Karine Schaal

Yann Le Meur

François Bieuzen

Odile Petit

Philippe Hellard

Jean-François Toussaint

Christophe Hausswirth
email: christophe.hausswirth@insep.fr

Effect of recovery mode on postexercise vagal reactivation in elite synchronized swimmers

Keywords: cryotherapy, female athlete, heart rate variability, maximal exercise, water immersion, active recovery cryothérapie globale, athlète féminine, variabilité du rythme cardiaque, exercice maximal, immersion dans l'eau, récupération active

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Introduction

Synchronized swimming is an aesthetic and highly technical judged sport that has become increasingly physically demanding over the past decades. In addition to developing artistic, technical, and acrobatic skills, elite synchronized swimmers must follow a high-volume, high-intensity regimen to optimize strength, flexibility, and aerobic and anaerobic exercise capacity [START_REF] Liang | Ulnar and tibial bending stiffness as an index of bone strength in synchronized swimmers and gymnasts[END_REF][START_REF] Mountjoy | Injuries and medical issues in synchronized Olympic sports[END_REF]) because these physiological parameters are directly correlated to performance scores in elite competition settings [START_REF] Yamamura | Physiological characteristics of well-trained synchronized swimmers in relation to performance scores[END_REF]. At the international level, synchronized swimmers usually perform 2 or more training sessions per day, with few days of rest [START_REF] Liang | Ulnar and tibial bending stiffness as an index of bone strength in synchronized swimmers and gymnasts[END_REF]. Hence, in the practical scheduling of so many training hours, the time spans allotted for physical recovery between training sessions can be fairly short. Likewise, during competitions, the various ballet performances are sometimes closely scheduled, and accelerating the postexercise return to resting physiological levels after each performance is particularly important to maintain a consistent level of execution throughout the competition.

Heart rate variability (HRV) analysis is an established method used to gauge the extent of autonomic recovery from intense exercise sessions [START_REF] Pichot | Relation between heart rate variability and training load in middledistance runners[END_REF][START_REF] Pichot | Autonomic adaptations to intensive and overload training periods: a laboratory study[END_REF]. During exercise, the intensity-dependent withdrawal of parasympathetic tone and the increase in sympathetic activity results in an increase in heart rate and a decrease in vagal-related HRV indices [START_REF] Cottin | Heart rate variability during exercise performed below and above ventilatory threshold[END_REF]). After completing a given exercise bout, a rapid decrease in sympathetic activity and the return of parasympathetic heart activity to resting levels suggests a relative systemic recovery from the physiological stress imposed by the workload [START_REF] Seiler | Quantifying training intensity distribution in elite endurance athletes: is there evidence for an "optimal" distribution?[END_REF][START_REF] Seiler | Autonomic recovery after exercise in trained athletes: intensity and duration effects[END_REF]. The amount of time necessary for full parasympathetic reactivation after exercise can be significantly influenced by several factors, including exercise intensity [START_REF] Seiler | Autonomic recovery after exercise in trained athletes: intensity and duration effects[END_REF][START_REF] Stuckey | Autonomic recovery following sprint interval exercise[END_REF]) and cardiorespiratory fitness [START_REF] Seiler | Autonomic recovery after exercise in trained athletes: intensity and duration effects[END_REF][START_REF] Sandercock | Effects of exercise on heart rate variability: inferences from meta-analysis[END_REF]. Acute high-intensity exercise and longer periods of intensified training have been shown to disrupt HRV indices at rest; in turn, these disruptions have been associated with increased fatigue, decreased sleep quality, and decreased performance [START_REF] Haddad | Nocturnal heart rate variability following supramaximal intermittent exercise[END_REF][START_REF] Hynynen | Heart rate variability during night sleep and after awakening in overtrained athletes[END_REF][START_REF] Hynynen | Effects of moderate and heavy endurance exercise on nocturnal HRV[END_REF][START_REF] Garet | Individual interdependence between nocturnal ANS activity and performance in swimmers[END_REF][START_REF] Plews | Heart rate variability in elite triathletes, is variation in variability the key to effective training? A case comparison[END_REF][START_REF] Uusitalo | Heart rate and blood pressure variability during heavy training and overtraining in the female athlete[END_REF]. A number of studies have demonstrated that different recovery techniques used in sports training, such as cold, contrasted, or thermoneutral water immersion, significantly aid parasympathetic reactivation after intense exercise in welltrained athletes (Buchheit et al. 2009a;Al Haddad et al. 2010a;[START_REF] Stanley | The effect of post-exercise hydrotherapy on subsequent exercise performance and heart rate variability[END_REF]. Cold exposure and water immersion have both been found to suppress cardiac sympathetic activity and augment parasympathetic modulation as a result of arterial baroreflex activation [START_REF] Pump | Cardiovascular effects of static carotid baroreceptor stimulation during water immersion in humans[END_REF]. Cold stimulation triggers peripheral vasoconstriction, leading to a shift in blood volume toward the core [START_REF] Shibahara | The responses of skin blood flow, mean arterial pressure and R-R interval induced by cold stimulation with cold wind and ice water[END_REF]). This increased central volume leads to increased cardiac output, stroke volume, and arterial pressure. This activates the arterial high-pressure and cardiopulmonary low-pressure baroreceptors, which are responsible for reducing sympathetic nerve activity while shifting autonomic heart rate control toward a parasympathetic dominance [START_REF] Pump | Cardiovascular effects of static carotid baroreceptor stimulation during water immersion in humans[END_REF]. During water immersion, regardless of temperature, the hydrostatic pressure exerted on the body provides a mechanical stimulus, which contributes to the central shift of blood volume and, hence, results in baroreflex activation [START_REF] Stanley | The effect of post-exercise hydrotherapy on subsequent exercise performance and heart rate variability[END_REF]Al Haddad et al. 2010a).

Although the effect of cold exposure on postexercise parasympathetic reactivation is usually studied using cold-water immersion protocols, the effect of dry-air whole-body cryostimulation (WBC; -110 °C) on postexercise autonomic recovery has not been documented, even though this recovery method is increasingly used in high-level sports [START_REF] Hausswirth | Effects of whole-body cryotherapy vs. far-infrared vs. passive modalities on recovery from exercise-induced muscle damage in highly-trained runners[END_REF][START_REF] Pournot | Time-course of changes in inflammatory response after whole-body cryotherapy multi exposures following severe exercise[END_REF]. Further, the influence of WBC on parameters of metabolic recovery and subsequent maximal exercise capacity has not been evaluated.

In light of the necessity to optimize short-term recovery in a sport as seldom studied as synchronized swimming, we aimed to describe the autonomic and metabolic responses of elite synchronized swimmers to 4 different recovery methods used in sports training between 2 closely scheduled competition ballets: dry-air WBC, contrast-water therapy (CWT), active recovery (ACT), and a passive (control) condition (PAS). We compared the effect of these 4 protocols on postexercise parasympathetic reactivation, on metabolic and subjective parameters of recovery, and on subsequent maximal aerobic and anaerobic work capacity. We hypothesized that WBC would yield a significantly larger increase in vagalrelated HRV indices than all other recovery modes, and that WBC, ACT, and CWT would all be associated with favorable changes in metabolic parameters of recovery, compared with PAS.

Materials and methods

Subjects

Eleven highly trained elite female athletes, composing the French synchronized swimming team, took part in the study 6 months before the 2011 World Swimming Championships in Shanghai, China. The mean age of the swimmers was 20.3 ± 1.8 years, mean height was 170.1 ± 4.8 cm, and mean body weight was 61.1 ± 4.6 kg. All participants were familiar with exercise testing and the site of the experiment, as all procedures took place at their usual training site. The study conformed to ethical guidelines of the Declaration of Helsinki, and the study was approved by a local human research ethics committee (Ile de France XI, France;Ref. 200978) prior to beginning the study. Every athlete on the team volunteered to participate and provided their informed consent in written form.

Experimental design

The testing period began after 10 consecutive days of recovery, following a 2-week training retreat, so that subjects were fit and well rested. After an initial 400-m freestyle swimming time trial to evaluate maximal oxygen consumption (V ˙ O 2max400 ) and its related swim velocity (V ˙ O 2max400 ) [START_REF] Lavoie | Applied physiology of swimming[END_REF][START_REF] Carré | Use of oxygen uptake recovery curve to predict peak oxygen uptake in upper body exercise[END_REF], each subject completed 4 test sessions, spread evenly over 5 weeks, during the precompetition period (end of January to early March). Each test session exposed the swimmers to a different recovery method between 2 identical maximal exercise bouts (their technical competition ballet, lasting approximately 3 min), performed 70 min apart. This recovery duration was chosen to mimic the time constraints that can occur during a competition for athletes taking part in multiple events (duo, solo, team, combination), and was the minimum duration that allowed us to perform all of the necessary tests and recovery interventions between the 2 ballets. All swimmers were tested on the same days each week, following a complete day of rest, and at the same time of day. The order in which each subject underwent the 4 different recovery protocols was randomized to reduce any possible effect of improved fitness or habituation to the protocol over these 5 weeks, and the number of subjects being tested with each recovery method was uniform throughout the experimentation period. Because all swimmers participating in the study were on a regular oral contraceptive treatment, we did not take menstrual cycle phases into consideration when planning the test sessions. Subjects were only allowed to consume water ad libitum during the test sessions. The volume of water consumed was calculated by weighing the water bottles with a food scale, and the subjects' weight fluctuations from the beginning to the end of each session were recorded.

