Moinecha Madi

Marie-Pierre Rols

Laure Gibot

Gene Electrotransfer in 3D Reconstructed Human Dermal Tissue

Keywords: Delivery, plasmid DNA, electroporation, extracellular matrix, gene therapy, skin, tissue engineering

Gene electrotransfer into the skin is of particular interest for the development of medical applications including DNA vaccination, cancer treatment, wound healing or treatment of local skin disorders. However, such clinical applications are currently limited due to poor understanding of the mechanisms governing DNA electrotransfer within human tissue. Nowadays, most studies are carried out in rodent models but rodent skin varies from human skin in terms of cell composition and architecture. We used a tissue-engineering approach to study gene electrotransfer mechanisms in a human tissue context. Primary human dermal fibroblasts were cultured according to the self-assembly method to produce 3D reconstructed human dermal tissue. In this study, we showed that cells of the reconstructed cutaneous tissue were efficiently electropermeabilized by applying millisecond electric pulses, without affecting their viability. A reporter gene was successfully electrotransferred into this human tissue and gene expression was detected for up to 48h. Interestingly, the transfected cells were solely located on the upper surface of the tissue, where they were in close contact with plasmid DNA solution. Furthermore, we report evidences that electrotransfection success depends on plasmid mobility within tissue-rich in collagens, but not on cell proliferation status. In conclusion, in addition to proposing a reliable alternative to animal experiments, tissue engineering produces valid biological tool for the in vitro study of gene electrotransfer mechanisms in human tissue.

INTRODUCTION

Since few decades, a physical method named electroporation has been developed to locally deliver therapeutic drugs (cytotoxic molecules and nucleic acids) to a large range of targeted tissues [START_REF] Gothelf | Electrochemotherapy: results of cancer treatment using enhanced delivery of bleomycin by electroporation[END_REF][START_REF] Rols | Gene transfer by electrical fields[END_REF][START_REF] Andre | Nucleic acids electrotransfer in vivo: mechanisms and practical aspects[END_REF][START_REF] Yarmush | Electroporation-based technologies for medicine: principles, applications, and challenges[END_REF][START_REF] Mir | High-efficiency gene transfer into skeletal muscle mediated by electric pulses[END_REF]. This promising method is currently applied in human clinics. The major fields of application of DNA electrotransfer are vaccination and gene therapy in oncology [START_REF] Sersa | Electrochemotherapy of tumors as in situ vaccination boosted by immunogene electrotransfer[END_REF][START_REF] Vandermeulen | Optimisation of intradermal DNA electrotransfer for immunisation[END_REF][START_REF] Vandermeulen | The site of administration influences both the type and the magnitude of the immune response induced by DNA vaccine electroporation[END_REF]. Clinical applications have already began in veterinary and human oncology [START_REF] Cemazar | Cancer electrogene therapy with interleukin-12[END_REF][START_REF] Heller | Electroporation gene therapy preclinical and clinical trials for melanoma[END_REF][START_REF] Escoffre | Electrochemotherapy: progress and prospects[END_REF]. Hopeful results have been obtained with the first published phase-I trial on interleukin-12 electroporation, conducted in a cohort of patients with metastatic melanomas [START_REF] Daud | Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma[END_REF]. However, while gene electrotransfer is highly efficient in a large panel of cell lines in vitro, gene expression is usually less efficient in vivo in some tissues such as tumors and the skin [START_REF] Vandermeulen | Optimisation of intradermal DNA electrotransfer for immunisation[END_REF][START_REF] Rols | In vivo electrically mediated protein and gene transfer in murine melanoma[END_REF]. Although the first study on DNA electrotransfer was initiated more than 30 years ago [START_REF] Neumann | Gene transfer into mouse lyoma cells by electroporation in high electric fields[END_REF], underlying mechanisms are not yet fully understood, in particular in in vivo situations [START_REF] Escoffre | What is (still not) known of the mechanism by which electroporation mediates gene transfer and expression in cells and tissues[END_REF]. It is therefore still mandatory, for further developing the method in clinics, to increase our knowledge of the process.

The skin is an appealing tissue target for gene electrotherapy because of its large surface area, its accessibility and its immunological properties with antigen presenting cells present both in the epidermis and in the dermis [START_REF] Rochard | Genetic immunization with plasmid DNA mediated by electrotransfer[END_REF]. Gene electrotransfer to muscle tissue is highly effective and leads to long-term transgene expression [START_REF] Mir | Long-term, high level in vivo gene expression after electric pulsemediated gene transfer into skeletal muscle[END_REF][START_REF] Muramatsu | In vivo gene electroporation in skeletal muscle with special reference to the duration of gene expression[END_REF]. With some plasmids and under optimized delivery conditions, it could achieve higher expression than in skin [START_REF] Gothelf | Gene electrotransfer to skin; review of existing literature and clinical perspectives[END_REF]. Nevertheless, high and/or long-term transfection are not always required [START_REF] Shirley | Controlled gene delivery can enhance therapeutic outcome for cancer immune therapy for melanoma[END_REF]. For example, in wound healing, there is no need for therapeutic growth factor transgene expression when the wound has closed [START_REF] Cupp | Gene therapy, electroporation, and the future of wound-healing therapies[END_REF][START_REF] Ferguson | Wound healing enhancement: electroporation to address a classic problem of military medicine[END_REF]. Similarly, DNA vaccination does not require a continuous production of antigens once the immune system has responded to the vaccine [START_REF] Yarmush | Electroporation-based technologies for medicine: principles, applications, and challenges[END_REF][START_REF] Gothelf | What you always needed to know about electroporation based DNA vaccines[END_REF]. Donate and collaborators gave evidence that vaccine to the skin is as, if not more, efficient than delivery to muscle [START_REF] Donate | Assessment of delivery parameters with the multi-electrode array for development of a DNA vaccine against Bacillus anthracis[END_REF]. Gene electrotransfer to the skin can be as efficient as muscle as reported by Heller et al showing over 30% efficiency delivering to the skin [START_REF] Heller | Optimization of cutaneous electrically mediated plasmid DNA delivery using novel electrode[END_REF].

Skin consists of distinct cell types, mainly keratinocytes, melanocytes, Langerhans cells, fibroblasts and endothelial cells. This diversity of cell types can differently respond to gene transfection depending on the plasmid construction (i.e., tissue-specific promoters), on the electrical parameters and/or on the type of electrodes used.

*Address correspondence to this author at IPBS-CNRS, 205 route de Narbonne, 31077 Toulouse, France; Tel/Fax: +33-561-175-827, +33-561-175-994; E-mails: gibot@ipbs.fr; rols@ipbs.fr

Please provide corresponding author(s) photograph

In order to understand the role of each parameter of the experimental setup on the distinct cell types present within the skin during the gene electrotransfer process, relevant biological models of human skin tissue have to be developed.

Using the tissue engineering self-assembly approach [START_REF] Athanasiou | Self-organization and the self-assembling process in tissue engineering[END_REF], we produced reconstructed human dermal tissue, devoid of any exogenous material, and rich in native extracellular matrix [START_REF] Gibot | A preexisting microvascular network benefits in vivo revascularization of a microvascularized tissue-engineered skin substitute[END_REF][START_REF] Gibot | Development of a tridimensional microvascularized human skin substitute to study melanoma biology[END_REF]. In this context, primary human dermal fibroblasts reproduce a native environment, by secreting and organizing a 3D extracellular matrix dense in collagens. These abundant collagen fibrils were clearly detected by second harmonic generation (SHG) [START_REF] Madi | Efficient In Vitro Electropermeabilization of Reconstructed Human Dermal Tissue[END_REF]. In a previous study, we showed that this tissue-engineered model is efficiently electropermeabilized with millisecond electric pulses [START_REF] Madi | Efficient In Vitro Electropermeabilization of Reconstructed Human Dermal Tissue[END_REF]. Thus, we propose here to further examine gene electrotransfer mechanisms in vitro in human tissue by using this relevant and useful biological model.

MATERIALS AND METHODS

Cell culture. Primary human dermal fibroblasts were extracted from human child foreskin after posthectomy as previously described [START_REF] Gibot | A preexisting microvascular network benefits in vivo revascularization of a microvascularized tissue-engineered skin substitute[END_REF]. Primary cells were grown at 37°C in Dulbecco's Modified Eagles Medium (Gibco-Invitrogen, Carlsbad, USA) supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin.

Reconstruction of human 3D dermal tissue. Human dermal substitutes, devoid of any exogenous material, were reconstructed in vitro according to the self-assembly approach. In a few words, primary human dermal fibroblasts were grown for three weeks in cell culture medium supplemented with 50 µg /mL of ascorbic acid (Sigma-Aldrich) as already published [START_REF] Gibot | Development of a tridimensional microvascularized human skin substitute to study melanoma biology[END_REF][START_REF] Madi | Efficient In Vitro Electropermeabilization of Reconstructed Human Dermal Tissue[END_REF]. After three weeks, enough extracellular matrix was secreted to produce a cell sheet that is easy to manipulate. Since the cells are primary dermal fibroblasts, extracellular matrix is comparable to human dermis.

Collagen detection by second harmonic generation (SHG).

Stacks of 425x425x60 μm were acquired using a 7MP multiphoton microscope (Carl Zeiss, Jena, Germany) coupled to a Ti-Sapphire laser, Chameleon Ultra 2 (Coherent Inc) settled to 800 nm. Organized fibrillar collagens were detected by second harmonic generation at a wavelength of 400 nm. Imaris software (Bitplane AG) was used to analyzed the acquired 3D images.

Electropermeabilization and gene electrotransfer in 3D

human dermal tissue. Two flat parallel stainless steel electrodes were placed on the top of a cell sheet and immersed in pulsing buffer (10 mM K2HPO4/ KH2PO4 buffer, 1 mM MgCl2, 250 mM sucrose, pH 7.4) containing or not 100µM propidium iodide (PI) (Sigma-Aldrich) or 2µg of 4.7 kb pEGFP-C1 DNA plasmid (Clontech, #6084-1). Electropulsation was performed with an ELECTRO cell S20 generator (βTech, Toulouse, France) which delivered squarewave electric pulses. The electrical conditions were the following: 8 pulses lasting 5 ms at a frequency of 1 Hz applied from 0 to 800V/cm at room temperature.

Electropermeabilization detection and quantification.

Cell membrane permeabilization was analyzed on living tissues by multiphoton microscopy. Electropermeabilization efficiency was investigated by adding PI to the pulsation buffer during electric field application. IMARIS software was used to draw an isosurface and to isolate the electropermeabilized cells (i.e. cells in which PI was present).

Cell viability. Tissue viability was determined by adding PI 24h after application of the electric field. Viability was also quantified using PrestoBlue assay (Invitrogen) according the manufacturer's protocol. Briefly, 24h after electric field application, the cell sheet was incubated for 30 min at 37°C in a PrestoBlue solution. Absorbance (excitation 570 nm, emission 600 nm) was then read using a plate reader (Clariostar, BMG). Controls, named "Dead" were cell sheets subjected to 3 freeze/thaw cycles).

Reporter gene electrotransfer efficiency. 3D stacks showing both SHG and efficiently electrotransfected GFPpositive cells were acquired using a multiphoton laser scanning microscope. Pictures were analysed with IMARIS software to show both top and side views of the cell sheet. Image J software (NIH, Bethesda, USA) was used to quantify the GFP-positive areas within total pictures (425x425 μm).

Cell proliferation within dermal tissue. Cell proliferation was assessed with Click-it® EdU Alexa Fluor® imaging kit (Molecular Probes). In a few words, cell sheet was incubated for 24h with 10µM EdU before being processed according to the manufacturer's recommendations. Reconstructed dermal tissues were observed with a 7MP multiphoton laser scanning microscope.

DNA staining and gene electrotransfer. pEGFP-C1 plasmid was stoichiometrically labelled with TOTO-1 (Molecular Probes), a DNA intercalating dye as previously described [START_REF] Golzio | Direct visualization at the single-cell level of electrically mediated gene delivery[END_REF]. The plasmid was stained with 2.3x10-4 M dye at a DNA concentration of 1 μg/μl for 60 min on ice. Cell sheet was incubated in labeled plasmid-containing pulsation buffer and exposed to 8 pulses each lasting 5 ms at a frequency of 1 Hz at 800V/cm at room temperature. Cell sheets were observed 1 minute after the pulses with the 7MP multiphoton laser scanning microscope.

