôme Gay-Queheillard, Philippe Glorennec, Thierry Guérin, Laurence Guldner, Laïla Lakhal, Claude Lambré, Raphaëlle Le Garrec, Catherine Leclerc, Eric Marchioni, César Mattéi, André Mazur, Sakina Mhaouty-Kodja, Fabrice Nesslany, Alain-Claude Roudot, Patrick Sauvegrain, Rémy Slama, Karine Tack, Eric Verdon and Jean-Paul Vernoux.

The infant TDS was supported by the Ministry for food, agriculture and fisheries, the Ministry for health the Ministry for ecology and sustainable development and the French Agency for Food, Environmental and Occupational Health & Safety (ANSES).


Elodie Martinez
email: elodie.martinez@ird.fr

Dionysios E Raitsos

David Antoine

Warmer, deeper and greener mixed layers in the north Atlantic subpolar gyre over the last 50 years

Keywords: phytoplankton decadal variability, bottom-up processes and top-down control, North Atlantic subpolar gyre, climate cycles, chlorophyll, Continuous Plankton Recorder (CPR), plankton climate links, NAO, AMO

Shifts in global climate resonate in plankton dynamics, biogeochemical cycles, and marine food webs. We studied these linkages in the North Atlantic Subpolar Gyre (NASG), which hosts extensive phytoplankton blooms. We show that phytoplankton abundance increased

Introduction

Phytoplankton (the microalgae that populate the upper lit layers of the ocean) fuel the oceanic food web and affect oceanic and atmospheric carbon dioxide levels through photosynthetic carbon fixation. The North Atlantic Subpolar Gyre (NASG) is home of the most widespread phytoplankton blooms globally, and is currently one of the largest oceanic sinks of atmospheric CO 2 [START_REF] Sabine | The oceanic sink for anthropogenic CO2[END_REF]. Observations in this area were used by [START_REF] Sverdrup | On conditions for the vernal blooming of phytoplankton[END_REF] to elucidate the conditions leading phytoplankton to bloom. Later on, the role of upper ocean stratification and dynamics (bottom-up processes) on the seasonal to interannual variability of phytoplankton in this region has been largely investigated using

Accepted Article

This article is protected by copyright. All rights reserved. recent (< 15 years) satellite ocean color observations [START_REF] Dutkiewicz | Interannual variability of phytoplankton abundances in the North Atlantic[END_REF][START_REF] Follows | Meteorological modulation of the North Atlantic spring bloom[END_REF][START_REF] Siegel | The North Atlantic spring bloom and Sverdrup's critical depth hypothesis[END_REF][START_REF] Behrenfeld | Abandoning Sverdrup's Critical Depth Hypothesis on phytoplankton blooms[END_REF]. Top-down control, i.e., zooplankton grazing, has been investigated on longer time scales using sustained biological observations mainly in the northeastern Atlantic and adjacent coastal seas [START_REF] Beaugrand | Long-term changes in phytoplankton, zooplankton and salmon related to climate[END_REF]. In these regions, observations revealed that phytoplankton changes over 1950-2000 were related to both top-down control and sea surface temperature variability [START_REF] Edwards | Ocean climate anomalies and the ecology of the North Sea[END_REF][START_REF] Barton | The Continuous Plankton Recorder survey and the North Atlantic Oscillation: Interannual-to multidecadal-scale patterns of phytoplankton variability in the North Atlantic Ocean[END_REF].

Coupled bio-physical models include the mechanisms of these controls, yet they diverge in reproducing decadal observations (Henson et al., 2009;[START_REF] Patara | Marine biogeochemical responses to the North Atlantic Oscillation in a coupled climate model[END_REF], in particular phytoplankton regime shifts (Henson et al., 2009b). Phytoplankton grow as a result of the combined supply of nutrients and light in the upper layers of the ocean. Vertical mixing of the water column can upwell nutrients up to the euphotic layer, resulting in a positive relationship between mixing and nutrient concentrations. In contrast, vertical mixing could also entrain phytoplankton down to depths where sunlight does not penetrate, generating light-limitation of photosynthesis and a negative relation between mixing and average available light. Both effects coexist in subpolar regions such as the NASG, where phytoplankton are light-limited in winter and subsequently bloom in spring when light increases and the mixed layer (ML) shallows. This bloom ends when phytoplankton are either starved of nutrients and/or grazed by zooplankton. On time scales from inter-annual to decadal, a decrease of winter mixing in subpolar regions due to global warming would reduce the time during which the ML is deeper than the illuminated upper layer. The consequence of such a shallower mixed layer depth would be an earlier start of the phytoplankton spring bloom and enhanced phytoplankton biomass [START_REF] Doney | Plankton in a warmer world[END_REF]. Contrary to this scheme, however, an increase of phytoplankton biomass has been reported in the NASG over the last decades [START_REF] Reid | Phytoplankton change in the North Atlantic[END_REF][START_REF] Raitsos | Extending the SeaWiFS chlorophyll data set back 50 years in the northeast Atlantic[END_REF]Henson et al., 2009b) although the ML has deepened (Carton et

