Bongu Charlotte Bodin

Chandra Sekhar

Mathieu Deschanels

Dr Sylvain Catrouillet

Dr Steven Le Vot

Dr Frédéric Favier

Agrégats Olivier Fontaine
email: olivier.fontaine@umontpellier.fr

Chandra Sekhar Bongur

Dr M Deschanel

Dr S Bongur

Dr F Le Vot

Dr O Favier

Fontaine

Shuttle Effect Quantification for Redox Ionic Liquid Electrolyte Correlated to the Coulombic Efficiency of Supercapacitors

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

Introduction

In their field, supercapacitors have a lower energy density than batteries (20 times lower) but a much greater maximum power (10 times). [START_REF] Dunn | [END_REF] However, the search for devices with both high energy density and high maximum power remains an important challenge for all scientists in this field. In the field of electrochemical double layer capacitor (EDLC), the state of the art offers an alternative based on redox molecules dissolved in the electrolyte to enhance the storage of electrical charges. As far as we know, the pioneering work is that of Frackowiak et al. devoted on the development of a redox active aqueous electrolyte with potassium iodide (KI) and vanadyl sulfate (VOSO4). [2] Device performance was great but the high-cost ion selective membrane (Nafion) used to avoid the crossover of redox species somehow tempered the success of this proof of concept. More recently, Boettcher et al. investigated redox-based EDLC supercapacitor using viologen and halides couples in an aqueous electrolyte for the negative and the positive electrodes, respectively. [3] After testing various halides and viologens, reported performance was beyond the state-of-the-art, including a low self-discharge despite the absence of any ion-selective membrane. The redox active electrolyte approach, although very attractive, has two identified limitations: (1) the selfdischarge is usually strong and (2) shuttle effects affect the performances. [4] Redox ionic liquids are good opportunities to address these two issues. [5] Some of us were the first to develop redox ionic liquids while not considering them for operation in devices. It is only from the work of Rochefort et al. that the first applications of redox ionic liquids are considered for batteries and supercapacitors. [6] However, depending on the nature of the redox ionic liquid, the shuttle effect may still remain, especially if the molecule has no particular adsorption or confinement within the porosity of the electrode material. In such a case, the scientific question is: how many times does the redox shuttle have to go back and forth before highly impacting the coulombic efficiency? To answer this question, we propose the study of the shuttle effect of a redox ionic liquid, 4-methylimidazolium-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOIm) + TFSI -. In this study, the number of shuttles of the redox molecule is correlated to the decrease of coulombic efficiency of the device. To calculate the number of shuttles, the Fick's law is used, which means that the redox molecule diffuses linearly in a continuous medium. First, the diffusion of TEMPO moieties is studied to confirm the diffusional behaviour of the molecules. Then the diffusion is correlated with the properties of the electrolyte that depend on its viscosity and ionic conductivity.

Results and Discussion

Diffusion coefficient of TEMPO moieties

(TEMPOIm) + TFSI -is electrochemically characterized and compared to commercial TEMPO. Figure 2 exhibits the cyclic voltammetry (CV) profiles of these molecules solubilized in NEt 4 + BF4 -at 0.5 mol•l -1 in acetonitrile (Figure 2a) and in BMIm + TFSI -ionic liquid (Figure 2c). In both electrolytes, the TEMPO (black line) is oxidized as TEMPO + at the potential of 0.29 V vs Fc/Fc + , then reduced back as TEMPO at 0.22 V vs Fc/Fc + by a reversible single electron transfer step. Similarly, the (TEMPOIm) + TFSI -(dashed line) is also oxidized as (TEMPOIm) 2+ TFSI -at 0.33 V vs Fc/Fc + and reduced back as (TEMPOIm) + TFSI -at 0.26 V vs Fc/Fc + . The difference of redox potential between TEMPO and (TEMPOIm) + TFSI -about 0.04V could be explained by an impact of the imidazolium grafted on the TEMPO. The imidazolium is positively charge so the addition of a second positive charge by the oxidation of (TEMPOIm) + needs a greater energy leading to a higher potential. CV shapes are characteristics of the diffusional behavior of the redox molecules. [7] The potential difference between current peaks of oxidation and reduction, ΔEEp, is 70 mV for all CVs, proving the reversibility of these redox systems. [8] Figures 2b and2d compare the values of current peak in oxidation depending on the scan rate, for NEt4 + BF4 -at 0.5 mol•l -1 in acetonitrile and BMIm + TFSI -respectively, with TEMPO (plain square) and (TEMPOIm) + TFSI -(open square). Current values form linear lines between 2.5•10 -2 V•s -1 and 1 V•s -1 . The apparent diffusion coefficient, Dapp, proportional to the line slope, is greater for TEMPO compared to (TEMPOIm) + TFSI -in both electrolytes. This discrepency can be assigned to molecule size difference. TEMPO is a smaller molecule than (TEMPOIm) + TFSI -, and , as such, diffuses faster than the larger one. [9]

 1/2 (V 1/2 s -1/2 )
The size of the molecule is not the sole factor influencing the diffusion coefficient of redox molecules. Indeed, the physico-chemical properties of the electrolyte (conductivity, viscosity) are critical parameters for characterizing the diffusion properties of redox molecules. [10]

Influence of viscosity and molar conductivity on the apparent diffusion coefficient

To characterize the properties of bulk electrolytes, five concentrations of BMIm + TFSI -were chosen from 1 mol•l -1 in acetonitrile to pure ionic liquid (which corresponds to 3.4 mol•l -1 ). The concentration of (TEMPOIm) + TFSI -was increased (compared to 2•10 -3 mol•l -1 previously) to observe the Faradaic contribution of the redox ionic liquid in the device. All solutions include (TEMPOIm) + TFSI -at 0.1 mol•l -1 .

To characterize these electrolytes, an ultramicroelectrode (UME) is used as working electrode. [11] This electrode has a much smaller diameter than an usual macroelectrode (10 µm in our case against 3 mm). With UME, the EDLC contribution is drastically reduced and does not interfere with measured signal. Then, at the potential of the redox couple, an increase in current is visible up to a plateau due to the steady state (all species in the diffusion layer have reacted), Figure 3a. From this plateau, the limit current is extracted for each concentration of BMIm + TFSI -and used to calculate the apparent diffusion coefficient of the (TEMPOIm) + using the Stokes-Einstein equation (Eq. 1). [12] i l =4 . z . F . D app .C . r (Eq. 1)

n corresponds to the number of electrons exchanged, F is the Faraday constant in C •mol -1 , Dapp is the apparent diffusion coefficient in m 2 •s -1 , C represents the concentration of redox ionic liquid in the electrolyte in mol•m -3 and r is the radius of the ultramicroelectrode in m. The Figure 3a shows the CV measured with a platinum UME at 0.1 V•s -1 for each electrolyte. The current is zero for the applied potential from 0.2 V to 0.6 V vs Ag/Ag + for tested solutions. This potential zone corresponds to the formation of the electrochemical double layer. Then, in the whole electrolyte series, the current increases with a significant slope between 0.6 V and 0.8 V vs Ag/Ag + . This step corresponds to the electronic transfer during the oxidation of (TEMPOIm) + . Above 0.8 V vs Ag/Ag + , the slope is decreasing and the curve could be considered as a plateau. It corresponds to the mass transfer of redox molecules to the surface of the UME. Since the current is not quite constant, the regime is not fully stationary. Limit current of the plateau (measured at the potential of 1.3 V vs Ag/Ag + ) increases when BMIm + TFSI -concentration decreases. Apparent diffusion coefficients, Dapp, were calculated from these limit current values using (Eq. 1).

As such, the apparent diffusion coefficient decreases while increasing BMIm + TFSI -concentration, as shown in Figure 3b. The viscosity gradually increases from 0.7 and 4.6 mPa•s for diluted solutions with salt concentrations from 1 to 2.5 mol•l -1 ). The viscosity for pure BMIm + TFSI -is 44 mPa•s. When the diffusion coefficient is expressed as a function of the inverse of viscosity, Figure 3d, a straight line with a slope of 3.6 •10 -7 cm 2 •mPa is obtained. The diffusion coefficient is therefore correlated with both viscosity and molar conductivity. Table 1 summarizes the physical characteristics of each BMIm + TFSI -solutions depending on its concentration. The solvation radius (rsol) is calculated from apparent diffusion coefficient values determined at corresponding limit current values (UME), (Eq. 2), where kB is the Boltzmann constant and T (K) the temperature. [12]

r sol = k B • T 6 • π • η • D app (Eq. 2)
Overall, the value of the rsol decreases with the diffusion coefficient and the increase in viscosity. However, the order of magnitude of the solvation radius is the same (about 4 nm). In this section, the critical correlation between diffusion coefficient and viscosity is highlighted. The next section is about the charge storage with respect to the characteristics of prepared electrolytes. The objective is to determine if one of them is leading to better charge storage (in the sense of a larger quantity of charges). For this purpose, the parameter studied is the electrical charge Q normalized by the electrode mass.

Impact of the diffusion coefficient of (TEMPOIm) + TFSI -on the electrical charge of a porous electrode

The electrical charge of the working electrode is measured in a three-electrode cell to limit the redox shuttle effect by applying a large distance between the working electrode and the counter electrode to measure the impact of the physical and chemical properties of the electrolyte on the electrical charge. The study of redox shuttles is reported in the next section using two-electrode cell with controlled thickness separating the electrodes. CVs are performed under inert atmosphere with a cell composed of self-supporting porous carbon electrodes. The counter electrode has a loading at least three times greater than that of the working electrode. Figure 4a details the CV measured in 1 mol•l -1 of BMIm + TFSI -in acetonitrile (dotted black) electrolyte before and after addition of 0.1 mol•l -1 of (TEMPOIm) + TFSI -(light green). The Faradaic contribution (0.6 V to 1.2 V vs Ag/Ag + ) comes along with the capacitive contribution (0 V to 0.6 V vs Ag/Ag + ). This superposition is supported by considering the purely capacitive curve when no redox molecule is present in the solution (dashed black). An increase of 0.7 mA is observed thanks to the Faradaic contribution. CVs corresponding to (TEMPOIm) + TFSI -at 0.1 mol•l -1 solutions with the various BMIm + TFSI -concentrations are shown in Figure 4b. The current is normalized by the mass of active material in the working electrode. At concentrations of 1 and 1.5 mol•l -1 of BMIm + TFSI -, CVs are almost identical and the Faradaic contribution is supposed to be the same. A slight shift in redox potential about 60 mV is observed for the electrolyte at 2 mol•l -1 (blue curve), Figure 4b. The fact that there is only one curve with a redox potential shift suggest a shift of the reference Ag/Ag + during the measure. On the other hand, when the concentration of BMIm + TFSI - was increased, the current peak and the peak-to-peak separation corresponding to the Faradaic contribution decreased. For pure ionic liquid, no current peak is visible (Figure 4a dashed curve). The charge calculated from CV measurements in the three-electrode cell is plotted in Figure 4c as a function of BMIm + TFSI -concentration and compared to the apparent diffusion coefficient. The observed charge and the diffusion coefficient decrease while BMIm + TSFI -concentration increases that corresponds to an increase in electrolyte viscosity (Figure 4d). The Faradaic contribution in the most viscous electrolyte (BMIm + TFSI -containing (TEMPOIm) + TFSI -) is almost zero. However, it is in this electrolyte that the diffusion coefficient is the lowest, and therefore also the one for which the redox shuttle effect should be the lowest. The effect of the redox shuttle can be quantified by calculating the number of shuttles back and forth between the two opposite electrodes. The objective is to correlate the number of shuttle back and forth made by the redox active ion with the coulombic efficiency of the device. It should allow to estimate the charge loss by the device because of these shuttles.

