P Monneyron

A De

La Guardia

M H Manero

E Oliveros

M T Maurette

F Benoit-Marquié

Co-treatment of industrial air streams using A.O.P. and adsorption processes

Coupling adsorption with two different photochemical oxidation methods was investigated, in order to treat industrial gaseous effluents containing a low concentration of Volatile Organic Compounds (VOC). Prior to adsorption/photocatalysis cyclic study, new TiO 2 based catalysts were prepared by impregnating commercial zeolites in pellets form using a sol-gel technique. Scanning Electronic Microscopy observations linked to BET and adsorption equilibrium measurements of two VOC (1-butanol or BuOH and methyl-ethyl-ketone or MEK) showed that TiO 2 happened to fix on the inert binder, without significant modification of support properties. A specific diffusion cell was used to produce dilute polluted air streams for each VOC in dynamic experiments, in which adsorption and photodegradation phases were alternatively carried out. Adsorption/photocatalysis co-treatment unable the total degradation of BuOH, and the regeneration of adsorbent. When using V-UV photolysis, pure zeolites were used, irradiated at 172 nm with an xenon excimer lamp. Excellent results were observed, since a total and rapid degradation was obtained for both VOC, especially thanks to a determining role of ozone.

INTRODUCTION

Volatile Organic Compounds (VOC) are major air pollutants coming largely from industrial processes. Thus, purification of effluents is of great necessity, and one of the main technique available towards dilute polluted airstreams is adsorption onto a porous material. This process is usually conducted in two-steps, since the saturated material requires regeneration [START_REF] Benoit-Marquié | Les composés organiques volatils dans l'environnement[END_REF].

During the last decade, developments in the area of chemical water and air treatment have led to an improvement of organic pollutants oxidative techniques, in particular when using photochemical methods. They are generally referred to as Advanced Oxidation Processes (AOP). Bearing in mind the industrial applications objectives, researchers have focused their work on design and development of new light sources and photochemical reactors, but also on the preparation of efficient photocatalysts. AOP are entirely based upon oxidative degradation reactions, in which organic radicals are generated through photolysis of the organic substrate or consecutively to a reaction with hydroxyl and/or oxygen radicals [START_REF] Legrini | [END_REF]. In this study, the combination of adsorption with two different photooxidation methods was investigated. They are photocatalysis and V-UV photolysis.

Photocatalysis of air pollutants has been largely developed using the classical UV-sensible catalyst, titanium dioxide (TiO 2 ) [START_REF] Ollis | Photocatalytic Purification and Treatment of Water and Air[END_REF]. However, since TiO 2 is mainly produced in powder form, which is technologically impracticable in continuous engineering processes, many attempts have been made to prepare supported catalysts, using fibber glass [START_REF] Robert | [END_REF], stainless steel [5], fibber textile [6], quartz beads [7], honeycomb [8], activated carbon [9], and zeolites [10]. Besides a large adsorption capacity, High Silica Zeolites (HSZ) exhibit notably a very low dependence on effluent humidity, contrary to activated carbon [11], and on compound's polarity, which may be of particular interest since nonpolar molecules hardly adsorb on pure TiO 2 [12]. In order to develop enhanced photoactivity of titanium dioxide, investigators elaborated very different methods to support active titanium on zeolites, from simple amalgam of powders through mechanical mixing [13] or with papermaking techniques [START_REF] Ichiura | Proceeding on "Environmentally Friendly and Emerging Technologies for a suitable Pulp and Paper Industry[END_REF], to chemical vapour deposition [START_REF] Ding | [END_REF], cation exchange [16], or direct synthesis [17] which tend to create Ti -O -Si bonds. In this study, zeolite based catalysts were prepared using a sol-gel method, and specific behaviour of adsorption and photocatalysis of daily used industrial solvents, 1butanol (BuOH) and 2-butanone, known as methyl ethyl ketone (MEK), were determined.

V-UV photolysis is the vacuum ultra-violet irradiation of pollutants [18]. This method is very efficient for water treatment [19] since it leads to water molecules photolysis and therefore to an important hydroxyl radicals production. Until now, it was scarcely used for gaseous stream treatment [20], and never used for adsorbent regeneration.

EXPERIMENTAL

Chemicals and zeolites supports. Titanium

(IV) butoxide (99%), 2,4-pentanedione (99%), 1-butanol 1 summarises the adsorbent main properties. [21]. The catalysts were prepared by im- pregnation of supports with a TiO 2 Sol-Gel. The synthesis method is summarised here. 0.1 mole of titanium (IV) butoxide was dissolved in 1 mole of absolute ethanol in an argon atmosphere and refluxed for 12 hours. 0.05 mole of 2,4-pentanedione and 0.01 mole of double distilled water were added dropwise under vigorous stirring. After refluxing for several hours, the clear solution was concentrated by solvent distillation. An amorphous and translucid gel was obtained. It contains enough alcohol to avoid ageing and could therefore be stored unchanged for several weeks. The TiO 2 coating was made by completely immersing the different supports in the gel. The wet materials could then undergo a two steps hot air treatment. In the first stage the catalyst system was dried at 100 • C, whereas in the second part the desired crystalline anatase phase was obtained at 450 • C. The synthesised catalyst are referred to as DAY * and DAZ * , respectively for DAY and DAZ based solids.

Preparation of supported titanium dioxide catalysts

Characterisation of catalysts. Elemental

analysis was performed by the analysis central center of the Centre National de la Recherche Scientifique (CNRS) using flame emission spectrometry. The specific surface area and porous distribution of adsorbents were determined using argon adsorption-desorption experiments at 77 K, using a conventional BET adsorption apparatus (ASAP 2010, Micrometrics). Degassing of samples was performed at 120 • C overnight. This equipment allowed the description of both microporosity (pore size smaller than 2 nm) and mesoporosity (between 2 and 50 nm). A scanning electron microscopy (JSM-840 A, JEOL) was operated at 20 keV, providing information on local properties together with chemical composition of material activated by electronic bombardment (EDS facility). Prior to analysis, the granulates were coated with gold to ensure conductivity. Both external and cross-section surfaces of samples were analysed. In order to characterise the influence of impregnation on adsorption capacities of zeolites, batch adsorption isotherms were performed using a volumetric method [START_REF] Monneyron | Air Pollution[END_REF].

Gas phase adsorption/photodegradation experiments.

