Daniele O Simão

Thalita D Honorato

Graciely G Gobo

Henrique L Piva

Patrícia L Goto

Larissa Araújo Rolim

Cédric-Olivier Turrin

Muriel Blanzat

Antonio C Tedesco

Marigilson P Siqueira-Moura
email: marigilson.moura@univasf.edu.br

Prof Pontes Marigilson

Siqueira De

Ph.D Moura

Preparation and cytotoxicity of lipid nanocarriers containing a hydrophobic flavanone

Keywords: Hesperitin, Nanostructured lipid carriers, PIT method, Characterization, Glioblastoma, Cytotoxicity

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

Introduction

Malignant gliomas are the most common primary brain tumors responsible for 80 % of diagnosed cases and glioblastoma multiforme, which is a subcategory of malignant gliomas, is the most aggressive and lethal brain cancer. The poor prognosis of glioblastoma is mainly related to cell drug-resistance and low bioavailability of drugs for brain [START_REF] Miranda | Breaching barriers in glioblastoma. Part II: Targeted drug delivery and lipid nanoparticles[END_REF][START_REF] Haar | Drug Resistance in Glioblastoma: A Mini Review[END_REF]. In this regard, researches in order to discover new drugs against glioblastoma become an urgent need in the treatment of brain cancer.

Polyphenolic compounds are ubiquitous in plants and classified as flavonoids, phenolic acids, coumarinea, lignans, tannins, and stilbenes [START_REF] Faridi Esfanjani | Improving the bioavailability of phenolic compounds by loading them within lipid-based nanocarriers[END_REF][START_REF] Khan | A comprehensive review on flavanones, the major citrus polyphenols[END_REF]. Hesperitin (3',5,7-trihydroxy-4'methoxyflavanone) is a bioflavonoid belonging to the chemical sub-class flavanone, which is widely found in citrus fruits (grapefruit, orange, and lemon). Hesperitin and its glycoside (hesperidin) have demonstrated several biological activities confirmed by in vivo and in vitro studies [START_REF] Khan | A comprehensive review on flavanones, the major citrus polyphenols[END_REF]. Among therapeutic effects attributed to hesperetin, neuroprotection in experimental models of Alzheimer's disease, reduction of blood plasma glucose, lipid levels and antioxidant activity, anti-inflammatory, and antineoplastic activity are the most common effects observed [START_REF] Chandrika | Hesperetin and Naringenin sensitize HER2 positive cancer cells to death by serving as HER2 Tyrosine Kinase inhibitors[END_REF][START_REF] Jayaraman | Antihyperglycemic effect of hesperetin, a citrus flavonoid, extenuates hyperglycemia and exploring the potential role in antioxidant and antihyperlipidemic in streptozotocin-induced diabetic rats[END_REF][START_REF] Kheradmand | Neuroprotective effect of hesperetin and nano-hesperetin on recognition memory impairment and the elevated oxygen stress in rat model of Alzheimer's disease[END_REF][START_REF] Sassi | Immunomodulatory potential of hesperetin and chrysin through the cellular and humoral response[END_REF]. Recently, the potential of antiproliferative and antitumor activities for hesperitin and hesperidin on in vitro and in vivo cancer models has been related to mechanisms of cell cycle arrest and apoptosis [START_REF] Ferreira De Oliveira | Therapeutic potential of hesperidin and its aglycone hesperetin: Cell cycle regulation and apoptosis induction in cancer models[END_REF]. However, in spite of several pharmacological activities of these flavanones, hesperidin and hesperetin have low solubility in water (< 0.01 %) decreasing biological absorption due to limited dissolution rate and consequently low bioavailability [START_REF] Faridi Esfanjani | Improving the bioavailability of phenolic compounds by loading them within lipid-based nanocarriers[END_REF][START_REF] Krishnan | Hesperetin conjugated PEGylated gold nanoparticles exploring the potential role in anti-inflammation and anti-proliferation during diethylnitrosamineinduced hepatocarcinogenesis in rats[END_REF][START_REF] Tsirigotis-Maniecka | Microencapsulation of hesperidin in polyelectrolyte complex microbeads: Physicochemical evaluation and release behavior[END_REF][START_REF] Ersoz | Comparative evaluation of hesperetin loaded nanoparticles for anticancer activity against C6 glioma cancer cells[END_REF].

Another factor that influences their absorption is the presence of microorganisms and enzymes in the small intestine and first-pass liver metabolism, which are able to degrade the active substances into inactive molecules. In addition, environmental factors such as pH changes, light, and temperature may affect their physical-chemical stability [START_REF] Bilia | Flavonoids Loaded in Nanocarriers: An Opportunity to Increase Oral Bioavailability and Bioefficacy[END_REF][START_REF] Duranoğlu | Synthesis of hesperetin-loaded PLGA nanoparticles by two different experimental design methods and biological evaluation of optimized nanoparticles[END_REF].

A possible solution to overcome these problems, increasing flavonoid stability and bioavailability, is the entrapment of such compounds in nanostructures well designed lipid nanoparticles. These colloidal particles are formed by solid lipid core at body temperature stabilized by surfactant(s) and dispersed in aqueous phase [START_REF] Faridi Esfanjani | Improving the bioavailability of phenolic compounds by loading them within lipid-based nanocarriers[END_REF][START_REF] Estanqueiro | Nanotechnological carriers for cancer chemotherapy: The state of the art, Colloids Surf[END_REF][START_REF] Gordillo-Galeano | Solid lipid nanoparticles and nanostructured lipid carriers: A review emphasizing on particle structure and drug release[END_REF][START_REF] Khosa | Nanostructured lipid carriers for site-specific drug delivery[END_REF][START_REF] Salvi | Nanostructured lipid carriers (NLC) system: A novel drug targeting carrier[END_REF]. Lipid nanocarrier systems are composed of submicron particles designed from biocompatible and biodegradable lipids. Solid lipid nanoparticles (SLN) represent the first-generation of lipid particles and are characterized by a core formed by solid lipid(s) at room and body temperature. On the other hand, nanostructured lipid carriers (NLC), which are considered the second-generation of lipid nanocarriers, are formed by unstructured solid core containing a mixture of liquid and solid lipids at room and body temperature [START_REF] Estanqueiro | Nanotechnological carriers for cancer chemotherapy: The state of the art, Colloids Surf[END_REF][START_REF] Gordillo-Galeano | Solid lipid nanoparticles and nanostructured lipid carriers: A review emphasizing on particle structure and drug release[END_REF][START_REF] Salvi | Nanostructured lipid carriers (NLC) system: A novel drug targeting carrier[END_REF][START_REF] Beloqui | Nanostructured lipid carriers: Promising drug delivery systems for future clinics[END_REF]. The main advantage of NLC is its higher drug loading capacity as compared to SLN, i.e. a lipid core formed by blend of solid and liquid lipids results in low expulsion of encapsulated drug due to formation of more irregular crystals of solid lipid(s). Furthermore, NLC are also able to protect the entrapped active compounds against chemical degradation in physiological pH [START_REF] Khosa | Nanostructured lipid carriers for site-specific drug delivery[END_REF][START_REF] Salvi | Nanostructured lipid carriers (NLC) system: A novel drug targeting carrier[END_REF][START_REF] Beloqui | Nanostructured lipid carriers: Promising drug delivery systems for future clinics[END_REF]. Several administration routes have been used to NLC, for instance, oral, topical, ocular, and directly to the brain. SLN and NLC have shown to be effective as brain drug delivery systems promoting the penetration of active substances into the nervous tissue [START_REF] Khosa | Nanostructured lipid carriers for site-specific drug delivery[END_REF][START_REF] Beloqui | Nanostructured lipid carriers: Promising drug delivery systems for future clinics[END_REF].

Lipid nanoparticles have been usually produced by high-energy or low-energy

emulsification methods [START_REF] Anton | Design and production of nanoparticles formulated from nano-emulsion templates-A review[END_REF][START_REF] Anton | The universality of low-energy nano-emulsification[END_REF][START_REF] Komaiko | Formation of Food-Grade Nanoemulsions Using Low-Energy Preparation Methods: A Review of Available Methods[END_REF][START_REF] Gao | Formation and stability of solid lipid nanoparticles fabricated using phase inversion temperature method[END_REF][START_REF] Mcclements | Delivery by Design (DbD): A Standardized Approach to the Development of Efficacious Nanoparticle-and Microparticle-Based Delivery Systems[END_REF]. High-energy approaches are able to generate intense disruptive forces using mechanical devices such as high-shear stirrers, high-pressure homogenizers, and ultrasound generators. However, high-energy methods require very high energy level to produce fine particles and most of the mechanical energy generated (99.9 %) is dissipated as heat [START_REF] Tadros | Formation and stability of nanoemulsions[END_REF][START_REF] Solans | Nano-emulsions: Formation by low-energy methods[END_REF]. On the other hand, low-energy emulsification approaches are more energy efficient producing small particles by modification of the internal chemical energy from system due to temperature or composition change [START_REF] Anton | Design and production of nanoparticles formulated from nano-emulsion templates-A review[END_REF][START_REF] Solans | Nano-emulsions: Formation by low-energy methods[END_REF][START_REF] Mcclements | Edible nanoemulsions: fabrication, properties, and functional performance[END_REF]. Low-energy emulsification methods are classified as spontaneous emulsification (SE), phase inversion composition (PIC), emulsion inversion point (EIP), and phase inversion temperature (PIT) [START_REF] Komaiko | Formation of Food-Grade Nanoemulsions Using Low-Energy Preparation Methods: A Review of Available Methods[END_REF][START_REF] Mcclements | Edible nanoemulsions: fabrication, properties, and functional performance[END_REF]. The main advantages to obtain lipid nanoparticles using PIT method are very easy production process through simple stirring and heating as well as this method does not require the use of expensive equipment [START_REF] Anton | Design and production of nanoparticles formulated from nano-emulsion templates-A review[END_REF][START_REF] Komaiko | Formation of Food-Grade Nanoemulsions Using Low-Energy Preparation Methods: A Review of Available Methods[END_REF][START_REF] Mcclements | Edible nanoemulsions: fabrication, properties, and functional performance[END_REF].

Different drug delivery systems have been developed to encapsulate hesperitin or hesperidin, such as polymeric nanoparticles [START_REF] Ersoz | Comparative evaluation of hesperetin loaded nanoparticles for anticancer activity against C6 glioma cancer cells[END_REF][START_REF] Duranoğlu | Synthesis of hesperetin-loaded PLGA nanoparticles by two different experimental design methods and biological evaluation of optimized nanoparticles[END_REF] and lipid nanoparticles [START_REF] Fathi | Hesperetin-Loaded Solid Lipid Nanoparticles and Nanostructure Lipid Carriers for Food Fortification: Preparation, Characterization, and Modeling[END_REF][START_REF] Ott | Exploitation of amaranth oil fractions enriched in squalene for dual delivery of hydrophilic and lipophilic actives[END_REF][START_REF] Saad | Improved cardioprotective effects of hesperidin solid lipid nanoparticles prepared by supercritical antisolvent technology[END_REF]. Even if in these studies using high-energy emulsification methods have been commonly reported the successful application of nanocarriers containing flavanones, low-energy methods are more rarely applied to produce lipid nanoparticles although low-energy approaches are considered simple and reproducible methods without using device and/or organic solvent. Thereby, we described hesperitin encapsulation into lipid nanoparticles, which was carried out through thermal low-energy method, and the in vitro potential of this hesperitin-loaded nanocarrier in the inactivation of neoplastic cells from brain cancer.

