N Huby

L Hirsch

L Aubouy

Ph Gerbier

A Van Der Lee

F Amy

A Kahn

Evidence of environmental strains on charge injection in silole based organic light emitting diodes

Keywords: 72.80.Le, 73.50.-h, 71.15.Mb, 73.40.Sx

Using density functional theory (DFT) computations, we have demonstrated a substantial skeletal relaxation when the structure of 2,5-[bis-(4-anthracene-9-yl-phenyl]-1,1-dimethyl-3,4-diphenyl-silole (BAS) is optimized in the gas-phase comparing with the molecular structure determined from monocrystal X-ray diffraction. The origin of such a relaxation is explained by a strong environmental strains induced by the presence of anthracene entities.

Moreover, the estimation of the frontier orbital levels showed that this structural relaxation affects mainly the LUMO that is lowered of 190 meV in the gas-phase. To check if these theoretical findings would be confirmed for thin films of BAS, we turned to ultraviolet photoemission spectroscopy / inverse photoemission spectroscopy and electro-optical measurements. Interestingly, the study of the current density / voltage and luminance / voltage characteristics of an ITO/PEDOT/BAS/Au device clearly demonstrated a very unusual temperature-dependant behavior. Using a thermally-assisted tunnel transfer model, we found that this behavior likely originated from the variation of the electronic affinity of the silole derivative with the temperature. The thermal agitation relaxes the molecular strains in thin films as it is shown when passing from the crystalline to the gas-phase. The relaxation of the intramolecular thus induces an increase of the electronic affinity and, as a consequence, the more efficient electron injection in organic light-emitting diodes.

Introduction

The wide range of organic semiconductors used as emissive layers in organic lightemitting diodes (OLED) for displays or lighting applications implies specific investigations of each molecular family. Understanding phenomena related to the molecular properties is crucial to these applications, and usually involves fundamental investigations. Conjugated molecules in solid state are weakly bond by van der Waals interactions or hydrogen bonds.

Although the binding energy is low, the molecular environment can play a fundamental role when it induces steric strains 1 . Bond strain is accompanied by a loss of molecular symmetry, which induces an unusual electron distribution and a variation of the band gap.

The investigations presented in this paper are carried out with a new silole derivative: 2,5-[bis-(4-anthracene-9-yl-phenyl]-1,1-dimethyl-3,4-diphenyl-silole, herein named BAS (see Figure 1). Silole-based compounds have been proposed as a new class of electroluminescent materials 2,3 . They are mainly electron transport semiconductors 4 , and have a low-lying lowest unoccupied molecular orbital (LUMO) due to an effective interaction between the σ*orbital of the silicon atom and the π*-orbital of the butadiene fragment 5,[START_REF] Yamaguchi | Chemistry of Organic Silicon Compounds[END_REF] . Two anthracene groups, well-known as hole-transport groups in molecular films [START_REF] Danel | [END_REF]8 , are attached to the central silole ring. By combining the electron-transport properties of the silole and the hole transport ability of the anthracene, the intra-molecular balance of charge carriers should be improved in single-layer devices 9 .

Density Functional Theory (DFT) computations and crystallographic structure analyses were carried out. Geometrical parameters of BAS have been measured in the single crystal and compared to the ones obtained from the optimization of the molecular geometry in the gasphase. In contrast with what we had previously observed with others silole derivatives using the same methodology, some significant discrepancies are found between the as-found and the optimized structures [9][10][11][12][13] . The origin of these discrepancies may be explained by a relaxation phenomenon reflecting strong environmental strains in the crystal lattice. This observation prompted us to look at the role of supramolecular environmental strain and its consequences on electron injection in a layer of silole-derivative semiconductor. Indeed, the balance of charge carriers is a fundamental parameter to optimize the performances of emissive layers.

Electro-optical study, Ultraviolet Photoemission Spectroscopy / Inverse Photoemission Spectroscopy (UPS/IPES) measurements are carried out to investigate the relaxation of environmental strains on BAS molecules in the solid state with increasing the temperature.

The consequence of that relaxation results in an increase of the electron affinity (EA), which plays a fundamental role in the electron injection probability.

Results and discussion

Structural characterization

BAS was synthesized by an adaptation of the general procedure reported by Tamao et al. involving the intramolecular reductive cyclization of dimethyl-bis(phenylethynyl)silane followed by the coupling reaction with 9-(4-bromophenyl)anthracene 14 . BAS crystallizes in the P -1 triclinic space group with a = 10.094(2) Å, b = 10.998(2) Å, c = 18.808(2) Å, α = 85.90(1) °, β = 84.34(1) °, γ = 88.44(2)° and Z = 2. The diffraction intensities for BAS were collected at the joint X-ray Scattering Service of the Institut Européen des Membranes and the Institut Charles Gerhardt of the Université de Montpellier II, France, at 175 K using an Oxford Diffraction Xcalibur I diffractometer and Mo-Kα photons (λ=0.71073 Å). The structure of BAS, C 58 H 42 Si, was solved by direct methods using SIR2002 15 and refined by least-squares methods using CRYSTALS 16,[START_REF]data and 532 parameters leading to the usual crystallographic reliability factors R 1 =0[END_REF] . As shown in figure 2, the molecule displays a propeller-like arrangement of the four benzene rings as usually observed with other tetraarylsiloles. The dihedral angles (φ) between the mean planes of the central silole (S, see Figure 1 for labeling of the rings), the 2,5-benzene rings (P 1 and P 2 ) and the anthracenyl groups (A 1 and A 2 ) are given in Table 1. While the first three values fall within the range of those generally reported for related structural units, it is worthy of note that the forth one (φ(P 2 -A 2 ) = 64.2(3)°), is substantially lower than the smallest value encountered for 9arylanthracene derivatives (ca. 70°), and induces appreciable intramolecular steric interactions. Significant π-π stacking interactions are found between A 1 anthracenyl groups of adjacent BAS molecules that in fact determine the cohesion of the three-dimensional intermolecular structure (Figure 3). The lowest distance between two phenyl centroids is 3.711(4) Å with 23.20° and 20.81° for the angles between the ring normals and the centroid vectors. These values are in accordance within the accepted range of values for appreciable ππ interactions [START_REF] Janiak | [END_REF] .

The geometry optimization of BAS in the gas phase was carried out using density functional theory (DFT) calculations with the B3LYP functional. Due to the size of the molecule, geometry optimization without symmetry constrains was performed with the 6-31G basis set to the standard convergence criteria as implemented in Gaussian G03w software 19 .

In the gas phase, the geometry of BAS approaches the C 2 symmetry by relaxing the geometrical constraints between the different rings in order to minimize the steric interactions and to increase the conjugation throughout the π-system. Thus, the values of the torsion angles between the silole and the adjacent phenyl rings are decreased (Table 1) whereas the values of the torsion angles between the phenyl rings and the anthracenyl groups are increased. Among the siloles we have already studied these results are quite unusual (see above). For instance, in the case of 1,1-dimethyl-2,5-bis(p-2,2'-dipyridylaminophenyl)-3,4diphenylsilole (DMPPS, Figure 1) for which we have already described the mechanism of injection, both the crystal and the optimized structures are very similar 9,20,21 . In the DMPPS molecule, the anthracene side-groups have been replaced by dipyridylaminophenyl groups, and for that reason, no structural constraint is observed in the solid state. Therefore, it is likely that the propensity of anthracene to adopt a π-stacking structure induces sufficient environmental strain to bring about the BAS molecules to adopt such an energetically costly conformation in the crystal lattice (see Figure 3).

Electronic level characterization

Ultraviolet photoemission spectroscopy was carried out using He I (21.2 eV) and He II (40.8 eV) sources in conjunction with a double-pass cylindrical mirror analyzer. The resolution of the photoemission system was 150 meV as determined from the width of the Fermi edge on an electrode of freshly deposited Au. Inverse photoemission spectroscopy was carried out using an electron energy scan from 5 to 15 eV with a system resolution of 500 meV. UPS and IPES measurements (Figure 4) were carried out on an 8 nm BAS vapordeposited thin film on a gold substrate to estimate the energies of its filled and empty orbitals.

The measured ionization potential (IP) and EA, defined as the energy difference between vacuum level and onset of occupied and unoccupied states, are 6 eV and 1.69 eV, respectively. The single particle energy gap between the onsets of LUMO and HOMO equals 4.31 eV. To complete this study, DFT single point calculations were performed on both the X-ray and optimized geometries of BAS using the B3LYP functional with a 6-31G* split valence plus polarization basis set. The calculated HOMOs and LUMOs (Figure 5) do not much vary in appearance. The major changes in the electronic distribution reflect the symmetrization of the geometry when going from the solid to the gas phase (optimized geometry). On the other hand, the variation of the geometry of BAS from gas-phase to solid (crystalline) phase has a strong influence on the energy parameters, in particular the electron affinity (Table 2). It is noteworthy that the HOMO level is not much affected from the solid state to the relaxed gas-phase, contrary to the LUMO level. The latter is clearly stabilized in the relaxed gas-phase. The EA increases from -1.81 eV in the solid state to -2.00 eV in the gas-phase. Simultaneously, the IP varies only slightly, from -5.37 eV in the solid state to -5.39 eV in the gas phase.

Thermally assisted electron tunneling injection

Thus, structural modifications induce significant variation of the electronic structure of the molecule. At low temperature (90 K), the solid state is frozen. When the temperature increases, thermal agitation should relax the environmental strains and thus increase EA. To further probe this important mechanism, we built a device based on the following structure: ITO/PEDOT/BAS (80nm)/Au. The gold cathode is chosen for its high work function in order to be sure to characterize the BAS/Au interface. Variation in EA with temperature should have a strong effect on the electron injection at this interface. Details of device manufacturing can be found in reference 20 .

