L Calarge

B Lanson
email: bruno.lanson@obs.ujf-grenoble.fr

A Meunier

M L Formoso

THE SMECTITIC MINERALS IN A BENTONITE DEPOSIT FROM MELO (URUGUAY)

Keywords: Clay minerals, Bentonite, Montmorillonite, Mixed-layered minerals, Interstratification, Layer charge, Simulation, XRD

A nearly monomineralic 1.5 m thick bentonite bed sampled in Melo (Uruguay) appears to be a pure high-charge montmorillonite: [Si 3.94 Al 0.06 ] (Al 1.40 Fe 3+ 0.11 Ti 0.02 Mg 0.49 Mn 0.01 ) O 10 (OH) 2 Na 0.01 K 0.08 Ca 0.18 . However, contrasting swelling behaviors have been evidenced by fitting the experimental X-ray diffraction patterns recorded on oriented preparations of the same sample in different saturation states. According to the expandability of the layers in the Ca-, K-and K-Ca-saturated (that is saturated first with K + and subsequently with Ca 2+ ) states, three "layer types" were defined. Low-, intermediate-, and high-charge layers are fully, partly, and not expandable, respectively, after K-saturation. Collapse of high-charge layers is not reversible after subsequent Ca-saturation, most likely because of tetrahedral substitutions.

These three different layer types are segregated in two distinct randomly interstratified mixedlayer phases. TSA and CEC are shown to depend on the interlayer cation composition.

INTRODUCTION

The structural features of smectites and illite-smectite mixed-layers (IS) from bentonite deposits have been largely studied and their expandable interlayers have consistently shown charge heterogeneities as revealed by potassium or alkylammonium saturation tests [START_REF] Howard | Lithium and potassium saturation of illite/smectite clays from interlaminated shales and sandstones[END_REF][START_REF] Talibudeen | Charge heterogeneity in smectites[END_REF][START_REF] Cetin | Layer charge of the expandable component illite/smectite in Kbentonite as determined by alkylammonium ion exchange[END_REF]. These heterogeneities are likely induced by contrasting chemical composition of octahedral and tetrahedral sheets from one layer to the other (Cuadros & Altaner, 1998a). In turn, these heterogeneities may also influence the expandability and total surface area of expandable layer silicates as shown by [START_REF] Laird | Layer charge influences on the hydration of expandable 2:1 phyllosilicates[END_REF]. Specifically, the distribution of high-and low-charge layers in the stacking may lead to mixed-layered sequences involving more than two layer types [START_REF] Foscolos | Diagenesis of clay minerals from Cretaceous shales of north eastern British Columbia[END_REF][START_REF] Cradwick | Calculated X-ray diffraction curves for the interpretation of a three-component interstratified system[END_REF]. In addition to non-expandable layers (no ethylene glycol adsorption or 0EG: non expandable layers include illite and collapsed high-charge smectite layers when K-saturated), expandable layers may incorporate 2 or 1 ethylene glycol layers (2EG or 1EG, respectively). The existence of such layers having contrasting expandabilities is supported by the description of IS mixed-layered minerals (MLM) from diagenetic series using profile fitting of X-ray diffraction (XRD) patterns which most often can be satisfactorily achieved only using three-component systems [START_REF] Drits | Sequence structure transformation of illite-smectite-vermiculite during diagenesis of Upper Jurassic shales, North Sea[END_REF][START_REF] Sakharov | Determination of illitesmectite structures using multispecimen X-ray diffraction profile fitting[END_REF].

To investigate the crystal structure of a smectite phase, and more specifically its layer charge heterogeneity, an almost monomineralic smectite was selected in a 1.5 m thick bentonite bed from Melo, Uruguay [START_REF] Calarge | A bentonite bed in the Acegua (RS,Brazil) and Melo (Uruguay) areas: a highly crystallized montmorillonite[END_REF]. In this deposit, amounts of non clay minerals do not exceed 5% in weight. This smectite was shown to be a nearly pure montmorillonite whose layer charge is about -0.45 per O 10 (OH) 2 . Layer charge heterogeneities were assessed using XRD patterns obtained from this sample in different saturation states, together with chemical composition, cation exchange capacity (CEC), total surface area data (TSA), and Fourier-transformed infrared (FTIR) spectroscopy.

GEOLOGICAL SETTING AND SAMPLING

The studied bentonite bed outcrops in a small quarry 50 m on the left side of the Melo to Montevideo road (Uruguay), 1 km aside from the R7 and R6 road intersection at 20 km from Melo (Fig. 1). It belongs to the Permian Yaguari Formation of the Paraná basin [START_REF] Andreis | El Sistema Pérmico en la República Argentina y en la República Oriental del Uruguay[END_REF]. The outcropping rocks are fluviatile and aeolian sandstones interlaminated with pelitic deposits (red beds) typical of the lagunar environments induced by the sea regression during the late Permian [START_REF] Andreis | El Sistema Pérmico en la República Argentina y en la República Oriental del Uruguay[END_REF]. During this period, the volcanic activity increased in the northern part of the Argentinian Patagonia, the maximum activity being attained during the Triassic and early Jurassic [START_REF] Andreis | El Sistema Pérmico en la República Argentina y en la República Oriental del Uruguay[END_REF]. The magmatic eruptions were highly explosive leaving large silica-rich ash deposits [START_REF] Axelrod | Role of volcanism in climate and evolution[END_REF]. The sampled bentonite is a 1.5 m thick bed interlaminated in sandstone formations. The studied sample was taken from the middle of this massive pink soft rock bed to avoid contamination from surrounding sandstones.

METHODS

Analytical procedures

The bentonite sample was gently ground after drying at 60°C. The powder was then dispersed in distilled water using an ultrasound probe. The < 1 µm size fraction was separated using centrifugation and then Ca-, K-, Li-, Mg-, or NH 4 -saturated with 1N CaCl 2 , KCl, LiCl, MgCl 2 , or CH 3 COONH 4 , respectively. Suspensions were kept in contact with saline solutions for 4-12 hours at room temperature to ensure a complete cation exchange. After three replications of this procedure, the chloride in excess was rinsed out using ethylic alcohol until no precipitate formed with AgNO 3 . After NH 4 -saturation, the presence of NH 4 + in solution was checked using Nessler reactive. Part of the K-saturated samples was subsequently Caexchanged using the same experimental procedure (K-Ca treatment). The initial K-saturation aimed at irreversibly collapsing high-charge "expandable" layers, whereas the subsequent Caexchange was performed to totally re-expand layers having lower charges.

XRD patterns were collected from randomly oriented powders of natural, Ca-, K-and K-Ca-saturated samples, as well as from oriented preparations of Ca-, K-and K-Ca-saturated samples in the air-dried (AD) and ethylene glycol solvated (EG) states. These patterns were recorded using a Siemens D500 diffractometer (Cu Kα radiation generated at 40 kV and 40 mA), equipped with a SOCABIM DACO® data collection system and a Kevex PSI solid state detector. The analytical conditions for oriented preparations were 2-35°2θ as angular range, 0.025°2θ as scanning step size and 4 s as counting time. Powder XRD patterns were recorded in the same conditions except for angular range (2-75°2θ) and counting time (8 s).

In addition, the octahedral charge of the sample was neutralized using the Hoffman-Klemen treatment (Hoffman & Klemen, 1950): Li-saturation and heating at 300°C for 12 hours. Untreated and treated samples were NH 4 -saturated to determine the respective contributions of octahedral and tetrahedral substitutions to the layer charge by comparing the surface areas of the IR NH 4 + absorption band [START_REF] Petit | Layer charge estimation of smectites using infrared spectroscopy[END_REF]. IR spectra were recorded using a Nicolet 510 FTIR spectrometer in the transmission mode. The spectrometer was continuously purged with dry CO 2 -depleted air. Pressed pellets (4 mg of samples mixed with 300 mg of KBr) were analyzed with a 4 cm -1 resolution over the 4000-400 cm -1 range.

The major elements were analyzed from a carbon-coated pressed pellet made from the < 1 µm size fraction of the sample in its natural state and after Ca-, K-and K-Ca-saturation using a CAMECA SX50 microprobe equipped with wavelength dispersive spectrometers. The analytical conditions were 4-20 nA, 15 kV, and a spot size diameter of 10 µm; the instrument was calibrated using natural silicates. An average composition was calculated for the natural sample from 50 analyses of the same pellet of the sample in its natural state. In addition, relative amounts of interlayer cations (Ca, and K) were calculated from 15 analyses made on the three saturated samples.

Cation exchange capacity (CEC) was measured on Ca-, K-, and K-Ca samples. These samples were first Mg-saturated and subsequently NH 4 -exchanged using the experimental procedure described above. The amount of exchanged Mg was measured in solution using a Perkin-Elmer atomic absorption spectrometer (AAS). Measurements of total surface area (external + internal surfaces) were performed on the Ca-, K-and K-Ca samples using the adsorption of ethylene glycol monoethyl ether (EGME) according to the method recommended by [START_REF] Heilman | The ethylene glycol monoethyl ether (EGME) technique for determining soil-surface area[END_REF]. The total surface area (TSA) was calculated from the measured EGME mass divided by the monolayer EGME mass adsorbed per surface unit (3.77 10 -4 g m -2 ).

XRD pattern calculation

Calculations of XRD patterns were performed using the MLM3C software developed by [START_REF] Plançon | Phase analysis of clays using an expert system and calculation programs for X-ray diffraction by two-and three-component mixed-layer minerals[END_REF]. Atomic coordinates for the different layer types were set as recommended by [START_REF] Moore | X-ray diffraction and the identification and analysis of clay minerals[END_REF], and the structural formula determined from chemical analyses (Table 1) was used to define the layer composition. The d 001 of the different layer types was varied as needed within the limits given by [START_REF] Sato | Effects of layer charge, charge location, and energy change on expansion properties of dioctahedral smectites[END_REF] to improve the quality of fit. Specifically, the following types of layers have been considered in the mixed-layering:

-AD and EG states: Collapsed smectite (0 water or EG layer: 0W, 0EG), d 001 ~ 10.0 Å, -AD state: 1 water layer smectite (1W), 12.1 < d 001 < 12.9 Å 2 water layer smectite (2W), 14.7 < d 001 < 15.5 Å -EG state: 1EG layer smectite, 12.7 < d 001 < 13.9 Å 2EG layer smectite, 16.5 < d 001 < 17.3 Å.

Among the other parameters which had to be adjusted in the trial-and-error fitting procedure, a special attention was paid to the proportions of each layer type, and to the coherent scattering domain size (N). The Reichweite parameter [START_REF] Jagodzinski | Eindimensionale Fehlordnung in Kristallen und ihr Einfluss auf die Röntgeninterferenzen: I. Berechnung des Fehlordnungsgrades aus der Röntgenintensitaten[END_REF] for these three-component MLM was limited to R = 0 (random interstratification). Because only the main features of experimental XRD patterns were reproduced, precision on the proportion of the different layer types is about ±10%.

RESULTS

Chemical composition

The average structural formula calculated from the 50 microprobe analyses of the smectite in its natural state is that of a montmorillonite: [Si 3.94 Al 0.06 ] (Al 1.40 Fe 3+ 0.11 Ti 0.02 Mg 0.49 Mn 0.01 ) O 10 (OH) 2 Na 0.01 K 0.08 Ca 0.18 (Table 1a). The octahedral occupation (2.03) is typical of dioctahedral structures. The average layer charge is relatively high: 0.45 per O 10 (OH) 2 , and is mostly compensated for by interlayer Ca 2+ and K + cations in the natural state. These cations seem to be entirely exchangeable as shown by the interlayer composition determined from microprobe analyses after Ca-and K-saturations: the 2Ca/(2Ca+K) ratios are 0.98 and 0.00 respectively (Table 1b). After K-Ca treatment, this ratio has an intermediate value (0.59), because some of the K + could not be exchanged any more after a complete K-saturation, which indicates the presence of high-charge layers [START_REF] Howard | Lithium and potassium saturation of illite/smectite clays from interlaminated shales and sandstones[END_REF].

