N Finck

M L Schlegel

A Bauer

Structural iron in dioctahedral and trioctahedral smectites: A polarized XAS study

Keywords: Montmorillonite, Hectorite, Iron, polarized XAS

The chemical form of structural Fe in smectites influences many physicochemical properties of these clay minerals. Powder EXAFS data for structural Fe in smectites have been reported, however, the preferred orientation of clay platelets with respect to the X-ray beam may lead to erroneous conclusions on the local chemical environment. Dioctahedral montmorillonite and for the first time trioctahedral hectorite were prepared as textured samples and the Fe local environment was probed by analysis of the X-ray absorption pre-edge peaks at the magic angle and by polarized EXAFS (P-EXAFS) spectroscopy. Compared to powder measurements, overlapping contributions from shells with distinct orientations can be filtered more easily by P-EXAFS, thus decreasing uncertainties on structural parameters.

The pre-edge spectrum for montmorillonite is similar to spectra commonly reported for dioctahedral smectites. In contrast, the pre-edge spectrum of hectorite is notably distinct, and hints to either differences in the site symmetry and/or in covalence. In both smectites, Fe is surrounded by a first O shell at a distance consistent with sixfold coordinated Fe(III), suggesting that Fe(III) is located in the smectite octahedral sheet. This is corroborated by the distances and orientations of neighboring cationic shells, such as in-plane (Mg,Al) and outof-plane Si shells. For montmorillonite, the results indicate Fe substitution for Al/Mg in the octahedral sheet, and a number of Fe neighbors consistent with random distribution in the octahedral sheet. For hectorite, results indicate a slight tendency for Fe atoms to form pairs in octahedral sheets; however, low numbers of neighboring cations were obtained, presumably a consequence of the presence of vacancies and/or Li in the vicinity of Fe, or of the coexistence of Fe and Mg neighbors with mutually cancelling EXAFS waves. Consistent with pre-edge data, the coordination numbers can also indicate some incoherency in Fe-cation interatomic distances in hectorite as a consequence of site distortion. These results suggest that Fe 3+ in hectorite locally distorts the structure of the trioctahedral phyllosilicate, and tends to aggregate charge-deficient (i.e. vacant or Li + -containing) octahedral sites.

Introduction

Smectites are widespread clay minerals forming under surface and subsurface conditions [START_REF] Meunier | Clays[END_REF] and widely occur in weathering formations and sediments [START_REF] Güven | Smectites. In Hydrous Phyllosilicates[END_REF]. These small, flat minerals possess a large surface area and permanent layer charge. As a consequence, they can absorb and retain a variety of ions or molecules and thus they dominate the physicochemical properties in the systems in which they are present. Their high reactivity has also spurred important industrial applications such as catalysis (e.g., [START_REF] Swarnakar | Catalytic activity of Ti-and Al-pillared montmorillonite and beidellite for cumene cracking and hydrocracking [A3411[END_REF][START_REF] Güven | Bentonites -clay for molecular engineering[END_REF] and health [START_REF] Williams | Bentonite, Bandaids, and Borborygmi[END_REF]. Smectites are also constituents of bentonites and confer to these materials cost-efficient mechanical, hydraulic and chemical properties. Bentonites are extensively used as constituents of engineered barriers for the isolation of pollutants, and could be used as backfill in high-level nuclear waste repositories [START_REF] Gates | Bentonite clay keeps pollutants at bay[END_REF]. Finally smectites such as hectorite can form as secondary minerals during the alteration of nuclear glass containing high-level waste [START_REF] Thien | Structural identification of a trioctahedral smectite formed by the aqueous alteration of a nuclear glass[END_REF].

These newly formed minerals can further react with hazardous radionuclides released by alteration of nuclear glass. Thus, it is important to understand the smectite crystallochemical properties which ultimately control their reactivity.

Smectites can exhibit a large variation in chemical compositions as a result of their versatile structure and of cationic substitution in crystal sites. For example, montmorillonite, the common aluminum smectite in soils and sediments, is made by the condensation of one octahedral sheet sandwiched between two tetrahedral sheets (Figure 1), forming a TOT structure. Two-third of the octahedral sites are occupied mostly by Al(III), forming a dioctahedral framework in which Al(III) can be substituted by cations such as Mg(II) or Fe(II,III) [START_REF] Stucki | Structural iron in smectites[END_REF]. In contrast, in trioctahedral hectorite all octahedral sites are filled, mostly by Mg(II), which can be substituted by Li(I) or trace amounts of Fe(II,III). Not only the extent, but also the distribution of chemical substitution can affect the chemical reactivity of smectite [START_REF] Mering | Sur le rôle de la valence des cations échangeables dans la montmorillonite[END_REF]. For example, reduction or oxidation of redox sensitive cations will modify the layer charge and thus alter properties such as swelling and/or cation exchange capacity [START_REF] Gates | Structural properties of reduced Upton Montmorillonite[END_REF][START_REF] Gates | Swelling and texture of iron-bearing smectites reduced by bacteria[END_REF], which in turn may affect the retention capacities of bentonites.

Structural Fe in clay minerals can significantly impact the redox properties in the environments where they are present. The reduction of Fe(III) in smectite can be achieved by microbial (e.g. [START_REF] Pentráková | A review of microbial redox interactions with structural Fe in clay minerals[END_REF] and by chemical treatments (e.g. [START_REF] Komadel | Structural Fe(III) reduction in smectites[END_REF][START_REF] Stucki | Chapter 8 Properties and Behaviour of Iron in Clay Minerals[END_REF]. For example, ferruginous smectite can be reduced by dithionite within hours and the reduction proceeds from the basal surfaces rather than from the particle edges [START_REF] Komadel | Structural Fe(III) reduction in smectites[END_REF]. Fe(II) in clay minerals is able to reduce heavy metals [START_REF] Bishop | Reduction and immobilization of hexavalent chromium by microbially reduced Fe-bearing clay minerals[END_REF] and radionuclides [START_REF] Bishop | Bioreduction of Fe-bearing clay minerals and their reactivity toward pertechnetate (Tc-99)[END_REF][START_REF] Yang | Effects of redox cycling of iron in nontronite on reduction of technetium[END_REF], thereby modifying their mobility and availability. In contrast, Fe(III) may act as terminal electron acceptor for iron-reducing bacteria, which provides a mechanism of structural Fe(II) (re)generation [START_REF] Ernstsen | Microbial Reduction of Structural Iron in Clays-A Renewable Source of Reduction Capacity[END_REF]. The magnitude and reversibility of these reactions largely depend on the Fe local environment in the octahedral sheet, i.e., the formation of a solid solution or the occurrence of Fe-rich clusters [START_REF] Drits | A model for the mechanism of Fe3+ to Fe2+ reduction in dioctahedral smectites[END_REF] which can be controlled in part by the smectite structure. For example, in dioctahedral smectites, two octahedral sites of distinct sizes can be identified, and Fe(III) would preferentially enter the largest one [START_REF] Tsipursky | The distribution of octahedral cations in the 2-1 layers of dioctahedral smectites studied by oblique-texture electron-diffraction[END_REF], and thus be relatively dispersed. In contrast, preferential clustering of Fe was observed in trioctahedral micas, i.e., non-swelling phyllosilicates with TOT layers as in smectites [START_REF] Manceau | Distribution of Fe in the octahedral sheet of trioctahedral micas by polarized EXAFS[END_REF]. This suggests that Fe distribution may differ depending on the octahedral sheet framework (dioctahedral or trioctahedral) of smectites. Unfortunately, no study reported unambiguous evidence for random Fe distribution in trioctahedral smectites so far.

X-ray absorption spectroscopy (XAS), and more specifically extended X-ray absorption fine structure (EXAFS) spectroscopy, have proven to be reliable techniques to probe the molecular environment of transition metals. Powder EXAFS spectroscopy was used to investigate the Fe environment in montmorillonite (SWy-1) [START_REF] Vantelon | Iron distribution in the octahedral sheet of dioctahedral smectites. An Fe K-edge X-ray absorption spectroscopy study[END_REF]. No Fe-Fe pair was detected, suggesting that the structure has an ordered Fe distribution obeying an exclusion rule in the octahedral sheet. The spectroscopic analysis of such powder spectra is however complicated by textural effects which are difficult to avoid during sample preparation of minerals having a layered structure (Manceau, 1990b). Powder EXAFS spectra for such anisotropic samples should be recorded with the sample plane oriented at the magic angle (at which any angular dependence is extinguished), otherwise XAS analysis will provide an inaccurate description of the local environment. Unfortunately, information concerning sample preparation and orientation with respect to the in-coming X-ray beam is most of the time missing. Additionally, EXAFS analysis for such samples is complicated by interferences between EXAFS waves scattered by neighboring shells located at similar distances from the absorber. This complication can be overcome by polarized EXAFS (P-EXAFS) spectroscopy which can discriminate the contributions of absorberbackscatterer pairs with distinct crystallographic orientations. This is of high interest in investigations on clay minerals where cationic shells from the tetrahedral and octahedral sheets are separated by < 0.25 Å. The theoretical background and principle of P-EXAFS applied to smectite preparation is well documented (e.g. [START_REF] Manceau | Polarized EXAFS, distance-valence least-squares modeling (DVLS) and quantitative texture analysis approaches to the structural refinement of Garfield nontronite[END_REF][START_REF] Schlegel | Sorption of metal ions on clay minerals. I. Polarized EXAFS evidence for the adsorption of cobalt on the edges of hectorite particles[END_REF]Manceau et al., 2000a). Thus P-EXAFS can provide unique information on the nature, number, distance and orientation of neighboring shells. For Fe, this information can be complemented by the analysis of pre-edge peaks which are related to quadrupolar and (forbidden) dipolar transitions from the 1s to d orbitals. The position and amplitude of these peaks is known to be sensitive to the oxidation state and the site symmetry of Fe [START_REF] Manceau | Surface structural model for ferrihydrite[END_REF][START_REF] Wilke | Oxidation state and coordination of Fe in minerals: an Fe K-XANES spectroscopic study[END_REF]. The combination of pre-edge peaks and P-EXAFS spectroscopy was powerful in determining the Fe short-range environment in Ferich smectites such as nontronites (Manceau et al., 2000a;Manceau et al., 2000b;[START_REF] Gates | Site occupancies by iron in nontronites[END_REF].

In this study, the Fe chemical state and distribution in dioctahedral montmorillonite (SWy-1) and trioctahedral hectorite (SHCa-1) were investigated by XAS. These smectites are taken as representative of dioctahedral and trioctahedral clay minerals. In addition, these minerals are also model systems for material present in the backfill of a nuclear waste repository site and as a secondary phase frequently detected in alteration experiments of nuclear glass, respectively. As such, thorough determination of their crystallochemical properties is useful to improve the robustness of long-term models of waste confinement. In this study, oriented self-supporting films were prepared in order to probe the Fe K-edge by P-EXAFS spectroscopy for SWy-1 and, for the first time, SHCa-1. Also, P-XAS data collected and analyzed to understand how Fe can affect the local structural properties of dioctahedral or trioctahedral smectites.

