Sophie Gentès
email: so.gentes@gmail.com

Brice Löhrer

Alexia Legeay

Agnès Feurtet Mazel

Pierre Anschutz

Céline Charbonnier

Emmanuel Tessier

Régine Maury-Brachet

Drivers of variability in mercury and methylmercury bioaccumulation and biomagnification in temperate freshwater lakes

Keywords: methylmercury, fish, invertebrates, epiphyton, stable isotope, lakes

The four largest freshwater lakes in southern France are of both ecological and economic importance. However, some of them are subjected to mercury (Hg) contamination, resulting in the ban of human consumption of piscivorous fish. Moreover, beyond predatory fish, little information exist regarding Hg levels in other species of these ecosystems. In this context, we used a food web analytical approach to investigate Hg bioaccumulation and biomagnification in relation to the trophic structure of these four lakes. More specifically, various organisms (macrophytes, epiphyton, invertebrates and fish) were collected at the four lakes and analysed for carbon and nitrogen stable isotopes as well as for total Hg (THg) and methylmercury (MeHg). A spatial variability of bioaccumulation in organisms was observed, particularly in carnivorous fish, with higher Hg levels being found in the two more northern lakes (median±SE: 3491±474 and 1113±209 ng THg.g -1 dw in lakes HC and L, respectively) than in the southern pair (600±117 and 911±117 ng THg.g -1 dw in lakes CS and PB, respectively). Methylmercury biomagnification was observed through the food webs of all four lakes, with different trophic magnification slopes (HC=0.16; L=0.33; CS=0.27; PB=0.27), even though the length of the food chains was similar between the lakes. Our results suggest that rather than the food web structure, anthropogenic inputs (sulfate in northern lakes and phosphorus inputs in southern ones) may have a strong impact, more or less directly, on Hg methylation in freshwater environments, and lead to concentrations exceeding environmental recommendations despite low mercury backgrounds in sediment and water.

Introduction

Mercury (Hg) is a toxic trace metal found naturally in the environment, and whose contemporary releases mainly come from anthropogenic activities [START_REF] Driscoll | Mercury as a global pollutant: sources, pathways, and effects[END_REF][START_REF] Lindberg | A synthesis of progress and uncertainties in attributing the sources of mercury in deposition[END_REF]. Methylmercury (MeHg) is the most toxic Hg species, mostly produced within aquatic ecosystems by prokaryotes (e.g. [START_REF] Bridou | Simultaneous determination of mercury methylation and demethylation capacities of various sulfate-reducing bacteria using species-specific isotopic tracers[END_REF][START_REF] Hamelin | Methanogens: Principal methylators of mercury in lake periphyton[END_REF][START_REF] Gilmour | Mercury methylation by novel microorganisms from new environments[END_REF], bioaccumulated in biota mainly by trophic pathway, and biomagnified through aquatic food webs [START_REF] Clarkson | The toxicology of mercury and its chemical compounds[END_REF][START_REF] Watras | Bioaccumulation of mercury in pelagic freshwater food webs[END_REF]. Nonetheless, wide disparities that exist within and between ecosystems in measured Hg concentrations point to the complexity of the Hg biogeochemical cycle. Indeed, MeHg formation and transfer in food webs are influenced by a multitude of factors, such as levels of environmental inorganic Hg, the activity of prokaryotes carrying out most of the methylation processes, the bioavailability of inorganic Hg to these prokaryotes, the bioavailability of MeHg at the base of the food web, as well as the ecological mechanisms affecting the efficiency of biomagnification among which are primary productivity, habitat use, bioenergetics and food web structure [START_REF] Arcagni | Species-and habitat-specific bioaccumulation of total mercury and methylmercury in the food web of a deep oligotrophic lake[END_REF][START_REF] Braaten | Five decades of declining methylmercury concentrations in boreal foodwebs suggest pivotal role for sulphate deposition[END_REF][START_REF] Clayden | Mercury biomagnification through food webs is affected by physical and chemical characteristics of lakes[END_REF][START_REF] Eagles-Smith | Modulators of mercury risk to wildlife and humans in the context of rapid global change[END_REF][START_REF] Lavoie | Biomagnification of mercury in aquatic food webs: a worldwide meta-analysis[END_REF][START_REF] Ward | Bioaccumulation syndrome: identifying factors that make some stream food webs prone to elevated mercury bioaccumulation[END_REF]Wyn et al. 2009). Thus, identifying environmental factors influencing Hg bioaccumulation and biomagnification within specific aquatic ecosystems is essential for predicting where risks may be high for humans and wildlife, and to find remedial solutions.

The four largest freshwater lakes (Hourtin-Carcans, Lacanau, Cazaux-Sanguinet and Parentis-Biscarrosse) in southern France, are recognized as emblematic aquatic systems for both ecological and economic reasons. Recently, in the two more northern lakes (Hourtin-Carcans and Lacanau), high Hg concentrations exceeding the World Health Organization consumption recommendation (0.5 μg.g-¹ wet weight, equivalent to 2.5 μg.g-¹ dry weight (dw) WHO, 1990) were detected in the carnivorous fish zander (Sander lucioperca) and northern pike (Esox lucius) (ANSES, 2003). Significantly in this context, no local Hg emission sources have been identified in Southwestern France, nor is the region a localized 'hot spot' for Hg atmospheric emissions [START_REF] Colette | Air pollution trends in the EMEP region between 1990 and[END_REF] with Hg deposition measurements revealing normal Hg fluxes (Roustan et al. 2006 and data not shown).

Understanding these spatial differences is therefore important for the conservation of these ecosystems. On this basis, the first objective of the present study was to determine and contrast the concentrations of Hg and MeHg in primary producers, invertebrates and fishes across the four lakes. The second objective was to assess the food web structure in each lake using stable C and N isotopes. The third objective was to determine and contrast Hg biomagnification across these four lakes and investigate underlying factors to the higher Hg concentrations measured in northern lakes.

To this end, we used a food web analytical approach combining carbon and nitrogen stable isotopes, which are useful tools to reveal feeding relationships among consumers. This approach is also used to understand Hg bioaccumulation in ecosystems because of the importance of diet as a route of exposure for Hg (Hall et al. 1997). Indeed, carbon and nitrogen stable isotopes can explain variability in Hg concentrations of different animal populations [START_REF] Jardine | Applications, considerations, and sources of uncertainty when using stable isotope analysis in ecotoxicology[END_REF]. The relatively low enrichment of δ 13 C along food chains (1‰ between two trophic levels) enables discriminating the different sources of organic carbon [START_REF] Peterson | Stable isotopes as tracers of organic matter input and transfer in benthic food webs: a review[END_REF][START_REF] Hobson | A stable isotope (δ13C, δ15N) model for the North Water food web: implications for evaluating trophodynamics and the flow of energy and contaminants[END_REF]. Thereby, it may be possible to link the various consumers to the various primary producers located at the base of food chains and to locate the entry point of matter and xenobiotics into food webs [START_REF] Cabana | Modelling food chain structure and contaminant bioaccumulation using stable nitrogen isotopes[END_REF][START_REF] Vizzini | The role and contribution of the seagrass Posidonia oceanica (L.) Delile organic matter for secondary consumers as revealed by carbon and nitrogen stable isotope analysis[END_REF]. In contrast to δ 13 C, nitrogen incorporation leads to an enrichment in δ 15 N of approximately 3 to 5‰ between each trophic level [START_REF] Peterson | Stable isotopes as tracers of organic matter input and transfer in benthic food webs: a review[END_REF][START_REF] Cabana | Modelling food chain structure and contaminant bioaccumulation using stable nitrogen isotopes[END_REF], allowing the trophic position of species to be inferred. Thus, nitrogen stable isotope allow assessing simultaneously the importance of food chain length and dietary pathway in Hg biomagnification processes [START_REF] Jardine | Applications, considerations, and sources of uncertainty when using stable isotope analysis in ecotoxicology[END_REF]). In addition, the use of isotopic mixing models, based on δ 15 N and δ 13 C values in prey and predators, complements these analyses by enabling the feeding relationships between organisms to be more precisely estimated [START_REF] Phillips | Combining sources in stable isotope mixing models: alternative methods[END_REF].

Methods

Study area

The four lakes under study are located in the Nouvelle Aquitaine region (Southwestern France) (Figure 1). Lake Hourtin-Carcan (hereafter termed 'HC'; 45°06'00.4"N 1°07'05.1"W) flows into lake Lacanau ('L'; 44°58'26.6"N 1°07'18.7"W), whereas lake Cazaux-Sanguinet ('CS'; 44°29'22.1"N 1°09'35.6"W) flows into lake Parentis-Biscarosse ('PB'; 44°21'05.3"N 1°09'58.6"W). The waterway connection between each pair is via a canal with locks that control lake water level. The watershed surfaces of the lakes, which belong to a common type of ecoregion [START_REF] Carvalho | Chlorophyll Reference Conditions for European Lake Types Used for Intercalibration of Ecological Status[END_REF], are between 200 to 360 km 2 (Table S1). Thermal stratification has already been observed in the two most southern lakes, but this is not systematic and the waters do not reach complete anoxia. Cyanobacteria blooms appear frequently in the eutrophic PB lake due to high amounts of sedimentary phosphorus [START_REF] Cellamare | Ecological assessment of French Atlantic lakes based on phytoplankton, phytobenthos and macrophytes[END_REF]). The lakes have been subjected to increasing anthropogenic pressure for several years, mainly due to recreational activities, especially angling and boating. They have also been exposed to the development of invasive aquatic macrophytes such as Lagarosiphon major and Egeria densa, particularly for the L and PB lakes [START_REF] Bertrin | Effects of wind-induced sediment resuspension on distribution and morphological traits of aquatic weeds in shallow lakes[END_REF]. The PB lake presents the highest colonized surface of invasive macrophytes (4.17 km 2 ), followed by L (1.19 km 2 ), HC (0.94 km 2 ) and CS (0.17 km 2 ; surface area of dense stands > 50 g.m -2 dw; [START_REF] Bertrin | Effects of wind-induced sediment resuspension on distribution and morphological traits of aquatic weeds in shallow lakes[END_REF]). Asiatic clams, crayfish (Procambarus clarkii) and stems of aquatic macrophytes (Lagarosiphon major, Egeria densa and Phragmites australis) were collected by hand.

Sampling and sample preparation

Special attention was given to the macrophytes to preserve their epiphyton during collection.

Epiphyton fixed on submerged reed stems (Phragmites australis, a common plant in the four lakes) was collected by scraping and washing with field water, maintained at 4°C for 24 h, then centrifuged for 3 min at 10 000 g. An extensive fishing sampling campaign (with both nets and lines) provided 490 individuals of various fish species. Five species common to the four lakes were targeted (n = 353) in order to compare the bioaccumulation and biomagnification of Hg between lakes: zander, northern pike, European perch (Perca fluviatilis), common bream (Abramis brama) and common roach (Rutilus rutilus). Based on the fact that size is correlated with the age of individuals, a size class (Table S2) was chosen in order to compare total Hg concentrations per lake for the five species (n = 128, Table 1).

For Hg speciation and nitrogen and carbon stable isotope analyses, five individuals of each species and lake were selected. Extended fishing facilities at lake CS only (permitted by a collaboration with a fishing sampling campaign organized by the French Agency for Biodiversity) resulted in the capture of additional fish species, namely bleak (Alburnus alburnus), white bream (Blicca bjoerkna), gudgeon (Gobio gobio), ruffe (Gymnocephalus cernua) and black bullhead (Ameiurus melas) (see Table 1 for details). Dorsal muscle tissues free of skin were dissected from fish, while muscle free of shell and soft tissues were dissected from crayfish and molluscs, respectively. A single tissue sample from molluscs comprised 10 pooled individuals. Samples were freeze-dried and homogenised by automatic grinding using Teflon® balls and mortars, washed with 3% HCl and rinsed with ultrapure water between each sample. The ground material obtained was stored in ultraclean amber glass tubes at 4°C.