CEC and TSA values depend on the saturating cations (Table 1b). CEC value decreases from 108.6 ceq.kg -1 for the Ca-saturated sample to 78.5 ceq.kg -1 for the K-saturated one. Part of this decrease is related to the collapse of high-charge layers after K-saturation. However, because the CEC increases to 94.2 ceq.kg -1 after subsequent Ca-saturation, another part of the observed decrease is linked to the nature of the interlayer cation. TSA value decreases from 605.63 m 2 g -1 for the Ca-saturated sample to 367.12 m 2 g -1 for the K-saturated one to indicate that about 40% of the layers in K-saturated sample are not accessible to EGME. This is consistent with the interlayer K + cation effect depicted by [START_REF] Cuadros | Interlayer cation effects on the hydration state of smectite[END_REF] who described Ksaturated clays as MLM of one water layer and dehydrated interlayers. Only 5% layers remain inaccessible to EGME after subsequent Ca-exchange (577.33 m 2 g -1 ). Thus, according to TSA measurements, three types of layers can be distinguished: 60% are always accessible to EGME whatever the interlayer cation; among the remaining 40% layers, 35% are inaccessible only in this K-saturated state, whereas 5% remain inaccessible after subsequent Ca-exchange.

XRD patterns

Powder XRD patterns. Together with small amounts of quartz, smectite is at large the major component of the bulk rock in its natural state (Fig. 2a). The d 060 spacing at 1.504Å confirms the dioctahedral character of the smectite. The high intensity of this peak indicates that the size of the coherent scattering domains is large in the a-b plane. The un-modulated asymmetrical (20,13) band at 34-39°2θ Cu-Kα for the Ca-and K-Ca-saturated samples is typical of a fully turbostratic stacking mode [START_REF] Mamy | Les phénomènes de diffraction de rayonnements X et électroniques par les réseaux atomiques. Application à l'étude de l'ordre cristallin dans les minéraux argileux. II. Evolution structurale de la montmorillonite associée au phénomène de fixation irréversible du potassium[END_REF][START_REF] Reynolds | X-ray diffractions studies of illite/smectite from rocks, <1µm randomly oriented powders, and <1µm oriented powder aggregates: The absence of laboratory-induced artifacts[END_REF]. The contribution of high order d 00l peaks in this angular range has been checked on oriented preparations and is reduced to weak modulations which become non-measurable on randomly oriented preparations. As a consequence, any change of the shape of this broad band is related to a modification of the stacking order. The K-saturated sample shows such a modulation (intense peak at 2.58 Å and weak peak at about 2.47 Å), indicating a decreasing proportion of random stacking faults with increasing amounts of interlayer K + (Fig. 2b).

Oriented preparations of the Ca-saturated sample. The XRD patterns in the AD and EG states are typical of a nearly pure smectite (Fig. 3a,b) with almost harmonic series of peaks.

However, the lack of strict harmonicity (d 001 ≠ l•d 00l ) indicates possible interstratification effects of layers having different thicknesses.

Oriented preparations of K-and K-Ca-saturated samples. The XRD patterns of K-saturated samples in the AD and EG states exhibit a broad peak at 11.96 and 14.12 Å respectively (Fig. 3). In the EG state, the shift of the d 001 value from 17.06 Å for Ca-saturated samples to 14.12 Å for the K-saturated one is due to the presence of 1 and/or 0EG layers in the stacking sequence. After K-Ca treatment in the EG state, the d 001 shifts back from 14.12 to 16.96 Å (Fig. 3d,f). Concomitantly, the (002), ( 003) and (005) peaks shift to 8.86, 5.56 and 3.37 Å respectively and become more intense. Positions, and more especially their non-rationality, contrasting widths, and asymmetry of these peaks indicate the interstratification of several layer types with variable amounts of ethylene glycol. Summarizing, both AD and EG XRD patterns recorded for the three saturation states show that the peak sequences do not form a purely harmonic series indicating that this smectite behaves as a MLM.

Calculation of XRD pattern from oriented preparations. The experimental XRD patterns of Ca-, K-and K-Ca-saturated samples in the AD and EG states were compared to calculated ones using a trial-and-error method to reproduce the main features of the experimental traces over the 2 to 35°2θ Cu-Kα angular range. Even though the agreement between experimental and calculated patterns is not perfect, calculations may be considered satisfactory approximations (Fig. 3).

For the Ca-saturated sample, no collapsed layers are detected in the AD or EG states. The two XRD patterns of this sample may be reproduced by using a unique MLM (MLM1) containing two different layer types. One may note in Table 2 that the proportion of layers with 1EG in the EG state corresponds to that of layers with 1W in the AD state.

After K-saturation, part of the layers are collapsed to 10.0 Å. In addition, collapsed layers seem to form clusters as experimental patterns could only be reproduced assuming the presence of two MLM, one containing about 30% of collapsed layers (MLM1) whereas the other contains 80-90% of these 10.0 Å layers (MLM2). The expandable:collapsed layers ratio in the two MLM components, as well as their relative proportions are similar for K-, and K-Ca-saturated samples in both AD and EG states. However, in contrast to the results obtained for the Ca-saturated sample, the proportion of layers with 1EG in the EG state does not correspond systematically with that of layers with 1W in the AD state. For the K-saturated sample, these two proportions are similar (55% of 1W layers and 60% of 1EG layers), but they differ significantly for the K-Ca-saturated sample (15% of 1W layers and 0% of 1EG layers).

FTIR spectra

The integrated intensity of the NH 4 + υ 4 absorption band at 1400 cm -1 was shown to be proportional to the quantity of NH 4 + ions exchanged for interlayer cations [START_REF] Petit | Layer charge estimation of smectites using infrared spectroscopy[END_REF], that is Ca 2+ in the present case. This quantity decreases drastically when the octahedral charge has been previously neutralized using the Hoffman-Klemen treatment (Fig. 4). This indicates that most of the layer charge originates from octahedral R 2+ for R 3+ substitutions with only a small part of the layer charge located in the tetrahedral sheet (Al for Si substitutions).

According to the respective areas of the 1400 cm -1 bands, the tetrahedral charge is estimated to represent about 8% of the total layer charge. This value is similar although lower than the one obtained using the structural formula (13%). As uncertainties in the surface area measurements of weak absorption bands are unavoidable, we think the tetrahedral charge calculated from the structural formula more accurate.

DISCUSSION

Variation of expandability

The number of expandable layers is much lower in K-and K-Ca-saturated samples than in the Ca-saturated samples in which expandable layers largely predominate. In addition, relative proportions of the different layer types are comparable in both AD and EG states for these two samples indicating that part of K + ions is irreversibly fixed during K-saturation thus reducing the expandability. As expandability is known to depend on the layer charge [START_REF] Malla | Identification of expanding layer silicates: layer charge vs. expansion properties[END_REF][START_REF] Sato | Effects of layer charge, charge location, and energy change on expansion properties of dioctahedral smectites[END_REF][START_REF] Laird | Layer charge influences on the hydration of expandable 2:1 phyllosilicates[END_REF], the differences between Ca-, K-and K-Casaturated samples observed here suggest that the layer charge is not similar for all smectite layers. If so, some properties like CEC or TSA would also vary as a function of the nature of interlayer cations.

For example, it is possible to study the relationships between crystal structure, physical (TSA, total amount of 2EG layers) and chemical (CEC) properties by using the contrasting amounts of interlayer K + ions in Ca-, K-and K-Ca-saturated samples as a variable. From the linear relations shown in Fig. 5 it is possible to assume that the relative proportion of 2EG layers, TSA and CEC are interdependent parameters controlled by the amount of K + in the interlayers. The steady decrease of the relative proportion of 2EG layers with increasing content of K + indicates that these cations are preferentially adsorbed by some layers which then collapse to 1EG or 0EG. Accordingly, in the K-saturated sample the TSA and CEC values are 40 and 30% lower, respectively, than the values measured for the Ca-saturated sample. However, it should be noted that whereas part of the K + cations is irreversibly fixed (~40% -Table1), in agreement with the observed irreversible collapse of smectite layers (3 5%), part of the CEC and most of the TSA are restored by subsequent Ca-saturation (K-Ca samples). This implies that CEC and TSA strongly depend on the nature of interlayer cation and not only on the amount of layer charge.

Layer charge heterogeneity [START_REF] Malla | Identification of expanding layer silicates: layer charge vs. expansion properties[END_REF] showed that the layer charge scatters from 0.30 to 0.63 per O 10 (OH) 2 for fully expandable (2EG) and collapsed (0EG) smectite layers, respectively, in the K-saturated state. Using the detailed description of the layer swelling behavior coming from the trial-and-error fitting of experimental XRD profiles (Table 2) it is possible to refine the description made by these authors and to differentiate three different layer types, and to estimate their relative proportions:

1) Full expandability after K-saturation -Low charge: 2EG in both Ca-and K-saturated states (~15%);

2) Partial expandability after K-saturation -Intermediate charge: collapse from 2EG in Casaturated state to 1EG after K-saturation (~50%); these layers are fully expandable after subsequent Ca-saturation (K-Ca-saturation);

3) No expandability after K-saturation -High charge: collapse from 2EG in Ca-saturated state to 0EG in K-saturated states (~35%); the collapse of these layers is not reversible after subsequent Ca-saturation.

From the 0.30 and 0.63 layer charges (per O 10 (OH) 2 ) defined by [START_REF] Malla | Identification of expanding layer silicates: layer charge vs. expansion properties[END_REF] for K-saturated 2EG and 0EG layers, respectively, it is possible to calculate that of the 1EG layers (0.37 per O 10 (OH) 2 = (0.45 -(15% x 0.30 + 35% x 0.63)) / 50%) from the average layer charge (0.45 per O 10 (OH) 2 ). On the other hand, from the average chemical composition (Table 1a) and the FTIR data (Fig. 4) which indicate that about 10% of the layer charge originates from the tetrahedral sheets, the average octahedral charge is 0.40 per O 10 (OH) 2 .

It is then possible to hypothetically distribute layer charges between octahedral and tetrahedral sheets for the different layer types. For low-and intermediate-charge layers, the total layer charge (0.30 and 0.37 per O 10 (OH) 2 , respectively) is lower than the average octahedral charge, and likely originates only from octahedral substitutions. In turn, it is possible to estimate for high-charge layers the relative contributions of octahedral (0.49 per O 10 (OH) 2 ) and tetrahedral (0.14 per O 10 (OH) 2 ) substitutions to the total layer charge. The former value is consistent with the results obtained by [START_REF] Calarge | A bentonite bed in the Acegua (RS,Brazil) and Melo (Uruguay) areas: a highly crystallized montmorillonite[END_REF] using FTIR which showed no Mg-OH-Mg absorption bands. In addition, the presence of tetrahedral charge is assumed to favor the irreversible collapse of smectite layers after K-saturation.

CONCLUSION

The exceptional montmorillonite sampled in Melo (Uruguay) is composed of layers whose composition varies from low to high-charge smectite. This variation results from the heterogeneous distribution of tetrahedral charges in polarized 2:1 layers. In turn, these layers, which present contrasting swelling behaviors are stacked in two distinct mixed-layered structures. The first one contains mostly (~80%) fully expandable (2EG) low-charge layers, whereas the second one contains mostly (~80%) high-charge layers which collapse irreversibly after K-saturation.

This layer charge heterogeneity, both at the layer and at the crystal scales, rises the problem for the definition of montmorillonite as a mineral phase, as it would make difficult (meaningless?) the measurement of thermodynamic parameters through dissolution or microcalorimetric experiments. From a theoretical point of view, the coexistence of two 2. patterns (Fig. 3). MLM1 and MLM2 refer to the two populations of crystallites containing layers with contrasting contents of interlayer water or ethylene glycol.

Layers with 2, 1, and 0 water layers are referred to as 2W, 1W, and 0W, respectively.

Layers with 2, 1, and 0 ethylene glycol layers are referred to as 2EG, 1EG, and 0EG, respectively. For each population of crystallites, the thickness of these layers is

FIGURE CAPTIONS Figure 1 .

 CAPTIONS1 FIGURE CAPTIONS

Figure 2 .

 2 Figure 2. Powder XRD patterns of Melo bentonite. Qz = quartz. a) Bulk rock in its natural state. b) <1 µm size fraction after Ca-, K-, and K-Ca-saturation. The 20, 13 band is shown to illustrate the contrasting degree of layer stacking disorder as a function of the interlayer cation.

Figure 3 .

