Sylvain Deville
email: sylvain.deville@saint-gobain.com

Éric Maire

Audrey Lasalle

Agnès Bogner

Catherine Gauthier

Jérôme Leloup

Christian Guizard

Eric Maire

In Situ X-Ray Radiography and Tomography Observations of the Solidification of Aqueous Alumina Particles Suspensions. Part II: Steady State

Keywords: directional solidification, X-ray tomography, synchrotron radiation, ceramics, crystal growth

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The behaviour of a propagating solidification interface in a suspension of particles is of major interest for its implications in a very large number of theoretical and practical issues. If the physical mechanisms controlling the interactions are relatively well understood for single large particles, the solidification behaviour of colloidal suspensions is a matter of great interest, but highly challenging. Conclusions derived from single large particles experiments [START_REF] Asthana | Engulfment of foreign particles by a freezing interface[END_REF][START_REF] Korber | Interaction of particles and a moving iceliquid interface[END_REF][START_REF] Lipp | Critical growth rates of advancing ice-water interfaces for particle encapsulation[END_REF][START_REF] Lipp | On the engulfment of spherical particles by a moving solid liquid interface[END_REF][START_REF] Omenyi | Attraction and repulsion of solid particles by solidification fronts. II. Dimensional analysis[END_REF][START_REF] Rempel | The interaction between a particle and an advancing solidification front[END_REF] can hardly be extrapolated to smaller particles (<1 microns) where Brownian motion is dominating and segregation effects are negligible. The analysis is further complicated by the necessity to take into account the various interactions between particles, which could be of different natures: electrostatic, Van der Waals, steric, etc… Additional deviations from the ideal situation, which could be modelled, such as the distribution of particle size, their surface state, charge and roughness, could have a major influence over the general behaviour and stability of the system but are difficult to take into account theoretically. We focus here on the behaviour of an interface propagating in a steady state (in terms of velocity), in a concentrated suspension of colloidal particles. A convenient approach to tackle the problem is to apply the formalism developed for the solidification of alloys, considering that particles can diffuse like atoms (although with much slower diffusion kinetics), with a dual-phase microstructure formation, one phase being the pure ice and the other one the concentrated particles entrapped in ice. If some common characteristics between these two systems are encountered, such as the relationship between the structural wavelength and the interface velocity [START_REF] Zhang | Aligned two-and threedimensional structures by directional freezing of polymers and nanoparticles[END_REF], it is nevertheless necessary to incorporate the specificities associated to particles in the analysis. Such an effort is under way, under the so-called "colloidal alloys" designation [START_REF] Peppin | Pressure and relative motion in colloidal suspensions[END_REF][START_REF] Peppin | Solidification of colloidal suspensions[END_REF][START_REF] Peppin | Experimental verification of morphological instability in freezing aqueous colloidal suspensions[END_REF][START_REF] Peppin | Morphological instability in freezing colloidal suspensions[END_REF]. Experimental results gathered so far concentrated on low interface velocities systems (<1 micron/s), which are hardly relevant to the conditions encountered in the materials processing route called freeze-casting. Conclusions were nonetheless derived for these systems, with a particular importance given to the diffusion of particles, which in turn might create conditions of constitutional supercooling, with consequences on crystals growth kinetics and particles redistribution.

It has previously been shown that during steady state, particles are repelled by the propagating interface and are concentrated between the growing crystals. Particles concentration increases up to the breakthrough concentration, where the interface moves into the interparticle space [START_REF] Shanti | Particle Redistribution During Dendritic Solidification of Particle Suspensions[END_REF]. This situation corresponds to the propagation of a solidification interface in a porous medium and has been greatly investigated, both theoretically and experimentally [START_REF] Jackson | Freezing of liquids in porous media with special reference to frost heave in soils[END_REF][START_REF] Boer | Phase Transitions in Gas-and Liquid-Saturated Porous Solids[END_REF].

A common conclusion to all the existing studies of such systems is the existence of a critical interface velocity (or correspondingly a critical particle size for a given velocity) above which particles are engulfed by the moving interface before the maximum particle concentration can be reached.