Total mercury analysis

Total Hg (THg) concentrations in samples were determined by flameless atomic absorption spectrometry (Altec AMA 254). The detection limit of this method is 0.01 ng and the limit for quantification is 0.010 µg.g -1 dw. Reference material IAEA 436 (International Atomic Energy Agency, Tuna Fish Flesh Homogenate) was used every ten samples to control analytical accuracy, which averaged 101.5%.

Mercury speciation analysis

Plants and epiphyton were digested with 6N nitric acid, whereas fish and crayfish muscle and mollusc soft tissues were digested with TMAH (Tetramethylammonium hydroxide), under microwave radiation. All samples were analysed by GC-ICP-MS (gas chromatography-inductively coupled plasma-mass spectrometry; Focus GC and ICPMS X2 series, Thermo Electron) as described elsewhere [START_REF] Martin-Doimeadios | Application of isotopically labeled methylmercury for isotope dilution analysis of biological samples using gas chromatography/ICPMS[END_REF][START_REF] Clémens | Mercury speciation analysis in seafood by species-specific isotope dilution: method validation and occurrence data[END_REF]. Quantification of Hg species was performed by species-specific isotope dilution, by adding the appropriate amount of isotopically enriched Hg standards ( 199 iHg and 201 MeHg) [START_REF] Martin-Doimeadios | Application of isotopically labeled methylmercury for isotope dilution analysis of biological samples using gas chromatography/ICPMS[END_REF]. Each assay was analyzed three times, with the measurement error for MeHg and IHg being <2%. Data quality was checked by blanks and by IAEA 407 reference material (Fish Homogenate) with a recovery rate of 93.4% for MeHg and 106.6% for IHg. The limits of quantification are 0.02 ng.L -1 for IHg and 0.005 ng.L -1 for MeHg. Hg concentrations are expressed in ng.g -1 on a dry weight basis.

Stable isotope analysis

Homogenized powder samples were weighed into tin capsules using a microbalance (XPE26, Mettler Toledo®). Isotopic analyses were performed by the platform 'Spectrométrie Isotopique' (LIENSs laboratory, La Rochelle) using a Thermo Scientific Delta V Advantage isotope ratio mass spectrometer [START_REF] Chartier | In french. Analyses isotopiques par spectrométrie de masse -Méthodes et applications[END_REF]). The 13 C/ 12 C (denoted δ 13 C) and 15 N/ 14 N (denoted δ 15 N) ratios, expressed in ‰, were calculated as the relative differences between the sample and the conventional standard following [START_REF] Peterson | Stable isotopes in ecosystem studies[END_REF]. Standards were run in duplicate every twenty measurements. The analytical precision for δ 13 C and δ 15 N were 0.2‰ and 0.3‰, respectively. According to [START_REF] Post | Getting to the fat of the matter: models, methods and assumption for dealing with lipids in stable isotope analyses[END_REF], organisms with a C/N ratio of > 4 (solely molluscs and bleak) were systematically lipid extracted. Lipids were removed by successive cyclohexane washing (addition of 4 ml cyclohexane to 15 mg of homogenized powder samples, placed 5 min in an ultrasonic bath, 10 min vortex, and centrifugation 5 min at 4500 rpm). The supernatant containing lipids was removed and cyclohexane washings were performed on the pellet until the supernatant was clear, then the pellet was dried at 45°C. Carbonate extraction was also tested for macrophytes and epiphyton from the four lakes.

Carbonates were removed prior to elemental analyses by adding HCl 0.5 N on samples (until cessation of bubbling) and placed for 3 min in an ultrasonic bath. Subsequently, samples were dried at 50 °C, homogenized using an ultrasonic bath after addition of milliQ water, and freeze-dried before ground again. No between lake differences were observed, except for reed epiphyton from HC where the δ 13 C value after carbonate extraction was kept. where TPi represents the average trophic position of species i; δ 15 Ni the average δ 15 N value of species i; δ 15 Npc the average δ 15 N value of primary consumers; 3.4 the mean δ 15 N trophic enrichment occurring per trophic level [START_REF] Post | Using stable isotopes to estimate trophic position: models, methods, and assumptions[END_REF]; and 2, the trophic position of the baseline organism (filter feeders in our study). Because of our inability to find a filter feeder in lake HC and since HC waters flow into lake L, calculations of an organism's trophic position in the former lake were based on the δ 15 N value for filter feeders from the latter.

Data analyses

Isotopic mixing model

The relative isotopic contributions of the different food sources to the three carnivorous fish species studied were estimated by incorporating C and N isotope data into a Bayesian stable isotope mixing model (SIMMR package in R software, [START_REF] Parnell | Bayesian stable isotope mixing models[END_REF].

Potential prey for each of these species were chosen according to the literature [START_REF] Bruslé | Biologie des poissons d'eau douce européens[END_REF][START_REF] Keith | Les poissons d'eau douce de France[END_REF][START_REF] Schlumberger | Poissons des lacs naturels français: Ecologie des espèces et évolution des peuplements[END_REF] and validated with stomach content observations: crayfish, common roach and common bream were identified as common food sources for European perch, northern pike and zander; juvenile and small European perch were considered as additional prey for northern pike and zander. We used trophic enrichment factors of 2.90 ±0.32‰ for nitrogen and 1.30 ±0.30‰ for carbon between prey and predator muscles, as previously advised in the study of Mc Cutchan et al. (2003). The degree of uncertainty associated with the mixing-model outputs was usually close to 10%.

Mercury biomagnification factors

Biomagnification factor (BMF)

Biomagnification factor (BMF) is a magnification factor between a predator and its main preys. To quantify the proportion of sources (PS) consumed by a predator (consumer, C), we used our results obtained from the stable isotope mixing models in the following equation modified according to [START_REF] Lavoie | Trophic structure and mercury distribution in a Gulf of St. Lawrence (Canada) food web using stable isotope analysis[END_REF]:

BMFPSC = [Hg] predator / ( ∑ ([Hg] prey i × fprey i )) (Eq 2)
where [Hg]prey i is the Hg concentration of prey i and fprey i is the proportion of prey i in the diet of its predator. Proportions of sources are provided in Table S3.

Trophic Magnification Slope (TMS)

The biomagnification potential throughout the entire food web for each lake was assessed using the slope (b), termed Trophic Magnification Slope (TMS), of the simple linear regression that included all organisms (Eq 3):

Log10[Hg] = b (δ 15 N) + a (Eq 3)
where a is the intercept. As for the TP calculation (Eq 1), we used δ 15 N values from filter feeders taken from lake L to calculate the TMS for HC.

Food web magnification factor (FWMF)

In order to compare different ecosystems, the biomagnification potential through each lake's entire food web was corrected by taking into account the different trophic enrichment factors and the baseline differences and was calculated as follow [START_REF] Fisk | Influence of chemical and biological factors on trophic transfer of persistent organic pollutants in the northwater polynya marine food web[END_REF]:

FWMF = 10 b (Eq 4)
where b is the TMS from Eq (3) using TL instead of δ 15 N.

Statistical analyses

Factorial ANOVAs were used to study differences in species THg concentrations for each site. Assumptions of normality and homoscedasticity of the error term were tested. If the assumption was satisfied, the parametric post hoc LSD Fisher test was applied; if not, the nonparametric Kruskal-Wallis test was used. Comparisons of means within the same lake for two different groups of individuals (e.g. carnivorous versus omnivorous) were performed using Student's t-test when the assumption was met or the non-parametric Wilcoxon test when it was not met. An ANCOVA followed by a post hoc Tukey's test was run to check if the difference in TMS differed significantly between lakes. In each test, p < 0.05 was considered significant. All statistical analyses were performed using STATISTICA version 6.1 software (Statsoft, USA).

Results

Mercury concentrations in biota

Total Hg concentrations measured in macrophytes, epiphyton, invertebrates and low-trophic level fish (both herbivorous and omnivorous) in all four lakes ranged from 5.5 ng. g -1 (plant Egeria densa, PB) to 982 ± 325 ng. g -1 (common roach, CS, Table 1). Carnivorous fish (European perch, northern pike, zander) expressed the highest THg concentrations and MeHg percentages in all lakes, particularly in HC (maximum value: 7384 ± 1536 ng. g -1 , 94% MeHg in zander, Table 1). Total Hg and MeHg concentrations were also averaged for omnivorous and carnivorous fish to compare Hg levels between lakes (Figure 2). In omnivorous species, a significant lake effect on THg concentrations was observed (ANOVA, F3,89=3.2, p=0.03).

Post hoc LSD Fisher's multiple comparison test indicated that THg concentrations were significantly lower in PB than in the other three lakes (all p<0.05). However, no significant effect of lake with respect to MeHg concentrations was observed (F3,27=1.4, p=0.26). In carnivorous species, a significant lake effect was observed for both THg (F3,124=25.04, p<0.001) and MeHg (F3,40=14.1, p<0.001) concentrations. Both THg and MeHg concentrations were significantly higher in HC than in the other lakes (all p<0.001).

Moreover, MeHg concentrations were also significantly higher at L and CS than at PB (both p<0.05).

3.2 Food web structure and feeding behavior of top predators Carbon (δ 13 C) and nitrogen (δ 15 N) stable isotopes mean values for the different species analysed are summarised in Table 1 and plotted for each lake in Figure 3 in order to visualize the food web relationships among the species. For three of the four lakes (L, CS and PB), the S3. Overall, these species had a similar diet, both within and between lakes. However, some variability was observed: at HC, L and CS: northern pike and European perch had higher isotopic contribution from benthic prey in their diet (for northern pike, 23 to 51% crayfish and 18 to 53% common bream; for European perch, 44 to 50% crayfish and 24 to 25% common bream) than of pelagic prey (for northern pike, 8 to 18% common roach and 13 to 15% European perch; for European perch, 15 to 26% common roach). Conversely, zander had approximately equal isotopic contribution of the different prey species (see Table S3 for details).

δ

Mercury biomagnification factors

The significant and positive linear relationships between log10[MeHg] and δ 15 N values (Figure 4) indicated that MeHg biomagnified along the food web of each lake (HC: F1,23=7.5, R 2 =0.21, p=0.01; L: F1,24=39.2, R 2 =0.60, p<0.01; CS: F1,45=184, R 2 =0.80, p<0.001; PB: F1,30=136.4, R 2 =0.81, p<0.001). Trophic Magnification Slopes (TMS) and Food Web Magnification Factors (FWMF) were calculated based on MeHg and THg concentrations measured in organisms from each lake (Table 2). The TMS for THg and MeHg ranged from 0.13 (PB) to 0.23 (L) and from 0.16 (HC) to 0.33 (PB), respectively. The TMS for MeHg was significantly steeper than for the THg of each lake (L: F52,53=4.13; p<0.05; CS: F100,101=11.13; p<0.001; PB: F68,69=20.86; p<0.001), except for HC (HC: F51,52=0.03; p=0.86). A significant lake effect on the TMS for MeHg was observed (ANCOVA, F3,122 = 2.8, p = 0.045), with lower TMS values measured at HC than the three other lakes (Post hoc Tukey's test, all p<0.01). Food web magnification factors ranged from 2.96 (PB) to 5.35 (L) for THg and from 3.48 (HC) to 12.87 (L) for MeHg. For carnivorous fish, the highest BMFpsc was observed for European perch from HC for both THg and MeHg (9.5), whereas the lowest BMFpsc was observed for zander from L for THg (2.0) and for northern pikes from L and CS for MeHg (2.9) (Table 1). For lakes L, CS and PB, the BMFpsc for carnivorous fish were systematically higher than the FWMF for MeHg, which was not the case for lake HC (Tables 1 and2).

