Marie Desnos-Ollivier

Olivier Lortholary

Françoise Stéphane Bretagne

Françoise Dromer

Marie Desnos Ollivier

Azoles susceptibility profiles of more than 9,000 clinical yeast isolates belonging to 40 common and rare species

Introduction

Invasive fungal infections (IFIs) represent a major worldwide public health issue with an incidence of 5.9 to 20.3 cases/100 000 patients per year in France, up to 27.2 / 100 000 in USA and 14.1/100 000 in the United Kingdom [START_REF] Lortholary | Worrisome trends in incidence and mortality of candidemia in intensive care units (Paris area, 2002-2010)[END_REF][START_REF] Bitar | Population-based analysis of invasive fungal infections, France, 2001-2010[END_REF][START_REF] Gangneux | Life program tSfdmmS-sg. 2016. An estimation of burden of serious fungal infections in France[END_REF][START_REF] Pegorie | Estimating the burden of invasive and serious fungal disease in the United Kingdom[END_REF][START_REF] Webb | Epidemiology and Clinical Features of Invasive Fungal Infection in a US Health Care Network[END_REF]. Despite treatment, the mortality remains high, ranging from 7.5 to 27.6%, with an estimation of 1.5 million deaths annually [START_REF] Brown | Hidden killers: human fungal infections[END_REF]. Yeasts are the main causative agents of these infections and only few antifungals are effective against the most common Ascomycetous yeasts, and even less against Basidiomycetous yeasts. The azoles are the largest class of antifungal agents used for treatment of IFIs, especially fluconazole for treatment of candidemia [START_REF] Arendrup | ESCMID and ECMM joint clinical guidelines for the diagnosis and management of rare invasive yeast infections[END_REF][START_REF] Pappas | Clinical Practice Guideline for the Management of Candidiasis: 2016 Update by the Infectious Diseases Society of America[END_REF]. Azoles inhibit the 14-alpha-lanosterol demethylase (Cyp51), resulting in depletion of ergosterol synthesis, a major component of the fungal cell wall and in accumulation of toxic 14-alpha-demethylated sterols in the membrane [START_REF] Bhattacharya | Candidiasis and Mechanisms of Antifungal Resistance[END_REF]. The determination of in vitro antifungal susceptibility allows determination of wild type population and those with acquired or mutational resistance to the drug. Therefore, epidemiological surveys and standardized methods of antifungal susceptibility testing are important to confirm or not that the antifungal susceptibility of a given species remains unchanged/stable. Among internationally recognized standards, two broth microdilution over the world, mono-or multicentric studies provide reports adding to the knowledge on antifungal susceptibility profiles, mostly of common yeast species [START_REF] Borman | MIC distributions for amphotericin B, fluconazole, itraconazole, voriconazole, flucytosine and anidulafungin and 35 uncommon pathogenic yeast species from the UK determined using the CLSI broth microdilution method[END_REF][START_REF] Diaz-Garcia | Susceptibility of uncommon Candida species to systemic antifungals by the EUCAST methodology[END_REF][START_REF] Guinea | Molecular identification and antifungal susceptibility of yeast isolates causing fungemia collected in a population-based study in Spain in 2010 and 2011[END_REF][START_REF] Jung | A multicenter retrospective analysis of the antifungal susceptibility patterns of Candida species and the predictive factors of mortality in South Korean patients with candidemia[END_REF][START_REF] Pfaller | In vitro antifungal susceptibilities of isolates of Candida spp. and Aspergillus spp. from China to nine systemically active antifungal agents: data from the SENTRY antifungal surveillance program, 2010 through 2012[END_REF][START_REF] Pfaller | Twenty Years of the SENTRY Antifungal Surveillance Program: Results for Candida Species From 1997-2016[END_REF][START_REF] Pfaller | Antifungal susceptibilities of Candida, Cryptococcus neoformans and Aspergillus fumigatus from the Asia and Western Pacific region: data from the SENTRY antifungal surveillance program (2010-2012)[END_REF][START_REF] Xiao | Distribution and Antifungal Susceptibility of Candida Species Causing Candidemia in China: An Update From the CHIF-NET Study[END_REF][START_REF] Xiao | Five-Year National Surveillance of Invasive Candidiasis: Species Distribution and Azole Susceptibility from the China Hospital Invasive Fungal Surveillance Net (CHIF-NET) Study[END_REF]. For rare species however, obtaining robust data is difficult given the low number of reported cases [START_REF] Borman | MIC distributions for amphotericin B, fluconazole, itraconazole, voriconazole, flucytosine and anidulafungin and 35 uncommon pathogenic yeast species from the UK determined using the CLSI broth microdilution method[END_REF].

Centralized data are therefore important for these species to determine their normal susceptibility profile [START_REF] Berkow | Antifungal Susceptibility Testing: Current Approaches[END_REF].

The French National Reference Center for Invasive Fungal Infections and Antifungals (NRCMA) provides antifungal susceptibility testing results using the EUCAST method for all the isolates collected through its missions of expertise on pathogenic fungi and surveillance of IFIs in France. We here report the results obtained for azoles on more than 9,000 clinical isolates belonging to 40 different species, collected nationwide in France between 2003 and 2019. These data provide an overview of the susceptibility to azoles of frequent and rare pathogenic yeast species in France.

Results

A total of 9,319 yeast isolates were included in the analysis. These isolates, received at the NRCMA between the ATCC6258) were always in the range of acceptable MIC defined by EUCAST-AFST for fluconazole and voriconazole, they were one dilution higher for posaconazole between 2011 and 2015. However, the proportion of posaconazole-resistant (MIC>0.06mg/L) isolates was not higher at that time (Figure 1).

We first compared the MIC distributions recorded at the NRCMA to those available in the EUCAST dataset. tropicalis and M. guilliermondii (Supplemental Figure 1) with a median MIC value for the NRCMA dataset 2 dilutions higher than for the EUCAST dataset.

Among the Candida parapsilosis complex, the three species had similar MIC distribution for posaconazole while C. metapsilosis isolates had lower MICs for voriconazole and fluconazole but a higher MIC50 for fluconazole than C. parapsilosis and C. orthopsilosis. Using EUCAST BP for fluconazole (R>4mg/L), C. orthopsilosis has the highest number of resistant isolates (12.2%) (Table 1, Figure 2).

For Cr. neoformans, range of MICs, MIC50 and MIC90 were determined for serotype A, serotype D and AD hybrids. The distribution of MICs was comparable for the three species but for the three azoles, serotype D isolates exhibited lower MIC50 and MIC90 than serotypes A and AD hybrids (Table1 and Table S1).

Using the EUCAST breakpoints (BP) for fluconazole, 32.4% C. glabrata isolates were resistant (R > 16mg/L) compared to less than 7.5% of the isolates for all the other common species (R>4 mg/L) (Table 1 2). The majority (>80%) of isolates resistant to at least one azole were resistant to posaconazole. The proportion of posaconazole-resistant isolates varied according to the year of isolation for C. tropicalis and C. parapsilosis (Figure 1). This variation was correlated with variation of voriconazoleresistance for C. tropicalis.

Discussion

In the present study, we report the azoles susceptibility profiles for [START_REF] Castanheira | Monitoring Antifungal Resistance in a Global Collection of Invasive Yeasts and Molds: Application of CLSI Epidemiological Cutoff Values and Whole-Genome Sequencing Analysis for Detection of Azole Resistance in Candida albicans[END_REF] [START_REF] Gago | Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis virulence in the nonconventional host Galleria mellonella[END_REF]. Finally, we confirmed that very rare species L. elongisporus and Cy. fabianii were susceptible to the three azoles while W. anomalus, K.

ohmeri and Cy. jadinii were less susceptible to fluconazole with "intermediate" MICs [START_REF] Borman | MIC distributions for amphotericin B, fluconazole, itraconazole, voriconazole, flucytosine and anidulafungin and 35 uncommon pathogenic yeast species from the UK determined using the CLSI broth microdilution method[END_REF][START_REF] Al-Obaid | Lodderomyces elongisporus: a bloodstream pathogen of greater clinical significance[END_REF][START_REF] Al-Sweih | Cyberlindnera fabianii fungaemia outbreak in preterm neonates in Kuwait and literature review[END_REF][START_REF] Bougnoux | Resolutive Candida utilis fungemia in a nonneutropenic patient[END_REF][START_REF] Dutra | Fatal Case of Fungemia by Wickerhamomyces anomalus in a Pediatric Patient Diagnosed in a Teaching Hospital from Brazil[END_REF][START_REF] Jung | Investigation of a nosocomial outbreak of fungemia caused by Candida pelliculosa (Pichia anomala) in a Korean tertiary care center[END_REF][START_REF] Lin | Reporting an outbreak of Candida pelliculosa fungemia in a neonatal intensive care unit[END_REF][START_REF] Park | Identification and Antifungal Susceptibility Profiles of Cyberlindnera fabianii in Korea[END_REF][START_REF] Zhou | Identification and antifungal susceptibility profiles of Kodamaea ohmeri based on a seven-year multicenter surveillance study[END_REF].

Despite reports of azole resistance acquired after treatment, in common and rare yeast species, and description of an increasing percentage of resistant isolates [START_REF] Arastehfar | The Quiet and Underappreciated Rise of Drug-Resistant Invasive Fungal Pathogens[END_REF], this phenomenon remains rare [START_REF] Lortholary | Worrisome trends in incidence and mortality of candidemia in intensive care units (Paris area, 2002-2010)[END_REF][START_REF] Pfaller | In vitro antifungal susceptibilities of isolates of Candida spp. and Aspergillus spp. from China to nine systemically active antifungal agents: data from the SENTRY antifungal surveillance program, 2010 through 2012[END_REF][START_REF] Castanheira | Monitoring Antifungal Resistance in a Global Collection of Invasive Yeasts and Molds: Application of CLSI Epidemiological Cutoff Values and Whole-Genome Sequencing Analysis for Detection of Azole Resistance in Candida albicans[END_REF][START_REF] Lass-Florl | A nationwide passive surveillance on fungal infections shows a low burden of azole resistance in molds and yeasts in Tyrol, Austria[END_REF] or has geographical specificity [START_REF] Tacconelli | Surveillance for control of antimicrobial resistance[END_REF][START_REF] Trouve | Epidemiology and reporting of candidaemia in Belgium: a multi-centre study[END_REF], and seems to concern especially C. tropicalis, C. glabrata and C. parapsilosis [START_REF] Pfaller | Twenty Years of the SENTRY Antifungal Surveillance Program: Results for Candida Species From 1997-2016[END_REF]. In the present study, we observed a proportion of fluconazole-resistant isolates among C. albicans and C. parapsilosis isolates similar to that reported in international surveys [START_REF] Pfaller | Twenty Years of the SENTRY Antifungal Surveillance Program: Results for Candida Species From 1997-2016[END_REF][START_REF] Xiao | Distribution and Antifungal Susceptibility of Candida Species Causing Candidemia in China: An Update From the CHIF-NET Study[END_REF][START_REF] Xiao | Five-Year National Surveillance of Invasive Candidiasis: Species Distribution and Azole Susceptibility from the China Hospital Invasive Fungal Surveillance Net (CHIF-NET) Study[END_REF][START_REF] Borman | Fluconazole Resistance in Isolates of Uncommon Pathogenic Yeast Species from the United Kingdom[END_REF][START_REF] Pfaller | Bacterial and fungal pathogens isolated from patients with bloodstream infection: frequency of occurrence and antimicrobial susceptibility patterns from the SENTRY Antimicrobial Surveillance Program (2012-2017)[END_REF]. We observed posaconazole-resistant isolates of C. tropicalis and C. parapsilosis complex (23.9 and 27.3%, respectively), with a variable proportion, according to the year of isolation. The increased proportion of posaconazole-resistant C. tropicalis isolates correlated with an increased proportion of voriconazole-resistance of those isolates. This heterogeneous percentage of resistant isolates in our collection remained unexplained. Indeed, we raised a few hypotheses:

(i) Technical issues related to the batch of antifungal powder or RPMI medium that translated into a higher median posaconazole and not the other azoles MIC for the QC strains, but this was not associated with an increase in the proportion of posaconazole-resistant isolates in the related series; (ii) Geographic specificity and/or local outbreak could be responsible as described for instance in the multicentric CHIF-Net study [START_REF] Xiao | Distribution and Antifungal Susceptibility of Candida Species Causing Candidemia in China: An Update From the CHIF-NET Study[END_REF] and in a national survey in Belgium [START_REF] Trouve | Epidemiology and reporting of candidaemia in Belgium: a multi-centre study[END_REF], but none was reported to our knowledge; (iii) Bias related to the isolates analyzed knowing that 22% of the isolates were sent for expertise following therapeutic failure and not part of the epidemiological surveys [START_REF] Bitar | Population-based analysis of invasive fungal infections, France, 2001-2010[END_REF][START_REF] Lortholary | The risk and clinical outcome of candidemia depending on underlying malignancy[END_REF]. However, posaconazole-resistance was not restricted to the isolates sent for expertise.