Quantification of the number of shuttles of the (TEMPOIm) + and its impact on coulombic efficiency

It has been shown that the redox molecule (TEMPOIm) + has a diffusional behavior (Figure 2). When the molecule is oxidized at, a concentration gradient of oxidized and reduced molecules is established from the electrode-electrolyte interface toward the electrolyte bulk. This concentration gradient is limited to a certain distance from the electrode assigned as the diffusion layer δ. The time needed for molecules to diffuse from one electrode to the other one can be calculated. In this case, the diffuse layer corresponds to the distance between the electrodes i.e. the thickness of the separator. From the diffusion coefficient of the redox molecule and the thickness of the separator, the time, t1, to diffuse from one side of electrode to the other side is calculated with the (Eq. 3):

δ = √ π • D app • t 1 (Eq. 3)
Knowing the time of diffusion from one electrode to the other, the number of shuttle back and forth made by the redox cation (TEMPOIm) + can be calculated and correlated to the performance of the device. Among the main performance indicators for a supercapacitor are Coulombic efficiency, power, energy, energy efficiency and lifetime. Coulombic efficiency is particularly affected by the redox shuttle effect, so it is on this parameter that we focused. The setup used is a Swagelok cell with two porous carbon electrodes and a separator in between of a defined thickness about 130 µm.

Figure 5. (a)

The time of a single back and forth shuttle is calculated according to the diffusion coefficient (log-log scale) for various separator thicknesses. The orange rectangle illustrates the value scales for laboratory systems of diffusion coefficients and separator sizes used. [13] (b) Electrolyte diffusion coefficient as a function of BMIm + TFSI -concentration diluted in acetonitrile with respect to the corresponding time for a single shuttle (one back and forth) of the redox molecules, calculated for a separator thickness of 130 µm (experimental value).

The relationship between the diffusion time, t2 (t2 = 2 * t1), required for a back and forth shuttle of the redox molecules and the apparent diffusion coefficient is depicted on Figure 5a as calculated from Eq. 2. It shows in log-log scale the time of a single back and forth shuttle depending on diffusion coefficient values and separator thicknesses. For example, for a diffusion coefficient of 10 -8 cm 2 •s -1 and a separator of 130 µm, the time for a back and forth shuttle of the molecule between the electrodes is 1.10 4 s (t2 = 2*(130•10 -4 ) 2 / (π*10 - 8 )). As the relationship (Eq. 2) shows, the time for a back and forth shuttle is inversely proportional to the diffusion coefficient. The thicker the separator, (i.e. the distance between the two electrodes), the longer the time required for a back and forth shuttle. In this figure, the orange rectangle corresponds to the usual thicknesses of separators used for supercapacitors: from 25 µm (Celgard) to 500 µm (Whatman glass fiber) and typical values of diffusion coefficient observed for a redox molecule (as an example for TEMPO) in ionic liquid media such as BMIm + TFSI -or solvent such as ACN. [13][14] Figure 5b shows the time for a back and forth shuttle through a 130 µm thick separator versus diffusion coefficients in the usually reported range. The greater the BMIm + TFSI -concentration, the longer the diffusion time. For pure BMIm + TFSI -, a calculated duration of almost 7000 s is out of scope when using a supercapacitor device. By performing galvanostatic measurements, it is possible to distinguish the Faradaic contribution from the purely capacitive contribution and to extract a characteristic time from it. This time corresponds to the total time of diffusion of the redox molecule tc. From this time and the time required for a single back and forth shuttle of a redox molecule, t2, the number of back and forth shuttle of the molecule can be calculated. Calculation details are given in the supplementary information section. This number of shuttles has been correlated to the Coulombic efficiency of the device.

Correlation between the Coulombic efficiency of a supercapacitor and the number of back and forth shuttles of a redox shuttle

To determine the impact of the redox shuttle effect on the performance of a supercapacitor, galvanostatic measurements were conducted using a two-electrode Swagelok device. For this purpose, porous carbon electrodes coated on an aluminum current collector (see details in experimental part) and a 130 µm thick cellulose separator were used. Measurements in galvanostatic mode were performed for currents between 1.5 A•g -1 and 0.01 A•g -1 . [15] Figure 6 shows the galvanostatic curves for current densities at 0.5 A•g -1 , 0.05 A•g -1 , 0.02 A•g -1 and 0.01 A•g -1 .

Figure 6a (i = 0.5 A•g -1 ) shows that all electrolytes containing 0.1 mol•l -1 (TEMPOIm) + TFSI -and BMIm + TFSI -diluted in acetonitrile have a similar charge/discharge time. The electrolyte of pure BMIm + TFSI -has a shorter charge/discharge time than diluted electrolytes. The galvanostatic curves have a triangular shape, typical of a supercapacitor. For a lower current at 0.05 A•g -1 (Figure 6b) limited variations of charge/discharge times are visible depending on the concentration. There is a difference of 130 s on charging time between the measurement for pure BMIm + TFSI -and electrolytes at 2.5 and 2 mol•l -1 . Again, a difference of 200 s in charging time is observed between electrolytes at BMIm + TFSI -2.5 and 2 mol•l -1 and those at 1.5 and 1 mol•l -1 . When the applied current was further decreased, the differences in charging time between the electrolytes increase, Figure 6c and6d. As the current decreases, a change in slope appears on the galvanostatic curves as the voltage approaches 2.8 V. This is observable for the application of a current of 0.05 A•g -1 , Figure 6b. The phenomenon is more pronounced with the decrease in applied current (0.02 A•g -1 ), Figure 6c, until plateaus are obtained when the current decreases down to 0.01 A•g -1 , Figure 6d. The cell voltage required (2.8 V) is never reached for the BMIm + TFSI -at 1 mol•l -1 (Figure 6d, light green curve). The observation of different slopes in galvanostatic mode on a supercapacitor indicates Faradaic reactions occuring. The total diffusion time, tc, considered for calculating the number of back and forth shuttles of the redox ion is the charging time between 1.4 V and 2.8 V, Figure 7a. The voltage of 1.4 V corresponds to the change in slope on the galvanostatic curves, the Figure 7a is given as an example of it. The number of redox shuttles is determined and reported in Figure 7b for the BMIm + TFSI -electrolyte diluted to 1 mol•l -1 . In this electrolyte, a single shuttle is observed for an applied current density of 0.2 A•g -1 . The applied current is quite low for operating a supercapacitor: the tests reported in the literature can go up to more than 10 A•g -1 , however for such high currents, Faradaic reactions do not have time to occur in the present case. [16] For a current density of 0.03 A•g -1 , the number of calculated shuttles is 11. Coulombic efficiency is the ratio of discharge time to charging time. Less than 100% means that a part of the charges stored are not recovered during discharge. Coulombic efficiencies of the devices are shown as a function of the applied current in Figure 7 for electrolytes with different concentrations of BMIm + TFSI -(7d : "without redox") and with the addition of 0.1 mol•l -1 of (TEMPOIm) + TFSI -(7c). The loss of Coulombic efficiency begins at greater currents in the presence of redox molecules, 70% at 0.06 A•g -1 for the 1 mol•l -1 BMIm + TFSI -electrolyte, Figure 7d, than without redox molecule, 73% at 0.03 A•g -1 for the 1.5 mol•l -1 BMIm + TFSI -electrolyte. The addition of (TEMPOIm) + TFSI -speed-up the Coulombic efficiency fading. For all applied currents corresponding to 4 shuttles, the Coulombic efficiency dropped down to 70 % ± 5 %. This means that a 30 % loss of Coulombic efficiency was observed after 4 shuttles of the redox molecules. This observation could be extended to any redox molecule with diffusional behavior (no adsorption at the electrode surface as an example).

To go further, the different impacts of the shuttle effect on the device were categorizing: (i) protection (ii) degradation (iii) or competition favourable or (iv) unfavourable to the device. These impacts are depicted on Figure 7. The term protection is used to mean that the coulombic efficiency of the device is not degraded at all with the addition of redox ionic liquid compare to a degradation with redox free electrolyte (CE redox = 100%). The redox ionic liquid provides a full protection by avoiding degradation of electrolyte at same potential. The second case presented is the opposite: the addition of redox shuttle degrades the electrolyte but no degradation is observed with redox free electrolyte (CEno redox = 100%). When these two phenomena are observed, there is a competition. If the coulombic efficiency is better with the addition of redox ionic liquid, what means there is degradation of the electrolyte but the RIL protects in part the device (CE redox < 100% and CEredox >> CEno redox). In the other case, the RIL precipitates the degradation of the RIL, a situation to avoid (CEno redox < 100% and CEredox << CEno redox).. The last case that could be observed is no effect of the addition of RIL (CEredox ≈ CEno redox)., the coulombic efficiency is similar with and without RIL. Statistically speaking, our case is more corresponding to a decrease of the coulombic efficiency with the addition of RIL but with also a degradation of the electrolyte (18 measures on 31 in totality). So far, the redox shuttle is damaging the device in our conditions. One step further is to find which conditions are needed to protect the device with the addition of RIL. The protection of overcharge by using RIL has been study for batteries [17] but could be a great advantage also for the use of supercapacitor containing electroactive species.

Conclusions

In this study, the TEMPO molecule functionalized with an imidazolium ion has a diffusional behavior leading to a loss of stored redox charges because of a shuttle effect between electrodes during the charge of the device. The impact of the shuttle effect on the Coulombic efficiency is quantifiable. In this case, 4 shuttles result in a 30% loss of Coulombic efficiency. This observation can be generalized. The redox shuttle effect is highly dependent on the diffusion coefficient of the redox molecule. The diffusion coefficient reflects the viscosity and molar conductivity of the electrolyte. It is therefore useful to determine the conditions needed to limit the redox shuttle effect and the loss of Coulombic efficiency. This also allows applying our conclusions to the use of other redox molecules and other supporting electrolytes as long as there are no specific interactions between the molecule and the electrode. The charging (or diffusion) times given are for a diffusion/separator size layer of 130 µm. The use of a viscous medium such as BMIm + TFSI -avoids the redox shuttle effect. The diffusion coefficient is limited (10 -8 cm 2 •s -1 ) which generates a very long diffusion time (6840 s for a distance of 130 µm). This time constant is way too long to be realistic in a supercapacitor and any redox shuttle effect will never be encountered. However, the use of a viscous electrolyte is detrimental to the power of the device.

A diffusion coefficient of 10 -7 cm 2 •s -1 is a good compromise, the charging times are faster than in the present case and the current density limit is 0.03 A•g -1 to avoid the Coulombic efficiency fading. For diffusion coefficients from 2•10 -7 to 5•10 -7 cm 2 •s -1 , it is necessary to avoid slow cycling (current density < 0.08 A•g -1 ) which would allow the diffusion of redox molecules and the loss in Coulombic efficiency.