The polluted air stream used in dynamic photodegradation experiments was generated with a diffusion cell [START_REF] Mckelvey | [END_REF], which can produce dilute gas mixtures accurately for very long period of time. The diffusion rate initially expressed by the Altshuller & Cohen equation was used in a previous study [24] for the prediction of the experimental temperature dependent relation, using Antoine equation to estimate the vapour pressure of liquid, and Fuller relation for diffusion coefficient calculation. Here, a 80 : 20 N 2 /O 2 mixture, supplied by Air Liquide, continuously flushed the mixing reservoir, regulated by a mass flowmeter (Brooks 58505, 10-50 mL • min -1 ), leading to a VOC concentration ranging between 300 and 5000 mg • m -3 , thermally controlled. Both adsorption and irradiation phases were carried out in a continuous flow multilamp annular reactor (length: 15 cm, optical path length: 3 mm, total volume: 60 cm 3 ) shifting from adsorption to oxidation phase just by turning the lamp on once breakthrough curve was totally described and adsorbent saturation completed. The VOC inlet concentration was kept unchanged during irradiation, which was interrupted as soon as the outlet concentration of VOC and by-products became still. This choice lead one to analyse final adsorption/photochemical steady-state instead of complete regeneration time which may be uneasy to characterise considering the complex mechanisms involved in this process. Efficiency of degradation was then estimated in terms of outlet to inlet concentration ratio. For photocatalytic experiments, a medium pressure mercury arc (Phillips HPK 125 W, photonic rate: 6.2 × 10 18 photon • s -1 [25]) was positioned in the internal cylinder, entirely made of Pyrex to avoid photolysis of compounds, and cooled with an air stream. For V-UV photolysis experiments, an quasimonochromatic 172 nm Xenon excimer lamp was used (photonic rate: 8.3(±0.5) × 10 18 photons • s -1 [26]), as shown in Figure 1. The fixed bed of catalysts was supported by means of a non porous quartz cloth inserted in the annular space and, with a typical mass of 2 g, an homogeneous bed height of about 1 cm was obtained. The on-line reactor outlet analysis was performed using a Chrompack CP900 gas chromatograph, and a Perkin-Elmer 1760-x Infra Red spectrograph equipped with a DTGS detector for CO 2 , O 3 and H 2 O detection. In IR analysis, the gas sample continuously flushed a 90 mL NaCl cell.

ADSORPTION/PHOTOCATALYSIS RESULTS

Characterisation of TiO 2 -Zeolites catalysts.

The reproducibility of impregnation was checked by evaluating the gain of mass from TiO 2 coating through a simple and rapid weighting method. The TiO 2 mass content of DAY * and DAZ * was 15% ±1%, confirmed by elemental quantification, with a titanium content of 9% for DAY * and 10% for DAZ * . X-Ray analysis have clearly demonstrated that the TiO 2 deposited on zeolites was totally in the anatase crystalline form [27].

Intimacy and homogeneity of TiO 2 loading-SEM-EDS analysis.

Results of external and cross-section surfaces of samples where very similar in terms of mixing and titanium content, determined through Si/Ti ratio calculations. Figure 2 shows SEM pictures of crosssection surfaces for the two catalysts.

On the DAZ * sample, the zeolites crystallites can be easily isolated via their rectangular shape. Using the microprobe elemental analysis (EDS), no titanium was detected on the zeolites crystals whereas a high content of titanium was found in the clearer irregular parts, mixing TiO 2 microcrystals and binder. The DAY * structure is quite different from DAZ * . The zeolites crystals are much smaller and they could not be distinguished from binder or TiO 2 , showing a greater intimacy of loading during sol-gel method. Homogeneity of catalysts was found to be fairly satisfactory, and strongly determined by the initial material, and zeolite crystals size.

Specific surface area modification-BET analysis. BET adsorption isotherms on the different sam-

ples were all of type Ia of IUPAC classification [28], corresponding to a microporous adsorbent with a small hysteresis coming from multilayer adsorption/desorption in mesopores. Microporous distribution was checked using Horvath-Kawazoe model extended by Saïto & Foley for a cylindrical pore geometry. Structure of zeolites were confirmed, with a monomodal porous distribution for faujasite Y, at 13.8 Å and bimodal for ZSM-5, at 10.0 and 12.6 Å. The modification of support's surface due to TiO 2 loading was investigated by two means. First, the surface area of catalysts where determined with Langmuir approach as shown in Table 2. This model was preferred to BET or Dubinin-Radushkevich equation considering the monomolecular adsorption taking place in microporous adsorbent.

The very good agreement between experimental and calculated surface areas confirm that TiO 2 loading does not have a strong impact on support's physical properties. The BJH model was applied to hysteresis parts of adsorption isotherms, indicating that on both adsorbents, the TiO 2 coating developed a noticeable mesoporous volume. These observations stand promising for photocatalytic experiments, mesopores being predominant sites for photocatalysis.

VOC adsorption properties of catalysts-Volumetric batch experiments.

Single component adsorp- tion isotherms of BuOH and MEK were carried out on naked and coated adsorbents. Representative results are given in Figure 3, p 0 being vapour pressure of VOC.

For the two VOC used in this study, no steric effect happened, and both compounds had very similar adsorption mechanisms. As can be seen on the left part of Figure 3, the maximum adsorption capacity was reduced by TiO 2 loading, and was then directly proportional to the available pore volume of zeolites given in Table 1. Similar results where found for DAY * materials, demonstrating that VOC adsorption on synthesised catalysts was almost completely driven by the properties of zeolite, whose micropores remained totally unobstructed by TiO 2 loading, contrary to the BET results obtained by Hsien, et al. [29] when impregnating zeolite powders. As shown in the right part of Figure 3, adsorption mechanism of both VOCs on TiO 2 anatase was also clarified thanks to this experimental technique. Very different behaviour were observed and quantified using Langmuir model. VOC adsorption maximum capacities (Qmax) given by the model were quite close in favour to BuOH, respectively 0.28 mmol • g -1 for BuOH and 0.23 mmol • g -1 for MEK, but adsorption energy parameter (b) differed largely: 1.3×10 2 for BuOH and 6.9×10 4 for MEK, pointing at the great affinity between TiO 2 and hydroxyl functional groups. It is interesting to note that, dealing with Qmax, similar tendencies between alcohols and ketones was found by Alberici [30] but with values being half of the ones found here, whereas same order values were obtained by Raupp [31]. Several preliminary remarks are to be made. Due to a great temperature dependence of the adsorption mechanism, fluctuations can be observed in each of the three adsorption periods, coming from laboratory temperature change during night and day, of about ±2 • C. In the same way, a thermodesorption was observed when initialising irradiation phase. Photocatalysis lead to significant degradation of BuOH, consecutively 94 and 93%. Regeneration of adsorption capacity was estimated by comparing the saturation time of adsorbent before and after the irradiation. In both cases, a partial regeneration was found of about 30%. This value is totally dependent on the experimental conditions (experimental set up design, irradiation conditions, inlet concentration and flow) but also on the competitive mechanisms of photochemical reactions, adsorption and desorption of VOC. Still, this value remaining constant indicates that no poisoning of catalysts occurred when irradiated. The concentration of BuOH oxidation sub-products (C2 to C4 alcohols and corresponding aldehydes and butanoic acid) was also determined in the outlet, after individual identification through direct injection of chemical's vapour. The evolutions of their respective concentration were very close to those obtained in a previous study on loaded silica beads in similar conditions by Benoit-Marquié, et al. [25], the aldehydes outlet concentration being greater than the corresponding alcohol. This could be directly attributed to the greater affinity of TiO 2 toward alcohols mentioned in the single component adsorption isotherms paragraph, that can lead to a selective TiO 2 -adsorption, and therefore a selective degradation, when dealing with the dynamic mixture. These findings tend to indicate that the zeolite used in this study does not interfere with the oxidative mechanisms that occur on the active TiO 2 UV-sensible sites; the unloaded zeolites had no catalytic properties when irradiated in identical conditions. Supporting TiO 2 on zeolites may have two different impacts on photocatalysis reactions: firstly it is clear that by creating a high concentration of compounds around TiO 2 sites, kinetic rates are to increase significantly. This phenomenon was observed in batch experiments by many researchers [32,33]. Secondly, the zeolites, being acid material, may influence selectivity of reactions through chemisorption on Lewis or Brönsted strong acid sites, induced by aluminium atoms [34]. Here, the impact of acidity on oxidation rate remains unclear since the zeolites used here had a very high Si/Al ratio, but it may tend to reduce mass transfer between external adsorption sites, and TiO 2 sites. The same study was performed with a BuOH inlet concentration of 2000 mg • m -3 , leading to similar sub-products curves, but to a different degradation steady state of 75%, which can be easily explained by the competitive adsorption and photodegradation mechanisms. Finally, the DAZ * catalyst was studied with a 2000 mg • m -3 inlet concentration: irradiation lead very rapidly to a steady state of 40% of degradation, but only a very small adsorbent regeneration was obtained (about 5%).