 3 Figure 3. XRD patterns of oriented preparations of the <1 µm size fraction after Ca-, K-, and K-Ca-saturation in the air dried (AD) and ethylene glycol (EG) solvated states. Experimental and calculated profiles are shown as crosses and solid lines, respectively. Peak positions determined on experimental XRD patterns are compared to those determined on calculated profiles. Calculated positions are given in parentheses. All parameters used for the calculation of XRD patterns are given inTable 2.

Figure 4 .

 4 Figure 4. FTIR spectra showing the typical υ 4 absorption band of NH 4 + ions for NH 4 +saturated samples. Untreated and Hoffman-Klemen treated samples are shown as solid and dashed lines, respectively.

Figure 5 .

 5 Figure 5. Evolution of expandability (a), CEC (b), and TSA (c) as a function of the interlayer cation composition expressed as 2Ca/(2Ca+K). Expandability is characterized by the

  indicated as well as their proportion. Only random interstratification was considered. N is the number of layers stacked coherently. Ab. is indicative of the relative abundance of the two populations of crystallites. a, and b refer to the calculations made for the AD and the EG patterns, respectively.

Table 1 .

 1 Chemical characteristics. a) Bulk chemical composition of the smectite in its natural state (average from 50 microprobe analyses) and structural formula calculated for O 10 (OH)

	). Expandability is characterized by the

2 . b) Cation exchange capacity (CEC) determined after Mg-exchange of the samples, average interlayer composition from 15 micropobe analyses (2Ca/(2Ca+K) ratio) and total surface area (TSA) measured by EGME absorption for Ca-, K-and K-Ca-saturated samples.

Table 2 .

 2 Composition of the smectitic minerals obtained by fitting the experimental XRD

MLMs with different compositions means that thermodynamic conditions should be controlled along a tie-line until one of them disappears. In the present case, the diagenetic environment would likely destabilize the more expandable MLM1, and MLM2 should be considered as a precursor of illite-smectite MLMs.

ACKNOWLEDGEMENTS

Javier Cuadros and Boris A. Sakharov are thanked for reviewing the initial version of this article with great care and for their extremely pertinent suggestions. Anne-Marie Karpoff is thanked for her editorial assistance. Financial support has been provided by the CAPES-COFECUB program 349/01.


Christophe Chalons
email: christophe.chalons@uvsq.fr

Maria Laura

Delle Monache

M L Delle Monache

P Goatin
email: paola.goatin@inria.fr

A conservative

Keywords: Scalar conservation laws with local moving constraints, Traffic flow modeling, PDE-ODE coupling, Conservative finite volume schemes. 35L65, 90B20

come

Introduction

We consider a coupled PDE-ODE model describing a large, slow moving vehicle (that we will refer to as "the bus") interacting with the sourrounding traffic flow. The system consists of the Lighthill-Whitham-Richards [START_REF] Lighthill | On kinematic waves. II. A theory of traffic flow on long crowded roads[END_REF][START_REF] Richards | Shock waves on the highway[END_REF] scalar conservation law, which describes the evolution in time of the mean traffic density on a road, and by an ODE accounting for the slower vehicle trajectory. The loss of road capacity due to the presence of the bus is expressed by an inequality constraint on the vehicles flow at the bus position.

The model was first introduced and studied in an engineering setting [START_REF] Giorgi | Prise en compte des transports en commun de surface dans la modélisation macroscopique de l'écoulement du trafic[END_REF][START_REF] Lebacque | Introducing buses into first-order macroscopic traffic flow models[END_REF] (see also [START_REF] Daganzo | Moving bottlenecks: A numerical method that converges in flows[END_REF][START_REF] Daganzo | On the numerical treatement of moving bottlenecks[END_REF] for a numerical treatment). A rigorous mathematical framework and an existence result were later proposed in [START_REF] Delle Monache | Scalar conservation laws with moving constraints arising in traffic flow modeling: an existence result[END_REF]. Other systems modeling moving bottlenecks on roads are proposed in the applied mathematics literature: in [START_REF] Gasser | Vehicular traffic flow dynamics on a bus route[END_REF][START_REF] Lattanzio | Moving bottlenecks in car traffic flow: a PDE-ODE coupled model[END_REF] the flux constraint is modeled by a mollifier, thus preserving the smoothness of the flux function; a 2 × 2 system modeling traffic flow coupled with a second order ODE is presented in [START_REF] Borsche | Mixed systems: Odes -balance laws[END_REF]. In these works, standard numerical methods can be applied.

Conservation laws with locally constrained flux function were first introduced in [START_REF] Colombo | A well posed conservation law with a variable unilateral constraint[END_REF] to model fixed constraints like toll gates. Well-posedness results in the BV and L ∞ settings, as well as the convergence of adapted finite volume schemes are given in [START_REF] Andreianov | Finite volume schemes for locally constrained conservation laws[END_REF][START_REF] Colombo | A well posed conservation law with a variable unilateral constraint[END_REF]. Extensions to non-concave fluxes and systems were also proposed in [START_REF] Chalons | General constrained conservation laws. application to pedestrian flow modeling[END_REF] and [START_REF] Garavello | The Aw-Rascle traffic model with locally constrained flow[END_REF] respectively. In those papers, anyway, the constraint position is not affected by the solution of the conservation law, and the analytical and numerical treatments are therefore easier than in the present case. In particular, no stability result with respect to initial data is currently available for the PDE-ODE system considered in this paper. A first numerical strategy to compute approximate solutions was proposed in [START_REF] Delle Monache | A front tracking method for a strongly coupled PDE-ODE system with moving density constraints in traffic flow[END_REF], based on locally non-uniform moving meshes to track the bus position.

In this paper we follow another approach to avoid non-uniform moving meshes. The main point here is related to the presence of non-classical shocks in the solutions of the model under consideration. It is well-known that, in this context, standard conservative finite volume methods cannot be applied and fail in producing good numerical results. Glimm's scheme can be used but it is not strictly conservative. In order to propose a numerical scheme which is conservative on fixed meshes and able to compute non-classical solutions, we propose to adapt to the present context of strongly coupled PDE-ODE system a reconstruction strategy approach introduced in [START_REF] Boutin | Convergent and conservative schemes for nonclassical solutions based on kinetic relations[END_REF], which allows to precisely capture moving non-classical discontinuities on fixed meshes still guaranteeing conservation, unlike Glimm's scheme. An important feature of the proposed method is to be exact for isolated classical and non-classical shocks, which means in particular only one point of numerical diffusion (on each cell the approximate value corresponds to the value of the average of the exact solution). In the general case, shocks are still computed without numerical diffusion and convergence is proved numerically. So far, however, no rigorous convergence result is available. Several test cases, some of them being significant from the modeling point of view, are proposed to illustrate these features.

Mathematical model

The coupled PDE-ODE model in [START_REF] Delle Monache | Scalar conservation laws with moving constraints arising in traffic flow modeling: an existence result[END_REF] reads

∂ t ρ + ∂ x f (ρ) = 0, (t,x) ∈ R + × R, (2.1a) ρ(0,x) = ρ 0 (x), x ∈ R, (2.1b) f (ρ(t,y(t))) -ẏ(t)ρ(t,y(t)) ≤ αR 4V (V -ẏ(t)) 2 t ∈ R + , (2.1c)
ẏ(t) = ω(ρ(t,y(t)+)), t ∈ R + , (2.1d)
y(0) = y 0 , (2.1e)
where ω(ρ(t,y(t)+)) denotes the right-hand limit of ω(ρ(t,x)) at y(t). Above, ρ = ρ(t,x) ∈ [0,R] is the scalar conserved quantity and represents the traffic density, whose maximum attainable value is R. The flux function f : [0,R] → R + is assumed to be strictly concave and such that f (0) = f (R) = 0. In this paper, we will take f (ρ) = ρv(ρ), where v(ρ) = V (1 -ρ/R) is the mean traffic speed, V being the maximal velocity allowed on the road. The time-dependent variable y denotes the bus position. If the traffic is not too congested, the bus moves at its own maximal speed V b < V . When the surrounding traffic density is too high, the bus adapts its velocity accordingly. In particular, it is not possible for the bus to overtake the cars. From a mathematical point of view, the velocity of the bus can be described by the following traffic density dependent function (see Figure 2.1) The bus acts as a moving obstacle, thus hindering the traffic flow as expressed by the flux constraint (2.1c). There, α ∈]0,1[ is the reduction rate of the road capacity due to the presence of the bus. The inequality (2.1c) is derived by studying the problem in the bus reference frame, i.e., setting X = xy(t) and rewriting the conservation law (2.1a) as

ω(ρ) = V b if ρ ≤ ρ * . = R(1 -V b /V ), v(ρ) otherwise. (2.2) ρ R ρ * ω(ρ) Vb v(ρ)
∂ t ρ + ∂ X F (ρ) = 0, F (ρ) = f (ρ) -ẏρ. (2.3)
In fact, let f α : [0,αR] → R + be the rescaled flux function describing the reduced flow at

x = y(t), i.e. f α (ρ) = V ρ 1 -ρ αR , and ρ α ∈ ]0,αR/2[ such that F ′ α (ρ α ) = 0 ⇔ f ′ α (ρ α ) = ẏ with F α (ρ) = f α (ρ) -ẏρ, i.e. ρ α = αR 2 1 -ẏ V , see Figure 2.2.
Then observing that 2 , and imposing that in the bus reference frame the flux F should be less than the maximum value of the flux of the reduced flow, at the bus position one gets (2.1c). Note that the inequality is always satisfied if ẏ(t) = v(ρ), since the left-hand side is 0. Moreover, it is well defined even if ρ has a jump at y(t) because of the Rankine-Hugoniot conditions.

F α = F α (ρ α ) = f α (ρ α ) -ẏρ α = αR 4V (V -ẏ(t))

The Riemann Problem

We recall here the definition of the Riemann Solver for problem (2.1) given in [START_REF] Delle Monache | Scalar conservation laws with moving constraints arising in traffic flow modeling: an existence result[END_REF]. Let us consider a Riemann type initial datum

ρ 0 (x) = ρ L if x < 0, ρ R if x > 0, y 0 = 0. (2.4)
Denote by R the standard (i.e., without the constraint (2.1c)) Riemann solver for (2.1a)-(2.1b)-(2.4), i.e., the (right continuous) map (t,x) → R(ρ L ,ρ R )(x/t) given by the standard weak entropy solution. Moreover, let ρα and ρα , with ρα ≤ ρα , be the intersection points of the flux function f (ρ) with the line

f α (ρ α ) + V b (ρ -ρ α ) (see Figure 2.2(a)). Definition 2.1. The constrained Riemann solver R α : [0,R] 2 → L 1 loc (R;[0,R]) is de- fined as follows. 1. If f (R(ρ L ,ρ R )(V b )) > F α + V b R(ρ L ,ρ R )(V b ), then R α (ρ L ,ρ R )(x/t) = R(ρ L , ρα )(x/t) if x < V b t, R(ρ α ,ρ R )(x/t) if x ≥ V b t, and
y(t) = V b t. 2. If V b R(ρ L ,ρ R )(V b ) ≤ f (R(ρ L ,ρ R )(V b )) ≤ F α + V b R(ρ L ,ρ R )(V b ), then R α (ρ L ,ρ R ) = R(ρ L ,ρ R ) and y(t) = V b t. 3. If f (R(ρ L ,ρ R )(V b )) < V b R(ρ L ,ρ R )(V b ), i.e., v(R(ρ L ,ρ R )(V b )) < V b then R α (ρ L ,ρ R ) = R(ρ L ,ρ R ) and y(t) = v(ρ R )t.
Note that, when the constraint is enforced (point 1. in the above definition), a non-classical shock arises between ρα and ρα , which satisfies the Rankine-Hugoniot condition but violates the Lax entropy condition.

The existence of solutions for the general Cauchy problem with BV initial data is proved in [START_REF] Delle Monache | Scalar conservation laws with moving constraints arising in traffic flow modeling: an existence result[END_REF]. In particular, solutions to (2.1d)-(2.1e) are to be intended in the Carathéodory sense.

Numerical method

Our aim is to design a scheme that is able to approximate numerically on a fixed mesh the non-classical shock and to track at the same time the bus position. Let ∆x and ∆t be the fixed space and time steps, and set x j+1/2 = j∆x, x j = (j -1/2)∆x for j ∈ Z and t n = n∆t for n ∈ N.

