Pascal Kintz
email: pascal.kintz@wanadoo.fr

The forensic response after an adverse analytical finding (doping) involving a selective androgen receptor modulator (SARM) in human athlete

Keywords: SARMs, doping, hair, urine, dietary supplements, liquid chromatography

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

Introduction

Selective androgen receptor modulators (SARMs) are a class of drugs presenting identical anabolic properties to classic anabolic steroids, such as testosterone, stanozolol, or nandrolone but with reduced androgenic effects. This last property is very important as it decreases the usual side-effects of steroids, such as liver toxicity. This is because SARMs selectively bind to androgen receptors in some specific tissues, but not in others [START_REF] Zhang | Deciphering the selective androgen receptor modulators paradigm[END_REF][START_REF] Bhasin | Selective androgen receptor modulators (SARMs) as function promoting therapies[END_REF][START_REF] Furuya | Mechanism of the tissue-specific action of the selective androgen receptor modulator S-101479[END_REF].

Initially, SARMs were proposed by pharmaceutical companies as therapeutic drugs to treat osteoporosis, hypogonadism or muscle-wasting pathology such as muscular dystrophy but none has been yet approved by the health authorities in any country of the world [START_REF] Chen | Discovery and therapeutic promise of selective androgen receptor modulators[END_REF].

SARMs have emerged in the doping area within the early 2000's. Ligandrol, ostarine (enobosarm), RAD 140 and andarine are the most popular agents belonging to this class.

These drugs are also known by a code, such as LGD-4033 for ligandrol, S-22 or MK-2866 for ostarine or S-4 for andarine. From the dozen of compounds listed as SARMs, ostarine and ligandrol are the more frequently identified.

According to the world anti-doping agency (WADA) prohibited list, SARMs are prohibited at all times (i.e. in and out-of-competition) and are listed under the section S1.2 (other anabolic agents), a section that also contains clenbuterol (5).

It is in 2010, that the Swiss laboratory for doping analyses reported the first case of SARMs abuse (it was andarine) during in-competition testing [START_REF] Grata | SARM-4 and metabolites detection in sports drug testing: a case report[END_REF]. SARMs have the potential to be misused for performance enhancement in sport due to their anabolic properties. In the recent years, SARMs have been increasingly observed in anti-doping rules violation (ADRV) as they can be purchased very easily on the Internet. Table 1 lists some statistics about adverse analytical findings (AAF) involving SARMs. Animal doping, mostly equine doping, has also been described with SARMs [START_REF] Hansson | Characterization of equine urinary metabolites of selective androgen receptor modulators (SARMs) S1, S4 and S22 for doping control purposes[END_REF][START_REF] Ventura | Development and validation of a semi-quantitative ultra-high performance liquid chromatography-tandem mass spectrometry method for screening of selective androgen receptor modulators in urine[END_REF].

In case of challenging an AAF, an athlete (who has the burden of proof in doping control) is in his right to produce evidence for his/her defence. This has prompted athletes to get closer to the forensic side to produce alternative admissible elements to confirm their claims of unintentional doping. Very recently, in 2020, a series of publications [START_REF] Trevorrow | Memorable findings: Suspected and purported cases of performance manipulation[END_REF], presenting investigations performed by WADA accredited laboratories have demonstrated that the forensic approach can be useful to document unusual adverse finding. For example, it was retrospectively demonstrated that the AAF of Dieter Baumann, in 1999, could be the consequence of a contaminated toothpaste with norandrostenedione, a prohormone of the anabolic drug nandrolone [START_REF] Schänzer | Tainted toothpaste -Analytical investigation into an unusual adverse finding[END_REF]. This clearly indicates that there is room for additional proofs in sports, despite a very strong regulation of evidence admissibility.

The strategy to document an adverse analytical finding

From the author's point of view, based on 25 years of experience with anti-doping rules violation, including the 1998 Tour de France, acceptance by sport federations or the Court of Arbitration for Sports (CAS) in Lausanne (Switzerland) of third-party contamination to account for an AAF has some prerequisites [START_REF] Kintz | What are the prerequisites to account for "no fault" in doping control after an adverse analytical finding possibly due to drug contamination? Perspective from a hair testing analyst[END_REF], which include, at least, the following points:

1. To support incidental exposure, only minute amounts of prohibited drug or metabolite(s) must be present in the urine collected for control. High concentrations that are observed after recreational abuse or for doping purposes cannot account for contamination. The possible dose, estimated after indication by the athlete of the moment of the exposure, has to be too small to have any recreational or performance-enhancing effect.

2. The athlete head hair test, which can support either 1. a negative results (with a suitable limit of detection) to confirm incidental exposure or 2. a result that can support external or environmental contamination, must be presented. This hair test must cover the period of time where the specimen was collected for doping control (alternatively, body hair or nail clippings have to fit to the requested window of detection).

The athlete's legal team must present verified circumstances of contamination or

evidence of drug possession. The source of contamination must be established, as, on a balance of probability, the indicted athlete bears the burden of persuading the judging body that the occurrence of the circumstances on which he / she relies is more probable than their non-occurrence or more probable than other possible explanations of the doping offence.

4. There must be verified claims of the athlete about the fact that he / she did not knowingly take the prohibited substance, i.e. that the violation was not intentional, although no test exists that can positively and definitively exclude the intentional use of a drug prior to the collection of a specimen for doping control 5. An experienced pharmacologist or toxicologist has to perform a pharmacological verification of the athlete's claims. This means that pharmacokinetic and pharmacodynamics parameters of the specific drug must fit with the submitted scenario and the athlete must establish how the prohibited substance entered his or her system. 6. A third-party positive hair test to support the claims of drug use by the partner and therefore being the source of contamination during an intimate moment, must be presented when the "kissing scenario" or the "sex scenario" is advocated (12-14).

As indicated in this protocol, the source of contamination must be identified, the dose to which the athlete was exposed should be estimated and a hair test must confirm the absence of long-term SARM exposure. These 3 items will be developed in the following lines.

Dietary supplements contaminated by SARMs

To the best of the author's knowledge, up to now, there is no case where an athlete or his/her legal team has challenged an AAF due to meat contamination. This has been described after animal administration of anabolic steroids (15) or ractopamine, zeranol and clenbuterol (16), but does not seem applicable for SARMs.

In the recent years, it has been increasingly published that dietary supplements can be falsified [START_REF] Fabresse | Analysis of pharmaceutical products and dietary supplements seized from the black market among bodybuilders[END_REF]. They may contain no active ingredient or other active ingredient(s). In addition, the announced active ingredient can be associated to other active substances or present a variable doses. These supplements can produce numerous adverse events, ranging from absence of therapeutic efficacy up to serious health effects, which can involve deaths [START_REF] Pena-Acevedo | A counterfeit multivitamin product inducing severe bleeding disorders in humans[END_REF]. In sport, contaminated supplements can lead to adverse analytical findings, with complicated consequences for the athletes to justify their behaviour [START_REF] Walpurgis | Dietary supplement and food contaminations and their implications for doping controls[END_REF][START_REF] Martinez-Sanz | Intended or unintended doping? A review of the presence of doping substances in dietary supplements used in sports[END_REF].

Although theoretically all doping agents can contaminate supplements, in most cases, anabolic steroids, stimulants, selective androgen receptor modulators, clenbuterol or diuretics have been identified. Low doses of SARMs have been found in pre-workout, fat burner/thermogenic, brain/cognitive booster, and hormone/testosterone booster. Back in 2008, Thevis et al [START_REF] Thevis | Detection of the arylpropionamidederived selective androgen receptor modulator (SARM) S-4 (Andarine) in a black market product[END_REF] reported for the first time the presence of a contaminated supplement with the identification of andarine.

In their paper [START_REF] Walpurgis | Elimination profiles of microdosed ostarine mimicking contaminated products ingestion[END_REF], Walpurgis et al have demonstrated that an oral administration of 10 µg (which is minimal, less than one grain of salt) of ostarine can produce a urine concentration up to 5.77 ng/mL, which is of course a concentration that constitutes a doping offence. Given WADA accredited laboratories can have limits of detection of 10 pg/mL for SARMs, these micro doses can influence the future activities of any athlete. The same authors have reported that 1 µg of ostarine can be detected for up to 9 days by monitoring ostarine glucuronide.