Experimental Section

Synthesis of ionic liquids

The 4-hydroxy-TEMPO, bromotrimethylsilane (TMSBr) and methylimidazole (MIm) were provided by Sigma Aldrich. The precursors 1-butyl-3-methylimidazolium chloride and lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) supplied by Iolitec. For the synthesis of 1-methyl-3imidazolium-TEMPO bis(trifluoromethanesulfonyl)imide, Figure 1, 4-hydroxy-TEMPO was dissolved in a minimum volume of anhydrous acetone under argon atmosphere. The bromotrimethylsilane (5 eq.) was added drop per drop. The mixture was stirring during 24 h at room temperature. The excess of bromotrimethylsilane was removed as well as the sub-product trimethylsilanol under vacuum. The obtained product was a brown batter. It was solubilized in dichloromethane before the addition of the methylimidazole. The slurry was stirring during 24 h at 45 °C. The solvent was removed with rotating evaporator. Afterwards, the metathesis was performed by solubilizing separately the obtained product and LiTFSI (1.2 eq.) in water. The two solutions were mixed and stirred during 2h at room temperature. Then, liquid-liquid extraction was performed to collect the organic phase. The aqueous phase was washed with dichloromethane three times. The organic layer was dried over magnesium sulfate and the solvent was removed with rotating evaporator. The obtained product was a little viscous red liquid. The (TEMPOIm) + TFSI -is characterized by DSC, TGA and IR in supplementary information.

The butylmethylimidazolium bis(trifluoromethanesulfonyl)imide were also synthesized. The components are separately dissolved in water then mixed and stirred during 12 h. The result ionic liquid is extracted with dichloromethane. The organic phase is washed with pure water three times to remove the sub product LiCl and dried with magnesium sulfate before removing the solvent with a rotary evaporator. The obtained product is still a transparent liquid.

Conductivity and viscosity measurement

To determine the molar ionic conductivity, a conductivity cell with a cell constant is used to measure the impedance of each solution. The cell constant, k, is 1.8 cm -1 . To determine the ionic conductivity, σ, in S•cm - 1 , of the solutions, the calculation σ =k /R is carried out with the electrical resistance R (Ohms) extracted from the impedance measurements. The molar conductivity Λ corresponds to the ionic conductivity divided by the concentration of the solution. All measurements are made in a glove box at 20 °C with an Ametek Princeton Applied Research potentiostat.

The viscosity of the solutions is measured with a Couette rheometer (cylinder) at a controlled temperature of 20 °C. The viscosity measurements correspond to an average of 4 measurements made for the shear gradients of 10, 20, 40 and 50 s -1 .

Electrochemical characterization

Electrochemical characterizations with three-electrode cell were performed in a glove box using an Ametek Princeton Applied Research potentiostat. The working electrode used was glassy carbon, the counter electrode was a platinum rod and the reference electrode was a silver wire. The working electrode was polished with alumina 0.3 µm and cleaned with water then ethanol before each experiment. The apparent diffusion coefficient was determined by using ultramicroelectrode (UME) in platinum with a diameter of 10 µm. The reference and counter electrode were a silver wire and a platinum wire respectively. The UME was polished with alumina 0.3 µm then rinsed with ultrapure water and ethanol before each experiment. The cyclic voltammetry were performed at 100 mV•s -1 with a sweep potential from 0.2 V to 1.3 V vs Ag/Ag + .

For cyclic voltammetry presented figure 3, three-electrode cell composed of self-supporting porous carbon electrodes were used. The electrodes were constituted of PICACTIF as activated material, acetylene black and polytetrafluoroethylene (60 wt % dispersion in water) as binder with a ratio of 75:15:10 respectively. Firstly, powders were dispersed in ethanol, the polytetrafluoroethylene was added. The ethanol was removed at 50 °C, then the mixture is spread out on a glass plate until the thickness reach 150 µm. Diameter of the self-supported film (electrodes) was 6 mm for the working electrode and 10 mm for the counter electrode. Electrodes were pressed into a stainless steel grid at 10 T.

For device experimentation, a two-electrode Swagelok was used with porous carbon electrode. The electrodes were constituted of PICACTIF as activated material, acetylene black and polyvinylidene fluoride as binder with a ratio of 75:15:10 respectively. Firstly, an ink was formulated by dissolving the polymer PVdF in NMP during 1 h at room temperature. Then the two carbons were added and the ink was stirred during 1 h at room temperature followed by 1 h in ultrasonic bath. Secondly, the ink was coated on a current collector. For the working electrode, the ink was coated on aluminium foil with a doctor blade at 200 µm. The dried thickness is about 60 µm (± 10µm). For the counter electrode, the ink was coated on copper foil with a doctor blade at 500 µm. The dried thickness is about 130 µm (± 10µm). Finally, the coating was dried at 90 °C during 12 h. The coating was punched at a diameter of 9.5 mm to make electrodes. The separator used is Whatmann cellulose paper previously dried under vacuum at 80 °C during 12 h. The thickness of the separator Is about 130 µm.

Figure 1 .

 1 Figure 1. Scheme of shuttle effect (a) formation of electrochemical double layer at electrodes surface (b) electronic transfer from redox ionic liquid to positive electrode (c) formation of gradient diffusion of redox ionic liquid, reduced form to the positive electrode and oxidized form to the negative electrode (d) shuttle effect: the molecule previously oxidized is reduced at the negative electrode (e) galvanostatic measurement.

Figure 2 .

 2 Figure 2. (a) and (c) Cyclic voltammetry at 0.1 V•s -1 of an electrolyte containing TEMPO (solid line) or (TEMPOIm) + TFSI -(dot line) at 2•10 -3 mol•l -1 on a glassy carbon electrode (Ø 3 mm). (b) and (d) Oxidation peak current as a function of the square root of the scan rate for TEMPO (plain square) and (TEMPOIm) + TFSI -(open square). The electrolytes are (a) and (b) NEt4 + BF4 -at 0.5 mol•l -1 in ACN and (c) and (d) BMIm + TFSI -. -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 -4 -2 0 2 4 6 (c)

Figure 3 .

 3 Figure 3. (a) CV with Pt UME (Ø 10 µm) at 0.1 V•s -1 , a Pt wire as counter electrode and a silver wire as reference. The electrolytes are composed of (TEMPOIm) + TFSI -at 0.1 mol•l -1 with various concentrations of BMIm + TFSI -in acetonitrile. (b) Apparent diffusion coefficient (solid squares) and molar ionic conductivity (open circle) depending on the BMIm + TFSI -concentration. (c) Apparent diffusion coefficient and viscosity of the solutions (white circle) depending on the BMIm + TFSI -concentration. (d) Apparent diffusion coefficient as a function of the inverse of viscosity.

  Figure 3b and Figure 3c depicts the diffusion coefficients as a function of the BMIm + TFSI - concentration. Figure 3b also shows that Dapp and the molar conductivity (open squares) which decrease the same way as the salt concentration increases. Figure 3c compares Dapp with the viscosity of the electrolyte.

Figure 4 .

 4 Figure 4. (a) CVs with porous carbon based electrodes and a silver wire as reference. The supporting electrolyte is BMIm + TFSI -at 1 mol•l -1 in acetonitrile (dashed line) and with the addition of 0.1 mol•l -1 of (TEMPOIm) + TFSI -(green line). (b) CVs with porous carbon based electrodes with various concentrations of BMIm + TFSI -in acetonitrile. The electrolytes contain (TEMPOIm) + TFSI -at 0.1 mol•l -1 . (c) Apparent diffusion coefficient (solid squares) and electrical charge (open circle) depending on the BMIm + TFSI - concentration. (d) Viscosity (solid squares) and electrical charge (open circles) of the solutions depending on the BMIm + TFSI -concentration.

Figure 6 .

 6 Figure 6. Galvanostatic measurements for a device with two porous carbon electrodes. The electrolyte is composed of 0.1 mol•l -1 (TEMPOIm) + TFSI -with different concentrations of BMIm + TFSI -in acetonitrile. The current density applied is (a) 0.5 A•g -1 and (b) 0.05 A•g -1 (c) 0.02 A•g -1 (d) 0.01 A•g -1 .

Figure 7 .

 7 Figure 7. (a) and (b) Galvanostatic measurements for a device with two porous carbon electrodes. The electrolyte is containing (TEMPOIm) + TFSI -at 0.1 mol•l -1 in BMIm + TFSI -at 1 mol•l -1 in acetonitrile with current densities of (a) 0.03 A•g -1 and (b) 0.03, 0.05, 0.07, 0.1 and 0.2 A•g -1 . (c) and (d) Coulombic efficiency associated to the galvanostatic measurements (c) with and (d) without (TEMPOIm) + TFSI -at 0.1 mol•l -1 .

Figure 8 .

 8 Figure 8. Classification of different impacts of the redox shuttle on the coulombic efficiency (CE) of supercapacitor and statistics of measurements realized. The proportion (y/x) on the left are: (x) the number of coulombic efficiencies calculated for different current density and (y) electrolyte and the number of CE corresponding to the aligned case.

Table 1 .

 1 Physical properties of electrolytes at different concentrations of BMIm + TFSI -in acetonitrile

	[BMIm + TFSI -]	[BMIm + TFSI -] (%wt)	Dapp (cm 2 •s -1)	η [a] (mPa•s)	Λ [a] (S•m 2 •mol -1 )	rsol (nm)
	1 mol.l -1	30	5.1•10 -7	0.75	4.0•10 -5	5.6
	1.5 mol.l -1	45	3.0•10 -7	1.2	2.6•10 -5	5.9
	2 mol.l -1	60	2.3•10 -7	2.1	1.7•10 -5	4.5
	2.5 mol.l -1	75	1.1•10 -7	4.6	8.3•10 -6	4.1
	3.4 mol.l -1 (pur)	100	1.6•10 -8	44	1.2•10 -6	3.1
	[a] Measurement at 20 °C.

Acknowledgements

This work was conducted within the framework of RS2E and the Direction Générale de l'Armement (DGA). C. B. is grateful to the DGA for financial support.


