Nassim Ben Brahim
email: nassim.benbrahim.fsm@gmail.com

Naim Bel

Haj Mohamed

Mosaab Echabaane

Mohamed Haouari

Ben Rafik

Michel Chaâbane

Hafedh Ben Negrerie

Ouada

Rafik Ben Chaâbane

Michel Negrerie
email: michel.negrerie@polytechnique.fr

Hafedh Ben Ouada

Thioglycerol-functionalized CdSe quantum dots detecting cadmium ions

Keywords:

published or not. The documents may come

Introduction

Quantum dots (QDs), or semiconductor nanocrystals, are nowadays inestimable tools for fundamental studies as well as for potential applications such as biological probes [START_REF] Zheng | Synthesis and cell-imaging applications of glutathione-capped CdTe quantum dots[END_REF][START_REF] Moussodia | Biocompatible and stable ZnO quantum dots generated by functionalization with siloxane-core PAMAM dendrons[END_REF], fluorescent biosensor [START_REF] Costa-Fernandez | The use of luminescent quantum dots for optical sensing[END_REF], light-emitting diodes (LEDs) [START_REF] Lim | Preparation of highly luminescent nanocrystals and their application to light-emitting diodes[END_REF] and solar cells [START_REF] Robel | Quantum dot solar cells. Harvesting light energy with CdSe nanocrystals molecularly linked to mesoscopic TiO 2 films[END_REF]. Compared to conventional organic dyes, QDs possess unique properties including continuous absorption band, high photoluminescence (PL) quantum yields, photobleaching stability and tunability of the PL emission as a function of their size [START_REF] Jamieson | Biological applications of quantum dots[END_REF][START_REF] Aldeek | Enhanced optical properties of core/shell/shell CdTe/CdS/ZnO quantum dots prepared in aqueous solution[END_REF]. To date, two different routes have been reported for the synthesis of CdSe QDs, one being the most conventional organometallic approach [START_REF] Murray | Synthesis and characterization of nearly monodisperse CdE (E = S Se Te) semiconductor nanocrytallites[END_REF][START_REF] Peng | Green chemical approaches toward high-quality semiconductor nanocrystals[END_REF]. As prepared, the CdSe nanocrystals have remarkable properties such as narrow width of the PL spectrum, high PL quantum yield, negligible photobleaching and excellent size homogeneity. This non-aqueous synthesis of capped QDs requires high temperature (360 °C) [START_REF] Qu | Alternative routes toward high quality CdSe nanocrystals[END_REF], use of hydrophobic ligands [START_REF] Khanna | The processing of CdSe/polymer nanocomposites via solution organometallic chemistry[END_REF] and solubilization strategies for their direct use in biological systems [START_REF] Yu | Water-soluble quantum dots for biomedical applications[END_REF]. Alternatively, the aqueous synthesis route produces QDs with other advantages, including control of the size by the pH [START_REF] Spanhel | Photochemistry of colloidal semiconductors .20. Surface modification and stability of strong luminescing CdS particles[END_REF], water solubility, biological compatibility and stability [START_REF] Gaponik | Thiol-capping of CdTe nanocrystals: An aLernative to organometallic synthetic routes[END_REF][START_REF] Rogach | Synthesis and characterization of a size series of extremely small thiol-stabilized CdSe nanocrystals[END_REF]. However, the aqueous method has several drawbacks such as lower emission efficiency and a wider PL spectrum compared to the organic method. The design and synthesis of high quality water-soluble CdSe QDs disclosing specific and tunable properties to target biochemical analytes or biomolecular systems is a major issue, mainly reached by the choice of the functionalizing ligand coupled to CdSe. For example, Xue et al. reported highly luminescent water-soluble CdSe QDs prepared using thioglycolic acid as a capping ligand, which were covalently coupled to bacteria [START_REF] Xue | Fluorescence detection of total count of Escherichia coli and Staphylococcus aureus on water-soluble CdSe quantum dots coupled with bacteria[END_REF].

Alternatively, particular capped CdSe QDs could be prepared at the polarizable interface between water and 1,2-dichloroethane electrolyte solutions by an electrochemical method [START_REF] Su | Adsorption and photoreactivity of CdSe nanoparticles at liquid/liquid interfaces[END_REF]. Triethanolamine-capped CdSe QDs were synthesized and used as fluorescent sensors for reciprocal recognition of mercury and iodide in aqueous solution [START_REF] Shang | Triethanolamine-capped CdSe quantum dots as fluorescent sensors for reciprocal recognition of mercury [II] and iodide in aqueous solution[END_REF] and CdSe nanocrystals incorporated in thin films were shown to interact with gazeous compounds such as benzylamine and triethylamine [START_REF] Nazzal | Photoactivated CdSe nanocrystals as nanosensors for gases[END_REF].

Here, we focus on the detection of cadmium ions, which is an extremely toxic metal widely found in plastics, fossil fuel combustion, phosphate fertilizers and other various chemicals [START_REF] Chaney | Cadmium in soils and plants[END_REF]. These sources of cadmium often lead to contaminations in water, soil and eventually in food [START_REF] Nordberg | Cadmium in the human environment[END_REF][START_REF] Friberg | World Health Organization Report[END_REF], causing serious environmental and health problems such as lung, prostate damage and kidney cancer [START_REF] Goyer | Cadmium and cancer of prostate and testis[END_REF][START_REF] Satarug | A global perspective on cadmium pollution and toxicity in nonoccupationally exposed population[END_REF]. In this study, we report the synthesis of thioglycerol-capped CdSe QDs and its interaction with cadmium in water. A facile preparation through a seed-assisted method at room temperature yields water-soluble CdSe QDs capped with thioglycerol (TG) which was used for the detection of Cd 2+ ion by UV-Visible spectroscopy. These CdSe functionalized nanoparticles are characterized by a simple and lowcost preparation and a remarkably high selectivity for Cd 2+ ions.

Experimental procedures

Synthesis of TG-capped CdSe QDs

We have followed the method previously described for the synthesis of CdS (cadmium sulfure) QDs capped with thiol derivatives [START_REF] Haj | Time resolved and temperature dependence of the radiative properties of thiolcapped CdS nanoparticles films[END_REF] with some modifications. The chemicals were purchased from Sigma, Aldrich and Fluka. Briefly, an aqueous solution was obtained by mixing cadmium acetate dehydrate with thioglycerol (TG) as the stabilizer in deionized water with continuous stirring under nitrogen atmosphere. The pH of the resultant mixture was adjusted to 11.2 with NaOH solution. Separately, an aqueous solution of Na 2 SeO 3 was prepared by introducing SeO 2 into a NaOH solution and injected into the pH-controled mixture of Cd 2+ and stabilizer under vigorous stirring. The molar ratio Cd 2+ /TG/Se 2-was set at 1/2/0.5. Then, a solution of the reducing agent NaBH 4 was injected with a syringe to the final solution under continuous stirring at 100 °C under N 2 until the solution became light yellow. The TG capped CdSe quantum dots were obtained at this stage. The particles were extracted by precipitation in isopropanol. The solution was stirred for one hour and the precipitate was filtered then dried in a desiccator under vacuum. Fig. 1 summarizes the mechanism of formation of TG-capped CdSe QDs.

Instrumentation

X-ray diffraction (XRD) powder spectra were taken by XPERT PRO MPD Panalytical X-ray generator using the Kα radiation of Cu at a wavelength of 1.542 Å whose integration time was monitored to improve the signal-to-noise ratio. The diffraction angle 2θ was scanned in the range 20 -70° with a speed of 0.02 °/s. To detect the presence of TG on the surface of QDs Fourier-transform infrared (FTIR) spectra were measured using a Perkin Elmer FTIR spectrophotometer in the range 600 -4000 cm -1 . For FTIR analysis, powder samples of QDs were mixed with anhydrous potassium bromide (KBr) pelletized.

For performing transmission electron microscopy (TEM) and energy dispersive X-ray (EDX) we have employed a JEOL 2010 FEG apparatus. Point EDX measurement was performed in scanning TEM mode.

Optical measurements

The UV-visible absorption spectra were collected either on a DR 5000 Hach Lange UV-vis spectrophotometer or a Shimadzu UV-1700. Fluorescence experiments were carried out on a Cary Eclipse spectrometer and spectra were collected in the range 400 -800 nm with an excitation at λ ex = 360 nm. Absorption and emission spectra were measured at room temperature (20°C) by preparing a colloide solution of TG-CdSe quantum dots at 1.5 mg•L -1 in pH 8.6 buffer solution. To increase step-wise the concentration of cadmium or other transition metal ions from 1 to 24 µM, aliquots (2 -4 µL) of a stock solution (10 -3 M) were added into a volume of 2 mL of CdSe QDs solution and mixed thoroughly. The final dilution factor (1.024) was negligible. The temperature-dependent measurements (Supplementary Material) were carried out with a home-designed 1-mm cell holder connected to a circulating bath. The optical path-length of the quartz cuvette was 1 cm for fluorescence and 1 mm for absorption measurements. The pH measurements were carried out with a Model Hanna 8519N pH-meter and a combined glass electrode.

Results and discussion

Characterization of TG-capped CdSe QDs by X-ray and electronic microscopy

The XRD spectrum of TG-CdSe QDs (Fig. 2A) shows several diffraction peaks which appeared at 2θ = 25.20°, 41.90°, 49.70°, 67.96° and 77.09° corresponding to the diffraction planes (111), ( 220), (311), ( 331) and (422) respectively, according to the standard JCPDS data (card 19-0191) of bulk cubic CdSe. The peak broadening in the XRD pattern is due to a smaller size nanoparticles population. The average diameter D of the CdSe nanoparticle can be estimated from the full width at half maximum (FWHM) of the (111) diffraction peak by means of Debye-Scherrer's [START_REF] Deng | A new route to zinc-blende CdSe nanocrystals: mechanism and synthesis[END_REF] formula:

D = 0.9λ / βcosθ (1)
where λ is the wavelength of the incident X-rays, β is the FWHM of the (111) peak in radians and θ is the Bragg diffraction angle. The average crystal size of the CdSe QDs thus estimated is 2.5 nm ± 0.02 nm.

An EDX analysis allowed to ascertain the composition of functionalized CdSe QDs whose spectrum (Fig. 2B Transmission electron microscopy (TEM) was employed to verify the morphology of the TG-CdSe QDs and to obtain a direct measurement of their size. The image (Fig. 3A) reveals a cluster of nanoparticles which are almost spherical and whose size population has a very small dispersion. The average diameter of nanoparticles was directly measured on high resolution TEM images (Fig. 3B) with the JEOL software and have a value of ~2.5 nm, remarkably close to the diameter calculated from the edge absorption spectrum and X-ray diffraction, which yielded 2.8 nm and 2.5 nm, respectively.

