Simon Pouil

Paco Bustamante

Michel Warnau

François Oberhänsli

Jean-Louis Teyssié

Dr Marc Metian
email: m.metian@iaea.org

Delineation of 134 Cs uptake pathways (seawater and food) in the variegated scallop Mimachlamys varia

Keywords: Bivalve, Scallop, Cesium, Kinetics, Depuration

Among bivalves, scallops have been shown to be good bioindicator species for radionuclide monitoring. The present paper looked at the Cs bioaccumulation capacities of the variegated scallop Mimachlamys varia exposed separately via seawater and food under laboratory conditions. Results were compared with data previously obtained for the king scallop Pecten maximus, the only Pectinid species for which Cs accumulation has been studied in laboratory. Results indicated that M. varia has higher uptake capacity (CF: 1.86 ± 0.08) but lower absorption efficiency (A0l: 33 ± 5%) than P. maximus when exposed to waterborne Cs (CF of P. maximus: 0.94 ± 0.05 and A0l: 45 ± 3%). When scallops were fed radiolabeled phytoplankton, the assimilation efficiency of Cs was similar for the two species (AE: 24 ± 3% for M. varia and 28 ± 4% for P. maximus). Interspecific differences in terms of accumulation and retention, can be explained by physiological factors (including size of individuals) and/or difference in storage mecha-nisms. Indeed, organotropism differed between the two scallop species, suggesting the occurrence of specific redistribution mechanisms towards the tissues involved in Cs storage, excretion and detoxification. Finally, the present study examined the relative contribution of the different exposure pathways (seawater and food) to global 134 Cs bioaccumulation for M. varia. Results showed that food constitutes the main accumulation pathway, contributing for 77% of the global 134 Cs bioaccumulation.

Introduction

Tracking radionuclide contamination using organisms (i.e. biomonitoring) helps understanding the levels as well as the variation (in time and space) of radionuclides that are bioavailable in the environment (Philips and Rainbow, 1993). Since 1950, marine ecosystems were sporadically subjected to radionuclide contamination from industries, nuclear accidents and fallout from nuclear weapon testing and uses. Recently, the accident that occurred in the Daiichi Nuclear Power Plant at Fukushima in Japan, released massive amount of radioactive Cs in the marine environment [START_REF] Chino | Preliminary estimation of release amounts of 131 I and 137 Cs accidentally discharged from the Fukushima Daiichi nuclear power plant into the atmosphere[END_REF][START_REF] Bailly Du Bois | Estimation of marine source-term following Fukushima Dai-ichi accident[END_REF].

Among marine organisms, Pectinids have been proposed as good bioindicator candidates of radionuclide contamination [START_REF] Marzano | Anthropogenic radionuclides bioaccumulation in Antarctic marine fauna and its ecological relevance[END_REF][START_REF] Bustamante | Concentration and distribution of 210 Po in the tissues of the scallop Chlamys varia and the mussel Mytilus edulis from the coasts of Charente-Maritime (France)[END_REF]. This has been confirmed experimentally for the king scallop Pecten maximus [START_REF] Metian | Characterization of 241 Am and 134 Cs bioaccumulation in the king scallop Pecten maximus: investigation via three exposure pathways[END_REF]. Field surveys (e.g. [START_REF] Marzano | Anthropogenic radionuclides bioaccumulation in Antarctic marine fauna and its ecological relevance[END_REF] have also documented the capacity of the family of Pectinids to concentrate radionuclides, in particular Cs. However, levels of radionuclides in organisms are influenced by various factors such as geographical origin, season, sexual maturity or species considered [START_REF] Bryan | The accumulation of radioactive caesium by marine invertebrates[END_REF][START_REF] Mauri | Heavy metals in the Antarctic scallop Adamussium colbecki[END_REF]Bustamante andMiramand, 2004, 2005).

Laboratory characterization of Cs bioaccumulation by Pectinids is currently limited to one species, P. maximus [START_REF] Metian | Characterization of 241 Am and 134 Cs bioaccumulation in the king scallop Pecten maximus: investigation via three exposure pathways[END_REF] and since phylogenetically-close species may have different accumulation capacities (as shown for metals in scallops, e.g. [START_REF] Metian | Interspecific comparison of Cd bioaccumulation in European Pectinidae (Chlamys varia and Pecten maximus)[END_REF]in oysters, Hedouin et al., 2010a), it is worth comparing Cs bioaccumulation capacities among scallops species.

Therefore, the objective of the present study was to investigate 134 Cs bioaccumulation capacities of the variegated scallop Mimachlamys varia, exposed via the dissolved and trophic pathways.

This species is an interesting model to assess Cs contamination in coastal area given the fact that it settles, with its byssus, on rocky substrates [START_REF] Shumway | Scallops: Biology, ecology and aquaculture, 2nd Edition[END_REF] whereas P. maximus lives buried in the sediment. The same protocol as described in [START_REF] Metian | Characterization of 241 Am and 134 Cs bioaccumulation in the king scallop Pecten maximus: investigation via three exposure pathways[END_REF] was used in order to allow easy comparison between the two scallop species. In addition, the relative contribution of the different exposure pathways (dissolved vs. dietary) of Cs bioaccumulation in M. varia was determined.

Materials and methods

Origin and acclimation of organisms

In spring 2004 and 2005, one hundred variegated scallops M. varia were collected on the French Atlantic coast (Pertuis Breton, Charente-Maritime) by SCUBA diving. They were transported to IAEA-EL premises in Monaco and were acclimated to laboratory conditions for 4 weeks (constantly aerated, open-circuit 700 L aquarium; flux: 50 L.h -1 ; salinity: 36 p.s.u.; temperature: 17 ± 0.5°C; pH: 8.0 ± 0.1; light/dark cycle: 12 h/12 h) prior to experimentation. During this period, scallops were fed daily an algal diet (Isochrysis galbana).

Radiotracer and counting

Uptake and depuration kinetics of 134 Cs in scallops were determined using high-specific activity 134 Cs purchased from Isotope Product Lab ( 134 CsCl 0.1 N, T½ = 2 years). 134 Cs was counted using a high-resolution γ-spectrometer system composed of four Germanium (N-or P-type) detectors (EGNC 33-195-R, Canberra ® and Eurysis ® ) connected to a multi-channel analyzer and a computer equipped with a spectra analysis software (Interwinner ® 6). The radioactivity was determined by comparison with standards of known activity and of appropriate geometry.