MD Jebrane Bouaoud
email: jebrane.bouaoud@gmail.com

PhD Mohamed El Beheiry

Eve Jablon

Thomas Schouman

Chloé Bertolus

MD Arnaud Picard

Jean-Baptiste Masson

MD PhD Roman H Khonsari

MD ; Chloé Bertolus

DIVA, a 3D virtual reality platform, improves undergraduate craniofacial trauma education

Keywords: 72 virtual reality, data visualization, image processing, craniofacial, traumatology, education 73

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

Craniofacial trauma is difficult to teach due to the complex anatomy of the skull [1]. Three-75 dimensional (3D) CT-scans help in visualizing fracture lines based on exploration of the 3D 76 objects on 2D screens. Virtual reality (VR) is a technology that uses real-world visual 77 perception within entirely artificial computer-generated environments. VR allows users to 78 efficiently explore 3D environments in an entirely immersive manner, providing a natural and 79 spatially realistic experience [START_REF] El Beheiry | Virtual 195 Reality: Beyond Visualization[END_REF]. 80

The benefits of VR in craniofacial surgery have been assessed in previous studies [START_REF] Ayoub | The application of virtual reality and augmented reality in Oral & 198 Maxillofacial Surgery[END_REF]. Almost 81 all studies reported the use of VR for pre-operative planning [START_REF] Von Sternberg | 201 Learning by doing virtually[END_REF] and intra-operative assistance 82 [START_REF] Bosc | Intraoperative augmented 204 reality with heads-up displays in maxillofacial surgery: a systematic review of the 205 literature and a classification of relevant technologies[END_REF], notably in implantology, orthognathic surgery and maxillofacial reconstruction [START_REF] Ayoub | The application of virtual reality and augmented reality in Oral & 198 Maxillofacial Surgery[END_REF]. Few 83 studies have reported the benefits of VR in surgical education. Within the educational 84 literature, radiology [START_REF] Soltanimehr | Effect of 208 virtual versus traditional education on theoretical knowledge and reporting skills of 209 dental students in radiographic interpretation of bony lesions of the jaw[END_REF] and implantology [START_REF] Durham | Digitally Augmented 212 Learning in Implant Dentistry[END_REF] applications have been evaluated but no study 85 has been dedicated to craniofacial trauma to date. 86 DIVA (Data Integration and Visualization in Augmented and Virtual Environments, Pasteur 87 Institute, Paris) is a software allowing a fast and user-friendly visualization of raw CT-scan 88 data with immediate immersion [START_REF] Masson -Maxime Dahan | EN) Fabien Reyal -(EN) Bassam Hajj -215 mohamed el beheiry -DIVA Data Integration and Visualisation in Augmented and 216 Virtual Environments[END_REF][START_REF] El Beheiry | DIVA: Natural 220 Navigation inside 3D Images Using Virtual Reality[END_REF]. DIVA allows users to intuitively navigate in a flexible 89 3D environment with virtual handheld tools that can be used to interact with 3D images in 90 real-time including landmarking, clipping and highlighting tools. 91

Here we assess DIVA as an educational tool for teaching craniofacial trauma using a 92 dedicated questionnaire. We furthermore assess the anatomical precision of this tool using 93 anatomical landmark placement. 94

Materials and Methods 95 96

VR in craniofacial trauma education: benefits and tolerance 97

Three craniofacial trauma cases (jaw fracture, naso-orbital-ethmoid complex fracture and Le 98 Fort 3 fracture) were submitted to 50 fourth-year undergraduate medical students from the 99 University of Paris, who had to provide a diagnosis and a treatment plan. For each student, 100 pre-and post-operative CT-scans were assessed using both a freeware DICOM viewer 101 (RadiAnt, Medixant, Poznan, Poland) and DIVA (Figure 1). 102

The DIVA interface offers two visualization tools: (1) a desktop tool for standard 3D 103 visualization and (2) the VR environment. The desktop tool was used to reconstruct the skull 104 in 3D based on pre-defined transfer functions. This same visualization is seamlessly switched 105 to the VR viewing context where it can be physically interacted with (Figure 2 The landmarks selected were: (1) anterior nasal spine, (2) posterior nasal spine, (3) distal 117 palatal cusp of tooth 17 (right second maxillary molar), (4) distal palatal cusp of tooth 27 (left 118 second maxillary second molar), ( 5) apex of the crista galli process and (6) apex of the 119 odontoid process of C2. Landmarks on both rendering were positioned 3 successive times (3 120 trials) by the 4 subjects and 3D coordinates were extracted. Geometric morphometrics was 121 performed using MorphoJ (Apache License, Version 2.0) [START_REF] Klingenberg | MorphoJ: an integrated software package for geometric 223 morphometrics[END_REF]. After screening for outliers 122 and Procrustes superimposition, canonical variate analysis on Procrustes coordinates using 123 both trials and subjects as classifier variables allowed to compute Procrustes distances 124 between groups (trials and subjects) [START_REF] Berge | Procrustes problems[END_REF]. Permutation test for pairwise distances with 10000 125 iterations allowed obtaining p-values accounting for the significance of Procrustes distances 126 between trials 1-3 and subjects 1-4. 127

We furthermore evaluated the variability in landmark positioning by assessing inter-and 128 intra-individual error rates, defined as the variance of the position divided by N-1, with N 129 being the number of points. 130

Results 131 132

VR in craniofacial trauma education: benefits and tolerance 133

Almost all undergraduate medical students (92%) reported a high degree of satisfaction 134 (question 1, responses defined as very satisfied and more than satisfied ) when using 135 DIVA to explore and analyze craniofacial trauma cases (Table 1). The use of DIVA was 136 deemed easy, intuitive (88%) and well tolerated (86%) (questions 2 and 3, Table 1). 137 Furthermore, DIVA allowed students to visualize some lesions not seen on the usual 2D and 138 3D renderings (82%), such as skull base and pterygoid process fractures (questions 4 and 5, 139 Table 1). Almost all students strongly recommended the use of DIVA for educational 140 purposes in traumatology (questions 6-8, Table 1). 141 142

Anatomical precision of VR 143

Geometric morphometrics analyses showed that all p-values accounting for the significance 144 of Procrustes distances between trials 1-3 and subjects 1-4 were > 0.05: landmarking using 145 standard 3D CT-scan visualization and landmarking using DIVA provided a similar 3D 146 display of landmarks, without significant distortion. All mean 3D errors, exploring the 147 variability in landmark positioning, were lower than 1 millimeter, ranging from 0.37 to 0.84 148 millimeters (Figure 3 and Supplemental Digital Contents 4). 149 Discussion 150 151 Craniofacial anatomy is challenging to teach to undergraduate medical students and common 152 misunderstandings lead to pitfalls in diagnosis and treating head and neck trauma. While the 153 teaching of traditional anatomy usually involves books and cadaveric dissection, which 154 requires significant amounts of time and effort, other teaching methods can be beneficial [12 155 14]. 156 VR has been recently re-introduced in medical schools as an easy-to-access technology due to 157 the availability of low-cost VR headsets. Through the integration of stereoscopy, motion 158 tracking technologies and total 3D immersion, VR aims at providing a more natural means to 159 visualize 3D structures. VR headsets are bundled with 3D controllers (that act as 3D computer 160 mice) allowing manipulation and interaction with data within the virtual environment. 161

Interactions with the VR controller are rather intuitive as they are performed as if the data 162 were physically present in front of the user. 163 DIVA is a software that automatically generates detailed 3D reconstructions of tomographic 164 medical images such as CT-scans. The avatar generated by DIVA based on raw DICOM CT-165 scan images is generated through volumetric reconstruction without pretreatment (e.g. 166 segmentation). DIVA is optimized to ensure high refresh rate (at least 60 Hz) and a high 167 quality of volume rendering and thus provides a fluid experience. Users are free from latency 168 issues that induce negative experience within VR environments [START_REF] El Beheiry | DIVA: Natural 220 Navigation inside 3D Images Using Virtual Reality[END_REF]. 169 170 Here we showed that using DIVA to explore and analyze 3D CT-scans [START_REF] Masson -Maxime Dahan | EN) Fabien Reyal -(EN) Bassam Hajj -215 mohamed el beheiry -DIVA Data Integration and Visualisation in Augmented and 216 Virtual Environments[END_REF] improved the 171 understanding craniofacial trauma. Furthermore, we found a high degree of interobserver 172 reproducibility reflecting the precision and accuracy of this tool to explore head and neck 3D 173 anatomy and landmarking. 174

As outlined by Alsabbagh et al., craniofacial fractures may be better interpreted using VR 175 compare to usual CT-scan renderings [START_REF] Alsabbagh | Comparison of CT, 3D Reconstruction, and 237 Virtual Reality in the Interpretation of Maxillofacial Fractures[END_REF]. We furthermore found that 82% of students 176 reported spotting lesions using VR that they had missed on usual 3D renderings, thus 177 confirming that VR is beneficial for exploring complex 3D objects [START_REF] Trelease | Anatomical informatics: Millennial perspectives on a newer frontier[END_REF]. 178 179

The value of VR in understanding fractures has recently been reported in orthopedics [START_REF] Brouwers | What is the value of 3D virtual reality in understanding acetabular 243 fractures?[END_REF] and 180 in pre-surgical planification of maxillofacial cases [START_REF] Zhang | Virtual surgical system in reduction of 246 maxillary fracture[END_REF], as well as in dental education [START_REF] Huang | Augmented reality (AR) and 249 virtual reality (VR) applied in dentistry[END_REF]. 181

Roessel et al. have recently reported a systematic literature review supporting the using of VR 182 in surgical education and training [START_REF] Roessel | A systematic literature review of practical 252 virtual and augmented reality solutions in surgery[END_REF]. 183 184 In brief, VR solutions such as DIVA can complement conventional teaching methods (2D 185 sketches, 3D renderings, cadaveric dissections) when complex 3D structures such as the skull 186 are involved [ 21 24]. High satisfaction and tolerance degrees could be obtained using VR 187 tools such as DIVA. Beyond traumatology, similar tools may be used to explore craniofacial 188 malformations and head and neck cancers, by combining CT-scan and MRI data. 189 190 No n =9

Figures legends 272

Fracture of the pterygoid processes were especially well apprehended and visualized using DIVA.

5. Does the use of DIVA allow you to better visualize some lesions seen on usual 2D and 3D renderings?

Yes n=49

No n =1

Fractures lines were better visualized and understood using DIVA.

Financial Disclosure Statement: 27

 27 We acknowledge funding from Institut Pasteur, Institut Curie, Paris Sciences Lettres and the 28 sponsorship of CRPCEN, Gilead-Sciences, and fondation EDF, the programme 29dinvestissement d avenir supported by the agence nationale de la recherche ANR-19-30 P3IA-0001 (PRAIRIE 3IA Institute), INCEPTION project (PIA/ANR-16-CONV-0005, 31 OG)), and TRamWAy ANR-17-CE23-0016. 32 33 Conflict of interest 34 Mohamed El Beheiry and Jean-Baptiste Masson are cofounders, shareholders and scientific 35 advisors of Avatar Medical. 36 Jean-Baptiste Masson is one of the scientific advisors of the Robeauté startup. 37 Introduction 74

2 .