Characterization of TG-capped CdSe QDs by FT-IR and UV-visible spectroscopies

The bonding between the stabilizer thioglycerol molecules and CdSe QDs was confirmed by FT-IR measurement in the range 600 -4000 cm -1 (Fig. 4). The assignments of vibrational modes are given in Table 1. This spectrum is similar to that of thioglycerol except for the absence of the S-H vibration band which appears usually at 2557 cm -1 , indicating that thiolate functions are connected to the Cd 2+ sites of the CdSe nanocrystals surface [START_REF] Tang | Preparation and characterization of watersoluble US nanocrystals by surface modification of ethylene diamine[END_REF], as observed for core-shell CdSe/CdS QDs [START_REF] Silva | Shell thickness modulation in ultrasmall CdSe/CdS x Se 1-x /CdS core/shell quantum dots via 1-thioglycerol[END_REF], implying that thiol-assisted capping of CdSe quantum dots has occurred. We can thus infer the presence of the organic layer coating on CdSe on the basis of the FTIR spectrum.

The UV-vis absorption spectrum is similar to that obtained for a film of TG-CdSe QDs synthesized via an organic route [START_REF] Artemyev | Spectroscopic study of electronic states in an ensemble of close-packed CdSe nanocrystals[END_REF]. Both the absorption profile and first peak position (400 nm; 3.01 eV) depend upon the particle size [START_REF] Jasieniak | Re-examination of the Ssize-dependent absorption properties of CdSe quantum dots[END_REF] and readily indicate the small size of these CdSe nanocrystals. The absorption spectra and fluorescence spectra (Fig. 5) of TG-CdSe QDs differ from that of bulk CdSe, with an absorption edge around 400 nm clearly blue shifted as compared to CdSe (1.74 eV, 700 nm). These values indicate an increase in the band gap of the QDs after TG treatment and witness about the quantum confinement effect which becomes more pronounced when the particle size becomes less or equal to the Bohr radius of the exciton in the corresponding bulk material [START_REF] Cao | One-Pot Synthesis of high-quality zinc-blende CdS nanocrystals[END_REF].

The absorption edge threshold (λ thres ) of the UV-vis absorption spectrum has been correlated with the particle diameter (D) by Henglein's [START_REF] Spanhel | Photochemistry of colloidal semiconductors. 20. surface modification and stability of strong luminescing CdS particles[END_REF] whose experimental curve was described by an empirical relation [START_REF] Moffitt | Size control of nanoparticles in semiconductor-polymer composites. 1. control via multiplet aggregation numbers in styrene-based random ionomers[END_REF]:

D CdSe = [1.338 -0.002345 λ thres ] -1 in nm (2)
One must note that this relation, which is a fit of experimental data [START_REF] Moffitt | Size control of nanoparticles in semiconductor-polymer composites. 1. control via multiplet aggregation numbers in styrene-based random ionomers[END_REF], is valide only in the range ~1.5 -6.5 nm. The absorption edge threshold (λ thres ) is defined as the wavelength at the inflection point of the sharply decreasing side absorption (crossing of the spectrum and its tangent), as described in Fig. 5B. This yielded the value λ thres = 420 ± 1 nm, allowing to estimate the particles average diameter to be 2.83 ± 0.02 nm, very close to that calculated from the X-ray diffraction measurement (2.5 nm).

To estimate the band gap energy, the effective mass model can be used since the radius of the nanoparticles is lower than the Bohr radius of bulk exciton [START_REF] Brus | Electron-electron and electron-hole interactions in small semiconductor crytallites: the size dependence of the lowest excited electronic state[END_REF] (5.6 nm). If one neglects the energy of coulombic interaction between electron and hole which varies as 1/R, the band gap energy of QDs can be deduced from the following equation:

eff g E = E g + (h 2 /8µR 2 ) ( 3
)
where R is the particle radius, μ the effective reduced mass, E g the bulk band gap energy (1.74 eV), the effective band gap energy and h is the Plank's constant. Since the effective mass of electron is much smaller than that of the holes ( = 0.

Detection of cadmium ion and response of TG-capped CdSe nanoparticles

The optical response of TG-CdSe QDs in buffer solution (pH 8.6) has been evaluated as a function of cadmium concentration (Fig. 6A). The absorbance of the first band, initially located at 400 nm, increases with the addition of Cd 2+ ions together with a shift of its position to 409 nm. We also observed that the band located at 365 nm in the absence of Cd 2+ disappears as the concentration increases, while the long-tailed absorption (> 450 nm)

increases. This latter change is not due to scattering from particles since the absorption increase at 450 nm is larger than at 300 nm and does not follow the proportionality with 1/λ 4 .

This long-tailed absorption increase was also observed in the case of functionalized CdS QDs interacting with Co 2+ ions [START_REF] Gore | Highly Selective and sensitive recognition of cobalt[II] ions directly in aqueous solution using carboxyl-functionalized CdS quantum dots as a naked eye colorimetric probe: applications to environmental analysis[END_REF]. These spectral changes are a direct consequence of the metal ions interaction with TG-CdSe QDs. To visualize the real spectral evolution, we plotted the difference spectra at a given Cd 2+ concentration minus the spectrum in the absence of Cd 2+ (Fig. 6B) which reveals the appearance of an absorption band centered at 427 nm and the shift of the small band at 373 nm to 385 nm.

The enhancement of absorbance of TG-CdSe QDs varies linearly at 427 nm with the Cd 2+ ion concentration within the range 5.0 -22 μM with a slope of K = 0.0278 µM -1 (Fig. 7) yielding a limit of detection LOD = 0.32 µM (37 µg•L -1 of Cd 2+ ) defined as LOD = 3σ/K [START_REF] Chen | Functionalized US quantum dots-based luminescence probe for detection of heavy and transition metal ions in aqueous solution[END_REF] where σ is the standard deviation. This value is very similar to that measured for the detection of Hg 2+ and I -by fluorescence quenching of triethanolamine-capped CdSe QDs [START_REF] Nazzal | Photoactivated CdSe nanocrystals as nanosensors for gases[END_REF] which, however, did not detect Cd 2+ , whereas the present TG-CdSe QDs do not interact with Hg 2+ , remarkably emphasizing the dependence of metal binding upon the nature of the capping chain.

The pH may affect the optical properties of functionalized QDs [START_REF] Skoog | Principles of instrumental analysis 7 th ed[END_REF]. We indeed observed the pH-dependence of the UV-vis absorption spectra of TG-CdSe QDs in the presence of 15 μM Cd 2+ ions over the pH range 2.0 -12 (Fig. 7A). These measurements were expressed as the difference in absorbance of the TG-CdSe QDs in the presence and absence of Cd 2+ at 400 nm. The maximum response A -A 0 at 400 nm was obtained at pH = 8.6 (Fig. 7A) whereas for pH < 6 the absorbance was too weak and vanished at pH = 2. This may be attributed to the dissociation of the TG modified CdSe nanoparticles by protonation of the surface-binding thiolates [START_REF] Biju | Temperaturesensitive photoluminescence of CdSe quantum dot clusters[END_REF]. On the other hand, for pH larger than 8.6, the absorbance decreases with a simultaneous precipitation of Cd(OH) 2 . Consequently, a standard pH of 8.6

was chosen for performing all experiments.

The response time of Cd 2+ binding to TG-CdSe should depend on the concentration since the process can be described as a diffusion dependent receptor-ligand interaction. The kinetics of the absorbance change at 400 nm (Fig. 7B) after the addition of 15 μM Cd 2+ to the TG-CdSe QDs solution shows that the induced change (A-A 0 ) has reached 75% of its maximum after 90 s. In order to standardize the measurements, we have chosen a 90-s response time after the addition of any metal cations at varying concentration to further characterize the TG-CdSe cation interaction.

The fluorescence intensity decreased when increasing the concentration of Cd 2+ (Fig. 8) but without noticeable changes in the position of maximum and shape of the fluorescence spectrum. Consequently, the presence of Cd 2+ gave rise to a quenching of the emission of QDs, which should be more pronounced than apparently observed if we consider the increase of the absorption coefficient at 360 nm, the excitation wavelength. This apparent quenching could be partly linked to reabsorption of emission due to the increase of absorption at 570 nm and suggests a strong interaction between Cd 2+ and TG-CdSe QDs [START_REF] Lakowicz | Luminescence spectral properties of CdS nanoparticles[END_REF]. The decrease of fluorescence observed here is at variance with the increase induced by Cd 2+ (5 -50 µM range) binding to CdSe/ZnS core/shell QDS caped with carnitine [START_REF] Li | L-Carnitine capped quantum dots as luminescent probes for cadmium ions[END_REF], suggesting different mechanisms for the two kinds of QDs.

As the thiol groups of TG are covalently bound to the QDs core, leaving the hydroxyl groups free, they can provide selective coordination sites for Cd 2+ ions. On this basis, we propose in Fig. 9 a model for a working hypothesis where the cadmium cations interact with the hydroxyl groups of TG and forms cadmium oxide on the surface of CdSe QDs. We have retained the hypothesis of Cd 2+ interacting with the non-ionized OH groups of TG rather than with their ionized form, because their pKa = 9.43 [START_REF] Xu | pH-sensitive photoluminescence for aqueous thiol-capped CdTe nanocrystals[END_REF] and we have observed the maximal response for pH = 8.6.

We have also investigated the influence of the buffer composition, QDs concentration and temperature on the spectral properties and binding of Cd 2+ to TG-CdSe QDs. The corresponding data are given in Supplementary Material. The various buffers used had no influence on the spectra whereas variations of the QDs concentration do not change the spectral shape and do not shift the bands. At large QDs concentration, a limited aggregation leads to an absorbance increase which is not proportional to the concentration. The absorbance edge of TG-CdSe QDs in the absence of Cd 2+ has very slightly shifted as the temperature increased from 20 °C to 50 °C (Fig. S3). In the presence of 15 µM Cd 2+ , there is a decrease of the absorbance from 10 to 50 °C (Fig. S3B), while disclosing the same shift of the first band maximum position to 409 nm due to Cd 2+ binding. This dependence is less pronounced between 10 and 20 °C and has a larger slope at 50 °C. We readily assigned this decrease to the displacement of the equilibrium of Cd 2+ interacting with the OH groups of TG, as it must be observed for any ligand-receptor system (Fig. S3C andD). This does not affect the capability of detecting Cd 2+ , but agrees with the origin of the absorption spectral changes assigned to Cd 2+ binding.