Despite their lack of safety approval, SARMs are often illegally marketed as dietary supplements, available for consumers after online purchase. In these products, discrepancies can range from a supplement in which no active ingredient was found to supplements containing undeclared prohibited agents. Sometimes, discrepancies were observed between the concentrations measured and those detailed on the product packaging [START_REF] Leaney | Analysis of supplements available to UK consumers purporting to contain selective androgen receptor modulators[END_REF]. In the laboratory of the author, a wide range of items, from various companies, were tested and in most cases a lower dosage was found when compared to what was labelled. It also can happen that a specific SARM can be replaced by another one, without informing the customer. For example, we bought liquid ostarine that was, after analysis, andarine [START_REF] Kintz | LGD-4033, S-4 and MK-2866 -Testing for SARMs in hair: about 2 doping cases[END_REF]. This can have an influence on the claims of an athlete as he can confirm not knowingly taking one product while in fact his/her supplement has been switched to another agent. Of course, cheaters can use this situation, but the final outcome, evidenced by a hair test, remains the same, as it constitutes a doping violation.

Because the use of dietary supplements can be risky, several national anti-doping agencies have advertised, on several occasions that safety can be breached due to contamination (25). For example, Supplement 411 is USADA's dietary supplement safety education and awareness resource (26).

Very recently, Lee et al [START_REF] Lee | Development and validation of liquid chromatography-tandem mass spectrometry method for screening six selective androgen receptor modulators in dietary supplements, Food addit[END_REF] tested 60 dietary supplements obtained in 2020 by on-line and direct purchase from international vendors. Various SARMs were identified in 20 products at high concentration, including for example one product containing andarine at 7.2 %. The authors concluded that a continuous control and supervision of dietary supplements is necessary.

On an analytical point of view, as these products are generally presented as tablets, capsules or oral liquid solutions, a simple dilute (with methanol) and shot approach is generally achieved.

Urine testing for SARMs

Urine is the standard specimen for anti-doping control. It can be easily collected by trained collectors but must be stored at low temperature to avoid drug degradation. Ventura et al [START_REF] Ventura | Investigation of stability of selective androgen receptor modulators in urine[END_REF] have published that the stability of SARMs in urine at + 4 °C (2 weeks) and -20 °C (4 weeks) has been verified. These authors concluded that the long-term stability testing data ensure confidence in findings from anti-doping processes.

If it was complicated to obtained authentic reference material for SARMs in the early 2010, this is no more the case today. One can easily purchase pure standards from suitable manufacturers without buying material from the Internet. However, this is not the case for SARMs metabolites. On numerous occasions, the Anti-Doping Administration and Management System (ADAMS) has issued an anti-doping rules violation based on the identification of a metabolite, for example di-OH-ligandrol. Generally, these specific metabolites have a longer window of detection when compared to the parent compound.

Unfortunately, metabolite(s) are not available for forensic laboratories for a reasonable delay as they are only available for WADA accredited laboratories. For some metabolites, it can take more than 6-9 months to get the permission of purchase, after tedious administrative investigations that verify the final use of the material. This is to avoid internal cheating approaches by verifying that the urine of an athlete is clean.

As SARMs have not been investigated in controlled large studies due to the lack of clinical interest, most data about metabolism are available after experimental tests involving human liver microsomes (HLMs) or administration of a specific drug to a single subject followed by urine collection over a long period [START_REF] Fragkaki | Human in vivo metabolism study of LGD-4033[END_REF].

All methods for SARMs identification in human urine involve liquid chromatography coupled to tandem mass spectrometry or high-resolution mass spectrometry. Briefly, methods involve hydrolysis with ß-glucuronidase and sometimes arylsulfatase, liquid-liquid extraction, separation on a reverse phase and mass spectrometric detection [START_REF] Gagaj | Enhanced UHPLC-MS/MS screening of selective androgen receptor modulators following urine hydrolysis[END_REF]. Typical limits of quantitation are lower than 1 ng/mL.

Hair testing for SARMs

In 2013, the President of the World Anti-Doping Agency (WADA), Sir Craig Reedie, wished for new approaches other than urine and blood in doping control. Indeed, he declared that hair testing "will create tremendous opportunities for advances in anti-doping and allow our scientists to look at alternative approaches to sample testing for banned substances" (31).

Although hair is not a routine specimen for the WADA, its use as a specimen of investigation is accepted in most criminal courts of justice in the world.

Hair testing is a useful measure of drug intake of an individual, in any situation in which a history of past rather than recent drug use is expected, as it reflects consumption over a long period of time. For practical purposes, it is commonly accepted that each cm of head hair represents the growth, and therefore drug accumulation, for one month. Using segmental hair analysis, statements about the course of drug intake and chronological correlations are possible. While a constant regular profile along the hair shaft is in favour of permanent drug use, any variation of concentration indicates a change in drug intake [START_REF] Kintz | Hair analysis in forensic toxicology: an update review with a special focus on pitfalls[END_REF].

The Society of Hair Testing (SoHT) has established in 1999 [START_REF] Sachs | Consensus of the Society of Hair Testing on hair testing for doping agents[END_REF] a consensus on hair testing for doping agents. This statement includes that hair specimens are not suitable for general routine doping control and that a negative hair test result cannot exclude the administration of the detected drug and cannot overrule an adverse analytical finding (AAF), identified in blood or in urine.

The World Anti-Doping Code has already addressed how the "no fault" plea should be considered. In the 2021 version (34), article 10.6 "reduction of the period of ineligibility based on no significant fault or negligence" and its explanatory footnotes, describe what is acceptable or not. In particular, at footnote 66 [Comment to Article 10.6.1.2: "In order to receive the benefit of this Article, the Athlete or other Person must establish not only that the detected Prohibited Substance came from a Contaminated Product, but must also separately establish No Significant Fault or Negligence".

Applications and limitations of hair tests in doping control have been extensively reviewed in several papers during the last years. In particular, it must be emphasized that a single exposure to some drugs, such as anabolic steroids, SARMs or diuretics is not detectable in hair [START_REF] Kintz | Hair testing for doping agents. What is known and what remains to do[END_REF]. The advantages of hair testing for active substances over blood and/or urine testing include non-invasive and ease of collection to prevent adulteration or substitution.

Hair test results allow establishing a retrospective calendar of drug exposure over weeks to months based on a standard head hair growth rate of 1 cm per month.

Given the author has 30 years experience in hair testing, particularly for doping agents, it has been suggested to test for SARMs in this alternative specimen [START_REF] Kintz | LGD-4033, S-4 and MK-2866 -Testing for SARMs in hair: about 2 doping cases[END_REF][START_REF] Kintz | Identification of S-22 (ostarine) in human nails and hair using LC-HRMS. Application to two authentic cases[END_REF]37). A comprehensive review of papers published with the key words "SARMs" and "hair" only produced one additional citation with respect to the identification of SARMs in human hair (38).

The first paper dealing with SARMs identification in human hair was published in 2019 [START_REF] Kintz | LGD-4033, S-4 and MK-2866 -Testing for SARMs in hair: about 2 doping cases[END_REF]. Since this initial method by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), the authors have implemented a confirmation procedure by liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) for both hair and nail clippings [START_REF] Kintz | Identification of S-22 (ostarine) in human nails and hair using LC-HRMS. Application to two authentic cases[END_REF].

Briefly, the method involves decontamination with dichloromethane, segmentation as per request, incubation in pH 9.5 buffer in presence of bicalutamide-d4, used as internal standard and extraction in a mixture of organic solvents. Separation is achieved on an Acquity HSS C18 column and detection performed by MS/MS on a Waters XEVO TQS micro.

Rading et al (38) identified GSK2881078 at 1.7 pg/mg in human hair collected 3 weeks after a single 1.5 mg oral administration. After cutting the hair into pieces of approx. 1 mm size, these authors incubated 50 mg in 3 mL of methanol followed by an ultrasonic disintegration for 6 hours at approx. 50 °C. After evaporation of the methanol and solubilization of the residue in an organic solvent, the drug was separated on a Kinetex C18 column and detected on a QTRAP 6500+ from Sciex. The limit of detection was 0.1 pg/mg. Kintz et al [START_REF] Kintz | LGD-4033, S-4 and MK-2866 -Testing for SARMs in hair: about 2 doping cases[END_REF] identified ostarine at 12 and 138 pg/mg, in the 2 x 3 cm hair segments of a male athlete (mixed martial fighter) who returned an AAF for ostarine. In this case, the hair test results confirmed the ADRV. In the hair of a subject arrested by the customs for trafficking, Kintz et al [START_REF] Kintz | Identification of S-22 (ostarine) in human nails and hair using LC-HRMS. Application to two authentic cases[END_REF] identified ostarine at 146, 168, 93, and 101 pg/mg in the 4 x 3 cm sections, clearly demonstrating long-term use.

Finally, the same group (37) , in 3 separate cases, identified ligandrol (14 to 42 pg/mg), andarine (0.1 to 0.7 pg/mg) and ostarine (3 to 21 pg/mg), offering new biological perspectives in SARMs abuse detection.