Cyclic adsorption/photocatalytic oxidation dynamic experiments.

In this study, two main parameters are distinguishing DAY * and DAZ * catalysts that can explain the differences observed in photocatalysis experiments. Firstly, it is obvious that the zeolite framework structure has a decisive impact, intracristalline diffusion being faster in faujasite Y than in ZSM-5. Secondly, SEM measurements showed that DAY crystals were smaller than DAZ ones, creating a greater intimacy of mixing and a larger mesoporous volume. Therefore, zeolite crystals size is likely to limit mass transfer in the catalyst. It was not possible to determine here their respective influence, but in both cases, intracristalline diffusion is the microscopic decisive parameter. These dynamic results confirm the work of Yoneyama and coworkers [35] who pointed at the major influence of diffusion in adsorbent used as a support for TiO 2 by studying the effects of adsorption strength of adsorbent supports on the rate of photodegradation.

Preliminary experiments with MEK were carried out on DAY * catalyst using a 1700 mg • m -3 inlet concentration. Irradiation lead very rapidly to a steady-state of only 5% of degradation, and logically no adsorbent regeneration was obtained. When comparing photocatalytic experiments towards MEK and BuOH, it is clear that their differentiating chemical functional groups play a determining role. Since in single component adsorption experiments, comparable behaviour were obtained for both VOC on zeolites, the greatly different affinities toward pure TiO 2 powder (SGP) noticed is to explain the larger conversion of BuOH. Keller-Spitzer [START_REF] Keller-Spitzer | 2nd European meeting on solar-chemistry and photocatalysis: environmental applications[END_REF] obtained a similar poor MEK degradation using a thin TiO 2 film, but reached a much higher conversion efficiency when doping the TiO 2 , likely by inducing a greater adsorption energy.

ADSORPTION/V-UV PHOTOLYSIS RESULTS

The degradation of several VOC (BuOH, 1-butylamine, benzene, toluene, and phenol) using V-UV photolysis has been studied in the last few years in our laboratory [18,24]. This method lead to total mineralization of compounds whatever flow and concentration were chosen up to a concentration of 5000 mg • m -3 . No organic compounds were detected in gas phase chromatography analysis, and IR spectrometry was used to look at mineralization products (H 2 O and CO 2 ) and at the ozone formed by air oxygen photolysis. Here, co-treatment by adsorption and photolysis V-UV was studied using zeolite as a pollutant reservoir, bearing in mind that zeolite would stand as an obstacle to photonmolecule contact. As shown in Figure 5, the vibrations characteristic band of C -H bond (∼ 3000 cm -1 ), that allowed us to follow the pollutant, disappeared as soon as that the lamp was switched on, indicating that total mineralization of BuOH was obtained. This irradiation is characterised by the appearance of the ozone band around 1000 cm -1 . The mineralization was confirmed by the presence of the CO 2 typical band (2350 cm -1 )being very large due to its strong molar absorptivityas well as two water characteristic bands in the 1300-2000 cm -1 and 3500-4000 cm -1 .

In case of BuOH irradiation conducted with saturated DAY-zeolite, the BuOH C -H band (3000 cm -1 ) completely disappeared after one hour of illumination. Here, degradation intermediates like aldehyde (band at 1740 cm -1 ) were detected but not yet identified. They disappear almost at the same time as BuOH. After one hour of irradiation, the steady-state was reached and the quantities of water corresponded to the mineralization of the on-going BuOH entering the reactor. Carbon monoxide was also formed during the first hour (bands at ∼ 2150 cm -1 ), indicating that, at the beginning of irradiation, the pollutant concentration was so high that a lack of oxygen occurred. Oxygen presence has been demonstrated to be essential to get total mineralization [24]. Investigations of alternative adsorption/photolysis V-UV phases confirmed that the adsorbent was then totally regenerated. It is interesting to note that in the experiments carried out on a zeolite, the ozone quantity detected remained stable and was smaller compared to the one obtained during direct photolysis. This tend to indicate that the ozone formed is either consumed in gas oxidation of pollutants, or adsorbed on zeolite surface. Ozone was reported to have a high affinity with acid sites of zeolites [START_REF] Mariey | [END_REF], and its basic character was found to be similar to CO. This phenomenon was observed when irradiating unpolluted zeolite: the ozone formed was first consumed by the zeolite, and then its concentration increased up to its initial value. From this observation, it is clear that the two phenomena mentioned earlier were happening simultaneously.

Very similar results were obtained when irradiating MEK-fully saturated DAY, as presented in Figure 7. MEK is characterised by bands at 1797 cm -1 (ketone), and in the 2700 cm -1 to 3000 cm -1 range (C -H). These bands disappear totally and water gets to a steady state after one hour of irradiation. At the same time, ozone is also present in very small quantity and CO band is present during the first hour. The great efficiency of this technique comes from the combination of several oxidation paths. Firstly, depending on their specific absorbance spectrum, VOC can be directly photolysed at 172 nm. At the same time, photodissociation of dioxygen lead to two single atoms of oxygen, which react afterwards with dioxygen and produce ozone [19]. Ozone may then act directly as an oxidative specie but can also be photolysed, in spite of its low absorbance at 172 nm, and produce oxygen atoms at an excited state [38]. Falkenstein's findings confirmed this statement [20], showing that this radical was one of the active species in the degradation of isopropanol by V-UV photolysis.

CONCLUSION

This study aimed at regenerating zeolite adsorbents using oxidative techniques. Firstly, two zeolites with very different crystalline framework were used as supports for TiO 2 impregnation. Physical characterisation of materials showed that a great intimacy of loading was obtained without significant modification of adsorbent. Using these new catalysts in dynamic photocatalysis experiments, it was clarified that the zeolite did not interfere with oxidation mechanism of BuOH, and that intracrystalline diffusion was likely to be the limiting kinetic parameter. In a second approach, V-UV photolysis was used to regenerate commercial zeolite, used here as a pollutant reservoir. Total degradation of pollutants, at relatively high concentrations, and complete and rapid regeneration of adsorbent was obtained, indicating a major influence of ozone.