Influence of foreign metal Ions nature and specificity for Cd 2+

The presence of interfering ions may affect the optical response due to the Cd 2+ -TG-CdSe interaction and we have therefore examined the selectivity of this binding. Fig. 10 shows the effect of particular metal cations at fixed concentration (20 μM) on the absorption of QDs at 400 nm. Saliently, only the addition of Cd 2+ ions led to a drastic change of the absorbance whereas no spectral change was observed after the addition of the following metal cations alone: K + , Na + , Li + , Pb 2+ , Ni 2+ , Mn 2+ , Mo 2+ , Hg 2+ , Zn 2+ , Ca 2+ , Mg 2+ , Fe 2+ , Cu 2+ , Co 2+ , Ba 2+ , Al 3+ and Fe 3+ . Moreover, the simultaneous presence of these cations with Cd 2+ in solution of TG-CdSe QDs induced a similar increase of absorption as observed for Cd 2+ alone (Fig. 10). Therefore, there is no competition between these common ions and Cd 2+ for the binding to TG-CdSe QDs which can thus be used as a selective probe for the Cd 2+ ions in aqueous solution at room temperature, whatever the absence or presence of other cations.

The binding properties of TG-CdSe QDs must be compared to those of other systems.

Indeed, QDs made with different materials and capped with various ligands were shown to interact with various metal cations such as Hg 2+ [START_REF] Nazzal | Photoactivated CdSe nanocrystals as nanosensors for gases[END_REF][START_REF] Chen | Synthesis of novel nanocrystals as fluorescent sensors for Hg 2+ ions[END_REF] ,Cu 2+ [START_REF] Yang | Phenol formaldehyde resin nanoparticles loaded with CdTe quantum dots: a fluorescence resonance energy transfer probe for optical visual detection of copper(II) ions[END_REF], Ba 2+ [START_REF]Functionalized ME-capped CdSe quantum dots based luminescence probe for detection of Ba 2+ ions[END_REF], and Pb 2+ [START_REF] Wu | A novel method for the determination of Pb 2+ based on the quenching of the fluorescence of CdTe quantum dots[END_REF].

When functionalized with 2-mercaptoethanol, CdSe QDs have been shown [START_REF]Functionalized ME-capped CdSe quantum dots based luminescence probe for detection of Ba 2+ ions[END_REF] to bind Ba 2+

but not Cd 2+ (no result was reported for Fe 2+ and Cu 2+ , which are common interfering cations). Such a difference is remarkable and may provide a future direction to investigate the mechanism of selectivity in these systems. Not only the functionalization, but also the nature of QD itself determines the selectivity. For example, CdS QDs functionalized with carboxyl [START_REF] Gore | Highly Selective and sensitive recognition of cobalt[II] ions directly in aqueous solution using carboxyl-functionalized CdS quantum dots as a naked eye colorimetric probe: applications to environmental analysis[END_REF] is selective for Co 2+ , whereas CdTe QDs coupled with phenol-formaldehyde resin nanoparticles detected the binding of Cu 2+ by means of fluorescence energy transfer.

We must note that neither CdSe QDs synthesized via the organic route at high T and functionalized with bovine serum albumin [START_REF] Liang | Functionalized CdSe quantum dots as selective silver ion chemodosimeter[END_REF], nor CdS QDs functionalized with carboxyl [START_REF] Brus | Electron-electron and electron-hole interactions in small semiconductor crytallites: the size dependence of the lowest excited electronic state[END_REF], nor CdTe QDs capped with phenol-formaldehyde [START_REF] Yang | Phenol formaldehyde resin nanoparticles loaded with CdTe quantum dots: a fluorescence resonance energy transfer probe for optical visual detection of copper(II) ions[END_REF] can bind Cd 2+ ions. The large number of cations that we tested here emphasizes the selectivity of the TG-CdSe sustem as a potential optical sensor for Cd 2+ ions, whose molecular properties and mechanism at the origin of this selectivity remain to be investigated.

Conclusion

In the present work, TG-capped CdSe QDs were successfully synthesized in aqueous medium, in an easy and highly reproducible way, by using safe and low cost materials. These functionalized QDs selectively detect Cd 2+ ions even in the presence of other cations which are physiologically relevant or can be present in water as pollutants. Under defined conditions (pH = 7 -9), this system shows sensitivity for Cd 2+ ions in the concentration range 1.0 -22 μM with a detection limit of 0.32 µM (37 µg•L -1 ). Together with a facile and low cost synthesis, its sensitivity and remarkable selectivity make the thioglycerol-capped CdSe a promising candidate for designing an optical tool to probe Cd 2+ ions in solution, possibly to investigate its interaction with cultured cells.

  ) shows strong atomic peaks which originate from Cd and Se of QDs and peaks from thioglycerol ligand (S, C and O); nickel from the grid is also detected. The integration of Cd and Se signals indicates an average atomic percentage ratio Cd/Se = 32.2/23.2 = 1.39, showing that the synthesized QDs are enriched in cadmium.

  affects the energetic level of the electrons. By using the estimated particle size from absorption edge threshold (2.83 nm), the calculated effective band gap of CdSe QDs is 2.21 eV.

Fig. 1 .

 1 Fig. 1. Summary of TG-capped CdSe QDs synthesis via the aqueous route.

Fig. 2 .

 2 Fig. 2. (A) X-ray diffraction pattern for the TG-CdSe QDs. The positions of the

Fig. 3 .

 3 Fig. 3. (A) TEM image of a cluster of TG-CdSe QDs. (B) High resolution and large

Fig. 4 .

 4 Fig. 4. Fourier transform infrared spectrum of TG-CdSe QDs mixed with anhydrous KBr. The

Fig. 5 .

 5 Fig. 5. (A) UV-visible absorption and fluorescence spectra of functionalized TG-CdSe QDs in

Fig. 6 .Fig. 7 .Fig. 8 .

 678 Fig. 6. (A) UV-vis absorption spectra of the TG-CdSe QDs (5 × 10 -4 M) solution with various concentrations of Cd 2+ ions at equilibrium. (B) Difference absorption spectra at increasing Cd 2+ concentration minus the spectrum of TG-CdSe QDs in absence of Cd 2+ . pH = 8.6.

Table

Table 1 .

 1 1. Assignment of the bands in the FT-IR spectrum of TG-CdSe QDs.

	Wavenumber (cm -1 )	Assignment
	746	Cd-Se bond stretching
	958, 1083 and 1239	C-C stretching modes
	1419	C-H stretching mode
	1649	O-H bending mode of water
	2862	C-H stretching mode
	2937	CH 2 symmetric stretching
	3400	O-H stretching mode

Acknowledgements

N.B.B. acknowledges a travel research fellowship "Bourse d'Alternance" from the Tunisian

Government. We thank Mélanie Poggi (Laboratoire de Physique de la Matière Condensée, Ecole Polytechnique) for her expert assistance in recording TEM images.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2015.07.049 Abbrevations TG-CdSe QDs: Cadmium-Selenium Quantum Dots functionalized with Thio-Glycerol capping. TEM: Transmission Electronic Microscopy. EDX: Energy Dispersive X-ray.


Agustina Ferrando
email: ferrando@cenpat-conicet.gob.ar

Emilia Gonzalez

Marcos Franco

Marta Commendatore

Marina Nievas

Cécile Militon

Georges Stora

Franck Gilbert

José Luis Esteves

Philippe Cuny

: M Commendatore

: M Nievas

Oil spill effects on

Keywords: Macrobenthic communities, Oil pollution, Bioturbation, Resistance capacities, Pristine marine sediments, Patagonia

