Clément Lambert
email: clement.lambert@univ-ubs.fr

Aurélie Penaud

Clément Poirier

Evelyne Goubert

Distribution of modern dinocysts in surface sediments of southern Brittany (NW France) in relation to environmental parameters: Implications for paleoreconstructions

Keywords: dinoflagellate cysts, marine palynology, taphonomic issues, southern Brittany shelf, paleoenvironmental reconstructions

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

Introduction

Dinoflagellates (currently about 2,377 species known; Gomez, 2012) are eukaryotic unicellulars found in most aquatic environments, mainly in marine waters and in the upper part of the water column, and play an important role in the trophic network [START_REF] Dale | Dinoflagellate cyst ecology: modeling and geological applications[END_REF]. Half of them are heterotrophic and feed on other dinoflagellates, microalgae, diatoms and organic debris in the water column [START_REF] Evitt | Sporopollenin Dinoflagellate Cysts: Their Morphology and Interpretation[END_REF][START_REF] Dale | Dinoflagellate cyst ecology: modeling and geological applications[END_REF]; the other half possesses chloroplasts (Gomez, 2012). As the vegetative growth of heterotrophic dinoflagellates is likely to be enhanced by prey availability (e.g. diatoms, autotrophic dinoflagellates, phytodetritus), they are commonly used as : i) productivity tracers in the marine realm (i.e. upwelling areas, e.g. [START_REF] Radi | Dinocyst distribution in surface sediments from the northeastern Pacific margin (40-60 N) in relation to hydrographic conditions, productivity and upwelling[END_REF][START_REF] Penaud | Palaeohydrological changes over the last 50 ky in the central Gulf of Cadiz: complex forcing mechanisms mixing multiscale processes[END_REF][START_REF] Hardy | Quantification of last glacial-Holocene net primary productivity and upwelling activity in the equatorial eastern Atlantic with a revised modern dinocyst database[END_REF] as well as ii) eutrophication tracers in coastal and estuarine areas (e.g. [START_REF] Dale | Dinoflagellate cysts as indicators of cultural eutrophication in the Oslofjord, Norway[END_REF][START_REF] Matsuoka | Eutrophication process recorded in dinoflagellate cyst assemblages -a case of Yokohama Port, Tokyo Bay, Japan[END_REF][START_REF] Sangiorgi | Reconstructing 150 years of eutrophication in the northwestern Adriatic Sea (Italy) using dinoflagellate cysts, pollen and spores[END_REF][START_REF] Pospelova | Dinoflagellate cysts in recent estuarine sediments from aquaculture sites of southern South Korea[END_REF][START_REF] Price | Effect of nutrient pollution on dinoflagellate cyst assemblages across estuaries of the NW Atlantic[END_REF][START_REF] Price | Dinoflagellate cysts track eutrophication in the Northern Gulf of Mexico[END_REF]Garcìa-Moreiras et al., 2018).

Fossilizable organic-walled dinoflagellate cysts (i.e., resting cysts corresponding to "dormant stages" mainly produced during sexual reproduction; e.g., von [START_REF] Stosch | Observations on vegetative reproduction and sexual life cycles of two freshwater dinoflagellates, Gymnodinium pseudopalustre Schiller and Woloszynskia apiculate sp[END_REF] show their highest concentrations in neritic and coastal areas (i.e. where dinoflagellate blooms can occur; [START_REF] Taylor | The Biology of Dinoflagellates[END_REF]. Organic-walled dinoflagellate cysts (dinocysts) thus represent an important group of microfossils well preserved in sediments. Previous studies carried out on modern marine sediments showed their worldwide distribution as being mainly driven by sea-surface environmental parameters such as temperature (SST for Sea-Surface Temperature), salinity (SSS for Sea-Surface Salinity), sea-ice cover duration in high latitudes, nutrient concentrations and related primary productivity regimes, as well as inshore-offshore gradients, allowing their use as powerful paleoceanographic tracers (e.g., [START_REF] Dodge | The distribution of planktonic dinoflagellates and their cysts in the eastern and northeastern Atlantic Ocean[END_REF]Mudie et al., 2001;[START_REF] Marret | Atlas of modern organic-walled dinoflagellate cyst distribution[END_REF][START_REF] De Vernal | Dinocyst-based reconstructions of sea ice cover concentration during the Holocene in the Arctic Ocean, the northern North Atlantic Ocean and its adjacent seas[END_REF][START_REF] De Vernal | Distribution of common modern dinoflagellate cyst taxa in surface sediments of the Northern Hemisphere in relation to environmental parameters: The new n= 1968 database[END_REF][START_REF] Zonneveld | Atlas of modern dinoflagellate cyst distribution based on 2405 data points[END_REF][START_REF] Marret | From bi-polar to regional distribution of modern dinoflagellate cysts, an overview of their biogeography[END_REF][START_REF] Van Nieuwenhove | An overview and brief description of common marine organic-walled dinoflagellate cyst taxa occurring in surface sediments of the Northern Hemisphere[END_REF]. However, studies on the current dinocyst diversity, concentration and distribution in coastal environments are scarce, especially for the French coasts. Along the Brittany's coasts, first steps towards understanding of the modern distribution of coastal to oceanic dinocyst taxa were initiated by [START_REF] Reid | Dinoflagellate cyst distribution around the British Isles[END_REF], [START_REF] Morzadec-Kerfourn | Les Kystes de dinoflagellés dans les sédiments récents le long des côtes Bretonnes[END_REF], [START_REF] Wall | The environmental and climatic distribution of dinoflagellate cysts in the North and South Atlantic Oceans and adjacent seas[END_REF], [START_REF] Larrazabal | Kystes modernes de dinoflagellés en baie de Vilaine-Bretagne sud (France)[END_REF], recently complemented by [START_REF] Ganne | Present-day palynomorph deposits in an estuarine context: The case of the Loire Estuary[END_REF] for the Loire estuary and [START_REF] Lambert | Modern palynological record in the Bay of Brest (NW France): Signal calibration for palaeo-reconstructions[END_REF] for the Bay of Brest. A recent spatio-temporal (i.e. in space and time) Holocene study discussed the nearshore-offshore dinocyst distribution on both sides of the freshwater front in the northern Bay of Biscay shelf [START_REF] Penaud | Oceanic versus continental influences over the last 7 kyrs from a mid-shelf record in the northern Bay of Biscay (NE Atlantic)[END_REF]. Dinocyst species were then classified in four groups accounting for different hydrological contexts (i.e, estuarine; shallow bay to inner neritic; Iroise Sea or neritic; outer neritic to full oceanic) due to a marine (i.e. distal or offshore) to coastal (i.e. proximal or onshore) transect ranging from 2,174 m to 8 m water depth [START_REF] Penaud | Oceanic versus continental influences over the last 7 kyrs from a mid-shelf record in the northern Bay of Biscay (NE Atlantic)[END_REF].

In this study, we investigated 15 new surface sediment samples collected in the Bay of Quiberon (BQ), which will improve the discussion of the modern spatial dinocyst distribution in a shallow bay environment, especially focusing on rarely addressed taphonomic processes.

Then, these data were compared to top cores retrieved in the southern Brittany shelf [START_REF] Naughton | Long-term and millennial-scale climate variability in northwestern France during the last 8850 years[END_REF][START_REF] Zumaque | Holocene paleoceanography of the Bay of Biscay: evidence for West-East linkages in the North Atlantic based on dinocyst data[END_REF][START_REF] Penaud | Oceanic versus continental influences over the last 7 kyrs from a mid-shelf record in the northern Bay of Biscay (NE Atlantic)[END_REF] and to surface sediments of the Loire estuary mouth [START_REF] Ganne | Present-day palynomorph deposits in an estuarine context: The case of the Loire Estuary[END_REF] in order to improve the understanding of the modern spatial dinocyst distribution along a macro-regional inshore-offshore transect.

Environmental and geographical settings of the Bay of

Quiberon

The 'Mor Bras' (Fig. 1) is a bathymetric depression bordering the southern coast of Brittany.