We also report an important percentage of C. glabrata isolates resistant to fluconazole (32.4%). The proportion of C. glabrata resistant to fluconazole seems to be very heterogeneous according to the country, the center and the year of isolation. In fact, [START_REF] Borman | Fluconazole Resistance in Isolates of Uncommon Pathogenic Yeast Species from the United Kingdom[END_REF][START_REF] Trouve | Epidemiology and reporting of candidaemia in Belgium: a multi-centre study[END_REF]. Of note, the BP values for C. glabrata was modified by EUCAST-AFST committee in 2020, from R>32mg/L to R>16mg/L [START_REF] Arendrup | How to interpret MICs of antifungal compounds according to the revised clinical breakpoints v. 10.0 European committee on antimicrobial susceptibility testing (EUCAST)[END_REF]. When using the previous BP (MIC>32mg/L), 18.8% of C. glabrata in our collection was identified as resistant to fluconazole, which is comparable to the studies published earlier before the change of the BP threshold.

Our data confirm azole susceptibility profiles for frequent species and, to our knowledge, it is one of the only studies to assemble MIC data for more than 40 pathogenic yeast species, mainly involved in invasive infection, including big samples of isolates, even for rare and emerging species such as C. lusitaniae (n=221), S. clavata (n=184), M. guilliermondii complex (n=150), C. haemulonii complex (n=87), R. mucilaginosa (n=55), W. anomalus (n=36) [START_REF] Borman | MIC distributions for amphotericin B, fluconazole, itraconazole, voriconazole, flucytosine and anidulafungin and 35 uncommon pathogenic yeast species from the UK determined using the CLSI broth microdilution method[END_REF]. This emphasizes the importance of centralization of isolates for collection and analysis by National Reference Centers. Our results confirm that an antifungal susceptibility profile is associated with each species, hence the importance of accurate yeast species identification as soon as possible to infer the susceptibility profile. Of course, it goes without saying that the determination of MICs, using standardized methods, remains important for rare species and the monitoring of potential emergence of resistance including in the context of therapeutic failure.

Materials & Methods

Isolates

Between the 1 st January of 2003 and the 31 st December of 2019, a total of 9,449 yeast were sent to the National Reference Center for Mycosis & Antifungal agents (NRCMA). We analyzed MICs data for 9,319 isolates belonging to the species for which at least 5 isolates were available. Majority of the isolates were sent for expertise (7358/9,319; 78.8%; i.e.

species identification, antifungal susceptibility testing) and/or as participation to epidemiological surveys. For rare species represented by 10 or less isolates, all isolates were recovered from different patients.

Species identification

For all isolates, purity was checked on chromogenic medium (BBL and reported only the percentage of resistant or non-wild-type isolates in our collection.

  methods are well standardized by the EUCAST (European Committee on Antimicrobial Susceptibility Testing) and the CLSI (Clinical and Laboratory Standards Institute) for determining minimal inhibitory concentrations (MICs). Expert committees in both instances establish breakpoints (BPs) and epidemiological cut-off (abbreviated ECOFFs for EUCAST, and ECVs for CLSI) values in order to easily distinguish between susceptible and resistant and wild-type and non-wild-type isolates, respectively. BPs are defined for a limited number of species and antifungal agents based on MICs distributions, clinical data, pharmacodynamics and pharmacokinetics of the drugs while ECOFFs are determined based only on MICs distributions. An ECOFF corresponds to the MIC that separates a population of isolates into wild-type and non-wild type. Unlike BP, ECOFF does not necessarily predict clinical failure but it can be helpful for clinical decision (SOP10.1 https://eucast.org/documents/sops/ (10)). The EUCAST Antifungal Susceptibility Testing subcommittee (EUCAST-AFST) regularly updates data concerning MICs distributions, and ECOFFs for frequent species involved in human infections based on data generated by more than 15 European centers (http://www.eucast.org/astoffungi/clinicalbreakpointsforantifungals/, (11)). Furthermore, all

  For fluconazole and voriconazole, ranges of MICs were similar or with a maximum variation of 2 dilutions. Medians of MICs were equal or with a maximum of one dilution difference for C. albicans, C. dubliniensis, C. glabrata, C parapsilosis, C. tropicalis, P. kudriavzevii, W. anomalus, K. marxianus, C. lusitaniae and M. guilliermondii (data not shown). For posaconazole, MIC distributions were also similar except for C. parapsilosis, C.

Figure 1 .

 1 Figure 1. Percentage of fluconazole, voriconazole and posaconazole -resistant isolates,

  , C. auris) had more than 90% of resistant isolates while three others (C. metapsilosis, C. orthopsilosis and C. nivariensis) had 2.2%, 12.2% and 7.7% of fluconazoleresistant isolates, respectively. According to the BPs for posaconazole (R>0.25 mg/L) and

	voriconazole (R>0.06 mg/L), the percentage of resistant isolates was 23.9% and 2.2% for C.
	parapsilosis and 27.3% and 9.5% for C. tropicalis, respectively (Table 1).
	According to the EUCAST ECOFF, 26.8% of P. kudriavzevii (ECOFF=128mg/L) and 20% of
	M. guilliermondii (ECOFF=16mg/L) isolates were considered as non-wild type for
	fluconazole. Likewise, 9.6% of C. glabrata (ECOFF=1mg/L), 4.5% of C. lusitaniae
	(ECOFF=0.06mg/L), 13.9% of M. guilliermondii (ECOFF=0.25mg/L) and 2.6% of P.

). Using the non-species related BP defined by EUCAST for fluconazole (R>4mg/L), four species (C. haemulonii, C. duobushaemulonii, C. palmioleophilakudriavzevii (ECOFF=1mg/L) isolates were considered as non-wild type for voriconazole (Table

1

).

Among the population of C. glabrata isolates, 7.9 % (101/1274) were simultaneously resistant to fluconazole and considered non-wild-type for voriconazole. Among the 155 C. albicans isolates resistant to at least one azole, 49 (31.6%) were resistant to the three azoles. Cross-resistance to the three azoles concerned 0/8 C. dubliniensis, 29/192 (15.1%) C. tropicalis, and 7/236 (3.0%) C. parapsilosis (Supplemental Figure

  yeast species involved in IFIs based on the MIC distribution of 9,319 clinical isolates recovered in France between 2003 and 2019. We provide azole profiles for common, emerging and rare species thanks to our varied and important collection of isolates. Our results confirm that C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis complex, K. marxianus and C. lusitaniae can be considered as susceptible in vitro to fluconazole, voriconazole and posaconazole[START_REF] Guinea | Molecular identification and antifungal susceptibility of yeast isolates causing fungemia collected in a population-based study in Spain in 2010 and 2011[END_REF][START_REF] Pfaller | In vitro antifungal susceptibilities of isolates of Candida spp. and Aspergillus spp. from China to nine systemically active antifungal agents: data from the SENTRY antifungal surveillance program, 2010 through 2012[END_REF][START_REF] Pfaller | Antifungal susceptibilities of Candida, Cryptococcus neoformans and Aspergillus fumigatus from the Asia and Western Pacific region: data from the SENTRY antifungal surveillance program (2010-2012)[END_REF][START_REF] Xiao | Distribution and Antifungal Susceptibility of Candida Species Causing Candidemia in China: An Update From the CHIF-NET Study[END_REF][START_REF] Astvad | Evaluation of the in vitro activity of isavuconazole and comparator voriconazole against 2635 contemporary clinical Candida and Aspergillus isolates[END_REF], while (i) C. neoformans complex has decreased in vitro susceptibility to fluconazole, (ii) P. kudriavzevii and uncommon species C. haemulonii complex, M. guilliermondii complex, P. norvegensis, S. clavata, G. candidus and R. mucilaginosa can be considered as intrinsically resistant to fluconazole (MIC90≥ 64mg/L)[START_REF] Pfaller | Twenty Years of the SENTRY Antifungal Surveillance Program: Results for Candida Species From 1997-2016[END_REF][START_REF] Xiao | Distribution and Antifungal Susceptibility of Candida Species Causing Candidemia in China: An Update From the CHIF-NET Study[END_REF][START_REF] Xiao | Five-Year National Surveillance of Invasive Candidiasis: Species Distribution and Azole Susceptibility from the China Hospital Invasive Fungal Surveillance Net (CHIF-NET) Study[END_REF][START_REF] Perez-Hansen | Antifungal susceptibility profiles of rare ascomycetous yeasts[END_REF][START_REF] Xiao | Five-year China Hospital Invasive Fungal Surveillance Net (CHIF-NET) study of invasive fungal infections caused by noncandidal yeasts: species distribution and azole susceptibility[END_REF][START_REF] Jamiu | Update on Candida krusei, a potential multidrug-resistant pathogen[END_REF]. Of note, C. haemulonii and C. duobushaemulonii, which belong to the same species complex and are closely related to the emerging C. auris, can also be considered intrinsically resistant to voriconazole and posaconazole[START_REF] Xiao | Distribution and Antifungal Susceptibility of Candida Species Causing Candidemia in China: An Update From the CHIF-NET Study[END_REF][START_REF] Bretagne | Predisposing factors and outcome of uncommon yeast species-related fungaemia based on an exhaustive surveillance programme (2002-14)[END_REF][START_REF] Desnos-Ollivier | Antifungal susceptibility profiles of 1698 yeast reference strains revealing potential emerging human pathogens[END_REF]. Other uncommon species such as S. cerevisiae,

K. ohmeri, C. palmioleophila, P. cactophila, M. capitatus, Y. lipolityca, Cr. gattii and T. asahii should be considered as less susceptible in vitro to fluconazole, with "intermediate" MIC value (MIC90≥16mg/L)

[START_REF] Fernandez-Ruiz | Fungemia due to rare opportunistic yeasts: data from a population-based surveillance in Spain[END_REF][START_REF] Borman | Fluconazole Resistance in Isolates of Uncommon Pathogenic Yeast Species from the United Kingdom[END_REF]

while voriconazole seems to be the most potent azole in vitro, which is already known for Trichosporon spp.

