Hengtong Liu

Amaia Lamarins

Jacques Labonne

Mathilde Monperrus

Pascale Coste

Emmanuel Huchet

Jacques Rives

Iban Seiliez

Valérie Bolliet
email: valerie.bolliet@inrae.fr

New insights into methylmercury induced behavioral and energy-related gene transcriptional responses in European glass eel (Anguilla anguilla)

Keywords: Glass eel, Anguilla anguilla, Migration behavior, Methylmercury, Metabolism

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

Introduction

The status of European eel (Anguilla anguilla) remains critical since the recruitment of glass eels strongly declined from 1980 to about 2010, and have remained at a low level since, which has urged Euro-wide European eel regulation (council regulation (EC) no. 1100/2007) for the protection and recovery of the stock (ICES, 2018). The European eel is a facultative catadromous species that crosses the Atlantic Ocean twice during its life. The present knowledge clearly indicate that the species reproduces in the Sargasso Sea, where the leptocephalus larval stage is first observed [START_REF] Schmidt | Breeding places and migrations of the eel[END_REF][START_REF] Van Ginneken | The European eel (Anguilla Anguilla, Linnaeus), its Lifecycle, Evolution and Reproduction: A Literature Review[END_REF]. The larvae move with ocean currents more than 5000 km toward the European continent, after which they transform into a juvenile stage, the glass eel. Then, glass eels migrate up estuary to reach freshwater habitats where they grow and develop into yellow eels. At an age of 5 to 20 years they commence the silvering process and become silver eels at which point they begin their migration back to the Sargasso Sea to reproduce and probably die [START_REF] Miller | A century of research on the larval distributions of the Atlantic eels: a re-examination of the data[END_REF][START_REF] Righton | Empirical observations of the spawning migration of European eels: The long and dangerous road to the Sargasso Sea[END_REF][START_REF] Schmidt | Breeding places and migrations of the eel[END_REF][START_REF] Tesch | Occurrence of eel Anguilla anguilla larvae west of the European continental shelf, 1971-1977[END_REF][START_REF] Tesch | The Eel[END_REF].

Glass eels migrate up estuaries to ascend rivers using selective flood transport: during flood tide, glass eels move up in the water column and migrate with the current while they go down and remain on or in the substratum during ebb tide [START_REF] Forward | Selective tidal-stream transport of marine animals[END_REF][START_REF] Gascuel | Flow-carried and active swimming migration of the glass eel (Anguilla anguilla) in the tidal area of a small estuary on the French Atlantic coast[END_REF][START_REF] Jellyman | Upstream migration of glass-eels (Anguilla spp.) in the Waikato River[END_REF]. They are also synchronized to the photoperiod, avoiding light and swimming mainly during the night when the water is clear. However, a high degree of geographical dispersion crossing marine and riverine water has been documented regarding to different migratory patterns of settlement and river colonization [START_REF] Daverat | Phenotypic plasticity of habitat use by three temperate eel species, Anguilla anguilla, A. japonica and A. rostrata[END_REF][START_REF] Secor | Can otolith microchemistry chart patterns of migration and habitat utilization in anadromous fishes?[END_REF][START_REF] Tsukamoto | Facultative catadromy of the eel Anguilla japonica between freshwater and seawater habitats[END_REF][START_REF] Tsukamoto | Do all freshwater eels migrate?[END_REF][START_REF] Tzeng | Effects of salinity and ontogenetic movements on strontium:calcium ratios in the otoliths of the Japanese eel, Anguilla japonica Temminck and Schlegel[END_REF]. These different patterns of migration could have a strong impact on the fate of the population because of the environmental sex determinism in eels, [START_REF] Geffroy | Sex differentiation and sex determination in eels: consequences for management[END_REF][START_REF] Krueger | Evidence for environmental sex determination in the American eel, Anguilla rostrata[END_REF]. Briefly, in European eels, males are generally observed to dominate in high-density environments, often associated with estuarine or lower river reaches, whereas individuals that migrated upstream to the river tend to become mostly females [START_REF] Adam | L'anguille EUROPÉENNE: indicateurs d'abondance et de colonisation[END_REF][START_REF] Davey | Sex determination in freshwater eels and management options for manipulation of sex[END_REF][START_REF] Harrison | A review of glass eel migratory behaviour, sampling techniques and abundance estimates in estuaries: implications for assessing recruitment, local production and exploitation[END_REF][START_REF] Laffaille | Patterns of silver eel (Anguilla anguilla L.) sex ratio in a catchment[END_REF][START_REF] Parsons | Relationship between elver recruitment and changes in sex ratio of silver eels Anguilla anguilla L. migrating from Lough Neagh, Northern Ireland[END_REF].

Although there is no consensus on the reason for the diverse migratory patterns, it has been suggested that early settlement in estuary or coast, if taking place, is partly due to low energy condition [START_REF] Bureau Du Colombier | Energy and migratory behavior in glass eels (Anguilla anguilla)[END_REF][START_REF] Edeline | Adaptive phenotypic plasticity of eel diadromy[END_REF][START_REF] Edeline | Effects of body condition and water temperature on Anguilla anguilla glass eel migratory behavior[END_REF][START_REF] Liu | Looking at the complex relationships between migration behavior and conditional strategy based on energy metabolism in the European glass eel (Anguilla anguilla)[END_REF]. Indeed, most glass eels do not feed throughout estuarine migration and they depend on energy reserves accumulated by the leptocephalus larvae during oceanic migration to reach the river [START_REF] Kawakami | Immigration patterns of glass-eels Anguilla japonica entering river in northern Kyushu, Japan[END_REF][START_REF] Tesch | The Eel[END_REF][START_REF] Bardonnet | Feeding of glass eels (Anguilla anguilla) in the course of their estuarine migration: new insights from stable isotope analysis[END_REF]. Moreover, estuary is a highly stressful ecosystem, where a combination of different stressors including large variations in the temperature, hydraulic conditions, salinities, oxygen availability or contaminants may influence the energy metabolic processes of glass eels during migration. Due to its proximity to urbanized and industrial areas, estuary represents a major sink for various contaminants including various forms of mercury (Hg), both inorganic (Hg(II)) and methylated (MeHg), this latest form being recognized to adversely affect fish physiology, growth, health and behavior [START_REF] Cambier | At environmental doses, dietary methylmercury inhibits mitochondrial energy metabolism in skeletal muscles of the zebra fish (Danio rerio)[END_REF][START_REF] Cambier | Effects of dietary methylmercury on the zebrafish brain: histological, mitochondrial, and gene transcription analyses[END_REF][START_REF] Lee | Effects of dietary methylmercury on growth performance and tissue burden in juvenile green (Acipenser medirostris) and white sturgeon (A. transmontanus)[END_REF][START_REF] Murphy | Modeling larval fish behavior: Scaling the sublethal effects of methylmercury to population-relevant endpoints[END_REF][START_REF] Scheulhammer | Effects of environmental methylmercury on the health of wild birds, mammals, and fish[END_REF].

Behavioral effects of MeHg exposure in fish have been largely documented, with a lot of concerns on prey capture ability [START_REF] Weis | Effects of mercury on the feeding behavior of the mummichog, Fundulus heteroclitus from a polluted habitat[END_REF][START_REF] Zhou | Effects of methylmercury on ontogeny of prey capture ability and growth in three populations of larval Fundulus heteroclitus[END_REF], predator avoidance [START_REF] Webber | Mercury effects on predator avoidance behavior of a forage fish, golden shiner (Notemigonus crysoleucas)[END_REF], reproduction [START_REF] Sandheinrich | Effects of dietary methylmercury on reproductive behavior of fathead minnows (Pimephales promelas)[END_REF], habitat selection [START_REF] Sampaio | Habitat selection disruption and lateralization impairment of cryptic flatfish in a warm, acid, and contaminated ocean[END_REF] and more recently on memory and aggressiveness [START_REF] Strungaru | Acute exposure to methylmercury chloride induces fast changes in swimming performance, cognitive processes and oxidative stress of zebrafish (Danio rerio) as reference model for fish community[END_REF].

The underlying molecular mechanisms of MeHg toxicity are predominantly related with oxidative stress, which in turn may induce lipid peroxidation and DNA damages [START_REF] Berntssen | Chronic dietary mercury exposure causes oxidative stress, brain lesions, and altered behaviour in Atlantic salmon (Salmo salar) parr[END_REF][START_REF] Gonzalez | Comparative effects of dietary methylmercury on gene expression in liver, skeletal muscle, and brain of the zebrafish (Danio rerio)[END_REF]. In glass eels, MeHg toxicity could increase energy expenditure through the processes of cellular repairment and detoxification which may thereby reduce glass eels fitness and their migratory success through estuary. [START_REF] Claveau | Methylmercury effects on migratory behaviour in glass eels (Anguilla anguilla): An experimental study using isotopic tracers[END_REF] reported that glass eels exposed to MeHg (50 ng L -1 ) for 11 days exhibited some perturbations of mitochondrial structures and metabolism associated to an activation of antioxidative defence systems. However, the effect of MeHg differed between behavioral phenotypes of glass eels, those displaying a low propensity to migrate up estuaries being more affected than their 'migrant' counterpart. These findings suggested the existence of differences in sensitivity to MeHg exposure among glass eels that remained to be elucidated.