Experimental

Samples

Montmorillonite (SWy-1) and hectorite (SHCa-1) were purchased from the Source Clays Repository of the Source Clay Mineral Project (SCMP). Montmorillonite was purified according to a standard procedure described in [START_REF] Golubev | Effect of pH and organic ligands on the kinetics of smectite dissolution at 25 °C[END_REF] and the fraction < 1 µm was isolated. For hectorite the fraction < 2 µm was isolated by sedimentation and purified following the procedure described in [START_REF] Schlegel | Sorption of metal ions on clay minerals. I. Polarized EXAFS evidence for the adsorption of cobalt on the edges of hectorite particles[END_REF]. The purified montmorillonite and hectorite fractions were saturated with Na. The elemental compositions of the purified fractioned samples were determined by acid digestion and ICP-MS (Thermo X-Series II) analysis of the resulting solutions. The results are presented in Table 1. Besides the elements listed in Table 1, hectorite also contains large amounts of fluorine (2.7 wt% for the SCMP hectorite, [START_REF] Thomas | FLUORIDE CONTENT OF CLAY-MINERALS AND ARGILLACEOUS EARTH MATERIALS[END_REF], but this element was not quantified because the analysis involved acid digestion using concentrated HF.

To remove possible X-ray amorphous ferric oxides during the purification, hectorite underwent a treatment with dithionite-citrate-bicarbonate that imposes strongly reducing conditions, followed by treatment with hydrogen peroxide that imposes strongly oxidizing conditions. With dithionite, a complete reduction of structural Fe from a trivalent to a divalent oxidation state can be achieved in smectites, such as reported for the Fe-rich SWa-1 (Gorski et al., 2012) and for the montmorillonite SWy-2 [START_REF] Neumann | Evaluation of redox-active iron sites in smectites using middle and near infrared spectroscopy[END_REF], and subsequent re-oxidation can be achieved with hydrogen peroxide [START_REF] Gorski | Redox Properties of Structural Fe in Clay Minerals: 3. Relationships between Smectite Redox and Structural Properties[END_REF]. Furthermore, reported spectroscopic data showed that structural changes in the Fe coordination environment and in the silicate lattice can be caused by the Fe reduction and that these are not fully reversible (e.g., SWy-2 and SWa-1, [START_REF] Neumann | Evaluation of redox-active iron sites in smectites using middle and near infrared spectroscopy[END_REF][START_REF] Gorski | Redox Properties of Structural Fe in Clay Minerals: 3. Relationships between Smectite Redox and Structural Properties[END_REF]). Similarly to the reported montmorillonite data, Fe in hectorite may also have underwent a reduction-oxidation cycle, resulting in partly irreversible structural modifications. However, our infrared spectra do not reveal any strong perturbation of the hectorite lattice, certainly due to the low Fe content.

Purified Na-saturated smectites were first characterized by powder X-ray diffraction and Fouriertransform infrared (FTIR) spectroscopy. The X-ray diffractograms were collected on oriented samples with a D8 Advance (Bruker) diffractometer (Cu K α radiation) equipped with an energy dispersive detector (Sol-X). The FTIR spectra were obtained with a Bruker IFS 55 spectrometer, equipped with an ATR (Attenuated Total Reflectance) cell and an MCT (Mercury Cadmium Telluride) detector. Self-standing films were prepared by slow filtration of the clay suspensions on a 0.025 µm pore size filter (Millipore) [START_REF] Schlegel | Sorption of metal ions on clay minerals. I. Polarized EXAFS evidence for the adsorption of cobalt on the edges of hectorite particles[END_REF]. This protocol readily provided highly textured self-supporting films to perform polarized XAS experiments, with a and b layer axes of the crystallites randomly oriented in the plane of the film.

X-ray absorption spectroscopy (XAS)

Polarized Fe K-edge XAS data of the oriented clay samples were collected at the FAME beamline [START_REF] Proux | FAME: a new beamline for X-ray absorption investigation of very-diluted systems of environmental materials and biological interest[END_REF] at the ESRF (Grenoble, France) with a ring energy of 6 GeV and a ring current of 50-90 mA.

The data were collected in fluorescence-yield detection mode using a 30-element Ge solid state detector (Canberra) at angles (α) between the electric field of the X-ray beam and the clay layer plane of 10, 35, 55 and 80°. The energy calibration was done by setting the first inflection point of the Fe K-edge of an iron foil at 7112.0 eV.

The pre-edge spectra were modeled by pseudo-Voigt line shapes [START_REF] Westre | A multiplet analysis of Fe K-edge 1s->3d pre-edge features of iron complexes[END_REF] after subtraction of the baseline with an exponential function. The energy position, the full width at half-maximum and the peak height were fit using a fixed 50:50 Gaussian:Lorentzian ratio for the peaks. The pre-peak intensity was calculated as the integrated areas of the pseudo-Voigt functions used for peak modeling.

Analysis of the EXAFS data was performed following standard procedures by using the Athena and Artemis interfaces to the Ifeffit software [START_REF] Ravel | HEPHAESTUS: data analysis for X-ray absorption spectroscopy using IFEFFIT[END_REF]. The spectra were extracted from the raw data and the Fourier transforms (FTs) were obtained from the k 3 ×χ(k) functions (3.2 -10.2 Å -1 for both samples at all angles). The spectral data were fit in R space (1.3 -3.8 Å for both samples at all angles) using phase and amplitude functions calculated with feff8.4 [START_REF] Ankudinov | Real-space multiple-scattering calculation and interpretation of x-ray-absorption near-edge structure[END_REF]. The amplitude reduction factor (S 0

2 ) was set to 0.70, a value well suited to properly fit the data of α-Fe (data not shown). In order to generate theoretical paths, the published structures of montmorillonite [START_REF] Tsipursky | The distribution of octahedral cations in the 2-1 layers of dioctahedral smectites studied by oblique-texture electron-diffraction[END_REF] and hectorite [START_REF] Breu | Fehlordnung bei Smectiten in Abhängigkeit vom Zwischenschichtkation[END_REF] were used with the absorber (Fe) located at octahedral position. Since the differences in backscattering amplitude between neighboring Mg, Al and Si are limited, the XAS fitting models were simplified. In montmorillonite, octahedral and tetrahedral contributions were fit by using only Al and Si backscatterers, respectively. In hectorite, only Mg (octahedral) and Si (tetrahedral) contributions were considered. Similarly, O and F only differ by Z ± 1 so that F neighbors in hectorite can be fitted with EXAFS phase and amplitude functions for O with only minimal discrepancy. Thus the first coordination shell of Fe in hectorite can be considered to be made of O atoms only for fitting purposes.

For a given film, the data were fit simultaneously at all angles using a single value of ΔE, and for a given shell a common bond length and mean square displacement ("Debye-Waller" factor). The Fe:Al (montmorillonite) and Fe:Mg (hectorite) ratios were kept equal at all angles. The uncertainties are typically ± 0.02 Å on the distances for well-resolved atomic shells and ± 20 % on the coordination numbers. The experimental uncertainty on α is estimated to ± 1°. The fit quality was quantified by the R f factor representing the absolute misfit between theory and data [START_REF] Ravel | EXAFS Analysis with Feff and Feffit[END_REF].

Results and discussions

X-ray diffraction and infrared spectroscopy

X-ray diffractograms for the two smectites are presented in Figure 2. The basal spacing for montmorillonite (d(001) = 12.2(2) Å) and hectorite (d(001) = 14.8(2) Å) are typical of smectite with one or two interlayer water molecules, respectively [START_REF] Meunier | Clays[END_REF]. Quartz (peak at 26.6 °2θ) is also present in small quantities in montmorillonite, but could not be detected in hectorite. Quartz does not contain Fe and thus will not affect XAS measurements. No other crystalline phase could be detected and the flat backgrounds of the diffractograms indicate the absence of additional X-ray amorphous phase.

The IR spectrum of montmorillonite (Figure 2) matches reported data [START_REF] Madejova | Baseline studies of The Clay Minerals Society Source Clays: Infrared methods[END_REF], with an -OH stretching band of structural hydroxyl groups at 3623 cm -1 and Si-O stretching bands at 989 cm -1 and 1116 cm -1 (longitudinal mode). The spectrum also contains AlAl-OH, AlFe(III)-OH and AlMg-OH deformation bands at 914 cm -1 , 882 cm -1 and 848 cm -1 , respectively. The spectrum of hectorite also matches reported data [START_REF] Madejova | Baseline studies of The Clay Minerals Society Source Clays: Infrared methods[END_REF], with -OH stretching bands of structural hydroxyl groups (3674 cm -1 ) and bonded water (3621 cm -1 ). The presence of adsorbed water is further indicated by two broad bands at 3380 cm -1 (-OH stretching) and ~3265 cm -1 (overtone of -OH deformation of water). This finding indicates that the interlayer of hectorite contains more water than that of montmorillonite for which these bands are absent, what is consistent with 2-layer and 1-layer hydrate state, respectively, determined by XRD. In hectorite, the bands at 960 cm -1 and 689 cm -1 correspond to Si-O stretching and the band at 651 cm -1 to Mg 3 -OH deformation.

The trioctahedral character of this smectite is revealed by the Mg 3 -OH band and the position of the structural hydroxyl group. The Si-O stretching bands at 780 cm -1 and 797 cm -1 can be related to the presence of quartz in both samples [START_REF] Madejova | Baseline studies of The Clay Minerals Society Source Clays: Infrared methods[END_REF]. The two bands are of similar intensity in hectorite, but not in montmorillonite, possibly because the band at 797 cm -1 in montmorillonite can also contain contribution from the Fe(III)Mg-OH bending mode [START_REF] Gates | Cation mass-valence sum (CM-VS) approach to assigning OH-bending bands in dioctahedral smectites[END_REF]. No other phases could be detected.

The XRD and IR data indicate that the smectite preparations were free from Fe-containing ancillary phases, attesting to the success of the purification procedures. Also, -OH bands where the hydroxyl groups are connected to Fe octahedra were detected in the IR spectrum of montmorillonite, but not in hectorite, probably due to the low Fe content of this mineral. FeFe-OH deformation bands were absent in the two spectra, excluding Fe clustering and thus hinting at a random Fe distribution, at least in montmorillonite.