Measurements were corrected for counting efficiency and physical radioactive decay. The counting time was adjusted to obtain a propagated counting error less than 5% (Rodriguez y [START_REF] Rodriguez Y Baena | Experimental evidence for 234 Th bioaccumulation in three Antarctic crustaceans: Potential implications for particle flux studies[END_REF].

Seawater exposure

Twenty three M. varia (30 ± 7g) were placed in a 70-L aquarium (constantly aerated, closedcircuit aquarium; salinity: 36 p.s.u.; temperature: 17°C ± 0.5°C; pH: 8.0 ± 0.1; light/dark cycle: 12 h/12 h) and exposed for 7 d to 134 Cs dissolved in 0.45 µm filtered seawater (1.4 kBq L -1 ).

Spiked seawater was renewed regularly in order to keep radioactivity constant. The 134 Cs in seawater was checked before and after each spike renewal. Nine tag-identified scallops were collected at different time intervals and were whole-body radioanalyzed alive. At the end of the 7 d exposure period, four scallops were collected and dissected into their body compartments (digestive gland, gills, kidneys, intestine, gonad, foot, mantle, adductor muscle and remaining tissues). Samples were radioanalyzed in order to assess the radionuclide body distribution. The remaining scallops were then placed in uncontaminated water in the same conditions for 36 d and the nine identified individuals were regularly radioanalyzed alive in order to follow the depuration kinetics of 134 Cs. Three non-exposed individuals were introduced into the aquarium in order to control possible tracer recycling from the contaminated scallops. During the 36 d depuration period, scallops were fed daily with I. galbana (5.10 4 cells mL -1 ). At the end of the depuration period, four contaminated scallops were collected and dissected.

Dietary exposure

134 Cs transfer to scallops through their diet was studied using the Haptophyceae Isochrysis galbana. Phytoplankton cells were exposed to 7 kBq 134 Cs L -1 during their exponential growing phase (7 d). After that period, the phytoplankton medium was filtered (1 µm-mesh size; Osmonic filters), and the phytoplankton cells re-suspended in a 70 L aquarium in the same conditions as previously described for the scallops. Cell density was 5.10 4 cells mL -1 to avoid pseudofeces production by the mollusks. Six M. varia (17 ± 5g) had been placed in the aquarium for one week before the start of feeding experiment. Scallops were fed with radiolabelled phytoplankton for 2 h in closed-circuit. After the feeding period, all scallops were γ-counted and flow restored in the aquarium. Depuration was followed during 30 d as described previously (see section 2.3). Three non-exposed individuals were used as controls of tracer recycling.

During the 30-d depuration period, scallops were fed daily with I. galbana (5.10 4 cells mL -1 ).

Four contaminated individuals were collected after 30 d and dissected to determine the radionuclide body distribution.

Data analysis

Whole-body uptake and depuration kinetics were fitted using nonlinear regression routines and iterative adjustment (Statistica ® 7) and statistical methods described by Warnau et al. (1996aWarnau et al. ( , 1996b) ) and [START_REF] Metian | Characterization of 241 Am and 134 Cs bioaccumulation in the king scallop Pecten maximus: investigation via three exposure pathways[END_REF]. The level of significance for statistical analyses was always set at α = 0.05. The relative contribution of each pathway (seawater and food) was determined using a global bioaccumulation model described in Hédouin et al (2010a). In addition to the parameters obtained from each biokinetic models, we have used the ingestion rate of M. varia (g g -1 d -1 , wet wt basis) and the Kdf, partition coefficients for phytoplankton calculated experimentally (Metian et al., 2009).

Results

Seawater exposure

Whole-body uptake of 134 Cs in M. varia followed exponential kinetics, reaching a steady-state equilibrium after approximately 4 days (R 2 = 0.79; Fig. 1A and Table 1). The kinetic parameters and their statistics are shown in Table 1. The whole-body concentration factor of 134 Cs ( i.e. ratio between the radioisotope activity in organisms -Bq g -1 wet wt -and time-integrated activity in the seawater -Bq g -1 ) estimated at steady state (CFss) was 1.86 ± 0.08 (Fig. 1A and Table 1). Calculated CF7d for the different body compartments are shown in Table 2. Among the tissues and organs, the foot displayed the highest CF closely followed by the kidney and the digestive gland (Table 2). In term of distribution, Cs was mainly located in the adductor muscle, the digestive gland and the mantle with, respectively, 26 ± 3, 24 ± 10 and 23 ± 5% of the total Cs body burden.

After the exposure period, non-contaminating conditions were restored and depuration of 134 Cs was followed for 36 d. The whole-body depuration kinetics of 134 Cs was best described by a two-component exponential model (Fig. 1B and Table 1). An important part of 134 Cs was efficiently absorbed (A0l: 33 ± 4.6%) with an associated long-term biological half-life (Tb1/2l) of 178 d (not significantly different from infinite; Table 1). After 36 d of the depuration, 134 Cs was mainly located in the soft body parts (72 ± 7%; Table 2). Distribution of 134 Cs among the soft tissues displayed a different pattern than that observed at the end of the exposure period (Table 2). Indeed, the digestive gland contained most of 134 Cs (59 ± 13%) and the relative proportion of the total Cs found in the intestine was much higher than the one in the same organ after the exposure phase (10 vs. 41%).

Dietary exposure

The depuration kinetics of the 134 Cs ingested with food in M. varia was best fitted by a double exponential model (Fig. 1C and Table 1). M. varia displayed an assimilation efficiency of 24 ± 3%. The depuration rate constant, kel (0.006 ± 0.006 d -1 ) was not significantly different from 0 (p>0.05), and therefore the derived Tb½l is not significantly different from infinite (115 d). At the end of the depuration period, the digestive gland contained the major part of 134 Cs (57 ± 16%, Table 2).

Bioaccumulation model

The relative contribution of each exposure pathway to the global bioaccumulation of 134 Cs was determined using the different kinetic parameters determined in the two experiments, as well as other parameters such as the 134 Cs Kdf in phytoplankton (1.32 x 10 2 for I. galbana; present study) and the ingestion rate of phytoplankton in scallops (IR = 0.0869 g g -1 d -1 ; Metian et al., 2009). Results of the computations indicated that feeding pathway was the major contributor (77%) to the global bioaccumulation of 134 Cs in M. varia whereas seawater contributed for 23%.