Statistical analyses. Statistical significance in cell viability

after different electrical treatments was determined using 1way ANOVA with a Dunnett's post-test. Statistical analyses used GraphPad Prism software, version 4.02 (GraphPad Software, San Diego, USA). All plotted data were expressed as means ± SEM.

RESULTS AND DISCUSSION

The aim of our study was to examine the relevance of reconstructed human dermal tissue as a model to address gene electrotransfer processes. To do so, we used standard electrical parameters for gene electrotransfer (i.e, 8 pulses lasting 5 ms at 1 Hz frequency with field intensities from 0V/cm to 800V/cm). Multiphoton imaging allowed visualization of membrane permeabilization, gene transfer and expression at the single cell level within tissue, on fresh human models. Cell uptake of propidium iodide was used to detect membrane permeabilization. A plasmid carrying the Green Fluorescent Protein gene allowed detection of gene transfer and expression. This methodology addresses the following issues: (i) Can the cells within reconstructed dermal tissue be efficiently electropermeabilized and transfected? (ii) Does transfection affect cell viability? (iii) Does the location of the cells within the tissue and their proliferative status affect the process? Cells within reconstructed human dermal tissue are efficiently electropermeabilized by millisecond electric pulses. Electropermeabilization was assessed by adding propidium iodide during electric field application. Images obtained by microscopy were analyzed by IMARIS software which allows cell sheets to be visualized both from top and side views (Fig. 1). In the control condition (0V/cm), a few permeable cells that correspond to dead cells were observed in the cell sheet. Indeed, cell renewal takes place in the reconstructed human dermal tissue model, and dead cells are entrapped within the extracellular matrix. By applying the electric pulses and increasing the electric field intensity, cells located both on the surface and in depth were efficiently and homogeneously electropermeabilized. Thus, the whole cells embedded within the tissue model can be electropermeabilized. We then checked whether membrane permeabilization was transient or not. Irreversible electroporation (IRE) offers great promise in interventional oncology with thermal or non-thermal tumor ablation [START_REF] Yarmush | Electroporation-based technologies for medicine: principles, applications, and challenges[END_REF][START_REF] Deipolyi | Irreversible electroporation: evolution of a laboratory technique in interventional oncology[END_REF]. However, IRE has to be avoided in gene therapy since it is not compatible with gene expression.

Cell viability within tissue is preserved after application of millisecond electric pulses. Membrane resealing occurs in about 20 minutes after application of millisecond pulses [START_REF] Rols | Electropermeabilization of mammalian cells. Quantitative analysis of the phenomenon[END_REF][START_REF] Saulis | Size of the pores created by an electric pulse: microsecond vs millisecond pulses[END_REF]. Thus, when added 24h after the electric pulses, PI only enters into irreversibly permeabilized cells i.e. dead cells. Control tissues revealed only a few positive cells while "dead" tissue was highly fluorescent. Images showed no significant increase in the number of dead cells after applying the electric field up to 600 V/cm (Fig. 2A). A resazurin-based functional test involving viable cell metabolic activity (Fig. 2B), was used to quantify tissue viability and showed no statistical difference between control cells (0 V/cm) and cells pulsed up to 600 V/cm. A statistically non-significant decrease was observed at 800V/cm, as confirmed by PI-positive cells increase on fluorescent pictures. At 800V/cm, side views indicated that dead cells were situated on the top of the cell sheet (data not shown). Previous histological analyses showed that several thousand cells are embedded in multiple layers within the 50 µm thick sheet [START_REF] Madi | Efficient In Vitro Electropermeabilization of Reconstructed Human Dermal Tissue[END_REF]. Thus, even though some cells appeared positive to PI at 800V/cm, their number was not significant.

Accordingly, millisecond electric pulses up to 800 V/cm did not significantly affect cell viability within the dermal tissue.

Gene electrotransfer is spatially and temporally limited within the tissue. GFP reporter gene plasmid was electrotransferred within 3D reconstructed dermal tissue (Fig. 3). No transfected cells were detected when no electric field was applied (0V/cm) or when no plasmid DNA was added (data not shown). GFP expression was observed 6h after gene electrotransfer and lasted up to 72h (data not shown). Electrotransfection was observed for electric field intensities above 200 V/cm, i.e. under electric field conditions leading to cell electropermeabilization. GFPexpressing cells increased with the electric field intensity. Cross-sectional views showed that only cells located at the top of the cell sheet were efficiently transfected (Fig. 3A), whatever the electric field intensity. This could be due to the fact that only the top of the sheet was in contact with the plasmids during application of the electric field. Quantification of GFP-positive area 24h and 48h after gene electrotransfer revealed that the most efficient electric field intensity was 800 V/cm, leading to a 8% rate of transfection (Fig. 3B). This percentage is quite low compared to in vivo studies achieving 30% efficiency delivery to the skin [START_REF] Heller | Optimization of cutaneous electrically mediated plasmid DNA delivery using novel electrode[END_REF]. Further optimization of both electrodes geometry and placement could improve electric field distribution and increase plasmid mobility within our tissue model, leading to a better gene electrotransfer efficiency.

Reporter gene expression depends on plasmid mobility within the tissue but not on cell proliferation status. A hypothesis to explain why transfected cells where solely located at the tissue surface is that only these cells are proliferative. Proliferative cells are usually more efficiently electrotransfected because, as previously reported [START_REF] Golzio | Cell synchronization effect on mammalian cell permeabilization and gene delivery by electric field[END_REF], the disintegration of the nuclear membrane during mitosis helps the direct access of DNA plasmid to the nucleus, enhancing gene expression. In order to characterize cell proliferation within our 3D model, we aimed to visualize cells engaged in S-phase replication [START_REF] Laurent | Multicellular tumor spheroid models to explore cell cycle checkpoints in 3D[END_REF]. A nucleoside analog of thymidine was added to the culture medium. Proliferative cells were homogenously distributed through the 3 dimensions within the reconstructed dermal tissue (Fig. 4A). This constant cell renewal cannot explain the localization of the transfected cells but could explain why transgene expression lasted for less than 72h.

We then aimed to determine if the accessibility of plasmid DNA to cells could explain why only cells in the upper part of the dermal tissue were transfected. To do so, cells were pulsed in the presence of a fluorescently labeled-plasmid DNA that allowed its 3D distribution within dermal tissue to be visualized (Fig. 4B). Under electrical parameters that induce efficient cell permeabilization and transfection (i.e 800 V/cm), plasmid DNA was showed to interact with the cells. Interestingly, this interaction was limited to the cell side facing the cathode as previously described on cells grown in monolayers and in suspension [START_REF] Golzio | Direct visualization at the single-cell level of electrically mediated gene delivery[END_REF]. Thus, for the first time we were able to observe DNA-membrane interactions in a healthy tissue. Interestingly, by DNA spot patterns we were able to recognize fibroblast typical morphology. Indeed, these stromal cells are elongated cells growing oriented in parallel. Analysis of tissue crosssectional views revealed that fluorescent plasmid DNA was confined to the tissue surface. It has been reported that DNA plasmid electrophoretic transport is inversely correlated with the amount of collagen in tumor tissue [START_REF] Zaharoff | Electromobility of plasmid DNA in tumor tissues during electric field-mediated gene delivery[END_REF]. Thus, tumors containing only some proteoglycans and collagens were found to be more efficiently electrotransfected in vivo than solid tumors with a rich extracellular matrix [START_REF] Cemazar | Effective gene transfer to solid tumors using different nonviral gene delivery techniques: electroporation, liposomes, and integrin-targeted vector[END_REF][START_REF] Mesojednik | The effect of the histological properties of tumors on transfection efficiency of electrically assisted gene delivery to solid tumors in mice[END_REF]. Collagen content modulates tumor tissue resistance to macromolecule diffusion by linking and stabilizing glycosaminoglycans [START_REF] Netti | Role of extracellular matrix assembly in interstitial transport in solid tumors[END_REF]. ]. Since the reconstructed human dermal tissue is highly rich in collagens [START_REF] Madi | Efficient In Vitro Electropermeabilization of Reconstructed Human Dermal Tissue[END_REF], it seems that this observation is also correct for solid healthy tissue.

We are now optimizing the electrical parameters to improve gene electrotransfer efficiency in a healthy tissue context. Indeed, some authors previously showed that an association of high voltage with low voltage pulses enhanced gene expression in skin [START_REF] Vandermeulen | Optimisation of intradermal DNA electrotransfer for immunisation[END_REF][START_REF] Andre | Efficiency of high-and low-voltage pulse combinations for gene electrotransfer in muscle, liver, tumor, and skin[END_REF][START_REF] Gothelf | Duration and level of transgene expression after gene electrotransfer to skin in mice[END_REF][START_REF] Pavselj | DNA electrotransfer into the skin using a combination of one high-and one low-voltage pulse[END_REF]. Additionally, skin is a complex tissue composed of distinct cell types. Theoretically cell types with different morphological characteristics can be targeted and transfected by specific electrical parameters [START_REF] Rols | Electropermeabilization of mammalian cells. Quantitative analysis of the phenomenon[END_REF]. Thus, we plan to refine our skin tissue-engineered model by adding an epidermis compartment as previously described [START_REF] Gibot | A preexisting microvascular network benefits in vivo revascularization of a microvascularized tissue-engineered skin substitute[END_REF][START_REF] Gibot | Development of a tridimensional microvascularized human skin substitute to study melanoma biology[END_REF] in order to study cell targeting. Finally, we would like to investigate the role of plasmid construction on gene electrotransfer efficiency in skin. Indeed, tissue-specific promoters lead to smaller transfection rates but specific cells can be better targeted [START_REF] Kos | Improved Specificity of Gene Electrotransfer to Skin Using pDNA Under the Control of Collagen Tissue-Specific Promoter[END_REF][START_REF] Vandermeulen | Skin-specific promoters for genetic immunisation by DNA electroporation[END_REF].

CONCLUSION

Reconstructed human dermal tissue appears to be a relevant tool to mimic and assess gene electrotransfer in human skin. Using this model, we will gain a better comprehension of the mechanisms involved in gene electrotransfer and its limits in tissues and propose strategies to improve its efficiency, while maintaining its safety, or to specifically target a particular cutaneous cell type.

CONFLICT OF INTEREST

Authors declare they have no competing financial interests. This work was supported by the Centre National de la Recherche Scientifique (CNRS) (MPR), the Agence Nationale de la Recherche (ANR) (MPR and LG), Projet PIERGEN ANR-12-ASTR-0039, and Mayotte department (MM).

Current Gene Therapy, 2015, Vol. 0, No. 0 Madi et al.

ACKNOWLEDGEMENTS

The Research was conducted in the scope of the EBAM European Associated Laboratory (LEA) and is a result of networking efforts within COST TD1104 action.

Microscopy experiments were carried out at the Plateforme Genotoul Toulouse RIO Imaging. The authors would like to gratefully acknowledge Elisabeth Bellard (IPBS) for her technical assistance in imaging analysis and Peter Winterton, a native English scientist, for his careful proofreading of this manuscript.


Sylvie Crasquin
email: sylvie.crasquin@upmc.fr.2

Francesco Sciuto
email: fsciuto@unict.it.tel.39957195769.

