Ibn Ferjani

M Jeguirim
email: mejdi.jeguirim@uha.fr

S Jellali

L Limousy

C Courson

H Akrout

L Ruidavets

A Audrey

S Bennici

The use of exhausted grape marc to produce biofuels and biofertilizers: Effect of pyrolysis temperatures on biochars properties

Keywords: Exhausted Grape marc, pyrolysis, biochar, nutrients availability, textural properties

INTRODUCTION

In the Mediterranean countries, agriculture and food processing activities produce huge amounts of by-products and wastes. These residues are generally not basically harmful, but their seasonal production and their high organic matter contents could represent serious environmental concerns. Thus, an efficient recovery of these wastes is very important for global sustainability, higher profitability of agriculture and agri-food industry and promotion of the circular economy concept.

Grape marc (GM) is one of the main food processing industries residues available worldwide since about 70 million tons of wine grape are annually produced [START_REF] Oiv | Organisation Internationale de la Vigne et du Vin[END_REF]. Up to 20 % of the harvested wine grape becomes waste during wine production [START_REF] Xu | Flash pyrolysis of grape residues into biofuel in a bubbling fluid bed[END_REF]. This residue originating from vineyard includes the skins, stalks and seeds grape. The implementation of environmental strategies for the grape marc treatment, in order to avoid various ecologic high design and manufacturing costs. Alternatively, thermochemical conversion through combustion was tested since GM can be burned on-site at the winery for heat generation that could be used in the winemaking process itself. However, heterogeneity, high humidity, and high mineral contents have strongly diminished the combustion efficiency and also caused important emissions of particulate matters in the exhaust gas [START_REF] Kraiem | Energy recovery from Tunisian agrifood wastes: Evaluation of combustion performance and emissions characteristics of green pellets prepared from tomato residues and grape marc[END_REF]. Therefore, examining the recovery of grape marc through an optimized pyrolysis processes could be an interesting alternative. In fact, this technique is well adapted to heterogeneous feedstock or substrates with high mineral contents, such as grape marc. Furthermore, it has the advantages to produce three different fractions (gas, liquid, solid) that could be valorized separately. The gaseous and liquid fractions could be used as biofuels in the pyrolysis plant for heating or electricity production. The solid fraction (char) could be used directly for agricultural soils amendment (called biochar) or activated in order to produce efficient adsorbents for pollutants removal from aqueous and gaseous effluents [START_REF] Deiana | Use of grape stalk, a waste of the viticulture industry, to obtain activated carbon[END_REF].

Only few investigations have examined the recovery of grape marc through pyrolysis techniques. Xu et al. [START_REF] Xu | Flash pyrolysis of grape residues into biofuel in a bubbling fluid bed[END_REF] have examined the flash pyrolysis (from 300 to 600°C) at different vapor residence times (2.5, 5, and 20 s) of grape skins and the mixture of grape skins and seeds for bio-oil production. Authors showed that the optimum pyrolysis temperature for grape skin was 550°C, and 450°C for the mixture [START_REF] Xu | Flash pyrolysis of grape residues into biofuel in a bubbling fluid bed[END_REF]. Khiari and Jeguirim [START_REF] Khiari | Pyrolysis of grape marc from Tunisian wine industry: Feedstock characterization, thermal degradation and kinetic analysis[END_REF] have examined the pyrolysis of grape marc provided by the Tunisian wine industry. The authors found that biochar production yield was close to 40 %. Such value was not mentioned in previous investigations for agricultural biomasses during slow pyrolysis experiments. Authors have attributed this result to high lignin and ash contents and affirmed the good potential of the use of grape marc for biochar production [START_REF] Khiari | Pyrolysis of grape marc from Tunisian wine industry: Feedstock characterization, thermal degradation and kinetic analysis[END_REF].

Biochars recovery for soil amendment has received widespread attention since it contributes to carbon sequestration, therefore decreasing the CO 2 emissions into the atmosphere [START_REF] Lehmann | Biochar effects on soil biota-a review[END_REF].

Furthermore, benefits of soils amendment with biochars were not only limited to climate change mitigation, but also to agronomic soils' physical, chemical, hydrodynamic and biological properties improvements [START_REF] Ahmad | Chemosphere Biochar as a sorbent for contaminant management in soil and water : A review[END_REF][START_REF] Lehmann | Bio-char sequestration in terrestrial ecosystemsa review[END_REF]. Indeed, biochars are highly rich in carbon (C), but contain also significant amounts of oxygen (O), hydrogen (H), sulfur (S), nitrogen (N), and non-negligible concentrations of various inorganic elements such as calcium (Ca), potassium (K), phosphorus (P) that are very useful for plants growth. A wide range of raw and pre-treated biomass feedstocks were pyrolysed for biochars production (Table 1). These feedstocks include woody biomass resources, agriculture residues, food processing residues, green waste, wastewater sludge, and dairy manures [START_REF] Casazza | Pyrolysis of grape marc before and after the recovery of polyphenol fraction[END_REF][START_REF] Karaosmanoglu | Biochar from the straw-stalk of rapeseed plant[END_REF][START_REF] Kloss | Characterization of slow pyrolysis biochars: effects of feedstocks and pyrolysis temperature on biochar properties[END_REF][START_REF] Liu | Characterization and application of chars produced from pinewood pyrolysis and hydrothermal treatment[END_REF][START_REF] Sun Cha | Production and utilization of biochar: a review[END_REF][START_REF] Dai | Science of the Total Environment Potential Role of Biochars in Decreasing Soil Acidi Fi Cation -A Critical Review[END_REF][START_REF] Dai | Increased Agronomic and Environmental Value Provided by Biochars with Varied Physiochemical Properties Derived from Swine Manure Blended with Rice Straw[END_REF]. During these investigations, biochar properties including pH, ash contents, elemental composition, specific surface area and micropores volume were generally determined. These properties were found to be very dependent on the feedstock type as well as on the operating parameters such as the final temperature, the residence time, and the heating rate. Indeed, these investigations showed that with increasing the pyrolysis temperature, the aliphatic components of the feedstock are progressively decomposed and transformed into aromatic compounds [START_REF] Dai | Science of the Total Environment Potential Role of Biochars in Decreasing Soil Acidi Fi Cation -A Critical Review[END_REF]. Furthermore, the biochar yields, acidic functional groups concentration, volatile matter content, total oxygen content, and cation exchange capacity (CEC) decreased with the increasing of the applied temperatures. In contrast, the aromatic character, the basic functional groups, the pH, the fixed carbon, the carbon stability, the ash contents, the exchangeable and soluble cations, and the specific surface area of biochars increased with the applied temperature [START_REF] Dai | Science of the Total Environment Potential Role of Biochars in Decreasing Soil Acidi Fi Cation -A Critical Review[END_REF][START_REF] Dai | Increased Agronomic and Environmental Value Provided by Biochars with Varied Physiochemical Properties Derived from Swine Manure Blended with Rice Straw[END_REF].

Generally, the biochars pH values increase by increasing the pyrolysis temperature due to the elimination of oxygen surface groups including -COOH and -OH [START_REF] Enders | Characterization of biochars to evaluate recalcitrance and agronomic performance[END_REF]. These basic properties allow the use of biochars as an alternative of lime for the improvement of acidic-soils quality [START_REF] Verheijen | Biochar Application to Soils. A Critical Scientific Review of Effects on Soil Properties[END_REF].

The heating rate and the residence time play also an important role during biochar production [START_REF] Sun Cha | Production and utilization of biochar: a review[END_REF]. The residence time not only influences the biochar production yields, but also their textural properties. Tsai et al. [START_REF] Tsai | Preparation and characterization of activated carbons from corn cob[END_REF] indicated that the residence time increase leads to an increase in the biochar pore sizes distribution. Besides, the heating rate strongly affects the composition and the nature of the pyrolysis products, including biochars. In fact, low heating rates during biochar production allows the limitation of the thermal cracking of biomass and therefore higher biochar yields are obtained. In contrast, a high heating rate promotes the fragmentation of biomass and increases the gaseous and liquid yields, limiting the biochar formation [START_REF] Tripathi | Effect of process parameters on production of biochar from biomass waste through pyrolysis: A review[END_REF].

The biochars production yields and properties also depend on the biomass composition.

Generally, high biochar yields are obtained for pyrolysis of solid wastes and animal litters compared to those obtained from crop residues and woody biomasses [START_REF] Enders | Characterization of biochars to evaluate recalcitrance and agronomic performance[END_REF]. These higher yields are related to the presence of the main inorganic constituents of the feedstock [START_REF] Xu | Flash pyrolysis of grape residues into biofuel in a bubbling fluid bed[END_REF]. Furthermore, the raw lignocellulosic biomass structure and its composition in terms of cellulose, hemicellulose and lignin could also significantly influence the pyrolysis products.

In particular, cellulose and particularly lignin considerably increase the biochars production yields [START_REF] Tsai | Preparation and characterization of activated carbons from corn cob[END_REF].

This study aims to examine the potential of grape marc as a feedstock for biochar production. Therefore, its slow pyrolysis was performed at different temperatures ranging between 300 and 700°C. Then, the produced biochars' physicochemical properties including their surface chemistry, as well as their textural and structural properties were analyzed using different analytical techniques. The determined features are essential to decide about the suitability of the produced chars for agronomic soil amendment.

MATERIALS AND METHODS

Samples preparation

The grape marc selected for this study is a mixture of different grape varieties available in the Alsace Region (France). It was provided from the Sigolsheim distillery (France), after ethanol extraction by distillation process. This residue of distillation is called exhausted grape marc (EGM). Prior to its use, the EGM was manually sorted to remove the branches. Then, EGM was grounded using a laboratory crusher and sieved to obtain a uniform sample with an average particle size ranging between 0.25 and 0.4 mm.

Exhausted Grape marc Characterization

The elemental analysis of EGM was carried out by a CHONS analyzer (FlashEA 1112, Thermo Scientific, Villebon, France) for the major elements, according to standard NF EN ISO 16994 for the chlorine determination. The inorganic elements concentrations were determined by ICP (720 ES, Varian, Les Ulis, France) according to standard NF EN ISO 17294-2.