come

Introduction

In quantitative terms, crude oil is the most important organic contaminant in the marine environment [START_REF] Spormann | Metabolism of alkylbenzenes, alkanes, and other hydrocarbons in anaerobic bacteria[END_REF][START_REF] Head | Marine microorganisms make a meal of oil[END_REF]. Despite the implementation of active and preventative policies, inputs are still increasing due to the rise of the world consumption and transportation, mainly via maritime routes, of petroleum products [START_REF] Dargay | World oil demand's shift toward faster growing and less price-responsive products and regions[END_REF]. Oil hydrocarbons are either chronically or accidentally introduced in many marine areas around the world. They can affect coastal and marine ecosystems in several ways. For instance, toxic effects of some hydrocarbons, particularly polycyclic aromatic hydrocarbons, on feeding, growth, development, and reproduction cascade across trophic levels affecting plankton, fish, marine birds and mammals, as well as benthic organisms [START_REF] Fleeger | Indirect effects of contaminants in aquatic ecosystems[END_REF]NRC 2003;[START_REF] Wiese | The extent of chronic marine oil pollution in southeastern Newfoundland waters assessed through beached bird surveys 1984-1999[END_REF][START_REF] Alonso-Alvarez | Effects of acute exposure to heavy fuel oil from the Prestige spill on a seabird[END_REF][START_REF] González | Effect of a simulated oil spill on natural assemblages of marine phytoplankton enclosed in microcosms[END_REF][START_REF] Engraff | Aquatic toxicity of PAHs and PAH mixtures at saturation to benthic amphipods: linking toxic effects to chemical activity[END_REF][START_REF] Almeda | Interactions between zooplankton and crude oil: toxic effects and bioaccumulation of polycyclic aromatic hydrocarbons[END_REF][START_REF] Bellas | Evaluation of artificially-weathered standard fuel oil toxicity by marine invertebrate embryogenesis bioassays[END_REF][START_REF] Lee | Effects of an oil spill on benthic community production and respiration on subtropical intertidal sandflats[END_REF]. The deposition of hydrocarbons or oil/weathered oil at the sediment-water interface can considerably alter the biogeochemistry of benthic systems and impact inhabiting organisms that maintain a close relationship with these substrates [START_REF] Rivero | First survey of macroinfauna in the Mar del Plata Harbor (Argentina), and the use of polychaetes as pollution indicators[END_REF][START_REF] Paletto | Hydrocarbon levels in sediments and bivalve mollusks from Bahía Nueva (Patagonia, Argentina): an assessment of probable origin and bioaccumulation factors[END_REF]. In particular, reduced diversity, retrogression to opportunistic species, and reduced size of individuals have been reported for macrobenthic organisms in contaminated sediments [START_REF] Lee | Petroleum hydrocarbons and their effects in subtidal regions after major oil spills[END_REF][START_REF] Mc | Effects of petroleum and its derivatives in benthic communities at Baía de Todos os Santos/Todos os Santos Bay, Bahia, Brazil[END_REF][START_REF] Blanchard | Long-term investigation of benthic fauna and influence of treated ballast water disposal in Port Valdez, Alaska[END_REF][START_REF] Belan | Benthos abundance pattern and species composition in conditions of pollution in Amursky Bay (the Peter the Great Bay, the Sea of Japan)[END_REF][START_REF] Je | Changes in benthic communities along a presumed pollution gradient in Vancouver Harbour[END_REF][START_REF] Lu | Relationship between soft-bottom macrobenthic communities and environment variables in Singaporean waters[END_REF][START_REF] Gomez Gesteira | Impact of the Aegean Sea oil spill on the Rîa of Ares and Betanzos fine sand community (Northwest Spain)[END_REF][START_REF] Lu | A field experimental study on recolonization and succession of macrobenthic infauna in defaunated sediment contaminated with petroleum hydrocarbons[END_REF][START_REF] Di Leonardo | Possible impacts of Hg and PAH contamination on benthic foraminiferal assemblages: an example from the Sicilian coast, central Mediterranean[END_REF][START_REF] Venturini | Petroleum contamination impact on macrobenthic communities under the influence of an oil refinery: integrating chemical and biological multivariate data[END_REF][START_REF] Gonzalez Egres | Effects of an experimental in situ diesel oil spill on the benthic community of unvegetated tidal flats in a subtropical estuary (Paranagua Bay, Brazil)[END_REF][START_REF] Yu | Initial impacts of the Hebei Spirit oil spill on the sandy beach macrobenthic community west coast of Korea[END_REF][START_REF] Seo | The macrofaunal communities in the shallow subtidal areas for the first 3 years after the Hebei Spirit oil spill[END_REF][START_REF] Zabbey | Community responses of intertidal soft-bottom macrozoobenthos to oil pollution in a tropical mangrove ecosystem, Niger Delta, Nigeria[END_REF]). Yet, soft-bottom macrobenthic organisms play a key role in processes such as organic matter degradation and nutrient cycling, dissolved oxygen diffusion, dispersion and burial of sediment particles, and secondary production [START_REF] Michaud | The functional group approach to bioturbation: II. The effects of the Macoma balthica community on fluxes of nutrients and dissolved organic carbon across the sediment-water interface[END_REF][START_REF] Gilbert | The influence of macrofaunal burrow spacing and diffusive scaling on sedimentary nitrification and denitrification: an experimental and model approach[END_REF]. This is mainly due to excavation or ingestion of mud and particulate organic matter [START_REF] Rhoads | Disturbance and Production on the Estuarine Seafloor: Dredge-spoil disposal in estuaries such as Long Island Sound can be managed in ways that enhance productivity rather than diminish it[END_REF][START_REF] Aller | The effects of macrobenthos on chemical properties of marine sediment and overlying water[END_REF][START_REF] Kennish | What is bioturbation? The need for a precise definition for fauna in aquatic sciences[END_REF]. Bioturbation was recently redefined by [START_REF] Kennish | What is bioturbation? The need for a precise definition for fauna in aquatic sciences[END_REF] as the displacement of particles (sediment reworking) and solutes (ventilation) due to the infauna activity. These bioturbating organisms, through the construction of tunnels and canals, particularly alter the flow of dissolved oxygen within marine sediments [START_REF] Pischedda | Imaging oxygen distribution in marine sediments. The importance of bioturbation and sediment heterogeneity[END_REF][START_REF] Pischedda | Spatial oxygen heterogeneity in a Hediste diversicolor irrigated burrow[END_REF]) and thereby positively influence microbial activities [START_REF] Arndt | Quantifying the degradation of organic matter in marine sediments: A review and synthesis[END_REF]. This subsequently affects significantly early diagenesis and, more particularly, biodegradation rates and pathways of organic pollutants [START_REF] Gilbert | In situ bioturbation and hydrocarbon fate in an experimental contaminated Mediterranean coastal ecosystem[END_REF][START_REF] Christensen | Effects of the polychaetes Nereis diversicolor and Arenicola marina on the fate and distribution of pyrene in sediments[END_REF][START_REF] Banta | Bioturbation and the fate of sediment pollutants[END_REF][START_REF] Granberg | Relative importance of macrofaunal burrows for the microbial mineralization of pyrene in marine sediments: impact of macrofaunal species and organic matter quality[END_REF][START_REF] Mermillod-Blondin | Ecosystem engineering: the impact of bioturbation on biogeochemical processes in marine and freshwater benthic hábitats[END_REF][START_REF] Cuny | Influence of bioturbation by the polychaete Nereis diversicolor on the structure of bacterial communities in oil contaminated coastal sediments[END_REF][START_REF] Miralles | Alkane biodegradation and dynamics of phylogenetic subgroups of sulfate reducing bacteria in an anoxic coastal marine sediment artificially contaminated with oil[END_REF][START_REF] Cuny | Biodegradation of hydrocarbons in bioturbated marine sediments[END_REF][START_REF] Stauffert | Impact of oil on bacterial community structure in bioturbated sediments[END_REF]. Conversely, bioturbation may be responsible for a large release of contaminant from the sediment into the water column [START_REF] Thibodeaux | The bioturbation driven chemical release process[END_REF][START_REF] Cuny | Biodegradation of hydrocarbons in bioturbated marine sediments[END_REF].

In case of contamination, the effects of oil on macrobenthic communities may thus in turn strongly affect the natural attenuation and, overall, the resistance and resilience capacities of the benthic system. Sediments chronically contaminated generally exhibit tolerant or opportunistic species populations, usually polychaetes worms, which can resist to relatively elevated concentrations of hydrocarbons [START_REF] Gilbert | In situ bioturbation and hydrocarbon fate in an experimental contaminated Mediterranean coastal ecosystem[END_REF][START_REF] Mc | Effects of petroleum and its derivatives in benthic communities at Baía de Todos os Santos/Todos os Santos Bay, Bahia, Brazil[END_REF][START_REF] Grossi | Burial, exportation and degradation of acyclic petroleum hydrocarbons following a simulated oil spill in bioturbated Mediterranean coastal sediments[END_REF][START_REF] Dauvin | Polychaete/amphipod ratio revisited[END_REF][START_REF] Miralles | Alkane biodegradation and dynamics of phylogenetic subgroups of sulfate reducing bacteria in an anoxic coastal marine sediment artificially contaminated with oil[END_REF][START_REF] Seo | The macrofaunal communities in the shallow subtidal areas for the first 3 years after the Hebei Spirit oil spill[END_REF][START_REF] Gilbert | Functional response of an adapted subtidal macrobenthic community to an oil spill: macrobenthic structure and bioturbation activity over time throughout an 18 months field experiment[END_REF]. In contrast, pristine benthic communities without historical exposure to hydrocarbons seem to be particularly sensitive to contamination [START_REF] Carman | Does historical exposure to hydrocarbon contamination alter the response of benthic communities to diesel contamination?[END_REF]. To date, the literature related to oil effects on pristine areas is still scarce, probably because more and more areas around the world are exposed to chronic or massive hydrocarbon spillages. Yet, in this kind of oil-sensitive marine area, a single large spill of crude oil or petroleum products may produce environmental damages that can amount to billions of dollars [START_REF] Dalton | Extent and frequency of vessel oil spills in US marine protected areas[END_REF].

Human activities in coastal areas involve port and industrial activities and maritime transport. Patagonian coast extends over more than 3000 km between 40°and 55°S and oil production is one of the most important economic activities in the region. There are two important oil basins named San Jorge and Austral basins, which produce petroleum in the order of 17.8 million m 3 per year, constituting nearly 50 % of the Argentina total annual production [START_REF] Nievas | Survey of the activities related with the oil exploitation in the patagonian coastal zone and preliminary data about waste of hydrocarbon in harbors. Project ARG/02/G31-PNUD-GEF[END_REF]. The main risk of Patagonian marine ecosystems contamination comes from crude oil loading in buoys and ports and maritime transport from exploitation sites in the south to the refineries in the north of the Country. Although hydrocarbon pollution was found in some punctual zones of the Patagonian coastal sediments [START_REF] Comendatore | Hydrocarbons in coastal sediments of Patagonia, Argentina: levels and probable sources[END_REF][START_REF] Esteves | Hydrocarbon pollution in coastal sediments of Tierra del Fuego islands, Patagonia Argentina[END_REF][START_REF] Commendatore | An assessment of oil pollution in the coastal zone of Patagonia, Argentina[END_REF][START_REF] Commendatore | Sources and distribution of aliphatic and polyaromatic hydrocarbons in coastal sediments from the Ushuaia Bay (Tierra del Fuego, Patagonia, Argentina)[END_REF], certain areas such as Caleta Valdés (CV; east of Península Valdés; Chubut province) have been never affected by massive or chronic pollution and remain pristine (unpublished data, Commendatore and Esteves). This ecosystem, established as Humanity Mundial Patrimony (UNESCO 1999) because it is a sensitive zone that constitutes a valuable environment for the reproduction and feeding of birds and marine mammalians, offers a relatively unique opportunity of assessing the response of the macrobenthic communities in case of petroleum contamination.

The objective of this work was to experimentally ex situ study the effects of a simulated oil spill at acute and chronic concentration levels on the macrobenthic community structure and the bioturbation activity of sediments sampled in a pristine site located in Caleta Valdés. Pollution-induced changes in macrobenthic community with a special interest for the determination of indicator species, as well as changes of the reworking activity, were assessed after 30 days of incubation.

Materials and methods

Study area

Caleta Valdés (CV) is a north-south oriented creek located at the eastern side of the Peninsula Valdés and connected to the Atlantic Ocean by a southern mouth. The sampling site for the experimentation was chosen in the muddy north continental zone of CV (42°15′53″ S, 63°40′50″ W) because of the absence of anthropogenic hydrocarbon pollution and it macrofauna richness (Fig. 1).

Bioturbation experimental assay conditions

Sampling

Sediment samples were collected by hand in April 2012 using 10 cm diameter and 25 cm length tubes, in agreement with previous works on bioturbation and reworking studies [START_REF] Timmermann | Modeling of advective solute transport in sandy sediments inhabited by the lugworm Arenicola marina[END_REF][START_REF] Quintana | Simultaneous study of particle reworking, irrigation transport and reaction rates in sediment bioturbated by the polychaetes Heteromastus and Marenzelleria[END_REF][START_REF] Hedman | Particle reworking and solute transport by the sediment-living polychaetes Marenzelleria neglecta and Hediste diversicolor[END_REF]. Twelve PVC corers were vertically introduced down to 20 cm sediment depth approximately. Then, they were carefully withdrawn and carried immediately to the laboratory avoiding disturbing the vertical structure of the sediments. Surficial sediment (first centimeter; 4 kg) was also collected for the laboratory experimentation. In addition, 20 L of seawater were also collected in plastic containers.

Laboratory experimental design and incubation conditions

With the aim to assess the oil impact on macrobenthic community structure and activity, three experimental conditions were set up: a control condition without oil addition (E0; 4 sediment cores), a low hydrocarbon concentration condition (E1; 4 sediment cores), and a high hydrocarbon concentration condition (E2; 4 sediment cores). These conditions were carried out by depositing at the surface of the respective cores, a layer of surficial CV sediments (height: 1 cm) either noncontaminated (E0), or contaminated by Escalante crude oil (Chubut) at final concentrations of 1000 ppm (1 g/kg; E1) and 20000 ppm (20 g/kg; E2). The different amounts of added oil were defined to simulate chronic and acute polluted sites, respectively (UNEP/IOC 1992). Then, the three groups of four cores were respectively distributed in three individual 56.1 L tanks. Tanks were filled with seawater, previously collected in CV, until cores were covered. They were then kept aerated (air bubbling) and monitored periodically during experimentation. In order to assess biological reworking activity, in each sediment core, 2 g of luminophores (63-355 μm particulate inert tracers, [START_REF] Duport | Benthic macrofauna and sediment reworking quantification in contrasted environment in the Thau Lagoon[END_REF] were homogeneously spread at the sediment surface at two different experimental times: green particles at initial time (t=0 days) and pink particles 15 days after the start of the experiment (t=15 days). Luminophores size was selected according to the sediment granulometry. Cores were incubated statically for 30 days at ambient temperature, after which they were manually collected and sliced in order to provide 0.5 cm thick sediment layers from the surface to 2 cm depth and 1 cm thick layers from 2 to 18 cm. Each sediment slice was then separated in four equal parts that were randomly distributed to perform the different further analysis.