Agatino Reitano

Late Carnian (Tuvalian, Tropites dilleri zone) ostracods (Crustacea) from the Mufara Formation (Monte Scalpello, Central-Eastern Sicily, Italy)

Keywords: Bairdia scaliai n. sp., Acratia maugerii n. sp., "Anchistrocheles" gemmellaroi n. sp. et Judahella? montanarii n. sp. D' Ostracods: Late Triassic, Neo-Tethys, Central-Eastern Sicily, Mount Scalpello, Mufara Formation, palaeoenvironmental reconstruction, new species Trias supérieur, Néotéthys, Sicile centre-orientale, Mont Scalpello, Formation Mufara, paléoenvironnement, nouvelles espèces

Ostracod association from the Upper Triassic (Tropites dilleri zone of the Carnian stage) of the sedimentary succession (Mufara Formation) exposed along the east side of Monte Scalpello (Catenanuova, central eastern Sicily) have been studied for the first time. The specimens, silicified, are rare but well preserved and often consist of complete carapaces. They belonging to eight families: Healdiidae, Cavellinidae, Bairdiidae, Acratiidae, Bythocyprididae, Pontocyprididae, Judahellidae, Glorianellidae. Twenty -three taxa have been listed; the family Bairdiidae is the most represented among all the others with fifteen species. Four species are new: Bairdia scaliae n. sp., Acratia maugerii n. sp., "Anchistrocheles"gemmellaroi n. sp. and Judahella? montanarii n. sp. Other species are left in open nomenclature because of the lack of specimens. Résumé L'association d'ostracodes de la succession sédimentaire du Trias supérieur (Carnien, Tropites dilleri zone, Mufara Formation) affleurant du côté oriental du Monte Scalpello (Catenanuova, Sicile centre-orientale) a été étudiée pour la première fois. Les spécimens, silicifiés, sont rares mais bien conservés et se composent souvent de carapaces complètes. Les espèces appartiennent à huit familles: Healdiidae, Cavellinidae, Bairdiidae, Acratiidae, Bythocyprididae,Pontocyprididae, Judahellidae, Glorianellidae. La famille Bairdiidae est la plus représentée avec quinze espèces sur les vingt-trois répertoriées. Quatre espèces sont nouvelles

INTRODUCTION

After the first studies on Mesozoic sediments cropping out in the Monte Scalpello area carried out since the early 19th century [START_REF] Calcara | Monografie dei generi Clausilia e Bulimo, coll'aggiunta di alcune nuove specie di conchiglie siciliane esistenti nella collezione della Sig[END_REF][START_REF] Calcara | Cenno sui molluschi viventi e fossili della Sicilia da servire di supplement ed insieme di critiche osservazioni all'opera di R. A. Philippi[END_REF]Nelli, 1899a, b;[START_REF] Marinelli | Osservazioni geologiche sopra i terreni secondari del gruppo del M. Judica in Sicilia[END_REF][START_REF] Gemmellaro | Cenno sopra taluni organici fossili del Turoniano e Nummulitico di Judica[END_REF][START_REF] Gemmellaro | I Cefalopodi del Trias Superiore della regione occidentale della Sicilia[END_REF], the Upper Triassic fauna of "Clay-calcareous-arenaceous alternation" ("Flysch Carnico" Auct.), best referring to the "Mufara Formation" (Schmidt di [START_REF] Schmidt Di Friedberg | Contribution à l'étude structurale du groupe du Monte Judica (Sicile orientale)[END_REF] of the "Monte Judica Group" [START_REF] Scalia | Il gruppo di M. Judica[END_REF] have been the subject of numerous palaeontological detailed studies on invertebrates: the molluscs have been studied by Scalia (1907[START_REF] Scalia | Il gruppo di M. Judica[END_REF]Scalia ( , 1910Scalia ( , 1912Scalia ( , 1914)), the cnidaria by Maugeri [START_REF] Maugeri Patanè | Sopra alcuni coralli del Trias superiore di M. Scalpello (Enna)[END_REF], the ammonites by [START_REF] Lentini | Caratteri stratigrafici e strutturali della zona di Monte Judica (Sicilia Orientale)[END_REF], the foraminifers, chaetetids, sphinctozoans and other organisms by [START_REF] Carrillat | Palaeoenvironmental reconstruction of the Mufara Formation (Upper Triassic, Sicily): High resolution sedimentology, biostratigraphy and sea-level changes Palaeogeography[END_REF].

So far, ostracods (Crustacea) coming from the Mufara Formation in the Monte Scalpello area never have been studied; the only references to these organisms are found in [START_REF] Lentini | Caratteri stratigrafici e strutturali della zona di Monte Judica (Sicilia Orientale)[END_REF] which refers about a high level of the Mufara Formation rich in Bairdiidae and in [START_REF] Carrillat | Palaeoenvironmental reconstruction of the Mufara Formation (Upper Triassic, Sicily): High resolution sedimentology, biostratigraphy and sea-level changes Palaeogeography[END_REF] which cite, only the presence of this organisms.

The present paper, therefore, wants to be a first contribution to the knowledge of the ostracod fauna of the Mufara Formation clays outcropping in the Monte Scalpello area.

Furthermore, this analysis is an important step in the knowledge of the recovery of ostracod fauna after the Permian -Triassic massive extinction. The data on Middle and early Late Triassic are rare.

MATERIALS AND METHODS

Monte Scalpello is located (Fig. 1) in central eastern Sicily in the right side of Dittaino River to the south east of the town of Catenanuova (EN), 40 Km west of Catania (F 633 IV NO of the Carta d'Italia alla scala 1: 25.000). It is inserted, as part of the "Monte Judica Units" [START_REF] Lentini | Introduzione alla geologia della Sicilia e guida all'escursione[END_REF], along the northern margin of the Gela Foredeep, in the geodynamic context (Fig. 2) of the southern end of Fig. 2. Structural scheme of the central Mediterranean area with the location of the study area (after Grasso, 2001 modified). Schéma structural de la Méditerranée centrale avec la localisation de la zone d'étude (d'après Grasso, 2001 modifié).

the Maghrebian -Sicilian-Southern Apennine nappes and as such, it would dwarf the mildly folded foreland (Hyblean Plateau) [START_REF] Lentini | Introduzione alla geologia della Sicilia e guida all'escursione[END_REF]Grasso, 2001 inter alias).

The Monte Scalpello area is lithologically characterized by a Mesozoic-Cainozoic sediments complex belonging to the so-called Imerese Succession [START_REF] Lentini | Introduzione alla geologia della Sicilia e guida all'escursione[END_REF][START_REF] Montanari | Lineamenti stratigrafico -paleogeografici della Sicilia durante il Ciclo Alpino[END_REF]inter alias) or Imerese-Sicano Succession [START_REF] Carrillat | Palaeoenvironmental reconstruction of the Mufara Formation (Upper Triassic, Sicily): High resolution sedimentology, biostratigraphy and sea-level changes Palaeogeography[END_REF][START_REF] Di Paolo | Maximum burial and unroofing of Mt. Judica recess area in Sicily: implication for the Apenninic-Maghrebian wedge dynamics[END_REF]. This sedimentary succession settled inside the pelagic Imerese Basin which had formed during the Meso-Cenozoic extensional phases and which was delimited by the Panormide Carbonate Platform to the west and the Trapanese Carbonate Platform to the east and to the south (Fig. 3) [START_REF] Catalano | Guida alla geologia della Sicilia occidentale. Guide geologiche regionali[END_REF][START_REF] Montanari | Lineamenti stratigrafico -paleogeografici della Sicilia durante il Ciclo Alpino[END_REF][START_REF] Speranza | Ultrathick Triassic dolomites control the rupture behavior of the central Apennine seismicity: Evidence from magnetic modeling of the L'Aquila fault zone[END_REF]. [START_REF] Speranza | Ultrathick Triassic dolomites control the rupture behavior of the central Apennine seismicity: Evidence from magnetic modeling of the L'Aquila fault zone[END_REF], [START_REF] Golonka | Late Triassic and Early Jurassic palaeogeography of the world[END_REF]Preto et al., 2010 modified). Reconstruction paléogéographique schématique de la Pangée et de la Néo-Téthys à la fin du Trias (d'après [START_REF] Speranza | Ultrathick Triassic dolomites control the rupture behavior of the central Apennine seismicity: Evidence from magnetic modeling of the L'Aquila fault zone[END_REF][START_REF] Golonka | Late Triassic and Early Jurassic palaeogeography of the world[END_REF]et Preto et al., 2010 modifié).

The Imerese Basin, therefore, is represented by the transitional pelagic facies between the Panormide and Trapanese shelf facies, on the one hand, and the bathyal facies of the Neo-Tethys, on the other (Fig. 3).

The Imerese succession of Monte Scalpello (Fig. 4) starts with the "Carnian Flysh" (Auctorum) or, according to Schmidt di [START_REF] Schmidt Di Friedberg | Contribution à l'étude structurale du groupe du Monte Judica (Sicile orientale)[END_REF], the Mufara Formation. This unit, outcropping in the southern slopes of the mount, is prevalently constituted by dark grey clays rich in ammonites with rare interbedded levels of fossiliferous calcarenites and fibrous calcite with Halobia spp. imprints. [START_REF] Lentini | Caratteri stratigrafici e strutturali della zona di Monte Judica (Sicilia Orientale)[END_REF]Di Paolo et al. 2012, modified). Carte géologique de la région de Monte Scalpello (d'après [START_REF] Lentini | Caratteri stratigrafici e strutturali della zona di Monte Judica (Sicilia Orientale)[END_REF]Di Paolo et al. 2012, modifié).

The detailed stratigraphic attribution of Mufara Formation has been the subject of different opinions since the beginning of the twentieth century; Calcara, 1841[START_REF] Calcara | Cenno sui molluschi viventi e fossili della Sicilia da servire di supplement ed insieme di critiche osservazioni all'opera di R. A. Philippi[END_REF]Nelli (1899a, b), [START_REF] Gemmellaro | I Cefalopodi del Trias Superiore della regione occidentale della Sicilia[END_REF]), Scalia (1907;[START_REF] Scalia | Il gruppo di M. Judica[END_REF]1910-1914[START_REF] Maugeri Patanè | Sopra alcuni coralli del Trias superiore di M. Scalpello (Enna)[END_REF], [START_REF] Lentini | Caratteri stratigrafici e strutturali della zona di Monte Judica (Sicilia Orientale)[END_REF] and Dal Sasso et al. ( 2014) assigned these sediments to the Late Triassic (Carnian). [START_REF] Lentini | Caratteri stratigrafici e strutturali della zona di Monte Judica (Sicilia Orientale)[END_REF], on the basis of the ammonites fauna found, attributes the lowest levels of Monte Scalpello section, to the Trachyceras aonoides ammonite zone of the Early Carnian; nevertheless the presence of Tropitidae among the specimens coming out from this levels, excludes that this formation belongs to the Early Carnian [START_REF] Lucas | The Triassic timescale: an introduction[END_REF][START_REF] Jenks | Ammonoid Paleobiology: From macroevolution to paleogeography[END_REF].

The subsequent sedimentary layers are attributed, again by [START_REF] Lentini | Caratteri stratigrafici e strutturali della zona di Monte Judica (Sicilia Orientale)[END_REF], to the Tropites subbullatus zone; nevertheless the contemporary presence of specimens of Trachyceratidae (?Neoprotrachyceras, Trachysagenites, Pamphagosirenites) found by the authors of the present paper, and specimens of Tropitidae, suggests that both this fossiliferous horizons can be referred to the Tropites dilleri zone [START_REF] Lucas | The Triassic timescale: an introduction[END_REF][START_REF] Jenks | Ammonoid Paleobiology: From macroevolution to paleogeography[END_REF]. A confirmation of this datum comes from [START_REF] Carrillat | Palaeoenvironmental reconstruction of the Mufara Formation (Upper Triassic, Sicily): High resolution sedimentology, biostratigraphy and sea-level changes Palaeogeography[END_REF] who, mostly on the basis of the foraminifers, refers the same clays of Mufara Formation outcropping at Monte Gambanera, nine kilometers to the south of the studied area, to the upper part of the Late Carnian (Tropites subbullatus/Anatropites spinosus zones).

According to [START_REF] Jenks | Ammonoid Paleobiology: From macroevolution to paleogeography[END_REF] the base of Tuvalian Substage (upper part of the Carnian Stage) is characterized by the appearance and radiation of the Tropitidae. Therefore the presence of Tropitidae reported already by [START_REF] Gemmellaro | I Cefalopodi del Trias Superiore della regione occidentale della Sicilia[END_REF], [START_REF] Scalia | Il gruppo di M. Judica[END_REF] and [START_REF] Lentini | Caratteri stratigrafici e strutturali della zona di Monte Judica (Sicilia Orientale)[END_REF] remove all doubts about the attribution of this sedimentary levels out cropping, to the Tuvalian substage (Late Carnian, Tropites dilleri zone) (Fig. 5).

Over the Mufara Formation follow conformably the Upper Triassic cherty limestone with

Halobia, the Jurassic -Cretaceous radiolarian cherts and unconformably the Eocene -Oligocene limestone ("Scaglia"). The stratigraphic sequence is finally closed by Oligocene -Miocene clays and glauconitic sandstones.