Typically, transport over a huge barrier is analyzed in terms of tunnel injection 22 . Figure 6 shows the current density-voltage (J-V) and luminance-voltage (L-V) characteristics for temperatures ranging from 90 K to 235 K carried out in a liquid nitrogen cryostat chamber under rotary pump vacuum (10 -3 mbar) and recorded with a Semiconductor Characterization System Keithley 4200 with a monitored photodiode. The first bias window (Domain I) is associated with hole-only injection and transport, because electron injection is ruled out by the large Au-LUMO potential barrier. The energy barrier between the onset of the empty states and the Au Fermi level is 2.64 eV (Figure 4). No luminescence is detected in this regime. The luminance appears in the second bias window (Domain II), which is the sign of significant injection of both types of charge carriers. The associated current in Domain II is much more dependent on the applied voltage than in Domain I, indicating that it is dominated by electrons. Considering the 2.64 eV electron injection barrier at the BAS/Au interface (see Figure 4), thermionic injection is not possible and only tunnel injection at high electric field needs to be considered. In the case of DMPPS, J-V and L-V characteristics were temperatureindependent and described using the Fowler-Nordheim tunnel transfer model 20 . On the contrary, in the case of BAS, J-V as well as L-V curves are thermally activated (Figure 6). The origin of this phenomenon is likely due to the relaxation of the environmental strains when increasing the temperature i.e. the higher the temperature, the lower the environmental strain pressure. DFT calculations summarized in Table 2 highlight that the LUMO and HOMO are strongly influenced by the torsion angles. Raghunath et al. recently obtained similar results in anthracene based molecule 8 . In the following model, we will consider that the environmental strain relaxation is Maxwell-Boltzmann temperature dependent.

We now introduce a thermally assisted tunnel injection model across a triangular potential barrier ψ(x). The Fermi level is taken as the reference level. The potential barrier thickness is a, with ψ(a) = 0. The potential barrier height at the interface, i.e. LUMO -E F (Au), is ψ(0) = φ c . Both a and ψ(0) are temperature dependant. ψ(x) and a are defined in equations 1 and 2:

Fx T k W x B c - ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ - - = exp 1 ) ( φ ψ (1) ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ - - = T k W F a B c exp 1 φ (2)
where F = V ap / d is the electric field, V ap the applied voltage, d the thickness of the BAS layer, φ c = φ m (Au) -EA (BAS) = ψ(0), W is the activation energy to relax the molecule of its environmental strain, E is the energy of the electrons and k B = 1.38×10 -23 J/K is the Boltzmann's constant. A gold cathode was chosen to have a high barrier φ c . Thus, the current density is assumed to be directly proportional to the tunnel transfer probability P 23,[START_REF] Sze | Semiconductor devices[END_REF] expressed by the equation (3) i.e. J = K × P:

( ) ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ - - = ∫ dx E x m P a 2 / 1 0 2 ) ( 2 2 exp ψ h (3)
Incorporating ψ(x), ψ(0) and a into equation (3) yields after integration:

⎟ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎜ ⎝ ⎛ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ - - + - - ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ - = 2 / 3 2 / 1 2 / 3 2 / 1 exp 1 3 2 ) ( 4 exp 3 2 ) ( 4 exp T k qW E V d mq V d E mq P B c φ h h (4)
Where m = 9.1×10 -31 kg and q = 1.6×10 -19 C being the mass and the charge of the electron respectively, d = 80×10 -9 m is the thickness of the BAS layer and h = h / 2π = 1.05×10 -34 J.s the reduced Planck constant.

In order to analytically solve equation ( 4), we have supposed that E → 0 i.e. electrons are supposed to be close to the Fermi level. This approximation is supported by two reasons: i) the probability to find electrons 100 meV over the Fermi level at T < 250 K is very low (less than 1%), and ii) if thermally dependent tunneling transport was resulting from the Fermi-Dirac statistic occupation of electrons over E F with increasing the temperature, such phenomenon should occur whatever the material. Actually, in that case of DMPPS-based devices, no thermally dependent tunneling transport was observed 20 . As a result, thermally effect originating from the occupation of electrons over the Fermi level in the device with a gold cathode can be neglected. Equally, we neglect any variation of the interfacial dipole with the temperature. Consequently, substituting the numerical values in equation ( 4) yields:

⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ × - - ≈ T W V P c 11601 exp 1 546 exp φ (5)
Figure 7 shows the J-V characteristics and the corresponding Arrhenius plots for Domain II in Figure 6. The experimental data are represented by the full symbols. The theoretical values (full curves) have been calculated from equation (5) using W = 35 meV and φ c = 1.3 eV. A thermal energy of 35 meV corresponds to a temperature of about 400 K, which is almost the BAS melting point. Above the fusion temperature, molecular environmental strain is inexistent. The weak value of φ c compared to the difference φ m (Au) -EA (BAS) indicates the presence of an electrical dipole at the interface BAS/Au [START_REF] Hill | [END_REF]26 and/or a possible diffusion of gold atoms in the BAS layer. The model of thermally assisted tunnel transfer fits experimental data. The higher the temperature, the lower the environmental strain on the BAS molecule.

The consequence is an increase in EA and an enhancement of electron injection in BAS-based devices. The effective variation of EA can be calculated from equation (1) using the fitting parameters. The variation of EA from 90 to 235 K is around 0.17 eV which is almost in accordance with that calculated with DFT.

Optical absorption or photoluminescence or electroluminescence (EL) does not directly provide data on LUMO and HOMO levels because in organic compounds, exciton binding energy is roughly 1 eV. However, a red-shift should be observed in the EL spectra by increasing the temperature of the sample. As a consequence, we recorded EL spectra for temperature varying from 83 K to 297 K on ITO/PEDOT-PSS/BAS (80 nm)/Ca devices.

Electroluminescence spectra were collected using an optic fiber connected to an OceanOptics HR2000 spectrometer while OLEDs were submitted to a constant current density of 10 mA.cm -2 . OLEDs were biased for less than one second to reduce any heating effect during the measurement. Results are presented in figure 8. A slight red-shift, of almost 70 meV, is observed when the temperature is increased from 83 K to 297 K. Actually, two phenomena are in competition: a red-shift resulting from the stabilization of the LUMO and a blue-shift (commonly observed in polymers and small molecules) due to a statistical occupation of higher excited states at high temperature [27][28][29] . Thus, this observed red-shift qualitatively confirm the relaxation of environmental strains with increasing the temperature.

Conclusion

In summary, we have demonstrated that the π-stacking of anthracene entities induces environmental strains on the BAS molecules affecting the molecular electronic levels. DFT calculations are supported by electrical measurements and by electro-optical study. A thermally assisted tunnel transfer was developed. All data are consistent with a modification of the EA with the temperature. Environmental strains on BAS molecule relax when increasing the temperature. It means that the energy optimization of the solid state induce torsion angles which put the BAS molecule of its stable energy configuration. This phenomenon is highlighted by the increase of the electron affinity and an enhancement of the electron injection at the interface Organic/cathode.

Table and figure captions

Table 1: Dihedral angles (φ) between the mean planes of the central silole (S), the 2,5benzene rings (P 1 and P 2 ) and the anthracenyl groups (A 1 and A 2 ).

Table 2: Calculated HOMOs and LUMOs energy levels on both the X-ray and optimized geometries of BAS using the B3LYP functional with a 6-31G* split valence plus polarization basis set.

Huby et al.

Figure 1 :

 1 Figure 1: 2,5-[bis-(4-anthracene-9-yl-phenyl]-1,1-dimethyl-3,4-diphenyl-silole (BAS). S is

Figure 2 :

 2 Figure 2: Three-dimensional structure of BAS such as found from the analysis of the X-ray

Figure 3 :

 3 Figure 3: π-π aromatic interaction between anthracenyl moieties of adjacent BAS molecules.

Figure 4 :

 4 Figure 4: UPS and IPES spectra performed on a BAS thin film (80 nm thick) deposited under

Figure 5 :

 5 Figure 5: DFT computed frontier orbitals of BAS in the crystal lattice and in the gas phase

Figure 6 :

 6 Figure 6: Current density -voltage and luminance -voltage characteristics of the device

Figure 7 :

 7 Figure 7: Experimental (symbols) and theoretical (lines) J-V curves (a) and the

Figure 8 :

 8 Figure 8: Normalized electroluminescent spectra recorded at 87 K, 148 K and 297 K on

  FIGURE 6Huby et al.

Table 2

 2 Huby et al.

		3)	59.3(3)	72.7(3)	64.2(2)
	BAS optimized	49.1	49.1	77.7	77.6

Acknowledgments :

This work was partially supported by the "Région Aquitaine", the "Région Languedoc-Roussillon" and the STII Department of French CNRS, and the National Science Foundation (DMR-0408589).

Si


Shaun Ferdous
email: ferdousjahan.shaun@esiee.fr

Elyes Nefzaoui
email: elyes.nefzaoui@esiee.fr

Frederic Marty

William Cesar

Tarik Bourouina

Micro-fabricated thermal flow-rate sensors: the substrate material impact on the device performance and power consumption

Keywords: Sensor, MEMS, Flow-rate, Thermal sensor, Micromachined sensor, Water Network Monitoring 1

We report on micro-machined flow-rate sensors as part of autonomous multi-parameter sensing devices for water network monitoring. Three different prototypes of the flow-rate sensors have been designed, fabricated and experimentally characterized. Those sensors are made of identical micrometric platinum resistors deposited on different substrates, made of glass, silicon and micro-structured silicon, with and without insulation layers. The sensors are tested under the anemometric operating scheme. They are experimentally characterized under a water velocity range from 0 to 0.91 m/s. We show that the glass substrate device is more sensitive and less power-consuming under identical operating condition. We also show that, when silicon is needed as the substrate material, further optimization and design strategies are required. Experimental results are analyzed with respect to Computational Fluid Dynamics simulations with the Finite Element Method.