Discussion

Pectinids have been used in biomonitoring programmes to assess radionuclide contamination in the marine environment (JCAC, 2002) given their high accumulation capacity shown during field surveys [START_REF] Marzano | Anthropogenic radionuclides bioaccumulation in Antarctic marine fauna and its ecological relevance[END_REF][START_REF] Bustamante | Concentration and distribution of 210 Po in the tissues of the scallop Chlamys varia and the mussel Mytilus edulis from the coasts of Charente-Maritime (France)[END_REF]. However, the behavior of radionuclides in scallops and their mode of uptake and depuration are less known in comparison to other bivalve families such as Ostreidae or Mytilidae [START_REF] Cranmore | Loss of 137 Cs and 60 Co from the oyster Crassostrea gigas[END_REF]Ke et al., 2000;[START_REF] Ryan | Transuranic biokinetic parameters for marine invertebrates: a review[END_REF]. To the best of our knowledge, experimental investigations of 134 Cs bioaccumulation have been limited to one species P. maximus exposed to three pathways. Unlike P. maximus, which lives buried in the bottom sediment, M. varia settles, with its byssus, on rocky substrates [START_REF] Shumway | Scallops: Biology, ecology and aquaculture, 2nd Edition[END_REF]; therefore, the exposure of M. varia to radionuclides occurs through seawater and food pathways.

Our results show that there are interspecific differences in Pectinids in terms of accumulation and retention of dissolved Cs. Indeed, the concentration factor (CFss) in M. varia was two times higher than CFss of P. maximus [START_REF] Metian | Characterization of 241 Am and 134 Cs bioaccumulation in the king scallop Pecten maximus: investigation via three exposure pathways[END_REF]. Difference can be attributed to variation of physiological factors (including size of individuals) and/or difference in storage mechanisms (as highlighted by the contrasting body distribution patterns of Cs in both species at the end of exposure; [START_REF] Metian | Characterization of 241 Am and 134 Cs bioaccumulation in the king scallop Pecten maximus: investigation via three exposure pathways[END_REF]. Similar interspecific differences of CFss between these two species have already been highlighted for other radionuclides ( 110m Ag, 109 Cd, 54 Mn, 210 Pb, 65 Zn; [START_REF] Metian | Interspecific comparison of Cd bioaccumulation in European Pectinidae (Chlamys varia and Pecten maximus)[END_REF][START_REF] Metian | Investigation of Ag in the king scallop Pecten maximus using field and laboratory approaches[END_REF]Metian et al., 2009aMetian et al., , 2009bMetian et al., , 2009c)). Interspecific differences also concerned Cs depuration. Indeed, the percentage of long-term retention of 134 Cs absorbed by M. varia after 36 d of depuration (following a 134 Cs exposure through seawater) did not exceed 33 ± 4.6% (Table 1) whereas 45 ± 2.9% of 134 Cs was retained by P. maximus [START_REF] Metian | Characterization of 241 Am and 134 Cs bioaccumulation in the king scallop Pecten maximus: investigation via three exposure pathways[END_REF]. Differences observed in term of retention were radionuclide-dependent: A0l were higher in P. maximus for 110m Ag, 57 Co, 109 Cd and 65 Zn than in M. varia whereas no differences were observed between the two species for 54 Mn and 210 Pb [START_REF] Metian | Interspecific comparison of Cd bioaccumulation in European Pectinidae (Chlamys varia and Pecten maximus)[END_REF][START_REF] Metian | Investigation of Ag in the king scallop Pecten maximus using field and laboratory approaches[END_REF]Metian et al., 2009aMetian et al., , 2009bMetian et al., , 2009c)). For Cs, difference in CF between the two species exposed to the same contamination conditions is related to a higher Cs uptake rate (uptake rate constant: 1.77 d -1 vs. 0.55 d -1 ) in M. varia compared to P. maximus [START_REF] Metian | Characterization of 241 Am and 134 Cs bioaccumulation in the king scallop Pecten maximus: investigation via three exposure pathways[END_REF]; Table 1) Interestingly, interspecific differences can be identified upon a dissolved exposure but not when Pectinids are exposed to 134 Cs through food. The present results show that the retention of Cs from food is similar for both scallop's species. Indeed, when M. varia were fed with radiolabeled phytoplankton cells (I. galbana), 134 Cs was similarly assimilated (AE: 24.5 ± 2.9%, Table 1) compared to AE of 28.1 ± 4.4% obtained for P. maximus fed in the same conditions [START_REF] Metian | Characterization of 241 Am and 134 Cs bioaccumulation in the king scallop Pecten maximus: investigation via three exposure pathways[END_REF]. This similarity in Cs AE between these two Pectinids was not observed for other elements: the AEs of 110m Ag, 57 Co, and 210 Pb were higher in M. varia whereas 54 Mn AE was higher for the P. maximus [START_REF] Metian | Interspecific comparison of Cd bioaccumulation in European Pectinidae (Chlamys varia and Pecten maximus)[END_REF][START_REF] Metian | Investigation of Ag in the king scallop Pecten maximus using field and laboratory approaches[END_REF]Metian et al., 2009aMetian et al., , 2009bMetian et al., , 2009c)). To confirm our results for 134 Cs, it would be appropriate to test the AE between these two scallop species with other radiolabelled phytoplankton species since Hédouin et al. (2010b) have already demonstrated that the quality of food may affect radionuclide accumulation ( 57 Co, 54 Mn and 65 Zn) in bivalves.

Globally, our results confirm that Cs is well retained in Pectinids. Figure 2 put these results in perspectives with previous studies on Cs accumulation in bivalves. Whereas 134 Cs CF in Pectinids is similar (or lower in some cases) than other bivalve species, retention is always higher in Pectinids (Figure 2). One has however to keep in mind that some of the observed differences in CF can be partly due to others factor than interspecific bioaccumulation properties, such as difference in ambient salinity. Indeed it is well documented that CF for a given species generally increases with decreasing salinity (e.g. [START_REF] Ke | Uptake and depuration of cesium in the green mussel Perna viridis[END_REF]. However, most of the species compared here (see Figure 2) were living in a narrow range of salinity (between 35 and 38 p.s.u.; [START_REF] Nolan | Accumulation of metal radiotracers by Mytilusedulis[END_REF][START_REF] Bustamante | Assessment of the exposure pathway in the uptake and distribution of americium and cesium in cuttlefish (Sepia officinalis) at different stages of its life cycle[END_REF][START_REF] Qureshi | Bioaccumulation of 137 Cs, Zn and Cr[VI] in the green mussel Perna viridis: influence of salinity and temperature[END_REF][START_REF] Lacoue-Labarthe | Contrasting accumulation biokinetics and distribution of 241Am, Co, Cs, Mn and Zn during the whole development time of the eggs of the common cuttlefish, Sepia officinalis[END_REF][START_REF] Metian | Characterization of 241 Am and 134 Cs bioaccumulation in the king scallop Pecten maximus: investigation via three exposure pathways[END_REF].

