Thomas Milinkovitch
email: t.milinkovitch@hotmail.fr

Perrine Geraudie
email: perrine.geraudie@akvaplan.niva.no

Lionel Camus
email: lionel.camus@akvaplan.niva.no

Valérie Huet
email: valerie.huet@univ-lr.fr

Hélène Thomas- Guyon

Biomarker modulation associated with marine diesel contamination in the iceland scallop (Chlamys islandica)

Keywords: marine diesel, Iceland scallops, Glutathione-S-transferase, oxidative stress, hemolymph metabolites, digestive gland

The decrease of ice cover in the Arctic will lead to an increase of ship traffic in the upcoming decades. Consequently, oil pollution is expected. In this context, the goals of this study were to evaluate the biological impact of marine diesel contamination and, on this basis, to determine analytical tools of interest (biomarkers) for future biomonitoring of diesel spills.

Using a seven days contamination protocol, this study investigated biochemical modulations in the digestive gland of the Iceland scallop (Chlamys islandica). Incorporation of contaminants was verified assessing hemolymph metabolites. Results showed a response of glutathione-S-transferase to contamination suggesting detoxification processes and the suitability of such a tool for diesel spills biomonitoring. The lack of modulation of superoxide dismutase activity and lipid peroxidation suggests no oxidative stress and the unsuitability of these molecular tools for biomonitoring.

Introduction

In Arctic, the decrease of ice cover will permit oil and gas prospecting as well as an increase of ship traffic. This will lead to operational discharges and spills of oil at sea. In order to minimize the impact of this pollution, the government of Norway recently banned the use of heavy fuel oils in parts of the Svalbard territorial waters and permitted only the use of marine diesel (Ministry of the Environment of Norway 2009). This decision is based on the fact that heavier oils are considered to be the more persistent ones [START_REF] Markarian | A Critical Review of Toxicity Values and an Evaluation of the Persistence of Petroleum Products for Use in Natural Resource Damage Assessments[END_REF]. However, even though lighter oils, such as marine diesel, tend to be less persistent, they are potentially more toxic to aquatic organisms since their PAHs (polycyclic aromatic hydrocarbons) concentrations are higher [START_REF] Neff | Polycyclic aromatic hydrocarbons in the aquatic environment: sources, fates and biological effects[END_REF]. While some ecotoxicological studies were conducted in temperate [START_REF] Lotufo | Toxicity of Sediment-Associated PAHs to an Estuarine Copepod: Effects on Survival, Feeding, Reproduction and Behaviour[END_REF][START_REF] Rodrigues | Deleterious effects of water-soluble fraction of petroleum, diesel and gasoline on marine pejerrey Odontesthes argentinensis larvae[END_REF]) and tropical [START_REF] Stirling | Effects of a spill of marine diesel oil on the rocky shore fauna of Lamma island, Hong Kong[END_REF][START_REF] Lüchmann | Biochemical biomarkers and hydrocarbons concentrations in the mangrove oyster Crassostrea brasiliana following exposure to diesel fuel water-accommodated fraction[END_REF]) marine organisms to investigate marine diesel toxicity, only one study to our knowledge investigated the toxicological effects of marine diesel on an Arctic species, the copepod Calanus glacialis [START_REF] Hansen | Acute exposure of water soluble fractions of marine diesel on Arctic Calanus glacialis and Calanus finmarchicus: Effects on survival and biomarker response[END_REF]. Thus, taking into account the likelihood of diesel oil spills in the Arctic waters, its potential ecotoxicity and the lack of investigation in this field of research, ecotoxicological studies are needed in order to (i) highlight the biological impact of this emerging environmental threat and (ii) propose valuable biological indicators for future biomonitoring.

Thus, this study aims to assess biomarker modulation in the digestive gland of the Iceland scallop (C. islandica) exposed to diesel. This species was chosen since (i) filter-feeding molluscs are considered valuable sentinel species of ecosystems due to their capacity to bioaccumulate and respond to contaminant exposure [START_REF] Viarengo | Cu, Zn and Cd content in different tissues of the Antarctic scallop Adamussium colbecki : role of the metallothionein in heavy metal homeostasis and detoxification[END_REF][START_REF] Solé | Bioaccumulation and biochemical responses in mussels exposed to the water-accommodated fraction of the Prestige fuel oil[END_REF]);

(ii) C. islandica is sessile and globally distributed in Arctic waters so that results of this study could be used for several future monitoring programs.

Glutathione-S-transferase (GST) activity was measured, since this enzyme is considered a valuable and sensitive biomarker of hydrocarbons exposure in bivalve molluscs and is consequently commonly used (reviewed in [START_REF] Amiard | Les biomarqueurs dans l'évaluation de l'état écologique des milieux aquatiques[END_REF]. More specifically, GST has been shown to be a reliable biomarker for marine diesel contamination [START_REF] Lüchmann | Biochemical biomarkers and hydrocarbons concentrations in the mangrove oyster Crassostrea brasiliana following exposure to diesel fuel water-accommodated fraction[END_REF].

A biomarker of oxidative stress, lipid peroxidation, has been chosen since it is a suitable biological indicator of hydrocarbon contamination and it reflects a degradation of the functional integrity [START_REF] Halliwell | Free radicals in biology and medecine[END_REF]. Additionally, activity of the superoxide dismutase (SOD) has been measured considering its important role in antioxidant protection [START_REF] Valavanidis | Molecular biomarkers of oxidative stress in aquatic organisms in relation to toxic environmental pollutants Ecotox[END_REF] as well as the precocity and the sensitivity of this enzyme response to hydrocarbon contamination [START_REF] Oliveira | Organ specific antioxidant responses in Golden grey mullet (Liza aurata) following a short-term exposure to phenanthrene[END_REF][START_REF] Hannam | Immunotoxicity and oxidative stress in the Arctic scallop Chlamys islandica: Effects of acute oil exposure[END_REF][START_REF] Sun | Bioaccumulation, depuration and oxidative stress in fish Carassius auratus under phenanthrene exposure[END_REF]Oliva et al. 2011;Milinkovitch et al. 2013a).

Finally, hydrocarbon incorporation was verified by assessing metabolites of hydrocarbon in the hemolymph of scallops.

Materials and methods

All Iceland scallops were sampled the third week of April in the fjord of the Norwegian sea close to the city of Tromsø (69° 35.060' N,18° 55.701' E), by dredging between 10 and 20 m depth (temperature varying between 4 and 6°C). The dredging did not damage the scallops, and all the animals used for the experiment had no sign of exterior diseases or damages. Then, the scallops were acclimatized at the Akvaplan-niva marine laboratory for 6 weeks in tanks supplied with flow through seawater from the fjord (5.14 ± 0.03 °C). Scallops were under natural photoperiod and fed daily with Shellfish Diet 1800 (Reed Mariculture Inc.), a diet made of five microalgae (Isochrysis, Pavlova, Tetraselmis, Chaetocerous calcitrans, Thalassiosira weissflogii), suitable for bivalves such as oysters, mussels and scallops. The average length of the shells, at the end of the acclimatization period, was 66.59 ± 0.41 mm (mean ± standard error of the mean) and the average weight was 34.50 ± 5.3 g. The soft tissues weight was 15.80 ± 3.30 g.

The experimental device is a static water system adapted from Milinkovitch et al. (2011a) and already used in several other studies investigating dispersed oil (e.g. [START_REF] Claireaux | Effects of oil exposure and dispersant use upon environmental adaptation performance and fitness in the European sea bass, Dicentrarchus labrax[END_REF]Milinkovitch et al. 2013aMilinkovitch et al. , 2013b)). It is made of two 120-L tanks stocked in a 5 °C temperature-controlled room. Each tank is composed of a funnel (at the water surface) connected to a 12-V water pump (on the floor of the tank). The marine diesel is constantly drawn up through the funnel into the pump so that it is maintained homogenously as droplets in the water column, i.e. physically dispersed, and no coalescence of oil slick at the water surface is possible.

Marine diesel (obtained from a BP® ship fuel station in Tromsø) was added to 110 L of filtered seawater in the contaminated tank (described above) to obtain a nominal concentration of 29 ppm. In parallel, a similar 120-L control (C) tank with 110 L of uncontaminated seawater was set up. Hydrocarbons concentration matches with values observed in situ in the water column during some oil spills: for instance, from 1 to 100 mg/L of total petroleum hydrocarbons were measured in coastal waters around Shetland during the Braer oil spill, as reported by [START_REF] Lunel | The Braer oil spill: oil fate governed by dispersion[END_REF]. After addition of contaminants, 10 scallops with a 50% sex ratio (sexed according to the colour of the gonads) were randomly distributed into each tank (i.e. each treatment) and exposed for 7 days without being fed. Physico-chemical parameters were measured daily for the 2 tanks and remained constant during all the experiment: temperature was 5.2 ± 0.08°C, pH was 7.85 ± 0.012 and dissolved oxygen was higher than 97 %.

At the end of the exposure period, the hemolymph was sampled and the digestive glands were removed and stored at -80°C.

Hemolymph was diluted (1/16 or 1/40, depending on the metabolites concentration) in absolute ethanol in quartz cuvettes. Fixed wavelength fluorescence (FF) was then measured for control and contaminated organisms on a spectrofluorimeter. Excitation-emission wavelength pair 290:335 was employed to detect naphthalene-derived metabolites [START_REF] Aas | PAH metabolites in bile, cytochrome P4501A and DNA adducts as environmental risk parameters for chronic oil exposure: a laboratory experiment with Atlantic cod[END_REF]. The FF values are expressed as arbitrary units of fluorescence and provide an estimation of the relative concentration of metabolites between control and the contaminated condition.

For all biochemical analysis, ten digestive glands were considered for control organisms as well as for exposed organisms. They were homogenised in ice-cold phosphate buffer (100 mM, pH 7.2, 1100 mOsm) containing ethylenediaminetetraacetic acid (EDTA) free protease inhibitors (Halt protease inhibitor cocktail, Thermo Scientific). The homogenates were centrifuged at 12500 g at 4 °C for 15 min and the post-mitochondrial fractions (PMF) were used for assays.

Total protein concentrations were determined using the method of [START_REF] Bradford | A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] with a BioRad assay kit (500-0002) containing bovine serum albumin as a standard and Brilliant Blue G 250 as a reactant. The reaction was measured spectrophotometrically at 570 nm.