In the present study we aimed to identify the MeHg-induced repercussions in synchronization of glass eels to the water current reversal, their level of swimming activity and the transcriptomic profiles of energy-related genes. This was achieved by transcriptome analyses related to energy metabolism and direct observations of swimming behavior.

Materials and methods

Ethics

Procedures used in this study have been validated by the ethics committee N°073 (ref: 2017012015086652). The experiment was carried out in strict accordance with the EU legal frameworks, specifically those relating to the protection of animals used for scientific purposes (i.e., Directive 2010/63/EU), and under the French legislation governing the ethical treatment of animals (Decret no. 2013-118, February 1st, 2013).

Primary test for MeHg accumulation kinetics over 30 days 2.2.1 Fish collection

The glass eels were collected at the mouth of a small estuary (courant d'Huchet) located 40 km north of the mouth of the Adour estuary, France (43° 55′N, 1° 23′W). They were sampled using a dip-net at night and during flood tide in February 2018, and were then transferred to the laboratory and maintained in a tank containing water from the fishing site.

During the next 48 h, the water was continuously aerated and progressively diluted with fresh water. Fish were kept under 12 °C and a photoperiod of 12 L / 12 D with a very low light intensity during the photophase (0.2-0.3 μW/cm 2 ).

MeHg exposure and kinetic sampling

After acclimation, glass eels were randomly selected and allocated by groups of three into a series of seven aquariums (1.5 L of aerated fresh water) with initial compartment concentration of 100 ng L -1 of MeHg. Glass eels were exposed to this single spike of MeHg prepared as follow:

The stock solution of MeHg at 1000 mg L -1 was prepared by dissolving MeHg chloride obtained from Strem Chemicals (Newburyport, MA, USA) in methanol. The spiking solution at 100 µg L - 1 was then prepared by diluting the stock solution in 1% hydrochloric acid. Three glass eels were collected in the beginning of this test as controls. All the aquariums were sealed with a transparent cover to prevent water volatilization and to guarantee the photoperiod. The aeration, temperature and photoperiod were maintained as described for the acclimation period. During the exposure period, no mortality neither erratic swimming (abrupt change of direction or swimming speed) were observed.

The MeHg-exposed fish were sampled at 1, 2, 3, 7, 11, 18 and 30 days. One aquarium was recovered on each sampling time point and the three fish in this aquarium were considered to be three replicates. Sampled fish were killed by anesthetic and quickly washed with distilled water.

After biometry, all the fish were immediately frozen into liquid nitrogen and stored at -80 ˚C for future mercury speciation analysis.

Mercury speciation analysis

Each glass eel was lyophilized, mashed using an agate mortar and then submitted to microwave extraction according to the procedure previously published by [START_REF] Navarro | Simultaneous determination of mercury and Butyltin species using a multiple species-specific isotope dilution methodology on the European, Anguilla anguilla glass eel and yellow eel[END_REF].

The supernatant was spiked with known amounts of standard solution of MeHg and Hg(II) and submitted to propylation. Mercury speciation analysis was performed by GC-ICPMS using the method with parameters, which are detailed by [START_REF] Navarro | Simultaneous determination of mercury and Butyltin species using a multiple species-specific isotope dilution methodology on the European, Anguilla anguilla glass eel and yellow eel[END_REF]. Mercury species concentrations were determined by speciated isotope dilution (Monperrus et al., 2005).

Analytical performances were evaluated using the Certified Reference Material DOLT-4 (Dogfish Liver, NRCC, Ottawa, Canada). Good agreement with certified values was obtained with recoveries of 105±8% and 95±7% for MeHg and Hg(II), respectively. Detection limits of 0.1 and 0.2 ng.g -1 were found for MeHg and Hg(II), respectively. All concentrations were expressed in ng Hg.g -1 dry weight.

Seven-day MeHg exposure

The kinetics experiment ran over 30 days showed that glass eels exposed to MeHg presented an increase in MeHg concentration measured in whole body for up to seven days which then reached a plateau (Fig. 1). The increase in Hg(II) concentration remained low and stabilized very quickly. According to these results, we chose 7 days as exposure duration to investigate the effect of MeHg on migration behavior and energy metabolism.

Fish collection and tagging

140 glass eels were collected in March 2018 at the same fishing site and using the same capture method described above (see 2.2.1). They were transferred to the laboratory and maintained at 12 °C overnight in an aerated tank containing water from the fishing site. In the morning following their capture, all glass eels were anesthetized (Benzocaine, 0.01 mg L -1 ) and individually tagged using Visible Implant Elastomer (VIE Tag) (combinations of one or two hypodermic spots of different colors as described by [START_REF] Imbert | Relationships between locomotor behavior, morphometric characters and thyroid hormone levels give evidence of stage dependent mechanisms in European eel upstream migration[END_REF] in order to trace the swimming behavior individually. Once tagged, glass eels were released to wake up in the water from fishing site. During the next 48 h, the water was continuously aerated and progressively diluted with fresh water.

MeHg exposure

After acclimation, fish were randomly divided into two groups: control group without Hg addition and MeHg-group exposed to an initial MeHg concentration of 100 ng L -1 . Seven glass aquariums (5 L of aerated fresh water) were used for each group, with 10 fish in each aquarium.

The exposure lasted for seven days. During the entire experimental period, both control and MeHg-exposed fish were kept under the same conditions as those during the acclimation period.

No mortality neither erractic swimming were observed during the experiment.

Observations of the post-exposure swimming behavior

After seven-day exposure, one aquarium of 10 glass eels from the control group and one aquarium of 10 specimens from the MeHg-exposed group were recovered for mercury speciation analysis. Procedures of sample preparation and mercury measurement were the same as primary test (see 2.2.3). All the other control and MeHg-exposed glass eels (n = 6 x 10 glass eels in each group) were transferred into two annular tanks (30 glass eels of both the control and MeHg groups in each tank) installed in two temperature-controlled rooms. The rooms were kept under the same conditions as describes above, except that we added a constant UV light (0.6 µW/cm²) in order to see the VIE Tag. The water temperature was kept at 12 ± 0.5 °C and continuously recorded by thermistors placed in the tank. The annular tank system was specially designed to mimic tidal rhythm by being equipped with two pumps at its opposite ends [START_REF] Liu | Looking at the complex relationships between migration behavior and conditional strategy based on energy metabolism in the European glass eel (Anguilla anguilla)[END_REF] Supplementary Fig. S1). The two pumps were programmed to alternately work to generate clockwise or counterclockwise water flow every 6.2 h. In each tank, the swimming activity of glass eels was traced continuously during 10 days by a camera programed to record 15 seconds every 40 min. The UV light allowed the identification of each glass eel during the light and dark phases by its elastomer mark. During the 10 days, a total of 360 sessions of 15 seconds were obtained.

Glass eels use selective tidal-stream transport to migrate up estuary, wherein individuals synchronize their swimming activity to tidal current, but they also have to sustain the level of swimming activity. Thus, in our experimental conditions, the propensity of glass eels to migrate was firstly evaluated by their capacity to synchronize the swimming activity to the change in water current direction by a period of 12.4 h. Then, their activity levels were quantitatively analyzed by counting the total number of observations of each elastomer mark in the water column in the 360 sessions of 15 seconds videos.

Gene expression analysis

Sampling procedure

After swimming test, all the glass eels in annular tank were recovered, anaesthetized and then killed by a lethal bath of anaesthesia (Benzocaine, 0.05 mg L -1 ), flash-frozen in liquid nitrogen, and stored at -80 ˚C.

RNA extraction and quantitative real-time PCR

Frozen glass eels were cut in three sections, containing (i) the head including the gills, (ii) the heart, the liver, the spleen and the stomach and (iii) most of the muscle tissue with the intestine (hereafter referred to as head, viscera and muscle, respectively, Fig. 2). Then, each section was immediately stored in TRIzol reagent for total RNA extraction.