It is partially isolated from the general oceanic circulation of the Bay of Biscay and from the high energetic Atlantic swells due to a belt of shoals in the extension of the Quiberon peninsula (Fig. 1). The Bay of Quiberon (BQ), which occupies the western part of the Mor Bras, is a shallow coastal embayment (15 m depth in average) that has been submerged during the Holocene [START_REF] Baltzer | Climatically-driven impacts on sedimentation processes in the Bay of Quiberon (south Brittany, France) over the last 10,000 years[END_REF][START_REF] Menier | Les réseaux fluviatiles anciens du plateau continental de Bretagne Sud[END_REF]. A vast network of flooded valleys converges into the Teignouse Strait (TS on Fig. 1) which separates the Quiberon Peninsula from the Houat Island [START_REF] Vanney | Étude sédimentologique du Mor Bras, Bretagne[END_REF][START_REF] Menier | Les réseaux fluviatiles anciens du plateau continental de Bretagne Sud[END_REF]Fig. 1). This strait is burrowed by strong tidal currents joining the inlet of the Gulf of Morbihan and contributing to the partial isolation of the BQ from the rest of the Mor Bras by the establishment of tidal gyres (yellow arrows on Fig. 1; [START_REF] Vanney | Étude sédimentologique du Mor Bras, Bretagne[END_REF][START_REF] Lemoine | Étude sédimentaire de la Baie de Quiberon : la zone ostréicole en eau profonde et ses abords[END_REF][START_REF] Tessier | Caractérisation et dynamique des turbidités en zone côtière : l'exemple de la région marine Bretagne Sud[END_REF].

Recent works carried out in the BQ, following high summer mortality of oysters (Mazurié et al., 2013a, b;[START_REF] Stanisière | Les risques conchylicoles en Baie de Quiberon[END_REF], have shown that the shallower parts of the BQ (between 0 and 6 m deep) are mainly composed of sandy sediments, due to strong wind-forced water column mixing, while the deepest parts of the BQ are covered with muddy sediments. This sediment distribution is associated with a heterogeneous distribution of benthic foraminiferal species, analyzed in the framework of the RISCO project ('Comité régional de la conchyliculture Bretagne Sud ' and Ifremer;[START_REF] Pospelova | Dinoflagellate cysts in recent estuarine sediments from aquaculture sites of southern South Korea[END_REF]-2013;coord. J. Mazurié;Mazurié et al., 2013a, b;[START_REF] Stanisière | Les risques conchylicoles en Baie de Quiberon[END_REF]. Among the high foraminiferal diversity studied, some taxa are characteristic of the deepest and fine grain-sized BQ areas (i.e. Cribroelphidium gerthi, Elphidium earlandi, Gavelinopsis nitida, Lagena sulcata, Lagena semistriata, Lamarckina haliotidea, Planorbulina mediterranensis, Trifarina angulosa, and Textularia truncata). They form the 'deepest foraminiferal assemblage', noted 'DeepForam' hereafter.

Methods

Sampling and environmental parameters

Data collection

The sampling campaign was carried out in 2010 in the framework of the RISCO project in 15 sites within the BQ using a 50 x 50 cm Van Veen grab for the optimal preservation of surface sediments (Mazurié et al., 2013a, b;Stanisère et al., 2013;Fig. 1). The first cm of each site was sampled (Table 1) for grain-size and microfossil analyses. All samples were stored in sealed vials with ethanol.

For grain-size analyses, sediments were passed through a column of sieves with different sizes of apertures (i.e. 2 mm, 500 µm, 125 µm and 45 µm). Averaged percentages of the fine sediment fraction (i.e. <125 µm, referred to as 'granulo<125' hereafter; Table 2) were considered in this study (Mazurié et al., 2013a, b;Stanisère et al., 2013).

In addition, we compiled the monthly measurements of the bottom water physico-chemical parameters measured at each station to obtain averaged values for the year 2010 (Table 2).

Bottom water temperature (referred to as 'Temp' hereafter), bottom water salinity ('Sal'), dissolved oxygen concentration in bottom waters ('O2'), bottom water turbidity ('Turb') and Chlorophyll a concentration in bottom water ('Chla') were measured with an MP6 probe (NKE). In addition, bottom water samples were taken from each station to calculate the suspended matter concentration ('SM') using vacuum pump filtration. Annual averaged values will be considered in this study (Table 2).

Correlation matrix of variables

A correlation matrix of variables was performed with the "Analysis Toolpack" of Microsoft Excel 2016, to assess the relationship between: i) sedimentological ('granulo<125', subsection 3.1.1) and foraminiferal ('DeepForam', section 2) data, ii) dinocyst data ('Cdino',subsection 3.2.1.;and 'Srdino','Hdino',subsection 3.2.2.) and iii) the measured environmental variables ('Depth','Temp','Sal','O2','Chla','Turb','SM',subsection 3.1.1).

The matrix consists of Pearson correlation coefficients (r) between each variable that range from -1 to 1. If r=0, no linear correlation exists between compared variables. The greater the absolute value of r, the stronger the correlation: positive when approaching 1 and negative when approaching -1. Statistical significance is expressed by the p-value. P-value < 0.05 indicates a strong presumption against the null hypothesis and a good confidence in the statistical correlation expressed by the Pearson coefficients.

Dinoflagellate cyst analysis

Laboratory procedure for palynological slides

Dinocyst extraction was performed at the UMR 6538 CNRS (LGO-Laboratoire Géosciences Océan, UBO-University of Brest) from the 10-150 μm sediment fraction. Three cm 3 of fifteen BQ surface sediment samples (samples from December 2010) were analyzed following a standard protocol described by de Vernal et al. (1999) that includes 10% cold HCl and 70% cold HF to remove carbonate and siliceous fractions, respectively. The final residues were mounted between slides and coverslips with glycerol. Dinocyst concentrations ('Cdino';

Table 2), expressed in number of cysts per cm 3 of dry sediments (cysts/cm 3 ) were calculated thanks to the marker grain method [START_REF] Stockmarr | Tabletes with spores used in absolute pollen analysis[END_REF][START_REF] De Vernal | Techniques de préparation et d'analyse en micropaléontologie[END_REF][START_REF] Mertens | Determining the absolute abundance of dinoflagellate cysts in recent marine sediments: the Lycopodium marker-grain method put to the test[END_REF]. This method consists in adding aliquot volumes of Lycopodium spores before the palynological treatment, these exotic spores being counted in parallel with fossilized palynomorphs.

Dinocyst identification, diversity indexes and statistical analysis

For each analyzed sample, except BQ sample n°3 (cf. Fig. 1), a minimum of 150 dinocysts was reached, using an Olympus CH-2 optical microscope (at magnifications ×630 and ×1000), in order to provide robust assemblages from a statistical point of view. The threshold of 100 individuals is indeed required to identify 99% of major (>5%) species [START_REF] Fatela | Confidence limits of species proportions in microfossil assemblages[END_REF] and thus robust to discuss the proportion of the two large groups of dinocysts (i.e. the strict heterotrophic taxa vs. the other dinocysts). The BQ sample n°3, mainly characterized by coarse sediments (i.e. 63% of grain sizes exceed 125µm), must be interpreted with caution since only 13 specimens were counted. Taxa identification followed [START_REF] Rochon | Distribution of recent dinoflagellate cysts in surface sediments from the North Atlantic Ocean and adjacent seas in relation to sea-surface parameters[END_REF], [START_REF] Zonneveld | A determination key for modern dinoflagellate cysts[END_REF] and [START_REF] Van Nieuwenhove | An overview and brief description of common marine organic-walled dinoflagellate cyst taxa occurring in surface sediments of the Northern Hemisphere[END_REF]. Dinocyst percentages were calculated for each taxon (list of identified taxa in Table 3), on a main sum including all taxa and excluding non-identified ones (always less than three undetermined specimens per analyzed level).