Accepted Article

This article is protected by copyright. All rights reserved. al., 2008) and has become warmer [START_REF] Sutton | Atlantic Ocean forcing of North American and European summer climate[END_REF]. The comparison of historical and contemporaneous satellite ocean color observations has also revealed that phytoplankton biomass was higher and ML was deeper in the North Atlantic Ocean in the 2000s as compared to the 1980s [START_REF] Martinez | Phytoplankton spring and fall blooms in the North Atlantic in the 1980s and 2000s[END_REF].

Therefore uncertainties on interactions among phytoplankton, zooplankton, and the physical environment at decadal scales are still large. These interactions are sensitive to climate [START_REF] Behrenfeld | Climate-driven trends in contemporary ocean productivity[END_REF][START_REF] Martinez | Climate-driven basin-scale decadal oscillations of oceanic phytoplankton[END_REF][START_REF] Boyce | Global phytoplankton decline over the past century[END_REF], and need to be better understood in order to forecast the future evolution of phytoplankton. Studies of these linkages are, however, usually limited by the short duration of available observations with respect to decadal time scales.

The aim of this study is to assess relationships between phytoplankton decadal variability and bio-physical dynamics in the NASG (40°-15°W; 50°-60°N; blue and red boxes on Fig. 1). This study addresses three questions: 1) how are stratification and phytoplankton biomass variability related at decadal time-scales? 2) How does top-down control influence phytoplankton decadal variability? 3) To which climate modes are decadal changes in bottom-up and top-down processes related to?

Materials and methods

Plankton datasets (phytoplankton and zooplankton) are acquired from the Continuous Plankton Recorder (CPR) survey, which consistently samples the upper ocean in the North Atlantic Ocean and North Sea using a virtually unchanged methodology since 1948 [START_REF] Richardson | Using continuous plankton recorder data[END_REF]. The Phytoplankton Color Index (PCI), a proxy of phytoplankton biomass has been extensively used to describe major spatio-temporal patterns of phytoplankton [START_REF] Reid | Phytoplankton change in the North Atlantic[END_REF][START_REF] Head | Spatial and inter-decadal variability in plankton abundance and composition in the Northwest Atlantic (1958-2006[END_REF], and has been validated with in situ and remotely sensed datasets [START_REF] Raitsos | A 60 year ocean colour dataset from the Continuous Plankton Recorder[END_REF]. Here, phytoplankton biomass is expressed

Accepted Article

This article is protected by copyright. All rights reserved. in terms of chlorophyll concentration (Chl, mg.m -3 ), which was derived from a calibration of the PCI using satellite ocean color observations [START_REF] Raitsos | Extending the SeaWiFS chlorophyll data set back 50 years in the northeast Atlantic[END_REF]Henson et al., 2009b).

In addition to PCI, taxonomic information (i.e. diatoms and dinoflagellates abundances) is also acquired from the CPR database to investigate changes in community structures.

Abundances of these phytoplankton functional groups are number of counts per sample, which represents ~3 m 3 of filtered seawater.

Zooplankton are key components in the marine food-web of the subarctic North Atlantic.

To account part of the top-down control, the abundance of the CPR total Calanus and total copepods was acquired. These zooplankton taxonomic groups include Calanus Finmarchicus which is one of the most commonly found species of zooplankton, accounting for the majority of copepod abundance in the subarctic waters of the North Atlantic.

We have used the CPR datasets over 1960-2010 in three well-sampled regions based on Henson et al. (2009b). However, the western and eastern NASG areas have been slightly reduced in the present study (Fig. 1) as compared to Henson et al. (2009b;their Mixed-layer depth (MLD) and sea-surface temperature (SST) are issued from the Simple Ocean Data Assimilation (SODA, version 2.1.6; http://soda.tamu.edu; [START_REF] Carton | A reanalysis of ocean climate using Simple Ocean Data Assimilation (SODA)[END_REF] over 1960-2008 on a 0.5° grid. MLD is determined as being the depth where the potential density is 0.03 kg.m -3 higher than its value at 10 m (de Boyer [START_REF] De Boyer Montégut | Mixed layer depth over the global ocean: An examination of profile data and a profile-based climatology[END_REF].