Discussion

Mercury concentrations in biota

Total Hg concentrations in epiphyton and macrophytes (Table 1) were relatively similar to those previously measured (Gentès et al. 2013a) in all four lakes, except PB where Hg levels were six times higher in epiphyton associated with Egeria densa than in Egeria densa itself. This could be explained by the fact that epiphyton is known to be a trap for pollutants such as Hg, due to their rich organic matter [START_REF] Ortelli | Micropollutants in fish and freshwater crayfish from Geneva Lake[END_REF]). However, THg levels in the carnivorous fish were close to, or exceeded, the WHO guideline, especially in the two northern lakes (HC and L), where THg levels were two to six times higher (in fish of similar standard length) than in fish from similar ecosystems [START_REF] Babut | In french. Contamination des poissons d'eau douce par les PCB et d'autres contaminants persistants dans le bassin Rhône-Méditerranée[END_REF]Luczynska et al. 2018), but were close to THg levels found in disturbed temperate freshwater lakes [START_REF] Gorski | Factors affecting enhanced mercury bioaccumulation in inland lakes of Isle Royale National Park, USA[END_REF][START_REF] Chasar | Mercury cycling in stream ecosystems. 3. Trophic Dynamics and Methylmercury Bioaccumulation[END_REF] or tropical ecosystems [START_REF] Berzas Nevado | Mercury in the Tapajós River basin, Brazilian Amazon: a review[END_REF].

Moreover, a spatial variability in MeHg concentrations between lakes was observed in the three carnivorous fish, highlighting a South-North positive gradient for MeHg, although this was less evident than for THg. A similar gradient for MeHg has been previously observed in the organic sediment of these lakes [START_REF] Canredon | Lake sediment mercury biogeochemistry controlled by sulphate input from drainage basin[END_REF], and also in epiphyton and crayfish, although statistical testing was lacking due to a paucity of sampled organisms.

Food web structure

In our study, carbon and nitrogen isotopic data suggest that the food web structures were not significantly different between the four studied lakes (despite that less organisms were sampled in lakes HC and L). Indeed, we found few differences between trophic levels of organisms (i.e., the same trophic guilds), and equivalent food web lengths (even if food web length of the two most southern lakes were slightly longer than the two northern ones).

However, significantly higher nitrogen ratios found in carnivorous fish of lake PB than at three other lakes show higher enrichment in nitrogen, but could be explained by the presence of high biomass of invasive macrophyte species [START_REF] Bertrin | Effects of wind-induced sediment resuspension on distribution and morphological traits of aquatic weeds in shallow lakes[END_REF] and regular cyanobacteria bloom [START_REF] Cellamare | Ecological assessment of French Atlantic lakes based on phytoplankton, phytobenthos and macrophytes[END_REF]) in this eutrophic system, leading to a higher incorporation of atmospheric nitrogen.

Methyl Hg trophic transfer efficiency in carnivorous fishes is influenced by the composition of food web and feeding relationships [START_REF] Vander Zanden | A Trophic Position Model of Pelagic Food Webs: Impact on Contaminant Bioaccumulation in Lake Trout[END_REF][START_REF] Cabana | Modelling food chain structure and contaminant bioaccumulation using stable nitrogen isotopes[END_REF]. Here, it is thus unlikely that food web structure was the main factor explaining the observed differences in Hg bioaccumulation in top predatory fishes. On the other hand, processes at the base of food webs that influence MeHg availability are generally the principal contributors to bioaccumulation and biomagnification of Hg in ecosystems [START_REF] Gorski | Factors affecting enhanced mercury bioaccumulation in inland lakes of Isle Royale National Park, USA[END_REF][START_REF] Chasar | Mercury cycling in stream ecosystems. 3. Trophic Dynamics and Methylmercury Bioaccumulation[END_REF][START_REF] Molina | Transfer of mercury and methylmercury along macroinvertebrate food chains in a floodplain lake of the Beni River, Bolivian Amazonia[END_REF]). Significant differences observed in δ 13 C values between invasive macrophytes and other organisms indicated that macrophytes do not represent a major feeding resource for grazing fish (common roach) or detritivorous (crayfish) in the four lakes. Conversely, their epiphyton (epiE, epiphyton Egeria) and epiphyton associated with endemic plants (epiR, epiphyton reed) appears to be a food source for these organisms. Previous studies have highlighted the relative importance of epiphytic algae compared to macrophytes as a carbon source in benthic food webs (France, 1995a). In addition to allochtonous inputs, therefore, other autochthonous sources of carbon need to be investigated within the trophic chain, such as phytoplankton, other macrophyte species and their associated epiphyton, as well as particulate organic matter, in order to assess more precisely food web functioning [START_REF] France | Stable isotopic survey of the role of macrophytes in the carbon flow of aquatic foodwebs[END_REF].

Methyl Hg biomagnification

A significant MeHg biomagnification was observed along the food webs of all lakes (Figure 4) with TMS values similar to those observed in other temperate freshwater lakes [START_REF] Lavoie | Biomagnification of mercury in aquatic food webs: a worldwide meta-analysis[END_REF]. Food Web Magnification Factor (FWMF) were similar to equivalent data from other studies [START_REF] Lavoie | Trophic structure and mercury distribution in a Gulf of St. Lawrence (Canada) food web using stable isotope analysis[END_REF][START_REF] Fisk | Influence of chemical and biological factors on trophic transfer of persistent organic pollutants in the northwater polynya marine food web[END_REF]) and were higher for MeHg than for THg in all the lakes, but this was not systematically the case for the BMFpsc of carnivorous fish, which showed the greatest bioavailability and the largest proportion of MeHg in organisms. The elevated BMFpsc calculated for European perch from HC could be explained by the greater weight of individuals sampled from this lake.

The trophic transfer efficiency of Hg depends on local geochemical and biological factors [START_REF] Luoma | Why is metal bioaccumulation so variable?[END_REF][START_REF] Veltman | Metal bioaccumulation in aquatic species: quantification of uptake and elimination kinetics using physicochemical properties of metals[END_REF]. In a related aspect of the current project, an extensive mapping of the sediment characteristics and their Hg rates was conducted in the four lakes [START_REF] Canredon | Lake sediment mercury biogeochemistry controlled by sulphate input from drainage basin[END_REF], which revealed no sediment contamination and similar THg concentrations (averaged concentration in organic sediment for the four lakes: 213 µg THg.g -1 dw). However, the MeHg proportion in organic sediment was higher in HC than the other three lakes (2.5± 1% of THg for lake HC, 1.7± 0.7% for L, 0.5± 0.1% for CS and PB; [START_REF] Canredon | Lake sediment mercury biogeochemistry controlled by sulphate input from drainage basin[END_REF], following the same South-North positive gradient as that observed in the present study for carnivorous fish and crayfish. Since the origin of carbon in HC seems to be more benthic than in the other lakes, HC's organic sediment and the epifauna living on this substrate (such as crayfish) could be the main entry point of MeHg into the food web. The MeHg trophic transfer efficiency associated with benthos is generally considered to be less than that associated with the pelagic compartment [START_REF] Cossa | Mercury speciation in the Lower St. Lawrence Estuary[END_REF], thus explaining the lowest TMS observed for HC. Here, the greater input of MeHg in sediment would offset the latter's lower trophic transfer efficiency, thereby providing an explanation for the highest biota contamination in this lake compared to the others.

In their study, [START_REF] Canredon | Lake sediment mercury biogeochemistry controlled by sulphate input from drainage basin[END_REF] also highlighted a South-North positive gradient of sulfate in the water column (PB = 114 µM, CS = 126 µM, L = 276 µM and HC = 392 µM), with optimal concentrations for the development of sulfate reducing microorganisms (SRM)

occurring in the HC water column. Sulfate reducers are considered as the main source of MeHg production in the environment [START_REF] King | A quantitative relationship that demonstrates mercury methylation rates in marine sediments are based on the community composition and activity of sulfate-reducing bacteria[END_REF][START_REF] Bridou | Simultaneous determination of mercury methylation and demethylation capacities of various sulfate-reducing bacteria using species-specific isotopic tracers[END_REF]. Moreover, a laboratory incubation with sulfate-enrichment of epiphyton on floating macrophyte roots (Ludwigia sp.) from CS and two other ecosystems of the region showed that Hg transformation processes were mainly due to SRM activity (Gentès et al. 2013b).

Consequently, in these lakes, the sulfate concentration gradient in water could entail a biotic MeHg production gradient in epiphyton as well as in organic sediment. A natural origin of sulfate is unlikely since (i) the geological formation of these lakes and their respective watersheds are similar, and (ii) the distances between them are relatively small. Agricultural activity has been identified as a source of sulfate for these lakes, especially HC, due to the addition of lime to soils to control crop pH [START_REF] Canredon | Lake sediment mercury biogeochemistry controlled by sulphate input from drainage basin[END_REF]. A survey on the use of sulfate by local farmers should therefore be conducted to further examine this hypothesis. If confirmed, solutions could be proposed to reduce these inputs and thus lower Hg concentrations in the biota of the more northern lakes [START_REF] Braaten | Five decades of declining methylmercury concentrations in boreal foodwebs suggest pivotal role for sulphate deposition[END_REF].

Parentis-Biscarrosse is the lake with the lowest THg and MeHg concentrations measured in biota in parallel with the lowest sulfate concentrations measured in its water [START_REF] Canredon | Lake sediment mercury biogeochemistry controlled by sulphate input from drainage basin[END_REF]. However, in addition to the presence of invasive macrophytes [START_REF] Bertrin | Effects of wind-induced sediment resuspension on distribution and morphological traits of aquatic weeds in shallow lakes[END_REF], cyanobacterial blooms occur regularly in PB [START_REF] Cellamare | Ecological assessment of French Atlantic lakes based on phytoplankton, phytobenthos and macrophytes[END_REF]). Another explanation for such low Hg levels in PB biota could be the presence of multiple carbon sources that in turn enhance the dilution factor for Hg bioavailability at the base of the food chain [START_REF] Chen | High plankton densities reduce mercury biomagnification[END_REF]. As a consequence, biodilution would be an indirect beneficial effect of the eutrophication on Hg dynamics in this lake, reducing bioaccumulation and the biomagnification factor in top predators.

Seasonal variation can affect TMS, for example through changes in δ 15 N values in lower TP organisms, with a resultant influence on the entire food web (Borgå et al. 2011). To assess Hg biomagnification variability during the course of a year, different seasonal sampling of biota should be considered for these lakes, and particularly PB.

Conclusion

In this study, THg and MeHg concentrations were measured in the biota of the four largest lakes of southwestern France, in relation to their trophic structure. High Hg concentrations in piscivorous fish from the two northern lakes were confirmed, whereas fish from the two more southern lakes were below the WHO consumption recommendation. Methyl Hg biomagnification was observed in all four lakes, and surprisingly, the most contaminated lake displayed the lowest TMS. Food web structure alone could not explain this difference, although sulfate concentrations in the water column seems to be the main driver. In addition, the lake with the lowest Hg concentrations in biota was eutrophic as a consequence of preceding phosphorus inputs, and was subjected to regular algal blooms. Eutrophication could therefore have an indirect beneficial (reducing) effect on Hg bioaccumulation. This study therefore highlights the view that even in environments with very low mercury backgrounds in sediment and water, and beyond the immediate interest of controlling mercury release, other anthropogenic inputs (here, sulfate and phosphorus) may have a strong impact, more or less directly, on Hg methylation in freshwater environments.

Future studies of these ecosystems will aim to assess the methylation and demethylation potentials in different compartments at the base of the food web (organic sediment, epiphyton associated with invasive and endemic plants) using enriched stable isotopes of Hg to identify the role played by each compartment in Hg transformations. In addition, microorganism diversity associated with each compartment will be studied, as well as sulfate reduction rates, since MeHg production in these aquatic compartments seems to be linked to the activity of sulfate reducers. Additionally, important information would derive from characterizing the Hg sources in these lacustrine ecosystems using Hg stable isotopes (isotopic fractionation).