The GST activity was measured using a commercially available kit (GST assay kit, Sigma-Aldrich) adapted for this study (i.e. samples were diluted to 1/20 in kit sample buffer and analyzed in triplicates). The chromogenic substrate 1-Chloro-2,4-dinitrobenzene (CNDB), conjugated with the reduced glutathione by the GST, was measured at 340 nm. GST is expressed in U/mg of protein.

Lipid peroxidation was evaluated via malondialdehyde (MDA) concentration using a commercially available MDA assay kit (Oxis International). The method was based on the reaction of a chromogenic reagent (n-methyl-2-phenylindole) with MDA measured at 586 nm [START_REF] Gérard-Monnier | Reactions of 1-Methyl-2-phenylindole with Malondialdehyde and 4-Hydroxyalkenals. Analytical Applications to a Colorimetric Assay of Lipid Peroxidation[END_REF]. The results are presented in nmol of MDA/mg of protein.

The SOD activity was measured using the assay developed by [START_REF] Paoletti | A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts[END_REF] adapted for use in microplate. The assay involves EDTA, MnCl 2 and mercaptoethanol and measures the decrease of nicotinamide adenine dinucleotide (NADH) oxidation. The results were presented in U of SOD/mg of protein.

The statistical analysis was carried out using Statistica software. Equality of variance (homoscedasticity) was demonstrated for FF, SOD, GST activities as well as MDA content using a Levene test. Consequently, for all variables, Student tests were conducted to highlight significant differences (for P value <0.05) due to the contamination. The results were expressed as mean ± standard error of the means.

Results and discussion

By investigating the modulation of several chosen biomarkers in C. islandica, the goals of this study were to evaluate the biological impact of the marine diesel and, on this basis, to validate new analytical tools of interest for future biomonitoring of diesel spills in the Arctic.

Hydrocarbon concentration is expressed as nominal concentration and was not measured in the water column, so that any decrease of hydrocarbon concentration during the exposure period (already observed and reported in Milinkovitch et al., 2011a, b, c;2012;2013a, b) cannot be measured. However, the relative concentration of metabolites (estimated through FF measurement) insures the incorporation of hydrocarbons contaminants by the organisms since the relative concentration of biliary metabolites was significantly higher in the hemolymph of scallops exposed to marine diesel until the last day of exposure (when compared to control, Figure 1A).

Regarding GST activity (Figure 1B), results showed values of 0.526 ± 0.021 U/mg of protein for control scallops. This is in accordance with a recent study conducted by [START_REF] Nahrgang | Seasonal variation in biomarkers in blue mussel (Mytilus edulis), Iceland scallop (Chlamys islandica) and Atlantic cod (Gadus morhua)-Implications for environmental monitoring in the Barents Sea[END_REF], who showed that the mean of the GST activity (measured at the same season in the digestive gland of C. islandica) was 0.5 U/mg of protein. In our study, the activity of the enzyme was significantly higher in contaminated organisms (compared to uncontaminated ones), which is in accordance with two studies conducted by [START_REF] Pan | Effects of benzo(k)fluoranthene exposure on the biomarkers of scallop Chlamys farreri[END_REF][START_REF] Pan | Effects of benzo(a)pyrene exposure on the antioxidant enzyme activity of scallop Chlamys farreri[END_REF] who showed an increase of GST activity in the digestive glands of Pectinidae contaminated with PAHs. The induction of GST activity by environmentally relevant concentrations of marine diesel showed the sensitivity of this biomarker. Thus, when measured in the digestive glands of C. islandica, GST activity appears to be a suitable tool, sensitively predicting the detoxification response to marine diesel.

In order to evaluate the degradation of the health status due to marine diesel exposure, oxidative stress was evaluated through lipid peroxidation (Figure 1C). The MDA content was 1.439 ± 0.083 nmol/mg of protein in the digestive glands of scallops exposed to uncontaminated water. This order of magnitude is in accordance with a recent study conducted by Liu et al. (2014) who assessed MDA contents ranging from 0.9 to 2.2 nmol/mg of protein in the digestive glands of uncontaminated and hydrocarbon contaminated scallops Chlamys farreri. Although marine diesel contamination seemed to induce an increase of lipid peroxidation, statistical analyses did not indicate significant differences due to these contaminations (P = 0.13). These results are in accordance with two recent studies: (i) a study conducted by [START_REF] Lüchmann | Biochemical biomarkers and hydrocarbons concentrations in the mangrove oyster Crassostrea brasiliana following exposure to diesel fuel water-accommodated fraction[END_REF], which did not highlight lipid peroxidation in digestive glands of Crassostrea brasiliana exposed to water accommodation fractions of marine diesel for 96h, and (ii) a study conducted by [START_REF] Hannam | Immunotoxicity and oxidative stress in the Arctic scallop Chlamys islandica: Effects of acute oil exposure[END_REF] that showed no modulation of MDA contents in the haemocytes of C. islandica after an 1 week exposure to crude oil. On another hand, our results disagree with previous studies that showed an increase of MDA in the digestive glands of C. farreri exposed to PAHs [START_REF] Pan | Effects of benzo(k)fluoranthene exposure on the biomarkers of scallop Chlamys farreri[END_REF][START_REF] Pan | Effects of benzo(a)pyrene exposure on the antioxidant enzyme activity of scallop Chlamys farreri[END_REF]. This discrepancy could be due to the fact that, in contrast to our study, these previous studies exposed Pectinidae to high concentrations of a single PAH such as benzo(k)fluoranthene or benzo[a]pyrene [START_REF] Pan | Effects of benzo(k)fluoranthene exposure on the biomarkers of scallop Chlamys farreri[END_REF]2009, respectively).

In parallel, regarding the antioxidant system, our study investigated the activity of the superoxide dismutase (SOD) (Figure 1D). In control scallops, this activity was 32.68 ± 4.00 U/mg of protein which agrees with a previous study conducted in the digestive glands of Chlamys varia [START_REF] Milinkovitch | In situ evaluation of oxidative stress and immunological parameters as ecotoxicological biomarkers in a novel sentinel species (Mimachlamys varia)[END_REF]) since these authors reported activity values of 29.4 U/mg of protein. Regarding the effects of marine diesel contamination, our results did not reveal modulation of SOD activity.

The absence of antioxidant enzyme reactivity (SOD) as well as of lipid peroxidation (MDA content) could be due to the fact that no increase of ROS was induced by the contamination.

Probably the efficiency of upstream detoxification systems (such as GST) may have eliminated most of the hydrocarbons incorporated and consequently no ROS were produced downstream, leading to no increase of SOD activity and MDA content.

The lack of significance regarding MDA content and SOD activity seems to indicate that these biomarkers cannot be considered valuable analytical tools for biomonitoring diesel spills. However, such a conclusion should be cautious since this lack of significance could also be due to a decrease of hydrocarbons exposure in the last days of experiment: a lower level of hydrocarbons exposure could have (i) permitted a recovery process leading to the absence of lipid peroxidation; (ii) induced no increase of SOD activity.

In conclusion, although this study clearly exposes the effects of marine diesel on GST activity, no effect was observed for oxidative stress and antioxidant response (MDA content and SOD activity, respectively). Altogether, these results are in accordance with the literature since GST, commonly claimed as a sensitive biomarker of detoxification, was modulated while MDA, a biomarker of damages claimed as less sensitive, was not modulated (see [START_REF] Amiard | Les biomarqueurs dans l'évaluation de l'état écologique des milieux aquatiques[END_REF] for an extensive review). Further studies, investigating several levels of biological organisation (sub-individual, individual and population levels) and including several species of the ecosystems, are required in order to evaluate the ecological impact of marine diesel pollution upon Arctic ecosystems and to set up analytical tools of interest for diesel spill monitoring.

Figure 1 :

 1 Figure 1: Biomarkers assessed after 7 days of exposure to Control solution (Ctrl) and marine

Acknowledgements

Postdoctoral fellow, mobility grants and experimentation costs for this work were supported by grants from Ambassade Royale de Norvège and Ministère des Affaires Etrangères (France). Parts of the experimentation costs were supported by Svalbard Miljøvernfund, the FearIce project of the Fram centre, Akvaplan-Niva, Université de La Rochelle, Centre

National de la Recherche Scientifique, FEDER, Conseil Régional Poitou-Charentes (France) and the Government of Svalbard. The authors thank Eric Rosenfeld for his helpful advices.


domain: chim sde

text:
Maria Dronova

Eric Lécolier
email: eric.lecolier@ifpen.fr

Loïc Barré

Laurent J Michot

Phase diagram, structure and rheology of boehmite dispersions: role of electrostatic interactions

Keywords:

Improving industrial processes such as filtration of colloidal suspensions requires an in depth understanding of colloidal stability that is linked to interparticle interactions. One way of investigating such phenomena is to determine colloidal phase diagrams and to characterize structure and rheology in various regions of the phase diagrams. Such a strategy was applied in the present paper to the case of boehmite dispersions. The phase diagram of this material, obtained from osmotic stress experiments shows a non trivial behavior, as the sol/gel transition line of boehmite displays a positive slope at low ionic strength and a negative one at higher ionic strength. Combining structural (SAXS) and rheological measurements, it appears that such a behavior can be linked to the ratio between particle anisotropy and Debye length. Such an interpretation is confirmed by modeling the evolution of elastic moduli in various regions of the phase diagram. The model developed takes into account the anisometric nature of particles and provides reasonably accurate predictions of the electrostatic interaction potential in the system.

Introduction

Boehmite has been studied for decades owing to its essential industrial importance. In particular, boehmite being a precursor of γ-alumina is widely found in alumina related industries: catalyst carriers, ceramics, metallurgy, treatment of Al-rich radioactive waste. Most boehmite properties are related to its particular structure. Its building structural unit is composed of octahedral double sheets of edge-shared AlO 4 (OH) 2 aluminum oxy-hydroxide chains. Two surface oxygen of these layers are linked via hydrogen bonding, which provides a highly polarized, stable hydroxyl-functional surface [START_REF] Russell | Lattice vibrations of boehmite (γ-AlOOH): evidence for a C122v rather than a D172h space group[END_REF].

Boehmite particles display a wide range of morphology evolving from elongated fibers towards diamond-shaped platelets. As far as synthetic boehmite is concerned, these various particle shapes can be obtained by varying the pH used during synthesis [START_REF] Jolivet | Design of oxide nanoparticles by aqueous chemistry[END_REF][START_REF] Zhang | Size and morphology controlled synthesis of boehmite nanoplates and crystal growth mechanisms[END_REF]. In contrast to clay minerals, boehmite does not present any atomic substitutions yielding structural charge [START_REF] Conroy | Importance of interlayer H bonding structure to the stability of layered minerals[END_REF] and, consequently the interlayer region between two sheets is free of charge balancing counterions and layer cohesion does not involve electrostatic forces. Despite these structural differences, the rheological properties (shear-thinning, thixotropy,…) of aqueous boehmite dispersions and its phase behavior are somewhat similar to those of clay minerals.