The proximate analysis were determined using various methods [START_REF] Jeguirim | Devolatilization behavior and pyrolysis kinetics of potential Tunisian biomass fuels[END_REF]. The moisture, ash contents, and volatile matters were determined according to NF EN ISO 18134-3, NF EN 14775 and NF ENISO/IEC 17025:2005 standards, respectively. Fixed carbon were then deduced from the previous analyses.

In order to follow the thermal behavior of the EGM under pyrolysis conditions, a thermogravimetric analyzer TGA/DSC3+ apparatus ((Mettler-Toledo, Columbus, OH, USA) was used. During these tests, 10 mg of EGM were inserted in an alumina crucible and introduced inside the thermobalance, then heated under N 2 at a flow rate of 6NL/h. This heating process occurred from 20 to 800°C at a constant heating rate of 5 °C min -1 .

Biochar Production

The biochar production was realized in a vertical tubular furnace already described in previous investigation [START_REF] Haddad | Olive Mill Wastewater: From a Pollutant to Green Fuels, Agricultural Water Source and Biofertilizer[END_REF]. During each test, 2 kg of grape marc was distributed in the different pyrolyzer drawers. N 2 flow of 5 NL h -1 was constantly fed at ambient temperature during 30 minutes to eliminate the residual oxygen. Then, the temperature was increased to the settled value (300-700°C), at a constant heating rate of 5°C min -1 . Once stabilized, the temperature was maintained constant during 1 h, and then natural cooling was performed under N 2 flow. During these experiments, the exhausted gases passed through a condensing system constituted of refrigerant connected to a liquid collector. The exhausted gases were cooled by circulating cold water in order to recover the liquid fraction (bio-oil).

Biochar characterization

The physico-chemical, textural and structural properties of biochars were assessed using the different following analytical methods.

The particle size distribution was determined on a water suspension of biochars using a laser granulometer (Mastersizer Malvern Instrument, Worcestershire, UK).

The produced biochars' pH values were determined by mixing 1 g of biochar with 10 mL of deionized water for 1 h by using a magnetic stirrer. The pH of the filtered aqueous solution was measured with a pH-meter (HI2211, HANNA Instrument, Woonsocket, RI, USA).

The elemental and proximate analyses of the biochars were determined according to the same procedures of the EGM (see section 2.2).

Scanning Electron Microscopy (SEM) was used in order to assess the produced biochars morphology. SEM images were carried out using a Philips model FEI model Quanta 400 apparatus (Amsterdam, The Netherlands). Prior to the observation, the samples were covered with a tinny gold layer by using an Agar automatic sputter coater. During the analyses, three magnification levels (x1000, x5000 and x20000) were selected.

Energy Dispersive X-Ray (EDX) (Oxford Inca Si, Oxford Instruments, Concord, MA, USA)

analyses have been also performed for the identification of the main mineral species and for the observation of their dispersion through the biochar matrix.

The specific area and porosity of the EGM biochars were determined by carbon dioxide adsorption using gas adsorption analyzer (ASAP 2020, Micrometrics, Norcross, GA, USA).

Before the measurements, biochars were out-gassed under vacuum during 24h at 150°C. This step is required to eliminate the surface water as well as the volatile organic compounds. During these analyses, the CO 2 adsorption was performed at a controlled temperature (0°C) by using an icewater bath.

X-ray Photoelectron Spectroscopy (XPS) also known as Electron Spectroscopy for Chemical Analysis (ESCA), which is a surface-sensitive quantitative spectroscopic technique was used to determine the elemental composition of the produced biochars. The average depth of analyses for the XPS measurements was approximately 5 nm. The measurements were made with a vacuum (UHV) spectrometer (Thermo-VG, Thermo Fisher Scientific, Dreieich, Germany) equipped with a CLAM4 hemispherical electron analyzer (CDM).

The structural properties of the different produced EGM chars were analyzed by Raman spectroscopy. During this analysis, the Raman spectra were recorded with a BX40 LabRam, Jobin Yvon/Horiba spectrometer (Kyoto, Japan). Raman spectra were obtained in a backscattered configuration with an excitation laser at 632.8 nm. The analyses parameters were selected as follows: microscope objective 50x, acquisition time 300 s, 1 accumulation. During this analysis, different parameters related to the biochar structure could be obtained. Details on their calculations and significance could be found in previous investigations [START_REF] Guizani | New insights on the structural evolution of biomass char upon pyrolysis as revealed by the Raman spectroscopy and elemental analysis[END_REF][START_REF] Guizani | Biomass chars: the effects of pyrolysis conditions on their morphology, structure, chemical properties and reactivity[END_REF].

RESULTS AND DISCUSSION

Characterization of the Exhausted Grape Marc

Table 2 displays the ultimate and proximate analyses as well as the inorganic elements concentrations in the EGM. The obtained values are in the range of typical composition of agriculture and food processing residues reported in literature [START_REF] Kraiem | Impregnation of olive mill wastewater on dry biomasses: Impact on chemical properties and combustion performances[END_REF][START_REF] Kraiem | Energy Recovery from Tunisian Agri-food wastes: Evaluation of Combustion Performance and Emissions Characteristics of green pellets prepared from Tomato Residues and Grape Marc[END_REF][START_REF] Jeguirim | Pyrolysis kinetics and physicochemical properties of agropellets produced from spent ground coffee blended with conventional biomass[END_REF][START_REF] Jeguirim | Devolatilization behavior and pyrolysis kinetics of potential Tunisian biomass fuels[END_REF].

The elemental composition analysis shows that carbon content (50.75%) is higher than the mean values reported in the literature. In addition, the fixed carbon value (24.35%) is also greater than the ones obtained for various lignocellulosic biomasses [START_REF] Haddad | Combined NMR structural characterization and thermogravimetric analyses for the assessment of the AAEM effect during lignocellulosic biomass pyrolysis[END_REF]. The high fixed carbon content found in this biomass sample can be explained by the exhaustion of sugars in the ethanol production, remaining a residue with higher content of lignin. Lignin is the component in biomass which provides a higher yield of solids after pyrolysis and therefore high char yields during the slow pyrolysis of EGM. The analysis of the inorganic elements indicates that potassium (K), calcium (Ca) and phosphorus (P) exist in EGM with relatively high contents compared to other biomasses (Table 2). Indeed, the contents of K, Ca and P were assessed to 0.677%, 0.622% and 0.257%, respectively (Table 2). These elements, if still present in the produced chars after the slow pyrolysis of EGM, they would be very useful for plants growth and development. The thermal degradation behavior of the used EGM was examined under inert atmosphere at a heating rate of 5°C min -1 . Figure 1 presents the Normalized Anhydrous Weight (NAW) and the Normalized Anhydrous Weight Loss Rate (NAWLR) of EGM. The degradation of dry EGM begins around 150°C lower than the initial degradation temperature of the most of the lignocellulosic biomasses. Such behavior is attributed to the degradation of lignin that starts at low temperature and occurs in a large temperature range [START_REF] Yang | Characteristics of hemicellulose, cellulose and lignin pyrolysis[END_REF]. Furthermore, alkali metals, like potassium, may catalyze the thermal degradation of EGM.

In the temperature interval ranging between 210 and 290 °C, two small shoulders are observed at 225 and 266 °C, respectively. Such observation was discussed in details in previous investigation and was attributed to the hemicellulose decomposition. In particular, the curve shape looks generally as a more or less pronounced shoulder instead of an intense peak depending on the hemicellulose content in the raw biomass. During the EGM thermal degradation, the presence of a first shoulder at 225 °C may be attributed to different hemicellulose compounds or to the catalytic effect of alkali elements on its decomposition [START_REF] Haddad | Combined NMR structural characterization and thermogravimetric analyses for the assessment of the AAEM effect during lignocellulosic biomass pyrolysis[END_REF].

The NAWLR curve shows a sharp peak observed at 320 °C with maximum loss rate of 0.039 % s - 1 . This well-known peak is attributed to the cellulose decomposition. Various peak temperatures and maximum loss rates are reported in the literature depending on the raw biomass characteristics. In this study, the maximum peak temperature of EGM is higher than that reported in the literature for grape marc (288 °C), while the maximum mass loss rate is lower (0.039 % s -1 against 0.055 % s -1 in the literature) [START_REF] Khiari | Pyrolysis of grape marc from Tunisian wine industry: Feedstock characterization, thermal degradation and kinetic analysis[END_REF]. Such results indicate that EGM reactivity is lower than that of the raw grape marc, which can be related to the difference in the inorganics content. In fact, the higher reactivity was obtained for grape marc with an ash content of 13 wt.%, while the EGM ash content is 3.25 wt.% (see Table 2). The distillation process may decrease the mineral contents of the grape marc and therefore affect the biochar production.

formation and rearrangement. At a temperature of 800°C, the char percentage for EGM was assessed to 28.9 wt% (30.9 wt.% at 700 °C) which is relatively higher than that of woody biomasses and several agriculture residues. This observation is very important for the economic viability of the biochar production from exhausted grape marc. However, the obtained value is lower than that obtained for Tunisian grape marc (not distillated) [START_REF] Khiari | Pyrolysis of grape marc from Tunisian wine industry: Feedstock characterization, thermal degradation and kinetic analysis[END_REF]. Such results suggest that an economic study for the selection between exhausted grape marc and raw grape marc for biochar production needs to be undertaken. EGM biochars were elaborated at various temperatures, ranging from 300 to 700°C, in a pilot scale pyrolyzer, according to the experimental protocol given in section 2.3. The obtained biochar yields are presented in Figure 2.