WHO (World

  were mainly collected during the autumn of 2015. Macrophytes and Asiatic clams (Corbicula fluminea) were also collected during the fall of 2017 and 2018 (Asiatic clams: 2017 in lake L, 2018 in lake PB; Lagarosiphon: 2016 in lake CS, 2017 in lakes PB and HC; Egeria: 2017 in lake PB). Isotopic and Hg data (see below) were pooled in assuming similar food web structures and baselines in both sampling years. As part of this study, sediment sampling in the four lakes has been previously conducted and reported (collected in April 2014 and in January 2015 for the two northern lakes, and in spring and summer 2016 for the two southern lakes; Canredon et al. 2019).

  2.6.1 Trophic position calculationThe trophic position (TP) of each organism was calculated based on δ 15 N values and following the equation of[START_REF] Bergamino | Food web structure of sandy beaches: Temporal and spatial variation using stable isotope analysis[END_REF]: TPi = [(δ 15 Niδ 15 Npc) / 3.4] + 2 (Eq 1)

Figure 1 .

 1 Figure 1. Location of the four sampled freshwater lakes in southwestern France.

Figure 2 .

 2 Figure 2. Boxplots of total mercury (left) and methylmercury (right) concentrations in muscle tissue of carnivorous (zander, northern pike and European perch) and omnivorous fish species (common roach and common bream) in lakes Hourtin-Carcans (HC), Lacanau (L), Cazaux-Sanguinet (CS) and Parentis-Biscarrosse (PB). n: number of samples. Letters indicate statistical differences (p < 0.05).

Figure 3 .

 3 Figure 3. Relationships between δ 15 N and δ 13 C mean values (‰) of species in the four lakes. Bars represent standard error. lag: Lagarosiphon, ege: Egeria, epiR: epiphyton reed, epiE:epiphyton Egeria, epiL: epiphyton Ludwigia, cray: crayfish, roa: common roach, bre: common bream, per: European perch, pik: northern pike, zan: zander, clam: Asiatic clams, ble: bleak, wbre:white bream, gud:gudgeon, ruf: ruffe, bbh: black bullhead.

Figure 4 .

 4 Figure 4. Averages of log10 MeHg concentrations versus trophic levels (δ 15 N) measured in biota of the four lakes. Bars represent standard errors for δ 15 N and [MeHg]. The linear regression and its correlation coefficient (R 2 ) were calculated for all individual species. lag: Lagarosiphon, ege: Egeria, epiR: epiphyton reed, epiE: epiphyton Egeria, epiL: epiphyton Ludwigia, cray: crayfish, roa: common roach, bre: common bream, per: European perch, pik: northern pike, zan: zander, clam: Asiatic clams, ble: bleak, wbre:white bream, gud:gudgeon, ruf: ruffe, bbh: black bullhead.

  13 C values were significantly different between species (Kruskal-Wallis test, L: H = 19.00; CS: H = 36.22; PB: H = 30.20, all p<0.01) except between the Asiatic clams and European perch in lake L, between gudgeon and ruffe and gudgeon and Asiatic clams in lake CS, and between the Asiatic clams and northern pike in PB (all p < 0.05). Carbon values were not

	fish were significantly different between lakes (Kruskal-Wallis test, H = 47.28, p < 0.001)
	with higher values in PB than in the other lakes p < 0.05). Trophic positions (TP, Table 1)
	ranged from 1.4 ± 0.1 for crayfish (HC) to 4.1 ± 0.1 for zander (PB). Trophic positions of
	zanders (considered as the top predator) were not statistically different between lakes

significantly different between species for HC (ANOVA, F8,22=0.91, p = 0.53). Carbon isotopic ratios in carnivorous fish were significantly different between lakes (ANOVA, F3,51=0.91, p = 0.15) with higher values occurring in HC than in the other three lakes (Post hoc LSD Fisher test, p < 0.001). Few significant differences in δ 15 N values were found between species within each lake (Kruskal-Wallis test, HC: H = 24.76; L: H = 23.11; CS: H = 47.00; PB: H = 40.56, all p<0.01), and when present, were mainly between species of low-and high-trophic levels (HC: zander vs common roach / zander vs crayfish; L: clam vs northern pike; CS: zander vs clam / zander vs gudgeon; PB: zander vs clam, all p < 0.05). Nitrogen isotopic ratios in carnivorous (Kruskal-Wallis test, H = 9.98, p = 0.018). The diets of zander, northern pike and European perch are presented in Table

Table 1 .

 1 Average mercury concentrations (Mean THg, ng.g -1 dry weight), percentages of MeHg (%), stable isotope ratios (δ 15 N and δ 13 C, ‰) and trophic positions (TP) of organisms sampled from the four lakes. Biomagnification Factor (BMFPSC) were calculated in zander,

	Lakes	Species	Common name (code) Trophic guild Body weight	Standard length	N/n	Mean δ 13 C	Mean δ 15 N	TP	Mean [THg]	% MeHg	BMF PSC THg	BMF PSC MeHg
	Hourtin-	Lagarosiphon major	Lagarosiphon (lag)	PP			1/1	-12.8	10.3		187.1	7.5
	Carcans	Epiphyton Phragmites australis	(epiR)				1/1	-17.6	8.4		35.8	53.7
	(HC)	Procambarus clarkii	Crayfish (cray)	Det			5/4	-19.5±1.2	5.7±0.4	1.4±0.1	523.9±79.6	90.7±3.2
		Rutilus rutilus	Common roach (roa)	Omni-Herbi	281±135	22±4	5/4	-15.7±1.3	11.3±0.5	3.1±0.1	803±218.4	97.2±3.2
		Abramis brama	Common bream (bre)	Omni-Benth	427±387	25±8	5/4	-21.7±0.8	8.8±0.9	2.4±0.3	440.1±110.6	90.4±1.5
		Perca fluviatilis	European perch (per)	Carni-Pisci	1660±416	36±3	5/4	-19±0.8	10.3±0.9	2.8±0.3	5463.9±1023.3 96.3±0.6	9.5	9.5
		Esox spp.	Northern pike (pik)	Carni	2030±416	56±3	5/4	-19.9±1.1	10.4±1.1	2.8±0.3	3518.5±1317.5 96.9±1.5	3.1	3.2
		Sander lucioperca	Zander (zan)	Carni-Pisci	2914±429	60±5	3/3	-18.9±0.7	12.7±0.1	3.5±0	7383.8±1536.1 93.9±2.7	3.6	4.3
	Lacanau	Epiphyton Phragmites australis Corbicula fluminea	(epiR) Asiatic Clam (clam)	PP FF	0.95±0.05	32±3	1/1 5/5	-23.5 -26.6±0.1	6.4 7.6±0.2	2	30.8 138.1±13.9	43 8.8±1.3
	(L)	Procambarus clarkii	Crayfish (cray)	Det			5/4	-23.1±2.1	8.6±0.6	2.3±0.2	260.1±64.9	87.4±2.5
		Rutilus rutilus	Common roach (roa)	Omni-Herbi	89-421	15-25	2/2	-25.2; -17.1	10.5-5.5	2.1±1.1	414.3-206.7	93.1-100.0
		Abramis brama	Common bream (bre)	Omni-Benth	1454±186	40±2	5/4	-26.5±1.6	9±1.2	2.4±0.3	753.4±216.6	93±1.3
		Perca fluviatilis	European perch (per)	Carni-Pisci	910±421	33±5	5/4	-23±0.7	11.3±0.8	3.1±0.2	1961.2±1500.7 96.2±1.1	4.2	5.0
		Esox spp.	Northern pike (pik)	Carni	2415±452	62±4	5/4	-24±0.5	11.9±0.6	3.3±0.2	2160.5±834.9	94.5±1.1	2.8	2.9
		Sander lucioperca	Zander (zan)	Carni-Pisci	2400-5100	56-71	2/2	-25.5; -22.5	11.9-13.7	3.5±0.4	4809.3-2642.9	96.4-96.3 2.0	4.9
	Cazaux-	Lagarosiphon major	Lagarosiphon (lag)	PP			1/1	-15.6	5.4		41.3	5.5
	Sanguinet	Epiphyton Phragmites australis	(epiR)				1/1	-21.5	5.1		23.3	35.8
	(CS)	Corbicula fluminea	Asiatic Clam (clam)	FF	0.5±0.1	20±2	3/3	-24±0.1	8.3±0.2	2	295.8±18.2	8.8±0.6
		Procambarus clarkii	Crayfish (cray)	Det			2/2	-23.1; -25.6	9.9-9.5	2.4±0.1	181.8-240.4	79.0-89.6
		Rutilus rutilus	Common roach (roa)	Omni-Herbi	270±146	23±4	5/5	-23.4±0.5	12.9±1	3.3±0.3	982.5±325.5	91.5±3
		Abramis brama	Common bream (bre)	Omni-Benth	1146±496	37±5	5/4	-23.4±1.5	12.8±0.7	3.3±0.2	234.7±110.4	83.9±7.1
		Perca fluviatilis	European perch (per)	Carni-Pisci	950±294	34±3	5/3	-22.6±0.7	14.1±0.3	3.7±0.1	2582.7±1210.3 92.8±2.3	6.2	5.2
		Esox spp.	Northern pike (pik)	Carni	2220±396	59±4	5/4	-22.8±0.7	14.2±0.4	3.7±0.1	1920.1±476.9	95.6±0.5	2.9	2.9
		Sander lucioperca	Zander (zan)	Carni-Pisci	3443±527	63±3	5/4	-22.9±0.6	15.2±0.4	4±0.1	3533.6±1044	96.3±1.8	4.0	4.2
		Alburnus alburnus	Bleak (ble)	Omni	39±8	14±1	5/4	-22.9±0.6	12.3±0.3	3.2±0.1	747.3±466	90.9±1.3
		Blicca bjoerkna	White bream (wbre)		183±41	20±2	5/4	-22.9±0.3	12.5±0.6	3.2±0.2	336.2±82.3	91.1±2.9
		Gobio gobio	Gudgeon (gud)		7±1	9±1	5/4	-14.6±1.5	11.4±0.4	2.9±0.1	261.7±56	83.2±6.7
		Gymnocephalus cernua	Ruffe (ruf)		18±3	10±1	5/4	-26.6±2.9	13±1.3	3.2±0.3	377.1±232.2	82.1±5.8
		Ameiurus melas	Black bullhead (bbh)		123±49	18±3	5/4	-23±0.9	12.5±0.9	3.4±0.4	239.4±67.7	85.2±4.7
	Parentis-Biscarrosse	Lagarosiphon major Egeria densa Epiphyton Egeria densa	Lagarosiphon (lag) Egeria (ege) (epiE)	PP			1/1 1/1 1/1	-11.3 -14.8 -22.6	4.7 10.8 8.2		149.9 5.5 33	4.2 3.7 2.7
	(PB)	Epiphyton Ludwigia sp.	(epiL)				1/1	-29.8	8.8		43	7
		Epiphyton Phragmites australis	(epiR)				1	-24.9	6.8		20.7
		Corbicula fluminea	Asiatic Clam (clam)	FF	0.8±0.1	20.9±0.8	5/5	-25.6±0.2	11.3±0.1	2	199.7±9.9	9.8±2.7
		Procambarus clarkii	Crayfish (cray)	Det			5/4	-22.4±0.7	14.1±1.1	2.8±0.3	116.7±20.7	75.6±13.4
		Rutilus rutilus	Common roach (roa)	Omni-Herbi	311±197	25±4	5/4	-22.5±1.5	15.5±0.8	3.2±0.2	531.7±151.7	90.6±6
		Abramis brama	Common bream (bre)	Omni-Benth	395±46	27±2	5/4	-22.8±1.8	16.1±0.3	3.4±0.1	275.2±91.1	86.4±4.7
		Perca fluviatilis	European perch (per)	Carni-Pisci	826±224	32±2	5/4	-22.6±0.2	17.8±0.1	3.9±0	1156.9±317.9	93.8±1.5	3.9	4.5
		Esox spp.	Northern pike (pik)	Carni	2460±451	65±3	5/4	-21.1±0.6	18.3±0.4	4±0.1	1459.4±332.5	95±2.3	3.4	3.9
		Sander lucioperca	Zander (zan)	Carni-Pisci	1890±284	55±3	5/4	-22.9±0.4	18.5±0.2	4.1±0.1	2065.7±258.1	92.5±1.4	4.8	5.1

northern pike and European perch for MeHg and THg for each lake. Biometric characteristics of organisms are also indicated: total weight (g) and standard length (cm). Values are means ± standard error; N: number of samples for δ 15 N, δ 13 C and THg analysis, n: number of samples for MeHg analysis. Table 2. Trophic Magnification Slope (TMS) and Food Web Magnification factor (FWMF) calculated for THg and MeHg in each lake. PP: Primary producers; FF:Filter-feeders; Det: detritivorous; Omni: Omnivorous; Herbi: Herbivorous; Benthophagous: Benth; Carni: Carnivorous; Pisci: Piscivorous *: ANCOVA, p <0.05