Concerning the phase behavior of boehmite, an isotropic-nematic (I-N) phase transition was observed by [START_REF] Buining | Isotropic-nematic phase separation of a dispersion of organophilic boehmite rods[END_REF] in the case of highly anisotropic charged boehmite rods [START_REF] Buining | Isotropic-nematic phase separation of a dispersion of organophilic boehmite rods[END_REF], whereas gelation with shear induced birefringence was found instead in platelike boehmite [START_REF] Fukasawa | Higher-order structure formation of ultrafine boehmite particles in sols, gels, and dried materials[END_REF]. Apart from the study of Fukasawa and Tsujii [START_REF] Fukasawa | Higher-order structure formation of ultrafine boehmite particles in sols, gels, and dried materials[END_REF], the phase behavior of boehmite platelets has scarcely been studied, which appears rather surprising considering the wide use of suspensions of this material in various industrial applications.

In contrast, rheological studies on boehmite dispersions have received more attention [START_REF] Evanko | Influence of aqueous solution chemistry on the surface charge, viscosity and stability of concentrated alumina dispersions in water[END_REF][START_REF] Weston | Connecting particle interactions to agglomerate morphology and rheology of boehmite nanocrystal suspensions[END_REF][START_REF] Ramsay | Structure and stability of concentrated boehmite sols, Faraday Discuss[END_REF][START_REF] Beattie | Anomalous aggregation behaviour of aluminium oxyhydroxides[END_REF][START_REF] Strenge | A rheological investigation of peptized boehmite suspensions[END_REF][START_REF] Cristiani | Study of the physico-chemical characteristics and rheological behaviour of boehmite dispersions for dip-coating applications[END_REF][START_REF] Drouin | Rheology and structure of peptized boehmite pastes[END_REF]. One of the earliest attempt to interpret the exceptional stability and elasticity of boehmite dispersions on the basis of the interaction forces between platelets dates back to the 70′s and was carried out by Ramsay et al. [START_REF] Ramsay | Structure and stability of concentrated boehmite sols, Faraday Discuss[END_REF]. Stable boehmite dispersions were found highly elastic and thixotropic, which was assigned to shortrange interparticle repulsions. This was indeed evidenced in the work of [START_REF] Karaman | A comparison of the interaction forces between model alumina surfaces and their colloidal properties[END_REF] [START_REF] Karaman | A comparison of the interaction forces between model alumina surfaces and their colloidal properties[END_REF] who studied the interactions between alumina coated silica spheres and flat alumina substrates using atomic force microscopy (AFM). In this latter work, the observed repulsion was assigned to natural hydration forces combined with the observation of a gel layer at the alumina surface. Interestingly, the formation of such hydrated gel layer was earlier mentioned by [START_REF] Beattie | Anomalous aggregation behaviour of aluminium oxyhydroxides[END_REF] [START_REF] Beattie | Anomalous aggregation behaviour of aluminium oxyhydroxides[END_REF] who linked it to the presence of mono-or polymeric ionic species that condense at the surface and provide repulsion at small interparticle separation distances (< 5 nm).

On the basis of rheometric measurement carried out at different pH and ionic strengths, [START_REF] Evanko | Influence of aqueous solution chemistry on the surface charge, viscosity and stability of concentrated alumina dispersions in water[END_REF] [START_REF] Evanko | Influence of aqueous solution chemistry on the surface charge, viscosity and stability of concentrated alumina dispersions in water[END_REF] also analyzed the effect of enhanced electrostatic repulsion on the viscosity of concentrated boehmite dispersions. It then appears that particle-particle interactions and bulk rheological behavior are intimately linked and that studying them in parallel could provide viable insight into structure-property relationships.

The present work focuses on platelet-shaped boehmite dispersions and investigates the effect of electrostatic interactions on the phase behavior, structural and rheological properties of these aqueous suspensions. In a first step, boehmite dispersions were equilibrated at fixed ionic strength by osmotic stress experiments to yield the equation of state (osmotic pressure curves vs. solid volume fraction) and phase behavior as a function of both solid volume fraction and ionic strength. The rheological features of the suspensions (in particular yielding and elasticity) were then measured in various areas of the previously determined phase diagrams. In parallel the structure of the suspensions was investigated by small angle X-Ray scattering (SAXS). Based on such information, we attempted to rationalize high frequency elastic moduli on the basis of electrostatic effects in the systems and took into account the anisotropic nature of boehmite particles by using effective volume fractions. In a last step, electrostatic interaction potentials between boehmite particles have been derived.

Materials and methods

The present study focuses on an industrial boehmite powder, Pural SB provided by Sasol group (Germany). This is a high purity microcrystalline powder (45 ppm of Fe, 450 ppm of Ti, <40 ppm of S, <50 ppm of Cl, <20 ppm of Na and 0.2 wt% of C) obtained via hydrolysis of aluminum alcoholate in water. Previous X-ray diffraction and small angle X-ray studies [START_REF] Mange | A microstructural investigation of nanostructured boehmite films prepared by the sol-gel route[END_REF] revealed the presence of platelet-shaped particles with average length and thickness of 10 nm and 3 nm, respectively. A representative TEM micrograph of the studied boehmite particles is displayed in SP.1 of supplementary information (see Appendix A) revealing aggregates of slightly anisometric platelets.

Boehmite sols were obtained by peptizing the powder in an aqueous solution containing 0.186 mol•L -1 of nitric acid at a total solid concentration of 143 g•L -1 . A mixing step using a high-speed disperser (Ultra-Turrax™) was carried out to ensure mechanical breakage of large aggregates into smaller units. Peptization mechanisms of boehmite in the presence of inorganic acids have been largely studied by several research groups [START_REF] Cristiani | Effect of ageing time on chemical and rheological evolution in γ-Al2O3 slurries for dip-coating[END_REF][START_REF] Fauchadour | Peptization mechanisms of boehmite used as precursors for catalysts[END_REF][START_REF] Zheng | Peptization mechanism of boehmite and its effect on the preparation of a fluid catalytic cracking catalyst[END_REF] revealing a complex interaction chemistry at the boehmiteacid interface that results in the formation of colloidal sol. Detailed information about peptization can be found elsewhere [START_REF] Cristiani | Effect of ageing time on chemical and rheological evolution in γ-Al2O3 slurries for dip-coating[END_REF][START_REF] Fauchadour | Peptization mechanisms of boehmite used as precursors for catalysts[END_REF][START_REF] Zheng | Peptization mechanism of boehmite and its effect on the preparation of a fluid catalytic cracking catalyst[END_REF].

The translucent sols thus obtained were then centrifuged at 4 000 g during 4 h in order to eliminate remaining large agglomerates. The resulting supernatant (pH 1.3) was then placed in a porous cellulose membrane with a cut off of 6-8 kDa for dialysis in order to remove ionic species. A buffer reservoir was renewed 1-2 times per week to accelerate the process. The pH of the reservoir was fixed at 5.0-5.2 by adding a few droplets of nitric acid (6.8 wt% HNO 3 ). The conductivity of the added solution is around 2-3 μS/cm and the conductivity in the reservoir was then monitored. Renewal by fresh reservoir solution is stopped once the conductivity of the reservoir displays an equilibrium value of 5 μS/cm. Typical total time for such a procedure was around 1-2 months. The volume fraction of the resulting boehmite dispersions after dialysis is ϕ = 0.4 vol% (= 12.7 g•L -1 ), and the pH is equal to 6.2.

In order to vary the ionic strength of the suspensions, various amounts of KNO 3 were added into the buffer reservoir at concentrations of 0.0809 g•L -1 and 1.110 g•L -1 that corresponds to calculated values of ionic strength 8•10 -4 M and 1•10 -2 M, respectively according to [START_REF] Russell | Lattice vibrations of boehmite (γ-AlOOH): evidence for a C122v rather than a D172h space group[END_REF] where c i is the molar concentration of ions and z i the number of charges on the ion. The composition of the aqueous phase was analyzed in the following way. Assuming equilibrium inside the dialysis bag and in the buffer reservoir, the latter was used for the analysis of nitrate, potassium and aluminum ions. Measurements were performed by inductively coupled plasma emission spectrometry (ICP-AES) to trace potassium and aluminum and by ion chromatography for nitrate determination.

Suspensions at various ionic strengths were then subjected to osmotic stress experiments. In these tests, cellulose dialysis tubes with a molecular weight cut off of 6-8 kDa were filled with 50 mL of boehmite dispersions and placed in 500 mL tubes containing PEG (MW = 20, 000) solutions at a fixed ionic strength. In such conditions, the relationship between PEG concentration, φ PEG , and osmotic pressure, Π, is given by [19].

(2)

The duration of the experiment was fixed at 1-2 weeks for boehmite systems, as such duration likely allows reaching osmotic equilibrium. The solid content of the osmotically stressed concentrated dispersions was evaluated by mass balance after drying at 140 °C during 24 h.

Optical transparency of diluted boehmite sols after dialysis (ϕ = 0.4 vol%) enables performing size distribution tests via Dynamic Light Scattering (DLS). These measurements have been carried out on a DLS setup of Cordouan Technologies. The incident beam of a He-Ne laser (λ = 658 nm) passes through the sample in a 10 mm Hellma quartz cuvette. The backscattered light is recorded at 90°by an Avalanche PhotoDiode (APD) detector and used after to build an intensity autocorrelation function G 2 (Q,t) (with wave vector Q and time t) to describe fluctuations in scattered light. G 2 (Q,t) is related to the modulus of the normalized field autocorrelation function g 1 (Q,t) by the Siegert equation [START_REF] Zhang | Size and morphology controlled synthesis of boehmite nanoplates and crystal growth mechanisms[END_REF] where α is a baseline and β is the coherence factor. The time dependence of g 1 (Q,t) is essentially linked to the dynamical properties of the given dispersion. In the case of Brownian monodisperse systems, it is described by a single exponential decay, where Γ is the decay rate (4) For a polydisperse system g 1 (Q,t) is written as a sum of exponential decaying functions as [START_REF] Buining | Isotropic-nematic phase separation of a dispersion of organophilic boehmite rods[END_REF] From the sum of exponential decaying functions, a standard Levenberg-Marquardt is used to adjust the weight of each exponential decaying function. If the studied dispersion is composed by Brownian particles, the diffusion coefficient can be further defined via , which enables determining the hydrodynamic radius R h of the hard sphere-equivalent using Stokes-Einstein equation [START_REF] Fukasawa | Higher-order structure formation of ultrafine boehmite particles in sols, gels, and dried materials[END_REF] where k B is the Boltzmann constant and η is the dynamic viscosity of the continuous phase.