As expected, the biochar yields were relatively important below 500 °C. The obtained values were 65.8% and 59.1% at 300 and 400 °C, respectively. These values are in agreement with EGM thermal degradation behavior during thermogravimetric analysis. In fact, the devolatilization step

was not ended at these temperatures. When the pyrolysis temperature was further increased, the biochar yields production dramatically decreased and reached 33.8%, 32.6% and 30.8% for temperatures of 500°C, 600°C and 700°C, respectively. Such results indicate that a pyrolysis temperature of 500 °C may be suitable for biochar production at large scale since the devolatilization step is finished and no significant EGM mass loss occurs above. Similar results for biochar yields were observed for various agricultural biomass such as peanutshell (36.80%-25.80%), corn-straw (35.50%-24.90%), and wheat-straw (32.40%-22.8%) in the temperature interval of 400 to 700 °C [START_REF] Gai | Effects of feedstock and pyrolysis temperature on biochar adsorption of ammonium and nitrate[END_REF]. The EGM char yield obtained in the present study are higher than those reported in literature for pyrolysis performed in similar conditions. This observation could be related to the high lignin content in grape marc. In fact, in the pyrolysis process, lignin is the most important constituent responsible for higher char formation, while hemicelluloses and cellulose contribute more to volatile compounds emissions [START_REF] Jeguirim | Thermal characterization and pyrolysis kinetics of tropical biomass feedstocks for energy recovery[END_REF]. The high content of lignin in the exhausted grape marc is attributed to the exhaustion of sugars during the ethanol production. Furthermore, the relatively high presence of alkali metals (see Table 2) tends to increase the char yields during the pyrolysis process, as abundantly discussed in the literature [START_REF] Haddad | Combined NMR structural characterization and thermogravimetric analyses for the assessment of the AAEM effect during lignocellulosic biomass pyrolysis[END_REF].

Figure.2. Effect of pyrolysis temperature on biochar yields

Biochars characterization

Figure 3 shows the distribution curves (logarithmic scale) of the biochars prepared at different temperatures. It can be clearly seen that the average particle sizes is ranging from 120 µm to 150 µm, and an increase of the smallest particles ratio (average 25 µm) was observed at higher pyrolysis temperatures. A small variation of the average particle size is also observable with the pyrolysis temperature increase (Table 3), with a maximum value for the biochar prepared at 600 °C (146 µm) and a strong decrease for the biochar prepared at 700 °C (129 µm). 4) show that all biochars samples have volatiles matters contents decreasing from 64.7 % to 15.7 % when the temperature increases from 300 to 600°C. The higher fixed carbon amounts are observed for pyrolysis temperature of 500 and 600 °C, when compared to the other pyrolysis temperature tested. In similar way, ash contents increase with the increase of pyrolysis temperature, except for the biochar obtained at 600 °C. Such result is attributed to the high mineral and lignin contents in EGM. It is well known that lignin is difficult to decompose during biomass pyrolysis, thus generating a high amount of solid residue [START_REF] Jeguirim | Thermal characterization and pyrolysis kinetics of tropical biomass feedstocks for energy recovery[END_REF]. The knowledge of the fixed carbon and volatile matter contents is very important for the selection of the most suitable thermochemical conversion process. Therefore, biomass with high fixed carbon engender higher production of biochars [START_REF] Jeguirim | Thermal characterization and pyrolysis kinetics of tropical biomass feedstocks for energy recovery[END_REF]. The chemical composition of the grape marc biochars are reported in the Table 5. It shows that the biochar gets richer in C and poorer in H and O as the pyrolysis temperature increases. Indeed, the C content increased from about 60.21% to 72.89 % with increasing temperature from 300 to 700°C. The obtained values are in the same order of magnitude as the ones experimentally and numerically obtained by Debiagi et al for peanut shell and wood waste chars [START_REF] Debiagi | A predictive model of biochar formation and characterization[END_REF].

The O and H contents significantly decrease from about 26.20% and 6.04% to 15.00% and 1.23%

for the biochars produced at 300 °C and 700 °C, respectively. This behavior can be attributed to the dehydration and decarboxylation reactions that become more important when the applied temperature increases. Therefore, the H/C ratios of the EGM biochars decrease with the increase of the pyrolysis temperature. This decrease is closely related to the augmentation of the hydrophilic character of the biochar and therefore to the enhanced water retention capacity. A similar trend was observed for the O/C ratio, up to 500 °C. The lower values obtained at higher temperatures indicate the presence of aromatic rings forming very stable graphite-like structures.

The low percentage of S observed after pyrolysis at 300 °C and its absence at higher temperatures can be probably assigned to the decomposition of sulfur compounds into volatile SO 2 [START_REF] Sugumaran | Production and characterization of activated carbon from banana empty fruit bunch and Delonix regia fruit pod[END_REF][START_REF] Volker | Thermokinetic investigation of cellulose pyrolysis -impact of initial and final mass on kinetic results[END_REF].

Most of the other elements studied (P, Ca, Mg, Na, and K) are still present after pyrolysis at different temperatures. Such behavior is attributed to the low volatility of the corresponding oxides. Indeed, compared to the raw EGM (see Table 2), the nutrients (Ca, K and P) contents have significantly increased reaching values of 1.84%, 2.13% and 0.83%, respectively (Table 5). This observation highlights the opportunity offered by these biochars to be used as amendments in agriculture and supports the choice of pyrolysis instead of combustion for EGM thermochemical conversion. The measured pH of the produced biochars increased with the increase of the pyrolysis temperature (Table 6). Indeed, these values rose from a relatively neutral pH (7.2) to a very alkaline one (10.1) for the biochars produced at 300 °C and 600 °C, respectively. This behavior is due to removal of acidic function groups with increasing the thermal treatment. Similar results

were reported in the literature for bagasse and straw [START_REF] Casazza | Pyrolysis of grape marc before and after the recovery of polyphenol fraction[END_REF][START_REF] Sun Cha | Production and utilization of biochar: a review[END_REF]. The BET specific surface area (SSA) of the produced biochars from EGM significantly increased with the increase of the pyrolysis temperature. Indeed, the measured specific surface areas increased from about 25 m 2 g -1 to more than 253 m 2 g -1 when the pyrolysis temperature increased from 300°C to 600°C (Table 7). A similar trend was obtained also for the micropores volume

where its value at a temperature of 400 °C (0.03 cm 3 g -1 increased by a factor of more than 2.6

when the applied temperature passed to 600°C. This improvement of the biochars textural properties can be attributed to the fact that, at higher temperature, volatilization is more important, and consequently the biochar obtained is more porous, due to the creation of empty spaces into the carbon matrix. Similar results have been reported by several authors [START_REF] Tsai | Fast pyrolysis of rice husk: Product yields and compositions[END_REF][START_REF] Cui | Potential mechanisms of cadmium removal from aqueous solution by Canna indica derived biochar[END_REF]. For example, Cui et al. [START_REF] Cui | Potential mechanisms of cadmium removal from aqueous solution by Canna indica derived biochar[END_REF] have characterized biochars obtained from the pyrolysis of Canna Indica biomass (China) at pyrolysis temperatures of 300, 400, 500 and 600°C, a residence time of 2 hours and a constant heating rate of 5°C min -1 . They demonstrated that the increase of the pyrolysis temperature from 300°C to 600°C enhanced their specific surface area from 3.46 to 10.4 m 2 g -1 due to the development of the microporous structure [START_REF] Cui | Potential mechanisms of cadmium removal from aqueous solution by Canna indica derived biochar[END_REF]. Besides, Kim et al. [START_REF] Kim | Characterization of cadmium removal from aqueous solution by biochar produced from a giant Miscanthus at different pyrolytic temperatures[END_REF] reported that increasing the pyrolysis temperature of a local Korean biomass (Miscanthus sacchariflorus) from 300 to 600°C, has considerably increased the BET surface areas of the produced biochars from 0.56 to about 381 m 2 g -1 [START_REF] Kim | Characterization of cadmium removal from aqueous solution by biochar produced from a giant Miscanthus at different pyrolytic temperatures[END_REF].

It is worth mentioning that the relatively high measured surface areas of the EGM biochars produced at temperatures of 500 and 600°C is a real advantage for the use of these chars in environmental applications, and especially for the removal of toxic pollutants from aqueous and gaseous effluents. 4 gives the SEM images at the three magnifications of 1000, 5000 and 20000 times of the four EGM biochars. The images obtained at 1000 and 5000 magnifications show that, when the pyrolysis temperature increases, the degradation of the biochars is more pronounced and the related porosity becomes more important. Indeed, the biochar produced at 300°C presents a smooth surface with some pores that are not clearly defined. At the opposite, the biochars obtained at 500 and 600°C present irregular surfaces with well-developed porosities, characterized by the presence of tubular structures that were clearly visible in the related images at a magnification of 5000. These results confirm the BET analyses that proved the development of microporosity with the increase of temperature. EDX analyses (data not shown) indicated the presence of various minerals, mainly potassium and calcium, while phosphorous and silicon are also present in the biochars. We can also observe the appearance of small needles on the surface of biochar produced at 400, 500 and 600°C (x20000), and associated to the decomposition of calcium tartrate that, as well as potassium tartrate, is naturally present in grapes. This observation is supported by the shape of the crystals which is characteristic of calcium tartrate and oxalate [START_REF] Cui | Potential mechanisms of cadmium removal from aqueous solution by Canna indica derived biochar[END_REF]. Figure 5 gives the XPS spectra of the four biochars generated by the pyrolysis of EGM performed at various (300, 400, 500, 600 and 700°C) temperatures. For a pyrolysis temperature of 300 °C, only carbon and oxygen were detected with respective percentages of 57.1% and 42.9%. These values decrease to 15.3 % and 38.5 % for the biochar produced at a temperature of 700°C. Indeed, with the increase of the applied temperature, other elements were detected on the surface of the biochar, particularly Ca and K whose percentages at 700°C reached 21.5 and 22.3 %, respectively.

This result is attributed to the removal of some volatile organic compounds from the EGM by increasing the pyrolysis temperature, leading to higher concentrations of Ca and K. Moreover, Ca and K are not completely transferred in the gas phase during the thermal treatment, even at relatively high temperatures (700°C). These relatively high nutrients contents are very important if these chars are valorized as biofertilizers in agricultural soils.