[START_REF] Arendrup | ESCMID and ECMM joint clinical guidelines for the diagnosis and management of rare invasive yeast infections[END_REF][START_REF] Xiao | Five-year China Hospital Invasive Fungal Surveillance Net (CHIF-NET) study of invasive fungal infections caused by noncandidal yeasts: species distribution and azole susceptibility[END_REF]

. When considering the C. parapsilosis complex, C. metapsilosis was the species the most sensitive in vitro to azoles with the lowest percentage of fluconazole-resistant isolates while C. orthopsilosis seemed to be more resistant than the other species of the complex with the highest percentage of fluconazole-resistant isolates and the highest MIC90 for fluconazole, voriconazole and posaconazole

[START_REF] Borman | MIC distributions for amphotericin B, fluconazole, itraconazole, voriconazole, flucytosine and anidulafungin and 35 uncommon pathogenic yeast species from the UK determined using the CLSI broth microdilution method[END_REF][START_REF] Xiao | Distribution and Antifungal Susceptibility of Candida Species Causing Candidemia in China: An Update From the CHIF-NET Study[END_REF]

. There may be a link between the low frequency of C. metapsilosis recovered from IFIs in the literature

[START_REF] Canton | Prospective multicenter study of the epidemiology, molecular identification, and antifungal susceptibility of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis isolated from patients with candidemia[END_REF]

and this highest azole sensitivity. Another hypothesis proposed by Gago et al. is that C. metapsilosis has a reduced ability to produce some of the known virulence factors

  published 0 to 28.6%, according to the year of isolation in the SENTRY study[START_REF] Pfaller | Bacterial and fungal pathogens isolated from patients with bloodstream infection: frequency of occurrence and antimicrobial susceptibility patterns from the SENTRY Antimicrobial Surveillance Program (2012-2017)[END_REF], Bassetti et al., reported a percentage of 0 to 21.2% in the SENTRY and ARTEMIS studies depending on the continent (46), Borman et al. and Trouvé et al. reported 12.7% and 11.3% of resistance, respectively in national studies

Xiao et al. reported 10.3 to 19% of resistant isolates in China between 2009-2017 (19, 20, 23), based on the CHIF-Net study, while Pfaller et al.

  TM Chromagar TM Candida Medium, BD, GmbH) or Niger seed agar for Cryptococcus spp.. Phenotypic identification Darmstadt, Germany) and Alsachim (Shimadzu Group Company, France); voriconazole provided by Pfizer and Alsachim and posaconazole provided by SheringPlough (Merck & Co. Reference 92096). The concentrations tested ranged between 0.0015 mg/L to 8 mg/L for posaconazole and voriconazole and between 0.125 mg/L to 64 mg/L for fluconazole.QC strains (ATCC22019, ATCC6258) were included in each set. The concentrations corresponding to the MIC that inhibited 50% (MIC50) and 90% (MIC90) of the isolates were determined for species having 10 or more isolates.Our dataset (MIC distribution) was compared with that available on the EUCAST website (https://mic.eucast.org/Eucast2/SearchController/search.jsp?action=init, June 2020).The BP or ECOFF values determined by EUCAST for some species and some antifungal agents (https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/AFST/Clinical_breakpoint s/AFST_BP_v10.0_200204.pdf) were used to calculate percentage of resistant (R) isolates and non-wild-type (NWT), respectively. Non-species related BP for fluconazole, defined by EUCAST, (MIC >16 mg/L) for Candida were also used to calculate percentage of resistant isolate for C. orthopsilosis, C. metapsilosis, C. nivariensis, C. haemulonii, C. duobushaemulonii and C. palmioleophila. Since not all isolates were collected through unbiased epidemiological survey (7358/9,319; 78.8%), we did not determine local ECOFF,

	Inc., USA) and Alsachim) by using the standardized broth microdilution method EUCAST
	following	the	procedure	E.	DEF	7.3.2
	(https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/AFST/Files/EUCAST_E_
	Def_7.3.2_Yeast_testing_definitive_revised_2020.pdf), in 96 plate (sterile Tissue culture
	plates, 96 well flat bottom in clear polystyrene, TPP® Techno Plastic Products AG,
	Switzerland,
	was performed using carbon assimilation profiles (ID32C, BioMérieux, Marcy-l'Etoile,
	France) before 2014, and matrix assisted laser desorption ionisation-time of flight mass
	spectrometry (MALDI-TOF, MALDI Biotyper, Bruker Daltonik, GmbH) since 2014. Duplex
	PCR was performed to differentiate Candida dubliniensis and Candida albicans (47). For all
	isolates, PCR and sequencing of ITS1-5.8S-ITS2 and D1D2 regions were performed, except
	for C. glabrata, C. tropicalis, P.kudriavzevii and K. marxianus, using V9D/LS266 (48, 49)
	and NL1/NL4 (50) primers, respectively. In addition, part of the actin gene (for C. lusitaniae
	(51) and Debaryomyces species (52)), part of the RPBI gene (for M. guilliermondii, M.
	caribbica, C. carpophila (53)) or the IGS1 region (for Trichosporon species (54)) were
	sequenced. Sequences were compared to sequences of the type strain of each species and of
	the closely related species. Serotype was determined for Cr. neoformans isolates by PCR
	amplification of part of GPA1 and PAK1 genes using a triplex PCR with primers specific of
	the serotype previously described (55).

Antifungal susceptibility

Minimal inhibitory concentrations (MICs) were determined for all isolates, for 3 antifungal agents (fluconazole provided by Pfizer Inc (NewYork, USA), Sigma-Aldrich (Merck KGaA,

Acknowledgments

Members of the French Mycoses Study group who contributed to the data are in alphabetical order of the cities, all the French microbiologists and mycologists who sent isolates for characterization of unusual antifungal susceptibility profiles or to contribute to the ongoing surveillance program on the epidemiology of invasive fungal infections in France (YEASTS and RESSIF programs): N. Brieu (CH Aix); T. Chouaki, C. Damiani, A. Totet (CHU Amiens; J. P. Bouchara, M. Pihet (CHU Angers); S. Bland (CH Annecy); V. Blanc (CH Antibes); S. Branger (CH Avignon); A. P. Bellanger, L. Million (CHU Besançon); C. Plassart (CH Beauvais); I. Poilane (hôpital Jean Verdier, Bondy); I. Accoceberry, L. Delhaes, F. Gabriel (CH Bordeaux); A. L. Roux, V. Sivadon-Tardy (hôpital Ambroise Paré, Boulogne Billancourt); F. Laurent (CH, Bourg en Bresse); S. Legal, E. Moalic, G. Nevez, D. Quinio (CHU Brest); M. Cariou (CH Bretagne Sud); J. Bonhomme (CHU, Caen); B. Podac (CH, Chalon sur Saône); S. Lechatch (CH, Charleville-Mézières); C. Soler (hopital d'Instruction des armées, Clamart); P. Poirier, C. Nourrisson (CHU, Clermont Ferrand); O. Augereau (CH, Colmar); N. Fauchet (CHIC, Créteil); F. Dalles (CHU, Dijon); P. Cahen (CMC, Foch); N. Desbois, C. Miossec (CHU, Fort de France); J. L. Hermann (hôpital Raymond Poincaré, Garches) ; M. Cornet, D. Maubon, H. Pelloux (CHU, Grenoble); M. Nicolas (CHU, Guadeloupe); C. Aznar, D. Blanchet, J. F. Carod, M. Demar, (CHU, Guyane); A. Angoulvant (hôpital Bicêtre, le Kremlin Bicêtre); C. Ciupek (CH, Le Mans); A. Gigandon (hôpital Marie Lannelongue, Le Plessis Robinson); B. Bouteille (CH Limoges); E. Frealle, B. Sendid (CHU Lille); D. Dupont, J. Menotti, F. Persat, M. Wallon (CHU, Lyon); C. Cassagne, S. Ranque (CHU, Marseille); T. Benoit-Cattin, L. Collet (CH Mayotte); A. Fiacre (CH Meaux); N. Bourgeois, L. Lachaud (CHU, Montpellier); M. Machouart (CHU, Nancy); P. Lepape, F. Morio (CHU, Nantes) ; O. Moquet (CH, Nevers) ; S. Lefrançois (hôpital Américain, Neuilly) ; M. Sasso (CHU, Nimes) ; F. Reibel (GH, Nord-Essone) ; M. Gari-Toussaint, L.


Amina Annane

Mouna Kamel

Cassia Trojahn

Nathalie Aussenac-Gilles

Catherine Comparot

Christophe Baehr

SYNOP Data Evaluation Using FAIR Maturity Model

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

Introduction

SYNOP dataset is one of the open meteorological datasets privided by Météo-France on its data portal. The dataset includes observation data from international surface observation messages circulating on the Global Telecommunication System (GTS) of the World Meteorological Organization (WMO) . The choice of this dataset is motivated by the fact that these data are open and free, they concern several atmospheric parameters measured (temperature, humidity, wind direction and force, atmospheric pressure, precipitation height, etc.). These parameters are important for many scientific studies, particularly because SYNOP dataset are published by all WMO member states. SYNOP data is published as open data. On its web page 1 , it is described by seven items: (i) description : natural language summary that describes the content of the dataset, (ii) conditions of access : Etalab license 2 for the data, (iii) means of access : specifies that the data can be accessed via direct download, (iv) download : possibility offered to the user to download the data in csv format for a given date, (v) download archived data : similar to the previous item, but for a given month, (vi) station information : list of stations (station id, name) accompanied by a map displaying the location of these stations, and (vii) documentation: three links that respectively reference (a) a pdf file that explains the acronyms (label, type, unit of measurement) present in the header of the SYNOP data files, (b) a csv file that describes the different meteorological stations of Météo-France (id_station, name, latitude, longitude, altitude), and (c) a JSON file containing the same information as the previous csv file. Table 1 shows an extract of the downloadable SYNOP data. The file contains 59 columns, the first two correspond to the station number and the date of the measurements made, the other 57 columns to the meteorological measurements.

FAIR Data Maturity Model

It has been developed by the RDA 3 FAIR Data Maturity Model working group [START_REF] Rda | FAIR Data Maturity Model. Specification and Guidelines[END_REF]. The model is based on three main components: (i) indicators, (ii) priorities, and (iii) evaluation methods. Indicators: the individual aspects of FAIRness that are evaluated. An indicator aims to measure the state or level of a digital resource with regard to a specific FAIR principle (e.g., F1, A2). Indicators are derived from the FAIR principles [START_REF] Wilkinson | The fair guiding principles for scientific data management and stewardship[END_REF]. The approach for developing the indicators was to create an indicator for each aspect that could be distinguished in the description of each principle. For instance from the principle "F1. (Meta)data are assigned a globally unique and persistent identifier", four indicators are extracted:

• RDA-F1-01M Metadata is identified by a persistent identifier • RDA-F1-01D Data is identified by a persistent identifier • RDA-F1-02M Metadata is identified by a globally unique identifier • RDA-F1-02D Data is identified by a globally unique identifier Following this approach, 41 indicators have been extracted (see Tables 2 and3).

Priorities: It consists in assigning the degree of importance to each indicator. Three levels are defined so that an indicator may be:

• Essential: FAIRness would be practically impossible to achieve if the indicator were not satisfied. e.g., "Metadata is identified by a persistent identifier"

• Important: its satisfaction, if at all possible, would substantially increase FAIRness. e.g. "Metadata uses machine-understandable knowledge representation" From the 41 indicators, 20 (48.8%) are essential, 14 (34.1%) are important and 7 (17.1%) are useful. Classifying indicators into essential, important and useful may be changed from one domain to another as highlighted by the authors.