Conservative scheme for hyperbolic PDEs with constraint

To approximate the conservation law we use a conservative finite volume scheme. We approximate the initial datum by a piecewise constant function given by its average on the discretization cells

C j = [x j-1 2 ,x j+ 1 2 [, ρ 0 j = 1 ∆x x j+ 1 2 x j-1 2
ρ 0 (x)dx, j ∈ Z. To compute the approximation ρ n j of the solution ρ at time t n and on the cell C j , for j ∈ Z and n ≥ 1, we look for a recursive update procedure of the classical form

ρ n+1 j = ρ n j - ∆t ∆x F n j+ 1 2 -F n j-1 2 , (3.1)
where the numerical fluxes F n j+ 1 2 at the cell interfaces x j+ 1 2 have to be suitably defined in order to capture non-classical shocks.

Following [START_REF] Boutin | Convergent and conservative schemes for nonclassical solutions based on kinetic relations[END_REF], we propose an algorithm that is able to recreate the discontinuity of the non-classical shock using a reconstruction technique. Indeed, whenever a nonclassical shock appears, the classical finite volume schemes, like Godunov's [START_REF] Godunov | A finite difference method for the numerical computation of discontinuous solutions of the equations of fluid dynamics[END_REF], fail to generate good approximations of the exact solution, see [START_REF] Boutin | Convergent and conservative schemes for nonclassical solutions based on kinetic relations[END_REF]. We proceed as follows. Assume that at time t n , the bus position y n is located in the cell C m for a given m. Whenever condition

f (ρ n m ) > F α + V b ρ n m , (3.2)
is satisfied, we consider that a non-classical shock is expected to appear locally around the bus. Hence, we propose to introduce in the cell C m the left and right states ρ n m,l = ρα and ρ n m,r = ρα of the non-classical shock, which is expected to be present in the Riemann solution associated with ρ n m-1 and ρ n m+1 in case also inequality

f (R(ρ n m-1 ,ρ n m+1 )(V b )) > F α + V b R(ρ n m-1 ,ρ n m+1 )(V b ), (3.3)
x n m-1

x n m+1

x for some d m defined such that the reconstruction procedure is conservative, which means

d n m ρ n m,l + (1 -d n m )ρ n m,r = ρ n m , (3.5)
or equivalently,

d n m = ρ n m,r -ρ n m ρ n m,r -ρ n m,l . (3.6)
Clearly, it is possible to reconstruct the discontinuity provided that

0 ≤ d n m ≤ 1. (3.7)
To conclude the definition of the reconstruction strategy, let us mention that the non-classical discontinuity moves with speed V b > 0, so that a natural definition of the numerical flux at x m+ 1 2 is given by ∆tF n

m+ 1 2 = min(∆t m+ 1 2 ,∆t)f (ρ n m,r ) + max(∆t -∆t m+ 1 2 ,0)f (ρ n m,l ) (3.8)
where ∆t

m+ 1 2 = 1 -d n m V b
∆x is the time needed by the discontinuity to reach the interface x m+ 1 2 . This definition of the flux is used provided that (3.2),(3.3), (3.7), (3.5), (3.6) are satisfied. Otherwise, and also for defining F n j+ 1 2 for j = m, a monotone and consistent numerical flux, like Godunov's, is used under the classical CFL condition:

∆t max j∈Z f ′ (ρ n j ) ≤ 1 2 ∆x. (3.9)
Following [START_REF] Boutin | Convergent and conservative schemes for nonclassical solutions based on kinetic relations[END_REF], it can be easily proved that isolated non-classical shocks are exactly captured with such a strategy (see also Case 0 in Section 4 below) and contain no numerical diffusion. Nevertheless, at this stage, classical shocks will suffer some numerical diffusion since classical Godunov's method is used away from the bus position, and this might be problematic in the validation of the solution on the constraint (3.3).

In fact, if we choose initial data in the neighborhoods of the values ρα , ρα , we observed that the approximate solution generated by this method may not be correct.

To overcome this problem, we apply the reconstruction technique also to classical shocks. Numerical diffusion is thus avoided also for classical shocks and we get correct solutions. In the spirit of [START_REF] Lagoutière | Stability of reconstruction schemes for scalar hyperbolic conservation laws[END_REF], we proceed as follows: given a cell C j for some j ∈ Z, j = m, such that ρ n j-1 < ρ n j+1 , we introduce the left and right traces ρ n l = ρ n j-1 and ρ n r = ρ n j+1 of a reconstructed discontinuity and we define d n j , by

d n j = ρ n r -ρ n j ρ n r -ρ n l . (3.10)
Let us denote by λ(ρ n l ,ρ n r ) the speed of the discontinuity given by the Rankine-Hugoniot condition, i.e.,

λ(ρ n l ,ρ n r ) = f (ρ n l ) -f (ρ n r ) ρ n l -ρ n r .
Then, if it is actually possible to reconstruct such a discontinuity within the cell C j , that is to say if d n j ∈ [0,1], the numerical fluxes at x j± 1 2 are defined by

• if λ(ρ n l ,ρ n r ) ≥ 0, ∆tF n j+ 1 2 = min(∆t j+ 1 2 ,∆t)f (ρ n r ) + max(∆t -∆t j+ 1 2 ,0)f (ρ n l ), (3.11)
with ∆t

j+ 1 2 = 1 -d n j λ(ρ n l ,ρ n r )
∆x.

• if λ(ρ n l ,ρ n r ) ≤ 0, ∆tF n j-1 2 = min(∆t j-1 2 ,∆t)f (ρ n l ) + max(∆t -∆t j-1 2 ,0)f (ρ n r ), (3.12)
with ∆t j-

1 2 = d n j -λ(ρ n l ,ρ n r ) ∆x,
where, with some abuse of notation, we mean that if λ(ρ n l ,ρ n r ) = 0 then

F n j+ 1 2 = f (ρ n r ) and F n j-1 2 = f (ρ n l )
. This additional reconstruction guarantees that also isolated classical shocks are exactly captured, as shown by the numerical tests presented in Section 4. Moreover, with this supplementary reconstruction we are able to handle correctly all the interactions among shocks, classical and non-classical. Remark 3.1. In the event of an ambiguity, for instance j = m + 1 and λ ≤ 0 above, the non-classical discontinuity reconstruction is always preferred.

Numerical method for the ODE

To precisely track the bus trajectory we adapt the algorithm introduced in [START_REF] Bretti | A tracking algorithm for car paths on road networks[END_REF]. At each time step, we update the position y n of the bus by studying interactions between the bus trajectory and the density waves within the corresponding cell. We distinguish two cases:

• Inequality (3.2) is satisfied. Then the bus moves at velocity V b and we update the bus position as y n+1 = V b ∆t n + y n . • Condition (3.2) is not satisfied. In this case the solution is classical and we implement the tracking algorithm introduced in [START_REF] Bretti | A tracking algorithm for car paths on road networks[END_REF]. We have to distinguish two situations: one when y n ∈ [x m-1 2 ,x n j [ and one when y n ∈ [x n j ,x m+ 1 2

[. In both cases, we check if the wave starting at the cell interface is a shock or a rarefaction and compute the time of interaction between the wave and the bus trajectory. The cell index m is updated according to the new position of the bus.

Conclusions

In this paper, we introduced a reconstruction based numerical method that is able to capture numerically non-classical shocks for a coupled PDE-ODE problem with moving constraints. The algorithm is conservative on a fixed mesh and isolated classical and non-classical shocks are propagated exactly. We showed several test cases, including shock interactions and tests that are more significant from a modeling point of view. The convergence of the method was shown numerically.

Fig. 2 . 1 :

 21 Fig. 2.1: Bus and cars speed.

Fig. 2 . 2 :

 22 Fig. 2.2: Flux functions for ẏ = V b .

Fig. 3 . 1 :

 31 Fig. 3.1: Reconstruction of a non-classical shock.

Fig. 4 . 1 :

 41 Fig. 4.1: Evolution in time of the density corresponding to initial data (4.1) (left) with ∆x = 0.001 compared with the exact solution.

Fig. 4 . 2 :

 42 Fig. 4.2: Evolution in time of the density corresponding to initial data (4.2) (left) and (4.3) (right) with ∆x = 0.001 compared with the exact solution.

Fig. 4 . 3 :

 43 Fig. 4.3: Evolution in time of the density at different times corresponding to initial data (4.4) and ∆x = 0.001.

Fig. 4 . 4 :

 44 Fig. 4.4: Evolution in time of the density at different times corresponding to initial data (4.5) and ∆x = 0.001.

  Fig. 4.5: L 1 convergence.

Table 4 .

 4 1: Order of convergence for the reconstruction scheme, corresponding to initial data (4.2) and (4.3) .

* This research was partially supported by the European Research Council under the European Union's Seventh Framework Program (FP/2007-2013) / ERC Grant Agreement n. 257661. -Quentin-en-Yvelines,

Numerical results

In this section we present some numerical tests performed with the scheme previously described. We deal with a road of length 1 parameterized by the interval [0,[START_REF] Andreianov | Finite volume schemes for locally constrained conservation laws[END_REF]. In all the simulations we fix V b = 0.3, α = 0.6, V = R = 1. Case 0: We consider Riemann type initial data with

The solution is given by a non-classical shock, as illustrated in Figure 4.1. It is possible to notice in this case that the numerical solution is exact everywhere but in the single cell containing the non-classical shock. However, as already said, the value in this cell coincides with the average of the corresponding exact solution and using (3.6) we can recover the exact location of the discontinuity.

Case I: We consider Riemann type initial data with

2

The solution is given by two classical shocks separated by a non-classical discontinuity, as illustrated in Figure 4.2, left.

Case II: We consider Riemann type initial data with

The values of the initial conditions create a rarefaction wave followed by non-classical and classical shocks, as illustrated in Figure 4.2, right.

Case III: We consider the following initial data

In this case, the bus initial position is not aligned with the discontinuity. We can see that the values of the initial conditions generate a rarefaction wave followed by nonclassical and classical shocks on the density that are created when the bus approaches the rarefaction and initiates a moving bottleneck, as illustrated in The solution is given by a non-classical shock and a classical one that collide. After the collision a classical shock is generated, as illustrated in Figure 4.4.

For cases I and II we also show the convergence curves in Figure 4.5. It represents the log-log L 1 error between the numerical solution and the exact one versus mesh size. The numerical order of convergence can be found in Table 4.1.


Grégory Genta-Jouve

Nadja Cachet

François Oberhänsli

Charlotte Noyer

Jean-Louis Teyssié

Olivier P Thomas

Thomas Lacoue-Labarthe

Comparative bioaccumulation kinetics of trace elements in Mediterranean marine sponges

Keywords:

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The coastal environment is continuously loaded with foreign chemicals such as metals originated from the anthropogenic activities, i.e. by urban communities and industries, and released through the river inputs. Therefore, the fate and effects of these contaminants in aquatic ecosystems have been extensively studied by environmental toxicologists (van der Oost et al., 2003, Johnston and[START_REF] Johnston | Contaminants reduce the richness and evenness of marine communities: A review and meta-analysis[END_REF]. In order to ensure the survey of the marine environment submitted to this anthropogenic contamination, researchers have developed a large array of biomonitoring tools that allow time-and biological integrated measures of the contaminants levels and their potential effects on biota (Philipps, 1990;Rainbow & Philipps, 1993). Three kinds of biomonitors were proposed, requiring the selection of organisms according to criteria such as sedentary, easy identification, dense population, wide dispersion, and longevity (e.g. [START_REF] Rainbow | Biomonitoring of heavy metal availability in the marine environment[END_REF]. The presence/absence of a "sentinel" species is first used as a clue of long-term effects of contamination on populations and communities. The survey of biological responses at the infra-individual scale can also be used as a biomarker of exposure or effect at short-term scale [START_REF] Lyons | Using biological effects tools to define good environmental status under the European Union Marine Strategy Framework Directive[END_REF]. Finally, organisms that are able to accumulate chemicals are used to monitor the bioavailable fraction of contaminants in the environment [START_REF] Zhou | Biomonitoring: An appealing tool for assessment of metal pollution in the aquatic ecosystem[END_REF]. An efficient characterization of the contamination state of an ecosystem must rely on several species that could reflect the potential sources of pollutants, i.e. waterborne, particular or trophic and sediments [START_REF] Rainbow | Biomonitoring of heavy metal availability in the marine environment[END_REF]. Consequently, there is a continuous need for new marine biomonitors in order to encompass the most significantly an ecosystem [START_REF] Conti | Marine organisms as biomonitors[END_REF].