Acknowledgements:

The authors acknowledge the financial support from the Agence de l'Eau Adour Garonne and the Nouvelle Aquitaine region through the CLAQH project (Cluster of Excellence Labex COTE, Bordeaux University). The authors are also grateful to the French Agency for Biodiversity (OFB), the Federation de Pêche des Landes et de la Gironde and AAPPMA (Association Agrée de Pêche et de Protection des Milieux Aquatiques) for their help in aquatic organism sampling. The authors are grateful to the platform 'Spectrométrie Isotopique' (LIENSS laboratory, CNRS-University of La Rochelle), especially Benoit Lebreton and Gaël Guillou for their expertise and advice. The authors also acknowledge Axel Canredon and Damien Buquet (UMR EPOC 5805, CNRS-Bordeaux University) for the collection of some data on the physico-chemical parameters of the lakes. Finally, thanks to Dr. John Simmers for proofreading the English.


Williams Lefebvre

N V Skiba

F Chabanais

M Yu Gutkin

Lorenzo Rigutti

M Yu Murashkin

T S Orlova

M Yu

Murashkin

Vacancy release upon heating of an ultrafine grain Al-Zr alloy: In-situ observations and theoretical modeling

Keywords: cavities, pore, UFG, vacancy, grain boundary, severe plastic deformation, in-situ, annealing, disclination

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

excess vacancies during the early stage of heat treatment is consequently identified as a phenomenon responsible for accelerated atomic mobility. This work hence provides a new perspective for understanding the accelerated precipitation kinetics observed in severely deformed alloys.

Introduction.

Ultrafine grain (UFG) microstructure are known to lead to very high strength often combined with significant ductility [START_REF] Valiev | Bulk nanostructured materials from severe plastic deformation[END_REF][START_REF] Valiev | Paradox of strength and ductility in metals processed by severe plastic deformation[END_REF][START_REF] Sauvage | Grain boundaries in ultrafine grained materials processed by severe plastic deformation and related phenomena[END_REF]. Such a combination of properties can even be improved by means of the fine scale precipitation of a second phase [START_REF] Zhao | Microstructures and mechanical properties of ultrafine grained 7075 Al alloy processed by ECAP and their evolutions during annealing[END_REF][START_REF] Deschamps | In-situ evaluation of dynamic precipitation during plastic straining of an Al-Zn-Mg-Cu alloy[END_REF][START_REF] Sha | Influence of equal-channel angular pressing on precipitation in an Al-Zn-Mg-Cu alloy[END_REF]. Furthermore, faster precipitation kinetics is often observed if precipitation hardening treatments are applied to alloys processed by severe plastic deformation (SPD) [START_REF] Deschamps | In-situ evaluation of dynamic precipitation during plastic straining of an Al-Zn-Mg-Cu alloy[END_REF][START_REF] Sha | Influence of equal-channel angular pressing on precipitation in an Al-Zn-Mg-Cu alloy[END_REF][START_REF] Wang | Accelerated precipitation and growth of phases in an Al-Zn-Mg-Cu alloy processed by surface abrasion[END_REF]. To explain this, numerous effects and interactions between defects can be considered and their influence is potentially variable from one alloy system to the other. For example, UFG can promote solute segregation and/or heterogeneous precipitation [START_REF] Sauvage | Grain boundaries in ultrafine grained materials processed by severe plastic deformation and related phenomena[END_REF][START_REF] Sauvage | Strain Induced Segregations in Severely Deformed Materials[END_REF][START_REF] Semenova | Enhanced strength and ductility of ultrafine-grained Ti processed by severe plastic deformation[END_REF][START_REF] Liddicoat | Nanostructural hierarchy increases the strength of aluminium alloys[END_REF][START_REF] Reddy | Crystal-plastic deformation of zircon: A defect in the assumption of chemical robustness[END_REF][START_REF] Sauvage | Grain Boundary Segregation in UFG Alloys Processed by Severe Plastic Deformation[END_REF], whereas the importance of such phenomena would be negligible in a coarse grain structure. In addition, SPD is known to produce large quantities of excess vacancies [START_REF] Saada | Les défauts ponctuels produits par écrouissage dans les métaux[END_REF][START_REF] Ruoff | On strain-enhanced diffusion in metals. III. Interpretation of recent experiments[END_REF][START_REF] Würschum | Interfacial free volumes in ultra-fine grained metals prepared by severe plastic deformation, by spark erosion, or by crystallization of amorphous alloys[END_REF][START_REF] Van Petegem | Free volume in nanostructured Ni[END_REF][START_REF] Mecking | The effect of vacancy generation on plastic deformation[END_REF][START_REF] Setman | The presence and nature of vacancy type defects in nanometals detained by severe plastic deformation[END_REF][START_REF] Huang | The formation of nanograin structures and accelerated room-temperature theta precipitation in a severely deformed Al-4 wt.% Cu alloy[END_REF][START_REF] Lechner | Microstructure and vacancytype defects in high-pressure torsion deformed Al-Cu-Mg-Mn alloy[END_REF][START_REF] Kazantzis | Deformation mechanism of aluminummagnesium alloys at elevated temperatures[END_REF]. However, giving an experimental evidence of the effect of vacancies on precipitation is a very challenging task. Previously, Deschamps et al. [START_REF] Deschamps | In-situ evaluation of dynamic precipitation during plastic straining of an Al-Zn-Mg-Cu alloy[END_REF] suggested that deformation induced non-equilibrium vacancies may contribute to nucleation, rapid growth and coarsening of precipitates formed along deformation induced dislocations and grain boundaries (GBs). In a paper by Wang et al. [START_REF] Wang | Accelerated precipitation and growth of phases in an Al-Zn-Mg-Cu alloy processed by surface abrasion[END_REF], who studied the precipitation in an Al-Zn-Mg-Cu alloy processed by surface abrasion, it was proposed that accelerated precipitation kinetics and selection of phases were driven by two phenomena. First, accelerated diffusivity along GBs and sub-GBs were calculated and found responsible for the accelerated precipitation and growth of phases. But to explain how solutes could be transported to these boundaries, these authors proposed that certain solute-vacancies complex could form, as observed by positron annihilation spectroscopy [START_REF] Lechner | Microstructure and vacancytype defects in high-pressure torsion deformed Al-Cu-Mg-Mn alloy[END_REF], and promote the selected transport of some solutes to the boundaries.

The purpose of the present work is to help elucidating the mechanisms at stake in the very early stages of precipitation in an alloy which has first undergone a severe plastic deformation. Instead of investigating the material at the scale of precipitation (i.e. nanometer scale), it has been chosen to follow the microstructure evolution at a larger scale, in order to reveal the behaviour and potential influence of GBs and dislocations. To this aim, it is proposed to follow in-situ, in a scanning transmission electron microscope (STEM), the evolution of the severely deformed microstructure heated to the precipitation annealing temperature of a model alloy system. Such a protocol allows to follow the eventual motion of GBs or sub-GBs, of dislocations as well the nucleation and growth of precipitates.

In the selected model alloy (Al-0.4wt%Zr), in absence of SPD, significant precipitation hardening is typically obtained for annealing temperatures superior to 350 °C [START_REF] Knipling | Precipitation evolution in Al-Zr and Al-Zr-Ti alloys during aging at 450-600 ??C[END_REF][START_REF] Knipling | Precipitation evolution in AlZr and Al Zr Ti alloys during isothermal aging at 375 -425[END_REF][START_REF] Knipling | Criteria for Developing Castable, Creep-Resistant Aluminum-Based Alloys A Review[END_REF][START_REF] Knipling | Nucleation and Precipitation Strengthening in Dilute Al-Ti and Al-Zr Alloys[END_REF]. On the contrary, the same alloy system exhibits a drastically increased precipitation kinetics if previously submitted to severe plastic deformation [START_REF] Lefebvre | Tracking the path of dislocations across ordered Al3Zr nano-precipitates in three dimensions[END_REF][START_REF] Masquelier | Caractérisation et modélisation de transformations microstructurales pour la mise au point d'une nouvelle génération d'alliages d'aluminium pour conducteurs électriques[END_REF][START_REF] Orlova | Influence of severe plastic deformation on microstructure, strength and electrical conductivity of aged Al-0.4 Zr (wt.%) alloy[END_REF][START_REF] Orlova | Effect of annealing on microstructure, strength and electrical conductivity of the pre-aged and HPT-processed Al-0.4Zr alloy[END_REF][START_REF] Latynina | The effect of hardening by annealing in ultrafine-grained Al-0.4Zr alloy: influence of Zr microadditives[END_REF]. Previous works have indeed demonstrated that in the UFG state, annealing temperature of 230 °C is sufficient to cause a precipitation to produce a dense precipitation of Al3Zr phase [START_REF] Orlova | Influence of severe plastic deformation on microstructure, strength and electrical conductivity of aged Al-0.4 Zr (wt.%) alloy[END_REF][START_REF] Orlova | Effect of annealing on microstructure, strength and electrical conductivity of the pre-aged and HPT-processed Al-0.4Zr alloy[END_REF][START_REF] Latynina | The effect of hardening by annealing in ultrafine-grained Al-0.4Zr alloy: influence of Zr microadditives[END_REF]. An UFG structure has hence been obtained by means of high pressure torsion (HPT) [START_REF] Valiev | Nanostructured materials from severe plastic deformation[END_REF][START_REF] Zhilyaev | Using high-pressure torsion for metal processing: Fundamentals and applications[END_REF]. Then, the alloy has been exposed to in-situ heating at 230°C in a scanning transmission electron microscope (STEM). This original procedure reveals the rapid formation of nanopores at GBs. The release of large amounts of vacancies induced by the rearrangement of near equilibrium GBs is the mechanism identified as responsible for the formation of pores. A physical model is proposed to describe both the formation and the evolution of these nanopores during in-situ annealing.

Material and experimental procedures

The Al-0.4Zr (wt.%) alloy with the chemical composition 99.25 Al, 0.393 Zr, 0.023 Si, 0.242 Fe, 0.018 Zn, 0.026 V, 0.05 -balance (wt.%) produced by combined casting and rolling (C-R) [START_REF] Belov | Effect of Zr additions and annealing temperature on electrical conductivity and hardness of hot rolled Al sheets[END_REF]34] was investigated. The material was supplied by UC RUSAL (Moscow, Russia).

The alloy was subjected to long term annealing at 375 °C for 60 h. Then the samples in the shape of disc were processed by high pressure torsion (HPT) under a hydrostatic pressure of 6 GPa to ten revolutions at room temperature (RT) [START_REF] Zhilyaev | Using high-pressure torsion for metal processing: Fundamentals and applications[END_REF]. As a result of such treatment the discshaped samples with a diameter of 20 mm and a thickness of 1.2 mm were obtained. The true strain at a distance of 5 mm from the disc center was ε ≈ 6.6. More details of the sample preparation are presented in [START_REF] Orlova | Effect of annealing on microstructure, strength and electrical conductivity of the pre-aged and HPT-processed Al-0.4Zr alloy[END_REF].

