Ellyn A King

Sabyasachi Sen

Wataru Takeda

Catherine Boussard-Pledel

Bruno Bureau

Jean-Pierre Guin

Pierre Lucas
email: pierre@arizona.edu

Extended aging of Ge-Se glasses below the glass transition temperature

Germanium selenide glasses of compositions spanning the whole glass-formation range are aged at room temperature for up to twenty years. A prominent enthalpy relaxation process is observed in all glasses and its structural origin is analyzed by Raman spectroscopy. The structural relaxation is manifested in the Raman spectra as a decrease in the ratio of edge to corner-sharing GeSe 4/2 tetrahedral units. This structural evolution can be explained in terms of configurational entropy and density changes. Changes in Raman features and enthalpy follow an identical stretched exponential relaxation function characteristic of ageing in glasses. The compositional dependence of enthalpy relaxation after twenty years is in agreement with kinetic considerations based on the glass transition temperature of each glass. The relaxation behavior and heat-capacity curves are consistent with standard glass relaxation models for all compositions. These results indicate that the non-reversing enthalpy obtained by modulated differential scanning calorimetry (MDSC) which suggests the existence of non-aging glasses is not a reliable measure of the ability of a glass to relax. Instead, it is suggested that an interpretation of MDSC data in terms of complex heat capacity provides a more complete and reliable assessment of the relaxation properties of glasses.

I.

INTRODUCTION

Sub-T g relaxation is an important problem in glass technology due to the unwanted property changes that can take place in glassy components over time. Volumetric changes are detrimental for glass substrates used in consumer electronics [START_REF] Welch | Dynamics of Glass Relaxation at Room Temperature[END_REF] while changes in refractive index are harmful

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for optical applications [START_REF] Wang | Structural relaxation and optical properties in amorphous Ge33As12Se55 films[END_REF] . Chalcogenide glasses are particularly exposed to these issues due to their relatively low glass transition temperature (T g ) which can lead to significant relaxation even at room temperature [START_REF] Lucas | Correlation between thermal and mechanical relaxation in chalcogenide glass fibers[END_REF][START_REF] Golovchak | Experimental verification of the reversibility window concept in binary As-Se glasses subjected to a long-term physical aging[END_REF] . Among these amorphous chalcogenides, germanium selenide glasses are of interest due to their complex structural dynamic [START_REF] Lucas | Bimodal phase percolation model for the structure of Ge-Se glasses and the existence of the intermediate phase[END_REF][START_REF] Gjersing | Mechanistic understanding of the effect of rigidity percolation on structural relaxation in supercooled germanium selenide liquids[END_REF][START_REF] Sen | 77Se Nuclear Spin-Lattice Relaxation in Binary Ge-Se Glasses: Insights into Floppy Versus Rigid Behavior of Structural Units[END_REF] and their precedent as testbeds for topological constraint theories [START_REF] Phillips | Topology of covalent non-crystalline solids. I. Short-range order in chalcogenide alloys[END_REF][START_REF] Bhosle | Meeting experimental challenges to physics of network glasses: Assessing the role of sample homogeneity[END_REF] . The structure of Ge-Se glasses is composed of Se chains and GeSe 4 tetrahedra that exhibit notably distinct dynamics as shown by Nuclear Magnetic Resonance (NMR) [START_REF] Lucas | Bimodal phase percolation model for the structure of Ge-Se glasses and the existence of the intermediate phase[END_REF][START_REF] Gjersing | Mechanistic understanding of the effect of rigidity percolation on structural relaxation in supercooled germanium selenide liquids[END_REF][START_REF] Sen | 77Se Nuclear Spin-Lattice Relaxation in Binary Ge-Se Glasses: Insights into Floppy Versus Rigid Behavior of Structural Units[END_REF] . These two structural elements can in turn be related to the floppy and rigid domains that constitute the basis of topological constraint theories [START_REF] Phillips | Topology of covalent non-crystalline solids. I. Short-range order in chalcogenide alloys[END_REF][START_REF] Thorpe | Continuous deformations in random networks[END_REF][START_REF] He | Elastic properties of glasses[END_REF] . Indeed, Ge-Se glasses have long been used to test topological constraint theories due to their covalent network structure. The well-defined number of bonds and bond angles provides a convenient testbed for constraint counting principles which are thought to help predict some physical properties of the glass. For example, it has been observed that some properties such as density show a broad maximum near the so-called rigidity percolation threshold where the number of atomic degrees of freedom (d=3) is exactly compensated by angular and bond constraints [START_REF] Senapati | Structure-property inter-relations in chalcogenide glasses and their practical implications[END_REF][START_REF] Wang | Observation of two elastic thresholds in GexAsySe1-x-y glasses[END_REF] . In this region the glass network is expected to undergo a floppy (under-coordinated)-to-rigid (over-coordinated) transition. Constraint counting shows that this transition should occur when the average coordination number (average number of bonds per atom) is <r>=2.4 [START_REF] He | Elastic properties of glasses[END_REF] .

More recently this theory has been extended to suggest that covalent networks can be optimally constrained over a broader compositional region where they are stress-free [START_REF] Thorpe | Self-organization in network glasses[END_REF] . Experimental observation of this compositional window has been claimed mainly by invoking a quantity called the non-reversing enthalpy ΔH nr measured by Modulated Scanning Differential Calorimetry (MDSC) [START_REF] Bhosle | Meeting experimental challenges to physics of network glasses: Assessing the role of sample homogeneity[END_REF][START_REF] Chakravarty | Ageing, fragility and the reversibility window in bulk alloy glasses[END_REF][START_REF] Boolchand | Self-organization and the physics of glassy networks[END_REF][START_REF] Vempati | The thermally reversing window in ternary GexPxS1-2x glasses[END_REF] . In MDSC, the sample is subjected to a linear heating ramp superimposed with a sinusoidal oscillation. The resultant heat flow is often decomposed into a "reversing" heat capacity component and a "non-reversing" kinetic component permitting to separate nonreversible processes such as cold crystallization from the total heat flow [START_REF] Verdonck | A discussion of the principles and applications of Modulated Temperature DSC (MTDSC)[END_REF] . This method was in turn applied to analysis of the glass transition with the goal of characterizing relaxation processes [START_REF] Hutchinson | Aging of polycarbonate studied by temperature modulated differential scanning calorimetry[END_REF] . In chalcogenide systems, it was observed that the non-reversing enthalpy decreases to zero in certain compositional ranges (0.20 to 0.25 Ge% in Ge-Se); thus, dubbed "reversibility window". It was further suggested that the self-organized nature of the network within this window would lead to glasses that do not age [START_REF] Bhosle | Meeting experimental challenges to physics of network glasses: Assessing the role of sample homogeneity[END_REF][START_REF] Chakravarty | Ageing, fragility and the reversibility window in bulk alloy glasses[END_REF][START_REF] Boolchand | Self-organization and the physics of glassy networks[END_REF][START_REF] Vempati | The thermally reversing window in ternary GexPxS1-2x glasses[END_REF] . This observation was derived from measurements of ΔH nr on glasses annealed at room temperature for up to several weeks. It was found that chalcogen-rich glasses exhibited significant change in ΔH nr , while glasses in the "reversibility window" with lower chalcogen content exhibited minimal or no change [START_REF] Bhosle | Meeting experimental challenges to physics of network glasses: Assessing the role of sample homogeneity[END_REF][START_REF] Chakravarty | Ageing, fragility and the reversibility window in bulk alloy glasses[END_REF][START_REF] Boolchand | Self-organization and the physics of glassy networks[END_REF][START_REF] Vempati | The thermally reversing window in ternary GexPxS1-2x glasses[END_REF] . However, one issue with this interpretation is that glasses with broadly different T g could have relaxation times spanning multiple orders of magnitude since the enthalpy relaxation time is exponentially dependent on T g [START_REF] Debolt | Analysis of structural relaxation in glass using rate heating data[END_REF][START_REF] Hodge | Calculation of the effects of annealing on Tg endotherms[END_REF] . Hence, annealing series of glasses with different T g at a common temperature for the same duration cannot provide a systematic measure of their ability to relax but merely a broad assessment of their relative relaxation times. In other words, low-T g glasses may relax within a few weeks even at room temperature while relaxation may be too slow to observe for high-T g glasses within the "reversibility window". Therefore, more extensive

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analyses are needed to assess the ability of glasses from the so-called "reversibility window" to undergo aging.

In this study we analyze glasses across the floppy to rigid range that have been aged over 20 years at room temperature. Furthermore we analyze the time dependent relaxation of two glasses, one outside and one inside the "reversibility window", for up to five years at room temperature.

It is found that all glass compositions exhibit significant relaxation in a way consistent with their relative T g . Glasses outside and inside the "reversibility window" undergo a large stretchedexponential enthalpy relaxation characteristic of traditional glassy systems. Furthermore, the structural origin of this relaxation process is found to be identical in all glasses and shown to involve a conversion from edge to corner-sharing tetrahedral units that is consistent with a decrease in configurational entropy and an increase in density, as expected from conventional glass relaxation.

II. BACKGROUND

Sub-T g glass relaxation -also called aging -has been extensively studied over the years [START_REF] Scherer | Volume Relaxation Far from Equilibrium[END_REF][START_REF] Ma | Relaxation near room temperature in tellurium chalcohalide glasses[END_REF][START_REF] Scherer | Theory of relaxation[END_REF][START_REF] Hodge | Physical aging in polymer glasses[END_REF][START_REF] Chen | Sub-Sub-T g Enthalpy Relaxation in a B 2 0 3 Glass[END_REF][START_REF] Sanghera | Shrinkage of fluorozirconate glass fibers at low temperatures[END_REF][START_REF] Moynihan | Structural relaxation in fluoride glasses[END_REF][START_REF] Ya | Enthalpy and Anisotropy Relaxation of Glass Fibers[END_REF][START_REF] Hu | Secondary Relaxation in Metallic Glass Formers: Its Correlation with the Genuine Johari-Goldstein Relaxation[END_REF][START_REF] Yu | Suppression of beta Relaxation in Vapor-Deposited Ultrastable Glasses[END_REF][START_REF] Zheng | Variability in the relaxation behavior of glass: Impact of thermal history fluctuations and fragility[END_REF][START_REF] Gulbiten | Relaxation of enthalpy fluctuations during sub-Tg annealing of glassy selenium[END_REF][START_REF] Guo | Predictive model for the composition dependence of glassy dynamics[END_REF] and it has been shown that the relaxation process depends on multiple factors including the nonexponentiality β, the non-linearity x, the fictive temperature T f and the fragility index m. The fragility is of particular interest as it affects the relaxation process in multiple ways that may appear contradictory. For example glasses located within the "reversibility window" are generally found to correspond to compositions exhibiting lower fragility index [START_REF] Zeidler | Topological Ordering and Viscosity in the Glass-Forming Ge-Se System: The Search for a Structural or Dynamical Signature of the Intermediate Phase[END_REF] so that prior suggestions that these glasses do not age, may appear to indicate that low fragility is associated with a resistance to relaxation. On the other hand, recent energy landscape simulations have shown that high-fragility glasses are more resistant to relaxation during linear heat treatments due to the higher free energy barriers below the glass transition [START_REF] Zheng | Variability in the relaxation behavior of glass: Impact of thermal history fluctuations and fragility[END_REF] . This apparent contradiction stems from the fact that fragility affects the relaxation process in two distinct ways: (i) higher fragility increases the thermodynamic driving force for relaxation and leads to greater relaxed enthalpy, (ii) higher fragility increases the relaxation time and leads to slower relaxation process.

