Laurent Erades

Céline Nayral

Katerina Soulantica

Bruno Chaudret

André Maisonnat

Didier Grandjean

Philippe Menini

Frederic Parret

Organometallic Approach for Platinum and Palladium doping of Tin and Tin Oxide Nanoparticles: Structural Characterisation and Gas Sensor Investigations

Decomposition of the organometallic precursors [Pt2(dba)3] or [Pd(dba)2] in a colloidal suspension containing tin/tin oxide nanocomposite core shell particles in anisole under 3 bars of dihydrogen at room temperature leads to surface doped tin particles which can be isolated as pure solids and for which elemental analyses show doping yields of 62% for Pt and 77%

for Pd and effective doping ratios [Pt]/[Sn] and [Pd]/[Sn] of 2.5 and 3.1 % respectively. These particles were characterized by means of transmission electron and high-resolution transmission electron microscopies (TEM and HRTEM), Energy Dispersive X-Ray spectroscopy (EDX), photoelectron spectroscopy (XPS) and X-ray absorption spectroscopy (EXAFS and XANES). The TEM micrographs show spherical nanoparticles, the size and size distribution of which are essentially similar to those of the initial Sn/SnOx material. HRTEM, EDX, XPS and X-ray absorption studies at the Pd and/or Sn-K edge indicate conservation of the composite nature of the particles that consist of a tin (0) core covered with a layer of tin oxides on which the doping element is deposited under the form of crystalline metallic platelets of size near 2 nm. The thermal oxidation of these Pt-or Pd-doped tin materials yields nanoparticles of crystallized SnO2 covered with crystallites of Pt or Pd oxides, as demonstrated by XRD, XPS and X-ray absorption experiments, without coalescence or size change. The thermal oxidation process can be easily achieved onto the silicon platform of a micro machined device after integration of the Pt-or Pd-doped Sn/SnOx colloidal suspension by drop deposition. The first electrical measurements indicate remarkable behaviours of the as-obtained micro sensors when exposed to traces of carbon monoxide: a surprising inversion of sensitivity from undoped and Pt-and Pd-doped sensors in dry atmosphere and an important increase of sensitivity relatively to undoped sensitive layer in humid atmosphere.

Introduction

Metal oxides are widely employed as sensitive element in gas sensors [1][START_REF] Williams | Solid State Gas Sensor[END_REF][START_REF] Lantto | Gas sensors, Principle, Operation and developments[END_REF][START_REF] Ihokura | The Stannic Oxide Gas Sensor: Principles and Applications[END_REF] and commercial devices produced by a typical ceramic manufacturing process have been available for a long time. [START_REF]Technical information for carbon monoxide sensors[END_REF] Such devices are able of detecting low concentration of flammable or toxic gases at a ppm level but their lack of selectivity and long term stability remains their weak points, and improve the cross sensitivities between gases like CO, H2, hydrocarbons and NO2 remains a major problem.

Even though modulations of the sensor response to a given gas in a mixture can be achieved through optimization of the working temperature of the sensitive layer, [START_REF] Heilig | [END_REF] of the operating mode and the signal processing [7][8][9] an accurate control of morphology of the sensing materials, in the nanometre range, is well known as an inescapable prerequisite for improvement of sensor performances. [7,[10][START_REF] Göpel | Gas sensors: principles, operation and development[END_REF][START_REF] Göpel | [END_REF][13] If the addition of small amounts of a noble metal has been shown to improve the detection of various kind of gases, [10,[14][START_REF] Norris | Solid State Gas Sensor[END_REF][START_REF] Demarne | Gas sensors, Principles, operation and developments[END_REF][START_REF] Schweizer-Berberich | [END_REF][18][19][20][21] sensitivity and selectivity again depend on morphology of the as-obtained doped material, in term of distribution and crystal size of the added noble metals. [22][23][24][25] In previous studies, we have evidenced the interest of organometallic complexes for the preparation of metal nanoparticles of controlled size and shape and organization. [26][27][28][29][30][31][32][33] We have shown that this methodology can be extended to the synthesis of nanoparticles of metal oxides with a precise control of morphology. Thermolysis of [ZnCy2], (Cy = C6H11), or [{Sn(NMe2)2}2] in anisole or methanolysis of InCp, (Cp = η 5 -C5H5), in toluene indeed lead to the formation of zinc, tin or indium nanoparticles of low size dispersity [34][35][36][37] which can be oxidized, in a second step, into well crystallized nanoparticles of the corresponding oxides, without coalescence. We recently reported a direct organometallic route to ZnO colloidal solution containing nanoparticles of controlled size and shape. [38] We have also shown that dibenzylideneacetone derivatives of palladium and platinum can be used as precursors to yield metal nanoparticles of uniform sizes in the range 1.2 -2.5 nm by submitting solutions of these compounds to CO or H2 and in the presence of a stabilizing agent as polymer or organic ligands. [39][40][41] We demonstrate in this paper that these Pd and Pt organometallic complexes can effectively be used as precursors for a surface doping of tin nanoparticles through very mild conditions and with an accurate control of morphology. The reaction with CO of [Pt2(dba)3] or [Pd(dba)3] in anisole containing tin nanoparticles indeed leads to the formation of tin/platinum or tin/palladium nano composites. The characterization of these nano composites by elemental analysis, TEM, HRTEM, EDX, EXAFS and XPS is described. Their oxidation by thermal treatment under air, leading to nano composites characterized as tin dioxide nanoparticles covered with platinum or palladium oxide, is also described as well as their implementation as sensitive layer on the silicon platform of a gas micro sensor and their electrical properties under carbon monoxide.

Experimental section

Materials and reagents

All compounds used in this work were sensitive to oxygen and moisture and were manipulated in a standard vacuum line or in argon atmosphere by using a MBraun Inert Gas System. Preparation of the platinum and palladium precursors, ([Pt2(dba)3] and [Pd(dba)2] respectively) follows the routes described earlier [42] . Anisole was purchased from Aldrich and its water content was checked by Karl-Fisher coulometric titration by using Metrohm equipment.

Preparation of the [Sn/SnOx] colloidal suspension

Preparation of the tin colloid follows the route described previously. [35,[START_REF] Fau | European patent[END_REF] The precursor [Sn(NMe2)2]2 (174 mg, 0.42 mmol; 0.84 10-3 eq. Sn), in anisole (degassed under vacuum, at liquid nitrogen temperature; 15 mL) containing traces of water (0.4 mg/mL of anisole; 0.33 mmol: [H2O]/[Sn] = 0.4) were heated at 135 °C under magnetic stirring in a Fischer-Porter bottle. The initial yellow solution darkened without 10 mn and a black solid progressively precipitated. After 3hours, the solution was removed by filtration, and the black precipitate was washed with anisole (3x40 mL). The black solid was then re-dispersed in 15 mL of degassed anisole and the Fischer-Porter bottle was then pressurized to 3 bars of dihydrogen.

After stirring for 3 hours, the resulting suspension was sampled for TEM analysis: nanoparticles, size 7-2O nm (Gaussian fit, mean size: 13.2 (2.2) nm).