The steady state behaviour of a system is investigated here, where the particle size is relatively large (0.2-3.4 microns) in comparison to the typical colloidal systems (<0.1 microns), but smaller than previous studies (>1 microns) and the interface velocity is relatively large too (>20 microns/s). This corresponds to what is encountered during the processing of porous materials by the so-called freeze-casting process [START_REF] Fukasawa | Pore structure of porous ceramics synthesized from water-based slurry by freeze-dry process[END_REF][START_REF] Deville | Freeze-Casting of Porous Ceramics: A Review of Current Achievements and Issues[END_REF], or solvent-induced phase separation. Under these conditions, diffusion cannot take place, the interface velocity being too high, and the particles redistribution is obtained by the direct interaction with the solidifying interface, which considerably simplify the analysis. The growth kinetics were directly measured (although the in-plane growth anisotropy between the a and c axis was not accessible), and several parameters controlling the redistribution of particles were identified, in agreement to those identified in the first companion paper.

Experimental methods

All the experimental methods have been described in the first companion paper, "In situ Xray radiography and tomography observations of the solidification of alumina suspensions, Part I: Initial Stages". In the present part, additional powders were also used; their characteristics are given in table 1. The steady state regime observations were performed in the area 4 to 6 mm from the nucleation surface.

Results

Steady-state solidification

Images of the steady state solidification regime of the 1.3 microns particles suspension, obtained by radiography, are shown in Figure 1. The displacement of the interface with time can be clearly observed in the pictures. Using these sequences, it was possible to track the position of the interface with time, to obtain a value for the interface velocity; data are plotted in figure 2. The interface exhibits a smooth displacement, with a small but noticeable decrease of the interface velocity with time, which can be explained through the imposed temperature conditions (linear cooling ramp). The average interface velocity was around 39 microns/s in this area of the sample.

Two peaks in the displacement and velocity data can be noticed, respectively, called A and B in figure 2. Similar to what was observed in figure 5 of part I, these peaks indeed correspond to instabilities but will be discussed in a separate paper.

A full tomography acquisition of the same part of the sample was performed once solidification was completed, providing after reconstruction a three-dimensional picture of the ice and particles phases, as shown in figure 3 and4. The vertical cross-section reveals continuous crystals, running from the bottom to the top of the area. Under these solidification conditions, crystals are growing continuously. The corresponding horizontal cross-sections shown in figure 3 b to e reveal the arrangement of the ice crystals in the direction perpendicular to the solidification direction. Crystals are lamellar, as previously discussed, but arranged in domains with similar orientations (figure 3 f to i). The orientation of each domain can be related to the original nucleation conditions, as discussed in the previous paper. The arrangement of the domains is retained over large distances, as seen on the sequence of cross-sections. The displacement of the domains boundaries can be partially explained by the small tilt of the crystals (a few degrees, visible in figure 3a).

Quantitative information can be extracted from these images, so that the particle rich phase fraction (as discussed previously) can be plotted versus the position of the interface (figure 5), along with the corresponding interface velocity obtained through the radiography data. The interface velocity is slowly decreasing, while the particle fraction is constant, with variations within the measurement error range (estimated at 0.5%). Hence the particle fraction is not dependent on the interface velocity, under these conditions.

Using the horizontal cross-sections, the structural wavelength of the frozen structure can be measured; this parameter corresponds to the average crystals + entrapped particles length measured perpendicular to the c-axis of the crystals, as measured before [START_REF] Deville | Ice-templated porous alumina structures[END_REF]. The structural wavelength is varying with the interface velocity (figure 6), from 41 to 35 microns, as shown before in such systems [START_REF] Zhang | Aligned two-and threedimensional structures by directional freezing of polymers and nanoparticles[END_REF][START_REF] Shanti | Particle Redistribution During Dendritic Solidification of Particle Suspensions[END_REF], although we have here a direct and local measurement of the relationship, which is not the case when measured after sublimation and sintering, with the corresponding shrinkage accompanying densification.

High-resolution observations

High-resolution observations were performed on the same systems (0.4 microns particles) with the same temperature conditions, to provide direct observations of the crystals growth and particles displacement. Such sequence is shown in figure 7 and movie 1. The first observation is the confirmation of the lamellar morphology of the ice crystals, as reported previously. The liquid-solid transformation of water into ice is accompanied by a 4 vol.% increase. This increase leads to a displacement of the suspension placed above the ice crystals. This displacement can clearly be observed in movie 1, with a vertical translation of the suspension as the solid-liquid interface is progressing.