We use the Shannon Diversity index to estimate the dinocyst diversity as an additional ecological indicator (noted 'Srdino' hereafter for Species Richness; Table 2). The Shannon index provides information both on specific richness and on the structure of populations. The higher the value, the greater the taxa diversity and the more homogeneous the relative abundances. In addition, we calculated the total percentages of brown cysts from heterotrophic dinoflagellates, or heterotrophic dinocysts ('Hdino'; Table 2). Cluster analysis was also conducted on the 15 samples, using PAST v.1.75b [START_REF] Hammer | PAST: Paleontological statistics software package for education and data analysis[END_REF], according to dinocyst assemblages to better highlight similarities and differences between analyzed samples.

Inshore-offshore transect

In order to discuss dinocyst diversity changes along an inshore-offshore gradient on a largerscale, we also compared dinocyst assemblages from surface sediments and core tops taken in the northern Bay of Biscay, in different environmental and bathymetric contexts: i) two samples taken in the downstream Loire Estuary [START_REF] Ganne | Present-day palynomorph deposits in an estuarine context: The case of the Loire Estuary[END_REF] are representative of the Loire river mouth, ii) the averaged 15 surface samples of this study account for the shallow coastal BQ, iii) a sample from the top core section (level 47 cm) of the CBT-CS11 core (73 m water depth) is representative of the shelf under mixed oceanic and winter fluvial influences [START_REF] Penaud | Oceanic versus continental influences over the last 7 kyrs from a mid-shelf record in the northern Bay of Biscay (NE Atlantic)[END_REF], iv) a sample from top core section (level 4 cm) of the VK03-58bis core (97 m water depth) is representative of the deeper shelf [START_REF] Naughton | Long-term and millennial-scale climate variability in northwestern France during the last 8850 years[END_REF]. Regarding these last two sites, we targeted the two "modern" samples on the basis of palynological evidence unambiguously signaling for their contemporaneity (i.e., presence of pollen grains of Zea mays). The distal MD95-2002 core, taken off the Armorican margin at 2,200 m deep, has finally been added to the transect [START_REF] Zumaque | Holocene paleoceanography of the Bay of Biscay: evidence for West-East linkages in the North Atlantic based on dinocyst data[END_REF]. The top core sample (0 cm) is estimated at ca. 1,000 Cal. years BP and is therefore representative of late Holocene conditions [START_REF] Zumaque | Holocene paleoceanography of the Bay of Biscay: evidence for West-East linkages in the North Atlantic based on dinocyst data[END_REF]. Such a gradient in dinocyst distribution as a function of the distance to the coast therefore provides a baseline reference for inferring dinocyst-derived past environmental changes on the temperate north-eastern Atlantic margin.

Results

Cluster analysis between study samples

All studied stations are represented within the BQ with a colour code corresponding to the result of the cluster analysis (Figs. 2a,b).

Station n°3 stands out from the others with a low similarity coefficient (i.e. around 0.65; Fig. 2a). The coarse grain size (Fig. 2h) and the related very low dinocyst concentration (Figs. 2c and3a) characterizing this station set it apart from the others. With the exception of station n°5, all stations located below the 6 m isobath (n°2, 10, 15, 11, 13, 12, 8, and 14) are grouped together in a same cluster (brown colour, Figs. 2a,b) with a high coefficient of similarity (> 0.825; Fig. 2a), and stations located above the 6 m bathymetric threshold (n°4, 6, and 9) are grouped in a same cluster (green colour, Figs. 2a,b) with the additional sites n°7 and 1. All these shallowest sites are characterized by generally coarser grain-size values (Fig. 2h).

Dinoflagellate cyst results

Dinocyst concentrations of the 15 surface sediment samples range from ca. 300 to 18,300 cysts/cm 3 with a mean of ca. 8,100 cysts/cm 3 (standard deviation of ca. 5,300 cysts/cm 3 ) (Fig. 3a). The lowest values correspond to the shallowest sites located to the north and west of the BQ whereas the highest ones are recorded at the deepest sites, below the 6 m isobath (Figs. 2c and3a).

A total of 26 different dinocyst taxa was identified (12 autotrophic and 14 heterotrophic taxa; cf. Table 3) with an average of 14 different taxa per sample (Fig. 3a). Assemblages (Figs. 3a,b) are largely dominated by Lingulodinium machaerophorum (45 % in average; Fig. 3b), commonly found dominant in areas with strong fluvial influences (Morzadec-Kerfourne, 1977;[START_REF] Ganne | Present-day palynomorph deposits in an estuarine context: The case of the Loire Estuary[END_REF][START_REF] Lambert | Modern palynological record in the Bay of Brest (NW France): Signal calibration for palaeo-reconstructions[END_REF][START_REF] Penaud | Oceanic versus continental influences over the last 7 kyrs from a mid-shelf record in the northern Bay of Biscay (NE Atlantic)[END_REF]. The other major autotrophic taxa are Spiniferites bentorii (11 % in average; Fig. 3b), Spiniferites membranaceus (6 % in average; Fig. 3b) and cysts of Pentapharsodinium dalei (5 % in average; Fig. 3b).

The heterotrophic rate is on average 20 % over the whole BQ (Fig. 3b) and is particularly represented by Brigantedinium spp. species (i.e. "round brown" cysts produced by Protoperidinium dinoflagellate species) (12 % in average; Fig. 3b). The highest relative abundances of heterotrophic cysts (Figs. 2e, 3a) are generally observed in the deepest areas of the BQ (Fig. 2), between 6 and 10 m, which are characterized by the lowest bottom water oxygen concentrations (Fig. 2g) and the lowest sediment grain-size values (Fig. 2h). The total dinocyst concentrations (Figs. 2c,3a) and in a lesser extent the specific richness (Fig. 2d) follow the same trend. The 6 m isobath therefore seems to constitute a limit below which dinocyst concentrations are greater.

Correlation matrix of variables

The correlation matrix of variables (Fig. 4) shows strong correlations between the different variables here used to explain the dinocyst distribution. Among these, a good positive correlation (r > 0.5) is noted between 'Hdino' and three variables: 'depth', 'granulo<125' and 'DeepForam' (Fig. 4). Heterotrophic dinocysts therefore increase with the bathymetry, in parallel with finer sediments and increasing occurrences of the deepest foraminiferal taxa. Also, a similar correlation with 'depth', 'granulo<125' and 'DeepForam' is observed with 'Cdino' and 'Srdino' (Fig. 4), likely caused by the greater occurrences of heterotrophics in the deepest zones of the BQ (> 6 m). Finally, a good positive correlation is noted between 'Srdino', 'Cdino', and to a lesser extent 'Hdino', and on the other hand 'O2', while 'Hdino' is little or not correlated with "Chla" (Fig. 4).

Discussion

Taphonomic issues and dinocyst preservation in the Bay of Quiberon

Some discrepancies in dinocyst distribution are observed within the BQ, especially for the heterotrophic taxa, showing slightly higher occurrences in the deepest areas (below the 6 m bathymetric threshold; Figs. 2e and3a; Fig. 4). Furthermore, we assume that the small BQ (i.e. 40 km²), characterized by strong tidal currents (cf. gyratory circulation in Fig. 1) which homogenize surface water properties including nutrients [START_REF] Tessier | Caractérisation et dynamique des turbidités en zone côtière : l'exemple de la région marine Bretagne Sud[END_REF], cannot explain the differential distribution of heterotrophic dinocysts in the BQ (Figs. 2e and3a).