Evaluation methods the way that the results of the evaluation of the indicators can be interpreted/given a value. The model defines two evaluation methods:

1. Measuring progress: this method attempts to answer the question "How the FAIRness of this data can be improved?", hence it is suitable for data providers that want to improve the FAIRness of their data. It consists in assigning a maturity level ( value from 0 to 4) for each indicator such that each value has the following meaning:

• 0: not applicable • 1: not being considered this yet

• 2: under consideration or in planning phase

• 3: in implementation phase

• 4: fully implemented 2. Measuring pass or fail: this method evaluates how a resource performs on meeting the indicators across the FAIR area. A binary answer (0 or 1) is assigned on each of the indicators. Finally, the method affects to each principle a FAIRness level taking into account the priorities as shown in Figure 1. This method is particularly suitable for external evaluators.

SYNOP data evaluation

We present the evaluation of SYNOP dataset against the indicators listed in Table 2 according to the RDA maturity model described previously. We adopt the pass/fail method since we are an external evaluator. More particularly, we present the value of each indicator (i.e., 0 or 1) before the generation of semantic metadata, and after their generation with a comment to explain the attributed value each time.

Findability

RDA-F1-01M. Metadata is identified by a persistent identifier

• (Before: 0) The URL of the home page may be considered as an identifier, but URLs are not persistent.

• (After: 0) The generation of persistent identifiers for Synop metadata should be managed by Météo-France using a third-party.

RDA-F1-01D. Data is identified by a persistent identifier

• (Before: 0) No identifier is assigned to the dataset, unless we consider the home page URL which is not persistent.

• (After: 0) The generation of persistent identifiers for Synop data should be performed by Météo-France

RDA-F1-02M. Metadata is identified by a globally unique identifier

• (Before: 0) No metadata records.

• (After: 0) The generation of unique identifiers for metadata records should be managed by Météo-France.

RDA-F1-02D. Data is identified by a globally unique identifier

• (Before: 0) The URL of the Synop data, which can be considered as an identifier, offers a form to select one dataset from all the Synop datasets (one dataset per month), hence it is not unique.

• (After: 0) The generation of unique identifiers for metadata records should be managed by Météo-France.

RDA-F2-01M. Rich metadata is provided to allow discovery

• (Before: 0) To assess this indicator, we have to define what rich metadata is. Note that we are evaluating the findability, hence we are interested in metadata that supports the findability such as keywords, theme, spatial coverage, etc. Most of these metadata are not available, hence we gave the value of zero. In addition, metadata are not machine actionable and therefore they are not indexed by search engines like google dataset search, which compromises the findability of the dataset.

• (After: 1) We use GeoDCAT-AP vocabulary to describe SYNOP datasets. This vocabulary offers a plurality of metadata that promotes their findability.

RDA-F3-01M. Metadata includes the identifier for the data

• (Before: 0) Synop datasets have no identifier.

• (After: 0) GeoDCAT-AP has a metadata element that points the data identifier, but Synop datasets have no identifier.

RDA-F4-01M. Metadata is offered in such a way that it can be harvested and indexed

• (Before: 0) No machine-actionable metadata are available.

• (After: 0*) We have generated machine-actionable metadata, but we have not yet published them on the WEB.

Accessibility

3.2.1 RDA-A1-01M. Metadata contains information to enable the user to get access to the data • (Before: 1*) Download form is available on the landing page, but no machine actionable metadata with the download link.

• (After: 1) A dcat:Distribution instance to describe each Synop monthly file, with the download link.

RDA-A1-02M. Metadata can be accessed manually (i.e. with human intervention)

• (Before: 1) Few metadata are available in natural language and can be accessed manually on the landing page • (After: 1) We have generated richer semantic metadata, but we have not published them on the web

RDA-A1-02D. Data can be accessed manually (i.e. with human intervention)

• (Before: 1) Synop data can be download manually using the form on the landing page.

• (After: 1 )

RDA-A1-03M. Metadata identifier resolves to a metadata record

• (Before: 0) No metadata record.

• (After: 0) Generation of metadata identifiers should managed by Météo-France.

RDA-A1-03D. Data identifier resolves to a digital object

• (Before: 0 ) Synop datasets do not have unique identifiers, they are accessible using a form on the landing page.

• (After: 0 ) Data identifiers should be generated for Synop datasets, eventually using a third-party to ensure that identifiers are persistent and globally unique.

RDA-A1-04M. Metadata is accessed through standardised protocol

• (Before: 0) No metadata record for each Synop dataset

• (After: 0) we have not published the generated semantic metadata yet

RDA-A1-04D. Data is accessible through standardised protocol

• (Before: 1 ) Data is accessible via the form on the landing page using http protocol

• (After: 1 ) We have represented the download link of each synop dataset, which allows humans and machine to access data.

RDA-A1-05D. Data can be accessed automatically (i.e. by a computer program)

• (Before: 0 ) the user has to manually select the day or the month for which he wants SYNOP data.

• (After: 1 ) semantic metadata that we have generated allow to access data automatically.

RDA-A1.1-01M. Metadata is accessible through a free access protocol

• (Before: 0 ) No metadata records

• (After: 0 ) We have not published the semantic-metadata yet.

RDA-A1.1-01D. Data is accessible through a free access protocol

• (Before: 1 ) Data is accessible to download with the form on the landing page using the free access protocol http.

• (After: 1 )

RDA-A1.2-01D. Data is accessible through an access protocol that supports authentication and authorisation

• (Before: 1 ) as explained previously data is accessible using http that supports authentication and authorisation

• (After: 1 ) No change

RDA-A2-01M . Metadata is guaranteed to remain available after data is no longer available

• (Before: 0) machine-actionable metadata record does not exist

• (After: 0 ) semantic metadata are generated, but we have not deposited it in a warehouse that guarantees its persistence yet.

Interoperability

RDA-I1-01M . Metadata uses knowledge representation expressed in standardised format

• (Before: 0) machine-actionable metadata record does not exist

• (After: 1 ) we have generated machine-actionable metadata record with FAIR vocabularies, mainly GeoDCAT-AP, using RDF format.

RDA-I1-01D . Data uses knowledge representation expressed in standardised format

• (Before: 0 ) Data is in CSV format without any knowledge representation

• (After: 1 ) Data schema is represented using FAIR ontologies, mainly RDF data cube, and many other domain ontologies.

RDA-I1-02M . Metadata uses machine-understandable knowledge representation

• (Before:0 ) No semantic metadata

• (After: 1 ) We have generated semantic metadata using FAIR ontologies in RDF format

RDA-I1-02D . Data uses machine-understandable knowledge representation

• (Before: 0 ) CSV files with no machine-understandable knowledge representation of SYNOP datasets

• (After: 1 ) Data schema is represented using ontologies, hence the meaning of data is represented in a machine-understandable format.

RDA-I2-01M . Metadata uses FAIR-compliant vocabularies

• (Before: 0 ) semantic metadata.

• (After: 1 ) Semantic metadata using FAIR vocabularies, namely DCAT.

RDA-I2-01D . Data uses FAIR-compliant vocabularies

• (Before: 0 ) No knowledge representation of data • (After: 1 ) Data schema is represented using FAIR ontologies, mainly RDF data cube.

RDA-I3-01M. Metadata includes references to other metadata

• (Before: 0 ) No references to other metadata.

• (After: 1 ) Usage of multiple vocabularies for metadata properties.

RDA-I3-01D . Data includes references to other data

• (Before: 1* ) "numer_sta" column references the station dataset, but no semantic referencing

• (After: 1+ ) the "numer_sta" has been represented in a semantic manner as a foreign key.

RDA-I3-02M . Metadata includes references to other data

• (Before: 0 ) No references to other data • (After: 1 ) references the station dataset with the new property ":require"

RDA-I3-02D . Data includes qualified references to other data

• (Before: 0 ) No qualified references, the single reference is "numer_sta" but not explicit relation as a foreign key.

• (After: 1 ) explicit qualified reference with csvw vocabulary property "csvw:foreignkey"

RDA-I3-03M . Metadata includes qualified references to other metadata

• (Before: 0 ) No references.

• (After: 1 ) multiple qualified references to other vocabularies specified by GeoDCAT-AP. In addition, "qb:concept" references to domain ontologies.

RDA-I3-04M. Metadata include qualified references to other data

• (Before: 0 ) no references to other data.

• (After: 1 ) qualified reference ":requires" to the station dataset. • (Before: 0 ) The description of the data columns, given as a PDF document, is not clear, nor precise. For instance, "t" acronym is explained to be "temperature" but which kind of temperature : air temperature?, soil temperature?

Reusablility

• (After: 1 ) a semantic documentation of the different measures included in the dataset using RDF data cube and domain ontologies. Plus, a semantic representation of Distribution structure using csvw vocabulary.

3.4.2 RDA-R1.1-01M . Metadata includes information about the licence under which the data can be reused

• (Before: 1 ) A link to the license under which the data may be used is available on the landing page.

• (After: 1+) a semantic qualified reference to the used license

RDA-R1.3-01M . Metadata complies with a community standard

• (Before: 0 ) No structured or semantic metadata following a standard schema, unless few natural language metadata.

• (After: 1 ) Usage of FAIR metadata vocabulary DCAT which is a community standard.

RDA-R1.3-01D Data complies with a community standard

• (Before: 1) SYNOP data are standard data for meteorologists at the international level

• (After: 1 ) No change.

3.4.9 RDA-R1.3-02M. Metadata is expressed in compliance with a machine-understandable community standard

• (Before: 0 ) No machine understandable metadata.

• (After: 1 ) Yes, metadata is expressed with GeoDCAT-AP.

RDA-R1.3-02D. Data is expressed in compliance with a machine-understandable community standard

• (Before: 0 ) No machine understandable format.

• (After: 1 ) Data is not transformed into RDF, but the semantic model of data is represented using RDF Data Cube and domain ontologies.

Evaluation Results

Figure 2 5 shows the summary of the pre-evaluation as suggested by the RDA WG. As we can see, the evaluation gives the level 0 for the three principles 'F', 'A' and 'R' since one or more essential indicators are not satisfied for each of them. 'I' is the single principle that has the level 1 while it doesn't have any satisfied indicator. This is because there is no essential indicator for the I principle. Finally, and based on our evaluation using the FAIR maturity model detailed hereafter, we may conclude that the SYNOP dataset has a very low FAIRness degree. Figure 3, suggested as a visualisation of the measuring progress method, is more informative. It shows the different levels of maturity per principle. Here, we have only used the two values: not being considered this yet (1) and fully implemented (4). As can be seen, most accessibility indicators are met, but no interoperability indicator is met. The SYNOP dataset has been re-evaluated after generating the semantic metadata that describe it . This metadata significantly improves the FAIRness level, especially for the "I" and "R" principles (see Fig. 4 and5). However, the "F" and "A" principles require the generation of persistent and globally unique identifiers, and the publication of the generated metadata on the web.

The goal of our work is to describe existing datasets with semantic metadata, in order to make them FAIR without making changes to the datasets. However, modifications are necessary to allow the reuse of published data. For example, in the station dataset provided in the SYNOP data documentation, only one location (latitude, longitude, and altitude) is provided for each station. However, in the PDF sheets describing the stations (one sheet per station)6 , we find the history of the stations' locations. These sheets are in PDF format and contain a lot of information, which makes it difficult to use them or to combine them with other data. We therefore recommend that Météo-France improves the station dataset -provided in the documentation -by enriching it with the history of locations so that users can have the precise location of each measurement over time. The history of the instruments used during the measurements is also included in the data sheets, which represents relevant metadata. Another recommendation would be to provide an instrument dataset per station and date interval.