Therefore, the main aim of this study was to develop a promising formulation based on NLC containing hesperitin using PIT method as a low-energy emulsification approach and to assess in vitro cytotoxicity of this encapsulated flavanone on T98G glioblastoma cells.

Materials and methods

Materials

Hesperitin (≥ 95%, W431300), sorbitan monooleate (SMO), medium chain triglycerides (MCT), and dialysis tubing cellulose membrane flat (MW cut-off 14,000) were purchased from Sigma-Aldrich Co. (St. Louis, MO). POE-40 hydrogenated castor oil (HCO-40) was kindly supplied from Croda Brasil Ltda (Campinas-SP, Brazil) and glyceryl behenate (GB) solid lipid was a gift from Brasquim (Porto Alegre-RS, Brazil).

Nanostructured lipid carriers (NLC) preparation

Phase inversion temperature (PIT) method was used as a low-energy approach to prepare nanostructured lipid carrier formulations [START_REF] Goto | Application of aluminum chloride phthalocyanine-loaded solid lipid nanoparticles for photodynamic inactivation of melanoma cells[END_REF]. Briefly, organic phase was composed of oil, solid lipid, and surfactants while aqueous phase consisted of purified water or phosphate buffer solution pH 7.4 (10 mM). The phases were gradually heated at 80 (±5 °C) using a water bath and aqueous phase was added to organic phase under magnetic stirring and constant temperature (80 °C). After this temperature was reached, the lipid dispersion obtained was held for 5 min and then cooled down under continuous stirring.

Both liquid lipid-to-solid lipid and surfactant-to-lipid weight ratios kept constant at 1.0 (Table 1) and sorbitan monooleate (SMO) and polyoxyethylene-40 hydrogenated castor oil (HCO-40) surfactants were mixed at constant ratio of 3.2:6.8 (wt %), respectively. All formulations were prepared in triplicate (n=18) and stored at 4 (±2 °C). Hesperitin-loaded NLC were prepared by adding different amounts of this flavanone in organic phase and using same procedure in order to prepare unloaded NLC.

Particle size and polydispersity index analysis

Hydrodynamic diameter of the NLC particles and polydispersity index (PdI) of the formulations were determined by photon correlation spectroscopy (PCS) at 25 °C and scattering angle of 173° (Zetasizer ® Nano ZS, Malvern PCS Instruments, UK). Samples were obtained by diluting 20 μL with 2 mL ultrapure water. The reported values are average ±SEM of three different batches of each colloidal dispersion.

Zeta potential measurement

Zeta (ζ) potential of NLC was measured by electrophoretic mobility using a Zetasizer ® Nano ZS apparatus (Malvern PCS Instruments, UK). The Smoluchowski model was used to estimate ζ potential from electrophoretic mobility. The analyses were conducted at 25 °C, and the samples were appropriately diluted (1/100) with ultrapurified water and pH measured. Values reported are average ±SEM of three different batches of each colloidal dispersion.

Stability study

Stability study was performed by using the analytical centrifugation method with photometric detection at 865 nm (LUMiSizer ® 611, LUM GmbH, Germany). The samples of unloaded NLC (F2), F4, and F6 formulations were incorporated into polycarbonate cell and equilibrated to 25 °C before analyses. Measurements were carried out at 3,618 rpm for 4.5 h resulting in accelerated migration of the dispersed particles and integration of transmission profiles within a selected region of the holding cell allowed to get information about dispersion stability.

Thermal analysis of NLC formulation

Differential Scanning Calorimetry (DSC) analysis was performed using a DSC Q200 ® Scanning Calorimeter (TA instruments ® ). For this, 5.0 mg of bulk lipid or freeze-dried samples of hesperitin-loaded NLC (F4 and F6) and unloaded NLC (F2) were weighed separately in a standard aluminum pan and hermetically sealed. An empty pan was used as the reference. The analyses were conducted under inert nitrogen atmospheric conditions at a flow rate of 50 mL min -1 and the samples were scanned at a constant rate of 5 °C min -1 from 20 to 120 °C. The crystallinity degree (CD) was calculated as shown in Eq. ( 1) [START_REF] Ott | Exploitation of amaranth oil fractions enriched in squalene for dual delivery of hydrophilic and lipophilic actives[END_REF][START_REF] Saad | Improved cardioprotective effects of hesperidin solid lipid nanoparticles prepared by supercritical antisolvent technology[END_REF]: CD (%) = (NLC melting enthalpy/melting enthalpy of the bulk lipid x lipid concentration in the NLC) x 100 (1)

Electron microscopy analyses

NLC formulations were analyzed using transmission electron microscopy (TEM)

technique. Aliquots (100 µL) of hesperitin-loaded NLC (F4) were put onto copper TEM grids and excess sample was removed by filter paper. After that, the samples were dried at room condition before analyses. Transmission electron micrographs of the samples were taken using a Microscope HT7700 Hitachi (Fukuoka, Japan) operated at an accelerating voltage of 80 KV and emission 12 µA.

Hesperitin quantification in NLC, entrapment efficiency, and drug loading

Hesperitin concentration was determined using a UV spectrophotometric method. In order to establish linearity of the proposed method three calibration curves were constructed at seven concentrations levels within the range of 3.0-15.0 µg mL -1 (n = 21).

Least square regression analysis was done for the data (Optical density = 0.074[hesperitin, µg mL -1 ] -0.027; r = 0.9987) and for all spectrophotometric analyses the Beer's law was obeyed. Hesperitin amount in NLC was assessed by dispersing a known amount of formulation in ethanol followed by centrifugation at 2,000 rpm for 10 min. Afterwards, hesperitin concentration in the supernatant was determined by UV spectroscopy conducted on an Even UV/VIS spectrophotometer (Ionlab, Paraná, Brazil) with detection at 285 nm. Free hesperitin content was determined by measuring the non-incorporated drug present in a clear ultrafiltrate obtained through separation of aqueous phase using an ultrafiltration/ultracentrifugation procedure (Microcon Ultracel YM-100, Millipore, Ireland) at 10,000 rpm for 1 h, at 4 °C (Eppendorf, Centrifuge 5430 R, Hamburg, Germany). All analyses were performed in triplicate and the entrapment efficiency (E.E.)

of hesperitin into NLC and drug loading (D.L.) were calculated from following equations:

E.E. (%) = (THet -FHet/ThHet) x 100 (2)
Where, THet is the total hesperitin concentration; FHet, free hesperitin concentration;

ThHet, theoretical hesperitin concentration. D.L. (%) = (Het encapsulated into NLC/amount of weighed NLC) x 100 (3)

In vitro release of hesperitin from NLC

In vitro release experiments were carried out using the dialysis technique and under sink conditions. An aliquot (4.0 mL) of hesperitin-loaded NLC (F4) or free hesperitin (hydroethanolic dispersion 10 % v/v) was placed inside a dialysis bag (cellulose membrane, molecular weight cut-off 14,000, Sigma-Aldrich Co., St. Louis, MO), sealed, and immersed in a vessel containing 125 mL of 10 mM phosphate buffer solution (PBS pH 7.4). The releasing system was maintained at 37 ± 1 ºC, under magnetic stirring (100 rpm). One-milliliter aliquots were taken out of the dissolution medium at pre-determined time intervals, replaced with fresh buffer solution (PBS), and analyzed for hesperitin release by spectrophotometry UV at 285 nm. Release profile of hesperitin was expressed as cumulative amount of released hesperitin (average ±SEM) and plotted versus time. The experiments were carried out in triplicate for both free and encapsulated hesperitin.

Cell culture and cytotoxicity assay

Human T98G glioblastoma cell line (ATCC CRL-1690) was cultivated in Dulbecco's Modified Eagle's Medium-Low (DMEM-LG; Gibco BRL, USA) supplemented with 2 mmol L -1 glutamine, 100 U mL -1 penicillin, 100 µg mL -1 streptomycin, 2.5 µg mL -1 amphotericin B, and 10 % fetal bovine serum (FBS) at 37 °C and 5 % CO2. In order to assess in vitro cytotoxic effect of free and nanoencapsulated hesperitin (F4 formulation) on glioblastoma cells, different concentrations of this flavanone were tested between 1.65 and 36.39 µM. T98G cells were seeded in 96-well plate at density of 5×10 3 cells per well.

After overnight adhesion, cells were treated with free hesperitin or hesperetin-loaded NLC for 24 h, following the cell treatment every well was washed with PBS (pH 7.4) and proliferation MTT method was applied.

Cell MTT proliferation method [START_REF] Denizot | Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability[END_REF] was employed for assessment of T98G cell viability after cytotoxicity assay. Briefly, aliquots of 20 μL MTT solution (5 mg mL -1 ) and 180 μL medium without phenol red were added to each well. The cells were then incubated for 4 h, at 37 °C and 5 % CO2 to allow the formazan-forming reaction. Following incubation, the medium containing MTT solution was removed, and formed formazan crystals were dissolved in 2-propanol. Optical density was measured at 570 and 690 nm using a Biotek Cytation 5 -Cell Imaging Multi-Mode Reader (Winooski, VT, USA). As positive controls T98G cells were incubated with unloaded NLC diluted at 2.0 % (v/v) or cell culture medium containing DMSO 0.5 % (v/v), while as a negative control, T98G cells were incubated only with cell culture medium. Results are presented as percentage of survival, taking the negative control (untreated cells) as 100 %.

Statistical analysis

All the experiments were performed in triplicate and data are expressed as average value (±SEM). Statistical analysis was performed by One-way ANOVA followed by Tukey's pairwise comparisons using the Prism GraphPad Software. The statistical significance was set at p < 0.05.

Results and discussion

Phenolic compounds present in different plants (fruits, vegetables, legumes, etc.) are the main bioactive molecules related to functional properties such as antioxidant, anti-inflammatory, antimicrobial, antiviral, and antineoplastic. However, biomedical application of these molecules has been adversely affected by their low aqueous solubility and poor stability in physiological environment resulting in a limited bioavailability. In order to overcome such limitations, several drug delivery systems have been developed

to encapsulate flavonoids, coumarinea, stilbenes, tannins and among these systems are nanoemulsions, microemulsions, liposomes, polymeric nanoparticles, and lipid nanoparticles [START_REF] Faridi Esfanjani | Improving the bioavailability of phenolic compounds by loading them within lipid-based nanocarriers[END_REF][START_REF] Estanqueiro | Nanotechnological carriers for cancer chemotherapy: The state of the art, Colloids Surf[END_REF][START_REF] Mcclements | Delivery by Design (DbD): A Standardized Approach to the Development of Efficacious Nanoparticle-and Microparticle-Based Delivery Systems[END_REF][START_REF] Bonifácio | Nanotechnology-based drug delivery systems and herbal medicines: a review[END_REF]. In this study, we prepared nanostructured lipid carriers containing hesperitin using a relatively simple and low-cost technique based low-energy emulsification approach, i.e. PIT method. Nanostructured lipid carrier formulations were characterized and the in vitro cytotoxic effect of NLC containing hesperitin was assessed on T98G glioblastoma cells.