According to the WADA's international standard for laboratories (39) hair tests are approved, but must not be used to counter analytical findings from urine or blood.

A potential use of hair analysis when challenging a urine result is to verify accidental or unintentional ingestion of food or liquids that has been contaminated with drugs. However, in case of a single use, the hair will not test positive for SARMs as these compounds are poorly incorporated into hair (38). While analysis of urine specimens cannot distinguish between repetitive use and single exposure, hair analysis can make this distinction, when several segments of hair, irrespective of their length, are positive.

The toxicological significance of the measured concentrations is difficult to determine because there is no controlled study about SARMs incorporation into hair and single dose detection tests have produced controversial results. It is therefore not possible to interpret the data in terms of dosage and frequency of abuse.

It is accepted by the authors that some parameters that must be evaluated during the validation phase are not yet assessed: dose necessary to give a positive result, influence of gender, variable incorporation into hair according to hair colour, external factors that may have an impact, cosmetic treatment(s), etc. The minimum dose that had to be ingested by the athlete in order to get a "positive" detection in one of his/her hair sections is unknown for ostarine, ligandrol and other SARMs, although there are abused by many athletes.

Given in hair, the target analyte is the parent compound, identification of SARMs administration is obviously facilitaed in this matrix when compared to urine, a specimen which mostly contains metabolites.

Nail testing for SARMs

Nails can stably accumulate substances for long periods of time, thus providing retrospective information regarding drugs of abuse and pharmaceutical use. As it is the case for hair, nails have several advantages over conventional matrices, including a longer detection window, non-invasive sample collection, and easy storage. These aspects make nails a very interesting matrix for forensic and clinical toxicology.

Incorporation of substances into nails mainly occurs through diffusion from the rich blood supply, which deposits substances to both the germinal matrix and the nail bed on the underside of the nail plate, thus allowing incorporation in both a vertical and horizontal way during nail formation. Other mechanisms of incorporation that have been proposed are incorporation through diffusion from biological fluids such as sweat, sebum, and saliva, and incorporation through diffusion from the external environment. Distribution patterns have indicated that drugs are not only deposited at the germinal matrix, but also along the length of the nail bed.

Nails grow at a continuous rate. The average growth rate of fingernails is 3.0 mm per month, while toenails grow at an average rate of 1.1 mm per month. The regeneration time (i.e., the time to grow from the germinal matrix to the nail's free edge) is 3-5 and 8-16 months for finger-and toenails, respectively. Compared to blood and urine, nails have a longer window of detection from which information on xenobiotic exposure or ingestion can be retrieved. There is no agreement on the exact time frame nail clipping can represent and each laboratory has its own window of detection.

Interestingly, nails provide some additional advantages over hair. Firstly, when hair is not (sufficiently) available (e.g., subjects with short hair, in case of alopecia, during chemotherapy, or during the first weeks or months after birth), nail analysis can be an important tool to gain retrospective information on xenobiotic use. Secondly, in contrast to hair, nails do not contain melanin. Since drug incorporation may be influenced by melanin concentrations, hair pigmentation can be an important source of bias when interpreting detected drug concentrations. Thirdly, nails grow slower than hair, which provides the opportunity to detect smaller exposure levels and/or to investigate longer periods of time.

Fourthly, while hair is characterized by a cyclic growth rate with different stages, nails grow at a constant rate, which facilitates the interpretation of results.

Finally, compared to hair sampling, nail collection is aesthetically more acceptable, easier, and less intrusive. Taken together, these advantages underline the potential of nails as an interesting and useful matrix for the retrospective detection of drug and pharmaceutical use.

To date, only one publication has presented some results for the identification of ostarine in human nail clippings [START_REF] Kintz | Identification of S-22 (ostarine) in human nails and hair using LC-HRMS. Application to two authentic cases[END_REF].

A famous example: the case of Laurence Vincent Lapointe

To illustrate the forensic approach of an AAF involving a SARM (here ligandrol), it is possible to summarize the case of the Canadian canoe star Laurence Vincent Lapointe. This is possible because the athlete has given all details during numerous press releases (40). Briefly, late in July 2019, the athlete failed an anti-doping control when minute amounts (4 pg/mL) of ligandrol, a SARM, were identified in her urine. She strongly denied any use of the molecule. A first set of dietary supplements analyses was unable to find any trace of ligandrol. Hair specimens were collected mid-September, and tested for ligandrol in 6 x 1 cm segments. This covered the period of the urine control. The hair test results were negative (limit of detection at 5 pg/mg). Further investigations by her legal team revealed that her partner was repetitively using a product manufactured under the trade name SR9011. It is a liquid, containing a Rev-ErbA agonist (a protein that regulates the circadian rhythm, modulates glucose and lipid metabolism and regulates the inflammatory response). However, when analyzed, ligandrol was identified in the liquid at approximately 6 mg/mL in addition to traces of ostarine, another SARM. The hair test of the partner was positive, both in head hair and chest hair. Later, he confessed having used the product some hours before intimate moments at the time of the urine control. After taking into consideration scientific expert evidence provided by Ms Vincent Lapointe's legal team, and also the miniscule traces of ligandrol found in the athlete's sample, an anti-doping panel convened by the International Federation (ICF) has accepted that Ms Vincent Lapointe did not knowingly take the illegal substance. The ICF has accepted Laurence Vincent Lapointe's evidence, which supports that she was the victim of third-party contamination, and has cleared her to return to training and competition immediately (41).

The final decision was based on investigations that support a forensic approach, including minute amounts of the drug in urine, negative hair test, no deliberate exposure to the drug, and identification of the source of contamination.

Conclusion

The recent widespread of new drugs all over the world via Internet has demonstrated that there is a need of analytical approaches to document these new habits of consumption. Therefore, implementing tests for SARMs seems of great importance, not only for antidoping purposes, but also for clinical toxicologists or poison centres, in order to correctly establish a diagnosis of addiction or to document unusual side-effects. Given the increasing popularity of SARMs abuse, vigilance and identification of new cases are required. As hair and nail clippings allow retrospective and long-term investigations, these specimens present all suitable properties to be qualified for SARMs detection.

The difficulties in interpreting results of hair analysis for new drugs, such as SARMs, must be highlighted. In particular, little is known about the incorporation of SARMs into the keratin matrix after intake and the correlation between dose, frequency of use and hair or nail clippings concentrations. For example, answering to the following question remains, to date, impossible: if one were to have a hair analysis performed to detect traces of a specific SARM and the results were negative, what conclusion can be made with respect to the amount of SARM he/she had been exposed to? Is it possible to conclusively say that he/she had consumed less than (for example) one dose of SARM? Therefore, hair or nail clippings tests results for SARMs should be interpreted with caution by experienced forensic toxicologists.


Annelise Cousture
email: annelise.cousture@cyu.fr

Norbert Renault
email: norbert.renault@cyu.fr

Jean-Louis Gallias
email: jean-louis.gallias@cyu.fr

Khadim Ndiaye
email: khadim.ndiaye@cyu.fr

Study of a binder based on alkaline activated limestone

Keywords: alkali-activation, limestone, pirssonite, portlandite, thermal analysis Alkaline Solution to limestone ratio, DTA: Differential Thermal Analysis, EDX: Energy-Dispersive X-ray spectroscopy, FTIR: Fourier Transform Infrared spectroscopy, SEM: Scanning Electron Microscope, S/L: Sand to Limestone ratio, TGA: Thermogravimetric Analysis

Binders based on the alkaline activation of raw materials is a subject of increasing importance for construction. This study focuses on a new non-hydraulic binder based on the alkaliactivation of limestone by an aqueous solution of sodium hydroxide. An optimized mortar of about 20 MPa compressive strength, made with siliceous sand, was studied by XRD, FTIR and SEM-EDX analyses showing that crystalline pirssonite and portlandite are the reaction products responsible of the cohesive capacity of the binder. A methodology based on thermal gravimetric analysis was applied to quantify the products formed and the consumption of reagents with high reliability.