Pre-edge spectroscopy

The Fe K-edge XAS spectra have a weak pre-edge feature ~15 eV below the main absorption edge arising predominantly from 1s → 3d (quadrupolar) transitions. The 1s → 3d transition is electric dipoleforbidden by parity considerations in a centrosymmetric octahedral environment (e.g., O h symmetry) [START_REF] Westre | A multiplet analysis of Fe K-edge 1s->3d pre-edge features of iron complexes[END_REF]. However, a very weak pre-edge feature, split into t 2g -and e g -like components, is still experimentally observed, the intensity depending on the local symmetry and on the cation electronic properties. The pre-peak intensity can thus be used to evaluate the Fe oxidation state and coordination geometry [START_REF] Manceau | Surface structural model for ferrihydrite[END_REF]Manceau et al., 2000a;[START_REF] Wilke | Oxidation state and coordination of Fe in minerals: an Fe K-XANES spectroscopic study[END_REF]. For example, a higher pre-edge amplitude is observed for fourfold coordinated Fe compared to sixfold coordinated Fe, a consequence of mixing 3d and 4p atomic orbitals in T d symmetry [START_REF] Westre | A multiplet analysis of Fe K-edge 1s->3d pre-edge features of iron complexes[END_REF].

The pre-edge is located at around 7114.5 eV for both montmorillonite and hectorite (Figure 3), indicating the Fe is in an oxidation state of +III in both clays. The intensity of the pre-edges is weak (~1.5 % of the main edge), suggesting that Fe atoms are predominantly sixfold coordinated. The pre-edge features were modeled with pseudo-Voigt line shapes [START_REF] Westre | A multiplet analysis of Fe K-edge 1s->3d pre-edge features of iron complexes[END_REF] to gain additional information on their amplitude and position (Figure 4). At α = 35° (polarized and powder XAS data are identical at this angle), the difference in energy (Table 2) of the components for montmorillonite (1.49 eV) compared to hectorite (1.45 eV) is below the uncertainty associated to the position of the features (~0.1 eV). The total pre-peak intensities (i.e., total integrated areas, Table 2) equal 0.097 and 0.094 for montmorillonite and hectorite, respectively. These low values compare with those obtained for sixfold coordinated Fe(III) [START_REF] Wilke | Oxidation state and coordination of Fe in minerals: an Fe K-XANES spectroscopic study[END_REF]. Also, the total pre-peak intensities are similar in both smectites. Interestingly, the ratio between the intensity of the contributions at lower to higher energies equals 1.31 for montmorillonite and 1.00 for hectorite. The intensity ratio of montmorillonite is close to expectation for octahedral high-spin ferric Fe (3:2) [START_REF] Westre | A multiplet analysis of Fe K-edge 1s->3d pre-edge features of iron complexes[END_REF]. The lower value for hectorite may best be explained by site distortion for Fe substituting for Mg/Li in the trioctahedral smectite. However, the low pre-peak intensity compared to the main edge suggested that the site is centrosymmetric and that such site distortion is actually limited. Alternatively, the observed difference may be explained by distinct covalence of the chemical bonds between Fe and the surrounding ligands [START_REF] Westre | A multiplet analysis of Fe K-edge 1s->3d pre-edge features of iron complexes[END_REF]. The e g set of the 3d orbitals is more covalent than the t 2g set due to σ bonding with the ligands. Hectorite contains significant amount of fluorine substituting for OH groups, and because F has an electronegativity higher than O, the observed differences may also be attributed to Fe binding to F in hectorite. Regardless of the correct interpretation, these dissimilarities in pre-peak intensities indicate slight differences in Fe local environments.

The pre-peak intensity ratios do not exhibit a monotonic trend with α, consistent with previous observations on Fe-rich phyllosilicate {Dyar, 2001 #74}. For montmorillonite, the ratio increases from 10° to 55° and then decreases whereas for hectorite it increases from 10° to 35° and then decreases. Since a dipole transition would be dichroic in nature, this complex angular behavior suggests that the transition is predominantly quadrupole [START_REF] Hahn | Observation of an electric quadrupole transition in the X-ray absorption spectrum of a Cu(II) complex[END_REF]. The weakness of the dipole contribution corroborates the predominantly octahedral nature of the coordination polyhedra {Munoz, 2013 #75}.

EXAFS spectroscopy

EXAFS spectra. Both sets of P-EXAFS spectra exhibit a significant angular dependence over the whole k range and well-defined isosbestic points are observed, attesting to the high degree of particle orientations in the self-supporting films (Figure 5). The variation in amplitude and position of the maxima at ~4.2, 5.3, 6.5, 8.0 and 9.1 Å -1 are consistent with the presence of atomic shells with distinct orientations, e.g., in-plane Mg/Al/Fe and out-of-plane Si atoms. The large dependence on polarization is also consistent with the successful preparation of highly oriented clay films.

The powder EXAFS spectra recorded at α = 35° for montmorillonite and hectorite show subtle differences in the position and amplitude of oscillation maxima, e.g., at k ~5.0, 6.3 and 8.0 Å -1 (Figure 3). The presence of distinct frequencies suggests that Fe has distinct molecular environments in terms of nature and number of backscattering atoms, consistent with the dioctahedral nature of montmorillonite and the trioctahedral nature of hectorite. This is also clearly seen on the corresponding Fourier transforms (FT) where the contributions from the cationic shells differ.

Fourier transforms. The FTs contain several peaks (Figure 5). Peak A (R + ΔR ~1.6 Å) corresponds to the first oxygen shell (O1) and its magnitude is higher in montmorillonite than in hectorite. Best fits (Figures 6 and7) were obtained for Fe-O1 interatomic distances of R Fe-O1 = 2.01(1) Å in montmorillonite and 2.00(1) Å in hectorite (Tables 3 and4). These bond lengths are characteristic of sixfold-coordinated Fe(III) and match reported values for Fe located at clay octahedral sites [START_REF] Manceau | Polarized EXAFS, distance-valence least-squares modeling (DVLS) and quantitative texture analysis approaches to the structural refinement of Garfield nontronite[END_REF]Manceau et al., 2000a;[START_REF] Vantelon | Iron distribution in the octahedral sheet of dioctahedral smectites. An Fe K-edge X-ray absorption spectroscopy study[END_REF]. The greater amplitude of peak A for montmorillonite correlates with a greater number of detected O1

neighbors, e.g., at α = 35°, N O1 = 5.8(5) for montmorillonite and 5.1(5) for hectorite (Tables 3 and4). The slightly lower N O1 in hectorite can be related to a larger structural disorder resulting in a broader distribution of Fe-(O,F) bond lengths (Manceau et al., 2000a) around a mean value, which is consistent with the pre-edge results.

The amplitude of peak A decreases for both smectites, and the decrease is more pronounced in montmorillonite than in hectorite (Figure 5). Fits (Figures 6 and7) indicate that N O1 decreases from 6.3(5) at α = 10° to 4.9(4) at α = 80° for montmorillonite and from 5.4(5) to 4.6(4) for hectorite (Tables 3 and4). Using the relationship between N as a function of α and the angle β between the X-ray absorber-backscatterer pair and the normal to the film plane [START_REF] Schlegel | Sorption of metal ions on clay minerals. I. Polarized EXAFS evidence for the adsorption of cobalt on the edges of hectorite particles[END_REF], the orientation of the O1 shell can be estimated. The calculated values of β O1 equal 58.1(1.5)° and 56.9(7)° for montmorillonite and hectorite, respectively. Though the value obtained for montmorillonite deviates significantly from the value expected for fully symmetric octahedra (54.7°), they are consistent with the octahedral flattening observed in most hydrous TOT phyllosilicates [START_REF] Güven | Smectites. In Hydrous Phyllosilicates[END_REF].

Peak B (R + ΔR ~2.75 Å) corresponds to the sum of contributions from the nearest octahedral (Mg,Al,Fe) shell (montmorillonite) or (Mg,Li) shell (hectorite), the nearest tetrahedral Si shell and the nextnearest O shell. The position, amplitude and angular dependences of this peak depend on the interferences between the EXAFS contributions from the octahedral and tetrahedral sheets. For example, peak B for montmorillonite displays a complex polarization dependence: its amplitude decreases from α = 10° to α = 35° and then increases with α, and the position and imaginary parts shift to higher R + ΔR values with α (Figure 5).

In smectites, this angular behavior is diagnostic of in-plane predominant contributions from (Mg,Al) cations in the octahedral sheet, and out-of-plane contributions from tetrahedral Si [START_REF] Schlegel | Sorption of metal ions on clay minerals. I. Polarized EXAFS evidence for the adsorption of cobalt on the edges of hectorite particles[END_REF]. This peak was fit assuming Fe, Al and Si contributions at R Fe-Fe1 = 3.02(1) Å, R Fe-Al1 = 3.04(1) Å and R Fe-Si1 = 3.22(1) Å, respectively (Table 3). Best-fit bond distances match values obtained from diffraction data [START_REF] Tsipursky | The distribution of octahedral cations in the 2-1 layers of dioctahedral smectites studied by oblique-texture electron-diffraction[END_REF]. For increasing α values, the coordination numbers decrease from 0.5(1) to 0.1(1) for the Fe shell, from 3.4(3) to 0.6(1) for the Al shell, and increase from 1.4(7) to 6.6(7) for the Si shell (Table 3). These angular dependences are consistent with Al and Fe shells oriented predominantly in the in-plane dimension while the Si shell is oriented in the out-of-plane direction. The values number of neighboring atoms obtained at α = 35° (N Fe1 = 0.4(1), N Al1 = 2.6(3) and N Si1 = 3.4(3)) are in reasonable agreement with coordination numbers for montmorillonite (3 (Al,Fe) neighbors in-plane and 4 (Si,Al) neighbors out-of-plane). The fit results indicate a small but significant contribution from neighboring Fe and thus the presence of Fe-Fe pairs. This finding is based on the ability of P-EXAFS to filter out overlapping contributions from shells with distinct orientations, thereby decreasing uncertainties on structural parameters. This can explain, at least partly, why our results slightly differ from earlier conclusions based on the analysis of powder EXAFS spectra [START_REF] Vantelon | Iron distribution in the octahedral sheet of dioctahedral smectites. An Fe K-edge X-ray absorption spectroscopy study[END_REF].

In contrast to montmorillonite, the amplitude of peak B in hectorite increases with α and its position varies only slightly (Figure 5). Peak B was modelled considering Mg, Fe and Si shells at R Fe-Mg1 = 3.03(1) Å, R Fe- Fe1 = 3.03(3) Å and R Fe-Si1 = 3.24(1) Å, respectively (Table 4). The bond distances are consistent with values obtained from diffraction data for Mg and Si shells [START_REF] Breu | Fehlordnung bei Smectiten in Abhängigkeit vom Zwischenschichtkation[END_REF] and R Fe-Fe1 is typical of edge-sharing Fe octahedra. The coordination numbers of octahedral Mg and Fe decrease from 2.8(2) to 0.6(1) and from 0.3(1) to 0.1(1), respectively, and increase for Si from 2.1(2) to 6.9(7), for increasing α values (Table 4). The angular dependences are consistent with Mg and Fe shells located in-plane and Si located out-of-plane, but the in-plane number of neighboring Mg+Fe are much lower than expected from the smectite composition. For a trioctahedral structure, six octahedral and four tetrahedral cationic neighbors would be expected. The detected number of tetrahedral neighbors equals 3.4(6), reasonably close to the theoretical value of four, but the number of octahedral neighbors equals ~2, much lower than the expected value of six (Table 4). Several explanations could possibly explain the deviation from the ideal values. First, hectorite contains Li substituting for Mg (Table 1) and

Li is too light to be detected by EXAFS spectroscopy. The impact of this substitution on N Mg can be estimated from the Li:Mg ratio of ~1:10 in hectorite. Assuming random distributions of Mg and Li in the octahedral sheet, the detected number of Mg neighbors should be close to 5.4, a value still significantly higher than the experimental value. Second, as Fe(III) substitutes for Mg(II), the increase in local charge may be balanced by vacancies or, more likely by Li(I) preferential insertion in adjacent octahedral sites, thereby lowering the number of detected octahedral neighbors. A third hypothesis is that electronic waves backscattered by octahedral Fe and Mg/Al are out of phase (Manceau, 1990a) leading to destructive interferences as can be seen in Figures 6 and7.