Variable assessment

Temperature, dissolved oxygen, and pH were assesses periodically in the microcosm sea water during the experiments using a multiparameter probe YSI-556.

Analytical procedures

Macrobenthic communities

The 75 % of each layer (i.e., three parts) obtained from of each corer was fixed with 4 % formaldehyde to perform macrobenthic community analysis, storing the one resting parts at -20 °C for chemical analysis. The fixed samples were first sieved with a 500 μm mesh to retain macroorganisms and then with a 44 μm mesh to collect the sediment fraction containing the luminophores particles. The sediment retained on the 500 μm mesh was preserved in alcohol 70 % for the counting and sorting of the organisms. The macrofauna was identified to the major taxonomic level possible with stereoscopic and optic microscopes using reference keys [START_REF] Hartman | Atlas of the errantiate polychaetous annelids from California[END_REF][START_REF] Hartman | Atlas of the sedentariate polychaetous annelids from California[END_REF][START_REF] Banse | Benthic errantiate polychaetes of British Columbia and Washington[END_REF][START_REF] Blake | Polychaeta. In: Carlton TJ (ed) Intertidal invertebrates from California to Oregon[END_REF]Orensanz et al. inedited, among others). Whole organisms and anterior fragments of each taxon were only registered. In order to provide more information that could support further studies, some classification parameters usually used in taxonomic identification and photographs of the three major undetermined species or group of species are presented in the Online Resource 1.

Bioturbation

The vertical luminophores profile of each sediment column at final time of experimentation (30 days) was obtained from the percentages of the luminophores found in each layer with respect to the total amount in the core. The sediment retained on the 44 μm mesh was homogenized and subsampled to quantify luminophores using a microplate reader (Biotek Synergy Mx) at λex/λem: 565/602 and 460/500 nm for the green and pink luminophores, respectively. According to the respective time of deposition of the tracers at the surface of the cores, data from green particles reflected the reworking activity from the whole experimentation period (30 days), while pink particles indicate activity during the last 15 days.

The quantification of sediment reworking was evaluated from the distributions of luminophores by the gallerydiffusor model [START_REF] François | Functional approach to sediment reworking by gallery-forming macrobenthic organisms: modeling and application with the polychaete Nereis diversicolor[END_REF]. This model allows to describe both the biodiffusion-like transport (D b coefficient) due to the continuous displacement of the tracers and the nonlocal advective displacement of the tracers (r coefficient). The best fit between observed and modeled tracer distribution with depth (i.e., producing the best Db and r coefficients couple) was estimated by the least squares method [START_REF] Gilbert | Sediment reworking by marine benthic species from the Gullmar Fjord (Western Sweden): importance of faunal biovolume[END_REF].

Statistical data analysis

Data analyses were performed by corer using the software package PRIMER 5.0 (Plymouth Marine Laboratory, UK). Specific richness (S), total abundance (expressed as densi-ty=number of individuals/m 2 ), the Shannon diversity index (H′), and Pielou's evenness index (J′) were calculated for each treatment. Differences in each biological variable were evaluated through one-way ANOVA considering conditions as fixed factors (n=12) (Statistica, version 7). The Tukey test for multiple mean comparisons was used to determine statistically significant differences between conditions.

The ordination and sorting using the total abundance data was carried out with a non-metric multidimensional scaling (nMDS) and CLUSTER analysis (Bray-Curtis index; group average link; square root), respectively. The differences between the macrobenthic communities composition were tested by ANOSIM (Similitude analysis) while SIMPER (Similitude Percentage) was applied to determine the species that most contributed to the observed differences (PRIMER 5.0 program).

Finally, the dominant and subdominant species per treatment were calculated according to [START_REF] Picard | Qualitative research on marine biotic communities of dragables soft substrates of the Marseille region[END_REF]. The species were considered dominant or subdominant when the mean dominance index was maximum or ≥5 %, respectively.

A comparison of the biodiffusion (D b ) and bioadvection (r) coefficients was performed to assess differences in the sediment transport along the sediment column between the experimental conditions by means of the non parametric Kruskal-Wallis one-way analysis of variance by ranks [START_REF] Kruskal | Use of ranks in one-criterion variance analysis[END_REF]. When significant, it was followed by a Mann-Whitney pairwise comparison test to evaluate the significance of the differences between conditions after a Bonferroni adjustment for multiple comparisons [START_REF] Siegel | Nonparametric statistics for the behavioral sciences[END_REF].

Results

Physico-chemical parameters

The seawater temperature, dissolved oxygen, and pH showed little changes throughout the incubation with mean values of 17.7±0.6 °C, 7.9±0.4 mg/L, and 7.8±0.3, respectively (n=7) for all the experimental conditions. There were no statistically significant differences of means found between conditions (E0, E1, E2) for these three variables.

Macrobenthic communities

Experiments with two levels of oil addition affected macrobenthic community structure in comparison with the control experiment, being these changes mainly evidenced in density and specific richness (Fig. 2a, b, Online Resource 2). The contamination caused a progressive reduction in both parameters with the added oil amount showing a negative tendency. Particularly, the addition of 20 g/kg of oil (E2) resulted in a decrease of approximately 40 and 55 % of density and specific richness, respectively. On the other hand, diversity and evenness showed little changes being diversity only significantly lower for E2 condition in comparison with E1 treatment. The low level contamination condition (E1) did not differ from control for these two parameters (Fig. 2c,d,Online Resource 2).

The density, specific richness, diversity, and evenness profiles of organisms vs. depth layers are shown in Fig. 3. In a general way, the maximum values of these parameters were found in the first 2 cm, and then they gradually decreased with depth. Density was mainly affected by oil addition in the superficial layer of the sediments (0-0.5 cm) where a decrease of approximately 54 and 78 % was observed for the lowly (E1) and highly (E2) contaminated cores, respectively. Specific richness, diversity, and evenness markedly decreased with depth for the highly contaminated cores compared to controls and lowly contaminated cores.

The nMDS and CLUSTER analysis showed that the E1 samples were close to the E0 samples with a 72 % of similarity while the E2 samples, which were located on the opposite side of the diagram, presented a 58 % of similarity with E0 and E1 (Fig. 4, Online Resource 3). The nMDS representation was satisfactory as attested by the low stress value (0.11).

The ANOSIM showed highly significant differences among conditions with a global R of 0.775 (p<0.02). The SIMPER results showed which species contributed in greater extent to the differences observed. Higher proportions of Ostracoda sp. 1, Oligochaeta sp. 1, Pseudocumatidae sp. 1, Leuroleberis poulseni and Cirolanidae sp. 1 were observed in noncontaminated sediments. Low and high contaminated sediments were characterized by the presence of Ostracoda sp. 2, Phoxocephalopsis sp., Ceratocephale sp., Anacalliax argentinensis and Gammaridae sp. 2, and Paranebalia sp., respectively (Table 1).

Lastly, a list of the dominant and subdominant species found in the oil pollution simulation experiments is given in Table 2. Ostracoda sp. 1 was the dominant species in the E0 (uncontaminated) and E2 (highly contaminated) conditions. This species was the most subdominant species in the E1 experience where Ostracoda sp. 2 predominated over there. Finally, Oligochaeta sp. 1 was the most subdominant species in the E2 experience.

Sediment reworking: luminophores vertical distribution

The vertical distribution of the luminophores showed an exponential decrease of the tracers with depth for the three experimental conditions (Fig. 5). Maximal penetration depth for the luminophores was of 14 cm for all conditions (data not shown) with more than 96 % of tracers located in the first 5 cm (Fig. 5). After 30 days of incubation, nearly all the green luminophores remained at surface (98 %) for the highly contaminated cores (E2) while for low contaminated (E1) and uncontaminated (E0) only 54 and 43 % of the initially deposited luminophores were detected in the deposit zone, respectively (Fig. 5a). The effects of oil addition were even more marked during the last 15 days of the experiments (Fig. 5b). Indeed, the percentages of buried pink luminophores were only 26 and 1 % for E1 and E2, respectively, while 47 % were moved down from the surface deposition layer for the controls. Quantification of sediment reworking showed significant differences in the biodiffusion coefficient (D b ) between the control and the highly contaminated condition (p<0.05) for both the full experience time (30 days) and the last 15 days in comparison with controls (Fig. 6a, b, Online Resource 2). The same pattern was found with the bioadvection coefficient (r) (Fig. 6c,d, Online Resource 2). Both coefficients indicated a decrease of sediment reworking intensity with oil concentration increase with almost null values for the highly contaminated sediments. Indeed, in the presence of the high contamination (E2), the 0.1 values given by the model for both Db and r are the lowest limit values of the model (i.e., they vary from 0 to 0.1).

Discussion

Caleta Valdés is a pristine zone in the Patagonian coast having no oil contamination history. Simulated ex situ oil spills were conducted to study the impact of oil on the structure and activity of the macrobenthic community at two levels of contamination, low and high, simulating a chronic pollution and an acute oil spill situation, respectively. Throughout the whole experimentation, water temperature, dissolved oxygen, and pH within experimental tanks were stable for all the incubation conditions, suggesting that these were not discriminating parameters. According to [START_REF] Puente | Medium-term assessment of the effects of the Prestige oil spill on estuarine benthic communities in Cantabria (Northern Spain, Bay of Biscay)[END_REF], fine-grained substrates characterized by low permeability and water-saturated sediments, such as the one used in this study, substantially prevents depth penetration of oil. Indeed, the high surface/ volume ratio of the sedimentary matrix grains facilitates oil absorption and retention in the superficial layers preventing oil penetration to deep layers. This phenomenon was probably reinforced by our experimental setup, i.e., no simulation of sediment re-suspension due to tidal currents. Such conditions may explain why the density of macrobenthic organisms was mainly affected in the most superficial layer of contaminated sediment (i.e., where the oil was applied). At the same time, no deeper burial of the organisms in order to avoid contamination was detected [START_REF] Bolam | Burial survival of benthic macrofauna following deposition of simulated dredged material[END_REF], suggesting a direct toxic impact of the oil compounds on the organisms. According to [START_REF] Baker | The effects of oils on plants[END_REF], low molecular weight compounds present in oil can dissolve the membranes and alter the biota respiration mechanisms. Particularly, the oil water-accommodated fraction (WAF) containing the most polar and low molecular weight compounds from oil, mainly aromatic hydrocarbons (PAHs), have been extensively described to be toxic. For example, LC 50 on benthic organisms of oil WAF were found to be as low as 1.2-3 ppm of total aromatic hydrocarbon for some amphipods, shrimps, crabs, and snails [START_REF] Holdway | The acute and chronic effects of wastes associated with offshore oil and gas production on temperate and tropical marine ecological processes[END_REF], while [START_REF] Echols | Acute aquatic toxicity studies of Gulf of Mexico water samples collected following the Deepwater Horizon incident (May 12, 2010 to[END_REF] reported >20 % mysid mortality for Gulf of Mexico crude oil WAF in a concentration range of 0.015-0.89 ppm of total PAHs. Escalante crude oil used in our experiments is classified as medium petroleum having low molecular weight compounds aromatic hydrocarbons. A concentration of 1.38 ppm of total BTEXs and 0.23 ppm of total PAHs from naphthalene up to traces of four aromatic ring compounds like pyrene were measured in the Escalante oil WAF in a 90:1 (seawater:oil) ratio volume mixture [START_REF] Marino | Determination of aromatic hydrocarbon of the soluble fraction in oil water using microextraction in solid phase with CG-EM[END_REF]). Thus, a top-down gradient of these compounds in the water phase of the oiled experimental cores may be expected due to diffusion processes. This fact may also explain the mortality found in the top layer, in addition to the direct contact of organisms with the organic oil phase.