Brigantedinium species, which dominate the heterotrophic assemblages (Fig. 3), as well as Echinidinium species, are sensitive to oxidation [START_REF] Zonneveld | Preservation of organic-walled dinoflagellate cysts in different oxygen regimes: a 10,000-year natural experiment[END_REF][START_REF] Kodrans-Nsiah | A natural exposure experiment on short-term species-selective aerobic degradation of dinoflagellate cysts[END_REF][START_REF] Bogus | Differences in the chemical composition of organic-walled dinoflagellate resting cysts from phototrophic and heterotrophic dinoflagellates[END_REF]. Their occurrences even decrease logarithmically with increasing bottom water oxygen concentrations [START_REF] Zonneveld | Palaeoproductivity and postdepositional aerobic organic matter decay reflected by dinoflagellate cyst assemblages of the Eastern Mediterranean S1 sapropel[END_REF][START_REF] Zonneveld | Selective preservation of organicwalled dinoflagellate cysts as a tool to quantify past net primary production and bottom water oxygen concentrations[END_REF][START_REF] Zonneveld | Preservation and organic chemistry of Late Cenozoic organic-walled dinoflagellate cysts: A review[END_REF]. Double-walled organic dinocysts (i.e. dormant resting stages with a mandatory dormancy period; Anderson and Wall, 1978) are composed of a macromolecule, whose chemical composition consists in a cellulose-like glucan in autotrophic, and a nitrogen-rich glycan in heterotrophic forms [START_REF] Bogus | Differences in the chemical composition of organic-walled dinoflagellate resting cysts from phototrophic and heterotrophic dinoflagellates[END_REF]. This difference in dinocyst chemical composition explains the greater vulnerability of heterotrophics to oxidation and a differential preservation during diagenetic processes [START_REF] Bogus | Differences in the chemical composition of organic-walled dinoflagellate resting cysts from phototrophic and heterotrophic dinoflagellates[END_REF].

In our study, statistical correlation between the percentages of heterotrophic dinocysts and measured environmental parameters allowed us to discuss the significance of the heterotrophic signature in the BQ (Fig. 4). A lack of correlation between 'Chla' and 'Hdino' (r = -0.06; Fig. 4) suggests that productivity is not a main factor explaining differences in the BQ spatial distribution of heterotrophic dinocysts. The weak negative correlation observed between heterotrophic cysts ('Hdino') and 'O2' (r = -0.25; Fig. 4) is mitigated by a nonobvious statistical correlation (p-value=0.369; Fig. 4). Moreover, dissolved oxygen values ('O2'), are derived from occasional measurements and are not representative of an averaged state of the BQ bottom water oxygenation. Indeed, seasonal hypoxia have been recognized in the deepest areas of the BQ and related to the establishment of a seasonal water column stratification, with oyster mortality below the bathymetric threshold of 6 m [START_REF] Stanisière | Les risques conchylicoles en Baie de Quiberon[END_REF]. Therefore, to test the effect of heterotrophic degradation(/preservation) under oxic(/anoxic) conditions, benthic foraminiferal data have been additionally considered. The deepest benthic foraminiferal assemblage considered ('DeepForam'; Fig. 2f) is mainly constituted by opportunistic species and characterize areas subjected to seasonal hypoxia, confirming the modern environmental assessment [START_REF] Stanisière | Les risques conchylicoles en Baie de Quiberon[END_REF]. It is also worth noting that the water column stratification, and its effects on the seasonal bottom water oxygen depletion from the 6 m isobath, is also confirmed by i) the negative correlation between 'depth' and 'Temp' (r = -0.79; Fig. 4), as well as between 'depth' and 'O2' (r = -0.70; Fig. 4), and ii) the positive correlation between 'depth' and 'DeepForam' (r = 0.95; Fig. 4).

The positive correlations we observed between 'DeepForam' and 'Hdino' (r = 0.76; Fig. 4), 'DeepForam' and 'Srdino' (r = 0.49; Fig. 4) and 'DeepForam' and 'Cdino' (r = 0.57; Fig. 4) therefore suggest that depleted oxygen concentrations may be involved in the higher dinocyst preservation by increasing heterotrophic taxa percentages and dinocyst concentrations. The heterotrophic signature also seems to be correlated with grain-size values. The distribution map (Fig. 2h) and the Pearson correlation coefficient (Fig. 4) show that the proportion of the finest sediments increases with depth. A significant correlation is highlighted between 'granulo<125' and 'Hdino' (r = 0.56; Fig. 4), 'granulo<125' and 'Srdino' (r = 0.61; Fig. 4) and 'granulo<125' and 'Cdino' (r = 0.75; Fig. 4). It is well-known that coarser sediments are not conducive to a good preservation of palynomorphs (cf. example of sampling site n°3 in the BQ; Fig. 3a) due to interstitial fluid circulation and higher oxygenation in a porous sediment. The coarser the sediment, the lower the proportion of heterotrophics (Figs. 2e,h).

Variations in the proportions of heterotrophics may thus be related to the effect of oxidation processes acting on species-selective degradation after cyst deposition in the BQ.

A conceptual model allows gathering environmental conditions prevailing in the BQ to explain the differential dinocyst distribution pattern (Fig. 5). At the scale of the study area, we postulate that "autotrophic/heterotrophic" dinocyst fluxes to the sediments are almost identical at all points of the BQ (red arrows on Fig. 5), due to a bay-wide homogenization of sea-surface properties through tidal currents. Beyond 6 m deep, the water column stratification results in an oxygen bottom-water consumption which exceeds its renewal and thus in seasonal bottom-water hypoxia. Because of this stratification, the weakening of coastal and tidal currents in the deepest areas of the BQ enhanced the sedimentation of finer sediments. The combination of seasonal bottom-water oxygen depletion and higher proportion of finer sediments may explain the greater occurrences of heterotrophics below the 6 m isobath.

Such considerations lead us to consider with great caution, in shallow-bay paleoecological reconstructions, the use of heterotrophic-derived productivity indexes. In this context, [START_REF] Zonneveld | Selective preservation of organicwalled dinoflagellate cysts as a tool to quantify past net primary production and bottom water oxygen concentrations[END_REF] suggested the use of the absolute concentrations of autotrophic dinocysts rather than total or heterotrophic concentrations to discuss varying productivity conditions. Also, since the sediment granulometry plays a crucial role in the dinocyst preservation, it appears essential to carry out grain-size analyses along core or to work in homogeneous sediment contexts.

Inshore-Offshore dinocyst assemblages across the Southern Brittany shelf

The influence of environmental parameters on the dinocyst distribution is increasingly understood thanks to the gradual improvement of the world marine current databases and atlases (e.g. [START_REF] Zonneveld | Atlas of modern dinoflagellate cyst distribution based on 2405 data points[END_REF][START_REF] Marret | From bi-polar to regional distribution of modern dinoflagellate cysts, an overview of their biogeography[END_REF][START_REF] De Vernal | Distribution of common modern dinoflagellate cyst taxa in surface sediments of the Northern Hemisphere in relation to environmental parameters: The new n= 1968 database[END_REF][START_REF] Van Nieuwenhove | An overview and brief description of common marine organic-walled dinoflagellate cyst taxa occurring in surface sediments of the Northern Hemisphere[END_REF]. However, although the highest dinocyst concentrations are observed in neritic and coastal areas, studies on current dinocyst distributions in French coastal environments are still scarce (e.g. [START_REF] Morzadec-Kerfourn | Les Kystes de dinoflagellés dans les sédiments récents le long des côtes Bretonnes[END_REF][START_REF] Larrazabal | Kystes modernes de dinoflagellés en baie de Vilaine-Bretagne sud (France)[END_REF][START_REF] Ganne | Present-day palynomorph deposits in an estuarine context: The case of the Loire Estuary[END_REF][START_REF] Lambert | Modern palynological record in the Bay of Brest (NW France): Signal calibration for palaeo-reconstructions[END_REF]. From previous studies (e.g. [START_REF] Williams | The distribution of marine dinoflagellates in relation to physical and chemical conditions[END_REF][START_REF] Morzadec-Kerfourn | Les Kystes de dinoflagellés dans les sédiments récents le long des côtes Bretonnes[END_REF][START_REF] Penaud | Oceanic versus continental influences over the last 7 kyrs from a mid-shelf record in the northern Bay of Biscay (NE Atlantic)[END_REF], dinocyst groups have been established: i) the oceanic zone (>100-150 m water depth, outer-neritic assemblage) appears characterized by Impagidinium aculeatum, O. centrocarpum, S. mirabilis, and S. elongatus (with S. ramosus and S. bulloideus in addition), I. aculeatum being restricted to full-oceanic waters, ii) the coastal zone (inner-neritic) is characterized by the association S. ramosus-S. bulloideus-S. bentorii, and iii) the estuarine-coastal zone, under fluvial-derived major influence, is characterized by L. machaerophorum, a species tolerant to large drops in salinity and mainly proliferating in brackish environments [START_REF] Reid | A regional sub-division of dinoflagellate cysts around the British Isles[END_REF][START_REF] Morzadec-Kerfourn | Les Kystes de dinoflagellés dans les sédiments récents le long des côtes Bretonnes[END_REF][START_REF] Morzadec-Kerfourn | Dinoflagellate cysts and the paleoenvironment of Late-Pliocene early-pleistocene deposits of Brittany, Northwest France[END_REF][START_REF] Mudie | Atlas of modern dinoflagellate cyst distributions in the Black Sea Corridor: from Aegean to Aral Seas, including Marmara, Black, Azov and Caspian Seas[END_REF][START_REF] Penaud | Oceanic versus continental influences over the last 7 kyrs from a mid-shelf record in the northern Bay of Biscay (NE Atlantic)[END_REF].