The tip of the ice crystals is particularly visible, and a zone of concentrated particles can be seen around the interface (dark area). A detail is shown in figure 8, where the tip of the ice crystals can be seen more clearly, just emerging from the concentrated particles zone. It is quite clear that under these conditions the particles are not concentrated ahead of the tip of the crystals, but inbetween the crystals.

Similar observations with larger particles (1.3 microns particles) were performed, to investigate the influence of particle size. The sequence is shown in figure 9 and movie 2. A similar behaviour is observed, with one noticeable difference: the presence of a concentrated particles zone ahead of the interface. The crystals tips are not emerging from this concentrated zone. In addition, the height of this zone is increasing as the interface is advancing. Segregation of the particles can also be observed in movie 2, with particles progressively falling towards the bottom of the suspension; which is not surprising considering the relatively larger size of the particles.

The position of the interface and its velocity can be measured from these observations; their values are plotted in figure 10, for the two types of particles (0.4 and 1.3 microns). As shown before (figure 4 and5), the interface velocity is slowly decreasing with time, from 24 to, respectively, 17 and 12 microns/s for small and large particles (faster decrease for larger particles).

Discussion

Crystals growth

Several hypotheses were made concerning the morphology of the ice crystals growing in colloidal suspensions of particles, mostly based on the anisotropy of growth kinetics. From observations of materials obtained through solidification of colloidal suspension, the observed lamellar porous morphology seems to indicate a lamellar shape for the ice crystals, in good agreement with the strong anisotropy of growth kinetics of the a-and c-axis of hexagonal ice [START_REF] Truby | Hexagonal microstructure of ice crystals grown from the melt[END_REF][START_REF] Nagashima | Nonequilibrium effect of anisotropic interface kinetics on the directional growth of ice crystals[END_REF]. The particles arrangement can nevertheless be expected to be affected by the high-temperature densification step (sintering). Such lamellar morphology can be directly observed here, in particular on the high resolution 2D observations in figure 7 and in 3D in figure 11, confirming the previous hypotheses. Additional growth behaviours usually associated with dendritic growth, such as tip splitting leading to twin crystals and protruding dendrites turning into primary crystal can be observed in figure 4a (arrows). Such instabilities seem to rarely yield stable dendrites here. By the time such protruding dendrites turn into stable dendrites growing along the z-axis, the surrounding zcrystals have already grown and induced concentration of particles ahead of the newly created dendrites. The remaining space available for their growth is therefore very limited; they rapidly come to a halt, either by stopping halfway in between the lamellar crystals (Figure 12 a), or by reaching the next adjacent crystals (Figure 12 b).

From the radiography sequences, where the evolution of the crystals and particles redistribution below the interface is visible, the growth kinetics along the a-and c-axis (Vaz and Vaxy) can also be estimated. The various interface velocities are schematically illustrated in Figure 13. Three different velocities exist in this situation, as a consequence of the crystal growth anisotropy and the thermal conditions of directional solidification: the Vcxy and Vaxy respectively correspond to the growth velocities along the c and x axis, in the xy plane, while Vaz corresponds to the growth velocity along the a axis perpendicular to the xy plane. Combined with the crystals dimensions measured on cross-sections obtained through tomography, the c-axis growth rate Vcxy can be estimated around 14 microns/s, for an a-axis growth rate Vaz of 18 microns/s in the solidification direction, with large particles (1.3 microns). The c-axis and a-axis growth rates Vcxy and Vaz were estimated, respectively, around 14 microns/s and 20 microns/s with small particles. No noticeable effect of the particle size can be observed here. The a-and c-axis growth kinetics are very similar, and more similar than expected. What we measure here is not the actual anisotropy of growth kinetics (defined as Vaxy/Vcxy), as the growth rate measured in the z-direction is dictated by the temperature gradient, which is not the case for the growth in the x-or y-direction (figure 13). To access the anisotropy of growth kinetics, one should measure the growth rate for the a-and c-axis within the xy-plane (Vaxy and Vcxy), where the temperature conditions are homogeneous, so that the measured growth rate are not dependent on the temperature gradient. Such measurement cannot be performed with these types of experiments, until full tomography acquisitions can be performed within a much shorter time to obtain in situ three-dimensional evolution of the crystals growth.