Owing to this interference, the apparent numbers of Fe and Mg neighbors are mutually lowered and so actual number of neighboring Fe (and Mg) cations. Please note that such interference would also occur in powder spectra, and would be further compounded by the EXAFS contribution of out-of-plane Si. Peak B for hectorite also contains contribution from the next nearest oxygen shell (N O2 = 0.6(1.1)) located on the basal plane (R Fe-O2 = 3.48(1) Å). This shell is not detected in the dioctahedral smectite because of corrugation of the basal oxygen planes that increases the average spread of the Fe-O2 distances (Manceau et al., 2000b). From the increase of N O2 with α (Table 4) this shell is clearly located out-of-plane, matching expectation for a shell located at the basal plane.

Two additional O shells were also used to improve the fit of peak B at higher distances in both smectites (Tables 3 and4). The O3 shell originates from O atoms of adjacent octahedra (R Fe-O3 = 3.78-3.79 Å) and the O4 shell from atoms of the basal planes of the silicate sheet (R Fe-O4 = 4.01(1) Å), and the bond distances are close to reported values [START_REF] Tsipursky | The distribution of octahedral cations in the 2-1 layers of dioctahedral smectites studied by oblique-texture electron-diffraction[END_REF][START_REF] Breu | Fehlordnung bei Smectiten in Abhängigkeit vom Zwischenschichtkation[END_REF]. For montmorillonite, the number of detected backscatterers at α = 35° (N O3 = 5.8(1.3) and N O4 = 4.2(1.9)) match coordination numbers expected for smectites (N O3 = 6 and N O4 = 4), whereas they are much lower than expected for hectorite (N O3 = 2.8(1.5) and N O4 = 1.0(1.3)). This again suggests that the phyllosilicate local structure around Fe(III) is distorted in hectorite.

For montmorillonite and hectorite, N O4 increases from 2.4(2.4) to 8.0(1.3) and from 0.1(1) to 5.5(5), respectively, for α increasing from 10° to 80°. These variations match expectations for the O4 shell located at the smectite basal planes, i.e., in out-of-plane direction. The O3 coordination number is invariant within uncertainty both for montmorillonite (N O3 = 5.4(1.6) to 6.1(9) for  = 10 to 80°, respectively) and hectorite (N O3 = 2.4(1.3) to 4.1(1.9) for  = 10 to 80°, respectively). Thus no clear angular dependence can be obtained from these values but they are compatible with the known orientation of the O3 shell inclined toward the octahedral sheet{Schlegel, 2001 #76}.

Discussion

X-ray diffraction and infrared data showed that both purified smectites are free from Fe-containing impurities, implying that Fe can only be located in the smectite structure. Infrared data are also consistent with a dioctahedral structure for montmorillonite and a trioctahedral structure for hectorite. The pre-edge and EXAFS spectroscopic data indicate that Fe is in trivalent oxidation state and located in the octahedral sheet but that the Fe local environments in the two clays are dissimilar. The bond lengths and the angular dependences of the neighboring atomic shells confirm this structural assignment and indicate that the amount of Fe in the tetrahedral sheet is marginal.

In montmorillonite, the number of cationic neighbors detected in-plane (N Fe1 + N Al1 = 3) is in agreement with a dioctahedral structure (N = 3). Fits to the data are consistent with Fe substituting for Al/Mg in the octahedral layer. Fe-Fe pairs were detected, but with a low number of backscatterers (N Fe1 = 0.4(1)). The elemental composition (Table 1) indicates a moderate Fe content (2.3 wt%) and the corresponding (Al+Mg):Fe molar ratio equals 9.4. Considering a fully random Fe distribution with all Al located in the octahedral sheet, about 0.3 neighboring Fe atom would be expected. This calculated value is similar to the fit results within uncertainties. This finding excludes Fe clustering and suggests that Fe is randomly distributed in montmorillonite.

Earlier powder EXAFS analysis [START_REF] Vantelon | Iron distribution in the octahedral sheet of dioctahedral smectites. An Fe K-edge X-ray absorption spectroscopy study[END_REF] also indicated an ordered Fe distribution in this montmorillonite but obeying an exclusion rule. However, powder XAS applied to sheet silicates is affected by larger uncertainties due to the interferences between waves backscattered by shells located at similar distances {Manceau, 1990 #55}. This limitation was overcome in the present study by using the improved sensitivity of P-EXAFS spectroscopy, and low number of Fe backscatterers could be unambiguously detected. This improved sensitivity of P-EXAFS spectroscopy was subsequently applied to investigate in detail and for the first time, the Fe distribution in the trioctahedral hectorite.

In hectorite all octahedral sites are filled by various cations such as Li, Mg, Al and Fe, and in addition some F substitutes for OH groups. This complex chemical composition of the octahedral sheet could somewhat complicate the powder EXAFS investigation of the Fe environment, thus requiring the use of P-EXAFS spectroscopy. The pre-edge is particularly sensitive to the electronic and geometric structure of the Fe site. The analysis of the hectorite pre-edge suggests differences in the binding environment compared to montmorillonite that may be attributed to some F substitution for OH groups. This substitution possibly distorted the site geometry and/or modified the ligand field. Although OH and F have similar ionic radii (r(OH) = 1.32 Å, r(F) = 1.28 Å in II-coordination; [START_REF] Shannon | Revised effective ionic radii and systematic studies of interatomic distances in halides and chalcogenides[END_REF], F is more electronegative than O, which can decrease the length of the Fe-F bond compared to the Fe-O one. This is confirmed by reported R Mg-O and R Mg-F distances in hectorite [START_REF] Breu | Fehlordnung bei Smectiten in Abhängigkeit vom Zwischenschichtkation[END_REF] differing by ~0.06 Å. The Fe-(O,F) 6 coordination octahedron is thus probably slightly distorted, as corroborated by the slightly larger mean square displacement of the O1 shell in hectorite (σ 2 = 0.006 Å 2 ) compared to montmorillonite (σ 2 = 0.005 Å 2 ). The distribution of interatomic distances around a mean value dampens the EXAFS oscillatory contribution from the (O,F) 1 shell, thus leading to a slightly lower number of neighboring (O,F) atoms. Furthermore, the mean square displacement is also larger for octahedral neighbors in hectorite (Mg, Fe) compared to montmorillonite (Al, Fe). However, previous NMR data have demonstrated Fe-F avoidance in phyllosilicate, i.e., F is usually not associated to Fe octahedra {Sanz, 1983 #79}. Also, NMR investigation on hectorite failed to report evidence for Fe-F association, although this could be due to the minute amount of structural Fe (0.2 %wt) compared to the high F content (up to 5.18 wt% in some preparations) {Thomas, 1977 #4008}. In addition, any distortion due to the presence of F is unlikely to result in a large incoherency of the distances between Fe and the neighboring cations. Indeed, the limited nature of this disorder is attested by the ability of conventional diffraction methods to derive a structural model for hectorite without the need of complex corrections {Oberlin, 1966 #77;Breu, 2003 #7}. The benign nature of this distortion is corroborated by the detection of similar numbers of Si atoms at very similar distances and comparable mean square displacement in both smectites.

Hectorite has a low Fe content (0.2 wt%) and the corresponding (Al+Mg):Fe molar ratio equals 130.

Considering a fully random distribution of Fe in the octahedral sheet, the number of neighboring Fe detected by EXAFS would be about 0.06, slightly smaller than the detected value of 0.2(1), values close to the lower limit of detection of EXAFS. This would suggest that Fe atoms within the octahedral sheet may preferentially form some pairs, as reported for trioctahedral micas [START_REF] Manceau | Distribution of Fe in the octahedral sheet of trioctahedral micas by polarized EXAFS[END_REF]. However, the extended formation of Fe(III) clusters within the octahedral sheet is ruled out because it would have led to much greater N Fe values.

Fe is also surrounded by Mg neighbors, but the number of detected Mg is much smaller than the theoretical number for random distribution in hectorite (N Mg = 5.4), which cannot be explained by structural disorder alone. In hectorite, almost all octahedral sites are filled with cations of similar sizes when sixfold coordinated by oxygen atoms (r VI (Mg(II)) = 0.72 Å and r VI (Li(I)) = 0.76 Å [START_REF] Shannon | Revised effective ionic radii and systematic studies of interatomic distances in halides and chalcogenides[END_REF]), but of different charge. The sixfold coordinated Fe(III) is slightly smaller (r VI (Fe(III) = 0.65 Å) than Mg or Li so that the substitution would not result in significant lattice strain. However, Fe(III) insertion in octahedral sites may be Figure 4. Normalized pre-edge spectra (solid black line) and best fits (dashed red lines) with individual components (blue and green thin lines) for montmorillonite (left) and hectorite (right).

Figure 5. Experimental P-EXAFS spectra (left) with the corresponding Fourier transforms (right) for montmorillonite and hectorite. Figure 6. Experimental (solid black lines) and modeled (dashed red lines) polarized Fourier transforms for the montmorillonite data. The right panel indicates the contributions at α = 35° of the single atomic shells. Figure 7. Experimental (solid black lines) and modeled (dashed red lines) polarized Fourier transforms for the hectorite data. The right panel indicates the contributions at α = 35° of the single atomic shells. a The data were fit over the entire range considering the data from both tables: N is the coordination number, R is the interatomic distance, σ 2 is the mean squared displacement.

The number in parentheses indicates the uncertainty. b Shift in ionization energy, threshold energy E 0 taken as zero crossing of the second derivative. c Figure of merit of the fit [START_REF] Ravel | EXAFS Analysis with Feff and Feffit[END_REF]. a The data were fit over the entire range considering the data from both tables: N is the coordination number, R is the interatomic distance, σ 2 is the mean squared displacement.