  .2-01D Data is accessible through an access protocol that supports authentication and au-Plurality of accurate and relevant attributes are provided to allow reuse Essential 33 RDA-R1.1-01M Metadata includes information about the licence under which the data can be reused Essential 34 RDA-R1.1-02M Metadata refers to a standard reuse licence Important 35 RDA-R1.1-03M Metadata refers to a machine-understandable reuse licence Important 36 RDA-R1.2-01M Metadata includes provenance information according to community-specific standards Important 37 RDA-R1.2-02M Metadata includes provenance information according to a cross-community language Useful 38 RDA-R1.3-01M Metadata complies with a community standard Essential 39 RDA-R1.3-01D Data complies with a community standard Essential 40 RDA-R1.3-02M Metadata is expressed in compliance with a machine-understandable community standard Essential 41 RDA-R1.3-02D Data is expressed in compliance with a machine-understandable community standard Important • Useful: nice to have but is not necessarily indispensable. e.g., "Data uses FAIR-compliant vocabularies"

Figure 1 :

 1 Figure 1: Fail or pass method: FAIRness levels

3. 4

 4 .3 RDA-R1.1-02M . Metadata refers to a standard reuse licence • (Before: 1 ) the data refers to Etalab license which is a standard license • (After: 1 ) No change. metadata using PROV-O properties. PROV-O ontology is a W3C recommendation since April 2013.

Figure 2 :

 2 Figure 2: SYNOP dataset FAIRness levels per area (pre-evaluation i.e., with Before values)

Figure 3 :

 3 Figure 3: SYNOP dataset FAIRness progress per indicator (pre-evaluation)

Table 2 :

 2 FAIR maturity model indicators (1)

FAIRNESS LEVELS PER AREA What level describes your digital object?

		Level 0	Not FAIR
		Level 1	FAIR essential criteria only
		Level 2	FAIR essential criteria + 50 % of important criteria
		Level 3	FAIR essential criteria + 100% of important criteria
		Level 4	FAIR essential criteria + 100% of important criteria + 50% of useful criteria
		Level 5	FAIR essential criteria + 100% of important criteria + 100% of useful criteria
				Level 5
				Level 4
				Level 3
				Level 2
				Level 1
				Level 0
				Value
				FAIRness
	FINDABLE	ACCESSIBLE	INTEROPERABLE	REUSABLE
	Figure 4: SYNOP dataset FAIRness levels per area (post-evaluation i.e., with
	After values)

https://www.etalab.gouv.fr/wp-content/uploads/2014/05/Licence_Ouverte.pdf

Figure 2, and Figure 3 are generated with the RDA maturity level tool.

https://donneespubliques.meteofrance.fr/?fond=contenu&id_contenu=37

FAIRNESS PROGRESS PER INDICATOR

Maturity level per indicator (per FAIR area)

0 -not applicable 1 -not being considered this yet 2 -under consideration or in planning phase 3 -in implementation phase 4 -fully implemented


T Vilarinho-Franco

R Chiriac

R Tenu

J J Counioux

J Delmas

P Capron

C Goutaudier

Polythermal diagrams of aqueous boundary ternary systems involving sodium borohydride hydrolysis for hydrogen generation

Keywords: solid-liquid equilibria, polythermal diagrams, DSC applied to ternary mixtures

The comprehension of solid-liquid equilibria involved in the quaternary system NaBH4-NaBO2-NaOH-H2O allows us to follow the evolution of mixtures during the sodium borohydride hydrolysis reaction for hydrogen generation. The addition of sodium hydroxide on saturated solutions of sodium borohydride and metaborate have been studied in a temperature range of -10 °C up to 50 °C which is the operational temperature range of a hydrogen generator for consumer electronics application. The solubility data was monitored at low temperatures by Differential Scanning Calorimetry, DSC analyses. Given the large number of solid phases that may crystallize in a stable or metastable state, the definition of a specific protocol was required in order to evaluate a ternary mixture via DSC analyses presenting a good repeatability. The nature of different solid phases present in this system was confirmed by powder X-ray analyses. Finally, two polythermal diagram outlines of aqueous boundary ternary systems involving water, sodium borohydride, sodium hydroxide and sodium borate were proposed. The results obtained in the water-rich area show that the sodium hydroxide hydrates are relatively soluble and the risk of precipitation is mainly due to the sodium borohydride dihydrate (NaBH4.2H2O) and sodium metaborate tetrahydrate (NaBO2.4H2O). The crystallization areas were defined as function of temperature and composition.

Introduction

For several years now, the hydrogen generation via hydrolysis of sodium borohydride is considered as having a real technological potential for consumer applications, as presented by Demirci in an overview article with over 280 references about this technology, highlighting the scientific and technical critical points of the hydrogen cycle via sodium borohydride hydrolysis [START_REF] Demirci | The hydrogen cycle with the hydrolysis of sodium borohydride: A statistical approach for highlighting the scientific/technical issues to prioritize in the field[END_REF].

NaBH4 has been attracting distinct interest because of its high gravimetric percentage of hydrogen, 10.8 w% of hydrogen released by hydrolysis [START_REF] Laversenne | Hydrogen storage in borohydrides: Comparison of hydrolysis conditions of LiBH4, NaBH4 and KBH4[END_REF]. NaBH4 alkaline solution can be transported safely and this hydrolysis reaction provides hydrogen with a good purity [START_REF] Minkina | Long-term stability of sodium borohydrides for hydrogen Generation[END_REF][START_REF] Çakanyildirim | Hydrogen cycle with sodium borohydride[END_REF][START_REF] Liu | A review: Hydrogen generation from borohydride hydrolysis reaction[END_REF][START_REF] Damjanović | A direct measurement of the heat evolved during the sodium and potassium borohydride catalytic hydrolysis[END_REF]. In aqueous solution, however, the hydrogen generation via sodium borohydride hydrolysis presents a major drawback concerning its by-products, hydrated sodium borate compounds in a saturated solution of sodium metaborate, NaBO2.yH2O, with pseudo hydration degree y = 0, 1/3, 2/3, 2 or 4 [START_REF] Andrieux | Revision of the NaBO2-H2O phase diagram for optimized yield in the H2 generation through NaBH4 hydrolysis[END_REF]. This metaborate hydrate formation significantly modifies the density and viscosity of the solution during hydrolysis reaction.

One of the main challenges to improve the energy density of this hydrogen generator system, is to increase the NaBH4 concentration in aqueous solution, thus avoiding the drawback induced by metaborate saturated solution and consequently metaborate hydrate precipitation in the operation temperature range of the system i.e. around ambient conditions.

Sodium borohydride decomposes spontaneously in water to produce hydrogen and by-products: hydrated sodium borate compounds. Sodium hydroxide is added to the solution to limit this self-decomposition, thus stabilizing the system [START_REF] Andrieux | Spontaneous hydrolysis of sodium borohydride in harsh conditions[END_REF], but the aqueous solution of sodium borohydride can be stabilized by making it alkaline through the addition of hydroxide ions, generally sodium hydroxide (NaOH) [START_REF] Liu | A review: Hydrogen generation from borohydride hydrolysis reaction[END_REF][START_REF] Suda | Catalytic generation of hydrogen by applying fluorinated-metal hydrides as catalysts[END_REF][START_REF] Mikheeva | Temperature variation of solubility in the NaBH4-H2O system and solubility isotherms at 0°, 18°, 30° and 50° C for the NaBH4-NaOH-H2O system[END_REF][START_REF] Jensen | A study on sodium borohydride with special reference to its analytical application in organic chemistry[END_REF]. In these conditions the aqueous solutions of stabilized sodium borohydride are particularly well adapted to on-board applications, because they provide a high volumetric storage capacity.

The optimization of key operational parameters of this portable hydrogen generator is directly linked to the hydrolysis reaction yield as a function of the initial concentration of sodium borohydride, operational temperature as well as a function of by-product hydrates and final solution solubility [START_REF] Vilarinho-Franco | Lifetime analysis of a hydrogen generator by hydrolysis of sodium borohydride[END_REF]. Therefore, the delimitation of the homogeneous liquid phase domain in the phase diagram quaternary system NaBH4-NaBO2-NaOH-H2O is fundamental.

In order to understand the behavior of the aqueous solution of sodium borohydride hydrolysis, the main challenge is to describe the solid-liquid equilibria involved in the quaternary system NaBH4-NaBO2-NaOH-H2O, which represents the evolution of the mixture during the hydrolysis reaction. In a previous work, the solubility limit of the hydrolysis's by-products, NaBO2.yH2O, has been investigated and the experimental results for the solubility limit in the ternary system NaBO2-NaOH-H2O under ambient pressure at 10, 25 and 50 °C have been presented [START_REF] Vilarinho-Franco | Solid-Liquid Equilibria in the ternary system NaBO2-NaOH-H2O. Fluid Phase Equilibria[END_REF]. In this present work, polythermal diagrams of the two aqueous boundary ternaries NaBH4-NaOH-H2O (corresponding to 0 % of hydrolysis reaction conversion) and NaBO2-NaOH-H2O (100 % of conversion) will be proposed from critical analysis of the description of several isotherm sections of each system [START_REF] Vilarinho-Franco | Solid-Liquid Equilibria in the ternary system NaBO2-NaOH-H2O. Fluid Phase Equilibria[END_REF][START_REF] Churikov | Phase Diagrams of the Ternary Systems NaBH4-NaOH-H2O, KBH4-KOH-H2O, NaBO2-NaOH-H2O and KBO2-KOH-H2O at -10[END_REF][START_REF] Churikov | Temperature-Induced Transformation of the Phase Diagrams of Ternary Systems NaBO2-NaOH-H2O and KBO2-KOH-H2O[END_REF][START_REF] Mikheeva | Temperature variation of solubility in the NaBH4-H2O system and solubility isotherms at 0°, 18°, 30° and 50° C for the NaBH4-NaOH-H2O system[END_REF] and will be completed by DSC analysis with the intent to give more precision about invariant transformations of the system. The third aqueous boundary ternary system NaBH4-NaBO2-H2O cannot be experimentally evaluated because of the spontaneous decomposition of NaBH4 in water, i.e. without hydroxide as a stabilizer. The quaternary mixture NaBH4-NaBO2-NaOH-H2O corresponds to the hydrolysis solution itself, and will be published subsequently as a subject of a thorough experimental work.

In favor of a good comprehension of the complexity of these two aqueous boundary ternary systems, NaBH4-NaOH-H2O and NaBO2-NaOH-H2O, three well-known aqueous boundary binary systems, NaBO2-H2O [START_REF] Andrieux | Revision of the NaBO2-H2O phase diagram for optimized yield in the H2 generation through NaBH4 hydrolysis[END_REF][START_REF] Nies | Solubility isotherms in the system sodium oxide-boric oxide-water. Revised solubility-temperature curves of boric acid, borax, sodium pentaborate, and sodium metaborate[END_REF][START_REF] Tolédano | presented by Champetier G. Sur un appareil d'analyse thermique différentielle sous pression. Etude du système métaborate de sodium-eau[END_REF][17]; NaBH4-H2O [START_REF] Mikheeva | Temperature variation of solubility in the NaBH4-H2O system and solubility isotherms at 0°, 18°, 30° and 50° C for the NaBH4-NaOH-H2O system[END_REF][START_REF] Jensen | A study on sodium borohydride with special reference to its analytical application in organic chemistry[END_REF] and NaOH-H2O [START_REF] Rollet | Les systèmes eau-hydroxyde alcalin Revue Chimie Minérale[END_REF] have been also taken into account. Note that the binary system NaBH4-H2O needs to be stabilized (NaOH as stabilize agent) in order to avoid Sodium borohydride spontaneous decomposition in water (see Table 1).

The Tables 1, 2 and 3 list the invariant transformations of each aqueous binary system. The three-phase equilibria are depicted on cooling direction, by convention.