Along the inshore-offshore gradient (Fig. 6a), estuarine to inner-neritic samples under strong fluvial influences exhibit an almost monospecific assemblage of L. machaerophorum (more than 80 %; Fig. 6b). L. machaerophorum is becoming less and less abundant with the distance from the coast and is rarely observed in the full-oceanic environment (i.e. around 0.3% in the MD95-2002 core; Fig. 6b). The offshore sea-surface conditions are indeed influenced by the North Atlantic general circulation, while the circulation on the continental shelf is influenced by a mixed river / tidal current limited to the coast [START_REF] Penaud | Oceanic versus continental influences over the last 7 kyrs from a mid-shelf record in the northern Bay of Biscay (NE Atlantic)[END_REF]. In shelf sediments (CBT-CS11 and VK03-58bis cores), the prevalence of the L. machaerophorum species over neritic to oceanic cyst taxa thus suggests seasonally coastal stratified waters subjected to strong continental influence [START_REF] Penaud | Oceanic versus continental influences over the last 7 kyrs from a mid-shelf record in the northern Bay of Biscay (NE Atlantic)[END_REF]. Indeed, from mid-autumn to early spring, strong river flows (i.e. Gironde, Loire, Vilaine) associated with sustained northeastward wind activity maintain this region under the influence of northward freshwater plumes and associated low salinities between the coast and the 100 m isobath [START_REF] Lazure | Circulation on the Armorican shelf (Bay of Biscay) in autumn[END_REF][START_REF] Charria | Surface layer circulation derived from Lagrangian drifters in the Bay of Biscay[END_REF][START_REF] Costoya | Changes in sea surface temperature seasonality in the Bay of Biscay over the last decades (1982-2014)[END_REF][START_REF] Costoya | Modulation of sea surface temperature warming in the Bay of Biscay by Loire and Gironde Rivers[END_REF]. The resulting density gradients explain water mass stratification and thermohaline front at the 100 m isobath, and thus the prevalence of L. machaerophorum until this limit [START_REF] Penaud | Oceanic versus continental influences over the last 7 kyrs from a mid-shelf record in the northern Bay of Biscay (NE Atlantic)[END_REF].

The transect confirms the ecological preferences of S. bentorii and S. ramosus for the coastal environments. However, while S. bentorii seems to be a good marker of the strict proximal domain (i.e. strong occurrence in BQ: 11.5 %; Fig. 6b), being almost absent from the shelf (i.e. VK03-58bis and CBT-CS11 cores: 0.3 and 2 %, respectively), S. ramosus exhibits an inverse distribution and becomes more and more present away from the coast (Fig. 6b): S. ramosus reaches 5% in the full-oceanic domain (MD95-2002 core) while S. bentorii is totally absent. S. ramosus and S. bentorii can therefore be considered as excellent markers of the proximal neritic environment under moderate fluvial influences. Cysts of P. dalei show a fairly homogeneous distribution and low percentages all along the transect (i.e. 3 to 10%; Fig. 6b), being more dominant in northern latitudes of the North Atlantic Ocean and in fjords [START_REF] Mudie | Distribution of dinoflagellate cysts in the Canadian Arctic marine region[END_REF][START_REF] Zonneveld | Atlas of modern dinoflagellate cyst distribution based on 2405 data points[END_REF][START_REF] Heikkilä | Surface sediment dinoflagellate cysts from the Hudson Bay system and their relation to freshwater and nutrient cycling[END_REF][START_REF] Marret | From bi-polar to regional distribution of modern dinoflagellate cysts, an overview of their biogeography[END_REF].

This species also occurs in environments where upper water salinities are reduced as a result of meltwater or river inputs [START_REF] Zonneveld | Atlas of modern dinoflagellate cyst distribution based on 2405 data points[END_REF]. The neritic zone of the southern Brittany shelf, marked by a winter salinity front, may explain its greater occurrence in the proximal zone of the continental shelf (i.e. CBT-CS11 and BQ; 12 % and 5 %, respectively). In temperate waters, cysts of P. dalei may likely be used as indicators of cooling and/or continental influences. S. mirabilis and O. centrocarpum are not significant in the Loire Estuary and in the other three coastal sites (i.e. BQ, CBT-CS11 and VK03-58bis; Fig. 6b), whereas both taxa respectively reach 53 and 21% in the deepest site (i.e. MD95-2002; Fig. 6b). These results confirm their affinity for full-oceanic North Atlantic areas. The distribution of heterotrophic dinocysts along this transect is more difficult to interpret. Although we cannot exclude an influence of productivity conditions on heterotrophics, their virtual absence from the Loire Estuary (LE; Fig. 6b) raises questions as it is the most productive area of the transect [START_REF] Gohin | Twenty years of satellite and in situ observations of surface chlorophyll-a from the northern Bay of Biscay to the eastern English Channel. Is the water quality improving?[END_REF]. As previously mentioned in section 5.1, conditions of oxygenation may result in differences between heterotrophic preservation. Comparing heterotrophics would also require to consider mean bottom oxygen conditions and grain-size values for each analyzed site.

Conclusion

The Bay of Quiberon (BQ), characterized by seasonal hypoxia, appears appropriate to investigate the effects of the different degrees of bottom water oxygenation on dinocyst taphonomic processes (i.e. species-selective degradation after cyst deposition). Among dinocyst assemblages, heterotrophic taxa, well-known to be sensitive to degradation processes, here show a spatial distribution that appears mainly controlled by bottom-water oxygen conditions and sediment granulometry; lower heterotrophic percentages and dinocyst diversity are observed under "higher O2-coarser sediments". Heterotrophic occurrences in coastal sediments are here mainly associated to oxidation processes, leading to caution about their use as productivity indexes in paleoecological reconstructions carried out in these kinds of shallow environments, unless high-resolution grain-size analyses are at least conducted in parallel with palynological studies to avoid misinterpretations.

The comparison of the averaged dinocyst results acquired in the BQ with surface sediments and top cores, issued from already published data, made it possible to improve our knowledge regarding the dinocyst distribution along a northern Bay of Biscay (southern Brittany) inshore-offshore gradient. We highlighted clear different ecological groups according to the hydrological and bathymetric contexts: i) an estuarine assemblage at the outlet of the Loire river strongly dominated by L. machaerophorum, ii) a proximal coastal assemblage in the shallow BQ still dominated by L. machaerophorum with, to a lesser extent, S. bentorii and S. membranaceus, iii) a neritic assemblage dominated by L. machaerophorum, S. ramosus and cysts of P. dalei, and iv) an oceanic group dominated by S. mirabilis and O. centrocarpum and in which L. machaerophorum is rare or absent.

Acknowledgment

The sediment sample collect as well as the environmental parameter measurements were carried out within the framework of the RISCO project [START_REF] Pospelova | Dinoflagellate cysts in recent estuarine sediments from aquaculture sites of southern South Korea[END_REF]-2013;coord: J. Mazurié) coordinated by the 'Comité régional de la conchyliculture Bretagne Sud', labeled 'Pôle Mer Bretagne' and set up jointed by the 'Laboratoire Environnement Ressources' (Ifremer). represent the residual gyratory tidal currents [START_REF] Vanney | Étude sédimentologique du Mor Bras, Bretagne[END_REF][START_REF] Lemoine | Étude sédimentaire de la Baie de Quiberon : la zone ostréicole en eau profonde et ses abords[END_REF][START_REF] Tessier | Caractérisation et dynamique des turbidités en zone côtière : l'exemple de la région marine Bretagne Sud[END_REF]. Table 1: Geographic coordinates of sampling sites. Sampling depths ('depth' in the correlation matrix of variables) are provided according to the chart datum ('lowest astronomical tides').