 2 and 106 Supplemental Digital Contents 1-3). 107 After using the DICOM viewer and DIVA, each undergraduate medical student filled a 108 questionnaire based on 8 items assessing satisfaction, potential benefit, comfort of use and 109 tolerance. 110 111 Anatomical precision of VR 112 4 postgraduate students were requested to explore these cases and to place 6 anatomical 113 landmarks on two types of 3D renderings: (1) 3D reconstructions from CT-scan using Avizo 114 (ThermoFisher Scientific, Waltham, MA, USA) and (2) 3D renderings of the same DICOM 115 data using the DIVA VR interface. 116

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. 3D renderings provided by a freeware (RadiAnt, Medixant, Poznan, Poland) (A, B) 274

Figure 1

 1 Figure 1

Figure 2 3

 23 Figure 2

Table legends 288 289 Table 1 .

 legends2891 Assessment of the use of VR in craniofacial trauma education by 5O undergraduate 290 medical students from the University of Paris. 291

d investissement d avenir supported by the agence nationale de la recherche ANR-19-

Acknowledgments 305

We acknowledge funding from Institut Pasteur, Institut Curie, Paris Sciences Lettres and the 306 sponsorship of CRPCEN, Gilead-Sciences, and fondation EDF, the programme

This study was approved by Institutional review board and performed in accordance with the 49 Helsinki declaration. Collection of data and analysis were in accordance with guidelines of 50 the French National Committee for the Protection of Personal Data (Commission Nationale

Author's role and participation on the manuscript 38

All authors have participated to the conception and design, acquisition of data, or analysis and 39


Sylvain Recoquillon

Jean-Louis Pépin

Bruno Vielle

Ramaroson Andriantsitohaina

Vanessa Bironneau

Frédérique Chouet-Girard

Bernard Fleury

François Goupil

Sandrine Launois

M Carmen Martinez

Nicole Meslier

Xuan-Lan Nguyen

Audrey Paris

Pascaline Priou

Renaud Tamisier

Wojciech Trzepizur

Frédéric Gagnadoux
email: frgagnadoux@chu-angers.fr

Effect of mandibular advancement therapy on inflammatory and metabolic biomarkers in patients with severe obstructive sleep apnoea: A randomized controlled trial

Keywords: Results, NCT01426607 obstructive sleep apnoea syndrome, mandibular advancement device, inflammation, cytokines

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

Effect of mandibular advancement therapy on inflammatory and metabolic biomarkers in patients with

severe obstructive sleep apnoea: a randomised controlled trial

Sylvain Recoquillon, Jean-Louis Pépin, Bruno Vielle, Ramaroson Andriantsitohaina, Vanessa Bironneau, Frédérique Chouet-Girard, Bernard H.

Fleury, François Goupil, Sandrine H. Launois, M Carmen Martinez, et al.

To cite this version:

Sylvain Recoquillon, Jean-Louis Pépin, Bruno Vielle, Ramaroson Andriantsitohaina, Vanessa Bironneau, et al.. Effect of mandibular advancement therapy on inflammatory and metabolic biomarkers in patients with severe obstructive sleep apnoea: a randomised controlled trial. Thorax, 2019, 74 [START_REF] Bratton | CPAP vs Mandibular Advancement Devices and Blood Pressure in Patients With Obstructive Sleep Apnea: A Systematic Review and Metaanalysis[END_REF] linking obstructive sleep apnoea (OSA) with cardiovascular diseases (CVD). [START_REF] Drager | Sleep Apnea and Cardiovascular Disease: Lessons From Recent Trials and Need for Team Science[END_REF] Even still debated owing to confounding factors, many studies have established an association between inflammatory cytokines and indices of OSA severity. [START_REF] Gaines | Gender differences in the association of sleep apnea and inflammation[END_REF] Exposure of rodents to intermittent hypoxia, a hallmark of OSA, stimulates inflammatory pathways and leads to cardiovascular or metabolic disorders. [START_REF] Unnikrishnan | Inflammation in sleep apnea: an update[END_REF] Mandibular advancement devices (MAD) have emerged as the main therapeutic alternative to continuous positive airway pressure (CPAP) for OSA. Despite the superior efficacy of CPAP in reducing OSA severity, most trials comparing MAD and CPAP have reported similar health outcomes. [START_REF] Sharples | Meta-analysis of randomised controlled trials of oral mandibular advancement devices and continuous positive airway pressure for obstructive sleep apnoea-hypopnoea[END_REF][START_REF] Bratton | CPAP vs Mandibular Advancement Devices and Blood Pressure in Patients With Obstructive Sleep Apnea: A Systematic Review and Metaanalysis[END_REF] In a recent multicentre randomized controlled trial, our group evaluated the impact of 2 months of effective MAD therapy versus a sham device on endothelial function in patients with severe OSA. [START_REF] Gagnadoux | Impact of Mandibular Advancement Therapy on Endothelial Function in Severe Obstructive Sleep Apnea[END_REF] The aim of the present ancillary study was to investigate the effects of MAD therapy on circulating levels of inflammatory and metabolic biomarkers.

Methods

Study Design and Interventions

As described previously, [START_REF] Gagnadoux | Impact of Mandibular Advancement Therapy on Endothelial Function in Severe Obstructive Sleep Apnea[END_REF] patients with severe OSA (Apnoea-Hypopnoea Index [AHI] > 30)

and no overt CVD, were randomly assigned to receive 2 months of treatment with either a custom-made effective MAD or a sham device with objective measurement of treatment adherence by an embedded microsensor (see online supplement Figure E1). Overnight in-lab polysomnography and blood sample collections were performed at baseline and after the

Results

Study flow and baseline characteristics

Among 150 randomized patients, 109 had available biomarkers before and after 2 months of effective MAD (n=55) or sham device (n=54), and were included in the analysis (Figure 1).

There were no differences between baseline characteristics of all randomized patients, patients included and not included in the present analysis (see online supplemental Table E1).

As shown in table

Discussion

Only few studies flawed by small sample sizes and the absence of placebo group have evaluated the impact of MAD therapy on circulating biomarkers, and reported discrepant findings. [START_REF] Galic | Effects of mandibular advancement device treatment on arterial stiffness and glucose metabolism in patients with mild to moderate obstructive sleep apnea: a prospective 1 year study[END_REF][START_REF] Niżankowska-Jędrzejczyk | Modulation of inflammatory and hemostatic markers in obstructive sleep apnea patients treated with mandibular advancement splints: a parallel, controlled trial[END_REF][START_REF] Yalamanchali | Impact of mandibular advancement devices on C-reactive protein levels in patients with obstructive sleep apnea[END_REF] In this randomized controlled trial, 2 months of effective MAD therapy had no effect on inflammatory and metabolic biomarkers in patients with severe OSA and no overt CVD, despite high objective device adherence and a significant reduction in OSA severity.

As expected, the mean reduction in AHI obtained in the effective MAD group (about 53%)

was lower than that is usually obtained with CPAP. [START_REF] Sharples | Meta-analysis of randomised controlled trials of oral mandibular advancement devices and continuous positive airway pressure for obstructive sleep apnoea-hypopnoea[END_REF] However, the hypothesis of an insufficient reduction of AHI with MAD is unlikely, as no significant changes in biomarkers were observed when the analysis was restricted to complete and partial responders. It also cannot be formally excluded that the 2-month intervention was too short. Previous studies have reported improvements of inflammatory and metabolic profiles after 3 months to 1 year of MAD therapy, but these studies were uncontrolled with no sham device group. [START_REF] Galic | Effects of mandibular advancement device treatment on arterial stiffness and glucose metabolism in patients with mild to moderate obstructive sleep apnea: a prospective 1 year study[END_REF][START_REF] Niżankowska-Jędrzejczyk | Modulation of inflammatory and hemostatic markers in obstructive sleep apnea patients treated with mandibular advancement splints: a parallel, controlled trial[END_REF][START_REF] Yalamanchali | Impact of mandibular advancement devices on C-reactive protein levels in patients with obstructive sleep apnea[END_REF] Conversely, recent randomized trials showed no impact of 6 months to 1 year of CPAP therapy on biomarkers despite a complete suppression of apneic events.[10,11] We observed a higher proportion of women in the effective MAD group. It has been reported that female patients with OSA exhibit a less severe inflammatory profile than men. [START_REF] Gaines | Gender differences in the association of sleep apnea and inflammation[END_REF] Interestingly, we found that effective MAD therapy was associated with a significant improvement of TNF-R1 levels only in male patients. Between-group differences were also observed for baseline levels of adiponectin, C-reactive protein and TNF-α, but adjustments for baseline values were performed to mitigate this potential bias.

We acknowledge that our ancillary study may have been underpowered to detect small changes in biomarkers, as the sample size was based on endothelial function, the primary outcome of the main study. [START_REF] Gagnadoux | Impact of Mandibular Advancement Therapy on Endothelial Function in Severe Obstructive Sleep Apnea[END_REF] Authors: Sylvain Recoquillon 1 , Jean-Louis Pépin 2,3 , Bruno Vielle 4 , Ramaroson Andriantsitohaina 1 , Vanessa Bironneau 5 ; Frédérique Chouet-Girard 6 , Bernard Fleury 7 , François Goupil 8 , Sandrine Launois 7 , M. Carmen Martinez 1 , Nicole Meslier 1,9 , Xuan-Lan Nguyen 7 , Audrey Paris 8 , Pascaline Priou 1,9 , Renaud Tamisier 2,3 , Wojciech Trzepizur 1,9 , Frédéric Gagnadoux

Methods (expanded version)

Study Design and Patients

This randomized, single-blind, parallel-group trial conducted in five French sleep centres was approved by our local ethics committee (Comité de Protection des Personnes, Ouest II, Angers; No. 2010/14), and registered with ClinicalTrial.gov (NCT01426607). Patients with severe obstructive sleep apnoea (OSA; Apnoea-Hypopnoea Index [AHI] > 30), aged 18-70 years, for whom mandibular advancement device (MAD) therapy was considered as secondline therapy because of continuous positive airway pressure (CPAP) intolerance, were assessed for eligibility. Exclusion criteria were body mass index (BMI) greater than or equal to 32 kg/m 2 ; history of cardiovascular disease (CVD) including coronary heart disease, heart failure, arrhythmias, and stroke; coexisting sleep disorders other than OSA; central sleep apnoea defined by a central apnoea index greater than or equal to 5; severe daytime sleepiness defined by an Epworth Sleepiness Scale greater than or equal to 16; and inadequate dental structure or temporomandibular joint disease contraindicating MAD treatment as assessed by a dentist. All patients provided their written informed consent to participate in the study.

Randomization

Patients were randomly assigned to receive 2 months of treatment with either effective MAD or a sham device according to a 1:1 allocation using a computer-generated randomization list stratified by site with permuted blocks of random sizes. The effective MAD was custommade, consisting of an adjustable two-piece acrylic oral appliance (AMO; Orthosom, Beaucouzé, France) with attachments of various sizes allowing adjustment of mandibular advancement (Figure E1). The sham device consisted of the upper appliance only and did not advance the mandible. As previously described, [START_REF] Drager | Sleep Apnea and Cardiovascular Disease: Lessons From Recent Trials and Need for Team Science[END_REF] Handelsagentur Gschladt, Hargelsberg, Austria). With ethics committee approval, patient blinding was achieved by concealing the less effective nature of the sham device. [START_REF] Drager | Sleep Apnea and Cardiovascular Disease: Lessons From Recent Trials and Need for Team Science[END_REF][START_REF] Gaines | Gender differences in the association of sleep apnea and inflammation[END_REF]

Interventions

At baseline, patients underwent clinical assessment, overnight in-lab polysomnography (PSG) followed by collection of blood samples. All patients underwent a 6-week MAD acclimatization period, during which the mandible was incrementally advanced by 1-mm steps every 1 or 2 weeks until symptom relief or until adverse effects prevented further advancement. Patients were then submitted to a one-week washout period, after which they were allocated to receive 2 months of treatment with either effective MAD or the sham device. Clinical assessment, PSG with effective MAD or sham device, and blood sample collection were repeated after the 2-month treatment period. Objective treatment adherence was also calculated after the 2-month treatment period.