The number in parentheses indicates the uncertainty. b Shift in ionization energy, threshold energy E 0 taken as zero crossing of the second derivative. c Figure of merit of the fit [START_REF] Ravel | EXAFS Analysis with Feff and Feffit[END_REF].

  locally charge-balanced by octahedral vacancies and/or by Li(I) filling neighboring octahedra. Because Li cannot be detected by EXAFS spectroscopy, these two hypotheses cannot be discriminated, but both of them would result in a low number of detected octahedral neighbors. In fact, the presence of vacancies surrounding Fe(III) would correspond to the local formation of a dioctahedral-like environment around Fe(III), a little similar to the Fe(III) local environment in montmorillonite. However, it would probably lead to a large deficit of local charge.Interestingly, in dioctahedral smectites, deficit of local charge can be decreased by thermal diffusion of Li(I) cations in vacant octahedra {Komadel, 2005 #80}. In the case of hectorite, such a diffusion would not be driven by heat, though, but would readily occur during crystallization. Other charge-balance mechanisms include the deprotonation of hydroxyl groups of the octahedral sheet close to Fe(III), but this would probably lead to severe distortion of the Fe coordination sphere. Still another mechanism of local charge compensation would be the substitution of Si(IV) by Al(III) in the tetrahedral sheet. From our EXAFS results, however, we surmise that local charge compensation by Li .ConclusionsThe local chemical environment around Fe in the dioctahedral smectite montmorillonite and, for the first time, in the trioctahedral smectite hectorite, was characterized by P-EXAFS spectroscopy. Iron(III) in these smectites is surrounded by in-plane (Mg,Al,Fe) atoms and out-of-plane Si atoms at distances in agreement with previous studies. Furthermore, Fe-Fe pairs in limited number could be unambiguously detected, owing to the ability of P-EXAFS to filter out overlapping contributions from shells with distinct orientations. Low numbers of neighboring Fe were detected, and no extended clustering could be observed in either of the minerals, indicating a next-to-random distribution of Fe in the octahedral sheet. Furthermore, the XAS data indicate that the substitution of Fe for Al in the dioctahedral montmorillonite does only marginally affect the local site symmetry.In contrast, the substitution of Mg(II) by Fe(III) creates an excess of charge, which is balanced by a deficit of charge in the neighboring octahedra, either in the form of vacancies or as Mg(II) substitutions by Li(I). If confirmed, this result would provide a nice example on how impurities in clay minerals can drive heterogeneities in the charge distributions at clay mineral surfaces. The additional amount of lattice flexibility offered by the presence of vacant sites would also explain the ability of hectorite to incorporate large cations such as lanthanides {Finck, 2009 #81}, and, possibly, actinides.

Figure captions Figure 1 .

 captions1 Figure captions

Figure 1 .

 1 Figure 1. Top: Structure of smectite. T: tetrahedral sheet, O: octahedral sheet. Middle: projection down c* of trioctahedral framework. Bottom: projection down c* of dioctahedral framework. Blue triangles are (Si,Al)O 4 tetrahedra, one tetrahedral sheet is not shown. M1 denotes trans sites, and M2 cis sites.

Figure 2 .

 2 Figure 2. X-ray diffractogram (basal spacing or d(001) is indexed) and ATR-FTIR spectrum of the clay fractions investigated in this study. Top: Montmorillonite. Bottom: Hectorite.

Figure 3 .

 3 Figure 3. Comparison of experimental Fe K-edge XANES (left), and EXAFS spectra (middle) recorded at α = 35° for montmorillonite and hectorite with the corresponding Fourier transforms (right).

Figure 4 .

 4 Figure 4. Normalized pre-edge spectra (solid black line) and best fits (dashed red lines) with individual components (blue and green thin lines) for montmorillonite (left) and hectorite (right).

Figure 5 .

 5 Figure 5. Experimental P-EXAFS spectra (left) with the corresponding Fourier transforms (right) for montmorillonite and hectorite.

Figure 6 .

 6 Figure 6. Experimental (solid black lines) and modeled (dashed red lines) polarized Fourier transforms for the montmorillonite data. The right panel indicates the contributions at α = 35° of the single atomic shells.

Figure 7 .

 7 Figure 7. Experimental (solid black lines) and modeled (dashed red lines) polarized Fourier transforms for the hectorite data. The right panel indicates the contributions at α = 35° of the single atomic shells.

Table captions Table 1 .

 captions1 Main cations content of montmorillonite (SWy-1) and hectorite (SHCa-1) determined by chemical analysis (Other includes oxygen). Table2. Results from the pre-edge fitting (uncertainties on the energies are estimated to 0.1 eV and 10 % on the intensities). Table3. Quantitative EXAFS analysis for montmorillonite a Table4. Quantitative EXAFS analysis for hectorite a

Table 1 .

 1 Main cations content of montmorillonite (SWy-1) and hectorite (SHCa-1) determined by chemical analysis (Other includes oxygen).

	(wt %)	Li	Mg	Al	Si	Fe	Other	Total
	SWy-1	<0.01	1.36 (±0.01)	8.93 (±0.07)	25.84 (±3.30)	2.30 (±0.14)	61.57	100.00
	SHCa-1	0.39 (0.01)	12.15 (±0.09)	0.40 (±0.01)	21.08 (±2.69)	0.24 (±0.01)	65.74	100.00

Table 2 .

 2 Results from the pre-edge fitting (uncertainties on the energies are estimated to 0.1 eV and 10 % on the intensities).

	Sample	α	Pre-edge peak energy [eV]	Pre-edge peak intensity 1	Peak energy difference 2	Peak intensity ratio 3
		10°	7113.83 7115.31	0.050 0.048	1.48	1.04
		35°	7113.81 7115.30	0.055 0.042	1.49	1.31
	Montmorillonite	55°	7113.81 7115.27	0.055 0.040	1.46	1.38
		80°	7113.81 7115.25	0.051 0.050	1.44	1.02
		10°	7113.82 7115.23	0.042 0.048	1.41	0.88
		35°	7113.76 7115.21	0.047 0.047	1.45	1.00
	Hectorite	55°	7113.77 7115.24	0.048 0.052	1.47	0.92
		80°	7113.81 7115.26	0.044 0.061	1.45	0.72

Table 3 .

 3 Quantitative EXAFS analysis for montmorillonite a

	α	N	Fe ↔ O1 R [Å]	σ 2 [Å 2 ]	N	Fe ↔ Fe1 R [Å]	σ 2 2 ]	N	Fe ↔ Al1 R [Å]	σ 2 [Å 2 ]
	10°	6.3(5)			0.5(1)			3.4(3)
	35° 55°	5.8(5) 5.5(5)	2.01(1)	0.005	0.4(1) 0.2(1)	3.02(1)	0.004	2.6(3) 1.4(5)	3.04(1)	0.004
	80°	4.9(4)			0.1(1)			0.6(1)
	α	N	Fe ↔ Si1 R [Å]	σ 2 [Å 2 ]	N	Fe ↔ O3 R [Å]	σ 2 [Å 2 ]	N	Fe ↔ O4 R [Å]	σ 2 [Å 2 ]	ΔE 0 [eV] b	c (×10 3 ) R f
	10°	1.4(7)			5.4(1.6)			2.4(2.4)				5.6
	35° 55°	3.4(3) 5.2(5)	3.22(1)	0.007	5.8(1.3) 6.0(1.1)	3.79(1)	0.004	4.2(1.9) 6.3(1.6)	4.01(2)	0.007	6.8(4)	5.4 5.6
	80°	6.6(7)			6.1(9)			8.0(1.3)				8.3

Table 4 .

 4 Quantitative EXAFS analysis for hectorite a

	α	N	Fe ↔ O1 R [Å]	σ 2 [Å 2 ]	N	Fe ↔ Mg1 R [Å]	σ 2 [Å 2 ]	N	Fe ↔ Fe1 R [Å]	σ 2 [Å 2 ]	N	Fe ↔ Si1 R [Å]	σ 2 [Å 2 ]
	10°	5.4(5)			2.8(2)				0.3(1)					2.1(2)
	35° 55°	5.1(5) 4.9(4)	2.00(1)	0.006	1.7(6) 1.1(6)		3.03(1)	0.008	0.2(1) 0.1(1)	3.03(3)		0.008	3.4(6) 5.1(6)	3.24(1)	0.007
	80°	4.6(4)			0.6(1)				0.1(1)					6.9(7)
	α	N	Fe ↔ O2 R [Å]	σ 2 [Å 2 ]	N		Fe ↔ O3 R [Å]	σ 2 [Å 2 ]	N		Fe ↔ O4 R [Å]	σ 2 [Å 2 ]	ΔE 0 [eV] b	c (×10 3 ) R f
	10°	0.1(1)			2.4(1.3)			0.1(1)				4.7
	35° 55°	0.6(1.1) 1.4(1.1)	3.48(1)	0.006	2.8(1.5) 3.9(1.6)	3.78(1)	0.008	1.0(1.3) 3.7(1.4)	4.01(1)	0.008	6.6(2)	3.6 2.9
	80°	1.4(6)			4.1(1.9)			5.5(5)				4.4

peak intensities correspond to integrated areas.

peak energy differences correspond to energy differences between the lowest energy and the highest energy pre-edge features.

ratio of the lowest energy peak intensity to the highest energy peak intensity.

Acknowledgements

The European Synchrotron Radiation Facility is acknowledged for provision of synchrotron radiation beamtime.

We thank O. Proux for assistance during XAS data collection at the FAME beamline.


Maria-Lorena Gonzalez

Stéphane Blain

Ingrid Obernosterer
email: ingrid.obernosterer@obs-banyuls.fr

Seasonal freshening of NW Mediterranean surface water impacts microbial heterotrophic activity and dissolved organic matter

Keywords: Low salinity waters, microbial heterotrophic activity, dissolved organic matter, NW Mediterranean Sea

The Rhone river represents the most important source of freshwater, nutrients and organic matter to the northwestern (NW) Mediterranean Sea and riverine input markedly affects biogeochemistry and ecosystem functioning in the estuarine and coastal zone. Structures of low salinity waters (LSW) originating near the river plume can also be transported along the continental shelf and offshore. The objective of the present study was to investigate the influence of LSW distant from their source, focusing on dissolved organic matter (DOM) and related microbial processes during two annual cycles (2007 and 2008) at a time series site characterized by the regular occurrence of LSW in spring (Microbial Observatory Laboratoire Arago). We observed enhanced bacterial heterotrophic production and community respiration and specific DOM features within these LSW, concurrently with low net community production. Our results suggest that LSW represent a mechanism of labile DOM supply, thereby sustaining enhanced heterotrophic microbial metabolism.