Decomposition of [Pt2(dba)3] in [Sn/SnOx] colloidal suspension

To 7.5 mL of the suspension of [Sn/SnOx] in anisole (0.21 10 -3 eq Sn) was added the platinum precursor (1 mL of a solution containing 46 mg of [Pt2(dba)3] in 10 mL of anisole; 0.0042 mmol; [Pt]/[Sn] = 4%). The mixture was exposed to 1 bar of CO in a Fisher-Porter bottle.

After 20 mn of stirring, the Fisher-Porter bottle was evacuated and the resulting black suspension was sampled for TEM, HRTEM, EDX and XPS experiments. The supernatant solution was then removed by filtration from the remaining suspension and the precipitate was washed with anisole (3x15 mL) and dried under vacuum. Elemental analysis (%): Sn 79.

Thermal oxidation of tin/platinum and tin/palladium products

The black solids resulting from decomposition of [Pt2(dba)3] and [Pd(dba)2] in [Sn/SnOx] colloidal suspensions were heat-treated in air in an oven for a period of 135 min at temperatures ranging from 50 to 525°C according to the optimized multi step process already used for the oxidation of [Sn/SnOx] nanoparticles [35] Electron microscopy experiments Samples for TEM and HRTEM studies were prepared in a glove-box by slow evaporation of a drop of suitably diluted colloidal suspensions deposited on holey carbon-covered grids. The TEM experiments were performed at the "Service Commun de Microscopies de l'Université Paul Sabatier" on a JEOL JEM 200CXT-T electron microscope working at 200 kV and a Philips CM12 electron microscope working at 120 kV while the high resolution images were obtained with a JEOL JEM 2010 electron microscope working at 200 kV. The size distribution of the particles was determined from enlarged photographs by measuring a minimum of 200 particles for each example.

X-ray photoelectron spectroscopy experiments

Samples for XPS studies were prepared in a glove-box by slow evaporation of a nanoparticle suspension deposited on a 1 cm 2 silicon support. The spectra were recorded on a VG ESCALAB MKII spectrometer with Mg-Kα radiation.

X-ray absorption spectroscopy experiments

X-ray absorption data were collected on beamline X1 (Hasylab, DESY, Hamburg, Germany), operating under beam conditions of 4.4 GeV, 150 mA using a Si (311) double-crystal monochromator. The monochromator was detuned on its rocking curve to approximately 60% of the maximum transmitted X-ray intensity to reduce the harmonic content of the beam at a minimum level. Ionisation chambers were filled with standard N2/Ar mixtures. Appropriate amounts of sample were finely ground with boron nitride and pressed into a self-supporting wafer producing a suitable edge jump. The spectra were recorded at 80 K by cooling the samples with liquid-nitrogen. XAS signals were measured in transmission mode at the Pd K edge (24350.3 eV) with 4 datasets summed to improve signal/noise ratio. PdO powder (Aldrich 99.999%) was used as reference sample.

Data reduction of experimental X-ray absorption spectra was performed using the program EXBROOK. [START_REF] Fletcher | the United Kingdom Chemical Database Service[END_REF] Pre-edge background subtraction and normalisation was carried out by fitting a linear polynomial to the pre-edge region and cubic splines to the post-edge region of the absorption spectrum. A smooth atomic background was then obtained. EXAFS refinements were performed with the EXCURV98 package. [START_REF] Fletcher | the United Kingdom Chemical Database Service[END_REF] Phase shifts and backscattering factors were calculated ab initio using Hedin-Lundqvist potentials. Refinements were carried out using k 3 weighting in the range 2.5-15 Å -1 for the reference compounds and k 2 in the range 3.0 to 12.6 Å -1 for the Pd doped SnO2 nanomaterial. In order to improve the quality of the signal for a better quantitative analysis and to reduce the number of fitting parameters, the data for all the samples (except for bulk Pd) were refined using Fourier filtering techniques. Fourier transform (FT's) peaks were isolated with a Gaussian window (1.0-4.9 Å) and the contribution of this group of shells was extracted by inverse transformation.

Implementation of colloidal suspensions of platinum and palladium doped tin/tin oxide nanoparticles on silicon platform and conductivity measurements under carbon monoxide.

A drop of the doped colloidal tin/tin oxide suspension in anisole is deposited on the heating element and electrical contacts of a silicon platform of a chemical sensor device integrating all structures that are needed to receive and make operational a sensing layer (heater, metal interconnects, membrane, insulating layer…) according to a procedure previously described. [45,46] The integrated micro heater is used to in situ oxidize the doped tin/tin oxide colloid layer at a temperature close to 500°C according to the multi step process previously described [35,45] to form fully oxidized platinum and palladium doped tin dioxide structures.

The sensor heating was controlled by a constant-voltage power supply. The conductivity measurements on the sensitive layers were carried out under controlled atmosphere in a flow chamber at a flow rate of 1 lmn -1 , temperature 21°C and variable relative humidity on a test bench by using interface hardware. Humidity concentration was controlled by mixing appropriate fractions of dry and saturated synthetic air. The data acquisition was controlled through Labwindows software (National Instruments).

Results and discussion

Synthesis and characterization of [Sn/SnOx/Pt] nano composite:

We recently demonstrated that the reaction of [Pt2(dba)3] with carbon monoxide in THF leads to the formation of well crystallized platinum nanoparticles of uniform size centred near 1.2 nm [41] which can be easily functionalized through coordination of various ligands at their surface. This organometallic complex then appears as a convenient potential precursor for the doping of tin nanoparticles, and we observed that [Pt2(dba)3] indeed also decomposed in the presence of colloidal tin/tin oxide nano composite suspended in anisole, when exposed to 1 bar of carbon monoxide. Elemental analysis of the black product isolated after the reaction indicates that 62.5% of the platinum initially introduced in the medium has been incorporated to the tin/tin oxide colloid. Transmission Electron Microscopy (TEM) observation and Energy Dispersive X-Ray spectroscopy (EDX) of this black product revealed spherical nanoparticles of uniform size centred near 13.4 nm, as the starting [Sn/SnOx] colloid (13.2 (2.2) nm) (Figure 1a andb), and homogeneous distribution of both Sn and Pt in the sample, the [Pt]/[Sn] atom ratio determined by this method, ie 2.3%, being in good agreement with the composition obtained from elemental analysis within the limits of instrument sensitivity. HRTEM image shows small crystallites of diameters in the range 2-3 nm at the surface of the particles (Figure 2). If the core-shell morphology observed for the starting tin colloidal material [34,35] is not apparent on this image, surface analysis by X-ray photo-emission spectroscopy (XPS) however indicates a composition basically similar consisting of a core of tin(0) surrounded with a tin oxide layer on which the added platinum is deposited. The Sn 3 d5/2 peak indeed splits into two components with binding energies of 484.7 and 486.4 eV indicative of both tin oxides and tin(0) (Figure 3). A Pt 4 f7/2 peak appears at 69.5 eV and corresponds clearly to zero-valent platinum. [START_REF]Handbook of X-Ray Photoelectron Spectroscopy[END_REF] Tin (0) component at 484.