The first case can be illustrated using a configurational entropy plot as in Fig. 1. As demonstrated by Martinez & Angell [START_REF] Martinez | A thermodynamic connection to the fragility of glass-forming liquids[END_REF] , the scaled entropy of glass-forming liquids follows the same pattern as the viscosity on a fragility plot and constitute a thermodynamic signature of fragility. The scaled entropy of three hypothetical glass-formers is therefore plotted in Fig. 1(a) using a modified Vogel-Tamman-Fulcher equation with fragility index m = 16, 55 and 200. The equilibrium lines are extended below T g while the glassy state is represented by the thick dashed line where excess entropy is frozen in. The part of the curve below T g is of particular interest here and is replotted in Fig. 1(b) as the normalized entropy versus normal temperature in a way similar to a Kauzmann plot 37 but normalized by T g . It is clear from Fig. 1(b) that the entropy of the fragile glass diverges much more rapidly from the equilibrium line than the strong one. Consequently, a much greater thermodynamic driving force for relaxation builds up, while that of the strong

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system remains minimal for an equivalent departure from T g . This indicates that fragile systems are prone to greater structural relaxation, once sufficient time is allowed to reach equilibrium. The second case can be simply derived from the Tool-Narayanaswamy-Moynihan (TNM) equation for the structural relaxation time τ [START_REF] Debolt | Analysis of structural relaxation in glass using rate heating data[END_REF][START_REF] Hodge | Physical aging in polymer glasses[END_REF] and the equation for the fragility index m [START_REF] Wang | Direct determination of kinetic fragility indices of glassforming liquids by differential scanning calorimetry: kinetic versus thermodynamic fragilities[END_REF] where the activation energy for enthalpy relaxation ΔH* is considered equivalent to the activation for viscous flow as suggested by Moynihan [START_REF] Moynihan | Estimation of activation energies for structural relaxation and viscous flow from DTA and DSC experiments[END_REF] .

𝜏𝜏 = 𝐴𝐴 exp � 𝑥𝑥∆𝐻𝐻 * 𝑅𝑅𝑅𝑅 + (1 -𝑥𝑥)∆𝐻𝐻 * 𝑅𝑅𝑅𝑅 𝑓𝑓 � (1)
𝑚𝑚 = ∆𝐻𝐻 * 𝑅𝑅𝑅𝑅 𝑔𝑔 • 𝑙𝑙𝑙𝑙10 (2)
It is then obvious that when the fragility index m increases, the relaxation time τ increases exponentially. Therefore, when all other parameters are kept constant, relaxation is considerably slower in the fragile systems than in the strong ones.

In summary, fragile glasses have a greater thermodynamic potential for relaxation during aging, but their relaxation rate is much slower compared to that of the strong ones.

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III.

EXPERIMENT A. Glass samples:

Two sets of Ge-Se glasses were produced for this study. All Ge-Se samples were high purity glasses used for the preparation of optical elements. Syntheses were performed in evacuated silica tubes in a rocking furnace as detailed in ref [START_REF] Melscoet | Synthese, Characterization et Optimization de Verre Optiques pour des Application Infrarouge dans la fenetre 8-12 micorns[END_REF] and [START_REF] Lucas | Structural and chemical homogeneity of chalcogenide glass prepared by melt-rocking[END_REF]. As shown in ref [START_REF] Lucas | Structural and chemical homogeneity of chalcogenide glass prepared by melt-rocking[END_REF] these glasses were fully homogenized and were free of impurities such as water, which may affect their structural and dynamic behavior as implied in ref [START_REF] Bhosle | Meeting experimental challenges to physics of network glasses: Assessing the role of sample homogeneity[END_REF]. The first set was a series of bulk glasses produced in 1999 with composition Ge 10 Se 90 , Ge 15 Se 85 , Ge 20 Se 80 , Ge 25 Se 75 and Ge 30 Se 70 [START_REF] Melscoet | Synthese, Characterization et Optimization de Verre Optiques pour des Application Infrarouge dans la fenetre 8-12 micorns[END_REF][START_REF] Guin | Hardness, toughness, and scratchability of germanium-selenium chalcogenide glasses[END_REF] .

This set of glass was stored in air in a thermostated room (22⁰C±2⁰) for 20 years in the LARMAUR lab at the University of Rennes with no light exposure until it was characterized.

The second set included samples of two compositions that were drawn into fibers. The first composition was the Se-rich GeSe 9 which lies well outside of the "reversibility window". The second composition was the archetypical GeSe 4 stoichiometry which corresponds to the rigidity percolation threshold at <r> = 2.4 and lies within the "reversibility window". For this study, GeSe 9 and GeSe 4 fibers were fabricated at various times over the course of five years. The fibers were drawn in a He atmosphere at a speed of 1.22 m/min and a tension of 20 g. At this speed and tension, no measurable structural anisotropy could be observed by 77 Se Nuclear Magnetic Resonance, polarized Raman or X-ray Diffraction along the axis of the fibers. In these conditions, the quench rate was estimated at ∼500°C/min 3 . After fabrication, the fibers were removed from their storage drums and stored in 15 cm long strands in silica tubes, in the dark at room temperature in the laboratory. Small pieces of the stored fibers were then removed from the silica tubes when DSC and Raman experiments were ready to be performed.

B. Raman spectroscopy:

Raman spectroscopy was performed on 20 year old bulk samples of each composition before and after rejuvenation in a DSC (See section C). Raman spectra were collected using a Renishaw InVia spectrometer with a 1200 lines/grating and a diode laser at a wavelength of 785 nm focused with a 10X objective at a power of 15mW to avoid any photostructural changes. Spectra were collected for 120 seconds and averaged over three scans. Raman spectroscopy was also performed on small sections of GeSe 9 and GeSe 4 glass fibers from the same batch as that used for calorimetric measurements. Prior to each measurement, the sample surface was cleaned with alcohol and the fibers were lightly crushed and pressed into aluminum sample holders. Unpolarized Raman spectra were collected at ambient temperature and in backscattering geometry using a Bruker RFS 100/S Fourier-Transform Raman spectrometer equipped with a Nd:YAG laser operating at 1064 nm and a liquid nitrogen cooled solid-state Ge detector. A laser power level of 50 mW was used, and spectra were collected using a resolution of 2 cm -1 . Approximately 100 scans were collected and averaged to obtain each Raman spectrum. The low photon energy and the low power level used to collect the Raman spectra, permitted to avoid any photostructural damage during data collection.

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C. Differential Scanning Calorimetry

Enthalpy relaxation measurements were performed on 20 year old bulk samples of each composition using a TA Instrument DSC25 equipped with a refrigerated cooling system RCS90 and calibrated with an indium standard. Small bulk glass pieces were lightly polished to obtain a flat surface to optimize thermal contact with the DSC pan. Samples of mass ∼10 mg were then hermetically sealed in a Tzero ® aluminum pan. An identical empty pan was used as the reference. Samples were first heated at 10°C/min up to 100°C above T g to measure the heat capacity of the relaxed glass. The sample was then rejuvenated by subjecting it to the same heattreatment as the original glass through equilibration at T g (up to 4h) and slowly cooling at 1°C/min to room temperature [START_REF] Melscoet | Synthese, Characterization et Optimization de Verre Optiques pour des Application Infrarouge dans la fenetre 8-12 micorns[END_REF] . A run of the rejuvenated glass was then immediately collected at 10°C/min over the same temperature range. The two thermograms were integrated and subtracted to calculate the relaxed enthalpy of the 20 year old sample. Enthalpy relaxation measurements were also performed on glass fibers using a Mettler Toledo DSC1 calorimeter equipped with a FRS5 sensor and sub-ambient cooling unit. DSC was performed on both sets of GeSe 9 and GeSe 4 fibers of varying ages, in standard (non-modulated) mode. Prior to any experiments, both the temperatures and the heat flow were calibrated using indium and zinc standards. Heat capacity values were calculated from the heat flow using the molar mass of the samples. Tested samples consisted of 3 mm long pieces of fiber, placed side by side in the center of an aluminum DSC pan and hermetically sealed. Empty aluminum pans were used as a reference, and sample masses were in the range of 5 to 7 mg. Enthalpy change calculations were completed using two consecutive heating scans of each sample. To perform such a calculation, each fiber sample was placed in the DSC and heated at 10 C min -1 to a temperature well above T g , allowed to equilibrate at this temperature, and subsequently cooled in the DSC using the same 10 C min -1 rate to well below T g . This first temperature up-scan captures the enthalpy of the fiber due to relaxation. Upon completion of cooling, the sample is then reheated, again at 10 C min -1 , to obtain the reference DSC curve. Enthalpy change is then determined by the integration of the area between the two heat flow curves.

IV. RESULTS

A. 20 year old bulk samples:

Calorimetry

The DSC curves of all Ge-Se bulk glasses after 20 year aging at room temperature are shown in Fig. 2. Each curve is compared with that of the same glass after rejuvenation. Glasses with lower T g exhibit large endothermic peaks corresponding to the regain of relaxed enthalpy during reheating. The large relaxed enthalpy is consistent with the shorter relaxation time associated with the lower T g since the A term in the TNM equation (Eq. 1) is exponentially dependent on T g . Moreover, the large relaxed enthalpy (>300 J/mol) is also consistent with the higher fragility m Accepted Manuscript of these glasses [START_REF] Zeidler | Topological Ordering and Viscosity in the Glass-Forming Ge-Se System: The Search for a Structural or Dynamical Signature of the Intermediate Phase[END_REF] . As shown in Fig. 1b, higher fragility leads to steeper departure from equilibrium and greater build up for relaxation. Interestingly, glasses within the so-called "reversibility window" also show rather large relaxed enthalpy (up to 167.5 J/mol) despite their higher T g and the reduced thermodynamic potential for relaxation associated with their lower fragility. Finally, glasses with T g values far above room temperature exhibit small but still measurable enthalpy relaxation as shown for Ge 30 Se 70 . Overall, these data clearly indicate that all glasses, including those in the "reversibility window" can undergo significant structural relaxation. As shown in the inset of Fig. 2, the ability to relax at room temperature is directly dependent on the T g of the glass and therefore appears to be mainly limited by kinetic factors.

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Fig. 2: DSC curves of bulk Ge-Se glasses after 20 year annealing at room temperature (295 K). The relaxed enthalpy ∆H R corresponds to the difference between the integrated curves of the relaxed and rejuvenated glass. The inset shows the dependence of ∆H R on the glass transition temperature T g . (endothermic is up)

Raman spectroscopy

As shown in Fig. 3, the Raman spectra of all Ge-Se glasses exhibit the characteristic three main spectral lines located at 255 cm -1 , 213 cm -1 and 195 cm -1 which corresponds to the Se-chain modes and the edge-sharing and corner-sharing tetrahedral modes, respectively [START_REF] Lucas | Bimodal phase percolation model for the structure of Ge-Se glasses and the existence of the intermediate phase[END_REF][START_REF] Edwards | Structure and Relaxation in Germanium Selenide Glasses and Supercooled Liquids: A Raman Spectroscopic Study[END_REF] . The spectra normalized to the Se-chain mode show a systematic decrease in edge-sharing tetrahedra modes with annealing while the corner-sharing mode concomitantly increases. This indicates that a conversion from edge-to corner-sharing tetrahedra is a structural marker of aging in these glasses. In order to further investigate a correlation between this structural evolution and enthalpy relaxation, the aging process was monitored over time in a series of Ge-Se glass fibers as detailed in the following section.