Exercise testing

Figure 1 outlines the procedures followed during each test session. After their usual precompetition warm-up (45 min) and mental preparation (5 min), each subject performed their technical ballet routine individually (B1, 3 min in duration, with phases of apnea adding up to 64% of the total time). The second ballet (B2, identical routine) was preceded by a shorter warm-up period (15 min). At the end of each ballet, after performing the final underwater figure, the subjects remained under water as they swam to the edge of the pool, where they started breathing directly into the mask of the Cosmed K4b 2 portable telemetry system (Rome, Italy); this method has been previously validated [START_REF] Hausswirth | The Cosmed K4 telemetry system as an accurate device for oxygen uptake measurements during exercise[END_REF]. Ventilatory and gas-exchange variables were collected over 4 breathing cycles, and peak oxygen consumption (V ˙ O 2peak ) was calculated using backward extrapolation of the O 2 recovery curve [START_REF] Lavoie | Applied physiology of swimming[END_REF][START_REF] Carré | Use of oxygen uptake recovery curve to predict peak oxygen uptake in upper body exercise[END_REF][START_REF] Montpetit | V ˙ O 2 peak during free swimming using the backward extrapolation of the O 2 recovery curve[END_REF]. Immediately before and after each ballet, subjects provided their global rating of perceived exertion (RPE g ) and muscle aches (RPE m ) on a 6-to 20-point Borg Fig. 1. Schema of the testing protocol followed during each session, with the order of events listed from top to bottom, then left to right. B1, B2, simulated competition ballets; ACT, active recovery; CWT, contrast-water therapy; HRV, heart rate variability; La -, lactate concentration; PAS, passive condition; RPE g , global rating of perceived exertion; RPE m , rating of perceived exertion for muscle aches; V ˙ O 2 , oxygen consumption; WBC, whole-body cryostimulation.

scale [START_REF] Borg | Perceived exertion as an indicator of somatic stress[END_REF]. To encourage maximal effort during each ballet, the coaches scored performance using 4 different 20-point scales commonly used in their practices to assess the swimmers' level of performance: precision, height, energy-displacement, and homogeneity.

Recovery protocols

Subjects performed 1 of the following 4 recovery methods during each testing session.

Passive condition

For the PAS protocol, subjects were instructed to lie quietly in a lounge chair placed near the pool for 30 min.

Contrast-water therapy

For the CWT protocol, subjects walked to the hydrotherapy unit, located in the same building as the pool, where they performed alternating cycles of cold and warm water immersion: 1 min standing in a cold-water bath (9° C) and 1 min sitting in a warm-water bath (39° C), immersed to the midsternal level for both, and beginning and ending with cold immersion (8 cold immersions and 7 warm immersions).

Active recovery

For the ACT protocol, 15 min of freestyle swimming at a speed corresponding to 40% of V ˙ O 2max (feedback on speed was given every 50 m by a coach) was followed by 15 min of light-intensity technical synchronized swimming exercises in the water (RPE g remaining below 13 on the Borg scale [START_REF] Borg | Perceived exertion as an indicator of somatic stress[END_REF][START_REF] Toubekis | Swimming performance after passive and active recovery of various durations[END_REF].

Whole-body cryostimulation

For the WBC protocol, sessions were administered onsite in a specially built WBC unit (Zimmer MedizinSysteme GmbH, Ulm, Germany), consisting of 3 consecutive temperature-controlled chambers (-10, -60, and -110 °C). Preliminary medical clearance was obtained to ensure that none of the subjects had any contraindications to performing the WBC protocol. All swimmers performed at least 1 session of WBC before the study began, and were therefore familiar with the procedure. After all PostB1 measurements were obtained, subjects thoroughly dried their body and hair and dressed warmly before being taken by car (�800 m) to the WBC unit; this was done to eliminate the walk from the pool in cold weather. They then changed into a dry swimsuit, were fitted with a mask covering the nose and mouth to protect the airways, and donned an ear band, gloves, socks, and slippers to protect the extremities. Subjects passed through the 2 warmer rooms to reach the therapy room, where they remained for exactly 3 min, walking and moving their arms slowly while being supervised by a technician.

At the end of each recovery period, just before B2, subjects marked, on a 10-cm visual analog scale, their perception of the recovery period's effectiveness, relative to their readiness to perform the second ballet (0, completely ineffective; 10, extremely effective).

HRV analysis

Subjects were fitted with heart rate monitors (Suunto Oy, Vantaa, Finland), and heart rate was collected in R-R interval mode for 6 min while the subject sat quietly in an isolated room at 4 distinct time points for each protocol: upon arrival before beginning their initial warm-up (PreB1); 6 min after the end of B1 (PostB1); 55 min after the end of B1, following the recovery protocol (PreB2); and 6 min after the end of B2 (Post B2). Data from the last 256 s of each sampling period were used for analysis, to allow the heart rate to stabilize once subjects assumed the seated position. To avoid influencing heart rate recovery after exercise, no particular breathing frequency was imposed [START_REF] Buchheit | Parasympathetic reactivation after repeated sprint exercise[END_REF]. For all HRV samples, it was subsequently verified that the respiration rate was always in the high-frequency (HF) range (>0.15-0.50 Hz).

Data were transferred to a computer, using the Suunto Training Manager software, and were analyzed using specialized HRV analysis software (Nevrokard aHRV, Izola, Slovenia). Data were visually inspected to identify artifacts and occasional ectopic beats, which were removed manually. All HRV data were processed by the same individual. The time-varying HRV indices kept for analysis were the root-mean square difference of successive normal R-R intervals (rMSSD), and the short-term R-R interval variability index, SD1. The standard deviation around the minor axis of the Poincaré scatterplot obtained by plotting the length of the nth R-R interval against the length of its preceding (n-1)th R-R interval. Mean heart rate (HR mean ) was also analyzed. Power spectral density analysis was then performed using a fast Fourier transform with a nonparametric algorithm. The power density of HF (>0.15-0.50 Hz, reflecting parasympathetic activity) and lowfrequency (LF; 0.04-0.15 Hz, reflecting mixed sympathetic and parasympathetic activity) components of the spectrum were calculated by integrating the power density within their respective frequency bands. The ratio of LF to HF, an indicator of relative sympathovagal balance, was retained for analysis.

.

Metabolite analysis

Blood samples were obtained from the finger tip, using a portable device (Lactate Pro LT-1710), to measure blood lactate concentration ([La -] b ) (Lactate Pro, Arkray, Kyoto, Japan) at 6 time points: before B1 (PreB1); 4 min after B1 (PostB1 4 =); before B2 (PreB2); and 4, 10, and 15 min after B2 (PostB2 4 =, PostB2 10 =, and PostB2 15 =, respectively). For the 400m swim time trial, [La -] b was measured 4 min after completing the swim.

Statistical analysis

Because this study involved a relatively small number of subjects and the data obtained did not always meet the assumptions of normality, as assessed visually by normal probability plot and by the Shapiro-Wilk test, nonparametric statistical analyses were conducted. To evaluate the effect of each exercise bout and recovery, within-protocol time differences in metabolic variables and HRV indices were evaluated using Friedman's ANOVA. If a time effect was significant, Wilcoxon's test was applied in a post hoc analysis to determine which specific time points differed from the others. Between-protocol comparisons were performed, using Wilcoxon's test, to determine whether there were any differences during the recovery period between the ACT, CWT, WBC, and PAS protocols. To isolate the effect of the recovery intervention, this analysis was performed on the difference between PreB2 and PostB1, represented as il"variable" rec (i.e., ilHF rec , il[La -] b rec , ilRPE m rec , etc.). Results are reported as the means ± SD for the parameters with normal distribution (ilV ˙ O 2peak , il[La -] b rec ); otherwise, results are expressed as the median and the values of the lower and upper quartiles. Spearman's coefficient of rank correlation was used to determine whether there were correlations between physiological, subjective, and autonomic variables of recovery and repeated maximal exercise. All analyses were performed using Statistica (version7.1, StatSoft France). The level of significance was set at p < 0.05 for all analyses.

Results

Exercise description

Mean completion time for the 400 m swimming time trial was 308 ± 8 s, with a mean V ˙ O 2max400 of 62.1 ± 3.0 mL•kg -1 •min -1 . During the 5 weeks of experimentation, no changes in any baseline or exercise variables were detected according to the week of testing. There were no differences in the amount of water consumed (885 ± 388 mL) or in the subjects' weight fluctuations (0.3 ± 0.4 kg) over the course of the study.