The evolution with pH of the zeta potential ζ of suspensions for ionic strengths of 8•10 -5 M, 8•10 -4 M and 1•93•10 -3 M was measured on a Malvern Nano-ZS zetasizer at ambient temperature. Measurements were carried out without any sample dilution (boehmite concentration of 12.7 g•L -1 ). The dispersions were equilibrated for 1 min prior to each measurement. Analysis was repeated 3 times, each measurement cycle was based on over 50-100 runs and averaged.

In the present work, rheological measurements were performed using a Modular Compact rheometer, MCR 302 from Anton Paar instruments equipped with parallel plates geometry. Two plates with a crosshatched surface were used to avoid wall slip during measurements. The gap between plates was adjusted by controlling the normal force between 2 and 10 N depending on the volume fraction of the samples. Prior to the measurements, the rest interval at the start of the test was set to enable sample relaxation from accumulated internal stresses after setting the gap. Measurements were performed at fixed temperature (20 °C). The elastic G′ and viscous G″ moduli were measured again in an oscillatory frequency sweep in the range 0.01-100 rad•s -1 . A strain of γ = 0.01% was chosen that corresponds to the linear viscoelastic range (LVE), where the storage moduli display constant plateau values. Steady-state viscosity curves were also recorded under controlled shear rate from 1•10 -5 to 0.1 s -1 with a measuring point duration of 5 min.

Small angle X-ray scattering (SAXS) measurements were carried out on boehmite dispersions at room temperature (at 20 °C) and without vacuum insulation on beamline SWING at synchrotron SOLEIL. Samples were conditioned between two 50 µm Kapton foils. The beam energy was fixed at 12 keV (λ ≈ 0.1 nm) and two sample to detector distances (6.2 m and 0.50 m) were used yielding a Q range between 1•10 -2 and 10 nm -1 . The beam size on the sample was approximately 400 µm wide and 100 µm high. Two-dimensional scattering patterns (typical acquisition times around 1 s) were recorded on an Eiger detector. The curves of scattering intensity versus scattering vector Q, were deduced from azimuthal angular integration of the SAXS patterns. Curves are provided in arbitrary intensity as the thickness of the various samples was not controlled precisely.

Results and discussion

Ionic composition of aqueous phase

The interactions between particles are highly affected by the ionic environment that directly influences the magnitude of electrostatic repulsion. Thus, ionic composition of dialyzed boehmite sols was examined to obtain an accurate determination of ionic strength. The composition of this aqueous phase is defined by (i) dissociation of added salts and (ii) dissolution reactions of alumina species (arising from peptization procedure), whose amount by the end of dialysis becomes negligible. Accordingly, ionic strength can be determined by the concentration of nitrate or potassium ions and the pH given in the exponent, which determines its (ionic strength) order of magnitude (in M) [START_REF] Evanko | Influence of aqueous solution chemistry on the surface charge, viscosity and stability of concentrated alumina dispersions in water[END_REF] The concentration of H + ions determined by pH 5.0-5.1 in the buffer dialysis reservoir is 10 -5 mol•L -1 . The concentration of Al ions is found to be 7•10 -5 mol•L -1 , which results in the lowest ionic strength considered in the present study I 1 = 4•10 -5 mol•L -1 . Increase of ionic strength in this "mother" solution was therefore achieved by adding KNO 3 into the buffer reservoir at a given pH. The expression for ionic strength can be rewritten as [START_REF] Weston | Connecting particle interactions to agglomerate morphology and rheology of boehmite nanocrystal suspensions[END_REF] The detected concentration of ions for intermediate ionic strength is found to be 7•10 -4 mol•L -1 for K + ions and 7.5•10 -4 mol•L -1 of ions, resulting in an experimental value of 8•10 -4 mol•L -1 (ionic strength I 2 hereinafter). For the highest ionic strength considered in this study the measured concentration of ions is 1•10 -2 mol•L -1 for K + and ions, leading to a value of 1•10 -2 mol•L -1 (ionic strength I 3 hereinafter).

Osmotic pressure curves and phase diagram

The osmotic pressure curves and phase diagrams both provide valuable information about the role of interparticle interactions on the resulting structure of particles assembly. Fig. 1 shows the results obtained by osmotic equilibration of dilute boehmite dispersions against PEG solutions of fixed ionic strength at pH = 5.0-5.2. Upon salt addition, the induced electrostatic screening results in a lower osmotic pressure, which is consistent with predictions from DLVO theory. Such an effect is observed for colloidal dispersions whatever the particle shape. In contrast, the presence of a plateau for medium volume fractions and osmotic pressure that is observed in the present case appears to be typical of some plate-like particles. Indeed, it was observed for Na-Wyoming montmorillonite [START_REF] Michot | Phase diagrams of Wyoming Na-montmorillonite clay. Influence of particle anisotropy[END_REF] and laponite [START_REF] Mourchid | Phase diagram of colloidal dispersions of anisotropic charged particles: equilibrium properties, structure, and rheology of laponite suspensions[END_REF], whereas no plateau was reported in the case of nontronite [START_REF] Michot | Sol/gel and isotropic/nematic transitions in aqueous suspensions of natural nontronite clay. Influence of particle anisotropy. 1. Features of the i/n transition[END_REF] and beidellite [START_REF] Paineau | Liquid-crystalline nematic phase in aqueous suspensions of a disk-shaped natural beidellite clay[END_REF] (at least for ionic strengths lower than 10 -3 M).

The presence of such a plateau might indicate the coexistence of two phases. Indeed, if a phase transition occurs, the osmotic pressure is expected to be equal in both phases which would lead to the appearance of a plateau at the volume fractions separating both phases. This was for instance evidenced in the study of Pages et al. ( 2008) [START_REF] Page | Osmotic pressure and phase boundary determination of multiphase systems by analytical ultracentrifugation[END_REF] who suggested that the plateau observed in osmotic pressure curves for an ionic strength of 10 -3 M can be assigned to the formation of a gel that is kinetically trapped during osmotic stress compression and equilibration. Such a micro-phase separated state was also experimentally evidenced on laponite dispersions using ultra-small angle X-ray scattering experiments (USAXS), displaying a Q -3 dependence at low angles [START_REF] Saunders | A small-angle X-ray scattering study of the structure of aqueous laponite dispersions[END_REF]. The formation of this latter phase was assigned to the onset of an isotropic/ nematic phase transition (Onsager transition), in agreement with numerical predictions for the phase diagram of charged disk-like colloids [START_REF] Jabbari-Farouji | On phase behavior and dynamical signatures of charged colloidal platelets[END_REF].

To further investigate the system, it is relevant to plot a phase diagram obtained on the basis of rheological measurements that will be detailed later in this paper. Basically at this point, the aim is to distinguish between a free-flowing sol phase and an arrested solid-like phase (which can be named gel as well). A solid-like state refers to a soft solid exhibiting a typical dominance of the storage modulus G′ over the loss modulus G″ over the tested frequencies (0.01-100 rad•s -1 ) and in the linear viscoelastic (LVE) range (see Supplementary Materials SP.2). By applying an increasing mechanical stress, the soft solid starts to flow. On this basis, the resulting phase diagram is plotted in Fig. 2. The dot- ted line in this diagram corresponds to the sol-gel transition defined as an average between two distinct points separating the two states. Osmotic pressure with a step of 10 Pa was imposed to ensure the accuracy window accounting for the error of 11-12%. One may notice that the evolution of the sol-gel transition with ionic strength is far from being trivial. Indeed, the sol-gel transition line first exhibits a positive slope up to 8•10 -4 M, indicating a system dominated by electrostatic repulsion whereas in the high ionic strength region, i.e. from 8•10 -4 M to 1•10 -2 M, the slope is reversed. By interpolation, it can be foreseen that this line will join the flocculation line located at high ionic strength (between 0.1 M and 0.15 M, according to [START_REF] Fauchadour | Etude de la peptisation de la boehmite[END_REF]). Similar features with a "nose" in the phase diagram have already been observed in other charged anisotropic systems, e.g. synthetic compound laponite [START_REF] Martin | Dissociation of thixotropic clay gels[END_REF] or natural clays such as nontronite [START_REF] Michot | Sol-gel and isotropic/nematic transitions in aqueous suspensions of natural nontronite clay. Influence of particle anisotropy. 2[END_REF] or beidellite [START_REF] Paineau | Liquid-crystalline nematic phase in aqueous suspensions of a disk-shaped natural beidellite clay[END_REF].

It must be pointed out that the sol/gel transition in Fig. 2 might coincide with the onset of the plateau in the osmotic pressure curves up to I 2 = 8•10 -4 M, as already observed for laponite [START_REF] Mourchid | On viscoelastic, birefringent, and swelling properties of laponite clay suspensions: revisited phase diagram[END_REF]. In contrast, such correlation significantly diverges for an ionic strength of I 3 = 1•10 -2 M. It is tempting to attribute this discrepancy to aggregation processes that become apparent at such salinity as will be discussed in more details further in the text.

As already mentioned, the shape of the phase diagram displayed in Fig. 2 is close to the one observed for laponite. In this latter case, the evolution of the gelation mechanism with ionic strength has been highly debated and remains somehow controversial. At very low ionic strength (in the range of the phase diagram with a positive slope) Levitz et al. [START_REF] Levitz | Liquid-solid transition of Laponite suspensions at very low ionic strength: Long-range electrostatic stabilisation of anisotropic colloids[END_REF] have shown that the sol-gel transition could be interpreted as a repulsive glass transition (Wigner glass transition) of spherical like objects. According to Wigner predictions, this arises from the longrange Coulombic interactions that block the particles at distances from each other that are much higher than the particle diameter resulting in a solid-like behavior for very small densities as compared to ordinary solids. Along similar lines, based on light scattering tests, Bonn et al. [START_REF] Bonn | Aging of a colloidal "Wigner" glass[END_REF] rationalized the dynamics of laponite dispersions at I ∼ 10 -4 M (i.e. just at the "nose" of the transition line) as corresponding to a glassy-like state (which is characterized by no long-range position order and by non-ergodicity), where particles are trapped in cages formed by their neighbors.