Particles redistribution

From the various observations shown here, it appears that redistribution of particles in front of the advancing interface is dependent on several variables. Under certain conditions, no particles are found ahead of the interface, while under other conditions, a layer of concentrated particles is clearly visible ahead of the interface. Several parameters have been identified here, and are discussed below.

• Particles redistribution depends on the interface velocity. This dependency seems rather obvious, and can be understood when interpreting the system in terms of colloidal particles and diffusion. However, when the interface is moving fast enough, particles cannot migrate by Brownian diffusion, so that the problem cannot be treated in terms of diffusion. Particles are directly repelled by the moving solidification front. For moderate to slow velocities (1 microns/s), enough time is available for the particles to migrate by Brownian diffusion, so that a layer of concentrated particles can build up ahead of the interface. This situation has already been deeply investigated [START_REF] Peppin | Solidification of colloidal suspensions[END_REF][START_REF] Peppin | Experimental verification of morphological instability in freezing aqueous colloidal suspensions[END_REF]. In such case, the concentration of particles ahead of the interface can potentially lead to a situation of constitutional supercooling, where ice crystals will be able to nucleate within the concentrated zone, leading to a different morphology of particles arrangement in the solidified body. This was observed here for very slow growth rate, when the crystals' growth was just about to stop (figure 14). In situ tomography acquisitions, only possible for very slow growth rates, reveal the nucleation of crystals within the concentrated particles zone. This situation could be detrimental when used for processing of materials, leading to large size defects and discontinuities in the final structure. Yet, such conditions are never used, for practical reasons. The structural wavelength of the structures obtained with such slow interface velocities are not of interest so far. Under the temperature conditions typically used, it is quite clear that diffusion is never taking place, and thus the diffusion approach cannot be used to determine the concentration profile and particles redistribution behaviour in this case.

• Particles redistribution depends on particle size, or, more precisely, on the ratio of particle size and intercrystals spacing. Although the relationship between particle size and diffusion is quite clear (see for example Fig. 3 in reference [START_REF] Peppin | Morphological instability in freezing colloidal suspensions[END_REF]), additional effects are associated to the particle size, under conditions where no diffusion is possible. As shown in figure 7 and9, particles redistribution is more difficult with larger particles. A zone of concentrated particles builds up when larger particles are used, a situation that cannot be explained through diffusion since this mechanism is less likely to occur as particle size is increasing. It is proposed here that this concentration ahead of the interface is related to the difficulty of obtaining the maximum packing density of particles between the crystals when the particle size falls within the same range of order as the crystals separation distance, as mentioned and schematically illustrated in figure 8 in the companion paper. The presence of a concentrated particles zone can affect the growth of the ice crystals, their growth kinetics was found to slow down faster when concentrated particles were present (figure 10). Again, this could be detrimental for the final materials, as this interface deceleration is associated with an increase of the structural wavelength. The resulting materials will be less homogeneous than those obtained with smaller particles.

• Particles redistribution depends on the shape and the orientation relationship between the crystals and the temperature gradient direction. This has been shown in the companion paper (part I: Initial instants), and is confirmed here. Under steady state conditions, the crystals are growing along the temperature gradient direction; the measured particle rich phase fraction in this direction is constant.

In addition, local instabilities can be observed, leading to disruption of the crystals continuity along the temperature gradient direction. Disruptions are shown in figure 15a. They are detrimental for the materials processed through this technique, as they are not healed with the densification stage, so that they turn into crack-like defects in the sintered pieces (figure 15b). Mechanical properties of these highly cracked structures are dramatically lower than their defect-free counterparts. The underlying reason for this behaviour is not clear at this time. It is dependent on the formulation, as a proper one can provide defect-free sintered bodies with excellent mechanical properties [START_REF] Deville | Freezing as a path to build complex composites[END_REF].