Because physiological parameters measured at PostB1 (V ˙ O 2peak , [La -] b ) were similar for all protocols, values were pooled for comparison with 400 m time-trial values. During the ballet (182 s), V ˙ O 2peak was slightly but not significantly lower than V ˙ O 2max400 (60.4 ± 2.0 mL•kg -1 •min -1 , p = 0.08), and [La -] b was significantly higher at PostB1 4 = than at the end of the 400 m time trial (11.0 ± 1.9 mmol•L -1 vs. 7.6 ± 2.0 mmol•L -1 , respectively, p = 0.004).

Heart rate variability

The impact of exercise on all HRV indices and HR mean was similar across all protocols, and there were no significant differences in these variables at PostB1 and PostB2. rMSSD, SD1, HF, and LF all decreased significantly with exercise, whereas HR mean increased and recovered to resting levels during the 70-min recovery period (SD1, and HR mean are shown in Fig. 2). LF/HF was the only index that did not show clear trends with exercise. WBC was the only protocol to yield a significant increase in vagal-related HRV indices and a decrease in HR mean at PreB2, compared with resting values at PreB1 (rMMSD, 178% ± 69% of PreB1 value, p = 0.012; SD1, 240% ± 144%, p = 0.009; and HR mean , 91% ± 14%, p = 0.047). ACT was the only protocol to result in a higher HR mean at PreB2 than at PreB1 (p = 0.01). Although some HRV indices appeared not to fully return to PreB1 levels with ACT, this difference did not reach significance (SD1, p = 0.09; HF, p = 0.11). Despite these differences, all HRV indices and HR mean values were similar at PostB2 and PostB1, and there were no differences between protocols. When the evolution of HRV indices during the recovery period were analyzed, WBC was the only protocol to yield a significantly larger change in HR mean and all HRV indices (ilHF rec , ilLF rec , ilLF/HF rec are displayed in Fig. 3). CWT did not differ from PAS on any of the HRV indices analyzed.

Metabolic parameters

WBC and ACT were the only protocols to yield significantly higher V ˙ O 2peak values at B2 than at B1, with gains of 5.4% ± 3.2% and 3.4% ± 2.9%, respectively (Fig. 4). Every swimmer reached a higher V ˙ O 2peak during B2 after using WBC in recovery, and all except 1 swimmer did so with ACT. In the CWT protocol, V ˙ O 2peak did not differ significantly between B1 and B2. PAS was the only protocol to yield a significant decrease in V ˙ O 2peak (-3.6% ± 2.1%); this was observed in every subject. [La -] b reached similar levels at PostB1 4 = and PostB2 4 =, and there was no significant protocol effect (all protocols combined, 11.2 ± 2.6 mmol•L -1 and 10.4 ± 2.1 mmol•L -1 , respectively; p = 0.14). il[La -] b rec was significantly larger, however, during ACT than PAS (p = 0.04), and nearly so for WBC than for PAS (p = 0.06), as shown in Fig. 5. After B2, there was

Subjective ratings

RPE m values peaked at PostB1 and PostB2, and returned to resting levels at the end of recovery (PreB2) for all protocols except WBC, during which RPE m remained slightly but significantly elevated, compared with PreB1 (13.4 ± 2.0 vs. 11.9 ± 2.5). PAS was the only protocol associated with significantly higher RPE m at PostB2 than at PostB1 (17.4 ± 1.4 vs. 16.5 ± 1.3); all other protocols showed similar RPE m after each ballet. The exercise and recovery trends for RPE g were similar to those for RPE m , except that RPE g values were significantly lower at PreB2 than at PreB1 after a shorter second warm-up for all protocols except WBC (p = 0.07). PAS was also associated with significantly lower ratings on the visual analog scale (lower perceived effectiveness of recovery) than all other protocols (PAS, 4.7 ± 2.1; CWT, 6.5 ± 1.6; ACT, 7.5 ± 1.1; WBC, 6.2 ± 1.3; p < 0.05).

Subjective performance scores

Mean scores obtained for B1 and B2 are shown in Table 1. Precision, energy-displacement, homogeneity, and overall scores were slightly but significantly lower at B2 than at B1, with a mean decrease of one-tenth of 1 point on the 20-point scale (-0.5% change). There was no significant effect of protocol on the difference in scores between B1 and B2.

Discussion

This study is the first to describe the physiological response of highly trained elite synchronized swimmers to 2 full-length competition ballets separated by a short recovery period. To our knowledge, this is the first time that WBC was investigated as a recovery technique between 2 closely scheduled maximal exercise bouts. The 2 most important findings of this study are that using WBC shortly after a full-length ballet resulted in a strong parasympathetic reactivation in elite swimmers, yielding 2-to 4-fold increases in vagal-related HRV indices, compared with pre-exercise values, within only 1 h; and that WBC exerted a significant influence on the metabolic parameters of recovery and subsequent exercise, with a larger clearance of plasma lactate and an increase in maximal aerobic work output during the second ballet. The latter was only matched by the effects of active recovery.

Effect of repeated exercise and recovery on HRV

Executing an elite-level synchronized swimming ballet demands a very large physical effort, requiring maximal aerobic work production with large anaerobic contributions. Rates of aerobic energy production at the end of each ballet were similar to those obtained after the 400-m swimming time trial; however, anaerobic contribution was significantly greater at the end of each ballet than after the 400-m swimming time trial, a test considered to be an accurate evaluation of maximal aerobic capacity in swimmers [START_REF] Lavoie | Applied physiology of swimming[END_REF]. Furthermore, RPE g and RPE m values obtained at the end of B1 and B2 were very high, indicating that the swimmers perceived these performances to be very difficult [START_REF] Borg | Perceived exertion as an indicator of somatic stress[END_REF]. Research on the metabolic toll of synchronized swimming ballets is scarce, especially at the elite level. Peak [La -] b values (mean, 11.0 ± 1.9 mmol•L -1 ) obtained at the end of each ballet surpassed those reported more than a decade ago by Table 1. Difference in the scores obtained between B1 and B2 for each protocol.

et al. 2012), greater cardiorespiratory fitness is associated with a significantly faster recovery of resting vagal tone [START_REF] Seiler | Autonomic recovery after exercise in trained athletes: intensity and duration effects[END_REF][START_REF] Sandercock | Effects of exercise on heart rate variability: inferences from meta-analysis[END_REF].

In our swimmers, HRV indices obtained after the completion of B2 indicated that the sympathetic influence over heart rate control remaining 10 min after exercise was no greater than that 10 min after B1, suggesting that autonomic response to repeating the same exercise in this short time span is similar. This finding, together with the similar [La -] b , RPE g , and RPE m , and equal or greater V ˙ O 2peak attained for all protocols (except V ˙ O 2peak after PAS), demonstrates that with WBC, ACT, and CWT, the swimmers were able to repeat the same maximal workload with similar autonomic, metabolic, and subjective responses.

Effect of specific recovery techniques on HRV

The augmentation of pre-exercise HRV values observed in synchronized swimmers after WBC reflects a strong parasympathetic reactivation at the cardiac level, with values largely surpassing those measured at rest as early as 60 min after maximal exercise. To our knowledge, this was the first study to describe such a large increase in vagal-related HRV indices from any cold exposure recovery technique used after exercise, with mean increases ranging from 78% for rMSSD and 140% for SD1 to 296% for HF. [START_REF] Westerlund | Heart rate variability in women exposed to very cold air (-110 °C) during whole-body cryotherapy[END_REF] investigated the HRV response to WBC in the resting state in nonathletic women, and reported that 2 min of WBC (-110 °C) augmented HRV indices of parasympathetic activity by 53% for rMSSD and 47% for SD1. In the case of highly trained swimmers, we showed that this significant effect of WBC occurred even when the treatment was performed shortly after maximal exercise, in a context of heightened cardiac sympathetic activity and suppressed vagal tone.

Other conventional cryostimulation methods used in the con-

Height Precision

Energydisplacement Homogeneity

Overall score text of recovery from physical training, such as shoulder-deep cold-water immersion (5 min in 11 to 14 °C water) have been found to significantly aid postexercise parasympathetic reactivation (Al Haddad et al. 2010a;Buchheit et al. 2009a;[START_REF] Stanley | The effect of post-exercise hydrotherapy on subsequent exercise performance and heart rate variability[END_REF]) to pre-exercise levels. The additional increase in vagal modulation observed with WBC, compared with typical cold-water immersion Note: Overall score, mean of all 4 protocols. B1, B2, simulated competition ballets; ACT, active recovery; CWT, contrast-water therapy; PAS, passive condition; WBC, whole-body cryostimulation. [START_REF] Yamamura | Physiological loads in the team technical and free routines of synchronized swimmers[END_REF], who found that [La -] b in Japan National finalists only reached 4.7 ± 1.1 mmol•L -1 , or 46% ± 11% of their previously measured peak [La -] b . In addition, we found that the swimmers' high V ˙ O 2max (62.1 ± 3.0 mL•kg -1 •min -1 ) supports their highly trained status and conveys that this sport has evolved over the years to become more physically demanding. Indeed, reported aerobic capacities of elite synchronized swimmers have evolved from 44 ± 4 mL•kg -1 •min -1 in 1980 [START_REF] Poole | Physiological characteristics of elite synchronized swimmers[END_REF]) to 53 ± 5 mL•kg -1 •min -1 in 1999 [START_REF] Chatard | Performance and physiological responses to a 5-week synchronized swimming technical training programme in humans[END_REF].