Introduction

During at least the last 20 years the study of alkaline activation has grown exponentially, even though research in this field began in the 1940s [START_REF] Ao | The action of alkalis on blast furnace slag[END_REF]. In the absence of clinker, blast furnace slag was activated by sodium hydroxide to form hydrated calcium silicate (CSH) and hydrated aluminates after the dissolution of silica and alumina and combination with sodium hydroxide. This alkali-activated binder has a compressive strength similar to that obtained with Portland cement. So far, an important knowledge has been accumulating on the subject and several books [START_REF] Davidovits | Geopolymer. Chemistry and Applications[END_REF][START_REF] Shi | Alkali-activated cements and concretes[END_REF][START_REF] Provis | Geopolymers, structure, processing, properties and industrial applications[END_REF][START_REF] Provis | Alkali activated Materials. State of the art Report[END_REF] and reviews [START_REF] Pacheco-Torgal | Alkali-activated binders: A review Part 1. Historical background, terminology, reaction mechanisms and hydration products[END_REF][START_REF] Palomoa | A review on alkaline activation: new analytical perspectives[END_REF][START_REF] Luukkonen | One-part alkali-activated materials: A review[END_REF][START_REF] Tang | Review on designs and properties of multifunctional alkali-activated materials (AAMs)[END_REF] have already been published on the subject. The common feature of all this research is the reaction between various raw materials and industrial wastes with alkalis-based reagents [START_REF] Xu | Microstructural characterization of geopolymers synthesized from kaolinite/stilbite mixtures using XRD, MAS-NMR, SEM/EDX, TEM/EDX, and HREM[END_REF][START_REF] Hafid | Influence of NaOH concentration on microstructure and properties of cured alkali-activated calcined clay[END_REF][START_REF] Fernández-Jiménez | Alkaline activation of metakaolin fly ash mixtures: Obtain of Zeoceramics and Zeocements[END_REF][START_REF] Chaipanich | Thermogravimetric analysis and microstructure of alkali-activated metakaolin cement pastes[END_REF][START_REF] Pouhet | Influence of the initial water content in flash calcined metakaolinbased geopolymer[END_REF][START_REF] Rakhimova | Marl-based geopolymers incorporated with limestone: A feasibility study[END_REF][START_REF] Bernal | Performance of alkali-activated slag mortars exposed to acids[END_REF][START_REF] Deb | The effects of ground granulated blast-furnace slag blending with fly ash and activator content on the workability and strength properties of geopolymer concrete cured at ambient temperature[END_REF][START_REF] Rangan | Properties and application of flyash-based concrete[END_REF][START_REF] Burciaga-Díaz | Structure, mechanisms of reaction, and strength of an alkali-activated blast-furnace slag[END_REF][START_REF] Martinez-Lopez | Alkali activated composite binders of waste silica soda lime glass and blast furnace slag: Strength as a function of the composition[END_REF][START_REF] Ramagiri | Effect of high-temperature on the microstructure of alkali-activated Binder[END_REF][START_REF] Bernal | Gel nanostructure in alkali-activated binders based on slag and fly ash, and effects of accelerated carbonation[END_REF][START_REF] Ahmari | Production of geopolymeric binder from blended waste concrete powder and fly ash[END_REF][START_REF] Cyr | Properties of inorganic polymer (geopolymer) mortars made of glass cullet[END_REF][START_REF] Idira | Investigations on the durability of alkali-activated recycled glass[END_REF][START_REF] Payá | A new geopolymeric binder from hydrated-carbonated cement[END_REF]. Depending on the 2 composition of precusors (i.e. aluminium, silicium and calcium contents) and those of activators (alkali oxydes or alkali hydroxydes and/or silicates) different chemistry and mechanisms occurs [START_REF] Van Deventer | Chemical research and climate change as drivers in the commercial adoption of alkali activated materials[END_REF].

The precursor (solid phase) react with the alkaline salt (solid or dissolved) to form structures with differetns degree of crystallinity (i.e. zeolitic network, inorganic polymer or characterictic chains of calcium silicate hydrates). Despite the growing interest in alkali-activated binders, very little research has focused on the alkaline activation of precursor with very high calcium content (higher than in portland based cement) availiable for reaction and particularly as calcium carbonates. The main studies about alkaline activated binders with calcium carbonate (i.e limestone) were related to mixtures containing active silicate compounds as precursor [START_REF] Avila-López | Investigation of novel waste glass and limestone binders using statistical methods[END_REF][START_REF] Menchaca-Ballinas | Limestone as aggregate and precursor in binders of waste glass activated by CaO and NaOH[END_REF] or as activator [START_REF] Ortega-Zavala | An initial study on alkali activated limestone binders[END_REF]. The presence of limestone in these binders modify the molecular structure of the reaction products and enhance the stability underwater [START_REF] Menchaca-Ballinas | Limestone as aggregate and precursor in binders of waste glass activated by CaO and NaOH[END_REF]. It leads to formation of cristalline carbonate phases such as pirssonite (Na2Ca(CO3)2.2H2O), gaylussite (Na2Ca(CO3)2.5H2O), portlandite (Ca(OH)2) or thermonatrite (Na2CO3, H2O) finely intermixed with amorphous calcium silicate hydrates (CSH), silica gel or calcium silicate gels (CSH), calcium-silicate gels incorporating sodium (NCSH), silica gels, sodium silicate gels (NSH), respectively. Depending on formulation parameters, these binders present a wide range of mechnical strength from 2 MPa to 32 MPa at 28 days with setting during conservation at 60°C for 24h followed by hardening at 20°C and relative humidity of 80-90% or hardening at 40°C, respectively. According to the same studies, the presence of cristalline phases in these binders could have differents effects on the matrix. They could cause a reduction in porosity, improving the compressive strength, but as they occupy a larger volume than the original hydroxides, they could generate internal stresses and loss of mechanical strength in the presence of moisture. The use of limestone as precusor and a solution of sodium hydroxide as activator without the addition of active silicate phases has only been investigated by one innovation patent filed in 2006 [START_REF] Pichat | Fabrication d'un matériau solide à partir d'un hydroxyde alcalin[END_REF] and previous work carried out in our laboratory [START_REF] Gallias | Etude de l'activation alcaline du filler calcaire par l'hydroxyde de sodium[END_REF]. This study, firstly, focuses on the optimization of workability and mechanical strengths of mortar made with limestone filler and non reactive siliceous sand activated by a concentrate solution of sodium hydroxyde, secondly, in the identification of the reaction products and their microstructure using XRD, FTIR and SEM-EDX analyses and, finally, in the reliable quantification of the reaction products by a methodology based on thermal gravimetric analysis. This study is a first step for undestanding and manage the reactional process with the aim to apply in the stabilisation and valorisation on wastes with high calcium carbonate content as wastes from food-processing industry (eggshells, crustacean shells) or from marble or cutstone and decorative stone quarries.

Materials

Limestone filler

The limestone used as a precursor in this study was a filler BETOCARB P2-MX supplied by OMYA France, commonly used as a mineral additive in concrete. Calcite (CaCO3 98.4%) was the only crystalline mineral identified by XRD (JCPDS 05-0586). The granulometry (determined by the supplier with a Metasizer 2000 laser particle sizer from Malvern Instruments) shows that 96% of the particles were smaller than 63 μm. Figure 1 shows their typical morphology.

Figure 1-Morphology of limestone particles.

2 Sand

Standardized sand in accordance with NF EN 196-1 [START_REF]NF EN 196-1 Methods of testing cement-part1: determination of strength[END_REF] and ISO 679 [START_REF] Afnor | ISO 679 Cement-Test methods-Determination of strength[END_REF] and corresponding to ASTM C778 [START_REF]C778 Standard specification for standard sand[END_REF], purchased from the SNL company (Leucate, France) was used for preparation of the mortars. Its composition was mainly siliceous (SiO2 96.2%). Identification by XRD revealed a mineralogical composition based on well crystallized quartz (JCPDS 46-1045), which is considered to be very weakly reactive with sodium hydroxide at room temperature. Particles are round shaped and their size distribution is regular between 0 and 2 mm (characteristics given by the supplier).

3 Alkaline activator

The activator used in this study was a solution of sodium hydroxide (NaOH) at 12 and 13 mol/L obtained by the dilution of 50 %wt concentrated soda solution in water, with a density of 1.515, from the Honeywell company. The pH of the activator solution was mesured with a pH-meter (Hanna Instrument HI 2221) equiped with a specific electrode for alkaline solutions and was around 15.

Mortar mixing, placing and curing

Before mixing, all materials were held in a climatic chamber at 20°C for at least 24 hours. Mortars were made of one part of limestone and three parts of sand (S/L =3), by mass, as in french standard cementitious mortars [START_REF]NF EN 196-1 Methods of testing cement-part1: determination of strength[END_REF]. With the aim to obtain acceptable workability fot the mixtures and satisfactory compressive strength a preliminary series of mortars was prepared with varying alkalinesolution to limestone ratio (AS/L) between 0.52 and 0.68 mL/g and two moralities of soda solution of 12 and 13 mol/L. In these conditions, the water to binder ratio (W/B) was varying between 0.42 to 0.52 (Table 2). W was the mass of water (including water from the soda solution) and B was the mass of limestone and mass of sodium oxide (from the soda solution). This optimization phase made possible to select a mortar with interesting results for fruther characterization in order to evaluate reactivity and quantify reaction products.