NLC characterization, stability study, electron microscopy, and thermal analysis

Average size, PdI, zeta potential, and pH values of NLC formulations tested are reported in Table 2. Both hesperitin-loaded NLC and unloaded NLC formulations varied in appearance from translucent to slightly turbid with bluish reflection typical of very fine colloidal dispersions [START_REF] Mcclements | Nanoemulsions versus microemulsions: terminology, differences, and similarities[END_REF]. This appearance could be attributed to poor light scattering by the relatively small lipid particles (r < 100 nm) in formulations since all the NLC dispersions tested showed an average particle diameter below 80 nm (Table 2). In addition, both hesperitin entrapment into NLC and different dispersant phases used (PW or PBS) here had only a minor effect on particle size even at the highest concentration of this flavanone (p < 0.05). Likewise, loaded and unloaded NLC formulations showed low PdI values (< 0.2) with a single peak in particle size distribution characteristic of homogeneous dispersions. In fact, F4 formulation showed a PdI value about 0.1 characterized by unimodal narrow size distribution (Fig. 1) confirming the highest quality of this colloidal dispersion. As reported by Bhattacharjee [START_REF] Bhattacharjee | DLS and zeta potential -What they are and what they are not?[END_REF], colloidal dispersions with PdI values lower or equal to 0.1 are considered highly monodisperse while those with PdI values between 0.1 and 0.4 are moderately polydisperse. Therefore, the combination of SMO and HCO-40 nonionic surfactants resulted in achievement of very small homogeneous lipid particles (< 100 nm) using a thermal method of emulsification. The formation of small lipid particles using PIT method is closely associated with changes in the nonionic surfactant structure as well as in its physicochemical characteristics, which occur during heating. At low temperatures, surfactant head-groups are highly hydrated and become gradually dehydrated under heating. All these temperature-dependent changes result in alterations of surfactant water solubility, along with its optimum curvature at interfacial monolayer. Above the PIT, the surfactant is mainly located in the organic phase and around the PIT there is similar affinity of surfactant between organic phase and aqueous phase, leading formation of bicontinuous microemulsion with very low interfacial curvature. The rapid cooling (< PIT) of emulsified system results in microemulsion disruption and consequent surfactant movement from organic phase to aqueous phase forming very fine lipid particles [START_REF] Anton | The universality of low-energy nano-emulsification[END_REF][START_REF] Gao | Formation and stability of solid lipid nanoparticles fabricated using phase inversion temperature method[END_REF][START_REF] Carbone | Mediterranean essential oils as precious matrix components and active ingredients of lipid nanoparticles[END_REF][START_REF] Chuesiang | Optimization of cinnamon oil nanoemulsions using phase inversion temperature method: Impact of oil phase composition and surfactant concentration[END_REF]. colloid particles preventing rapid agglomeration and sedimentation or flotation [START_REF] Bhattacharjee | DLS and zeta potential -What they are and what they are not?[END_REF].

Stability studies were carried out for unloaded NLC (F2), F4, and F6 formulations using analytical photocentrifugation method. This method is based on measurement of particle migration and aggregation from colloid dispersions by application of a centrifugal force and photometric detection. The kinetics of the particle separation process is characterized by shape and progression of light transmission profiles measured as a function of time and position across the entire sample length. The first and the last registered profiles are shown in red and in green, respectively. A total of 255 transmission profiles were recorded in intervals of 65 s. As can be observed in Fig. 1, there were no significant differences in light transmission profiles among lipid dispersions (F2, F4, and F6) during all analysis time and throughout sample. Moreover, high transmittances (about 85 %) for NLC samples were maintained during analysis, i.e. there were no instability signals such as particle agglomeration, sedimentation/flotation of dispersed phase, or visible phase separation. Moreover, F4 formulation presented distribution (D) of particle migration velocity at D10 %, D50 %, and D90 % of 9.169, 9.391, and 10.163 µm s -1 , respectively, and mean velocity 9.591 µm s -1 (±0.392). Such narrow velocity distribution, i.e. only slight speed variation of particle migration between D10 % and D90 %, indicates good physical stability of hesperitin-loaded NLC. According to these results, it could be to claim that encapsulation of this flavanone into NLC was not able to alter physical stability of lipid formulations even at the highest flavanone concentration. Therefore, results obtained from NLC stability study could be mainly explained due to gravitational stability of nanoparticles, i.e. smaller particles (< 90 nm) in these dispersions may be under influence of Brownian motion only [START_REF] Mcclements | Delivery by Design (DbD): A Standardized Approach to the Development of Efficacious Nanoparticle-and Microparticle-Based Delivery Systems[END_REF][START_REF] Mcclements | Nanoemulsions versus microemulsions: terminology, differences, and similarities[END_REF]. In general, NLC produced herein could remain stable for 12 months when stored at 25 °C.

DSC analyses were performed for bulk glyceryl behenate powder and freeze-dried formulation samples (F2, F4, and F6). Fig. 2 shows the DSC heating thermographs ranging from 20 to 120 °C and Table 3 summarizes the data extracted from calorimetric thermograms. During heating, for all samples, main peaks were centered between 67.0 and 72.0 °C. Bulk glyceryl behenate exhibited an endothermic peak at 71.9 °C, this melting temperature is characteristic of the most stable form of glyceryl behenate, i.e. β' polymorphic form [START_REF] Goto | Application of aluminum chloride phthalocyanine-loaded solid lipid nanoparticles for photodynamic inactivation of melanoma cells[END_REF][START_REF] Carvalho | Optimization of -tocopherol loaded solid lipid nanoparticles by central composite design[END_REF]. On the other hand, for lipid nanocarriers (F2, F4, and F6) were observed less intense endothermic peaks with lower melting temperatures than the bulk lipid. Therefore, the main components of NLC formulation (lipids, surfactants, and hesperitin) altered crystallinity degree of solid lipid matrix as compared to pure glyceryl behenate (Table 3). It has been widely reported that the addition of surfactant(s) and liquid lipid(s) to NLC formulation as well as when transforming bulk lipid in nanoparticle might result in melting point depression and broadening peaks. These events could occur due to interactions between solid lipid(s) and liquid lipid(s) and/or surfactants [START_REF] Gordillo-Galeano | Solid lipid nanoparticles and nanostructured lipid carriers: A review emphasizing on particle structure and drug release[END_REF][START_REF] Goto | Application of aluminum chloride phthalocyanine-loaded solid lipid nanoparticles for photodynamic inactivation of melanoma cells[END_REF][START_REF] Carvalho | Optimization of -tocopherol loaded solid lipid nanoparticles by central composite design[END_REF].

Moreover, the reduction of onset, enthalpy, melting temperature, and crystallinity degree from F2, F4, and F6 formulations suggests also that hesperitin was mostly entrapped into lipid core of nanocarriers affecting crystallization process of solid lipid. It is worth mentioning that this change in lipid matrix from NLC might enhance drug entrapment efficiency decreasing expulsion of drug during storage when compared to SLN [START_REF] Gordillo-Galeano | Solid lipid nanoparticles and nanostructured lipid carriers: A review emphasizing on particle structure and drug release[END_REF][START_REF] Khosa | Nanostructured lipid carriers for site-specific drug delivery[END_REF][START_REF] Salvi | Nanostructured lipid carriers (NLC) system: A novel drug targeting carrier[END_REF].

In view of results from physicochemical characterization, stability study, and thermal analysis described above, F4 NLC formulation was chosen to further assays due to its better stability profile as well as this formulation was prepared with PBS (pH7.4) as aqueous phase, which is an isotonic solution and non-toxic to most cells. Transmission electron microscopy (TEM) has been widely applied to characterize particle matrix structure and diameter of nanostructures [START_REF] Salvi | Nanostructured lipid carriers (NLC) system: A novel drug targeting carrier[END_REF]. TEM analyses were carried out for F4 NLC formulation and micrographs are shown in Fig. 3. According to photomicrographs, there was no particle aggregation and lipid nanoparticles had almost spherical shapes with a particle size of less 100 nm and homogeneous size distribution.

These findings are in accord with earlier results from particle size analyses using photon correlation spectroscopy. Moreover, the matrix from lipid core was highly heterogeneous presenting lipid cluster domains compatible with multiple oil-in-solid fat-in-water type of NLC [START_REF] Khosa | Nanostructured lipid carriers for site-specific drug delivery[END_REF][START_REF] Salvi | Nanostructured lipid carriers (NLC) system: A novel drug targeting carrier[END_REF]. It is worth noting that the drying process and imperfect crystallization of lipids might result in sample deformation.

Hesperitin quantification, entrapment efficiency, and in vitro release study

Quantitative analyses of hesperitin into NLC formulation were carried out using UV spectrophotometric method. F4 NLC formulation was assessed for total amount, entrapment efficiency, drug loading, and in vitro release of hesperitin. Total concentration of this flavanone found for NLC formulation was 0.55 mg mL -1 (±0.01). Meanwhile, entrapment efficiency of hesperitin into NLC and drug loading reached 72.7 % (±0.92) and 3.48 % (±0.17), respectively, confirming a better capacity of this kind lipid dispersion for encapsulation of hydrophobic drugs.

Dialysis bag diffusion technique was chosen to assess and compare hesperitin release from hydroethanolic dispersion (free flavanone) and NLC formulation. Release profiles of free and encapsulated hesperitin in PBS pH 7.4 are shown in Fig. 4. Comparing both release profiles, free hesperitin was released 2.5-fold faster than flavanone entrapped into lipid nanocarriers, and thus it was observed a sustained release profile of hesperitin from NLC formulation. Almost all content of free hesperitin (90 %) was released in the first 24 h of essay while from NLC after 72 h only 63 % (±2.0) of hesperitin were released.

Moreover, it was not noticed a burst release of hesperitin from NLC at initial phase demonstrating that there was no drug adsorbed to particle surface. These results propose that hesperitin might be entrapped into lipid core of NLC, which results in slow drug release from formulation. The release profile of hesperitin from NLC in PBS pH 7.

In vitro cytotoxicity assessment

The in vitro cytotoxic effect of free hesperitin and hesperitin-loaded NLC was assessed on T98G glioblastoma cells following 24 h exposure to flavanone concentrations ranging from 1.65 to 36.39 µM. The results are presented in Fig. 5.