Synthesis and characterization of [Sn/SnOx/Pd] nano composite:

Similarly, the decomposition under CO atmosphere of Pd(dba)2 in the presence of colloidal [Sn/SnOx] suspended in anisole yields [Sn/SnOx/Pd] nano composite as a black solid. The elemental analysis of this solid indicates that 77% of the palladium initially introduced in the starting suspension has been incorporated to the tin/tin oxide colloid. TEM observation indicates again similar spherical morphology with similar mean diameter as in the starting tin/tin oxide material (figure 1c) and HRTEM images (Figure 4) shows small crystallites at the surface of the particles. XPS surface analysis (Figure 5) again is consistent with conservation of a core-shell structure consisting in a core of tin(0) surrounded with a tin oxide layer on which the added palladium is deposited, the atom ratio [Pd]/[Sn] decreasing from 1.9% to 1.4% after argon bombardment of the sample for 1 mn. If the palladium oxidation state can not be deduced from XPS experiment, the Pd 3 d5/2 peak at 335.8 eV being close to those reported for Pd as for PdO, [START_REF] Wagner | NIST Standard reference Database 20[END_REF] XANES and EXAFS analyses shows that the palladium is zerovalent and has a structure close to the metal but with very small particle size and/or amorphous character [START_REF] Grandjean | [END_REF] ….. Didier, à completer et illustrer Pd was found to be surrounded by 2.6 Sn at short distance of ca 2.58 Å, and by a second coordination shell of ca 2.3 Pd atoms located at 2.77 Å. This result identifies two metallic phases in the as-prepared sample, ie a mixed Sn/Pd phase in the metallic core of the nanocomposite accounting for 80% of the Pd atoms and a pure Pd metal one. This demonstrates the absence of the homogeneous mixing of the Pd atoms in the Sn matrix already observed in the case of volume Pd-doped tin/tin oxide nanocomposites, [START_REF] Grandjean | [END_REF] and is on the contrary consistent with their localization as small crystalline platelets embedded in the amorphous tin oxide outer layer of the tin/tin oxide nanoparticles as suggested by XPS and as observed with HRTEM.

Oxidation of [Sn/SnOx/Pt] and [Sn/SnOx/Pd] nanocomposites

The [Sn/SnOx/Pt] and [Sn/SnOx/Pd] nanocomposites can be fully oxidized in air in an oven, at temperatures ranging from 50 to 525°C according to the optimized multi step process already used for the oxidation of [Sn/SnOx] nanoparticles, [35] to yield black solids. This black solid is essentially constituted of crystallized tin dioxide as shown by their XRD patterns (which exhibit the classical quadratic phase of SnO2 and, to a lesser extent, the high pressure orthorhombic phase of SnO2). The apparent black colour of this solid is then consistent with the presence of palladium or platinum oxide at the surface of the material. The micrograph of platinum doped material (Figure 6) shows individual agglomerated particles and displays the same type of morphology as the non-oxidized particles. Their mean-size, around 13 nm, and the size distribution are unchanged and we did not observe any sign of coalescence. The HRTEM micrograph shows well crystallized domains at the surface of the particles.

Figure 6. TEM (insert) and HRTEM images of platinum doped tin/tin oxide particles after oxidation

For confirmation of these results, XPS study of the surface of the oxidized platinum doped tin particles (Figure 7) and EXAFS study of the oxidized palladium doped tin particles were performed. XPS spectra of the oxidized Pt-doped tin particles provides evidence of the presence of oxidized tin insofar as they feature one single symmetrical peak at 486.4eV for the Sn 3 d5/2 electrons, and no Sn 0 was observed even after argon bombardment. The position of the Pt 4 f7/2 emission peak, ie 75.0 eV, is indicative of the presence of an oxidized PtO2 phase at the surface of the particles. [START_REF] Wagner | NIST Standard reference Database 20[END_REF] Side peaks at lower binding energies between 71.5 and 74.0 eV are observed and can be assigned to Pt(OH)2, PtO and/or Pt-O-Sn phases by reference to previous studies. [50] Moreover, the Pt 4 f7/2 peak is strongly shifted, after argon bombardment for 1 mn, towards a position at lower binding energy, ie 72.1 eV, very close to that previously reported for Pt-O-Sn surface species [51] which are supposed to model the platinum chemisorption on tin dioxide. Our observations suggest a core shell structure for the nano composite SnO2/PtO2 in which platinum dioxide lies at the surface of tin dioxide particles through an interface constituted of SnO2-bonded Pt species and rule out the presence of metallic platinum at the surface of tin oxide grains, in agreement with previous observations [52]

EXAFS studies of the fully oxidized Pd-doped Sn/SnOx nano particles

EXAFS studies of the oxidized Pd-doped tin particles suggest that the transformation occurring during the oxidation process of the Pd doped tin nanomaterial consists in the oxidation of the Pd metallic platelets into a pure Pd3O4 phase whilst the mixed metallic phase present in the core of the particle is transformed into two phases: a mixed Pd/Sn oxide having a PdO type structure and a SnO2 phase in which some Sn are substituted by Pd atoms.

Results of the EXAFS structural refinements are summarised in Table 1. The EXAFS and Fourier transform best fit are respectively presented in Figure 8 and Figure 9 The low intensity of the second and third peaks already shows that the local environment around Pd atoms is relatively disordered and that the Pd phase is part of a rather amorphous phase and/or that this phase is composed of very small particles.

The results of the EXAFS refinement presented in Table 1 show that the first peak could be fitted with two oxygen shells of respectively 3.5 atoms located at 1.997 Å and 1.7 oxygen atoms located at 2.486 Å. The second peak could be fitted with 2.2 tin atoms located at 2.648 Å. The higher R part of the FT corresponds to a Pd shell of 3.1 atoms located at 3.24 Å and two additional shells of around 0.7 Pd at 3.037 and 0.6 Sn at 3.653 Å.

The first oxygen bond distance is slightly smaller than the corresponding first Pd-O bond distance both in the PdO (2.018 Å) and the Pd3O4 (2.035 Å) phases whilst the second oxygen shell corresponds almost exactly to the O bond distance of the Pd3O4 phase (2.492 Å). The oxygen shells already suggest that we are in presence of two different Pd oxide phases. The relatively high Debye-Waller factor associated with this second shell O shell indicates an important level of disorder in this phase.

The general fit was greatly improved by fitting the second shell with Sn atoms instead of the Pd that would be normally expected as second neighbours in a Pd oxide phase. The distance of this Sn shell is very close to the typical Pd-Pd bond of the PdO phase (2.665 Å) but with a slightly smaller value (2.648 Å). This could correspond to a Sn atom substituting a Pd atom in a PdO type phase. This shorter bond distance is not surprising if one considers the smaller size of the Sn 4+ ion (55pm 4fold coordinated) compared to the Pd 2+ ion (64 pm 4 fourfold coordinated). Unusually short Pd-Sn bond distances belonging to a metallic phase were also observed in the as-prepared material before its full oxidation. The next shell of the FT was fitted with 0.6 Pd atoms located at a distance of 3.037 Å that is very close to the third Pd-Pd bond distance (3.030 Å) observed in the PdO phase. The formation of a relatively disordered and/or nanocrystalline mixed Pd/Sn oxide phase with a structure close to the PdO structure seems to be the best explanation to account for these results.