Figure 1 .

 1 Figure 1. Annular reactor during V-UV photolysis experiments.

Figure 2 .

 2 Figure 2. SEM pictures (G = 1200) of internal surface. DAZ * (top), DAY * (bottom).

( a )Figure 3 .

 a3 Figure 3. Single component adsorption isotherms, on DAZ ( * ) adsorbents (left), on SGP (right).

Figure 4

 4 presents the out- let concentration of the reactor versus time during the cyclic study of adsorption breakthrough curves (A) followed by photocatalytic phase (P) of BuOH on DAY * for an inlet concentration of 550 mg • m -3 .

Figure 4 .

 4 Figure 4. Cyclic study of adsorption/photodegradation of BuOH on DAY * .

Figure 5 .

 5 Figure 5. Time evolution of outlet effluent IR spectrum during gas BuOH photolysis at 172 nm (total irradiation time: 2 hours).

Figure 6 .

 6 Figure 6. Time evolution of outlet effluent IR spectrum during DAY-zeolites saturated by BuOH irradiation at 172 nm (total irradiation time: 2 hours).

Figure 7 .

 7 Figure 7. Time evolution of outlet effluent IR spectrum during DAY-zeolites saturated by MEK irradiation at 172 nm. (total irradiation time: 2 hours)

Table 1 .

 1 Main properties of adsorbents.

	Symbol	Crystalline type (Pore structure)	Si/Al ratio	Available porous volume (cm

  3

Table 2 .

 2 Specific surface area of catalysts.

	Adsorbent	Specific surface area of adsorbent (a) (m 2 /g)	Specific surface area of TiO 2 -loaded adsorbent (b) (m 2 /g)	Calculated surface area of mixture (c) (m 2 /g)
	SGP (d)	65
	DAY	645	568	566
	DAZ	379	337	336

ACKNOWLEDGEMENTS

The authors are grateful to the "Conseil Régional de Midi Pyrénées" for financial support of this work, Prof. A. M. Braun for discussions and material support and to Dr. M. T. Boisdon and G. Tayebi for technical support.


Pierre Miramand
email: pierre.miramand@univ-lr.fr

Paco Bustamante

Daniel Bentley

Noussithé Kouéta

Variation of heavy metal concentrations (Ag, Cd, Co, Cu, Fe, Pb, V, and Zn) during the life cycle of the common cuttlefish Sepia officinalis

Keywords: Trace element, bioaccumulation, detoxification, cephalopods, sexual maturity, embryogenesis

The evolution of the concentration of 8 essential and non essential heavy metals (Ag, Cd, Cu, Co, Fe, Pb, V, Zn) in the tissues (digestive gland, cuttlebone and whole animal) of the common cuttlefish Sepia officinalis collected in the Bay of Seine has been tracked since the end of the embryogenesis until the reproduction period. Compared to the embryos, the juveniles after hatchling display much higher concentrations of Ag, Cu, Fe and Zn suggesting an efficient incorporation from seawater. Conversely, the amounts of Cd, Pb and V in hatchlings remain suggesting that these metals are barely bioavailable for the juveniles. Once the juveniles start to feed, the digestive gland appears to play a major role in the storage of all metals. After only one month of benthic life, the digestive gland already contains up to 90% of the total metal body burden, indicating that digestive gland plays a major role in the storage and presumed detoxification of the selected metals. Metal concentrations in the digestive gland increase in a logarithmic fashion with age during the entire life of cuttlefish, except for Ag which decreases as soon as cuttlefish migrate to open sea. This strongly suggests that (1) Ag is depurated from the digestive gland in relation to supposedly lower exposure in less contaminated environments compare to coastal waters and (2) the digestive gland of cephalopod could be a very good indicator of Ag contamination in the marine environment.

INTRODUCTION

As other cephalopods, the cuttlefish Sepia officinalis has a very short life cycle during which migrations related to growth and reproduction take place. In the English Channel and the Atlantic areas, the reproduction period after which these organisms die, occurs between April and August of their second year of life, when they are aged between 14 and 18 months (Boucaud-Camou et al., 1991;[START_REF] Legoff | Etude des déplacements de la seiche commune Sepia officinalis L. dans le Golfe du Morbihan au cours de la période de reproduction : premiers résultats[END_REF]. Only some male individuals not attaining their sexual maturity during this period can survive for another year. Therefore, the life of a cuttlefish never exceeds 2 years in those areas [START_REF] Richard | Contribution à l'étude expérimentale de la croissance et de la maturation sexuelle de Sepia officinalis L. (Mollusque céphalopode[END_REF]. During this period, the growth rate of cuttlefish is very high. Their weight can even be multiplied by a factor of 2,000, cuttlefish weighing 0.25 g after hatching and more than 600 g at the sexual maturity [START_REF] Richard | Contribution à l'étude expérimentale de la croissance et de la maturation sexuelle de Sepia officinalis L. (Mollusque céphalopode[END_REF][START_REF] Pascual | Crecimiento y alimentación de tres generaciones de Sepia officinalis in cultivo[END_REF][START_REF] Forsythe | Growth, reproduction and life of sepia officinalis (Cephalopoda: Mollusca) cultured through seven consecutive generations[END_REF]. This exceptional growth rate can be explained in terms of their active metabolism owing to their carnivore diet [START_REF] Mangold | Reproduction, croissance et durée de vie[END_REF].

Regardless of such a short life cycle, the strong capability of cuttlefish to concentrate a large number of metals in their tissues has been previously shown, as well as the major role of the digestive gland in the bioaccumulation processes (e.g. [START_REF] Decleir | Determination of protein-bound copper and zinc in some organs of the cuttlefish Sepia officinalis L[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 | Cadmium, copper and zinc in octopuses from Kerguelen Islands, Southern Indian Ocean[END_REF]2002a). This capability seems to be shared by many other species of cephalopods, octopodidae, teuthoidae or nautilidae [START_REF] Rocca | Copper distribution in Octopus vulgaris Lam. Hepatopancreas[END_REF][START_REF] Nardi | Ferritin in the hepatopancreas of Octopus vulgaris Lam[END_REF][START_REF] Schipp | Distribution of copper and iron in some central organs of Sepia officinalis (Cephalopoda). A comparative study by flameless atomic absorption and electron microscopy[END_REF]Miramand and Guary, 1980;Smith et al., 1984;[START_REF] Finger | Molecular association of Cu, Zn, Cd, and 210 Po in the digestive gland of the squid Nototodarus gouldi[END_REF][START_REF] Bustamante | Etude des processus de bioaccumulation et de détoxication d'éléments traces (métaux lourds et terres rares) chez les mollusques céphalopodes et bivalves pectinidés. Implication de leur biodisponibilité pour le transfert vers les prédateurs[END_REF][START_REF] Bustamante | Cadmium, copper and zinc in octopuses from Kerguelen Islands, Southern Indian Ocean[END_REF][START_REF] Bustamante | Bioaccumulation of 12 trace elements in the tissues of the nautilus Nautilus macromphalus from New Caledonia[END_REF]. More recently, radiotracer experimental studies have shown the relative importance of the transfer pathways (water, food, sediments) in the bioaccumulation of 4 heavy metals (Ag, Cd, Co, Zn) in juvenile and adult cuttlefish (Bustamante et al., 2002b;[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], confirming the storing nature of the digestive gland regardless of the uptake pathway.