Resistance of macrobenthic community to oil contamination

Regarding oil effect on the structure of macrobenthic community, both the density and specific richness of organisms decreased with regard to the uncontaminated controls with a progressive impact with the increasing oil contamination. Harmful effects of crude oil on different macrobenthic communities have been previously reported. For instance, [START_REF] Hartwick | Effects of a crude oil and an oil dispersant (Corexit 9527) on population of the littleneck clam (Protothaca stamined)[END_REF] carried out field and laboratory studies about the effect of Alberta crude oil on behavior and survival of Fig. 6 Sediment reworking biodiffusion (D b ) and bioadvection (r) coefficients after 30 days of incubation and for the last 15 days. E0 control (no contamination); E1 low oil contamination (1 g/kg); E2 high oil contamination (20 g/kg). Note that, in presence of the high contamination (E2), the 0.1 values given by the model for both Db and r are the lowest limit values of the model (i.e., they vary from 0 to 0.1). Lowercase letters indicate significant differences between experimental conditions natural littleneck clam (Protothaca staminea) populations from uncontaminated sites during 231 and 10 days, respectively. In both studies, superficial sediments were contaminated with different oil concentrations and no mortality was found through the experimental period when the clams were treated with crude oil at 1000 ppm, the same concentration used in our study for the low concentration experiment (E1). Nevertheless, siphon activities were impaired at this contamination level; this functional inhibitory effect would probably render the animals more vulnerable to natural mortality factors (e.g., predation) in their habitat. In another study, carried out in Singapore coasts chronically contaminated by discharges from industry, sewage, and shipping, a significantly negative correlation between the specific richness, abundance, and diversity of macrobenthic organisms and total petroleum hydrocarbons was found (for oil added at concentration>800 ppm) [START_REF] Lu | Relationship between soft-bottom macrobenthic communities and environment variables in Singaporean waters[END_REF].

Response of the different macrobenthic species to oil contamination

Benthic organisms with little or no mobility have been widely used in marine environmental impact assessment and monitoring. As a matter of fact, they are sensitive sensors of physical and chemical changes undergone by benthic systems (e. g., [START_REF] Dauer | Biological criteria, environmental health and estuarine macrobenthic community structure[END_REF][START_REF] Poulton | Effects of an oil spill on leafpack-inhabiting macroinvertebrates in the Chariton River, Missouri[END_REF][START_REF] Muniz | Testing the applicability of a Marine Biotic Index (AMBI) to assessing the ecological quality of soft-bottom benthic communities, in the South America Atlantic region[END_REF][START_REF] Gomez Gesteira | Impact of the Aegean Sea oil spill on the Rîa of Ares and Betanzos fine sand community (Northwest Spain)[END_REF][START_REF] Borja | Assessing the environmental quality status in estuarine and coastal systems: comparing methodologies and indices[END_REF][START_REF] Ocon | Evaluation of zoobenthic assemblages and recovery following petroleum spill in a coastal area of Rıo de la Plata estuarine system, South America[END_REF][START_REF] Dauvin | Benthic indicators: from subjectivity to objectivity-where is the line?[END_REF]). Because they are closely associated with the seafloor they can hardly avoid deteriorated conditions of water and/or sediment quality. Furthermore, they have relatively long life cycles and exhibit different stress tolerance [START_REF] Bilyard | The value of benthic infauna in marine pollution monitoring studies[END_REF][START_REF] Dauer | Biological criteria, environmental health and estuarine macrobenthic community structure[END_REF]. According to their response to stress, species can be divided into Bsensitive species^(i.e., able to only survive within a narrow range of environment conditions and disappear from polluted areas); Btolerant species( i.e., being not sensitive to a particular stress and/or pollution); Bopportunistic species^(i.e., able to quickly exploit new recourses or ecological niches as they become available, characterized by early reproduction, high reproduction rates, rapid development, small body size, and an uncertain adult survival rate), and Bindifferent species^(without real affinity for any particular community and showing no response to pollution) [START_REF] Dauvin | Benthic indicators: from subjectivity to objectivity-where is the line?[END_REF]. In Caleta Valdés sediments and with regard to our experimental oil contamination, the three first types of organisms were found. Opportunistic species could be represented by Oligochaeta sp. 1 that reached high densities under the highly contaminated conditions (E2). Different opportunistic species of oligochaetes have been shown to be favored by the organic enrichment of sediments due to discharge of sewage outfall [START_REF] Gamito | Three main stressors acting on the Ria Formosa lagoonal system (Southern Portugal): physical stress, organic matter pollution and the landeocean gradient[END_REF], untreated abattoir wastes [START_REF] Arimoro | The impact of sawmill wood wastes on the water quality and fish communities of Benin River, Niger Delta Area, Nigeria[END_REF]) and livestock effluents [START_REF] Solimi | The benthic community structure in mountain ponds affected by livestock watering in nature reserves of central Italy[END_REF]. These substrates are mostly covered by bacteria and sewage fungi, which are the main food source for the most part of oligochaetes [START_REF] Rueda | Effect of episodic and regular sewage discharges on the water chemistry and macroinvertebrate fauna of a Mediterranean stream[END_REF]. Species such as Limnodrilus hoffmeisteri and Tubifex tubifex have been used as indicators of this kind of environment [START_REF] Brinkhurst | Aquatic Oligochaeta of the world[END_REF][START_REF] Aston | Field and experimental studies on the effects of a power station effluent on Tubificidae (Oligochaeta, Annelida)[END_REF][START_REF] Miserendino | Macroinvertebrates of a fluvial system in Patagonia: altitudinal zonation and functional structure[END_REF]. Particularly, Gonzalez Egress et al. ( 2012) found a marked decrease in the density of oligochaetes shortly after the in situ diesel oil spill impact but a recovery trend with a gradual increase from day 2, behaving as tolerant or resilient species to the oil impact [START_REF] Gomez Gesteira | Amphipods are good bioindicators of the impact of oil spills on soft-bottom macrobenthic communities[END_REF][START_REF] Ocon | Evaluation of zoobenthic assemblages and recovery following petroleum spill in a coastal area of Rıo de la Plata estuarine system, South America[END_REF]. Also, Paranebalia sp. (Crustacea: Phyllocarida: Leptostraca) was dominant in the most contaminated sediments 30 days after the oil addition and seems to be a tolerant species. Even if it was known as a primarily tropical-subtropical genus, it was recorded for the first time in the northern Patagonia during the years 2003 to 2007 on sediments and among holdfast of Macrocystis pyrifera and Undaria pinnatifida [START_REF] Roccatagliata | On the occurrence of the genus Paranebalia (Crustacea: Phyllocarida: Leptostraca) in northern Patagonia, Argentina[END_REF].

Leptostracans are known to occur in a variety of habitats, from hydrothermal vents and marine caves to the intertidal zone showing a wide resistance to adverse environmental conditions [START_REF] Haney | A new genus and species of leptostracan (Crustacea: Malacostraca: Phyllocarida) from Guana Island, British Virgin Islands, and a review of leptostracan genera[END_REF]. Under mild contaminated conditions (E1) Ostracoda sp. 2 was the dominant species at the end of the incubation. Yet, previous field and laboratory studies demonstrated that ostracods seem particularly sensitive to oil [START_REF] Carman | Does historical exposure to hydrocarbon contamination alter the response of benthic communities to diesel contamination?[END_REF][START_REF] Ruiz | Marine and brackish-water ostracods as sentinels of anthropogenic impacts[END_REF]. Nevertheless, after a high mortality event due to the introduction of oil, it has also been observed a high recovery capacity of ostracods communities characterized by the development of high densities, 2 days after the contamination (Gonzalez Egress et al. 2012), or by changes in the trophic structure of the community [START_REF] Carman | Does historical exposure to hydrocarbon contamination alter the response of benthic communities to diesel contamination?[END_REF]. Clearly, the response of the different species of ostracods may vary. Indeed, in the present study, Ostracoda sp. 1 predominated both under uncontaminated and highly contaminated conditions but its abundance decreased with the oil addition. On the other side, some taxa known for their sensibility to contamination such as Phoxocephalopsis sp.

(Gomez Gesteira and Dauvin 2000) also disappeared when superficial sediments were highly contaminated (E2). The present study constitutes the first characterization of the macrobenthic communities of Caleta Valdés sediments both subject or not to oil contamination. This perturbation led to the selection or disappearance of particular organisms which may serve as indicator species for this kind of environment in case of oil pollution. However, although our results do provide a first indication about potential oil pollution indicators, further studies are needed in order to identify the macrobenthic organisms to species level (e.g., ostracods) but also characterize their functional traits. This information is so far clearly missing for the Patagonian coastal zone where exploitation and sea transportation of oil are rapidly expanding.