The next shell corresponds to 3.1 Pd atoms located at 3.24 Å is very close to the Pd-Pd bond distance of the Pd3O4 phase (3.218 Å). The presence of this shell together with the O shell at Bulk Pd: fcc a=3.8898 Å 12Pd at 2.751 Å, 6Pd at 3.890 Å [START_REF] Fletcher | the United Kingdom Chemical Database Service[END_REF] Bulk PdO: P42/mmc a=b=3.03 Å, c=5. 2.49 Å confirms that a Pd3O4 type phase is present in these samples. Contrarily to the other oxide phase this Pd3O4 seems to a pure Pd phase without any trace Sn atom in its structure.

The last shell that could be included in the fitting model is a Sn shell at 3.653 Å that does not correspond to any atomic shell in any of the existing Pd oxide phases. This distance could correspond to a Sn-Sn distance found in the SnO2 phase (3.590 Å) meaning that a small part of the Pd atoms may be incorporated in the major SnO2 phase that composed these nanocomposites.

Because the oxygen shell located at 2.489 Å only belongs to the Pd3O4 phase, a rough evaluation of the relative ratio of the Pd atoms in this phase can be simply done by using the corresponding coordination number. The number of the oxygen located at this distance in the Pd doped nanomaterial is 1.7 compared to 4 atoms in the Pd3O4 phase which gives a ratio of ca. 1.7/4 x 100 = 42% of Pd atoms in the Pd3O4 phase in the nanomaterial.

A previous XAFS study [START_REF] Grandjean | [END_REF] could identify two metallic phases in the as-prepared sample: a mixed Sn/Pd phase in the metallic core of the nanocomposite accounting for ~80% of the Pd atoms and a pure Pd metal one forming the small crystalline platelets of Pd metal embedded in the amorphous tin oxide outer layer of the particle that could be observed with TEM. A possible interpretation of the changes occurring during the full oxidation of this nanomaterial is a transformation of the pure Pd metallic platelets into a pure Pd3O4 phase whilst the mixed metallic phase present in the core of the particle is transformed into two phases: a mixed Pd/Sn oxide having a PdO type structure and a SnO2 phase in which some Sn are substituted by Pd atoms. The discrepancy between the relative amounts of the Pd3O4 phases (ca. 40%) in the oxidised sample compared to ca. 20% of Pd metal in the as-prepared material may be explained by a higher amount of Pd metal in the as-prepared sample. Indeed, the evaluation was made using as a reference, a Pd coordination number of 12 corresponding to the bulk Pd metal; this figure could be too large for a highly dispersed nanomaterial. An additional phenomenon of atom migration at the interface core/outer layer/ Pd platelets may also be involved in the oxidation process explaining this discrepancy.

Resistance measurements and gas sensing properties

Gas sensitivity measurements were performed in order to compare systematically the influence of doping and of the humidity content on the sensor responses to carbon monoxide.

Resistance measurements were performed after deposition of a drop of the tin nanoparticles suspensions, doped or not, in anisole between the electrodes of a gas sensing device integrated onto a silicon platform, evaporation of the solvent, and in-situ oxidation of the nanoparticles according to a multi step process similar to that used for the oxidation in the oven.

Figure 11 shows the resistance variations of undoped and palladium-doped tin dioxide sensitive layers working near 450°C when exposed to increasing amounts of carbon monoxide, eg from 50 to 500 ppm, in dry air. Surprisingly, the responses of these two types of sensors to carbon monoxide are characterized by opposite variations of the resistances, ie, a decrease of resistance for the undoped sensitive layer, which is the normal behaviour commonly observed toward reducing gas, and a sharp increase of resistance for the palladium-doped sensitive layers. Quite similar behaviours have also been observed for platinum-doped sensitive layers. The response to CO of palladium-doped sensitive layers is in fact dramatically dependant of humidity and Figure 12 shows that the introduction of a small amount of humidity is accompanied by opposite variations of resistances under CO, whereas Figure 13 The accepted mechanism of detection of reducing gases such as CO involves the partial reduction of the surface of the sensitive layer. The result of this reduction is an increase of the conductivity of the sensitive layer. The inversion of sensitivity toward CO from undoped and Pd-and Pt-doped sensors is therefore surprising and presently under investigation.

Conclusions

The aim of this study was to demonstrate that the organometallic route is a powerful tool for an accurate doping of tin dioxide nanoparticles and can be easily implemented in micro sensors technology. For this purpose, we have achieved a high yield doping process consisting first in the doping of the nano composite [Sn/SnOx] through the decomposition of Pd or Pt organometallic precursors, followed by the thermal oxidation under air of the as-obtained Pdor Pt-doped material.

The first interesting aspect of this approach is that neither the doping process nor the subsequent thermal treatments affect the morphology of the starting tin/tin oxide materials, all nanoparticles isolated at each step of the process having mean sizes close to 13 nm with narrow size distributions, as observed for the starting material. The doping element is homogeneously localized at the surface of the Sn/SnOx particles under the form of crystalline metallic platelets having sizes close to 2 nm, whereas the full oxidation of this material yields composite nanoparticles consisting of a core of crystalline tin dioxide surrounded by crystalline platelets of Pt or Pd oxides.

The second interesting aspect is that, as the undoped Sn/SnOx nanoparticles, Pd-or Pd-doped Sn/SnOx can be easily deposited onto silicon platforms and implemented as sensitive layer of a micro machined silicon gas sensor. Finally, the as-obtained gas sensors show remarkable behaviours when exposed to carbon monoxide: i) a surprising inversion of sensitivity toward carbon monoxide from undoped and Pd-or Pt-doped sensors, in dry atmosphere (RH<5%), and ii) an important increase of sensitivity to CO relatively to undoped sensitive layer under humidity level in the range 15-55 %. Current and future studies are oriented toward the understanding of this unprecedented behaviour which is believed to be basically related to the detection phenomena at the surface of the material. This includes behavioural studies of the doped sensitive layers toward other reducing gases like H2 or hydrocarbons and toward oxidizing gases like NO2 and O3. ______________________________

Figure 1 .

 1 Figure 1. TEM image of tin/tin oxide nanoparticles a) before doping, b) after platinum and c) palladium dopings

  7 eV moreover considerably increased relatively to tin oxide component at 486.4 eV after argon bombardment of the sample for 1 mn whereas the atom ratio [Pt]/[Sn] decrease from 2.3% to 1.4%.

Figure 2 .Figure 3 .

 23 Figure 2. HRTEM image of platinum doped tin/tin oxide nanoparticles

Figure 4 .Figure 5 .