For the present work twenty kilograms of sediments were collected from one stratigraphic level of the Mufara Formation cropping out in contrada Paraspora, two hundred meters southwest of Masseria Mannino (37°32'56.86''N;14°40'30.64''E) along the east side of Monte Scalpello, (Figs. 1,4). Sediments were routinely washed, dried in oven and sieved. Then, ostracod specimens were picked out from the > 63µm fraction. The ostracod specimens were examined and measured under a stereomicroscope, then photographed under an LMU Tescan Vega II SEM. The material is housed in the Palaeontological Museum of the University of Catania. The repository number of the holotypes and the paratypes are given in the systematic descriptions.

RESULTS

The sample taken results to be constituted mostly by pelitic sediments, indeed almost the entire sediment is passed over the 63µm sieve. The poor washing residue (about 100 grams) provided in order of abundance, gastropods, brachiopods, bivalves and crinoids, echinoids skeletal fragments and small size ammonites. Microfauna is very poor in foraminifers represented prevalently by the genera Glomospira and Hemidiscus; ostracods, the majority of which is constituted by juvenile forms, are relatively abundant.

Systematic palaeontology

Ostracods from a sample of clay of the Mufara Formation taken at the base of Monte Scalpello are systematically listed. The specimens belong to the Order Metacopida Sylvester- [START_REF] Sylvester-Bradley | Suborder Metacopina Sylvester-Bradley, n. suborder[END_REF]and Podocopida Sars, 1866, and eight families Healdiidae Harlton, 1933, Cavellinidae Egorov, 1950, Bairdiidae Sars, 1866, Acratiidae Gründel, 1962, Bythocyprididae Maddocks, 1969, Pontocyprididae Müller, 1894, Judahellidae Sohn, 1968, Glorianellidae Snejder, 1960. Abbreviations. L: length; H: height; T: thickness; RV: right valve; LV: left valve; DB: dorsal border; VB: ventral border; AB: anterior border; PB: posterior border; PVB: postero-ventral border; AVB: antero-ventral border; PDB: postero-dorsal border; PVB: postero-ventral border.

Class Ostracoda [START_REF] Latreille | Genera crustaceorum et insectorum secundum ordinem naturalem in familias disposita, iconibus exemplisque plurimis explicata[END_REF]Order Metacopida Sylvester-Bradley, 1961Suborder Metacopina Sylvester-Bradley, 1961Superfamily Healdioidea Harlton, 1933Family Healdiidae Harlton, 1933 Genus Ogmoconchella [START_REF] Gründel | Zur Gattung Healdia (Ostracoda) und zu einigen verwandten Formen aus dem unter Jura[END_REF]emend Michelsen, 1975 Type species: Healdia aspinifera [START_REF] Drexler | Foraminiferen und Ostracoden aus dem Lias von Siebeldingen/Pfalz[END_REF] Ogmoconchella felsooersensis [START_REF] Kozur | Neue Ostracoden-Arten aus dem Obersten Anis des Bakonyhochlandes (Ungarn)[END_REF] Fig. 6 a -b 1970 Healdia (Healdia) felsooersensis n. sp. - Kozur, p. 409, pl. 3, fig. 13, pl.4, figs;1 -6 1995"Hungarella" felsooersensis (Kozur, 1970) -Monostori, p. 40 -41, pl. 1, figs. 2 -4 1996 Healdia (Healdia) felsoorensis Kozur, 1970 -Crasquin-Soleau and Gradinaru, pl. 9, fig. 14 2013 Healdia (Healdia) felsooersensis Kozur, 1970 -Sebe et al., pl. 4, fig. 14 2013 Hungarella problematica (Méhes, 1911) -Monostori and Toth, p. 308, Pl. 4, Figs. 14 -17 Material: 2 complete carapaces.

Dimensions (figured specimen): L= 790-810 µm; H= 580 µm; T= 380 µm.

Stratigraphic and geographic range:

Early Anisian of Felsőörs, Balaton Highland, Hungary [START_REF] Kozur | Neue Ostracoden-Arten aus dem Obersten Anis des Bakonyhochlandes (Ungarn)[END_REF][START_REF] Monostori | Environmental significance of the Anisian Ostracoda fauna from the Forras Hill near Felsoors (Balaton Highland, Transdanubia, Hungary)[END_REF], Early Anisian of Dobrogea, Romania [START_REF] Sebe | Early and Middle Anisian (Triassic) deep-water ostracods (Crustacea) from North Dobrogea (Romania)[END_REF], Late Carnian (Tropites dilleri zone), Mufara Formation outcropping in the east side of Monte Scalpello (37°32'56.86''N; 14°40'30.64''E), Sicily, Italy (this work).

Remarks: A great confusion exists in the systematics of Late Permian -Triassic Healdiidae genera Hungarella -Ogmoconcha -Ogmochonchella. Some authors consider Hungarella [START_REF] Méhes | Über Trias-Ostrakoden aus dem Bakony. Resultate der Wissenschaftlichen[END_REF](which has not type material -Monostori, 1995, p. 40) and Ogmoconcha Triebel, 1941 as synonyms [START_REF] Moore | Treatise of Invertebrate Paleontology. Part Q. Arthropoda 3, Crustacea, Ostracoda[END_REF][START_REF] Anderson | Rhaetic ostracoda[END_REF]. Shaver (in [START_REF] Moore | Treatise of Invertebrate Paleontology. Part Q. Arthropoda 3, Crustacea, Ostracoda[END_REF] and [START_REF] Sohn | Triassic Ostracodes from Makhtesh Ramon, Israel[END_REF] don't agree with this synonymy. In fact the two genera are extremely close. The third genus Ogmoconchella was introduced by [START_REF] Gründel | Zur Gattung Healdia (Ostracoda) und zu einigen verwandten Formen aus dem unter Jura[END_REF] and emended later by [START_REF] Michelsen | Lower Jurassic biostratigraphy and ostracods of the Danish Embayment[END_REF] mainly on the presence of a spine at PVB. We use here this character to attribute the species felsooersensis to Ogmoconchella.

Genus Healdia [START_REF] Roundy | Mississippian formations of the Saba County, New York and its fauna[END_REF] Type species Healdia simplex [START_REF] Roundy | Mississippian formations of the Saba County, New York and its fauna[END_REF] Healdia? sp.

Remarks:

In right lateral view, the carapace has all the characteristics of the genus Healdia.

However the dorsal view is very far from this genus. Indeed, here the carapace is biconvex with a maximum of thickness at mid-L (in Healdia genus the posterior part of the carapace is truncated) and here there is only one spine at RV (in Healdia genus spines are present at PVB of both valves)

As far as the muscle scars cannot be observed, the figured specimens are dubiously attributed to the genus Healdia.

Superfamily Cavellinoidea [START_REF] Egorov | Ostrakody franskogo iarusa Russkoi platformy, 1. Kloedenellidae: Moscow-Leningrad Vses[END_REF]Family Cavellinidae Egorov, 1950 Genus Bektasia Özdikmen, 2010 Type species. Reubenella avnimelechi [START_REF] Sohn | Triassic Ostracodes from Makhtesh Ramon, Israel[END_REF] Bektasia sp.1 Remarks: this specimen could be compared to Bektasia subcynlindrica [START_REF] Sandberg | Die Stellung der Raibler Schichten in dem frankisichen und schwabischen Keuper[END_REF] in [START_REF] Kristan-Tollmann | Zur phylogenetischen und stratigraphischen Stellung der triadischen Healdiiden (Ostracoda) II[END_REF] from the Carnian of North Italy (Kristan- [START_REF] Kristan-Tollmann | Zur phylogenetischen und stratigraphischen Stellung der triadischen Healdiiden (Ostracoda) II[END_REF] which has a similar lateral outline but has a shorter ADB and deeper sulci.

Order Podocopida Sars, 1866Suborder Bairdiocopina Gründel, 1967 Superfamily Bairdioidea [START_REF] Sars | Oversight af Norges marine Ostracoder[END_REF] Family Bairdiidae [START_REF] Sars | Oversight af Norges marine Ostracoder[END_REF] Genus Hiatobairdia [START_REF] Kristan-Tollmann | Einige neue Bairdien (Ostracoda) aus der alpinen Trias[END_REF] Type species Hiatobairdia subsymmetrica [START_REF] Kristan-Tollmann | Einige neue Bairdien (Ostracoda) aus der alpinen Trias[END_REF] Hiatobairdia subsymmetrica [START_REF] Kristan-Tollmann | Einige neue Bairdien (Ostracoda) aus der alpinen Trias[END_REF]Fig. 6 f -h 1970Hiatobairdia subsymmetrica n. gen. n. sp. -Kristan-Tollmann, p. 268, pl. 35, figs. 1 -3. 1976 Hiatobairdia subsymmetrica [START_REF] Kristan-Tollmann | Einige neue Bairdien (Ostracoda) aus der alpinen Trias[END_REF][START_REF] Tollmann | Analyse des klasschen nordalpinen Mesozoikums[END_REF], p. 276, pl. 163, fig. 14. 1978 Hiatobairdia subsymmetrica deformis n. sp. - Kristan-Tollmann, p. 83, pl. 4, figs. 1 -7 1979Hiatobairdia subsymmetrica Kristan-Tollmann, 1970 -Kristan-Tollmann et al., p. 147, pl. 6, fig. 4. [START_REF] Tollmann | Analyse des klasschen nordalpinen Mesozoikums[END_REF][START_REF] Kristan-Tollmann | Bairdiidae (Ostracoda) aus den obertriadischen Cassianer Schichten der Ruones-Wiesen bei Corvara in Südtirol[END_REF], Late Carnian (Tropites dilleri zone; this work), Rhaetian of Austrian Alps [START_REF] Kristan-Tollmann | Einige neue Bairdien (Ostracoda) aus der alpinen Trias[END_REF] and Central Iran [START_REF] Kristan-Tollmann | Beitrage zur Kenntnis der Trias von Persien. I. Revision der Triasgliederung, Rhäfazies im Raum von Isfahan und Kössener Fazieseinschlag bei Waliabad SE Abadeh[END_REF].

Material

Remarks: the Triassic genus Hiatobairdia Kristan-Tollmann, 1970 and the Permian genus Kempfina Crasquin, 2010[START_REF] Crasquin | Ostracods (Crustacea) through Permian-Triassic boundary in South China: the Meishan stratotype (Zhejiang Province)[END_REF] differ from other bairdiidae by the "acratian beak" at AVB, and the two symmetric valves. The surface of the carapace is often punctuated. [START_REF] Crasquin-Soleau | Early Anisian ostracode fauna from the Tulcea unit (Cimmerian Notrh Dobrogean orogeny, Romania)[END_REF]; Late Anisian of Balaton Highland, Hungary [START_REF] Monostori | Environmental significance of the Anisian Ostracoda fauna from the Forras Hill near Felsoors (Balaton Highland, Transdanubia, Hungary)[END_REF], Ladinian of Balaton Highland (Monostori and Tóth, 2013), Early Carnian of Southern Alps, Italy (Reuss, 1868[START_REF] Gümbel | Uber Foraminiferen, Ostracoden und mikroskopische Tier-Uberreste in den St. Cassianer und Raibler Schichten[END_REF][START_REF] Ulrichs | Variability of some Ostracods from the Cassian beds (Alpine Triassic) depending on the ecology[END_REF][START_REF] Kristan-Tollmann | Bairdiidae (Ostracoda) aus den obertriadischen Cassianer Schichten der Ruones-Wiesen bei Corvara in Südtirol[END_REF] [START_REF] Forel | In the aftermath of the Permian-Triassic boundary mass extinction: ostracod new species and genus from South Tibet[END_REF], Anisian [START_REF] Monostori | Environmental significance of the Anisian Ostracoda fauna from the Forras Hill near Felsoors (Balaton Highland, Transdanubia, Hungary)[END_REF], Ladinian (Monostori and Tóth, 2013) and Early Carnian [START_REF] Méhes | Über Trias-Ostrakoden aus dem Bakony. Resultate der Wissenschaftlichen[END_REF][START_REF] Széles | Ostracodåk a Bakonyi Nosztori-Volgy felsokarni rétegeibol[END_REF]) and ADB in a regular arc of a circle at LV, ADB slightly concave at RV; AB with large radius of curvature with maximum of curvature located a little below mid -H, a very fine ridge underlines the AB; VB quite horizontal to very slightly concave; PB short, with very small radius of curvature and maximum of curvature located around lower 1/3 of H, presence of the very thin ridge along the PB; bairdiid beak poorly expressed but present; strong overlap of LV on RV along all the dorsal margin with maximum at DB; overlap weak on all other parts of the carapace; surface smooth.