Introduction

Riverine input represent approximately 40% of fresh water entering the ocean [START_REF] Dagg | Transformation of dissolved and particulate materials on continental shelves influenced by large rivers: plume processes[END_REF], and thereby plays a key role in linking terrestrial and marine environments. Rivers deliver a large amount of inorganic nutrients to the adjacent coastal area, stimulating rates of primary production and associated food web processes [START_REF] Frame | Does the Columbia River plume influence phytoplankton community structure along the Washington and Oregon coasts?[END_REF][START_REF] Iriarte | Primary production and plankton carbon biomass in a river-influenced upwelling area off Concepción, Chile[END_REF][START_REF] Liu | Picophytoplankton and bacterioplankton in the Mississippi River plume and its adjacent waters[END_REF]. Riverine discharge also transports terrigenous particulate (POM) and dissolved organic matter (DOM), mainly from terrestrial vegetation, to the coastal ocean [START_REF] Bianchi | Temporal variability in sources of dissolved organic carbon in the lower Mississippi River[END_REF][START_REF] Medeiros | Export of terrigenous dissolved organic matter in a broad continental shelf[END_REF][START_REF] Opsahl | Distribution and cycling of terrigenous dissolved organic matter in the ocean[END_REF]. DOM from freshwater input of the 30 major rivers, accounts for 90,2 Tg dissolved organic carbon (DOC) per year (Raymond and ocean is not well quantified due to multiple biotic and abiotic processes involved, and the spatiotemporal heterogeneity of river-influenced ocean margins [START_REF] Bianchi | The role of terrestrially derived organic carbon in the coastal ocean: A changing paradigm and the priming effect[END_REF]. In large river systems, such as for example the Mississippi-Atchafalaya river, biological mineralization results in the removal of about 40% of terrestrial DOM in shelf waters [START_REF] Fichot | The fate of terrigenous dissolved organic carbon in a riverinfluenced ocean margin[END_REF].

Export of terrigenous DOC from the Amazon river varies on a seasonal scale and accounts for 50% to 76% [START_REF] Medeiros | Fate of the Amazon River dissolved organic matter in the tropical Atlantic Ocean[END_REF]. How riverine input of nutrients and organic matter affect marine ecosystems has been investigated in a range of estuarine and coastal environments (Cole et al., 2007, Dagg et al., 2004 andreference therein). The influence of terrestrial input at varying distances from shore is, however, far less understood.

The Mediterranean Sea is a mid-latitude semi-enclosed sea, divided in an eastern extreme oligotrophic basin (<0.1 µg Chlorophyll a L -1 ) and a moderately-oligotrophic western basin (0.1 to 3 µg Chlorophyll a L -1 ) [START_REF] Bosc | Seasonal and interannual variability in algal biomass and primary production in the Mediterranean Sea, as derived from 4 years of SeaWiFS observations[END_REF], mainly due to P-limitation [START_REF] Krom | Phosphorus limitation of primary productivity in the eastern Mediterranean Sea[END_REF][START_REF] Mcgill | The relative supplies of phosphate, nitrate and silicate in the Mediterranean sea[END_REF]. The Rhone River discharge represents the most important freshwater source to the Mediterranean Sea [START_REF] Naudin | Transfer mechanisms and biogeochemical implications in the bottom nepheloid layer. A case study of the coastal zone off the Rhone River (France)[END_REF]. The average runoff of 1750 m 3 s -1 varies strongly with season and is highest during the frequent precipitation events in spring and autumn (Ludwig et al., 2009, Naudin and[START_REF] Naudin | Transfer mechanisms and biogeochemical implications in the bottom nepheloid layer. A case study of the coastal zone off the Rhone River (France)[END_REF]. The Rhone River represents an important source of inorganic and organic matter of continental origin to the Mediterranean Sea (de [START_REF] De Madron | Marine ecosystems' responses to climatic and anthropogenic forcings in the Mediterranean[END_REF]. Increased inorganic and organic nutrient concentrations are reported near the Rhone river freshwater plume in shelf waters (100m depth, up to about 20 nautical miles offshore) in the Gulf of Lion [START_REF] Diaz | Biogeochemical and ecological functioning of the low-salinity water lenses in the region of the Rhone River freshwater influence, NW Mediterranean Sea[END_REF][START_REF] Sempéré | Microbial decomposition of large organic particles in the northwestern Mediterranean Sea: an experimental approach[END_REF] and the associated enhanced biological activity in the estuary and adjacent waters has been documented during several previous field studies [START_REF] Christaki | Community structure and trophic role of ciliates and heterotrophic nanoflagellates in Rhone River diluted mesoscale structures (NW Mediterranean Sea)[END_REF], Joux et al., 2009[START_REF] Lefevre | Review of gross community production, primary production, net community production and dark community respiration in the Gulf of Lions[END_REF][START_REF] Pujo-Pay | Impact of phytoplankton and bacterial production on nutrient and DOM uptake in the Rhône River plume (NW Mediterranean)[END_REF].

The hydrodynamics of the Rhone river plume are governed by atmospheric conditions, discharge volume and larger scale water mass circulation. When south-easterly winds predominate, the plume remains close to the coast, while north-westerly winds result in the extension of the Rhone rive plume in a south-western direction [START_REF] Demarcq | La dynamique superficielle du panache du Rhône d'après l'imagerie infrarouge satellitaire[END_REF].

Intrusions of Rhone river water were detected in 5-20% of a 4-year bi-monthly survey in the Bay of Marseille [START_REF] Fraysse | Intrusion of Rhone River diluted water into the Bay of Marseille: Generation processes and impacts on ecosystem functioning[END_REF], indicating that these events are frequent phenomena in the coastal Mediterranean Sea. Diluted structures with low salinity waters (LSW) detached from the Rhone river plume can be transferred to open waters [START_REF] Barrier | Strong intrusions of the Northern Mediterranean Current on the eastern Gulf of Lion: insights from in-situ observations and high resolution numerical modelling[END_REF][START_REF] Diaz | Biogeochemical and ecological functioning of the low-salinity water lenses in the region of the Rhone River freshwater influence, NW Mediterranean Sea[END_REF][START_REF] Nencioli | Surface coastal circulation patterns by in-situ detection of Lagrangian coherent structures[END_REF][START_REF] Petrenko | A review of the LATEX project: mesoscale to submesoscale processes in a coastal environment[END_REF]. Under conditions of north-westerly winds and in combination with a persistent slope current along the shelf break of the Gulf of Lion (Northern Current; [START_REF] Raimbault | Research activities in the Gulf of Lion (NW Mediterranean) within the 1997-2001 PNEC project[END_REF]), these LSW can be directed south-west (Fig. 1). The transit of these buoyant water masses can last up to 3 weeks, and they can be transported over relatively long distances [START_REF] Diaz | Biogeochemical and ecological functioning of the low-salinity water lenses in the region of the Rhone River freshwater influence, NW Mediterranean Sea[END_REF], rendering the Gulf of Lion an appropriate site for the investigation of cross-shelf exchange [START_REF] Petrenko | A review of the LATEX project: mesoscale to submesoscale processes in a coastal environment[END_REF].

The objective of the present study was to investigate whether these structures, referred to as lower salinity waters (LSW), represent a supply of biologically labile DOM to the offshore NW Mediterranean Sea. We addressed this question by observations at the time-series site MOLA (Microbial Observatory of the Laboratoire Arago) that is characterized by the recurrence of LSW at surface in spring (Fig. 1). The combined investigation of the temporal changes in environmental characteristics and microbial processes at Station MOLA provided insight on how freshwater input through these physical features could affect biogeochemistry in the offshore 90 Mediterranean Sea. S1) that sustain the spring phytoplankton bloom. During the two years considered here, maximum concentrations of Chlorophyll a (Chl a; 0.75 and 0.85 µg L - 1 ) (Fig. S3; Table S1) were observed in the upper 40m in March 2007 and February 2008, respectively [START_REF] Laghdass | Impact of lower salinity waters on bacterial heterotrophic production and community structure in the offshore NW Mediterranean Sea[END_REF]. During summer, the water column is thermally stratified (Fig. S4) and concentrations of inorganic nutrients are low (<0.35 µM NO 3 -+ NO 2 -, ~ <0.03

µM PO 4 3-) in the surface mixed layer (10 to 30m) and Chl a concentrations in surface waters remain <0.2 µg L -1 [START_REF] Laghdass | Impact of lower salinity waters on bacterial heterotrophic production and community structure in the offshore NW Mediterranean Sea[END_REF] (Table S1).

Sample collection

Seawater was sampled between February 2007 and October 2008 on a monthly time scale except for the autumn-winter period when the sampling frequency was reduced due to bad weather conditions. Samples were collected in the upper 150m water column with 12 liter Niskin bottles mounted on a rosette equipped with a SeaBird SBE 19 CTD. For bacterial heterotrophic production, and the characterization of dissolved organic matter 8 depths in the upper 150m were sampled. For the determination of microbial community respiration and net community production 6 depths in the euphotic layer were sampled (upper 80m).

Bacterial heterotrophic production, microbial community respiration and net community production

We used ( 3 H) leucine incorporation and the centrifugation method to determine bacterial heterotrophic production (BP) [START_REF] Smith | A simple, economical method for measuring bacterial protein synthesis rates in seawater using 3H-leucine[END_REF]. The detailed protocol and data are reported in [START_REF] Obernosterer | Rapid bacterial mineralization of organic carbon produced during a phytoplankton bloom induced by natural iron fertilization in the Southern Ocean[END_REF][START_REF] Laghdass | Impact of lower salinity waters on bacterial heterotrophic production and community structure in the offshore NW Mediterranean Sea[END_REF]. Bacterial growth rates (BGR)

were determined by dividing BP by cell biomass derived from bacterial abundance. To convert abundance to biomass, we applied a conversion factor of 12.4 fg C cell -1 [START_REF] Fukuda | Direct determination of carbon and nitrogen contents of natural bacterial assemblages in marine environments[END_REF]). We measured microbial community respiration (CR) and net community production (NCP) from changes in the concentration of dissolved oxygen (O 2 ) in 24h light and dark incubations as described in [START_REF] Lefèvre | The metabolic balance at two contrasting sites in the Southern Ocean: The iron-fertilized Kerguelen area and HNLC waters[END_REF]. Incubations were performed at Photosynthetically Active Radiation (PAR) levels of 1%, 4%, 8%, 25%, 50% and 100% of surface values, using optical density filters (Nickel screens). The outdoor incubators were connected with a running seawater system to maintain the incubation bottles at the temperature of the mixed layer. The 1% light depth was below the mixed layer depth during spring and summer with differences in temperature ranging between 2°C in May-June and up to 5° in August (Table S1). The measurements at time zero and all incubations were done in 8 replicate 125 mL borosilicate glass bottles. Dissolved O 2 concentration was determined by spectrophotometric detection of iodine following the Winkler reaction, using a Hitachi U-3010 Spectrophotometer equipped with a sipper system [START_REF] Labasque | Spectrophotometric Winkler determination of dissolved oxygen: re-examination of critical factors and reliability[END_REF].

