De Bandt

Prof L Cynober
email: luc.cynober@parisdescartes.fr

Effect of citrulline on muscle protein turnover in an in vitro model of muscle catabolism

Keywords: Protein turnover, Glucocorticoids, Cytokines, Muscle hypercatabolism, Citrulline

Muscle net catabolism, as seen after severe trauma or sepsis or in post-operative situations, is mediated by hormones, such as cortisol, and pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α). Specific amino acids (AAs) may be able to limit this muscle mass loss. Citrulline (Cit) stimulates muscle protein synthesis in various situations, but there are few data on hypercatabolic situations, and its effects on protein breakdown are unknown. Our aim was to assess the effect of Cit on protein turnover in an in vitro model of muscle hypercatabolism.

Myotubes derived from C2C12 myoblasts were treated with 150 nM dexamethasone (DEX), 10 ng/ml TNF-α, or 0.006% ethanol (as control, CON) for 24 h. Myotubes were then incubated with or without 5 mM Cit for 6 h. Muscle protein synthesis rate (PSR) was evaluated by the surface sensing of translation (SUnSET) method and by L-[3,5-3 H]tyrosine (Tyr) incorporation. Muscle protein breakdown rate (PBR) was evaluated from Tyr release into culture medium. Cit action was analyzed by non-parametric Kruskal-Wallis and Mann-Whitney tests. Cit treatment significantly increased PSR compared with the DEX group or TNF-α group (SUnSET method; DEX+CIT vs. DEX, p = 0.03 and TNF-α+CIT vs. TNF-α, p = 0.05), and significantly decreased PBR in the CON and DEX groups (CON+CIT vs. CON p = 0.05 and DEX + Cit vs. DEX, p = 0.05). Cit treatment regulated muscle protein turnover in an in vitro model of muscle net catabolism. It would be of interest to explore the underlying mechanisms.

Introduction

Muscle wasting occurs in net catabolic situations such as heavy surgery, trauma and sepsis [START_REF] Golden | Protein turnover, synthesis and breakdown before and after recovery from protein-energy malnutrition[END_REF][START_REF] Lynch | Therapeutic approaches for muscle wasting disorders[END_REF]. It is a consequence of increased glucose metabolism in the metabolic response to injury: since glycogen stores are very limited, and our bodies are unable to convert lipids into carbohydrates, most of the glucose required for energy supply comes from amino acids (AAs) [START_REF] Cynober | Amino acid metabolism[END_REF], which in turn come from proteins, especially muscle proteins. In a healthy state, maintenance of muscle mass depends on a dynamic balance between anabolic and catabolic reactions. Conversely, in stress situations, there is an imbalance, with a relative or absolute reduction in protein synthesis and/or increase in protein breakdown [START_REF] Rittig | Amino acid supplementation is anabolic during the acute phase of endotoxin-induced inflammation: A human randomized crossover trial[END_REF]. The mobilization of muscle AAs is mainly driven by catabolic hormones (e.g. cortisol) [START_REF] Warner | Effect of Catabolic Hormone Infusion on Protein Turnover and Amino Acid Uptake in Skeletal Muscle[END_REF] and mediators such as tumor necrosis factor alpha (TNF-α), interleukin-1, or interleukin-6 [START_REF] Zamir | Effect of tumor necrosis factor or interleukin-1 on muscle amino acid uptake and the role of glucocorticoids[END_REF].

Severe prolonged net catabolism is a factor of morbidity and mortality among injured patients, and an appropriate nutritional support for these patients may be essential to limit the protein losses leading to muscle wasting and loss of muscle function. However, the effects of standard enteral or parenteral nutrition are limited by what is termed "resistance to renutrition" [START_REF] Charrueau | Metabolic response and nutritional support in traumatic brain injury: evidence for resistance to renutrition[END_REF][START_REF] Rennie | Anabolic resistance in critically ill patients[END_REF]. Attention has accordingly focused on targeted nutritional strategies using specific nutrients such as leucine (Leu), which has been shown to regulate protein synthesis [START_REF] Cynober | Leucine and Citrulline: Two Major Regulators of Protein Turnover[END_REF]. However, results on the use of Leu in intensive care unit (ICU) patients are disappointing [START_REF] Ginguay | Indications and contraindications for infusing specific amino acids (leucine, glutamine, arginine, citrulline, and taurine) in critical illness[END_REF]. More recently, another AA, citrulline (Cit) was shown to modulate nitrogen homeostasis (see [START_REF] Cynober | Leucine and Citrulline: Two Major Regulators of Protein Turnover[END_REF][START_REF] Kaore | Citrulline: pharmacological perspectives and its role as an emerging biomarker in future[END_REF] for recent reviews) and protein synthesis. Osowska et al. [START_REF] Osowska | Citrulline increases arginine pools and restores nitrogen balance after massive intestinal resection[END_REF], in a model of short bowel syndrome, demonstrated that Cit supplementation of enteral feeding was associated with an 80% increase in muscle protein synthesis rate and a 20% net muscle protein gain relative to rats fed a standard diet. These results have since been confirmed in various experimental models, and appear to be dependent on the mTOR (mammalian/mechanistic target of rapamycin) signaling pathway [START_REF] Plénier | Effects of leucine and citrulline versus non-essential amino acids on muscle protein synthesis in fasted rat: a common activation pathway?[END_REF][START_REF] Faure | Citrulline enhances myofibrillar constituents expression of skeletal muscle and induces a switch in muscle energy metabolism in malnourished aged rats[END_REF]. Furthermore, supplementation with Cit stimulated muscle protein synthesis in young adults fed a hypoproteic diet [START_REF] Jourdan | Citrulline stimulates muscle protein synthesis in the post-absorptive state in healthy people fed a low-protein diet -A pilot study[END_REF]. There are few data on the effects of Cit in net catabolic situations: besides the already mentioned study on short bowel syndrome [START_REF] Osowska | Citrulline increases arginine pools and restores nitrogen balance after massive intestinal resection[END_REF], only three studies have yet been published: Wijnands et al. [START_REF] Wijnands | Citrulline a more suitable substrate than arginine to restore nitric oxide production and the microcirculation during endotoxemia[END_REF], in a model of endotoxemia in mice, showed improved intestinal microcirculation in animals supplemented with Cit; Batista et al. [START_REF] Batista | Pretreatment with citrulline improves gut barrier after intestinal obstruction in mice[END_REF] showed that Cit preserved the integrity of the intestinal barrier, and also decreased the production of pro-inflammatory cytokines, such as IFNγ, and maintained that of anti-inflammatory cytokines such as IL-10; finally, in vitro in isolated myotubes, Ham et al. [START_REF] Ham | L-Citrulline Protects Skeletal Muscle Cells from Cachectic Stimuli through an iNOS Dependent Mechanism[END_REF] showed that Cit maintained myotube protein synthesis in catabolic conditions induced by hydrogen peroxide or LPS. Whereas in vivo and in vitro studies yield evidence that Cit stimulates protein synthesis, no study has evaluated the effect of Cit on muscle protein breakdown. In addition, a large majority of the studies on protein synthesis were performed in vivo, which does not tell us whether the action is direct (i.e. Cit-mediated) or indirect (e.g. through Cit conversion into arginine) [START_REF] Deutz | ESPEN Sir David Cuthbertson Lecture: Amino acids between and within organs. The glutamate-glutamine-citrulline-arginine pathway[END_REF]. Finally, the one in vitro study [START_REF] Ham | L-Citrulline Protects Skeletal Muscle Cells from Cachectic Stimuli through an iNOS Dependent Mechanism[END_REF] did not consider hormones and mediators, which directly control muscle turnover.

The literature data being scant, the aim of our study was to evaluate the effect of Cit on the balance between protein synthesis and breakdown in hypercatabolic situations by an in vitro approach.

Myotubes derived from C2C12 myoblasts were treated with glucocorticoids (GCs, e.g. dexamethasone, DEX) or TNF-α. GCs play a major role in muscle wasting by decreasing protein synthesis while increasing protein breakdown [START_REF] Wang | Dexamethasone stimulates proteasome-and calciumdependent proteolysis in cultured L6 myotubes[END_REF]. The net catabolic effect of GCs results mainly from two mechanisms. First, they inhibit the transport of AAs into muscle cells, which could limit the synthesis by reduction in substrate availability. Second, they exert an inhibitory effect on key effectors (e.g. Akt) of the initiation of protein translation. In addition, the catabolic effect of GCs results from the activation of the major proteolytic systems such as the ubiquitin/proteasome system (MuRF1 and Atrogin-1) [START_REF] Schakman | Glucocorticoid-induced skeletal muscle atrophy[END_REF]. TNF-α is a pro-inflammatory cytokine stimulating muscle catabolism, inducing a contractile dysfunction in muscle cells [START_REF] Reid | Cytokines and oxidative signalling in skeletal muscle[END_REF], and potentiating cortisol/corticosterone action [START_REF] Zamir | Tumour necrosis factor (TNF) and interleukin-1 (IL-1) induce muscle proteolysis through different mechanisms[END_REF].

Materials and Methods

Materials: C2C12 (CRL-1172), high glucose-DMEM (Dulbecco's modified Eagle medium); 0.6 g/l L-Gln, 4.5 g/l D-glucose, 1 mM sodium pyruvate, and 1.5 g/l bicarbonate were purchased from LGC Standards-ATCC (Molsheim, France). Fetal calf serum, antibiotics, horse serum, cytosine arabinoside, dexamethasone, TNF-α, bovine serum albumin and L-tyrosine were from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Citrulline was a gift from Citrage (Créteil, France). Trypsin-EDTA solution was from Fisher Bioreagent TM (Illkirch, France). Matrigel and PBS were purchased from Dominique Dutscher (Paris, France). Puromycin and anti-puromycin antibody (clone 12D10) were purchased from Merck Millipore (Yvelines, France). Lysis buffer was purchased from Roche (Meylan, France). DC protein assay kit and mini-protean TGX TM precast protein gels were from Bio-Rad Laboratories (Marnes-la-Coquette, France). Horseradish peroxidase-conjugated anti-mouse antibody was from Jackson ImmunoResearch Laboratories Inc. (Newmarket, Suffolk, UK), and L-[3,5-3 H]tyrosine Ci/mmol), was from PerkinElmer Life Sciences (Boston, MA, USA).