Remarks:

This species is quite different from all the other species recognized in the Triassic. It could be compared to Bairdia jeancharlesi Forel, 2011 from the Smithian -Anisian of Tibet [START_REF] Forel | In the aftermath of the Permian-Triassic boundary mass extinction: ostracod new species and genus from South Tibet[END_REF] but here the PB is shorter and the AB has a larger radius of curvature. Bairdiacypris triassica [START_REF] Kozur | Die Bairdiacea der Trias. Teil 3: Einige neue Arten triassischer Bairdiacea und Bemerkungen zur Herkunft der Macrocyprididae (Cypridacea)[END_REF] from Late Anisian of Hungary [START_REF] Kozur | Die Bairdiacea der Trias. Teil 3: Einige neue Arten triassischer Bairdiacea und Bemerkungen zur Herkunft der Macrocyprididae (Cypridacea)[END_REF] has the same radius of curvature at PB and AB but L is really greater.

Remarks:

The present specimens are very close to Bairdia finalyi [START_REF] Méhes | Über Trias-Ostrakoden aus dem Bakony. Resultate der Wissenschaftlichen[END_REF] from the Smithian -Ladinian of Balaton Highland, Hungary [START_REF] Méhes | Über Trias-Ostrakoden aus dem Bakony. Resultate der Wissenschaftlichen[END_REF][START_REF] Kozur | Die Bairdiacea der Trias. Teil 3: Einige neue Arten triassischer Bairdiacea und Bemerkungen zur Herkunft der Macrocyprididae (Cypridacea)[END_REF]Monostori, 1995), West Carpathians (Salaj and[START_REF] Salag | Ecology and facial relation of some groups of Triassic foraminifers and ostracods of stratigraphic importance[END_REF], Guangxi, South China [START_REF] Crasquin-Soleau | Early Triassic ostracods from South China[END_REF], Tibet [START_REF] Forel | In the aftermath of the Permian-Triassic boundary mass extinction: ostracod new species and genus from South Tibet[END_REF]. They differ only by a larger radius of curvature of AB here. Remark: this specimen has a remarkable and characteristic AB truncated in ventral part but there is not enough material to go further in the description. Remarks: these specimens are very close to the specimens presented by Mette and Mohat-Aghai (1999;Pl. 4;fig .4 -5) as Bairdia sp. 7 from the Rhaetian of Tyrol in Austria. All these specimens could belong to a new species. The generic attribution is just at the limit between the two genera Bairdia and Urobairdia. As the ventral "blade" from AB to PB, characteristic of Urobairdia, is not clearly shown, we chose to let it in genus Bairdia. Genus Ptychobairdia [START_REF] Kollmann | Ostracoden aus der alpinen Trias Österreichs. I Parabairdia n. g. und Ptychobairdia n. g. (Bairdiidae)[END_REF] Type species Ptychobairdia kuepperi [START_REF] Kollmann | Ostracoden aus der alpinen Trias Österreichs. I Parabairdia n. g. und Ptychobairdia n. g. (Bairdiidae)[END_REF] Ptychobairdia kristanae Kollmann, 1960 Formation outcropping in the east side of Monte Scalpello (37°32'56.86''N; 14°40'30.64''E) (this work), Rhaetian -Early Jurassic of Austria [START_REF] Kollmann | Ostracoden aus der alpinen Trias Österreichs. I Parabairdia n. g. und Ptychobairdia n. g. (Bairdiidae)[END_REF][START_REF] Kollmann | Ostracoden aus der alpinenTrias. II.-Weitere Bairdiidae[END_REF].

Bairdia sp. 3

Remarks:

The types figured by Kollmann (1960, Pl. 25, figs 6 -9) have a ventral bulge more pronounced than here but intraspecific variations are very common in bairdiids Genus Bairdiacypris [START_REF] Bradfield | Pennsylvanian ostracods of the Ardmore Basin, Oklahoma[END_REF] Type species Bairdiacypris deloi [START_REF] Bradfield | Pennsylvanian ostracods of the Ardmore Basin, Oklahoma[END_REF] Bairdiacypris cf. aequisymmetrica Mette, Honigstein et Crasquin .,2014 Remarks: the specimen discovered here is quite close in general outline to Bairdiacypris mirautaae Crasquin-Soleau and Gradinaru, 1996 from the Early Anisian of Dobrogea (Romania; [START_REF] Crasquin-Soleau | Early Anisian ostracode fauna from the Tulcea unit (Cimmerian Notrh Dobrogean orogeny, Romania)[END_REF] and Ladinian of Hungary (Monostori and Tóth, 2013). Here the carapace is shorter and the PB is less tapering.

Genus Anchistrocheles Brady and Norman, 1889

Type species Anchistrocheles fumata Brady, 1890 "Anchistrocheles" gemmellaroi n. sp.

Fig. 7 d -f

Etymology: The species is named in memory of the palaeontologistoGaetano Giorgio Gemmellaro (1832[START_REF] Gemmellaro | I Cefalopodi del Trias Superiore della regione occidentale della Sicilia[END_REF].

Material: Seven complete carapaces (three adults and four juveniles). Diagnosis: A species attributed to "Anchistrocheles", with bairdioid carapace sub trapezoidal, with rows of spines at AB, PVB and PB, flanks parallel in dorsal view.

Description: Bairdioid carapace sub trapezoidal in lateral view, with DB, PDB and ADB straights at both valves; AB with large radius of curvature; ventral margin concave at both valves; PB short and with a small radius of curvature; AB, PVB and PB strongly compressed laterally, without reticulation and ornamented with marginal spines at both valves; LV overlaps RV all around the carapace with maximum at dorsal parts; In dorsal view, flanks parallel (like in Orthobairdia genus), extremities strongly compressed laterally, a small sulcus is observable in front of mid-L; surface reticulated and small tubercles are observed on the surface in ADP and DP.

Remarks: This species is attributed to the genus "Anchistrocheles". It is clear that is not this genus which is a recent one and for which the specific determinations are made on soft parts. This generic attribution with "?" was used in the past, particularly for Triassic -Jurassic specimens exhibiting row of spines along AB and PB (Carnian, Late Triassic of Turkey (Forelet al., 2018); Rhaetian, Late Triassic of Austria [START_REF] Bolz | Die Zlambach-Schichten (alpine Obertrias) unter besonderer Berücksichtigung der Ostrakoden, 1: Ostrakoden der Zlambach-Schichten, besonders Bairdiidae[END_REF], Sinemurian, Early Jurassic of Germany and Switzerland [START_REF] Beher | Zur Ostracodenfauna des Ober-Sinemurium und untersten Unter-Pliensbachium im Gebiet von Tuningen (Baden-Württemberg)[END_REF][START_REF] Beher | Ostracodenfauna und Biostratigraphie im Ober-Sinemurium von Süddeutschland und der Nordschweiz[END_REF], Bathonian, Early Jurassic of France [START_REF] Sheppard | Middle Jurassic Ostracoda from Southern England and Northern France[END_REF] PhD MS). A revision of all these species and of the definition of a new genus is in preparation (Crasquin and Forel, work in progress).

"Anchistrocheles" gemmellaroi n. sp. could be compared to Bairdia marginosulcata [START_REF] Bolz | Die Zlambach-Schichten (alpine Obertrias) unter besonderer Berücksichtigung der Ostrakoden, 1: Ostrakoden der Zlambach-Schichten, besonders Bairdiidae[END_REF] from the Late Norian -Rhaetian of Austria [START_REF] Bolz | Die Zlambach-Schichten (alpine Obertrias) unter besonderer Berücksichtigung der Ostrakoden, 1: Ostrakoden der Zlambach-Schichten, besonders Bairdiidae[END_REF] which presents a biconvex carapace in dorsal view and a smaller radius of curvature at AB. The new species is very close from Anchistrocheles? spinosa from the Bathonian, Middle Jurassic of North-Western France with the flanks parallel and compressed extremities in dorsal view, with spines along AB and PVB and PB. This species was described and illustrated in the PhD thesis of [START_REF] Sheppard | Middle Jurassic Ostracoda from Southern England and Northern France[END_REF]. It seems that this species was never published and is considered here as unvalid. "Anchistrocheles" gemmellaroi n.

sp. has a AB with larger radius of curvature, and dorsal parts straighter.

Genus Nodobairdia [START_REF] Kollmann | Ostracoden aus der alpinenTrias. II.-Weitere Bairdiidae[END_REF] Type species Nodobairdia mammillata [START_REF] Kollmann | Ostracoden aus der alpinenTrias. II.-Weitere Bairdiidae[END_REF] Nodobairdia mammillata [START_REF] Kollmann | Ostracoden aus der alpinenTrias. II.-Weitere Bairdiidae[END_REF]Fig.7 g 1963Nodobairdia mammillata n. g. n. sp. -Kollmann, p. 174, pl. 7, figs. 6 -15 1971Nodobairdia mammillata Kollmann, 1963-Kristan-Tollmann, p. 63, pl. 1, fig. 1 1971 Triebelina (Nodobairdia) triassica n. sp. [START_REF] Bolz | Die Zlambach-Schichten (alpine Obertrias) unter besonderer Berücksichtigung der Ostrakoden, 1: Ostrakoden der Zlambach-Schichten, besonders Bairdiidae[END_REF], p. 216, pl. 16, figs. 229 -232 1978Nodobairdia mammillata Kollmann, 1963 -Kristan-Tollmann, p. 84, pl. 8, figs. 1 -6 Stratigraphic and geographic range: Late Ladinian of Alps, Austria [START_REF] Kollmann | Ostracoden aus der alpinenTrias. II.-Weitere Bairdiidae[END_REF][START_REF] Kristan-Tollmann | Weitere Beobachtungen an skulptierten Bairdiidae (Ostracoda) der alpinen Trias[END_REF], Late Carnian of Alps, Austria [START_REF] Kristan-Tollmann | Bairdiidae (Ostracoda) aus den obertriadischen Cassianer Schichten der Ruones-Wiesen bei Corvara in Südtirol[END_REF]. Tropites dilleri zone of the Mufara Formation outcropping in the east side of Monte Scalpello (37°32'56.86''N; 14°40'30.64''E) Sicily, Italy (this work); Norian -Rhaetian of Alps, Austria [START_REF] Bolz | Die Zlambach-Schichten (alpine Obertrias) unter besonderer Berücksichtigung der Ostrakoden, 1: Ostrakoden der Zlambach-Schichten, besonders Bairdiidae[END_REF].

Family Acratiidae [START_REF] Gründel | Zur Taxionomie der Ostracoden der Gattendorfia Stufe Thüringens[END_REF] Genus Acratia [START_REF] Delo | Some Upper Carboniferous ostracods from the Shale Basin of Western Texas[END_REF] Type species -Acratia typica [START_REF] Delo | Some Upper Carboniferous ostracods from the Shale Basin of Western Texas[END_REF] Acratia maugerii n. sp. Remarks: the figured specimens are put in synonymy with Acratia sp., from the Late Ladinian from NE Iran (Kristan-Tollmann,1991) and one specimen figured by Monostori and Tóth (2013) from Ladinian of Balaton Highland, Hungary. Acratia triassica Kozur, 1970 (figs. 2-5 only, not fig. 1) is very close from the new species. However here the carapace is more rounded in dorsal part with a PB less acuminate.

Family Bythocyprididae [START_REF] Maddocks | Revision of Recent Bairdiidae[END_REF] Genus Bythocypris [START_REF] Brady | Report on the Ostracoda dredged by H.M.S. Challenger during the Years 1873-1876[END_REF] Type species Bythocypris reniformis [START_REF] Brady | Report on the Ostracoda dredged by H.M.S. Challenger during the Years 1873-1876[END_REF] Bythocypris? sp. 1 Stratigraphic and geographic range: Late Carnian of Balaton Highland, Hungary [START_REF] Méhes | Über Trias-Ostrakoden aus dem Bakony. Resultate der Wissenschaftlichen[END_REF] and Monte Scalpello (Tropites dilleri zone), (37°32'56.86''N, 14°40'30.64''E) Sicily, Italy (this work).