For both free and encapsulated hesperitin, T98G cell viability decreased in a dose dependent manner. After 24 h of treatment of glioblastoma cells, there was no evidence of cytotoxic effect for free hesperitin and hesperitin-loaded NLC at flavanone concentrations below 33.08 µM and 26.47 µM, respectively, as compared with negative control cells (NC). However, at higher concentrations (33.08 and 36.39 µM) of encapsulated hesperitin, the T98G cell viability was significantly affected with a reduction in viable cell percentage levels for less than 10 % in comparison untreated cells. In addition, hesperitin-loaded NLC demonstrated also higher cytotoxic activity as compared to free hesperitin at 29.77, 33.08, and 36.39 µM. Therefore, the NLC formulation containing hesperitin led to a great decrease in cell viability at the highest flavanone concentrations tested. Yoo et al. [START_REF] Yoo | Constituents of the Fruits and Leaves of Euodia daniellii[END_REF] assessed in vitro cytotoxicty of hesperidin isolated from Euodia daniellii on XF-498 cell line (human CNS solid tumor) and IC50 found was higher than 30 µM following 48 h exposure to this glycoside. Recently, Ersoz et al. [START_REF] Ersoz | Comparative evaluation of hesperetin loaded nanoparticles for anticancer activity against C6 glioma cancer cells[END_REF] assessed anticancer activity of hesperitin-loaded polymeric nanoparticles against C6 glioma cells and IC50 of hesperitin loaded nanoparticles was 2.0-fold lower than free hesperitin as well as dose-and time-dependent. Thus all these findings revel that encapsulation of hesperitin into nanoparticles could enhance cytotoxic effect on neoplastic cells. This improved antitumor efficacy of hesperitin combination in polymeric or lipid nanoparticles might be mainly related to better internalization of these nanocarriers by cells [START_REF] Ersoz | Comparative evaluation of hesperetin loaded nanoparticles for anticancer activity against C6 glioma cancer cells[END_REF].

Conclusions

In summary, we have successfully encapsulated hesperitin into NLC formulation using PIT method, which is a low-energy emulsification approach. TEM analyses were in accord with PCS results showing that the nanoparticles obtained had almost spherical shapes with a heterogeneous lipid matrix structure and diameter below 100 nm.

Accelerated stability study confirmed the good physical stability of F4 NLC formulation with minimal variation in lipid particle migration rate. In addition, F4 formulation reached an encapsulation efficiency of hesperitin about 70 % as well as the in vitro release assay demonstrated that this flavanone could be released in a sustained manner from NLC.

Cytotoxicity study revealed that hesperitin-loaded NLC was able significantly to reduce cell viability of T98G cells at 33 µM of flavanone when compared to free hesperitin.

Therefore, these results might confirm NLC formulation as an effective flavanone delivery system for application in different treatments. Moreover, such findings encourage further studies on NLC containing flavonoids for use in the therapeutics protocols against other neoplastic cell lines.

Figure 1 .

 1 Figure 1.

Figure 2 .

 2 Figure 2.

Figure 3 .

 3 Figure 3.

  [START_REF] Khan | A comprehensive review on flavanones, the major citrus polyphenols[END_REF] was kinetically evaluated by zero-order (r 2 = 0.9616), first-order (r 2 = 0.7613), Higuchi (r 2 = 0.9964), and Rigter-Peppas (r 2 = 0.8488) models. The best-fit model was Higuchi that describes the hesperitin release from NLC formulation as a process controlled by Fickian diffusion mechanism.

Figure 4 .

 4 Figure 4.

Figure 5 .

 5 Figure 5.

Table 1 . Composition (wt %) of NLC formulations.

 1

	NLCs	MCT	GB	Het	PW	PBS
	F1	5.0	5.0	-	80	-
	F2	5.0	5.0	-	-	80
	F3	5.0	5.0	0.0625	80	-
	F4	5.0	5.0	0.0625	-	80
	F5	5.0	5.0	0.1875	80	-
	F6	5.0	5.0	0.1875	-	80
	MCT: medium chain triglycerides; GB: glyceryl behenate; Het: theoretical hesperitin
	concentration; PW: purified water; PBS: 10 mM phosphate buffer solution pH 7.4.

Table 2 . Particle size, polydispersity index (PdI), zeta-potential, and pH of NLC formulations (average  SEM, n = 3).

 2

			ζ potential
	NLCs	Size (nm)	PdI	pH
			(mV)
	F1	77.52 (±1.07)	0.131 (±0.005) -19.70 (±1.14) 7.94 (±0.08)
	F2	69.75 (±2.19)	0.122 (±0.030) -24.80 (±0.15) 7.93 (±0.09)
	F3	66.82 (±0.58)	0.187 (±0.017) -17.80 (±1.69) 6.25 (±0.05)
	F4	66.17 (±1.34)	0.099 (±0.004) -20.43 (±0.49) 6.34 (±0.11)
	F5	76.74 (±0.99)	0.140 (±0.003) -17.20 (±0.44) 7.51 (±0.04)
	F6	63.90 (±0.49)	0.079 (±0.009) -19.07 (±0.92) 6.98 (±0.09)

Table 3 . Parameters extracted from the DSC thermograms of the bulk glyceryl behenate and NLC dispersions.

 3

	Samples	Melting temperature (ºC)	Onset (ºC)	Enthalpy (J g -1 )	Crystallinity degree (%)
	bulk GB	71.9	68.8	-136.3	100.0
	F2	67.8	64.0	-83.1	61.1
	F4	67.7	65.3	-80.7	59.2
	F6	66.9	63.0	-22.1	16.2

Acknowledgements

This study was supported by CAPES/COFECUB agreement (#Ph-C 859-15). M.P.S.M. and T.D.H. thank Science and Technology Support Foundation of Pernambuco State (FACEPE #BIC-3173-4.03/16). A.C.T. thanks the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES), the State of São Paulo Research Foundation (FAPESP) Thematic project #2013/50181-1 & Project FINEP 01.10.0758.01, and the National Council for Scientific and Technological Development (CNPq) for their fi013/501 support Project PRONON-SIPAR #25000.077093/2015-86. H.L.P thanks FAPESP project #2017/16356-0 and Project PRONON-SIPAR #25000.077093/2015-86. We also thank the National Institute of Science and Technology (INCT) of Nanobiotechnology project #573880/2008-5 for their financial support.


Vincent Rey
email: rey@univ-tln.fr

Christiane Dufresne

Jean-Luc Fuda

Didier Mallarino

Tathy Missamou

Caroline Paugam

Gilles Rougier

Isabelle Taupier- Letage

On the use of long term observation of water level and temperature along the shore for a better understanding of the dynamics: Example of Toulon area, France

Keywords: Observation network, water level, temperature, tide, extreme water level, seiching, upwelling, water mass mixing

A dense network of instruments has been deployed within harbours along the Mediterranean coast, in the Toulon Metropole area, between the Hyères islands and the Sanary Bay in the framework of the observation network HTM-NET. Each station is equipped with two piezometric sensors, the first immersed and the second emerged, which allows the calculation of the water level. Both piezometric sensors are also equipped with a temperature sensor. Water level and temperature data are analyzed and discussed, also considering meteorological data provided by Météo-France stations. The tide gauges provide information about tide harmonic components, extreme water level and seiching. Moreover, significant differences are observed between sheltered zones in enclosed bays and offshore zones, such as between the back of the Bay of Toulon and at the Port-Cros Island. Differences in water level up to 0.10m are indeed observed under windy conditions, of same order as the tidal range (order of 0.20m) or the annual level variability due to the volumetric expansion (order of 0.10m). Water level variations, up to about 1m, are found to be mainly due to atmospheric effects, with a more or less isostatic behavior according to the weather events. In addition, seiching with an amplitude of few centimeters is observed within the Little Bay of Toulon, for east wind conditions. The near-surface water temperature is measured at the submerged piezometer location (depth of immersion range 0.10m -1.80m according to the station and to the water level). The analysis of the temperature associated with the weather conditions allows to detail a strong variability of the upwelling intensity under Mistral wind conditions in summer, leading to more or less pronounced temperature drops according to the shore configuration. The Bay of Toulon is more prone to the generation of upwellings than the neighboring bays. During winter, water exchanges between the Little Bay of Toulon and offshore are also clearly observed during windy conditions. The HTM-NET long-term observation network thus provides useful insights to increase our knowledge of the hydrodynamics and mass fluxes, and therefore enhances our modeling capacity and risk assessment at the scale of a bay.

Introduction

The Mediterranean and in particular its coastal zones are exposed to natural and man-made hazards. Coastal vulnerability is exacerbated by climate change, leading to the development of risk analysis plans (see e.g. [START_REF] Nicholls | The Mediterranean: vulnerability to coastal implications of climate change[END_REF][START_REF] Satta | Assessment of coastal risks to climate change related impacts at the regional scale: The case of Mediterranean region[END_REF]. The study of coastal dynamics is often complex, and must integrate both circulation at the basin scale and local forcing. Classical numerical approaches use nested models, which allow a detailed description of the dynamics at the bay scale with a high-resolution model, while offshore forcing at the open boundary is provided by a large scale model. This was done for instance for the Gulf of Taranto in Italy [START_REF] Gaeta | A coupled wave-3D hydrodynamics model of the Taranto Sea (Italy): a multiple-nesting approach[END_REF][START_REF] Federico | Coastal ocean forecasting with an unstructured grid model in the southern Adriatic and northern Ionian seas[END_REF], or the Bay of Toulon [START_REF] Dufresne | Wind forced circulation in the Bay of Toulon and water exchanges at the Little Bay fairway[END_REF]. Such numerical approaches are validated in the coastal zone of interest by the use of data including velocity fields, surface current, water temperature profiles, sea level, often collected at the seasonal scale. Water level at the shore, in the same way as the dynamics at the Western Mediterranean scale, are characterized however by a pluri-annual variability, due to both weather conditions at the Mediterranean basin scale and the North Atlantic dynamics [START_REF] Tsimplis | The forcing of mean sea level variability around Europe[END_REF]. Coastal water levels have been measured for many decades by tide gauges, supplemented at the scale of the ocean basins by satellite measurements (see e.g [START_REF] Vigo | Mediterranean Sea level variations: Analysis of the satellite altimetric data, 1992-2008[END_REF]. While satellite measurements allow a detailed study of the water level variability at seasonal or multiannual scales, measurements in the coastal fringe remain a challenge despite recent improvement in the spatial resolution, that allows small-scale observation of ocean dynamics [START_REF] Morrow | Observability of fine-scale ocean dynamics in the northwestern Mediterranean Sea[END_REF]. Sea level at the coast have then to be validated with a few tide gauge, located in major ports, most often several hundred kilometers apart in the Western Mediterranean [START_REF] Bonaduce | Water level variability in the Mediterranean sea from altimetry and tide gauges[END_REF].