In accordance with metabolic and subjective indicators of maximal work output during the ballets, the large decrease in all vagal-related HRV indices observed from 6 to 10min after the end of each ballet reflected a significant reduction in the parasympathetic modulation of heart rate after the completion of intense exercise [START_REF] Cottin | Heart rate variability during exercise performed below and above ventilatory threshold[END_REF]). Even though we did not measure heart rate response during exercise, previous data obtained during competitions from the French elite synchronized swimming team revealed that the swimmers reached their maximal heart rate over the course of the routine [START_REF] Hausswirth | Proceedings of the 14th Annual Congress of the ECSS. 24-27[END_REF]. In spite of the large exercise-induced shift in autonomic heart rate control, complete recovery of all vagal-related HRV indices occurred within 1hour of the end of the first ballet, attesting to the high fitness of these athletes [START_REF] Seiler | Autonomic recovery after exercise in trained athletes: intensity and duration effects[END_REF]. Even though the time span required for full postexercise parasympathetic reactivation (to pre-exercise levels) is known to increase with the relative intensity of exercise [START_REF] Seiler | Autonomic recovery after exercise in trained athletes: intensity and duration effects[END_REF]; Stuckey methods, could potentially be explained by the fact that the very low temperatures in WBC imposed a larger thermal stress than immersion in 14 °C water. However, studies quantifying relative thermal stress and changes in core temperature resulting from various modes of cryostimulation are lacking. Additionally, during WBC, the entire body is exposed to cold, including the face and neck. It has been shown that the direct effect of cold on the head alone, using face immersion in cold water (without breathholding), aids parasympathetic reactivation significantly after exercise (Al Haddad et al. 2010b). This increase in vagal tone is thought to be principally mediated by trigeminal brain stem pathways, rather than by the arterial baroreflex [START_REF] Khurana | The cold face test: a non-baroreflex mediated test of cardiac vagal function[END_REF]. Further, [START_REF] Eckberg | Trigeminal-baroreceptor reflex interactions modulate human cardiac vagal efferent activity[END_REF] showed that stimulating trigeminal cutaneous receptors using cold-water face immersion augments the magnitude of the vagal response induced by arterial baroreflex activation alone.

CWT did not stand out as being particularly beneficial to autonomic recovery in our subjects, compared with PAS; both protocols resulted in a return to resting HRV indices. The short intermittent exposure to cold during CWT might have provided insufficient thermal stress to significantly boost parasympathetic activity beyond pre-exercise levels. [START_REF] Stanley | The effect of post-exercise hydrotherapy on subsequent exercise performance and heart rate variability[END_REF] reported that cold-water immersion exerts a larger effect on parasympathetic reactivation than contrast-water therapy, suggesting that the more important cold stimulus augments the effectiveness of water immersion in this respect. Further, the swimmers' habituation to spending several hours each day immersed in a thermoneutral pool for their training could have reduced the impact of this recovery method in this specific population. .

The workload performed during ACT, despite its low intensity, could be expected to slow autonomic recovery after maximal exercise, because even a light exercise load would maintain a relative intensity-dependent degree of parasympathetic withdrawal and sympathetic nerve activity. However, in the case of highly aerobically trained individuals, our results demonstrate that a subsequent low-intensity active recovery does not significantly hinder parasympathetic reactivation. Five minutes after the completion of active recovery, all HRV parameters (except LF) had returned to near-baseline levels; only HR mean remained slightly elevated. These findings remain specific to active recovery performed in water; the effect of immersion, together with the horizontal body position during swimming, probably aided parasympathetic reactivation (Buchheit et al. 2009a[START_REF] Buchheit | Effect of in-versus out-of-water recovery on repeated swimming sprint performance[END_REF]. In this respect, the effects of the active recovery used here may not be comparable to active recovery protocols performed on land.

Finally, during PAS, complete recovery to pre-exercise HRV indices occurred at least twice as fast as that observed by [START_REF] Stanley | The effect of post-exercise hydrotherapy on subsequent exercise performance and heart rate variability[END_REF], who showed that HRV indices returned to pre-exercise levels after 130 min of passive recovery. This discrepancy could be attributed to a higher level of cardiorespiratory fitness [START_REF] Seiler | Autonomic recovery after exercise in trained athletes: intensity and duration effects[END_REF]), but could also be attributed to different body positions. Our subjects adopted a supine position for 30 min, whereas the subjects of [START_REF] Stanley | The effect of post-exercise hydrotherapy on subsequent exercise performance and heart rate variability[END_REF] remained in a seated position for 10 min. Buchheit et al. (2009b) demonstrated that lying supine led to a faster heart rate recovery after exercise than sitting.

Parasympathetic reactivation and metabolic parameters of recovery and repeated exercise

Despite the large systemic sympathetic response necessary to support maximal work production rates during exercise [START_REF] Brooks | Balance of carbohydrate and lipid utilization during exercise: the "crossover" concept[END_REF], we showed that in these highly trained athletes, autonomic function at the cardiac level recovered fully within 70 min, and that poor parasympathetic reactivation after maximal exercise was therefore not a limiting factor to recovery and subsequent exercise capacity. No link was found between the extent of parasympathetic reactivation and any metabolic or subjective parameters of recovery or subsequent exercise capacity. Specifically, no differences in parasympathetic reactivation were found between PAS, ACT, and CWT; however, aerobic work output decreased significantly from B1 to B2 after PAS (-3.6% ± 2.1%), was maintained with CWT (+1.0% ± 3.4%), and increased with ACT (+3.4% ± 2.9%). To our knowledge, only a few studies have investigated whether a larger postexercise parasympathetic reactivation, occurring as a result of cold-water immersion, is associated with improved performance in the short term (Buchheit et al. 2009a;[START_REF] Stanley | The effect of post-exercise hydrotherapy on subsequent exercise performance and heart rate variability[END_REF]). Even though in both studies cold-water immersion enhanced parasympathetic reactivation and yielded greater subjective ratings of recovery than passive conditions, no associations were found between HRV recovery and absolute performance measures, suggesting that, in the case of healthy highly trained individuals, this aspect of recovery may not affect subsequent exercise capacity. From a medical safety perspective, however, optimizing parasympathetic reactivation might help reduce the occurrence of dangerous arrhythmias, which tend to occur more readily in situations with a strong sympathetic background, such as immediately after intense exercise in the heat [START_REF] Billman | Aerobic exercise conditioning: a nonpharmacological antiarrhythmic intervention[END_REF].

To our knowledge, this was the first study to describe the effect of WBC as a short-term (i.e., �1 h) recovery aid. It showed that elite athletes respond favorably to this technique in the context of repeated maximal aerobic exercise. WBC yielded results nearly similar to ACT on the parameters of metabolic recovery, aiding blood lactate clearance, and enabling a greater aerobic work output during B2, compared with PAS. It is well established that, compared with passive conditions, active recovery increases the clearance of lactate and other metabolic byproducts, such as hydrogen ions, ammonium ions, and inorganic phosphate [START_REF] Banfi | Whole-body cryotherapy in athletes[END_REF][START_REF] Fairchild | Glycogen synthesis in muscle fibers during active recovery from intense exercise[END_REF]). An increased blood lactate clearance has also been reported after cold exposure, compared with passive recovery [START_REF] Heyman | Effects of four recovery methods on repeated maximal rock climbing performance[END_REF]. Because the removal of these ions from the bloodstream is associated with a greater ability to repeat maximal aerobic and anaerobic performance in the short term [START_REF] Neric | Comparison of swim recovery and muscle stimulation on lactate removal after sprint swimming[END_REF], our findings support the reduced effectiveness of passive recovery in this regard, confirming active recovery and cold exposure to be more appropriate options between 2 synchronized swimming performances.

In spite of the desirable effects of WBC, ACT, and CWT on the metabolic aspects of recovery and subsequent exercise capacity, these benefits were not accompanied by differences in performance scores attributed by the coaches. Even though the technical and aesthetic merit of a given ballet performance does not solely depend on maximal exercise capacity (they also depend on several aesthetic and technical characteristics), an increase in aerobic work output during the second ballet could have favorably impacted the scores attributed by coaches (such as the increased height of compulsory figures, the amplitude of movement, and the overall energy level and homogeneity of the performance). In this respect, [START_REF] Yamamura | Physiological characteristics of well-trained synchronized swimmers in relation to performance scores[END_REF] found a significant correlation between performance scores and the physiological attributes of synchronized swimmers, including aerobic and anaerobic work capacity and the amplitude of leg movements. Thus, in spite of the sensitivity and reproducibility limitations inherent to subjective evaluations of performance in judged sports, [START_REF] Damisch | Olympic medals as fruits of comparison? Assimilation and contrast in sequential performance judgments[END_REF][START_REF] Ansorge | Bias in judging women's gymnastics induced by expectations of within-team order[END_REF][START_REF] Ste-Marie | Prior processing effects on gymnastic judging[END_REF], the physiological evidence pointing to an optimized recovery with WBC and ACT encourage further investigations of sports events in which a 3% to 5% increase in V ˙ O 2peak , as was seen here, is usually associated with improved absolute performance (such as during a cycling or running time trial [START_REF] Saunders | Physiological measures tracking seasonal changes in peak running speed[END_REF]).