Family Judahellidae [START_REF] Sohn | Triassic Ostracodes from Makhtesh Ramon, Israel[END_REF] Genus Judahella [START_REF] Sohn | Triassic Ostracodes from Makhtesh Ramon, Israel[END_REF] Type species Judahella tsorfatia [START_REF] Sohn | Triassic Ostracodes from Makhtesh Ramon, Israel[END_REF] Judahella? montanarii n. sp.

Remarks:

The new species is attributed to Judahella? The doubt comes from the presence of the posterior caudal process never described in the genus. The present specimens could be compared to Judahella tsorfatia [START_REF] Sohn | Triassic Ostracodes from Makhtesh Ramon, Israel[END_REF] from the Ladinian of Eastern France [START_REF] Sohn | Triassic Ostracodes from Makhtesh Ramon, Israel[END_REF]) by their similar subrectangular lateral outline. But here the nodes are gathered in ridges and there is a caudal process. Judahella tuberculifera [START_REF] Gümbel | Uber Foraminiferen, Ostracoden und mikroskopische Tier-Uberreste in den St. Cassianer und Raibler Schichten[END_REF] from Carnian of Italian Alps [START_REF] Gümbel | Uber Foraminiferen, Ostracoden und mikroskopische Tier-Uberreste in den St. Cassianer und Raibler Schichten[END_REF][START_REF] Lieberman | Die Bivalven-und Ostracodenfauna von Raibl und ihr stratigraphischer[END_REF], Anisian -Ladinian of Poland [START_REF] Styk | Triassic microfauna in the neighbourhood of Chrzanow and the northwestern part of the Mesozoic periphery of the Swiety Kryzyz Mts[END_REF], Ladinian of France [START_REF] Sohn | Triassic Ostracodes from Makhtesh Ramon, Israel[END_REF], Anisian of Germany (Knüpfer and [START_REF] Knupfer | Eine neue Ostracodengattung aus der Trias des germanischen Binnenbeckens[END_REF], Ladinian of Spain [START_REF] Kozur | Contribution to the Triassic ostracode faunas of the Betic Zone (southern Spain)[END_REF] and Anisian of South China [START_REF] Kristan-Tollmann | Ostracoden aus dem Oberanis von Leidapo bei Guiyang in Süd China[END_REF]) has a general dorsal outline which can evoke the new species.

Family Glorianellidae Snejder, 1960emend. Kozur 1970 Genus Mockella [START_REF] Bunza | Beiträge zur Ostracodenfauna der tethyalen Trias[END_REF] Type species Mockella marinae [START_REF] Kozur | Neue Ostracoden-Arten aus dem Obersten Anis des Bakonyhochlandes (Ungarn)[END_REF] Mockella muelleri [START_REF] Bunza | Beiträge zur Ostracodenfauna der tethyalen Trias[END_REF] Fig. 7 o -p 1971 Mockella muelleri n. sp. - Bunza and Kozur,pl. 1,fig. 12 Material: 2 carapaces, 1 figured.

Dimensions: L = 390 µm; H = 235 µm (figured specimen).

Stratigraphic and geographic range: Late Carnian of Tyrol, Austria [START_REF] Bunza | Beiträge zur Ostracodenfauna der tethyalen Trias[END_REF] and east side of Monte Scalpello (37°32'56.86''N, 14°40'30.64''E) Sicily, Italy (this work).

Conclusion.

Carnian Ostracods are known worldwide, from Alaska [START_REF] Sohn | Triassic Ostracodes from Makhtesh Ramon, Israel[END_REF], Alps [START_REF] Kristan-Tollmann | Zur stratigraphischen Reichweite der Ptychobairdien und Anisobairdien (Ostracoda) in der alpinen Trias[END_REF][START_REF] Kristan-Tollmann | Bairdiidae (Ostracoda) aus den obertriadischen Cassianer Schichten der Ruones-Wiesen bei Corvara in Südtirol[END_REF][START_REF] Kollmann | Ostracoden aus der alpinenTrias. II.-Weitere Bairdiidae[END_REF]), Austria (Kristan-Tollmann, 1971[START_REF] Kristan-Tollmann | Zur phylogenetischen und stratigraphischen Stellung der triadischen Healdiiden (Ostracoda) II[END_REF][START_REF] Kristan-Tollmann | Coccolithen aus den Älteren Allgäuschichten (Alpiner Lias, Sinemur) von Timor, Indonesien[END_REF][START_REF] Bunza | Beiträge zur Ostracodenfauna der tethyalen Trias[END_REF], British Columbia [START_REF] Arias | Upper Trias marine ostracoda from the Queen Charlotte Islands British Columbia Canada[END_REF], Germany [START_REF] Gründel | Zwei neue Ostracoden-Arten aus dem unteren Keuper Thueringens[END_REF][START_REF] Bunza | Beiträge zur Ostracodenfauna der tethyalen Trias[END_REF], Hungary [START_REF] Kristan-Tollmann | Ostracods from the Middle Triassic Sina Formation (Aghdarband Group) in NE-lran[END_REF][START_REF] Kozur | Die Bairdiacea der Trias. Teil 3: Einige neue Arten triassischer Bairdiacea und Bemerkungen zur Herkunft der Macrocyprididae (Cypridacea)[END_REF][START_REF] Kozur | Die Bedeutung triassischer Ostracoden fur stratigraphische und palaookologische Untersuchungen[END_REF][START_REF] Bunza | Beiträge zur Ostracodenfauna der tethyalen Trias[END_REF], Indonesia [START_REF] Kristan-Tollmann | Ostracoden aus der Obertrias von Misol (Indonesien)[END_REF], Israel [START_REF] Sohn | Triassic Ostracodes from Makhtesh Ramon, Israel[END_REF][START_REF] Gerry | The Carnian salinity crisis: ostracods and palynomorphs as indicators of palaeoenvironment[END_REF][START_REF] Bunza | Beiträge zur Ostracodenfauna der tethyalen Trias[END_REF]; Italy [START_REF] Kristan-Tollmann | Weitere Beobachtungen an skulptierten Bairdiidae (Ostracoda) der alpinen Trias[END_REF][START_REF] Kristan-Tollmann | Zur phylogenetischen und stratigraphischen Stellung der triadischen Healdiiden (Ostracoda) II[END_REF][START_REF] Kristan-Tollmann | Bemerkungen zur triadischen Ostracoden-Gattung Mostlerella[END_REF][START_REF] Kristan-Tollmann | Ostracoden aus dem Oberanis von Leidapo bei Guiyang in Süd China[END_REF]Liebermann, 1979), Slovenia (Kolar-Jurkovsek ,1990) and Turkey [START_REF] Forel | Discovery of a long-term refuge for ostracods (Crustacea) after the end-Permian extinction: a unique Carnian (Late Triassic) fauna from the Mersin Melange, southern Turkey[END_REF]. The turn-over between Palaeozoic and Mesozoic ostracod fauna is long, at least 14 My, began in the early Late Permian (in the Early Wuchiapingian) and is considered to be ended in the Anisian [START_REF] Crasquin | Ostracods (Crustacea) through Permian -Triassic events[END_REF]. However, some Palaeozoic genera are still present in the Carnian: three Kirkbyidae (Carinaknightina, Kirkbya, Tubulikirkbyia?) in Turkey deep environment [START_REF] Forel | Discovery of a long-term refuge for ostracods (Crustacea) after the end-Permian extinction: a unique Carnian (Late Triassic) fauna from the Mersin Melange, southern Turkey[END_REF] and Acratia, discovered as well in Turkey [START_REF] Forel | Discovery of a long-term refuge for ostracods (Crustacea) after the end-Permian extinction: a unique Carnian (Late Triassic) fauna from the Mersin Melange, southern Turkey[END_REF] whose presence is confirmed here.

The paleoecological interpretation of the Carnian ostracods association of Monte Scalpello is not easy. It is necessary to consider both the structure, composition and taphonomic characteristics of the association found, as well as the sedimentological characteristics of the sedimentary Unit and its position in the palaeogeographic context at that time.

First of all the strong shelled forms and absence of typical deep water species it would seem to indicate a not deep waters association. From the taphonomic point of view, it is very interesting to note that the ostracods specimens are predominantly represented by integer carapaces and rare valves. This could suggest a rapid burial in situ in a high sedimentation rates environment, since the valves, after the death of the specimens, tend to open in a few hours [START_REF] Guernet | Ostracodes et recherche des milieux anciens: possibilités et limites[END_REF]. Similar taphonomic characteristics were also found by [START_REF] Pokorny | Some paleoecological problems in marine ostracode faunas, demonstrated on the Upper Cretaceous ostracodes of Bohemia[END_REF] and [START_REF] Oertli | The aspect of ostracode faunas -a possible new tool in petyroleum sedimentology[END_REF] for pelitic layer associations deposited in extremely rapid distal sedimentation basins.

Another important datum concerns the size of all the found skeletal remains. Gastropods, bivalves, crinoids and brachiopods remains are all of the same little size; also the ostracod thanatocoenosis structure is characterized by abundance of juveniles and rare adults; according to [START_REF] Harten | Use of ostracodes to recognize dowslope contamination in paleobathymetry and a preliminary reappraisal of the paleodepth of Prasas Marls (Pliocene), Crete, Greece[END_REF], all that would suggest strong phenomena of dimensional selection and, therefore, displaced of faunas and sediments ().

From the sedimentological point of view the study area is characterized by the extreme abundance of pelitic sediments with interbedded sandy levels sometimes rich in oolites. Sometimes this sediments shown in the outcrop, particular sedimentary structures as parallel lamination, wavy lamination and vortices structures, all features that make one think of a high-energy sedimentation environment.

From a palaeogeographic point of view, the Mufara Formation outcropping in the studied area, is located inside the pelagic Imerese Basin that is delimited by the carbonate platforms of Complesso Panormide to the west and the Complesso Trapanese to the east and to the south (Fig. 3) [START_REF] Catalano | Guida alla geologia della Sicilia occidentale. Guide geologiche regionali[END_REF][START_REF] Montanari | Lineamenti stratigrafico -paleogeografici della Sicilia durante il Ciclo Alpino[END_REF][START_REF] Speranza | Ultrathick Triassic dolomites control the rupture behavior of the central Apennine seismicity: Evidence from magnetic modeling of the L'Aquila fault zone[END_REF]. All the materials produced by the erosion of the carbonate platforms were conveyed in this basin (Carillat and Martini, 2009).

On the basis of all these data it is possible to hypothesize for the ostracods association an environment characterized by high sedimentation rates and high depositional energy, features that controls strongly, today as in the past, the colonization of the bottom as well as the composition and structure of the populations (cf. Di Geronimo & Robba, 1989;[START_REF] Sciuto | Initial report on ostracod fauna from the Messina Straits (Ionian Sea, northeastern Sicily)[END_REF]. According to Carillat and Martini (2009), the Monte Scalpello area, it could be a probably, distal turbiditic sedimentary environment not necessarily very deep (Fig. 8). The collection of further faunistic and sedimentological data (whose finding and observation is made extremely difficult by the intense agricultural activity) can lead to more detailed information on the Triassic palaeo-environment of the Monte Scalpello area. [START_REF] Golonka | Late Triassic and Early Jurassic palaeogeography of the world[END_REF]Preto et al., 2010 modified). Reconstruction paléogéographique schématique de la Pangée et de la Néo-Téthys à la fin du Trias (d'après [START_REF] Speranza | Ultrathick Triassic dolomites control the rupture behavior of the central Apennine seismicity: Evidence from magnetic modeling of the L'Aquila fault zone[END_REF][START_REF] Golonka | Late Triassic and Early Jurassic palaeogeography of the world[END_REF]et Preto et al., 2010 modifié). Fig. 4. Geological map of the Monte Scalpello area (after [START_REF] Lentini | Caratteri stratigrafici e strutturali della zona di Monte Judica (Sicilia Orientale)[END_REF]Di Paolo et al. 2012, modified). Carte géologique de la région de Monte Scalpello (d'après [START_REF] Lentini | Caratteri stratigrafici e strutturali della zona di Monte Judica (Sicilia Orientale)[END_REF]et Di Paolo et al. 2012, modifié) p. Bairdia balatonica [START_REF] Méhes | Über Trias-Ostrakoden aus dem Bakony. Resultate der Wissenschaftlichen[END_REF]. Complete carapace, right external lateral view. q -r. Bairdia scaliai n. sp. q. Paratype, complete carapace, left external lateral view; r. Holotype, complete carapace, right external lateral view. s. Bairdia cf. finalyi [START_REF] Méhes | Über Trias-Ostrakoden aus dem Bakony. Resultate der Wissenschaftlichen[END_REF]. Complete carapace, right external lateral view.