For a symmetric monovalent electrolyte, such as KNO 3 , the Debye length κ -1 evolves with ionic strength, I, as [START_REF] Ramsay | Structure and stability of concentrated boehmite sols, Faraday Discuss[END_REF] In the case of boehmite particles, considering the size of the platelets, i.e. 10 nm, at the lowest ionic strength used in the present work (8•10 -5 M), the corresponding Debye length (≈ 34 nm) is much higher than the average particle size. The particles can then be assimilated to repulsive spheres with a radius of 34 nm forming a gel, once their ionic clouds start interpenetrating each other. Numerical simulations carried out by Jabbari-Farouji et al. [START_REF] Jabbari-Farouji | On phase behavior and dynamical signatures of charged colloidal platelets[END_REF] suggest that particles immobilized by strong repulsive double-layer interaction (= in the limit of low ionic strengths) can indeed reach a solid-like state with ordered regions. These results are in accordance with data on high-aspect ratio boehmite rods [START_REF] Wierenga | Aqueous dispersions of colloidal boehmite: structure, dynamics, and yield stress of rod gels[END_REF] revealing the existence of glassy state that is formed way before the isotropic-nematic (I-N) transition occurs. Moreover, neither the glassy state nor the phase separation was relevant for dispersions containing shorter particles, similarly to our results, suggesting that these processes are presumably driven by excluded volume effects.

In the limit of high ionic strengths, i.e. I 3 = 1•10 -2 M, the Debye length is reduced down to ∼3 nm in thickness, electrostatic repulsions are strongly screened and Van der Waals forces start playing a major role. Even if the structure of the gel formed in such regions of the phase diagram remains debated, attractive forces likely start playing a role providing an argument in favor of a percolation of stacks of platelets [START_REF] Michot | Phase diagrams of Wyoming Na-montmorillonite clay. Influence of particle anisotropy[END_REF][START_REF] Ruzicka | A fresh look at the Laponite phase diagram[END_REF] governed by attraction between edges and faces that results in the so-called "house of cards" configuration. The formation of objects resulting from attraction appears likely in the case of the boehmite suspensions investigated in the present work as (i) suspensions in this region of the phase diagram appear slightly less translucent than those at lower ionic strength, which could well indicate the occurrence of aggregation processes; (ii) these suspensions evolve with time in the long term. Indeed, gel-like behavior appears with time for volume fractions as low as 0.8% but the sample can be reversibly transformed back to sol after gentle shaking. Theoretical works (i.e. [START_REF] Delhorme | Monte Carlo simulations of a clay inspired model suspension: the role of rim charge[END_REF]) associated with numerical simulations on model clay dispersions suggest various modes of association between platelets when sufficiently high salinity is reached.

In the intermediate region of the phase diagram (8•10 -4 M) the Debye length κ -1 is comparable to the length of platelet (∼ 10 nm). In this situation, particle anisotropy starts playing a role and long-range interaction with anisotropic short-range excluded interactions both participate in a complex way to the sol-gel transition. Norrish [START_REF] Norrish | The swelling of montmorillonite[END_REF] proposed in the 1950's that gel formation results from the mutual repulsion of particles resulting from interactions between their double layers. Evidence for such processes was provided, for instance, by viscosity tests of Evanko et al. [START_REF] Evanko | Influence of aqueous solution chemistry on the surface charge, viscosity and stability of concentrated alumina dispersions in water[END_REF] on concentrated alumina dispersions, or by shear stresses and shear moduli measurements on montmorillonite dispersions acquired by Rand et al. [START_REF] Rand | Investigation into the existence of edge-face coagulated structures in Na-montmorillonite suspensions[END_REF].

To conclude this section, it appears that a possible interpretation of boehmite phase behavior can be linked to the interplay between particle anisotropy and Debye length resulting in a characteristic sol-gel mechanism given by its delayed appearance at I 2 = 8•10 -4 M as compared to the low and high limits of tested ionic strengths. Therefore, it is tempting to attribute the status of reentrant phase transition at high salt concentration to substantial effect of attraction forces causing clustering effects, as observed by dispersion's turbidity and time-dependent gelation.

Structure and particle organization

The structure of dispersions in various zones of the phase diagram was examined by small-angle X-ray scattering in a Q range 1•10 -2 and 10 nm -1 . The total scattering intensity I(Q) per unit particle volume V is usually expressed as U M. Dronova et al. [START_REF] Beattie | Anomalous aggregation behaviour of aluminium oxyhydroxides[END_REF] where Q is the scattering vector, A e is the amplitude scattered by one electron, Δρ 2 is the electronic density difference between particle and solvent, P(Q) is the form factor and S(Q) the structure factor. In very dilute dispersions, particle interactions are minimized and, in this case, S(Q) is close to unity and the scattering pattern then corresponds to the form factor P(Q). It should be noted, however, that Eq. ( 10) is valid for identical particles and if particle positions and particle orientations are independent.

Owing to the small anisotropy of boehmite particles, we assume, as first approximation, that the structure factor can be calculated by dividing the scattered intensity by the form factor of the boehmite particle. This calculated structure factor is referred to as an effective structure factor.

A curve corresponding to such a situation (ϕ = 0.2 vol%) is shown in Fig. 3. At short distances (high Q), the asymptotic decrease in intensity follows a Q -4 power law, which is described as the Porod law region dependent on the interfacial area of the solid-liquid interface. At intermediate scattering vector, the intensity decreases as ≈ Q -2 over a narrow range of Q, suggesting the presence of flat objects characterized by low anisotropy [START_REF] Mange | A microstructural investigation of nanostructured boehmite films prepared by the sol-gel route[END_REF]. A similar dependence, but rather more pronounced was constantly reported on several mineral clays, such as lathshaped nontronite [START_REF] Michot | Sol/gel and isotropic/nematic transitions in aqueous suspensions of natural nontronite clay. Influence of particle anisotropy. 1. Features of the i/n transition[END_REF] or disk-shaped laponite, which is consistent with scattering expected from the scattering objects displaying bidimensional nature. Interestingly, whatever the ionic strength, at low Q, the intensity keeps on rising, which reveals the presence in the dispersion of a few primary aggregates that dominate the signal at low Q. Note, however, that the curves are not plotted in absolute scale due samples preparation procedure, which prevents a correct fitting of the form factor. Still, previous work on similar particles were plotted in cm -1 and were fitted by a form factor corresponding to average particle sizes of 3 × 32 × 32 nm [START_REF] Morin | Préparation d′alumine à porosité contrôlée: étude de l′interaction de la boehmite dans des solvants et des propriétés fonctionnelles des matériaux résultants[END_REF]. Comparison between both curves shows that the system studied in the present work is very close to the one already studied.

SAXS patterns were also acquired on boehmite gels concentrated by osmotic stress procedures (also presented in Fig. 3). In such cases, the structure factor S eff (Q) cannot be neglected and can be experimentally estimated by dividing the intensity obtained for the concentrated dispersion I(Q) conc by the intensity measured for a dilute non-interacting dispersion I(Q) dil (volume fraction of 0.2% in the present study), that is to say the single-particle function. The measured intensities were di- vided by the known concentration in both cases in order to exclude the effect of different particle concentrations as [START_REF] Strenge | A rheological investigation of peptized boehmite suspensions[END_REF] Therefore, Eq. ( 11) contains an adjustable parameter f to account for the effects linked to the non-constant thickness of the gels analyzed by SAXS.

An example of derived S(Q) in the Q range comprised between 10 -1 and 1 nm -1 is shown in Fig. 4 for boehmite gels prepared at an ionic strength of 8•10 -4 M. The positive values of S eff (Q) regardless of concentration and ionic strength imply a repulsive nature of interparticle interactions in the given boehmite dispersions. Furthermore, the curves display at low Q the presence of a peak, the position of which shifts with volume fraction whereas at high Q, S(Q) approaches unity. The maximum observed is rather broad, which can be linked to the fact that the investigated samples are gels and consequently, rather disordered in terms of particles positions. Still, the position of the maximum of this peak can be used to provide an estimate of the interparticle distance d* according to [START_REF] Cristiani | Study of the physico-chemical characteristics and rheological behaviour of boehmite dispersions for dip-coating applications[END_REF] Fig. 5 displays the evolution with volume fraction of the correlation distances d*. Various trends can be deduced from this plot. (i) The correlation distances decrease with increasing ionic strength. This is expected and reflects the screening effects of the double layer that results in a shorter distance between particles. Though the data are limited at low volume fractions, it is likely a scaling exponent approaching the value of -1/3, which is a typical of an isotropic state. A similar behavior was observed on several clay minerals, such as beidellite and montmorillonite [START_REF] Paineau | Aqueous suspensions of natural swelling clay minerals. 1. Structure and electrostatic interactions[END_REF], or nontronite [START_REF] Michot | Sol-gel and isotropic/nematic transitions in aqueous suspensions of natural nontronite clay. Influence of particle anisotropy. 2[END_REF]. At higher volume fractions, the curves for the I 1 = 8•10 -5 M and I 2 = 8•10 -4 M scale as ϕ -0.96 and ϕ -0.8 , respectively, suggesting a local lamellar ordering of the platelets. In such a case, the swelling law should be d* = t/ϕ, where t is the thickness of individual platelets. Since the thickness of elementary particle is accurately known from earlier studies [START_REF] Mange | A microstructural investigation of nanostructured boehmite films prepared by the sol-gel route[END_REF], its integration in the slope fitting in Fig. 5 might suggest the fact of a certain degree of layers exfoliation in the investigated boehmite gels. The transition between both swelling regimes that could indicate the position of a potential isotropic/nematic phase transition is located at volume fractions ≈ 8% and 6% for I 1 = 8•10 -5 M and I 2 = 8•10 -4 M, respectively, i.e. far in the gel region according to the phase diagram of Fig. 2. Consequently, the I/N transition, if present is totally hindered by the sol/ gel transition in the boehmite suspensions used in the present study. Notably, several studies on clay mineral have demonstrated that the order-disorder transition does not display a trivial dependence with increasing repulsion. For instance, occurrence in the gel phase of flowinduced birefrigence reminiscent of nematic texture has been reported on laponite [START_REF] Mourchid | On viscoelastic, birefringent, and swelling properties of laponite clay suspensions: revisited phase diagram[END_REF] at lower volume fractions when lowering the ionic strength (in the range of 10 -2 -10 -4 M). Also, aqueous dispersions of gibbsite platelets have revealed that the I-N transition (not hindered by gelation, as in our case) is shifted to higher volume fraction when increasing ionic strength [START_REF] Van Der Beek | Nematic ordering vs. gelation in suspensions of charged platelets[END_REF]. Another example is the phase diagram of beidellite displaying the similar sol/gel transition line as in our study. However, the narrow regions of a two-phase system and the nematic phase were additionally observed by authors [START_REF] Paineau | Liquid-crystalline nematic phase in aqueous suspensions of a disk-shaped natural beidellite clay[END_REF] showing dependence on the particle size only, whereas effect of ionic strength was found negligible. Similar trends have been therefore reported on the phase diagram of nontronite [START_REF] Michot | Sol-gel and isotropic/nematic transitions in aqueous suspensions of natural nontronite clay. Influence of particle anisotropy. 2[END_REF].