INTRODUCTION

Flow-rate measurement is widely used in various fields including biological and bio-medical applications [START_REF] Silvestri | Micromachined Flow Sensors in Biomedical Applications[END_REF]- [START_REF] Hsiai | Micro Sensors: Linking Real-Time Oscillatory Shear Stress with Vascular Inflammatory Responses[END_REF], industrial processes [START_REF] Billmann | Leak detection methods for pipelines[END_REF], environmental [START_REF] Bartlett | Modeling of Occupant-Generated CO2 Dynamics in Naturally Ventilated Classrooms[END_REF], [START_REF] Zhu | 2-D Micromachined Thermal Wind Sensors #x2014;A Review[END_REF] monitoring and weather forecasting [START_REF] Shen | A FCOB packaged thermal wind sensor with compensation[END_REF]. For instance, flow-rate measurement is particularly important for water distribution systems monitoring not only for measuring water consumption but also for leakage detection. Generally, a water distribution system at an urban scale is a vast network. Consequently, a finely meshed monitoring system based on a sensor network can be quite expensive. Micro-fabrication technologies offer solutions for such a problem with a good meshing/cost tradeoff through cheap lab on chip devices, which can be deployed in large number. During the last decades, the advancement of micro-fabrication techniques enabled the design of low-cost miniaturized sensors optimized for different applications. Extensive reviews can be found in [START_REF] Nguyen | Micromachined flow sensors-a review[END_REF], [START_REF] Kuo | Micromachined Thermal Flow Sensors-A Review[END_REF]. Such devices offer several advantages: compactness, low cost and low power consumption. In addition, mass production can be considered thanks to the wide range of flow-rates that can be covered by these sensors, from µl/min to liter/min [START_REF] Vilares | Fabrication and testing of a SU-8 thermal flow sensor[END_REF], [START_REF] Lin | Low-power micro-fabricated liquid flow-rate sensor[END_REF]. Flow-rate sensors are mainly based on mechanical and thermal transduction phenomena. The device described in the present work belongs to the second category. Thermal flow-rate sensors can be sorted into three categories: calorimetric, anemometric (hot-wire) and time-of-flight (TOF) and () [START_REF] Nguyen | Micromachined flow sensors-a review[END_REF], [START_REF] Lin | Low-power micro-fabricated liquid flow-rate sensor[END_REF]. A Calorimetric sensor detects the fluid flow by measuring temperature distribution around the heater by at least one temperature sensor. A hotwire flow-rate sensors is made of a resistive wire, which transfers heat to the surrounding fluid. The hot wire also acts as a temperature probe. Flow-rate is deduced from the convective cooling rate of the hot wire due to the fluid flow [START_REF] Hsiai | Micro Sensors: Linking Real-Time Oscillatory Shear Stress with Vascular Inflammatory Responses[END_REF], [START_REF] Bartlett | Modeling of Occupant-Generated CO2 Dynamics in Naturally Ventilated Classrooms[END_REF].The TOF method is based on measuring the travel time of a thermal pulse from a heater to a temperature sensor. Accordingly, it is obvious that the largest the convective heat flow compared to other heat flows, the largest the sensor sensitivity. Indeed, when convection is the dominating thermal channel, a variation of the fluid velocity and flow-rate will have the largest effect on the heater temperature. Consequently, conductive heat leakage through the substrate should be reduced in order to increase the convective transfer weight, hence the sensor sensitivity and reduce the power consumption. In general, low thermal conductivity substrates are used for this purpose [START_REF] Lin | Low-power micro-fabricated liquid flow-rate sensor[END_REF]. In some cases, the substrate material cannot be optimized with regard to thermal considerations due to external constraints such as the compatibility of the device with the operating medium or with system integration constraints.

In this work, we report on a comparative study of three micro-machined thermal flow-rate sensors made on different substrates and operated in the anemometric mode. We focus on the triple constraint of a large thermal conductivity substrate, silicon for instance, low power consumption and a large sensitivity. We report in particular an experimental and numerical comparison of the three devices.

The paper is organized as follows: first, the devices design and fabrication process are presented. Then, the experimental setup and numerical methods are described. Finally, the major results regarding the devices performances, sensitivity and power consumption in particular, are reported and discussed. The advantages and weaknesses of each configuration are highlighted and further improvements are suggested.

METHODS

2.1

Sensors Design and Fabrication The three sensors have in common the sensing element made of three 4-probe platinum resistors. Platinum wires are 106-µm long, 10-µm wide and 340-nm thick. Platinum is chosen as a material for its high TCR, which is 2.218×10 - 3 °C-1 in our case, and the excellent linearity of its resistivity temperature dependence. There are some other materials that have higher TCR value than platinum (Pt), but none of them show high sensitivity, reproducibility and chemical stability at high temperature [START_REF] Mailly | Anemometer with hot platinum thin film[END_REF]. Besides, this material has corrosion resistance and outstanding accuracy with approximately one order of magnitude uncertainty [START_REF] Nguyen | Micromachined flow sensors-a review[END_REF], [START_REF] Van Honschoten | Application of a microflown as a low-cost level sensor[END_REF]- [START_REF] Kreider | Platinum/palladium thin-film thermocouples for temperature measurements on silicon wafers[END_REF]. The use of Platinum as a resistor material is predominant in this research field [START_REF] Ma | A MEMS-Based Flow Rate and Flow Direction Sensing Platform with Integrated Temperature Compensation Scheme[END_REF], along with the employment of several other metals and mixtures such as platinum/palladium [START_REF] Zanini | Fabrication and properties of a Si-based high-sensitivity microcalorimetric gas sensor[END_REF], platinum/titanium [START_REF] Viard | A robust thermal microstructure for mass flow rate measurement in steady and unsteady flows[END_REF], platinum/chromium [START_REF] Götz | A micromachined solid state integrated gas sensor for the detection of aromatic hydrocarbons[END_REF], platinum/aluminum [START_REF] Norlin | A chemical micro analysis system for the measurement of pressure, flow rate, temperature, conductivity, UV-absorption and fluorescence[END_REF] and chromium/gold [START_REF] Lammerink | Micro-liquid flow sensor[END_REF]. These metals are used to fabricate the heater resistor for a variety of flow-rate sensors for different operation media.

The sensors under consideration are fabricated on three different substrates: glass, silicon and a silicon membrane. These three configurations will be referred to as Gl-sensor, Si-sensor and Mb-sensor, respectively. Three resistors are deposited on each substrate to enable the different operating modes previously mentioned in the introduction: calorimetric, anemometric and TOF (Time-of-Flight). Only one resistor is used in the present study. we operate according to the anemometric mode. The geometric properties of the three configurations are presented in Fig. 1. When the substrate is not made of glass (Fig. 1-a,c), a 0.45 µm-thick SiO2 insulation layer is introduced between the resistors and the 500 µm-thick silicon substrate or the silicon membrane to reduce the overall device thermal conductance. The SiO2 layer is produced by thermal wet oxidation. The Pt resistors are patterned by a lift-off process following sputter-deposition. For Mb-sensor (Fig. 1-c), the Pt resistors are mounted on a silicon membrane resulting from backside silicon etching. The cavity depth of the membrane is nearly 430 µm. Glass is used as a support under the silicon membrane.

In order to perform the experiments, the sensor chip is connected to an electric circuit for power supply and the sensor's response reading. A T-shaped PCB is designed for this purpose. On the top of the PCB, a 1 × 1 cm space is created to place the chip and the electrical wire bonds are built using gold between the sensor's connection pads and the PCB. The height of the PCB including the base is 7.2 cm and the width of the body part excluding the base is 1.18 cm. The height and width of the base itself is 0.89 cm and 3.82 cm, respectively, as shown in Fig. 2. An adhesive for glass and plastic materials is used to attach the sensor chip and the PCB. Finally, the wire bonds are hermitically protected using UV-cured epoxy against water to avoid electrical short-circuit.

Experimental Setup

The sensor is inserted into a 25-mm diameter PVC pipe. The pipe length is 10 times larger than its diameter to ensure an established flow regime at the sensor's position and avoid edge effects. The fluid flow is created by a variable speed pump, which is submerged in a 20-liter water reservoir and operated in closed loop. A schematic and picture of the experimental setup are shown in Fig. 3a andb, respectively. Hot-wire anemometric operation scheme is employed to extract the sensor's response to fluid velocity. To ensure a proper operation under the anemometric mode, a constant current source is used to supply the current to the 4-probe heater-resistor. LabView software interface is used for data acquisition. For each flow-rate value, the resistance drop due to the temperature variation caused by flow cooling is measured. Comparison of the resistance value with zerovelocity situation provides access to the fluid velocity using tabulated data obtained through a calibration process.

2.3

Simulation A computational fluid dynamics numerical analysis based on finite elements method is carried out to extract the effect of different parameters, of both geometry and used materials, on the sensor performances. COMSOL Multiphysics is used to perform the numerical study using conjugate heat transfer module; to account for heat transfer both in solid and fluid media as well as the fluid transport. The geometry as presented in Fig. 1a, b andc is used to build the numerical simulation domain shown in Fig. 4. All used materials physical properties are imported from COMSOL Material Library. A parametric study is conducted for each configuration in order to study the temperature variation under a water flow at different velocities for a given supplied power. The considered sensors are sensitive to the velocity. Therefore, they are placed in the middle of the simulation domain to experience the maximum velocity. The fluidic domain is 6 times larger in width and 3 times larger in height than the chip size to minimize edge effects and the effect of boundary conditions on the sensor.

RESULTS AND DISCUSSION

An anemometric flow rate sensor measures a temperature drop due to a fluid flow. The velocity is then deduced from the cooling magnitude. Consequently, the sensor Joule self-heating under non-flowing fluid is a critical parameter. Joule self-heating experiment enables the prediction of the sensor behavior and sensitivity without performing further experiments.