In the Western Mediterranean, the tidal range is generally low, and most of the water level variations at the scale of several days are then related to the atmospheric pressure variations associated to weather events. The barometric factor, which is the ratio between the water level and the atmospheric pressure variations is generally of -1cm/hPa. Significant differences may however exist. They can be due to long wave propagation over continental shelves, as pointed out by Hamon (1956). They are also observed in semi-enclosed basins [START_REF] Garrett | Variable water level and straight flow in the Mediterranean : A theoretical study of the response to meteorological forcing[END_REF][START_REF] Tsimplis | Meteorological forcing and water level variability in the Aegean Sea[END_REF]. In the Mediterranean for instance, the water level variation may not follow the atmospheric pressure variations without the conjunction of currents within straights or passages. Such level variations may lead to flooding and erosion, with possible damages in anthropized areas. Many studies have been carried-out to better understand the sea-level variation at the scale of the Mediterranean basin, including tide gauges and altimetric data and numerical modelling [START_REF] Tsimplis | A two-dimensional tidal model for the Mediterranean sea[END_REF][START_REF] Fenoglio-Marc | Long-term water level change in the Mediterranean Sea from multi-satellite altimetry and tide gauges[END_REF]Vigo et al, 2005;[START_REF] Oddo | Sensitivity of the Mediterranean water level to atmospheric pressure and free surface elevation numerical formulation in NEMO[END_REF][START_REF] Bonaduce | Water level variability in the Mediterranean sea from altimetry and tide gauges[END_REF]. For a better insight of coastal risks such as flooding or erosion, nested models including global and regional circulation, representing atmospheric and oceanic dynamics are proposed. They may be used to produce sea-level rise hazard scenarios as done by [START_REF] Torresan | Assessment of Climate Change Impacts in the North Adriatic Coastal Area. Part I: A Multi-Model Chain for the Definition of Climate Change Hazard Scenarios[END_REF] for the North Adriatic Sea coast, characterized by a large tide range and prone to frequent storm surge flooding.

The North Western Mediterranean circulation along the coast is governed by the Northern Current, with a flow rate of about 1 Sverdrup (10 6 m 3 •s -1 ) [START_REF] Millot | Circulation in the Mediterranean sea[END_REF]. Originating between North Corsica and the gulf of Genoa, it follows the continental slope up the Hyères islands, where its dynamics becomes more complex due to both a complex bathymetry and the beginning of the continental shelf of the Gulf of Lions at the west of Toulon [START_REF] Guihou | Étude de la dynamique du Courant Nord au large de Toulon, à l'aide de modèle[END_REF][START_REF] Guihou | A case study of the mesoscale dynamics in the North-Western Mediterranean Sea : combined data-model approach[END_REF][START_REF] Taupier-Letage | Les îles d'Hyères dans le système de circulation marine et atmosphérique de la Méditerranée[END_REF][START_REF] Berta | Wind-induced variability in the Northern Current (northwestern Mediterranean Sea) as depicted by a multi-platform observing system[END_REF]. The tidal current is weak since the mean tidal range is low, approximately 0.20 m [START_REF] Albérola | Tidal currents in the Western Mediterranean Sea[END_REF]. Its variability is due to both the dynamics at the scale of the Western Mediterranean basin and the weather conditions on the northern coasts. The low Rossby number, of the order of 5 to 15 km and variable according to the stratification [START_REF] Grilli | The computation of Rossby radii of deformation for the Mediterranean Sea[END_REF], requires a refinement of the large scale models and the observation of fine-scale dynamics for its validation. Morrow et al (2018) recently studied small-scale circulation in the Northwestern Mediterranean Sea from long-term observations of the sea level and the sea surface temperature (SST) from satellite data, and coastal surface currents from VHF radar, associated with salinity and velocity measurements in the water column from gliders deployed offshore. Promising results were obtained concerning the capture of fine-scale dynamics, even if further developments were proposed in the coastal band to separate geostrophic and ageostrophic current components. Indeed, wind conditions play an important role in the variability of the upper part of the Northern Current as observed by [START_REF] Berta | Wind-induced variability in the Northern Current (northwestern Mediterranean Sea) as depicted by a multi-platform observing system[END_REF] during strong westerly wind events. During summertime (stratified conditions), upwelling cells develop in some specific places of the gulf of Lions, which induce episodically a strong cooling of water near the coast, as already reported fourty years ago by [START_REF] Millot | Wind induced upwellings in the Gulf of Lions[END_REF]. These water mass dynamics play an important role in the water mass exchanges along the coast, especially within the bays, as reported by [START_REF] Dufresne | Wind forced circulation in the Bay of Toulon and water exchanges at the Little Bay fairway[END_REF]2018) for the Bay of Toulon, an urbanized and industrial center with pollution hazards. Water temperature is also a key parameter to monitor, since its evolution (increase) with climate change is expected to impact the marine life. Large-scale positive temperature anomalies already occurred in the northwestern Mediterranean Sea in 1999[START_REF] Albérola | Circulation in the French mediterranean coastal zone near Marseilles: the influence of wind and the Northern Current[END_REF]and 2006(Pairaud et al, 2014 and references cited). These anomalies were associated with mass mortality events of macrobenthic species in coastal areas (0-40 m in depth). In addition, recent studies highlight the significant contribution of the SST to heavy precipitation events and heat waves in Europe, as mentioned by [START_REF] Pastor | Sea Surface Temperature in the Mediterranean: Trends and Spatial Patterns (1982-2016[END_REF], in their detailed analysis of daily SST data series derived from satellites. However, such a data only gives information on the surface layer, and are noisy and hardly exploitable in the nearshore. The deployment of CTD probes allows measurements of vertical temperature and salinity profiles. They are generally used during field campaigns, and adapted to either ocean or coastal measurements. Measurements in deep water conditions have been also provided by drifting autonomous instruments since the 1980's, as in the framework of the MedArgo program [START_REF] Poulain | MedArgo: a drifting profiler program in the Mediterranean Sea[END_REF]. Glider transects have also been used more recently in the Western Mediterranean, as reported for instance by [START_REF] Berta | Wind-induced variability in the Northern Current (northwestern Mediterranean Sea) as depicted by a multi-platform observing system[END_REF]. In the nearshore, temperature sensors are often available on wave buoys or currentmeters. Characterization of local temperature and stratification is very important for a better understanding and modelling of the interannual variability in the nearshore. A high resolution temperature time series is provided by the T-MEDNet data base [START_REF] Bensoussan | T-MEDNet observation network and resource platform on climate change effects in Mediterranean coastal ecosystems[END_REF] for coastal waters in the range 5-40m spanning more than 10 years, mainly along the shores of the northern half of the Mediterranean. The seasonal and multiannual variability observed from the large scale circulation to the scale of the bay demonstrates the need to collect long term time series.

Toulon is located on the north coast of the Western Mediterranean (Fig. 1). The Toulon area is exposed to the threat of the sea level increase with climate change, particularly towards the peninsula of Giens [START_REF] Mavromatidi | Mapping and analyzing socio-environmental vulnerability to coastal hazards induced by climate change: An application to coastal Mediterranean cities in France[END_REF]. It is also an urbanized zone, with varied socio-economic activities. Recent studies have focused on the dynamics of the Bay of Toulon for socio-economic purposes [START_REF] Dufresne | Wind forced circulation in the Bay of Toulon and water exchanges at the Little Bay fairway[END_REF]2018). During these latest studies, some questions arose about the possible effects of the water level differences at the scale of the bays on induced flows. Indeed, if wind-forced mass-flux between the Little Bay of Toulon and offshore were clearly observed and modelled, significant currents at the entrance of the Little Bay are sometimes also observed under calm wind conditions [START_REF] Dufresne | Compréhension et analyse des processus hydro-sédimentaires de la baie de Toulon. Apport à la modélisation de la dispersion des radionucléides[END_REF]. If sea level data has been collected for a long time by the tide gauge of Toulon, located at the back of the Little Bay (see Fig. 1), no other information on the water level variability was available in the area of interest, since tide gauges are only few and restricted to the Mediterranean main harbors (see e.g. [START_REF] Bonaduce | Water level variability in the Mediterranean sea from altimetry and tide gauges[END_REF]. Since strong upwelling events are also observed in the area of Toulon, information on sea surface temperature also appears as a key parameter to better understand the water circulation and mass exchanges at the bay scale. It was then decided to develop a dense network of instruments for the long term measurement of water level and near-surface temperature within and around the Bay of Toulon.

The aim of the present paper is to give an overview of the potentiality of such a long-term dataset to provide insights on the variability of the water level and of the near-surface temperature, from one bay to another, on time scales ranging from the weather episode to the season, and up to the interannual variability. The area of study is presented in section 2, the network in section 3. Water level and nearsurface temperature changes are presented and analyzed in sections 4 and 5 respectively. Discussion and conclusion are given in section 6.

Description of the study area

East of the Sanary bay the continental shelf is very narrow (Fig. 1), so that the Northern Current, guided by the continental slope (upper part at ~200m), flows close to the coast. Westward, the Northern Current follows the continental slope off the Gulf of Lions, while a superficial branch often continues inside the gulf and follows the coastline. Mesoscale dynamics is mainly forced by the Northern Current instability, more intense in wintertime (e.g. [START_REF] Millot | The structure of mesoscale phenomena in the Ligurian Sea inferred from the DYOME Experiment[END_REF]. Its width can vary due to the meanders it generates, and that propagate downstream (see Fig. 8 in [START_REF] Taupier-Letage | Les îles d'Hyères dans le système de circulation marine et atmosphérique de la Méditerranée[END_REF]Fig. 7 in Van Haren et al., 2011). In addition, due to the abrupt bathymetry near the Hyères islands, meanders may form downstream the islands [START_REF] Berta | Wind-induced variability in the Northern Current (northwestern Mediterranean Sea) as depicted by a multi-platform observing system[END_REF], which may affect the nearshore circulation. The coastline in the vicinity of Toulon, France, presents many types of configurations including open or semi-enclosed bays of intermediate or shallow water, peninsulas, and islands. The strongly anthropized area of Toulon Metropole neighbors the preserved area of the National Park of Port-Cros. Socioeconomic activities and interests are various and sometimes conflicting, with fisheries and a strongly developed tourism on one hand, and military, Toulon being France's largest naval port, or civilian maritime economic activities on the other hand.

There are two main prevailing winds: the Mistral, a cold and dry north-westerly wind which blows 40% of the time, and the East Wind, which is warmer and more humid. Calm weather with low wind speed (< 5m•s -1 ) occurs 20% of the time. Originating offshore, the East wind is usually accompanied by clouds, rain and swell, it blows less frequently during summer and corresponds to low atmospheric pressure conditions. The fall and winter seasons are often characterized by a succession of strong Mistral and East wind events. As described by [START_REF] Dufresne | Wind forced circulation in the Bay of Toulon and water exchanges at the Little Bay fairway[END_REF], the circulation within the Bay of Toulon is mainly wind-driven, with several episodes of either upwelling or downwelling according to the wind direction. Such a water mass dynamics has also been described at other locations on the French Riviera such as Cassis [START_REF] Albérola | Circulation in the French mediterranean coastal zone near Marseilles: the influence of wind and the Northern Current[END_REF] or Marseille (Pairaud et al. 2011). Toulon is surrounded by rugged hills that influence weather conditions, particularly rainfall and wind speeds and directions. Several weather stations, operated by Météo-France, the French national meteorological and climatological service, are therefore installed in the area of interest. The wind data of the stations of Cépet, Toulon, Porquerolles and Levant (see locations in Fig. 1) are used in this study.