Monthly wind speeds over 1960-2009 are issued from the Woods Hole Oceanographic Institute Objectively Analysed Air-Sea Fluxes-V3 (WHOI OAFlux) available on a 1° grid (http://oaflux.whoi.edu; [START_REF] Yu | Multidecade Global Flux Datasets from the Objectively Analyzed Air-sea Fluxes (OAFlux) Project: Latent and sensible heat fluxes, ocean evaporation, and related surface meteorological variables[END_REF]. Monthly net heat losses from the ocean are issued

Accepted Article

This article is protected by copyright. All rights reserved. from the National Center for Environmental Prediction / National Center for Atmospheric Research (NCEP/NCAR) 40-year reanalysis, available on a T62 Gaussian grid (~1.9° grid) over 1960-2010 (http://www.esrl.noaa.gov/psd; [START_REF] Kalnay | The NCEP/NCAR 40-year reanalysis project[END_REF].

The monthly NAO index since January 1950 is derived through a rotated principal component analysis of monthly standardized 500-mb height anomalies over 20°N to 90°N and is provided by the Climate Prediction Centre (http://www.cpc.ncep.noaa.gov). The monthly, unsmoothed AMO index since 1948 is provided by the National Oceanic and Atmospheric Administration (NOAA) Earth System Research Laboratory. The time series is the area-weighted average over the North Atlantic (0°N-70°N) derived from the Kaplan SST dataset (http://esrl.noaa.gov).

Biological and physical parameters have been spatially averaged over each region.

Available raw monthly CPR data can be seen on Figure 2 (colored plate). Years with more than four missing months and with three consecutive missing months were removed from the analysis. Linear interpolations in time were carried out and annual means were calculated. A linear interpolation in time has been performed on the annual mean of CPR data to fill the 1year gap in 1982 in the W-NASG, and in 1996 and 2009 in the E-NASG. As the time series of the monthly physical parameters had full time-coverage, their annual means were calculated without any statistical treatment. Anomalies are computed as the difference between the average of a given year minus the mean of the entire series. By removing the seasonality from the data set and computing correlations on the residuals, temporalautocorrelation is reduced. Cumulative sum of these anomalies are derived to detect changes of a given parameter, as well as the intensity and duration of its decadal variability [START_REF] Leterme | Decadal basin-scale changes in diatoms, dinoflagellates, and phytoplankton color across the North Atlantic[END_REF][START_REF] Leterme | Decadal fluctuations in North Atlantic water inflow in the North Sea between 1958-2003: impacts on temperature and phytoplankton populations[END_REF]. Successive positive residuals are observed when annual values are consistently above the climatological average, producing an increasing slope, and conversely.

No slope shows a succession of values similar to the mean. Note that the cumulative sum

Accepted Article

This article is protected by copyright. All rights reserved.

method is sensitive to missing data, thus, due to excess data gap in the CPR dataset over 1986-1990, this method was not applicable to W-NASG.

In order to quantify the spatial and temporal decadal variability of physical parameters (i.e., MLD, wind speed, SST, net heat flux) in the NASG over the last five decades, Empirical Orthogonal Function analyses (EOFs) have been performed on annual anomaly fields (see supplemental figures).

Results

Phytoplankton decadal variability in the NASG

A sharp Chl increase over 1985-1991 delineates a regime with lower Chl concentrations before 1985 and a regime with higher concentrations after 1990 in the N-Seas (Fig. 2a). This change has been previously reported (e.g., [START_REF] Edwards | Long-term and regional variability of phytoplankton biomass in the Northeast Atlantic (1960-1995)[END_REF][START_REF] Raitsos | From silk to satellite: half a century of ocean colour anomalies in the Northeast Atlantic[END_REF]. The corresponding anomaly of annual Chl (Chl ano ) and the corresponding cumulative sum are displayed as the black and red curves, respectively, to highlight the timing and duration of decadal changes. The descending (ascending) slope of the cumulative curve before (after) the mid-1980s corresponds to the period with below-average (above-average) Chl ano . These patterns are partly observed in the W-NASG (Fig. 2b), where Chl values are also higher after the mid-1980s in comparison to their values before that time. Chl reacts differently in the E-NASG (Fig. 2c). A sharp increase occurs from the mid-1980s to the mid-1990s. Then, Chl decreases and the cumulative sum shows values close to average (i.e., no slope) vs. lower than average values (descending slope) before 1985. These Chl trends were reported till 2004 by Henson et al. (2009b) in the NASG and N-Seas, and they appear to be maintained till

Accepted Article

This article is protected by copyright. All rights reserved.

Changes in community structures

Diatoms and dinoflagellates are phytoplankton functional types key to the classic North Atlantic spring bloom scenario. Thus, they could be used to better understand the processes leading to the decadal variability observed in Chl. In the NASG, diatoms bloom in spring, followed by dinoflagellates and other small phytoplankton in summer [START_REF] Mcquatters-Gollop | Spatial patterns of diatom and dinoflagellate seasonal cycles in the NE Atlantic Ocean[END_REF]. The interannual variability of Chl ano in the W-NASG is well correlated with anomalies of diatom and dinoflagellate abundance (r=0.52 and 0.5, respectively, p<0.01; Fig. 3a).