Percentage of Cs retention from seawater (A0l) and food (AE) is higher in Pectinids than other bivalves. For example, Mytilidae showed low AEs for Cs with values (ranging between 3 and 13% for Perna viridis; Table 2, e.g. Wang et al., 2000). However, Figure 2 also shows the reported AEs of Cs in other mollusks (predators) and they can be higher than Pectinids (ranging between 19 and 55% for gastropods and cephalopods; e.g. Wang et al., 2000;[START_REF] Bustamante | Assessment of the exposure pathway in the uptake and distribution of americium and cesium in cuttlefish (Sepia officinalis) at different stages of its life cycle[END_REF]. These results suggest that food source is key pathway for the transfer of Cs towards molluscs.

In the natural environment, M. varia may be exposed to radioactive Cs through seawater and food and each pathway contribute simultaneously to the global bioaccumulation of 134 Cs in the organism. Using the kinetic parameters that we obtained experimentally and Kdf and IR that we measured, the relative contributions of the two exposure pathways were delineated, using the model developed by [START_REF] Landrum | Toxicokinetics in aquatic systems: Model comparisons and use in hazard assessment[END_REF] and adapted more recently (Hédouin et al., 2010a).

Results indicate that food has a major role in Cs accumulation in M. varia in the field (relative contribution of 77% in average). The importance of the trophic pathway in the Cs accumulation has been suggested in taxa such as cephalopods [START_REF] Bustamante | Assessment of the exposure pathway in the uptake and distribution of americium and cesium in cuttlefish (Sepia officinalis) at different stages of its life cycle[END_REF] and fish [START_REF] Pentreath | The Uptake of Radionuclides by I-Group Plaice (Pleuronectes platessa) off the Cumberland Coast, Irish Sea[END_REF][START_REF] Hewett | The accumulation of radioactive caesium from food by the plaice (Pleuronectes platessa) and the brown trout (Salmo trutta)[END_REF] although without direct evidences. In Pectinids, food has been shown to be the major contamination pathway for 109 Cd (M. varia and P. maximus respectively >99% and 84% of the total contribution; [START_REF] Metian | Interspecific comparison of Cd bioaccumulation in European Pectinidae (Chlamys varia and Pecten maximus)[END_REF] and for 110m Ag (P. maximus: contribution over 98%; [START_REF] Metian | Investigation of Ag in the king scallop Pecten maximus using field and laboratory approaches[END_REF] although there is a non-negligible effect of the type of food used in the model.

Conclusion

The pectinids, Mimachlamys varia included, stand out from bivalves group when one considers their ability to retain 134 Cs. This characteristic makes them very good candidates to be used in biomonitoring programmes. However, our study highlighted interspecific differences among Pectinids regarding accumulation of waterborne radionuclides: M. varia shown a higher capacity to concentrate 134 Cs but lower absorption efficiency than P. maximus. A global bioaccumulation model indicated that ingestion with food is the major uptake pathway of Cs in M. varia. In light with the general recommendation to use multiple species in biomonitoring (e.g. Philips and Rainbow, 1993;Warnau et al., 1996), the present work strongly suggests that the use of M. varia in such programmes would be an excellent way to assess the trophicallyavailable Cs and would ideally complement information provided by other species accumulation Cs predominantly from the dissolved phase. a Present study and [START_REF] Metian | Characterization of 241 Am and 134 Cs bioaccumulation in the king scallop Pecten maximus: investigation via three exposure pathways[END_REF] b [START_REF] Ueda | Comparison of influences of sediments and sea water on accumulation of radionuclides by marine organisms[END_REF][START_REF] Nolan | Accumulation of metal radiotracers by Mytilusedulis[END_REF][START_REF] Hutchins | Effects of arctic temperatures on distribution and retention of the nuclear waste radionuclides 241 Am, 57 Co, and 137 Cs in the bioindicator bivalve Macoma balthica[END_REF][START_REF] Ke | Uptake and depuration of cesium in the green mussel Perna viridis[END_REF]Wang et al., 2000 ;[START_REF] Güngör | Experimental studies on the biokinetics of 134 Cs and 241 Am in mussels (Mytilus galloprovincialis)[END_REF][START_REF] Qureshi | Bioaccumulation of 137 Cs, Zn and Cr[VI] in the green mussel Perna viridis: influence of salinity and temperature[END_REF][START_REF] Kalaycı | Investigation of radiocesium biokinetics in Manila clam (Ruditapes philippinarum)[END_REF] c [START_REF] Bryan | The accumulation of radioactive caesium by marine invertebrates[END_REF][START_REF] Bustamante | Assessment of the exposure pathway in the uptake and distribution of americium and cesium in cuttlefish (Sepia officinalis) at different stages of its life cycle[END_REF][START_REF] Lacoue-Labarthe | Contrasting accumulation biokinetics and distribution of 241Am, Co, Cs, Mn and Zn during the whole development time of the eggs of the common cuttlefish, Sepia officinalis[END_REF] Table 1. Parameters of whole-body uptake and depuration kinetics of 134 Cs in M.varia 1) exposed for 7 d to waterborne radionuclide (n = 9) and followed by 36 d of depuration (n = 9) and 2) after a 2-h feeding on radiolabelled I. galbana followed by 30 d of depuration (n = 6).

Figure 2 .

 2 Figure 2. Comparison of concentration factors (CF) and percentages of long-term component

Acknowledgments

The IAEA is grateful for the support provided to its Environment Laboratories by the Government of the Principality of Monaco. MW is an Honorary Senior Research Associate of the National Fund for Scientific Research (NFSR, Belgium). The authors thank L. Hédouin (CNRS-LABEX) for assistance on the contribution model.

Uptake parameters: CFss: concentration factors at steady state; ku: uptake rate constant (d -1 ).