Fig. 3: Raman spectra of bulk Ge-Se glasses after 20 year aging at room temperature (295 K) compared with the rejuvenated glass. The peak intensity ratio between corner-and edge-sharing tetrahedra systematically increases with annealing.

B. 5 year old fiber samples

Calorimetry

Figure 4 presents the heat capacity curves of GeSe 9 and GeSe 4 glass fibers collected at several time intervals as they were aging at room temperature for up to five years. The DSC curves reveal notably different evolutions between the two fibers which correspond to the expected behavior of glasses annealed at different temperatures relative to T g (for GeSe 9 : T g -T a ∼ 70°C while for GeSe 4 : T g -T a ∼ 140°C). The GeSe 9 shows a large C p overshoot while the GeSe 4 shows the development of a pre-T g endotherm that slowly grows with aging. The main DSC features of these two glasses can be predictably reproduced using the TNM modeling procedure of Hodge [START_REF] Hodge | Effects of annealing and prior history on enthalpy relaxation in glassy polymers. 2[END_REF] as shown in Fig. 5. The GeSe 9 and GeSe 4 glasses were modeled using a cooling rate of 500°C/min 3 and a reheating rate of 10°C/min after annealing at 25°C (room temperature) for increasing time periods. Modeling parameters were estimated from literature for fragility index m [START_REF] Zeidler | Topological Ordering and Viscosity in the Glass-Forming Ge-Se System: The Search for a Structural or Dynamical Signature of the Intermediate Phase[END_REF] , non-exponentiality β [START_REF] Boehmer | Nonexponential relaxations in strong and fragile glass formers[END_REF] and non-linearity x [START_REF] Hodge | Adam-Gibbs formulation of nonlinear enthalpy relaxation[END_REF] . The modeled and experimental C p curves show good qualitative match. Figure 4a & 5a shows that, as a result of its low T g , the GeSe 9 glass can relax rapidly at room temperature and exhibits a large overshoot of C p after only 3.5 years. This overshoot is indicative of a large relaxed enthalpy enhanced by its higher fragility, as predicted from Fig. 1. In contrast, the C p of the GeSe 4 glass in Fig. 4b & 5b shows the development of a pre-T g endotherm that shifts to higher temperatures upon longer annealing. This endotherm is the well-known signature of fast relaxing domains regaining enthalpy upon reheating after a long anneal. Such behaviors have been described in great detail by Hodge both experimentally and theoretically [START_REF] Hodge | Effects of annealing and prior history on enthalpy relaxation in glassy polymers. 2[END_REF][START_REF] Berens | Effect of annealing and prior history on enthalpy relaxation in glassy polymers. 1. Experimental study on poly(vinyl chloride)[END_REF][START_REF] Hodge | Effects of annealing and prior history on enthalpy relaxation in glassy polymers. 3. Experimental and modeling studies of polystyrene[END_REF][START_REF] Hodge | Effects of annealing and prior history on enthalpy relaxation in glassy polymers. 4. Comparison of five polymers[END_REF][START_REF] Hodge | Effects of annealing and prior history on enthalpy relaxation in glassy polymers. 5. Mathematical modeling of nonthermal preaging perturbations[END_REF][START_REF] Hodge | Effects of annealing and prior history on enthalpy relaxation in glassy polymers. 6. Adam-Gibbs formulation of nonlinearity[END_REF] . Such pre-T g endotherms are only observable for a specific range of annealing temperatures far below T g after a fast quench [START_REF] Hodge | Effects of annealing and prior history on enthalpy relaxation in glassy polymers. 2[END_REF][START_REF] Berens | Effect of annealing and prior history on enthalpy relaxation in glassy polymers. 1. Experimental study on poly(vinyl chloride)[END_REF] , which is precisely the case for the GeSe 4 fibers. For glasses with T g closer to their annealing temperature like GeSe 9 , the entire distribution of relaxation times is sufficiently short that relaxation occurs quickly at room temperature, and upon reheating the enthalpy relaxation appears as the conventional overshoot superimposed on the T g , as shown in Fig. 4a & 5a. Overall, both glasses show the characteristic relaxation behavior predicted by the TNM model.

Raman spectroscopy

Figure 6 shows the spectra of the GeSe 4 glass fibers collected by FT-Raman. The spectra show the characteristic Se-chain and tetrahedral modes near 250 cm -1 and 200 cm -1 respectively as well as the low frequency broadband Se bending modes near 100 cm -1 43 (Fig. 6a). Upon close inspection of the tetrahedral modes, it is found that the relative intensity of the edge-sharing mode progressively decreases as a function of aging, as depicted in Fig. 6b. This change is

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observed in both glasses. A similar evolution of edge-sharing mode intensity as a function of temperature has been previously observed in the supercooled liquid of GeSe 4 [START_REF] Edwards | Structure and Relaxation in Germanium Selenide Glasses and Supercooled Liquids: A Raman Spectroscopic Study[END_REF] . The edgesharing mode was shown to decrease with decreasing temperature of the liquid. In the present case the edge-sharing mode intensity is found to decrease with decreasing fictive temperature of the glass as it relaxes towards equilibrium upon aging. This indicates that the structural evolution observed by Raman is related to changes in thermal properties (enthalpy) of the glass. Indeed, a strong correlation between the structural and thermal properties can be revealed by plotting the time evolution of the enthalpy and Raman features as shown in Fig. 7 for GeSe 4 . In Fig. 7(a) the ratio of the intensities of the edge-and the corner-sharing tetrahedral modes is normalized and plotted as a function of aging time. The decay of the spectral ratio is found to follow a stretched exponential function (Kohlrausch function). In Fig. 7(b), the relaxed enthalpy calculated from data in Fig. 4(b) is similarly normalized and plotted as function of time over the same period. It is found that the enthalpy decay can be fitted with the same stretched exponential function with a relaxation time τ = 294 days and a stretching exponent β = 0.686. This result confirms that the two relaxation processes are strongly correlated. The same construction can be performed for the GeSe 9 glass as shown in Fig. 8. Again, Raman features and enthalpy both follow a stretched exponential decay that can be fitted with the same function. In the case of GeSe 9 the relaxation time is τ = 118 days and the stretching exponent factor is β = 0.424. As expected, the lower T g glass has a shorter relaxation time at room temperature consistent with results of Fig. 4(a). The stretching exponent values also show that the non-exponential factor β is notably smaller for the fragile glass (GeSe 9 m∼38) than the strong glass (GeSe 4 m∼27). The two glasses are likely still far from equilibrium, hence, these values of β are only estimates, nevertheless they are consistent with expected trend between fragility and non-exponentiality, and the β values obtained in this study are in good agreement with previously reported data [START_REF] Boehmer | Nonexponential relaxations in strong and fragile glass formers[END_REF] . Overall, the results of Fig. 2 & 5 reveal that given enough time, all Ge-Se glasses undergo a sizeable enthalpy relaxation even at room temperature. The relative extent of relaxation of all glasses is consistent with expectations for samples annealed at different temperatures relative to Accepted Manuscript T g . Hence, it can be concluded that glasses from the so-called "reversibility window" do not exhibit any unusual resistance to relaxation, granted sufficient observation time is allowed for the measurement. The features of the DSC curves and their evolution with aging are also well predicted by the TNM model for glasses inside and outside the so-called "reversibility window". The multiple features of the DSC curves are fully consistent with their relative fragility and T g values. Furthermore, the time dependence of the enthalpy relaxation for both glasses exhibit a stretched exponential decay characteristic of conventional amorphous systems during sub-T g relaxation [START_REF] Scherer | Theory of relaxation[END_REF] . The non-exponential factors β describing the decay are also found to be in good agreement with expectations from glasses with their relative fragilities [START_REF] Boehmer | Nonexponential relaxations in strong and fragile glass formers[END_REF] . Hence, all Ge-Se glasses exhibit a rather conventional relaxation behavior and no abnormal relaxation properties are observed for glasses within the "reversibility window", in fact these glasses behave exactly as expected from model predictions.

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B. Structural origin of relaxation in Ge-Se glasses.

As shown in Fig. 3 & 6, aging of Ge-Se glasses is systematically associated with a conversion of edge-sharing tetrahedra to corner-sharing tetrahedra. The magnitude and kinetics of this structural change is closely related to enthalpy relaxation for each composition studied. This behavior can be explained in two ways, based on considerations of density and configurational entropy. In terms of density, it is expected that the reduced structural dimensionality imparted by edge-sharing tetrahedra leads to poorer packing efficiency and in turn to lower volumetric density. For example, the corner-sharing 3D crystalline polymorph of GeSe 2 is 9.6% denser than the corner-and edge-sharing 2D polymorph consisting of layers [START_REF] Grande | Structural Properties of GeSe2 at High Pressures[END_REF] . This trend is also broadly observed in tetrahedral liquids such as GeSe 4 and ZnSe 2 where the fraction of edge-sharing units decreases with lower temperature and higher density [START_REF] Edwards | Structure and Relaxation in Germanium Selenide Glasses and Supercooled Liquids: A Raman Spectroscopic Study[END_REF][START_REF] Zeidler | Structure of the network glass-former ZnCl 2 : From the boiling point to the glass[END_REF][START_REF] Lucas | Structure of ZnCl2 Melt. Part II: Fragileto-Strong Transition in a Tetrahedral Liquid[END_REF] . In the case of Ge-Se glasses, the decrease in edge-sharing units is therefore consistent with the volume contraction associated with lowering of the fictive temperature during aging. In terms of configurational entropy, the relaxation process is associated with a decrease in entropy towards the equilibrium liquid line as illustrated schematically in Fig. 1(b). This entropy loss upon aging can indeed be observed experimentally in the heat capacity curves of Fig. 2 & 4 where the C p overshoot is representative of the regain in lost entropy upon reheating. This decrease in entropy can also be qualitatively linked to the structural changes observed by Raman using the constraint counting formalism [START_REF] Gupta | Composition dependence of glass transition temperature and fragility. I. A topological model incorporating temperature-dependent constraints[END_REF][START_REF] Naumis | Energy landscape and rigidity[END_REF][START_REF] Mauro | Viscosity of glass-forming liquids[END_REF] .

As previously described [START_REF] Lucas | Structure of ZnCl2 Melt. Part II: Fragileto-Strong Transition in a Tetrahedral Liquid[END_REF] , a pair of edge-sharing tetrahedra possess two fewer angular constraints than a pair of corner-sharing tetrahedra. Hence, a decrease in edge-sharing tetrahedra will lead to a decrease in network degrees of freedom f and in turn to a decrease in configurational entropy S c according to:

𝑆𝑆 𝐶𝐶 = 𝑓𝑓𝑓𝑓𝑓𝑓𝑙𝑙𝑙𝑙𝛺𝛺 ( 3
)
where Ω is the number of accessible microstates per atomic degree of freedom (independent of f) [START_REF] Gupta | Composition dependence of glass transition temperature and fragility. I. A topological model incorporating temperature-dependent constraints[END_REF][START_REF] Naumis | Energy landscape and rigidity[END_REF] . The structural mechanism for entropy relaxation therefore appears to be related to the backbone of tetrahedral units in the glassy structure. Interestingly, that mechanism is identical in

Accepted Manuscript

both glasses, as shown in Figs. 7 &8. This further confirms that glasses from the "reversibility window" do not exhibit any abnormal relaxation behavior relative to other compositions from the same system.