Toulon's port is equipped with a tide-gauge RONIM since March 1998, using a Radar gauge since 2008, located at the back of the Little Bay of Toulon (see Fig. 1). The observatory is managed by the hydrographic service of the Navy (Shom).

The HTM-NET network

Ten instruments compose the HTM-NET network in the Toulon Metropole area (HTM-NET network https://htmnet.mio.osupytheas.fr). Instruments are installed within harbors from the Island of Port-Cros to the Sanary Bay (see Fig. 1). The sites and their date of installation are given in Tab. 1. Two piezometric sensors, manufactured by the society Keller, measure both pressure and temperature. One sensor is immersed all the time (series 36XiW), the other one is aerial (series ARC1) (Fig. 2). The aerial sensor houses the GSM transmission system and the data storage unit, to prevent data loss in the event of the GSM malfunction. As of now, the memory capacity allows 65 days at a 10-minute sampling period. To date, 8 of the 10 stations are equipped with a GSM transmitter, which makes data available within few hours on the network's web site. Sensors are fixed on both ends and inside of a cylindrical tube made of PVC, fixed inside of a protection tube of diameter 0.15m made of aluminum (see Fig. 2). The PVC cylinder is removable to allow maintenance operations. The aluminium cylinder is fixed vertically next to piers or pontoons. Cylindrical tubes are 2m long, their depth of immersion slightly varies from one site to another according to the harbor pier height. The depth of immersion of the sensor is in the range 0.10m -1.80m according to the water level and the pier height.

Neglecting the pressure variation in the air column, the water level  relative to the lower pressure sensor location is given by = p/g, p being the difference between the pressure measured by both sensors,  the water density and g=9.81m.s -2 the acceleration due to gravity. For the processing, water density is fixed at 1025kg.m -3 , which corresponds to a salinity S=35 PSU and a temperature T=19°C. The variations of density from S=32 PSU to S=38 PSU, or of temperature from 14°C to 25°C, result in less than 2% (about 1mm) of uncertainty on the water level, which corresponds to the precision of the pressure measurements. Each piezometer is equipped with a temperature gauge (requested for the computation of the pressure), having a precision of 0.5°C. Data was collected every 10 min up to the beginning of the year 2019. Biofouling impact on the measurements is found to remain weak even on long periods, instruments are however cleaned at least once a year. The proximity between the La Seyne/Ifremer station and the Shom tide gauge initially made it possible to readjust the HTM-NET station data with respect to the French hydrographic reference NGF by comparing them with those of the Shom tide gauge, the latter being provided with respect to the zero NGF. For a precise sea level reference, HTM-NET station level data are referenced with respect to the French hydrographic reference NGF thanks to the French Navy hydrographic service Shom. Water level data are also available on the Shom REFMAR web site (tide reference, www.refmar.shom.fr).

Fig. 2. Stations configuration

As an example, time series for the hourly averaged water level (with respect to the NGF reference) and the hourly averaged temperature are presented in Fig. 3 for the station La Seyne/Ifremer. The observed water level and temperature variations at the network stations are discussed in detail in the next sections.

Site

Date of installation

Water level variations

Histograms of the water level data collected at stations La Seyne/Ifremer and Port-Cros are presented in Fig. 4. The maximum of occurrence corresponds more or less to the mean water level, 0.42m with respect to the NGF reference at the location of the tide gauge of the Shom in the harbor of Toulon (see Fig. 1). The difference between the lower and upper water levels is about 1m. The origins of such variations are discussed in the following subsections.

Fig. 5: Spectral analyses of water level series at La Seyne/Ifremer (IF)

Components K2, S2, M2, N2 for the semi-diurnal group and components K1, P1, O1, Q1 for the diurnal group are then respectively identified. Both amplitude and phase are then given by the Fourier coefficients for each of the corresponding frequencies from sufficiently long temporal series. Calculations are done for time series of 181.26 days, in order to distinguish components K2 and S2, on a moving window from 2014 to 2018. Tidal constituent coefficients and the standard deviation are reported in Tab. 2 for the stations of La Seyne/Ifremer and Port-Cros. Coefficients for the tide gauge of Toulon are also given, using the same time intervals for the calculations. Results from [START_REF] Wöppelmann | Rescue of the historal water level record of Marseille (France) from 1885 to 1988 and its extension back to 1849-1851[END_REF] for the tide gauge of Marseilles are also listed with a 95% confidence. These later results are based on the years 1941-1956, corresponding to the longest continuous period of time with highly stable yearly values of tidal amplitude. The rather high standard deviation for the results in the area of Toulon is mainly due to gaps in the time series. The amplitudes are similar in both sites, with a maximal tidal range of about 0.20 m, in phase matching conditions for the tidal constituents. Constituents M2, S2, N2 and K1 were reported as the main constituents by Alberola et al (1995). O1 is of however slightly higher than component N2, and [START_REF] Tsimplis | A two-dimensional tidal model for the Mediterranean sea[END_REF] have considered constituents O1, M2, S2 and K1 for their two-dimensional tidal model of the Mediterranean. The tidal signal measurement at the scale of the network does not exhibit any phase lags between the stations. It is due to the presence of standing waves in the western basin for both M2, S2 and N2 modes (Alberola et al, 1995;[START_REF] Lozano | The M2 tide in the Mediterranean Sea : dynamic analysis and data assimilation[END_REF][START_REF] Tsimplis | A two-dimensional tidal model for the Mediterranean sea[END_REF].

Amplitude (in cm)

Steric dilatation

As observed in Fig. 3, the water level gradually increases every year from March to November and then decreases more steeply during wintertime. These changes present some significant variations from one year to the other. If they are mainly due to steric dilatation, they are considered as semi-annual and annual harmonics tides, characterized by harmonic constants Ssa and Sa respectively. A rough estimate of the steric factor can then be determined from the station of La Seyne/Ifremer for which data span more than 4 years. The amplitude of the semi-annual and annual harmonics are 0.015 m and 0.058 m, respectively. This corresponds more or less to the annual evolution observed in Fig. 3 which is highly variable at the yearly scale. It is of the same order as yielded by the measurements from Perez et al (2014) at the old and the new stations of Ibiza, Spain (respectively (0.0194 m; 0.0526 m) and (0.0221 m; 0.0764 m) for (Ssa; Sa)).

Large scale meteorological forcing and water level anomalies

As shown in Figs. 3 and4, maximum water level variations are of order 1m, much higher than the tidal range of the Mediterranean in this area. As mentioned in the introduction, the barometric factor, the ratio between the water level and the atmospheric pressure variations, is generally of -1cm.hPa -1 , which corresponds to isostatic variations. Between low and high atmospheric pressure conditions, respectively of about 985 and 1035 hPa in this area, water level changes may be of about 0.50m. However, significant differences may exist. The Fig. 6 illustrates cases of isostatic and non isostatic behaviors of the water level in line with atmospheric pressure evolutions are presented in Fig. 6. Time series of both the atmospheric pressure and the water level are as recorded at the station La Seyne/Ifremer. Daily averaged data are presented on both schemes. We can observe an isostatic response of the water level from November 16 th to December 20 th , when an increase of the atmospheric pressure of about 30 hPa resulted in a decrease of the water level by 30cm. On the contrary, non isostatic behavior is observed afterwards, since atmospheric pressure variations of about 35 hPa result in a water level change of about 20cm. In order to quantify the co-variation of the water level with the atmospheric pressure, histograms of the water level anomaly are calculated for the water level series after filtering out both the tide and the steric contributions (see Fig. 7). Zero values correspond to isostatic variations. The residual level observed in Fig. 7 confirms that the isostatic conditions are not always fulfilled. We can conclude that high water level values corresponding to low pressure conditions can be roughly estimated, with an uncertainty of about 0.25m in the present, corresponding to the maximum of the residual level (see Fig. 7). One may notice that for low atmospheric conditions, the high water level is enhanced in autumn due to the steric effects discussed in section 4.2.

Meteorological forcing at local scale

Since HTM-NET stations are located in harbors, which are by definition sheltered, the water level variations do not include the wave set-up. However, they provide information about the wind influence on the water level at the scale of the network, including locations within enclosed bays (e. g. La Seyne/Ifremer, in the Little Bay of Toulon) and "offshore" in the Hyères islands. Wind conditions are provided by the meteorological stations of Cépet, Porquerolles and Levant, from west to east. (see Fig. 1). Corresponding three hours averaged wind speed and direction are presented in Figs. 8a and8.b respectively. Fig. 8c shows the comparison of daily averaged water levels at stations La Seyne/Ifremer and Port-Cros for the period ranging between August, 8 th and December, 12 th , 2016. Solid line rectangles indicate Mistral (northwesterly) episodes, and dotted line rectangles indicate East wind episodes. Mistral blows offshoreward in the Bay of Toulon, whereas the east wind blows shoreward. We can observe water level differences of few centimeters under both wind conditions between the two stations. Under Mistral conditions (resp. easterly), the water level at La Seyne/Ifremer is systematically lower (resp. higher) than in Port-Cros. This corresponds to additional water dragged by wind stress under offshoreward-(resp. shoreward-) blowing conditions. Wind conditions and hourly and daily averaged water levels are presented in Figs. 9 and 10 for the last Mistral and East wind events respectively. Differences of level can reach 0.10m as shown in Figs. 9c and10c for the last events of Mistral and East wind. However, the wind effect at the scale of the network, which is of the order of that of the tide, as shown by the hourly averaged level variations, remains small compared to the atmospheric pressure effect (~0.5m). Indeed, as shown in Fig. 8.c, the water level is always higher (resp. lower) for easterly (resp. Mistral) conditions, since they correspond to low (resp. high) atmospheric pressure conditions. This level difference is not always significant, as shown by the lack of variation during the east wind episode of September 28 th . As a general trend, this difference in water level is observed during windy periods, and results in a higher level in Port-Cros for Mistral events and a higher level in the Little Bay of Toulon (station La Seyne/Ifremer) during east wind events. The daily averaged water level difference (PC-IF) versus the mean wind speed measured at station Cépet is presented in Fig. 11, for wind events lasting at least two days for the period summer 2014 -autumn 2018. We can observe water level differences of few centimeters for both wind directions, up to almost 10cm for stronger wind conditions. Let us note that wind speed and direction may vary from west (station Cépet) to east (station Levant), and that the wind speed at Cépet is not fully representative of the averaged wind field over the entire study area. Hourly averaged water level differences may be higher depending on the wind event duration and regularity, as the values greater than 10cm that can be observed in Figs. 9.c and 10.c.