Measurements of inflammatory and metabolic biomarkers

After overnight fasting, blood samples were collected in EDTA tubes (Vacutainers, Becton Dickinson, Le Pont de Claix, France) from a peripheral vein using a 21-gauge needle to minimize platelet activation, and were processed for assays within 2 hours. Samples were centrifuged for 20 minutes at 250 g. Platelet-rich plasma was harvested and centrifuged 20 minutes at 1500 g to obtain platelet-free plasma (PFP). PFP was aliquoted and stored at 80°C for subsequent use.

The assessment of inflammatory biomarkers was performed on PFP with an enzyme-linked immunosorbent assay (ELISA) using MESO QuickPlex SQ 120 assay (MSD, Rockville, Md., USA). Each assay was performed in duplicate in order to verify the intra-assay variability using samples randomly prepared. Then, if the coefficient of variability was less than 10%, the mean of each duplicate was calculated. Measures with a coefficient of variability > 10% were considered as unavailable biological data (n=5). A single assay was used to measure plasma levels of adiponectin, C-reactive protein (CRP) and leptin. Multiplex assays were used for the assessment of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), N-terminal probrain natriuretic peptide (NT-ProBNP), tumor necrosis factor receptor 1 and 2 (TNF-R1 and TNF-R2) and P-selectin. Both assays depended on the electro chemiluminescent compound SULFO-TAG™ linked to a detection antibody. All the solutions were supplied by MSD and experiments were performed according to manufacturer's protocol.

For single assays, PFP was first 1000-fold diluted for adiponectin and CRP in an assay diluent whereas detection of leptin did not require any dilution. Then, the samples were incubated in 96-wells plate coated by a capture antibody directed against the protein of interest. After 2 hours of incubation at room temperature with vigorous shaking, the plate was washed three times with PBS supplemented with 0.05% of Tween-20. Then, SULFO-TAG-labelled detection antibodies were added for another 2 hours at room temperature with vigorous shaking. At the end, three other washes were performed before addition of a read solution.

Then, voltage stimulation of plate electrodes induced chemilumescence read by the instrument (MESO QuickPlex SQ 120, USA). The level of the protein of interest is proportional to the emitted light present in the PFP and is calculated thanks to a calibration curve. The limits of detection (LOD) of CRP, adiponectin and leptin were 1.33 pg/mL, 0.005 ng/mL and 43 pg/mL respectively. None of the measured values were outside of these limits.

For the multiplex assay, samples were 2-fold diluted and then incubated for 2 hours with vigorous shaking in a 96-wells plate. Each well is seeded in 6 distinct spot coated with capture antibody specific of the protein of interest. After three washes with PBS supplemented with 0.05% of Tween-20, SULFO-TAG-labelled detection antibodies were added for another 2 hours at room temperature with vigorous shaking. At the end, three other washes were performed before addition of a read solution. Then, voltage stimulation of plate electrodes induced chemilumescence read by the instrument (MESO QuickPlex SQ 120, USA). The level of the protein of interest is proportional to the emitted light present in the PFP and is calculated thanks to a calibration curve. The calculated LOD of IL-6, TNF-α, TNF-RI, TNF-RII, P-selectin and NT-proBNP were 0.06 pg/mL, 0.04 pg/mL, 0.569 pg/mL, 0.102 pg/mL, 29.9 pg/mL and 0.311 pg/mL respectively. None of the measured values were outside of these limits.

Plasma glucose, insulin, triglycerides, total serum cholesterol, and high-density lipoprotein serum cholesterol (HDL-c) were directly measured in accredited laboratories using standard techniques. Low-density lipoprotein serum cholesterol (LDL-c) was calculated. The homeostasis model assessment resistance index (HOMA-IR.) was calculated from fasting glucose and insulin concentrations, as follows: insulin (mIU/l) * glucose (mmol/l)/22.5.

Statistics

A sample size calculation was performed for the primary endpoints of the main study. [START_REF] Drager | Sleep Apnea and Cardiovascular Disease: Lessons From Recent Trials and Need for Team Science[END_REF] In the present ancillary study, we performed a per-protocol analysis including patients with available biomarkers before after 2 months of effective MAD or sham device. Continuous variables were described as mean (SD) or mean (95% confidence interval [CI]) for variables with a normal distribution and as median (interquartile range) for variables with a non-normal distribution. Normality of distribution was assessed using the Kolmogorov-Smirnov test.

Normal variables were analysed using an unpaired Student's t test for intergroup difference and a paired t test for intragroup difference. Linear regression analysis was used to adjust for baseline values and potential covariates. Non-normal variables were analysed using the

Figure 1 :

 1 Figure 1: Flow diagram showing trial allocation Abbreviation: MAD, mandibular advancement device

Figure E1 :Figure E1 :

 E1E1 Figure E1: The effective mandibular advancement device used in the study. Full-coverage

Table 1 :

 1 Baseline characteristics of the study population

	Thorax

1, only gender was significantly different between the effective and the sham device groups (p=0.04). https://mc.manuscriptcentral.com/thorax

Table 3 :

 3 Impact of effective mandibular advancement device (MAD) versus sham device on

	apnoea-

inflammatory, metabolic biomarkers and N-terminal pro-brain natriuretic peptide Data are expressed as mean (standard deviation) or mean (95% confidence interval [CI]) Abbreviations: CRP, C-reactive protein; IL-6, interleukin-6; TNF-α, tumour necrosis factor-α; TNF-R1, tumour necrosis factor receptor 1; TNF-R2, tumour necrosis factor receptor 2; HDLc, high-density lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol; HOMA-IR, homeostasis model assessment-insulin resistance index; NT-ProBNP, N-terminal probrain natriuretic peptide. * Adjusted for baseline value, age, gender and body mass index and baseline https://mc.manuscriptcentral.com/thorax

  effect of the intervention, including a relatively small proportion of metabolic disorders, no overt CVD, and moderate daytime sleepiness at baseline. Further studies are required to determine whether MAD therapy for OSA can improve circulating biomarkers in patients who exhibit more severe inflammatory and metabolic dysfunction at baseline. However, recent randomized trials and a meta-analysis have shown remarkably small effects of CPAP on inflammatory and metabolic biomarkers, even in patients at high cardio-metabolic risk.[10-12]ConclusionTwo months of MAD therapy in patients with severe OSA reduced OSA severity, but had no effect on inflammatory and metabolic biomarkers despite high treatment adherence.

	Page 9 of 24 Page 13 of 24	Thorax Thorax Thorax Thorax Thorax Thorax
	Laryngoscope 2015;125:1733-6. Legends
	10 Figure 1: Flow diagram showing trial allocation Chirinos JA, Gurubhagavatula I, Teff K, et al. CPAP, weight loss, or both for
	obstructive sleep apnea. N Engl J Med 2014;370:2265-75. Abbreviation: MAD, mandibular advancement device
	11	Thunström E, Glantz H, Yucel-Lindberg T, et al. CPAP Does Not Reduce
	C o n f C o n f C o n f C o n f Inflammatory Biomarkers in Patients With Coronary Artery Disease and Nonsleepy C o C o Obstructive Sleep Apnea: A Randomized Controlled Trial. Sleep 2017;40. 12 Ning Y, Zhang T-S, Wen W-W, et al. Effects of continuous positive airway pressure n f n f
		i i i i i i
	d e d e d e d e on cardiovascular biomarkers in patients with obstructive sleep apnea: a meta-analysis of randomized controlled trials. Sleep Breath Published Online First: 22 April 2018. d e d e
	n t n t n t n t n n doi:10.1007/s11325-018-1662-2 t t
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Furthermore, our study population presented several characteristics that may have contributed to a lower cardiovascular response and/or a floor https://mc.manuscriptcentral.com/thorax https://mc.manuscriptcentral.com/thorax https://mc.manuscriptcentral.com/thorax https://mc.manuscriptcentral.com/thorax

  1,9 1 INSERM UMR 1063, Angers, France; 2 Université Grenoble Alpes, HP2, INSERM UMR 1042, Grenoble, France; 3 CHU de Grenoble, Laboratoire EFCR, Clinique Universitaire de Physiologie, Grenoble, France; 4 Centre de Recherche Clinique, CHU d'Angers, Angers, France ; 5 Université de Poitiers, CHU, Service de Pneumologie, Poitiers ; 6 Service de Chirurgie Maxillo-faciale et Stomatologie, Centre Hospitalier, Le Mans, France; 7 Université Paris VI, Hôpital Saint-Antoine, Unité de Sommeil, Paris, France; 8 Service de Pneumologie,

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  treatment adherence with the effective MAD and the sham device was objectively measured by a validated embedded microsensor thermometer (TheraMon ® , IFT Handels-und Entwicklungsgesellschaft GmbH,

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Table E1 :

 E1 Mann-Whitney test for intergroup difference and the Wilcoxon signed rank test for intragroup difference. The Chi-square test and Fisher's exact test were used for categorical variables, as appropriate. All reported p values are two-sided. A p value less than or equal to 0.05 was considered to indicate statistical significance. All analyses were performed using STATA version 13.1 (STATA Corp., College Station, TX). Baseline characteristics of all patients randomized in the main study[START_REF] Drager | Sleep Apnea and Cardiovascular Disease: Lessons From Recent Trials and Need for Team Science[END_REF] with patients included and not included in the present ancillary study.

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	Results
	C o C o	All randomized patients	Patients included in the ancillary study	Patients not included in ancillary study	p value
	n f Age, years n n f	150 53.8 (10.2)	109 53.6 (10.1)	41 54.7 (10.7)	0.97
	i d e n t 27.0 (3.2) i 14.4 Hypertension, % BMI, kg/m² Women, % 20.7 Diabetes, % 5.1 d e Dyslipidemia, % 11.7 ESS 9.3 (4.2) n t	27.1 (3.3) 12.1 20.9 5.7 11.2 9.1 (4.2)	26.56 (2.9) 23.1 17.8 3.6 14.3 9.8 (4.3)	0.71 0.48 0.51 0.71 0.64 0.76
	AHI, n ODI, n	i a 41.0 [35.0-53.0] 31.9 (17.9) i a	41.0 [34.0-52.0] 42.0 [36.0-53.0] 31.7 (17.1) 32.2 (19.9)	0.78 0.99
	SBP, mmHg DBP, mmHg		l : 125.7 (14.4) 126.4 (15.1) 77.7 (10.9) 77.6 (11.4) l :	123.6 (11.4) 78.21 (9.0)	0.66 0.97
	F o r Data are expressed as mean (standard deviation), median [IQR] or percentages. Abbreviations: BMI, body mass index; AHI, apnoea-hypopnoea index; ESS, Epworth sleepiness score; ODI, 3% oxygen desaturation index, SBP, office systolic blood pressure; F o r
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Table E2 :

 E2 Impact of effective mandibular advancement device (MAD) versus sham device on Gagnadoux F, Pépin J-L, Vielle B, et al. Impact of Mandibular Advancement Therapy on Endothelial Function in Severe Obstructive Sleep Apnea. Am J Respir Crit Care Med 2017;195:1244-52. 2 Gotsopoulos H, Kelly JJ, Cistulli PA. Oral appliance therapy reduces blood pressure in obstructive sleep apnea: a randomized, controlled trial. Sleep 2004;27:934-41.