Similar relationships hold in the E-NASG (r=0.62 and 0.47, p<0.01; Fig. 3b). The decadal trends of Chl ano in the W-NASG and E-NASG are also observed for diatom and dinoflagellate abundance. In the W-NASG, the post-1980s increase of Chl is associated with an increase of diatoms from March to September and of dinoflagellates from May to October (Fig. 3c). In the E-NASG, the sharp increase of Chl over 1986-1995 corresponds to a large increase in diatom and dinoflagellate abundance (Fig. 3b) from April to October and June to November, respectively (Fig. 3d, vertical bars with horizontal hatched).

There is a good correspondence between changes in abundance of diatoms and dinoflagellates and changes in Chl in the W-and E-NASG (except after 1996 in the E-NASG where abundance of diatoms and dinoflagellates decreases while Chl ano is moderately above the average). Although part of the phytoplankton community that contributes to Chl is not captured by the CPR (e.g. picophytoplankton), this result suggests Chl as a good indicator of phytoplankton biomass in the sub-polar gyre, contrary to more oligotrophic areas where phytoplankton photo-adaptation may jeopardize the Chl-biomass relationship [START_REF] Siegel | Regional to global assessments of phytoplankton dynamics from the SeaWiFS mission[END_REF].

Accepted Article

This article is protected by copyright. All rights reserved.

Decadal variability in stratification, and associated climate modes

We investigated the role of MLD, winds, heat loss from the ocean and SST in forcing the decadal variability of plankton. These parameters are issued from models or objectively The decadal variability in winds, heat loss from the ocean, and MLD, follows the low frequency signal of the NAO in both E-and W-NASG (black line vs. shaded area on Fig. 4a,b; Fig. S1d,e) [START_REF] Eden | Mechanism of interannual to decadal variability of the North Atlantic Oscillation[END_REF][START_REF] Visbeck | The North Atlantic Oscillation: climatic significance and environmental impact[END_REF]. Changes in the slopes of the cumulative sums highlight that, before 1980, the negative phase of the NAO (NAO-) resulted in weaker westerlies and weaker heat loss from the ocean, leading to shallower MLD (downward slope). Then, the NAO turned positive (NAO+) in the early 1980s and further strengthened in the mid-1980s, associated with stronger winds, stronger heat loss from the ocean, and deeper ML (upward slope). In the mid-1990s the NAO turned from a strong to a moderate NAO+ (horizontal slope). Figures 4a andS1b illustrate these wind speed changes between strong and moderate NAO+ conditions, homogenous across the NASG. Our results

Accepted Article

This article is protected by copyright. All rights reserved. highlight that the observed changes in physics are related to the broader climate (as seen by climate indices).

Bottom-up processes

Weaker mixing is associated with weaker (below average) phytoplankton biomass before 1980 under NAO-conditions, and vice-versa after the mid-1980s, in both E-and W-NASG (Fig. 4a,b respectively, black vs. green lines and shaded areas). Stronger winds and heat losses from the ocean in winter under NAO+ conditions lead to deeper MLs. The wintertime MLD maximum is about 25 m and 45 m (up to 150 m some years) deeper on average after the mid-1980s in the W-and E-NASG, respectively. This ML deepening possibly induced a larger nutrient input to the surface, creating favorable conditions for phytoplankton increase in spring and summer. Consistently, higher Chl are associated with higher abundances of diatoms and dinoflagellates in the W-NASG (E-NASG) over 1986-2010 (1986-1995) than prior to 1985. The increase of diatoms during NAO+ events could be related with nutrient richer waters in early spring due to deeper MLs but also to their ability to grow quickly under turbulent conditions [START_REF] Margalef | Assessment of the effects on plankton[END_REF][START_REF] Margalef | Turbulence and marine life[END_REF]. Then, a larger pool of recycled nutrients could be available in summer, allowing the increase of dinoflagellates and secondary production.

Deeper MLs during winter could be expected to extend the duration of the period when phytoplankton growth is light limited. Consequently, the initiation of phytoplankton blooms would be delayed, the growth season shortened, and the annual phytoplankton biomass decreased as shown in the modeling study by Henson et al. (2009). On the other hand, modeling has also shown that deeper mixing occurring during NAO+ conditions at subpolar latitudes can cause a decrease in late winter Chl due to light limitation, which is however overcome by an increase in spring Chl due to higher nutrient availability [START_REF] Patara | Marine biogeochemical responses to the North Atlantic Oscillation in a coupled climate model[END_REF].