C. Non-reversing enthalpy and MDSC

The results described above conclusively indicate that glasses within the "reversibility window" can undergo significant enthalpy relaxation. This raises questions regarding the validity of the non-reversing enthalpy ΔH nr as a means of assessing the relaxation behavior of glasses. Indeed it has been broadly suggested that vanishing values of ΔH nr are indicative of a resistance to aging [START_REF] Bhosle | Meeting experimental challenges to physics of network glasses: Assessing the role of sample homogeneity[END_REF][START_REF] Chakravarty | Ageing, fragility and the reversibility window in bulk alloy glasses[END_REF][START_REF] Boolchand | Self-organization and the physics of glassy networks[END_REF][START_REF] Vempati | The thermally reversing window in ternary GexPxS1-2x glasses[END_REF] . However, the present results clearly indicate that this is not the case. The physical interpretation of ΔH nr therefore remains unclear and in fact it has been highly controversial since the early development of MDSC. In particular, Schawe [START_REF] Schawe | A comparison of different evaluation methods in modulated temperature DSC[END_REF] emphasizes that ΔH nr is the difference between two quantities that are measured with two different time constants: the underlying heat flow associated with the time constant of the average heating rate and the reversible heat flow associated with the time constant of the superimposed oscillation. Schawe then concludes that "the non-reversing component is determined by subtracting two logically different quantities that are not directly comparable" [START_REF] Schawe | A comparison of different evaluation methods in modulated temperature DSC[END_REF] . Nevertheless, ΔH nr has been tentatively equated to the enthalpy relaxation upon annealing [START_REF] Boller | Modulated differential scanning calorimetry in the glass transition region[END_REF] , but Hutchinson et al. [START_REF] Hutchinson | Aging of polycarbonate studied by temperature modulated differential scanning calorimetry[END_REF] showed experimentally that this equality does not actually hold. Instead, MDSC signals can be effectively interpreted in terms of complex, in-phase and out-of-phase heat capacities with a real part C' (storage heat capacity) and an imaginary part C" (loss heat capacity) [START_REF] Gulbiten | Relaxation of enthalpy fluctuations during sub-Tg annealing of glassy selenium[END_REF][START_REF] Schawe | Principles for the interpretation of modulated temperature DSC measurements. Part 1. Glass transition[END_REF][START_REF] Bustin | Slow structural relaxations of glass-forming Maltitol by modulated DSC calorimetry[END_REF][START_REF] Yang | Fragile-strong behavior in the As x Se 1-x glass forming system in relation to structural dimensionality[END_REF] . It should be noted that the use of the complex heat capacity is contingent upon a linear response between the heating rate and heat flow which should be ascertained through construction of a Lissajous plot [START_REF] Gulbiten | Relaxation of enthalpy fluctuations during sub-Tg annealing of glassy selenium[END_REF][START_REF] Bechgaard | Temperaturemodulated differential scanning calorimetry analysis of high-temperature silicate glasses[END_REF] . When correctly implemented, this approach permits to extract the fragility by measuring the shift of the loss peak with oscillating frequency [START_REF] Yang | Fragile-strong behavior in the As x Se 1-x glass forming system in relation to structural dimensionality[END_REF] and can also provide estimates of the stretching exponent β 64 . These two parameters provide a more complete and reliable assessment of the relaxation properties of a glass-forming system than ΔH nr .

VI. CONCLUSIONS

In this study, Ge-Se glasses lying outside and inside the "reversibility window" were annealed at room temperature for up to twenty years. The purpose of this experiment was to test whether glasses from the "reversibility window" exhibit any resistance to physical aging as previously claimed. A sizable enthalpy relaxation was observed for all glasses including those within the socalled "reversibility window". Furthermore, the relaxation process obeys a stretched exponential decay characteristic of amorphous systems. The heat capacity curves of each glass followed the behavior expected from the TNM model for systems annealed far from T g . The values of stretching exponent β and relaxation time τ were also consistent with expectations. These results demonstrate that glasses from the "reversibility window" do not show any particular resistance to relaxation or aging. In addition, Raman spectra of both glasses reveal that structural relaxation is associated with a conversion of edge-sharing to corner-sharing tetrahedra. This structural

Accepted Manuscript

evolution is consistent with an increase in density and a decrease in configurational entropy. The structural mechanism is identical in both glasses and further confirms that glasses from the "reversibility window" do not show any unusual relaxation behavior relative to other compositions in the same system. Finally, these results invalidate the interpretation of a vanishing value of ΔH nr as indicative of a resistance to aging. This is an important consideration in view of the increasing popularity of MDSC and its extension to the characterization of high temperature oxide glasses [START_REF] Bechgaard | Temperaturemodulated differential scanning calorimetry analysis of high-temperature silicate glasses[END_REF] .

Fig. 1 :

 1 Fig. 1: (a) Scaled entropy (inverse excess entropy normalized by its value at T g ) for three hypothetical glass-formers with fragility ranging from m = 16 to 200. Thick dashed lines indicate the idealized glass state where configurational entropy is frozen in. (b) Variation of the equilibrium entropy with temperature relative to the glassy state for the same three glass-formers.

Fig. 4 :

 4 Fig. 4: Experimental heat capacity curves for (a) GeSe 9 glass fibers annealed at room temperature for up to 3.5 years and (b) GeSe 4 glass fibers annealed at room temperature for up to 5 years.

Fig. 5 :

 5 Fig. 5: Calculated heat capacity curves as a function of annealing at room temperature following the TNM modeling procedure developed by Hodge 44 for (a) GeSe 9 glass using model parameters β = 0.54; x = 0.51; m = 38 and T g = 365 K; (b) GeSe 4 glass using model parameters β = 0.60; x = 0.57; m = 27 and T g = 435 K.

Fig. 6 :

 6 Fig. 6: (a) Typical unpolarized Raman spectrum of GeSe 4 glass showing the two modes associated with corner-sharing and edge-sharing tetrahedra at 195 cm -1 and 213 cm -1 respectively; (b) Increase of the relative intensity of the edge-sharing mode upon aging at room temperature for 0, 2 and 5 years (all spectra are normalized to the intensity of the corner-sharing mode).

Fig. 7 :

 7 Fig. 7: (a) Normalized ratio of the intensities of the edge-and the corner-sharing tetrahedral Raman modes of GeSe 4 glass as a function of aging time at room temperature; (b) Normalized enthalpy relaxation of GeSe 4 glass as a function of aging time at room temperature. The line in both graphs correspond to the same stretched exponential function with τ = 294 days and β = 0.686

Fig. 8 :

 8 Fig. 8: (a) Normalized ratio of the intensities of the edge-and the corner-sharing tetrahedral Raman modes of GeSe 9 glass as a function of aging time at room temperature; (b) Normalized enthalpy relaxation of GeSe 9 glass as a function of aging time at room temperature. The line in both graphs correspond to the same stretched exponential function with τ = 118 days and β = 0.424

ACKNOWLEDGEMENT

PL acknowledge financial support from NSF-DMR under grant#: 1832817. We thank Isabelle Melscoët for synthesizing the bulk Ge-Se glasses.

DATA AIVAILABILITY

The data that support the findings of this study are available from the corresponding author upon reasonable request.


Claudia Cosio
email: claudia.cosio@univ-reims.fr

Davide Degli-Esposti

Christine Almunia

Véronique Gaillet

Hervé Sartelet

Jean Armengaud

Arnaud Chaumot

Olivier Geffard

Alain Geffard

Subcellular distribution of dietary methyl-mercury in Gammarus fossarum and its impact on the amphipod proteome

Keywords: bioaccumulation, cell walls, cytosol, proteogenomics, subcellular distribution

The transfer of methyl-Hg (MeHg) from food is central for its effects in aquatic animals, but we still lack knowledge concerning its impact in invertebrate primary consumers. In aquatic environments, cell walls of plants are particularly recalcitrant to degradation and as such remain available as food source for long periods. Here, the impact at the proteomic level of dietary MeHg in Gammarus fossarum was established and linked to subcellular distribution of Hg. Individuals of G. fossarum were fed with MeHg in cell wall or intracellular compartments of Elodea nuttallii. Hg concentrations in subcellular fractions were 2 to 6x higher in animals fed with cell wall than intracellular compartments. At the higher concentrations tested, the proportion of Hg in metal sensitive fraction increased from 30.0±6.1% to 41.0±5.7% for individuals fed with intracellular compartment, while biologically detoxified metals fraction increased from 30.0±6.1% to 50.0±2.8% when fed with cell wall compartment. Data suggested that several thresholds of proteomic response are triggered by increased bioaccumulation in each subcellular fraction in correlation with Hg exclusively bound to the metal sensitive fraction, while the increase of biologically detoxified metal likely had a cost for fitness. Proteomics analysis supported that the different binding sites and speciation in shoots subsequently resulted in different fate and cellular toxicity pathways to consumers.

Our data confirmed that Hg bound in cell walls of plants can be assimilated by G. fossarum, which is consistent with its feeding strategy, hence pointing cell walls as a significant source for Hg transfers and toxicity in primary consumers. The high accumulation of Hg in macrophytes make them a risk for food web transfer in shallow ecosystems. The present results allowed gaining new insights on the effects and uptake mechanisms of MeHg in aquatic primary consumers.

Introduction

Mercury (Hg) is a global pollutant seriously threatening ecosystems and eventually health, due to the biomagnification of methyl-Hg (MeHg) through the food chain [START_REF] Boening | Ecological effects, transport, and fate of mercury: a general review[END_REF][START_REF] Bravo | Extremely elevated methyl mercury levels in water, sediment and organisms in a Romanian reservoir affected by release of mercury from a chloralkali plant[END_REF][START_REF] Lawrence | Factors controlling the bioaccumulation of mercury and methylmercury by the estuarine amphipod Leptocheirus plumulosus[END_REF] . Past Hg deposits of anthropogenic sources continue to cycle through compartments, forming up to 60% of current Hg emissions [START_REF]Global Mercury Assessment 2018: Sources, Emissions, Releases and Environmental Transport[END_REF] . Consequently, even if new emissions of Hg are kept constant, Hg deposition will increase in the future [START_REF] Amos | Legacy impacts of alltime anthropogenic emissions on the global mercury cycle[END_REF][START_REF] Obrist | A review of global environmental mercury processes in response to human and natural perturbations: Changes of emissions, climate, and land use[END_REF][START_REF] Branfireun | Mercury cycling in freshwater systems -An updated conceptual model[END_REF] . In this context, understanding how Hg is bioaccumulated and biomagnified in food webs is central to increase the effectiveness of environmental management programs for biota and human health. For example, aquatic plants are key species in shallow aquatic environment in which MeHg is formed [START_REF] Regier | Hg bioaccumulation in the macrophyte Elodea nuttallii in the field and in microcosm: Hg in shoots accumulated from the water might involve Cu transporters[END_REF] . The high accumulation of Hg in aquatic plants make them considered a model species for phytoremediation programs, biomonitoring or to perform ecotoxicological tests [START_REF] Regier | Analysis of the Elodea nuttallii transcriptome in response to mercury and cadmium pollution: development of sensitive tools for rapid ecotoxicological testing[END_REF] , but also at risk for food web transfer [START_REF] Beauvais-Fluck | Role of cellular compartmentalization in the trophic transfer of mercury species in a freshwater plant-crustacean food chain[END_REF][START_REF] Beauvais-Fluck | Trophic fate of inorganic and methyl-mercury in a macrophyte-chironomid food chain[END_REF] . A current research priority is to establish the sources of dietary Hg having a detrimental impact in aquatic plant-based food webs for mitigation programs.