The mortars were mixed according to standard NF EN 196-1 [START_REF]NF EN 196-1 Methods of testing cement-part1: determination of strength[END_REF], close to ASTM C305 [START_REF]C305 Standard specification for mortar of unit masonry[END_REF] and three 40x40x160 mm prismatic specimens were cast in two layers, vibrated for 30 seconds each to release residual air bubbles. To minimize water evaporation at early ages, the molds were sealed in plastic bags. After 3 days the specimens were demolded. They were then cured for 11 days sealed in plastic bags at 20°C and then dried at 45°C for 14 days with the aim of eliminating free water in the mortar.

Free water could be responsible of partial dissolution of reaction products [START_REF] Cousture | Etude de l'activation alcaline de composés calcaires et siliceux[END_REF] or internal stresses [START_REF] Ortega-Zavala | An initial study on alkali activated limestone binders[END_REF]. In our case, the elimination of free water of the mortar improves the mechanical strength [START_REF] Gallias | Etude de l'activation alcaline du filler calcaire par l'hydroxyde de sodium[END_REF].

Tests methods

The mortars have been characterized in different ways. For the optimization phase, the workability and mechanical strengths were measured according the methods given below. On the composition judged to be the most efficient (optimized), more tests were carried out: weight loss (during drying), porosity (at 32 days), thermal analysis (DTA-TGA at 32 days), FTIR (at 35 days), XRD (at 40 days), and SEM-EDX (at 52 days) according to the following methods.

Workability

The workability of fresh mortars was characterized according to NF P 18-452 [START_REF] Afnor | NF P 18-452 Concretes -Measuring the flow time of concretes and mortars using a workabilitymeter[END_REF], the processing method is similar to ASTM C 230 [START_REF]C230 Standard specification for flow table for use in tests of hydraulic cement[END_REF], French standard using a workability meter. The device is composed of a prismatic receiver that is divided into two parts, separated by a removable entity, and this prismatic receiver is put under vibration using an electric vibrator. The mortar is casted in one of the prismatic cavities in 4 successive layers in less than 2 minutes and 30 seconds. Each layer is tamped six times with a metal rod. The workability meter is put in vibration by taking off the removable entity that was used to retain the mortar. The mortar can flow freely under the action of the vibration, and the time (in seconds) is measured from the beginning of the flow until the mortar reaches a defined height on the second prismatic cavity.

Weight loss

The weight loss of the specimens, expressed as a percentage of the initial mass, was monitored throughout the curing time (28 days) by weighing once a day. This made it possible to check that the mass of the specimens had stabilized at the end of drying, before the following tests were made.

Mechanical strengths

The flexural and compressive strengths of the cured specimens were measured at 28 days according to standard NF EN 196-1 [START_REF]NF EN 196-1 Methods of testing cement-part1: determination of strength[END_REF], close to ASTM C348 [START_REF]C348 Standard test method for flexural strength of hydraulic-cement mortars[END_REF] and C349 [41], using 3R Quantech apparatus with loading speeds of 50 and 2400 N/s for flexural and compressive strength tests, respectively. The resulting flexural and compressive strength values were the means of three and six individual values, respectively.

Porosity

Porosity measurements in accordance with standards EN NF 1936 [START_REF] Afnor | NF EN 1936 Natural stone test methods: determination of real density and apparent density, and of total and open porosity[END_REF] and NF P18-459 [START_REF]NF P18-459 Test for hardened concrete: Porosity and density test[END_REF] (processing method similar to ASTM C830 [START_REF]C830 Standard test method for apparent porosity, liquid absorption, apparent specific gravity, and bulk density of refractory shapes by vacuum pressure[END_REF]) were made on representative samples after the mechanical tests, using absolute ethanol instead of water in order to avoid the possible dissolution of alkali-activation products. Porosity (P) was calculated from equation ( 1) and the dry density of mortars

(d) from equation (2). = 100 ( - ) ( - ) ⁄ (1) = d ( - ⁄ ) (2)
where mdry was the dry mass of the samples at the end of curing, msat: the mass in air of ethanolsaturated surface-dry samples, mEth: the mass of ethanol-saturated samples and dEth: the density of ethanol.

FTIR

The FTIR spectra were recorded on ground samples (powders less than 0.315mm, sealed in plastics bags between grinding and testing) using an Alpha spectrometer with ATR cell (Bruker) in the range of 4000 to 600 cm -1 . Baseline correction, provided by the FTIR software, was applied to all spectra.

XRD

XRD analysis was carried out, on representative samples used for mechanical test, after grinding to of less than 0.315 mm powder and sealed in plastics bags until testing. The scans were run on an X-Pert 3

Powder instrument (Malvern Panalytical) with a copper anode (λ(Kα)=1.540598 Å, with a step size of 0.0167° and a counting time of 0.5715 s/step from 2θ = 5° to 2θ = 70°.

Thermal analysis

Thermal analysis were carried out, on representative samples of the 3 prismatic specimens, used for mechanical test, after grinding to of less than 0.315 mm powder and sealed in plastics bags until testing. DTA-TGA tests were performed simultaneously with a STA 449 F1 Jupiter analyzer (Netzsch) from 25°C to 1000°C with a heating rate of 10°C/min under dynamic nitrogen atmosphere (50 mL/min). All the tests were performed using approximately 110 mg of powder. The results enabled us to identify and quantify the products formed in the mortar.

SEM-EDX

The microstructure of mortars was examined by scanning electron microscopy (SEM) on platinumcoated fractured pieces of cured specimen using a Gemini 300 (ZEISS) scanning electron microscope equipped with the Quantax 10 (Bruker) X-ray microanalysis detector. A working distance of 8.7 mm was fixed and two acceleration voltages of 10 kV for surface imaging and 15 kV for elementary analysis were applied. The highest compressive strength was obtained for the smallest AS/L ratio (0.52 mL/g) and was around 10 MPa but, on the other hand, this mortar reached the limit of acceptable workability to ensure the homogeneity of the mortar under vibration (two times 30 seconds). The increase of the AS/L ratio, from 0.52 to 0.68, affects slightly the flexural strength of mortar, however, the compressive strength decreases of around 25% while the workability of the fresh mortar increases.

Results and discussion

Influence of composition parameters on workability and mechanical strength

These variations can be explained by the increase of the water relative amount (Table 2) in the mortar with the AS/L ratio which leads to a better wettability of particules but, as in cementitious materials, to an increase of porosity and a decrese of mechanical strength. The increase of the water amount seems to have greater influence than the proportional increase of the sodium hydroxide relative amount.

Mortars with AS/L ratio higher than 0.68 mL/g with high workability have not sufficient compressive strength for applications in the field of construction.

With the aim to improve the mechanical strength of mortar maintaining the workability in acceptable values, the AS/L ratio of 0.60 mL/g was chosen for determine the influence of sodium hydroxide molarity on the properties of the mortar. Figure 3 presents the comparison of workability and mechanical strengths of mortars with the same AS/L ratio and varying sodium hydroxide molarity. almost that of the mortar with AS/L ratio equal to 0.52 ml/g (figure 2). Same behaviour, in terms of workability, was observed on alkali-activated natural pozzolan and limestone powder mortars made with sodium hydroxide solution above 10 to 14 M. It was attributed to the low water content in theses solutions that is not enough to wet all the particules [START_REF] Adewumi | Effect of sodium hydroxide concentration on strength and microstructure of alkali-activated natural pozzolan and limestone powder mortar[END_REF].

The optimized formulation in terms of compressive strength with AS/L ratio equal to 0.60 mL/g and molarity equal to 13 M has been further studied. Its initial composition is presented in

Weight loss

Figure 4 shows the weight loss kinetics of the optimized mortar from demolding through to the end of drying. Figure 4-Weight loss of optimized formulation of mortar during 28 days curing.

For all specimens, a slight loss of weight (less than 0.5% of the initial mass) was observed during storage in sealed bags at 20°C for 14 days. It was followed by a great loss of weight during the first days of drying at 45°C. For all specimens the weight loss at the end of curing was around 7.5%. The standard deviation for weight loss between the different specimens during 28 days curing was very low (< 0.3%). This result indicates the great homogeneity of the mortar. As will be discussed below, the amount of water removed by drying is significantly lower than the free water introduced into the mortar's composition (10.22%, Table 3).