Elasticity of boehmite gels

The use of oscillatory rheology provides detailed information about the material structure and further illustrates the substantial effect of bulk solution chemistry on interparticles forces. In order to obtain additional information on the suspensions, dynamic moduli were also measured as a function of angular frequency (see Supplementary Materials SP.3). The evolution of the high frequency elastic modulus G′ for different volume fractions and ionic strengths displays a single master curve that follows a power law with a (ϕ -ϕ sol-gel ) 1.9 dependence (see Supplementary Materials SP.4). Data points are more scattered at low volume fractions, which is related to the accuracy of the determination of the critical volume fraction corresponding to the sol-gel transition. The data obtained here are similar to those obtained on various clay minerals (laponite [START_REF] Mourchid | Phase diagram of colloidal dispersions of anisotropic charged particles: equilibrium properties, structure, and rheology of laponite suspensions[END_REF], nontronite [START_REF] Michot | Sol-gel and isotropic/nematic transitions in aqueous suspensions of natural nontronite clay. Influence of particle anisotropy. 2[END_REF], montmorillonite and beidellite [START_REF] Paineau | Aqueous suspensions of natural swelling clay minerals. 2. Rheological characterization[END_REF]) where an exponent of 2.35 was determined.

Rheological determination of the interaction potential energy

As shown in different publications, the elastic modulus can be used to obtain information about electrostatic interactions in various systems characterized by an elastic response to small deformations, e.g. lattices [START_REF] Buscall | Viscoelastic properties of concentrated latices. Part 2.-Theoretical analysis[END_REF], aqueous clay dispersions [START_REF] Baravian | Rheological determination of interaction potential energy for aqueous clay suspensions[END_REF], sterically stabilized [START_REF] Bergenholtz | Colloidal charge determination in concentrated liquid dispersions using torsional resonance oscillation[END_REF] or weakly flocculated systems [START_REF] Goodwin | The elasticity of weakly flocculated suspensions[END_REF]. Buscall et al. [START_REF] Buscall | Viscoelastic properties of concentrated latices. Part 2.-Theoretical analysis[END_REF] were the first ones to propose a formula linking the high frequency modulus of a slightly deformed lattice with face-centered cubic symmetry (fcc) and the interaction potential that writes [START_REF] Drouin | Rheology and structure of peptized boehmite pastes[END_REF] where ψ is the interaction potential, R the center-to-center separation distance between particles, N the coordination number (assuming a f.c.c. packing configuration N = 12) and ϕ max the maximal packing (ϕ max = 0.74 for f.c.c.).

Eq. ( 13) therefore illustrates an increasing elastic response originating from enhanced electrostatic interactions (via deformation of the charged ionic clouds surrounding particles) that are governed by the fluctuations of interparticle separation distances during high frequency oscillatory shearing. Since the electrostatic potential increases steeply with decreasing separation distance, this thereby leads to deepen interaction potentials via increasing second derivative of the potential in the vicinity of its minimum (= at the nearest neighbor in contact).

In the case of a mainly repulsive system, neglecting attractive interactions, and in the limit of weak surface potentials and small κa < 3 the repulsive potential ψ(R) can be written as [START_REF] Buscall | Viscoelastic properties of concentrated latices. Part 2.-Theoretical analysis[END_REF] (14

)
where a is the sphere radius, ε r the relative permittivity of the electrolyte solution, ε 0 the permittivity in the vacuum and ψ 0 the diffuse layer potential defined from ζ-potential measurements. After its differentiation twice and combining with Eq. ( 13), one then obtains the resulting expression for elastic moduli [START_REF] Mange | A microstructural investigation of nanostructured boehmite films prepared by the sol-gel route[END_REF] Increasing concentration results in shorter distances between particles and, for spherical objects, the relation between R, a and ϕ can be written as [START_REF] Cristiani | Effect of ageing time on chemical and rheological evolution in γ-Al2O3 slurries for dip-coating[END_REF] Since elasticity is linked to a Brownian contribution that acts between particles like elastic springs to restore the microstructure, it is best investigated when the suspension is probed at high frequencies ω (ω is linked to the inverse characteristic relaxation time τ). Therefore, the G′ value that must be considered in the above described analysis is the high frequency limit of G′.

In the case of boehmite suspensions, we applied such a procedure to low and intermediate ionic strengths, as for the highest ionic strength (I 3 = 1•10 -2 M), van der Waals attraction is most likely involved, which impairs the analysis.

Two additional points must be considered. (i) charged colloidal particles cannot be considered as hard spheres due to the presence of double layers and (ii) boehmite particles are not spheres. Indeed, the SAXS analysis presented above clearly shows the existence of anisotropic features for boehmite suspensions. Consequently, some additional calculations must be performed before being able to apply Eq. ( 15) to boehmite dispersions.

Let us first introduce an effective volume fraction ϕ eff defined as [START_REF] Fauchadour | Peptization mechanisms of boehmite used as precursors for catalysts[END_REF] that depends on the range of electrostatic repulsion through κa and in which α is a scaling parameter accounting for the overlap of double layers. Whereas κa is known, α must be determined. To do this, we assume a classical dependence of viscosity for concentrated suspensions [START_REF] Quemada | Rheology of concentrated disperse systems and minimum energy dissipation principle[END_REF] as [START_REF] Zheng | Peptization mechanism of boehmite and its effect on the preparation of a fluid catalytic cracking catalyst[END_REF] where η r is the relative viscosity of the suspension defined as the ratio of its viscosity to the viscosity of the suspending medium and ϕ* is the maximum packing fraction determined experimentally from rheological tests. According to this equation, for increasing volume fraction, the viscosity of hard spheres dispersions should follow a power law with an exponent of -2 and finally diverge at maximum packing ϕ* . Fig. 6 presents the evolution of the viscosity of boehmite suspensions obtained for various volume fractions at low and intermediate ionic strengths. The extrapolation of data at zero relative viscosity provides estimates of ϕ* of 9.6 vol% and 12.1 vol% for ionic strengths of 8•10 -5 M and 8•10 -4 M, respectively (as shown in Fig. 7).

Further assuming that for particles such as the boehmite used in our study, the maximum volume fraction can be assimilated to ϕ* eff = 1 [START_REF] Baravian | Rheological determination of interaction potential energy for aqueous clay suspensions[END_REF], it then easy to determine the parameter α in Eq. [START_REF] Fauchadour | Peptization mechanisms of boehmite used as precursors for catalysts[END_REF].

Furthermore, for non-spherical particles, the evolution of the interparticle distance R as a function of volume fraction cannot be described by Eq. ( 16) and it was shown recently [START_REF] Baravian | An effective geometrical approach to the structure of colloidal suspensions of very anisometric particles[END_REF] that the following expression applies well to repulsive anisometric particles The term in the denominator in Eq. ( 19) provides the harmonic average between the isotropic state at low volume fractions and the organized state in the limit of high concentrations. The parameter n can be directly obtained from SAXS data (fitting slopes from Fig. 5) and in our case, values of 0.96 and 0.8 were taken for ionic strengths of 8•10 -5 M and 8•10 -4 M, respectively. Fig. 8 compares the experimental data obtained for G′ with the predictions made on the basis of Eq. ( 13). An increase in ionic strength leads to a decrease of G′, a trend that was already observed for PBMA systems [START_REF] Bergenholtz | Colloidal charge determination in concentrated liquid dispersions using torsional resonance oscillation[END_REF] and polystyrene lattices [START_REF] Benzing | The viscoelastic properties of ordered latices: experiments[END_REF]. It is worth noting that for the disordered state, the maximum packing number N is no longer valid. Horn et al. [START_REF] Horn | Hydrodynamic and colloidal interactions in concentrated charge-stabilized polymer dispersions[END_REF] has applied instead the perturbation theory of Bergenholtz et al. [START_REF] Bergenholtz | Colloidal charge determination in concentrated liquid dispersions using torsional resonance oscillation[END_REF] by considering a pair distribution function g(r) that reflects the spatial configuration of particles, displaying a pronounced sharp maximum at the nearest neighbor distance. In a similar way, since the plateau moduli were measured for highly concentrated gels, where particles display locally 2D stacking (Fig. 5), in this case, as a first approximation, N can be fixed to 6 by assuming the repulsive contribution of all six face-face platelet configurations.

Using radii of 30 -20 nm for I 1 = 8•10 -5 M and I 2 = 8•10 -4 M, respectively, a reasonable fit of G′ data is obtained (Fig. 8). Therefore, such values of a appear in close accordance with DLS measurements, presented in supplementary information (Appendix E, SP.6). It must be pointed out though that such values are average values whereas the actual particles likely display significant polydispersity.

Remarkably, for the lowest ionic strength it can be seen that the zeta values are over predicted by a factor of 3 in comparison with experimental ζ data acquired for diluted suspensions (see Supplementary Materials in Appendix D, SP.5). This may be well a consequence of the constraints of Eq. ( 14) for repulsive interaction potential ψ(R), which is applicable only in the limit of weak surface potentials (ψ 0 < 50 mV) [START_REF] Buscall | Viscoelastic properties of concentrated latices. Part 2.-Theoretical analysis[END_REF].

Further tests of the values obtained by fitting G′ values can be obtained by comparing the evolution with volume fractions of distance <R> obtained by SAXS with those derived from applying Eq. ( 19) as displayed in Fig. 9. The results agree fairly well, values calculated from Eq. ( 19) being higher than experimental data by a few %. Considering the complexity1 of the system under investigation, such a result is truly convincing. The systematic slight overestimation of calculated values could be due to an overestimation of the effective volume fraction that could be related to the choice that was made to fix ϕ* eff at 1.