Data availability Figures

Table 2: Values of the environmental quantitative parameters used in the correlation matrix of variables for each sampling station (cf. Fig. 4).

Table 3: List of dinocyst taxa (and their thecate name) identified in this study, abbreviations and percentages recorded in the averaged fifteen BQ surface sediment samples.

  RISCO project was funded by the 'Conseil Régional de Bretagne'. Analysis also benefited credits by a CNRS-INSU project HCOG2 'Forçages climatiques Holocène et répercussions Côtières et Océaniques dans le Golfe de Gascogne' (2013-2014 ; coord. A. Penaud) in the context of the LEFE-IMAGO research axis.

Figure

  Figure 2

  Figure 2

  641

Figure 3 Figure 4

 34 Figure 3

  Figure 5:

Figure

  Figure 6

Figure 1 :

 1 Figure 1: a) Location of the study area in NW France. b) Zoom on the S Brittany area identified in a): main bays and rivers are highlighted in the map, as well as the investigated Bay of Quiberon (BQ). The 'Mor Bras' is a bathymetric depression bordering the southern coast of Brittany. c) Distribution of the 15 sampling sites within the BQ. The yellow arrows

Figure 2 :

 2 Figure 2: Distribution maps of various palynological and environmental parameters within the BQ. a) Clustering of sampling stations according to the relative abundances of dinocyst taxa (using PAST v.1.75b; Hammer et al., 2001); b) Colorization of sampling stations according to their membership group in the cluster; c) Dinocyst concentrations (number of specimens/cm 3 ); d) Dinocyst diversity (Shannon diversity index); e) Heteretrophic dinocyst taxa percentages; f) Proportion of the 'deep foraminiferal assemblage' regarding the total benthic foraminifera taxa (as described in section 2.); g) Bottom water oxygen concentrations; h) Percentages of grain-size classes.

Figure 3 :

 3 Figure 3: a) Diagram representing the percentages of the main dinocyst taxa (greater than 2% on at least one study site), as well as the number of taxa and the total dinocyst concentration, for each BQ study sample. Sampling sites are classified according to water depth. Sample n°3 (with only 13 individuals counted) is highlighted in grey. The station numbers (to the left of

Figure 4 :

 4 Figure 4: Correlation matrix of the different environmental, foraminiferal and dinocyst taxa presented in Tables2 and 3. A color gradient from blue to red is used to represent the correlation coefficients (blue is used for negative values and red for positive ones; the higher the absolute value (i.e. when it tends to 1), the darker the color). Regarding the other half of the matrix, only p-values below 0.1 are highlighted in light (0.1<p-value<0.01) and dark (p-value<0.1) green.

Figure 5 :

 5 Figure 5: Conceptual model presenting the taphonomic preservation bias impacting dinocysts (especially heterotrophics and dinocyst concentrations) in the shallow Bay of Quiberon, on either side of the 6 m bathymetric threshold. The red arrows represent dinocyst fluxes to the sediments. H/A: ratio Heterotrophic to Autotrophic dinocysts. Due to homogenized hydrological parameters of the surface waters within the BQ, we consider that the H/A ratio and nutrient concentrations remain stable at the BQ scale. Gradients for particle size, oxygen concentration, and dinocyst preservation are highlighted under the figure with horizontal thicker or thinner lines on either side of the 6m isobath, and gradients for oxygen concentration, sea-surface temperature and water column mixing are highlighted to the right of the figure with vertical thicker or thinner lines according to the BQ water depth.

Figure 6 :

 6 Figure 6: a) Location of modern samples and core tops used in the inshore-offshore transect at the southern Brittany-scale. Map performed using the SHOM bathymetric data (SHOM, 2015. 'MNT Bathymétrique de façade Atlantique' (Homonim Project). http://dx.doi.org/10.17183/MNT_ATL100m_HOMONIM_WGS84) and depths are given according to the chart datum. b) Selected dinocyst taxa percentages: heterotrophics vs.autotrophics for the first line and percentages of the major taxa based on a main autotrophic sum (i.e. excluding heterotrophics). Sites are presented, from right to left, according to their

  6

	Site	Longitude	Latitude	Sampling depth
				(m)
		-3.0501	47.5592979	5.24
		-3.02359375	47.5560063	7.57
		-3.09542143	47.5507071	3.37
		-3.06407917	47.5513313	4.82
		-3.03639167	47.5489833	9.83
		-3.10814524	47.5394214	4.58
		-3.09032917	47.5399396	5.55
		-3.0630375	47.5434083	7.77
		-3.11331927	47.5170971	4.4
	10	-3.08969323	47.5264333	8.3
	11	-3.0739	47.5334896	8.52
	12	-3.06528125	47.52605	8.69
	13	-3.04997917	47.5341708	9.6
	14	-3.10756875	47.5167521	6.75
	15	-3.07979245	47.5159747	9.74

Table 655

 655

	Site depth	Srdino	Cdino	Hdino	Temp	Sal	O2	Chla	Turb	SM	DeepF	granulo
		(m)		(nb./cm 3 )	(%)	(°C)	(‰)	(mg/l)	(µg/l)	(NTU)	(mg/l)	oram	<125
												(%)	(%)
	1	5.24	1.78	1504.49	7.6	14.5	34.3	93.55	1.95	2.04	6.23	6	54
	2	7.57	1.94	13979.95	22.7	14.5	34.4	94.77	1.56	2.40	7.06	12	72

Table 3 659

 3 Figure captions:


Atourya Mhoumadi

Mohamed Elkhashab

Sylvain Prillieux

Jean-Bernard Dumas

Franck Collas

Nicolas Louvain

Bernard Fraisse

Philippe Espeau
email: philippe.espeau@parisdescartes.fr

Characterization of the heat behavior of amiodarone hydrochloride

Keywords: amiodarone hydrochloride, melting degradation, thermal analyses, flash scanning calorimetry, X-ray powder diffraction, TGA-FTIR

Introduction

Amiodarone hydrochloride (2-butyl-3-benzofuranyl) [4-[2-(diethylamino)ethoxy]-3,5diiodophenyl]methanone hydrochloride, C25H30ClI2NO3) is the hydrochloride salt of an iodine-rich benzofuran derivative known for its antiarrhythmic and vasodilatory activities. It is a class III antiarrhythmic agent that blocks myocardial calcium, potassium and sodium channels in heart tissue. In addition, this molecule is also known for its properties of inhibiting alpha and beta-adrenergic receptors (also called adrenoreceptors).

Regarding the solid state of amiodarone hydrochloride, its crystal structure was previously determined and showed that the related compound crystallizes in a monoclinic system, P21/c, with four molecules in the unit cell [START_REF] Cody | Structure-activity Relationships of Antiarrhythmic Agents: Crystal Structure of Amiodarone Hydroehlodde and Two Derivatives, and Their Conformational Comparison with Thyroxine[END_REF]. On the other hand, this substance is reported to degrade on melting. As far as the melting characteristics of this compound are concerned, some data are reported in the literature [START_REF] O'neil | The Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals[END_REF][START_REF] Ledeţi | Solid-state preformulation studies of amiodarone hydrochloride[END_REF][START_REF] Fasihi | Thermodynamic approaches for the prediction of oral drug absorption[END_REF][START_REF] Yoshida | Thermal behavior study and decomposition kinetics of amiodarone hydrochloride under isothermal conditions[END_REF][START_REF] Creţeanu | Thermal stability assessment of amiodarone hydrochloride in polymeric matrix tablets[END_REF][START_REF] Vadlapatla | Thermal characterization of amiodarone hydrochloride and its interactions with excipients[END_REF], but most of the melting temperatures are obtained at DSC scan rates not high enough to bypass the degradation of the compound.