Benthic species such as bivalves, seagrasses and macroalgae have been commonly used as biomonitors of a metal contamination [START_REF] Roberts | Biomonitors and the assessment of ecological impacts: Distribution of herbivorous epifauna in contaminated macroalgal beds[END_REF]. Considering the renowned characteristics of appropriate biomonitors, marine sponges are undoubtedly candidates for more detailed investigations. Sponges are sessile marine invertebrates that are able to filter large volumes of seawater and this characteristic will undoubtedly favor metal accumulation from dissolved and associated trace elements [START_REF] Pérez | Evaluation de la qualité des milieux côtiers par les spongiaires: état de l'art[END_REF]. Indeed, previous studies demonstrated their high capacities to accumulate and to concentrate metals from the field and they were already proposed as biomonitors of a metal contamination [START_REF] Hansen | Accumulation of copper, zinc, cadmium and chromium by the marine sponge Halichondria panicea pallas and the implications for biomonitoring[END_REF][START_REF] Pérez | Effects of 12 years' operation of a sewage treatment plant on trace metal occurrence within a Mediterranean commercial sponge (Spongia officinalis, Demospongiae)[END_REF][START_REF] Cebrian | Sponges as biomonitors of heavy metals in spatial and temporal surveys in northwestern Mediterranean: Multispecies comparison[END_REF][START_REF] Venkateswara Rao | The use of marine sponge, Haliclona tenuiramosa as bioindicator to monitor heavy metal pollution in the coasts of Gulf of Mannar, India[END_REF][START_REF] Pan | Sponges and sediments as monitoring tools of metal contamination in the eastern coast of the Red Sea, Saudi Arabia[END_REF][START_REF] De Mestre | Sponges as sentinels: Patterns of spatial and intra-individual variation in trace metal concentration[END_REF]. The high bioaccumulation factors measured for some species rendered them suitable for the tracing of elements at very low concentration in the water column.

Sponges are widely distributed in the sublittoral area and they can dominate both in terms of diversity and biomass benthic communities from temperate rocky bottoms to polar continental shelf. Furthermore, they are known to tolerate a high level of contaminations in most cases.

For these reasons, they have been proposed for a 'Sponge Watch Program', additional to the 'mussel watch' [START_REF] Patel | Sponge "sentinel" of heavy metals[END_REF][START_REF] Hansen | Accumulation of copper, zinc, cadmium and chromium by the marine sponge Halichondria panicea pallas and the implications for biomonitoring[END_REF][START_REF] Pérez | Evaluation de la qualité des milieux côtiers par les spongiaires: état de l'art[END_REF]. Surprisingly, the uptake and loss kinetics of trace metal elements have been poorly studied in sponges even if these data are essential for several reasons [START_REF] Patel | The chemical form of cobalt-60 in the marine sponge Spirastrella cuspidifera[END_REF][START_REF] Patel | Sponge "sentinel" of heavy metals[END_REF][START_REF] Hansen | Accumulation of copper, zinc, cadmium and chromium by the marine sponge Halichondria panicea pallas and the implications for biomonitoring[END_REF]. A high concentration factor is often required for a reliable monitoring of a contamination and the uptake and elimination rates of a trace element are complementary data to assess the efficiency of an organism to "register" an environmental contamination. Furthermore, these rates give access to the exposure time required to obtain this information, but also to the "storage" period during which the information is present in the organism.

Consequently, these kinetics data will give valuable information for the use of a sponge species as a trace element biomonitor. High uptake rates will be required for the use of sponges as sentinel to quickly identify a contamination. Furthermore, low loss rates will leave traces of an old incident while high loss rates would rather be needed to follow the decontamination process of the water medium. Although it is well known that sponges can accumulate metal efficiently, very little information is available on the contrasting bioaccumulation capacities among species from the same area [START_REF] Pérez | In situ comparative study of several Mediterranean sponges as potential biomonitors of heavy metals[END_REF][START_REF] Pan | Sponges and sediments as monitoring tools of metal contamination in the eastern coast of the Red Sea, Saudi Arabia[END_REF]. The primary objective of the present work was to investigate and to compare the uptake and loss kinetics of trace elements from waterborne pathway by six taxonomically diverse sponge species, highly represented in the coralligenous of the Mediterranean Sea, namely Acanthella acuta, Cymbaxinella damicornis (previously Axinella damicornis), Cymbaxinella verrucosa (previously Axinella verrucosa), Agelas oroides, Chondrosia reniformis and Ircinia variabilis, and then to identify their main features as potential biomonitors. Trace elements, Ag, Mn, Zn, Se, Cd, Co, Cs and Am, were chosen among essential and non essential elements using γ-radiotracers in order to follow the bioaccumulation of these elements in a highly sensitive manner. Bioaccumulation studies have already been reported on some of these sponge species, mainly with the aim to obtain the concentration factors of trace elements comparing with the concentration of these elements in the water column or the sediments [START_REF] Cebrian | Sublethal effects of contamination on the Mediterranean sponge Crambe crambe: metal accumulation and biological responses[END_REF], 2006, 2007[START_REF] Pérez | In situ comparative study of several Mediterranean sponges as potential biomonitors of heavy metals[END_REF]). These results showed contrasting results relative to accumulation and suitability for the assessment of marine environmental quality. In our study we decided to reinvestigate some of these species as biomonitors of a trace element contamination but most importantly in order to give important kinetic data on the uptake and loss properties of these trace elements.

Materials and methods

Target species

The target species were six Mediterranean sponges, namely Acanthella acuta (Schmidt, 1862); Agelas oroides (Schmidt, 1864); Cymbaxinella damicornis (Esper, 1794); Cymbaxinella verrucosa (Esper, 1794); Chondrosia reniformis (Nardo, 1847), and Ircinia variabilis (Schmidt, 1864). These sponges were selected because of a wide sublittoral or circalittoral distribution in the western Mediterranean Sea. They were also chosen as sufficiently diverse from a taxonomical point of view and well described with regard to their morphology and cytology as summarized below [START_REF] Hooper | Systema Porifera: a guide to the classification of Sponges[END_REF]: a) Chondrosia reniformis (Class: Demospongiae, Order: Chondrosida, Family: Chondrillidae) is a thick encrusting species predominantly composed of collagen and lacking a fiber skeleton (mineral or spongin). Thus, the mesohyl is thick and well developed, whereas the aquiferous system is reduced. This species is also considered to be a sensitive organism that has difficulty to survive in polluted habitats [START_REF] Cebrian | Response of the Mediterranean sponge Chondrosia reniformis Nardo to copper pollution[END_REF]. b) Agelas oroides (Class: Demospongiae, Order: Agelasida, Family Agelasidae) is a massive sponge with irregular lobes. Oscules are large, circular, and distributed at the top of the lobes and its spicules attached in a well-developed spongin fiber skeleton. c) Ircinia variabilis (Class: Demospongiae, Order: Dictyoceratida, Family: Irciniidae) is a massive sponge. Oscules are large and located at the top of the conical lobes. This species does not have mineral spicules but a dense fiber skeleton structurally diverse from classical spongins [START_REF] Junqua | Biochemical and morphological studies on collagens of horny sponges. Ircinia filaments compared to spongines[END_REF]. d) Acanthella acuta (Class: Demospongiae, Order: Axinellida, Family: Dictyonellidae) is a small erect sponge with rather small oscules and a skeleton of silica spicules responsible for its fibrous quality. e) Cymbaxinella damicornis (Class: Demospongiae, Order: Axinellida, Family: Axinellidae) is a rather small branching-erect sponge with small and compressed branches.

Oscules are small and are located on the apices of the branches, partially surrounded by a small triangular 'flap' of tissue. The skeleton is constituted of silica spicules that are axially condensed and embedded in spongin fibers. f) Cymbaxinella verrucosa (Class: Demospongiae, Order: Axinellida, Family: Axinellidae) is a branching-erect sponge with an arborescent shape and cylindrical branches. Oscules are small and rather circular and its skeleton is composed of silica spicules that are axially condensed and embedded in spongin fibers. Individuals were selected of a homogenous size, with an average length of 7 ± 2 cm for the branch-erect sponges (A. acuta, C. damicornis and C. verrucosa) and an average diameter of 5 ± 2 cm for the massive sponges (A. oroides, I. variabilis and C. reniformis), Immediately after collection, sponges were kept in seawater at constant temperature during their transfer to the aquaria within 30 minutes.

At the laboratory, sponges were acclimated during 2 weeks in the aquarium conditions with flowing seawater. Then, individuals were transferred into a 70 L closed-circuit tank with stabilized environmental conditions: 0.45 µm filtered and U.V sterilized seawater; temperature 19 ± 1 °C; salinity 38 g L -1 ; pH 8.02; light/dark cycle, 12h/12h. In these conditions, they were further exposed to dissolved radiotracers for 170 hours. Sponges were fed daily with Isochrysis galbana at a concentration of 10 5 cells l -1 . No mortality was observed during these experimental conditions.

Eight radiotracers ( 110m Ag, 241 Am, 109 Cd, 60 Co, 134 Cs, 54 Mn, 75 Se and 65 Zn) were used dissolved in seawater to follow the bioaccumulation behaviors of both anthropogenic radionuclides released from the nuclear facilities (e.g. 110m Ag, 241 Am, 60 Co, 134 Cs, 54 Mn) (e.g. [START_REF] Warnau | Biokinetics of radiocobalt in the asteroid Asterias rubens (Echinodermata): sea water and food exposures[END_REF][START_REF] Ke | Uptake and depuration of cesium in the green mussel Perna viridis[END_REF] and corresponding stable metals discharged in marine waters (e.g. [START_REF] Warnau | Radiotracer techniques: A unique tool in marine ecotoxicological studies[END_REF]. They were chosen because of their large spectrum of physico-chemical properties and their different contribution to a marine contamination. The radioisotopes were obtained from Amersham ( 241 Am, 109 Cd, 60 Co, 134 Cs), Isotope Products ( 54 Mn, 65 Zn) and Polatom ( 110m Ag, 75 Se). Isotopes were dissolved in 0.1 N HCl ( 109 Cd, 54 Mn, 60 Co), 0.5 N HCl ( 65 Zn), 0.1 N HNO 3 ( 110m Ag, 241 Am), or pure water ( 134 Cs). Isotopes were spiked by additions of microliters of these stock solutions in the seawater to obtain the following activities ( 110m Ag 0.5 kBq L -1 , 241 Am 0.2 kBq L -1 , 109 Cd 1.3 kBq L -1 , 60 Co 0.5 kBq L -1 , 134 Cs 1.0 kBq L -1 , 54 Mn 0.5 kBq L -1 , 75 Se 0.5 kBq L -1 , 65 Zn 0.5 kBq L -1 ). For trace elements, these additions increased the concentrations by 400 pM Ag, 4.6 fM 241 Am, 3.4 pM Cd, 0.1 pM Co, 1.4 nM 134 Cs, 83.4 pM Mn, 80 pM Se, and 10 pM Zn. The resulting concentrations stand below natural seawater concentrations of these elements [START_REF] Bruland | Trace elements in seawater[END_REF]. No change of pH in the tank seawater was measured after radioisotope addition. During the experiment, seawater and radiotracer spikes were renewed daily during the first 5 days of incubation to maintain water quality and a constant radiotracer concentration after each injection. Radiotracer activities in seawater were checked after 24, 48, 72, 98 and 170 hours of incubation, before and after each water renewal yielding timeintegrated activities in seawater for the 8 radiotracers of 0.33 ± 0.18 kBq 110m Ag L -1 , 0.16 ± 0.05 kBq 241 Am L -1 , 0.85 ± 0.11 kBq 109 Cd L -1 , 0.46 ± 0.05 kBq 60 Co L -1 , 1.04 ± 0.01 kBq 134 Cs L -1 , 0.44 ± 0.10 kBq 54 Mn L -1 , 0.46 ± 0.03 kBq 75 Se L -1 , 0.42 ± 0.05 kBq 65 Zn L -1 [START_REF] Warnau | Biokinetics of selected heavy metals and radionuclides in the common Mediterranean echinoid Paracentrotus lividus: seawater and food exposures[END_REF]. The metal uptake was followed by radiotracer activity counting in the tag-identified sponge sampled after 24, 48, 72, 98 and 170 hours of incubation. Each individual was sampled, rinsed in clean seawater to remove radiolabelled water from the aquifer system, weighted and radiocounted according to the method described below.