While our results do not show a shortened growth season, possibly due to the monthly resolution, they appear to be in agreement with an increase in spring Chl as in Patara et al.

Accepted Article

This article is protected by copyright. All rights reserved.

(2011) (Fig. 2b,c for Chl, Fig. 3c,d for community structures). Thus, stronger winds and deeper MLs possibly associated with a larger nutrient pool could enhance phytoplankton biomass in the NASG under NAO+ conditions.

Top-down control

We finally considered the top-down control (i.e., grazers controlling the abundance of phytoplankton) through the variability of total Calanus and total copepod abundances as well as their ratio in the NASG. Mesozooplankton are important consumers of phytoplankton, particularly in productive ecosystems [START_REF] Calbet | Mesozooplankton grazing effect on primary production: a global comparative analysis in marine ecosystems[END_REF].

Year-to-year fluctuations in Calanus follow that of total copepods in the NASG (not shown). In the W-NASG, annual anomalies of Calanus and copepod abundances are anticorrelated with Chl ano over 1960-1987 (r 1960-1987 = -0.46, p<0.01) and after 1996 (Fig. 4c, red vs. green line). Negative correlations between abundances of predator and prey highlight the impact of top-down control in this region as postulated by [START_REF] Frank | Trophic cascades in a formerly cod-dominated ecosystem[END_REF]. This copepod-Chl anti-correlation, which occurs over almost forty years, is temporarily disrupted from 1991 to 1995 with simultaneous positive anomalies in Chl and copepod abundances. This increase of copepods is not associated with a parallel drop of Chl, suggesting a temporary decoupling between phytoplankton and copepods. This decadal variability of Chl concentrations and copepod abundance could be related through at least two processes. First, changes in MLD may influence the disturbancerecovery cycle following concepts proposed by [START_REF] Behrenfeld | Annual cycles of ecological disturbance and recovery underlying the subarctic Atlantic spring plankton bloom[END_REF]. Second, changes in SST are known to influence zooplankton abundance and community structures.

The disturbance-recovery hypothesis proposes that deep winter mixing generates a spatial decoupling between phytoplankton and their predators (zooplankton). Pressure from grazers on the phytoplankton population would be relieved by decreasing their encounter rates.

Springtime stratification would enhance phytoplankton specific growth rates, while

Accepted Article

This article is protected by copyright. All rights reserved. simultaneously intensifying loss rates by predation, effectively 'recoupling' predators and prey when they become confined to a smaller water volume. Modeling has shown that deeper mixing would consistently yield greater winter decoupling between phytoplankton divisions and grazing losses and, consequently, higher phytoplankton inventories [START_REF] Behrenfeld | Annual cycles of ecological disturbance and recovery underlying the subarctic Atlantic spring plankton bloom[END_REF].

Copepods are known to be temperature sensitive [START_REF] Beaugrand | Reorganization of North Atlantic marine copepod biodiversity and climate[END_REF][START_REF] Beaugrand | Long-term changes in phytoplankton, zooplankton and salmon related to climate[END_REF][START_REF] Beaugrand | Unanticipated biological changes and global warming[END_REF], thus we investigated their changes in parallel to changes in SST. As mentioned in the bottom-up section, the spatial variability of the SST first component is homogenous (i.e., of the same sign everywhere in the domain) within each region (Fig. S2a), and is representative of the spatially averaged annual mean variations in the NASG (Fig. S2b, black vs. colored lines). The AMO index well describes SST decadal variability in the NASG.

Colder SST and a cold phase of the AMO can be observed prior to 1996 in the NASG (shaded area on Fig. S2b and Fig. 4c). Then, a shift to a warm phase occurs. In the mid-1990s when warmer SST associated with the AMO warm phase takes place, zooplankton abundance decreases (r sst-ttcop = -0.43, p<0.01; 43 cells per sample on average over 1960-1987vs. 30 over 1996[START_REF] Behrenfeld | Abandoning Sverdrup's Critical Depth Hypothesis on phytoplankton blooms[END_REF]Fig 4c) as well as the abundance of total Calanus compared to total copepods decreases by up to 20% (r sst-cal/cop = -0.52, p<0.01; Fig. 4d).

Thus, before the mid-1980s, the NAO-regime provides unfavorable bottom-up and topdown conditions with low MLD leading to a low nutrient pool and possibly a weak decoupling between phytoplankton and grazers, reducing phytoplankton biomass. In addition, before the mid-1990s, the AMO-regime also provides unfavorable top-down conditions with cold SST leading to high copepod abundance also reducing phytoplankton biomass.