Conclusion

This study described the autonomic and metabolic responses of elite synchronized swimmers to 2 full-length competition ballets, and their adaptations to 4 different protocols performed during the recovery period separating them. We brought forth 2 novel findings, supporting the effectiveness of WBC in the context of postexercise autonomic and metabolic recovery and repeated exercise performance. First, we demonstrated that a single session of WBC performed shortly after a maximal exercise exerted a strong influence on parasympathetic reactivation in the context of a heightened sympathetic background. Second, our results showed that, similar to ACT, WBC is associated with improved metabolic recovery after maximal exercise. Future research should try to determine whether the regular use of WBC as a recovery technique confers additional benefits over longer periods of time, because fatigue accumulation during phases of intensified training has been associated with changes in the autonomic modulation of heart rate in athletes [START_REF] Pichot | Autonomic adaptations to intensive and overload training periods: a laboratory study[END_REF].

Fig. 2 .

 2 Fig. 2. Evolution of SD1 (A) and mean heart rate (HR mean ) (B) for each ballet and recovery period. Values are means ± SE. Box plots represent median interquartile range (IQR, Q25-Q75), and error bars are maximal and minimal observations within 1.5 × IQR. Squares represent maximum and minimum observations above or below 1.5 × IQR. *, Significantly different from PreB1 (p < 0.05); †, significantly different from PAS (p < 0.05). In all conditions, PostB1 and PostB2 values were different from PreB1 and PreB2 values, so symbols were omitted for clarity. From light grey to black: PAS, CWT, WBC, ACT. B1, B2, simulated competition ballets; ACT, active recovery; CWT, contrast-water therapy; PAS, passive condition; SD1, short-term variability of successive R-R intervals; WBC, whole-body cryostimulation.

Fig. 3 .

 3 Fig. 3. Change in power spectral density variables from PostB1 to PreB2, ilHF (A), ilLF (B), and LF/HF (C). Box plots represent median, interquartile range (IQR, Q25-Q75), and error bars are maximal and minimal observations within 1.5 × IQR. Squares represent maximum and minimum observations above or below 1.5 × IQR. *, Significantly different from PAS (p < 0.05). HF, high frequency; LF, low frequency; ACT, active recovery; CWT, contrast-water therapy; PAS, passive condition; WBC, whole-body cryostimulation.

Fig. 4 .

 4 Fig. 4. ilV ˙ O 2peak , change in peak oxygen consumption between B2 and B1. B1, B2, simulated competition ballets; ACT, active recovery; CWT, contrast-water therapy; PAS, passive condition; WBC, wholebody cryostimulation. *, Significantly different from baseline (p < 0.05); †, significantly different from PAS (p < 0.05).

Fig. 5 .

 5 Fig. 5. Mean difference in blood lactate concentration [La -] b rec ) for each protocol. ACT, active recovery; CWT, contrast-water therapy; PAS, passive condition; WBC, whole-body cryostimulation. †, Significantly different from PAS (p < 0.05). The difference between WBC and PAS nearly missed significance (p = 0.059).

Acknowledgements

The authors of this study thank the French synchronized swimming athletes for participating in this study, as well as their respective coaches -Charlotte Massardier, Pascale Meyet, Anne Capron, and Julie Fabrefor their valuable assistance in scheduling this protocol within the athletes' training schedules. They would also like to thank Dr. Philippe Le Van, Jean-Robert Filliard, Marielle Volondat, and Philippe Van de Cauter for supervising the cryostimulation sessions and, finally, INSEP and the French National Swimming Federation for their support in this study.


Natascha Schmidt
email: natascha.schmidt@mio.osupytheas.fr

Delphine Thibault
email: delphine.thibault@ird.fr

François Galgani
email: francois.galgani@ifremer.fr

Andrea Paluselli
email: andrea.paluselli@mio.osupytheas.fr

Richard Sempéré
email: richard.sempere@mio.osupytheas.fr

Occurrence of microplastics in surface waters of the Gulf of Lion (NW Mediterranean Sea)

Keywords: Marine litter, microplastic, Mediterranean Sea, Gulf of Lion, Marseille Bay

Between 2014 and 2016 a total of 43 microplastic samples were collected at six sampling stations in the eastern section of the Gulf of Lion (located in the northwestern Mediterranean Sea), as well as upstream of the Rhône River. Microplastics were found in every sample with highly variable concentrations and masses. Concentrations ranged from 6 10 3 items km -2 to 1 10 6 items km -2 (with an average of 112 10 3 items km -2 ), and mass ranged from 0.30 g km -2 to 1018 g km -2 DW (mean 61.92 ± 178.03 g km -2 ). The samples with the highest and lowest microplastic count originate both from the Bay of Marseille. For the Bay of Marseille, it is estimated that the total microplastic load consist of 519 10 3 -101 10 6 items weighing 0.07 -118 kg. Estimations for daily microplastic transport by the Northern Current and the Rhône River, two important hydrologic features of the northwestern Mediterranean Sea, range from 0.18 to 86.46 t and from 0.20 to 21.32 kg, respectively. Particles < 1 mm 2 clearly dominated sampling stations in the Northern Current, the Rhône River and its plume (52, 53 and 61 %, respectively), suggesting a long exposure time in the environment. Items between 1 mm 2 and 5 mm 2 in size were the most abundant microplastics in Marseille Bay (55 %), which suggests coastal pollution sources or the removal of smaller particles from surface waters e.g. by ballasting owing to the presence of epibionts.

Introduction

Plastic and its chemical compounds have played an important role in the Anthropocene and might threaten human health [START_REF] Kobrosly | Prenatal Phthalate Exposures and Neurobehavioral Development Scores in Boys and Girls at 6-10 Years of Age[END_REF][START_REF] Tranfo | Urinary phthalate monoesters concentration in couples with infertility problems[END_REF][START_REF] Sathyanarayana | Phthalates and Children's Health[END_REF][START_REF] Heudorf | Phthalates: Toxicology and Exposure[END_REF] and both terrestrial [START_REF] Zhao | Microscopic anthropogenic litter in terrestrial birds in Shanghai, China: Not only plastics but also natural fibers[END_REF][START_REF] Lwanga | Microplastics in the Terrestrial Ecosystem: for Lumbricus terrestris (Oligochaeta, Lumbricidae)[END_REF][START_REF] Oehlmann | A critical analysis of the biological impacts of plasticizers on wildlife[END_REF] and marine environments [START_REF] Przybylinska | Environmental Contamination with Phthalates and its Impact on Living Organisms[END_REF]Van Franeker & Law, 2015;[START_REF] Sigler | The Effects of Plastic Pollution on Aquatic Wildlife: Current Situations and Future Solutions[END_REF]. In 2014, 311 million tons of plastic were produced worldwide, 15 % of which were consumed in Europe (PlasticsEurope, 2015). The degradation of large plastic items into microplastics (≤ 5 mm) in the ocean is a slow and heterogeneous process, varying with respect to the quality, shape and size of the plastic. This process is driven by mechanical forcing (e.g., waves), salt water, and UV radiation [START_REF] Ter Halle | Understanding the Fragmentation Pattern of Marine Plastic Debris[END_REF]. Because of its small size, micro debris can easily be ingested (e.g., [START_REF] Desforges | Ingestion of Microplastics by Zooplankton in the Northeast Pacific Ocean[END_REF][START_REF] Neves | Ingestion of microplastics by commercial fish off the Portuguese coast[END_REF]. Approximately 270 10 3 tons of plastic are suspected to float in the world's oceans [START_REF] Eriksen | Plastic Pollution in the World's Oceans: More than 5 Trillion Plastic Pieces Weighing over 250,000 Tons Afloat at Sea[END_REF]. Estimates for floating microplastic loads range from 7 10 3 to 35 10 3 tons for global open-ocean surface waters [START_REF] Cózar | Plastic debris in the open ocean[END_REF] or from 93 10 3 to 236 10 3 tons depending on the model used [START_REF] Van Sebille | A global inventory of small floating plastic debris[END_REF]. Plastic accounts for 60 to 80 % of all marine litter, followed in quantity by glass and metal [START_REF] Unep | Marine Litter: A Global Challenge[END_REF]. About 370 10 9 plastic particles or 1,455 tons have been estimated to be floating on the surface of the Mediterranean Sea [START_REF] Ruiz-Orejón | Floating plastic debris in the Central and Western Mediterranean Sea[END_REF]. Other estimates range from 756 to 2,969 tons [START_REF] Cózar | Plastic Accumulation in the Mediterranean Sea[END_REF] and from 874 to 2,576 tons [START_REF] Suaria | The Mediterranean Plastic Soup: synthetic polymers in Mediterranean surface waters[END_REF].