Both inorganic Hg (IHg) and MeHg are bioaccumulated in biota. While MeHg generally represents 0.05 to 1% and up to 30% of total Hg (THg = IHg + MeHg) in water, MeHg forms up to 80-100% of the THg in fish muscles, resulting in very high toxic body burdens in fish, therefore threatening humans' health through fish consumption [START_REF] Bravo | Extremely elevated methyl mercury levels in water, sediment and organisms in a Romanian reservoir affected by release of mercury from a chloralkali plant[END_REF][START_REF] Lavoie | Mercury transport and human exposure from global marine fisheries[END_REF][START_REF] Wu | The importance of bioconcentration into the pelagic food web base for methylmercury biomagnification: A meta-analysis[END_REF] . Consequently, although both IHg and MeHg trigger toxicity in biota, in animals dietary MeHg is the main risk of Hg [START_REF] Bravo | Extremely elevated methyl mercury levels in water, sediment and organisms in a Romanian reservoir affected by release of mercury from a chloralkali plant[END_REF][START_REF] Lavoie | Mercury transport and human exposure from global marine fisheries[END_REF] . Nonetheless, a major gap in knowledge concerns the impact of dietary Hg uptake in invertebrate primary consumers. Data supported the oxidative stress to be central for MeHg toxicity in aquatic animals [START_REF] Graves | Response of oxidative stress transcripts in the brain of wild yellow perch (Perca flavescens) exposed to an environmental gradient of methylmercury[END_REF][START_REF] Lee | Adverse effects of methylmercury (MeHg) on life parameters, antioxidant systems, and MAPK signaling pathways in the copepod Tigriopus japonicus[END_REF] , but few studies concern effects through diet at environmental concentrations and none concern invertebrates [START_REF] Gentes | Specific effects of dietary methylmercury and inorganic mercury in zebrafish (Danio rerio) determined by genetic, histological, and metallothionein responses[END_REF] . In this context, the impact of MeHg in realistic environmental exposure scenarios and representative biota need to be studied to better anticipate its potential environmental impact. In aquatic invertebrates, toxicity and tolerance of metal accumulation can be explained by its subcellular partitioning [START_REF] Wallace | Subcellular compartmentalization of Cd and Zn in two bivalves. I. Significance of metal-sensitive fractions (MSF) and biologically detoxified metal (BDM)[END_REF] . For example, in biota the sequestration of toxic metal by metallothioneins and granules is known as biologically detoxified metals (BDM), while the partition in the metal sensitive fraction (MSF) is expected to cause the toxicity [START_REF] Wallace | Subcellular compartmentalization of Cd and Zn in two bivalves. I. Significance of metal-sensitive fractions (MSF) and biologically detoxified metal (BDM)[END_REF][START_REF] Monteiro | Subcellular metal distributions and metallothionein associations in roughtoothed dolphins (Steno bredanensis) from Southeastern Brazil[END_REF] . Moreover, linking intracellular concentration and distribution to molecular and cellular responses is a current research priority in biomonitoring, as it could participate in revealing innovative biomarkers for environmental risk assessment [START_REF] Regier | Analysis of the Elodea nuttallii transcriptome in response to mercury and cadmium pollution: development of sensitive tools for rapid ecotoxicological testing[END_REF][START_REF] Gouveia | Ecotoxico-proteomics for aquatic environmental monitoring: first in situ application of a new proteomics-based multibiomarker assay using caged amphipods[END_REF] .

Pioneer studies on diatoms suggested that intracellular MeHg was primarily transferred to copepods, while Hg from cell walls, consisting in silicate frustules, was not assimilated [START_REF] Mason | Uptake, toxicity, and trophic transfer of mercury in a coastal diatom[END_REF] . By contrast, we recently demonstrated that IHg and MeHg from cell walls -formed primarily of cellulose, hemicellulose and pectin-in shoots of the macrophyte Elodea nuttallii resulted in a higher transfer to gammarids than intracellular Hg [START_REF] Beauvais-Fluck | Role of cellular compartmentalization in the trophic transfer of mercury species in a freshwater plant-crustacean food chain[END_REF] . Similarly, chironomids fed with shopped macrophytes, to mimic incorporation in sediments of decaying tissues, revealed that MeHg was more transferable to sensitive subcellular targets and bioavailable fraction in primary consumers than IHg found in intracellular or cell wall compartments [START_REF] Beauvais-Fluck | Trophic fate of inorganic and methyl-mercury in a macrophyte-chironomid food chain[END_REF] . Data point that the composition of cell walls could affect the transfer rate of Hg to consumers [START_REF] Beauvais-Fluck | Role of cellular compartmentalization in the trophic transfer of mercury species in a freshwater plant-crustacean food chain[END_REF][START_REF] Beauvais-Fluck | Trophic fate of inorganic and methyl-mercury in a macrophyte-chironomid food chain[END_REF][START_REF] Lee | Bioaccumulation of methylmercury in a marine copepod[END_REF] .

The proportion of diatoms in phytoplankton is particularly high in nutrient-rich marine coastal and high latitudes ecosystems, but significantly less in freshwater rivers, lakes and other environments [START_REF] Malviya | Insights into global diatom distribution and diversity in the world's ocean[END_REF][START_REF] Tadonleke | Phytoplankton productivity increased in Lake Geneva despite phosphorus loading reduction[END_REF][START_REF] Descy | Phytoplankton of the River Loire, France: a biodiversity and modelling study[END_REF] . In this context, data concerning Hg food transfer in marine plankton might not be generalized to other animals with distinct feeding strategies or other ecosystems, including freshwater, brackish water or contaminated sites. Moreover, studies on effect of trophic Hg exposure in primary consumers, i.e. invertebrates are rare [START_REF] Beauvais-Fluck | Role of cellular compartmentalization in the trophic transfer of mercury species in a freshwater plant-crustacean food chain[END_REF][START_REF] Beauvais-Fluck | Trophic fate of inorganic and methyl-mercury in a macrophyte-chironomid food chain[END_REF][START_REF] Lee | Bioaccumulation of methylmercury in a marine copepod[END_REF] . At environmental concentrations, cell walls appeared as the main bioaccumulation compartment of Hg in macrophytes and because of their low degradability are expected to remain for long periods in the aquatic systems, likely serving as food source [START_REF] Castro | Accumulation, distribution and cellular partitioning of mercury in several halophytes of a contaminated salt marsh[END_REF][START_REF] Larras | Physiological and proteomic changes suggest an important role of cell walls in the high tolerance to metals of Elodea nuttallii[END_REF][START_REF] Valega | Mercury intracellular partitioning and chelation in a salt marsh plant, Halimione portulacoides (L.) Aellen: strategies underlying tolerance in environmental exposure[END_REF] . Here, we hypothesized that the biding of Hg to plant's cell walls results in a higher dietary transfer and subsequently in a higher toxicity in primary consumers than Hg bound in plant's intracellular compartment.

Gammarids as generalist herbivores are instrumental in European lotic ecosystems food webs because of their role in the breakdown of the leaf litter. They are also good bioaccumulators of metals and good candidates for ecotoxicological testing of freshwater ecosystems [START_REF] Gouveia | Ecotoxico-proteomics for aquatic environmental monitoring: first in situ application of a new proteomics-based multibiomarker assay using caged amphipods[END_REF][START_REF] Chaumot | Chapter 11 -Gammarids as reference species for freshwater monitoring[END_REF] .

Gammarus spp are found in E. nuttallii beds and were previously reported to feed on the latter 29, 30 . Here we aimed to address how the MeHg localization in E. nuttallii shoots (cell wall vs intracellular) defines its subcellular distribution in G. fossarum; and how dietary MeHg impacts the proteome of G. fossarum. Gammarids were fed for 7 days with either cell wall or intracellular compartment of the plant E. nuttallii previously exposed to MeHg and we analyzed the fate and effects of MeHg in animals through subcellular distribution and shotgun proteomics. Extensive proteome profiles further revealed the molecular toxicity pathway of dietary MeHg. This fundamental knowledge contributes to a better knowledge of the fate and impact of dietary MeHg in food webs.

Material and Methods

Labware cleaning

Glasswares and plastic consumables for exposures and sampling were washed for once in detergent, twice in 10% HNO 3 , once in 10% HCl, rinsed after each washing step with MilliQ water (Merck Millipore) and kept in a laminar flow hood until dry. Glass material was in addition heated overnight at 200ºC.

Elodea nuttallii and Gammarus fossarum exposure

Exposure protocols of E. nuttallii and G. fossarum were described in [START_REF] Beauvais-Fluck | Role of cellular compartmentalization in the trophic transfer of mercury species in a freshwater plant-crustacean food chain[END_REF] . Briefly, shoots were collected in Etournels pounds and acclimated 48h in laboratory conditions. Shoots (10 cm) without roots were exposed to MeHg (CH 3 HgCl; Alfa Aesar) or not exposed (control). The exposure concentrations in water were chosen based on previous studies to result in concentrations in shoots after 2h-long exposure spanning lowly to highly contaminated environments (Table S1) 2, 31, . After the exposure, shoots were washed to conserve the shoot morphology and retain Hg either from intracellular or cell wall compartment. Briefly, intracellular Hg was conserved in shoots by rinsing with 10 -3 M EDTA (AppliChem) and 10 -3 M cysteine (Sigma-Aldrich) to remove the extracellular loosely bound Hg [START_REF] Regier | Hg bioaccumulation in the macrophyte Elodea nuttallii in the field and in microcosm: Hg in shoots accumulated from the water might involve Cu transporters[END_REF] . Hg bound in the cell wall were obtained by soaking shoots in methanol-chloroform 3d (Sigma), resulting in lipids solubilization and consequently allowing to recover an intact insoluble cell wall matrix with a conserved structure [START_REF] Hart | Characterization of the transport and cellular compartmentation of paraquat in roots of intact maize seedlings[END_REF] . This latter method affects cell integrity and organic solvents could cause IHg and MeHg redistribution. As such, concentrations might not be strictly representative of initial concentrations in shoots but was performed to allow feeding animals with purified cell walls.

Gammarus fossarum were sampled in the Bourbre River (France) [START_REF] Coulaud | In situ feeding assay with Gammarus fossarum (Crustacea): Modelling the influence of confounding factors to improve water quality biomonitoring[END_REF][START_REF] Besse | Caged Gammarus fossarum (Crustacea) as a robust tool for the characterization of bioavailable contamination levels in continental waters: towards the determination of threshold values[END_REF] , and acclimatized three weeks to laboratory conditions. Nine 1 cm-long males were fed during 7 days with E. nuttallii intracellular (I) or the cell wall (CW) compartments pre-exposed to 4 and 20 µg L -1 MeHg concentrations, thereafter referred as 'low' and 'high' MeHg treatments in line with their concentrations corresponding to lowly and highly contaminated environments, respectively [START_REF] Bravo | Extremely elevated methyl mercury levels in water, sediment and organisms in a Romanian reservoir affected by release of mercury from a chloralkali plant[END_REF][START_REF] Beauvais-Fluck | Role of cellular compartmentalization in the trophic transfer of mercury species in a freshwater plant-crustacean food chain[END_REF][START_REF] Beauvais-Fluck | Trophic fate of inorganic and methyl-mercury in a macrophyte-chironomid food chain[END_REF][START_REF] Cosio | Effects of macrophytes on the fate of mercury in aquatic systems[END_REF] . To ensure exclusive exposure of gammarids through plant consumption, water renewal and daily removal of dead individuals was done to avoid exposure to Hg through water or cannibalism, respectively. We observed a weak (15%) mortality of gammarids during exposure in all conditions [START_REF] Beauvais-Fluck | Role of cellular compartmentalization in the trophic transfer of mercury species in a freshwater plant-crustacean food chain[END_REF] . This rate agrees with the criteria from the Afnor standard (XP T 90-722-3) for feeding rate measurement in the sentinel species G. fossarum [START_REF]Qualité de l'eau -Mesures moléculaires, physiologiques et comportementales chez le gammare (crustacé amphipode) -Partie 3 : mesure du taux d'alimentation[END_REF] . It was not correlated with MeHg concentration, or with the plant compartment (intracellular/cell wall) and supported an absence or a sublethal toxicity in these experimental conditions [START_REF] Beauvais-Fluck | Role of cellular compartmentalization in the trophic transfer of mercury species in a freshwater plant-crustacean food chain[END_REF] . No depuration was applied here, based on a former study, using the same experimental exposure conditions showed that a 7 day-long depuration didn't varied Hg concentrations [START_REF] Beauvais-Fluck | Role of cellular compartmentalization in the trophic transfer of mercury species in a freshwater plant-crustacean food chain[END_REF] .