The protocol conditions for quantitative RT-PCR have been previously published [START_REF] Lansard | Integration of insulin and amino acid signals that regulate hepatic metabolism-related gene expression in rainbow trout: role of TOR[END_REF]. The primers specific to 27 genes involved in antioxidant system, mitochondrial function and autophagy activity have been described in previous study by [START_REF] Liu | Looking at the complex relationships between migration behavior and conditional strategy based on energy metabolism in the European glass eel (Anguilla anguilla)[END_REF]. For the expression analysis, relative quantification of target gene expression was done using the ΔCT method described by [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF]. The relative expression of Luciferase was used for data normalization as described previously [START_REF] Marandel | Remodelling of the hepatic epigenetic landscape of glucose-intolerant rainbow trout (Oncorhynchus mykiss) by nutritional status and dietary carbohydrates[END_REF].

Statistical analyses 2.4.1 Propensity of glass eels to migrate

To characterize the propensity of glass eels to migrate, we first investigated the synchronization of glass eel's swimming activity to the change in water current direction every 6.2h (synchronized / non-synchronized). For this purpose, we used a modeling method to categorize all the recovered glass eels into synchronized ones and non-synchronized ones, which has been previously described by [START_REF] Liu | Looking at the complex relationships between migration behavior and conditional strategy based on energy metabolism in the European glass eel (Anguilla anguilla)[END_REF] (See Supplementary Text S1). Since it has been previously shown that glass eels display rhythmic swimming activity in response to current reversal [START_REF] Bolliet | Individual patterns of rhythmic swimming activity in Anguilla anguilla glass eels synchronised to water current reversal[END_REF], two parameters, the probability of being swimming of an individual i at time t, ( , ) and the periodicity of swimming occurrence of an individual i, ( ), were derived from the model. Fish having a P value above the mean of P meanwhile having an activity periodicity close to 12.4 h were considered synchronized, others were considered non-synchronized. Finally, the number of synchronized fish in the control and MeHgexposed groups was compared by chi-squared test.

The second aspect to investigate the propensity of glass eels to migrate was the level of swimming activity, expressed as the total number of observations of glass eels swimming in the water column during the 360 sessions of 15 seconds, regardless of synchronization. The comparison between control and MeHg-exposed groups were conducted using chi-squared test.

Principal Component Analysis (PCA) of gene transcriptional profiles

To determine the transcriptional profile of studied genes, PCA was used as a multivariate statistical approach to reduce the number of the variables considered. The data set of gene expression levels was compressed by PCA procedure without much loss of information. In detail, to evaluate the global transcriptional response in each pathway, we ran a PCA analysis for each cellular function, using a table providing the normalized gene expression levels for all individuals. The first axis of PCA performed on each function in each tissue explained 47% ~ 77% of the total variances of all the genes involved, making it an acceptable synthetic measure of gene expression level of each function (See Supplementary Table S1). Supplementary Fig. S2 ~ Fig. S4 showed the relevance of all the genes involved in each function to the first axis of PCA.

We then retrieved the score of individuals on the first axis of the PCA, and used these coordinates as a synthetic indicator of the individual level of expression for the cellular function.

All the statistical analyses relevant to gene transcriptions were examined using the first axis of PCA for each genomic function. Two-way ANOVA was used to analyze the varying gene transcriptional profiles in response to MeHg exposure and synchronization behavior. The interactions in the responses were also evaluated. Differences were considered significant at p < 0.05. The relationships of swimming activity to gene transcriptional profiles were estimated by Spearman's Rank Order Correlation test. The correlation was considered significant at p < 0.05 level. Then, the slope of the correlations observed in control and MeHg-exposed groups were compared by a Fisher's Z transformation method.

Results

MeHg exposure and accumulation in glass eels

After seven days of exposure, concentrations of MeHg measured in the whole body of glass eels were 3.6 times higher than those measured in the control fish (476 ± 58 and 132 ± 31 ng Hg g -1 dry weight, respectively, p < 0.001). Concentrations in Hg(II) remained similar with 9 ± 3 and 7 ± 2 ng Hg g -1 dry weight in the control and exposed group, respectively.

MeHg-induced behavioral changes

Two dead glass eels were found in both the control and the exposed groups after swimming test, which left a total of 58 glass eels in each group.

The propensity of glass eels to migrate was first evaluated by their ability to synchronize to the change in water current direction every 6.2 h. Glass eels exhibiting swimming activity with a period of approximately 12.4 h were considered as synchronized and the others as nonsynchronized. As shown in Fig. 3a, the number of synchronized fish was lower in MeHgexposed group relative to the control (30 and 35 individuals, respectively corresponding to 52% and 60% of the total group, respectively), although the difference was not significant using a chisquared test (X 2 = 0.56, p = 0.454).

The level of swimming activity was then expressed by the total number of observations of glass eels swimming in the water column during the 360 sessions of 15 seconds. MeHg treatment significantly decreased the total number of observations when compared to control (chi-squared test, X 2 = 55.55, p < 0.001, Fig. 3b). When analyzing separately the two behavioral phenotypes, the decrease in the total swimming activity level was significant in non-synchronized glass eels but not in synchronized ones (Table 1). In addition, the maximum number of observations per fish decreased in the exposed group when compared to the control one (222 and 163 observations during the 360 sessions, respectively).

MeHg-and behavior-induced gene transcriptional responses

The transcriptional profiles of 27 genes involved in five metabolic functions were analyzed. Some of these genes code for proteins involved in the mitochondrial respiratory chain complexes, mitochondrial catabolism and the antioxidant system. The other genes code for proteins involved in macroautophagy (the best-characterized autophagy subclass) and mitophagy (a macroautophagy-dependent specific degradation of mitochondria). As outlined in Materials and Methods, the data set of gene expression levels was compressed by PCA procedure to reduce the number of the variables considered. All the statistical analyses were then examined using the score of individuals on the first axis of the PCA for each function considered.

We first analyzed the transcriptional responses of the five metabolic functions to both MeHg exposure and synchronization behavior. As shown in Table 2, none of the functions considered appear to have been significantly affected by the treatment of glass eels with MeHg, regardless of the tissue examined. In contrast, significant different transcriptional responses were evidenced in head tissues between synchronized and non-synchronized glass eels. Of the five functions considered, only one (mitophagy) did not differ between the two behavioral groups. The expression of genes from the other four functions was significantly higher in synchronized glass eels than the non-synchronized ones (Table 2 and Supplementary Fig. S5).

In order to consider in our analysis the effect of glass eels swimming activity, which could mask (or at least influence) the effect of MeHg, we then performed a Spearman's Rank Order Correlation test between individual swimming activity level and the first axis of PCA for each function in both control and MeHg-exposed fish. As locomotor activity is expected to increase energy expenditure, correlations were tested using glass eels displaying similar range of activity.

Although no significant difference could be evidenced between correlations observed in the control and the contaminated groups (using a Fisher's Z transformation), Table 3 shows that all the functions considered, except mitophagy, presented in the head a significant positive correlation to swimming activity after MeHg exposure but not in the control group. In the viscera, except mitochondrial catabolism, all others functions displayed a positive correlation to swimming activity in the contaminated group but not in the control. Finally, in the muscle, no correlation could be observed for the antioxidant system, the mitochondrial chain and the mitochondrial catabolism, while genes involved in mitophagy and macroautophagy responded positively to the activity in both groups.

Discussion

To clarify the adverse behavioral and metabolic responses due to MeHg exposure, changes in migration behavior and tissue-level transcriptions related to energetics were assessed with MeHg treatment in glass eels.

After a 7-day exposure, average MeHg concentration in whole body of glass eels was 476±58 ng Hg g -1 dry weight, revealing the ability of the whole body to accumulate MeHg in a short-term exposure. In contrast, the concentrations of Hg(II) remained low, supporting a previous study using isotopic tracers and showing a low potential of demethylation in glass eels after 11 days of exposure to MeHg (50 ng L -1 ) [START_REF] Claveau | Methylmercury effects on migratory behaviour in glass eels (Anguilla anguilla): An experimental study using isotopic tracers[END_REF].

Effect of MeHg on glass eels swimming activity and energy-related genes expression

Exposure to MeHg significatively decreased the total number of observations of glass eels swimming in the water column and reduced the maximum number of observations per fish ( 222and 163 in the control and the exposed group, respectively). These results are consistent with previous studies conducted in Salmo salar and Diplodus sargus displaying lower swimming activity after dietary exposure to MeHg at 10 mg.kg -1 during 4 months and 8.7 µg.g -1 during seven days, respectively [START_REF] Berntssen | Chronic dietary mercury exposure causes oxidative stress, brain lesions, and altered behaviour in Atlantic salmon (Salmo salar) parr[END_REF][START_REF] Puga | Unveiling the neurotoxicity of methylmercury in fish (Diplodus sargus) through a regional morphometric analysis of brain and swimming behavior assessment[END_REF]. Interstingly, when analyzing separately the synchronized and non-synchronized glass eels, the decrease in swimming activity level after MeHg exposure was only significant in the non-synchronized group, which supports the idea that glass eels presenting a low propensity to migrate might be more vulnerable to stress than those displaying a high probability to migrate [START_REF] Bolliet | Migratory behavior, metabolism, oxidative stress and mercury concentrations in marine and estuarine European glass eels (Anguilla anguilla)[END_REF][START_REF] Claveau | Methylmercury effects on migratory behaviour in glass eels (Anguilla anguilla): An experimental study using isotopic tracers[END_REF].