However, little is known about the evolution of metal concentrations and burdens during the embryogenesis and growing of Sepia officinalis. The aim of this study was therefore to investigate the variation of the concentrations of 8 essential and non essential (Ag, Cd, Cu, Co, Fe, V, Pb, Zn) heavy metals during the growth of the cuttlefish from the embryogenesis to the reproduction period. Eggs, hatched juveniles, juveniles during their 2 first months of benthic life, immature individuals and mature adults returning to the coast to mate were analysed. Thus, both the physiological changes and the migrations have been considered. The former occur during the life cycle of cuttlefish and may modify the bioaccumulation processes of metals, whereas during the migrations the animals move from coastal areas (with metallic inputs of anthropogenic origin) to the open sea (supposed to be less polluted than the coast) hence potentially modifying the metal concentrations in their tissues. In this work, special attention has been paid to the digestive gland owing to its major role in the metabolism of metals. To this end, the zone of study chosen is particularly interesting since direct arrivals of metallic contaminants, especially Ag [START_REF] Rno | Surveillance du milieu marin, travaux du RNO-Edition[END_REF] and Cd [START_REF] Chiffoleau | Trace metal distribution, partition and fluxes in the Seine estuary (France) in low discharge regime[END_REF][START_REF] Chiffoleau | Distribution des contaminants métalliques dans l'estuaire de Seine en période de crue (Février 1995)[END_REF][START_REF] Miramand | Contamination of the biological compartment in the Seine estuary by Cd, Cu, Pb and Zn[END_REF] are poured by the Seine River. Furthermore, the bay of Seine constitutes a laying and nursery area for the common cuttlefish that is subjected to fishery activities of important economical impact in the region (Boucaud-Camou and Boismery, 1991).

MATERIALS AND METHODS

Biological material

1) Animals from the field

Between July 1989 and July 1991, 153 cuttlefish were collected in 6 times in the bay of Seine off the harbour of Ouistreham (Normandy, France). The age of animals was estimated according to the previous works of [START_REF] Medhioub | Etude de la croissance et du cycle sexuel de la seiche (Sepia officinalis) des côtes normandes[END_REF] and Boucaud-Camou et al. (1991) using the model of seasonal growth established by [START_REF] Pauly | A simple method for fitting oscillating growth data, with a program for pocket calculators[END_REF]. According to their mantle length (ML), the animals were pooled in estimated age classes for heavy metal analysis as follows:

-about 1-week old: 3 pools of 25 individuals (mean ML =10 ± 1 mm); -about 2-weeks old: 4 pools of 8 individuals (mean ML =18 ± 3 mm); -about 1-month old: 3 pools of 4 individuals (mean ML = 33 ± 4 mm); -about 2-months old: 4 pools of 4 individuals (mean ML =59 ± 6 mm); -about 12-months old (immature cuttlefish): 3 pools of 5 individuals (mean ML = 133 ± 19 mm); -about 18-months old (mature cuttlefish): 3 pools of 1 individual (mean ML = 215 ± 5 mm).

The cuttlefish specimens were immediately frozen on board and stored at -20° C. After a short frozen period (< 1 month), each individual was dissected in the laboratory: digestive gland and cuttlebone were removed from the remaining tissues and pooled to allow metal analyses in minute organs and tissues. Our sampling of cuttlefish is supposed to represent a single troop, at least for the individuals of the first year.

2) Eggs reared in the laboratory

Eggs collected in the same area and the same period as the cuttlefish were incubated at the laboratory in aquaria with an open circuit system. 4 pools of 25 eggs were dissected and embryos, yolk and eggshells were separated for analysis. The embryos collected from the eggs were about to hatching and were completely formed but still attached to the yolk sack, which was not completely resorbed. The rest of the eggs were maintained in aquaria until hatchling, and the former 25 juvenile hatched (ML = 10 ± 1 mm) were dissected as previously described.

Sample preparation and analytical procedure

All the samples were dried at 60°C for several days to constant weight and then reduced to powder using porcelain mortar and pestle. Aliquots ranging from 10 to 300 mg of the homogenised samples were digested with 4 ml of 14N ultrapur HNO 3 and 1 ml of 22N

ultrapur HClO 4 at 100°C on a hot plate during 3 days. After evaporation of the acids, the residues were taken up in 5 ml 0.3 N HNO 3. Ag, Cd, Co, Cu, Pb and V were analyzed by Zeeman graphite furnace atomic absorption spectrophotometry and Fe and Zn by flame atomic absorption spectrophotometry. Heavy metal concentrations in whole individuals were calculated for reconstructed individuals from analysis of remaining tissues, cuttlebone and digestive gland. However, the high Ca content in the cuttlebone hinder Co measures in this organ, not allowing calculations for the whole juveniles and adults.

Quality control was assay by heavy metal analyses in blanks and reference materials. Thus, Orchard-Leaves (National Bureau of Standards) and MA-A-1, MA-A-2, respectively copepods and fish flesh standard (IAEA) were treated and analysed in the same way as the samples. Our results for the standard reference materials were in good agreement with the certified values (Table 1). The detection limits were (µg.g -1 dry weight): 0.004 (Cd), 0.02 (Ag), 0.1 (Co, Pb), 0.5 (Cu, V, and Zn) and 2.5 (Fe). Results are also expressed in micrograms per gram of the dry tissue weight (µg.g -1 dwt).

Statistical analysis

Comparison of the metal concentrations between eggshell and embryos/yolk were assessed using t-test comparison of means. Differences between metal concentrations at the different ages were tested using the non-parametric Kruskall-Wallis test. Regressions between the concentrations measured in the cuttlefish digestive gland and mantle length were tested using regression procedures for linear and non linear fitted models. Accumulation of metals in the cuttlefish was described by linear and logarithmic fitted models, with the exception of Ag in the digestive gland which is described by a two-order linear component model. Statistical analyses were performed using XLStat Pro 7.0. The level of significance was always set at α=0.05.

RESULTS

Levels of concentration and distribution among compartments

The heavy metal concentrations in eggs, embryos, juveniles of different sizes, immature and mature Sepia officinalis are given in Table 2.

At the end of the embryo development, the eggs were dissected between eggshell, yolk and embryo. Heavy metal concentrations were similar between yolk and embryo, except for Cd and Cu. However, when compared metal concentrations of those internal compartments with the external one (i.e. the eggshell), results appear contrasted. For Co, Fe, Pb, and V, the concentrations in the eggshell were significantly (P t-test <0.0001 for the 4 metals) higher than in yolk and embryo whereas they were significantly lower for Ag (P t-test =0.015) and Cu (P t- test <0.0001). Overall, metal concentrations in the embryos extracted from the eggs were generally lower (Ag, Cu, Fe, and Zn) or in the same range (Cd, Pb and V) than in hatchlings. These concentrations were between 2 and 4 times lower than those measured in mature individuals.