After the incubation period, the radiolabelled sponges were placed in an open-circuit 70 L tank and held for 7 weeks in clean and flowing water (seawater flux: 50 L h -1 ; temperature: 19°C; salinity 38 p.s.u.; light/dark cycle 12h/12h). Sponges were sampled and counted every day during the first week and then weekly to establish the depuration kinetics of each radiotracer.

Radioanalyses and data treatments

Radioactivity in all samples was assessed using a high-resolution γ spectrometry system consisting of four coaxial Germanium (N-or P-type) detectors Intertechnique) connected to a multi-channel analyzer (Interwinner6, Intertechnique). The radioactivity of the samples was determined by comparison with the radioactivity in standards of appropriate geometry and was corrected for background and radioisotope physical decay.

Gamma emissions of 110m Ag, 241 Am, 109 Cd, 60 Co, 134 Cs, 54 Mn, 75 Se and 65 Zn were assayed at 657, 60, 88, 1332, 605, 835, 264 and 1116 keV, respectively. Counting time was adjusted to obtain counting errors less than 5% but was limited to a maximum of 15 min to avoid stress induction for the organisms [START_REF] Jeffree | Phylogenetic consistencies among chondrichthyan and teleost fishes in their bioaccumulation of multiple trace elements from seawater[END_REF].

The uptake of radiotracer was expressed as changes in concentration factors (CF), which is the ratio between radiotracer activity in the sponge -Bq g -1 -and the time-integrated activity in seawater -Bq g -1 [START_REF] Metian | Bioaccumulation of essential metals (Co, Mn and Zn) in the king scallop Pecten maximus: seawater, food and sediment exposures[END_REF]. This unitless term expressed the efficiency of the organism to accumulate and concentrate an element from the seawater after a determined time of exposure. Uptake kinetic was best described using either a linear equation (Eq. 1) or a saturation exponential equation (Eq. 2): CF t = k u t (Eq. 1)

CF t = CF ss (1-e -ke t ) (Eq. 2)

where CF t and CF ss are the concentration factors at time t (d) and at steady-state respectively,

and k e and k u are the biological depuration and uptake rate constants (d -1 ) [START_REF] Whicker | Radioecology: nuclear energy and the environment[END_REF].

Radiotracer depuration kinetics was expressed in terms of change in percentage of remaining activity (i.e., radioactivity at time t divided by the initial radioactivity measured in the individual at the beginning of the depuration period * 100) along with time.

The depuration kinetics was best fitted by either a single-(Eq. 3) or a double-(Eq. 4) exponential equation:

A t = A 0 e -ke t (Eq. 3)

A t = A 0s e -kes t + A 0l e -kel t (Eq. 4)

where A t and A 0 are the remaining activities at time t (d) and 0, respectively, k e is the biological depuration rate constant (d -1 ), and « s » and « l » subscripts refer to the short-and long-lived component of the depuration kinetics [START_REF] Warnau | Biokinetics of selected heavy metals and radionuclides in the common Mediterranean echinoid Paracentrotus lividus: seawater and food exposures[END_REF][START_REF] Warnau | Biokinetics of radiocobalt in the asteroid Asterias rubens (Echinodermata): sea water and food exposures[END_REF]. The determination of k e allows the calculation of the radiotracer biological half-life (d) according to the relation:

Tb 1/2 = ln2 / k e (Eq. 5)

Constants (and their statistics) of the best fitting uptake and depuration kinetic equations (decision based on ANOVA tables for two fitted model objects) were estimated by iterative adjustment of the models using the nls curve-fitting routine in R freeware. The level of significance for statistical analyses was always set at α = 0.05. We also realized HCA and PCA on both CF 170h and T b1/2 to clearly identify the different patterns in both uptake and loss rates.

Results

Radiotracer uptake kinetics in sponges

The uptake kinetics of 110m Ag, 241 Am, 109 Cd, 60 Co, 134 Cs, 54 Mn, 75 Se and 65 Zn in the six sponge species are shown in Figure 1. In our experimental conditions, these elements were taken up in the whole-body of sponges following contrasting bioaccumulation patterns. Most of the uptake kinetics followed an exponential model (see the trace element and species. Thus, Acanthella acuta showed the highest accumulation efficiency for 110m Ag (i.e. 11.9 ± 1.1 h -1 and 1895 ± 896 for k u and CF 170h , respectively; Table 1) whereas the lowest uptakes were observed for 109 Cd in Cymbaxinella verrucosa (i.e. 0.07 ± 0.01 h -1 and 12 ± 3 for k u and CF 170h , respectively), 75 Se in Acanthella acuta (i.e. 0.02 ± 0.001 h -1 and 4 ± 1 for k u and CF 170h , respectively) and 134 Cs in all species. For this last radionuclide, it is noteworthy that the accumulation reached a steady-state equilibrium in all species close to CF ss value ≈ 1, with the notable exception of a two-fold higher value observed in I. variabilis. (CF ss = 1.8 ± 0.1).

The multivariate analysis on the CF reached at the end of the waterborne exposure revealed that species could be gathered into two groups according to their bioaccumulation behaviors (Figure 3).

Radiotracer loss kinetics in sponges

The 134 Cs loss was not followed in all species because radiotracer activities in sponges dropped below the detection limit after 1-2 d of depuration in natural seawater. Loss behaviors are quite similar for all trace elements and sponge species with the notable exception of 110m Ag in A. acuta which has an extremely low kinetics of depuration (Figure 2).

Considering all species, it is noteworthy that most of the depuration kinetics were best fitted by a double exponential equation (

Discussion

The assessment of the quality of the marine environment usually can be inferred from the presence/absence of a contaminant in the water column, the sediments or a living organism, but also from the physiological state of organisms. The target species used as biomonitor has to obey strong criteria such as sedentary, easy recognition, dense population, wild dispersion, and longevity (e.g. Rainbow and Philipps, 1993). Some sponge species have been already studied for this purpose and the results have been usually found to be encouraging [START_REF] Cebrian | Sponges as biomonitors of heavy metals in spatial and temporal surveys in northwestern Mediterranean: Multispecies comparison[END_REF][START_REF] Pérez | Effects of 12 years' operation of a sewage treatment plant on trace metal occurrence within a Mediterranean commercial sponge (Spongia officinalis, Demospongiae)[END_REF][START_REF] Pan | Sponges and sediments as monitoring tools of metal contamination in the eastern coast of the Red Sea, Saudi Arabia[END_REF][START_REF] Venkateswara Rao | The use of marine sponge, Haliclona tenuiramosa as bioindicator to monitor heavy metal pollution in the coasts of Gulf of Mannar, India[END_REF]. Indeed, sponges are widely distributed and they may represent the most prominent group of macroinvertebrates in a given ecosystems where usual bioindicator (e.g. mussel) are missing [START_REF] Pérez | Effects of 12 years' operation of a sewage treatment plant on trace metal occurrence within a Mediterranean commercial sponge (Spongia officinalis, Demospongiae)[END_REF]. Moreover, their tolerance to physico-chemical fluctuations allows them to be present both in polluted and non-contaminated seawater (de Mestre et al. 2012). Their simple tissue organization and their biology as active filter favor the concentration and accumulation of contaminants from both dissolved and particulate phases. Basically, sponges from the field are therefore well known as bioaccumulator of hydrocarbons [START_REF] Féral | Evolution des teneurs en hydrocarbures chez quelques organismes marins du Nord Finistère depuis l'échouage de l'Amoco Cadiz et comparaison des méthodes de dosage en infrarouge et spectrofluorimétrie[END_REF], organochlorinated compounds (e.g. [START_REF] Verdenal | Pollutant levels in Mediterranean commercial sponges[END_REF][START_REF] Pérez | Marine sponges as biomonitor of polychlorobiphenyl contamination: concentration and fate of 24 congeners[END_REF] and metals (e.g. [START_REF] Verdenal | Pollutant levels in Mediterranean commercial sponges[END_REF][START_REF] Hansen | Accumulation of copper, zinc, cadmium and chromium by the marine sponge Halichondria panicea pallas and the implications for biomonitoring[END_REF][START_REF] Cebrian | Sublethal effects of contamination on the Mediterranean sponge Crambe crambe: metal accumulation and biological responses[END_REF][START_REF] Pérez | Effects of 12 years' operation of a sewage treatment plant on trace metal occurrence within a Mediterranean commercial sponge (Spongia officinalis, Demospongiae)[END_REF][START_REF] Pan | Sponges and sediments as monitoring tools of metal contamination in the eastern coast of the Red Sea, Saudi Arabia[END_REF]. Nevertheless, a "Sponge Watch program" has surprisingly been little used for field surveys of contamination level. Only few works delineated the bioaccumulation fate of contaminating metals in a limited number of sponge species [START_REF] Patel | Sponge "sentinel" of heavy metals[END_REF]Hansen et al., 1985;[START_REF] Cebrian | Sublethal effects of contamination on the Mediterranean sponge Crambe crambe: metal accumulation and biological responses[END_REF]. However, these values are of high interest to discriminate between a regulation and a net accumulation of the trace elements in the studied organism (Rainbow and Philipps, 1993). In this study, it must be emphasized that the bioaccumulation capacities of sponges are higher than those found for other marine biomonitors. In similar experimental conditions, CF values of the same order of magnitude were reached only after 40 days of exposure in mussels Mytilus edulis and Mytilus chilensis [START_REF] Hervé-Fernandez | Cadmium bioaccumulation and retention kinetics in the Chilean blue mussel Mytilus chilensis: Seawater and food exposure pathways[END_REF]unpublished data). It is also noteworthy that Ag CF in Acanthella acuta is two-fold higher that this determined in clams Gafrarium tumidum after two weeks of exposure (Hedouin et al., 2006).

Our comparative study on six diverse Mediterranean sponge species and eight trace elements revealed two major contrasting bioaccumulation trends. The massive sponges Agelas oroides, Chondrosia reniformis and Ircinia variabilis displayed the highest concentration factors after 170 h of exposure compared to the other three species (Figure 2) for most of the considered radiotracers with CF maximum values around 700 (Figure 1; Table 1). The separation into two groups is in accordance with previous studies showing that trace element accumulation vary between sponge species (Figure 3) [START_REF] Patel | Sponge "sentinel" of heavy metals[END_REF][START_REF] Pérez | In situ comparative study of several Mediterranean sponges as potential biomonitors of heavy metals[END_REF][START_REF] Cebrian | Sponges as biomonitors of heavy metals in spatial and temporal surveys in northwestern Mediterranean: Multispecies comparison[END_REF][START_REF] Venkateswara Rao | The use of marine sponge, Haliclona tenuiramosa as bioindicator to monitor heavy metal pollution in the coasts of Gulf of Mannar, India[END_REF][START_REF] Pan | Sponges and sediments as monitoring tools of metal contamination in the eastern coast of the Red Sea, Saudi Arabia[END_REF]. Metal specificity and species specific variability in the BC factors within sponges has been previously documented [START_REF] Patel | Sponge "sentinel" of heavy metals[END_REF][START_REF] Pérez | In situ comparative study of several Mediterranean sponges as potential biomonitors of heavy metals[END_REF][START_REF] Cebrian | Sponges as biomonitors of heavy metals in spatial and temporal surveys in northwestern Mediterranean: Multispecies comparison[END_REF][START_REF] Pan | Sponges and sediments as monitoring tools of metal contamination in the eastern coast of the Red Sea, Saudi Arabia[END_REF].