Subcellular fractionation of Hg in Gammarus fossarum

The fractionation procedure of the whole body (Figure 1) adapted from [START_REF] Wallace | Subcellular compartmentalization of Cd and Zn in two bivalves. I. Significance of metal-sensitive fractions (MSF) and biologically detoxified metal (BDM)[END_REF][START_REF] Geffard | Subcellular compartmentalization of cadmium, nickel, and lead in Gammarus fossarum: Comparison of methods[END_REF] was described elsewhere [START_REF] Gimbert | Mercury tissue residue approach in Chironomus riparius: Involvement of toxicokinetics and comparison of subcellular fractionation methods[END_REF] . Analyses were done in triplicates. Two frozen gammarids per conditions were homogenized in 1 mL of ice-cold buffer (0.02M Tris-HCl pH 7.5; Sigma-Aldrich, purity 99%). The homogenate (H) was centrifuged 30 min at 4°C at 10,000g. The pellet (P1) was hydrolyzed at 100°C 2 min and 1h with 1M NaOH 65°C and centrifuged 10 min at 10°C at 10,000g, resulting in a supernatant including gut content, cellular debris and likely part of mitochondria (S2), while the pellet contained metal-rich granules and exoskeleton (P2). The supernatant (S1) was ultracentrifuged 30 min at 4°C at 100,000 g forming a pellet (P3) including lysosomes and microsomes and a supernatant (S3) with the cytosol. However, no marker enzymes (i.e. [START_REF] Rosabal | Assessment of a subcellular metal partitioning protocol for aquatic invertebrates: preservation, homogenization, and subcellular fractionation[END_REF][START_REF] Cardon | Enzymatic validation of species-specific protocols for metal subcellular fractionation in freshwater animals[END_REF] ) was used to confirm further the nature of each subcellular fraction besides operational fractions according to [START_REF] Wallace | Subcellular compartmentalization of Cd and Zn in two bivalves. I. Significance of metal-sensitive fractions (MSF) and biologically detoxified metal (BDM)[END_REF] . All S3 was further filtered with a sterivex filter (millipore) at 0.45 µm, and thereafter named S4.

High-performance liquid chromatography (HPLC) was performed to fractionate S4 with a column using steric exclusion allowing (BIOSEP-SEC-S 2000; 1 -300 kDa; Figure S1).

Eluting fractions of 0.5 mL were collected up 18 mL, acidified at 0.5% with suprapur HCl (Merx) and kept at 4°C until analyzed. Three pools of metal-ligand were separated: (i) highmolecular weight (HMW; 20-255 kDa) eluting 9 to 15 min after injection, (ii) metallothionein-like protein eluting 16 to 24 min after injection (MTLPs-MW; 1.8-20 -kDa) and (iii) low-molecular weight (LMW; <1.8 kDa) eluting 25 to 35 min after injection. Using this protocol, MTLPs-MW are biologically detoxified metal in cytosol, while the heat sensitive protein (HSeP) or proteins in cytosol is the sum of HMW and LMW [START_REF] Geffard | Subcellular compartmentalization of cadmium, nickel, and lead in Gammarus fossarum: Comparison of methods[END_REF] . We also summed the area of peaks at 280 nm in these three fractions and normalized them by the biomass used in H. We eventually compared the sum of Hg amounts found in the different subcellular fractions with the fraction H, using H= P2 +S2 + P3 + HMW +MMW + LMW as a quality control, and observed a good agreement (n = 18: 93.4 ± 4.9%). We also injected periodically 1 mM EDTA using the same mobile phase to wash the HPLC column and to verify that Hg concentrations in eluting fractions remained at background level. For the biological interpretation, metal sensitive fraction (MSF) was calculated as P3 + HSeP and biologically detoxified metals (BDM) as P2 + MTLPs-MW. As such, MSF + BDM + S2 = H.

Determination of Hg in gammarids

Pellets, i.e. P2 and P3 fractions, were resuspended in 1 mL water and mineralized 12h at 60°C 30% HNO 3 (suprapur, Merck). THg concentration in P2, S2, P3, S4, MTPLs and HSeP were measured with a MERX-T analytical system (Brooks Rand instrumentsA) using THg method [START_REF] Usepa | Method 1631: Mercury in water by oxidation, purge and trap, and CVAFS[END_REF] showing a limit of detection (DL) of 0.03 ng•THg•L -1 , an error of 2% using certified reference materials (CRMs) ORMS-5 (95.0 ± 1.7 % recovery) and TORT-2 (100 ± 0.1% recovery). MeHg was analyzed in P2, S2, P3, S4, MTPLs and HSeP samples with a MERX-M (Brooks Rand instruments) using MeHg method 41,42 showing a DL of 0.002 ng•MeHg•L -1 , an error of 15% using the CRM TORT-2 (102.5 ± 2.4 % recovery).

Data are presented as arithmetic means ± standard deviations (SD). Normality and homoscedasticity were tested. An analysis of variance (ANOVA, p < 0.05) with a post-hoc Tukey's HSD test (p < 0.05) was used to perform pairwise comparisons of bioaccumulation in the software R version 3.1.3 [START_REF]R: a language and environment for statistical computing[END_REF] .

Protein extraction and Tandem mass spectrometry

Proteins were extracted (n= 6) from individuals of G. fossarum and identified following the same protocol as formerly described [START_REF] Cogne | Comparative proteomics in the wild: Accounting for intrapopulation variability improves describing proteome response in a Gammarus pulex field population exposed to cadmium[END_REF] . Briefly, each animal was subjected to a bead beating homogenization using a single 3.2 mm steel bead per tube in LDS buffer (20 μl•mg -1 ; Invitrogen). Cellular debris were eliminated after centrifugation for 3 min at 10,000 g. Proteins from the supernatant were denatured for 5 min at 99 °C. An aliquot of 20 μL was then loaded onto a SDS-PAGE gel for a brief (5 min) electrophoresis as recommended [START_REF] Hartmann | Taking the shortcut for high-throughput shotgun proteomic analysis of bacteria[END_REF] .

Each proteome sample was processed and proteolyzed with trypsin (Roche) in ProteaseMAX surfactant (0.01% , Promega) as recommended [START_REF] Gouveia | Shotgun proteomics datasets acquired on Gammarus pulex animals sampled from the wild[END_REF] . Peptides were analyzed with a Q-Exactive HF mass spectrometer (Thermo) operated in data-dependent mode with the same parameters as previously described [START_REF] Gouveia | Quick microbial molecular phenotyping by differential shotgun proteomics[END_REF] . Peptides were resolved at a flow rate of 0.2 μL•min -1 along a 90 min gradient of CH 3 CN in presence of 0.1% formic acid.

Peptide identification and proteomic data analysis

MS/MS spectra were interpreted against the GFOSS database, that is available through PRIDE (accession PXD024583 as GFOSS_db01.fasta) with the MASCOT software (Matrix Science) and the same parameters as those previously described [START_REF] Trapp | Proteogenomics of Gammarus fossarum to document the reproductive system of amphipods[END_REF] . Proteins were validated on the basis of at least two identified peptides, resulting in a false discovery rate (FDR) lower than 1% as estimated with the decoy database search option. Abundance of each peptide was measured by their Spectral Count (SC), which corresponds to the count of MS/MS spectra attributed to the same peptide. The protein abundance is the sum of the SC of all the peptides assigned to the protein. Protein abundances were compared between organisms fed with control plants or plants pre-exposed to MeHg. Abundance of Methionine and Cysteine residue content in proteins from each sample was calculated by extracting spectral counts of these residues as previously proposed [START_REF] Gallais | Heterogeneity of SARS-CoV-2 virus produced in cell culture revealed by shotgun proteomics and supported by genome sequencing[END_REF] , i.e. multiplying the spectral counts of each protein by its number of S-containing residues. The weighted relative abundance in S-containing residues was then obtained by dividing the sum of S-spectral counts by the sum of spectral counts of all residues.

Proteomics data have been deposited to the ProteomeXchange Consortium via PRIDE [START_REF] Perez-Riverol | The PRIDE database and related tools and resources in 2019: Improving support for quantification data[END_REF] with the dataset identifier PXD024583 and project DOI 10.6019/ PXD024583

Gene Ontology (GO term) functional annotation, classification and analysis

Identified proteins were annotated in April 2018 using Swissprot (https://www.uniprot.org/) and NCBInr (https://www.ncbi.nlm.nih.gov/protein/) as databases explored with Diamond, a free high-throughput sequence aligner [START_REF] Buchfink | Fast and sensitive protein alignment using DIAMOND[END_REF] . The NCBInr database was used to find the most probable protein homolog of the closest species to Gammarus fossarum for each protein with an e-value below 10 -10 . The Swissprot database was used to find the top 5 closest proteins in terms of sequence identity to define for each identified protein their biological processes and molecular functions as previously described [START_REF] Trapp | Digging deeper into the pyriproxyfen-response of the amphipod Gammarus fossarum with a next-generation ultra-high-field orbitrap analyser: new perspectives for environmental toxicoproteomics[END_REF] , selecting a bit score alignment above 20 and an e-value below 10 -4 .

Differential proteomics

The quantities of each protein in animals exposed to treated intracellular or cell wall compartments vs non-treated intracellular or cell wall compartments respectively were compared using Patternlab version 4.0 [START_REF] Carvalho | Integrated analysis of shotgun proteomic data with PatternLab for proteomics 4.0[END_REF] . The L-stringency was set at 0.6. At least four measurements out of the five replicates per condition were taken into account. The selected data were normalized with the total signal. Proteins were distinguished into 4 groups: the first one corresponds to proteins that satisfied both the F-stringency (<0.30) and statistical criterion (FDR < 0.05); the second one was filtered out by their L-stringency because of the low protein abundance; the third one groups proteins that meet only the fold criterion, and the fourth one comprises proteins that did not satisfy both criteria. Proteins from the first group were selected for the Gene Ontology (GO) slim differential analysis. The average percentage of S-containing residues in proteins significantly modulated, either over-detected or lessdetected, for each comparative test was calculated without specific weight.

GO slim differential analysis

GO slim analysis was performed taking the sum of SC of each GO slim related proteins for each sample. Univariate statistical analysis was performed to define, with the Ttest pValue, the reliability between conditions taking into account the six replicates. The ratio R for each GO slim was calculated for comparing treated conditions and non-treated conditions, either for the intracellular or the cell wall compartment, as the sum of SC of all replicates in the treated samples divided by the sum of SC of all replicates in the control samples. The ratio was compared using "prcomp" from the basic stats package of R to perform a Principal Component Analysis (PCA). From the PCA, significant GOSlim contributing for more than 50 % to distinguish each condition (contrib > 0.5) were extracted.