 45 Figure 4. HRTEM image of palladium doped tin/tin oxide nanoparticles

Figure 7 .

 7 Figure 7. Pt 4 f (a) and Sn 3 d5/2 (b) emission peaks of the XPS spectrum of platinum doped tin/tin oxide nanoparticles after oxidation (arbitrary y scales)

Figure 8 .

 8 Figure 8. Sn K-edge EXAFS spectra (after background substraction, k 2 -weighed) of the fully oxidized Pd surface-doped Sn/SnOx nano material. The solid lines are the experimental data and the dotted line is the best fit.

Figure 9 .

 9 Figure 9. Fourier transform of the Pd K-edge k 2 -weighted of the fully oxidized Pd surface-doped Sn/SnOx nano material. The solid lines are the experimental data, the dotted line is the best fit.

Figure 10 .

 10 Figure 10. Fourier transforms of the Pd K-edge k 3 -weighted or k 2 -weighted EXAFS of a) fully oxidized Pd surface-doped Sn/SnOx nano material and reference samples, b) bulk Pd, c) bulk PdO.

Ef

  = contribution of the wave vector of the zero photoelectron relative to the origin of k [eV] AFAC = amplitude reduction due to many-electron processes Ni = number of atom in the i th shell Ri = radial distance of atoms in the i th shell [Å] Ai = Debye-Waller term of the i th shell (A=2σ 2 with σ = Debye-Waller factor)[Å 2 ] R factor in % * these parameters were kept fixed during the refinement.

Figure 11 :

 11 Figure 11: Undoped and Pd-doped sensor responses to CO under dry air

  shows that the humidity dependence of the sensitivity S = [(Rgas -Rair)/Rgas] x 100 toward CO is quite different according as the sensitive layer is doped or not. For Pd-doped sensitive layers, a zero-sensitivity to 50 ppm of CO is observed for humidity content close to 5%. Then the sensitivity increases until values in the range [-50, -60%] when RH increases from 20 to 55%. Under similar conditions and within the same limits of humidity content, undopped tin oxide layers exhibit smaller sensitivity values stabilized around -10%.

Figure 12 .Figure 13 .

 1213 Figure 12. Pd-doped sensitive layers responses to CO in dry and humid atmospheres

Table 1 .

 1 Summary of structural results of Pd K-edge EXAFS refinements of the oxidised Pd doped Sn/SnOx nano-materials along with bulk Pd and bulk PdO.

		Bulk Pd PdO	Pd surface-doped
				Sn/SnOx
	Ef	-0.4(2)	4.7 (7) 1.4 (6)
	AFAC	0.80	0.80	0.80
	k	3	3	2
	k-range	3-16	3-15	2.6-12.6
	N1	12* Pd	4* O	3.5 (2) O
	R1	2.737 (1) 2.005 (1) 1.997 (3)
	A1	0.0100 (1) 0.003 (1) 0.004 (1)
	N2	6* Pd	4* Pd	1.7 (3) O
	R2	3.87 (1)	3.030 (2) 2.486 (9)
	A2	0.018 (4) 0.004 (1) 0.01 (1)
	N3	24* Pd	8* Pd	2.2 (3) Sn
	R3	4.741 (6) 3.416 (4) 2.648 (8)
	A3	0.016 (2) 0.005 (1) 0.016 (5)
	N4	12* Pd	-	0.7 (2) Pd
	R4	5.47 (2)	-	3.037(20)
	A4	0.023 (3) -	0.002(8)
	N5	24 Pd	-	2.4 (4) Pd
	R5	6.13 (3)	-	3.24 (2)
	A5	0.028 (1) -	0.021 (9)
	N6	-	-	0.6 (2) Sn
	R6	-	-	3.653 (20)
	A6	-	-	0.0001 (10)
	R (%)	15.3	17.7	10.1

The authors thank CNRS and European Commission for funding this work within the NANOSENSOFLEX project (GROWTH-G5RD-CT-2002-00722). We express our gratitude to MM. Vincent Collière, Lucien Datas, and UPS-TEMSCAN facility for TEM measurements, M. Gérard Chatainier, and INPT-ENSIASET for ESCA measurements, Dr François Senocq for XRD measurements, M. Ad van der Eerden (Utrecht University) and Dr Julia Wienbold (X1, Hasylab) for their assistance and advise during EXAFS measurement experiments.


Justyna Jońca

Andrey Ryzhikov

Audrey Chapelle

Philippe Menini

Katia Fajerwerg

Myrtil L Kahn
email: myrtil.kahn@lcc-toulouse.fr

Pierre Fau
email: pierre.fau@lcc-toulouse.fr

Ultra-sensitive SnO2 gas sensors based on hierarchical octahedra

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I. INTRODUCTION SnO 2 nanostructures including nanoparticles [1], nanowires

[2], nanobelts [3], and nanotubes [4, 5], have been widely used in many fields, such as gas sensors, solar cells and lithium batteries. Recently, hierarchical and/or hollow SnO 2 micro-and nanostructures have attracted much interest because of their widespread potential applications such as gas sensors [6]. We present here the formation of self-assembled tin oxohydroxide (Sn 3 O 2 (OH) 2 ) octahedral supercrystals obtained by an organometallic synthesis. These supercrystals have been characterized by transmission electron microscopy, and field-emission scanning electron microscopy. These octahedra have been used as gas sensitive layers deposited on silicon devices. After an in-situ annealing to 500°C, Sn 3 O 2 (OH) 2 oxidizes into SnO 2 while retaining the initial morphology. The response of the sensors to CO gas has been measured under a relative humidity (RH) of 50%. The sensors present an outstanding dynamic response to very low CO concentrations (0.25 to 20 ppm).

II. EXPERIMENTAL SECTION

A. Synthesis of Sn 3 O 2 (OH) 2

The synthesis of tin hydroxide is performed at room temperature and under argon atmosphere in standard Schlenk tubes. In a typical experiment, 2 mL of THF (Braun MB-SPS-800) containing degassed distilled water (72 µL, 8 eq.) is slowly added to a THF (4 mL) solution of [(Sn(NMe 2 ) 2 ] 2 (NanoMePS, 206.9 mg) with hexadecylamine (HDA, 12 mg, 0.1 eq.). After 16 h, the supernatant is removed by centrifugation (1000 rpm, 5 min, and 20°C) and the precipitated powder is washed 3 times with 5 mL of acetone. The resulting product is dried under low vacuum in a desiccator overnight. A white powder composed of Sn 3 O 2 (OH) 2 is obtained (Fig. 1).