This ability of sponges to highly bioaccumulate trace elements both dissolved in the seawater or adsorbed on particles was already reported and mainly inferred to their high pumping flow and filtering activity [START_REF] Turon | Clearance rates and aquiferous systems in two sponges with contrasting life-history strategies[END_REF]). The interspecific variability might be explained by different pumping ability of sponge species [START_REF] Cebrian | Sponges as biomonitors of heavy metals in spatial and temporal surveys in northwestern Mediterranean: Multispecies comparison[END_REF]. Indeed, massive sponges possess higher choanocyte chamber volumes and consequently a much higher area to bind trace elements. These interactions may often be of low energy and could explain the usual loss kinetic patterns of most of the studied elements. The role of the associated microorganisms is an important issue to be addressed in the context of an interspecific variation in the bioaccumulation processes [START_REF] Selvin | Sponge-associated marine bacteria as indicators of heavy metal pollution[END_REF][START_REF] Erwin | Phylogenetic diversity, host-specificity and community profiling of sponge-associated bacteria in the Northern Gulf of Mexico[END_REF]. Indeed, it is well known that marine sponges can be associated to a diverse array of microbial symbionts which appeared to be sponge-specific in some cases. These microorganisms should also play a role in the bioaccumulation processes of trace elements [START_REF] Gadd | Heavy metal accumulation by bacteria and other microorganisms[END_REF] and may explain the interspecific differences [START_REF] Selvin | Sponge-associated marine bacteria as indicators of heavy metal pollution[END_REF]. In our case, the six studied species are considered as HMA (High Microbial Abundance) sponge species [START_REF] Taylor | Sponge-associated microorganisms: evolution, ecology, and biotechnological potential[END_REF][START_REF] Weisz | Do associated microbial abundances impact marine demosponge pumping rates and tissue densities?[END_REF][START_REF] Erwin | Phylogenetic diversity, host-specificity and community profiling of sponge-associated bacteria in the Northern Gulf of Mexico[END_REF] and then the interspecific bioaccumulation differences cannot be inferred to microbial density but rather to distinct microbial populations into these species.

Further investigations are required to assess the exact role of the associated microorganisms into these processes.

Among the studied trace elements, americium is an actinide of high interest as an anthropogenic radionuclide. Our study demonstrated that massive sponges can serve equally as biomonitors of an Am contamination with very high CF values and rapid loss kinetics. As a transuranic radionuclide, americium (III) is strongly particulate reactive [START_REF] Ryan | Transuranic biokinetic parameters for marine invertebrates-a review[END_REF][START_REF] Metian | Characterization of 241 Am and 134 Cs bioaccumulation in the king scallop Pecten maximus: investigation via three exposure pathways[END_REF]. These three species can also serve as biomonitors for Zn, Mn and Co even if the CFs are two magnitude lower for this latter trace element. Cadmium is also very important to monitor in the marine environment as this trace element has been proven to induce toxicity in several phytoplanktonic micro-organisms [START_REF] Höss | Toxicity of ingested cadmium to the nematode Caenorhabditis elegans[END_REF]. Among our studied species, both Agelas oroides and Chondrosia reniformis were identified as the best sponge biomonitors in the Mediterranean Sea to follow Cd contamination.

This study also highlighted some interesting specificity within both massive and erect sponge groups concerning the concentration capacities of trace elements (Figure 3). Indeed, 1) among the "low uptake" species, Acanthella acuta displayed the highest uptake of Ag, whereas 2) the "high uptake" species, Chondrosia reniformis did not accumulate Se as much as Agelas oroides and Ircinia variabilis. In these cases low loss rates suggested a net accumulation mechanism instead of a simple regulation. In our study, Acanthella acuta differed from all other sponge species by a very high accumulation of dissolved Ag (i.e. 110m Ag CF = 1895 ± 896 at 170 h of exposure). Moreover, all elements (A 0l = 95%; Table 2) are also strongly retained by this sponge. During the observation period, the estimated T b½ was 1093 d (Table 2), suggesting that Ag is efficiently accumulated and tightly bound to specific sites of the sponge. Interestingly and unlike other studied species, A. acuta is well known to produce some secondary metabolites that include high concentrations of sulfurated compounds [START_REF] Ciminiello | Cis-eudesmane nitrogenous metabolites from the marine sponges Axinella cannabina and Acanthella acuta[END_REF] and isocyanates sesquiterpenes [START_REF] Mayol | Nitrogenous sesquiterpenes from the marine sponge Acanthella acuta: three new isocyanide-isothiocyanate pairs[END_REF] that could exhibit a high affinity for Ag [START_REF] Eisler | Silver hazards to fish, wildlife and invertebrates: a synoptic review, Contaminant Hazard Review: Biological report 32 US Fish and wildlife Service[END_REF]. This observation suggests a role of the biomolecules and especially the secondary metabolites in the bioaccumulation of trace elements by sponges but further studies need to be conducted to identify the putative complex between silver and these ligands.

A second atypical behaviour was observed for Selenium. This transition metal plays a very important role in the environment and for human health and then its concentration must be precisely followed in the marine environment [START_REF] Winkel | Environmental Selenium Research: from Microscopic Processes to Global Understanding[END_REF]). Among the three species that showed the highest accumulation efficiencies of trace elements, Chondrosia reniformis did not accumulate Se as efficiently as Agelas oroides and Ircinia variabilis (CF 170h = 12 ± 1 in C. reniformis vs. 225 ± 147 and 217 ± 62 in A. oroides and I. variabilis respectively). In these two latter species, the major fraction of accumulated Se (> 94%) is tightly bound to sponge tissue with estimated T b½ of 112 and 197 d, respectively. Selenium is an essential element but toxic in large amount, known for its function as an active co-factor or components of enzymes such as glutathione peroxidase or thioredoxin reductase [START_REF] Mertz | The essential trace elements[END_REF] which present a selenocysteine into their primary structure [START_REF] Stadtman | Selenocysteine[END_REF].

Moreover, Se was proved to stimulate the sponge biosilification of spicules but the mechanism is still unknown [START_REF] Müller | Selenium affects biosilica formation in the demosponge Suberites domuncula[END_REF][START_REF] Schwertner | Selenium-enriched liquid media for the cultivation of siliceous sponges and for biogenic silica production[END_REF]. A gene coding for a selenoprotein was found to be up-regulated in a Suberites sponge. Difference in the rate of the biosilification processes between our studied sponges may then explain the high incorporation and low depuration of this element in some species.

Among the analyzed elements, the uptake kinetics of 134 Cs in all species displayed a steady state equilibrium rapidly reached during the experiment duration (Table 1). Compared to the other element, CF ss values were very low and estimated close to 1, suggesting that this radionuclide was in equilibrium between the whole body of sponge and the surrounding water. This result has to be related to the physico-chemicals properties of Cs, an alkaline highly hydrosoluble and with very low coordination constants with any ligand. It is also assumed that low Cs bioaccumulation might be enhanced by competitive inhibition by the over-whelmingly high concentration of K + in the seawater [START_REF] Bryan | The accumulation of radioactive caesium by marine invertebrates[END_REF][START_REF] Wang | Modeling radiocesium bioaccumulation in a marine food chain[END_REF].

This element is rather known to follow the water movement from seawater to the tissues [START_REF] Lacoue-Labarthe | Contrasting accumulation biokinetics and distribution of 241 Am, Co, Cs, Mn and Zn during the whole development time of the eggs of the common cuttlefish, Sepia officinalis[END_REF][START_REF] Metian | Characterization of 241 Am and 134 Cs bioaccumulation in the king scallop Pecten maximus: investigation via three exposure pathways[END_REF]. Therefore, the 134 Cs activity detected in each individual might reflect the water volume contented in the whole-body of sponge.

Nevertheless, I. variabilis displayed a 2-fold higher Cs uptake efficiency with an estimated CF ss of 1.8 ± 0.1 and a CF 170h of 2.0 ± 0.4 (Table 1).

Our work contributed to confirm that trace element bioaccumulation efficiencies are species-specific among sponges [START_REF] Patel | Sponge "sentinel" of heavy metals[END_REF][START_REF] Pan | Sponges and sediments as monitoring tools of metal contamination in the eastern coast of the Red Sea, Saudi Arabia[END_REF]. Using radiotracers at relevant concentrations, the uptake and loss parameters observed in this work suggest that Agelas oroides, Chrondrosia reniformis and Ircinia variabilis present the adequate characteristics of a biomonitor, as they would readily reveal an environmental contamination by any of the studied elements, except for 134 Cs. Agelas oroides and Chondrosia reniformis have already been proposed for such purpose by [START_REF] Pérez | In situ comparative study of several Mediterranean sponges as potential biomonitors of heavy metals[END_REF]. The loss kinetics of all trace elements in Ircinia variabilis is around 40% longer comparing to other species in average which indicates that this species would allow a long-term integrated measure (T b½ > 66 d) of the bioavailable fraction from dissolved contaminants, especially for 110m Ag, 109 Cd, 60 Co, 54 Mn and 75 Se. Among all, Acanthella acuta is remarkable because of its high 110m Ag uptake capacity and the total retention of the quasi-totality of accumulated metal (A 0l = 95%).

As Ag is well known to be mainly accumulated from waterborne pathway, e.g. in bivalves [START_REF] Wang | Kinetic determination of trace element bioaccumulation in the mussel Mytilus edulis[END_REF][START_REF] Metian | Investigation of Ag in the king scallop Pecten maximus using field and laboratory approaches[END_REF], in crustacean [START_REF] Metian | Metal and metalloid bioaccumulation in the Pacific blue shrimp Litopenaeus stylirostris (Stimpson) from New Caledonia: Laboratory and field studies[END_REF], or in cephalopod [START_REF] Bustamante | Uptake, transfer and distribution of silver and cobalt in tissues of the common cuttlefish Sepia officinalis at different stages of its life cycle[END_REF], A. acuta appears therefore as a good biomonitor for a contamination by this element. This result underlines the need to perform the trace metal analysis on a large array of sponge species before choosing the right sponge sentinel. This also strongly suggests that several sponge species could be valuable candidates as sentinel of a trace metal contamination and that massive sponges appear as better candidate than brancherected congeners. It would also be interesting to extend this approach to encrusting sponges even if the experimental design would be much more complicated [START_REF] De Mestre | Sponges as sentinels: Patterns of spatial and intra-individual variation in trace metal concentration[END_REF].

In summary, the uptake and loss kinetic data of trace elements into the organism appear essential to fully characterized the bioaccumulation properties of marine species and to validate their role as biomonitor, reflecting a time-integrated picture of the contamination state of the environment. A net accumulation of silver in A. acuta and selenium in A. oroides and Ircinia variabilis strongly suggest an important role as chelators for the metabolites present in these species. Additional insights into the speciation of these elements into the water column and the tissue animals will also be of high interest to fully understand the processes behind trace metal bioaccumulations. These results also raise the question of the fate of the highly accumulated element into sponge tissues, in terms of storage, detoxification process and potential role in the metabolism. Another very important issue is related to the presence of associated microorganisms in these complex invertebrates, which can play an important role in the processes of trace element bioaccumulation.

Acknowledgement

The International Atomic Energy Agency is grateful to the Government of the Principality of Monaco for the support provided to its Environment Laboratories. We thank the Conseil Régional Provence Model parameters are reported in Table 1. L and E: linear and exponential models, respectively; CF ss : concentration factor at steady-state, k u (h - 1 ): uptake rate, respectively; SE: standard error; R 2 : determination coefficient. * 134 Cs uptake was probably underestimated in A. acuta because of the high accumulation of 110m Ag.

2.

  Collection site, radiotracer and experimental design Three specimens of each sponge species were sampled off the Fontvieille harbor at St Nicolas rocks in Monaco by SCUBA between 20 and 35 meters depth in June 2008.

Fig. 2 .

 2 Fig. 2. Metal and radionuclide loss kinetics (CF; mean ± SD; n = 3) in the whole body of Acanthella

Fig. 3 .