Role of the benthic organisms in the polluted sediments natural attenuation

Benthic biota has a marked role in microbial aerobic biodegradation of hydrocarbons in marine sediments [START_REF] Cuny | Biodegradation of hydrocarbons in bioturbated marine sediments[END_REF]. Indeed, bioturbation processes such as burrow construction and ventilation carried out mainly by macrofauna are key process to stimulate the availability of oxygen in superficial sediments [START_REF] Timmermann | Effects of the polychaetes Arenicola marina and Nereis diversicolor on microbial pyrene mineralization[END_REF]). In addition, some macrobenthic organisms can affect positively the biodegradation of hydrocarbons through the production of digestive compounds acting like surfactants that modify their dispersion state and improve their bioavailability [START_REF] Penry | Digestive determinants of benzo[a]pyrene and phenanthrene bioaccumulation by a deposit-feeding polychaete[END_REF][START_REF] Weston | In vitro digestive fluid extraction as a measure of the bioavailability of sediment-associated polycyclic aromatic hydrocarbons: sources of variation and implications for partitioning models[END_REF][START_REF] Gilbert | Alteration and release of aliphatic compounds by the polychaete Nereis virens (Sars) experimentally fed with hydrocarbons[END_REF] for hydrocarbon-degrading bacteria [START_REF] Bertrand | Biosurfactant production by marine microorganisms. Potential application to fighting hydrocarbon marine pollution[END_REF][START_REF] Bonin | Involvement of bioemulsifier in heptadecane uptake in Pseudomonas nautica[END_REF][START_REF] Cuny | Influence of bioturbation by the polychaete Nereis diversicolor on the structure of bacterial communities in oil contaminated coastal sediments[END_REF][START_REF] Cuny | Biodegradation of hydrocarbons in bioturbated marine sediments[END_REF]. Therefore, in sediments strongly affected by oil spills without active macrobenthic fauna, the microbial bioremediation of hydrocarbons would be markedly reduced [START_REF] Timmermann | Effects of bioturbation on the fate of oil in coastal sandy sediments -An in situ experiment[END_REF]).

In the frame of this study, oil affected the structure but also the activity of the Caleta Valdés macrobenthic community. Indeed, the quantification of the sediment reworking clearly showed a negative impact of oil contamination on the transport and mixing of particles by organisms. This impact was highlighted in highly contaminated conditions by the almost null values of the biodiffusion (D b ) and bioadvection (r) coefficients calculated for the full experience and for the last 15 days of incubation. These facts evidenced a general reduction of sediment reworking for both mixing modes with oil addition. This suggests that the activity of the community was inhibited throughout the sedimentary column, even in deeper sediments where community parameters were not strongly affected by oil. Similar observations have been reported for laboratory experiments where Nereis diversicolor reworking was found to be reduced in the presence of Arabian Light crude oil [START_REF] Gilbert | The in-vitro influence of the burrowing polychaete Nereis diversicolor on the fate of petroleumhydrocarbons in marine-sediments[END_REF]. But this is not always the rule, as other authors have found that Hediste diversicolor reworking activity may be enhanced in case of oil contamination [START_REF] Stauffert | Impact of oil on bacterial community structure in bioturbated sediments[END_REF]). These discrepancies underline the complexity of the response of the benthic system to oil contamination, which is controlled by several factors as mentioned above. Overall, the different responses to oil pollution will be given by the resistance capacity of the species according to their biological traits, physiological status, and stage of development.

Finally, the resistance capacity seems to be strongly determined by the different events of contamination that may have led to the selection of an adapted community. As highlighted by the weak resistance capacity to oil of the macrobenthic communities of Caleta Valdés, pristine environments without previous history of contamination would be probably strongly impacted in case of an oil spill [START_REF] Carman | Does historical exposure to hydrocarbon contamination alter the response of benthic communities to diesel contamination?[END_REF].

In conclusion, this study allowed for the first time to experimentally evaluate the effects of oil exploited in Patagonia on the structure and activity of the benthic macrofauna from a pristine site located in Caleta Valdés. Macrobenthic community structure, and above all, sediment reworking activity were impacted even at the lowest contamination level tested (level corresponding to chronically contaminated sites). On the other hand, in a high level of contamination, similar to those recorded in intertidal areas after an oil spill, results demonstrated an almost complete inhibition of macroorganisms reworking activity. It further causes severe structural changes in the macrobenthic community due to high mortality rates. These results clearly demonstrate the weak resistance capacity of the macrobenthic communities from a pristine environment in case of oil contamination. They also highlight the need to carry out longer-term experiments in order to test the resilience capacities of this type of benthic system. A special attention to the shifts in macrobenthic species functional traits due to oil contamination should also be paid as they can markedly affect biogeochemical cycling with cascading effects on the ecological functioning of the whole ecosystem [START_REF] Van Colen | Organism-sediment interactions govern posthypoxia recovery of ecosystem functioning[END_REF][START_REF] Hale | Characterizations of how species mediate ecosystem properties require more comprehensive functional effect descriptors[END_REF][START_REF] Kristensen | Influence of benthic macrofauna community shifts on ecosystem functioning in shallow estuaries[END_REF]). This knowledge is fundamental in developing an efficient environmental management strategy against pollutions particularly for sensitive pristine marine protected areas such as Caleta Valdés.

Fig. 1

 1 Fig. 1 Sampling site in Caleta Valdés (CV; Península de Valdés, Patagonia Argentina)

Fig. 2

 2 Fig. 2 Global community parameters measured for each experimental condition (mean values for the entire cores±standard deviation; n=4). E0 control (no contamination); E1 low oil contamination (1 g/kg); E2 high

Fig. 3

 3 Fig. 3 Community parameters measured at each depth layer for each condition (mean values; n= 4). E0 control (no contamination); E1 low oil contamination (1 g/kg); E2 high oil contamination (20 g/kg)

  The results clearly demonstrate the low resistance capacities to oil contamination of the macrobenthic communities of Caleta Valdés sediments. Castège et al. (in press) studied benthic community from Guéthary (Bay of Biscay, France), affected by the Prestige oil spill in 2002. These authors concluded that 5 years after the spill, the macrobenthic community still showed perturbation signs, such as proliferation of grazers, highlighting the complexity and slowness of the recovery process. The severity of oil impacts on the benthic macrofauna depends on many factors, most notably (1) oil amount; (2) chemical composition; (3) form (weathered or not, emulsified

Fig. 5

 5 Fig. 5 Vertical profiles of luminophores distribution after 30 days of experimentation and for the last 15 days. E0 control (no contamination); E1 low oil contamination (1 g/kg); E2 high oil contamination (20 g/kg)

Table 1

 1 Similarity percentage analysis (SIMPER) for oil pollution simulation experiments

	Species	Mean abundance
		E0	E1	E2
	Ostracoda sp. 1	44.50	16.75	15.25
	Oligochaeta sp. 1	14.25	5.50	12.00
	Axiothella sp.	10.25	10.25	0.75
	Ostracoda sp. 2	5.25	18.75	3.50
	Phoxocephalopsis sp.	4.00	4.25	0.00
	Pseudocumatidae sp.	3.50	0.75	0.75
	Paranebalia sp.	1.00	0.25	1.25
	Leuroleberis poulseni	1.00	0.50	0.00
	Cirolanidae sp. 1	0.75	0.25	0.00
	Ceratocephale sp.	0.50	0.75	0.25
	Anacalliax argentinensis	0.25	1.25	0.00
	Gammaridae sp. 2	0.25	1.50	0.00

E0 control (no contamination), E1 low oil contamination (1 g/kg), E2 high oil contamination (20 g/kg)

Table 2

 2 Dominant and subdominant species in oil pollution simulation experiments

	E0			E1			E2
	Species	P	D	Species	P	D	Species	P	D
	Ostracoda sp. 1	1112.50	51.74	Ostracoda sp. 2	468.75	30.86	Ostracoda sp. 1	381.25	44.85
	Oligochaeta sp. 1	356.25	16.57	Ostracoda sp. 1	418.75	27.57	Oligochaeta sp. 1	300.00	35.29
	Axiothella sp.	256.25	11.92	Axiothella sp.	256.25	16.87	Ostracoda sp. 2	87.50	10.29
	Ostracoda sp. 2	131.25	6.10	Oligochaeta sp. 1	137.50	9.05
				Phoxocephalopsis sp.	106.25	7.00
	E0 control (no contamination), E1 low oil contamination (1 g/kg), E2 high oil contamination (20 g/kg). P mean density, D mean dominance index

Acknowledgments This work was funded by the Evaluationorientation de la Coopération Scientifique Sud -Ministerio de Ciencia, Tecnología e Innovación Productiva (ECOS Sud -MINCYT) collaboration program (Proyecto Caleta; action no A10U02) and the French Agence Nationale de la Recherche (project DECAPAGE; ANR CESA-2011-006 01). We thank Anthony Maire for help in statistical analysis.


A Rajaraman

§ A R Sahoo

§ F Hild

C Fischmeister

M Achard

C Bruneau

Ruthenium(II) and Iridium(III) complexes featuring NHC-Sulfonate chelate

Three new complexes bearing a chelating (κ 2 C,O) NHC-SO 3 ligand have been prepared. An original method for the synthesis of the imidazolium-sulfonate NHC precursor is described. The 5-membered ruthena-and irida-cycle containing complexes were fully characterized and evaluated in a series of catalytic transformations involving hydrogen auto-transfer processes.

 More recently, pincer ligands have also permitted major advances in a broad diversity of catalytic transformations. 5

Our group investigated the potential of phosphine-sulfonate ligands 6,[START_REF] Drent | [END_REF] in a number of catalytic transformations such as allylic activations, 8 C-H bond functionalizations, 9 hydrogenations 10 including asymmetric hydrogenation of ketones and imines. 11,[START_REF] Jiang | New ruthenium and iridium catalysts for transformations involving hydrogen transfers[END_REF] Having in mind the tremendous impact of N-Heterocyclic Carbenes (NHCs) in many catalytic transformations 13 we became interested in the synthesis of metal complexes coordinated by chelating NHC-sulfonate ligands. Well-defined transition-metal catalysts bearing a chelating NHC ligand have already been described in a number of ways such as NHC-NHC, 14 NHC-oxazoline, 15 NHC-alkyl. 16 Bidentate NHCsulfonate ligands have only recently been reported in a very limited number of architectures. NHCarenesulfonate ligands were extensively described by Hoveyda in various catalytic transformations involving in situ generated catalysts 17 while Jordan 18 and Wang 19 reported on well-defined NHC-arenesulfonate metallacycles in polymerization reactions. NHC-alkylsulfonate ligands have also been reported, however the sulfonate functional group was used as a polar solvent (water, alcohol) solubilising tag. 20

Figure 1 New ruthenium and iridium complexes

To the best of our knowledge, only one example of bidentate NHC-alkylsulfonate ligand was reported by Nozaki in 2009. 21 In this article we report our results on the syntheses of ruthenium and iridium complexes bearing a NHC-methylenesulfonate ligand (Figure 1) and their use in alkylation of amines through hydrogen transfer processes and in alcohol etherification reaction. The synthesis of these three new complexes required the preliminary synthesis of the imidazolium salt 1 that proceeds via the synthesis of an imidazolium methylene halide salt such as 2 (Scheme 1). As outlined by several groups, this synthesis requires the utilization of expensive unsymmetrical ICH 2 Cl or BrCH 2 Cl in order to prevent the side formation of a bis-imidazolium salt.