3.1

Joule self-heating and Device turndown We plot in Fig. 5 the temperature ∆Tmax of a platinum resistor under no flow for different supply currents and for the different prototypes, where the ∆Tmax is the difference between the heater and the ambient temperature under noflow condition. For a 30 mA supply current, Gl-sensor exhibits a temperature increase by 11.66 °C larger than the two other devices. As a result, we expect Gl-sensor to be more sensitive to a wider flowrate range and less power consuming under the same operating conditions than the two other prototypes. The sensitivity difference is expected to be more critical when low power consumption is targeted. Furthermore, Joule self-heating results provide information regarding the minimal operating current of a given device. For instance, according to Fig. 5 results, Gl-sensor can be properly operated under supply currents as low as 15mA. On the other hand, operating the Si-sensor and Mb-sensor at a supply current lower than 25 mA would be difficult. The temperature increase due to Joule self-heating, ∆T 𝑚𝑎𝑥 , is close to zero for these two sensors at supply current smaller than 25 mA. As a consequence, the Si and Mb-sensor deliver inconsistent responses for velocities smaller than 0.6 m/s as shown in Fig. 6 where the sensors' relative temperature (RT) drop is plotted versus the flow velocity. The RT drop is defined as,

𝑹𝑻 = ∆𝑻 ∆𝑻 𝒎𝒂𝒙 (1)
Where, ∆𝑇 = 𝑇 ℎ,𝑣 -𝑇 𝑎 and ∆𝑇 𝑚𝑎𝑥 = 𝑇 ℎ,0 -𝑇 𝑎 . Here, 𝑇 ℎ,𝑣 and 𝑇 ℎ,0 is the heater temperature at velocity 𝑣 and at zero velocity respectively; and 𝑇 𝑎 is the ambient temperature. The value of ∆T/∆T max quantifies the fluid flow cooling effect which provides information on the sensor sensitivity level and turndown ratio as well. The smaller the value of ∆T/∆T max , the more sensitive the sensor. However, when ∆T/∆T max reaches zero for a given velocity, the sensor is saturated for larger velocities. In other words, ∆T/∆T max ranges between 1 and 0. For the former, the fluid velocity is not large enough to be sensed. For the latter, the sensor is saturated and any further increase of the velocity is not sensed. According to Fig. 6, we observe no significant variation of the sensors temperature when the velocity increases up to 0.6 m/s. At 10 mA, the problem persists for larger velocities. For a 15 mA supply current, a significant cooling of the devices is observed for velocities larger than 0.6 m/s. On the other hand, at 15 mA supply current, the membrane and silicon substrate based sensors are saturated at 0.42 m/s since no temperature variation is observed for larger velocities as shown in Fig. 7. This in not observed for Glsensor. Therefore, the sensors' behaviors expected from simple Joule heating experiments (Fig. 5) are in agreement with experiments under a variable velocity flow (Fig. 7).

Sensitivity

We performed numerical simulations for the three considered flow-rate sensors to enable a quantitative assessment of the devices' sensitivity, both numerically and experimentally. Results are plotted in Fig. 8. Numerical simulations are done over a wide velocity range at different power supply values. Experimental data are recorded for different supply currents under a velocity range from 0.33 m/s to 0.91 m/s because of experimental constraints on the reachable velocity values. For both cases, the sensors sensitivity was calculated at 10 mW power supply. The sensitivity is defined as the ratio between the non-dimensional temperature variations with respect to the velocity changes:

𝑆𝑒𝑛𝑠𝑖𝑡𝑖𝑣𝑖𝑡𝑦 = ( ∆𝑇 ∆𝑇𝑚𝑎𝑥 ) 𝑛 -( ∆𝑇 ∆𝑇𝑚𝑎𝑥 ) 𝑛+1 (𝑣𝑒𝑙𝑜𝑐𝑖𝑡𝑦) 𝑛+1 -(𝑣𝑒𝑙𝑜𝑐𝑖𝑡𝑦) 𝑛 (2)
Where n is the sequence number of the velocities and the corresponding relative temperature variations.

Sensitivity trends as a function of velocity obtained by experimental (Fig. 8) and numerical (Fig. 8 inset) data are consistent. The Gl-sensor shows a larger sensitivity for both low and high velocity values than the Si-sensor and Mbsensor. The main substrate material property that governs the sensor's response is thermal conductivity. Glass has low thermal conductivity compared to silicon, hence a better thermal insulation. As a result, numerical and experimental results show Gl-sensor is better than the silicon and membrane based sensors with respect to sensitivity and power consumption. If another insulator is used instead of glass then the sensor's response will depend on its thermal conductivity. For a larger thermal conductivity, the sensor's performance will decrease. On the opposite, for lower thermal conductivities, the sensor performance will be improved. A numerical study is conducted to assess this dependency, where glass is used as substrate material but its thermal conductivity is varied. The standard value of quartz glass thermal conductivity in COMSOL Materials Library is 1.4 W/m.K. Simulations are performed with two different thermal conductivity values , 0.014 W/m.K and 14 W/m.K; which can be considered as insulating materials 1 and 2 respectively. Numerical results are presented in Fig. 9. The glass substrate device with standard thermal conductivity (1.4 W/m.K) shows an intermediate response in comparison with insulator 1 and 2. The device with low thermal conductivity substrate (0.014 W/m.K) exhibits a high sensitivity in the whole considered velocity range. Besides, the RT of insulator 2 flow-rate sensor with as substrate goes to zero and reaches saturation very rapidly; which indicates a faster cooling of the heater and a small device's turn-down ratio. We also observe that, at a given velocity range, around 0.6 m/s, the sensitivity of the Gl-sensor is not following the monotonous decreasing trend (Fig. 8 inset). The same non-monotonic behavior can be observed in Fig. 7 and Fig. 9 for Gl-sensor on the normalized temperature decrease plot. This unexpected behavior is discussed in the following paragraph.

The response non-monotonicity and outliers

In Fig. 7, Fig. 8 inset and Fig. 9 a non-monotonic variation is observed of the Gl-sensor temperature when the fluid velocity increases around 0.6 m/s. Such a behavior is not expected since an increase of the flow velocity, induces a larger convective cooling, hence a smaller heater temperature.

The velocity field around the device is plotted in Fig. 10. The plotted field shows a reverse flow (opposite to the main flow velocity direction) at the vicinity of the resistor. This reverse flow carries back downstream hot fluid which, instead of cooling the resistor, heats it up. The same velocity field, with a reverse flow in particular, can be observed for Si-sensor and Mb-sensor. However, due to their lower Joule self-heating, downstream fluid brought back by the reverse flow is not hot enough in this case to induce an increase of the sensor temperature. In addition, the reverse flow is observed for velocities larger than 0.6 m/s while Si-sensor and Mb-sensor are saturated starting from velocities as small as 0.4 m/s. The reverse flow and the resulting non-monotonic evolution of Gl-sensor can be detrimental for an accurate measurement of the fluid velocity and flowrate. Indeed, the fluid velocity is deduced from the sensed temperature. A "wrong" temperature measurement leads to an error in the calculated velocity value. We can observe in Fig. 7 that the same temperature is measured for two different fluid velocities around 0.6 m/s. With no additional information, the two velocity values can-not be discriminated. Such problems can be avoided by a proper optimization of the sensor chip shape, the edges curvature for instance, which significantly reduces the reverse flow magnitude, hence the resulting flow-rate measurements discrepancies. This solution has been validated by numerical simulations. Results are not shown here.

3.4

Power consumption According to the previous paragraphs and the sensitivity assessment of the three sensors, Gl-sensor exhibits higher performances. In addition to large sensitivity, low power consumption is a critical criterion for the studied devices which are developed as components of low power sensor networks. A comparative study of the three sensors power consumption is conducted. Power consumption is calculated using Ohm's law: P = IR 2 . Supply current intensity is varied from 10 mA to 30 mA with step of 5 mA and the resultant Joule self-heating is assessed through the heating resistance value measurement for the three devices. The test is conducted under constant current, so the power consumption is proportional to the resistance value which varies due to the resistor's temperature variation. We plot in Fig. 11, power consumption of the three devices as a function of the supply current under zero velocity flow. The presented results show that the glass sensor is nearly 1.4 times more power consuming at maximum supply current, 30 mA for instance, than the membrane and silicon sensors. Such a result is surprising at first glance. Indeed, the three devices heaters are made of the same platinum resistances with identical geometries. Consequently, their power consumption under a constant current is expected to be the same. Such an expectation does not account for the platinum resistance value temperature dependence. Indeed, the larger consumption of Gl-sensor is mainly due to its better thermal insulation, hence to its larger Joule self-heating. For a given supply current, the glass substrate low thermal conductivity ensures larger insulation of the heater, hence a larger resistance temperature. Since the resistance value is temperature dependent, a larger temperature induces a larger resistance value, hence a larger power consumption. The drawback of the Gl-sensor larger power consumption at a given electric current can be balanced by the possibility of a proper operation of this sensor at lower supply currents, as low as 20 mA, which in turn reduces the power consumption. Results presented in Fig. 8 are obtained for a supply current slightly larger than 20 mA.

The numerical and experimental study presented in this paper shows that the substrate thermal conductance is the main parameter governing ∆T max and ∆T/∆T max for a given power supply value, hence the sensor sensitivity. Since, silicon thermal conductivity is much larger than that of glass, around 100 times larger for instance, Gl-sensor Joule self-heating and sensitivity overcome those of silicon based sensors. Although the introduced silicon membrane diminishes the conductive heat loss through the substrate, the sensor performances enhancement is not significant. Consequently, a possible solution to obtain a sensitive and low power anemometric flow-rate sensor on a silicon substrate is to further increase the thermal insulation of the heating/sensing resistor through a geometric optimization.

4

CONCLUSION Three micro-machined flow-rate sensors for autonomous water network monitoring devices have been designed, fabricated and experimentally characterized. Their main difference appears in the used substrate material: glass, bulk silicon and a silicon membrane. We show experimentally and numerically that the glass substrate device is far more sensitive and less power consuming. We also show that the key parameter for such performances is its low thermal conductivity. However, large thermal conductivity materials such as silicon can be needed for co-integration of different sensors in lab-on-chip devices which increases power consumption and reduces sensitivity. We show that a simple geometric optimization such as the use of a silicon membrane is insufficient to overcome this problem. More complex geometries and strategies for the overall device thermal conductance reduction are then needed to obtain more energy efficient and sensitive devices.

Fig. 1

 1 Fig. 1 Schematic of (a) silicon, (b) glass, and (c) membrane structure flow rate sensor; fabricated flow-rate sensor on (d) silicon substrate and (e) glass substrate.

Fig. 2

 2 Fig.2The PCB designed for the sensor chip connection.

a b Fig. 3

 3 Schematic (a) and picture (b) of the used experimental setup.

Fig. 4

 4 Fig. 4 Schematic representation of the numerical simulation domain.

Fig. 5

 5 Fig. 5 Heater-resistor's temperature under no-flow condition at different supply current intensities. Error bars are plotted but cannot be distinguished because of small uncertainties.