Identification of reaction products

Figure 5 shows the XRD pattern for the optimized alkali-activated mortar. Only the three or four most important peaks for each identified crystalline phase have been referenced. The crystalline phases present in the mortars resulting from alkali-activation of the limestone were pirssonite (Na2Ca(CO3)2.2H2O, JCPDS 24-1065) and portlandite (Ca(OH)2, JCPDS 44-1481). There was also quartz (SiO2, JCPDS 46-1045) from sand and a small amount of calcite (CaCO3, JCPSD 05-0586), essentially from the unreacted limestone. This first result showed that most of the limestone reacted with sodium hydroxide to form a calcium/sodium carbonate hydrate and portlandite after curing, according to the chemical reaction (equation 3) [START_REF] Gallias | Etude de l'activation alcaline du filler calcaire par l'hydroxyde de sodium[END_REF][START_REF] Cousture | Etude de l'activation alcaline de composés calcaires et siliceux[END_REF]:

2NaOH + 2CaCO3 + 2H2O Na2Ca(CO3)2.2H2O + Ca(OH)2 (3)
Figure 6 shows the FTIR spectra of initial materials (i.e. sand and limestone) in comparison with the spectra of alkali-activated limestone in mortar and in paste, prepared in the same conditions than the optimized mortar but without siliceous sand. The characteristic bands for siliceous sand and limestone were 1164, 1091, 1058, 796, 778, 694 cm -1 and 1795, 1396, 873, 713 cm -1 , respectively. The spectra of the paste and mortar samples present a split into two bands (1410 and 1480 cm -1 ) of the limestone strongest peak (1396 cm -1 ). Furthermore, six new peaks, at 3645, 3060, 1067, 903, 835 and 655 cm -1 , appears in the paste after alkali-activation and drying. Smallest characteristic peaks of limestone (1795, 875 and 711cm -1 ) were also present in the alkali-activated paste. The FTIR spectrum of the mortar present the same peaks as the spectrum of the paste in addition to the characteristic peaks of sand. Only the peak at 903 cm -1 was no longer visible due to the presence of sand. The changes observed for paste and mortar spectra (split and new peaks) are the result of reaction of the limestone with soda solution and could be attributed to formation of pirssonite and portlandite, as explicited behind.

Figure 6-FTIR spectra of initial materials and alkali-activated limestone in paste and mortar.

FTIR studies have been performed on natural hydrated double carbonates [START_REF] Adler | Infrared spectra, symmetry and structure relations of some carbonate minerals[END_REF][START_REF] Estep | Characterization of carbonate minerals from oil shales and coals by infrared spectroscopy[END_REF][START_REF] Böttcher | Dehydration of natural gaylussite (Na2Ca(CO3)2, 5H2O) and pirssonite (Na2Ca(CO3)2, 2H2O) as illustrated by FTIR spectroscopy[END_REF][START_REF] Frost | Hydrated double carbonates -A Raman and infrared spectroscopic study[END_REF] for years, and Table 4 shows their FTIR spectroscopic data. All the authors reported two vibration modes (υ1 and υ3) of the carbonate ion group in the natural pirssonite, even if it did not come from the same region of the world. They found that υ3 divided into two bands, but were able to find three by peak deconvolution/analysis [START_REF] Frost | Hydrated double carbonates -A Raman and infrared spectroscopic study[END_REF]. The other vibration modes (υ2 and υ4) show a single and a double band, respectively [START_REF] Adler | Infrared spectra, symmetry and structure relations of some carbonate minerals[END_REF][START_REF] Böttcher | Dehydration of natural gaylussite (Na2Ca(CO3)2, 5H2O) and pirssonite (Na2Ca(CO3)2, 2H2O) as illustrated by FTIR spectroscopy[END_REF], but Frost and Dickfos [START_REF] Frost | Hydrated double carbonates -A Raman and infrared spectroscopic study[END_REF] reported a single band for υ4. The wave numbers of the vibrations modes of the carbonate ion group differ slightly according to authors, but not the υ1 mode, which is located at around 1067 cm -1 . Pirssonite also contains water molecules, which show two vibration modes υ1 and υ3 between 2900 and 3400 cm -1 [START_REF] Böttcher | Dehydration of natural gaylussite (Na2Ca(CO3)2, 5H2O) and pirssonite (Na2Ca(CO3)2, 2H2O) as illustrated by FTIR spectroscopy[END_REF][START_REF] Frost | Hydrated double carbonates -A Raman and infrared spectroscopic study[END_REF].

Frequencies (cm - Our alkali-activated samples (paste and mortar) show almost all the characteristic vibration modes for pirssonite, as well for water molecules (3060 cm -1 ) and for carbonate ion groups (υ3 at 1480 and 1410 cm -1 , υ1 at 1067 cm -1 , υ2 at 835 cm -1 , υ4 at 710 and 655 cm -1 ) with some slight shifts compared to the bibliographic data. This can be explained by the possible deformation of carbonate units in the structure of our synthetic pirssonite. Furthermore, in our samples the bands at 875 and 711 cm -1 could be linked to the pirssonite and/or to the limestone contribution. The peak at 3645 cm -1 was attributed to the main band for portlandite [START_REF] Lafuente | The power of databases: the RRUFF project[END_REF] and was more or less visible in the alkali-activated samples.

Other characteristic bands [START_REF] Lafuente | The power of databases: the RRUFF project[END_REF] were in the same region as pirssonite or limestone bands, so it has not been possible to identify them clearly.

The FTIR results confirm the XRD results and show that the identification of pirssonite is possible in a mortar because the additional peaks due to sand contribution do not mask the main characteristic peaks of pirssonite.

Microstructure of optimized mortar

Figure 7 shows the microstructure of the alkali-activated mortar at different scales. Figure 7a shows the different constituents of the mortar: the alkali-activated paste and its porosity as well as a round shaped grains of sand. The sand grain has a clean/smooth surface, without presence of silicate gels, which means that the sodium hydroxide solution did not attack their silica. This conclusion will be reinforced, thereafter, by the quantification of reaction products and consumption of reactants ( §5.5), particularly, the total consumption of sodium hydroxide. In the paste, two types of microstructures were observed: one considered to be dense and one granular (grainy) (Figure 7b and7c). The distribution of phases seems to be random, but grainy areas were often on the periphery of dense zones, creating an intermediate or transition zone between the two types of microstructure where the grains become more and more joined (Figure 7d), as their coalescence leads to the formation of a dense zone. Both structures are three-dimensional as shown in Figure 7e. Porosity was also visible in all images: this comprised large pores of size between 50 and 10 µm and fine pores of less than 0.1 µm into the granular structures according to the spacing of the particles. This could explain the measured porosity on the optimized mortar: 20.09 ± 0.28% with a density of 1984 ± 24 kg/m 3 .

Furthermore, a point-counting procedure, with EDX analyses, was made, in ten different areas in the paste, on these two different microstructures to identify them. Such procedure, using SEM or SEM-EDX analyses, has already been used to estimate the degree of hydration in cement pastes [START_REF] Feng | Estimation of the degree of hydration of blended cement pastes by a scannng electron microscope point-counting procedure[END_REF][START_REF] Hu | Direct threedimensional observation of the microstructure and chemistry of C3S hydration[END_REF] or for chracaterization of building derived materials [START_REF] Suluguru | Experimental studies on the microstructural, physical and chemical characteristics of building derived materials to assess their suitability in ground improvement[END_REF] with good reliability. Figure 8 gathers together an example of results obtained in one of the ten paste areas analyzed. obtained by the standardless PB-ZAF method, using EDX software. The Na/Ca ratio was calculated to assign microstructure to the reaction product. The dense and the granular microstructures had Na/Ca ratios of 1.1 ± 0.1 and 0.1 ± 0.0, respectively. Theoretically, the Na/Ca ratios for pirssonite and portlandite or calcite are respectively 1.15 and 0.00. Thus, the compact microstructure was related to the pirssonite previously identified by XRD and FTIR experiments. The granular microstructure could be related to portlandite or calcite, but the shape and the size of this microstructure was closer to that of calcite in limestone (figure 1). No typical hexagonal plates of portlandite were observed. These observations confirm that the dissolution of filler grains by sodium hydroxide progressively leads to the crystallization of dense reaction products that explain the binding capacity of the mixture and the development of mechanical strength.