	Page 21 of 24	daytime polysomnographic indices E References 1 Page 22 of 24 Thorax Thorax
		Effective MAD	Sham device	Adjusted intergroup differences *
		Baseline 40.0 [34.0-51.0] 30.2 (18.5) C AHI, n ODI, n o C o	Follow-up 17.5 † [11.5-25.0] 15.2 (10.8) †	Baseline 44.5 [35.0;56.0] 33.2 (15.6)	Follow-up 38.5 † [19.0;51.0] 28.0 (17.4)	Mean (95%CI) -17.2 (-23.6;-10.8) ll -11.9 (-17.6;-6.1) ll
		TST, min N1sleep, n 402.7 (74.4) 40.9 (32.8) f n f min i i N2 sleep, min 200.3 (67.3) N3 sleep, 69.5 (45.7) d 397.9 (61.9) 32.4 (24.5) 194.9 (46.6) 78.1 (38.7) e d e min REM, 87.0 (30.7) 91.8 (28.6) n n min MAI, n 32.7 (16.9) 23.1 (11.9) ‡ t t i a i a Data are expressed as mean (standard deviation), median [interquartile range] or mean (95% 376.2 (67.8) 372.3 (71.2) 14.9 (-11.2;41.0) 36.6 (30.1) 29.6 (21.8) 0.2 (-7.4;7.8) 202.0 (69.9) 196.5 (58.7) -2.3 (-22.6;18.0) 61.4 (43.0) 65.8 (40.7) 11.4 (-2.1;24.9) 68.2 (36.0) 77.3 (34.7) § 6.0 (-6.3;18.2) 36.3 (12.8) 31.9 (13.9) § -7.2 (-12.5;-1.8) ** confidence interval [CI]) l : l :
		Abbreviations: AHI, apnoea-hypopnoea index; AI, apnoea index; ODI, 3% oxygen desaturation index; TST, total sleep time; REM, rapid eye movement sleep; MAI, micro-arousal index. * Adjusted for baseline value, age, gender and body mass index † p<0.001 versus baseline; ‡ p<0.01 versus baseline; § p<0.05 versus baseline F o r F o r
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Acknowledgements

The authors thank Jean-Marie Chrétien, Sonia Dias Domingo, Caroline Erignoux, Anaïg le Cam, Mathieu Lelay, and Marie Peeters for their help with trial administration, monitoring and coordination, as well as data management.

Funding

This study was supported by a grant from the French Ministry of Health (PHRC-I 2010-06) JLP and RT are supported supported by the French National Research Agency in the framework of the "Investissements d'avenir" program (ANR-15-IDEX-02).

Patient consent

Obtained

Contributors

SR, J-LP, BV, RA, FC-G, BF, FGou, SL, CM, NM, VB, X-LN, AP, PP, RT, WT, and FGag were substantially involved in the design of the study and critical revision of the paper for important intellectual content. SR, FGag, J-LP, BV, WT, were substantially involved in drafting the article.

All authors were substantially involved in data acquisition, data analysis, and/or interpretation of data. All authors critically revised the article for important intellectual content.

Competing interests

The authors declare no competing interests.

Ethics approval

Comité de Protection des Personnes (CPP), Ouest II, Angers, France; No. 2010/14


Marlène Rio

Christian Salles
email: christian.salles@umontpellier.fr

Patricia Licznar-Fajardo

Pierre Marchand

Mylène Toubiana

Claire Rodier

Flavie Cernesson

Patrick Monfort

Marie-George Tournoud

Fecal Microbial Inputs To Coastal Waters From An Urbanized Basin

Keywords: Fecal indicator bacteria, Mediterranean area, anthropogenic activities, bacterial load, urban area, regressive model

Continental human activities are mainly responsible for the contamination of coastal waters. Anthropogenic pressures and more specifically urban activities represent diffuse and point sources of pollution that release contaminants on impervious surfaces and into drainage and sewage networks. Surface leaching, land runoff and floods are the key factors driven by rain events causing the transfer of contaminants from the points of emission to receiving waters. The sharp characteristics of Mediterranean rainfall patterns, long dry periods cut by short rainfall events, strengthen the river dynamics and contaminant transfer. Since fast changes in urbanized areas are observed around the Mediterranean Sea, the contaminant inputs and the apportionment of the sources to coastal pollution are key issues for integrated coastal water management. This study deals with fecal pollution of surface waters through the use of two indicators of anthropogenic and land storage contamination. The fecal indicators bacteria (FIB) are Thermo-Tolerant Coliforms (TTC) and Intestinal Enterococci (IE). The study site is the Gulf of Aigues-Mortes, a very attractive site for summer seaside tourism and winter retirement in southern France.

Introduction

Within urbanised areas, anthropogenic activities generate a wide range and increasing levels of contaminants. Human and animals disseminate fecal bacteria on land surfaces and into inland waters. Surface leaching and land runoff drive contaminants to the drainage network [START_REF] Jeong | Identifying Pollutant Sources in Tidally Mixed Systems: Case Study of Fecal Indicator Bacteria from Marinas in Newport Bay, Southern California[END_REF] and dysfunctions of sewage network represent the main point source load of fecal pollution to the river network (Anh et al., 2005). Fecal Indicator Bacteria (FIB), such as thermo-tolerant coliforms (TTC) and intestinal enterococci (IE) are indicators of the hygienic status of an aquatic environment [START_REF] Cho | Modeling fate and transport of fecally-derived microorganisms at the watershed scale: State of the science and future opportunities[END_REF] and are commonly used across the world to assess the threat of pathogen contamination in coastal and surface waters [START_REF] Gronewold | An Assessment of Fecal Indicator Bacteria-Based Water Quality Standards[END_REF].

Rainfall events are the triggering events for the transfer of inland contaminants to the catchment outlet. Mediterranean rainfall patterns, short and intense rainfall events cutting long dry periods, strengthen contaminant dynamics in the hydrographic network. During those events, the abrupt contaminant load from the catchment can severely affect the quality of the receiving waters such as lagoons, estuaries or coastal waters. To face fecal contamination issues that affect fishing and recreational activities, the active management of coastal waters needs an accurate quantification of bacterial loadings and a detailed identification of the main inland activities contributing to contamination.

This study aims at assessing the FIB inputs from Mediterranean coastal urbanised catchments and provide a picture of the apportionment of FIB contaminant sources. The study site is the Lez basin, which is one of the watersheds of the Gulf of Aigues-Mortes, a very attractive site for summer seaside tourism and winter retirement in southern France. From measurements conducted at the catchment outlets, a statistical model provides daily TTC and IE loadings from the basin. Downstream of spots of anthropogenic activities, monitored sites for river discharge and water quality give insights into the apportionment of bacterial loads from the anthropogenic activities. The considered activities are a hospital zone, a densely populated area, a waste disposal landfill and a peri-urban area.

Materials and methods

Description of the study area

The Gulf of Aigues-Mortes (hereafter GAM), located in southern France, is a well-known seaside spot for recreational activities. Its sunny and mild climate is very attractive for summer seaside tourism and winter retirement. The average annual rainfall is 672 mm. The climate is semi-arid with the majority of rainfall observed in autumn. A high inter-annual variability of rainfall is characteristic of the Mediterranean climate, along with the occurrence of extreme rainstorms. Consequently, the hydrological regime is extremely variable and leads to flood events that yield contaminant loads. In this context, the major rainfall events can represent more than 98% of the annual bacterial load [START_REF] Reeves | Scaling and Management of Fecal Indicator Bacteria in Runoff from a Coastal Urban Watershed in Southern California[END_REF][START_REF] Chu | Faecal bacterial loads during flood events in Northwestern Mediterranean coastal rivers[END_REF].

The study area is the Lez-Mosson catchment that covers the urban area of Montpellier (410 000 inhabitants). The Lez river drains a 709 km 2 basin, the population density is 691 inhabitants/km 2 and the land use is mixed: 28% of agricultural area, 44% of natural vegetation and forests and 20% of urbanisation. The remainder areas are water courses, lagoons and wetlands. The Lez River is fed by a karstic aquifer and flows for 20 km to the GAM at Palavas-Les-Flots. Its main tributary is the Mosson River that flows directly into the sea during floods through the "Etangs Palavasiens" coastal lagoon system. This lagoon system is connected to the sea by the Prevost "Grau", Occitan term for a small channel between lagoon and sea. At the south end of Montpellier, a new wastewater treatment plant, MAERA, is operational since 2006 with a capacity of 470,000 population equivalent. The effluents are discharged through a sea outfall 10 km off the coast, 30 m deep.

The spatial observation network is composed by 14 sites selected within the drainage network along the main river and near spot of anthropogenic activities (Fig. 1). The sites L1 to L6 are located along the Lez River. L1, located downstream of the karstic spring gives the baseline status of river water. Then L2 to L6 scattered from the entrance of the city area to the vicinity of the outlet gradually integrate the urban pressures and tributaries flows. Sites along the tributaries are selected for spot activities in the surrounding. Two sites, FA1 and FA2, were located along the Font d'Aurelle tributary that is channelled under the area of the hospital. FA1 is upstream of the hospital and FA2 is at the outlet of the underground channel. Along the Verdanson tributary, 2 sites were selected upstream (V1) and downstream (V2) of a residential area. The site downstream of the Lantissargues tributary (Lant) is representative of a peri-urban catchment with mainly commercial activities. Along the Mosson River, the sites M1 and M2 are respectively downstream of a mainly agricultural and natural area and downstream of a disused landfill area, the Thot area (T). The Thot area was the sanitary landfilling used from 1975 to 2006 to eliminate municipal solid wastes of the City of Montpellier. The landfill leachates are collected and directly transferred to the wastewater treatment plant. The runoff water from the reclaimed area of the landfill area was collected in basins. The site labelled T corresponds to one of these basins. Main characteristics of the drained area for the 14 sampling sites are given in Table 1.

Loads simulation and data collection

For A first campaign took place on 9-May-2016 during a rainy day of spring and a second on 12-Dec-2016 during a clear and sunny day of autumn. At each site, river discharges were measured using an Acoustic Doppler Current Profiler (ADCP) for large river section and an electromagnetic current meter for tributaries with section not suited for ADCP measurements. Concurrently to discharge measurement water grab samples were collected in the river section and stored in suited bottles packaging for analysis. Water samples were processed at the laboratory using the reference methods ISO 9308-1 and ISO 7899-2 mentioned in the European bathing water directive 2006/7/EC to enumerate termo-tolerant coliforms (TTC) and intestinal enterococci (IE), respectively (European Parliament. Council of the European Union, 2006). Water volumes (0.1, 1, 10 and 100 mL) were filtered in duplicate on cellulose nitrate membrane (Whatman filters). TTC were quantified on Triphenyl Tetrazolium Chloride Tergitol agar incubated for 24h at 44.5°C, and intestinal enterococci were enumerated on Slanetz medium incubated for 48h at 37°C. The results are expressed as colony forming units per 100 mL (CFU/100mL).