Figure. 5. XPS spectra of the produced biochars from EGM at different pyrolysis temperatures

Raman spectrometry was used to further analyze the structure of the biochars produced at different temperatures. At 300 and 400°C, the Raman spectra exhibited a signal without distinguishable peaks (not shown). Such behavior is attributed to high proportion of amorphous carbon structures obtained during the pyrolysis at low temperatures. Above 500°C, the Raman spectra exhibited two relatively broad Raman bands at around 1330-1370 cm -1 and 1590-1610 cm -1 (Figure 6). The values derived from Figure 6 show that the IV/IG ratio decreases from 0.814 at 500°C to 0.682 at 600°C. Similar trend is also observed for the IV/ID ratio from 0.865 at 500°C to 0.715 at 600°C. Such behavior is attributed to the char evolution structure with pyrolysis severity from the amorphous carbon to an organized carbon (turbostratic char). Furthermore, the IG/ID ratio decreases from 1.063 at 500°C to 1.048 at 600°C indicating the increase of the proportion of condensed aromatic ring structures having defects. The D structures are created by the condensation of small aromatic amorphous carbon structures. These findings are in accordance with the ones obtained during the analysis of the structural parameters evolution of wood char under different thermal treatment [START_REF] Guizani | Biomass chars: the effects of pyrolysis conditions on their morphology, structure, chemical properties and reactivity[END_REF].

Practical Implication

Several information could be derived from the EGM biochar production and characterization. In fact, experimental results showed that the chars yields production were approximately constant for pyrolysis temperatures above 500°C with an average value close to 33% which is higher than the ones obtained for lignocellulosic biomass feedstocks. This behavior is attributed to the higher lignin and alkali minerals contents. The biochars characterization indicated that the biochar obtained at 500°C has the highest amount of potentially available minerals with an interesting value of surface area and microporosity. These properties make this biochar as a suitable candidate for soil amendments and also pollutants adsorption. However, further investigations regarding their agronomic potential use through controlled plant growth, soil structure improvements and nutrients release/adsorption tests are required.

CONCLUSION

In this study, slow pyrolysis of exhausted grape marc was carried out at temperatures of 300, 400, 500, 600 and 700 °C and a constant heating gradient rate of 5 °Cmin -1 in perspective of obtaining specifically featured biochars for potential uses in agriculture as soils amendment and/or in environmental applications as pollutant adsorbent. For this purpose, in depth characterization of the produced biochars including various complementary physico-chemical parameters was performed.

Obtained results indicated that the optimal temperature was 500°C with a chars yield value close to 33%. In addition, the characterization study confirmed the EGM biochar prepared at 500°C has an interesting minerals amount as well as a developed surface area and microposity. These properties make EGM biochar a promising material that could be used as biofertilizers as well as efficient adsorbents.

Figure. 1 .

 1 Figure.1. Normalized Anhydrous Weight (NAW) and Normalized Anhydrous Weight Loss Rate (NAWLR) of Exhausted Grape Marc (EGM) in N 2 atmosphere at heating rates of 5°C min -1 .

Figure. 3 .

 3 Figure. 3. Particle size distribution of biochars produced from EGM at different temperatures

Figure. 4 :

 4 Figure.4: SEM images of EGM biochars pyrolysed at different temperatures (300-600 °C).

Figure. 6 .

 6 Figure.6. Raman spectra of the EGM biochars produced at different temperatures

Table 1 .

 1 Main feedstocks, operating conditions and the produced biochars characteristics.

Feedstock Pyrolysis Temperature (°C) Heating rate (°C min -1 ) Residence time (h) Yield (%) pH Surface area (m 2 g -1 )

								Ash	C	H	C /H	C/O	Reference
								(%)	(%)	(%)
	Bagasse	500	10	1	43.7	9.3	-	8.57 85.59 -	30.35	8.17	[14]
	Rapeseed	800	5.0	-	28.2	-	19.0	15.3 79.51 0.72	-	-	[15]
	plant
	Poplar wood	400	8	-	32	9	3.0	3.5	67.30 4.42	-	-	[16]
	Pine wood	700	10	-	-	6.6	29.0	3.8	95.30 0.82	-	-	[17]
	Conocarpus	200	-	4	51.33	7.37	-	4.53	64.19	-	16.21	2.42	[14]
	wastes	400	-	4	31.86	9.67	-	5.27	76.83	-	27.15	5.43
		800	-	4	23.19	12.38	-	8.64	84.97	-	137.05	17.45
	Straw	200	_	1	84.95	5.34	-	11.9	45.57		7.90	1.19	[18]
		200		4	78.24	6.11		12.78	46.52		8.46	1.29
		400		1	37.30	10.82		25.74	57.59		17.14	3.46
		400		4	36.65	10.78		28.4	59.17		19.05	3.77
		600		1	32.48	10.93		32.33	59.09		38.42	5.36
		600		4	30.89	10.99		34.31	60.8		45.71	5.73

Table 2 .

 2 EGM

Proximate and Ultimate analyses (db : dry basis, ar : as received)

3.

2 Exhausted Grape Marc Thermal degradation Elemental composition db (%) Mineral composition db (mg/kg)

	C	50.75	Al	50
	H	6.40	Ca	6220
	N	1.18	Fe	110
	S	0.02	K	6770
	O	38.40	Mg	890
	Proximate analysis (%)	Na	40
	Moisture ar	7.55	Cl	140
	Volatile matter db	72.40	P	2570
	Fixed Carbon db	24.35	Si	330
	Ash db	3.25	Ni	-

Table 3 .

 3 Particle Size Distribution of the different biochars

	Pyrolysis	300 °C	400 °C	500 °C	600 °C	700 °C
	temperature
	d10 (µm)	33	28	44	32	22
	d50 (µm)	117	113	147	146	129
	d90 (µm)	219	221	238	254	230

Table 4 .

 4 Proximate analyses of the produced biochars (as received basis)

	Pyrolysis	300	400	500	600
	Temperature (°C)
	Moisture (%)	3.4	3.7	4.4	4.5
	Volatile matter (%)	64.7	45.2	27.9	15.7
	Fixed carbon (%)	27.0	43.4	58.2	71.3
	Ash (%)	4.9	7.7	9.5	8.5

Table 5 .

 5 Ultimate Analysis of EGM biochars produced at different temperatures (DL: detection

			limit)
	Pyrolysis	300	400	500	600	700
	Temperature
	(°C)
			Elemental composition (%)
	C	60.21	67.42	72.91	72.13	72.89
	H	6.04	5.07	3.15	2.16	1.23
	N	2.51	2.71	2.72	1.71	1.57
	S	1.09	<DL	<DL	<DL	<DL
	O	26.2	20.1	12.9	15.3	15.0
	H/C	0.10	0.08	0.04	0.03	0.02
	O/C	0.44	0.30	0.18	0.21	0.21
		Mineral composition (mg/kg)
	Al	554	433	471	326	357
	Ca	7010	10300	13400	17961	18381
	Fe	512	771	930	718	724
	K	11000	16400	21700	20095	21281

Table 6 .

 6 pH of biochars produced at different temperatures

	Pyrolysis	300	400	500	600
	Temperature (°C)
	EGM (This study)	7.2	8.7	9.9	10.1
	Straw [18]	*	10.8	*	10.9
	Bagasse [14]			9.3

Table 7 .

 7 Textural Properties of EGM biochars produced at different temperatures

	Pyrolysis	300	400	500	600
	Temperature (°C)
	SSA (m 2 g -1 )	25	130	205	253
	Micropores volume	-	0.03	0.06	0.08
	(cm 3 g -1 )
	Figure

Acknowledgements

The authors would like to thank the Carnot Institute MICA for supporting a part of this study in the frame of the Carbovit project and the Tunisian ministry of higher education and scientific research for financing the concerned "contrat-programme" of CERTE. Authors also would like to thank the Region Est for their financial support. The exhausted grape marc samples were provided by Sigolsheim Distillery (Grap'sud Group) and authors are very grateful.

The physico-chemical characterizations were performed on the technical platforms of IS2M, the authors are particularly grateful to L. Josien, L. Michelin, C. Vaulot for their technical help.


Jérôme Gattacceca

Pierre Rochette

Yoann Quesnel

Sounthone Singsoupho

Revisiting the paleomagnetism of Muong Nong layered tektites: implications for their formation process

Keywords: Muong Nong tektite, magnetic properties, Paleomagnetism, Australasian

10 Among Australasian tektites, the so-called Muong Nong tektites stand out for their peculiar 11 layering and blocky aspect. Although the source crater for the Australasian tektites is not 12 known, Muong Nong tektites are generally considered as a relatively proximal ejecta. The 13 mechanism responsible for the formation of the layering has been a matter of debate. In this 14 work, we revisit the paleomagnetism of Muong Nong tektites. They retain a thermoremanent 15 magnetization acquired during cooling below 585°C in the presence of the ambient 16 geomagnetic field, and carried magnetite in most samples, although at least one sample 17 containing metallic iron was detected. The inclination of the paleomagnetic direction with 18 respect to the layering plane clusters around 18±12°, compatible with the inclination of the 19 geomagnetic field for this latitude at the time of impact. This indicates that the layering of the 20 Muong Nong tektites was sub-horizontal while they were cooling below 585°C. The preferred 21 scenario for the formation of the layering of layered tektite is therefore by horizontal shear in 22 pools or sheets of molten material.

27 hypervelocity impacts at the surface of the Earth, and spread over extensive strewnfields (Glass, 28 1990). Five tektite fields are known: Australasian tektites, belizites, moldavites, ivoirites, 29 North-American tektites, by order of increasing age [START_REF] Koeberl | Tektite origin by hypervelocity asteroidal or cometary impact. Target rocks, 436 source craters, and mechanisms[END_REF]Rochette et al., 2021). All 30 can be associated to a known impact crater, except Australasian tektites whose source is yet not 31 firmly identified, although it has been recently proposed to be hidden under a volcanic plateau 32 in Southern Laos [START_REF] Sieh | 479 Australasian impact crater buried under the Bolaven volcanic field, Southern Laos[END_REF]. This is surprising in view of their relatively young age of 33 788±3 ka [START_REF] Nomade | Ultra-precise age and 427 temperature of formation of the Australasian tektites constrained by 40 Ar/ 39 Ar analyses[END_REF], the assumed large diameter of the putative crater and extensive 34 spread over at least 180 million km 2 (28% of the surface of the Earth) when including 35 microtektites found in deep sea sediments and Antarctica [START_REF] Folco | 409 Microtektites from Victoria Land Transantarctic Mountains[END_REF]Di Vincenzo al., 36 2021). A source crater located in Indochina is favored by most authors based on tektite and 37 microtektite distribution [START_REF] Glass | Geographic variation in Australasian microtektite 421 concentrations: Implications concerning the location and size of the source crater[END_REF][START_REF] Glass | Australasian microtektites and associated impact ejecta in 424 the South China Sea and the Middle Pleistocene supereruption of Toba[END_REF], despite 38 alternative locations proposed in Northern China [START_REF] Mizera | On a possible parent crater for Australasian tektites: 455 Geochemical, isotopic, geographical and other constraints[END_REF]. However, such a crater 39 has not been confirmed so far. In the absence of crater, competing hypotheses of formation by 40 multiple km-sized craters [START_REF] Wasson | Layered tektites: a multiple impact origin for the Australasian tektites[END_REF], or airburst without the need for an impact crater 41 have emerged for the Australasian tektites (e.g., Barnes, 1963;Wasson, 2003). Besides the 42 Australasian tektite case, this debate has far-reaching implications for the understanding of the 43 possible risk and environmental effects associated with large airbursts (e.g., Cavosie and 44 Koeberl, 2019).