Culture cells

The protocol used was that of Menconi et al. [START_REF] Menconi | Dexamethasone and corticosterone induce similar, but not identical, muscle wasting responses in cultured L6 and C2C12 myotubes[END_REF], and C2C12 mouse cells were used between P2 and P5. The model of using C2C12-derived myotubes as a model of catabolism by exposure to DEX or TNF is well established; see for example Kimura et al. [START_REF] Kimura | β-Hydroxy-β-methylbutyrate facilitates PI3K/Akt-dependent mammalian target of rapamycin and FoxO1/3a phosphorylations and alleviates tumor necrosis factor α/interferon γ-induced MuRF-1 expression in C2C12 cells[END_REF] and Mirza et al. [START_REF] Mirza | In vitro assessment of the combined effect of eicosapentaenoic acid, green tea extract and curcumin C3 on protein loss in C2C12 myotubes[END_REF].

Multiplication: After thawing, 310,000 myoblasts were incubated in a T25 culture flask in 7 ml of multiplication medium [high glucose-DMEM supplemented with 10% fetal calf serum (FCS) and 1% antibiotics (10,000 U.ml¯¹ of penicillin and 10 mg/ml of streptomycin)] for 4 days at 37 °C with a humidified atmosphere and 5% CO2.

Differentiation: At 85% confluence, myoblasts were collected after trypsin treatment (0.25% trypsin-0.53 mM ethylenediaminetetraacetic acid), and 350,000 myoblasts were seeded in six-well plates coated with Matrigel in 3 ml of multiplication medium for 24 h. The culture medium was then removed and replaced by 3 ml of differentiation medium [high glucose-DMEM supplemented with 2% horse serum (HS) and 1% antibiotics]. The culture medium was changed every day during which time the change in shape of the C2C12 was observed under optic microscopy. After 7 days, the culture medium was changed and supplemented with 10 µM cytosine arabinoside (CA) for 3 days. CA enabled removal of the undifferentiated myoblasts [START_REF] Menconi | Dexamethasone and corticosterone induce similar, but not identical, muscle wasting responses in cultured L6 and C2C12 myotubes[END_REF]. Catabolism in myotubes was induced by adding 150 nM dexamethasone (DEX) [START_REF] Menconi | Dexamethasone and corticosterone induce similar, but not identical, muscle wasting responses in cultured L6 and C2C12 myotubes[END_REF] or 10 ng/ml TNF-α [START_REF] Li | Skeletal muscle myocytes undergo protein loss and reactive oxygen-mediated NF-kappaB activation in response to tumor necrosis factor alpha[END_REF] to the medium for 24 h [START_REF] Li | NF-kB mediates the protein loss induced by TNF-α in differentiated skeletal muscle myotubes[END_REF] or 48 h [START_REF] Menconi | Dexamethasone and corticosterone induce similar, but not identical, muscle wasting responses in cultured L6 and C2C12 myotubes[END_REF] as shown in Figure 1. Control myotubes (CON group) were studied in parallel, and cells were incubated with 0.006% ethanol (vehicle).

For the study of Cit effects, cells were further incubated with/without 5 mM Cit for 6 h, as indicated below and in Figure 1.

Protein breakdown rate (PBR)

PBR was assessed by the measurement of Tyr [START_REF] Neveux | Measurement of amino acid concentrations in biological fluids and tissues using ion exchange chromatography[END_REF] release into cell medium: After 24 h of incubation with either DEX, TNF-α, or 0.006% ethanol, the medium was replaced, and the myotubes further incubated for 6 h with or without 5 mM Cit (Figure 1A). After 6 h, the culture medium was collected for the determination of tyrosine concentration. For this purpose, 1.5 ml of medium was deproteinized with 150 μl of sulfosalicylic acid (SSA 30%) and left for 10 minutes in ice, followed by 5 min centrifugation at 10,000 rpm at 4 °C. The supernatant was collected and kept at -80 °C until analysis by ion-exchange chromatography with post-column ninhydrin-based detection using a JLC-500/V AminoTac™ apparatus (Jeol, Tokyo, Japan).

Protein synthesis rate (PSR) PSR was evaluated using two different approaches: surface sensing of translation (SUnSET), described [START_REF] Schmidt | SUnSET, a nonradioactive method to monitor protein synthesis[END_REF][START_REF] Mobley | L-leucine, beta-hydroxy-betamethylbutyric acid (HMB) and creatine monohydrate prevent myostatin-induced Akirin-1/Mighty mRNA down-regulation and myotube atrophy[END_REF] as an alternative way to evaluate protein synthesis without using the radioactivity method, and cell incorporation of L-[3,5-3 H] Tyr [START_REF] Menconi | Dexamethasone and corticosterone induce similar, but not identical, muscle wasting responses in cultured L6 and C2C12 myotubes[END_REF].

SUnSET: This method is based on the capacity of puromycin, a structural analogue of aminoacyl tRNA, to be incorporated in newly synthesized proteins and be revealed by Western blot (WB). Each well was incubated with 1 µM of puromycin 30 min before the end of the protocol (Figure 1A). The plates were then placed in ice, and the wells rinsed once with 1 ml of cold PBS. Myotubes were lyzed with 120 µl ice-cold lysis buffer containing 40 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 25 mM sodium glycerophosphate, 25 mM sodium fluoride, 1 mM sodium orthovanadate, 10 µg/ml leupeptin, 1X concentrated anti-protease solution, and 0.5% Triton X-100. The samples were left at 4 °C for 15 minutes and centrifuged at 4 °C, 14,000 rpm for 20 min. Supernatants were collected and stored at -80 °C. Protein concentrations in the samples were determined with a DC protein assay kit, and the same amounts of proteins (5 µg) from each sample were dissolved in Laemmli buffer and subjected to electrophoretic separation on 4-15% SDS-PAGE acrylamide gels. Proteins were transferred to a nitrocellulose membrane. This step was verified by Ponceau red staining. The membrane was then incubated for 1 h with 5% powdered milk in Tris-buffered saline-0.05% Tween 20 (TBST). Nitrocellulose blots were then incubated overnight at 4 °C with anti-puromycin primary antibody (1:1000). After washing, membranes were incubated for 1 h at room temperature in 5% milk-TBST with horseradish peroxidase conjugated anti-mouse antibody (1:10,000). The signal was measured on an ImageQuant™ Las 4000 system using a CCD camera (GE-Healthcare, Vélizy-Villacoublay, France). Quantifications were performed using image analysis software (Image J). The optical density (OD) signal of puromycin was determined relative to the Ponceau red staining of the corresponding sample.

Cell incorporation of L-[3,5-3 H]tyrosine:

This measure was performed according to Menconi et al. [START_REF] Menconi | Dexamethasone and corticosterone induce similar, but not identical, muscle wasting responses in cultured L6 and C2C12 myotubes[END_REF].

Following their results, DEX significantly decreased the size of myotubes after 48 h of treatment, and so at the end of cell differentiation, myotubes were incubated for 48 h with either 150 nM DEX, 10 ng/ml TNF-α, or 0.006% ethanol (Figure 1B). After 48 h of incubation, the medium was replaced and the myotubes further incubated for 6 h with 5 mM Cit (Figure 1B). One hour before the end of the 6 h incubation period, the medium was removed in order to add L- [3,5 3 H]tyrosine (2 μCi/ml) and unlabeled L-tyrosine (2 mM) to the medium (Figure 1B). Thereafter, the cells were rinsed once with 1 ml ice-cold PBS followed by 5 min incubation with 1 ml 10% trichloroacetic acid (TCA). After washing three times with 1 ml 10 % TCA, the myotubes were lyzed with 120 µl of ice-cold lysis buffer containing 40 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 5 mM EGTA, 25 mM sodium glycerophosphate, 25 mM sodium fluoride, 1 mM sodium orthovanadate, 10 µg/ml leupeptin, 1X concentrated anti-protease solution, and 0.5% Triton X-100. The samples were left at 4 °C for 15 minutes and then centrifuged at 4 °C and 14,000 rpm for 20 min. Supernatants were collected in specific plastic flasks filled with 3 ml Ultima Gold® liquid scintillation cocktail (Solvable TM PerkinElmer Life Sciences, Boston, MA, USA). The radioactivity of each sample was counted on a scintillation counter (Tricarb®, PerkinElmer Life Sciences). PSR was expressed in dpm/µg protein.

Statistical analysis

Results are expressed as mean ± SEM. The action of Cit was analyzed by non-parametric tests: Kruskal-Wallis followed by Mann-Whitney. Statistical analyses were performed using Graphpad Prism (version 5.0; Graphpad Software Inc., San Diego, CA, USA). Differences were considered significant at p < 0.05.

Results

Protein breakdown rate

We did not observe any effect of TNF-α treatment on PBR, compared with CON. By contrast, Cit treatment significantly decreased PBR, vs. the CON group and vs. DEX-treated cells even thought that we did not observe any effect of DEX treatment on PBR, compared with CON (Figure 2).

Protein synthesis rate

Only the DEX treatment significantly decreased PSR compared with CON using both methods (Figure 3A and3B). Cit treatment significantly increased PSR in the DEX-treated group using both methods (Figure 3A and 3B). TNF-α had no significant action on PSR, whether or not the cells were treated with Cit.