A number of studies reported the toxic effects of MeHg, notably on the oxidative status, the mitochondrial function and the Calcium homeostasis in fish [START_REF] Cambier | At environmental doses, dietary methylmercury inhibits mitochondrial energy metabolism in skeletal muscles of the zebra fish (Danio rerio)[END_REF][START_REF] Cambier | Serial analysis of gene expression in the skeletal muscles of zebrafish fed with a methylmercury-contaminated diet[END_REF][START_REF] Claveau | Methylmercury effects on migratory behaviour in glass eels (Anguilla anguilla): An experimental study using isotopic tracers[END_REF][START_REF] Graves | Response of oxidative stress transcripts in the brain of wild yellow perch (Perca flavescens) exposed to an environmental gradient of methylmercury[END_REF][START_REF] Gonzalez | Comparative effects of dietary methylmercury on gene expression in liver, skeletal muscle, and brain of the zebrafish (Danio rerio)[END_REF]Nostbakken et al., 2012;[START_REF] Rasinger | Methylmercury induced neurotoxicity and the influence of selenium in the brains of adult zebrafish (Danio rerio)[END_REF][START_REF] Richter | Gene expression changes in female zebrafish (Danio rerio) brain in response to acute exposure to methylmercury[END_REF][START_REF] Yadetie | Quantitative analyses of the hepatic proteome of methylmercury-exposed Atlantic cod (Gadus morhua) suggest oxidative stress-mediated effects on cellular energy metabolism[END_REF]. Surprisingly, we did not observe any effect of MeHg on transcriptional profiles of the studied genes involved in mitochondrial metabolism, antioxidant system or autophagy. Our results contrast with a previous study in glass eels showing an activation of antioxidative defence system at the transcriptomic level after eleven days of exposure to MeHg (50ng/l) [START_REF] Claveau | Methylmercury effects on migratory behaviour in glass eels (Anguilla anguilla): An experimental study using isotopic tracers[END_REF]. However, in this last study, gene expression was analyzed just after exposure while in the present one analyses were conducted after the behavioral test, i.e., 10 days after the end of exposure. In addition, both studies focused on gene expression at a single time point that does not give a real picture of the dynamic aspect of the different events at play during complex and integrative processes such as antioxidant system, energy metabolism or autophagy. Furthermore, depending on the genes studied, the levels of transcripts do not always correlate with the amount or the activity of the corresponding proteins [START_REF] Vogel | Insights into the regulation of protein abundance from proteomic and trascriptomic analyses[END_REF][START_REF] Yadetie | Quantitative analyses of the hepatic proteome of methylmercury-exposed Atlantic cod (Gadus morhua) suggest oxidative stress-mediated effects on cellular energy metabolism[END_REF]. Future functional studies will therefore be necessary to draw definitive conclusions on the effect of MeHg in the functions monitored in glass eels.

However, interestingly, we evidenced a significant positive correlation between individual swimming activity levels and the expression of the genes studied. The expression of genes related to autophagy and mitophagy increased with activity in the muscle, both in the control and exposed groups, probably reflecting an increase in energy requirement related to activity. However, in the head and viscera, most of the genomic functions related to antioxidant system and metabolism showed a positive correlation in the contaminated group, but not in the control one. As mentioned above, oxydative stress and mitochondrial impairment are among the most studied effects of MeHg in fish. MeHg targets some specific thiol containing proteins such as gluthatione peroxydase involved in the anti-oxydant system, predisposing cell to oxidative stress and generation of Reactive Oxygen Species (known as ROS) [START_REF] Farina | Special Issue: Environmental Chemicals and Neurotoxicity Oxidative stress in MeHg-induced neurotoxicity[END_REF]. In addition, MeHg can also directly target specific thiol-containing enzymes of the respiratory chain complex and both effects may affect cellular energy pathways [START_REF] Farina | Special Issue: Environmental Chemicals and Neurotoxicity Oxidative stress in MeHg-induced neurotoxicity[END_REF][START_REF] Glaser | Oxidative stress-mediated inhibition of brain creatine kinase activity by methylmercury[END_REF]Yadieti et al., 2016). Thus, although our results must be taken with caution (because the differences between correlations obtained for the control and contaminated groups were not strong enough to be significant), they strongly suggest that contaminated glass eels were affected by MeHg and needed to increase energy metabolism more than non-contaminated fish to cope with increasing swimming activity.

It is also noteworthy that the head was the most affected section by MeHg, a positive correlation being observed between swimming activity and all genomic functions. Although the head section include not only the brain but also the gills, this result seems consistent with the literature reporting that the brain is a predominant target for MeHg in fish [START_REF] Gonzalez | Comparative effects of dietary methylmercury on gene expression in liver, skeletal muscle, and brain of the zebrafish (Danio rerio)[END_REF][START_REF] Graves | Response of oxidative stress transcripts in the brain of wild yellow perch (Perca flavescens) exposed to an environmental gradient of methylmercury[END_REF][START_REF] Pereira | Fish eyes and brain as primary targets for mercury accumulation -A new insight on environmental risk assessment[END_REF]Pereira et al., , for review 2019)). Indeed, MeHg has been reported to cross the blood-brain-barrier and accumulates in the brain having serious toxic effects including proteome changes related to oxidative stress and mitochondrial dysfunction, morpho-structural changes and dysfunction in neurotransmission processes [START_REF] Cariccio | Mercury involvement in neuronal damage and in neurodegenerative diseases[END_REF] for review see [START_REF] Pereira | A multidimensional concept for mercury neuronal and sensory toxicity in fish -From toxicokinetics and biochemistry to morphometry and behavior[END_REF][START_REF] Pletz | Dose-response analysis indicating timedependent neurotoxicity caused by organic and inorganic mercury -Implications for toxic effects in the developing brain[END_REF]. Considering also that in both control and exposed glass eels, the expression levels of metabolism-related genes were lower in non-synchronized glass eels than in synchronized ones in the head but not in the other sections, these results suggest that the head may represent the one to focus on for a better understanding of glass eel's migration and the effect of environmental stressors on this migration.

Effect of MeHg on the rhythmic swimming activity in glass eels

To migrate up estuary, glass eels use selective tidal stream transport and synchronize their swimming activity to the flood thanks to an endogenous clock [START_REF] Bolliet | Rhythmic swimming activity in Anguilla anguilla glass eels: Synchronisation to water current reversal under laboratory conditions[END_REF][START_REF] Forward | Selective tidal-stream transport of marine animals[END_REF][START_REF] Hickman | Densities and swimbladder development of juvenile American eels, Anguilla rostrata (Lesueur) as related to energetics of migration[END_REF][START_REF] Mccleave | Selective tidal stream transport in the estuarine migration of glass eels of the American eel (Anguilla rostrata)[END_REF][START_REF] Wippelhauser | Precision of behavior of migrating juvenile American eels (Anguilla rostrata) utilizing selective tidal stream transport[END_REF]. They are also known to avoid light and mainly migrate during the night, likely using a circadian clock [START_REF] Bolliet | Rhythmic swimming activity in Anguilla anguilla glass eels: Synchronisation to water current reversal under laboratory conditions[END_REF]. In our experimental conditions, a very low light intensity during photophase was used to avoid synchronization of glass eels activity to photoperiod that could have masked synchronization to the tidal cue. Thus, in the present study, synchronized glass eels corresponded to fish that synchronized their swimming activity to the change in water current direction with a period close to 12.4 h. A lower number of glass eels synchronizing to tidal period were observed in contaminated condition compared to control one, even though it was not statistically significant using a chi-squared test. Xenobiotics have been shown to disturb the circadian system in different fish species [START_REF] Prokkola | Circadian rhythms and environmental disturbancesunderexplored interactions[END_REF] and a couple of studies showed that MeHg disrupted circadian rhythms in rodents, the crayfish Astacus astacus and the freshwater crab Potamon potamios [START_REF] Arito | Effect of methylmercury chloride on sleep-waking rhythms in rats[END_REF][START_REF] Parmalee | Metals and circadian rhythms[END_REF][START_REF] Styrishave | Evaluation of mercury-induced changes in circadian heart rare rhythms in the freshwater crab, Potamon potamios and the crayfish, Astacus astacus as an early predictor of mortality[END_REF]. In addition, [START_REF] Depledge | Disruption of circulatory and respiratory activity in shore crabs (Carcinus maenas (L.)) exposed to heavy metal pollution[END_REF] evidenced an effect of mercury exposure on tidal rhythmicity in the heart rate of the shorecrab Carcinus maenas. Though the location of the circatidal clock is still unknown in fish, the pacemaker regulating circadian rhythms has been located in the pineal gland. Interestingly, [START_REF] Korbas | Chemical form matters: Differential accumulation of mercury following inorganic and organic mercury exposures in zebrafish larvae[END_REF][START_REF] Korbas | Methylmercury targets photoreceptor outer segments[END_REF] reported an accumulation of inorganic Hg in the pineal gland of zebrafish exposed to MeHg. Thus, the relationship between mercury species and the endogenous clock(s) driving the rhythmic swimming activity in glass eels appears as an interesting avenue to explore.