Results and discussion

Accumulation and subcellular distribution of Hg in G. fossarum

THg concentrations in subcellular fractions were 2 to 6x higher in animals fed with cell wall compartments than intracellular compartment of MeHg pre-exposed plants, and these differences were significant (p<0.05; Figure 2). For the high and low MeHg treatment respectively, THg in MSF was 2.9x and 2.0x higher, while BDM 5.8x and 2.3x higher for individuals fed with cell wall than intracellular compartments. To gain insights into transfer dynamics of MeHg in G. fossarum, we analyzed by differential centrifugation the subcellular distribution of THg and MeHg (see below) in 3 fractions (Figure 2). In controls and low MeHg treatments, 42.5±6.5% of THg was found in S2 and an equal concentration in MSF and BDM fractions (30.0±6.1%). For gammarids fed with the high MeHg intracellular compartment, the MSF increased (41.0±5.7%), while for the high MeHg cell wall treatment the proportion of THg significantly increased in the BDM (50.0±2.8%; p<0.05). We analyzed the spectrograms obtained by HPLC (Figure S1). For example, 5 peaks appeared visually increased and 4 decreased in high MeHg vs ctl cell wall compartments (Figure S1). Because individual peaks are poorly defined by HPLC method used here because bias are caused by the overlapping of peaks, we compared the area of peaks measured at 280 nm in HPLC eluting fractions normalized by the biomass of animals: the area of MTLPs-MW was increased significantly (p>0.05) by 1.7±0.1 vs controls in both cell wall treatments and low intracellular treatments. Despite visual differences in peaks (Figure S1), no significant differences were found for the sum of areas in HMW and LMW between treatments and control. Sequestration of toxic metal by MTs and granules is a very common strategy in biota to control intracellular metal speciation and thus toxicity [START_REF] Wallace | Subcellular compartmentalization of Cd and Zn in two bivalves. I. Significance of metal-sensitive fractions (MSF) and biologically detoxified metal (BDM)[END_REF][START_REF] Monteiro | Subcellular metal distributions and metallothionein associations in roughtoothed dolphins (Steno bredanensis) from Southeastern Brazil[END_REF] . MeHg in bivalves and several fish preys was hypothesized to be bound in the intracellular fraction to heat-stable-protein [START_REF] Znidaric | Induction of metallothionein-like proteins by mercury and distribution of mercury and selenium in the cells of hepatopancreas and gill tissues in mussel Mytilus galloprovincialis[END_REF][START_REF] Dang | Subcellular controls of mercury trophic transfer to a marine fish[END_REF] . THg partition between the sensitive (HeSP; 40%) and detoxified (MTLPs-MW; 60%) fractions was previously observed in the cytosol of C. riparius exposed to IHg spiked sediments [START_REF] Gimbert | Mercury tissue residue approach in Chironomus riparius: Involvement of toxicokinetics and comparison of subcellular fractionation methods[END_REF] . In the same line, during a 7 day-long depuration phase, both IHg and MeHg concentrations in G. fossarum feeding on IHg or MeHg pre-exposed E. nuttallii did not significantly vary [START_REF] Beauvais-Fluck | Role of cellular compartmentalization in the trophic transfer of mercury species in a freshwater plant-crustacean food chain[END_REF] , supporting that in gammarids the accumulated MeHg was bound strongly to biological ligands, i.e. MTs and granules [START_REF] Park | The phytochelatin transporters AtABCC1 and AtABCC2 mediate tolerance to cadmium and mercury[END_REF][START_REF] Gupta | Role of glutathione and phytochelatin in Hydrilla verticillata (lf) Royle and Vallisneria spiralis L. under mercury stress[END_REF][START_REF] Tai | Computational studies of the coordination stereochemistry, bonding, and metal selectivity of mercury[END_REF] . Here, this detoxification strategy could allow gammarids to reduce the toxicity of the MeHg entering their cytosol when the concentration increased.

Ratio of MeHg as THg was 111±17 % in S2 and S4, 54.3±3.7% in P2 and 60.0±3.8% in P3.

Consequently, MeHg proportion as THg increased from 68 to 95% similarly in BDM and MSF in control to high MeHg treatments, respectively (n=3; Table S2). In our previous study with similar feeding of gammarids with E. nuttallii 10 , bioaccumulation measured on whole individuals hindered to determine where the Hg demethylation took place, e.g. in plant tissues before feeding (for instance, because of plant material decay or biotransformation by microbes), or during digestion or metabolization in gammarids . Here, data supported the second hypothesis, in line with observations made in C. riparius of an intracellular demethylation in the insoluble fraction [START_REF] Beauvais-Fluck | Trophic fate of inorganic and methyl-mercury in a macrophyte-chironomid food chain[END_REF] . These observations are consistent with a generalized demethylation of MeHg in experimental food chains formed by algae (70%) and zooplankton (13%) [START_REF] Pickhardt | Impacts of zooplankton composition and algal enrichment on the accumulation of mercury in an experimental freshwater food web[END_REF] , but contrast in natural food chains with observed MeHg biomagnification [START_REF] Bravo | Extremely elevated methyl mercury levels in water, sediment and organisms in a Romanian reservoir affected by release of mercury from a chloralkali plant[END_REF][START_REF] Cosio | Effects of macrophytes on the fate of mercury in aquatic systems[END_REF] .

Impact of dietary MeHg on proteome in G. fossarum

Number and pattern of modulated proteins

We analyzed the whole proteome extracted from animals by shotgun proteomics and interpreted the high-resolution tandem mass spectra using a G. fossarum RNA-seq derived database. This proteogenomic approach led to 375,286 peptide-to-spectrum matches corresponding to 12,719 unique peptide sequences, resulting in 3,261 proteins identified with at least 2 peptides (Table S3). Globally, a total of 151 gammarid proteins were significantly modulated for the 4 treatment groups vs the control (Table S3). Among them, 78 were found more abundant, 67 were less abundant and 4 showed a higher abundance in intracellular but were less abundant in cell wall treatments. Four distinct profiles of differentially abundant proteins were identified for each treatment (Figure 3). However, a higher overlap (6-8% of proteins) was observed in the same compartment, while only few proteins were shared between intracellular and cell wall fed gammarids (Figure 3A). Despite inter-individual variability is the main component of proteome variability in the organism analyzed in this study (Supplementary Figure 1) and as previously discussed [START_REF] Cogne | Comparative proteomics in the wild: Accounting for intrapopulation variability improves describing proteome response in a Gammarus pulex field population exposed to cadmium[END_REF] , the differential analysis allowed the identification of rather distinct protein profiles for each treatment (Figure 3B).

The treatment affecting the higher number of proteins was the high MeHg intracellular treatment that modulated the abundance of 71 proteins, in line with the higher % of THg found in MSF. The low MeHg cell wall and intracellular compartments modulated 45 and 44 proteins respectively. The high MeHg cell wall treatment modulated 36 proteins, suggesting together with the higher proportion of THg in BDM, the occurrence of a higher detoxification.

Functional analysis of modulated proteins

The highest measured upregulation (Fold change 55x and 52x) and downregulation (-17x, -28x) were observed for proteins involved in 'cytoskeleton' for both cell wall treatments (Table 1). The Mass of these proteins are predicted between 42-35 kDa and could be involved in the visual increase of peaks in HMW on HPCL spectrograms (Figure S1), although because the area of HPLC eluting fractions is not strictly quantitative this comparison has to be made with caution. However, these proteins are known targets of both MeHg and IHg toxicity across taxa. For example, in copepods exposed to 50 µg•L -1 IHg in water, authors proposed that proteins assigned to this GO term (e.g. myosin) were involved in the cell reorganization to withstand Hg toxicity [START_REF] Xu | Comparative quantitative proteomics unveils putative mechanisms involved into mercury toxicity and tolerance in Tigriopus japonicus under multigenerational exposure scenario[END_REF] . Proteins involved in cytoskeleton (actin, tubulin, keratin) that are essential for vesicle trafficking were significantly modified in fish and mammals exposed to IHg and MeHg supporting cytoskeleton disruption due to metal exposures [START_REF] Keyvanshokooh | Proteome modifications of juvenile beluga (Huso huso) brain as an effect of dietary methylmercury[END_REF][START_REF] Berg | Responses in the brain proteome of Atlantic cod (Gadus morhua) exposed to methylmercury[END_REF][START_REF] Correa | Spinal cord neurodegeneration after inorganic mercury long-term exposure in adult rats: Ultrastructural, proteomic and biochemical damages associated with reduced neuronal density[END_REF][START_REF] Wang | Quantitative proteomic analysis reveals the mode-of-action for chronic mercury hepatotoxicity to marine medaka (Oryzias melastigma)[END_REF][START_REF] Quintá | Management of cytoskeleton architecture by molecular chaperones and immunophilins[END_REF] . In tuna muscle, binding of Hg to Beta-actin encompassed approximately 34% of Hg in the fish muscles [START_REF] Nong | Characterization of the mercury-binding proteins in tuna and salmon sashimi: Implications for health risk of mercury in food[END_REF] . Authors hypothesized that Hg binds to thiols in proteins. Here we observed that S represented 4.27 ± 0.08 % of residues in all samples (Table S4, Figure S3), not pointing to an increase of thiols in proteome of animals fed with Hg treatments.

Several modulated GO terms such as development/growth, energy metabolism, reproduction, locomotion/motility were common in all treatments (Table 2). To compare the impact of treatments on G. fossarum at a higher system level, we identified the most influent GOslim in the PCA as being autophagy (148), mitochondrion organization (18), ribosome biogenesis (164) and sulfur compound metabolic processes (1; Figure 4). Indeed, MeHg cell wall and intracellular treatment decreased and increased the level of those GO terms respectively (Table 3), suggesting a different impact of the cell wall treatment than intracellular treatment on G. fossarum cell metabolism. GOslim identified for gammarids fed with MeHg from the intracellular compartment supported the induction of the cellular machinery likely for replacement of damaged proteins and warranting cell functions, while in animals fed with the cell wall compartment GOslim pointed mitochondrial perturbations and the induction of autophagy, ligase and nuclease activities supporting a perturbation of cell organization triggering repair pathways. In the same line, the contrasted modulation of GO terms related to gene expression (ribosome, RNA, DNA), protein maturation (ligase and transferase activities) and growth/development supports a higher modulation of proteome by intracellular MeHg, but a more detrimental impact to the cell metabolism of the cell wall MeHg. As such, the tolerance mechanisms triggered by the high-MeHg cell wall treatment resulting in a higher proportion of THg in BDM appeared to have a cost on fitness for those animals. Data are congruent with studies on one wild G. fossarum population exposed historically to Cd , evidencing the fitness costs and modification of toxicological sensitivities [START_REF] Vigneron | Evolution of cadmium tolerance and associated costs in a Gammarus fossarum population inhabiting a low-level contaminated stream[END_REF] .