The degradation pathway as well as the identification of the degradation products was performed by coupling thermogravimetric analysis (TGA) and FTIR experiments. We have previously shown that, when substance degrades on melting, the melting temperature as well as the melting enthalpy depend on the DSC scan rate [START_REF] Corvis | The role of stearic acid in ascorbic acid protection fromdegradation: a heterogeneous system for homogeneous thermodynamic data[END_REF][START_REF] Rosa | Crystal structure determination and thermal behavior upon melting of p-synephrine[END_REF]. Since degradation phenomena occurring during melting can be bypassed at high DSC heating rates, DSC studies at various scan rates are then carried out on this compound in order to get correct values of temperature and enthalpy of fusion. Flash Scanning Calorimetry (FSC) experiments were also performed in order to confirm the melting temperature and to try to push the degradation even further towards high temperatures.

Experimental

2.1. Chemicals Amiodarone hydrochloride (scheme 1) was purchased from SAFIC ALCAN, with a purity of 99.7 %. It is a white or almost white fine crystalline powder which needs to be protected from light and stored at a temperature not exceeding 30 °C. Its molecular weight was found to be 681.8 g/mol. The compound was used without further purification. Temperature and enthalpy of the DSC device were carried out using melting point of indium.

Thermal analysis

An empty aluminum pan was used as a reference for all the DSC experiments. The onset of the corresponding endotherms was set as the melting temperature of the sample. Scan rates ranging from 1 to 100 K/min were used for the DSC experiments. Then, to avoid a calibration step for each heating rate, a tau lag calibration of the DSC device was performed beforehand [START_REF] Kehl | Multiple Thermocouple Sensor[END_REF]. This was made possible thanks to the multiple thermocouple sensor technology with which the device is equipped. The experiments were conducted using sample masses between 5 and 10 mg, weighed with a microbalance sensitive to 10 μg.

Flash Scanning Calorimetry was performed using a Flash DSC1 (METTLER TOLEDO) connected with a TC45 Intracooler (Huber) and an UFS-1 sensor. The starting temperature was set at 20 °C and the measuring cell was continuously purged with nitrogen gas at a flow rate of 10 mL/min. Temperature calibration was done using melting of high purity indium.

Since the sample was in powder form, a hair was used to place few grains on the surface of the sensor using the Leica microscope mounted on the Flash DSC. However, the contact between the sample and the sensor was therefore not optimal since the grains do not have a flat surface. To improve the contact, we applied a micro drop of silicone oil (Wacker AK 60 Pa.s) on both sides of the sensor. Then, to obtain a homogeneous oil film thickness, several heating ramps up to 300 °C were carried out before depositing one or more grains of the material. More details on the preparation of the sample and on the Flash DSC technique is reported in Ref. [START_REF] Schawe | Material characterization by fast scanning calorimetry: practice and applications[END_REF].

X-ray powder diffraction

Powder X-ray diffraction measurements were carried out on a Brucker D8 Discover diffractometer, using Cu-Kα1 radiation (1.5405980 Å) selected with a primary Johansson-type focusing (101) quartz crystal monochromator, and equipped with the LynxEye XE-T detector (mode 1D). Data as collected on a spinner-type (15 rpm) zerobackground sample holder from 4° to 65° in 2-θ with a step size of 0.015° and 5 seconds per step for a total collection time of 6 h 12 min 45 s. The white crystalline powder sample was used as received. Profile matching was performed with FullProf 2k (Version 7.40 -Jan2021-ILL JRC).

TGA/FTIR analysis

Thermogravimetric analysis experiments were performed using a Hi-res TGA 2950 analyzer (TA Instruments, USA). The TGA experiments were carried out in the 15-500 °C temperature range under a constant nitrogen flow of 100 mL/min. The TGA experiments were conducted at a scan rate of 30 K/min for the analyses coupled with FTIR. Isatherm, Nickel and Trafoperm were used for temperature calibration of the TGA device. The infrared analysis were performed with a Nexus model 470 FTIR device (Thermo/Nicolet, USA) with a heating transfer line and a heating TGA Interface from Thermo/Nicolet. The operating temperature for the transfer line and the TGA Interface was 250 °C. FT-IR analyses of evolved gas from TGA device were performed with 32 scans, a mirror speed of 2.5317 cm/s and a resolution of 4 cm -1 in the scan range of 450-4000 cm -1 .

The FTIR bench was permanently purged with dry air. The FTIR calibration is checked by using a polystyrene film for wave number accuracy and resolution performance. Sample masses between 3 and 35 mg were used for these experiments.

Results and discussion

X-ray powder diffraction confirms that the Amiodarone hydrochloride powder used in the present study is crystalline, as shown by the intense peaks observed at 5.2°, 7.3°, 10.3°, 11.6°, or 16.1°, corresponding to (100), ( 110), (020), (210), and (211) hkl planes, respectively (Fig. 1). An analysis of the experimental pattern with the ICDD and COD databases showed that the reference 00-045-1755 (PDF-2, ICDD), corresponding to the Amiodarone hydrochloride, was a close match, as displayed in Fig. S1 (in the Supporting Information), but some experimental low-intensity peaks were not indexed by the reference. The single crystal structure of the Amiodarone hydrochloride has already been reported [START_REF] Cody | Structure-activity Relationships of Antiarrhythmic Agents: Crystal Structure of Amiodarone Hydroehlodde and Two Derivatives, and Their Conformational Comparison with Thyroxine[END_REF], and the cell parameters and monoclinic space group were used as a starting point for a profile refinement.

The profile matching calculation of the XRD data, displayed on Fig. 1, confirms the analyzed crystalline powder is Amiodarone hydrochloride, in good agreement with the single crystal structure [START_REF] Cody | Structure-activity Relationships of Antiarrhythmic Agents: Crystal Structure of Amiodarone Hydroehlodde and Two Derivatives, and Their Conformational Comparison with Thyroxine[END_REF]. No crystalline impurity is evidenced by XRD. More information are given in the supporting information file. Fig. 1. XRD pattern for Amiodarone hydrochloride (red dots), its calculated profile (black line) and the difference between the experimental (red crosses) and calculated patterns (blue line). The hkl indexes of selected Bragg peaks are given in brackets (green dashes). More information is provided in the Supporting Information.

When Amiodarone hydrochloride is heated at different scan rates from 1 to 100 K/min, the resulting DSC thermograms undergo significant shifts through high temperatures (Fig. 2) indicating that the compound degrades on melting. Moreover, this hypothesis is confirmed when observing the sample itself. Indeed, samples turned from a white powder before analysis to a black viscous liquid. As a consequence, the onset of the ''apparent'' melting temperature increases with the scan rate to reach a plateau from v = 80 K/min, with a value equal to 165.9 ± 0.8 °C (Fig. 3A, Table 1). From Fig. 3A, it can be seen that the value of 156 °C, reported in Ref. 2 (Table 1), is obtained at very low scan rate (1 K/min).

We can also observe on the DSC thermograms an exothermic signal corresponding to degradation of amiodarone hydrochloride. It is interesting to note that at 1 K/min, the return to the baseline of the melting peak seems "ballistic", which could mean that the departure of the degradation compounds occurs concomitantly with fusion, probably linked to the fact that the heating rate is low. This phenomenon of rapid return to the baseline is less and less abrupt as the heating rate increases. As for the melting temperature, the "apparent" melting enthalpy increases with the scan rate to reach a plateau from v = 20 K/min, with a value equal to 54.9 ± 0.2 kJ/mol (Fig. 3B, Table 1). In ref. [START_REF] Vadlapatla | Thermal characterization of amiodarone hydrochloride and its interactions with excipients[END_REF], TGA experiments are carried also out on amiodarone hydrochloride. The authors report two thermal decomposition stages, the first one being between 250 and 415 °C with a mass loss of 61.94 %. But no more details are given about this decompostion. The degradation was demonstrated by simultaneously coupling DSC and ATG analyses, for a given scan rate. The experiments were carried out at different heating rates between 2 and 80 K/min. The experiments at 10 and 60 K/min are presented in Fig. 4 to illustrate the following statement. The melting temperature is located on the graphs in order to observe the temperature shift between melting and the beginning of degradation (indicated by an asterisk on the figure). Then, it is unquestionable that at low heating rate the product begins to degrade before melting (Fig. 4A). This is no longer the case when the heating rate increases where it can be observed that degradation takes place just after melting (Fig. 4B).