21,22

In order to decrease the cost of the ligand synthesis and because in our hands, the utilization of ICH 2 Cl did not prevent side products formation, we have developed a new synthetic pathway based on biphasic synthesis consisting in water and inexpensive dibromomethane. Indeed, we hypothesized that the halogenated imidazolium salt intermediate would readily migrate to the aqueous phase hence preventing bis-imidazolium side product formation. Thus, mesityl imidazole 3 was reacted with dibromethane in the presence of water for 16 hours. Analysis of the water layer highlighted the exclusive formation of the halogenated imidazolium salt 2 along with the protonated imidazole 4 arising from side dibromomethane decomposition. 23 Addition of sodium sulphite acting both as nucleophile and base resulted in the clean formation of imidazolinium sulfonate zwitterion 1 along with bromomethylsulfonate side product. Extraction and recrystallization, cleanly afforded 1 in 30-40% reproducible isolated yields thus competing with previously described methods. It should be noted that the excess of dibromomethane could be easily recycled and reused by distillation. The zwitterionic structure was confirmed by X-ray crystallography (Scheme 1). According to Nozaki protocol, further preparation of the polymeric silver complex Ag-1 was successfully achieved for further transmetallation processes (Scheme 2). With this silver complex in hand, we next investigated the coordination chemistry with various ruthenium and iridium metallic precursors (Scheme 2). Transmetallation of silver complex Ag-1 with 0.5 equivalent of the dimeric [RuCl 2 (p-cymene)] 2 [START_REF] Süss-Fink | For a recent update on arene ruthenium complexes see[END_REF] in dichloromethane afforded the expected half-sandwich chiral ruthenium(II) complex Ru-1 in 85% isolated yield. NMR analyses of the complex demonstrated the formation of sixmembered chelate by the presence of diastereotopic protons of the methylene bridge at 5.47 and 4.49 ppm (J H- H = 13.0 Hz). Analyses also demonstrated that the arene ligand remained bound to the ruthenium metallic centre highlighted by the presence of four distinct signals located at 5.75, 5.41, 5.24 and 3.30 ppm, respectively. It is noteworthy that these data are comparable to those reported by Albrecht and co-workers for the ruthenium complex Ru-3 analogue bearing a NHC-carboxylate chelate (Scheme 2).

Scheme 1 Preparation of the zwiterrionic imidazolium sulfonate

1 Replacement of [RuCl 2 (p-cymene)] 2 by [Ru(OAc) 2 (p-cymene)]
26 was also successful and the corresponding arene ruthenium(II) complex Ru-2 featuring the chelate ligand 1 was obtained in 40% yield. This methodology is not limited to ruthenium metallic precursors and the reaction with [Cp*IrCl 2 ] 2 cleanly afforded the corresponding three legs piano stool iridium(III) complex Ir-1. 2. The six-membered {C,O}Ru chelate adopts a half-chair conformation and the molecular structure features a pseudotetrahedral environment at the ruthenium center. The structure of Ru-1 was compared with the previously reported carboxylate Ru-3 25 and the phosphine-sulfonate analogs [RuCl(p-cymene)(DPPBS)] Ru-4 27 (Scheme 2)(Table 1). The Ru-Cl bond (2.405( 3)) is slightly shorter in Ru-1 than in the carboxylate analog (2.424(3)) but similar with the phosphine analog (2.3992(6)). Interestingly, the longer Ru-O bond lengths in Ru-1 and Ru-4 than in Ru-3 suggest lower stability of the resulting sulfonate chelates. The leaving ability/slipping hapticity of the arene ligand have a crucial impact toward catalysis in the corresponding ruthenium complexes.

28,29

Scheme 2 Preparation of the well-defined ruthenium and iridium complexes Thus, we next focused our attention on the reactivity of the well-defined Ru-1 complex toward arene displacement. As we previously reported, facile ligand exchange of arene ligand with acetonitrile occured in Ru-4 at 90 °C.

12 However, similar reaction in boiling acetonitrile with Ru-1 did not lead to ligand exchange and surprisingly even after two days reaction time, the starting complex remained intact. Higher temperature, gave partial decomposition of the starting material and no noticeable effect of light irradiation was observed with this complex. This result is in line with the reported high thermal stability of Ru-3. 25 It also tends to suggest that this stability of Ru-1 might impact in catalyses involving the loss of the arene ligand and thus would require higher reaction temperature. With this preliminary result in hand, we next focused our attention on the reactivity of the obtained well-defined complexes in hydrogen transfer reactions. Hydrogen autotransfers known as hydrogen borrowing or hydrogen shuttling have found interesting applications in synthesis for the preparation of various functionalized alcohols, amines and carbonyl derivatives. 30 In order to avoid side effects (positive or negative) of external additives or solvent, we examined the activities of the prepared complexes in the base free N-alkylation of amines with alcohols under solvent free reaction conditions (Table 2). Reaction of piperidine with benzyl alcohol at 130 °C under inert atmosphere of argon with a 100:1 substrate/catalyst ratio resulted in the formation of the N-benzylpiperidine 31 and side product arising from our previously reported N,C-dialkylation. 27 Good activity was obtained with Ru-1 and almost complete formation of 7aa was obtained in the presence of the iridium(III) precatalyst Ir-1 (Table 2, entry 1 and2). However, a slight conversion decrease was observed with Ru-2 precatalyst (Table2, entry 3). Comparison with our previously reported precatalyst [IrClCp*(DPPBS)] highlighted that the use of Ir-1 led to a lower amount of the side dialkylated product 8aa, which decreased from a 80:20 to 97/3 ratio of 7aa/8aa (Table 2, entries 6, 9) whereas this reaction was minimized with other complexes. Taken together, these results suggest that the NHC-sulfonate chelate is more stable in Ir-1 than the corresponding [IrClCp*(DPPBS)]. Having established our best reaction conditions with Ir-1, we next investigated the scope of the transformation with various alcohols and amines (Scheme 4). Reaction with benzyl alcohol and various aliphatic secondary cyclic amines such as pyrrolidine, piperidine, morpholine and piperazine cleanly afford the corresponding tertiary amines in up to 90% isolated yield after column chromatography over alumina gel.

Ru-1

Ir-1 The use of aniline resulted in lower conversion giving 7ea in 64% yield and confirmed the influence of amine basicity toward conversion. The use of aliphatic alcohol such as hexanol was also compatible with the optimized reaction conditions to afford the amines 7ab, 7bb, 7db and 7eb in 56-85% range isolated yield. Finally, secondary alcohol such as phenylethanol led to the corresponding racemic amines 7bc, 7ac, 7cc in moderate isolated yields. The catalytic activity of complexes Ru-1 and Ir-1 was next evaluated in the direct β-alkylation of tertiary cyclic amines with benzaldehyde through in situ generation of the corresponding enamine (Table 3). 10a As we previously reported, β-alkylation of N-phenylpiperidine 9a is difficult in regard to the direct endo dehydrogenation. As result the use of catalytic amount of Ru-1 at 150 °C only provided 18% of the corresponding amine 11a after complete reduction of the resulting alkylated enamine derivatives (Table 3, entry 1). Surprisingly, the use of precatalyst Ir-1 also afforded poor yield of 11a (Table 3, entry 2). In contrast reaction with our previously reported *IrClCp*(DPPBS)+ gave 93% of the expected β-alkylated amine 11a (Table3, entry 3). The activities of Ru-1 and Ir-1 were not restored with the use of N-methylpiperidine and decomposition of 9b mainly occurred yielding a maximum 18% yield with Ru-1 (entry 4). These results confirmed the higher stability of the new (Κ 2 C,O) chelate complexes. Given the electronic properties of the sulfonate ligand which acts as a strong electron withdrawing ligand hence increasing the overall electrophilicity of the metal centre of the resulting complexes, [START_REF] Drent | [END_REF] we next examined the relative electrophilicity of our synthesized complexes in the absence of organic bases such as amine. As depicted in Scheme 5, when the rac-phenylethanol was heated in the presence of Ru-1, selective etherification occurred affording the rac/meso ether 12 in 45% isolated yield along with traces of styrene whereas in the presence of Ir-1 the alcohol 6 remained unreacted. 32 Noteworthy that no β-alkylation occurred on 6c in the absence of base to give the corresponding dimeric alcohols.

Scheme 3 Arene ligand exchange

Scheme 4 N-alkylation of amines with alcohols

Conclusions

We have reported the straightforward synthesis of an imidazolium-sulfonate NHC precursor from symmetrical dibromomethane allowing the preparation of the corresponding well-defined ruthenium and iridium complexes. Preliminary investigation in catalysis highlighted its high thermal stability. Future efforts will be devoted to the use of more labile arene ligand to facilitate catalysis as well as the application to other catalytic transformations involving cooperative mechanisms. Considering the facile preparation of the bromomethylimidazolium, it might also open new insights for the preparation of diverse heterotopic prochelates and mixed bis-NHCs.

1 H

 1 NMR analyses confirmed the formation of the six-membered chelate supported by the two doublet signals located at 5.18 and 4.73 ppm in 1 H NMR. Successful crystallization by solvent diffusion technique (CH 2 Cl 2 /n-hexane) allowed the determination of the molecular structure of Ru-1 and Ir-1 which are represented in Figure

Figure 2

 2 Figure 2 Structure of pure complexes Ru-1, 0.5 CH 2 Cl 2 and Ir-1, 1 CH 2 Cl 2 . Thermal ellipsoids are drawn at the 50% probability level. Hydrogen atoms and solvents are omitted for clarity.

33 Scheme 5

 335 Scheme 5 Etherification of phenylethanol 6c

Table 1

 1 Selected bond lengths and angles of prepared complexes, comparison with reported complexes

		Ru-1	25 Ru-3 ref.	27 Ru-4 ref.	Ir-1
	M-Cl	2.405(3)	2.424(3)	2.3992(6)	2.374(1)
	M-O	2.157(5)	2.079(8)	2.118(1)	2.227(3)
	M-C centroid	1.685	1.701	1.693	1.806
	M-C NHC (P)	2.088(8)	2.033(9)	2.3570(8)	2.058(5)
	Cl-M-C NHC (P)	83.2(2)	83.5(3)	85.9	89.85(10)
	O-M-C NHC (P)	88.7(3)	86.7(3)	90.7	83.27(12)
	O-M-Cl	88.12(16)	88.5(2)	80.9	82.31(8)

Table 2 N

 2 -Alkylation of piperidine 5a with benzyl alcohol 6a

	a

b

Table 3 C

 3 -Alkylation of piperidines 9 with benzaldehyde 10

	a

b a

Experimental conditions : all reactions were performed under an inert atmosphere of argon and carried out with 9/10/HCO 2 H/precatalyst in 1/1.2/1.5/0.01 molar ratio at 150°C.

b conversion and yield were determined by GC analysis.

Ackowledgements:

The authors thank Dr. Thierry Roisnel and Dr. Vincent Dorcet for X-Ray analyses. F.H. thanks the ANR (MemChem project) for funding. A.R.S. thanks the CEFIPRA/IFCPAR (IFC/A/5105-4/2014 1777) for PhD fellowship.