Considering the satisfactory fit between experimental and calculated values displayed in Fig. 9, Eq. ( 19) can be used for determining the interaction potential according to Eq. ( 14). The results of such a calculation are displayed in Fig. 10. For all the regimes investigated in the present study, the electrostatic potential is higher than the energy k B T of thermal fluctuations by at least a factor of 20. The potential decays rapidly with increasing surface separation and increasing ionic strength decreases the electrostatic, which concurs with the electrical double layer concept. As this model was successfully applied to montmorillonite disk-like particles [START_REF] Baravian | Rheological determination of interaction potential energy for aqueous clay suspensions[END_REF], the results presented in our study strongly suggest that the methodology used here could really be fruitful for analyzing correlations between electrostatic effects and viscoelastic properties in colloidal suspensions of anisometric particles.

Conclusion and perspectives

Studying in details the phase diagram, rheology and structure of industrial boehmite suspensions comprised of plate-like particles has provided a consistent picture of the impact of interparticle interactions on gelation mechanisms, viscoelastic properties and structural features. In terms of phase diagram, distinct ranges of interactions modulated by salt addition have been revealed. Indeed, the transition line separating liquid suspensions from gel phases exhibits a change of slope from positive to negative when increasing ionic strength, a behavior similar to that observed for other plate-like charged colloids (i.e. laponite [START_REF] Levitz | Liquid-solid transition of Laponite suspensions at very low ionic strength: Long-range electrostatic stabilisation of anisotropic colloids[END_REF] or nontronite [START_REF] Michot | Sol-gel and isotropic/nematic transitions in aqueous suspensions of natural nontronite clay. Influence of particle anisotropy. 2[END_REF]). This slope inversion occurs at an ionic strength value (I = 8•10 -4 M), for which the corresponding Debye length becomes comparable with the platelet size. Structural analysis by small angle Xray scattering has therefore evidenced local nematic ordering far above the sol-gel transition, likewise on different clay minerals ( [START_REF] Michot | Sol-gel and isotropic/nematic transitions in aqueous suspensions of natural nontronite clay. Influence of particle anisotropy. 2[END_REF][START_REF] Paineau | Aqueous suspensions of natural swelling clay minerals. 1. Structure and electrostatic interactions[END_REF]). Still no isotropic/nematic transition was observed for boehmite, in contrast with what was found for aqueous dispersions of gibbsite [START_REF] Mourad | Sol-gel transitions and liquid crystal phase transitions in concentrated aqueous suspensions of colloidal gibbsite platelets[END_REF] or small aspect ratio nontronite [START_REF] Michot | Sol-gel and isotropic/nematic transitions in aqueous suspensions of natural nontronite clay. Influence of particle anisotropy. 2[END_REF]. The sol/gel transition appears to hinder the I/N transition, a feature that was also observed on numerous anisotropic systems such as montmorillonite [START_REF] Michot | Phase diagrams of Wyoming Na-montmorillonite clay. Influence of particle anisotropy[END_REF] or laponite [START_REF] Mourchid | Phase diagram of colloidal dispersions of anisotropic charged particles: equilibrium properties, structure, and rheology of laponite suspensions[END_REF]. The features revealed in the present paper may help in rationalizing these different situations on the basis of particle anisotropy and ionic strength. Lastly, by coupling rheological measurements with structural 14), using values of interparticle distance 〈R〉 determined from calculations, Eq. ( 19), experimentally measured zeta potentials ψ 0 and values of a as mentioned in the text. information obtained by SAXS measurements, we were able to extend the approach initially developed by Buscall et al. [START_REF] Buscall | Viscoelastic properties of concentrated latices. Part 2.-Theoretical analysis[END_REF] to the case of boehmite platelets displaying moderate polydispersity. An effective volume scaling accounting for excluded volume effects due to induced orientational and rotational motion of particles allows capturing interactions over a wide range of particle concentrations and in the limit of moderate ionic strengths before aggregation processes comes into effect. We could thus rationalize rheological data on the basis of the electrostatic interactions in the system, which provides a coherent picture of viscoelastic properties with regard to increasing volume fraction and ionic strength.

The use of such a model should be further examined on experimental data of other anisotropic systems to reinforce its validity. This could also be completed by taking into account electroviscous effects that were neglected in the present approach [START_REF] Quemada | Energy of interaction in colloids and its implications in rheological modeling[END_REF]. Additional static and dynamic birefringence measurements could also provide clearer information about the nematic ordering of platelets and future work could certainly focus on coupling rheological experiments with scattering measurements. This would strengthen the analysis of flow induced orienta-

Fig. 1 .

 1 Fig. 1. Equation of state for boehmite dispersions at various ionic strengths.

Fig. 2 .

 2 Fig. 2. Rheological phase diagram for boehmite dispersions showing the ">" shape of the sol-gel transition. Boundaries between the sol and gel phases were determined using dynamic oscillation shearing in LVE range.

Fig. 3 .

 3 Fig. 3. Example of small-angle X-ray spectra for boehmite dispersions at different volume fractions (ionic strength I 2 = 8•10 -4 M).

Fig. 4 .

 4 Fig. 4. Structure factor S eff (Q) for boehmite dispersions at I 2 = 8•10 -4 M and at different volume fractions.

Fig. 5 .

 5 Fig. 5. Evolution of the interparticle distances as a function of volume fraction of boehmite dispersions at various ionic strengths.

Fig. 6 .

 6 Fig. 6. Zero-shear viscosity curves for colloidal boehmite dispersions at ionic strengths of 8•10 -5 M and 8•10 -4 M (steady shear flow measurements).

Fig. 7 .

 7 Fig. 7. Determination of experimental maximum packing fraction for colloidal boehmite dispersions at ionic strengths of 8•10 -5 M and 8•10 -4 M.

Fig. 8 .

 8 Fig. 8. High frequency elastic modulus as a function of the volume fraction for boehmite gels at different ionic strengths. The solid lines correspond to the fitting as discussed in the text according to Eq. (15) with effective volume fraction ϕ eff given by Eq. (17). The effective maximum packing is taken 1. The zeta potentials are ψ 0 =187 mV for I 1 = 8•10 -5 M and ψ 0 = 56 mV for I 2 = 8•10 -4 M. The reciprocal Debye length κ -1 calculated from Eq. (9) is set 34 nm and 10.75 nm for I 1 = 8•10 -5 M and for I 2 = 8•10 -4 M, respectively. N = 6.

Fig. 9 .

 9 Fig. 9. Evolution of the interparticle distance with volume fraction of boehmite gels.

Fig. 10 .

 10 Fig. 10. Estimated electrostatic potential for boehmite dispersions at ionic strengths of 8•10 -5 M and 8•10 -4 M from Eq. (14), using values of interparticle distance 〈R〉 determined from calculations, Eq. (19), experimentally measured zeta potentials ψ 0 and values of a as mentioned in the text.

The complexity of the system stems from the particles polydispersity presented in Appendix A (TEM micrograph) and Appendix E (DLS measurements).

Acknowledgements

We thank Mrs Palazzo (IFPEN) for technical assistance in samples preparation, Dr D. Frot for DLS measurements and Dr V. Rouchon (IF-PEN) for TEM observations. We would like to thank synchrotron SOLEIL for beamtime allocation.

tion which might lead to changes in processing condition upon filtration of such "complex" colloidal suspensions.

CRediT authorship contribution statement

Maria Dronova: Investigation, Formal analysis, Conceptualization, Writingoriginal draft. Eric Lécolier: Conceptualization, Validation, Writingreview & editing, Supervision. Loïc Barré: Investigation, Supervision. Laurent J. Michot: Conceptualization, Investigation, Writingreview & editing, Supervision.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.colsurfa.2021.127564.


Thomas Milinkovitch
email: t.milinkovitch@hotmail.fr

Christel Lefrançois

Marie Durollet
email: marie.durollet@univ-lr.fr

Hélène Thomas-Guyon

Influence of temperature on age-related lipid peroxidation in a short-lived vertebrate (Nothobranchius furzeri)

Keywords: Aging, Temperature, Oxidative stress, Malondialdehyde, Nothobranchius furzeri, Vertebrates

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

Introduction

In the mid-50's, the "free radical theory" was proposed; this theory states that radical oxygen species (ROS) generated in cells result in damage which leads to an age-related decline in biological functions [START_REF] Harman | Aging: A theory based on free radical and radiation chemistry[END_REF]. Although subsequent research has emphasised that ageing is a multifactorial process, oxidative stress is still suggested to be a key factor in senescence [START_REF] Preedy | Aging: Oxidative stress and dietary antioxidants[END_REF]. While genetic determinism partly explains age-related oxidative stress, studies have also shown the effects of environmental insults (reviewed in [START_REF] Finkel | Oxidants, oxidative stress and the biology of ageing[END_REF].

For example, the influence of caloric restriction has been intensively researched (e.g., [START_REF] Sohal | Oxidative stress, caloric restriction, and aging[END_REF][START_REF] Mattison | Impact of caloric restriction on health and survival in rhesus monkeys from the NIA study[END_REF]. Conversely, although temperature modulates oxidative stress in ectotherms (reviewed in [START_REF] Lushchak | Environmentally induced oxidative stress in aquatic animals[END_REF], few studies have considered the influence of this factor on age-related oxidative stress [START_REF] Malek | The effects of temperature reduction on gene expression and oxidative stress in skeletal muscle from adult zebrafish[END_REF][START_REF] Valenzano | Temperature affects longevity and age-related locomotor and cognitive decay in the short-lived fish Nothobranchius furzeri[END_REF][START_REF] Hsu | Ambient temperature influences aging in an annual fish (Nothobranchius rachovii)[END_REF][START_REF] Hemmer-Brepson | Non-stressful temperature effect on oxidative balance and life history traits in adult fish (Oryziaslatipes)[END_REF][START_REF] Wang | Late-onset temperature reduction can retard the aging process in aged fish via a combined action of an anti-oxidant system and the insulin/insulin-like growth factor 1 signaling pathway[END_REF]. Moreover, these studies have investigated the influence of temperature on oxidative stress using a single measurement point in adult or senescent ectotherms, and thus, no approach has considered the effect of temperature on lipid peroxidation using two temperatures, and at several phases of an ectotherm's life.