Fig. 6

 6 Fig. 6 Silicon and membrane based flow-rate sensor's responses under different velocities at 10 and 15 mA supply current.

Fig. 7

 7 Fig. 7 Glass, membrane and silicon flow-rate sensors responses at 30 mA under different velocities.

Fig. 8

 8 Fig.8Sensitivity of the three sensors as a function of the flow velocity calculated with experimental (main plot) and numerical (inset) results under a 10 mW power supply. The velocity range of the numerical data is much larger that of the experimental ones due to experimental constraints on to the available pumps and circuitry.

Fig. 9

 9 Fig. 9 Normalized temperature variation of the flow-rate sensor at different velocities when different hypothetical insulating materials are used instead of glass as substrate.

Fig. 10

 10 Fig. 10 Reverse flow at the sensor's heater-resistor vicinity at 0.3 m/s and 0.4 m/s.

Fig. 11

 11 Fig. 11 Power consumption of the three sensors at zero velocity for different supply currents

ACKNOWLEDGEMENT

This work has received funding from the European Union's H2020 Programme for research, technological development and demonstration under grant agreement No644852. Part of this work had the support of the National Research Agency (ANR) in the frame of the EquipEx project SENSE-CITY of the Programme Investissement d'Avenir (PIA), involving IFSTTAR and ESIEE Paris as founding members of the consortium. Fabrication of the chips was done in the cleanroom facilities of ESIEE Paris, whose technical staff is deeply acknowledged. Authors would like to thank H. Regina and S. Sarkar for valuable help on numerical simulations.


P Bustamante

J.-L Teyssié

B Danis

S W Fowler

P Miramand

O Cotret

M Warnau

Uptake, transfer and distribution of silver and cobalt in tissues of the common cuttlefish Sepia officinalis at different stages of its life cycle

Keywords: Metal, Radiotracer, Bioaccumulation, Digestive gland, Cephalopods

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Uptake, transfer and distribution of silver and cobalt in tissues of the common cuttlefish Sepia officinalis at different stages of its life cycle

Paco Bustamante, Jean-Louis Teyssié, Bruno Danis, Scott W. Fowler, Pierre

Miramand, Olivier Cotret, Michel Warnau

To cite this version:

Paco Bustamante, Jean-Louis Teyssié, Bruno Danis, Scott W. Fowler, Pierre Miramand, et al.. Uptake, transfer and distribution of silver and cobalt in tissues of the common cuttlefish Sepia officinalis at different stages of its life cycle. Marine Ecology Progress Series, 2004, 269, pp.185-195. 10.3354/meps269185. hal-00186607

INTRODUCTION

Interest in the bioaccumulation of trace elements in cephalopods stems mainly from their high trophic position in the marine food web, and from their role as important prey for marine mammals, seabirds and fish [START_REF] Clarke | Cephalopods as prey. III. Cetaceans[END_REF][START_REF] Croxall | Cephalopods as prey. I. Seabirds[END_REF][START_REF] Klages | Cephalopods as prey. II. Seals[END_REF][START_REF] Smale | Cephalopods as prey. IV. Fishes[END_REF].

Furthermore, heavy metal levels in cephalopods, which are extensively fished and consumed by humans, are also of direct concern to public health. Various studies have investigated the concentrations and tissue distribution of several trace metals in cephalopods (e.g. [START_REF] Martin | High copper concentrations in squid livers in association with elevated levels of silver, cadmium, and zinc[END_REF][START_REF] Miramand | High concentrations of some heavy metals in tissues of the Mediterranean octopus[END_REF][START_REF] Miramand | Concentration and distribution of heavy metals in tissues of two cephalopods, Eledone cirrhosa and Sepia officinalis, from the French coast of the English Channel[END_REF][START_REF] Bustamante | Bioaccumulation of 12 trace elements in the tissues of the nautilus Nautilus macromphalus from New-Caledonia[END_REF], Ishihashi et al. 2001); however, most have been focused on essential trace elements or specific organs such as the digestive gland, the branchial hearts or the gills (see [START_REF] Miramand | Concentration and distribution of heavy metals in tissues of two cephalopods, Eledone cirrhosa and Sepia officinalis, from the French coast of the English Channel[END_REF]). Owing to the high feeding rates characteristic of cephalopods, food has often been cited as the main uptake pathway for explaining the high concentrations of metals found in cephalopod tissues (e.g. [START_REF] Martin | High copper concentrations in squid livers in association with elevated levels of silver, cadmium, and zinc[END_REF][START_REF] Bustamante | Cadmium, copper and zinc in octopuses from Kerguelen Islands, Southern Indian Ocean[END_REF], Gerpe et al. 2000).

However, there are very few published studies that determine the assimilation efficiencies and depuration rate constants of trace metals in cephalopods [START_REF] Suzuki | Accumulation of cesium-137 by useful Mollusca[END_REF][START_REF] Guary | Experimental studies on the biokinetics of plutonium and americium in the cephalopod Octopus vulgaris[END_REF][START_REF] Ueda | Accumulation of 65 Zn by Octopus Octopus vulgaris[END_REF], Bustamante et al. 2002a). These parameters are necessary in order to assess the degree of retention of waterborne and dietary metals and to assess the relative importance of both pathways. In addition, benthic cephalopods live in direct contact with the substratum, and many swimming species such as cuttlefish spend long periods buried into sediment [START_REF] Mangold | Toxicities of ten metals to Crassostrea gigas and Mytilus edulis embryos and Cancer magister larvae[END_REF]. Therefore sediments should also be considered as a possible pathway for metal transfer to cephalopods.

Ag and Co have been reported to be concentrated, sometimes to a very large extent, in cephalopods [START_REF] Martin | High copper concentrations in squid livers in association with elevated levels of silver, cadmium, and zinc[END_REF], Ueda et al. 1979); however, little is known about the metabolism of these 2 heavy metals. The digestive gland appears to contain most of the body burden of Ag and Co in squid (Ishihashi et al. 2001), in octopus and cuttlefish [START_REF] Miramand | Concentration and distribution of heavy metals in tissues of two cephalopods, Eledone cirrhosa and Sepia officinalis, from the French coast of the English Channel[END_REF]) and in nautilus [START_REF] Bustamante | Bioaccumulation of 12 trace elements in the tissues of the nautilus Nautilus macromphalus from New-Caledonia[END_REF]. A large proportion (>60%) of the Ag in digestive gland is located in the cytosolic fraction of the cells [START_REF] Tanaka | Subcellular distribution and binding of heavy metals in the untreated liver of the squid; comparison with data from the livers of cadmium and silver exposed rats[END_REF]. To the best of our knowledge, no data are available in the literature regarding pathway-specific bioaccumulation (viz., water vs food bioaccumulation) of Ag and Co in these organisms.

Therefore, the present work has focused on uptake and loss kinetics of Ag and Co in a cephalopod exposed via different routes in order to better characterise their bioaccumulation rates, the subsequent tissue distribution and their retention times depending on the uptake pathway. The common cuttlefish Sepia officinalis was selected as an experimental model, and contamination via seawater, food and sediment was studied in eggs/embryos, juvenile and adult cuttlefish.

MATERIAL AND METHODS

Experimental organisms. Eggs of the common cuttlefish Sepia officinalis L. were obtained from cultured adults and maintained in an aquarium with flowing seawater until hatching. their respective acids (0.1 N) to obtain radioactivities which would allow the use of spikes measuring only a few µl (typically 10 to 20 µl).

Ag and Co uptake in Sepia officinalis exposed via sediments. Sediments (2.5 kg dry wt) from the North Sea (Audresselles, Pas-de-Calais, France) were spiked for 4 d with 110m Ag and 57 Co using the rolling jar method [START_REF] Murdoch | Spiking sediment with organochlorines for toxicity testing[END_REF] Ag and Co bioaccumulation in Sepia officinalis from food. Different prey were selected to match as closely as possible to the size of the natural food of juveniles and adult cuttlefish [START_REF] Mangold | Toxicities of ten metals to Crassostrea gigas and Mytilus edulis embryos and Cancer magister larvae[END_REF]). To prepare radiolabelled food, mussels Mytilus galloprovincialis and brine shrimp Artemia salina were exposed for 7 d in plastic aquaria containing 4 l of natural seawater spiked with 110m Ag and 57 Co (6 kBq l -1 each). Radiolabelled seawater was renewed daily and the organisms were subsequently used as food for juvenile (brine shrimp) and adult (mussels) cuttlefish.

Juvenile cuttlefish (n = 8) were held in a 70 l glass aquarium (open circuit with constant aeration; seawater renewal: 20 l h -1 ; salinity: 36 psu; temperature: 16.5 ± 0.5°C; 12:12 h light:dark cycle). For identification purposes, each individual was enclosed in a separate compartment allowing free circulation of seawater. After 1 h of ingesting radiolabelled brine shrimp, each individual was immediately γ-counted. From that time on, cuttlefish were fed twice daily with non-contaminated Artemia salina and regularly γ-counted to determine radiotracer loss kinetics and assimilation efficiency. Throughout the depuration period (29 d), faeces were removed 3 times per day to reduce possible indirect contamination by radiotracer recycling through leaching from the faeces. At the end of the depuration period, 5 juveniles were dissected to determine the radiotracer distribution in their tissues.

Adult cuttlefish (n = 18) were held in a 3000 l aquarium (open circuit with constant aeration;

seawater renewal: 300 l h -1 ; salinity: 36 psu; temperature: 16.5 ± 0.5°C; 12:12 h light/dark cycle) and fed soft parts of the previously labelled mussels for 2 h. Immediately after ingestion, each individual was γ-counted and the same procedure was followed as for the juveniles. In addition, 3 adult cuttlefish were dissected at each counting time to determine the radiotracer distribution among their organs and tissues.