B. Sensors preparation

Sn 3 O 2 (OH) 2 powder is dispersed in ethanol (5 mg.mL -1 ) using an ultrasonic bath (45 kHz, 10 min). The suspension is deposited as a sensitive layer on a micro-machined silicon substrate using a drop deposition system. The silicon platform is prepared in the MICA group of Laboratoire d'Analyse et d'Architecture des Systèmes (LAAS-CNRS). The sensor die (2×2 mm) comprises a 1.4 µm thick dielectric membrane (SiN x /SiO 2 ), a spiral shaped platinum heater, and interdigitated electrodes which contact the sensitive layer. Tests under gas have been performed in a bench equipped with a humidity sensor. The gas sensors are conditioned by an in-situ heating the sensitive layer up to 500°C in ambient air. During this anneal, Sn 3 O 2 (OH) 2 transforms into SnO 2 while keeping the octahedra shape. Then, the sensitive layer resistance is stabilized during few hours at 500°C in synthetic air flow (1 L.min -1 , RH 50 %). The sensor performance is measured under to various ppm levels of CO. The normalized response of the sensors is defined as Rn(%) = (R air -R gas )/R air *100, where R air corresponds to the sensor resistance in synthetic air and R gas corresponds to the sensor resistance under CO exposure.

III. RESULTS

A SnO 2 octahedra characterization

The growing process of the octahedra in solution has been followed by TEM investigations. After 30 minutes, selfassembled triangular foils are present in solution (Fig. 2a, left). After 7 hours, the sample only displays octahedral structures (Fig. 2a, center). HRTEM imaging has revealed the hierarchical structuration of the octahedra's walls. Selfassembled triangular sheets and octahedral particles are built by the self-assembly of nanoparticles with a diameter of 3.9 ± 0.9 nm (Fig. 2a,right). This suggests that the octahedra Ultra-sensitive SnO 2 gas sensors based on hierarchical octahedra * Justyna Jońca 1 , Andrey Ryzhikov 1 , Audrey Chapelle 3 , Philippe Menini 3 , Katia Fajerwerg 1,2 , Myrtil L. Kahn 1 , Pierre Fau 1, 2 building process starts by the formation of Sn 3 O 2 (OH) 2 nanoparticles stabilized by HDA, which self-assemble by an oriented attachment process into triangular polycrystalline foils in solution. The subsequent assembly of the triangular sheets leads to the tridimensional construction of octahedra by an oriented attachment process (Fig. 2b).

B Gas sensing properties of SnO 2 microstructures (octahedra and thin film)

The sensing properties of the above described SnO 2 octahedra sensors have been compared with a standard SnO 2 made of sputtered thin films (grain diameter ca. 500 nm, thickness ca. 250 nm, commercial source) (Fig. 3). For all sensors, the maximum sensitivity to CO is obtained at an operating temperature of 500°C. The sensors have been exposed to increasing CO concentrations between 0.25 and 20 ppm. Normalized responses for all sensors are presented in table 1.

TABLE I. Comparison of sensor's responses to increasing CO amounts

The thin film SnO 2 sensors show no response at all at 0.25ppm CO, whereas the SnO 2 octahedra response Rn is 7%. Thin film sensors give very weak responses to CO in the concentration range of 1-10 ppm with resistance variations of ca. 3%-10%, respectively. In the same conditions, normalized responses of the SnO 2 octahedra gas sensors increase from 20% to 55%. Moreover, they exhibit an almost saturated gas response at 20 ppm CO only. These sensors present an unprecedented dynamic of their response in the low CO concentration range (0.25 to 20 ppm) (Fig. 4). In air at 500°C, the adsorption and dissociation of oxygen species on SnO 2 grains, and grain boundaries, generates an electron depletion layer which extends to few nanometres depth (L). CO molecules directly react with these adsorbed oxygen species during the sensing operation of the device. For nanosized SnO 2 grains, the depletion layer can extend to the whole grain volume oxide and therefore enhance the CO response. With SnO 2 thin films, a material with wide and densely packed grains, the gas sensing properties are mostly controlled by the grain boundary barriers. In the case of octahedral sensitive layers, the combination of a porous microstructure and of nanosized building blocks provides a high response to low CO contents. In addition, according to N. Barsan et al.[7], the gas sensor response also strongly depends on the effective contact area between neighboring grains in the sensitive layer. Their model suggest that the sensor signal can be improved by changing the shape or dimensions of grains of the sensitive layers, and the best responses are obtained by the reduction of the effective contact area parameter. Therefore, the superior performance of SnO 2 octahedra layers may also be the consequence of the reduced contact area between the large octahedra grains.

CO content (at 500°C)

ppm 1 ppm 10 ppm 20 ppm

Response Rn (%) SnO2 octahedra 7 20 55 67

Response Rn (%) SnO2 thin film 0 2.9 10 25

IV. CONCLUSION

In summary, the SnO 2 octahedra sensitive layers present a remarkable response to low CO amounts, and a very dynamic response between 1 and 20 ppm where the saturation threshold is almost reached. The very open structure of SnO 2 octahedra provides a large porosity for an effective gas diffusion, and small contact area between the grains are favorable to a high gas sensitivity. Such hierarchically assembled sensitive layer may be used for applications where ultra-sensitive CO gas sensors are required.

Figure 1 .

 1 Figure 1. Scheme of the synthesis of Sn3O2(OH)2 octahedra.

Figure 2 .

 2 Figure 2. (a) TEM image of the particles obtained in solution after, left: self-assembled triangles shapes obtained after 30 minutes, center: octahedra obtained after 7 hours, left: HRTEM image of the nanoparticles (yellow circles) inside the octahedra walls,(b) scheme of the hierarchical selfassembly process of the octahedra in solution.

Figure 3 :

 3 Figure 3: SEM image of SnO2 sputtered thin film (left), and octahedra (right).

Figure 4 .

 4 Figure 4. Response of the sensors at CO levels between 0.25 and 20 ppm.

ACKNOWLEDGMENTS

The authors thank the Université Fédérale de Toulouse, Paul Sabatier UT III, and CNRS for their support.

*Research funded by the French BPI ISI project "Object's World".


Héloïse Guillot

Xavier Bonnet

Paco Bustamante

Carine Churlaud

Jacques Trotignon

François Brischoux
email: françoisbrischoux@gmail.com

Trace element concentrations in European pond turtles (Emys orbicularis) from Brenne Natural Park, France

Keywords: trace elements, contamination, wetlands, Emys orbicularis

We assessed trace elements concentration in European pond turtle (Emys orbicularis) from Brenne Natural Park (France). We sampled road-killed turtles (N = 46) to measure the concentrations of 4 non-essential (Ag, Cd, Hg, and Pb) and 10 essential (As, Co, Cr, Cu, Fe, Mn, Ni, Se, V, and Zn) elements in muscle, skin, liver and claws. Body size or sex did not influence the concentrations of most elements; except for Hg (liver, skin and claws) and Zn (muscle) which increased with body size. We found relatively high concentrations of Hg and Zn, possibly linked to fish farming. This result deserves future investigations to evaluate possible ecotoxicological effects on E. orbicularis.