The Mediterranean Sea is a semi-enclosed basin subject to significant anthropogenic pressures (e.g., The MerMex group, 2011; [START_REF] Blanfuné | Response of rocky shore communities to anthropogenic pressures in Albania (Mediterranean Sea): Ecological status assessment through the CARLIT method[END_REF][START_REF] Hassoun | Acidification of the Mediterranean Sea from anthropogenic carbon penetration[END_REF][START_REF] Casale | Annual survival probabilities of juvenile loggerhead sea turtles indicate high anthropogenic impact on Mediterranean populations[END_REF]. Marine debris, including microplastics, are a particularly important concern in this region [START_REF] Deudero | Mediterranean marine biodiversity under threat: Reviewing influence of marine litter on species[END_REF][START_REF] Cózar | Plastic Accumulation in the Mediterranean Sea[END_REF][START_REF] Ioakeimidis | A comparative study of marine litter on the seafloor of coastal areas in the Eastern Mediterranean and Black Seas[END_REF][START_REF] Faure | An evaluation of surface micro-and mesoplastic pollution in pelagic ecosystems of the Western Mediterranean Sea[END_REF][START_REF] Pedrotti | Changes in the Floating Plastic Pollution of the Mediterranean Sea in Relation to the Distance to land[END_REF]. Concerns about marine litter in the Mediterranean Sea were first expressed in 1976 when the Barcelona Convention was signed with the goal of preventing and abating marine and coastal pollution (UNEP, 2009). In subsequent years, studies have been undertaken to better understand pollution sources and trajectories, through approaches as modeling the transport of floating marine debris [START_REF] Mansui | Modelling the transport and accumulation of floating marine debris in the Mediterranean basin[END_REF]. However, knowledges on the spatial and temporal microplastic distribution remain limited [START_REF] Ruiz-Orejón | Floating plastic debris in the Central and Western Mediterranean Sea[END_REF][START_REF] Suaria | The Mediterranean Plastic Soup: synthetic polymers in Mediterranean surface waters[END_REF][START_REF] Cózar | Plastic Accumulation in the Mediterranean Sea[END_REF]. Their contents are highly variable, although the sea surface circulation seems to be the main driver on the distribution of floating marine litter whatever their sizes. Currents affect timedependent movements that remain difficult to predict, and cause several non-trivial Lagrangian mechanisms [START_REF] Zambianchi | Marine litter in the Mediterranean Sea, An Oceanographic perspective[END_REF]. In semi-enclosed seas, such as the Mediterranean Sea, aggregation patterns are not permanent and high variability is observed at a small scale [START_REF] Suaria | The Mediterranean Plastic Soup: synthetic polymers in Mediterranean surface waters[END_REF]. Wind-induced effects on floating material and Stokes drift velocities require further investigation, such as refinement of regional models. Nevertheless, some available scenarios could be hypothesized with possible retention areas in the northwestern Mediterranean and the Tyrrhenian sub-basins [START_REF] Poullain | Surface Geostrophic Circulation of the Mediterranean Sea Derived from Drifter and Satellite Altimeter Data[END_REF][START_REF] Mansui | Modelling the transport and accumulation of floating marine debris in the Mediterranean basin[END_REF]. The Gulf of Lion is in the northwestern sector of the Mediterranean Sea, and its hydrodynamics are influenced by shallow water depths of the shelf, wind regimes (Mistral and Marin), the Northern Current (NC), and freshwater inputs from the Rhône River [START_REF] Gatti | The Rhone river dilution zone present in the northeastern shelf of the Gulf of Lion in December 2003[END_REF][START_REF] Fraysse | Intrusion of Rhone River diluted water into the Bay of Marseille: Generation processes and impacts on ecosystem functioning[END_REF]. The NC has a high seasonal variability: while a decrease in intensity is observed in summer, it becomes faster, deeper and narrower in winter [START_REF] Millot | Mesoscale and seasonal variabilities of the circulation in the western Mediterranean[END_REF]. Intrusion of the NC onto the shelf of the Gulf of Lion has been observed [START_REF] Ross | Impact of an intrusion by the Northern Current on the biogeochemistry in the eastern Gulf of Lion, NW Mediterranean[END_REF]Barrier et al., 2016 and references therein). This productive shelf is also highly exploited for commercial fishing [START_REF] Bănaru | Trophic structure in the Gulf of Lions marine ecosystem (north-western Mediterranean Sea) and fishing impacts[END_REF] and the coastal area is strongly influenced by tourism activities. Given this areas great economic, touristic and environmental significance, monitoring threats, such as pollution sources, is essential. Therefore, the primary goal of this study was to provide insight into the temporal and spatial distribution of microplastics in the eastern sector of the Gulf of Lion. Furthermore, we wanted to examine relationships between microplastic size distributions and possible pollution sources and transportation routes.

Materials and Methods

Following the framework of the Particule-MERMEX and PLASTOX projects, microplastic debris were collected at different times between February 2014 and April 2016 (Table S1) in three distinct areas with specific hydrodynamic characteristics (Figure 1) within the eastern sector of the Gulf of Lion (northwestern Mediterranean Sea). The first area is located 40 km offshore at the eastern part (station #1, also called 'Antares site') and is within the direct influence of the Northern Current, which runs east to west along the shelf break over 2,475 m of water [START_REF] Martini | Bacteria as part of bioluminescence emission at the deep ANTARES station (North-Western Mediterranean Sea) during a one-year survey[END_REF]. The second area includes the Bay of Marseille (stations #2, 3 and 4), which is significantly influenced by a population of approximately 1 million inhabitants and by the daily volume of about 250 10 3 m 3 of waste waters released from the Marseille-Cortiou wastewater treatment plant (WWTP) [START_REF] Savriama | Impact of sewage pollution on two species of sea urchins in the Mediterranean Sea (Cortiou, France): Radial asymmetry as a bioindicator of stress[END_REF][START_REF] Tedetti | Fluorescence properties of dissolved organic matter in coastal Mediterranean waters influenced by a municipal sewage effluent (Bay of Marseilles, France)[END_REF]. To the west, the third study area is the downstream part of the Rhône River (station #6, Arles, 48 km from the river mouth) and within the dilution plume (station #5, about 2.5 km from the mouth) [START_REF] Sempéré | Carbon inputs of the Rhône River to the Mediterranean Sea: Biogeochemical implications[END_REF]. Sampling dates, GPS coordinates, microplastic concentration, mean wind speed and wind direction are provided in the supplementary data (Table S1) along with information on precipitation. Surface current speeds and directions were extracted from the Mars 3D model (http://marc.ifremer.fr). Microplastic samples were collected using a Manta net (0.50 m x 0.15 m opening) mounted with a 780 µm mesh size and towed horizontally at the surface. Ten samples from March and April 2016 were collected (in Marseille at stations 3 and 4) with a 330 µm mesh size (Suppl. Table 1). Sampling was only conducted under low swell conditions (< 1 m). The net was towed for 20 minutes at an average speed of 2.5 knots approximately 50 m behind the research vessel. It was towed at a slight angle to avoid disturbances caused by the boat's wake. Samples from the Rhône River (station #6) were collected from a fixed location on the dock of the river. Sampled superficies at this station were calculated by comparing the flow rate during sampling with reference flow rates and river speeds. Lower-limit river speeds were used for estimates, since river speeds tend to be slower near the dock.

The net was carefully rinsed and the content of the cod-end was poured into a 1 L glass bottle, preserved with a buffered seawater formalin solution (final concentration 5 %), and kept in cold and dark conditions until further analysis. Samples were then sieved (mesh size 125 µm), and rinsed with ultrapure water (ISO 3696). Plastic debris were picked out with tweezers under a dissecting microscope. Fibers were not taken into account due to the high risk of contamination. No Fourier Transform Infrared Spectroscopy (FTIR) Analysis was performed to verify the nature of the items, so despite all efforts to maximize result reliability, it cannot be excluded that some non-plastic items were estimated to be microplastics.

The number, size and shape of each item was identified using a ZooScan © (HYDROPTIC SARL). Each item was placed on the screen of the ZooScan without any water. Surface area measurements in pixels were obtained using the ImageJ software and then converted into mm 2 and the Equivalent Spherical Diameter (ESD). Plastic items ≤ 5 mm were considered. All microplastics from each sample were then weighed (Mettler AE 240, reliability ± 0.1 mg). Microplastic abundance (items km -2 ) and dry weight (g km -2 ) were calculated for each sample using the towing distance and the net opening surface. Analysis of variance (oneway and two-way ANOVA) with a 0.05 level of significance was performed to assess whether the microplastic abundance and size distribution varied with space (stations) and time. The Tukey test was used whenever significant differences were detected. All statistical analyses were performed using R version 3.3.2.