Radioanalyses. Radioactivities of the tracers were measured using a high-resolution γspectrometry system consisting of coaxial Ge (N-or P-type) detectors (EGNC 33-195-R, Intertechnique) connected to a multichannel analyser and a computer with spectra analysis software (Interwinner). The detectors were calibrated with appropriated standards for each of the counting geometries used and measurements were corrected for background and physical decay of the radiotracers. Counting times were adapted to obtain relative propagated errors less than 5%. However, in a few cases, this counting precision could not be obtained even after 48 h of counting, due to the very low activity in extremely small organs. Counting times ranged from 10 min to 1 h for whole cuttlefish, mussel and brine shrimp, and from 10 min to 48 h for the dissected organs and tissues.

Data and statistical analyses. Uptake of 110m Ag and 57 Co from seawater and sediments was expressed, respectively, as change in whole-body concentration factors (CF) and transfer factors (TF) over time (Bq g -1 wet wt organism divided by the time-integrated Bq g -1 in seawater, CF, or sediments, TF). Uptake kinetics in the eggs were described using a singlecomponent first-order kinetic model (eq. 1):

CF t = CF ss (1 -e -k t ) (1)
where CF t and CF ss are concentration factors at time t (d) and steady state, respectively, and k is the depuration rate constant (d -1 ) [START_REF] Whicker | Radioecology: nuclear energy and the environment[END_REF].

Radiotracer loss was expressed in terms of percentage of remaining radioactivity over time, i.e. radioactivity at time t divided by initial radioactivity measured in the organisms at the beginning of the depuration period. Loss kinetics were described either by a single-component exponential model (Eq. 2) or by a 2-component exponential model (Eq. 3):

A t = A 0 e -k t
(2)

A t = A 0s e -k s t + A 0l e -k l t (3)
where A t and A 0 are the remaining activities (%) at time t (d) and 0, respectively; the 's' subscript refers to a short-lived component (s component) and the 'l' subscript refers to a long-lived component (l component) [START_REF] Hubbell | Radiotracer, gravimetric and calorimetric studies of ingestion and assimilation rates of an isopod[END_REF][START_REF] Whicker | Radioecology: nuclear energy and the environment[END_REF]. The exponential model showing the best fit (based on calculation of the determination coefficients, R², and examination of the residuals) was selected. In the case of radiotracer loss following ingestion of spiked food, the long-lived 'l' component is a model of the loss of the radiotracer fraction that is actually assimilated by the organism. Thus, the constant A 0l is an estimate of the fraction of radioactivity (or metal) assimilated from food, i.e. the assimilation efficiency (AE) [START_REF] Hubbell | Radiotracer, gravimetric and calorimetric studies of ingestion and assimilation rates of an isopod[END_REF][START_REF] Reinfelder | Trace element trophic transfer in aquatic organisms: a critique of the kinetic model approach[END_REF].

Depuration rate constant k allows the calculation of the radiotracer biological half-life (d) using the following equation:

T b½ = ln 2/k (4).
Constants of the models and their statistics were estimated by iterative adjustment of the model and Hessian matrix computation, respectively, using the non-linear curve-fitting routines in the Statistica 5.1 Software. Changes in radiotracer distribution among cuttlefish tissues were tested for significance by the G-procedure (adapted from the log-likelihood ratio test) for 2 × k contingency tables [START_REF] Zar | Biostatistical analysis[END_REF]. Changes in % of radioactivity in a single tissue during the depuration period were tested by 1-way ANOVA (after arcsin-transformation of data) followed by the Tukey's multiple comparison test. The significance level for statistical analyses was always set at α = 0.05.

RESULTS

Sediment exposure

Regular monitoring of radiotracer activities in sediment showed no significant variation during the experimental time course; time-integrated radioactivities were 14.7 ± 1.2 Bq g wet wt -1 for 110m Ag and 45.5 ± 3.4 Bq g wet wt -1 for 57 Co. Uptake of 110m Ag in juveniles cuttlefish displayed linear kinetics during the experiment period, with TF reaching 1 after 29 d of exposure (Fig. 1A). In contrast, very low 57 Co activities were recorded in the same individuals, with maximum TF values of 0.013 (Fig. 1B). Dissection of 3 individuals after 29 d of exposure showed that, for both metals, the digestive gland contained the highest proportion of the whole body burden, i.e. 57 ± 2% of 110m Ag and 79 ± 0.3% of 57 Co (Table 1).

Seawater exposure

Regular monitoring of radiotracer activities in seawater allowed calculation of timeintegrated radioactivities, viz. 8.3 ± 0.9 and 5.8 ± 0.3 kBq l -1 for 110m Ag and 57 Co, respectively.

Eggs

Despite the relatively short experimental exposure (11 d), the uptake of both radiotracers clearly displayed saturation kinetics (Fig. 2). Estimated steady-state concentration factors (CF ss ) in the eggs were 28.6 ± 1.5 for 110m Ag and 33.1 ± 1.7 for 57 Co. During the experiment, the greatest proportion of both radiotracers was associated with the egg capsule membrane, viz. 57 to 84% for 110m Ag and ≥ 99% for 57 Co (Fig. 3).

Juveniles

The activities measured in juveniles after 36 h of exposure via seawater were 2660 ± 1040 and 131 ± 24 Bq g wet wt -1 for 110m Ag and 57 Co, respectively. Mean calculated CF were elevated, viz. 320 ± 125 and 23 ± 4 for 110m Ag and 57 Co, respectively. Following transfer to non-contaminated seawater, loss kinetics of both radiotracers in juvenile cuttlefish were best described by a single-component model (Fig. 4A,B Table 2). Loss kinetics were characterised by T b½ of 1 wk for 110m Ag and 5 wk for 57 Co. At the end of the depuration period, both radiotracers were mainly associated with the digestive gland of the young cuttlefish (69 to 78% of whole-body activity), while the lowest fraction was deposited in the cuttlebone (<1% of the total activity) (Table 1).

Adults 110m

Ag and 57 Co activities recorded in the different organs and tissues of adult cuttlefish after 8 h of exposure and corresponding CFs are presented in Table 3. The highest activities of 110m Ag were found in the gills (1253 ± 465 Bq g -1 wet wt) followed by branchial hearts and skin (631 ± 398 and 335 ± 135 Bq g wet wt -1 , respectively). In the case of 57 Co, the digestive gland contained the highest activities, followed by branchial hearts and kidney (86 ± 77, 70 ± 12, and 66 ± 30 Bq g wet wt -1 , respectively). When considering the tissue distribution of the radiotracer activities, muscular tissues of adults (i.e. sum of the mantle muscles, skin and head) were found to contain the highest proportion of 110m Ag and 57 Co, 66 and 60%, respectively (Table 3). The second highest fraction was found in the gills (25% for 110m Ag)

and in the digestive gland (20% for 57 Co).

The radiotracer distribution among the tissues varied greatly between the beginning and the end of the depuration period (Table 3). Indeed, the fraction of 110m Ag and 57 Co increased significantly over time (p < 0.001) in the digestive gland (from 5 to 61% and from 20 to 64%, respectively). In contrast, tissues in direct contact with seawater generally exhibited a lower % of the radiotracer after depuration; this was particularly evident for 110m Ag in the gills, head and skin (p ≤ 0.005). It is noteworthy that radioactivity measured in the digestive gland increased from the beginning to the end of the depuration period (factor of 14 for 110m Ag and 3 for 57 Co).

Exposure via the food chain

In these experiments, juveniles (n = 8) were fed radiolabelled adult brine shrimp ad libitum for 1 h and adult cuttlefish (n = 18) ingested a total of 123 radiolabelled mussels within 2 h.

After feeding, the cuttlefish with their ingested prey were immediately γ-counted for determination of total radiotracer contents (Table 4).

Juveniles

The loss kinetics of ingested 110m Ag fitted a 2-component exponential model composed of a rapid-and a slow-loss compartment while loss kinetics of 57 Co was best described by a single component model (Fig. 4C,D, Table 2). The short-lived compartment contained 34% of the initially ingested 110m Ag activity (Table 2) and was characterised by a T b1/2s of 1 d; the longlived component, representing the proportion of radiotracer actually absorbed by the individuals, had a T b1/2l of 13 d. 57 Co was lost with a T b½ of 5 d (Table 2). Results showed that both radiotracers were readily assimilated in juveniles with assimilation efficiencies (AE) of 67% for 110m Ag and 99% for 57 Co (Table 2). Dissections performed 29 d after ingestion indicated that the highest proportion of remaining activity of both tracers occurred in the digestive gland (≥79%; Table 1).

Adults

The loss kinetics of both 110m Ag and 57 Co ingested with food were best described by a 2component exponential model in adults. As shown in Fig. 4E,F and in Table 2, 81 and 57% of the ingested activity of 110m Ag and 57 Co, respectively, were rapidly lost with T b1/2s of 6 and 13 h. The assimilated fraction of ingested 110m Ag and 57 Co was much lower in adults than in juveniles (AE = 19 vs 67% for 110m Ag and 43 vs 99% for 57 Co). Assimilated 57 Co was strongly retained within the tissues of adult cuttlefish; during the observation period, the estimated T b1/2l was 990 d (Table 2).

The tissue distribution of ingested radiotracers was determined on several occasions after feeding (Table 5). At the end of the depuration period, both 110m Ag and 57 Co were predominantly found in the digestive gland (i.e. 58 and 95%, respectively). The distribution of 57 Co among tissues remained unchanged for 29 d of observation. In contrast, some significant changes were observed for 110m Ag (G-test, p ≤ 0.01); for example, the proportion of 110m Ag activity decreased in the digestive tract and muscular tissues (mantle muscles and head), while it increased dramatically in the genital tract and ovaries (Table 5).