Depuration parameters: A0s and A0l: remaining activity (%) according to the short-and the longlived exponential component, respectively; Tb½: biological half-life (d). ASE: asymptotic standard error; R 2 : determination coefficient of the uptake or depuration kinetics.

a Probability of the model adjustment: p < 0.0001.


Sylvain Delchini

Abdeltif Lahfid

Claire Ramboz

Yannick Branquet

Lhou Maacha

Contribution of the RSCM geothermometry to understanding the thermal history of the Hajjar deposit (Guemassa massif, Morocco).

The knowledge of the thermal history of rocks is a key point for reconstructing the history of basins or mountain belts for mining or petroleum industries. Conventional techniques such as mineralogy, isotopic analysis, provide basic data concerning the maturity degree of organic matter. Recent new geothermometric approach based on the Raman Spectroscopy of Carbonaceous Materials (RSCM) has been developed. This approach allows successfully estimating peak temperatures of advanced diagenesis to high-grade metamorphic rocks. The aim of this study is mainly to apply the RSCM geothermometry for 3D paleotemperatures cartography in the Guemassa area, a Hercynian massif located at 35 Km SW of Marrakech, Morocco. This area composed of the carboniferous metasediments, underwent tectonic, metamorphic and hydrothermal events that explain the presence of several base metal deposits like Zn-Pb-Cu Hajjar mine. Combining RSCM data and classical methods of thermometry like fluid inclusions and chlorite thermometry will allow a good understanding of thermal history of Hajjar deposits. The samples used in this study were collected around the Hajjar mine and from different depths in the Hajjar body collected in the footwall and hangingwall of the massive ore. Our peak temperature estimates show values superior to 500°C. These temperatures differ from the ones obtained by other classical methods, which are not higher than 450°C. Nevertheless, fluid inclusion homogenization temperatures of 450°C represent minimum trapping temperature conditions, since the fluids were trapped above boiling conditions. Also, 450°C represents minimum thermic condition for the biotite isograd. Higher Raman temperatures obtained in this work confirm the hypothesis of a late heat flow related to a deep granitic intrusion. This intrusion could be closer to the Hajjar deposit which would explain the higher Raman temperature around the mineralization. It is important to properly evaluate the consequences of this high late heat flux on the Hajjar mineralization, as it may have caused the recrystallization of the ore, with an increase of the particle size related. This thermal event could also have generated new mineralizing fluids. That is why future work will include the acquisition of complementary geochemical, chronological and structural data to better explain these high temperatures and to analyse their impact on the mineralization and their possible link with different mineralization processes.


J Thiery

S Rodts

E Keita

X Chateau

P Faure

D Courtier-Murias

T E Kodger

P Coussot

Water transfer and crack regimes in nano-colloidal gels

Keywords: number(s): 47.56.+r, 68.03.Fg, 81.40.Np I

Direct observations of the surface and shape of model nano-colloidal gels associated with measurements of the spatial distribution of water content during drying show that air starts to significantly penetrate the sample when the material stops shrinking. We show that whether the material fractures or not during desiccation, as air penetrates the porous body, the water saturation decreases but remains almost homogeneous throughout the sample. This airinvasion is at the origin of another type of fracture due to capillary effects; these results provide a new insight in the liquid dynamics at the nano-scale.

INTRODUCTION

The evaporation of solvent from colloidal suspensions, gels or pastes often leads to shrinking, wrapping, and eventually cracking [1] which impacts the material. Cracking during drying raises tremendous technological and scientific concerns as it can drastically impair the structural properties of a concrete [2], the integrity of ceramic films [3], soil or clays [4], the functionalities of cosmetics, paints or coatings [5].

Among the different physical mechanisms [6] suggested to explain why a crack nucleates during drying the most common model relies on the framework of elastic fracture mechanics [7]. This tensile failure theory considers the propagation of cracks triggered once the near tip stress state which intensifies as desiccation proceeds if the material remains bound to its substrate reaches the failure criterion for brittle materials [4,[8][9]. By contrast, the opening of cracks during drying is considered to stem from large capillary stresses resulting from the penetration of irregular air-liquid interface into the material [6,10]. However, even though the examination of fracture patterns as a function of experimental conditions is considerable [11], no study bears direct witness of these effects at local length scale. Additionally, despite drying regimes are already accurately detailed with the spatial distribution of the liquid in time [12][13], the role of liquid dynamics on fracturing appear to be somewhat neglected so far notwithstanding that the tensile failure theory implicitly assumes the sample to remain saturated with a rather homogeneous distribution of water, whereas, its counterpart brings up the idea of a partially saturated medium, at least locally. Therefore, measurements of the spatial distribution with time of water during drying would greatly enhance the current understanding of crack nucleation.

In this paper we follow both the surface and internal characteristics, specifically the spatial distribution of water, of a drying model nano-colloidal gel with the help of proton NMR profiling (a 1D use of Magnetic Resonance Imaging) measurements with micrometre spatial resolution. We show that whether it develops widely open fractures or not during the process, the material remains saturated as it shrinks, and then starts to desaturate almost homogeneously, which induces thin fractures that do not span the gel. These results provide a general view of water transport during drying and cracking, and a new insight in the thin liquid film dynamics in nanopores.

II.

MATERIALS AND METHODS

We use aqueous dispersions of hydrophilic nano-sized silica particles (r=6 nm radius) obtained from Sigma Aldrich (Ludox HS-40) in which 0.5mol/L of Sodium chloride is added to trigger aggregation. The initial stable suspension of particles has a solid volume fraction of 23%. Since according to the DLVO theory the electrical double layer thickness around a single particle is governed by the concentration of the surface charge counterions in liquid phase of the suspension, the higher the salt concentration in this electrolyte, the higher the aggregation rate of colloids. Therefore, in order to obtain a fairly homogeneous final gel structure i.e avoiding substantial structural heterogeneity possibly caused by local gradients of rate of aggregation -sedimentation not applying here-we dissolved the quantity of salt needed into the additional necessary water (thus mixing two homogeneous phases together) to work at initial volume fraction of particles ( 0  ) ranging from 5% to 20%.

The mixture processed being firstly liquid, we poured it into glass Petri dishes of 90 mm diameter, therefore making the initial sample thickness ( 0 h ) either 5 or 10 mm, and further sealed the recipient and leave the mixture at rest for gelation time. The material structure in time is followed by rheometrical tests imposing oscillations at very low strain ( 4 10 .