Introduction

Wetlands are important habitats for biodiversity, yet they are among the most endangered ecosystems in the world, suffering from a drastic reduction of their surface and from a degradation of water quality [START_REF] Schneider | Hydrological threats to riparian wetlands of international importance-a global quantitative and qualitative analysis[END_REF]. Monitoring environmental contaminations in wetlands is difficult because they are connected to complex and large hydric networks composed by both surface and underground waters. As a consequence, the connectivity of aquatic systems can induce contamination in areas that otherwise appear unscathed from direct sources of pollution [START_REF] Baker | Introduction to nonpoint source pollution in the United States and prospects for wetland use[END_REF]). Among environmental contaminants, trace elements are wellknown for their ability to enter and move across aquatic environments [START_REF] Agarwal | Heavy Metal Pollution[END_REF]). These elements comprise both non-essential elements (that exhibit high toxicity at low concentrations) and essential elements (that may become toxic when they exceed normal levels) [START_REF] Förstner | Metal pollution in the aquatic environment[END_REF]. Overall, trace elements represent a threat to aquatic ecosystems because of their high toxicity, persistence, bioaccumulation in organisms and Biomagnification across trophic levels [START_REF] Agarwal | Heavy Metal Pollution[END_REF].

Freshwater turtles are suitable organisms to survey contamination levels in complex aquatic ecosystems [START_REF] Overmann | Snapping turtles (Chelydra serpentine) as biomonitors of lead contamination of the Big River in Missouri's old lead belt[END_REF][START_REF] Ayub | Determination of some heavy/trace metals in two species of turtles (Lissemys punctata and Trionyx Gangeticus) collected from different spots of Hadiara drain[END_REF][START_REF] Nagle | Accumulation and selective maternal transfer of contaminants in the turtle Trachemys scripta associated with coal ash deposition[END_REF][START_REF] Bergeron | Influence of feeding ecology on blood mercury concentrations in four species of turtles[END_REF][START_REF] Yu | Metal accumulation and evaluation of effects in a freshwater turtle[END_REF][START_REF] Malik | Trace metals in Ganges softshell turtle (Aspideretes gangeticus) from two barrage: Baloki and Rasul, Pakistan[END_REF][START_REF] Yadollahvand | Assessment trace elements concentrations in tissues in Caspian Pond Turtle (Mauremys caspica) from Golestan province, Iran[END_REF][START_REF] Allender | Select metal and metalloid surveillance of free-ranging Eastern box turtles from Illinois and Tennessee (Terrapene Carolina carolina)[END_REF][START_REF] Slimani | Large-scale geographic patterns of mercury contamination in Morocco revealed by freshwater turtles[END_REF]. First, they are widely distributed and occupy a variety of habitats. Second, they have a long life expectancy, which allows studying bioaccumulation processes and long-term trends of contaminants. Finally, most species are sedentary and provide information on the contamination at a precise location.

In this study, we used opportunistic sampling of roadkilled European pond turtles (Emys orbicularis) to study trace element contamination of one of the largest French wetlands: the Brenne Natural Park. The goals of our descriptive study were to document the concentrations of 4 nonessential trace elements (Ag, Cd, Hg, and Pb) and 10 essential trace elements (As, Co, Cr, Cu, Fe, Mn, Ni, Se, V, and Zn) in the muscle, skin, liver and claws of E. orbicularis; and to investigate the relationship between the concentrations of these elements and the sex and the size of the turtles.

Material and methods

Emys orbicularis is a small European freshwater turtle species [START_REF] Priol | Répartition de la cistude d'Europe (i) en aquitaine[END_REF].

Carcasses of accidentally killed pond turtles were found on roads situated in vicinity of the Réserve Naturelle Nationale de Chérine (46°47'25.23"N, 1°12'3.54"E) between 2008 and 2014. A total of 46 turtles were collected including 38 adults (17 females, 16 males, 5 unsexed individuals) and 8 juveniles. We used plastron length as an index of body size.

All individuals were dissected to take samples of the posterior right leg muscles and skin, liver and claws (Table 1). Because of the limiting mass of the claw samples, only Hg concentrations were measured in this tissue. The muscles, skin and liver samples were lyophilized and hand-ground with a porcelain mortar and pestle. Claws were washed three times with a mixture of 2:1 chloroform/methanol solution in an ultrasonic cleaner and rinsed in milli-Q quality water. Claws were then dried for 48 h at 50°C. The average weights of the tissue samples used for trace element analysis were 200 mg for muscles samples, 215 mg for skin samples, 119 mg for liver samples and 12 mg for claws.

Total Hg concentrations were measured using and atomic absorption spectrophotometer (Advanced Mercury Analyser-254, Altec) on dried tissue aliquots (ranging from 2 to 10 mg) as described by [START_REF] Chouvelon | Hg concentration and related risk assessment in coral reef crustaceans, molluscs and fish from New Caledonia[END_REF]. Accuracy was checked using TORT-2 Lobster Hepatopancreas (NRC, Canada; certified Hg concentration: 0.27 ± 0.06 μg g -1 dw) as certified reference material (CRM). Our measured values were 0.245 ± 0.003 μg g -1 dw (N = 4) showing a recovery of 91%.

Blanks were analysed at the beginning of each set of samples and the detection limit of the method was 0.05 ng.

Other elements (Table 1) were analysed using a Varian Vista-Pro ICP-OES and a Thermo Fisher Scientific X Series 2 ICP-MS (following [START_REF] Kojadinovic | Multielemental concentrations in the tissues of the oceanic squid Todarodes filippovae from Tasmania and the Southern Indian Ocean[END_REF]). CRM (DOLT-4 dogfish liver and TORT-3 Lobster Hepatopancreas, NRC, Canada) and blanks treated and analysed in the same way as the samples were included in each analytical batch. The recovery ratio were in good agreement with DOLT-4 [94% (Ag); 99% (As); 105% (Cd); 104% (Cu); 90% (Fe); 99% (Ni); 87% (Pb); 101% (Se) and 101% (Zn)] and TORT-3 [109% (As); 101% (Cd); 101% (Co); 95% (Cr); 103% (Cu); 86% (Fe); 96% (Mn); 97% (Ni); 93% (Pb); 101% (Se); 99% (V) and 96% (Zn)]. The limits of quantification (μg g-1) were 0.01 (Ag, Co, Cd, Mn, Pb); 0.12 (As, Cr); 0.02 (Cu); 5.03 (Fe, Zn); 0.05(Ni); 0.5 (Se); and 1.26 (V). Trace element concentrations are expressed in μg g -1 dw.

When trace elements concentrations were lower than the limit of detection (LoD) in more than 30% of the samples (Table 1), these samples were excluded from statistical analyses (EPA 2000). When trace elements concentrations were lower than LoD in less than 30% of the samples, values below LoD were replaced by (LoD) × 0.5 for statistical analyses (EPA 2000). To investigate the influence of sex on trace element concentrations, possible sexual size dimorphism was considered: plastron length was compared between sexes with a Kruskal-Wallis test (data were not normally distributed). Because females were larger than males (see results), we used ANCOVAs to test for sex effects in trace element concentrations, using plastron length as the covariate. To investigate the influence of body size (hence presumably age) on trace element concentrations, and thus the bioaccumulation of these elements through time, we used Spearman rank's correlation between trace elements and plastron length.