Discussion

The disturbance of protein turnover in injured patients leads to net catabolism, which causes a decrease in muscle mass, and if protracted, muscle wasting and loss of function [START_REF] Golden | Protein turnover, synthesis and breakdown before and after recovery from protein-energy malnutrition[END_REF]. DEX and TNF-α play an essential role in muscle wasting: during stress, plasma GC concentration rises to 3.0 µmol/l [START_REF] Voisin | Cytokine modulation by PX differently affects specific acute phase proteins during sepsis in rats[END_REF] and stimulates protein breakdown and/or decreases protein synthesis in muscle, and this leads to an increase in the rate of appearance of free AAs in the plasma [START_REF] Cynober | Plasma amino acid levels with a note on membrane transport: characteristics, regulation, and metabolic significance[END_REF]. Menconi et al. [START_REF] Menconi | Dexamethasone and corticosterone induce similar, but not identical, muscle wasting responses in cultured L6 and C2C12 myotubes[END_REF] confirmed the in vitro effect of GC on protein breakdown, something we did not observe in our model. We used the same range of DEX concentration as they did (Menconi et al. noted a peak effect at 100 nM of DEX, and we used 150 nM of DEX), and the same incubation time for PBR evaluation, but we did not use the same evaluation method. Using the HPLC method enabled us to evaluate only the release of Tyr into the culture medium. This can adversely affect the sensitivity of the method: the radioactivity-based method takes into account all cell compartments where free Tyr may occur. In our study, we did note a GC effect on PSR: we report the GC effect, testing two different times of exposure (24 h and 48 h) and using two different methods (SUnSET and radioactivity-based method) because the SUnSET method is relatively new, and its reliability needs confirmation. The effect of GC on protein synthesis is controversial: some studies have reported an effect of GC on protein synthesis [START_REF] Schakman | Mechanisms of glucocorticoid-induced myopathy[END_REF], other not [START_REF] Menconi | Dexamethasone and corticosterone induce similar, but not identical, muscle wasting responses in cultured L6 and C2C12 myotubes[END_REF].

Concerning PSR evaluation, Menconi et al. [START_REF] Menconi | Dexamethasone and corticosterone induce similar, but not identical, muscle wasting responses in cultured L6 and C2C12 myotubes[END_REF] and our study differ in the DEX concentration: they used a much higher DEX concentration than we did. The lack of effect of GCs in Menconi's study [START_REF] Menconi | Dexamethasone and corticosterone induce similar, but not identical, muscle wasting responses in cultured L6 and C2C12 myotubes[END_REF] may therefore be related to a cytotoxic effect induced by pharmacological levels of GCs.

Thus, taken as a whole, in our in vitro model, GC induces net catabolism by decreasing drastically PSR without effect on PBR. This indicates that our model reflects situation such as surgical stress rather than trauma, burn injury or sepsis [START_REF] Cynober | Understanding the pathological mechanisms underlying protein breakdown for new therapeutic strategies[END_REF].

A possible explanation of our results is that inhibition of PSR leads to an increase in intracellular free amino acid content which in turn blunts protein breakdown [START_REF] Cynober | Plasma amino acid levels with a note on membrane transport: characteristics, regulation, and metabolic significance[END_REF].

In general, muscle wasting appears in inflammatory conditions (sepsis, cancer, etc.). In these situations, TNF-α concentration can be as high as 6 ng/ml [START_REF] Li | NF-kB mediates the protein loss induced by TNF-α in differentiated skeletal muscle myotubes[END_REF]. Buck et al. [START_REF] Buck | Muscle wasting and dedifferentiation induced by oxidative stress in a murine model of cachexia is prevented by inhibitors of nitric oxide synthesis and antioxidants[END_REF] have reported muscle wasting in a murine model of cachexia induced by chronically elevated serum TNF-α concentrations.

Reid and Li, in a review paper [START_REF] Reid | Tumor necrosis factor-α and muscle wasting: a cellular perspective[END_REF], have reported that the action of TNF-α can be related to NFκB activation and to reactive oxygen species (ROS) production in the mitochondria of the cells. In our model, we did not observe any effect of TNF-α on protein turnover. The effects of TNF-α reported in the literature are generally from in vivo studies. It is likely that TNF-α effects are related to cross-talk between myocytes and macrophages and/or muscle adipocytes, and also collaboration with other mediators such as other cytokines or GCs. However, Li and Reid et al. [START_REF] Li | NF-kB mediates the protein loss induced by TNF-α in differentiated skeletal muscle myotubes[END_REF], in an in vitro model of transfected C2C12 myoblasts overexpressing I-κBα proteins (endogenous NFκB inhibitor), treated by TNF-α, demonstrated that TNF-α induced protein breakdown by activating NFκB. Our method differed from theirs in using cytosine arabinoside during the cell culture protocol, which enables removal of undifferentiated myoblasts. The presence in the culture cells of both differentiated and undifferentiated myoblasts may have an influence on the action of TNF-α.

Concerning the effects of Cit, only one earlier study is relevant to ours: Ham et al., [START_REF] Ham | L-Citrulline Protects Skeletal Muscle Cells from Cachectic Stimuli through an iNOS Dependent Mechanism[END_REF] reported the direct beneficial effect of Cit on protein synthesis on C2C12 using different catabolic stimuli (starvation, inflammation and oxidative stress). After 48 h of starvation, PSR fell by 26%, and 1 h of Cit treatment restored it to the CON level. These conditions differed from ours, and the concentration of Cit was different (2.5 mM). Despite these differences, we obtained the same effect of Cit on protein synthesis. This agreement with Ham's findings [START_REF] Ham | L-Citrulline Protects Skeletal Muscle Cells from Cachectic Stimuli through an iNOS Dependent Mechanism[END_REF], and the fact that we obtained the same results using our methods, is strong evidence that Cit improves myocyte protein synthesis in various net catabolic conditions, and is an important finding. We also examined the effect of Cit on protein breakdown, which Ham et al. [START_REF] Ham | L-Citrulline Protects Skeletal Muscle Cells from Cachectic Stimuli through an iNOS Dependent Mechanism[END_REF] did not evaluate. Cit treatment decreased slightly (but significantly) the PBR in the DEX group in C2C12 cells. Our study is the first to be interested in the effect of a pharmaconutrient in these catabolic conditions (DEX or TNF-α). Ham et al. [START_REF] Ham | L-Citrulline Protects Skeletal Muscle Cells from Cachectic Stimuli through an iNOS Dependent Mechanism[END_REF] recommend the use of a dose of Cit above 1 mM, which we did with a dose of 5 mM in our study.

12 Taken as a whole, our data point out that the action of citrulline on muscle protein turnover relates primarily to an increase in protein synthesis with a marginal effect on muscle protein breakdown.

The action of Cit can be direct (i.e. through its own anti-oxidant properties [START_REF] Sinha | Nitric oxide down regulates tumour necrosis factor mRNA in RAW 264.7 cells[END_REF]), or indirect (through its metabolism into arginine (Arg) and/or further metabolism into nitric oxide [START_REF] Breuillard | In vitro anti-inflammatory effects of citrulline on peritoneal macrophages in Zucker diabetic fatty rats[END_REF]). Our in vitro approach yields evidence that the action of Cit is direct, because it is known [START_REF] Wakabayashi | The glutamate crossway[END_REF] that myocytes do not contain argininosuccinate synthase and lyase, which are required to convert Cit into Arg [START_REF] Curis | Almost all about citrulline in mammals[END_REF].

Our study has two limitations: first, we did not study the effect of a combination of DEX and TNF-α, knowing that the latter acts primarily to modulate the action of the former [START_REF] Zamir | Tumour necrosis factor (TNF) and interleukin-1 (IL-1) induce muscle proteolysis through different mechanisms[END_REF], and second, we did not make any attempt to determine the underlying mechanism of Cit action. This point also deserves further study. However, we believe that our results taken as a whole represent a significant advance in the field.

In conclusion, our results indicate that Cit treatment leads to a regulation of muscle protein turnover, acting both on PSR and PBR, in an in vitro model of muscle catabolism. It will now be of interest to explore the underlying mechanisms of these effects.

Legends of figures Figure 1. Study design

C2C12 cells were at the myoblast state; we started by inducing the differentiation of myoblasts in myotubes (Figure 1A).

Myotubes were then treated with 150 nM of DEX or 10 ng/ ml of TNF-α or 0.006 % ethanol for 24 h, after which myotubes were treated with 5 mM of Cit for 6 h (Figure 1A). The Tyr released into the medium was measured by HPLC (PBR evaluation).

For the SUnSET protocol, myotubes were treated with 1 µM of puromycin 30 min before the end of the 6 h protocol (PSR evaluation) (Figure 1A). Concerning the evaluation of L-[3,5-3 H]Tyr (radioactivity protocol), myotubes were treated with 150 nM of DEX or 10 ng/ml of TNF-α or 0.006 % ethanol for 48 h (Figure 1B). Myotubes were then treated with 5mM of Cit, and 1 h before the end of the protocol with 2 µCi/ml labeled Tyr and 2 mM unlabeled Tyr (PSR evaluation). Each experiment was repeated at least four times, and each experimental point was run in duplicate within each experiment.

Figure 2 .

 2 Figure 2. Protein breakdown rate in C2C12 myotubes using HPLC method.

Figure 3 .

 3 Figure 3. Protein synthesis rate (PSR) in C2C12 myotubes (A) using SUnSET method and (B) using radioactivity-based method.

  Figure 1


Rachid Cherif
email: rachid.cherif@univ-lr.fr

Ameur El

Amine Hamami

Abdelkarim Aït-Mokhtar

Effects of leaching and chloride migration on the microstructure and pore solution of blended cement pastes during a migration test

Keywords: cement pastes, pore size distribution, chlorides, multispecies transfer, thermodynamic equilibrium, leaching

This paper investigates the influence of the multispecies diffusion and leaching phenomena on the thermodynamic equilibrium and microstructure of cementitious materials containing mineral additions during a chloride migration test. Because of a lack of data on these effects, the aim of this study was to quantify changes in the material's microstructure during an experimental program based on mercury intrusion porosimetry before and after chloride exposure, scanning electron microscopy and chemical analyses of the materials. The experimental results highlight a modification of pore size distribution and a reduction of up to 60% in total porosity after chloride transfer. This was due to chloride interactions which led to the formation of new compounds in the porosity range of 7 to 70 nm. These compounds were investigated by elemental analyses and quantified.