Quantification of the reaction products

Figure 9 shows the thermal analysis curves for standardized sand and the average thermal analysis curves for three samples of the three specimen of optimized mortar, after curing. For sand, no weight loss was observed and only one endothermic peak, at 568°C, associated with the allotropic transformation of quartz α into quartz β, was observed. For mortar samples, the following dissociation reactions were identified:

-Dehydration of pirssonite at 175°C (equation 4)

Na2Ca(CO3)2.2H2O Na2Ca(CO3)2 + 2H2O (4)
-Dehydration of portlandite at 435°C (equation 5)

Ca(OH)2 CaO + H2O (5)
-Decarbonatation of nyerereite at around 775°C (equation 6)

Na2Ca(CO3)2 Na2CO3 + CaO + CO2 (6)
-Decarbonatation of the calcite of the unreacted limestone above 800°C (equation 7)

CaCO3 CaO + CO2 (7) The dissociation temperatures of pirssonite and nyerereite were consistent with the literature [START_REF] Dheilly | Contribution à l'étude de la Gaylussite[END_REF]. The thermal analysis curves also presented two crystallographic transformations without any variation in mass. The first, around 386°C, corresponded to the transition from the low temperature form of nyerereite to the high temperature form [START_REF] Johnson | Gaylussite: Thermal Properties by simultaneous thermal analysis[END_REF]. The second, around 568°C, related to sand contribution.

These results confirmed those obtained by XRD and FTIR showing that no additional amorphous phase was present. The weight losses related to the dehydration of pirssonite and portlandite in dried specimens, denoted ∆mpirss (TGA) and ∆mport (TGA) respectively, were measured on TG curves between 25 and 335°C and between 335 and 560°C respectively (figure 9). As the sample's TG curves were not affected by the presence of sand (i.e. no interference noted) and the successive dissociation easily identified for this type of mortar, the measures on these curves could be considered reliable.

Consequently, the following assumptions were made in order to quantifying, reaction products and remaining reagents:

a) The sand and the limestone contained impurities; the accurate quantities of SiO2 and CaCO3 were therefore recalculated considering them.

b) Grinding of the samples for DTA-TGA testing did not change the composition of the mortars, particularly as regards the amount of pirssonite. However, as will be discussed below, it was possible that portlandite formed from the alkali-activation reaction was partially carbonated during curing, crushing and storage up to the DTA-TGA tests.

Balance of water and quantification of reaction products

Firstly, two types of water were distinguished in the hardened material: free or evaporable water (mfree w) and bound water (mbound w) in pirssonite and in portlandite. Secondly, the following assumptions were made: a) Even though storage of the molds and specimens in sealed bags for 14 days (after demolding)

prevents water evaporation, a very low loss of weight was observed (< 0.5%, Figure 4). This loss of weight is due to the free water in mortars and must be taken in account.

b) Drying at 45°C for 14 days leads to total evaporation of the rest of the free water in mortars (figure 3). Thus the relative mass of free water in the mortar after alkali-activation (expressed as %) was determined from the equation 8.

∆ = 100 ( - ! ) ⁄ " (8)
where m0 was the mass of the specimens after casting (0 day) and m28d was the mass of the specimens at the end of drying (28 days). c) Considering the consumption of NaOH by the reaction to be complete (the limiting reagent in the reaction (equation 3) is in this case the sodium hydroxide), the relative mass of bound water in mortars after alkali-activation expressed as a % of the initial mass of mortar (m $%&'( ) ,) came only from the bound water in the reaction products (i.e. pirssonite and portlandite) and can be determined from the following equation.

∆ *+,- = ∆ ./ + ∆ .+ 1 (9)
where ∆mpirss w and ∆mportl w were the relative mass of bound water in pirssonite and portlandite, respectively, calculated from the equations 10 and 11.

∆ ./ = ∆ ./ (234) (100 -∆ ) 100 ⁄ (10)
∆ .+ 1 = ∆ .+ 1 (234) (100 -∆ ) 100 ⁄ (11)
The total amount of water (∆mwater) in relation to the initial mass of mortar was calculated using equation ( 12):

∆ = ∆ *+,- + ∆ (12)
The water balance, corresponding to the difference between the initial amount of free and bound water in mixing with NaOH and the amount of water calculated from loss of weight and TGA (∆mwater, equation ( 12)) was also calculated. Table 5 shows the analytical results for all the previously determined quantities for each prismatic specimen tested. 9) to ( 12) amounts of water for all specimens.

The three specimens had very close values for all the quantities of water determined, ensuring the homogeneity of the mortar. However, the calculated amount of water is slightly lower than the initial amount of water in the mixture (about 2% of the initial value). This water difference is probably due to incomplete reaction (equation 3) in the alkali-activation process. Sodium hydroxide, the limiting reagent for this reaction, should not have been completely consumed. In order to go further, an indirect approach to quantifying the remaining reagents is proposed in the following paragraph.

From the previous quantities of water determined by DTA-TGA, the relative amount of pirssonite and portlandite in the specimens in relation to the initial mass of the mortar (m0) were determined according the following equations:

5677 (234) = ∆ ./ 8M :;<== 2M )? @< ⁄ A (13)
B6CD (234) = ∆ .+ 1 8M :%< E M )? @< ⁄ A (14)
where Mpirss, Mportl and Mwater were respectively the molar weights of pirssonite (242 g/mol), portlandite (74 g/mol) and water (18 g/mol).

On the other hand, it is also possible to determine the relative amount of portlandite (equation ( 15)) in the specimens indirectly, using the previous relative amount of pirssonite (Pirss (JKL) ) and the stoichiometric amount of both products from equation (3):

B6CD (./ ) = 5677 (234) 8M :%< E M :;<== ⁄ A (15)
This value does not take in account the possible carbonation of portlandite during curing and crushing of the specimens, and must be compared with the previous value Portl (JKL) determined directly from DTA-TGA tests to evaluate the degree of carbonation of the portlandite.

The calculated amount of pirssonite (Pirss (TGA)) was 18.50 ± 0.50 %wt. The portlandite quantities were 4.75 ± 0.08 %wt and 5.66 ± 0.15 %wt with direct DTA measurement (Portl (TGA)) and undirect measurement from pirssonite quantity (Portl (pirss)), respectively. The three specimens had very close values for all the quantities determined, ensuring that the mortar was homogeneous and the methods employed reliable. The relative amount of portlandite determined directly by DTA-TGA tests (Portl (TGA)) was about 16 % lower than that determined indirectly by pirssonite ((Portl (pirss)). This confirmed partial carbonation of the portlandite between the end of the activation reaction and the thermal analysis experiments.

Quantification of remaining reagents

Based on the relative amount of pirssonite ( 5677 (JKL) ), it is possible to determine the remaining relative amounts of sodium oxide (∆m < P? Q R ) from the remaining sodium hydroxide, and remaining limestone (∆m < S?SR T ) according to equations 17 and 18.

∆ U Q V = ∆ U Q V -5677 (234) 8M P? Q R M :;<== ⁄ A (17)
∆ W WV T = ∆ W WV T -5677 (234) 82M S?SR T M :;<== ⁄ A

where ∆m P? Q R and ∆m S?SRX were the initial relative quantities of solid sodium oxide (present in the sodium hydroxide solution) and limestone used for preparation of the mortar (shown in Table 3), M S?SR T and M P? Q R were the molar weights of limestone (100 g/mol) and sodium oxide (62 g/mol), respectively. Table 6 presents the composition of the mortar, taking into account the impurities from sand and limestone, before and after the alkali reaction. The composition balance after alkali-activation was very close to 100%, confirming the consistency and reproducibility of the proposed methodology for determining free water, new products and remaining reagents. It will be seen that a very small amount (0.23%) of sodium oxide remains present in the mortar composition as sodium hydroxide solution after alkali-activation corresponding to lesser than 5% of its initial quantity. This confirms that the alkali-activation process was almost complete, consuming more than 95% of the initial amount of alkali activator. This sodium oxide remains as sodium hydroxide in the hardened mortar and includes 0.07% of bound water that must be considered when reducing the water balance (Table 5). On the other hand, the consumption of limestone was not complete; leaving about 25.3% of the initial amount unreacted, showing that the limiting reagent for the reaction is sodium hydroxide.

Finally, the alkaline reaction of limestone leads to the formation of a mortar composed of 18.50% pirssonite, 5.66% portlandite, 5.18% of remaining calcite, 0.23% of remaining sodium oxide, 2.73% impurities (from limestone and sand) and 60.08% silicon oxide, leaving 7.53% of evaporable water.

Overall, we can consider that the binding capacity of the paste results from the pirssonite, portlandite and remaining calcite in a relative proportion of 3.5/1/1.

Comparison with published data in litterature

As seen above (section 5.1) the optimized mortar formulation present a flexural and compressive strenght of 4.1 ± 0.3 MPa and 19.9 ± 1.5 MPa, respectively. The compressive strength could be directly linked to pirssonite quantity if we take in account that the amount of pirssonite determined for one specimen (17.96 %) was lower than for the two others (18.59 and 18.69 %, respectively) and the compressive strength obtained for this specimen (17.5 and 16.2 MPa) was lower then that of the two others (21.6 MPa, 20.3MPa and 20.2 MPa, 19.9 MPa, respectively).