Consequently, the preliminary study reported here is important for aging research as it investigates the influence of temperature on oxidative stress at different life phases of a vertebrate ectotherm. The species chosen for investigation was the short-lived killifish Nothobranchius furzeri, given its suitability as a model species for aging studies (Lucas-Sanchez et al. 2014;[START_REF] Cellerino | From the bush to the bench: The annual Nothobranchius fishes as a new model system in biology[END_REF]. Age-related oxidative stress was estimated through lipid peroxidation in the liver and muscles, at two temperatures: 26°C, considered as the optimal temperature for the fish since it induces a higher growth rate [START_REF] Genade | Annual fishes of the genus Nothobranchius as a model system for aging research[END_REF]; and 22°C, considered as a suboptimal temperature.

Material and methods

During experiment, the life cycle of N. furzeri (strain MZM 0410, with a maximum lifespan of 32 weeks, as described in [START_REF] Terzibasi | Large differences in aging phenotype between strains of the short-lived annual fish Nothobranchius furzeri[END_REF]) was entirely controlled. From hatching to 10 days post-hatching (dph), juveniles were fed twice a day with nauplii of Artemia salina, and water (24°C) was renewed daily. At 10 dph, individuals were gently transferred to rearing tanks and acclimatised gradually (by mixing water) at 22.1 ± 0.8°C or 25.6 ± 0.5°C (mean ± standard error of the mean). In rearing tanks (0.50 x 0.30 x 0.25 m), fish were fed twice a day ad libitum with Chironomus larvae, and were exposed to an artificial light period of 12 hours per day. Seven fish were euthanized (using 0.008% eugenol) after seven weeks (juvenile), 18 weeks (adult) and 30 weeks (senescent) post-hatching, for each temperature of acclimatization. Fish weights were 0.69 ± 0.07 g and 0.85 ± 0.16 g at 7 weeks, 1.44 ± 0.07 g and 2.22 ± 0.45 g at 18 weeks and 1.71 ± 0.30 g and 2.48 ± 0.48 g at 30 weeks (respectively for 22.1°C and 25.6°C acclimatization temperatures). Due to high variability, no significant difference in average weights were observed between the acclimatization temperature groups. The liver and axial muscle were removed from each individual and stored at -80°C. Next, these tissues were homogenised in ice-cold phosphate buffer containing Halt protease inhibitor cocktail (Thermo Scientific). The homogenates were centrifuged at 12500 g at 4°C for 15 min, and the postmitochondrial fractions (PMF) were used for assays. Total protein concentrations were determined using the method of [START_REF] Bradford | A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] with a BioRad assay kit (500-0002) containing bovine serum albumin as a standard and Brilliant Blue G 250 as a reactant. The reaction was measured spectrophotometrically at 570 nm. Lipid peroxidation was then evaluated via malondialdehyde (MDA) concentration using a commercially available MDA assay kit (Oxis International). The method was based on the reaction of a chromogenic reagent (n-methyl-2phenylindole) with MDA measured at 586 nm [START_REF] Gérard-Monnier | Reactions of 1-methyl-2-phenylindole with malondialdehyde and 4-hydroxyalkenals. Analytical applications to a colorimetric assay of lipid peroxidation[END_REF]. The results are presented in µmol of MDA/g of protein, and significant differences between the two temperatures and between the three ages were investigated using non parametric Mann-Whitney and Kruskal-Wallis tests, respectively. A Bonferroni correction was applied, and thus, differences were considered statistically significant if P ˂ 0.01.

Results and Discussion

Regarding the effect of age on lipid peroxidation, our results showed higher hepatic MDA levels (Figure 1A) in juveniles than in adult fish, for both temperatures of acclimatization. This finding highlights potential oxidative stress during the early phase of the life of the fish. This agrees with the hypothesis of developmental-phase related oxidative stress (proposed by [START_REF] Monaghan | Oxidative stress as a mediator of life history tradeoffs: Mechanisms, measurements and interpretation[END_REF]). This hypothesis suggests that there is a link between high metabolic activities for growth and ROS generation. Given the lifecycle of our model species, such a hypothesis is even more plausible. Indeed, [START_REF] Terzibasi | The short-lived fish Nothobranchius furzeri as a new model system for aging studies[END_REF] described the lifecycle of N. furzeri as "tied with explosive growth and accelerated with sexual maturation". Since the liver is highly irrigated and involved in nutrient synthesis and storage, the metabolic demand on this organ during development may have been intense, and consequently, may have induced particularly high oxidative stress. When considering both organs (Figure 1A and 1B), our results indicate an increase in MDA in senescent organisms (30 weeks), when compared to the adult phase (18 weeks). Such a pattern, obtained in both tissues, is in line with the "free-radical theory" of aging [START_REF] Harman | Aging: A theory based on free radical and radiation chemistry[END_REF], and more particularly, with recent studies conducted by [START_REF] Hsu | Age-related markers assayed at different developmental stages of the annual fish Nothobranchius rachovii[END_REF] and [START_REF] Liu | Differential expression of aging biomarkers at different life stages of the annual fish Nothobranchius guentheri[END_REF], both of which observed an increase in oxidative stress linked with a decrease in the response of antioxidant systems in aged Nothobranchius rachovii and Nothobranchius guentheri (respectively). In the current study, measurement of antioxidant enzymes in muscles and liver would have been of interest to provide a better understanding of the cellular mechanisms leading to an increase in MDA in senescent Nothobranchius furzeri.

Regarding the effects of temperature, the results did not fit with our expectations. Indeed, it is generally admitted that (i) a higher acclimatization temperature in ectotherm vertebrates induces higher metabolic rates (up to a limit of temperature above the optimal temperature, Portner and Farell 2008), and (ii) metabolic activity is responsible for the production of freeradicals [START_REF] Turrens | Superoxide production by the mitochondrial respiratory chain[END_REF]). Based on these two statements, we expected that lipid peroxidation would have been positively modulated by temperature (i.e., temperature-dependent oxidative stress mediated through variation of metabolic rate). However, in our study, this phenomenon was not observed in muscles; no significant difference in lipid peroxidation was observed between 26°C and 22°C acclimatized fish. Similar results have been reported by [START_REF] Malek | The effects of temperature reduction on gene expression and oxidative stress in skeletal muscle from adult zebrafish[END_REF][START_REF] Hemmer-Brepson | Non-stressful temperature effect on oxidative balance and life history traits in adult fish (Oryziaslatipes)[END_REF], who suggested that antioxidant defence systems (such as superoxide dismutase) neutralize the increases in ROS due to temperature.

In the liver of senescent fish, the results were contrary to our expectations; the lowest (and sub-optimal) acclimatization temperature, i.e., 22°C, led to the highest oxidative stress. A study conducted by [START_REF] Valenzano | Temperature affects longevity and age-related locomotor and cognitive decay in the short-lived fish Nothobranchius furzeri[END_REF] showed a decrease in lipofuscin in the liver cells of senescent N. furzeri acclimatized to a lower temperature (22°C compared to 25°C). The authors suggested that oxidative stress varies positively with temperature, which is in direct contrast with our findings. Such a discrepancy could be due to the origin of this oxidative stress. Indeed, in our study, by measuring MDA we estimated the lipid peroxidation in polyunsaturated fatty acids (mainly contained in the cell membrane), while in the study of [START_REF] Valenzano | Temperature affects longevity and age-related locomotor and cognitive decay in the short-lived fish Nothobranchius furzeri[END_REF], the measurement of lipofuscin estimated oxidative stress in lysosomes of post-mitotic cells.

Consequently, we suggest that a lower (and sub-optimal) acclimatization temperature in senescent fish could lead to an increase in oxidative stress in the cell membrane and, on the other hand, to a decrease in oxidative stress in lysosomes, and potentially in other intra-cellular components [START_REF] Valenzano | Temperature affects longevity and age-related locomotor and cognitive decay in the short-lived fish Nothobranchius furzeri[END_REF]. Such a discrepancy could also be due to the husbandry and/or the fish strains used. Indeed, as previously described by Di [START_REF] Cicco | The short-lived annual fish Nothobranchius furzeri shows a typical teleost aging process reinforced by high incidence of agedependent neoplasias[END_REF], different strains (or rearing conditions) could cause differences of aging processes. For instance, the incidence of liver neoplasias was accelerated in a short-lived strain. While the fish strains and the husbandry conditions are not precisely described in [START_REF] Valenzano | Temperature affects longevity and age-related locomotor and cognitive decay in the short-lived fish Nothobranchius furzeri[END_REF], these authors showed a median lifespan of 9 to 10 weeks for their fish, while our study showed a median lifespan of 34 weeks, suggesting that the husbandry and/or fish strains were different.

To explain the higher oxidative stress in the liver of senescent fish acclimatized to 22°C, we consider that decreasing efficiency of the antioxidant systems may be responsible for oxidative stress in senescent killifish (in accordance with [START_REF] Hsu | Age-related markers assayed at different developmental stages of the annual fish Nothobranchius rachovii[END_REF]. We postulate that this decrease was more important in fish acclimatized to the sub-optimal temperature than in fish acclimatized to their optimal temperature.

In conclusion, as our study was conducted at three phases of an ectotherm's life, it gives a novel insight into the influence of temperature on age-related lipid peroxidation in ectotherms.

The increase in oxidative stress with aging in the two target tissues is consistent with the "free radical theory" of aging. The high level of lipid peroxidation in the liver of juveniles is in line with the hypothesis of a developmental-phase related oxidative stress. Finally, the results indicated higher lipid peroxidation in the liver of senescent fish acclimatized to the sub-optimal temperature, than in those acclimatized to the optimal temperature. This finding disagrees with the idea that oxidative stress varies positively with temperature, and consequently, is inconsistent with the idea that the variation in oxidative stress due to acclimatization temperature is mediated via the metabolic rate. However, these findings should be interpreted with caution since effects of oxidative stress were only investigated through lipid peroxidation in this study. Thus, further studies are required to provide a more comprehensive framework for understanding the influence of temperature on age-related oxidative stress. Indeed, cross-sectional and/or longitudinal approaches should be conducted in several ectotherms, using different indicators of oxidative stress (e.g., DNA damage, protein oxidation) in target organs.

Figures captions Fig. 1 :

 captions1 Figures captions

Acknowledgments

Postdoctoral fellowship, mobility grants and experimentation costs for this work were supported by the project ALIVE (Ageing and functional integrity: modulation by LIfe-extending treatments in a VErtebrate model with extremely short lifespan) funded by Agence Nationale de la Recherche (ANR-10-JCJC-1711).