Moreover, a chloride migration test was performed in order to investigate the leaching of pore solution ions in the two compartments of the migration cell. The results enabled us to: (i) quantify the concentration of ions that diffused into/from the material, and (ii) estimate the kinetics of calcium release and the quantity of portlandite dissolved in the material. The chloride diffusion coefficient of the materials ranged between 2.38x10 -12 and 5.96x10 -12 m²/s, the kinetics of calcium, expressed as a flux, were between 1.8x10 -2 and 1.2x10 -1 mmol.m -2 .s -1 .

Introduction

The durability of reinforced concrete structures is governed by the mechanical behavior of the materials used and vice-versa. Durability is also affected by environmental attacks, in particular by aggressive species present in the environment of the structures [START_REF] Shi | Durability of steel reinforced concrete in chloride environments: An overview[END_REF][START_REF] Zahedi | Evaluation of the mechanical properties and durability of cement mortars containing nanosilica and rice husk ash under chloride ion penetration[END_REF][START_REF] Balapour | An investigation on mechanical and durability properties of mortars containing nano and micro RHA[END_REF][START_REF] Weiss | Prediction of chloride ingress in saturated concrete using formation factor and chloride binding isotherm[END_REF][START_REF] Ramezanianpour | Modeling of chloride ions penetration in cracked concrete structures exposed to marine environments[END_REF][START_REF] Qiao | Damage in cement pastes exposed to NaCl solutions[END_REF]. This phenomenon leads to the corrosion of rebar steel and damage to concrete [START_REF] Apostolopoulos | Consequences of steel corrosion on the ductility properties of reinforcement bar[END_REF][START_REF] Franco-Luján | Chlorideinduced reinforcing steel corrosion in ternary concretes containing fly ash and untreated sugarcane bagasse ash[END_REF]. The aggressive species studied in the literature are mainly carbon dioxide, marine or de-icing salts and sulfates. The present work focused on chloride ion transport through cementitious materials.

During ionic transport, chlorides interact chemically with anhydrous compounds in cement (chemical interactions) [START_REF] Amiri | Modelling of chloride-binding isotherm by multi-species approach in cement mortars submitted to migration test[END_REF][START_REF] Sutter | Petrographic evidence of calcium oxychloride formation in mortars exposed to magnesium chloride solution[END_REF]. Chlorides can also be adsorbed by the C-S-H layers (electrochemical interactions and electrical double layer) [START_REF] Amiri | Electrochemical modelling of chloride migration in cement-based materials. Part I. Theoretical basis at microscopic scale[END_REF][START_REF] Friedmann | Physical modeling of the electrical double layer effects on multispecies ions transport in cement-based materials[END_REF]. Indeed, the chemical interactions produce compounds that precipitate in the pore solution and modify the porosity and pore size distribution of the material, as has been demonstrated in the carbonation of cementitious materials [START_REF] Metalssi | Consequences of carbonation on microstructure and drying shrinkage of a mortar with cellulose ether[END_REF][START_REF] Turcry | Analysis of an accelerated carbonation test with severe preconditioning[END_REF], and this can even induce cracking [START_REF] Mauroux | Study of cracking due to drying in coating mortars by digital image correlation[END_REF]. In terms of the chemical composition of the pore solution, external ions such as chlorides generate a thermodynamic imbalance in the hydrates/pore solution system, promote the dissolution of portlandite [START_REF] Chatterji | Mechanism of the CaCl2 attack on Portland cement concrete[END_REF], [START_REF] Glasser | Solubility reactions of cement components with NaCl solutions: I. Ca(OH) 2 and C-S-H[END_REF] and the precipitation of compounds such as Friedel's and Kuzel's salts [START_REF] Qiao | Damage in cement pastes exposed to NaCl solutions[END_REF][START_REF] Suryavanshi | Mechanism of Friedel's salt formation in cements rich in tri-calcium aluminate[END_REF][START_REF] Mesbah | Crystal structure of Kuzel's salt 3CaO•Al2O3•1/2CaSO4•1/2CaCl2•11H2O determined by synchrotron powder diffraction[END_REF].

Qiao et al. [START_REF] Qiao | Damage in cement pastes exposed to NaCl solutions[END_REF] quantified the Friedel's salt formed in the porosity of cement-based materials during chloride transfer and its influence on the mechanical properties (flexural strength) due to crystallization pressure. Furthermore, Midgley and Illston [START_REF] Midgley | The penetration of chlorides into hardened cement pastes[END_REF] and Kayyali [START_REF] Kayyali | Chloride penetration and the ratio of Cl -/OHin the pores of cement paste[END_REF] monitored the porosity of Portland cement pastes after 28 and 90 days of immersion in sodium chloride solutions (30 and 39 g/l respectively). They showed that chlorides reduced and refined the porosity due to the formation of calcium chloride on the surface of C-S-H. Regourd et al. [START_REF] Regourd | Microstructure of Concrete in Aggressive Environments, 1st Int. Conf. on Durability of Building Materials and Components[END_REF] reported that chlorides changed the initially fibrous C-S-H to a reticulated morphology. In addition, Koleva et al. [START_REF] Koleva | Microstructural analysis of plain and reinforced mortars under chloride-induced deterioration[END_REF] measured the pore size distribution of mortars partially immersed in a 7% NaCl solution. The results showed that chloride diffusion into the fully immersed part of the material decreased its total porosity and refined the pore network. Sutter et al. [START_REF] Sutter | Petrographic evidence of calcium oxychloride formation in mortars exposed to magnesium chloride solution[END_REF] reported a formation of calcium oxychloride in the porosity of mortar specimens immersed in a 15% MgCl2 solution. This modification of the microstructure influences the intrinsic permeability [START_REF] Koleva | Microstructural analysis of plain and reinforced mortars under chloride-induced deterioration[END_REF] of the material and its chloride diffusion coefficient. Finally, Hu et al. [START_REF] Hu | Influences of chloride immersion on zeta potential and chloride concentration index of cement-based materials[END_REF] immersed samples of cement in a NaCl solution (0.1 to 1.0 mol/l). Their investigations showed that the chloride concentration index and Zeta potential decrease with the concentration of the soaking solution.

More recently, Electrochemical Impedance Spectroscopy (EIS) has been used as a powerful technique to study the microstructure and mechanical properties of cementitious materials [START_REF] Aït-Mokhtar | Relationship between the transfer properties of the coating and impedance spectroscopy in reinforced cement-based materials[END_REF][START_REF] Hamami | Cement fraction effect on EIS response of chloride migration tests[END_REF][START_REF] Sànchez | Microstructural modifications in Portland cement concrete due to forced ionic migration tests. Study by impedance spectroscopy[END_REF]. This technique can be used to correlate the dielectric response and the characteristics of the material. Sanchez et al. [START_REF] Sànchez | Microstructural modifications in Portland cement concrete due to forced ionic migration tests. Study by impedance spectroscopy[END_REF] used EIS to monitor the microstructural changes of concrete in real time during a chloride migration test from the beginning (non-steady state) until the steady state was reached. The authors noted that the dielectric response of concrete was strongly modified during the chloride migration test and that these modifications were due to microstructural changes in the concrete. Indeed, during the first stages of chloride migration, resistance decreased to a minimum value (180 Ω) when the material was completely saturated with chlorides. In addition, the capacity values remained low and constant during the first 100 -200 hours. No modification in the microstructure was observed, only the pore solution changed. After an average of 200 hours, an increase in resistance and capacity was recorded.

This was explained by a reduction in total porosity and an increase in tortuosity due to the formation of new compounds in the porosity. Subsequently, these dielectric parameters became constant at the steady state.

In reality, chlorides penetrate into concrete by natural diffusion under total immersion in water or by diffusion and convection for partial immersion, e.g. in the tidal phenomenon. Diffusion is a very slow phenomenon for investigations in the laboratory compared to accelerated chloride migration. Several researchers [START_REF] Tang | Concentration dependence of diffusion and migration of chloride ions: Part 1. Theoretical considerations[END_REF][START_REF] Tang | Concentration dependence of diffusion and migration of chloride ions: Part 2. Experimental evaluations[END_REF][START_REF] Castellote | Measurements of the steady and non-steady-state chloride diffusion coefficients in a migration test by means of monitoring the conductivity in the anolyte chamber. Comparison with natural diffusion tests[END_REF] have discussed the difference between chloride diffusion (what is really happening) and accelerated diffusion (i.e. migration) due to the presence of a counter electrical field. In this sense, several theoretical relationships between chloride diffusion and migration coefficients have been proposed [START_REF] Tang | Concentration dependence of diffusion and migration of chloride ions: Part 1. Theoretical considerations[END_REF][START_REF] Chiang | Relation between the diffusion characteristic of concrete from salt ponding test and accelerated chloride migration test[END_REF]. In general, accelerated diffusion does not exist, but remains a simple and rapid way to estimate the chloride diffusion coefficient as well as the influence of chloride on microstructure in the lab [START_REF] Sànchez | Microstructural modifications in Portland cement concrete due to forced ionic migration tests. Study by impedance spectroscopy[END_REF][START_REF] Castellote | Changes in concrete pore size distribution due to electrochemical chloride migration trials[END_REF].