Fig. 11: Modulus of the daily averaged water level difference between stations of Port-Cros (PC) and La Seyne/Ifremer (IF) versus wind speed measured at the meteorological station of Cépet

(CEP); Water level is higher (resp. lower) at Port-Cros during Mistral (resp. east wind) events.

Seiching

A well-known source of water level variations within an enclosed bay or a harbor is seiching, due to long wave resonance. For semi-enclosed basins, the fundamental standing wave wavelength  is four times the basin length L from the entrance to the rear. This corresponds to a node at the open boundary and to an antinode at the impervious boundary. The fundamental wavelength is twice the basin length in the transverse direction, both boundaries being impervious. Since for long waves the celerity is 𝑐 = √𝑔ℎ, where h is the water depth, the wave period T =/c is of the order of several minutes for basins of several hundred meters length and of about 4m deep. It is of order of several tens of minutes for larger and deeper bay. [START_REF] Millot | Les oscillations propres de la Rade de Toulon[END_REF] have shown the presence of such oscillations within the Little Bay of Toulon, the main oscillations corresponding to periods of 20 and 45 min. In their study, the seiching of period 20min is ascribed to the oscillation within the Little Bay considered as a closed basin, while the period of 45min corresponds to the fundamental mode of oscillation of the whole Bay of Toulon. A spectral analysis of the data series of station La Seyne/Ifremer using a moving window of one day (not shown here) evidences signals at about 22min and 45 min. Since our sampling period is 10min, the lower period is at the limit of what we can describe (close to Nyquist frequency). Thus we consider here only the oscillations at the period T=45 min. After calculation of the amplitude of the corresponding Fourier coefficient on the above moving window (32 wave periods) versus time for the stations located within the Bay of Toulon, we can identify some events for which maxima amplitude of several centimeters are observed.

Results for the period ranging between December 17 th and 22 th , 2016 are presented in Fig. 12. Seiching appears on December, 19 th 2016 for easterly conditions. The oscillation amplitude is of same order of magnitude at the stations Ifremer, Tamaris and St Louis. This corresponds to the observations of [START_REF] Millot | Les oscillations propres de la Rade de Toulon[END_REF] since the amplitude is maximum at the western boundary of the Little Bay for this mode. No seiching is observed at the Lazaret station, which is on the southern boundary, rather far from the western side of the bay and actually not on the oscillation node.

Analysis of near-surface temperature variations

SST, measured either by satellites or in situ sensors, is a key parameter for upper-ocean circulation modeling at least. SST may significantly vary daily. [START_REF] Donlon | The global ocean data assimilation experiment high-resolution sea surface temperature pilot project[END_REF] distinguished skin and subskin temperature measurements from remote sensing techniques and in situ measurement and proposed typical near-surface vertical profiles according to solar radiation for light wind. The diurnal cycle of SST is necessary for accurately computing air-sea heat fluxes as underlined by Pimentel et al (2018). These authors modelled the fine-scale SST near-surface structure in the Mediterranean, allowing comparisons between SST characteristics such as foundation (insensitive to diurnal cycle), depth, subskin and skin. The net heat flux at the air-sea interface is the sum of four dominant terms, the net short wave radiation flux, the net long wave radiation flux, the latent heat flux of evaporation and the sensible heat flux. Averaged short wave and long wave radiation fluxes given in the ERA-40 data set are 162 W.m -2 and -79 W.m -2 , respectively, for the Mediterranean [START_REF] Pettenuzzo | On the corrections of ERA-40 surface flux products consistent with the Mediterranean heat and water budgets and the connection between basin surface total heat flux and NAO[END_REF]. Intense airsea exchanges favored by strong northerly and north-westerly dry wind situations (Mistral and Tramontane) characterize the northwestern Mediterranean, where latent and sensible heat fluxes reach up to -590 W.m -2 and -180 W.m -2 , respectively, for strong Mistral events [START_REF] Rainaud | Characterization of air-sea exchanges over the Western Mediterranean Sea during HyMeX SOP1 using the AROME-WMED Model[END_REF]. Such meteorological conditions in autumn and winter lead to significant evaporation and ocean heat loss. Offshore, if the mixing induced by these strong winds reaches the saline intermediate layer, this leads to deep convection and triggers the formation of deep water (Deep Water Formation, e.g. Lebeaupin Brossier et al, 2017). On the shelf (Gulf of Lion), where no mixing with the intermediate layer can take place, the same water is cooled and densified, up to a point when its density drives it to overflow and cascade down to more than 2000m possibly (Dense Shelf Water Cascading, e.g. Durrieu de [START_REF] Durrieu De Madron | Interaction of dense shelf water cascading and open-sea convection in the northwestern Mediterranean during winter 2012[END_REF]. Evaporation and water cooling during such north-westerly winds are also observed in lagoons. During Tramontane events, temperature drops of about 2°C mainly due to evaporation are observed in the Thau lagoon (mean water depth ~4m), which corresponds to turbulent heat fluxes in the range [-300 ; -500] W.m -2 [START_REF] Bouin | Long-term heat exchanges over a Mediterranean lagoon[END_REF]. Such a heat flux range is consistent with a vertical mixing of the whole water layer, since it corresponds to a cooling in the range [1.8 -3.0] °C, taking 4kJ.kg -1 .K -1 as the value of the specific heat for salt water. Earlier hydrological studies of the Thau lagoon showed a drop of the water level of about 10mm per day during Tramontane episodes [START_REF] Audouin | Hydrologie de l'étang de Thau[END_REF]. This drop in level corresponds to a latent flux of -270W.m -2 (taking 2400kJ.kg -1 as the value of the latent heat of evaporation for salt water), which is consistent with the turbulent heat flux in the range [-300 ; -500] W.m -2 since about 30% of the temperature drop is attributed to the sensible heat flux [START_REF] Bouin | Long-term heat exchanges over a Mediterranean lagoon[END_REF].

The temperatures measured by our immersed piezometer provide information on the sub-surface temperature, in the range 0.10m -1.80m deep, according to the immersion depth and the station. However, at a given time, water level differences between the stations remain less than about 10cm, as shown in section 4.4. So, the measuring depth, which also depends slightly on the immersion of the station cylinder (see section 3), remains fairly close from one station to the next. Since stations are located along the shore, in harbors of few meters depth, the relationship between SST at depth of several tens of centimeters and the SST skin may not follow the proposed temperature profiles for deep water conditions. In addition, even if recent progress in remote sensing techniques allow skin SST maps closer and closer to the coast, data are still not available at the coast. The confidence interval of the network's stations is of ~0.5 degrees, which is not precise enough to capture quantitatively heat fluxes and temperature gradients induced water flux. However, even if only the temperature at a given near surface depth is available in our case, we will consider the information precise enough to discuss water mass movements at the scale of the instrumented area. Summertime upwelling events and wintertime water mass exchanges between the enclosed bays and the coastal shelf circulation are given as examples and discussed in the following subsections.

Upwelling events during summertime

One can observe in Fig. 3b both seasonal variations of the temperatures and rapid variations of 5 to 10 °C in summertime, which are discussed below. Histograms for the stations installed before spring 2017 are presented in Figs. 13 and14, for the temperatures collected between June, 21 th and September, 21 th , for the years 2017 and 2018, respectively. The N data percentage indicates the temporal availability of the data. Except for the station of La Capte, all are close to 90%, which means a small number of missing or discarded data. Time series for the corresponding periods are presented in Figs. 15 and16 respectively. They include (a) wind speed and (b) direction at meteorological stations of Cépet, Porquerolles and Levant, and (c) hourly averaged temperatures at stations La Seyne/Ifremer, Giens and Port-Cros. The Mistral (northwesterly) is a prevailing wind in the region, even during summer. The summer 2017 was very windy, as shown in Fig. 15: 8 Mistral episodes lasting several days, with intensity >15m.s -1 , and one easterly episode >15m.s -1 . The impact of the Mistral event of the end of July 2017 in terms of daily averaged temperatures is presented and compared with neighboring bays in Fig. 17. Summer 2018 displayed a weak and variable wind regime (see Fig. 16): only one Mistral episode peaked above 15m.s -1 , beginning on August, 23 rd , followed by a second Mistral episode of a lesser intensity. Significant variations of the temperature can be observed in Figs. 15, 16 and 17. The diurnal oscillations, of the order of 1°C (Figs. 15 and 16), are due to the solar heating of the aluminum cylinder housing the sensors, as well as, in harbors with shallow depths, to the solar heating of water in the absence of mixing.

Steep drops of temperature of several degrees are also observed. Such drops cannot be due to cooling by evaporation, since they would amount to ~ 1.2°C per day for a net heat flux of about -500W.m -2 , assuming vertical mixing within a water column of 10m. They indicate the upwelling formation related to the Mistral events. These events are discussed hereafter for summers 2017 and 2018.

During summer 2017, one can observe that the temperatures recorded at the stations of Port-Cros, La Capte, Giens and Brusc remain the warmest overall (average temperature between 22.0°C and 23.2°C), with few events with temperature under 20°C, contrary to the stations of La Seyne/Ifremer, Saint Elme, Tamaris and Saint Louis (average temperature between 20.6°C and 21.1°C) (Fig. 13). In Fig. 15.c, the temperature time series show sudden drops of temperature, up to 10°C within less than 3 days. The station of St Elme, facing the sea, and stations of Ifremer, Tamaris, and St Louis, within the Bay of Toulon, are strongly impacted by the upwelling cells induced by the strong Mistral episodes, and marked by minima of ~ 15°C (on July, 1 st , 14 th and 27 th and on August, 13 th ). These upwelling events allow a rapid water mass renewal, especially in the Little Bay as already observed by [START_REF] Dufresne | Wind forced circulation in the Bay of Toulon and water exchanges at the Little Bay fairway[END_REF]. The temperature decrease is much less pronounced for stations Giens and Port Cros, further east. Note that the Mistral veers from northwest to west, as it progresses eastward from Cépet to Levant. Temperature variations within the Bay of Toulon and neighboring bays are detailed in Fig. 17 for the Mistral event of the end of July, 2017. We can observe that stations of Brusc, Giens and Lazaret are much less affected by the upwelling than stations of Tamaris, La Seyne/Ifremer and Port Saint Louis. Stations of Brusc and Giens are indeed located in semi-enclosed bays westerly oriented and exposed to the waves with Mistral conditions. The near-surface water mass is then pushed at the back of the bay. We can observe a regular decrease of the temperature of about 0.8°C per day during the Mistral event, lasting from July, 23 rd to July, 29 th . Cold water which appears later at the near surface is then mainly due to both cooling by evaporation and wind and wave induced water column mixing and possibly to the coldest water masses induced nearshore circulation. The station of Lazaret is located within a sheltered part of the Little Bay of Toulon, of shallow water. This may explain the more moderate drop in temperature. Station of St Elme, facing the sea, and stations of Ifremer, Tamaris, and St Louis, within the Bay of Toulon, are strongly impacted by the upwellings, with temperature drops down to 15°C.