This therefore favorably argues the fact that the product degrades on melting but that this degradation is bypassed when we increase the heating rate to be displaced at higher temperatures. In order to identify the evolved gas from amiodarone hydrochloride degradation, TGA experiments, carried out at 30 K/min, were coupled with FTIR analysis. Then, upon heating, one can observe that amiodarone hydrochloride starts to degrade before melting since between 135 and 159 °C, the departure of water and 2-fluoroethanol is observed on the FTIR spectrum (0.2 % mass loss, Fig. S3, Table S1). This corroborates the result we have found from DSC and TGA experiments carried out at 10 K/min (Figure 4A). From the FTIR analysis, triethylamine was identified as the following degradation product the departure of which is comprised between 159 and 190 °C, i.e. during and after melting (2.0 % mass loss, Fig. S3, Table S1). Regarding the departure of hydrogen chloride, it is not observed at the very beginning of the thermal decomposition but rather after 229 °C. This compound is not subject to a resolved event on the TGA thermogram since the departure of other degradation compounds such as hydrogen fluoride or hydrogen iodide is also observed in the same time (Fig. S3).

In figure S4 are shown the FTIR spectra of the main evolved gas obtained during TGA experiment carried out at 30 K/min.

With regard to the thermogram obtained for the highest heating rate by conventional DSC (80 K/min), although the degradation process is pushed back to higher temperatures, the exothermic signal remains. We therefore undertook thermal analysis measurements using the flash DSC technique in order to obtain much higher heating rates and thus ensure that the degradation process could be pushed towards higher temperatures. In order to facilitate the departure of the degradation compounds, a first series of measurements was made by directly depositing a small crystal of amiodarone hydrochloride on the sensor without silicone oil (Fig. 5). A first FSC experiment was carried out at the heating rate of 60 K/min (curve A on Fig. 5),

for comparison with the conventional DSC. One can observe that the behavior is the same.

However, for higher heating rates, the exothermic signal related to the departure of the degradation compounds is no longer visible at all (curves B and C on Fig. 5). It should be noted that the temperatures reported in Fig. 5 have no physical significance insofar as the contact between the measuring cell and the amiodarone sample is not optimal.

However, these experiments clearly show that the departure of the degradation compounds can be pushed to temperatures well above the melting point. No exothermic signal is no more detected on the thermograms, probably due to the quick departure of the products of degradation given the very high heating rates. The only exothermic signal which is observed on the thermograms is therefore necessarily linked to the melting of amiodarone.

Another set of experiments were carried by FDSC but using, this time, silicone oil in order to have a better contact between the sample and the measuring cell and then to get a better precision on the measurement of the melting temperatures.

Based on the value of the melting enthalpy found previously (54.9 kJ/mol or 80.4 J/g), we

were then able to determine the mass of the amiodarone hydrochloride samples subjected to the different temperature ramps. They were estimated at 183, 54 and 36 ng for the speeds of 100, 200 and 500 K/s respectively. Then, normalized curves could then be drawn taking into account the heating rate and the sample mass (Fig. 6) The melting temperatures thus measured confirm the one obtained by conventional DSC for high scanning rates (165.9 ± 0.8 °C). As for experiments realized without silicone oil, the departure of the degradation products seem to be pushed to higher temperatures but with better definition of the thermograms and better accuracy about melting temperatures.

Conclusion

As a first approach, the X-ray diffraction analysis confirmed that the product used in a pharmaceutical context was indeed of the same nature as that already published. In a second step, the accurate melting data of amiodarone hydrochloride were obtained by using high DSC scan rates, allowed separating melting and degradation. The use of FSC made it possible not only to confirm the melting temperature obtained by the "plateau method" but also to obtain a melting signal independent of the degradation, thus confirming our hypothesis that the disturbance of the melting signal was due to the loss of degradation compounds, as corroborated by TGA-FTIR experiments.

The knowledge of the thermal behavior of amiodarone hydrochloride is of importance from the point of view of the formulation of amiodarone hydrochloride as a drug. Indeed, the degradation of amiodarone hydrochloride on melting indicates that this active ingredient must not be heated alone to be formulated as a drug. In the case where this active ingredient is formulated as a solid dispersion to increase its aqueous solubility and its absorption, for example, a strategy would be to use ingredients with lower melting point and a sophisticated technology (like Hot Melt Extrusion with proper process conditions) to bypass degradation effect potentially observed.

CRediT authorship contribution statement

Declaration of Competing Interest

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

Scheme 1 .

 1 Scheme 1. Chemical structure of Amiodarone hydrochloride, C25H30ClI2NO3 (2-butyl-1benzofuran-3-yl)-[4-[2-(diethylamino) ethoxy]-3,5-diiodophenyl] methanone).

  Differential scanning calorimetry and thermogravimetric analyses experiments were carried out using an 822e thermal analyzer, equipped with a FRS5 sensor, and a TGA 850 from Mettler-Toledo (Switzerland). The DSC and TGA experiments were achieved in the 25 -250 °C temperature range under a constant dry air flow of 60 mL/min.

Fig. 2 .

 2 Fig. 2. Selected DSC thermograms of amiodarone hydrochloride as a function of the heating rate. From top to bottom: 1, 5, 20, 60 and 80 K/min. (The curves were shifted for clarity).

Fig. 3 .

 3 Fig. 3. ''Apparent'' melting temperature (A) and enthalpy (B) of amiodarone hydrochloride as a function of the DSC scan rate.

Fig. 4 .

 4 Fig. 4. Superposition of TGA and DSC curves for amiodarone hydrochloride at two different scanning rates (in K/min): (A): 10, (B): 60. The asterisk indicates the beginning of mass loss.

Fig. 5 .

 5 Fig. 5. FSC thermograms of amiodarone hydrochloride obtained without oil silicone as a function of the heating rate (in K/s): (A): 1; (B): 1000 and (C): 2000. The curves were shifted for clarity.

Fig. 6 .

 6 Fig. 6. FSC thermograms of amiodarone hydrochloride obtained with oil silicone as a function of the heating rate (in K/s): (A): 100; (B): 200 and (C): 500. (The curves were shifted for clarity).

  Elkhashab and Atourya Mhoumadi: DSC and TGA experiments. Franck Collas: FSC experiments and writing. Nicolas Louvain and Bernard Fraisse: XRPD experiments, profile matching and writing. Sylvain Prillieux and Jean-Bernard Dumas: supervision. Philippe Espeau: conception and design of the study, writing.

Table 1

 1 It is therefore surprising that some values may coincide with the values we have found pushing back the degradation at temperatures above the melting temperature.

	gathers the thermal data resulting from this study as well as the values found in the
	literature. As can be seen, discrepancies are observed both for temperatures and enthalpies of
	fusion. It should be noted that in reference 3, two sets of data are reported. One set is obtained
	from TG/DTG/HF experiments and another set from by DSC analysis. Both experiments were
	carried out at 10 K/min. The enthalpy of fusion obtained by DSC agrees well with our value

found for higher heating rates. Surprisingly, their value obtained by TG/DTG/HF is very different. The other experiments reported in the literature were carried out at heating rates of place during melting.

Table 1

 1 Experimental melting properties of amiodarone hydrochloride compared to literature data.

	Tfus (°C)	∆fusH (kJ/mol)	DSC scan rate (K/min)	Ref
	165.9 ± 0.8	54.9 ± 0.2	> 80	This work
	156			2
	149-157	44.6	10 (TG/DTG/HF)	3
	157	54.95	10 (DSC)	3
	169.1	49.159	20	4
	159	60.1	10	5
	166		10	6
	164		10	7

Acknowledgements

The "Réseau des Rayons X et Gamma" (Université Montpellier, France) is gratefully thanked for granting access to their XRD platform. The Grand Est region is sincerely thanked for supporting this project.