These studies only focused on the causes of microstructure changes during chloride transfer due to the precipitation of new compounds. The latter were not quantified. The physicochemical process of the dissolution/precipitation phenomena was not investigated to explain these microstructural changes. To our knowledge, there are few studies on the change in thermodynamic equilibrium induced by chloride diffusion, or the quantification of new species derived from the dissolution or precipitation of compounds to reach a new equilibrium in the thermodynamic system. Moreover, numerical models for simulating chloride transfer have been developed from Fick's law [START_REF] Majlbro | The complete solution of Fick's second law of diffusion with time dependent diffusion coefficient and surface concentration, Durab[END_REF][START_REF] Amiri | A complement to the discussion of A. Xu and S. Chandra about the paper «Calculation of chloride coefficient diffusion in concrete from ionic migration measurements[END_REF] and, more recently, from the Nernst-Planck equation [START_REF] Xia | Numerical simulation of ionic transport in cement paste under the action of externally applied electric field[END_REF][START_REF] Jiang | Modeling the chloride concentration profile in migration test based on general Poisson Nernst Planck equations and pore structure hypothesis[END_REF][START_REF] Jensen | Use of a multi-species reactive transport model to simulate chloride ingress in mortar exposed to NaCl solution or sea-water[END_REF][START_REF] Mao | Multi-phase modelling of electrochemical rehabilitation for ASR and chloride affected concrete composites[END_REF]. Often, these models do not take into account the porosity or pore size distribution induced by chloride transfer because of a lack of data and the complexity in modeling this phenomenon.

Therefore, the aim of this study was to investigate the changes in both microstructure and pore solution of cement paste samples exposed to a NaCl solution. These changes are investigated MIP before and after migration tests, along with image processing from scanning electron microscopy (SEM). The objective was to characterize pore size modification and then to identify and quantify the compounds responsible for the change in porosity, apart from known salts like Friedel's.

In addition, chemical analyses of the pore solution and upstream and downstream analyses of the migration cell were performed to identify the species diffusing into/out of the material during a migration test. Thus, we could quantify the species that leached from the sample, and monitor the kinetics and degree of portlandite dissolution, which is responsible for the stabilization of the thermodynamic equilibrium. These phenomena modify the pore solution and microstructure and affect chloride transport conditions.

Experimental approach

Materials

An ordinary European Standard (NF EN 197-1) Portland cement "CEMI 52.5 N" was used [START_REF]Ciment -Partie 1: Composition, spécifications et critères de conformité des ciments courants[END_REF]. The mass fractions of the principal clinker phases provided by the manufacturer (Calcia, Bussac-France) and based on the Bogue calculation method are 65% C3S, 13% C2S, 7% C3A, 13% C4AF and 4.9% gypsum. Furthermore, pozzolanic materials and limestone (limestone filler "LF", fly ash "FA", blast furnace slag "S" and silica fume "SF") were used as a mass substituent of the cement. The chemical composition, density and Blaine specific surface area (BSSA) of the cement and the different additions used are given in Table 1. Firstly, a reference concrete based on CEM I 52.5 N was formulated following the Dreux-Gorisse method, with an average compressive strength fcm of 38 MPa. A fixed amount of cement of 300 kg/m 3 was used. Then, we formulated other concretes containing pozzolanic materials and limestone using an approach similar to that used by Khokhar et al. [START_REF] Khokhar | Mix design of concrete with high content of mineral additions: Optimisation to improve early age strength[END_REF] and Younsi et al. [START_REF] Younsi | Performance-based design and carbonation of concrete with high fly ash content[END_REF], keeping the paste volume (Vp) constant and equal to that of the reference concrete (VP = 24.5% of the total volume). This approach is based on solving a system of equations for 5 unknowns: VG, VS, VC, VA and Vw that are volumes of gravel, sand, cement, additions and water, respectively. The total volume of concrete was 0.975 m 3 (considering a volume of air of 0.025 m 3 ). The system of equations is as follows:

+ + + + = 0.975 = + = + + = = 0.5 + -0.5" (1)
where (σ=52.5MPa) is the cement resistance, C, A and W are the masses in (kg) of cement, additions and water, respectively. The last equation (Bolomey relation) links fcm and the W/B (Water/Binder) ratios by taking into account the coefficient of chemical activity "k" of each addition #$ = + %. This causes a difference between the W/B of the formulated materials.

The properties of the formulated concrete pastes were used to manufacture the cement pastes studied in this work. Investigations on cement pastes are easier than on concretes, mainly for pore solution extraction, thanks to the higher porosity of the cement paste.

Five cement pastes were used: the reference cement paste without additions is called PCI, while those containing different pozzolanic materials and limestone are called LF25 (25% of Limestone Filler), FA30 (30% of fly ash), S75 (75% of blast furnace slag) and SF10 (10% of silica fume) respectively. Table 2 gives the W/B ratios of the cement pastes tested. The choice of these additions and their ratios of cement replacement are justified as follows:

75% of blast furnace slag brings major modifications to the microstructure of cementitious materials and chloride diffusivity, particularly the chemical and electrochemical fixation of chlorides on the C-S-H layers [START_REF] Dhir | Chloride binding in GGBS concrete[END_REF]. Fraay et al. [START_REF] Fraay | The reaction of fly ash in Concrete. A critical Examination[END_REF] demonstrated that for the mass replacement of cement by more than 30% of fly ash, only 50% of the latter reacts at the end of the first year.

According to Perraton et al. [START_REF] Perraton | Permeability of Silica Fume Concrete[END_REF], it is preferable to substitute between 5 and 10% of the mass of cement with silica fume; above 10%, the replacement is useless and the performance of the material decreases.

Cylindrical specimens (11cm diameter and 22cm height) were cast and demoulded 24 hours after manufacturing and cured for 365 days in an alkaline solution composed of 25 mmol/l NaOH and 83 mmol/l KOH to reduce leaching [START_REF] Hamami | Cement fraction effect on EIS response of chloride migration tests[END_REF][START_REF] Truc | A new way for determining the chloride diffusion coefficient in concrete from steady state migration test[END_REF]. Afterward, the samples used in the migration test were obtained from these specimens by coring samples from the heart and saw cutting to obtain discs 6.3 cm in diameter and 2.8 cm thick.

Tests and procedures

Cement paste samples, aged 1 year, were subjected to migration tests for 12 -14 days. This period corresponds to the time required to achieve a steady state of chloride migration. Pore size distribution was determined before and after each migration test. For a better representativeness, samples used for pore size distribution before chloride migration were taken in the neighborhood of the sample used for the migration test. At the end of the migration test, samples were cut off manually from the disc for an MIP test (mercury porosity, pore size distribution and critical pore diameter) and SEM analyses (see details of the performed tests in Fig. 1). A steady-state chloride migration test was used in order to quantify more precisely the complete change induced by the test. While in a non-steady state, modifications always take place while measurements are being made to calculate the apparent diffusion coefficient.

Quantifying these modifications is then difficult to achieve.

Water and mercury porosimetry

The water porosity of the cement pastes was measured by hydrostatic weighing according to the French NFP 18-459 standard [START_REF] Nf P | Concrete -Testing hardened concrete -Testing porosity and density[END_REF]. The weighing scale used was accurate down to 1 mg.

The water porosity was calculated using eq. ( 2).

& = ' ()* -' +,- ' ()* -' .)*/, × 100 (2)
where, Msat is the mass of the saturated sample, Mdry is the mass of the dried sample and Mwater is the mass of the saturated specimen immersed in water (obtained by hydrostatic weighing).

MIP measurements were performed with an Autopore III 9420 from Micrometrics ® whose pressure limit is more than 400 MPa; it covers pore diameters from 3 nm to 360 µm. Some authors [START_REF] Diamond | Aspects of concrete porosity revisited[END_REF][START_REF] Diamond | Mercury porosimetry. An inappropriate method for the measurement of pore size distributions in cement-based materials[END_REF] have suggested that this technique is not optimal for measuring pore size distribution. Nevertheless, it remains widely used [START_REF] Sànchez | Microstructural modifications in Portland cement concrete due to forced ionic migration tests. Study by impedance spectroscopy[END_REF][START_REF] Aït-Mokhtar | On the applicability of washburn law: study of mercury and water flow properties in cement-based materials[END_REF]. Prior to the test, the samples were first dried at 45°C under a vacuum until mass stabilization. The main advantage of using this temperature is that it avoids cracking and delays ettringites formation [START_REF] Divet | Delayed Ettringite Formation: The Effect of Temperature and Basicity on the Interaction of Sulphate and C-S-H Phase 1[END_REF]. The samples were cut off manually, avoiding the use of a saw, which can modify the sample's microstructure.

Migration test

The method developed by Aı̈t-Mokhtar et al. [START_REF] Aı̈t-Mokhtar | A new method for determination of chloride flux in cement-based materials from chronoamperometry[END_REF] was used. The objective was to determine the effective chloride diffusion coefficient in the steady state. An electrical field of 300 V/m was applied between the two sides of the saturated sample. This value was chosen to avoid the Joule effect, temperature increase and sample degradation [START_REF] Amiri | Modelling of chloride-binding isotherm by multi-species approach in cement mortars submitted to migration test[END_REF][START_REF] Sànchez | Microstructural modifications in Portland cement concrete due to forced ionic migration tests. Study by impedance spectroscopy[END_REF][START_REF] Castellote | Measurements of the steady and non-steady-state chloride diffusion coefficients in a migration test by means of monitoring the conductivity in the anolyte chamber. Comparison with natural diffusion tests[END_REF][START_REF] Andrade | Calculation of chloride diffusion coefficients in concrete from ionic migration measurements[END_REF]. A support solution based on 25 mmol/l of NaOH and 83 mmol/l of KOH was used in the upstream and downstream compartments of the migration cell to simulate the pore solution of cementitious materials [START_REF] Hamami | Cement fraction effect on EIS response of chloride migration tests[END_REF][START_REF] Truc | A new way for determining the chloride diffusion coefficient in concrete from steady state migration test[END_REF]. NaCl was added to the upstream solution at a concentration of 500 mmol/l. The compartment solutions were renewed every 24 hours, without taking into account changes in their concentration, in order to keep the same boundary conditions on both sides of the specimens during the migration test. The solutions in the compartments were analyzed before each renewal in order to monitor diffusion/leaching kinetics. The effective chloride diffusion coefficient Deff (m/s²) was calculated using eq.( 2) [START_REF] Amiri | Optimisation de l'exploitation de l'essai d'électrodiffusion d'ions chlorure dans le béton[END_REF].