3  ) (frequency: 1 Hz) that do not interfere with gelation. The storage modulus ( ' G ) increases by several orders of magnitude and finally, typically after one or two hours, reaches a plateau that we associate with the steady gel structure. The values of ' G at the plateau were typically in the range 5 10 -

Pa 10 6

, which means that we are dealing with initially rigid samples.

In order to avoid a possible effect on drying characteristics of the presence of salt in solution we removed 97% of the salt initially incorporated through a dialyse protocol. It consists to cyclically pour de-ionized water on top of the sample, then waiting the time needed for the sodium chloride to homogenize throughout this new two-phase system, and finally carefully removed the liquid. Each dialyze enabled us to remove roughly half the quantity previously contained in the gel within a very small amount of time which actually depends on the sample volume. The amount of NaCl extracted after each dialyze was evaluated performing conductivity measurements on dialysates. The remaining very low concentration of salt is expected to negligibly affect water transfer processes, and the diffusion of salt in water being much more rapid than advection due to liquid motion, no significant accumulation of salt crystals should play a role in the fracture processes.

In order to look at the impact of dialyze on gel strength we managed to dialyze the sample during rheometrical tests thanks to a special set up allowing a water bath around the geometry. We observed a drop of about 25% of the ' G value when water is first put in contact with the sample, likely due to a structure modification of the gel generated by the osmotic pressure of de-ionized water in the bath, but subsequent dialyzes did not further affect ' G . This means that dialyze negligibly affects the strength of the structure which relies on strong van der Waals links, and thus we keep a structure strength at a high level (see above).

Drying is performed by blowing 0% humidity air along the top surface of the sample at a given flow rate (0.3 m/s). In some cases the dish walls are either covered with a thin layer of perfluorinated grease (Krytox GPL 205, DuPont) allowing the sample to glide.

The distribution of apparent water along the sample axis is measured by a 0.5 T 1 H MRI spectrometer with a one-dimensional double spin-echo sequence (two first echoes of the so-called CPMG sequence [16] with exponential extrapolation to compensate spin-spin relaxation) (see details on the sequence and data analysis in Appendix A). Each measured value of this distribution corresponds to the total water amount in a thin crosssectional layer of 75 m thickness along the axis of the Petri dish. The total NMR signal in the sample is also measured separately, providing a reliable measure of the total water amount at each time down to very low values, in particular when NMR data become too noisy.

Finally, since only the presence of liquid water is detected on MRI profiles, our measurements cannot provide the effective sample thickness especially if some near free surface region is dry. We therefore place upon the central point of the sample a sealed tripod pot (3 cm diameter) containing a solution with a similar NMR behaviour; the sample to pot size ratio and the distance (3 cm) between the pot and the sample are sufficiently large to negligibly affect the drying characteristics; imaging the position of this pot provides the exact position of the sample free surface in time.

III. RESULTS

The direct observation of the sample makes it possible to identify different stages in the drying process (see Figure 1). Initially the gel adheres to the glass surface of the dish walls (adhesive samples). The observation of the samples from their upper side during drying reveals a first period (Regime A) where the gel starts to fracture along its periphery, which detaches it from the sidewalls of the container, then, additional open transverse fractures develop (see Fig. 1b), with exact patterns varying with the drying rate [14]. In this regime the gel pieces newly formed significantly shrink (compare Figs. 1b and1c) while gliding over the substrate. After a while this process visually stops, the sample scatters light (see Fig. 1c), the free surface is opalescent, and we then enter the second regime (Regime B).

The opalescence of the sample likely occurs at the beginning of desaturation because in this regime small patches of saturated region coexist with unsaturated regions around the free surface of the sample, at a length scale which scatters visible light (micrometer scale). Opalescence would thus disappear when further drying decreases the length scale of those patches down to the nanometric scale, so that our system reappears as homogeneous for visible light wavelength.

Shortly after, the pieces develop fractures (opening width of the order of 2 m, see inset of Fig. 3) without further significant shrinkage (see Fig. 1d upper), an effect reminiscent of that observed for confined colloidal suspensions drying in channel [15]. None of these fractures fully cross a piece which remains as a continuous section. At the end of this phase the gels become translucent again.

By contrast, once the dish walls are covered with a thin layer of grease, the gel is able to freely glide on its substrate Fig1.a',b',c' and d'. We observe no fracture during the first regime, that we will also call Regime A: the sample simply shrinks (compare Figs. 1a', 1b', 1c'). There is again an opalescence at the transition (see Fig. 1c') to a regime B, with similar fractures nucleating without apparent further shrinkage (see Fig. 1d').

The basic difference in the evolution of adhesive and non-adhesive samples is during Regime A, the absence of open fractures followed by further shrinkage. The fractures observed in regime A in the adhesive case are due to tensile stresses resulting from two opposite tendencies: radial shrinkage due to water withdrawing and bounding of the bottom interface of the sample. On the contrary in regime B the second type of fractures develops in a non-shrinking situation, which means that there is no macroscopic deformation in the sample and thus no macroscopic tensile stress. This means that these fractures occur as a result of local high stresses in relation with air penetration in the sample. The validity of this scheme is now investigated though NMR which yields clear information on the water saturation ( , water to void volume ratio) in the material during the drying process. The water content for adhesive and non-adhesive samples continuously decreases in time as shown by the decrease of the profile level (see Fig. 2). Crucially, the position of the sample free surface exactly follows the position of the end of the plateau in the water profiles (see Fig. 2) assuring that no significant dry region around the top of the sample exists. During a first period both the profile level and extent decrease, indicating a loss in water content in the radial and axial directions; furthermore, we observe the profiles holding parallel to each other, signifying water remains homogeneous distributed along a vertical axis. In a second regime (profiles starting from the thick line, see Fig. 2) the sample thickness remains approximately constant while the profile level goes on decreasing.

Note the linear ramp shape at bottom and top sides of profiles at the beginning of the experiment, and the non-zero water amount above the measured position of the free surface. Both are due to the bending (see Appendix B) of the sample which becomes slightly concave (with an almost constant radius of curvature of about 1 m) rapidly after the test beginning [17], so that squares in Figure 2 only indicate some average position of tripod's legs on the curved sample surface. For the adhesive samples the measurements correspond to the average water amount through different material pieces. This suggests that each of the pieces resulting from fractures behave in a similar way, but here the analysis of the water content profiles is not straightforward since the different pieces formed with different sizes, shapes and curvatures may evolve with somewhat different timings.