Results and Discussion

Microplastic abundance

Microplastic abundance ranged from 6 10 3 to 1 10 6 (mean 96 10 3 ) items km -2 in the Marseille Bay area, from 33 10 3 to 400 10 3 (mean 113 10 3 ) items km -2 in the Rhône River plume, from 7 10 3 to 69 10 3 (mean 34 10 3 ) items km -2 in the river itself and from 9 10 3 to 916 10 3 (mean 212 10 3 ) items km -2 off-shore (Fig. 2,top). The highest microplastic concentration (1 10 6 items km -2 ) was observed at station #2 (Marseille Bay area). The day this sample was collected was characterized by calm conditions with no noteworthy surface currents near the station. In contrast, the other two stations on the coast of Marseille, stations #3 and #4, showed very low particle concentrations (averages 20 10 3 and 10 10 3 items km -2 , respectively). While a comparison between these both stations and the station #2 is difficult, because the samples were collected in different years (2016 vs. 2014), some assumptions can still be considered. [START_REF] Goldstein | Scales of Spatial Heterogeneity of Plastic Marine Debris in the Northeast Pacific Ocean[END_REF] reported that a high spatial heterogeneity for microplastic concentrations could be found not only at a large scale but also on a smaller scale for samples taken at distances of 10 km from one another. Heterogeneous spatial debris distribution can be the result of currents, wave-and wind-driven turbulences, river inputs or hydrodynamic features such as upwelling, downwelling, gyres or fronts (e.g., [START_REF] Kukulka | The effect of wind mixing on the vertical distribution of buoyant plastic debris[END_REF][START_REF] Suaria | Floating debris in the Mediterranean Sea[END_REF][START_REF] Collignon | Neustonic microplastic and zooplankton in the North Western Mediterranean Sea[END_REF]. More generally, high concentrations of microplastics, especially small fragments, are found in coastal waters because of the proximity of densely populated areas, [START_REF] Pedrotti | Changes in the Floating Plastic Pollution of the Mediterranean Sea in Relation to the Distance to land[END_REF] and continental inputs from the atmosphere or rivers [START_REF] Collignon | Neustonic microplastic and zooplankton in the North Western Mediterranean Sea[END_REF]. Point pollutions could also play an important role in the Bay of Marseille, where the fierce northwestern Mistral wind can transport litter from city streets into coastal waters.

Another possible source of microplastics in the Bay of Marseille is the local sewage facility (Cortiou) where treated wastewater enters the sea in the southeastern part of the city. On March 17, 2016 a slight surface current coming from Cortiou at a speed of approximately 0.5 m s -1 entered the area of stations #3 and 4. The microplastic concentrations observed that day were the highest ever found at station #4 (15 10 3 items km -2 ) and the second highest for station #3 (27 10 3 items km -2 ). Interestingly, microplastic abundance was always higher at station #3 compared to station #4, in spite of their geographical proximity (p < 0.05).

Our median concentration (31 10 3 items km -2 ) was about one third of the mean value, highlighting potential surges in microplastic presence, possibly linked to climatic and hydrodynamic events. Hydrodynamic processes influencing microplastic distribution are e.g. vertical mixing or eddies and anticyclonic gyres. The latter of which are unsteady formations in the Mediterranean Sea [START_REF] Pedrotti | Changes in the Floating Plastic Pollution of the Mediterranean Sea in Relation to the Distance to land[END_REF], but could lead to punctual increases in regional microplastic abundances. Additionally, in our study area, there is the Northern Current, which varies greatly in intensity, depth, and position [START_REF] Millot | Mesoscale and seasonal variabilities of the circulation in the western Mediterranean[END_REF]. Data collected at station #1 showed temporal variability, with concentrations of microplastics being significantly higher on March 10, 2014 (p < 0.05) when the Northern Current was fast and narrow with maximum speeds of approximately 0.9 m s -1 . However, triplicated trawls exhibited a range of microplastic abundances from 103 10 3 to 916 10 3 items km -2 at this sampling date, implying that a nine fold difference in abundances can be observed in the same sampling area within two hours. This further highlights the strong temporal variability observed for microplastic concentrations. Overall no seasonal differences were detected (p > 0.05), but the low number of observations limits the strength of any comparison. [START_REF] Goldstein | Scales of Spatial Heterogeneity of Plastic Marine Debris in the Northeast Pacific Ocean[END_REF] observed seasonal heterogeneity at much larger scale in the northeastern Pacific Ocean between summer 2009 and fall 2010.

Floating debris transported by the NC could be transported to the Balearic Islands, where models calculated high beaching probabilities [START_REF] Mansui | Modelling the transport and accumulation of floating marine debris in the Mediterranean basin[END_REF], or to the seafloor which is known to be a (micro-) plastic sink [START_REF] Claessens | Occurrence and distribution of microplastics in marine sediments along the Belgian coast[END_REF][START_REF] Ioakeimidis | A comparative study of marine litter on the seafloor of coastal areas in the Eastern Mediterranean and Black Seas[END_REF][START_REF] Woodall | The deep sea is a major sink for microplastic debris[END_REF]. Reasons of microplastic sedimentation can be the nature of the plastic material, (if its density is higher than the one of seawater, [START_REF] Tekman | Marine litter on deep Arctic seafloor continues to increase and spreads to the North at the HAUSGARTEN observatory[END_REF], the biofouling accumulation on microplastic surfaces [START_REF] Woodall | The deep sea is a major sink for microplastic debris[END_REF], the incorporation of free microplastics into marine aggregates or the incorporation of microplastics into fast-sinking faecal pellets after ingestion by zooplanktons and fishes [START_REF] Cole | Microplastic Ingestion by Zooplankton[END_REF]. The overall average microplastic abundance for our samples was 112 10 3 items km -2 , which is in the same range as other areas in the northwestern basin, where mean densities have been estimated to 115 10 3 items km -2 [START_REF] Collignon | Neustonic microplastic and zooplankton in the North Western Mediterranean Sea[END_REF], 130 10 3 items km -2 [START_REF] Faure | An evaluation of surface micro-and mesoplastic pollution in pelagic ecosystems of the Western Mediterranean Sea[END_REF] and 150 10 3 items km -2 (De Lucia et al., 2014). Higher amounts were measured for the entire Mediterranean basin (243 10 3 items km -2 , [START_REF] Cózar | Plastic Accumulation in the Mediterranean Sea[END_REF], due to high concentrations in some Mediterranean areas. Densely populated areas as the semienclosed Adriatic Sea and the Levantine Basin were characterized by high densities of 1,050 10 3 (max: 4,600 10 3 ; [START_REF] Suaria | Neustonic microplastics in the Southern Adriatic Sea. Preliminary results[END_REF] and 1,518 10 3 (max: 65 10 6 ; Van der Hal, 2017) items km -2 , respectively. Our results are consistent with previous studies and indicate that the northwestern Mediterranean Sea contains similar mean microplastic concentrations as the Atlantic and Pacific Oceans (mean: 134 10 3 items km -2 and 124 10 3 items km -2 , respectively, [START_REF] Eriksen | Plastic Pollution in the World's Oceans: More than 5 Trillion Plastic Pieces Weighing over 250,000 Tons Afloat at Sea[END_REF]. Hereby it needs to be considered that the Atlantic and Pacific Oceans are also known to be highly heterogeneous, with microplastic accumulation and nonaccumulation zones. Examples for a heavily contaminated area are the East Asian Seas, where a mean microplastic abundance of 1,720 10 3 items km -2 was measured [START_REF] Isobe | East Asian seas: A hot spot of pelagic microplastics[END_REF].

Microplastic abundances in the Rhône River at Arles (station #6; 34 10 3 ± 19 10 3 items km -2 ; net size 0.50 m x 0.15 m, mesh size 780 µm) were relatively low, but were similar to values reported by De Alencastro (2014) upstream at Chancy (~ 52 10 3 items km -2 ; net size 0.60 m x 0.18 m, mesh size 300 µm). In comparison, a mean microplastic abundance of 893 10 3 items km -2 was found in the Rhine River, a watercourse flowing through highly industrialized areas, such as North-Rhine Westphalia (Germany), where many plastic factories are located [START_REF] Mani | Microplastics profile along the Rhine River[END_REF]. Concentrations observed in the Rhône River plume (station #5, up to 400 10 3 items km -2 ) were higher than in the river itself, suggesting that the Rhône River -sea interface may generate a temporal accumulation zone for debris. In general, however, the area covered by our six sampling stations is not considered to be a retention area, but can better be described as a "transit area". The size of the Mediterranean basin reduces the potential for formation of permanent gyres as in the Atlantic, Pacific and Indian Oceans, where plastic often concentrates [START_REF] Cózar | Plastic Accumulation in the Mediterranean Sea[END_REF].