10 2 /33 = 456 7 8 9 :; < = >1 -? @ AB 9 CD EF G (2)
where L (m) is the thickness of the sample; Cup (mol/m 3 ) the concentration of chloride ions in the upstream solution; U (V) the potential difference at the sample terminals; F, zCl -, R and T represent the Faraday constant, the valence of the chloride ion, the ideal gas constant and the absolute temperature (K); J is the chloride flux.

Analyzes of solutions with ionic chromatography (IC)

The upstream and downstream solutions of the migration cell were regularly analyzed. In addition, the initial concentration of calcium and sulfates in the pore solution of the material (before the migration test) were dosed and compared to those that leached into the two compartments during the migration test. Chemical analyses were performed using a "883Basic

Ion Chromatograph Plus" from Metrohm ® . Note that for the upstream solution, dilutions of 1:1800, 1:150, 1:40 and 1:1 were necessary to dose sodium, potassium, calcium and sulfates, respectively. The downstream solution required a 1:150 dilution to dose sodium and potassium and 1:1 for calcium and sulfates.

Extraction of the pore solution samples

The pore solution of the cement pastes was extracted using an OpiCAD ® device according to a procedure previously described [START_REF] Longuet | The liquid phase of hydrated cement[END_REF][START_REF] Barneyback | Expression and analysis of pore fluids from hardened cement pastes and mortars[END_REF]. The cement paste sample, with a volume of 137 cm 3 , was placed in a cylindrical chamber underneath a piston. The piston was loaded using a standard compression test machine. In order to obtain an optimal performance and take into account the creep effects, two steps were conducted. First, the samples were loaded at a constant loading velocity of about 0.02 MPa/s for 15 minutes. This first step was followed by a 5 minutes idle phase. Finally, the solution was collected from the bottom of the cylindrical chamber and analyzed by ionic chromatography.

Scanning Electron Microscopy (SEM)

The microstructure of the tested samples was analyzed by scanning electron microscopy (SEM) in environmental mode before and after chloride migration. The advantage of this mode is that no preparation is required and no coverage is deposited on the surface of the specimens, which avoids disturbing the morphology and the microstructure of the sample. Also, the MEB test was performed on manually fractured samples immediately at the end of the migration test. This avoids using a concrete saw and preserves the microstructure and chemical composition of the surface of the specimens. Furthermore, to measure the elemental distribution of precipitated compounds at the microscopic level, energy dispersive X-ray micro-analysis (EDX) was coupled to the SEM analyses.

Results and Discussion

Diffusion coefficient and water porosity

Fig. 2 shows the water porosity and its standard deviation from three measurements of the cement pastes. PCI had the highest porosity compared to the other pastes. This is mainly due to the higher W/B ratio of PCI compared to LF25, FA30, S75 and SF10 (see Table 2). Furthermore, the use of 10% silica fume should decrease the porosity as long as it is compared to pastes with the same W/B ratios [START_REF] Kdureković | Cement pastes of low water to solid ratio: An investigation of the porosity characteristics under the influence of a superplasticizer and silica fume[END_REF]. However, the W/B ratio of SF10 is higher than the other cement pastes with pozzolanic additions and a filler, which is why the total porosity of the cement pastes used was relatively similar. LF25 has a low W/B ratio and thus a relatively low porosity. Fig. 3 gives the chloride diffusion coefficient and its standard deviation from three measurements of the cement pastes. The results show that S75 had the lowest chloride diffusion coefficient compared to the other cement pastes. This is due to the replacement of Portland cement by 75% of blast furnace slag, which refines the microstructure. Indeed, a finer microstructure increases the electrochemical fixation of chlorides at the pore-solution interface of the material, which slows down diffusivity [START_REF] Dhir | Chloride binding in GGBS concrete[END_REF]. Despite the use of pozzolanic materials such as silica fume (SF), which are finer than cement and are supposed to reduce the diffusion coefficient [START_REF] Kong | Influence of nano-silica agglomeration on microstructure and properties of the hardened cement-based materials[END_REF], the results show that the chloride diffusion coefficients of PCI, LF25, FA30 and SF10 were of the same order of magnitude. The difference observed can be attributed to the mixing of the cement pastes without vibration and without a superplasticizer, which generates a non-optimized microstructure. The low W/B ratio of SF10 is a disadvantage for the non-optimization of the microstructure, taking into account the high-water demand for silica fumes.

Influence of chlorides on the microstructure of the cement pastes

Fig. 4 illustrates the mercury porosity values before and after the chloride migration tests. After migration, a reduction in porosity was noticed for all the materials, with ratios of 2.5, 2.1, 2.2, 1.6 and 1.4 for PCI, LF25, FA30, S75 and SF10, respectively. This reduction is due to the formation of new chloride-based compounds in the porosity such as Friedel's and Kuzel's salt [START_REF] Qiao | Damage in cement pastes exposed to NaCl solutions[END_REF][START_REF] Mesbah | Crystal structure of Kuzel's salt 3CaO•Al2O3•1/2CaSO4•1/2CaCl2•11H2O determined by synchrotron powder diffraction[END_REF], and calcium oxychloride [START_REF] Sutter | Petrographic evidence of calcium oxychloride formation in mortars exposed to magnesium chloride solution[END_REF]. In addition, Fig. 5 illustrates the pore size distribution obtained by MIP before and after the migration tests. Before the migration tests, the pore size distribution of the cement pastes was generally monomodal. The critical pore diameters were located around 69 nm for PCI, 50 nm for LF25, 45 nm for FA30 and 69 nm for SF10. However, with about 8 nm, the S75 critical pore diameter was the smallest. This is in agreement with previously published results [START_REF] Dhir | Chloride binding in GGBS concrete[END_REF].

After chloride migration, there were changes in pore size distribution and a shift in critical diameter peaks. number of large pores and the increase in the number of fine pores [START_REF] Castellote | Changes in concrete pore size distribution due to electrochemical chloride migration trials[END_REF][START_REF] Marcotte | The effect of the electrochemical chloride extraction treatment on steel-reinforced mortar. Part II. Microstructural characterization[END_REF]. However, S75 highlighted the changes in pore size distribution and porosity, but its critical diameter remained constant. The latter was too small to contain new compounds, which require more space [START_REF] Scherer | Crystallization in pores[END_REF].

These microstructure changes affect transport properties such as air or water permeability. In general, cement hydrates form a basic pore solution composed mainly of sodium, potassium and hydroxyl [START_REF] Andersson | Chemical composition of cement pore solutions[END_REF]. The exposure of cementitious materials to saline environments generates an imbalance in the hydrates/pore solution thermodynamic system due to chloride diffusion and leaching of the pore solution ions. This imbalance induces some hydrate dissolution to stabilize the system [START_REF] Damidot | Thermodynamic investigation of the CaO-Al2O3 CaSO4-H2O system at 50°C and 85°C[END_REF]. For instance, Chatterji [START_REF] Chatterji | Mechanism of the CaCl2 attack on Portland cement concrete[END_REF] showed that chloride transfer is accompanied by the formation of salts, providing compounds in the form of (C3A CaCl2 x H2O). Furthermore, Monosi and Collepardi [START_REF] Monosi | Research on 3CaO•CaCl2•15H2O identified in concretes damaged by CaCl2 attack[END_REF] and Sutter et al. [START_REF] Sutter | Petrographic evidence of calcium oxychloride formation in mortars exposed to magnesium chloride solution[END_REF] showed that chlorides induce the precipitation of hydrated calcium oxychloride (3CaO CaCl2 15H2O) in the porosity and a consumption of portlandite according to reaction [START_REF] Shi | Durability of steel reinforced concrete in chloride environments: An overview[END_REF]. They noted the precipitation of Friedel's salt

(3CaO•Al2O3•CaCl2•10H2O) in
In general, the refinement in porosity due to the precipitation of new salts during chloride transfer can influence the measurement of the diffusion coefficient, mainly for a migration test in a non-steady state, where the chemical interactions and the precipitation phenomena are not completed. The crystallization of these salts can generate cracks, principally in materials with low porosities [START_REF] Scherer | Crystallization in pores[END_REF]. Therefore, this influences the estimation of the diffusion coefficient. If the steady state test is used, the microstructure modification will have already taken place. There is a constant flow of chlorides downstream and the calculated diffusion coefficient is that of the modified material.

In order to quantify the chemical composition of these newly formed salts, apart from known ones such as Friedel's salt, we used SEM analyses to investigate the surface characteristics and microstructure of the hardened cement pastes, before and after the migration tests. A comparison between the SEM Images of healthy materials and those exposed to chloride migration is shown in Fig. 7 to Fig. 9 (For PCI, FA30 and S75, respectively). Figs 7a, 8a and 9a

show the healthy materials, and Figs 7b, 8b and 9b show the materials exposed to chlorides.

The corresponding EDX spectra, with a precision of 10 -2 , are also given. Note that the location of these investigated areas was chosen in wall pores where new chloride-based compounds are formed. This allowed us to quantify the species that make up these salts.

For PCI, X-ray analyses showed new chloride-based precipitates in the porosity after the migration tests. We also observed sulfides, which could confirm the possible formation of monosulfoaluminates (AFm) and trisulfoaluminates (AFt) in the porosity. Indeed, Famy et al. [START_REF] Famy | Effects of an early or a late heat treatment on the microstructure and composition of inner C-S-H products of Portland cement mortars[END_REF] showed that sulfates are physically adsorbed by the C-S-H layers. The presence of calcium and alkalis in the pore solution increases the quantity of sulfates adsorbed [START_REF] Divet | Delayed Ettringite Formation: The Effect of Temperature and Basicity on the Interaction of Sulphate and C-S-H Phase 1[END_REF]. That is why leaching alkalis and calcium affect the release of sulfates and then the precipitation/dissolution of AFm and AFt [START_REF] Damidot | Thermodynamic investigation of the CaO-Al2O3 CaSO4-H2O system at 50°C and 85°C[END_REF][START_REF] Glasser | The role of sulphate mineralogy cue temperature in delayed ettringite formation[END_REF]. Moreover, Marcotte et al. [START_REF] Marcotte | The effect of the electrochemical chloride extraction treatment on steel-reinforced mortar. Part II. Microstructural characterization[END_REF] showed that ionic migration through concrete leads to the partial decomposition of some hydrates such as C-S-H and AFm.