Fig. 1 .

 1 Fig. 1. Geographical location of the sampling area. Localisation géographique de la zone d'étude.

Fig. 3 .

 3 Fig. 3. Schematic palaeogeographic reconstruction of Pangaea and Neo-Tethys in Late Triassic times (after Speranza &

Fig. 4 .

 4 Fig. 4. Geological map of the Monte Scalpello area (after[START_REF] Lentini | Caratteri stratigrafici e strutturali della zona di Monte Judica (Sicilia Orientale)[END_REF] Di Paolo et al. 2012, modified). Carte

Fig. 5 .

 5 Fig. 5. Carnian Ammonoid zones. (After Lucas, 2010). Zones à Ammonoïdés du Carnien (d'après Lucas,

Fig

  Fig.6 c -d

Fig. 6 e

 6 Fig.6 e

  : 3 complete carapaces. Dimensions (specimen figured in fig. 6 f): L = 1040 µm; H = 520 µm. Stratigraphic and geographic range: Early Carnian of South Tirol, Italy (Kristan-

  Fig.6 i -j

  Fig.6 n -o

Fig

  Fig.6 q -r

Fig

  Fig. 6 s

Bairdia? sp. 2 Fig. 6 t

 26 Fig.6 t

Fig. 6 u

 6 Fig. 6 u

Figure 6 .

 6 Figure 6. Carnian ostracods from Monte Scalpello. Scale bar 200μm. Ostracodes du Carnien du Monte Scalpello. Barre d'échelle 200μm.

Fig. 7 a 1960

 1960 Fig. 7 a

Fig. 7 b

 7 Fig. 7 b

  Holotype: A complete carapace (fig. 7 e), PMC. O 18 H 10/4/2017 (L = 742 µm; H = 440µm). Paratypes: Two complete carapaces (fig. 7 d: L = 690 µm; H = 420 µm and fig. 7 f: L = 800 µm; H = 490) and other not figured (PMC. O 68-73 P 10/4/2017). Type locality: East side of Monte Scalpello (37°32'56.86''N; 14°40'30.64''E), Sicily, Italy Type level: Late Carnian (Tropites dilleri zone) in the dark grey clays of the Mufara Formation.

Fig1991

  Fig.7 h -j

Fig. 7 k

 7 Fig. 7 k

Fig

  Fig. 7 m -n

Fig. 7 .

 7 Fig. 7. Carnian ostracods from Monte Scalpello. Scale bar= 200µm (al); 100µm (mp). Ostracodes du Carnien du Monte Scalpello. Barre d'échelle 200μm (a-l), 100µm (m-p).

Fig. 1 .

 1 Fig. 1. Geographical location of the sampling area. Localisation géographique de la zone d'étude.

Fig. 2 .

 2 Fig. 2. Structural scheme of the central Mediterranean area with the location of the study area (after Grasso, 2001 modified). Schéma structural de la Méditerranée centrale avec la localisation de la zone d'étude (d'aprèsGrasso, 2001 modifié)

Fig. 5 .

 5 Fig. 5. Carnian Ammonoid zones (after Lucas 2010 modified). Zones à ammoinoidés du Carnien (d'après Lucas 2010, modifié).

Figure 6 .

 6 Figure 6. Carnian ostracods from Monte Scalpello. Scale bar 200μm. Ostracodes du Carnien

  t. Bairdia? sp. 2. Complete carapace, right external lateral view. u. Bairdia sp. 3. Complete carapace, right external lateral view.

Fig. 7 .

 7 Fig. 7. Carnian ostracods from Monte Scalpello. Scale bar = 200µm (al); 100 µm (mp).

Fig. 8 .

 8 Fig. 8. Schematic palaeoenvironmental model for the Late Carnian Mufara Formation basin

  of Hungary, Early Carnian of Southern Alps, Italy[START_REF] Kristan-Tollmann | Bairdiidae (Ostracoda) aus den obertriadischen Cassianer Schichten der Ruones-Wiesen bei Corvara in Südtirol[END_REF], Late Carnian (Tropites dilleri zone). Mufara Formation outcropping in the east side of MonteScalpello (37°32'56.86''N; 14°40'30.64''E), Sicily, Italy (this work)

	Bairdia scaliai n.sp.

Acknowledgements

The authors are grateful to the reviewers and the editors for detailed suggestions and comments to the manuscript. Thanks are due also to Mr. Alfio Viola (Electronic Microscopy Laboratory, University of Catania) for SEM photos assistance. Catania Palaeoecological Research Group contribution nº xxx.

The authors declare that they do not have any links of interest.


Yanan Yuan

Dan Zhao

Jinjun Li

Feng Wu
email: fengwu@whu.edu.cn

Marcello Brigante

Gilles Mailhot
email: gilles.mailhot@uca.fr

Rapid Oxidation of Paracetamol by Cobalt(II) Catalyzed Sulfite at Alkaline pH

Keywords: Sulfate radical, sulfite ion, cobalt ion, advanced oxidation processes, paracetamol

In this study we have investigated the efficiency Cobalt (II) (Co(II)) for the activation of sulfite ions following the oxidation of paracetamol used as model contaminants.

Physico-chemical parameters that can impact the paracetamol degradation (pH, initial paracetamol concentration, Co(II)/S(IV) molar ratio, oxygen concentration) and contribution of various radicals were investigated in order to elucidate the chemical mechanism. Main results show that the pH is a key factor controlling the efficiency in the system Co(II)/Sulfite. Higher efficiency is observed for pH between 9.0 and 10.0.

Increasing S(IV) concentrations, until 1 mM, slightly promoted the degradation of paracetamol. In fact, an excess of sulfite ions inhibits the reaction through the scavenging of SO 4 -and SO 5 -. Moreover, degradation efficiency drastically decreases from ~ 85% to less than 5% in absence of oxygen. SO 4 -was confirmed to be the main oxidant responsible for the paracetamol degradation. For the first time we determined the second order rate constant between SO 4 -and paracetamol (1.33 ± 0.79 × 10 9 M -1 s -1 (at pH 5) and 6.14 ± 0.99 × 10 8 M -1 s -1 (at pH 11.0)). Moreover, radical-scavenging experiments also suggest the possible implication of SO 5 -. Hence, this work provides a precise understanding of the overall mechanism and a new 2 promising strategy by using sulfite and transition metal such as Co(II) to promote organic compounds degradation in water under neutral and alkaline pH conditions.

Introduction

Advanced oxidation processes based on sulfate radical (SR-AOPs) have emerged as a promising method in the field of oxidative decontamination of polluted water and soil [1][2][3]. Sulfate radical (SO 4

-), a strong one-electron oxidant, has relatively high standard redox potential (E 0 = 2.6 V vs NHE) with an oxidation potential comparable to or even higher than that of hydroxyl radical [4]. Moreover, SO 4 -can react via electron transfer, by addition to C-C double bonds and H-abstraction [5,6], thus, it is able to oxidize a large number of pollutants such as phenol derivatives and aniline in water [7][8][9]. SO 4 -can be generated in homogeneous or heterogeneous systems via photolysis, thermolysis and radiolysis [10,11] or via transition metal activation of persulfate (S 2 O 8 2-, PS) [7,[12][13][14][15] and peroxymonosulfate (SO 5 2-, PMS) [16,17].

In fact, PMS can be activated by various transition metals such as Fe, Mn, Ni and Co in the homogenous systems [1,18]. Among them, Co and Fe are the most commonly used metal to promote radical formation due to their occurrence in natural media and low cost. Huang and Huang investigated the ability of Co(II) and PMS system to degrade Bisphenol A at pH 7, and achieved an efficient detoxification and mineralization method [19]. A process based on the sulfate radicals generation through iron (Fe(II), Fe(III)) activation of PMS or PS was studied for polychlorinated biphenyls degradation in aqueous system [20]. The high oxidation efficiency and slow rate of consumption of the oxidants make metal-mediated activation system a feasible strategy for degradation of recalcitrant organic compounds. Furthermore, PMS activation using cobalt oxide or cobalt-metal oxide as heterogeneous catalysts also gains significant relevance in water treatment applications [21]. The cobalt oxides such as CoO, CoO other metals were used as efficient heterogeneous catalysts for activation of PMS [22][23][24][25]. The most advantage of heterogeneous catalyst is that solid particles can be easily removed from liquid phase and, in some cases, reused.

Recently, sulfite ions were found to react with transition metals such as Fe(II), Fe(III) and Cr(VI) to generate SO 4 -and application for the azo dyes and amine compounds decontamination was tested [17,[26][27][28][29][30].

In our previous work, we reported some novel AOPs using Fe(II)-sulfite, Fe(III)-sulfite, photo-Fe(II)-sulfite system able to produce oxysulfur radicals (including SO 3 -, SO 4 -and SO 5 -). Combined with the work conducted by other researchers [31][32][33], the basic chain oxidation mechanisms of oxysulfur radicals generation has been investigated.

In this work, Co(II)-sulfite ions (S(IV)) system has been investigated to promote the paracetamol degradation in water. Paracetamol (PARA) is a widely used analgesic and antipyretic drug and an important material for the manufacturing of azo dyes.

PARA was chosen as a target contaminant in this work due to its presence in the environment from several emissions from manufacturing facilities, consumer use and disposal, and hospital waste [34,35]. The effect of pH, initial PARA concentration, Co(II)/S(IV) molar ratio, the presence of oxygen were investigated. Moreover, the activation mechanism and contribution of reactive oxygen and sulfur species were elucidated by using different kinds of radical scavengers and transient absorption spectroscopy.

Materials and Methods

Chemicals

Cobalt(II) sulfate (CoSO to determine the oxygen concentration in solution. During anoxic reaction, the DO was 0.01 mg L -1 while for O 2 saturated solution a concentration of 20 mg L -1 was determined. All experiments were carried at least two times.

Chemical analysis

The concentration of PARA was determined using a high-performance liquid chromatography (HPLC) Shimadzu LC-10A system equipped with UV-vis detector (SPD-10AV; Shimadzu) and an ODS-C18 column (25 cm × 4.6 mm, 5 μm; Shimadzu, Kyoto, Japan). The separation was warried out using methanol:water (25:75 v/v) as isocratic mobile phase at a flow rate of 1.0 mL min -1 . The detector was set at 241 nm.

The concentration of Co(II) in solution was determined by Methyl isobutyl ketone and Ammonium thiocyanate (see Supplementary material for the method description and Fig. S1) [36]. An optical-fiber coupled to a spectrophotometer (UV-1601 Shimadzu, Japan) was used to scan the UV-vis absorption spectra of Co(II)-SO 3 2-complex.

For SO 4 -reactivity a laser flash photolysis (LFP) system was used with 266 nm excitation following the procedure reported by Wu at al. [15]. Analysis of transient decay and second order rate constant determination (using PARA concentrations from 0 to 0.1 mM) were determined at 470 nm corresponding to the maximum absorption of SO 4 -. The second order rate constants were determined at pH 5.0 and 11.0 corresponding to the molecular and deprotonated forms respectively of PARA (pKa = 9.5).

Results and discussion

Effect of initial pH on paracetamol degradation

The effect of the initial pH on the Co(II)+S(IV) system oxidation of PARA was ) complex that is expected to precipitate in solution [37].

Moreover, it has been reported that deprotonated complexes (Co(II)-OH), which is more reactive toward SO 3 2-compared to HSO 3 -would be formed at alkaline pH values (R1) leading to the formation of a Co(II)-SO 3 complex [27]. Co(II)-SO 3 could be oxidized to Co(III)-SO 3 + complex in the presence of dissolved oxygen as reported in different works (R2 and R3) [38]. SO 3 -could be generated during the redox reaction between the Co(III)-SO 3 + complex and SO 3 2-(R4).