Conversely, after the mid-1980s, the NAO+ regime provides more favorable bottom-up and top-down conditions with deeper MLD leading to a higher nutrient pool and a greater decoupling between phytoplankton and grazers, enhancing phytoplankton biomass. In

Accepted Article

This article is protected by copyright. All rights reserved. addition, after the mid-1990s, the AMO+ regime also provides favorable top-down conditions with warmer SST leading to lower copepod abundance also enhancing phytoplankton biomass. From 1996 onwards, the abundance of total Calanus also decreases by up to 20% as compared to total copepods (Fig. 4d, blue line). Such a change in the zooplankton community structure possibly also modifies the grazing pressure on phytoplankton. Finally, the 1991-1996 temporary parallel increase of both phytoplankton and copepods occurs when the NAO and AMO low frequency regimes are out of phase (NAO+ vs. AMO-). Under AMO-, zooplankton abundance remains high and changes in community structures have not yet occurred. The expected prey-predator decoupling under NAO+ seems to be overcame by the bottom-up control. During this time period when wind, MLD and net heat flux undergone their strongest increase over the five decades, the associated strong input of nutrients would be the main driver of the strong Chl increase followed by a strong zooplankton increase.

In the E-NASG, Calanus and copepod mean abundance and standard deviation are lower than in the W-NASG (6.3 ± 4.4 vs. 41.2 ± 23.7 cells per sample, respectively). This weaker grazing pressure possibly explains the weak correlation between copepods and phytoplankton (Fig. S3), and a weaker top-down control contributing to higher phytoplankton abundance than in the western region. Moreover, according to the decoupling-recovery theory, and as shown in [START_REF] Behrenfeld | Annual cycles of ecological disturbance and recovery underlying the subarctic Atlantic spring plankton bloom[END_REF], the top-down control would be less efficient in this deep wintertime mixed layer maximum environment. In the E-NASG, the wintertime maximum MLD is on average 60 to 85 m deeper (before and after 1985, respectively) than in the W-NASG, possibly inducing a lower top-down control.

Accepted Article

This article is protected by copyright. All rights reserved.

DISCUSSION:

Decadal changes in phytoplankton abundance in the whole NASG have been shown here to be associated with the changes of bottom-up, and possibly top-down forcing, related to the low frequency regimes of the NAO. Our results show that phytoplankton has increased as the mixed layer became deeper. The expected paradigm would be that this increase of phytoplankton is observed when the ML becomes shallower due to surface warming in the subpolar region [START_REF] Doney | Plankton in a warmer world[END_REF]. We hypothesize that under NAO+ conditions, the observed stronger winds, stronger heat losses from the ocean, and increased mixing, have enhanced nitrate availability in the euphotic zone [START_REF] Patara | Marine biogeochemical responses to the North Atlantic Oscillation in a coupled climate model[END_REF][START_REF] Henson | Global patterns in efficiency of particulate organic carbon export and transfer to the deep ocean[END_REF], thus possibly explaining the higher Chl concentrations and higher diatom/dinoflagellate abundances observed after 1985. The decrease of the prey-predator encounter rate that can be expected when the mixed layer is deeper (Behrenfled et al., 2013) is another possible cause of the increased phytoplankton abundance. The possible influence of the AMO on top-down control of phytoplankton by copepods has been shown for the western region. In this area a lower grazing pressure due to higher SST under AMO+ conditions would also have provided better conditions for phytoplankton growth.

It is expected that warming in subpolar regions would lead to shallower MLs and an increase of phytoplankton due to less light limitation [START_REF] Doney | Plankton in a warmer world[END_REF]. Consistently with the first part of this paradigm concerning SST and MLD, we found shallower MLs associated with warmer SST during NAO-conditions in the 1960s-1980s, compared with deeper MLs and colder SST during strong NAO+ conditions over the mid 1980s-1990s. Then, this covariability breaks down after 1995 when the AMO turns into a warm phase while the NAO remains positive. Thus, ML remains deeper than before the 1980s while SST becomes warmer partly contradicting the paradigm. A switch over, from a cold subpolar gyre during a positive NAO index phase to a warmer than normal state, seems to occur if the positive phase

Accepted Article

This article is protected by copyright. All rights reserved. persists for more than 6-8 years [START_REF] Visbeck | The North Atlantic Oscillation: climatic significance and environmental impact[END_REF]. These changes in temperature are caused by the advection of warmer water from the subtropical gyre region, driven by a spinup of the Atlantic meridional overturning circulation and the effect of an anomalous wind stress curl in the north-eastern Atlantic, which counteracts the local buoyancy forcing of the subpolar gyre [START_REF] Lohmann | Response of the North Atlantic subpolar gyre to persistent North Atlantic oscillation like forcing[END_REF]. Finally, the deeper ML associated with warmer SST after 1995 shows that MLD and SST are not interchangeable indicators of the stratification level of the upper ocean in this region over the last decades. This is in accordance with recent observations in the North Atlantic subtropical gyre [START_REF] Lozier | On the relationship between stratification and primary productivity in the North Atlantic[END_REF], as well as in the transition zone near the subpolar gyre between the 1980s and 2000s [START_REF] Martinez | Phytoplankton spring and fall blooms in the North Atlantic in the 1980s and 2000s[END_REF].