Conclusion

Our results suggest that MeHg may affect the estuarine migration of glass eels by reducing their ability to synchronize to the tide and their level of swimming activity. They also support the idea that non-synchronized fish may be more vulnerable to stress and the first affected by contamination. A decrease in the propensity to migrate in natura would lead to an increase in glass eels settlement in estuary and consequently a decrease in population recruitment in upper reaches. Non-migrant glass eels becoming mostly males, such effect would change the fate of the population by influencing the sex ratio in this species. A better understanding of the effect of MeHg on the maximum swimming activity in glass eels and their biological clocks is now required to clearly assess the impact of this contaminant on their synchronization to environmental cues and migration.

The results also support the interest of focusing on the head to investigate facultative migration behavior in glass eels and the effect of environmental stressors on this rhythmic behavior. However, as head samples include the entire brain tissue, eyes but also the gills, they also question the relationships between the ability to migrate and osmoregulatory functions. Table 1. Assessment of the MeHg-induced effects on swimming activity within two behavioral phenotypes. Synchronized glass eels: Fish which synchronized their swimming activity to the change in water current direction every 6.2 h. Number of fish: Total number of synchronized or non-synchronized glass eels observed during the behavioral test (for a total of 58 glass eels in both groups). Number of observations: Total number of observations of glass eels swimming in the water column during the 360 sessions recorded. A tagged glass eel can be observed only once during a recorded session. Significant values are in bold.

Figure captions Fig. 1 .

 captions1 Figure captionsFig.1. Kinetics of MeHg (blue circles) and Hg(II) (red triangles) concentrations (mean ± sd) in glass eels along 0-30 days of exposure to MeHg. Data for 0 d are from control fish (n = 3).

Fig. 2 .

 2 Fig. 2. Sections of glass eel.

Fig. 3 .

 3 Fig. 3. Swimming behavior of control (blue bar, n = 58) and MeHg-exposed (orange bar, n = 58) glass eels over 10 days. (a) Bar chart showing the total number of glass eels which synchronized their swimming activity to the change in water current direction every 6.2 h, Pearson's chisquared test, X 2 =0.56, p=0.454 (b) Bar chart showing the total number of observations of all glass eels, Pearson's chi-squared test, X 2 =0.56, p<0.001.

Figure 1

 1 Figure 1

Figure 2 Figure 3

 23 Figure 2

Table 2 .

 2 Statistical results of the two-way ANOVA for detecting transcriptional profiles of each genomic function in response to MeHg exposure and synchronization behavior. P-values of ANOVA are presented in the table. Significant values are in bold. Factor 'Treatment' is MeHg treatment (control vs MeHg exposure), factor 'Behavior' is behavioral phenotype (nonsynchronized vs synchronized), 'Int' is interaction of both factors. H-head, V-viscera, Mmuscle.

	Pearson's chi-squared test

Table 3 .

 3 Spearman's Rank Order Correlation test between individual swimming activity level and the first axis of PCA for each function in both control and MeHg-exposed fish. R and p values of Spearman's correlation test are presented in the table. Significant values are in bold. H-head, V-viscera, M-muscle.

			Control		MeHg
	Function	Tissue	r	p-value	r	p-value
	Antioxidant system	H V M	0.32 0.05 0.33	0.169 0.826 0.156	0.65 0.53 0.32	<0.001 0.014 0.135
	Mitochondrial respiratory chain	H V M	0.22 0.30 0.27	0.346 0.193 0.251	0.57 0.43 0.27	0.005 0.049 0.205
	Mitochondrial catabolism	H V M	0.23 -0.10 -0.07	0.326 0.686 0.772	0.68 0.28 0.06	<0.001 0.223 0.774
		H	0.63	0.003	0.76	<0.001
	Mitophagy	V	0.40	0.078	0.51	0.019
		M	0.67	0.001	0.75	<0.001
		H	0.39	0.091	0.72	<0.001
	Macroautophagy	V	0.38	0.099	0.62	0.002
		M	0.75	<0.001	0.68	<0.001

Acknowledgements

We wish to thank the IE ECP for the technical support and the China Scholarship Council for the grant to Hengtong Liu.

Supplementary data

Text S1. Openbugs code, the prior values for parameters, the Markov chain initial values and the data for the swimming activity and the inference model.


Y Song
email: yu.song@univ-orleans.fr

F Foucher

The impact of EGR components on ozone decomposition under engine relevant conditions in a Rapid Compression Machine

Keywords: Ozone decomposition, EGR components, rapid compression machine

In this work, with the addition of 90 ppm ozone, the influence of the main EGR components, namely nitrogen, carbon dioxide, steam, and methane, along with the gasoline surrogate iso-octane, on ozone oxidation were investigated at 30 bar, 800 K using a rapid compression machine (RCM). Taking the case diluted with nitrogen as a reference, it was found that the chemical effects of CO2 on ozone decomposition accelerated with the increase in the carbon dioxide concentration. The promoting effect of steam on ozone decomposition remained constant regardless of the steam mole fraction added. The mutual effect of methane and ozone was detectable, but the enhancement of ozone decomposition was lower than when iso-octane was added. A comprehensive detailed kinetic model was employed to interpret the experimental data. The model performance was improved by replacing the rate coefficients of O3+O=2O2. Analysis of the rate of production showed that the IC8H18+O=CC8H17+OH reaction predominated over the chain terminating reaction O3+O=2O2, which can explain the significant promoting effect in the presence of iso-octane. When steam is added, two OH radicals are derived from the chain branching reaction H2O+O=2OH. When methane is added, only one OH radical, originating from the O radical attacking methane, is formed. Consequently, the promoting effect of methane addition is the lowest.

Introduction

Ozone (O3) is a popular plasma oxidizer which has attracted widespread attention during the last decade. When applied to the combustion field, it exhibits significant advantages in terms of ignition promotion especially in the ultra-lean combustion mode, which can provide better efficiency and attenuate pump losses [START_REF] Contino | CFD simulations using the TDAC method to model iso-octane combustion for a large range of ozone seeding and temperature conditions in a single cylinder HCCI engine[END_REF]. The byproducts of ozone decomposition are oxygen atoms (O) and molecular oxygen (O2), which are very environmentally friendly. The use of ozone-assisted combustion would therefore simultaneously fulfill the two requirements of high efficiency and low emissions. In particular, it could solve the issues associated with on-load vehicle auto-ignition in Homogeneous Charge Compression Ignition (HCCI) engines. Given the fact that commercial ozone generators are compact and economical, ozoneassisted combustion could serve as an alternative method to deal with the ignition process [START_REF] Masurier | Application of an Ozone Generator to Control the Homogeneous Charge Compression Ignition Combustion Process[END_REF].