UV-vis absorption spectra of Co(II), S(IV), Co(II) and S(IV) during the reaction (Fig. S1) were acquired to investigate the complexation between Co(II) and S(IV). The result showed that: only Co(II) or S(IV) at pH 9.0 did not show absorption in the range 250-600 nm. However, when S(IV) was added to the solution containing 0.1 mM Co(II)

an absorption band absorbing up to ~ 600 nm is present. The presence of this new absorption band demonstrates that the Co(II)-S(IV) complex could be formed and decreasing in the absorption during time proved the reaction between Co(II) and S(IV), and corresponding depletion of S(IV).

( ) ( )

Co II H O Co II OH    alkaline pH (R1) 2 3 3 2 ( ) ( ) Co II OH SO Co II SO H O       (R2) 3 2 3 ( ) ( ) Co II SO O Co III SO      (R3) 2 3 3 3 3 ( ) ( ) Co III SO SO Co II SO SO         (R4)
The pH represents also a key factor influencing the SO 3 -reactivity in water. In fact, the primary step is the oxygen-mediated oxidation of SO

S(IV) and Co(II) effect on the PARA degradation

Different amounts of Co(II) were used to investigate the effect of Co(II) concentration on the PARA removal at pH 9.0 (Fig. 2A). When experiments were conducted using a Co(II) concentration between 0.05 and 0.15 mM and a fixed sulfite concentration of 1.0 mM, no significant differences were observed on the PARA degradation profile. One of reasons explaining the slightly effect of Co(II) concentration may be the fast recycle of Co(II) and Co(III) in the presence of a stoichiometry excess of S(IV) concentrations in aerated solution (R3-R4). Moreover, when the concentration of Co(II) was monitored during the reaction (see the Supplementary material for the method description), it was found that its concentration was nearly stable (Fig. S3). Such results suggest that in the presence of enough sulfite in solution, the cobalt recycle could achieved very fast leading to the generation of SO 3 -at the same time. Even the addition of Co(II) didn't play a relevant role on the paracetamol degradation, but for the sake of feasibility and accuracy, 0.1 mM Co(II)

was employed during all experiments, as when the experiments were conducted under low concentration of Co(II) such as 0.05 mM, the repeatability could not be ensured every time.

The dependence of the PARA degradation on the initial concentration of S(IV) was also investigated (Fig. 2B). An increase in S(IV) concentration (between 0.5 to 1 mM) accelerate the PARA degradation being , through Co(II) reactivity, a source of SO 3 -. However, at higher sulfite ions concentrations (2 mM) there is no more enhancement of PARA degradation and this effect could be attributed to the reactivity competition of SO 4 -/SO 5 -between PARA and SO 3 2-(R8 and R10). Hence, the balance between S(IV) concentration and its catalytic character is a key factor for the oxidative system efficiency.

In all these experiments (Fig. 2) it is impossible to obtain more than 90% of PARA degradation. This observation is certainly due to a competition reaction of the sulfate radicals on PARA, the excess of sulfite and the degradation products of PARA. After a short period of time sulfate radicals react no more on paracetamol due to its low concentration.

Effect of O 2

The effect of oxygen concentration on the PARA degradation using 0.1 mM of Co(II) and 1 mM of S(IV) was investigated comparing results from aerated and nitrogen-purged solutions. When experiment was conducted in aerated solution (under air bubbling), a PARA degradation plateau was reached after first 10 min corresponding to ~82% of degradation (Fig. 3). However, under nitrogen-saturated solution, the efficiency dropped to less than 8% confirming that oxygen is a crucial parameter.

Oxygen strongly favors the degradation of pollutant through SO 3 -oxidation into SO 5

-

that undergo further reaction to form SO 4 -in the solution (R5-R7).

Radical species involvement

The second order rate constants of sulfate radical with molecular and deprotonated PARA (at pH 9.0, pH used in our experiments, about 25 % of PARA is under deprotonated form) were determined from the linear fit of pseudo-first order decay monitored at 470 nm (corresponding to the maximum absorption of sulfate radical) vs concentration of PARA in solution (Fig. S4). The second order rate constant was estimated to be 1.33 ± 0.79 × 10 9 M -1 s -1 (at pH 5) and 6.14 ± 0.99 × 10 8 M -1 s -1 (at pH 11.0) corresponding respectively to the molecular and deprotonated form, on the phenol group, of PARA.

To shed light onto radical mechanism involved in such system, EtOH and TBA were used as radical scavengers. This competition kinetic approach is based on the different second-order rate constant with HO  and SO 4 -( , TBA HO k  = 6.0 × 10 8 M -1 s -1

[44] which is nearly three orders of magnitude higher than 1 mM at pH 9.0. Indeed, if hydroxyl radical were produced in the system, addition of 1 mM TBA should be scavenged about 98 % HO  reducing drastically the PARA degradation. However, when EtOH was used as radical scavenger in solution (Fig. 4)

PARA degradation rate and efficiency (after 30 min) were modified. In the presence of 500 mM of EtOH, a complete inhibition of PARA degradation should be expected on the basis of second order rate constant reported before. But, PARA is still degraded with ~ 29% of disappearance after 30 min. Such trend could be explained considering the possible involvement of SO 5 -during PARA degradation. In fact, EtOH is not able to scavenge strongly SO 5 -due to the very low rate constant (

5 , EtOH SO k  < 10 3 M -1 s -1 [46]).
Such results suggest that SO 4 -represent the main radical leading to the degradation, but also possible implication of SO 5 -during degradation of PARA is possible. to react with PMS to oxidize different kinds of organic pollutants [24]. Experiments to prove the efficiency of sulfite activation by CoO were conducted at pH 9.0 with the paracetamol concentration of 10 μM and SO 3 2-at 1.0 mM. As shown in Fig. 5, when 0.1 mM and 1.0 mM CoO were added to the solution, the degradation efficiency were 32.9% and 71.7%, after 30 min respectively, which indicates that CoO exhibit a good catalytic activity. One of heterogeneous catalysts advantages belong to their stability and their reusability as catalyst. However, oxide can easily agglomerate during catalytic reaction, resulting in the reduction of catalytic performance [47], also cobalt ion leaching and dissolving problem can cause the same potential environmental and health problem as homogeneous catalysts do. As reported in Fig. 5 and Fig. 2A, the initial degradation rate is completely different in the two systems, very fast with soluble Co(II)

Heterogeneous reaction between CoO and SO

and much slower with CoO. This observation shows that the reactivity of sulfite with cobalt is efficient in homogeneous phase and so with cobalt oxide a first process of solubilisation seems necessary.

Sequential experiments for high concentration paracetamol

Sequential experiments were performed with multiple additions of S(IV) to enhance the degradation of high concentration of paracetamol in this Co(II)-S(IV) system. In the experiment, the concentration of paracetamol was 500 µM, Co(II) was 0.5 mM, and the initial pH value of solution was adjusted to 9.0 and then controlled during the whole reaction. 5 mM S(IV) was added to the solution at the beginning, while 0.5 mM were spiked every 20 min. After 200 min of reaction, (Fig. S6) nearly complete PARA degradation was archived. Compared to Co(II), which is recycled during the reaction, sulfite is consumed as transforms into SO 3 -and subsequently into SO 4 2-. So due to the depletion of S(IV), multiple additional sulfite is necessary to promote sequential treatment.

Conclusion

Our results showed the high efficiency of this Co(II)-S(IV) system using PARA as organic pollutant model in aqueous solution at alkaline pH. In this system, the degradation of paracetamol depend on initial concentrations of S(IV) and strongly the pH. In fact, it is clear that dissolved oxygen plays a crucial role allowing the oxysulfure Multiple additions of 0.5 mM S(IV) every 20 min.

investigated from 3 .

 3 0 to 11.0. The initial concentrations of Co(II) and SO 3 2-were 0.1 mM and 1.0 mM, respectively and during the reaction, air (otherwise stated) was constantly bubbled at a flow rate of 0.8 L min -1 . The PARA degradation shows a strong pH-dependence as illustrated in Fig. 1A. Despite a negligible degradation at relatively acidic values (pH 3.0 -5.0), PARA degradation is enhanced from pH 5.0 to pH 10.0 while a decrease is observed at pH 11.0. It is interesting to observe that degradation efficiency between pH 3.0 and 10 is strongly correlated to the HSO 3 -/SO 3 2-speciation in solution (pKa = 7.2) as shown in Fig. 1B in which degradation of PARA after 30 min at different pH values is correlated to the concentration of SO 3 2-in solution. However, at pH 11, the inhibition of PARA degradation can be attributed to the formation of insoluble Cobalt-hydroxide (Co(OH) 2

   = 5.6 × 10 7 M -1 s -1[41]). In the presence of TBA (up to 6 mM) no effect was observed on the PARA degradation [Fig.S5] indicating that hydroxyl radical are not generated using Co(II) 0.1 mM and SO 3 2-

3 2 -

 32 Cobalt oxide such as CoO, CoO 2 , CoO(OH), Co 2 O 3 and Co 3 O 4 were usually used

Figures Captions Fig. 1 :Fig. 2 :Fig. 3 :Fig. 4 :Fig. 5 :

 Captions12345 Figures Captions

Fig. 1 16 397Fig. 2 398Fig. 3 .Fig. 4 .

 16234 Fig. 1

Fig. S0 Fig. S3 .Fig. S5 .

 S0S3S5 Fig. 5

Fig. S6 .

 S6 Fig. S6. Changes in paracetamol concentration over time in sequential experiments for the oxidation of paracetamol at high concentration. Initial conditions: [PARA] 0 = 500 μM, [Co(II)] 0 = 0.5 mM, [Na 2 SO 3 ] 0 = 5.0 mM, pH 0 = 9.0, T =25°C, F air = 0.8 L min -1 .

  Co/MgO), on TiO 2 (Co/TiO 2 ) and Co 3 O 4 /TiO 2 and combined with

2 , CoO(OH), Co 2 O 3 and Co 3 O 4 , Fe-Co mixed oxide nanocatalysts, cobalt oxide supported on MgO (

  4

Acknowledgement

This work was supported by the National Natural Science Foundation of China (NSFC-CNRS_ PRC No. 21711530144 and CNRS No. 270437) .This work was also supported by the "Federation des Recherches en Environnement" through the CPER "Environnement" founded by the "Région Auvergne," the French government and FEDER from European community. The authors gratefully acknowledge financial support from China Scholarship Council provided to Yanan Yuan to study at the University Clermont Auvergne in Clermont-Ferrand, France.

Supplementary material

Rapid Oxidation of Organic Contaminants by Cobalt(II) Catalyzed

Sulfite at Alkaline pH

Yanan Yuan 1,2 , Dan Zhao 1 , Jinjun Li 1 , Feng Wu Corresponding Authors: Feng Wu (fengwu@whu.edu.cn) and Gilles Mailhot (gilles.mailhot@uca.fr)

Preliminary experiment using Co(II)-S(IV)

A typical degradation of Paracetamol by three different kinds of system (only Co(II), only sulfite(S(IV), Co(II)+S(IV)) is presented in Fig. S0. The result showed that when the experiment conducted in the presence of S(IV) without any catalyst, negligible change in paracetamol concentration was observed. A similar trend was also achieved in the experiment by only using Co(II) even after being centrifuged for 15 min at 9000 rpm. There was no precipitation occurred, which illustrated that there is no adsorption in this system and neither S(IV) nor Co(II) added to the reagent had an effect on Paracetamol. However, when both S(IV) and Co(II) were in solution, Paracetamol can be degraded to more than 80% in 10 min. These results suggest that Paracetamol degradation depends heavily on the reaction between S(IV) and Co(II), it proceeded only when S(IV) and Co(II) were present.

UV spectra of the reaction solution

Cobalt(II) quantification

The calibration of Cobalt(II) was measured by preparing a series of standard solutions, firstly , 5 mL cobalt(II) and 1 mL NH 4 SCN(566g/L) was added to a 10 mL centrifuge tube and hand shaken for 1 min, then another 4 mL pure MIK was added to the mixture, and ultrasonic shaking for 1min and equilibrating until stratication occurred. Using the UV-Vis spectrophotometer for the absorbance at 620 nm for the upper layer complex which containing Cobalt(II). The calibration curve is presented in Fig. S2a.