The phytoplankton step-wise increase in the mid-1980s has been widely reported in the N-Seas in parallel to changes in zooplankton community structures following the Northern Hemisphere Temperature warming since the 1980s [START_REF] Beaugrand | Reorganization of North Atlantic marine copepod biodiversity and climate[END_REF][START_REF] Beaugrand | Unanticipated biological changes and global warming[END_REF]. Interestingly, this warming occurs almost 10 years earlier in the N-Seas than in the Wand E-NASG (Fig. S4b purple line vs. Fig. S2b blue and red lines, respectively). The first PC derived from the SST EOFs represents most of the SST annual signal in the N-Seas (Fig. S4b black vs. purple line, respectively) as well as the post-1996 warming related with the AMO index. However, the 1988-1995 earlier warming in the N-Seas than in the NASG can be related with NAO+ conditions as shown on the second PC (9.5% of the total variance, Fig. S4c purple vs. black line and shaded area). The associated second spatial component illustrates how the NAO can simultaneously increase ocean temperatures in one part of the Atlantic basin, while reducing them in another (Fig. S4a). Although the NASG region is cooler during NAO+ conditions, the N-Seas becomes warmer [START_REF] Rodwell | Oceanic forcing of the wintertime North Atlantic Oscillation and European climate[END_REF][START_REF] Leterme | Decadal basin-scale changes in diatoms, dinoflagellates, and phytoplankton color across the North Atlantic[END_REF]. Thus, the decrease of total copepods and Calanus in the N-Seas follows a warming since the end of the 1980s (Fig. 4e) likely due to both NAO+ and AMO+ conditions. The increase of phytoplankton biomass is likely related to both a weaker top down control and

Accepted Article

This article is protected by copyright. All rights reserved. favorable bottom-up conditions (i.e., higher nutrients) partly associated with stronger winds and net heat loss from the ocean over the last 2.5 decades (Fig. 4e).

Our results illustrate how the different biomes of the North Atlantic [START_REF] Longhurst | Ecological Geography of the Sea, 2nd edn[END_REF] may react differently to climatic variations, although the intermingled bottom-up and topdown controls cannot be fully separated here. They reveal the complex effects of climate oscillations on bottom-up and top-down controls of phytoplankton variability. These results also emphasize the critical importance of historical datasets in our search for long-term (decadal) changes, trends and regime shifts that modify bottom-up and top-down forcings.

Supporting Information Legends

Accepted Article

This article is protected by copyright. All rights reserved.

(purple line). The AMO index is reported as the grey shaded area. (c) Principal component of the second EOF (black line) associated with (a). Anomaly of annual SST in the N-Seas is reported as the purple line as in (b). The NAO index is reported as the grey shaded area. [1960-1986] and [1991-2010] in the W-NASG. (d) as in (c) but for the E-NASG over , [1986][1987][1988][1989][1990][1991][1992][1993][1994][1995] and [1996][1997][1998][1999][2000][2001][2002][2003][2004][2005][2006][2007][2008][2009][2010]. and total copepod abundance (red curve) in the W-NASG. The cumulative sum of the AMO index is reported as the grey shaded area in (c) and (d). (d) Percentage contribution of total Calanus in total copepods in the W-NASG (blue curve). The scale on the right axis is inverted. (e) Cumulative sums of anomalies of annual Chl (green curve), total copepod abundance (red curve), SST (grey shaded area) and wind speed (black curve) for the N-Seas.

  Fig. 1) to ensure that the open-ocean NASG is considered separately from coastal regions: the Western part of the NASG (W-NASG; ~20000 samples), the Eastern part of the NASG (E-NASG, ~19500 samples) and the more coastal Northeastern Atlantic/ North Sea region (N-Seas; ~61000 samples).

  analyzed products. Their full spatial coverage along time allows the investigation of their spatio-temporal variability over the last five decades. EOFs performed on the different physical parameters all show that: 1) The spatial variability of the first component is homogenous (i.e., of the same sign everywhere in the domain) within each region (see wind speed and SST as examples on Fig. S1a and S2a, respectively); and 2) The associated first principal component (PC) closely follows the annual means averaged over each region (Fig. S1c and S2b, black line vs. colored lines). These results imply that the spatially averaged time-series of the physical parameters (which are ultimately compared with the biological data) are representative of the variability within each area. Winds, net heat fluxes and MLD variability at decadal scales are strongly related in the NASG (see Fig. S1d,e), the two formers being two main drivers of the MLD variability. Processes involved in the N-Seas are discussed in the last section.