Table 1. Invariant transformation in the binary system NaBH4-H2O (stabilize agent NaOH 0.1 mol.L -1 ) [START_REF] Mikheeva | Temperature variation of solubility in the NaBH4-H2O system and solubility isotherms at 0°, 18°, 30° and 50° C for the NaBH4-NaOH-H2O system[END_REF][START_REF] Jensen | A study on sodium borohydride with special reference to its analytical application in organic chemistry[END_REF] at ambient pressure. After the review of the ternary systems, differential scanning calorimetry (DSC) analyses were carried out in ternary aqueous mixtures in order to complete data and more particularly the temperature of some feature invariant transformations. Differential scanning calorimetry (DSC) is a commonly technique used to study and characterize thermal phenomena in complex media. As an example, Reyes-Labarta et al. applied DSC analyses to evaluate transitions and reactions undergone by polymers, such as melting, crystallization, and reordering in their structure [START_REF] Reyes-Labarta | DSC study of transitions involved in thermal treatment of foamable mixtures of PE and EVA copolymer with azodicarbonamide[END_REF]. Although, it is important to consider the complexity of DSC analyses concerning ternary mixtures, the transitions phenomena are dependent of the temperature scanning protocol and the proper calibration of DSC in order to attempt accurate results and a good repeatability. Powder X-ray diffraction analyses were performed for the structural identification of the solid phases present in some crystallization domains.

T [°C] w % NaBH

Material and Methods

Ternary mixture preparation

For the ternary mixture preparation, anhydrous NaOH (Sigma-Aldrich®, ≥ 99.99 % purity, pellets, n° CAS: 1310-73-2) is stored and handled in an argon-filled glove box in order to prevent hydration. Deionized water is introduced at room temperature. Commercial sodium borate, NaBO2.yH2O (ACROS Organics, 98.5 % purity, n° CAS: 10555-76-7) is submitted to heat treatment in order to obtain the anhydrous NaBO2. In this latter case, commercial borate (mixture of NaBO2.4H2O and NaBO2.2H2O) is heated at 300 °C for 72 hours in a tubular oven under argon flow. Afterwards, anhydrous borate is confirmed by powder X-ray diffraction analysis.

Differential scanning calorimetry (DSC) Analysis

Thermal analyses by DSC were performed to determine the ternary invariant transformations at low temperatures.

The tests were performed on a differential scanning calorimeter DSC820 (Mettler-Toledo), under nitrogen with a flow of 30 mL.min -1 . The calibration of the DSC equipment in terms of heat flow and temperature and tau lag was done by using the theoretical values of enthalpies and melting temperatures of two metal standards: mercury (Hg) and indium (In), for which the precise values of enthalpies and melting temperatures are well known (see Table 4).

After the experimental determination of DSC at two increasing temperature rate 0.5 K.min -1 and 5.0 K.min -1 , the errors do not exceed 0.1 % and 1.0 % for temperature and enthalpy values, respectively. The difference between the temperature of set point and the actual temperature measured on the sample was 0.02 K maximum. The software used to run the acquisition and to exploit the results was 9.20 STAR (Mettler-Toledo). DSC analyses applied to ternary mixtures exhibit a certain complexity particularly when the mixtures are viscous. It is the case in the NaBO2-NaOH-H2O system. In order to avoid the phenomenon of major segregation between the solid phases able to crystallize, the rapid cooling leads to the quenching of the solution. The definition of a protocol for temperature scanning in order to have repeatability demands much attention. To support this statement figure 1 reveals a thermogram with two consecutive runs and exemplifies the lack on repeatability concerning the phase transformation phenomena in the ternary mixture NaBO2-NaOH-H2O, exemplifying a non-reproducible thermogram and illustrating the difficulty encountered during the DSC measurements of the studied mixtures which are very viscous. It is clear that the thermograms on Fig. 1 are not usable. This is why many trials have been necessary to achieve a protocol leading to reproducible results. The selected protocol is presented in Figure 2 and has been successfully to determine the invariant phenomena presented in this work. ) to obtain a stable crystallization and a very slow heating rate (1 K.min -1 ) for endothermic events was finally defined after many tests at different cooling and heating rates. Indeed the determination of temperature of the equilibria is highly dependent of the kinetics. Three consecutive run analyses were then performed using this defined protocol in order to verify results repeatability.

Powder X-ray diffraction (PXRD)

In order to determine the crystallization domain in the polythermal diagrams, PXRD analyses were performed on the Bruker D8 Advance diffractometer, in air and at room temperature.

This diffractometer has a Bragg-Brentano configuration, used in reflection mode, with a θ-2θ installation and a copper anticathode tube (λ Kα1 = 1.5406 Å -λ Kα2 = 1.54439 Å). The sample is prepared by removing some solid phase from the diphasic solution. The solid mixture sample is then dried with a filter paper at ambient temperature and atmospheric pressure. Then the sample is placed on a specific holder. A 50 m Kapton® is used to prevent hydration and CO2 capture of the solid during sample handling and analysis. It is important to note that the solid phase is impregnated with saturated solution, and thus the spectrum background can be significant. To obtain an exploitable spectrum, the PXRD experiment time is about 2h for each experimental solid mixture in order to minimize the spectrum background.

Discussions on polythermal diagram of the ternary system NaBH4-NaOH-H2O | Literature data critical analyses

In a first step, a critical analysis of solubility data as a function of temperature from the literature was performed with the intent to give a qualitative representation of the polythermal diagrams for the NaBH4-NaOH-H2O and NaBO2-NaOH-H2O systems.

In a second step, a series of experimental investigations by XRD and DSC analysis was carried out in order to confirm the qualitative representation, to validate the nature of the crystallized solid phases present in different domains and to specify the characteristics of the invariant transformations.

The representation of a polythermal diagram consists of projecting the isothermal invariant points in the composition plane. One line connecting these points fixes the limits of two crystallization fields. The variance of these lines, which are only function of the temperature, is equal to 1. The intersection of two monovariant lines constitutes an invariant equilibrium point. The composition of the corresponding liquid phase, in equilibrium with three solid phases is independent of temperature at a given pressure.

Figure 3 shows the superposition of isothermal sections of the ternary system NaBH4-H2O-NaOH at -10 °C determined by Churikov et al. [START_REF] Churikov | Phase Diagrams of the Ternary Systems NaBH4-NaOH-H2O, KBH4-KOH-H2O, NaBO2-NaOH-H2O and KBO2-KOH-H2O at -10[END_REF] and at 0, 18 and 30 °C determined by Mikheeva and Breitsis [START_REF] Mikheeva | Temperature variation of solubility in the NaBH4-H2O system and solubility isotherms at 0°, 18°, 30° and 50° C for the NaBH4-NaOH-H2O system[END_REF] also illustrated by Shang and Chen [START_REF] Shang | Hydrogen Storage via the Hydrolysis of NaBH4 Basic Solution: Optimization of NaBH4 Concentration[END_REF]. The graphic on Figure 3 highlights the solid phase domains of different ternary invariant points of each isotherm. It also presents the temperature of the two invariant transformation of the binary system NaBH4-H2O (eutectic at -37.5 °C and a peritectic at 36.4 °C) and the temperature of the lower stable eutectic of the binary system NaOH-H2O (eutectic at -28.7 °C), as mentioned before in Tables 1 and2. [12] and Mikheeva and Breitsis [START_REF] Mikheeva | Temperature variation of solubility in the NaBH4-H2O system and solubility isotherms at 0°, 18°, 30° and 50° C for the NaBH4-NaOH-H2O system[END_REF].

Two monovariant lines can be drawn from these results illustrated at Figure 3: a first monovariant line connecting the points (1), ( 3), ( 5) and ( 7) which are invariant points of a three-phases equilibrium: liquid+NaBH4.2H2O+NaBH4; and a second one connecting the invariant points (2), ( 4), ( 6) and ( 8) of a three-phases equilibrium: liquid+NaOH.H2O+NaBH4.

Figure 4 presents the polythermal diagram of the ternary system NaBH4-NaOH-H2O with the two aqueous binary systems located on the edge: NaBH4-H2O and NaOH-H2O. Figure 4 also illustrates the projection of the invariants points (from 1 to 8 illustrated on Figure 3) on the composition plane of the two invariant lines just mentioned previously. Figure 4 depicts an extra invariant point from the isotherm at 50 °C of the ternary system NaBH4-NaOH-H2O determined by Mikheeva and Breitsis [START_REF] Mikheeva | Temperature variation of solubility in the NaBH4-H2O system and solubility isotherms at 0°, 18°, 30° and 50° C for the NaBH4-NaOH-H2O system[END_REF], integrating the second monovariant line. This picture provides a better view of the evolution of the entire system depending on the temperature. Polythermal representation of the ternary system NaBH4-NaOH-H2O, invariants points from isotherms at -10, 0, 18, 30 and 50 °C [START_REF] Churikov | Phase Diagrams of the Ternary Systems NaBH4-NaOH-H2O, KBH4-KOH-H2O, NaBO2-NaOH-H2O and KBO2-KOH-H2O at -10[END_REF][START_REF] Mikheeva | Temperature variation of solubility in the NaBH4-H2O system and solubility isotherms at 0°, 18°, 30° and 50° C for the NaBH4-NaOH-H2O system[END_REF] with the aqueous boundary binary systems on the edge: NaOH-H2O [START_REF] Rollet | Les systèmes eau-hydroxyde alcalin Revue Chimie Minérale[END_REF] and NaBH4-H2O [START_REF] Mikheeva | Temperature variation of solubility in the NaBH4-H2O system and solubility isotherms at 0°, 18°, 30° and 50° C for the NaBH4-NaOH-H2O system[END_REF][START_REF] Jensen | A study on sodium borohydride with special reference to its analytical application in organic chemistry[END_REF].

The intersection resulting from the extrapolation to lower temperatures of the two monovariant lines (based on experimental results from the literature, Figure 4) allows us to estimate the composition of the invariant liquid phase Figure 5a illustrates, firstly, the isobaric binary invariant transformations (represented by ■ on the axis) from which start the ternary monovariant lines and secondly, the intersections of these curves which result in eight ternary invariant transformations: 0, 1, 1, eqb2,  3, 4 and 2 all reported in Table 5.

The intersection of the two monovariant lines based on experimental results from the literature, extrapolated to lower temperatures, defines the invariant liquid phase 0 in equilibrium with 3 solid phases: NaBH4, NaBH4.2H2O and NaOH.H2O (see Figure 5a). The associate transformation is a transitory peritectic type, i.e., each composition point is composed by three solid phases and a liquid phase, each point is located in a convex quadrilateral. Figure 5b schematizes the four-phase equilibrium corresponding to the peritectic transformation between the liquid 0 and the solid NaBH4, NaBH4.2H2O and NaOH.H2O. Under constant pressure condition, the variance is zero, so the temperature is fixed and the convex quadrilateral is located in the plane of the compositions. An interesting observation is the isoplethic section NaBH4.2H2O-NaOH.7/2H2O which is indicated by a dashed blue line that intersects the monovariant line pi-0 to point Z, at TZ temperature. This isoplethic section explains why below TZ temperature, the isoplethic section provides a quasi-binary behavior, i.e., it behaves like a binary system, more precisely; all tie-lines are in the plane of this section. As a conclusion, the schematic polythermal diagram of the ternary system NaBH4-NaOH-H2O presented on Figure 5a is speculative, and the position of certain invariant points could be slightly different in the quantitative diagram. Accurate determination of the latter implies the knowledge of the compositions and temperatures of isobaric invariant transformations whose study remains to be done. The crystallization domains of ice, NaBH4.2H2O and anhydrous NaBH4 are much broader than those of NaOH with higher hydrates. The precipitation domain of NaOH.2H2O is not well defined because of the inaccuracy of the position of the line dividing the crystallization domains of NaOH.2H2O and anhydrous NaOH. The comparison of the experimental pattern with the ICDD file n° 00-036-1233 confirms the solid phase domain of NaBH4.2H2O. Effectively, X-ray diffractograms show a large continuous background and low intensity diffraction peaks. This is related to the fact that they are recorded on wet solids. In order to maintain the equilibrium state of the solid phase, some stock solution must be retained. However, there is no ambiguity in the identification of the solid phase in comparison with the ICDD files.