45 Of particular interest in this debate are the Muong-Nong tektites, a sub-category of Australasian 46 tektites characterized by their larger size (up to above 20 kg), blocky aspect without the typical 47 splashforms attesting solidification during flight, and marked layering with adjacent dark and 48 light layers showing differences in chemical composition (Koeberl, 1992). These objects, also 74 focused primarily on the paleointensity of the magnetic field recorded by the tektites, with the 75 objective of determining if tektites were of lunar or terrestrial origin, an active debate at that 76 time. The directional data were only briefly mentioned and discussed, and it was John Wasson 77 who wrote "I suggest that the remanence should be measured again…" (Wasson, 2003), and 78 initiated this second study of Muong Nong tektites paleomagnetism. 79 80 2. Samples 81 Tektites are usually characterized by a purely paramagnetic behavior [START_REF] Gattacceca | Magnetic properties of tektites and other related impact glasses[END_REF](Rochette et al. , 82 2019)), not adapted for paleomagnetic investigations that require at least 10 ppm of 83 ferromagnetic inclusions such as magnetite or metal. However, it appears that Muong Nong 84 tektites contain a significant amount of such inclusions (Kleinman et al., 1969;Glass and 85 Barlow, 1979;[START_REF] Gattacceca | Magnetic properties of tektites and other related impact glasses[END_REF], making them suitable for a paleomagnetic study (de 86 [START_REF] Gasparis | The demagnetization data are presented in Figure 6. Origin-trending 243 high-temperature (HT) and high coercivity (HC) components could be isolated in all samples. 244 All samples have relatively well-defined paleomagnetic directions with an average maximum 245 angular deviation (MAD, as defined by Kirschvink[END_REF].

87 We studied 32 samples taken from 18 Muong Nong tektites (Table 1, Figure 1). They were 88 selected for their marked layering (Figure 2). Nine tektites, with label starting with T or TS 89 were selected and characterized by John Wasson. Seven tektites, with label starting with N, 90 were recovered and selected by Pierre Rochette within five collection sites prospected during a 91 field trip in 2019 around the Muong Nong locality in Southern Laos. The larger samples were 92 bought directly from villagers who collected them while cultivating their fields, thus ensuring 93 that the coordinates given in Table 1 are reliable. Finally, two samples (MNP1, MNP2) were 94 obtained from a dealer in Thailand. Selection criteria were well-expressed planar unfolded 95 layering (apart from sample N16, see below), large enough size to allow cutting samples of a 96 few cm 3 lacking magnetic soil incrustations that often found in the porous parts of Muong Nong 97 tektites. We encountered a significant number of Muong Nong where folding of layering 98 prevented the definition of a consistent presumed paleo-horizontal plane for the whole tektite. 124 chosen based on the initial NRM measurement to aim for a NRM of at least 10 -6 Am 2 allowing 125 reliable remanence measurements. Cutting was also aimed at removing the external, weathered 126 part of the tektites, with particular attention to soil incrustations that may be highly magnetic 127 compared to the bulk tektite glass. Additional cleaning was performed using a non magnetic 128 metallic needle, and for some samples with an additional treatment with ethanolamine 129 thioglycolate (EATG) to dissolve remaining adhering terrestrial oxihydroxides. An oriented 130 thin slice was cut from most sample to better define the layering (Figure 2).

131

Magnetic measurements were performed at CEREGE. Stepwise alternating field (AF) and thermal demagnetization were used 138 to characterize the NRM. AF demagnetization was performed using the in-line three-axis 139 demagnetization of the 2G magnetometer, or off-line by three-axis demagnetization with a 140 LDA5 AF demagnetizer from Agico. Thermal demagnetization was performed with a zero-141 field oven from ASC. Saturation isothermal remanent magnetization (sIRM) was imparted in a 142 field of 3 T with MMPM9 pulse magnetizer. Anhysteretic remanent magnetization was 143 imparted using the LDA5AF instrument. Magnetization components were computed from 144 NRM demagnetization data by principal component analysis [START_REF] Kirschvink | The least-squares line and plane and the analysis of palaeomagnetic 430 data[END_REF], using the 145 Paleomac software [START_REF] Cogné | PaleoMac: a Macintosh TM application for treating paleomagnetic data and 389 making plate reconstructions[END_REF]. 1). Only 3 samples stand out with higher residual 177 IRM after AF demagnetization of 100 mT: T428 (24 %) whose hysteresis properties suggest 178 that it contains metal instead of magnetite, T490 (23%), and N19 (18%). We have no hysteresis 179 data for T490 and N19, but a sample of T490 was demagnetized thermally and shows a 180 unblocking temperature spectrum that is different from the other tektites, with a marked 181 decrease of NRM starting at 490°C rather than 560°C (Figure 4A). This may evidence the 182 presence of metal that is being destroyed rather than demagnetized during the thermal 183 treatment. 3.

146
187 5.1. Tektites affected by lightning-generated magnetic fields 188 The NRM intensities span 4 orders of magnitude, ranging from 10 -7 to 10 -3 Am 2 /kg. This is in 189 contrast with the more restricted range of 10 -7 to 10 -5 Am 2 /kg obtained by de Gasparis et al. 190 (1975). NRM/sIRM ratios of the ten samples with highest NRM are close to, or exceeds 0.1, 191 suggesting that these tektites were exposed to a strong field at some point in their history. This 192 is confirmed by the more refined comparison of AF demagnetization data of NRM and sIRM 193 (Figure 5), using the so-called REM' approach [START_REF] Verrier | Estimating peak currents at ground lightning impacts using 490 remanent magnetization[END_REF]Gattacceca and 194 Rochette, 2004). While most samples have REM' values below 0. 354 layering show that the folding was acquired at temperatures above 585°C. For samples with 355 simple planar layering, the inclination of the paleomagnetic direction with respect to the 356 layering plane is not random and clusters near horizontal, with an average of 18±12°, 357 compatible with the inclination of the geomagnetic field for this latitude at the time of impact.

358 This confirms, based on a more extensive dataset, previous paleomagnetic results ( de Gasparis 359 et al., 1975) showing that the layering of the Muong Nong tektites was sub-horizontal while 360 they were cooling below 585°C. Some tektites kept this position at least until they cooled below 361 100-200°C, while others were affected by movements suggesting a dynamic cooling 362 environment. Please find attached a manuscript called "Revisiting the paleomagnetism of Muong Nong layered tektites: implications for their formation process" by myself and co-authors that we would like to submit to Meteoritics and Planetary Sciences for the special issue honoring John Wasson.

Indeed, the topic of this study was suggested to us in 2014 by John Wasson who had a long time interest in tektites and in Muong Nong tektites in particular. He also selected the samples for the study. Unfortunately, I was not able to complete the study before he passed away.

The original scope of this paleomagnetic study was to confirm that the layering of Muong Nong tektites was sub-horizontal at the time these glasses were cooling. In addition to this confirmation, that provide constraints on the formation mechanism of these tektites, we provide interesting data about the magnetic mineralogy of this unusual material. 2 Table 1: List of studied samples.

Yours sincerely,

  glassy bodies formed by high-temperature melting of target rocks during

  results are summarized in Table

F

F

  remagnetized by lightning; £: demagnetized by AF to 18 mT before thermal demagnetization. MDF: median destructive field, I: angle between 15 the magnetization component and the layering plane, MAD: maximum angular deviation, n: numbers of steps used in the computation of the 16 magnetization component.

  Demagnetization of sIRM by AF (Figure4B) shows coercivity spectra in agreement with 174 pseudo-single domain magnetite, with median destructive fields of 21±8 mT. The median 175 destructive field of ARM is 31±9 mT. After AF demagnetization of 100 mT, less than 10% of 176 the sIRM is left in 14 out of 17 tektites (Table

	173
	F F
	o r o r
	P P
	e e r e e r
	R R
	e v i e w e v i e w
	O n l O n l
	y y
	8

147 4. Intrinsic magnetic properties

  the original polarity of the layering of the Muong Nong tektites, as the inclination 345 of the NRM with respect to the layering plane should be negative (i.e., the NRM should be Our results show that while some Muong Nong tektites have been remagnetized by strong 350 magnetic fields, likely lighting-generated, a significant fraction retains a thermoremanent 351 magnetization acquired during cooling below 585°C in the presence of the ambient 352 geomagnetic field. This magnetization is carried by magnetite in most samples, although at 353 least one sample containing metallic iron was detected. The study of a sample with a folded

	344 to determine 346 pointing up).
	347
	348 7. Conclusion
	349
	F F F F F F F
	o r o r o r o r o r o r o r
	P P P P P P P
	e e r e e r e e r e e r e e r e e r e e r
	R R R R R R R
	e v i e w e v i e w e v i e w e v i e w e v i e w e v i e w e v i e w
	O n l O n l O n l O n l O n l O n l O n l
	y y y y y y y
	15

1, five samples with strong 195 NRM/sIRM have REM' values well above 0.1 over the whole upper range of their coercivity 196 spectrum (samples N03A, N03B, N17, N20A, T419). It is noteworthy that the REM' values of

  The preferred scenario for the formation of the layering of layered tektite is 363 therefore by horizontal shear in pools or sheets of molten material. Paleomagnetism also 364 provide a way to determine the polarity of the layering of Muong Nong tektites.