IV. DISCUSSION AND ANALYSIS

The critical question is whether the samples are saturated (

1  
) or not during desiccation. In order to clarify this we use our measure of the total water amount in the sample ( water  ), the sample thickness ( h ) in time as measured from NMR profiles, and the horizontal apparent area (neglecting the impact of bending) of the sample ( S ) measured from videos taken from above during the test, which provides the apparent volume

Sh  
. We deduce the saturation:

) ( p water       , in which ) 0 ( 0     t p 
is the volume of particles, and the solid concentration:

   p 
. The reproducibility of the MRI data is excellent (see Appendix C) with an uncertainty of less than 2.5%. The main uncertainties on the present data result from slight variations of sample shape and the uncertainty on the estimation of its section area, which explains some saturation values larger than 1 through our above computations.

From the evolution of  as a function of  two regimes clearly appear (see Fig. 3). In a first regime  remains equal to 1, indicating that a negligible amount of air penetrates the gel, and the sample exhibits isotropic shrinking, i.e. its thickness to apparent radius (



S R 

) ratio remains constant. The material finally reaches a solid concentration which is not exactly the maximum packing fraction in a disordered configuration for noncolloidal particles (

% 60 
), but here the initial loose system was already an aggregated network. In a second regime  first slightly increases [18] then remains constant while the saturation drops to zero. The results are the same for a sample fracturing in regime A. This means that we would get the same overall characteristics (shape and water content) for a non-adhesive and an adhesive sample by gathering the pieces formed in the latter case. x is the distance from sample bottom) (see Fig. 4 top inset) from the water content profiles by scaling the NMR signal by that measured at the same position just before desaturation starts. After a short transient period the saturation profile keeps a constant shape, i.e. the profiles rescaled by the mean saturation at each time (

) , ( ) ( t x t   
) fall along a master curve (see Fig. 4 bottom inset). This slightly inclined curve shows that the saturation gently decreases from the bottom to the free surface.

In such a case, most of the air volumes inside the sample are saturated by vapor [12,19] so that evaporation mainly occurs around the air-liquid interfaces situated close to the sample free surface. Indeed a process of vapor diffusion from the interior of the sample cannot be significant since inside the sample the air volumes surrounded by liquid patches are saturated with vapor, which means that the vapor flux associated with such a diffusion process towards the sample free surface would be extremely slow. That also means that if the liquid was distributed in independent patches in the sample, the main vapor flux would come from the patches close to the free surface which would be rapidly dried. Since this process would start again at the next depth in the sample we would observe a drying front growing in depth. Our observation of the progressive homogeneous desaturation thus proves that there is a continuous liquid film coating the particles. Moreover if some dewetting process was possible somewhere it would also imply the possibility of a dewetting around the free surface of the sample thus leading to a growing dry front. As a consequence the only possibility is a continuous film coating all the particles. This implies that the liquid forms a continuous film coating the particles, which flows through the bead packing and evaporates around the free surface. Here the original situation is that this flow involves a continuous liquid layer of very small thickness (of the order of a few molecule sizes) which extends from the bottom to the top of the sample. Indeed if we assume that each particle is covered with a liquid layer of uniform thickness e , for a particle concentration of 50% we find 3 r e 



, which implies that for

5 . 0   we have nm 1  e
. In contrast, for larger particles, a dry front is usually observed after a first period of almost homogeneous desaturation (see for example [20]).

A liquid flow in such a nanoporous medium under the action of capillary effects would be extremely slow. The micron-sized cracks can only slightly help this flow. As soon as some water is removed from the free surface (over a thickness  ) evaporation then essentially occurs by diffusion from the first liquid-air interface at a distance  from the dry air flow, with a characteristic velocity

d V =      n D  ) 1 ( 1
, in which  is the liquid density, n the vapour density and D the coefficient of diffusion of vapour in air. A receding dry front of length  (increasing in time) should form so that d V balances e V , the vapour volume flux imposed by the external conditions (effective air flow along the sample free surface depending on sample shape and size), i.e. the drying velocity at the end of regime A. With our values of e V this would lead to a value for  of several millimetres, in contrast with our observations. This implies that for nano-colloids there is an effect which precludes the formation of a dry front. This is likely the disjoining pressure [21] which will tend to maintain a continuous film of liquid molecules at the particle surface even under evaporation. Under these drying conditions this effect finally precludes any further evaporation since the saturation tends to a finite value (see Fig. 4) which varies between 8 and 20% depending on exact experimental conditions.

The saturation profiles in time may be used to get precise information on the liquid dynamics in the regime B. The similarity of the profiles in time implies that we can write

) ( ) ( ) , ( x f t t x   
. Moreover the profiles obtained under different conditions are approximately similar when x is rescaled by h (see bottom inset of Fig. 4), which implies that for our tests f is a single function of h x . We can compute the average flow velocity of the liquid through the sample:

  ) ( ) ( ' ) 1 ( ) 1 ( ) , ( 0 h x hF t dx t t x V x            (1) with dy y f h x F h x   0 ) ( ) (
. The average velocity inside the liquid network along the sample axis writes

  ) ( ) ( ' ) 1 ( h x f h x F h V v        (2)
In particular at the top free surface of the sample we have  h

h v    ' ) (  where 1 . 0 5 . 1 ) 1 ( ) 1 (    f F  .
The variations of  in time thus governs the velocity variations. We find that  ln decreases linearly in time (see Fig. 4), which means that for a given test   ' is a constant. Thus the velocity profile ), i.e.

T t  

, we find a single curve:

    log
. Finally we deduce the general result:

e V h v 0.4) 4 . 3 ( ) (   (3)
which is valid as soon as an air path throughout the sample exists. Thus we have a liquid flow in a layer which thins in time but the average velocity in this layer is constant, independent of sample thickness and proportional to the external air flux. Under these conditions the evaporation is apparently governed by local phenomena at sample surface. This situation is completely different from that observed for usual porous media, with larger pores, for which the drying rate remains constant during most of the desaturation process as a result of fast capillary equilibration processes [12][13]22]. nano-particles there is an almost homogeneous desaturation process but drying rate is governed by the thickness of the liquid layer covering the particles, and not by the thickness of the dry region (which apparently does not exist). Thus, in this situation it seems that when the liquid film over the particles is of nanometric size it becomes able to ensure a relatively easy draining of the liquid towards the free surface, which avoids the formation of a dry front. This suggests that, in that case, both the mobility of the liquid is higher than usual (i.e. smaller apparent viscosity) and its ability to evaporate is lower, but further study is needed to understand the origin of these effects.