Reports associated with ozone-assisted combustion can be found in engine studies [START_REF] Contino | CFD simulations using the TDAC method to model iso-octane combustion for a large range of ozone seeding and temperature conditions in a single cylinder HCCI engine[END_REF][START_REF] Masurier | Application of an Ozone Generator to Control the Homogeneous Charge Compression Ignition Combustion Process[END_REF][START_REF] Kobashi | Ignition control in a gasoline compression ignition engine with ozone addition combined with a two-stage direct-injection strategy[END_REF][START_REF] Schönborn | Ignition control of homogeneous-charge compression ignition (HCCI) combustion through adaptation of the fuel molecular structure by reaction with ozone[END_REF][START_REF] Masurier | Effect of Additives on Combustion Characteristics of a Natural Gas Fueled HCCI Engine[END_REF][START_REF] Foucher | Influence of ozone on the combustion of n-heptane in a HCCI engine[END_REF][START_REF] Masurier | Ozone applied to the homogeneous charge compression ignition engine to control alcohol fuels combustion[END_REF][START_REF] Masurier | Investigation of iso-octane combustion in a homogeneous charge compression ignition engine seeded by ozone, nitric oxide and nitrogen dioxide[END_REF], jet-stirred reactors [START_REF] Rousso | Low-Temperature Oxidation of Ethylene by Ozone in a Jet-Stirred Reactor[END_REF][START_REF] Rousso | Identification of the Criegee intermediate reaction network in ethylene ozonolysis: impact on energy conversion strategies and atmospheric chemistry[END_REF], flow reactors [START_REF] Zhao | Kinetic studies of ozone assisted low temperature oxidation of dimethyl ether in a flow reactor using molecular-beam mass spectrometry[END_REF], shock tubes [START_REF] Jones | The thermal decomposition of ozone in a shock tube[END_REF][START_REF] Heimerl | The unimolecular ozone decomposition reaction[END_REF][START_REF] Michael | Thermal decomposition of ozone[END_REF][START_REF] Peukert | High Temperature Shock Tube Studies on the Thermal Decomposition of O3 and the Reaction of Dimethyl Carbonate with O-Atoms[END_REF], and flame studies [START_REF] Ombrello | Flame propagation enhancement by plasma excitation of oxygen. Part I: Effects of O3[END_REF][START_REF] Zhang | Ozone effect on the flammability limit and near-limit combustion of syngas/air flames with N2, CO2, and H2O dilutions[END_REF]. Among engine studies, the impact of ozone on various kinds of fuels has been investigated, ranging from light alkane (natural gas) [START_REF] Masurier | Effect of Additives on Combustion Characteristics of a Natural Gas Fueled HCCI Engine[END_REF] to primary reference fuel blends [START_REF] Foucher | Influence of ozone on the combustion of n-heptane in a HCCI engine[END_REF], with an extension to alcohol [START_REF] Masurier | Ozone applied to the homogeneous charge compression ignition engine to control alcohol fuels combustion[END_REF]. In general, ozone addition was found to have a promoting effect on fuel activity, apart from one case [START_REF] Masurier | Investigation of iso-octane combustion in a homogeneous charge compression ignition engine seeded by ozone, nitric oxide and nitrogen dioxide[END_REF] in which ozone and NO were injected simultaneously, where it delayed iso-octane combustion when the ozone concentration was less than half of the nitric oxide concentration seeded. This is attributed to the reaction between ozone and nitric oxide, yielding nitric dioxide and oxygen. Recently, in our group, Seignour et al. [START_REF] Seignour | Experimental Understanding of Ozone Decomposition inside a Low-Temperature Combustion Engine[END_REF] experimentally investigated the effect of different gas compositions, namely exhaust gas recirculation (EGR) components, on ozone decomposition in a diesel engine, since EGR is considered a promising method to overcome emission mitigation issues. However, as temperature stratification may exist inside the engine, a better controlled experimental apparatus relevant to engine studies is necessary to obtain more precise experimental results.

A rapid compression machine (RCM), which operates at low-intermediate temperatures and intermediate-high pressures, is a well-suited for engine relevant studies. Equipped with a well-designed crevice in the piston, an RCM can guarantee temperature homogeneity in the core region since the effect of the boundary layer is negligible. In a recent study on ozone-enhanced compressed ignition in an RCM [START_REF] Seignour | Measurements and modeling of ozone enhanced compression ignition in a rapid compression machine and optically-accessible engine[END_REF], it was found that ozone decomposition accelerated when the charge temperature was increased, oxygen concentration was reduced, or fuel was added. In the unfueled condition, the temporal prediction of O3 decomposition was generally retarded too much by the model adopted. It appears that the impact of the diluent on ozone decomposition is still unclear.

From the modelling point of view, Sun [START_REF] Sun | The effect of ozone addition on combustion: Kinetics and dynamics[END_REF] reviewed the kinetic models in terms of O3 oxidation.

Several elementary reactions included in the O3 sub-mechanism were highlighted. Various kinetic models associated with O3 oxidation have been put forward to predict the experimental results. In terms of ozone decomposition reactions, early in the 1960s, Benson and Axworthy [START_REF] Benson | Mechanism of the gas phase, thermal decomposition of ozone[END_REF] studied the O3+M=O2+O+M reaction, which is an initial step for ozone reactions. This was followed by numerical studies devoted to this elementary reaction [START_REF] Jones | The thermal decomposition of ozone in a shock tube[END_REF][START_REF] Michael | Thermal decomposition of ozone[END_REF][START_REF] Heimerl | The unimolecular ozone decomposition reaction[END_REF][START_REF] Thoma | Experiments on shock-heated ozone dissociation in oxygen/air using UV laser spectroscopy[END_REF]. In 2013, Peukert et al. [START_REF] Peukert | High Temperature Shock Tube Studies on the Thermal Decomposition of O3 and the Reaction of Dimethyl Carbonate with O-Atoms[END_REF] experimentally and numerically measured and validated the rate of O3+M=O2+O+M with the reflected shock tube technique using O-atom atomic resonance absorption spectrometry (ARAS) detection. Temperature and pressure dependent rate coefficients were proposed in this work, but the third-body role of triatomic molecules such as CO2 and H2O was not addressed. On the other hand, the rate constants of the O3+O=2O2 reaction remain more uncertain; the most common rate constants adopted in the O3 mechanism are derived from the evaluation by Atkinson et al. [START_REF] Atkinson | Evaluated kinetic and photochemical data for atmospheric chemistry: Volume I-gas phase reactions of Ox, HOx, NOx and SOx species[END_REF].

Hence, a validation of the current O3 kinetic model is needed.

The purpose of this study was to extend previous work [START_REF] Seignour | Experimental Understanding of Ozone Decomposition inside a Low-Temperature Combustion Engine[END_REF][START_REF] Seignour | Measurements and modeling of ozone enhanced compression ignition in a rapid compression machine and optically-accessible engine[END_REF] to an experimental and modelling study aiming to determine the impact of different EGR intermediate species on ozone decomposition with the aid of an RCM setup. A modified comprehensive kinetic model was used to interpret the experimental data. A rate of production (ROP) analysis was also conducted to study the chemical effects of the various additives on ozone decomposition.

Experimental setup

The experimental apparatus used in this work was a classical single piston RCM, developed and built by the University of Orléans. A detailed description of this setup can be found in [START_REF] Pochet | Experimental and numerical study, under LTC conditions, of ammonia ignition delay with and without hydrogen addition[END_REF]; here, only a brief description is given. Firstly, homogeneity inside the combustion chamber of the RCM is guaranteed thanks to the crevice design, which allows strong absorption of boundary layers. It was therefore assumed that compression in the core area of the chamber was isentropic. With the aid of a mass flow controller, the gases (CH4, O2, N2 and CO2) from the individual cylinders supplied by Messer (purities of 99.99%) passed through the manifold and entered the mixture tank (volume of 4.08 L) directly. Liquid iso-octane and deionized water were injected into the tank by the syringe with the desired value. Apart from the gas supply system, the main part of the RCM was equipped with several kinds of temperature and pressure sensors. A type K thermocouple with an accuracy of ± 1 K was used to measure the intake temperature.

An Anseros COM-AD-01 corona-discharge ozone generator was used to generate ozone in the RCM chamber. With the intake bypass connected to the compressed air, the ozone was generated at the outlet of the generator. An ozone analyzer (Anseros Ozomat MP6060) with a range of 0-200 ppm ozone was used to measure and monitor the desired ozone mole fraction.

Two opposed Suprasil windows were mounted on the sides of the RCM chamber head to provide The signal was recorded and analyzed over 20 shots by the RCM. Two independent samples were measured during each experiment. One is a "reference" sample in which no ozone was injected into the system to account for the light attenuation from the optical setup, and the other is a "target" sample with ozone seeded. The ozone mole fraction was calculated with the Beer-Lambert Law:

= ln

where Iref is the reference sample absorption rate, I is the target sample absorption rate, Kb is the Boltzmann constant, B is the bore diameter, denotes the absorption cross section at 254 nm as a function of temperature [START_REF] Astholz | Temperature Dependence of the Ozone Absorption Coefficient in the Hartley[END_REF], T and P are the in-cylinder temperature and pressure, respectively. In-cylinder temperature was deduced from the in-cylinder pressure trace.

Kinetic Model

The mechanism adopted in this work consists mainly of the recent comprehensive kinetic model used in [START_REF] Masurier | Investigation of iso-octane combustion in a homogeneous charge compression ignition engine seeded by ozone, nitric oxide and nitrogen dioxide[END_REF], in which the ozone decomposition reactions were updated. A modification to the O3+M=O2+O reaction was made [START_REF] Arnold | Temperature dependence of the reactions O (3P)+ O3→ 2O2 and O (3P)+ O2+ M→ O3+ M[END_REF], which turns out to predict the experimental data better. The third body coefficient of triatomic molecules (CO2 and H2O) was adopted following the modularity principle behind [START_REF] Benson | Mechanism of the gas phase, thermal decomposition of ozone[END_REF]. The current complete mechanism (1037 species and 4250 reactions) is provided in the supplementary material (SM) in CHEMKIN format, along with thermodynamic properties.