DISCUSSION

Published literature regarding Ag and Co concentrations in cephalopod tissues only concerns data from a limited number of field studies (e.g. [START_REF] Martin | High copper concentrations in squid livers in association with elevated levels of silver, cadmium, and zinc[END_REF], Ueda et al. 1979[START_REF] Miramand | Concentration and distribution of heavy metals in tissues of two cephalopods, Eledone cirrhosa and Sepia officinalis, from the French coast of the English Channel[END_REF][START_REF] Bustamante | Bioaccumulation of 12 trace elements in the tissues of the nautilus Nautilus macromphalus from New-Caledonia[END_REF][START_REF] Ishiashi | Multielemental analysis of purpleback flying squid using high resolution inductively coupled plasma-mass spectrometry (HR ICP-MS)[END_REF]. Cephalopods have been reported to concentrate Ag and Co, sometimes to high levels, with concentrations up to 84 µg Ag g dry wt -1 in the digestive gland of squid and 70 µg Co g dry wt -1 in the branchial hearts of octopus [START_REF] Martin | High copper concentrations in squid livers in association with elevated levels of silver, cadmium, and zinc[END_REF], Ueda et al. 1979). Field data also suggest that the digestive gland plays a important role in the bioaccumulation of these trace metals. Indeed, this organ contains more than 80% of the total body burden of Ag and Co in the octopus Eledone cirrhosa and in the cuttlefish Sepia officinalis from the English Channel [START_REF] Miramand | Concentration and distribution of heavy metals in tissues of two cephalopods, Eledone cirrhosa and Sepia officinalis, from the French coast of the English Channel[END_REF], and in the nautilus Nautilus macromphalus from New Caledonia [START_REF] Bustamante | Bioaccumulation of 12 trace elements in the tissues of the nautilus Nautilus macromphalus from New-Caledonia[END_REF]. However, virtually no information is available about the relative importance of the uptake pathways for these elements.

Many cephalopods like the common cuttlefish Sepia officinalis have rapid growth rates, implying intense metabolism and high food intake requirements. Hence, most of them are active predators and have high digestion efficiencies [START_REF] Boucher-Rodoni | Feeding and digestion[END_REF]. Therefore, food most probably constitutes an important source of trace element uptake for cephalopods.

This has been demonstrated experimentally for a few metals and radionuclides; e.g. Cd, Zn, 241 Am and 237 Pu [START_REF] Guary | Experimental studies on the biokinetics of plutonium and americium in the cephalopod Octopus vulgaris[END_REF][START_REF] Ueda | Accumulation of 65 Zn by Octopus Octopus vulgaris[END_REF][START_REF] Koyama | Bioaccumulation of waterborne and dietary cadmium by oval squid Sepioteuthis lessoniana, and its distribution among organs[END_REF], Bustamante et al. 2002a). Nevertheless, uptake of Ag and Co from seawater cannot be neglected, since the whole organism is in close and permanent contact with the ambient medium. In addition, cuttlefish spend most of the daylight period buried into sediment; hence transfer of heavy metals from the sediment should also be considered as a potential uptake pathway.

Juvenile cuttlefish exposed to spiked sediments for 1 mo accumulated Ag following linear uptake kinetics, suggesting that a steady-state in uptake would take a very long time to achieve under natural conditions. Moreover, uptake of Co from sediment was very low (TF ≤ 0.013). Examination of the tissue distribution of both accumulated metals suggests that an efficient translocation occurs from the tissues in direct contact with sediment to the digestive gland, but not to the cuttlebone.

During embryonic development, Ag and Co were efficiently taken up from seawater.

However, most of the metals taken up remained associated with the capsule membrane of the eggs. This membrane thus acts as an efficient shield protecting the embryo against direct metal exposure. Similar observations have been reported previously for Cd and Zn (Bustamante et al. 2002a). However, for Ag, the capsule-membrane shielding capacity appears to be limited, since after 6 d Ag was also found in relatively high concentrations within the embryos. In molluscs, it has been established that Ag is not highly toxic for adults (for a review, see [START_REF] Wood | Biological effects of silver[END_REF], whereas embryos and larvae are very sensitive to the metal [START_REF] Calabrese | The toxicity of heavy metals to embryos of the American oyster Crassostrea virginica[END_REF][START_REF] Mangold | Toxicities of ten metals to Crassostrea gigas and Mytilus edulis embryos and Cancer magister larvae[END_REF]. Such a functional shield for these toxic metals could be an important advantage for organisms that live and reproduce in coastal areas, which are often heavily contaminated by metals. For example, this could be the case in the bay receiving the outflow from the Seine River, where an important population of cuttlefish migrate in spring for mating and breeding. Indeed, very high inputs of Ag in the Bay of Seine were shown to result in high accumulation in the tissues of marine organisms such as the scallop Pecten maximus and the whelk Buccinum undatum (Miramand unpubl. data). Therefore, cephalopod ontogenic development could be affected under such particular environmental conditions.

After a short exposure to radiolabelled seawater (viz. 8 h), adult cuttlefish displayed high whole-body activities for both Ag and Co, indicating that both metals are rapidly accumulated from seawater. However, Ag appears to be more bioavailable to cuttlefish than Co, since Ag displayed 6 fold higher CFs. Following seawater exposure, the respiratory tissues, i.e. gills and branchial hearts, displayed the highest 110m Ag activities, whereas digestive gland, kidney and branchial hearts concentrated Co to a greater extent (Table 3). However, in terms of body burden, radiotracers were mainly found in tissues which are in direct contact with seawater (gills and skin accounted for 44% of the whole-body activity of 110m Ag) or which represent the main proportion of the total body weight (muscle and head contained 47% of the total 110m

Ag and 50% of the total 57 Co). Interestingly, the cuttlebone contained the lowest activity among tissues and was the only compartment that always displayed a CF less than one.

Similar results (CF ≤ 0.1) have been found for Cd and Zn in the cuttlebone of Sepia officinalis (Bustamante et al. 2002a).

After 6 d of depuration in clean, running seawater, the tissues in direct contact with seawater contained significantly less 110m Ag and 57 Co than at the beginning of the depuration period; gills and skin contained only 10% of the total 110m Ag, and the muscular parts contained 24% of the total radioactivity for both radiotracers (Table 3). These tissues also contained the lowest activities, indicating that previously incorporated tracers were rapidly lost and/or redistributed. In contrast, the digestive gland displayed higher activities of 110m Ag and 57 Co (14 and 3 times higher than at the beginning of the depuration period, respectively) and contained more than 60% of the whole body activity for both radiotracers, indicating an important internal metal translocation to the digestive gland. Therefore, this organ appears to be the main target organ for both Ag and Co. Due to its stronger retention capacity compared to other tissues, the digestive gland could act as a long-term biological indicator organ for Ag and Co contamination.

Branchial hearts also showed a specific affinity for Co as their 57 Co activity increased by a factor of 3 during the 6 d of depuration. These observations are consistent with those made for 60 Co in the branchial hearts of the octopus Octopus vulgaris [START_REF] Nakahara | Peculiar accumulation of cobalt-60 by the branchial hearts of Octopus[END_REF]). In addition, [START_REF] Nakahara | Peculiar accumulation of cobalt-60 by the branchial hearts of Octopus[END_REF] suggested that the increase in 60 Co in branchial hearts was due to transfer of the nuclide from other parts of the body.

When juveniles were exposed in seawater, subsequent 110m Ag and 57 Co release followed a single exponential loss model. Whole-body loss was rapid for 110m Ag but relatively slow for 57 Co (T b½ : 1 vs 5 wk). After 29 d of depuration, residual radioactivity of both Ag and Co was mainly located in the digestive gland, suggesting that, like in adults, metal translocations occur from other tissues to the digestive gland. Efficiency of the translocation processes for Ag and Co might be responsible of such different retention times noted in cuttlefish.

In the case of dietary exposure, less than 20% of ingested Ag was assimilated into the tissues of adult cuttlefish. In contrast, Ag was absorbed to a much greater extent in juveniles (AE up to 67%). Such a radically different degree of Ag assimilation might be due to differences in digestive metabolism between juvenile and adult cuttlefish [START_REF] Mangold | Toxicities of ten metals to Crassostrea gigas and Mytilus edulis embryos and Cancer magister larvae[END_REF]. In contrast, assimilated Ag was rapidly depurated with similar half-lives in juveniles and adults (i.e. 13 vs 9 d, respectively) which suggests that similar processes govern Ag elimination at both ages. In this context, it appears that difference in AE could also be related to variation in Ag bioavailability due to the different food types ingested by juveniles and adults (brine shrimp vs mussels). Indeed, different metal storage strategies in mussels and brine shrimp could partially control bioavailability owing to different physico-chemical speciation of the metals in the prey (e.g. [START_REF] Reinfelder | The assimilation of elements ingested by marine copepods[END_REF][START_REF] Wallace | Bioavailability of biologically sequestered cadmium and the implications of metal detoxification[END_REF].

The digestive gland clearly plays a major role in the storage of Ag in juveniles and adult cuttlefish. Indeed, regardless of the exposure route, the digestive gland retained most of the metal incorporated. Bivalves such as Pectinidae and Ostreidae have been shown to accumulate very high levels of Ag in their tissues and to store the contaminant mainly as Ag 2 S, a stable, non-toxic form of Ag [START_REF] Martoja | Effets de la contamination expérimentale par l'argent chez Chlamys varia L. (Bivalve Pectinidé). Données quantitatives, histologiques et microanalytiques[END_REF][START_REF] Berthet | Bioaccumulation, toxicity and physico-chemical speciation of silver in bivalve molluscs: ecotoxicological and health consequences[END_REF]). This detoxified form remains sequestered in the amoebocytes and in the basement membranes of various bivalve organs [START_REF] Berthet | Effets chimiques et histologiques de la décontamination de l'huître Crassostrea gigas Thunberg préalablement exposée à l'argent[END_REF][START_REF] Berthet | Bioaccumulation, toxicity and physico-chemical speciation of silver in bivalve molluscs: ecotoxicological and health consequences[END_REF]. The occurrence of sulphydryl-rich proteins such as metallothioneins have been found in the cephalopod digestive gland [START_REF] Tanaka | Subcellular distribution and binding of heavy metals in the untreated liver of the squid; comparison with data from the livers of cadmium and silver exposed rats[END_REF], Bustamante et al. 2002b). Histochemical and microanalytical investigations in the digestive gland of the cuttlefish Sepia officinalis have shown that Ag is associated with Cu and Zn in spherules accumulated in the basal cells, and these spherules are composed of metallothionein-like proteins [START_REF] Martoja | Localisation cytologique du cuivre et de quelques autres métaux dans la glande digestive de la seiche, Sepia officinalis L. (Mollusque Céphalopode)[END_REF]. Metalloproteins play a major role in the homeostasis of Cu and Zn and are well known for their capacity to bind Ag, Cd and Hg (see e.g. [START_REF] Cosson | Metallothioneins and detoxification. Is the use of detoxification proteins for MTs a language abuse?[END_REF][START_REF] George | Cadmium, zinc and copper accumulation in the squid Illex argentinus from the Southwest Atlantic Ocean[END_REF]. In the digestive gland of the squid Todarodes pacificus, ca. 60% of the whole-body burden of Ag is associated mainly with the low molecular weight cytosolic proteins (mol. wt < 20 kDa) [START_REF] Tanaka | Subcellular distribution and binding of heavy metals in the untreated liver of the squid; comparison with data from the livers of cadmium and silver exposed rats[END_REF].