	Jérôme Gattacceca
	e-mail:gattacceca@cerege.fr
		Editor of MAPS
		Aix-en-Provence, April 19, 2021
	Dear Editor,	F o r F o r F o r F o r F F o r o r
		P e e r P e e r P e e r P e e r P P e e r e e r
		R e v i e w R e v i e w R e v i e w R e v i e w R e R e v v i e w i e w
		O n l y O n l y O n l y O n l y O O n n l l y y

365 Acknowledgements 366 This work is dedicated to the memory of John T. Wasson, for his inspiring leadership in the 367 field of meteorite and tektite studies. John suggested the topic of this article to the first author

  : saturation magnetization, M RS : saturation remanence, B C : coercivity, B CR : coercivity of remanence,  HF : high-field (paramagnetic + 10 diamagnetic) susceptibility, : low-field susceptibility. 11

	Page 29 of 32 Page 31 of 32								Running Head Running Head Running Head							Page 30 of 32
	6 12 Table 3. Paleomagnetic results
	7 Table 2: Hysteresis properties						HC/HT component			MC/MT component
								MDF	MDF	
	sample sample MNP1 MNP2 N03A N03B N16 N17 N19 N20A N20B TS6 T157 T365 T417 T419 T420 T428 T482 9 M sample mass (g) sub-sample method latitude N mass 95 350 278 16°02'48.72" mass (g) 128 16°02'48.72" 210 16°17'37.5" 275 16°15'33.5" 36 16°26'32" 49 16°26'34.5" 36 16°26'34.5" 6000 179 ~16°22' 322 14°26'0" 288 14°53'12" 215 ~14°54' 348 ~14°54' 127 ~14°54' 546 ~14°54' 8 (g) N17 0.23 N20A 1.04 N20B 5 .14 T419 0.74 T428 1.22 TS6 1.92 MNP1 AF 1.81 MNP2 AF 16.1 N03A † a AF 9.83 N03B † a Th 10.69 N03B † b AF 4.65 N16 1 Th 17.9 N16 2 Th 14.71 N16 3 Th 14.86 N16 4 Th 11.58 N17 † a Th 10.75 N17 † b AF 5.85 N19 AF 10.47 N20A † a AF 6.98 N20A † b Th 6.14 N20B b AF 5.15 T157 B1 Th 5.89 T157 B2 AF 2.84 T365 A1 Th 0.44 T365 A2 AF 0.40 T417 A2 Th 2.90 T417 B2 AF 3.26 T419 † B3b Th 0.40 T419 † B2 AF 0.74 T420 a AF 11.12 T420 b Th £ 14.26 T428 AF 13.33	longitude E M S NRM (Am2/kg) 106°25'08" Muong Nong area, Southern Laos area M RS sIRM ARM sIRM (10 -9 (Am2/kg) (mT) (mT) m 3 /kg) range B C collection Thailand (?) CEREGE collection£ n Thailand (?) CEREGE collection£ CEREGE collection$ 106°25'08" Muong Nong area, Southern Laos CEREGE collection$ 106°33'33.2" Muong Nong area, Southern Laos CEREGE collection$ 106°34'40.4" Muong Nong area, Southern Laos CEREGE collection$ 106°28'06.3" Muong Nong area, Southern Laos CEREGE collection* 106°28'48.7" Muong Nong area, Southern Laos CEREGE collection* 106°28'48.7" Muong Nong area, Southern Laos CEREGE collection* SE of Ubon Ratchathani, Thailand Smithsonian Institution (°) B CR I ~106°30' Muong Nong area, Southern Laos UCLA collection 105'25'54" Ban Nong Paen, North East Thailand UCLA collection 105°25'54" Ban Huai Sai, North East Thailand UCLA collection ~105°25' Ban Huai Sai, North East Thailand UCLA collection ~105°25' Ban Huai Sai, North East Thailand UCLA collection ~105°25' Ban Huai Sai, North East Thailand UCLA collection ~105°25' Ban Huai Sai, North East Thailand F o r P e e r R e v i e w O n l y MAD (°) (10 -3 Am 2 /kg) (10 -3 Am 2 /kg) (mT) (mT) 8.3 2.24 27 43 16.6 6.47 29.4 45.1 0.8 0.120 16 35 1 0.080 9 30 13.1 0.052 0.8 30 1.5 0.105 9 27 2.3310 -6 3.8710 -5 28 17 81.9 24-70 mT 15 73.2 29.3 2.8710 -7 1.1210 -5 32 19 99.3 16-70 mT 12 -28 9.3 6.5210 -6 1.6210 -5 30 16.5 77.3 3.3410 -6 80.5 330-585 mT 10 51.9 2.6 5.1610 -6 2.1710 -5 13 13 80.5 14-100 mT 19 45.4 1.3 1.4910 -7 93.6 5.1410 -8 87.3 6.9310 -8 86.9 2.4610 -7 87.7 1.9710 -5 83.5 250-585°C 12 38.6 2.6 1.8510 -5 1.2810 -4 24 15 83.5 10-80mT 20 48.8 0.9 7.0110 -8 8.2610 -6 26 42 88.6 4-120 mT 23 16.9 21.3 5.7910 -4 1.9110 -3 40 34 147.3 8-70 mT 20 15.6 1.4 9.2710 -4 147.3 100-585 °C 16 15.1 1.1 1.3710 -5 1.2010 -4 43 24 86.6 46-100 mT 7 3.3 5.5 1.6810 -7 80.6 575-585 °C 2 26 8.9 2.0310 -7 1.4110 -4 32 21 81.3 13-90 mT 27 9.5 21.7 F o r P e e r R e v i e w 4.2410 -6 83.4 560-585 °C 4 -4.5 2.9 4.4610 -6 7.6110 -5 30 22 84.5 20-100 mT 16 5 10.7 2.0810 -7 89.9 540-580 °C 4 -32.6 2.9 7.4810 -7 3.5010 -5 29 17 88.1 15-100 mT 22 -7.3 7.5 5.2910 -6 108.3 500-585 °C 6 57.7 3.2 6.9710 -6 7.2310 -5 28 17 96.6 12-100 mT 25 58.6 3.1 3.2910 -6 8.0510 -5 31 14 94.8 22-120 mT 18 -46.4 6.3 O n l y 1.9610 -6 96.1 250-570 °C* 11 -76.7 6.3 UCLA collection 2.5110 -6 5.5910 -5 50 21 132.7 22-100 mT 18 8.5 7.3	 HF (10 -9 m 3 /kg) range n 60.1 61.1 54.4 81.9 54.8 65.1 5-20 mT 4 540-575 3 5-12 mT 10 100-560 °C 15 8-17 mT 9 240-540 °C 10 5-15 mT 11 8-20 mT 6	I (°) -7.2 65.6 65.9 23.5 18.6 -58.2 -61.9 5.1	MAD (°)  (10 -9 m 3 /kg) 133 205 147 108 133 106 3 4.3 9.1 5.5 4.9 19.8 3.3 8.4
		T490 T482	183 B2B1	~14°54' Th	1.11	~105°25' 3.2910 -6	Ban Huai Sai, North East Thailand 94.6	UCLA collection 100-585°C 18	15.7	8
	3	T482 T490	B2A a	AF AF	1.55 14.41	2.8010 -6 1.2610 -7	5.2310 -5 8.5410 -6	28 38	17 31	93.5 84.9	34-90 mT 18-120 mT	15 20	2.2 64.4	19.7 9.6	2-14 mT	7	3.6	2.4
	4 *collected in situ, $: bought from villagers, £: bought from a dealer T490 b Th 16.63 1.8410 -7	81.2	540-585 °C	4	63.6	4.1	25-520 °C	12	-8	9
	5	TS6 TS6	C1b B	Th AF	1.96 1.90	1.3910 -6 8.7410 -7	1.1610 -4	18	13	103.1 106.5	560-585 °C 22-100 mT	4 17	-5.3 32.5	6.1 20.6	280-560 °C 9-20 mT	10 11	-37.5 -44.6	7.6 17
	13
								Meteoritics & Planetary Science Meteoritics & Planetary Science Meteoritics & Planetary Science							2 3

S

Meteoritics & Planetary ScienceRunning Head


Faraj Zgheel

Stéphanie Perrier

Lamia Remila

Ursula Houngue

Jean-Philippe Mazzucotelli

Olivier Morel

Cyril Auger

Valerie Schini-Kerth
email: valerie.schini-kerth@unistra.fr

Jean- Philippe Mazzucotelli

Valérie B Schini- Kerth

PhD Valérie B Schini-Kerth

EPA:DHA 6:1 is a superior omega-3 PUFAs formulation attenuating platelets-induced contractile responses in porcine coronary and human internal mammary artery by targeting the serotonin pathway via an increased endothelial formation of nitric oxide

Keywords: Omega-3 polyunsaturated fatty acids, endothelium, nitric oxide, eNOS, platelets, serotonin

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

Introduction

Several epidemiological studies and randomized control trials have indicated an inverse relationship between the dietary intake of long-chain omega-3 polyunsaturated fatty acids (PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and the primary and secondary prevention of cardiovascular diseases [START_REF] Delgado-Lista | Long chain omega-3 fatty acids and cardiovascular disease: a systematic review[END_REF][START_REF] Dinicolantonio | Omega-3s and cardiovascular health[END_REF]. Indeed, in 1999 the GISSI-P trial showed that dietary supplementation with omega-3 PUFAs (1 g/day) significantly decreased the risk of cardiovascular death (GISSI-Prevenzione Investigators, 1999). Similarly, EPA supplementation in Japanese patients with a history of coronary artery diseases is associated with a significant reduction of major coronary events, especially non-fatal events [START_REF] Yokoyama | Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint analysis[END_REF]. The beneficial cardiovascular effect of omega-3 PUFAs has been attributed, at least in part, to their ability to improve the blood lipid profile, particularly by decreasing the triglyceride level [START_REF] Delgado-Lista | Long chain omega-3 fatty acids and cardiovascular disease: a systematic review[END_REF][START_REF] Shaikh | Efficacy of a unique omega-3 formulation on the correction of nutritional deficiency and its effects on cardiovascular disease risk factors in a randomized controlled VASCAZEN® REVEAL Trial[END_REF]. Additional mechanisms have also been suggested, including anti-atherosclerotic effects, antihypertensive effects, anti-inflammatory effects and inhibition of platelet aggregation [START_REF] Calder | Omega-3 fatty acids and inflammatory processes[END_REF][START_REF] Stone | Fish consumption, fish oil, lipids, and coronary heart disease[END_REF]. Moreover, the beneficial cardiovascular effect of omega-3 PUFAs may also be due to the improvement of the endothelial function. Indeed, our previous study demonstrated that amongst several omega-3 PUFAs formulations, EPA:DHA 6:1 was identified as a potent stimulator of the endothelial formation of nitric oxide (NO), and also, to a lesser extent, of endothelium-dependent hyperpolarization (EDH) in porcine coronary arteries [START_REF] Zgheel | Redox-Sensitive Induction of Src/PI3-kinase/Akt and MAPKs Pathways Activate eNOS in Response to EPA:DHA 6:1[END_REF]. The increased endothelial NO formation is mediated by a redoxsensitive activation of Src/PI3-kinase/Akt and MAPKs pathways leading to endothelial NO synthase (eNOS) activation, which is dependent on both ratio and purity of the EPA:DHA formulation [START_REF] Zgheel | Redox-Sensitive Induction of Src/PI3-kinase/Akt and MAPKs Pathways Activate eNOS in Response to EPA:DHA 6:1[END_REF].