Results and discussion

Ozone decomposition measurements were conducted in the presence of various amounts of N2, CO2, H2O, CH4 and the gasoline surrogate iso-octane in the 313-800 K temperature range and at a pressure of 30 bar. The experimental conditions investigated in this study are presented in Table 1. The initial experimental conditions selected are relevant to engine conditions. Numerical calculations were performed with CHEMKIN-PRO software [START_REF]CHEMKIN-PRO 15083[END_REF]. A closed homogenous reactor solver with variable volume profiles (see SM), which was derived from the aforementioned non-reactive pressure profiles by adopting the calculation methods proposed in [START_REF] Mittal | Computational fluid dynamics modeling of hydrogen ignition in a rapid compression machine[END_REF], was employed for ozone quantification.

Experimental results and comparison with simulations

The ozone conversions as a function of time under various conditions are presented in this section.

Simulation results are also shown to compare with the experimental data. Experimental results are represented by solid lines, and simulations by different types of dashed lines in all the figures depicted in this part.

Ozone decomposition diluted in nitrogen

Fig. 2 shows the ozone (90 ppm) decomposition diluted in nitrogen as a function of time, along with the corresponding temperature and pressure profiles. A small ozone consumption can be observed between t= -5 ms to t=-2 ms, followed by a steep ozone consumption until complete depletion is achieved. Note that the initial oscillation arises from the uncertainty and the vibrations caused by the UV technology and RCM apparatus, respectively. Two simulations were compared in this work. First, with the previous model (Model-1) used in [START_REF] Seignour | Experimental Understanding of Ozone Decomposition inside a Low-Temperature Combustion Engine[END_REF], a retarding effect was detected compared to the experimental trend. On replacing the rate coefficient of O3+O=2O2 [START_REF] Arnold | Temperature dependence of the reactions O (3P)+ O3→ 2O2 and O (3P)+ O2+ M→ O3+ M[END_REF], the prediction improved as the sharp consumption timing was better captured by the updated model (Model-2), although the model still failed to predict the initial slow ozone consumption zone (t= -5 to -2 ms). Overall, good agreement between the steep ozone oxidation experimental data and model output was observed. A possible explanation for the slow ozone consumption might be the surface effect when the temperature is above 550 K. 4.1.2 The impact of carbon dioxide Fig. 3 shows the ozone decomposition profile with different amounts of carbon dioxide (0, 9.875%, 19.75%, 39.5%) as a function of time. In these cases, the initial temperatures were kept the same (313 K).

The end pressures (Pc) was approximately 30 bar, which is consistent with the cases shown in Fig. 2. As for the following cases (steam, methane and iso-octane added), the Pc were all kept at 30 bar. The corresponding temperature profiles are given in Fig. 4. Overall, CO2 addition retarded the initiation of ozone decomposition.

In the presence of CO2, the inhibiting effect increased with the increase in the CO2 mole fraction. In particular, in the case of 39.5% CO2 addition, 30% of ozone still remained in the system compared to the nearly completed consumption case without CO2 addition at a time of 9 ms. Due to the high thermal capacity of CO2, the temperature profiles with CO2 addition were generally below that without CO2, as shown in Fig. 4.

In this case, the retarding effect might be ascribed to the thermal effect of CO2, which deteriorates the whole system.

The model predicted the experimental data well in the case of 9.875% and 19.75% CO2 addition, but a deviation between the experimental data and simulation was found with 39.5% CO2 addition. While it might be assumed that the main role of CO2 in ozone decomposition is participating in the O3+M=O2+O+M reaction as a third body, the introduction of the third body coefficient did not in fact impact the model behavior significantly. It could be ascribed to the predominantly thermal role of CO2 in this temperature range. In order to decouple the CO2 thermal effect, another set of experiments was conducted in which the initial temperature was increased to 60, 80, and 120 °C for the three carbon dioxide conditions (9.875%, 19.75%, 39.5%), respectively, to ensure a similar end of compression temperature (Tc). Fig. 5 shows the ozone decomposition profile with different amounts of carbon dioxide (0, 9.875%, 19.75%, 39.5%) at a similar end of compression temperature as a function of time. With a similar Tc, the ozone decomposition behavior is dramatically different from those shown in Fig. 3. In the case of 9.875% CO2 addition, ozone decomposition shows a similar trend compared to the one without CO2 addition, while in the presence of 19.75% CO2, a slight promoting effect can be observed. As CO2 is further increased to 39.7%, the accelerating effect becomes significant. Although the temperature profiles for the aforementioned cases are not exactly the same as the one without CO2 addition as shown in Fig. 6, the temperature is consistent among the 0, 9.875%, and 19.75% CO2 addition cases at t=3 ms. When taking a close look at Fig. 5 again at t= 3 ms, one may note that the ozone is consumed more in the presence of CO2 than without CO2 addition. This implies that the chemical effect of CO2 should not be neglected even at 800 K, 30 bar, under engine relevant conditions. The model reproduced the cases with different amounts of carbon dioxide (9.875% and 19.75%) reasonably well, but the deviation between the experimental and simulated results became larger when further increasing the carbon dioxide mole fraction to 39.5%. The reason for this poor model performance in the case of a relatively high amount of carbon dioxide is still unclear.

The impact of steam

Fig. 7 shows the ozone decomposition profile with different amounts of steam (0, 2.5%, 5%, 10%) as a function of time. It is interesting to note that different amounts of steam exhibit a very similar promoting trend compared to the one without steam addition, which is also observed in engine work [START_REF] Seignour | Experimental Understanding of Ozone Decomposition inside a Low-Temperature Combustion Engine[END_REF]. In addition, at around t= 0.4 ms, a short plateau (1 ms) was observed, after which it continued to decompose. The "plateau" phenomenon could be explained by the potential formation of H2O2 or HO2, which also show characteristic absorption at this wavelength [START_REF] Moortgat | Absorption spectrum and kinetics of reactions of the acetylperoxy radical[END_REF]. In general, Fig. 7 shows that ozone initiation advances by 2 ms in the presence of steam. In this case, the temperature profiles (Fig. 8) are almost the same among all the investigated conditions, except the one with 10% steam addition, whose temperature is 10 K lower. Hence the promoting effects can be attributed to the chemical effect of steam. While the model was able to capture the promoting trend, a retarded model performance was observed compared to the experimental data. A possible explanation for the deviation between the experimental results and numerical calculations could be the inaccuracy of the elementary reaction O3+H2O=H2O2+O2. Its reaction rate was adopted from the early experimental work by [START_REF] Hatakeyama | Rate constants for reactions between atmospheric reservoir species. 2. Water[END_REF]. By implementing this rate constant, the contribution of O3+H2O=H2O2+O2 is minor as shown in the following analysis (Fig. 9).

The rate of production (ROP) analysis (Fig. 9 and 10) showed that the main consumption channel for ozone is O3+M=O2+O+M, following O3+O=2O2. The ROP of the reaction O3+H2O=H2O2+O2 was negligible. For steam, H2O+O=2OH is the main consumption channel; the formation channels are HO2+OH=H2O+O2 and H2O2+OH=H2O+HO2. Once ozone has decomposed, the O atoms can react with either ozone or steam. Steam can serve as a promoter since the products of O radicals and steam are hydroxide radicals. On the other hand, O3+O=2O2 is a chain terminating reaction due to the stable oxygen products. In this case, the participation of steam in the system competes with the chain terminating reaction and yields two OH radicals, which may accelerate ozone decomposition. This could explain the advance of the experimental and model results in the presence of steam shown in Fig. 7. It is worth mentioning that different amounts of steam have a very similar promoting trend compared to the case without steam. In [START_REF] Seignour | Experimental Understanding of Ozone Decomposition inside a Low-Temperature Combustion Engine[END_REF], the effect of steam was attributed to the low activated energy required by O3+H2O=O2+O+H2O. However, the similar promoting effect in the presence of different amounts of steam was not clarified. In the present work, the ROP involved in steam evolves as shown in Fig. 10. The main consumption channel of steam is H2O+O=2OH. The function of this elementary reaction can be twofold: on the one hand, it competes with the chain terminating reaction O3+O=2O2; on the other hand, the two OH radicals are reactive radicals, which is beneficial to the whole system. It is reported in the review paper [START_REF] Sun | The effect of ozone addition on combustion: Kinetics and dynamics[END_REF] that the highly oxidative hydroxy radical attaching ozone is of importance. The main steam formation reactions are OH radical consuming ones, along with the yield of stable products. Note that the ROP of O3+H2O=O2+O+H2O increases with the increase of steam; accordingly, the ROP of the chain terminating reactions HO2+OH=H2O+O2 and H2O2+OH=H2O+HO2 also increase. In sum, the balance of the consumption and formation channels of steam results in the similar influence of various amounts of steam addition on ozone decomposition.