2.4. Dissolved organic carbon and total hydrolysable amino acids Samples for dissolved organic carbon (DOC) and dissolved total hydrolysable amino acids (THAA) were filtered through two pre-combusted glass fiber filters (Whatman GF/F 25mm, 0.7 µm nominal pore size). Samples for DOC were acidified (H 3 PO 4 , pH 2) and stored in pre-combusted glass tubes at room temperature in the dark until analysis. DOC was analyzed by high temperature catalytic oxidation (HTCO) technique [START_REF] Benner | A critical evaluation of the analytical blank associated with DOC measurements by high-temperature catalytic oxidation[END_REF]) using a Shimadzu TOC-5000 analyzer.

THAA were analyzed by high performance liquid chromatography (HPLC) after liquidphase acid hydrolysis according to a recent protocol described by [START_REF] Escoubeyrou | Quantification of free, dissolved combined, particulate, and total amino acid enantiomers using simple sample preparation and more robust chromatographic procedures[END_REF]. This protocol uses a high volume (100 µL) of undiluted samples injected on a robust hybrid C18 column. HPLC analyses were performed on an Ultimate 3000 Dionex system equipped with an autosampler and a fluorescence detector (excitation at 335 nm, emission at 450 nm). The separation was done with a Phenomenex Gemini C18 column

Results and Discussion

To investigate whether LSW revealed a specific biogeochemical signature at Station MOLA, we compared the depth profiles of microbial heterotrophic activity and of the DOM characteristics at the dates of the most pronounced salinity anomalies during the observation period (May 22 2007 and June 9 2008) with those of the annual medians (Fig. 2). Bacterial heterotrophic production (pmol Leu L -1 h -1 ), CR: Community Respiration (µM O 2 L -1 d -1 ), DOC: Dissolved Organic Carbon (µM), THAA: Total Hydrolizable Amino Acids (µM), DI:

Dauwe Degradation Index.

The extent of the salinity anomalies of about -0.3 was similar in both years, but lower salinity waters were present to 60m in 2007 and constrained to the upper 30m in 2008. The annual median values of BP varied between 10-20 pmol Leu L -1 h -1 in the upper 60 m water column and they were below 5 pmol Leu L -1 h -1 ≥ 100 m for both years. During LSW, rates of BP were 2-4 times higher than the annual medians in the upper 40 m water column, a pattern that was particularly pronounced in 2008. Annual medians of CR varied between 0.5 and 1 µmol O 2 L -1 d -1 in the upper 40 m water column and they were < 0.5 µmol O 2 L -1 d -1 below this depth.

During LSW in 2008, rates of CR were up to 3-fold higher than the annual median, while differences were minor in 2007. In addition, bacterial growth rates revealed highest values (0.17 to 0.26 d -1 ) during the LSW of both years (Table S1). These observations indicate a signature of enhanced microbial heterotrophic activity within LSW that was more pronounced in 2008 than in 2007, possibly due to a stronger dilution with surrounding water masses during our first year of observation.

To link these observations to the characteristics of DOM, we considered bulk DOC, the concentration of dissolved THAA and the DI (Fig. 2). Annual medians of bulk DOC concentrations varied between 60 µM and 80 µM in the upper 150 m water column. In surface waters, we did not observed any pronounced differences between the annual medians and LSW. This is most likely due to the high DOC concentrations in surface waters during summer observed at our study site [START_REF] Laghdass | Impact of lower salinity waters on bacterial heterotrophic production and community structure in the offshore NW Mediterranean Sea[END_REF]. The accumulation of DOC in stratified surface waters during summer is a recurrent phenomenon reported for Mediterranean waters [START_REF] Avril | DOC dynamics in the northwestern Mediterranean Sea (DYFAMED site)[END_REF][START_REF] Romera-Castillo | Production of chromophoric dissolved organic matter by marine phytoplankton[END_REF][START_REF] Sánchez-Pérez | Aerosol inputs affect the optical signatures of dissolved organic matter in NW Mediterranean coastal waters[END_REF] and other oligotrophic oceans (for BATS- [START_REF] Carlson | Effect of nutrient amendments on bacterioplankton production, community structure, and DOC utilization in the northwestern Sargasso Sea[END_REF]; for HOTS- [START_REF] Mopper | Fluorescence as a possible tool for studying the nature and water column distribution of DOC components[END_REF]). Inorganic nutrient limitation of heterotrophic microbes has been suggested as the underlying mechanism for the observed build-up of DOC in stratified surface waters [START_REF] Thingstad | Conceptual models for the biogeochemical role of the photic zone microbial food web, with particular reference to the Mediterranean Sea[END_REF].

This idea is supported by short-term (24h) incubation experiments performed at our study site revealing that labile organic carbon and phosphorus were the main limiting factors of BP during summer [START_REF] Laghdass | High contribution of SAR11 to microbial activity in the North West Mediterranean Sea[END_REF].

Amino acids represent a quantitatively significant and biologically labile fraction of DOM, and temporal or spatial changes of this pool can therefore provide insights to modifications of DOM characteristics [START_REF] Davis | Amino acid and amino sugar yields and compositions as indicators of dissolved organic matter diagenesis[END_REF]Benner, 2012). The concentrations of dissolved THAA varied between 262 and 1027 nM, with higher concentrations in the upper 40m. Within LSW in 2008, THAA concentrations were up to 2-fold higher than the annual median, a pattern that was absent in 2007 (Fig. 2 et al. 1999). Based on the amino acid composition of marine plankton and sediment organic matter from varying depth layers, the DI was initially proposed to characterize the degradation stage of particulate organic matter. Its application has been extended to DOM in a range of marine [START_REF] Amon | Linkages among the bioreactivity, chemical composition, and diagenetic state of marine dissolved organic matter[END_REF][START_REF] Davis | Amino acid and amino sugar yields and compositions as indicators of dissolved organic matter diagenesis[END_REF][START_REF] Tremblay | Origin and fate of particulate and dissolved organic matter in a naturally iron-fertilized region of the Southern Ocean[END_REF][START_REF] Kaiser | Biochemical composition and size distribution of organic matter at the Pacific and Atlantic time-series stations[END_REF] and freshwater environments [START_REF] Ylla | Organic matter characteristics in a Mediterranean stream through amino acid composition: changes driven by intermittency[END_REF][START_REF] Peter | Bioavailability and diagenetic state of dissolved organic matter in riparian groundwater[END_REF], with values ranging from -3 to +5. The observed decreases in the DI over the course of biodegradation experiments further suggest this index to reflect changes in DOM bioavailability [START_REF] Amon | Linkages among the bioreactivity, chemical composition, and diagenetic state of marine dissolved organic matter[END_REF][START_REF] Rochelle-Newall | Degradation of riverine dissolved organic matter by seawater bacteria[END_REF]). At Station MOLA, the annual median DI varied between 1 and 1.5 and no pronounced depth profile was detectable over the 150 m water column. These positive values are indicative of overall diagenetically fresh DOM [START_REF] Davis | Amino acid and amino sugar yields and compositions as indicators of dissolved organic matter diagenesis[END_REF], Kaiser and Benner, 2009[START_REF] Peter | Bioavailability and diagenetic state of dissolved organic matter in riparian groundwater[END_REF]. In LSW, the DI was noticeably higher in the upper 20m as compared to the respective average median values in both years (Fig. 2 This observation raises the question of the origin of the DOM within LSW. The LSW that we observe at Station MOLA most likely originates from the Rhone River and its plume (Fig. 1). The pronounced pattern observed within LSW could be due to an enrichment in compounds from recent primary production occurring in the vicinity of the estuary [START_REF] Diaz | Biogeochemical and ecological functioning of the low-salinity water lenses in the region of the Rhone River freshwater influence, NW Mediterranean Sea[END_REF] and in small amounts of riverine, terrigenous DOM. Besides these sources that can be considered allochthonous to our study site, in situ primary production within the LSW could fuel the observed enhanced microbial activity. To address this question we followed the pattern in S1) could be the limiting factor of autotrophic activity, while heterotrophs can utilize these elements in organic form. These results support the idea that heterotrophic microbial processes are favored as compared to autotrophic processes within these buoyant water masses, suggesting that external rather than in situ produced DOM sustains heterotrophic activity.

Fig 3

Our observations from a time-series site in the NW Mediterranean Sea extend previous studies within LSW lenses in close vicinity to the Rhone river estuary and they provide insights on how biological activity in these water masses could change with time and distance from its origin.

Direct measurements and satellite observations report increased autotrophic productivity in coastal LSW lenses as compared to surrounding marine waters [START_REF] Auger | Functioning of the planktonic ecosystem on the Gulf of Lions shelf (NW Mediterranean) during spring and its impact on the carbon deposition: a field data and 3-D modelling combined approach[END_REF][START_REF] Diaz | Biogeochemical and ecological functioning of the low-salinity water lenses in the region of the Rhone River freshwater influence, NW Mediterranean Sea[END_REF], and the high NCP determined in situ [START_REF] Joux | Ultraviolet radiation in the rhone river lenses of low salinity and in marine waters of the northwestern mediterranean sea: attenuation and effects on bacterial activities and net community production[END_REF] suggests net autotrophy of the system in the vicinity of the estuary. With increasing distance from the plume and time since formation, inorganic nutrients are depleted and primary production is based on regenerated nutrients [START_REF] Diaz | Biogeochemical and ecological functioning of the low-salinity water lenses in the region of the Rhone River freshwater influence, NW Mediterranean Sea[END_REF]. This suggests a scenario where autotrophic processes dominate in early stages of LSW and heterotrophic processes become more important with distance and time.

Primary production in coastal marine environments and export of terrigenous DOC are potential sources of organic carbon for the open oligotrophic ocean (Smith andMacKenzie 1987, Bauer and[START_REF] Bauer | Ocean margins as a significant source of organic matter to the deep open ocean[END_REF][START_REF] Medeiros | Fate of the Amazon River dissolved organic matter in the tropical Atlantic Ocean[END_REF] and proposed transport mechanisms are physical features such as for example mesoscale eddies, internal waves or currents. The resulting spatio-temporal decoupling between the origin of organic matter in the coastal zone and its respiration in open waters was suggested to explain in part the imbalance between primary production and respiration in the oligotrophic ocean [START_REF] Duarte | The oligotrophic ocean is heterotrophic[END_REF][START_REF] Williams | The oligotrophic ocean is autotrophic[END_REF].

The LSW could represent this linkage in the NW Mediterranean Sea. In addition to recent and in situ primary production, photochemical transformations of DOM could be an important mechanism in providing bioavailable substrates in these buoyant surface water masses in particular when compounds of terrigenous origin are present [START_REF] Abboudi | Effects of photochemical transformations of dissolved organic matter on bacterial metabolism and diversity in three contrasting coastal sites in the Northwestern Mediterranean Sea during summer[END_REF][START_REF] Amon | Photochemical and microbial consumption of dissolved organic carbon and dissolved oxygen in the Amazon River system[END_REF][START_REF] Obernosterer | Differences in the optical and biological reactivity of the humic and nonhumic dissolved organic carbon component in two contrasting coastal marine environments[END_REF]. The combination of a suite of tools including satellite-based and autonomous instrumentation offers the possibility for a more detailed characterization and adapted sampling strategy of these mesoscale features at different locations in the open ocean. Taken together, our study illustrates the potential of water masses of estuarine origin to affect biological activity in the offshore oligotrophic ocean. The extension, lifetime and occurrence of these types of buoyant water masses will govern their impact on marine biogeochemistry and ecosystem functioning at different scales and distances from shore.