The spontaneous contraction of arteries, such as the coronary artery in Prinzmetal angina, has been described as an independent risk factor of cardiovascular events [START_REF] Macalpin | Some observations on and controversies about coronary arterial spasm[END_REF].

Coronary artery spasms are generally more frequent at sites of atherosclerotic lesions, which are characterized by an endothelial dysfunction [START_REF] Zaya | Provocative Testing for Coronary Reactivity and Spasm[END_REF]. The local activation of platelets at sites of endothelial dysfunction has been suggested to contribute to vascular injury, in part, by releasing vasoactive factors including serotonin (5-HT) and by stimulating the formation of vasoconstrictor prostanoids [START_REF] Kim | Dietary omega-3 fatty acids and endotheliumdependent responses in porcine cerebral arteries[END_REF][START_REF] Komori | Endothelium-dependent relaxation in response to aggregating platelets in porcine femoral veins and its modulation by diet[END_REF]Shimokawa et al., 1988aShimokawa et al., , 1988b;;[START_REF] Shimokawa | Angiographic demonstration of hyperconstriction induced by serotonin and aggregating platelets in porcine coronary arteries with regenerated endothelium[END_REF].

The long-term consumption of fish oil has been shown to improve endothelium-dependent vasodilatations in patients with coronary artery diseases, and also endothelium-dependent relaxations of coronary artery rings from hypercholesterolemic and atherosclerotic pigs (Shimokawa and Vanhoutte, 1988;[START_REF] Tagawa | Long-Term Treatment with Eicosapentaenoic Acid Improves Exercise-Induced Vasodilation in Patients with Coronary Artery Disease[END_REF]. Moreover, EPA induced endothelium-dependent and -independent relaxations of isolated sheep pulmonary arteries by increasing the formation of NO and by blunting the calcium pathway leading to relaxation [START_REF] Singh | Eicosapentaenoic acid-induced endothelium-dependent and -independent relaxation of sheep pulmonary artery[END_REF]. Altogether, these findings suggest that omega-3 PUFAs could exert a beneficial effect against platelets-induced vasospasms. Therefore, the present study aims to determine the ability of different ratios of EPA and DHA to inhibit aggregating plateletsinduced contractions in porcine coronary artery and human internal mammary artery rings and, if so, to determine the role of the endothelium and the underlying mechanism. internal mammary artery segments not used for bypass surgery. IMA were immediately collected and placed in chilled Krebs solution at 4°C, cleaned and cut in rings of 4-5 mm in length before the evaluation of vascular reactivity. The vasorelaxant effect or acetylcholine, bradykinin and EPA:DHA 6:1 was determined by constructing a concentration-relaxation curve following contraction of IMA rings to about 70% of the maximal contraction with U46619. In some experiments, rings were exposed to an inhibitor for 30 min before contraction. Neither the removal of the endothelium nor the different pharmacological tools affected the basal tension (data not shown).

Platelets

Platelets freshly isolated from blood of healthy human donors and suspended in Tyrodealbumin buffer were kindly provided by the local Blood Transfusion Center (Etablissement Français du Sang-Alsace, Strasbourg). The suspension of platelets was adjusted to 600 000 platelets/µl. Thereafter, increasing numbers of platelets were added to the Krebs solution in the organ chamber triggering their subsequent aggregation and degranulation. In some cases, rings were incubated in the presence of a pharmacological agent for 30 min before the addition of either platelets or 5-HT.

Statistical analysis

All values are expressed as means ± S.E.M. of n different experiments. Statistical analysis was performed using Student's paired t-test or an analysis of variance test followed by Bonferroni's post-hoc test as appropriate using Graphpad Prism software (version 5.04 for Windows, GraphPad Software, INC, CA, USA). P < 0.05 was considered as statistically significant.

Results

EPA:DHA 6:1 inhibits platelets-induced contractions in coronary artery rings: role of 5-HT and endothelial NO

Addition of increasing numbers of washed human platelets to organ baths induced concentration-dependent contractions in coronary artery rings, which were similar in rings with or without endothelium (Fig. 1A). The platelets-induced contractions were abolished in the presence of ketanserin, a 5-HT2 receptor antagonist, but not in the presence of the cyclooxygenase inhibitor indomethacin, indicating a major role of platelet-derived 5-HT (Fig. 1A andB). Although the EPA:DHA 1:1 formulation at 0.4 % v/v did not affect plateletinduced contractile responses in rings either with or without endothelium, EPA:DHA 6:1 at 0.4 % v/v abolished the contractile response in rings with endothelium but not in those without endothelium (Fig. 1C andD). The inhibitory effect of EPA:DHA 6:1 was prevented in the presence of the eNOS inhibitor N G -nitro-L-arginine (NLA) whereas it was not affected by inhibitors of EDH (TRAM-34 plus apamin, inhibitors of intermediate (IKCa) and small (SKCa) conductance calcium-activated potassium channels, respectively) or cyclooxygenases (indomethacin, Fig. 1E). These findings indicate that EPA:DHA 6:1 is a superior omega-3 PUFAs formulation reducing the activated platelets-induced 5-HT-mediated contractile responses in coronary artery rings most likely through an increased endothelial formation of NO.

EPA:DHA 6:1 inhibits 5-HT-induced contractions in coronary artery rings by increasing endothelial NO

Next, experiments were performed to determine whether omega-3 PUFAs attenuate contractions induced by 5-HT in coronary artery rings, and to clarify the role of the EPA:DHA ratio and the endothelium. In rings with endothelium, the EPA:DHA 1:1 formulation (0.4 % v/v) slightly but significantly attenuated the 5-HT-induced contractions only at the highest concentration (10 -5 M, Fig. 2A). In contrast, EPA:DHA 6:1 strongly inhibited 5-HT-induced contractions in a concentration-dependent manner (Fig. 2B). Indeed, EPA:DHA 6:1 at concentrations of 0.2 and 0.4 % v/v significantly reduced the contractile responses assessed at 10 -5 M of 5-HT by 57.5±2.1 and 93.2±0.5 %, respectively (Fig. 2B).

Although, EPA:DHA 6:1 markedly reduced 5-HT-induced contractile responses in rings with endothelium, no such effect was observed in those without endothelium (Fig. 2C). In addition, EPA:DHA formulations at a ratio of either 1:1 or 6:1 did not affect contractions induced by the thromboxane A2 analogue U46619 (Fig. 3). These findings indicate that EPA:DHA 6:1 is a superior omega-3 PUFAs formulation that preferentially attenuates the 5-HT-induced contractile responses in intact coronary artery rings.

Since EPA:DHA 6:1 is a superior stimulator than EPA:DHA 1:1 of the endothelial formation of NO and also, to some extent, of EDH [START_REF] Zgheel | Redox-Sensitive Induction of Src/PI3-kinase/Akt and MAPKs Pathways Activate eNOS in Response to EPA:DHA 6:1[END_REF], the role of NO and EDH in the ability of EPA:DHA 6:1 to inhibit the 5-HT-induced contraction was investigated. In the presence of indomethacin, the inhibitory effect of EPA:DHA 6:1 on 5-HT-induced contractile responses was abolished in the presence of the eNOS inhibitor NLA and not affected by inhibitors of EDH (TRAM-34 plus apamin, inhibitors of IKCa and SKCa, respectively), indicating a major role of NO (Fig. 4A andB). To determine the sensitivity of the 5-HTinduced contractile response to the inhibitory effect of NO, rings without endothelium were exposed to a low concentration of the NO donor sodium nitroprusside (SNP, 100 nM) prior to the construction of a concentration-contraction curve. Preliminary studies have indicated that SNP at 100 nM induced about 20-30 % relaxation of porcine coronary artery rings (data not shown). SNP (100 nM) markedly attenuated 5-HT-induced contractile responses, whereas those to U46619 were reduced only slightly but significantly at concentrations greater than 1µM (Fig. 5A andB). Altogether, these findings indicate that EPA:DHA 6:1 effectively inhibits 5-HT-induced contractile responses mainly by increasing the endothelial formation of NO.

EPA:DHA 6:1 is a superior inhibitor of 5-HT-induced contractile responses in human internal mammary artery rings

To determine the clinical relevance of the present findings, experiments were performed with human internal mammary artery (IMA) rings obtained from patients undergoing bypass surgery. Like with porcine coronary artery rings, EPA:DHA 1:1 did not affect the 5-HTinduced contractile responses in IMA rings, whereas they were abolished by EPA:DHA 6:1 at 0.4 % v/v (Fig. 6A andB). Moreover, the inhibitory effect of EPA:DHA 6:1 was abolished by NLA and not affected by inhibitors of EDH, indicating a pivotal role of the NO pathway (Fig. 6C). In contrast to contractions induced by 5-HT, those induced by U46619 were not affected by EPA:DHA 6:1 at 0.4 % v/v (Fig. 6D). In addition, EPA:DHA 6:1 as well as bradykinin and acetylcholine, two physiological agonists, induced concentration-dependent relaxations in human IMA rings (Fig. 7). These relaxations were abolished by NLA and not affected by inhibitors of EDH (Fig. 7A-C). Moreover, the relaxation induced by EPA:DHA 6:1 was significantly more pronounced than that to either bradykinin or acetylcholine (Fig. 7 A-D).