 3 Fig. 3. Hierarchical Clustering on Principal Components for CF 170h values.

Figure 3

 3 Figure 3

  Table1) indicating that the accumulation of such trace elements in the sponge is limited by a steady-state equilibrium after 170 hours. On the contrary, the kinetics best fitted by a linear model indicated that optimal accumulation capacities for the species were not reached after time of exposure to dissolved radiotracers.Overall, the CF reached at the end of the exposure duration (CF 170h ) varied according to the element with relative values as 110m Ag ≈ 241 Am ≈ 65 Zn > 54 Mn > 109 Cd ≈ 60 Co ≈ 75 Se > 134 Cs

for all species. Considering morphological differences between sponge species, it is worth noting higher concentration factors for massive sponges (I. variabilis, C. reniformis and A. oroides) than for the erect ones (A. acuta, C. damicornis and C. verrucosa) with the exception of A. acuta for an extremely high CF of 110m Ag. More precisely, the analysis of the uptake rate (k u ) calculated from the models and the CF reached at the end of the exposure duration (CF 170h ) highlighted contrasting metal bioaccumulation efficiencies in sponges with respect to

  The first cluster includes the species C. verrucosa, C. damicornis and A. acuta that are characterized by low accumulation efficiencies for all the trace elements compared to species gathered in the second cluster, i.e. A. oroides, C. reniformis and I. variabilis.However, within the first group, A. acuta distinguished from C. damicornis and C. verrucosa by a higher 110m Ag accumulation. In the second group, C. reniformis displayed a peculiar low

uptake of

75

Se compared to A. oroides and I. variabilis (CF 170h = 12 ± 1 in C. reniformis compared to CF 170h = 225 ± 147 and 217 ± 63 in A. oroides and I. variabilis)

Table 2

 2

	) indicating that metals and radionuclides were

  Table 2). Interestingly, in A. acuta, 110m Ag was lost following a single component equation giving a very high estimated biological half-life (i.e. T b½ = 1100 d). This result suggests that the accumulated metal is strongly retained by this species. Similarly, A. oroides and I. variabilis that accumulated the most efficiently 75 Se (Table 1) released this metal following a simple equation and with a T b½ of 112 and 197 d, respectively.

  Alpes Côte d'Azur, the French ANR (www.ecimar.org, ANR-06-BDIV-001) and the European Union through the BIOCAPITAL UE grant (MRTN-CT-2004-512301) for financial supports (G.G.J and Chondrosia reniformis (■), and Ircinia variabilis (❋) exposed to dissolved radiotracer for 170 hours. Model parameters are reported in Table1. Please note the logarithmic scale for 110m Ag CF.

	Captions to Figures
	Fig. 1. Metal and radionuclide uptake kinetics (CF; mean ± SD; n = 3) in the whole body of
	Acanthella acuta (!), Agelas oroides (!), Cymbaxinella damicornis (❍), Cymbaxinella verrucosa
	(❏),

Table 1 .

 1 Parameters of metal uptake kinetics (CF; mean ± SD, n = 3) in the six species of sponge exposed for 170 hours to radiotracers dissolved in seawater (see Figure1):

	Metal	Species	Model	k u ± SE	CF ss ± SE	R 2	CF 170h
	110m Ag	A. acuta	L	11.9 ± 1.1	-	0.878	1895 ± 896
		A. oroides	L	5.6 ± 0.8	-	0.659	838 ± 657
		C. damicornis	E	4.1 ± 2.2	466 ± 127	0.771	351 ± 147
		C. verrucosa	E	1.2 ± 0.7	261 ± 92	0.963	137 ± 32
		C. reniformis	E	9.6 ± 1.3	829 ± 51	0.989	717 ± 52
		I. variabilis	L	3.3 ± 0.2	-	0.931	581 ± 147
	241 Am	A. acuta	E	3.2 ± 1.2	280 ± 50	0.920	240 ± 61
		A. oroides	E	8.9 ± 5.5	612 ± 159	0.723	550 ± 239
		C. damicornis	E	2.7 ± 0.8	237 ± 32	0.892	196 ± 46
		C. verrucosa	E	1.6 ± 0.6	119 ± 18	0.919	104 ± 31
		C. reniformis	E	6.7 ± 0.8	960 ± 66	0.996	658 ± 24
		I. variabilis	L	3.2 ± 0.2	-	0.950	540 ± 109
	109 Cd	A. acuta	L	0.2 ± 0.01	-	0.919	24 ± 3
		A. oroides	E	3.8 ± 2.4	238 ± 61	0.692	224 ± 94
		C. damicornis	E	0.5 ± 0.3	33 ± 8	0.687	28 ± 15
		C. verrucosa	L	0.07 ± 0.01	-	0.936	12 ± 3
		C. reniformis	E	5.2 ± 0.1	279 ± 11	0.989	276 ± 4
		I. variabilis	E	0.9 ± 0.5	172 ± 53	0.957	101 ±17
	60 Co	A. acuta	E	0.3 ± 0.2	32 ± 13	0.801	25 ± 12
		A. oroides	E	2.9 ± 1.6	250 ± 63	0.809	216 ± 79
		C. damicornis	E	0.7 ± 0.4	64 ± 19	0.633	54 ± 33
		C. verrucosa	E	0.3 ± 0.2	32 ± 10	0.839	27 ± 12
		C. reniformis	E	3.2 ± 0.2	366 ± 14	0.998	289 ± 5
		I. variabilis	L	1.7 ± 0.1	-	0.934	280 ± 81
	134 Cs	A. acuta*	E	0.03 ± 0.01	0.7 ± 0.1	0.725	0.6 ± 0.04
		A. oroides	E	0.06 ± 0.02	1.3 ± 0.1	0.914	1.4 ± 0.3
		C. damicornis	E	0.07 ± 0.04	1.0 ± 0.1	0.593	1.1 ± 0.5
		C. verrucosa	E	0.06 ± 0.01	1.0 ± 0.03	0.965	1.0 ± 0.2
		C. reniformis	E	0.02 ± 0.01	1.0 ± 0.2	0.793	0.8 ± 0.2
		I. variabilis	E	0.13 ± 0.05	1.8 ± 0.1	0.925	2.0 ± 0.4

Table 1 (

 1 following). Parameters of metal uptake kinetics (CF; mean ± SD, n = 3) in the six species of sponge exposed for 170 hours to radiotracers dissolved in seawater (see Figure1):

	Metal	Species	Model	k u ± SE	CF ss ± SE	R 2	CF 170h
	54 Mn	A. acuta	E	0.6 ± 0.3	107 ± 31	0.949	68 ± 13
		A. oroides	L	2.8 ± 0.3	-	0.787	437 ± 240
		C. damicornis	L	0.6 ± 0.1	-	0.744	94 ± 56
		C. verrucosa	L	0.4 ± 0.03	-	0.860	59 ± 25
		C. reniformis	E	3.7 ± 0.9	727 ± 101	0.991	421 ± 29
		I. variabilis	L	3.1 ± 0.2	-	0.929	553 ± 157
	75 Se	A. acuta	L	0.02 ± 0.001	-	0.926	4 ± 1
		A. oroides	E	3.4 ± 2.8	250 ± 90	0.629	225 ± 147
		C. damicornis	E	0.2 ± 0.1	16 ± 5	0.605	14 ± 8
		C. verrucosa	E	0.2 ± 0.1	13 ± 3	0.767	12 ± 6
		C. reniformis	E	0.1 ± 0.01	16 ± 1	0.996	12 ± 1
		I. variabilis	L	1.2 ± 0.1	-	0.931	217 ± 62
	65 Zn	A. acuta	L	1.4 ± 0.1	-	0.955	223 ± 51
		A. oroides	E	8.7 ± 8.2	951 ± 456	0.692	741 ± 469
		C. damicornis	E	2.9 ± 1.3	337 ± 79	0.817	254 ± 89
		C. verrucosa	E	1.2 ± 0.8	263 ± 100	0.954	142 ± 37
		C. reniformis	E	6.7 ± 0.8	610 ± 33	0.992	520 ± 29
		I. variabilis	L	3.3 ± 0.2	-	0.954	571 ± 113

Table 2 .

 2 Parameters and statistics of the depuration kinetics of radiotracers in the six species of sponge previously seawater exposed for 170 hours:

	Metal	Species	Model	A 0l ± SE (%)	k el	T b½ ± SE (d)	R 2
	110m Ag	A. acuta	O	95 ± 3	0.0006 ns	1093 ± 1773	0.085
		A. oroides	T	72 ± 32	0.020*	35 ± 17	0.955
		C. damicornis	T	76 ± 24	0.013 ns	55 ± 29	0.912
		C. verrucosa	T	83 ± 4	0.013***	54 ± 7	0.973
		C. reniformis	T	61 ± 2	0.012***	60 ± 6	0.990
		I. variabilis	T	39 ± 3	0.002 ns	285 ± 301	0.965
	241 Am	A. acuta	T	64 ± 3	0.016***	43 ± 5	0.901
		A. oroides	T	27 ± 11	0.015 ns	47 ± 38	0.920
		C. damicornis	T	50 ± 6	0.007*	99 ± 48	0.854
		C. verrucosa	T	42 ± 3	0.016***	43 ± 3	0.980
		C. reniformis	T	30 ± 3	0.020***	35 ± 6	0.994
		I. variabilis	T	52 ± 3	0.009***	77 ± 14	0.985
	109 Cd	A. acuta	-	-	-	-	-
		A. oroides	T	47 ± 3	0.006**	116 ± 49	0.920
		C. damicornis	T	67 ± 5	0.003 ns	249 ± 195	0.641
		C. verrucosa	T	74 ± 6	0.0004 ns	1950 ± 12890	0.544
		C. reniformis	T	63 ± 1	0.004***	167 ± 29	0.955
		I. variabilis	T	55 ± 3	0.008***	88 ± 23	0.887
	60 Co	A. acuta	T	79 ± 4	0.006**	110 ± 32	0.672
		A. oroides	T	84 ± 3	0.011***	66 ± 8	0.933
		C. damicornis	T	60 ± 13	0.004 ns	157 ± 188	0.641
		C. verrucosa	T	60 ± 14	0.006 ns	113 ± 99	0.949
		C. reniformis	T	80 ± 2	0.015***	46 ± 2	0.993
		I. variabilis	T	83 ± 3	0.006***	118 ± 25	0.866
	134 Cs	A. acuta	-	-	-	-	-
		A. oroides	-	-	-	-	-
		C. damicornis	-	-	-	-	-
		C. verrucosa	-	-	-	-	-
		C. reniformis	-	-	-	-	-
		I. variabilis	-	-	-	-	-

Table 2 (

 2 following). Parameters and statistics of the depuration kinetics of radiotracers in the six species of sponge previously seawater exposed for 170 hours: and T: 1-and 2-component exponential models, respectively; A 0l : assimilation efficiency of the long-lived component, respectively; SE: standard error; R 2 : determination coefficient; p-values: < 0.001 (***), < 0.01 (**), < 0.05 (*), > 0.5 (ns). In the case of T model, only the long-lived component parameters are shown (A 0l , k el and T b½ )

	Metal	Species	Model	A 0l ± SE (%)	k el	T b½l ± SE (d)	R 2
	54 Mn	A. acuta	T	84 ± 3	0.011***	61 ± 8	0.865
		A. oroides	T	85 ± 8	0.010***	67 ± 16	0.929
		C. damicornis	T	50 ± 17	0.004 ns	179 ± 338	0.862
		C. verrucosa	T	61 ± 14	0.010 ns	69 ± 34	0.976
		C. reniformis	T	75 ± 5	0.012***	60 ± 9	0.986
		I. variabilis	T	67 ± 14	0.003 ns	240 ± 349	0.945
	75 Se	A. acuta	T	71 ± 5	0.008**	93 ± 33	0.627
		A. oroides	O	94 ± 1	0.006***	112 ± 12	0.772
		C. damicornis	T	76 ± 6	0.011***	61 ± 14	0.753
		C. verrucosa	T	62 ± 6	0.014***	49 ± 11	0.937
		C. reniformis	T	74 ± 3	0.007***	99 ± 15	0.955
		I. variabilis	O	95 ± 1	0.004***	197 ± 37	0.612
	65 Zn	A. acuta	T	68 ± 24	0.010 ns	73 ± 57	0.898
		A. oroides	T	75 ± 7	0.017***	42 ± 8	0.937
		C. damicornis	T	71 ± 18	0.006 ns	112 ± 88	0.894
		C. verrucosa	T	73 ± 6	0.007**	104 ± 34	0.961
		C. reniformis	T	61 ± 2	0.018***	38 ± 2	0.994
		I. variabilis	T	55 ± 4	0.011***	66 ± 13	0.866

O

C.N.). T. Pérez (DIMAR, Marseille) is greatly acknowledged for his help in the sponge identification and fruitful discussions.

Figure 1

Figure 2