NaBO2-NaOH-H2O polythermal diagram proposition (based on experimental data from this author's previous work)

After the experimental work on isothermal of the ternary system NaBO2-NaOH-H2O published previously [START_REF] Vilarinho-Franco | Solid-Liquid Equilibria in the ternary system NaBO2-NaOH-H2O. Fluid Phase Equilibria[END_REF] an outline of polythermal diagram is proposed for this ternary system in Figure 7. The proposed monovariant lines are based on experimental results from the literature. It shows the invariant points from isotherm at -10 °C determined by Churikov et al. [START_REF] Churikov | Phase Diagrams of the Ternary Systems NaBH4-NaOH-H2O, KBH4-KOH-H2O, NaBO2-NaOH-H2O and KBO2-KOH-H2O at -10[END_REF] and from isothermal at 10, 25 and 50 °C determined in previous work [START_REF] Vilarinho-Franco | Solid-Liquid Equilibria in the ternary system NaBO2-NaOH-H2O. Fluid Phase Equilibria[END_REF]. This polythermal diagram consists of eight invariant or monovariant lines fairly well defined (solid lines) and three hypothetical lines drawn in dot lines. The monovariant lines delimitated the crystallization domains of ice, metaborate tetrahydrate NaBO2.4H2O, metaborate dihydrate NaBO2.2H2O, and superior hydrates of sodium hydroxide NaOH.7H2O, NaOH.5H2O and NaOH.4H2O.

The compound Na2BO2(OH) (ICDD reference 00-037-0114) mentioned in Figure 7 can be written in the form of a double compound NaOH.NaBO2 or in more general system of oxides as 2Na2O.B2O3.H2O. Compound Na2BO2(OH) equilibrium with a liquid phase had never been observed before and the determination of its solubility is unprecedented. The structure of the compound Na2BO2(OH) was defined by Menchetti and Sabelli in 1982 [START_REF] Menchetti | Structure of hydrated sodium borate Na2[BO2(OH)][END_REF].

Structural analysis of the double compound leading to the formula Na3[(B(OH)4)2OH] was performed by the author in a previous publication [START_REF] Vilarinho-Franco | Solid-Liquid Equilibria in the ternary system NaBO2-NaOH-H2O. Fluid Phase Equilibria[END_REF].

These monovariant lines intersect each other to give five isobaric invariant transformations. Figure 7 illustrates four of these isobaric invariant transformations 1, 2,  3, 1, reported in Table 6. All the solid phases listed in the literature regarding the ternary system NaBO2-NaOH-H2O are represented in Figure 7, although some are not involved in the equilibria studied. Considering the path followed by the sample (mixture M) during a slow cooling that serves the mixture to reach equilibrium, it is possible to observe two partially overlapping peaks between -13 and -22 °C corresponding to two neighboring transitional transformations Ʈ2 and Ʈ3. Both transformations are close enough to the intersection point of the corresponding line NaBO2.4H2O-M with the monovariant lineƮ2Ʈ3. Due to its location on the ternary diagram, the mixture M contains a large quantity of liquid phase with respect to solid phase, which explains the large amount of heat from this transformation; see Figure 10 (a). Their specific onset temperature determined by the intersection of the downward straight portion of peaks with the baseline, are respectively equal to -17.7 ± 0.2 °C for the ternary invariant Ʈ2 and -20.4 ± 0 2 °C for the invariant Ʈ3.

Figure 10 (b) shows the first endothermic peak, much less intense than the previous. It is due to the disappearance of solid phase ice at the isobaric ternary eutectic transformation Ɛ1 which explains the lower peak intensity caused by the reduction of the amount of material in this transformation. The ternary eutectic onset temperature of this was estimated to be -35.2 ± 0.2 °C. All results concerning the temperatures determinate by DSC analyses are summarized in Table 6.

The analyses of the evolution of monovariant lines led to propose four isobaric ternary invariants better defined, i.e. a ternary eutectic Ɛ1 and three transitional transformations Ʈ1; Ʈ 2; Ʈ3 and Ʈ4. The corresponding transition reactions are given in Table 6. Table 7 presents the equilibrium of invariant points L1 to L6 from previous experimental results of isothermal at 10, 25 and 50°C illustrated at polythermal diagram on Figure 7.

Conclusion

The phase equilibria study of aqueous boundary ternary system NaBH4-NaOH-H2O and NaBO2-NaOH-H2O was undertaken. A polythermal diagram of each ternary system was proposed according to experimental solubility data of these systems and their subsystems. Ternary system solubility data consider previous work from the author [START_REF] Vilarinho-Franco | Solid-Liquid Equilibria in the ternary system NaBO2-NaOH-H2O. Fluid Phase Equilibria[END_REF] and other authors [START_REF] Churikov | Phase Diagrams of the Ternary Systems NaBH4-NaOH-H2O, KBH4-KOH-H2O, NaBO2-NaOH-H2O and KBO2-KOH-H2O at -10[END_REF][START_REF] Churikov | Temperature-Induced Transformation of the Phase Diagrams of Ternary Systems NaBO2-NaOH-H2O and KBO2-KOH-H2O[END_REF][START_REF] Mikheeva | Temperature variation of solubility in the NaBH4-H2O system and solubility isotherms at 0°, 18°, 30° and 50° C for the NaBH4-NaOH-H2O system[END_REF].

A critical literature data review and analysis allowed the proposition of two polythermal diagram outlines including the definition of monovariant lines, several invariants transformations and, consequently, the definition of different crystallization domains from low temperatures up to 50 °C. Furthermore, powder XRD analyses have confirmed the nature of the solid phases present in different crystallization domains. At the end of this study, it was possible to clearly established that the risk of precipitation in these two ternary systems are primarily due to sodium metaborate hydrates and sodium borohydride hydrate, whose crystallization domains are fairly well-defined in the polythermal diagrams.

In the area of low concentration sodium borohydride or sodium metaborate solutions, all hydrates of the sodium hydroxide may be observed in a stable state below 50 °C. In this temperature range, the sodium borohydride and its hydrate, NaBH4.2H2O, present a stable equilibrium with the liquid phase.

The proposed polythermal diagram outline regarding the ternary system involving the hydrolysis by-product final solution, NaBO2-NaOH-H2O, was completed by DSC analyses in order to characterize, qualitatively, the nature of some invariant transformations and to specify their temperatures. The superior hydrates of sodium metaborate, NaBO2.4H2O and NaBO2.2H2O can easily precipitate in a stable state, although the inferior hydrates, NaBO2.2/3H2O and NaBO2.1/3H2O present metastable equilibria, which explains the necessity to establish an appropriate heat treatment during DSC analyses in order to obtain repeatable results. Moreover, the presence of double compounds presenting stable and metastable behavior, as Na2BO2(OH) or Na3[(B(OH)4)2OH] -discovery in a previous work, also emphasize the importance of a specific temperature scanning protocol for the application of DSC analysis to evaluate these ternary mixtures.

Figure 1 .

 1 Figure 1. DSC thermogram of an aqueous ternary mixture M (24.8 w% NaOH -5.16 w % NaBO2-70.04w% H2O) first results underscoring no repeatability on the phase transformation phenomena.

Figure 2

 2 Figure2illustrates the protocol of the temperature scanning profile of the DSC analyses. A sample of the aqueous ternary mixture M (39.03 mg) was introduced in a hermetic 120 L stainless steel pan and then placed into the DSC cell. A combination of a rapid cooling rate (10 K.min -1 ) to obtain a stable crystallization and a very slow heating rate (1 K.min -1 ) for endothermic events was finally defined after many tests at different cooling and heating rates. Indeed the determination of temperature of the equilibria is highly dependent of the kinetics. Three consecutive run analyses were then performed using this defined protocol in order to verify results repeatability.

Figure 2 .

 2 Figure 2. Temperature scanning profile adopted for DSC analyses.

Figure 3 .

 3 Figure3. Superposition of the isotherms at -10, 0, 18 and 30 °C of the ternary system NaBH4-H2O-NaOH. Literature data from Churikov et al.[START_REF] Churikov | Phase Diagrams of the Ternary Systems NaBH4-NaOH-H2O, KBH4-KOH-H2O, NaBO2-NaOH-H2O and KBO2-KOH-H2O at -10[END_REF] and Mikheeva and Breitsis[START_REF] Mikheeva | Temperature variation of solubility in the NaBH4-H2O system and solubility isotherms at 0°, 18°, 30° and 50° C for the NaBH4-NaOH-H2O system[END_REF].

Figure 4 .

 4 Figure 4. Polythermal representation of the ternary system NaBH4-NaOH-H2O, invariants points from isotherms at -10, 0, 18, 30 and 50 °C[START_REF] Churikov | Phase Diagrams of the Ternary Systems NaBH4-NaOH-H2O, KBH4-KOH-H2O, NaBO2-NaOH-H2O and KBO2-KOH-H2O at -10[END_REF][START_REF] Mikheeva | Temperature variation of solubility in the NaBH4-H2O system and solubility isotherms at 0°, 18°, 30° and 50° C for the NaBH4-NaOH-H2O system[END_REF] with the aqueous boundary binary systems on the edge: NaOH-H2O[START_REF] Rollet | Les systèmes eau-hydroxyde alcalin Revue Chimie Minérale[END_REF] and NaBH4-H2O[START_REF] Mikheeva | Temperature variation of solubility in the NaBH4-H2O system and solubility isotherms at 0°, 18°, 30° and 50° C for the NaBH4-NaOH-H2O system[END_REF][START_REF] Jensen | A study on sodium borohydride with special reference to its analytical application in organic chemistry[END_REF].

0 as: 9 .

 9 0 w% NaBH4, 40.0 w% NaOH and 51.0 w% NaOH. All other ternary invariants like 1, 1, eqb2,  3, 4 and 2 presented on the polythermal diagram outline are an attempt towards a representation based on thermodynamics and phase diagrams theory, respecting the rules of polythermal diagrams construction.

Figure 5a .

 5a Figure 5a. Polythermal diagram of the ternary system NaBH4-NaOH-H2O with isobaric binary invariant transformations (■), different hydrates (▲). Definition of different domains of crystallization with the isobaric invariants points. Proposition of monovariant lines based on literaturedata: isotherm at -10 °C by Churikov et al.[START_REF] Churikov | Phase Diagrams of the Ternary Systems NaBH4-NaOH-H2O, KBH4-KOH-H2O, NaBO2-NaOH-H2O and KBO2-KOH-H2O at -10[END_REF] and isotherms at 0, 18, 30 and 50 °C by Mikheeva and Breitsis[START_REF] Mikheeva | Temperature variation of solubility in the NaBH4-H2O system and solubility isotherms at 0°, 18°, 30° and 50° C for the NaBH4-NaOH-H2O system[END_REF].

Figure 5b .

 5b Figure 5b. Representation of the convex quadrilateral involving Ʈ 0, transitory peritectic transformation.

Figure 6

 6 Figure 6 present the experimental results of PXRD of a mixture located on the crystallization domain of the sodium borohydride dihydrate, NaBH4.2H2O (mixture point composition: 25 w% NaBH4; 10 w% NaOH and 65 w% H2O).The comparison of the experimental pattern with the ICDD file n° 00-036-1233 confirms the solid phase domain of NaBH4.2H2O. Effectively, X-ray diffractograms show a large continuous background and low intensity diffraction peaks. This is related to the fact that they are recorded on wet solids. In order to maintain the equilibrium state of the solid phase, some stock solution must be retained. However, there is no ambiguity in the identification of the solid phase in comparison with the ICDD files.