Results and discussion

At the catchment scale

The fecal bacterial loadings from the catchment assessed by the regressive model are reported Fig. 2a and 2b for TTC and IE loads, respectively. The model output is the daily load most probable value within a confidence interval. Fig. 2 illustrates the significant variability of the daily loads from the catchment. The loads variability is linked with the rainfall temporal variability. The base level of catchment loads are 10 12 cfu/day for TTC and IE. At the annual scale loads are in the range 3.6 10 15 to 1.9 10 17 cfu for TTC and 2.0 10 15 to 6.0 10 16 cfu for IE. The average MAERA loads, 2.3 10 14 cfu/day for TTC and 2.0 10 13 cfu/day for IE, are slightly larger than base loads from the catchment. But at the annual scale IE loads from MAERA are one tenth to one hundredth less than IE loads from the catchment. There is less agreement on the ratio MAERA vs catchment for the annual TTC loads. MAERA TTC inputs to the coastal area are twice than the catchment inputs for 2009 but are half to 1/60 for 2010 to 2012. From the catchment, the inter-annual load variation depends on the magnitude of the major rainfall event that occurs during the year. The major rainfall event can generate most of the annual load. As an example, for the hydrological years 2010 and 2011 the maximum load of 6 consecutive days accounts for at least 93% of the annual load. The magnitude and the abruptness of the loads during those short and intense events can be critical for water quality of the receiving waters. It demonstrates the critical importance of inland sources of fecal bacteria transferred through the drainage network. During dry periods, a large number of fecal bacteria accumulates on land surfaces and stores in the sediments. During intense rain events, runoff and sediments resuspension combined with sewer overflow release fecal bacteria and create major critical loads to the downstream waters. As a consequence, water quality is impacted during extreme rainstorms. Identifying the contribution of anthropogenic activities to fecal bacterial loads and their balance at the catchment scale is nowadays a key issue for the risk assessment and the management of water quality. The statistical regressive model assesses the global fecal bacterial load from the catchment but does not consider the load origin within the catchment. Indeed, considering changes in the hydrosystem in land use, land cover and anthropological activities, the parameters of the statistical model might misfit and make the load prediction unreliable.

Inland activities, fecal bacterial sources and load apportionment

For the 14 monitored sites located along the drainage network in the vicinity of anthropological activities, we collected water quality data and hydrological characteristics. Two spatial sampling campaigns were conducted, on 9-May-2016 and on 12-Dec-2016. For both the hydrological conditions along the hydrographic network correspond to low flow conditions. The first day on 9-May-2016 was a rainy day. A few short rain events occurred during the sample collection. The total daily rain amount was 25 mm. On 12-Dec-16, it was a late autumn sunny day. There was no rain, and the antecedent period was dry. The rain amount for the antecedent ten days was 1 mm. The last significant event last for more than 15 days, on the 24-Nov-2016. Fig. 3 and 4 are snapshot of the measurements. On Fig. 3, the concentrations of FIB in the water column are given; part 1 of the figure corresponds to the sites along the main drains: L1 to L6, from upstream to downstream of the Lez River that crosses the urban area of Montpellier, and sites M1 and M2, in the downstream part of the Mosson River that drains natural and agricultural areas. Part 2 of the figure is related to sites with specific anthropological activities: T (the disused landfill area), Lant (downstream of a peri-urban area), FA1 and FA2 (upstream and downstream of a hospital) and V1 and V2 (upstream and downstream of a housing area). For both, TTC and IE, the concentrations observed on 9-May-2016 and 12-Dec-2016 are given. Fig. 4 is drawn the same way as Fig. 3, but it describes the FIB load assessed from the product of concentration and discharge at the monitored site.

The concentrations observed along the drainage network are from 21 to 9 10 4 TTC/100 mL and from 50 to 9.7 10 4 IE/100 mL. The concentrations are higher for tributaries than in the Lez and Mosson rivers. The runoff water on the surface of the Thot disused landfill area shows the lowest TTC concentration. As there is no anthropic activity in the area, the main source for this contamination should be the wild rabbits and birds that have their habitats in this free zone as well as some roaming dogs. Along the Lez River, the TTC concentration slightly increases from the spring (L1) to downstream of the city (L4). For the 2 sites located downstream, L5 and L6, TTC concentration level off and slightly fall down. The combination of sunlight self-purification process and the presence of marine salt water can be the basis of the decrease. The TTC levels in the Mosson River are in the same range as in the Lez River. The level of FIB concentrations is significantly higher on the 9-May than on the 12-Dec for the tributaries and does not vary a lot for the two main rivers. The rainy event, on the 9-May, induces larger FIB concentrations in small tributaries for TTC as well as for IE. Actually, the small rain event of the 9-may leads to a flash variation in the discharge of the small tributaries in low flow period, but poorly affects the discharge of the Lez and Mosson rivers for which the time transfer of water is larger. The bacterial loads at the monitored sites are reported on Fig. 4. The TTC loads vary from 8 10 8 To 2 10 12 cfu/day. IE loads are in the range 5 10 8 to 3 10 12 cfu/day close to TTC loads. The loads are not assessed for sites L4 and L5 as no discharge measurements were processed in this sites. Neither, load is not associated to the site T that refers to the basin that collect runoff water from the Thot disused landfill surface. The general pattern of loads along the drainage network differs than concentration pattern. Loads are related to concentration and discharge. The discharges of the tributaries are an order of magnitude lower than discharges of the two rivers. Tributaries discharge range from 5 to 45 L/s while rivers discharge range from 400 to 4000 L/s. Thus, for the low flow conditions of the two sampling days, river loads are the same order or higher than tributaries loads. Loads at the outlets of the two rivers (M2 and L6) remain quite stable between the two sampling days, while the loadings in the Lantissargues and Verdanson sites vary by two order of magnitude in between 9-May and 12-Dec. The highest TTC and IE loads were observed the first day, on 9-May, downstream of the Lantissargues site. This tributary drain a peri-urban area that contains an equestrian centre very close to the sampling points. This site needs more investigation to identify the actual origin of the FIB load. The hospital area and the residential zone generate similar increase of FIB load that differs from less than one order of magnitude. The first data analysis shows that in low flow condition, the contribution of the various anthropogenic activities (residential, hospital and agricultural) are comparable, but tributary loads show a large variability.

Conclusions

This study analyses the fecal indicator bacteria loads at the global scale of the catchment and locally downstream of spots of anthropogenic activities. The fecal bacterial loading of the basin is assessed from a statistical linear regression model. Simulations over a four years period illustrate the balance between loads from the catchment and loads at the output of the wastewater treatment plant. The relevance of the major rainy event, that can provide in a few days more than 90 % of the annual load, is shown.

Within the basin, anthropogenic activities such as a residential area, a hospital zone, a former landfill area, a peri-urban area and an agricultural area are monitored for their FIB loads. The measurements, conducted during low flow periods, point out the great variability of FIB concentration within the tributaries and the comparable level of FIB loads from the various considered activities.

The main flaw of the statistical model is the hypothesis of temporal stationarity of the activities within the catchment. To better assess the contributions of anthropogenic activities to FIB load, a thoughtful analysis and monitoring during flood events is planned.

  the period, Aug 2009 to Sept 2013, daily simulation of thermo-tolerant coliforms (TTC) and intestinal enterococci (IE) loadings are provided at the basin outlet from a statistical linear regressive model. The model, described by Rio et al. (submitted), is based on explanatory relationships established between antecedent hydrometeorological conditions and measured loads of TTC and IE at the outlets of the Lez catchment. The developed model quantifies daily TTC and IE loadings at the catchment outlets in relation to the Antecedent Precipitation Index (API) derived from collected rain data within the catchment. The main hypothesis for model application is temporal stationarity in territorial dynamics and rainfall patterns, avoiding major changes during the modelling period due to land use planning or climate change. Confidence intervals of the model predictions are assessed. From three evenly distributed rain gauge stations, daily rainfall data from Aug-2009 to Sept-2013 were obtained from Météo-France in the study area. The daily rainfall amount over the Lez catchment was assessed applying Thiessen interpolation to the rain gauge network. Monthly records of TTC and IE concentrations at the sea outfall of the wastewater treatment plant were provided by the Montpellier-Méditerranée Métropole from 2009 to 2013. The catchment loadings assessed from the model outputs are compared to the FIB loads measured at the entrance of the sea outfall pipe of the wastewater treatment plant.

Fig. 1 :

 1 Fig. 1: Sampling stations on the Lez catchment. The black labeled points are the monitored sites along the hydrographic network. The sites L1 to L6 are located along the Lez River, FA1 and FA2, along the Font d'Aurelle tributary, V1 and V2, along the Verdanson tributary, Lant, along the Lantissargues tributary, and M1 and M2, along the Mosson River that is also a tributary of the Lez River. The site T is situated in one of the basins, which receive the landfill leachates of the Thot sanitary landfilling.

  Fig. 2a and 2b also report the loads of the MAERA wastewater treatment plant at the outlet of the sea outfall pipe. The loads are assessed from self-monitoring of discharge and concentration by MAERA manager and provided by the Montpellier Méditerranée Metropole. The self-monitoring is run twice a month. The results are shown for four hydrological years, from August 2009 to July 2013.

Fig. 2 :

 2 Fig. 2: Loads of fecal indicator bacteria. Daily simulated load from the linear model (most probable value: dotted line; interval of confidence of the prediction: thin line). Observed loads at the outlet of the wastewater treatment plant (bold line). The second axis refers to the daily rainfall (vertical bars) (a) reports for thermo-tolerant coliforms (TTC) and (b) reports for intestinal enterococci (IE).

Fig. 3 :

 3 Fig. 3: Thermo-tolerant coliforms (TTC) (a) and intestinal enterococci (IE) (b) concentrations, at the monitored sites along the drainage network. Black bars represent observation on 9-May-2016, right hatched bars are on 12-Dec-2016.

Fig. 4 :

 4 Fig. 4: Thermo-tolerant coliforms (TTC) (a) and intestinal enterococci (IE) (b) concentrations, at the monitored sites along the drainage network. Black bars represent observation on 9-May-2016, right hatched bars are on 12-Dec-2016.

Table 1 :

 1 Characteristics of the sub-catchments upstream of the monitored sites (based on Corine Land Cover 2012)

	Site name	Area (km²)	Natural area (%)	Agricultural area (%)	Artificial area (%)	Waterbodies (%)
	L2	127.8	53.1	32.0	14.9	0
	L3	141.2	48.4	30.7	20.9	0
	L4	147.6	46.5	29.7	23.8	0
	L5	150.9	45.5	29.3	25.2	0
	L6	519.8	48.5	30.5	20.9	0.2
	FA1	2.1	12.8	0.0	87.2	0

Acknowledgements

This work was supported by funding provided by the French Ministry of Ecology (Liteau III Grant No. 0001138; Programme MICROGAM), by the Human-Environment Observatory of the Mediterranean Coast (OHM Littoral Méditerrannéen), and by a doctoral fellowship (Marlène Rio) from the DRIIHM Labex, CNRS. We acknowledge Météo-France and the Montpellier Méditerranée Métropole for access to data regarding rainfall records and effluent quality of the wastewater treatment plant.