During the whole-life cycle of cuttlefish, metal concentrations showed significant variations (H-value ranges from 15.32 to 17.34, and P Krsukall-Wallis -value ranges from 0.004 to 0.009).

Overall, the highest metal concentrations correspond to those of the essential metals Cu, Fe and Zn (between 30 and 200 µg.g -1 dwt) and the lowest to non essential metals (between 0.13 and 2.4 µg.g -1 dwt).

The dissection of cuttlefish from 7 days old to 2 years old showed that the digestive gland exhibited about one order of magnitude higher metal concentrations compared to the whole animal (Table 3). Consequently, this organ generally contained a great proportion of the whole-body burden of the metals (Table 4). In contrast, the cuttlebone displayed very low non-essential metal concentrations (Table 5). In this organ, Pb and V concentrations remained close to the detection limit during the whole-life cycle and Ag and Cd concentrations were one order of magnitude lower than those measured in the whole animal. Therefore, the cuttlebone only contained a small fraction of the total non essential metal load (Table 6).

Finally, the concentrations of the essential metals measured in the cuttlebone are similar (Fe and Zn) or lower (Cu) than those found in the whole organisms (Table 5).

Variation of metal concentrations and burdens with age

Immediately after hatching, the concentrations of Cu, Fe, and Zn have strongly increased in cuttlefish (Table 2). Indeed, the very young cuttlefish displayed concentrations values of 1.5 to 2 (Cu and Zn) and 6 (Fe) times higher than those found in the embryos taken from the eggs immediately before hatching. However, considering the whole-body concentrations, Cu and Zn increased significantly with age following a logarithmic fashion (R=0.408, ddl=18, P<0.05 and R=0.865, ddl=18, P<0.0001, respectively) whereas Fe tended to decrease significantly (R=0.657, ddl=18, P<0.001). Among toxic elements, Ag was the only metal showing a significant tendency to decrease with age (R=0.558 ddl=18, P<0.01) whereas Cd, Pb and V globally increase (Table 2). However, such variations in the whole-body animals are likely to be due to the variations in the storage organs such as the digestive gland. These tissue-specific variations are actually faked by natural dilution by non-storage organs. Therefore, the tissues presently dissected at all stages of the life cycle, i.e. the digestive gland and the cuttlebone, were considered to investigate metal variations with age (Figure 1 & 2, respectively).

Regardless of the essential or non essential role of the metals, their concentrations in the digestive gland showed significant variations during the whole-life of cuttlefish (Table 3). The amounts of metals contained in the digestive gland relatively to the whole-body burden also show remarkable changes during the life cycle (Table 4). With the exception of Ag, all metal concentrations and burdens in the digestive gland increase significantly with age following a logarithmic fashion (Figures 1 &3). Ag concentrations increase linearly (R=0.812, ddl=13, P<0.001) until the age of 2 months (ML = 59 mm), then decrease linearly (R=0.855, ddl=9, P<0.001) when cuttlefish migrate to open ocean waters.

In the cuttlebone, the concentrations of Ag, Cu and Fe decrease with age following a logarithmic fashion (R=0.861, ddl=18, P<0.001, R=0.910, ddl=18, P<0.001, R=0.660, ddl=18, P<0.005, respectively) whereas Zn display a linear accumulation (R=0.989, ddl=18, P<0.001) (Figure 2). The other metals either varied randomly (i.e. Cd) or were below the detection limit (i.e. Pb and V).

Figure 3 shows the variation of metal burdens in relation to the mantle length (age) of the cuttlefish. Both essential and non essential element burdens clearly increase during the whole life cycle of the cuttlefish, with the exception of Ag whose burdens have decreased in mature individuals.

DISCUSSION

Metal bioaccumulation in eggs and hatchlings

At the end of the embryonic development, the embryos showed very low metal concentrations compared to older cuttlefish (Table 2). Such concentrations are close or even identical to those measured in the vitellus of the early spawned eggs, suggesting that the vitellus contains a sufficient amount of essential metals (Cu, Fe, and Zn) necessary for the development of the embryo Previous studies with radio-labelled metals have demonstrated that 65 Zn, 109 Cd and 57 Co were mainly retained on the eggshell (96% after 11d of exposure), acting as a protective barrier limiting/hindering the incorporation of waterborne metals. However, the eggshell the thickness of the eggshell changes during embryonic development. When spawned, its thickness is in the mm-region, i.e. ± 1.5 mm [START_REF] Lemaire | Etude du développement embryonnaire de Sepia officinalis L[END_REF] and is composed of albumin and other proteins, hardening when put into contact with seawater. Then, it becomes thinner during embryonic development, being almost transparent at the moment of eclosion (Wolf et al., 1985). Very weak concentrations of Pb and V in the embryos also suggest a specific limitation of their incorporation into the eggs, but this issue should be addressed specifically in the future.

Immediately after hatching, the rapid increase of Cu, Fe and Zn concentrations in cuttlefish tissues (Table 2) suggests that the hatchlings are highly dependent on the essential metals to fulfil their metabolic demands. It therefore follows that the metals are rapidly uptaked once they are in contact with seawater. At this stage, the accumulation only occurs through the dissolved pathways by branchial absorption and tegumental adsorption, the hatchlings still living only on their vitellian reserves for a week [START_REF] Mangold | L'appareil digestif et la digestion[END_REF]. High 65 Zn accumulation rate has been experimentally shown for juveniles exposed to radiolabelled seawater (Bustamante et al., 2002b). Similarly to essential elements, Ag concentrations are 10 times higher in hatchlings than in the embryos (Table 2), showing a very rapid bioaccumulation of the metal. Similarly to 65 Zn, exposure of hatchlings to 110m Ag via seawater leads to elevated concentration factors (i.e. 320) after a period of only 36h [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]. Therefore, the dissolved pathway appears as a very important route for essential elements and Ag, whereas its role appears rather limited for Cd (Bustamante et al., 2002b), or Pb and V (Miramand et al., 1980[START_REF] Miramand | Uptake, assimilation and excretion of vanadium in the shrimp Lysmata seticaudata (Risso) and the crab Carcinus maenas (L.)[END_REF] whose concentrations remained very low. Further investigations on the uptake of V and Pb are specifically needed to asses this hypothesis.

Metal bioaccumulation during growth

After a week of benthic life, the hatchlings use up their vitellian reserves and therefore need to eat. Once they start to feed, cuttlefish rapidly grown (data not shown) but at the same time, are exposed to a new source of metals. Despite dietary intakes, the concentrations of most of the metals in the whole organisms did not vary significantly and only a few metals show a significant increase of the concentrations in the whole cuttlefish (i.e. Cd, Fe, Table 2). This could be due either to 1) control of essential element concentrations through homeostatic regulation, or to 2) dilution of non essential accumulated metals related to very fast somatic growth. With the exception of Fe, the essential metals seem to be well regulated and Cu and Zn showed remarkably homogenous concentrations during the whole life (Table 2). Among non essential elements, Ag concentration is also slightly influenced by dietary supplies.