Scanning Transmission Electron Microscopy (STEM) observations were performed using a JEOL ARM 200F operating at 200 kV. The probe size was set to 0.11 nm. The half convergence angle was 22.5 mrad. Thin foils for STEM were prepared using a standard twin jet polishing procedure. The electrolyte (2/3 methanol, 1/3 nitric acid) was used and cooled at -30°C. In large angle annular dark field (LAADF) mode in STEM, the collection angle was in the range 20-80 mrad. In high angle annular dark field (HAADF) mode, the collection angle was in the range 80-180 mrad. The contrast in LAADF STEM is dominated by diffraction whereas images in HAADF STEM are dominated by Z-contrast and specimen thickness. For in-situ heating in STEM, the thin foil was placed in a Gatan heating double tilt specimen holder (model 652 with tantalum furnace). During in-situ heating, the temperature was directly set to 230 °C starting from room temperature. The 230 °C target temperature was reached after 30 s. The start of heating is the reference for the time recorded with each picture.

Results

Microstructure characterization

The characterization of the samples by X-ray diffraction (XRD) analysis, electron backscatter diffraction (EBSD) and transmission electron microscopy (TEM) was performed in our preliminary studies and the results were reported in [START_REF] Orlova | Effect of annealing on microstructure, strength and electrical conductivity of the pre-aged and HPT-processed Al-0.4Zr alloy[END_REF] and summarized in Table 1. In Table 1, the samples after aging and HPT processing before annealing are referred to as AG-HPT.

The samples after subsequent annealing at 230 oC for 1 h and 3 h are referred to as AG-HPT-AN(1h) and AG-HPT-AN(3h), respectively. As seen in Table 1, annealing of AG-HPT sample at 230 °C for 1 h results in only slight increase of the average grain size dav, whereas the continuation of annealing at the same temperature up to 3 h does not change dav.

According to the XRD data the annealing leads to 4-5 time decrease of dislocation density (Table 1) [START_REF] Orlova | Effect of annealing on microstructure, strength and electrical conductivity of the pre-aged and HPT-processed Al-0.4Zr alloy[END_REF].

Table 1 : Microstructure parameters of Al-0.4Zr samples obtained by XRD and EBSD (data of [START_REF] Orlova | Effect of annealing on microstructure, strength and electrical conductivity of the pre-aged and HPT-processed Al-0.4Zr alloy[END_REF]) are: dav the average grain size; θav the average GB misorientation angle; f≥15 the fraction of high-angle GBs with a misorientation angle θ ≥ 15 о ; a the lattice parameter; DXRD the average size of coherent scattering domains; <ε 2 > 1/2 the level of microdistortions of the crystalline lattice; Ldis the dislocation density.

State

Experiment on annealing at 230 °C for 3 h in-situ in STEM

Annealing of the HPT-processed Al-0.4Zr alloy was performed in-situ at 230 °C in STEM for a maximum duration of 3 h. Attention has been paid to the evolution of microstructure at the grain scale. In the initial state, contrast inside grains (shown by arrows in Figure 1a) corresponds to dislocations. After heat treatment a part of this contrast disappears that most probably points to annihilation of these dislocations. This finding is in good agreement with the decrease of integrated dislocation density after such annealing (Table 1, the XRD data [START_REF] Würschum | Interfacial free volumes in ultra-fine grained metals prepared by severe plastic deformation, by spark erosion, or by crystallization of amorphous alloys[END_REF]). It is important to note that no noticeable displacement of GBs has been observed during in-situ annealing. Nevertheless, an important detail is revealed by focusing on areas highlighted by circles in Figure 1. In these areas, annealing involves a slight rearrangement of GBs orientation. Indeed, some GBs are shown to straighten and the angles between them approach 120 degrees. This is all the more obvious for the GBs in the upper circle of Figure 1b. Such an observation is the experimental evidence that some GBs are relaxing their configuration during in-situ annealing. In some areas pointed by arrows, dislocations are not visible after annealing. White circles outline GBs which straighten during annealing.

In Figure 2, images of the same region acquired simultaneously are displayed with two distinct contrasts. It must be pointed out that for the magnification used for images of Figure 2 and Figure 3, the formation of nanometer scale precipitates enriched in Zr cannot be followed because of their too small dimension. On the right, the contrast in LAADF is dominated by diffraction. Hence, it is possible to distinguish grains, GBs and dislocations. On the left, a HAADF detector has been used to map the variation of specimen density. Indeed, the intensity in HAADF mode of probed volumes of identical thicknesses varies as a function of Z n , Z being the average atomic number in the volume scanned by the probe and ranging from 1.7 to 2 [START_REF] Jesson | Incoherent imaging of crystals using thermally scattered electrons[END_REF]. In cases when no significant changes of composition are involved in the specimen, HAADF intensity varies linearly with the thickness [START_REF] Jesson | Incoherent imaging of crystals using thermally scattered electrons[END_REF]. This corresponds to the conditions of observations, for which no large Al3Zr precipitates are present in the specimen.

Further discussion of the contrast associated to pores is provided in the supplemental information, together with additional data. After further holding at 230 °C, the size of pores decreases and some of them even disappear as seen in Figure 3(f) (see also the comment about pore's resorption in the supplemental information), which shows the same area after annealing for 8 800 s (~ 2 h 27').

A sequence of images recorded for the whole duration of the in-situ heat treatment is indeed displayed in Figure 3, where the pores are observed during an annealing time ranging from 75s to 8 800s. Table 2 : Metrics associated to the pores observed at the very beginning of the in-situ annealing in STEM (Figure 3a). Initial cross sections were measured on the image. An equivalent diameter was deduced from cross sections. An estimation of the volume of pores was calculated using the assumption of a spherical shape. Because of a slight shift of the specimen, pore number 5 could not be seen in all images. Attributing number to pores allows following their distinct evolution during annealing. As illustrated in Table 2, at the start of heat treatment, the diameter of pores ranges from 40 nm to 90 nm and their estimated volume is found to range from 0.5 10 5 nm 3 to 3.5 10 5 nm 3 . By looking at Figure 3, one notices that some pores disappear as the annealing time increases. Pore 7 is not visible after 3 640 s whereas only pores 3 and 6 are still visible after 8 800 s. In the following section, the formation and further dissolution of pores is discussed based on the literature and thanks to original models.

Pore

Discussion

Modeling of the formation of pores during annealing of HPT-processed Al-Zr alloy

Excess free volume released by non-equilibrium grain boundaries

Several kinds of solicitation can lead to the formation of pores (cavities) in alloys.

Among these solicitations are high temperature deformation and irradiation. In the former case, models have been proposed to predict the kinetics and thermodynamic of pore formation [START_REF] Hull | The growth of grain-boundary voids under stress[END_REF][START_REF] Raj | Intergranular fracture at elevated temperature[END_REF]. In the later case, pores may be the result of He condensation [START_REF] Lee | Precipitation and cavity formation in austenitic stainless steels during irradiation[END_REF]. Though these solicitations differ from the case studied in the present work, it is relevant to at least consider the physical model of cavity formation during high temperature deformation in order to later be able to distinguish it from the model we propose. In their paper [START_REF] Raj | Intergranular fracture at elevated temperature[END_REF], Raj and Ashby have developed a model to describe the nucleation kinetic of pores along GBs based on the classical nucleation theory. This model was developed to predict the kinetics of intergranular fracture at high temperature. It accounted for a tensile stress acting across a GB, which may result in the formation of pores that may later coalesce and therefore generate a fracture. Both the critical radius of pores and their nucleation barrier were found to depend on the amplitude of this tensile stress. These results were in agreement with the model proposed by Hull and Rimmer [START_REF] Hull | The growth of grain-boundary voids under stress[END_REF], who also studied theoretically the growth of GB pores under stress.

The physical model associated to the formation of pores in an alloy previously subjected to SPD is undoubtedly very different. The observed formation of pores in the beginning of annealing and their slow disappearance with further continuation of the heat treatment is more likely associated with a rapid agglomeration of vacancies at multiple GB junctions followed by a release of vacancies in a second stage. Working on a closer topic, Vetrano et al. [START_REF] Vetrano | Evidence for excess vacancies at sliding grain boundaries during superplastic deformation[END_REF] have shown that pores may form along GBs of Al-Mg-Mn alloys during a quench applied after superplastic deformation. The authors have concluded that pores result from the localized excess vacancies formed during GB sliding. They are also shown that cavities were not stable upon ageing at 175 °C.

In the present work, instead of considering a tensile stress acting on GBs, we have accounted for the structure of defects and their interactions in the GB plane. The GBs in metals and alloys subjected to SPD are often in non-equilibrium state [START_REF] Valiev | Bulk nanostructured materials from severe plastic deformation[END_REF][START_REF] Divinski | Ultra-fast diffusion channels in pure Ni severely deformed by equal-channel angular pressing[END_REF]. Non-equilibrium GBs (NEQ GBs) in materials either contain less uniformly distributed constituent structural units than in the equilibrium GBs (EQ GBs) with the same misorientation angle, and/or contain additional external dislocations, that leads to an excess free volume and an excess energy of GBs [START_REF] Nazarov | On the structure, stress fields and energy of nonequilibrium grain boundaries[END_REF][START_REF] Orlova | Effect of grain boundaries on the electron work function of ultrafine grained aluminum[END_REF][START_REF] Romanov | Nonequilibrium grain boundaries with excess energy in graphene[END_REF]. As was demonstrated in [START_REF] Orlova | Effect of grain boundaries on the electron work function of ultrafine grained aluminum[END_REF][START_REF] Orlova | Influence of grain boundary state on electrical resistivity of ultrafine grained aluminium[END_REF], GBs in commercial purity (CP) Al, processed by HPT with exactly the same regime as used for HPT processing Al-0.4Zr alloy, are mainly NEQ high-angle GBs.

The transition of GBs from a non-equilibrium state to a more equilibrium one should be accompanied by the loss of the excess free volume. Excess vacancies, from which the pores are formed during annealing, can consequently arise in the process of dissolution of the excess free volume contained in NEQ GBs during their relaxation under annealing. It is hence relevant to compare the predicted excess free volume with the dimensions of pores revealed experimentally.

Our experiments did show that the pores are formed mainly at GB junctions (Figure 2)

(see also the supplemental information for additional information about the location of pores).

We can estimate the summary excess free volume of three NEQ GBs which form a triple junction. According to literature data [START_REF] Musalimov | Dilatometric analysis of aluminium alloy with submicrometre grained structure[END_REF][START_REF] Musalimov | Dilatometric study of an aluminum alloy with submicrongrained structure[END_REF][START_REF] Chuvil | Non-equilibrium grain boundary in metals, Theory and applications[END_REF][START_REF] Chuvil | Micromechanisms of deformation-stimulated grain boundary self-diffusion. Communication 1. Influence of excess free volume on free energy and diffusion parameters of grain boundaries[END_REF][START_REF] Chuvil'deev | A theory of non-equilibrium grain boundaries and its applications to nano-and micro-crystalline materials processed by ECAP[END_REF][START_REF] Valiev | Bulk Nanostructured Materials[END_REF], the excess free volume of NEQ GBs is equal to 3-10 % of the equilibrium GB volume. Dilatometric investigation of aluminum alloys (Al-4%Cu-0.5%Zr) processed by HPT at RT was performed in [START_REF] Musalimov | Dilatometric analysis of aluminium alloy with submicrometre grained structure[END_REF][START_REF] Musalimov | Dilatometric study of an aluminum alloy with submicrongrained structure[END_REF]. The authors reported that the free volume of NEQ GBs exceeded the free volume of EQ GBs by at least 3 %. It was also concluded that the areas nearby to NEQ GBs were also characterized by increased free volume [START_REF] Valiev | Bulk Nanostructured Materials[END_REF]. An increased width of NEQ GBs compared with the width of EQ GBs was noted in a number of works [START_REF] Valiev | Bulk Nanostructured Materials[END_REF][START_REF] Valiev | Direction of a grain-boundary phase in submicrometre-grained iron[END_REF][START_REF] Islamgaliev | The Determination of the Grain Boundary Width of Ultrafine Grained Copper and Nickel from Electrical Resistivity Measurements[END_REF][START_REF] Mavlyutov | Effect of annealing on microhardness and electrical resistivity of nanostructured SPD aluminium[END_REF]. The increase in GB width is associated with the presence of defects introduced into the GB from the grain body, mainly lattice dislocations, which results in changes of physical properties of GBs, for example, charge scattering at the GB [START_REF] Orlova | Influence of grain boundary state on electrical resistivity of ultrafine grained aluminium[END_REF][START_REF] Mavlyutov | Effect of annealing on microhardness and electrical resistivity of nanostructured SPD aluminium[END_REF].