Altogether, these findings indicate that EPA:DHA 6:1 is a potent inhibitor of 5-HT-induced contractile responses in IMA rings most likely due to its ability to stimulate the endothelial formation of NO.

Discussion

The major novel findings of this study indicate that EPA:DHA 6:1, an omega-3 PUFAs formulation, effectively prevents activated platelets-induced 5-HT-mediated contractions in porcine coronary artery and 5-HT-mediated contractions in human internal mammary artery rings most likely by stimulating the endothelial formation of NO.

The inhibition of the activated platelets-and 5-HT-induced contractions in coronary arteries is dependent on the EPA:DHA ratio, with the 6:1 ratio being more potent than the 1:1 ratio.

These results are in line with our previous study indicating that the EPA:DHA 6:1 is a superior omega-3 PUFAs formulation for the induction of endothelium-dependent relaxations mediated by NO, and, to a lesser extent, EDH [START_REF] Zgheel | Redox-Sensitive Induction of Src/PI3-kinase/Akt and MAPKs Pathways Activate eNOS in Response to EPA:DHA 6:1[END_REF]. Indeed, although EPA and DHA alone evoked similar endothelium-dependent relaxations, their combination EPA:DHA 6:1 and 9:1 demonstrated superiority to the formulation EPA:DHA 1:1, 3:1, 1:3, 1:6 and 1:9.

Although the mechanism explaining such a synergistic effect remains unclear, it might involve differences in the integration of omega-3 PUFAs into membrane lipids and/or that the stability of the omega-3 PUFAs requires an optimal EPA:DHA ratio. EPA but not DHA has also been shown to cause cerebral microvascular vasodilatation involving arachidonic acid metabolites [START_REF] Ellis | Effect of fish oil n-3 fatty acids on cerebral microcirculation[END_REF], whereas DHA but not EPA supplementation improved forearm microvascular reactivity in overweight hyperlipidemic men [START_REF] Mori | Differential effects of eicosapentaenoic acid and docosahexaenoic acid on vascular reactivity of the forearm microcirculation in hyperlipidemic, overweight men[END_REF].

The inhibitory effect of EPA:DHA 6:1 requires the presence of a functional endothelium and since it is prevented by an inhibitor of eNOS, it implies a major role of the endothelial formation of NO. Similarly, EPA:DHA 6:1 also effectively inhibited the 5-HT-induced contractions in human internal mammary artery rings by an endothelium-dependent NOmediated mechanism. Previous studies have also shown that chronic ingestion of omega-3 PUFAs, in particular EPA, improved endothelium-dependent NO-mediated relaxations in porcine coronary artery rings as well as the forearm endothelium-dependent vasodilatation in humans [START_REF] Shimokawa | Effects of dietary supplementation with cod-liver oil on endothelium-dependent responses in porcine coronary arteries[END_REF][START_REF] Tagawa | Long-Term Treatment with Eicosapentaenoic Acid Improves Exercise-Induced Vasodilation in Patients with Coronary Artery Disease[END_REF].

In addition to the direct activation of the endothelial formation of NO, studies have suggested that omega-3 PUFAs might also exert beneficial effects by inhibiting the aggregation and activation of platelets [START_REF] Abeywardena | Longchain n-3 polyunsaturated fatty acids and blood vessel function[END_REF]. Indeed, dietary supplementation of human volunteers with fish oil rich in omega-3 PUFAs is associated ex vivo with a reduced susceptibility of platelets to aggregation [START_REF] Von Schacky | Long-term effects of dietary marine omega-3 fatty acids upon plasma and cellular lipids, platelet function, and eicosanoid formation in humans[END_REF]. Moreover, EPA and DHA inhibited platelet reactivity by a direct inhibition of the thromboxane receptor [START_REF] Swann | Eicosapentaenoic acid and docosahexaenoic acid are antagonists at the thromboxane A2/prostaglandin H2 receptor in human platelets[END_REF]. In the present study, platelets-induced contractions in porcine coronary artery rings involved mainly a 5-HT-mediated mechanism, as indicated by the prevention of the contractions in the presence of ketanserin, an antagonist of 5-HT2 receptors. Moreover, the contraction induced by aggregating platelets in coronary artery rings was not affected by indomethacin, ruling out the involvement of prostanoids.

Previous studies have indicated that in the rat mesenteric artery and aorta, EPA and DHA inhibited contractions in response to both a thromboxane A2 analogue and prostaglandin F2α involving TP and FP receptors, respectively (Shimokawa and Vanhoutte, 1988;[START_REF] Zellers | Heterogeneity of endothelium-dependent and endothelium-independent responses to aggregating platelets in porcine pulmonary arteries[END_REF]. However, the present findings indicate that although EPA:DHA 6:1 effectively inhibited the 5-HT-induced contraction in porcine coronary artery rings, the contraction induced by the thromboxane A2 analogue was not affected. Moreover, contractions induced by activated platelets in coronary artery rings, and the inhibitory effect of EPA:DHA 6:1 on platelet-induced contractions were both not affected by indomethacin, indicating that vasoactive prostanoids are not involved. Altogether, the present findings and the previous ones suggest that the role of prostanoids on the vascular effects of omega-3 PUFAs might depend on the species and/or the vascular bed.

To determine whether the observations in porcine coronary artery rings have a potential relevance for human blood vessels, the effect of EPA:DHA on 5-HT-induced contractions was assessed in human IMA rings. In cardiac surgery, the IMA is often used for coronary artery bypass graft, and is known to have a similar reactivity as the coronary artery (Shimokawa et al., 1988a). In addition, previous studies have indicated that the endothelium of the IMA expresses eNOS, releases NO and is subjected to potent NO-mediated relaxations in response to acetylcholine [START_REF] He | Expression and Function of Endothelial Nitric Oxide Synthase Messenger RNA and Protein Are Higher in Internal 16 Mammary Than in Radial Arteries[END_REF][START_REF] Shapira | Enhanced nitric oxide-mediated vascular relaxation in radial artery compared with internal mammary artery or saphenous vein[END_REF]. Moreover, the presence of 5-HT receptors has been demonstrated in IMA, and their activation by 5-HT has been suggested to contribute to vasospasms [START_REF] Rosenfeldt | Pharmacology of coronary artery bypass grafts[END_REF], in particular in arteries with an endothelial dysfunction [START_REF] Komori | Endothelium-dependent relaxation in response to aggregating platelets in porcine femoral veins and its modulation by diet[END_REF]. In the present study, 5-HT induced potent concentration-dependent contractions of IMA rings, which were significantly inhibited by EPA:DHA 6:1, but not by 1:1 at 0.4% v/v. In addition, EPA:DHA 6:1 also induced concentration-dependent NO-mediated relaxations in IMA rings and inhibited the 5-HTinduced contractions by a NO-dependent mechanism. Of interest, the ability of EPA:DHA 6:1 to induce endothelium-dependent NO-mediated relaxation in IMA rings was greater than that induced by physiological agonists including acetylcholine and bradykinin. Indeed, both physiological agonists induced only partial relaxations of IMA rings (less than 55%) indicating that IMAs obtained from patients undergoing coronary artery bypass graft surgery have an endothelial dysfunction. Taken together, these findings suggest that EPA:DHA 6:1 is a potent stimulator of the endothelial formation of NO, and, hence, may help to prevent platelet activation which can contribute to local vasospasms and possibly also to the arterial response to injury. Indeed, previous studies have indicated that platelets contribute to vascular remodeling via different mechanisms including recruitment of immune cells into the arterial wall, formation of inflammatory mediators, shedding of deleterious microparticles, chemokines release, and direct cell-cell interaction [START_REF] Lannan | Thrombosis, platelets, microparticles and PAH: more than a clot[END_REF][START_REF] Nording | Platelets in Inflammation and Atherogenesis[END_REF]. coronary artery remodeling in a model of left ventricular hypertrophy [START_REF] Yang | Coronary Artery Remodeling in a Model of Left Ventricular Pressure Overload Is Influenced by Platelets and Inflammatory Cells[END_REF].

Moreover, platelets promoted arterial remodeling through the GPIbα-mediated interaction with endothelial cells and the release of platelet dense granules [START_REF] Chandraratne | Critical role of platelet glycoprotein ibalpha in arterial remodeling[END_REF][START_REF] King | Platelet Dense-Granule Secretion Plays a Critical Role in Thrombosis and Subsequent Vascular Remodeling in Atherosclerotic Mice[END_REF].

In conclusion, the present findings indicate that EPA:DHA 6:1 is able to effectively prevent the platelets-induced 5-HT-mediated contractions in porcine coronary and human internal mammary artery rings most likely through an increased endothelial formation of NO, and, hence, may help to protect the cardiovascular system. The present findings using the human internal mammary artery further highlight the potential protective effect of omega-3 PUFAs on the human vasculature.

Acknowledgments

F.Z. was supported by a PhD fellowship from the Higher Education Ministry of Libya.

Conflict of interest

The authors report no conflict of interest.

Figure 1 .

 1 Figure 1. The platelets-induced 5-HT-mediated contraction of porcine coronary artery rings is

Figure 2 .

 2 Figure 2. Effects of EPA:DHA ratio and concentration on contractile responses to 5-HT in

Figure 3 .

 3 Figure 3. Effects of EPA:DHA ratio and concentration on contractile responses to a

Figure 4 .

 4 Figure 4. Characterization of the role of endothelium-derived relaxing factors in the inhibitory

Figure 5 .

 5 Figure 5. The presence of a low concentration of the NO donor sodium nitroprusside markedly

Figure 6 .

 6 Figure 6. EPA:DHA 6:1, but not 1:1 ratio, inhibits the 5-HT-induced contractions in human