Concentrations of Pb and V in the whole cuttlefish remain very low and will only be over the detection limit only after 1 and 2 months of benthic life, respectively. Therefore, in what concerns the whole animal, cuttlefish bioaccumulate poorly these 2 metals.

Because of the metal dilution with the somatic growth, it was necessary to consider the tissues separately and particularly the digestive gland as a storage organ of metals. With the first feedings, the immature digestive gland of the juveniles starts to evolve to be fully functional at the age of one month (Yim and Boucaud-Camou, 1980). The food intake provokes the beginning of the digestive processes, i.e. secretion of enzymes, absorption of nutrients, and excretion of digestive residues. In a few weeks, the digestive gland takes a dark colour due to the development of digestive cells called "Boules" cells (e.g. [START_REF] Boucaud-Camou | Etude de l'appareil digestif de Sepia officinalis L. (Mollusque Céphalopode). Essai d'analyse expérimentale des phénomènes digestifs[END_REF][START_REF] Boucaud-Camou | Feeding and digestion in cephalopods[END_REF][START_REF] Boucher-Rodoni | Feeding and digestion[END_REF]. The development of the digestive gland in the retention process of metals is very important and is reflected by the increased fraction of the whole body burdens of all metals in this organ (Table 4). This process is particularly significant in the case of Fe, for which the metal content stored in the digestive gland raises respectively, from 6 to about 50% before and after the digestive gland development. After only two months of benthic life, the digestive gland contains between 30 (Pb) and 90 % (Ag) of the total amount of metals (Table 4). These contents are identical to those previously measured in adult cuttlefish collected in the same area [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].

Thus, the digestive gland plays a major role in the metabolism of all metals in cuttlefish, and especially Cd (Figure 1). Indeed, during the period of development of the digestive gland (i.e. the first month of benthic life), Cd concentrations are increased 9 times compared to a factor 2 for the remaining metals. During this period, the digestive gland weight increased 30 times and the Cd amounts in this organ were multiplied by 230, i.e. 3 to 5 times higher than for the other metals (Figure 3). The digestive gland has an primarily role in the accumulation of Cd in cuttlefish, as also reported after both field and experimental studies [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 | Etude des processus de bioaccumulation et de détoxication d'éléments traces (métaux lourds et terres rares) chez les mollusques céphalopodes et bivalves pectinidés. Implication de leur biodisponibilité pour le transfert vers les prédateurs[END_REF]Bustamante et al., 2002b). Such a role of the digestive gland can be related both to the very efficient detoxification processes of the metal occurring in this organ (Bustamante et al., 2002a) and to the very high Cd assimilation efficiency (viz. 62 % for juvenile cuttlefish). Both processes lead to a strong retention of the metal in the digestive gland, which biological half-life (T b½ ) exceeds 8 months in juveniles (Bustamante et al., 2002b).

Influence of age

The process of bioaccumulation of all metals except Ag appears as age dependent in the digestive gland (Figure 1). Indeed, the digestive gland weight is increased by 3,500 between hatching and sexual maturity whereas its metal amounts is increased between 6,000 and 9,000 for the essential metals (Cu, Fe, Zn), more than 20,000 (Cd and V) and about 70,000 (Co) (Figure 3). Thus, the digestive gland contains after 2 years of life between 15 and 90 µg of Ag, Co, Pb and V, 165 µg of Cd and between 3 and 7 mg of Cu, Fe and Zn (Figure 3). Therefore, the digestive gland bioaccumulate large concentrations of the metals even those not mostly incorporated through the food pathway, such as for Ag. It is therefore noteworthy that different uptake pathway for metals, i.e. via seawater for Ag and via food for Cd, lead to large concentrations of both toxic metals in the digestive gland. Since the beginning of cuttlefish benthic life, the digestive gland shows 5 to 6 times higher Ag and Cd concentrations compared to those measured in the whole organisms (Tables 2 &3) and already accounts for 50 to 90% of the total amount of these two metals (Table 4).

Consequently to its storage capacities, all the metals concentrations increased following a logarithmic shape in the digestive gland. Ag is the only exception to this pattern (Figure 1) and its concentrations seem strongly influenced by the migration behaviour of the cephalopods. Indeed, after a few months in coastal areas, juveniles migrate to deeper ocean waters and return to coastal ground to mate only the following year [START_REF] Richard | Contribution à l'étude expérimentale de la croissance et de la maturation sexuelle de Sepia officinalis L. (Mollusque céphalopode[END_REF]. The migration to less polluted area will provoke a decrease in metal concentrations when they have a relatively rapid turn over. In cuttlefish, Ag has short biological half-life either following exposure from seawater or from food (less than 2 wks), as a consequence of its high depuration rates [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]. Our results are likely due to the equilibrium established between contamination (when the animals live by the coast) and decontamination (when the animals live in the open sea) episodes. Therefore, both the elevated bioaccumulation of Ag and its weak retention in the digestive gland of cephalopods will allow the use of these molluscs to monitor Ag concentrations in the marine environment.

The decontamination is actually much slower in the case of other metals as Cd or Co for which the biological ½ life measured in adults after incorporation of Cd or Co by the food pathway is largely higher than the lifetime of cuttlefish (Bustamante et al., 2002b;[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].

Owing to this, it is very likely that since the beginning of the migration to the open sea, the digestive gland of adults is strongly decontaminated in Ag and very little in Co and Cd. Since the main incorporation mechanism of V to the marine organisms seems to be the food pathway [START_REF] Miramand | Uptake, assimilation and excretion of vanadium in the shrimp Lysmata seticaudata (Risso) and the crab Carcinus maenas (L.)[END_REF][START_REF] Miramand | Bioaccumulation and transfer of vanadium in marine organisms[END_REF], the behaviour of V is similar to Cd and Co and is strongly retained by cuttlefish. Thus, it would be very interesting to confirm experimentally such hypothesis.

In contrast to the digestive gland, the concentrations measured in the cuttlebone of juveniles are very low, with most of the metals showing concentrations close to those observed in juvenile just after hatching (Table 2 &5). The shell in cuttlefish is internal, therefore not exposed to metal adsorption phenomena by direct contact with seawater, unlike other molluscs, especially mussels (Van Weers, 1973;[START_REF] Fowler | Accumulation and distribution of selenium in mussel and shrimp tissues[END_REF]Ünlü & Fowler, 1976;Miramand et al. 1980). As discussed above, albeit the significant penetration of the metals into the tissues, internal transfer of metals to the cuttlebone appear to be very slow and somatic growth of this organ lead to overall dilution of most metals. The only exception was Zn which concentrations significantly increase with age whereas the other analysed elements tended to decrease (Figure 2). Consequently, the metal amounts contained in the cuttlebone correspond in fact either to their weight importance (Cu, Pb, Fe) or are clearly lower (Ag, Cd, V and Zn) (Table 6). This observation is in good agreement with the experimental radiotracer results obtained for Ag, Cd and Zn (Bustamante et al. 2002b[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].