The estimates of the width δ of NEQ GBs based on the measurement of electrical resistance [START_REF] Islamgaliev | The Determination of the Grain Boundary Width of Ultrafine Grained Copper and Nickel from Electrical Resistivity Measurements[END_REF][START_REF] Mavlyutov | Effect of annealing on microhardness and electrical resistivity of nanostructured SPD aluminium[END_REF] and Mössbauer spectra [START_REF] Valiev | Direction of a grain-boundary phase in submicrometre-grained iron[END_REF] gave the following values: δ ≈ 2.1 and 3.7 nm for HPTprocessed high purity Cu and Ni, respectively [START_REF] Islamgaliev | The Determination of the Grain Boundary Width of Ultrafine Grained Copper and Nickel from Electrical Resistivity Measurements[END_REF], δ ≈ 9 nm for HPT-processed CP Al [START_REF] Mavlyutov | Effect of annealing on microhardness and electrical resistivity of nanostructured SPD aluminium[END_REF] and δ ≈ 8.4 nm for UFG Fe [START_REF] Valiev | Direction of a grain-boundary phase in submicrometre-grained iron[END_REF]. Thus, physical width of NEQ GBs in metals is much larger than their crystallographic width, the latter equaling to 2-3 interatomic distances [START_REF] Valiev | Bulk Nanostructured Materials[END_REF].

According to theoretical investigations, the relative free volume of the NEQ GB in Al is approximately 0.35 (relative to the perfect crystal without GB with the same number of atoms), and the relative excess free volume of the NEQ GB is 0.05-0.10 [START_REF] Chuvil'deev | A theory of non-equilibrium grain boundaries and its applications to nano-and micro-crystalline materials processed by ECAP[END_REF].

One can consequently estimate the total excess free volume of three NEQ GBs which form a triple junction. For a grain size of 1 000 nm and the physical width of NEQ GBs of about 9 nm [START_REF] Valiev | Bulk Nanostructured Materials[END_REF], the excess free volume of the three GBs varies in the limits of (0.9-2.7) 10 6 nm 3 .

As shown in Table 2, the typical diameter of pores is dp ~ 40-60 nm. However, larger pores with dp ~ 90 nm are also observed. The biggest observed pore of non-spherical shape has the largest dimension which does not exceed 120 nm (pore 1 in Figure 3b). Typical volumes of pores are estimated about 0.5 10 5 nm 3 to 3.5 10 5 nm 3 . Thus, the obtained estimates for the observed pore volume and the summary excess free volume of three GBs which form a triple junction are comparable, the latter being even greater (by a factor 10).

One must however keep in mind the fact that the thin foil geometry may alter the kinetics of microstructural evolution with respect to the bulk material. Indeed, the thin foil has a nearly two-dimensional geometry, with a small thickness (< 50 nm) compared the planar dimension of the specimen. As a consequence, free surfaces, which act as sinks for vacancies, are extremely close compared to a bulk state. The main impact in this study is that a significant fraction of vacancies is expected to annihilate on free surfaces during the in-situ annealing. The experimental hence implies that a significant fraction of excess vacancies may not contribute to the formation of pores. This may explain why the estimated volume of pores observed is an order of magnitude larger than the expected excess free volume of GBs.

The vacancies, formed as a result of such GB compaction, will go to the nearest sinks and form pore by their accumulation. Positive wedge disclinations at triple junctions and GBs [START_REF] Romanov | Disclinations in crystalline solids[END_REF] are obviously the most effective sinks. During the formation of pores, there is a relaxation of the GBs due to a decrease of the excess free volume of GBs. But, at the same time, not all external (extrinsic) GB dislocations (EGBDs) manage to annihilate. The part of EGBDs, the Burgers vectors of which do not allow to slide along the GBs, can only climb, and climbing is always slow and long process. It hence seems that the formation of pores on disclinations at triple junctions and the climb of EGBDs that form these disclinations should go with significantly different velocities. Pores will grow rapidly on disclinations, which do not have sufficient time to "dissolve" in this short while. Based on these considerations, a model accounting for the major role of disclinations in the formation of pore is presented hereafter.

Model accounting for the role of disclinations in the formation of pores

On one hand, the formation of such pores (cavities) decreases the strain energy of the crystal. However, on the other hand, it increases the crystal energy due to the formation of the pore free surface. The balance of these decrease (which is proportional to dp 3 ) and increase (which is proportional to dp 2 ) gives an estimate of the equilibrium pore diameter dp eq . To make

such an estimate, one can compare two elastically isotropic cylinders: a solid one and a hollow one, each of which contains a wedge disclination of strength ω on its axis. The strain energy (per unit length of the cylinder) of the solid disclinated cylinder of radius R is [54]:

8 2 2 R D W s ω = (1) where )], 1 ( 2 /[ ν π - = G D
G is the shear modulus and ν is the Poisson ratio. The total energy t W of the hollow disclinated cylinder of the same outer radius R and with the inner radius R0 is the sum of its strain energy h W which is given by [START_REF] Romanov | Disclinations in crystalline solids[END_REF]:

        - - - = 2 2 2 2 2 2 ) (ln 1 4 1 8 t t t t R D W h ω (2)
where

1 / 0 < = R R t
, and the surface energy γ W of the hole, which can be approximated as

γ π γ 0 2 R W ≈
with γ being the specific surface energy of the material. In its turn, this γ can be approximated as 2 / Db ≈ γ , where b is the interatomic distance. Then the total energy reads

DbRt t t t t R D W W W h t π ω γ +         - - - ≈ + = 2 2 2 2 2 2 ) (ln 1 4 1 8 . ( 3
)
Figure 4 shows the dependence

        - + - = - = ∆ 2 2 2 2 2 2 ) (ln 1 4 8 t t t t R D DbRt W W W s t ω π (4)
After some algebra, the energy change takes the form:

            - + - = ∆ 2 2 2 0 2 0 ) (ln 1 4 1 8 8 t t R b DR W ω π ( 5
)
The transformation is energetically favorable if 0 < ∆W . With Eq. ( 5), this inequality can be rewritten as follows:

2 2 2 ) (ln 1 4 8 t t t t R b - + < ω π . ( 6
)
Let us denote the right-hand side of this inequality as 5 for the aforementioned values of the system parameters. As is seen, in dependence on the disclination strength value, the inequality ( 6) can either have or not a solution. Indeed, for example, there is no solution for °= 5 ω , because in this case 2 1 f f < for any t. This means that the disclination strength is not high enough to make the formation of the pore energetically favorable. The same conclusion follows from the behavior of the corresponding curve in Figure 4, which monotonically raises with t. During the further annealing, the dissolution of the pores and the "disappearance" of the disclinations will go in parallel, stimulating each other. The model describing the concomitant dissolution of pores is introduced in the next section.

of the pore dissolution during annealing of HPT-processed Al-Zr alloy

Let us consider a model of consequent dissolution of pores located in triple junctions of GBs during the annealing. It is known that during annealing, NEQ GBs (GBs containing EGBDs) decrease their energy because of a decrease in the number of the EGBDs at these GBs. The process of decreasing the density of the EGBDs at the GBs is usually associated with their emission from GBs and/or moving of the EGBDs to sinks. The pores are the effective sinks for the EGBDs.

In the framework of the model, it is assumed that the driving force of the pore dissolution during annealing is the decrease in the density of the EGBDs as a result of their climb along the GBs towards the pores. The decrease in the number of the EGBDs leads to the decrease in the strain energy of GB disclination configurations characterized by wedge disclinations.

Thus, during the annealing, the dissolution of the triple junction pores and the climb of EGBDs along the GBs goes in parallel, stimulating each other. The decrease of the pore size (pore healing) occurs through the emission of vacancies which migrate mainly along the neighboring GBs due to their enhanced permeability for mass transport. The flux of emitted vacancies provides the climb of EGBDs which therefore effectively increase their mobility along the GBs. As a result, one part of EGBDs of opposite signs annihilates while the remaining part of them climbs towards triple junctions of GBs. Climbing closer to a disclinated triple junction, these EGBDs decrease the strain energy of the wedge disclinations, thus forming a positive feedback, because such a decrease in the strain energy of the disclination structure stimulates further dissolution of the triple junction pores.

Figure 6 shows a two-dimensional model of dissolution of a triple junction pore as a result of the climb of EGBDs along the GBs adjacent to the pore. In the initial state, there is a cylindrical pore of radius 0 r , which has formed at a positive wedge triple junction disclination (not shown in Figure 6a andb) of strength ω + ( ω + -disclination) created by three walls of EGBDs, which are shown as walls of climbing edge dislocations with lines parallel to the pore axis and spaced periodically with an average spacing s (Figure 6a).

Within the model, a symmetric situation is considered when the GBs adjacent to the pore make the same angles 120° between each other and are characterized by the same linear density s / 1 of the EGBDs. As the EGBDs climb toward the pore, their number and the pore radius n r decrease: 0 r r n < (Figure 6b). For the sake of simplicity, the periodicity sof the EGBD walls remains however constant within the model. For convenience of further calculations, the walls of EGBDs are modeled by dipoles of the wedge disclinations of 6c and Figure 6d). -disclination quadrupole) (Figure 6c). In this case, the climb of the EGBDs to the pore is equivalent to the decrease in the arm of this quadrupole, which leads to a decrease in its strain energy (Figure 6d).

strength 3 / ω ± ( 3 / ω ± -disclination dipole) (Figure
Let us consider the energy characteristics of the initial stage of the pore dissolution as a result of the decrease in the arm of the 3 / ω ± -disclination quadrupole. In this case, the assumption that 0 0 r L >> allows one not to take into account the effect of the pore free surface on the strain energy of the disclination quadrupole. The initial arm 0 L of the quadrupole is assumed to be smaller than the lengths of the GBs adjacent to the triple junction. However it is of the same order of magnitude as the average length of GBs in the area under consideration.

Let us calculate the change in the strain energy, which corresponds to the decrease of the arm of the disclination quadrupole by a distance ns p n = (where n is the number of the EGBDs absorbed by the pore from each GB), accompanied by the decrease in the pore radius

π 2 / 3 0 n n p r r - =
. In doing so, we study the defect system transformation from the To calculate the interaction energy ) (n i E between two disclination dipoles, we used the standard procedure of calculating the work spent to nucleate one defect in the stress field of another defect. It is given by the following expression: . sin 2

, 3 3 3 3 3 3 ) 1 ( ) 1 ( ) 1 ( ) 1 ( ) ( ) ( ) ( ) ( - - - - - - - - + + + = ∆ n GB n n i n s n GB n n i n s n E E E E E E E E W γ γ (7) where
) cos 2 1 ( 1 ln )] sin 2 ( ) cos 2 1 ( [ ] sin 2 ) cos 2 1 ( [ 1 ln ln 4 2 2 2 2 2 2 2 2 2 2 2 2 ) (           + - + + + - + + +       + - + + +     + = n n n n n n n n n n n i L RL R L L R R L L R R L L L L R R R D E α α α α α α θ (9)
The energies

s n n r E γ π γ ) 1 ( ) 1 ( 2 - -= , ( 10
) s n n r E γ π γ 2 ) ( = , (11)
where s γ is the specific surface energy of the pore.