During summer 2018, the average temperatures are much higher than during summer 2017, with only two events of temperature below 20°C, whatever the station. The coldest mean temperature was recorded at Tamaris for both years, with an average value of 20.3°C in 2017 and 23.5°C in 2018. The Port-Cros station exhibits the warmest mean temperatures, with 23.3°C in 2017 and 25.4°C in 2018. This correlates with the small number of Mistral events of significant duration (at least 2 days), observed that summer: only 2 were recorded, beginning on August, 23 rd and 31 st , respectively. The few short Mistral events of July were not long enough (no more than one-day duration) to allow the development of upwelling: the water temperature remains above 20°C at the station of La Seyne/Ifremer. On the contrary, the intense event of August (from August, 23 rd ) decreased the water temperature by almost 10°C in 3 days at the La Seyne/Ifremer station. As observed in 2017 and 2018, the summer water temperature at the coast has a high interannual variability. During the five last years, 2017 was the coldest and 2018 the warmest in terms of water temperature, as shown in Fig. 16 for the stations of La Seyne/Ifremer and Port-Cros. Late December, the temperature decreases slightly from ~16°C to ~15°C in Port-Cros, while it decreases sharply from ~15°C to 13°C at La Seyne/Ifremer. This temperature decrease, of about 1°C per day in the Little Bay of Toulon, is consistent to either net heat flux or water mass exchanges between the Little Bay and offshore. This latter temperature also corresponds to the mixed-layer wintertime average minimum temperature (dashed lines in Fig. 19.c). Water mass exchange seems the major reason of this sharp temperature decrease since one can observed that the strong Mistral episode beginning on January, 10 th , 2015 does not cool the water mass at the LaSeyne/Ifremer station as significantly. On both series there are short-lived minima of temperature, but they are not related, since the wind events were not homogeneous over the area (individual peaks of one color, in both wind speed and direction). As shown in Fig. 19d, the atmospheric pressure remains rather high during this period, and even the strong Mistral events beginning on January, 3 rd and on January, 9 th , respectively have little influence on the water temperature at both stations. Correlatively, there is no evident relation between the type and intensity of the wind event and the temperature at La Seyne/Ifremer: between January, 19 th and January, 29 th there is a decrease of ~1.5-2°C (min. on January, 20 th ), under both a Mistral and an easterly wind events. After January, 20 th , the atmospheric pressure gradually increases until the January, 25 th . By January, 28 th , there is a long and intense Mistral episode, along with a sudden decrease of the atmospheric pressure resulting in a sudden rise of the water level (see Fig. 19e). At the same time, the water temperature increases at station La Seyne/Ifremer, while it decreases at Port-Cros: both stations reach similar temperatures of ~13°C (January, 31 st ). The local weather conditions decreased the temperature at the shallow station of la Seyne below 13°C, while at the same time offshore the mixedlayer deepened. Under such intense Mistral conditions the water renewal in the Bay of Toulon must have advected ~13°C-offshore mixed-layer water. We have shown in section 5.1 that Mistral episodes are at the origin of the mass exchanges through the development of upwellings during summertime. During wintertime, the water temperature is lower within the Little Bay because of its small extent and its shallow water depth compared to offshore. The relative increase of the temperature within the Little Bay of Toulon (to 13°C) might correspond to an intrusion of the offshore water masses, warmer, through a sudden increase of the water level, following the flush of the wind-cooled water. They may be linked with the variability of the North Current, observed throughout the year (see e. g Millot, 1987;[START_REF] Carret | Synergy between in situ and altimetry data to observe and study Northern Current variations (NW Mediterranean Sea)[END_REF]. Alternately, at Port-Cros the Mistral induced a strong mixing, and decreased the temperature to that of the mixed-layer. The exact interplay of wind regimes, sea levels and temperatures in the bays has yet to be investigated in details. However, it is clear that even the collection of cost-effective lowresolution subsurface temperature at the network's stations helps to understand the dynamics in such complex nearshore systems.

Discussion and conclusion

We have shown with these examples and illustrations the potentiality of the HTM-NET network in the understanding of the nearshore circulation and water mass exchanges through a long term collection of data on the water level and the near surface temperature along the shore of Toulon area.

The collection of water level data have long been restricted to the main French ports (Marseille, Toulon and Nice along the Mediterranean). But the HTM-NET data show that even low-cost solutions can fill the gaps and achieve the important goal to monitor water level at such a fine scale. Indeed, unlike the level variations due to the tide and atmospheric pressure variations which are more or less the same in this area, some level differences, due to the influence of the wind for example, can exist between different stations in the study area. Seiching and its origin may also be better understood by such a network of data around the basins. If contributions of the wind or oscillations to the level change are found to be of several centimeters, they may however contribute to high water level and coastal flooding and erosion with potential disastrous socio-economic consequences. It is thus important to be able to quantify maximum water levels, either for the understanding of the dynamics or for socio-economical purposes. In addition, water level reconstruction models use a combination of coastal tide gauges with water level fields in the whole basin from altimetry data (see Adloff et al, 2018 and references cited). We have shown that differences in the order or 0.10 m could be observed between tide gauges in the rear of enclosed bays and in open boundaries such as on the Port-Cros island. This may induce significant bias in the technique of reconstruction.

Concerning the near-surface temperature, we could observe the strong variability of the temperatures at the scale of the network and from one bay to another, some places being strongly affected by upwellings. A strong interannual variability is also observed, directly related to the weather conditions in terms of number of wind events. The present network provides also complementary information to SST thematic products computed with satellite data, not only to improve the resolution in the coastal zones, but most importantly to correct the bias of the products where coastal upwelling areas are often flagged as bad data since way off the summertime climatology (Buongiorno [START_REF] Nardelli | High and Ultra-High resolution processing of satellite Sea Surface Temperature data over Southern European Seas in the framework of MyOcean project[END_REF].

In the present analysis, the sampling period is 10 min. This sampling period was prompted by the need to minimize the maintenance effort for downloading the data. It is presently being reduced to 2 min for the whole network along with the GSM-transmitter equipment of all stations. This new sampling period is motivated by the monitoring of seiching, which was previously limited to periods over 20min, and then inadequate for faster oscillations as observed also in the Little Bay of Toulon [START_REF] Millot | Les oscillations propres de la Rade de Toulon[END_REF] or potentially existing in bays or harbors of smaller extent. In addition, the sampling period of 10 min was also found inadequate in recording the amplitudes of the tsunami induced water level oscillations following the earthquake of Boumerdès-Zemmouri (Algeria) on 21 May 2003 [START_REF] Sahal | The tsunami triggered by the 21 May 2003 Boumerdès-Zemmouri (Algeria) earthquake: field investigations on the French Mediterranean coast and tsunami modelling[END_REF]. For more detailed analyses of the water level regarding either tide, storms or other hazards origins, the sampling period of 2 minutes appears as a good compromise between the technical constraints and the quality of the data. The data stored in the station spans now ~ 12 running days, which proves enough to plan the maintenance in case of a faulty GSM transmission.

The network is being extended westward up to the Cape Couronne (West of Marseilles), with 4 additional stations (see the updated map on the site https://htmnet.mio.osupytheas.fr). These stations are located at the eastern part of the Gulf of Lion, characterized by a strong variability of the Northern-Current (Alberola and [START_REF] Albérola | Circulation in the French mediterranean coastal zone near Marseilles: the influence of wind and the Northern Current[END_REF] and strong upwelling events [START_REF] Millot | Wind induced upwellings in the Gulf of Lions[END_REF]). The circulation is rather complex and still under investigation, not only for scientific purposes, but also to address socioeconomic issues. Long term data sets on both water level and near-surface temperature will thus provide important information for the understanding and the modelling of dynamics.

Data availability

Data on relative water levels of the HTM-NET network stations and the water level with respect to the reference level NGF (official altimetric reference in the French continental metropolitan territory)

Fig. 1 .

 1 Fig. 1. Studied area and location of the stations.

Fig. 3 .

 3 Fig. 3. Example of data series: (a) water level, (b) water temperature, site of La Seyne/Ifremer (IF).

Fig. 4 .

 4 Fig. 4. Water level histograms, (a) La Seyne/Ifremer (IF), (b) Port-Cros (PC)

Fig. 6 :

 6 Fig. 6: Atmospheric pressure induced water level variations, station La Seyne/Ifremer (IF).Daily averaged series of (a) atmospheric pressure and (b) water level.

Fig. 9 :

 9 Fig. 9: Wind induced water level variations the last Mistral event: (a) wind speed and (b) wind direction at meteorological stations Cépet, Porquerolles and Levant, (c) hourly (dashed lines) and daily (full lines) averaged water level at stations Port-Cros and La Seyne/Ifremer.

Fig. 12 .

 12 Fig. 12. Seiching analysis between December, 17 th and 22 th , 2016. (a) Wind speed and (b) wind direction at the meteorological station of Toulon, (c) seiching amplitude, T=45min, at stations of La Seyne/Ifremer (black), Lazaret (blue), Tamaris (red) and Port St Louis of Mourillon (green).

Fig. 13 .

 13 Fig. 13. Histograms of the temperatures, summer 2017.

Fig. 14 .

 14 Fig. 14. Histograms of the temperatures, summer 2018.

Fig. 15 .

 15 Fig. 15. Time series of the (a) wind speed and (b) wind direction at meteorological stations of Cépet, Porquerolles and Levant. (c) hourly averaged temperature time series at stations La Seyne/Ifremer, Port Cros and Giens, summer 2017.

Fig. 18 .

 18 Fig. 18. Mean temperature and standard deviation (error bars) during summertime from 2014 to 2018, stations La Seyne/Ifremer and Port-Cros.

Fig. 19 .

 19 Fig. 19. Evolution of near surface water temperature from the end of December 2014 till end of February 2015, Wind speed (a) and direction (b); (c) temperature, (d) atmospheric pressure, (e) daily averaged water level, Ifremer (black) and Port-Cros (blue).

of the tide gauge of Toulon, operated by the Shom, can be downloaded from the website DATA.SHOM.FR. Data from the last 60 days of temperature, water level and atmospheric pressure of the HTM-NET network stations are displayed on the website of the network: https://htmnet.mio.osupytheas.fr. Data of water and air temperature, and water and air pressure are available on request, on the catalogue of the Pytheas observatory https://dataset.osupytheas.fr/geonetwork/srv/fre/catalog.search#/metadata/7d538d6b-dd15-42af-9de7-c9eac27a840d.

Meteorological data are available on request on the dedicated website of Météo-France, the French national meteorological and climatological service, https://donneespubliques.meteofrance.fr/.

Acknowledgements

Financial supports from the Toulon Metropole (Contracts HTM-NET, OLBIA, CAPTILE) and from the French national scientific programs SOERE and DYNALIT-ILICO on the long term observation of the coastline are acknowledged. The French National park of Port-Cros (PNPC), Toulon Metropole and the city of Hyères are acknowledged for the installation of the stations, the French Navy service Shom for its collaboration and MétéoFrance for the meteorological data.