Influence of sexual maturation

In two years old individuals (sexually mature), Ag, Cd, Cu and Pb concentrations are lower than those in one-year old animals (immature) against those of Fe, V, and Zn (Table 2). This would be due to physiological changes related to the sexual maturation as well as to the growth of the gonads. These organs exhibit globally low metal levels in cephalopods [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 contribute to dilute the non-essential metal (Ag, Cd and Pb) concentrations in the whole animals. This is clearly exemplified by the fact that, with the exception of Zn, metal concentrations in the digestive gland do not significantly differ between mature and immature individuals (Table 3). High levels of Zn in the genital tract of the males and females of Sepia officinalis (190 ± 22 and 123 ± 3 µg.g -1 dwt, respectively) have been reported [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], which would explain that Zn concentrations still increased after sexual maturation. This 1.5-fold increase observed in the whole animals is in all likelihood due to a Zn accumulation in the digestive gland. This value represents a very significant concentration in the mature individuals and is 3 times and even 2 times higher than that measured in the juveniles and in the immature individuals, respectively (Table 3). During this period, the cuttlebone also showed a concentration of Zn higher than that measured in the juveniles and in the one-year old immature individuals. This observation could be translated by an increased flux of Zn in the haemolymphe of cuttlefish during this period of sexual maturity. This metal could therefore be partially accumulated by the cuttlebone. In this way, in the animals collected at the end of their sexual maturity the amounts of Zn associated to the cuttlebone (expressed as total Zn % contained in the organisms) are almost 2 times higher compared to the contents calculated in the juveniles and in the one-year old immatures. Interestingly enough, this phenomenon seems to be limited to Zn. The concentrations of the remaining 7 metals measured in the cuttlebone and the percentages they represent do not vary during this period (Tables 5 &6).

  Smith JD, Plues L, Heyraud M, Cherry RD. Concentrations of the elements Ag, Al, Ca, Cd, Cu, Fe, Mg, Mn, Pb and Zn and the radionuclides 210 Pb and 210 Po in the digestive gland of the squid Nototodarus gouldi. Marine Environmental Research 1984;13:55-68. Tanaka T, Hayashi Y, Ishizawa M. 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. Experientia (Basel) 1983;39:746-748. Ünlü MY, Fowler SW. Factors affecting the flux of arsenic trough the mussel Mytilus galloprovincialis. Marine Biology 1976;51:209-219. Van Weers AW. Uptake and loss of 65 Zn and 60 Co by the mussel Mytilus edulis L. In: Radioactive contamination of the marine environment, Vienna: International Atomic Energy Agency; 1973. p. 385-401. Wolf G, Verheyen E, Vlaeminck A, Lemaire J, Decleir W. Respiration of Sepia officinalis during embryonic and early juvenile life. Marine Biology 1985;90:35-39. Yim M, Boucaud-Camou E. Etude cytologique du développement post-embryonnaire de la glande digestive de Sepia officinalis L (Mollusque, Céphalopode). Archives d'Anatomie Microscopique et de Morphologie Expérimentale 1980;69(2):6-79

Figure 2 .Figure 3 .

 23 Figure 2. Variations of metal concentrations (µg g -1 dry wt) in the cuttlebone of Sepia officinalis in relation with the mantle length.

Table 1

 1 Comparison of elemental concentrations (µg.g -1 dry weight) of Orchard-leaves standard, SRM 1571 (National Bureau of Standards), copepods homogenate, MA-A-1 and fish flesh homogenate, MA-A-2 (International Agency of Atomic Energy) obtained in present study with certified values.

	Standard	Ag	Cd	Co	Cu	Fe	Pb	V	Zn
	Orchard leaves
	Present study	-	0.10 ± 0.05 0.17 ± 0.04	10 ± 1	272 ± 14	38 ± 2	0.5 ± 0.1	22 ± 6
	Certified values	-	0.11 ± 0.02	(0.2)	12 ± 1	300 ± 20	45 ± 3	(0.6)	25 ± 3
	MA-A-1
	Present study	0.2 ± 0.1	0.73 ± 0.06 0.12 ± 0.02 6.9 ± 0.4	61 ± 4	2.0 ± 0.5	-	161 ± 1
	Certified values 0.33 ± 0.06 0.75 ± 0.03 0.12 ± 0.01 7.6 ± 0.2	60 ± 2	2.1 ± 0.3	-	158 ± 2
	MA-A-2
	Present study	0.12 ± 0.01 0.07 ± 0.01 0.09 ± 0.04 3.4 ± 0.7	65 ± 5	0.43 ± 0.14	-	35 ± 4
	Certified values 0.10± 0.01 0.066 ± 0.004 0.08 ± 0.01 4.0 ± 0.1	54 ± 1	0.58 ± 0.07	-	33 ± 1
	() : recommended values

Table 3 :

 3 Heavy metal concentrations (Mean ± SD; µg g -1 dry wt) in the digestive gland of Sepia officinalis sampled in the bay of Seine at different stage of its life cycle. n: number of pool analysed. ( ): number of individuals in each pool. ML: mantle length.

	Mantle length	Age	n	Ag	Cd	Co	Cu	Fe	Pb	V	Zn
	(mm)	(estimation)
	Juveniles
	10 ± 1	7 days	3 (25) 10 ± 1	2.0 ± 0.1	0.3 ±0.1	175 ± 5	90 ± 10	< 0.1	0.3 ± 0.2	240 ± 5
	18 ± 3	15 days	4 (8)	18 ± 7	11 ± 1	1.3 ± 0.3	400 ± 10	160 ± 10	< 0.1	0.8 ± 0.1	400 ± 10
	33 ± 4	1 month	3 (4)	14 ± 1	15 ± 3	2.6 ± 0.5	450 ± 10	210 ± 10	0.5 ± 0.2	1.6 ± 0.4	500 ± 10
	59 ± 6	2 months	4 (4)	35 ± 7	7.5 ± 0.2	3.5 ± 0.5	450 ± 20	110 ± 10	0.5 ± 0.2	2.5 ± 0.7	400 ± 10
	Immature
	133 ± 19	12 months	3 (5)	19 ± 7	21.6 ± 1.5	6.8 ± 0.9	760 ± 80	340 ± 30	2.4 ± 0.5	2.9 ± 0.1	770 ± 40
	Mature adults
	215 ± 5	18 monthss	3 (1)	13 ± 2	25 ± 5	10 ± 2	600 ± 10	390 ± 10	2.2 ± 0.5	3.3 ± 0.1	1400 ± 500

Acknowledgements: We are grateful to Professor E. Boucaud-Camou for its fruitful advises on this work. Statistical analysis and writing up of this work was supported by the EC under the CEPHSTOCK project (QLRT-2001-00962).

Table 2. Heavy metal concentrations (Mean ± SD; µg g -1 dwt) in whole Sepia officinalis sampled in the bay of Seine at different stage of its life cycle. n: number of pool analysed. ( ): number of individuals in each pool. ML: mantle length; NM: not measured.

Life stage

Mantle