Fig. S1 :

 S1 Fig. S1: (a) Spatial patterns of the EOF first mode of the annual wind speed (25% of the total variance) and (c) its associated principal component (black line). Anomalies of annual wind speed averaged over the W-and E-NASG and the N-Seas are reported as blue, red and purple curves respectively. The NAO index is reported as the grey shaded area. (b) Wind speed changes between strong (1986-1995) and moderate (1996-2010) NAO+ conditions as the difference of their respective means. (d) Cumulative sums of anomalies of annual wind speed (continuous curve), net heat flux (dashed curve) and MLD (dotted curve) in the E-NASG. The cumulative sum of the NAO index is reported as the grey shaded area. (e) as in (d), but in the W-NASG.

Fig. S2 :

 S2 Fig. S2: (a) Spatial patterns of the EOF first mode of the annual SST (21% of the total variance) and (b) its associated principal component (black line). Anomalies of annual SST averaged over the W-and E-NASG are reported as blue and red curves, respectively. The AMO index is reported as the grey shaded area.

Fig. S3 :

 S3 Fig.S3: Anomalies of annual Chl (green curve) and total copepod abundance (red curve) in the E-NASG. The AMO index is reported as the grey shaded area.

Fig. S4 :

 S4 Fig. S4: (a) Spatial patterns of the EOF second mode of the annual SST (9,5% of the total variance) for the N-Seas region. (b) The first EOF principal component presented in Fig. S2 (black line) is reported here with the anomaly of annual SST averaged over the N-Seas

Fig. 1 :

 1 Fig. 1: Spatial distribution of the CPR data over 1960-2010 in the W-and E-NASG (blue and red boxes, respectively) and the N-Seas (purple box) regions.

Fig. 2 :

 2 Fig. 2: Monthly Chl for (a) the N-Seas, (b) the W-NASG and (c) the E-NASG regions over 1960-2010, and prior to linear interpolation. Months are reported on the left axis and years on the bottom axis. Interpolated anomalies of Chl annual means (mg.m -3 ) are reported on the right axis (black curves). The cumulative sums are reported as the red curve (red right axis), except in the W-NASG region due to 5-years missing.

Fig. 3 :

 3 Fig. 3: (a) and (b) Anomalies of annual Chl (green line; left axis), of diatom abundances (orange dashed line; right axis) and of dinoflagellate abundances (blue dashed-dotted line; right axis) for the W-NASG and E-NASG regions. (c) Monthly abundances of diatoms (orange vertical bars) and dinoflagellates (blue vertical bars) averaged over and[1991][1992][1993][1994][1995][1996][1997][1998][1999][2000][2001][2002][2003][2004][2005][2006][2007][2008][2009][2010] in the W-NASG. (d) as in (c) but for the E-NASG over,[1986- 1995] and[1996][1997][1998][1999][2000][2001][2002][2003][2004][2005][2006][2007][2008][2009][2010].

Fig. 4 :

 4 Fig. 4: (a) Cumulative sums of anomalies of annual Chl (green curve) and wind speed (black curve) in the E-NASG. The cumulative sum of the NAO index is reported as the grey shaded area in (a) and (b). (b) as in (a), but in the W-NASG. Anomalies of annual Chl are shown instead of cumulative sum due to 5-years missing. (c) Anomalies of annual Chl (green curve)and total copepod abundance (red curve) in the W-NASG. The cumulative sum of the AMO index is reported as the grey shaded area in (c) and (d). (d) Percentage contribution of total Calanus in total copepods in the W-NASG (blue curve). The scale on the right axis is inverted. (e) Cumulative sums of anomalies of annual Chl (green curve), total copepod abundance (red curve), SST (grey shaded area) and wind speed (black curve) for the N-Seas.

Acknowledgements

We are grateful to past and present staff of SAHFOS who have contributed to the maintenance of the CPR time series. Particular thanks to David Johns for his advice on CPR datasets and methods. The authors gratefully acknowledge the data sources for this study: the WHOI OAFlux project funded by the NOAA Climate Observations and Monitoring (COM) program, the NCEP Reanalysis Derived data provided by the NOAA/OAR/ESRL PSD, Boulder, Colorado, USA. This work was supported by a grant from the Changing Earth Science Network initiative funded by the STSE program of the European Space Agency (ESA). We thank H. Claustre, G. Beaugrand, and R.J. Brewin for the comments they provided on early versions of the manuscript. We also thank the two anonymous reviewers and the editor for their insightful comments which helped improve the manuscript.