V. CONCLUSION

Our results show that during most of the time in Regime B liquid coexists with significant air volumes and flows towards the sample free surface in layers which progressively thin down. Thus the internal stresses do not significantly evolve during this period. The situation differs from Regime B when air for the first time progressively penetrates the sample (see Fig. 4 top inset). High local stresses, induced by disjoining pressure effects when air fingers enter the thin liquid films, can push close particles against each other, and tend to further concentrate the system which is already well packed. Only some slight rearrangements extending over large distances are possible, leading to then allowing slight further concentrations in separate regions. Thin fractures will thus occur in the sample during the short transient period at the beginning of Regime B. This conclusion is consistent with our observations of the approximate time of appearance of these fractures (see Fig. 3).

These different observations show how the air penetrates through a porous medium and its impact on fractures. The direct analysis of the water dynamics at the molecular scale through a compacted sample show that liquid flows at the nanoscale have unexpected specific properties. The experiment was performed at the magnetic center of our gradient coil (a BGA26 by Bruker, 26cm inner diameter), which guaranties minimal profile distortion due to possible gradient non linearity (at least well under our space resolution). Finally, prior to the experiment, our magnetic field was shimmed so as to get a Hz 50  f large spectral line for our sample. The blurring to be expected on our NMR profiles due to field inhomogeneities is then:

μm 30 2    z blur G f x   
The effective space resolution on our data was then estimated as:

Figure 1 :

 1 Figure 1: Aspects of drying gel layers ( % 20 0  

Figure 2 :

 2 Figure 2: Distributions of water content in time inside a non-adhesive (a) and adhesive (b) gel ( 0  =20%) during drying. The continuous curves from top to bottom correspond to successive times (every 30 min. for (a) and every 55 min. for (b)) from the test beginning. The symbols are the corresponding positions of the sample free surface as measured (by NMR) from the position of the water pot. The thick line corresponds to the transition between the two regimes (see text).

Figure 3 :

 3 Figure 3: Saturation as a function of particle concentration for different



  Since in regime B the solid structure is fixed, we can deduce the saturation profiles through the sample (

  precisely, for our different tests, for  as a function of the time rescaled by the characteristic drying time (

Figure 4 :).

 4 Figure 4: Average saturation as a function of dimensionless time (same symbols as in Fig.3, crosses correspond to % 20 0   , mm 10 0  h ,

Figure A. 1 :,

 1 Figure A.1: (Color online) NMR sequence used in our experiments.

1 T

 1 Our measurements were not subjected to any weighting, because the recycling delay between sequences was set to 5 times the spin-lattice relaxation time in our sample at the beginning of the experiment (and this time was not prone to increase during sample drying).In order to remove the spurious contribution of spin-spin relaxation, we proceeded as follows. Assuming a mono-exponential relaxation in each layer of our sample, with a local relaxation time) density profile, the profile obtained from first and second echoes were modelled respectively as:

Figure C. 1 :Figure C. 2 :

 12 Figure C.1: Water content profiles in time for two identical tests under conditions of Figure 2a. Data of Figure 2a correspond to continuous lines, the other data to dotted lines.

Acknowledgments: This paper benefited from useful discussions with G. Scherer, E. Dufresne and F. Boulogne. We are grateful to David Hautemayou for his technical support. We warmly thank Max Zieringer et Adrian Pegoraro for their useful advices.

VI.

The profiles presented in the article were then calculated following the profile extrapolation formula:

which is supposed to remove the space-dependent ) ( 2 z T contribution. Note that this formula still provides a fair quantitative correction even if 2 T exhibits slight fluctuations inside each sample slice, and may be shown to correct for imperfections (if any) of  pulses.

The correctness of the (quasi-) exponential assumption for signal decay in each single pixel was checked experimentally. Using a sequence with a larger number of echoes, the evolution of NMR signal intensity for echo times ranging between 8 and 120 ms was followed for a few pixels, in the case of a partially desaturated sample which seems to us the most critical case. It was seen that for any given pixel, recorded data can be accurately fitted by a decreasing exponential function, within the noise level. It was also seen that the relaxation rate strongly evolves from one pixel to another (depending on local water amount), thus justifying the use of a position-dependent relaxation time in our theory. At last, the smallest observed value of 2 T value at the end of a drying experiment was about 10ms, which is still in the proper range -regarding our echo times -for a proper use of our extrapolation formula.

Appendix B. Impact of sample curvature

The impact of sample curvature on the NMR profiles is illustrated in Figure B.1 in the case of a spherical shape of the sample, with a large radius of curvature. In that case a simple mathematical integration of the total water volume in each cross-section shows that the resulting gradient in water content observed at the top and bottom of the sample is a constant, giving a straight inclined line at the beginning and end of the profile. In reality the sample shape is not exactly spherical, which explains a slight difference with regards to a straight line.

The effective sample shape evolution can depend on the rate of drying, as shown for the test of Figure B.2 which corresponds to the same conditions as in Figure 2a except that the air flow velocity was much lower. In that case there is a first stage during which the sample significantly bends but in a second stage its curvature tends to cancel. Despite these differences the evolution of the water content profiles are similar to those observed for a larger air flow velocity: we observe the same two regimes and a transition at almost the same solid volume fraction. ) and the squared symbols correspond to the sample free surface similarly as in Figure 2a.

Appendix C. Reproducibility of MRI data

The uncertainty on data mentioned in the text is extracted from identical tests. We observe that the set of water content profiles for two identical experiments evolve very close to each other all along the drying process; the two same drying regimes and position of the transition superimpose properly (see Figure C.1). This good correlation with time and water content enables us to validate a good control of the external conditions. Even if we acknowledge a difference in the effective drying rate for the 2 samples (see inset of Figure C.2), it is very light and could be due to slight differences in the sample surface shape originating from the initial state or during drying therefore leading to slight variations of the characteristics of the air flow along the sample surface. As the deformation (curvature) of the sample can be slightly different (maximum difference: 2%), this may also lead to slightly different shapes of the saturation profiles around the edges too. However despite these variations the uncertainty on the saturation and solid fraction remains very low, i.e. 2.5% (maximum relative difference between corresponding values on each profile).