Here, differentially detected GO terms are in line with several known effects of MeHg. As mentioned above, the cytoskeleton participates in the transport of molecules and signaling by vesicular trafficking involved in autophagy [START_REF] Amaya | Autophagy and proteins involved in vesicular trafficking[END_REF][START_REF] Kast | The cytoskeleton-autophagy connection[END_REF] . Autophagy is involved in elimination of aberrant or obsolete cellular structures. Mitochondria are known target of MeHg in animals [START_REF] Correa | Spinal cord neurodegeneration after inorganic mercury long-term exposure in adult rats: Ultrastructural, proteomic and biochemical damages associated with reduced neuronal density[END_REF][START_REF] Ferreira | MeHg causes ultrastructural changes in mitochondria and autophagy in the spinal cord cells of chicken embryo[END_REF] . For example, in shrimps 203 Hg exposure by diet resulted in Hg bioaccumulation in organelles (10%) and HeSP (40%), while in Crassostrea virginica a 4 week-long exposure to 0.056 μM Hg through water resulted in 16x higher concentration in organelles than MTLPs [START_REF] Seebaugh | Assimilation and subcellular partitioning of elements by grass shrimp collected along an impact gradient[END_REF][START_REF] Fitzgerald | Examining the relationship between metal exposure (Cd and Hg), subcellular accumulation, and physiology of juvenile Crassostrea virginica[END_REF] . Besides, MeHg was shown to depolarize mitochondrial membrane in synaptosomes [START_REF] Hare | Comparative action of methylmercury and divalent inorganic mercury on nerve terminal and intraterminal mitochondrial membrane potentials[END_REF] .

Here, bioaccumulation in mitochondria was not investigated, but this GO term supported an interaction of Hg with this organelle in the high MeHg cell wall treatment. The GO term "gene regulation" was also abundant in copepods exposed to 50 µg•L -1 IHg in water and in Medaka exposed to 1 and 10 µg•L -1 IHg in water [START_REF] Xu | Comparative quantitative proteomics unveils putative mechanisms involved into mercury toxicity and tolerance in Tigriopus japonicus under multigenerational exposure scenario[END_REF][START_REF] Wang | Quantitative proteomic analysis reveals the mode-of-action for chronic mercury hepatotoxicity to marine medaka (Oryzias melastigma)[END_REF] .

On the other hand, some expected GO terms were not found as significantly different, notably proteins involved in reduction-oxidation (redox) metabolism and MTs in opposition with observation of a predominant BDM distribution for the high MeHg cell wall treatment.

MeHg, IHg and other metals induce ROS production. Here, antioxidant enzymes that are often observed in response to IHg exposure, e.g. superoxide dismutases, peroxiredoxins and catalases [START_REF] Correa | Spinal cord neurodegeneration after inorganic mercury long-term exposure in adult rats: Ultrastructural, proteomic and biochemical damages associated with reduced neuronal density[END_REF][START_REF] Wang | Quantitative proteomic analysis reveals the mode-of-action for chronic mercury hepatotoxicity to marine medaka (Oryzias melastigma)[END_REF] , were not significantly modulated at the protein level. Similarly, a proteomic analysis of rats exposed to IHg by diet did not identify GO terms directly linked to redox, despite the fact that they measured significantly increased lipid peroxidation by classical biochemical analyses [START_REF] Correa | Spinal cord neurodegeneration after inorganic mercury long-term exposure in adult rats: Ultrastructural, proteomic and biochemical damages associated with reduced neuronal density[END_REF] . Nevertheless, here the GO term sulfur compound metabolic process related to all chemical reactions and pathways involving sulfur-containing compounds (e.g. methionine, cysteine, glutathione) strongly influenced the distribution in the PCA among intracellular samples, while the proportion of Cysteine and Methionine residues in modulated proteins didn't changed among treatments. This suggests the preexistence in the cytosol of a sufficient pool of MTLP-MW proteins that could be involved in Hg-chelation in cytosol, as also supported by the quite stable S proportion in residues of proteins for all treatments (Table S4, Figure S3). Similarly, G. fossarum specimens collected in situ from a clean and a contaminated river in Croatia showed increased soluble metal levels in gammarids in the contaminated site, but similar MT concentrations in both sites [START_REF] Filipovic Marijic | Investigation of the soluble metals in tissue as biological response pattern to environmental pollutants (Gammarus fossarum example)[END_REF] . In the same line, Caenorhabditis elegans chronically exposed to 60 mg•L -1 MeHg showed no increase in mt gene expression level after 15h exposure, while mt-gene loss-of-function mutants were markedly more sensitive than wildtype to MeHg [START_REF] Helmcke | Hormetic effect of methylmercury on Caenorhabditis elegans[END_REF] . Nevertheless, previous studies in fish showed that MTs correlated to Hg concentration in liver, but not muscles and gills [START_REF] Bebianno | Hg and metallothionein-like proteins in the black scabbardfish Aphanopus carbo[END_REF] . Here the proteomic analysis was conducted on whole organisms, potentially masking putative organspecific regulation. More investigation, outside the scope of the current study would be necessary to conclude on the role of MTs in the tolerance to dietary MeHg in our experimental conditions.

In conclusion, our analysis supported a sublethal stress likely coped by G. fossarum according to the relative low number of modulated proteins in our experimental conditions. The analysis revealed a correlation between protein abundance and Hg subcellular bioaccumulation in MSF, but a different protein profile for each concentration, suggesting the occurrence of several thresholds of regulation of the proteome. Data also suggested that in response to increasing doses of MeHg, G. fossarum activated and/or deactivated some biochemical processes to compensate the loss or impairment of other pathways caused by the toxicity of MeHg. As such, defense mechanisms triggered by dietary MeHg appeared to have a cost for the fitness of animals. Our experiment brought thus new insights on the specific effects of MeHg in diet at sublethal concentration.

Our data in addition confirmed that Hg bound in cell walls of plants can be assimilated by G. fossarum, which is consistent with its feeding strategy. The cell wall constituents are expected to remain for a long period in the environment because of their recalcitrant nature. Here, data point cell walls as a potential source of Hg transfers and toxicity to primary consumers.

Moreover, proteomic analysis revealed contrasted responses for intracellular and cell wall treatments, congruent with detoxification mechanisms and supporting that the different binding sites in shoots consequently caused different toxicity to consumers. In this context feeding behaviour of consumers appears instrumental for the fate and impact of Hg in food chains. In particular, the abundance of species and their feeding strategy need to be considered to adequately protect aquatic systems, as both will significantly influence the fate of Hg in local food webs. For example, the abundance of macrophytes in shallow water systems might favor the transfer of MeHg to food webs.

Aknowledgements

This research received financial support by Grand Reims in the frame of the Aquasurv chair.

Authors thanks Dr Rebecca Beauvais-Flück and Mr Hervé Quéau for their help during exposures. The authors also benefitted from the French GDR "Aquatic Ecotoxicology" framework which aims at fostering stimulating scientific discussions and collaborations for more integrative approaches.

Supporting Information.

Table S1: Hg concentrations in water, E. nuttallii and G. fossarum; Table S2: Hg concentration in subcellular fractions; Table S3: list of identified proteins; Tables S4 to S6

  : proportion of thiols in proteins; Figure S1: HPLC chromatograms, Figure S2: Principal Component Analysis and Hierarchical clustering of samples, Figure S3: proportion of thiols in proteins against fold changes.

Figure 2 .

 2 Figure 2. Concentration (ng•g -1 ; mean ± sd; A) and proportion (%; B) of THg measured in MSF, BDM and debris (S2) for G. fossarum feeding on the MeHg contaminated intracellular (I) or cell wall (C) compartment of E. nuttallii (*p<0.05 vs control; n = 3).

Figure 3 :

 3 Figure 3: Venn diagram (A) and heatmap (Log2 fold change) (B) of differentially abundant proteins in G. fossarum feeding 7 days on the low or high MeHg contaminated intracellular (I) or cell wall (C) vs control (p-value <0.05). Maximum of heatmap is set to 4 for visibility of smaller fold changes (see

Figure 4 .

 4 Figure 4. PCA of GOslim terms according to biological processes in G. fossarum feeding 7 days on the low or high MeHg contaminated intracellular (I) or cell wall (C) vs control.

Table 1 .

 1 List of the five most up and down-detected proteins in each treatment. Abundance vs control (Log2 fold change), annotation, % identities and e-value are listed. Values in bold are significantly different to control (p-value <0.05 calculated with univariate statistic: Ttest).

		% sequence	Expect
	Functional annotation	identity	value	I-low	I-high C-low C-high
	Cell structure
	actin, cytoplasmic 1	51.3	1.5e-58	1.1	-1.8	55.2	53.0
	myosin light chain alkali-like	53.8	5.9e-23	-2.3	-5.3	2.2	1.7
	tubulin alpha-3 chain	75.2	7.1e-135	5.1	18.1	-1.7	-1.7
	myosin heavy chain, muscle-like isoform X18	76.1	1.7e-70	1.1	6.1	-11.5	-4.9
	myosin heavy chain type b	58.7	2.6e-69	-1.0	5.9	-7.1	-7.0
	collagen alpha-1, IV, chain precursor, putative	63.5	1.2e-61	-1.7	-2.7	-2.3	-7.9
	myosin heavy chain, partial	83.8	2.9e-79	2.2	17.1	-17.3	-28.3
	Energy metabolism
	ADP/ATP translocase 3-like	94	1.4e-44	-8.0	-2.2	7.2	5.7
	fructose-bisphosphate aldolase	85.7	5.8e-31	7.0	2.7	1.1	-1.0
	sucrase-isomaltase, intestinal	59.8	9,00E-102	-6.4	-2.6	-2.1	-1.1
	glycogen phosphorylase	67.4	2.5e-98	6.4	8.6	-1.9	-1.2
	aconitate hydratase, mitochondrial	91.6	2.4e-168	3.9	6.5	1.4	-1.2
	endoglucanase E-4	72	5.1e-147	-4.4	-5.4	-3.0	-1.2
	malate dehydrogenase, cytoplasmic	87.8	4.1e-161	3.9	6.0	-1.1	-1.6
	enolase isoform X1	77.7	1.7e-113	-14.1	-3.0	-6.0	-5.1
	family 7 cellobiohydrolase	77.9	6.7e-61	1.4	-8.9	-2.1	-7.9
	Response to stress
	endoplasmin-like isoform X1	78.5	5.2e-177	3.4	2.2	6.8	7.1
	oplophorus-luciferin 2-monooxygenase non-catalytic subunit 34	4.4e-46	2.1	1.7	5.3	3.3
	glucosylceramidase-like	73.6	4.5e-56	-4.1	-6.2	2.1	2.1
	phosphate carrier protein, mitochondrial-like	81.3	2.5e-119	-2.0	-7.5	1.2	1.5
	hemolymph clottable protein-like isoform X1	31.6	1.1e-06	4.3	3.1	2.8	1. 3
	putative glucosylceramidase 3	63.8	7.9e-42	-1.3	-1.2	4.5	1.0

Table 2 .

 2 Ratio of GOslim terms for molecular function (MF), biological process (BP) and cellular compartment (CC) showing opposite trends for G. fossarum feeding on the low or high MeHg contaminated intracellular (I) or cell wall (C) compartment vs respective controls of E. nuttallii. Values in bold are significantly different to control (Log2 fold change, *pvalue <0.05 calculated with univariate statistic: Ttest).

	I-low I-high	C-low	C-high

Table 3 .

 3 Ratio of GO terms responding to concentration for G. fossarum feeding on the low and high MeHg contaminated intracellular (I) and cell wall compartment (C) vs respective controls of E. nuttallii (Log2 fold change; *p-value <0.05; #N/A GO term not found for the treatment).

	GO Term # on PCA	I-low I-high C-low C-high

Table 1

 1

for identification of most modulated proteins).