Figure 6 .

 6 Figure 6. Powder X-Ray pattern of solid phase of the crystallization domain of borohydride superior hydrate: NaBH4.2H2O, ICDD file n° 00-036-1233 represented in red (mixture point composition: 25 w% NaBH4; 10 w% NaOH and 65 w% H2O).

Figure 8

 8 Figure 8 presents the experimental PXRD patterns of a mixture located on the crystallization domain of the sodium metaborate tetrahydrate, NaBO2.4H2O (mixture point composition: 15 w% NaBH4; 10 w% NaOH and 75 w% H2O). The comparison of the experimental pattern with the ICDD file n° 04-011-5978 confirms the solid phase of NaBO2.4H2O.

Figure 7 .

 7 Figure 7. Polythermal diagram of the ternary system NaBO2-NaOH-H2O with binary invariant temperatures (■), different hydrates (▲). Definition of different domains of crystallization with the isobaric invariants points. Proposition of monovariant lines: isotherm at -10 °C by Churikov et al.[START_REF] Churikov | Phase Diagrams of the Ternary Systems NaBH4-NaOH-H2O, KBH4-KOH-H2O, NaBO2-NaOH-H2O and KBO2-KOH-H2O at -10[END_REF] and isothermal at 10, 25 and 50 °C from previous experimental work[START_REF] Vilarinho-Franco | Solid-Liquid Equilibria in the ternary system NaBO2-NaOH-H2O. Fluid Phase Equilibria[END_REF].

Figure 8 .

 8 Figure 8. Powder X-Ray pattern of solid phase of the crystallization domain of metaborate superior hydrate: NaBO2.4H2O, ICDD file n° 04-011-5978 represented in red (mixture point composition: 15 w% NaBH4; 10 w% NaOH and 75 w% H2O).

4. 3 .

 3 Temperature determination of ternary invariant points of the system NaBO2-NaOH-H2O. DSC analyses In order to accurately determine the temperature of the ternary invariants, a suitably chosen mixture sample M (24.8 w% NaOH and 5.16 w% NaBO2) located in the triangle of solid-phases NaBO2.4H2O-NaOH.7H2O-NaOH.5H2O was prepared and then analyzed by DSC.

Figure 9

 9 Figure9illustrates a close-up of the polythermal diagram already presented in the previous Figure7. This close-up shows the position of the mixture point M (purple) and the path followed by the liquid phase during cooling (in orange). The path starts from point M and continues toward the monovariant line Ʈ1Ʈ2 (on the tie-line NaBO2.4H2O-M), then follows the invariant lines Ʈ1 Ʈ2; Ʈ2 Ʈ3 and Ʈ3 until the ternary eutectic Ɛ1 where the liquid phase disappears.

Figure 9 .

 9 Figure 9. Close-up of polythermal diagram of the ternary system NaBO2-NaOH-H2O presented in Figure 8, mixture point M (24.8 w% NaOH and 5.16 w% NaBO2) sample located on solid-phase domain of NaBO2.4H2O and in the triangle of solid-phases NaBO2.4H2O-NaOH.7H2O-NaOH.5H2O.

Figure 10

 10 Figure10depicts the superposition of three consecutive DSC runs for the point mixture M. These curves are perfectly superimposable, presenting a very significant exothermic peak corresponding to the crystallization of species with temperature around -65 °C (onset) during the first heating. This peak is no longer manifested in the second heating attesting complete crystallization of the sample M. Three other endothermic peaks (surrounded by dotted line) appear during heating at temperatures remarkably reproducible. These three endothermic peaks were enlarged in Figure10(a) and (b). Temperature scanning protocol adopted for DSC analysis was presented in Figure2.

Figure 10 .

 10 Figure 10. DSC thermograms presenting three consecutive run analyses for mixture sample M, the temperature range: -80 °C to 25 °C. (a) zoom on the two last overlapped endothermic peaks; (b) zoom on the first endothermic peak.

  [START_REF] Çakanyildirim | Hydrogen cycle with sodium borohydride[END_REF]

Table 2 .

 2 Invariant transformation in the binary system NaOH-H2O (stable or metastable)[START_REF] Rollet | Les systèmes eau-hydroxyde alcalin Revue Chimie Minérale[END_REF] at ambient pressure.

	T [°C] w% NaOH	Transformation	Solid phases in equilibrium
	-28.7	18.6	stable eutetic (e1)	Ice + NaOH.7H 2 O
	-24.0	22.3	stable peritectic (p1)	NaOH.7H 2 O + NaOH.5H 2 O
	-18.0	24.7	stable peritectic (p2)	NaOH.5H 2 O + α-NaOH.4H 2 O
	5.1	32.4	stable peritectic (p3)	-NaOH.4H 2 O + 2NaOH.7H 2 O
	-1.3	35.7	metastable fusion	-NaOH.4H 2 O
	15.9	38.8	congruent fusion (fc1)	2NaOH.7H 2 O
	-2.7	37.2	metastable eutectic	β-NaOH.4H 2 O + NaOH.3H 2 O
	2,6	42.5	metastable fusion	NaOH.3H 2 O
	46.8	6.2	stable eutectic (e2)	2NaOH.7H 2 O + NaOH.2H 2 O
	3.0	48.5	metastable eutectic	2NaOH.7H 2 O + NaOH.H 2 O
	12.9	50.5	Stable peritectic (p4)	NaOH.2H 2 O + NaOH.H 2 O
	65.1	68.9	congruent fusion (fc2)	NaOH.H 2 O
	62.0	74.0	stable eutectic (e3)	NaOH.H 2 O + α-NaOH
	297.0	96.8	stable phase transition	α-NaOH + β-NaOH

Table 3 .

 3 Invariant transformation in the binary system NaBO2-H2O[START_REF] Andrieux | Revision of the NaBO2-H2O phase diagram for optimized yield in the H2 generation through NaBH4 hydrolysis[END_REF] at ambient pressure.

	T [°C]	w % NaBO 2 Transformation	Equilibrium
	-7.0 ± 2.0	12.3	eutectic (ei)	Liquid ⇋ Ice + NaBO 2 .4H 2 O
	55.0 ± 2.0	37.2	peritectic (pi)	Liquid + NaBO 2 .2H 2 O ⇋ NaBO 2 .4H 2 O
	103.5 ± 0.2	52.6	peritectic (pii)	Liquid + 3NaBO 2 .2H 2 O ⇋ NaBO 2 .2H 2 O
	131.6 ± 0.2	58.7	peritectic	Vapor + 3NaBO 2 .2H 2 O ⇋ Liquid
	163.2 ± 0.2	84.6	peritectic	Vapor + 3NaBO 2 .H 2 O ⇋ 3NaBO 2 .2H 2 O
	260.0 ± 0.7	91.6	peritectic	Vapor + NaBO 2 ⇋ 3NaBO 2 .H 2 O

Table 4 .

 4 Metal standards temperature and fusion enthalpy.

	Hg	234.35	11.4
	In	429.75	28.45

T fusion [K] ΔH° fusion @ Tfusion [J.g

-1 ]

Table 5 .

 5 Isobaric ternary invariant transformations of the system NaBH4-NaOH-H2O. The four-phase equilibria are represented on cooling direction, by convention.An outline of the complete polythermal diagram of the ternary system NaBH4-NaOH-H2O at low temperature is proposed in the composition plane on Figure5a. The proposed polythermal diagram is built based on few experimental results from the literature and on thermodynamics rules guiding the monovariant lines. All proposed monovariant lines define different crystallization domains and solid phases likely to occur in the ternary system: ice; NaOH.7H2O; NaOH.5H2O; NaOH.4H2O; NaOH.7/2H2O; NaOH.2H2O; NaOH.H2O; NaBH4.2H2O and NaBH4. Arrows at the monovariant lines indicate the direction of lower temperatures.

	Point	Transformation	Equilibrium
	Ʈ 0 Liquid Ʈ Ʈ 1 Transitory peritectic Transitory peritectic Liquid Ʈ 1 + NaOH.H 2 O ⇋ NaBH 4 .2H 2 O + NaOH.2H 2 O
	Ɛ1	Eutectic	Liquid Ԑ 1 ⇋ NaOH.2H 2 O + 2NaOH.7H 2 O +NaBH 4 .2H 2 O
	Ʈ 2	Transitory peritectic	Liquid Ʈ 2 + 2NaOH.7H 2 O ⇋ NaBH 4 .2H 2 O + NaOH.4H 2 O
	Ʈ 3	Transitory peritectic	Liquid Ʈ3 + NaOH.4H 2 O ⇋ NaBH 4 .2H 2 O + NaOH.5H 2 O
	Ʈ 4	Transitory peritectic	Liquid Ʈ4 + NaOH.5H 2 O ⇋ NaBH 4 .2H 2 O + NaOH.7H 2 O
	Ɛ 2	Eutectic	Liquid Ԑ 2 ⇋ Ice + NaBH 4 .2H 2 O + NaOH.7H 2 O
	Ɛ eqb	Quasi-binary eutectic Liquid Ԑ eqb ⇋ NaBH 4 .2H 2 O + 2NaOH.7H 2 O
	4. Experimental results
	4.1 NaBH4-NaOH-H2O solid phase characterization of solid crystallization domain

0 + NaBH 4 ⇋ NaBH 4 .2H 2 O + NaOH.H 2 O

Table 6 .

 6 Isobaric ternary invariant transformations of the ternary system NaBO2-NaOH-H2O. Experimental results of temperature of three isobaric invariant transformations determined by DSC analyses.

	Point	T [°C]	Transformation	Equilibrium
	Ʈ1	-	transitory peritectic	Liquid Ʈ 1 + NaBO 2 .2H 2 O ⇋ NaBO 2 .4H 2 O + NaOH.4H 2 O
	Ʈ 2	-17.7	transitory peritectic	Liquid Ʈ 2 + NaOH.4H 2 O ⇋ NaBO 2 .4H 2 O + NaOH.5H 2 O
	Ʈ 3	-20.4	transitory peritectic	Liquid Ʈ 3 + NaOH.5H 2 O ⇋ NaBO 2 .4H 2 O + NaOH.7H 2 O
	Ɛ 1	-35.2	eutectic	Liquid Ԑ 1 ⇋ Ice + NaBO 2 .4H 2 O + NaOH.7H 2 O

Table 7 .

 7 Equilibrium of invariant points L1 to L6 from previous experimental results of isothermal at 10, 25 and 50 °C illustrated at polythermal diagram on Figure 7. NaBO 2 .2H 2 O + 3NaBO 2 .H 2 O L5 Liquid ⇋ 3NaBO 2 .H 2 O + Na 2 BO 2 (OH) L6 Liquid ⇋ Na 2 BO 2 (OH) + NaOH.H 2 O

	Isotherm temperature [°C]	Invariants points	Equilibrium
	10	L1	Liquid ⇋ NaBO 2 .2H 2 O + Na3[(B(OH)4)2OH]
		L2	Liquid ⇋ Na3[(B(OH)4)2OH] + NaOH.H 2 O
	25	L3	Liquid ⇋ NaBO 2 .2H 2 O + NaOH.H 2 O
		L4	Liquid ⇋
	50

Acknowledgments

This research was supported by CEA-Liten (Commissariat à l'Energie Atomique et aux Energies Alternatives) and Université de Lyon, Université Claude Bernard Lyon 1, LMI department. We thank our colleagues from both institutions who provided insight and expertise that greatly assisted the research work.