Ingested Co was assimilated to a greater extent in juveniles (99%) than in adults (57%) (Table 2). Furthermore, depuration rate constants were very different between both stages, resulting in very different half-lives, viz. 5 d for juveniles and 990 d for adults. Thus, Co appears to be depurated and/or detoxified by completely different processes in juveniles and adults. Such a difference is difficult to explain, since Co is an essential element and has been shown to be easily redistributed to branchial hearts and digestive gland following seawater exposure to the metal (Nakahara et al. 1979, present study). In fact, the very elevated whole-body retention capacity of assimilated Co by adult cuttlefish concerned exclusively the digestive gland, which contained from 91 to 95% of the total Co body burden. Thus, once incorporated into the adult digestive gland, assimilated Co ingested with prey can be considered as definitively sequestrated. The lower retention efficiency for Co in juvenile cuttlefish could be due to the incomplete development of the digestive gland in early juveniles, as has been suggested for assimilated Cd (Bustamante et al. 2002a). However, although incompletely mature, the digestive gland also plays a role in the storage of Co in juveniles, given that this organ contained 79% of the whole-body burden of Ag after 29 d of loss. Further investigations are needed to better understand the metabolism of Co during the life cycle of cephalopods.

a FRIA doctoral grant; M.W. is a Honorary Research Associate of the FNRS, Belgium. We are grateful to the anonymous referees of this manuscript for their critical reviews.

Captions to Figs.

  These newly hatched juveniles (n = 25; 0.387 ± 0.071 g wet wt) were then used in the experiments. Adult cuttlefish (n = 18; 138 ± 40 g wet wt) were reared in the Oceanographic Museum (Principality of Monaco) from hatching to 1 yr old, or collected by net fishing off Monaco (n = 5; 253 ± 97 g wet wt). Prior to any experimentation, adults were anaesthetised in seawater containing 2% ethanol for making biometric measurements, sex determination and for the insertion of a numbered plastic tag into the mantle fin to identify each animal during the experiments. Radiotracers. Radiotracers 110m Ag [t ½ = 250.4 d] and 57 Co [t ½ = 271.8 d] were purchased from Amersham as nitrate and chloride salts, respectively. Stock solutions were prepared in Commentaire [NAB1] : Pleas e define. If this is the same as Tb1/2, please use consistently throughout.

Fig. 1 .Fig. 2 .Fig. 3 .

 123 Fig.1. Sepia officinalis. Whole-body uptake kinetics of (A) 110m Ag and (B)57 Co in juvenile cuttlefish exposed to radiolabelled sediments (transfer factor, TF; mean ± SD, n = 9 from Day 0 to 22, and n = 3 on Day 29)

Ag and Co uptake in Sepia officinalis exposed via seawater and subsequent loss. Eggs/embryos: Cuttlefish eggs were placed in

	110m Ag and 57 Co radioactivities were measured in the eggs on Days 1, 2, 3, 6, 9, and 11. At
	each time, 3 eggs were dissected to determine the distribution of the radiotracers among the
	capsule membrane, peri-embryonar liquid and embryo.
	Juveniles and adults: juvenile (n = 8) and adult (n = 5) cuttlefish were placed for 36 and 8 h,
	respectively, in 70 l glass aquaria containing seawater spiked with 110m Ag and 57 Co (6 kBq l -1
	. Before initiating the experiment, each). Cuttlefish were then radioanalyzed and transferred to another 70 l aquarium supplied
	radiolabelled sediments were held in flowing seawater overnight in order to leach weakly with natural flowing seawater (open circuit with constant aeration; seawater renewal: 20 l h -1 ;
	bound radiotracer. Sediments (50 g wet wt) were sampled at fixed intervals during the salinity: 36 psu; temperature: 16.5 ± 0.5°C; 12:12 h light:dark cycle). Juvenile cuttlefish were
	experiment to check for possible variations in radiotracer concentrations. Juvenile cuttlefish fed Artemia salina twice daily and were periodically γ-counted to follow radiotracer loss
	(n = 9) were exposed for 29 d in a 20 l plastic aquarium (constantly aerated open circuit kinetics over 29 d. At the end of the loss period, 4 juveniles were dissected to determine the
	radiotracer distribution among digestive gland, cuttlebone and remainder.
	Adults were fed daily with soft parts of the mussel Mytilus galloprovincialis. Three adults
	were dissected after 8 h and the 2 remaining ones were dissected after 6 d depuration. For
	maximise the time of contact with sediments. During the experiment, all juvenile cuttlefish each individual, the branchial heart appendages, branchial hearts, gills, digestive tract (after
	were fed twice daily with Artemia salina and were periodically γ-counted to follow the removal of the gut contents), genital tract, ovary or testes, ink sac, digestive gland, kidneys,
	radiotracer uptake kinetics over the 29 d. At the end of the uptake experiment, 3 individuals mantle skin, mantle muscle, head and cuttlebone were separated, weighed, and their
	were dissected to determine the distribution of the radiotracers among digestive gland, radiotracer content measured.
	cuttlebone and remainder (rest of the organs).
	a 70 l glass aquarium containing natural
	filtered (0.45 µm) seawater spiked with 6 kBq 110m Ag l -1 (57 pM) and 6 kBq 57 Co l -1 (0.68
	pM) for 11 d (constantly aerated closed circuit; salinity: 36 psu; temperature: 16.5 ± 0.5°C;
	12:12 h light:dark cycle). Radiotracers and seawater were renewed daily to maintain seawater
	quality and radiotracer concentrations as constant as possible. Radiotracer activities in
	seawater were checked daily (before and after seawater renewals). During the experiment,

aquarium; salinity: 36 psu; temperature: 16.5 ± 0.5°C; 12:12 h dark:light cycle) containing ca.

3 l of natural seawater running over a 4 cm layer of spiked sediments. The depth of seawater was maintained at a low level in order to minimise the movements required for feeding, and to

Table 2 Table 1 .

 21 Sepia officinalis. Radiotracer distribution (%; mean ± SD) among 3 body compartments of juvenile cuttlefish (a) after a 29 d exposure to spiked sediments, (b) after a 29 d depuration following a 36 h exposure to spiked seawater, and (c) after a 29 d depuration following ingestion of spiked food (brine shrimp)

	Exposure pathway	n	Body compartment
		Digestive gland	Cuttlebone	Remainder
	(a) Sediments	3
	110m Ag	57 ± 2		0.5 ± 0.4	43 ± 3
	57 Co	79 ± 0.3		1.8 ± 0.6	19 ± 1
	(b) Seawater	4
	110m Ag	69 ± 11		0.5 ± 0.1	31 ± 11
	57 Co	78 ± 1		0.4 ± 0.1	22 ± 1
	(c) Feeding	5
	110m Ag	83 ± 5		0.9 ± 0.7	16 ± 4
	57 Co	79 ± 6		0.8 ± 0.8	20 ± 6

Table 2 .

 2 Sepia officinalis. Parameters of the equations describing the loss kinetics of 110m Ag and 57 Co in whole cuttlefish previously exposed to the radiotracers via different pathways: (a) juveniles previously exposed for 36 h via seawater; (b) juveniles previously fed radiolabelled brine shrimp Artemia salina; (c) adults previously fed radiolabelled mussels Mytilus galloprovincialis. O and T: 1-and 2-exponential loss equations, respectively; ASE: asymptotic standard error; R²: determination coefficient; p: probability of the model adjustment. For abbreviation definitions, see 'Data and statistical analyses'

	Pathway	Model	A 0s (ASE)	k s (ASE)	T (d)	A 0l (ASE)	k l (ASE)	T b1/2l (d)	R²	p
	(a) Loss in juveniles after seawater
	exposure 110m Ag	O	100.8 (2.5) 0.103 (0.006)	6.7	-	-	-	0.97	<0.001
	57 Co	O	93.3 (2.0)	0.020 (0.003)	34	-	-	-	0.98	<0.001
	(b)

Loss in juveniles after a single feeding on brine shrimp

	110m Ag	T	33.8 (7.3)	0.668 (0.237)	1.0	67.2 (6.4)	0.053 (0.009)	13.2	0.82	<0.001
	57 Co	O	98.8 (3.1)	0.128 (0.100)	5.4	-	-	-	0.86	<0.001
	(c)

Loss in adults after a single feeding on mussels

	110m Ag	T	80.7 (3.7)	2.683 (0.667)	0.26	19.3 (3.1)	0.078 (0.027)	8.9	0.94	<0.001
	57 Co	T	56.9 (4.9)	1.290 (0.325)	0.54	43.2 (3.5)	0.001 (0.007)	990	0.66	<0.001

Acknowledgements. We thank Dr. N. Tevenin and Dr. P. Gilles from the Oceanographic Museum of the Principality of Monaco for supplying adult cuttlefish and Dr. S. von Boletzky from the Marine Station of Banyuls, France for providing the cuttlefish eggs. We are also grateful to Dr E. Boucaud-Camou for his advice on culturing cuttlefish. The Marine Environment Laboratory operates under a bipartite agreement between the International Atomic Energy Agency and the Government of the Principality of Monaco. B.D. is holder of

Table 3. Sepia officinalis. Concentration factors (CF), radiotracer activities (Bq g wet wt -1 ; mean ± SD) and tissue distribution of radioactivity (%; mean ± SD) in adult cuttlefish after 8 h of exposure via seawater (n = 3) and after 6 d of depuration (n = 2)