The impact of methane and iso-octane

With methane addition, surprisingly, the accelerating effect was also observed. In [START_REF] Sun | The effect of ozone addition on combustion: Kinetics and dynamics[END_REF], it was argued that the interaction between methane and ozone is very limited at room temperature. In contradiction with [START_REF] Sun | The effect of ozone addition on combustion: Kinetics and dynamics[END_REF], a promoting effect of methane addition was found here, demonstrating the mutual effect between methane and ozone under engine relevant conditions as shown in Fig. 11. In the presence of methane, the acceleration of ozone decomposition was observed after the time (t= 0 ms). Fig. 12 shows that the temperature profile was similar with and without methane addition. So the chemical effect of methane is noticeable during the ozone decomposition process. The agreement between the experimental data and simulation results was generally acceptable although a slight deviation was observed. The promoting effect of methane was also illustrated by ROP analysis. At the beginning of ozone decomposition as shown in Fig. 13, O3+M=O2+O+M is the main contributing reaction. The chain terminating reaction O3+O=2O2 also shows a significant effect on ozone decomposition. With regard to methane oxidation (Fig. 14), CH4+O=CH3+OH exhibits a similar production rate to the O3+O=2O2 reaction. Only one OH radical is released by the CH4+O=CH3+OH reaction. This could explain the relatively small promoting effect compared to the case with steam since two OH radicals are formed via H2O+O=2OH. On the other hand, CH4+OH=CH3+H2O is the main consumption channel of methane. When comparing the different cases (250 and 500 ppm addition), as shown in Fig. 14, it can be seen that the initiation timing of CH4+O=CH3+OH with 500 ppm methane addition is advanced compared to the one with 250 ppm addition. Moreover, the more methane is added, the higher the rate of production, which explains the more promoting effect with 500 ppm methane addition. The effect of iso-octane addition on ozone decomposition at 30 bar is shown in Fig. 15. Two different iso-octane mole fractions (250 and 500 ppm) were tested. As expected, ozone consumption started at early timing, indicating that the presence of iso-octane promotes ozone oxidation. In this case, at around t=1.4 ms, a small plateau also occurred. However, with the decrease in the iso-octane mole fraction, this plateau vanished. Note that the temperature profile (Fig. 16) shows a similar trend when the timing is below 2 ms. This is the timing range of ozone decomposition in the iso-octane addition cases.

The model captured the experimental results well regardless of the prediction failure for the "plateau" characteristic since it is the absorption signal of the HO2 or H2O2 species. The ROP analysis (Fig. 17 and18)

showed that the main ozone consumption channel is O3+M=O2+O+M, while other channels exhibited a minor effect in terms of ozone oxidation. On the other hand, upon the initial iso-octane oxidation, IC8H18+O=CC8H17+OH and IC8H18+OH=AC8H17+H2O played a similar role, while the other channels involving IC8H18+OH exhibited a minor effect compared to that of IC8H18+OH=AC8H17+H2O. Note that the yield of O atoms serves as a reactant in the branching reaction IC8H18+O=CC8H17+OH, as shown in the inset of Fig. 18. This reaction plays a dominant role compared to the chain terminating reaction O3+O=2O2, which shows a small effect on ozone decomposition, as depicted in Fig. 17. This is the reason why the promoting effect of iso-octane on ozone decomposition is greater than that of methane. The influence of different amounts of iso-octane on ozone decomposition exhibits the same trend as in the presence of methane. The insets present the ROP profile of the IC8H18+O reaction.

Conclusions

In this work, in the presence of 90 ppm ozone, the impact of the main EGR components, i.e. nitrogen, carbon dioxide, steam, and methane along with the gasoline surrogate iso-octane, on ozone oxidation were studied at 30 bar, 800 K with the aid of an RCM. Results reveal that the chemical effect of CO2 on ozone decomposition increases with the increase in the carbon dioxide mole fraction, whereas the promoting effect of steam on ozone decomposition remains constant regardless of the steam concentration added. The enhancement of ozone decomposition is less in the presence of methane compared to that of iso-octane. A comprehensive detailed kinetic model with rate modification of O3+O=2O2 was employed to interpret the experimental data. It is found that the reaction IC8H18+O=CC8H17+OH strongly dominates the chain terminating reaction O3+O=2O2 in the presence of iso-octane. As for steam additives, the chain branching reaction H2O+O=2OH shows a high production rate and is of particular importance since it competes with the chain terminating reaction O3+O=2O2 and two OH radicals are formed. The ROP of CH4+O=CH3+OH is similar to that of O3+O=2O2, and only one OH radical is released in the presence of methane.

  optical access. UV light was produced by an Ocean Optics Deuterium Lamp, and collimated by a lens into a Thorlab integrating sphere. A Hamamatsu photomultiplier tube (254 nm) was employed to collect the signals through the port. A schematic of the setup is shown in Fig.1.

Fig. 1 .

 1 Fig.1. Schematic of optical setup of the RCM. PM: photomultiplier

Fig. 2

 2 Fig.2 Ozone decomposition as a function of time during the compression stroke. Solid lines denote the experimental results and dashed lines represent the simulation. Temperature and pressure profiles are also provided as a reference.

Fig. 3 .Fig. 4 .

 34 Fig.3. Ozone decomposition in the presence of CO2 as a function of time during the compression stroke. Solid lines denote the experimental results and dashed lines represent the simulation.

Fig. 5 .

 5 Fig.5. Ozone decomposition in the presence of CO2 as a function of time during the compression stroke with a similar Tc. Solid lines denote the experimental results and dashed lines represent the simulation.

Fig. 6 .

 6 Fig.6. The temperature profiles corresponding to the conditions shown in Fig.5

Fig. 7 .

 7 Fig.7. Ozone decomposition in the presence of H2O as a function of time during the compression stroke. Solid lines denote the experimental results and dashed lines represent the simulation.

Fig. 8 .

 8 Fig.8. The temperature profiles corresponding to the conditions shown in Fig.7

Fig. 9 .

 9 Fig.9. The main rate of production (ROP) involved in ozone as a function of time in the presence of steam

Fig. 11 .

 11 Fig.11. Ozone decomposition in the presence of CH4 as a function of time during the compression stroke. Solid lines denote the experimental results and dashed lines represent the simulation.

Fig. 12 .

 12 Fig.12. The temperature profiles corresponding to the conditions shown in Fig.11

Fig. 13 .Fig. 14 .

 1314 Fig.13. The main rate of production (ROP) involved in ozone as a function of time in the presence of methane

Fig. 15 .

 15 Fig.15. Ozone decomposition in the presence of iso-octane as a function of time during the compression stroke. Solid lines denote the experimental results and dashed lines represent the simulation.

Fig. 16 .

 16 Fig.[START_REF] Ombrello | Flame propagation enhancement by plasma excitation of oxygen. Part I: Effects of O3[END_REF]. The temperature profiles corresponding to the conditions shown in Fig.15

Fig. 17

 17 Fig.17. The main rate of production (ROP) involved in ozone as a function of time in the presence of iso-octane

  Fig.17. The main rate of production (ROP) involved in ozone as a function of time in the presence of iso-octane

Table 1

 1 Experimental conditions (Inlet composition volume basis; ozone mole fraction: 90 ppm; End of compression pressure Pc = 30 bar).

	Exp.	Tin [K]	XN2	XO2	XCO2	XH2O XCH4 [ppm]	XIC8H18 [ppm]
	1	313	0.79	0.20991	-	-	-	-
	2	313	0.395	0.20991	0.395	-	-	-
	3	313	0.5925	0.20991	0.1975	-	-	-
	4	313	0.69125	0.20991	0.9875	-	-	-
	5	333	0.395	0.20991	0.395	-	-	-
	6	353	0.5925	0.20991	0.1975	-	-	-
	7	393	0.69125	0.20991	0.9875	-	-	-
	8	313	0.69	0.20991	-	0.1	-	-
	9	313	0.74	0.20991	-	0.05	-	-
	10	313	0.765	0.20991	-	0.025	-	-
	11	313	0.7895	0.20991	-	-	500	-
	12	313	0.78975	0.20991	-	-	250	-
	13	313	0.7895	0.20991	-	-	-	500
	14	313	0.78975	0.20991	-	-	-	250

Acknowledgements

The research leading to these results has received funding from the French Government's Investissement d'Avenir program: Laboratoire d'Excellence CAPRYSSES (Grant No ANR-11-LABX-0006-01).