At the river mouth, microplastic concentrations were either significantly greater (p < 0.05) or similar to those observed upstream in the Rhône River. Concerning the river plume, we should highlight the similitude in zooplankton composition of two samples collected with the same Manta trawl, first, on the 10/03/14 at the station #1 (NC) and then (18/03/14), at the Rhône River Plume station (station #5). High abundances (> 1,000 individuals per sample) of Velella velella, a free-floating hydrozoan, were observed at both dates (Thibault D. pers. com.), implying a potential intrusion of water masses from the NC onto the shelf. Such intrusions have already been observed before [START_REF] Barrier | Strong intrusions of the Northern Mediterranean Current on the eastern Gulf of Lion: insights from in-situ observations and high resolution numerical modelling[END_REF]. Salinity data from the Mars 3D model support the hypothesis: while the Rhône River plume was extended in all directions on 10/03/14 and the following days, saltier surface waters pushed from the eastern direction into the area from 16/03/14 on and thus, reduced the extension area of the river plume. During the period examined, the velocity of the NC flowing through station #1 was about 0.4 m s -1 (Suppl. Table 1), but currents leaving the main branch in northwestern directions flowed at reduced speeds of about 0.2 m s -1 . At this speed range (0.2-0.4 m s -1 ), water masses could have travelled about 140-275 km in eight days, which is consistent with the straight line distance (120 km) between both stations. However, we would like to point out that these are only indications, since an accurate model would be needed to simulate the exact trajectory of the water masses and microplastics in question.

Microplastic weight

Microplastic dry weight showed a similar variability, ranging from 0.30 g DW km -2 to 1018 g DW km -2 , with the maximum observed in Marseille Bay (Fig. 2,bottom). An average of 61.92 g DW km -2 (± 178.03 g DW km -2 ) was found in the study area. This value is similar to averages of 60 and 63 g DW km -2 reported for the western part of the northwestern Mediterranean Sea [START_REF] Collignon | Neustonic microplastic and zooplankton in the North Western Mediterranean Sea[END_REF] and the upstream part of the Rhône River (De Alencastro, 2014), respectively.

An estimated surface area of 87 km 2 of the Bay of Marseille would provide a total microplastic load of 0.07 to 118 kg (mean 9.94 kg), representing a range of concentrations from 0.5 10 6 to 101 10 6 (mean 8 10 6 ) microplastic pieces in surface waters. For the Rhône River, the flow rate used for calculations varied between 1,150 m 3 s -1 and 1,600 m 3 s -1 during the sampling period. Using minimum and maximum concentration and weight values, we calculated a daily microplastic spill of 0.20 -21.32 kg (dry weight), representing 10 10 6 -40 10 6 items discharged by the Rhône River into the Mediterranean Sea. Similarly, microplastic loads for the Northern Current were calculated using volumetric transport rates of 0.7 Sv [START_REF] Conan | Variability of the Northern Current off Marseilles, western Mediterranean Sea, from February to June 1992[END_REF] and 2 Sv [START_REF] Petrenko | Variability of circulation features in the Gulf of Lion NW Mediterranean Sea. Importance of inertial currents[END_REF] and the minimum and maximum concentration and weight values. This method provided an estimate of daily transport ranging from 0.18 to 86.46 tons (dry weight) of microplastic, representing 4 10 9 to 1 10 12 items. These calculations give minimum ranges, since they are based on the assumption that microplastics concentrate within 15 cm under the surface. Turbulences, especially in rivers, may however transfer microplastics through several meters of the water column. As interesting as they are, these extrapolations should be considered with caution, since microplastic abundances show a high amount of variability and are difficult to predict.

Microplastic size distribution

The mean size of microplastic was 1.48 ± 0.88 mm, however significant differences (p < 0.01) were observed between samples from the Bay of Marseille (stations # 2-4) and all other sampling stations. For better visualization of the size distribution of our samples, we calculated the equivalent spherical diameter (ESD) of each microplastic particle (Figure 3). A general exponential distribution curve was observed with the smallest items being the most important, except in the Bay of Marseille, where microplastics were more evenly distributed over the size range. The overall size distribution observed in this study closely resembles those observed for the Mediterranean Sea [START_REF] Ruiz-Orejón | Floating plastic debris in the Central and Western Mediterranean Sea[END_REF][START_REF] Cózar | Plastic Accumulation in the Mediterranean Sea[END_REF], open ocean waters [START_REF] Cózar | Plastic debris in the open ocean[END_REF] and the Northeast Pacific Ocean [START_REF] Goldstein | Scales of Spatial Heterogeneity of Plastic Marine Debris in the Northeast Pacific Ocean[END_REF]. Manta nets are the most commonly used sampling device for microplastic sampling in aquatic ecosystems and were also used in this study. The mesh size of the net can influence the size distribution as well as the speed of the tow, as smaller particles avoiding the net can be forced aside from the net opening or large particles can squeeze out through the mesh. This study used mainly a 780 µm mesh sized net and a 330 µm mesh sized net only for ten sampling events at stations # 3 and #4 (Suppl. Table 1). We expected to collect more 0.0-0.4 mm items at both concerned stations (#3 and #4) by using the 330 µm mesh sized net, but microplastics of this size class were observed neither in samples from the 330 µm mesh size, nor in samples from the 780 µm mesh size. No influence on the microplastic size distribution caused by the use of these different mesh sizes was hence observed. This was statistically confirmed by removing all samples collected with the 330 µm net from the dataset and repeating the one-way ANOVA with the following posthoc test and obtaining the same significant results.

Size distribution can be an indicator of the source of marine debris and of its distance to the shoreline. While [START_REF] Pedrotti | Changes in the Floating Plastic Pollution of the Mediterranean Sea in Relation to the Distance to land[END_REF] observed that small sized microplastics were more abundant within the first kilometre adjacent to the coastline, [START_REF] Isobe | East Asian seas: A hot spot of pelagic microplastics[END_REF] found that the percentage of larger plastic particles is typically greater in areas close to the pollution source. The surface area distributions of stations #1 (NC) and 6 (Rhône River) clearly resemble each other. Both are dominated by small particles (< 1 mm 2 : 52 and 53 %, respectively, Figure 4). This size class only represented 27 % for stations in the Bay of Marseille, but represented 61 % of microplastic particles at the Rhône River plume. The second size class (1-5 mm 2 ) was the most abundant in Marseille Bay (55 %). The largest pieces (> 10 mm 2 ) were poorly represented (< 5 %) at all stations. The size class distributions are likely related to the distance of the collected particles from pollution sources. In the case of station #1 (NC), it is likely that microplastics were transported by the Northern Current and may have originated in regions farther east, such as the Italian coast. At station #6 (Rhône River), the size distribution suggests that the collected microplastics were in the Rhône River watershed for some time and certainly originated from highly industrialized and/or populated regions higher upstream (e.g., Lyon with ~ 500,000 inhabitants). The position of the Rhône River plume varies based on wind and river flow; therefore, debris will be contributed from both the river itself and surrounding coastal areas in variable amounts. Since the smallest particles are most abundant here, it is probable that these microplastics, have also been transported by water masses for some time before collection. In the Bay of Marseille (stations #2-4) the dominance of larger particles (1-5 mm 2 ) suggests that the microplastics collected in this area were closer to their source and mainly originate from the urban area. A more efficient removal of the smallest floating particles in this region, via ballasting due to epiphytic growth, could also be a possible explanation [START_REF] Ryan | Does size and buoyancy affect the long-distance transport of floating debris[END_REF].

Conclusions

This study provides additional data on microplastic occurrence in the eastern Gulf of Lion. Our results revealed that surface water microplastic concentrations and size distributions in this area affected by anthropogenic impacts are consistent with those already published for the western Mediterranean Sea. Significant temporal and spatial heterogeneity was observed for microplastic abundances. Our results confirm that the Rhône River, large cities, such as Marseille, and the Northern Current act as sources and/or transportation routes of microplastics collected in the northwestern basin of the Mediterranean Sea. As our microplastics are floating, it was shown that it can be pertinent to study the zooplankton composition of samples additionally to currentology data, in order to improve our knowledge on microplastic transport in the sea.

Fig. 1 .

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Acknowledgments

This study was conducted as part of the PARTICULE-region PACA, MERMEX/MISTRALS and JPI Oceans PLASTOX projects. We acknowledge the technical support provided by the Service Atmosphere Mer (SAM) and the Microscopie et Imagerie (MIM) and Marine Environmental Chemistry (PACEM) M I O platforms. We sincerely thank the captain and crew of N. O. Antedon II and Thethys, as well as Sandrine Ruitton, who kindly allowed sampling time during her diving trips. We thank Maryvonne Henry and Anne Delmont for help with sample collection. Maria Luiza Pedrotti's work group from LOV laboratory in Villefranche-sur-Mer kindly shared their ZooScan expertise. Thanks to Javier Castro-Jimenez and Vincent Fauvelle for revising an earlier version of the manuscript. The project leading to this publication has received funding from European FEDER Fund under project 1166-39417. A PhD scholarship for N. Schmidt was provided by Agence de l'Eau.