Therefore, different ions such as Ca 2+ , OH -and SO4 2-can be released in the pore solution. These species may participate in the precipitation of salts in larger pores. This phenomenon could be projected to the transfer of chlorides in general. Furthermore, potassium in a solid form was observed in materials after the migration tests (Table 3). Unfortunately, we did not follow this reaction. This was probably due to a precipitation of KCl. However, at this stage, we have no justification for this phenomenon. More chemical investigations will be necessary to elucidate the reaction responsible for the potassium precipitation. Table 3 shows the mass fraction of elements corresponding to the spectra. It represents a comparison between the weights of the chemical elements in the solid part analyzed before and after the migration of chlorides. This gives information on the elemental distribution of the new compounds formed. The spectra in Fig. 9 give the chemical composition of the investigation area of S75 before and after the migration tests. After the tests, the spectrum of S75, whose microstructure has fine pores, highlights the low chloride content: 0.68% in weight for S75 (Table 5) compared to 2.41% and 1.71% for PCI and FA30, respectively. This result confirms that new chloride based compounds precipitate more easily in large pores [START_REF] Scherer | Crystallization in pores[END_REF]. The chemical composition of S75 presented in Table 5 is in agreement with that of PCI and FA30. Finally, the amount of carbon 20 noted for the three measurements is certainly due to the preparation and handling of the sample before MEB analyses (see section 2.2.5). There is no risk of carbonation in the MEB chamber with the environmental mode since the vacuum pump does not contain oil.

Changes to upstream and downstream solutions during the migration test

To avoid leaching of the material during the migration test, the two compartments of the migration cell were filled with a basic solution considered to be similar to the pore solution (see section 2.2.2). However, real pore solution chemistry is not so simple. It contains other divalent ions such as calcium and sulfates [START_REF] Andersson | Chemical composition of cement pore solutions[END_REF][START_REF] Xu | The influence of sulphates on chloride binding and pore solution chemistry[END_REF]. In all cases, free ions diffuse from the material to the solutions in the compartments and vice versa, under the effect of the concentration gradient and the electrical field. Chlorides and sulfates react with the remaining anhydrous cement, calcium, sodium and potassium to form products that modify porosity, as shown above. In order to quantify the ions that diffused to/from the sample, pore solution and compartment solutions were dosed regularly during the migration test before each renewal (see section 2.2.2). We present below the concentration of ions that diffused to/from upstream and downstream. The variation in sodium and potassium in the compartments (basic solution) can then be quantified, together with the calcium leached from the material to the solutions in the compartments, and therefore the amount of portlandite dissolved and the dissolution kinetics due to the thermodynamic imbalance generated can be assessed. The concentration of diffused ions as a function of time was linearly fitted in the steady state regime in order to determine the kinetics, given that it is a migration.

Change in the upstream solution

Chemical analyses showed that, for all cement pastes, the upstream sodium concentration decreased compared to the initial sodium concentration (525 mmol/l) with the kinetics expressed as a flux of 6.4x10 -2 ; 5.7x10 -2 ; 7.1x10 -2 ; 8.6x10 -2 and 9.6x10 -2 mmol.m -2 .s -1 for PCI, LF25, FA30, S75 and SF10, respectively (Fig. 10). Note that the sample surface was 3.1 x10 -3 m 2 and the compartment volume was 2l. Furthermore, the potassium concentration decreased at a flux of 5x10 -2 ; 6.4x10 -2 ; 5.9x10 -2 ; 7.7x10 -2 and 5.7x10 -2 mmol.m -2 .s -1 , respectively (Fig. 11).

For S75, a steady state was reached later than for the other materials (after about 100 hours of migration). This is explained by the capacity of S75 for the electrochemical fixation of chlorides, which delays the steady state (see section 3.1) [START_REF] Dhir | Chloride binding in GGBS concrete[END_REF]. Since sodium ions are attracted by chlorides, their steady state could be delayed until chloride fixation is stabilized. That is why a linear trend line was considered after 100 hours (flux in steady state). Also, for the Na flux of SF10, a disturbance of the upstream sodium concentration was noted. It is known for the SF10 microstructure that the dissolution of silica forms negative charges which are neutralized by alkalis [START_REF] Dent Glasser | The chemistry of "alkali-aggregate" reaction[END_REF]. Alkalis are adsorbed on the silica surface [START_REF] Urhan | Alkali silica and pozzolanic reactions in concrete. Part 1: Interpretation of published results and an hypothesis concerning the mechanism[END_REF]. During chloride migration and the thermodynamic disequilibrium, it seems that these alkalis could participate in the stabilization of the thermodynamic equilibrium. This could disturb the sodium concentration in the pore solution as well as its diffusivity and concentration upstream. More investigations will be necessary to confirm this explanation. An increase in upstream calcium concentration was noticed. In order to determine the source of this leached calcium, the initial calcium concentration of the material's pore solution was also dosed (after extraction) and compared to the cumulative concentration leached upstream during the migration test (Fig. 12). Note that leached downstream calcium was found to be negligible. Firstly, the quantity of calcium in PCI, LF25, FA30 and S75 was similar (~2 mmol/l). However, the quantity of calcium in SF10 was higher, despite the pozzolanic reaction of silica fume, Andersson et al. [START_REF] Andersson | Chemical composition of cement pore solutions[END_REF] showed that the calcium concentration in the pore solution of cement paste with silica fume is higher than those containing Portland cement, blast furnace slag or fly ash. This is in accordance with our results. Moreover, the results showed that the cumulative calcium concentration leached at the end of the test, i.e. after 12 -14 days, ranged from 32.2 to 209.4 mmol/l. These concentrations are higher than the initial ones in the pore solution for the corresponding pastes. They are about 115, 15, 89, 64 and 15 times higher for PCI, LF25, FA30, S75 and SF10, respectively. This excess calcium was due to the dissolution of portlandite caused by cation leaching under the electrical field and chloride migration to stabilize the thermodynamic system. Calcium ions leach subsequently upstream. The quantity of calcium in the upstream compartment, leached from LF25, was lower than those of the other cement pastes, PCI, FA30, S75 and SF10. The decalcification of cementitious materials generally involves the dissolution of Portlandite and part of C-S-H [START_REF] Catineaud | Influence of limestone addition on calcium leaching mechanisms in cement-based materials[END_REF]. Given that limestone filler is chemically inert when used in cementitious materials [START_REF] Ye | Influence of limestone powder used as filler in SCC on hydration and microstructure of cement pastes[END_REF], the low quantity of leached calcium can be assigned to, on the one hand, a smaller amount of hydrates (Portlandite and C-S-H) that can dissolve and release calcium, and on the other hand, to the use of limestone filler, where a high level (more than 20%), as in our case, can lead to the formation of monocarboaluminates and hemicarboaluminates with a consumption of Portlandite [START_REF] Catineaud | Influence of limestone addition on calcium leaching mechanisms in cement-based materials[END_REF] and thus a decrease in the amount of calcium that can leach from the material.

Moreover, the cumulative calcium concentration with time and the kinetics of the increase in upstream calcium during the migration test are given in Fig. 13. The results could account for the kinetics of portlandite dissolution, taking into account the sample size tested, the limited amount of portlandite and supposing that the excess calcium derived only from the portlandite, according to [START_REF] Tran | Modelisation of chloride reactive transport in concrete including thermodynamic equilibrium, kinetic control and surface complexation[END_REF]. The kinetics expressed as a flux were 1.2x10 -1 ; 1.8x10 -2 ; 1.1x10 -1 ; 9.1x10 -2 and 1.2x10 -1 mmol.m -2 .s -1 for PCI, LF25, FA30, S75 and SF10, respectively. Furthermore, the molar concentration of upstream calcium that was released from the material represented the molar concentration of portlandite dissolved during the migration test. Indeed, the calcium released was calculated from the cumulative calcium leached as shown in Fig. 13 and the upstream volume (2l). The amount of portlandite dissolved in the pore solution (for a sample volume of 87 cm 3 ) that can modify the microstructure can then be calculated (Table 6).

Calcium released (mmol) = Portlandite dissolved (mmol) = Cumulative calcium concentration upstream (mmol/l) x Upstream Volume (2l). In reality the total quantity of portlandite dissolved during the migration test was higher, taking into account the fact that calcium could react with the other species to form new compounds in the porosity. It would also be interesting to determine the quantity and volume of new compounds formed in the porosity during chloride migration in order to compare with porosity closure shown in section 3.2. Finally, it should be noted that the sulfate concentration upstream was very low compared to the other ions.

Change in the downstream solution

The same approach was followed for the downstream solution before each renewal. The calcium concentration remained very low compared to the other ions it was not analyzed, and this section focuses only on sodium, potassium and sulfates. A low variation in downstream sodium concentration during the migration test was noted, apart from LF25 (Fig. 14). For this material, sodium decreased at 1.1x10 -2 mmol.m -2 .s -1 (the downstream volume was 1l). The sodium that is supposed to migrate from downstream to upstream, under the electrical field, tends to be balanced by the sodium attracted downstream by chlorides under electrostatic forces.

However, downstream potassium concentration decreased compared to the initial concentration of 83 mmol/l. Indeed, potassium fluxes from downstream to the material under the electrical field were 1.8x10 -2 ; 3.2x10 -2 ; 2.3x10 -2 ; 3.4x10 -2 and 3.2x10 -2 mmol.m -2 .s -1 for PCI, LF25, FA30, S75 and SF10, respectively.