Comparatively to these value, the compressive strength observed by Ortega-Zavala [START_REF] Ortega-Zavala | An initial study on alkali activated limestone binders[END_REF] (less than 2

MPa obtained on mortars with W/B = 0,27 and setting at 60°C for 24h followed by a hardening at 20°C and a relative humidity of 80-90%), was significantly lower. This very low compressive strength was explained by the presence of moisture which is responsible of internal stresses in alkali-activated limestone and therfore leads to a loss of mechanical strength. In the case of mortar with similar composition developped by Avila-Lopez [START_REF] Avila-López | Investigation of novel waste glass and limestone binders using statistical methods[END_REF], the compressive strength obtained depend on curing temperature: 2,44MPa after curing at 20°C (6% Na2O, W/B = 0.3) and 19,10MPa after curing at 60°C (12% Na2O, W/B = 0.5). The increase in Na2O quantity and in the curing temperature leads to a higher compressive strength which is very close to that obtained on our optimized mortar. This result could be explained by the abscence of residual water when the mortars were cured at a temperaure upper than or equal to 45°C. Indeed, at 28 days the motars were totaly dryied (i.e. no moisture) and consequentely the internal stress could be eliminated.

As the main reaction products of the alkali-activate limestone are soluble in water like gypsum, the intended applications as building material must be considered only under indoor conditions. The elimination of free water by drying is necessary to obtain compressive strengh higher then that required for indoor building applications as plasters, screeds, partition walls, suspended ceilings (> 8

MPa).

The porosity and the density of the optimized mortar are 20.09 ± 0.28% and 1984 ± 24 kg/m 3 , respectively. Unfortunately, theses parameters were not studied by others researchers who have studied the alkanie activation of limestone with sodium hydroxide but can be compared to traditional cement based and gypsum mortars. The density of the optimized alkali activated mortar is similar to traditional cement based mortars, 1990 kg/m 3 [START_REF] Zeng | Effect of porosity on thermal expansion coefficient of cement pastes and mortars[END_REF], and 12,5% higher than gypsum mortars, 1764 kg/m 3 [57] but it's porosity is 34% higher compared to traditional cement based mortars, 15% [START_REF] Zeng | Effect of porosity on thermal expansion coefficient of cement pastes and mortars[END_REF] and 38% lower compared to gypsum mortars, 28% [START_REF] Krejsová | The influence of different aggregates on the behavior and properties of gypsum mortars[END_REF].

The reduction of porosity in the alkali activated limestone mortar, certainly, must leed to an increase of mechanichal strengths and this may be considered by the reaction of remained unreacted limestone.

Conclusion

This study presents a new type of binder obtained by reaction between limestone and sodium hydroxide solution. The investigation on physical properties (strenght vs workability) of mortars, made with siliceous sand, on characterization of reaction products for optimized formulation (identification, microstructure and quantification) allows the following conclusions:

• Limestone can be used as a precursor to produce alkali activated binders after drying at 45°C.

• The compressive strength is in the range of 8 to 20 MPa, while the flexural strength was 3-4 MPa. It depends on formulation parameters (water to binder ratio (W/B) or alkaline solution to limestone ratio (AS/L) and sodium hydroxide molarity of the solution). An optimized mortar with a 13 M soda solution and an W/B ratio equal to 0.46 (AS/L=0.60 mL/g) promotes the strength with an acceptable workability.

• The proposed method, based on weight loss and thermogravimetric analysis, for quantification of reaction products and remaining reagents have a high reliability and corroborate the balance of the initial amounts but also the mechanical behaviour of the mortar.

• The matrix of the optimized mortar is a mix of crystalline sodium/calcium carbonate hydrate (pirssonite), calcium hydroxide (portlandite) and unreacted limestone in a relative proportion of 3.5/1/1, without amorphous phase.

These results indicates that this new binder could be employed as an indoor construction material.

Further research is needed to improve knowledge of reaction mechanisms and the parameters that can influence them and assess durability.

Figure 2

 2 Figure 2 presents the mechanical strength and workability as a function of the alkaline-solution to limestone ratio (AS/L) of mortars made with 12 mol/L alkaline solution.

Figure 2 -

 2 Figure 2-Mechanical strength and workability of mortars obtained with a sodium hydroxide solution at 12 mol/L.

Figure 3 -

 3 Figure 3-Mechanical strength and workability of mortars obtained with 12 and 13 mol/L sodium hydroxide solution and AS/L ratio of 0.60 mL/g. It can be observed that the increase of 1 mol/L of NaOH in the solution more than doubles the compressive strength of the mortar, which reaches almost 20 MPa, when the water amount in the mortar remains constant. However, the flexural strength is not significantly affected by the variation of concentration of sodium hydroxide solution. Furthermore, the workability decreases and reaches

Figure 5 -

 5 Figure 5-XRD pattern of optimized mortar after curing.

Figure 7 -

 7 Figure 7-SEM images of alkali-activated mortar paste at different scales: (a) large field, (b) and (c) intermediate fields, (d) and (e) low field.

Figure 8 -

 8 Figure 8-SEM image of dense and grainy areas in the paste and their respective EDX analyzes. The same elements: C, O, Na, Ca were detected in both types of microstructures, but in different proportions. Al and Si were observed only in the granular zones. These were related to sand. Element quantification in the two different microstructures, in atomic percentages for sodium and calcium was

Figure 9 -

 9 Figure 9-TGA and DTA curves of standardized sand and average curve of mortar samples after 28 days curing (dash: TGA and solid: DTA).

Table 1 .

 1 Table 1 summarizes the main physical and chemical characteristics of the limestone filler. Physical and chemical properties of limestone filler BETOCARB P2-MX.

	Physical properties		Chemical composition
	Absolute density (kg.m -3 ) Blaine specific surface area (m 2 .g -1 ) 755 2600	CaCO3 (%) SiO2 (%)	98.4 0.2
	Fine particles < 63 µm (%)	96.1	Na2O (%)	0.009
	Activity index i28	0.80	S (%)	0.041

Table 2 .

 2 Parameters of preliminary mortars.

	[NaOH]	AS/L	S/L	W/B
	(mol/L)	(mL/g)		ratio
	12	0.52	3	0.42
	12	0.60	3	0.47
	12	0.68	3	0.52
	13	0.60	3	0.46

Table 3

 3

		Relative amount (%wt)
	SiO2 (from sand)	60.08
	CaCO3 (from limestone)	20.47
	Impurities (from sand and limestone)	2.73
	Na2O (from NaOH solution)	5.04
	Free water	10.22
	Bound water in NaOH	1.46
	Total water	11.68
	Total	100.00

Table 3 -

 3 Composition of the optimized formulation of mortar.

Table 4 -

 4

	1 )

FTIR spectroscopic data of pirssonite from Adler and al.

[START_REF] Adler | Infrared spectra, symmetry and structure relations of some carbonate minerals[END_REF]

, Estep and al.

[START_REF] Estep | Characterization of carbonate minerals from oil shales and coals by infrared spectroscopy[END_REF]

, Böttcher and al.

[START_REF] Böttcher | Dehydration of natural gaylussite (Na2Ca(CO3)2, 5H2O) and pirssonite (Na2Ca(CO3)2, 2H2O) as illustrated by FTIR spectroscopy[END_REF]

, Frost and al.

[START_REF] Frost | Hydrated double carbonates -A Raman and infrared spectroscopic study[END_REF]

.

Table 5 -

 5 ∆mfree w ∆mpirss (TGA) ∆mportl (TGA) ∆mpirss w ∆mportl w ∆mbound w ∆mwater Initial, measured by experiments and calculated by equations (

	Initial amount of water (%wt)	Measured amount of water (%wt)	Calculated amount of water (%wt)	Water balance (%wt)
	11.68 ± 0.00	7.53 ± 0.01	2.98 ± 0.08	1.25 ± 0.02	2.75 ± 0.08	1.16 ± 0.02	3.91 ± 0.07	11.44 ± 0.06	0.24 ± 0.06

Table 6 -

 6 Composition before and after alkali-activation of each specimen of mortars.

Acknowledgements

The X-ray diffraction tests were carried out in the Physics and Chemistry Forensics Division of IRCGN (Institut de Recherche Criminelle de la Gendarmerie Nationale Française). Its staff is warmly thanked.

The FTIR and the SEM-EDX analyses were carried out in Ermeece Laboratory (EA1391) and in the ″Microscopies & Analyses″ imaging facility, Federation I-Mat (FR4122) of the CY Cergy Paris Université (France), respectively.

Funding:

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