Fig. 1 .

 1 Fig. 1. Spatial and temporal distribution of salinity in the Gulf of Lion (NW Mediterranean Sea). Upper panels: Mean monthly surface salinity (4 km x 4 km) for February 2008 (left panel/a) and June 2008 (right panel/b) (February and May 2007 are shown in S1). Surface salinity distributions are provided by a circulation model overlaid with current speed and directions (yellow arrows), derived from altimetry for the date of sampling at Station MOLA (indicated by a white dot). Black lines represent isobaths (500 m, 1000 m and 2000 m). The white line denotes salinity of 37.85. Lower panel (c). Temporal changes of in situ salinity at Station MOLA

(

  250x4.6 mm, 5 µm, 110 Ǻ) and a Security Guard column (4 x 3 mm). We used the Dauwe Degradation Index (DI,[START_REF] Dauwe | Amino acids and hexosamines as indicators of organic matter degradation state in North Sea sediments[END_REF]) calculated based on the relative distribution of individual AA and adapted for DOM (as reported in[START_REF] Peter | Bioavailability and diagenetic state of dissolved organic matter in riparian groundwater[END_REF][START_REF] Davis | Amino acid and amino sugar yields and compositions as indicators of dissolved organic matter diagenesis[END_REF][START_REF] Kaiser | Biochemical composition and size distribution of organic matter at the Pacific and Atlantic time-series stations[END_REF] as an indicator for the degradation state of DOM. The original DI was calculated from Principal Component Analysis (PCA) applied to diagenetic altered sediments were DI = ∑ ((mol% amino acid -average mol % amino acid)/standard deviation mol%)* PCA amino acid score) .

Fig. 2 .

 2 Fig. 2. Depth profiles of salinity anomaly (blue line), microbial activity and DOM characteristics (box plots) at Station MOLA for the year 2008. The black lines across the boxes represent the median annual values. The ends of the boxes define the 25 th and 75 th percentile and the error bars represent the 10 th and 90 th percentiles. Dots represent the individual value for the date for which the salinity anomaly is shown (June 9 2008). Results for 2007 are shown in Fig. S5. BP:

  and Fig S5). The contribution of dissolved THAA to DOC, referred to as the amino acid yield, varied between 1.4-5.6% in the upper 150 m water column. The depth profiles of the amino acid yields during the LSW events were similar to those observed for dissolved THAA (data not shown). The DI is a chemical based indicator of the degradation state of organic matter, based on the relative distribution of individual amino acids (Dauwe and Middelburg 1998). Over the course of organic matter degradation, certain amino acids are selectively utilized resulting in a decrease of the DI (Dauwe

  and Fig S5). Thus, DOM in the uppermost water layer of LSW had a different chemical signature that is indicative of more bioavailable DOM. Complementary to the analyses illustrated in Fig.2, we carried out a Principal Component Analysis (PCA) with the combined biogeochemical and microbial parameters of the two years from surface waters (5m) (Fig.S6). The PCA analysis revealed one group consisting of samples from May and June from both years being explained by variables of microbial activity (BP, r = 0.85; CR, r = 0.77; BGR, r = 0.88) and features of DOM (dissolved THAA concentration, r = 0.48; amino acid yields, r = 0.21; DI, r = 0.52) that were positively correlated with PC1. By contrast, this group was also explained by salinity, which was negatively correlated with PC1 (r = -0.81). Taken together, these results suggest that LSW contains bioavailable DOM that sustains enhanced microbial heterotrophic activity in the offshore NW Mediterranean Sea.

  euphotic depth integrated fluxes of net community production (NCP) and CR. Highest fluxes of NCP were observed in March and April in both years, corresponding to the spring bloom period (Fig. 3). During the LSW in May and June 2007, NCP was negative and associated with highest depth-integrated fluxes of CR, illustrating a switch from net autotrophy in March and April to net heterotrophy in May and June 2007. During the LSW event in 2008, euphotic layer integrated fluxes of CR were again enhanced and associated with low positive (June 2008) and negative (July 2008) fluxes of NCP (Fig. 3). These NCP fluxes indicate low in situ primary production in LSW. The depletion in nitrate and phosphate in LSW (Table

Fig. 3 .

 3 Fig. 3. Euphotic depth integrated fluxes of Net Community Production (NCP) and Community Respiration (CR) and salinity at 5m depth. Results are shown for 2007 (February 14 to December 5) and 2008 (January 29 to October 6).

;[START_REF] Hedges | What happens to terrestrial organic matter in the ocean?[END_REF]. The fraction of terrigenous organic matter that is removed in the coastal

Acknowledgements.

We thank the captain and the crew of the R/V Nereis for their support aboard and the members of the Service d'Observation of the Observatoire Océanologique in Banyuls for their help in sample collection. We are grateful to Louise Oriol and Jocelyne Caparros for their enthusiastic participation to the cruises to Station MOLA. We thank Karine Escoubeyrou from the platform Bio2Mar for the amino acid analyses. L. Zudaire provided the CTD profiles for all cruises. We thank 3 anonymous reviewers for their insightful comments on a previous version of the manuscript. This work was conducted as part of the national project MEDEA (INSU-LEFE-CYBER). This work is part of the PhD thesis of M.L. Gonzalez. M.L. González was funded by the National Doctorate Scholarship (CONICYT-Chile), INCAR (FONDAP 15110027), and LIA-MORFUN (LIA 1035). We also thank to Patricio Torres and Angel Rain for their help in the construction of some supplementary figures. Salinity data are from Copernicus and in situ data are available online (http://www.obs-vlfr.fr/proof/index2.php).


Comments on the "Influence of the load modelling during gait on the stress distribution in a femoral implant" by Gervais et al.

Raphael Dumas

Univ Lyon, Université Claude Bernard Lyon 1, IFSTTAR, LBMC UMR_T9406, 69622 Lyon, France Gervais et al. [START_REF] Gervais | Influence of the load modelling during gait on the stress distribution in a femoral implant[END_REF] have recently published in the Multibody System Dynamics journal a study entitled "Influence of the load modelling during gait on the stress distribution in a femoral implant" where they put forward an interesting inverse dynamics method with flexible bony segments in order to estimate the loads applied to a femoral implant. They have compared different static and dynamic methods to estimate the joint loads required as boundary conditions for a finite element analysis of the implant. These joint loads are obtained either by considering the body weight in a static condition or by computing the inter-segmental forces and moments using inverse dynamics.

It seems that the authors did not really distinguish the inter-segmental forces and moments from the loads transmitted to the joints or to the bony segments. However, in biomechanics, the intersegmental forces and the forces transmitted to the joints (generally designated as joint reaction forces, joint or articular contact forces) are two different things.

Inter-segmental forces and moments

From an external point of view, the inter-segmental forces and moments represent the resultant of the contact actions of the body with the environment (typically at the foot-ground contact), as well as the gravitational, centrifugal and Coriolis actions arising from motion of the segments. These are the forces and moments computed by inverse dynamics, and in the paper of Gervais et al. [START_REF] Gervais | Influence of the load modelling during gait on the stress distribution in a femoral implant[END_REF], efficiently computed with flexible multibody dynamics.

Though, from an internal point of view, these inter-segmental forces and moments represent the resultant of the mechanical actions of all the anatomical structures crossing the joint: skin, fat, facias, bi-and mono-articular muscles, ligaments and articular surfaces. The loads transmitted to the joints correspond to the articular contact forces. That is, the loads transmitted to the joints represent a component of the inter-segmental forces and moments, a component which cannot be obtained without taking into account the actions of the muscles (and of the ligaments and other passive structures, but which are neglected most of the time).

Forces transmitted to the joints and bony segments

Not only 1 body weight (BW) but 2.5 BW have been considered an "improved static" loading conditions of the implant, as explained by the authors [START_REF] Gervais | Influence of the load modelling during gait on the stress distribution in a femoral implant[END_REF], based on in vivo instrumented prosthesis measurements [START_REF] Bergmann | Hip contact forces and gait patterns from routine activities[END_REF]. The same rationale could have been followed for the dynamic loading conditions.

For gait, in vivo measurements from instrumented implants [START_REF] Lu | Influence of muscle activity on the forces in the femur: An in vivo study[END_REF][START_REF] Taylor | The forces in the distal femur and the knee during walking and other activities measured by telemetry[END_REF] as well as simulation results from musculoskeletal models [START_REF] Duda | Internal forces and moments in the femur during walking[END_REF][START_REF] Moissenet | A 3D lower limb musculoskeletal model for simultaneous estimation of musculo-tendon, joint contact, ligament and bone forces during gait[END_REF] have reported 2 to 4 BW of compression of the femur. Similar measurements and results have reported hip and knee contact forces in the same range, e.g. [START_REF] Kinney | Update on Grand Challenge Competition to Predict in Vivo Knee Loads[END_REF][START_REF] Modenese | Prediction of hip contact forces and muscle activations during walking at different speeds[END_REF].

Furthermore, modelling studies have demonstrated that the forces transmitted to the joints and bony segments are mainly due to the contributions of the muscle actions (e.g. rectus femoris and hamstrings) [START_REF] Moissenet | Individual muscle contributions to ground reaction and to joint contact, ligament and bone forces during normal gait[END_REF][START_REF] Pandy | Muscle and joint function in human locomotion[END_REF] as opposed to gravitational, centrifugal and Coriolis actions. That is why musculotendon forces have to be taken into account, like in previous musculoskeletal models with flexible bony segments, e.g. [START_REF] Kłodowski | Flexible multibody approach in forward dynamic simulation of locomotive strains in human skeleton with flexible lower body bones[END_REF]. In other words, the muscle actions should not be omitted when estimating the stress or strain in the bone or the implant.

It is only in the case of lower limb amputees, e.g. transfemoral amputees with osteo-integrated implants measured in vivo using embedded sensors [START_REF] Lee | Magnitude and variability of loading on the osseointegrated implant of transfemoral amputees during walking[END_REF], that the forces transmitted to the bony segments remain of 1 BW during gait. In this case, the flexible inverse dynamics proposed by Gervais et al. [START_REF] Gervais | Influence of the load modelling during gait on the stress distribution in a femoral implant[END_REF] would perfectly apply, typically because the prosthetic component include deformable parts and because the classical inverse dynamics methods has been found inaccurate in this case [START_REF] Dumas | Gait Analysis of Transfemoral Amputees: Errors in Inverse Dynamics Are Substantial and Depend on Prosthetic Design[END_REF][START_REF] Schwarze | Loads on the prosthesis-socket interface of above-knee amputees during normal gait: Validation of a multi-body simulation[END_REF].