In a migration cell with only NaOH in the two compartments (unlike our migration test where there was both NaOH and KOH), Andrade [START_REF] Andrade | Calculation of chloride diffusion coefficients in concrete from ionic migration measurements[END_REF] noted an eventual potassium leaching from the material to the compartments under a concentration gradient. Furthermore, sodium (supposed to be in equilibrium between the material and the compartments) diffused from downstream (cathode) to upstream (anode) under the electrical field. In this work, we observed a reduction in alkalis (sodium and potassium) downstream, which was explained by their migration upstream under the electrical field. However, the same reduction upstream was also noted. This could be explained by an eventual alkali attraction by chlorides, which diffuse from upstream to downstream, under the electrostatic force. Finally, from these analyses and the principle of conservation (since the migration cell is a closed environment), these results highlight an increase in alkalis in the material, which participates in the precipitation of new compounds, as indicated above in the discussion of the MEB results and the EDX spectra. More investigations on the pore solution of the materials after the migration test should be performed to complete these results. due: (i) to the chemical composition of the silica fume used, which is high in sulfates compared to the pozzolanic and limestone additions (see Table 1); (ii) to the hydration mechanism of the cement and silica fume that slows or stops the initial formation of ettringite (sulfate consumption), although the quantity of anhydrous cement and SO4 2-in the pore solution remained significant [START_REF] Lobo | Hydration of type K expansive cement paste and the effect of silica fume: II. Pore solution analysis and proposed hydration mechanism[END_REF].

Conclusion

The objective of this study was to investigate the change in the microstructure and pore solution of cement pastes induced by chloride transport and the chemical interaction with anhydrous cement and hydrates during a migration test. It should be noted that the same physical and chemical interactions of chlorides with cementitious material, which influences the microstructure, can occur during the migration or natural diffusion of chloride, but with different kinetics. Additional investigations on materials immersed in a salt solution without the use of an electric field may complete the following conclusions:

-The porosity and critical pore diameter of all materials were significantly reduced, by up to 60% for porosity and 88% for critical pore diameter, except for S75, whose critical -During the migration test, chlorides attract sodium and potassium, with the kinetics expressed as a flux of 5.7x10 -2 to 9.6x10 -2 and 5x10 -2 to 7.7x10 -2 mmol.m -2 .s -1 , respectively, from the upstream to the sample, due to electrostatic forces. Furthermore, potassium flux from downstream to the sample ranged from 1.8x10 -2 to 3.4x10 -2 mmol.m -2 .s -1 for the tested materials.

-Calcium in the pore solution diffused to the upstream compartment at a flux ranging between 1.8x10 -2 and 1.2x10 -1 mmol.m -2 .s -1 . This investigation estimated the amount of portlandite dissolved in cement pastes exposed to chloride (12 -14 days of a chloride migration test), which was about 418.8, 64.4, 361, 279.4 and 357.4 mmol for a sample of 87 cm 3 of PCI, LF25, FA30, S75 and SF10, respectively.

-Sulfates in the pore solution diffused to the downstream compartment mainly for cement pastes with silica fumes that contained a high initial amount of sulfates (6.7 mmol/l in the pore solution). The amount of leached sulfates for SF10 was about 28% of the initial concentration in the pore solution.

These changes in microstructure and pore solution will in turn modify the multispecies diffusivity through cement-based materials induced by chloride transport. This highlights the importance of considering changes to the microstructure and pore solution as well as thermodynamic equilibria in modeling the transfer phenomena and diffusivity of reinforcement coatings for better accuracy in describing chloride transport and then predicting the durability of reinforced concrete structures. Finally, it would be interesting to dose the pore solution of the tested material before and after the migration test and compare with the quantity of ions that diffused to/from the material. Furthermore, a comparison between porosity changes shown in this paper and the volume of dissolved/formed compounds may be of great interest.

Declaration of interest:

This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.

Fig. 1 .

 1 Fig. 1. Schematic representation of specimens sampled for studying the influence of chloride migration on the microstructure of cement pastes.

Fig. 2 .Fig. 3 .

 23 Fig. 2. Porosity of cement pastes. Fig. 3. Chloride diffusion coefficient of cement pastes.

Fig. 4 .

 4 Fig. 4. Mercury porosity of cement pastes before and after the migration test.

Fig. 5 .

 5 Fig. 5. Pore size distribution before and after the chloride migration test. (a) PCI; (b) LF25; (c) FA 30; (d) S75 and (e) SF10.

Fig. 6

 6 Fig.6illustrates the reduction rate of the critical pore diameter. After the migration test, there

Fig. 6 .

 6 Fig.6. Critical pore diameter of cement pastes before and after the migration test.

Fig. 7 .

 7 Fig. 7. SEM images and EDX spectra of PCI: (a) before and (b) after the migration test.

Fig. 8 .

 8 Fig. 8.SEM images and EDX spectra of FA30: (a) before and (b) after the migration test.

Fig. 9 .

 9 Fig. 9.SEM images and EDX spectra of S75: (a) before and (b) after the migration test.

Fig. 10 .Fig. 11 .

 1011 Fig. 10. Sodium loss upstream during the migration test.

Fig. 12 .

 12 Fig. 12. Comparison between cumulative calcium concentration leached upstream during the migration test and the calcium concentration of the pore solutions.

  which consumes calcium by the silanol groups (chemisorption) or by silice to form C-S-H[START_REF] Greenberg | The chemisorption of calcium hydroxide by silica[END_REF].

Fig. 13 .

 13 Fig. 13. Cumulative calcium concentration leached upstream during the migration test and leaching kinetics.

Fig. 14 .

 14 Fig. 14. Variation in downstream sodium during the migration test.

Fig. 15 .

 15 Fig. 15. Potassium loss downstream during the migration test.

Fig. 16 shows

 16 Fig.16shows the cumulative concentration of downstream sulfates and pore solutions. The

Fig. 16 .

 16 Fig. 16. Comparison between the concentration of sulfates in pore solutions (before the migration test)and the cumulative concentration of sulfates leached downstream after the migration test.

  pore diameter was initially smaller than those of the other materials and remained broadly unchanged after the migration test.-During the chloride migration test, new compounds based on chlorides, potassium and sulfates were formed in the sample. According to the MIP results, these compounds occupied the porosity of the material in the range of 10 -69; 15 -50; 7 -45 and 8 -69 nm for PCI, LF25, FA30 and SF10, respectively.

Table 1 .

 1 Chemical composition and physical properties of materials used

	Composition	CaO	SiO2 Al2O3 Fe2O3 SO3 K2O Na2O Chlorides Density BSSA(m²/kg)
	CEM I (wt.%) 64.20 20.50	5.00	3.90	2.50 0.29	0.05	1.40	3.18	405
	LF (wt.%)	98.80	0.30	0	0	0.01		0.01	0.001	2.70	498
	FA (wt.%)	5.10		85.53		0.59 2.00	1.95	0.013	2.21	405
	Slag (wt.%)	41.50 33.30 12.50	0.40	0.50	0	0	0	2.89	450
	SF (wt.%)	1.00	89.00	0	0	2.00	0	1.00	0.10	2.24	-

Table 2 .

 2 W/B ratio of cement pastes used.

	Material	PCI	LF25	FA30	S75	SF10
	W/B	0.50	0.38	0.40	0.44	0.47

  the porosity of the same specimens and showed that calcium oxychloride and Friedel's salt can occur together.

	3Ca(OH)2 + CaCl2 + 12H2O→ 3CaO・CaCl2・15H2O
		0.08	0.069		Before migration test After migration test	0.069
	Critical diametr (µm)	0.02 0.04 0.06	0.01 (-86%)	0.05	0.015 (-70%)	0.045	0.007 (-84%)	0.008	0.008 (-0%)	0.008 (-88%)
		0
			PCI	LF25	FA30	S75	SF10

Table 3 .

 3 Chemical species detected in the vicinity of pores of PCI (% in weight)

	Element	C	O	Na	Mg	Al	Si	S	Ca	Cl	K
	Before migration test	7.17 33.88	0.78	0.84	1.72	13.43	0.50	40.14	0	0
	After migration test	9.33 38.40	0.42	0.43	2.13	16.98	1.08	26.88	2.41	0.70
	Fig. 8 highlights chlorides (1.71%), sulfide (0.63%), sodium (1.03%) and potassium (1.61%) in

solid form in the porosity of FA30 subjected to migration tests; these species were not present in solid form before the tests. The quantity of these species is given in Table

4

.

Table 4 .

 4 Chemical species detected in the vicinity of FA30 pores (% in weight)

	Element	C	O	Na	Mg	Al	Si	S	Ca	Cl	K
	Before migration test	17.89	45.60	0	3.18	4.47	4.66	0	16.35	0	0
	After migration test	44.60	27.45	1.03	0.32	1.05	1.75	0.63	19.21	1.71	1.61

Table 5 .

 5 Chemical composition of the solid phase in the vicinity of S75 pores (% in weight)

	Element	C	O	Na	Mg	Al	Si	S	Ca	Cl	K
	Before migration test	15.76	31.87	0.25	0.95	0.24	0.57	0.17	49.25	0	0
	After migration test	24.06	34.26	1.47	4.06	2.36	8.67	0.21	19.27	0.68	2.14

Table 6 .

 6 Quantity of portlandite dissolved during the migration test.

	Materials	Sample volume (cm 3 )	Cumulative calcium concentration (mmol/l)	Calcium released amount (mmol)	Portlandite dissolved amount (mmol)
	PCI	87	209.4	418.8	418.8
	LF25	87	32.2	64.4	64.4
	FA30	87	180.5	361	361
	S75	87	139.7	279.4	279.4
	SF10	87	178.7	357.4	357.4

Acknowledgments:

The authors would like to thank the Poitou-Charentes Region (now part of the Nouvelle Aquitaine Region) and the Operational Program CPER-FEDER ("Bâtiment durable", Axis 2 "MADUR") 2014-2020 for funding this work.