The energies

) 1 ( - n GB E and ) (n GB E are GB n n GB p E γ ) 1 ( ) 1 ( - -= , ( 12
) GB n n GB p E γ ) ( ) ( = , ( 13
)
where GB γ is the specific energy of the GB (for simplicity, we assume that GB γ is the same for all the three GBs adjacent to the pore).

Using Eqs. With decreasing the pore size, more vacancies are released, which assist the climb of EGBDs and, therefore, the quadrupole arm correspondingly decreases. As a result, the strain energy of the disclination quadrupole decreases too. This stimulates further dissolution of the pore. Thus, a positive feedback applies. Along with this process, the precipitation and growth of precipitates can occur, possibly at the GB disclinations of the opposite sign. By "cleaning" GBs from EGBDs during longer annealing, triple junction disclinations and pores reduce the role of GBs as sources of lattice dislocations under external loading, hence the hardening after annealing will take place. This is in agreement with the model proposed earlier to explain the hardening by annealing in HPT-processed CP Al [START_REF] Orlova | Hardening by annealing and implementation of high ductility of ultra-fine grained aluminum: Experiment and theory[END_REF][START_REF] Gutkin | Mechanism of Hardening of Ultrafine-Grained Aluminum after Annealing[END_REF].

Conclusion

In-situ annealing in STEM offers the possibility to reveal phenomena which are hardly accessible by conventional experimental protocols and post-processing observations. An original experiment was designed here to elucidate the mechanisms at stake during the early stages of annealing of a model aluminium alloy (Al-0.4wt%Zr) previously subjected to SPD.

Our study demonstrates that the rapid rearrangement of NEQ GBs provokes the liberation of a significant excess free volume, initially stored in the NEQ GBs, which results in the quasi immediate formation of pores (already observed after 75 s at 230 °C). This fast process is explained by the migration of vacancies to GB triple junctions. Though this change in the GB structure does not involve any GB migration, local change in the configuration of some GBs could be observed. Straightening of some of them, approaching the angles between them to 120 degrees was observed.

The typical pore size was 40-80 nm and the volume of pores is in perfect agreement with the values of excess free volume calculated. As annealing proceeds, the experiment reveals the subsequent slow resorption of these pores with the complete disappearance of some of them, which indicates a release of vacancies in a second stage.

To explain the aforementioned experimental observations, original theoretical models have been introduced. The first model describes the pore formation at disclinated triple junctions as a thermodynamically driven process of free volume dissolution through generation of vacancies which then migrate to the triple junctions and coagulate at them with growth of the pores diminishing the strain energy of the triple junction disclinations. Within the model, we have calculated the equilibrium pore radius eq R , 0 and the critical pore radii The revealed features of the GB structure evolution during low-temperature annealing of UFG Al-Zr alloys opens a new GB engineering approach to increase the strength of these materials at elevated temperatures. Moreover, this work will help understanding the accelerated precipitation kinetics observed in similar system, by accounting for the behavior and amount of vacancies available for low temperature annealing of such alloy systems.

Figure 1 :

 1 Figure 1 : LAADF STEM images of the material (a) before in-situ annealing and (b) after 8 800 s (~2h27') in-situ heating at 230 °C. Diffraction contrast allows distinguishing GBs and dislocations.

Figure 2a and b

  Figure2aand b display a large region of the STEM foil before the in-situ annealing. After annealing for 75 seconds, many pores are already visible in the same area (Figure2c and d) and their location is pointed by arrows in all images for an easy comparison of associated contrasts. The formation of pores is hence found to occur at the very beginning of the in-situ heat treatment. The pores are mostly located at GB junctions. Only few of them are revealed along simple GBs. The typical size of pores is about 40-80 nm and the largest dimension does not exceed 120 nm.

Figure 2 :

 2 Figure 2 : STEM observations of the UFG Al-0.4wt%Zr alloy before in-situ annealing (a,b) and after 75 s of annealing at 230 °C. (a) and (c) are HAADF STEM images whereas (b) and (d) are LAADF STEM images. White arrows indicate where nanopores are forming.

Figure 3 :

 3 Figure 3 : HAADF STEM images of the UFG Al-0.4w%Zr alloy recorded at different times of in-situ annealing at 230 °C: (a) 75 s, (b) 500 s, (c) 950 s, (d) 2 100 s, (e) 3 640 s, (f) 8 800 s. Numbers are placed to distinguish each nanopore.

  various values of ω . It is seen that at certain values of ω , there are equilibrium values eq t radius of the cylinder can be obtained. For example, for °= 6

Figure 4 :

 4 Figure 4 : Dependence Wt(t) for G=27 GPa, ν = 0.31, b ≈ 0.25 nm, R = 250 nm and various values of ω.

Figure 5 : 6 ω, the solution exists in the interval 2 9 ω,

 5629 Figure 5 : The functions f1(t) (blue curve) and f2(w) (red lines) for G = 27 GPa, ν = 0.31, b ≈ 0.25 nm, R = 250 nm and various values of ω.

Figure 6 :

 6 Figure 6: Model of the triple junction pore dissolution due to climb of EGBDs during annealing. (a) The initial configuration of the EGBDs near the pore. (b) Decrease of the pore size due to the climb of the EGBDs towards the pore along the GBs. (c) Modeling the walls of EGBDs by dipoles of wedge disclinations which form a disclination quadrupole. (d) Pore dissolution due to a decrease in the arm of the disclination quadrupole.

  n-th state with a total energy n W , corresponding to the disclination quadrupole with the arm unit length of the pore) is determined by the expression:

  n-th states of the defect system, respectively; n-th states of the defect system, respectively; n-th states of the defect system, respectively.

E

  γ are given by standard formulas, respectively:

( 7 )

 7 Figure 7 shows the predicted variations of ) ( n n p W ∆ for the case of UFG Al-Zr alloy at

Figure 7 :

 7 Figure 7: Dependence of the energy change ∆Wn on the distance pn for various values of the quadrupole arm L0 ≈ 500, 700 and 1000 nm.

  range of which the pore existence is energetically favorable at a triple junction disclination. We have shown that both the equilibrium pore radius eq R strength of the triple junction disclination. We have also demonstrated that the free volume stored in the NEQ GBs, adjacent to a triple junction, is large enough for creation of an observable pore at the triple junction during fast transformation of the NEQ GBs into more equilibrium GBs under fast annealing.The second model describes a further decrease of the triple junction disclination strain energy through the climb of EGBDs towards the hollow disclinated triple junctions, accompanied with dissolution of the triple junction pores by emission of vacancies which assist the dislocation climb in the course of long-term annealing. In the framework of the model, we have calculated the energy difference n W ∆ , caused by the shrinkage of the disclination structure around a hollow disclinated triple junction, and shown that this shrinkage is energetically favorable. Thus, we have proved that the process of the pore dissolution can be realized due to the successive climb of EGBDs along the GBs to the triple junction pore.

Acknowledgments

In situ STEM experiments have been performed on the GENESIS platform supported by the Région Normandie, the Métropole Rouen Normandie, the CNRS via LABEX EMC3 and the French National Research Agency as a part of the program "Investissements d'avenir" with the reference ANR-11-EQPX-0020. N.V.S. and M.Yu.G. are thankful to the Institute for Problems in Mechanical Engineering of the Russian Academy of Sciences, and T.S.O. is thankful to the Ioffe Institute of the Russian Academy of Sciences for their support (within the corresponding state tasks) in development of the theoretical models figuring in the paper.


Christophe Corpechot

Olivier Chazouillères

Lydie Humbert

Alexandra Rousseau

Antonia Le Gruyer

François Habersetzer

Philippe Mathurin

Odile Goria

Pascal Potier

Anne Minello

Christine Silvain

Armand Abergel

Marilyne Debette-Gratien

Dominique Larrey

Olivier Roux

Jean-Pierre Bronowicki

Jérôme Boursier

Victor De Ledhingen

Alexandra Heurgue-Berlot

Eric Nguyen-Khac

Fabien Zoulim

Isabelle Ollivier-Hourmand

Jean-Pierre Zarski

Gisèle Nkontchou

Farid Gaouar

Tabassome Simon

Raoul Poupon

Dominique Rainteau

Changes in serum bile acid levels associated with bezafibrate add-on therapy in patients with primary biliary cholangitis and inadequate biochemical response to ursodeoxycholic acid: results from the BEZURSO trial (NCT01654731)

Christophe Corpechot

Of the 100 patients participating in the trial, M0, M12, and M24 serum samples were available in 91, 82, and 79, respectively. Eightyone patients had at least 2 consecutive samples including M0. At baseline, both the BZF (n=45) and PLB (n=46) groups were similar for total and individual BA concentrations and C4 precursor levels (Table 1). Changes in serum bile acid levels associated with bezafibrate add-on therapy in patients with primary biliary cholangitis and inadequate biochemical response to ursodeoxycholic acid: results from the BEZURSO trial

INTRODUCTION RESULTS

We recently reported the results of a 24-month, multicenter, double-blind, randomized, placebocontrolled trial of bezafibrate (BZF) add-on therapy in patients with primary biliary cholangitis (PBC) and inadequate biochemical response to ursodeoxycholic acid (UDCA).

[1] (Figure 1)

These results showed that a combination of BZF with UDCA is associated with a significant improvement in the symptoms, standard biochemical liver tests, and prognostic markers of the disease. Here, we assessed the changes in serum bile acid (BA) profiles and C4 precursor levels associated with these therapeutic effects.

Furthermore, reductions from baseline to M24 in both endogenous BA concentration and percentage and in C4 precursor were significantly higher in the patients who reached the primary endpoint of the trial (i.e. normal total bilirubin, alkaline phosphatases, transaminases, albumin and prothrombin time), as opposed to the others (Figure 3). The concentrations of total, primary and secondary (i.e. endogenous) BA, UDCA, and C4 precursor were determined using a high-performance liquid chromatography-mass spectrometry method.

Concentrations, percentages, and relative changes in BA species were compared between the BZF and placebo groups, and according to the biochemical response to BZF using nonparametric tests. Analyses were performed on the intent-to-treat population.

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The overall changes in total BA at M24 did not differ significantly between the 2 groups despite a trend towards a reduction in cholic acid concentration (Table 2).

Figure 3 .

 3 Figure 3. Relative changes in C4 and endogenous BA according to primary endpoint

Figure 1 .

 1 Figure 1. Flowchart of the BEZURSO trialChanges in C4 precursor were significantly different between groups, with a 70% reduction in the BZF group and no modification in the placebo group (p=0.0011, Figure2).

Figure 2 .

 2 Figure 2. Changes from baseline in serum level of C4 bile acid precursor according to trial group

Table 1 .

 1 Serum levels of bile acids and C4 precursor at baseline according to trial group

	Placebo	Bezafibrate

UDCA -µmole/L 25.6 ± 37.7 28.1 ± 26.2 C4 precursor -ng/mL 8.4 ± 8.1 9.3 ± 8.4

Table 2 .

 2 Relative changes from baseline to 24 months in BA concentrations according to trial group Bezafibrate add-on therapy in patients with PBC and inadequate response to UDCA is associated with a marked decrease in serum level of C4 precursor.The beneficial effect of BZF on biochemical liver tests is associated with a parallel decrease in serum level of C4 precursor and endogenous BA concentration and percentage, suggesting it is, at least in part, mediated by a reduction in BA synthesis and hepatocellular overload.

	However, the percentage of endogenous BA
	was significantly reduced in the BZF compared
	to the PLB group both at M12 (21% vs. 29%;
	p=0.009) and M24 (18% vs. 25%; p=0.0260).

Christophe Corpechot, MD. Saint-Antoine Hospital, Paris, France. Email: christophe.corpechot@aphp.fr