Results and Discussion

The mean concentrations of each trace element are presented in Table 1. Only four elements (Cd in muscle and skin; Cr in liver; Ag in muscle, skin and liver and V in muscle, skin and liver) were below the LoD in > 30% of individuals (Table 1).

Although females were larger than males (Kruskal-Wallis χ²=19.7449, df = 1, p < 0.01), sex did not influence the concentrations of trace elements (all p > 0.12). This suggests that feeding, metabolism or growth rates were broadly similar for both genders [START_REF] Allender | Select metal and metalloid surveillance of free-ranging Eastern box turtles from Illinois and Tennessee (Terrapene Carolina carolina)[END_REF][START_REF] Yu | Metal accumulation and evaluation of effects in a freshwater turtle[END_REF] and that contrarily to what has been shown in another species [START_REF] Nagle | Accumulation and selective maternal transfer of contaminants in the turtle Trachemys scripta associated with coal ash deposition[END_REF] or in sea turtles [START_REF] Guirlet | Maternal transfer of trace elements in leatherback turtles (Dermochelys coriacea) of French Guiana[END_REF], eggs may not represent a major excretion pathway in female E. orbicularis.

Most trace elements concentrations did not correlate with body size suggesting that E. orbicularis does not bioaccumulate these contaminants with age as showed in other turtle species [START_REF] Allender | Select metal and metalloid surveillance of free-ranging Eastern box turtles from Illinois and Tennessee (Terrapene Carolina carolina)[END_REF][START_REF] Yadollahvand | Assessment trace elements concentrations in tissues in Caspian Pond Turtle (Mauremys caspica) from Golestan province, Iran[END_REF]). Yet, and similarly to other studies [START_REF] Overmann | Snapping turtles (Chelydra serpentine) as biomonitors of lead contamination of the Big River in Missouri's old lead belt[END_REF][START_REF] Nagle | Accumulation and selective maternal transfer of contaminants in the turtle Trachemys scripta associated with coal ash deposition[END_REF][START_REF] Bergeron | Influence of feeding ecology on blood mercury concentrations in four species of turtles[END_REF][START_REF] Yu | Metal accumulation and evaluation of effects in a freshwater turtle[END_REF], we found two notable exceptions to this trend: Zn concentrations (in muscle, rs= 0.49, p < 0.05; Fig. 1) and Hg concentrations (in liver, skin and claws) were correlated with body size (respectively rs = 0.56, p < 0.05; rs = 0.44, p < 0.05; rs = 0.40, p < 0.05; Fig. 2). These results suggest that these elements bioaccumulate over the life of the turtle. Interestingly, these two elements were found in relatively high concentrations (up to 208 μg g -1 dw for Zn and 4.451 μg g -1 dw for Hg), suggesting that excretion rates do not compensate for Zn and Hg exposure.

Bioaccumulation of Hg in its methylated form is a well-known process in vertebrates as this non-essential metal is not regulated and bound to proteins potentially leading to adverse effects [START_REF] Eagles-Smith | Scientific Opinion on safety and efficacy of zinc compounds (E6) as feed additive for all animal species: Zinc oxide, based on a dossier submitted by Grillo Zinkoxid GmbH/EMFEMA[END_REF]. It is noteworthy that the value of Hg we report in various tissues of E. orbicularis are among the highest reported for freshwater turtles [START_REF] Yu | Metal accumulation and evaluation of effects in a freshwater turtle[END_REF][START_REF] Zapata | Mercury concentrations in tissues of Colombian slider turtles, Trachemys callirostris, from northern Colombia[END_REF][START_REF] Slimani | Large-scale geographic patterns of mercury contamination in Morocco revealed by freshwater turtles[END_REF], and the consequences of such high values should be investigated.

Zn is an essential metal subjected to homeostatic regulation as it included in the functional groups of various enzymes, play a structural role in respiratory pigments and metalloenzymes, and can act as activating co-factor for various proteins (see e.g. [START_REF] Simkiss | Metal ions in cells[END_REF][START_REF] Williams | The Bakerian lecture, 1981: natural selection of the chemical elements[END_REF]. Accordingly, Zn concentrations measured here could be physiological and thus not a problem on the health status of E. orbicularis.

Nonetheless, the increase of Zn concentrations with age is likely due to its accumulation on specific metalloproteins such as metallothioneins, which serve as intracellular protein in metal homeostasis [START_REF] Vallee | Introduction to metallothionein[END_REF]. Future investigations on metallothioneins in E. orbicularis would be needed to validate this hypothesis. In addition, the values we report are substantially higher than those reported for other species of turtles [START_REF] Malik | Trace metals in Ganges softshell turtle (Aspideretes gangeticus) from two barrage: Baloki and Rasul, Pakistan[END_REF][START_REF] Yadollahvand | Assessment trace elements concentrations in tissues in Caspian Pond Turtle (Mauremys caspica) from Golestan province, Iran[END_REF], and the potential effects of such high values of Zn should be investigated.

Although the sources of Hg and Zn contamination remain unknown, several hypotheses can be proposed. For instance, the concentrations of Hg we detected may be related to a natural contamination due to the methylating activity of microorganisms in anoxic sediments rather than a direct anthropogenic source of contamination [START_REF] Morel | The chemical cycle and bioaccumulation of mercury[END_REF]. Alternatively, relatively high concentrations of both Hg and Zn could be linked to the composition of fish pellets used for fish farming that occur in many ponds of Brenne. Indeed, commercial fish pellets exhibit high Hg concentrations of marine origin [START_REF] Hansson | Transfer of marine mercury to mountain lakes[END_REF]; see also [START_REF] Lemaire | Determinants of mercury contamination in viperine snakes, Natrix maura, in Western Europe[END_REF]) and are enriched with Zn oxide (EFSA 2012). Future studies should usefully investigate both the origin of Hg and Zn as well as the physiological and toxicological consequences of elevated levels of these elements for E. orbicularis [START_REF] Meyer | Mercury Exposure associated with altered plasma thyroid hormones in the declining Western Pond Turtle (Emys marmorata) from California Mountain Streams[END_REF].

(Institut Universitaire de France) is also acknowledged for its support to PB as a Senior Member.

Figure 1 .

 1 Figure 1. Relationship between turtle size (plastron length) and concentrations of Zn in muscles. Symbols stand as follow: white squares for juveniles, black circles for adult males, light grey triangles for adult females and dark grey diamonds for unsexed adults

Figure 2 .

 2 Figure 2. Relationship between turtle size (plastron length) and concentrations of Hg in A liver, B skin and C claws. Symbols stand as follow: white squares for juveniles, black circles for adult males, light grey triangles for adult females and dark grey diamonds for unsexed adults

Acknowledgments

We warmly thank the staff from the Réserve Naturelle Nationale de Chérine and M.

Brault-Favrou for assistance in trace element analyses. The CPER (Contrat de Projet Etat-Région) for funding the ICPs and AMA. Funding was provided by the Région Poitou-Charentes, the Conseil Départemental des Deux-Sèvres, the CNRS, the Agence de l'Eau Loire-Bretagne and the Agence de l'eau Adour-Garonne. The IUF