Analogies and disparities with alloys solidification and solidification in porous media

Previous experimental and theoretical investigations regarding the solidification of suspensions have revealed two extreme cases, depending on the particle size: the problem can either be treated using Fick's law, which is similar to what is done in the case of the solidification of alloys, or using Darcy's law, considering the propagation of a solidifying interface in a porous media, when particles of larger size are used. For alloys, transport properties exhibit a linear concentration dependence, a characteristic that greatly simplify the analysis. Further improvements were performed when the interactions between particles were taken into account, to characterize the behaviour of the system from a colloidal suspension point of view. In this situation, particles redistribution can still occur by diffusion, but the system is characterized by a strongly nonlinear concentration dependence of the transport properties, and in particular a nonlinear freezing temperature curve [START_REF] Peppin | Experimental verification of morphological instability in freezing aqueous colloidal suspensions[END_REF]. The interface velocities investigated were nevertheless much lower (0.1-0.8 microns/s) that the ones observed here (20-40 microns/s). The other extreme situation is the propagation of a solidifying interface in a porous media. In such case, no particles redistribution can take place and obviously no diffusion can occur. This extreme is too different from the current situation when particles redistribution by the moving interface begins, considering the range of particle size and concentration used here, with the major consequence that particles redistribution induced by the moving interface plays a major role in the structural arrangement of the frozen body.

However, when the lateral growth of crystals is large enough, particles concentration increases up to the maximum possible concentration, corresponding to a situation where the movement of individual particles is hardly possible, typical particles concentration at this point being close to the maximum packing. In this situation, the particles are engulfed by the propagation of the interface in the interparticle space, without movements of particles; Darcy's law is in this case relevant to describe the behaviour of the system.

The mechanisms involved here (before particles concentration get close to its maximum) are similar to the solidification of alloys, although several major differences are observed. The particle size are in a range (0.2-3.4 microns) where diffusion could occur, but the interface velocities applied here are too fast for diffusion to occur. No particles redistribution can occur by diffusion, as observed directly on high resolution radiography results. Therefore, the influence of concentration dependent properties have less influence than for colloidal alloys investigated both theoretically and experimentally. The first consequence is that no situation of constitutional supercooling can occur.

The second consequence is that the redistribution of particles in directions parallel and perpendicular to the interface displacement direction is not dictated by diffusion, but rather by the orientation of the growing crystals and the efficiency of particles packing when the particles concentration is increasing between the moving interfaces. As a consequence, one should refer to this situation as a metastable steady state, dictated by the applied temperature conditions (cooling rate). Given more time, particles would diffuse away from the interface and reach an equilibrium situation, as observed experimentally [START_REF] Peppin | Experimental verification of morphological instability in freezing aqueous colloidal suspensions[END_REF][START_REF] Mashl | Dynamics of solidification in 2% corn starch-water mixtures: Effect of variations in freezing rate on product homogeneity[END_REF][START_REF] Muldrew | Cryobiology of articular cartilage: Ice morphology and recovery of chondrocytes[END_REF]. Providing that the crystals orientation is constant and the intercrystals distance large enough (compare to the particle size), particles redistribution occurs homogeneously in the xy-plane (with a constant packing efficiency) and does not occur in the z-direction. The fraction of entrapped particles is therefore constant, resulting in homogeneous materials after removal of the solvent.

Conclusions

Based on in situ experiments by X-ray radiography and tomography of the controlled solidification of concentrated alumina particles suspensions, the following conclusions can be drawn, regarding the steady state regime (in terms of velocity):

-Due to the relatively rapid interface velocities (20-40 microns/s), particles redistribution cannot occur by diffusion. Situation of constitutional supercooling cannot be involved here.

-Particles redistribution is obtained through direct interactions with the solidification front, particles being pushed by the moving interface. The critical parameters to be considered here are therefore the orientation of the growing crystals and the efficiency of particles packing when the particles concentration increases between the moving interfaces, efficiency directly related to the particle size and intercrystals spacing.

Figure 1 :

 1 Figure 1: Solidification sequence in the steady state, radiography, 1.3 microns particles. Time picture was taken at: a=t0, b= t0+6s, c= t0+13.7s, d= t0+22.3s, e= t0+27.3s, f= t0+34.7s. Scale bar: 500 microns.

Figure 2 :

 2 Figure 2: Interface location () and velocity (▲, dashed line: linear fit) vs. time, steady state, 1.3 microns particles. The average velocity of the interface is 39 microns/s. The z=0 position corresponds to the position of the interface in Fig.1a (time t0). The velocities peaks noted A and B are significant, but will be discussed in a separate paper.

Figure 3 :

 3 Figure 3: Reconstruction from tomography acquisition, 1.3 microns particles. Vertical (a) and horizontal (b-f) cross-sections of the frozen arrangement. The corresponding orientation domains, corresponding to domains of long range order of the crystals alignment, are given in (f)-(g). Scale bars : a : 300 microns, b-e : 150 microns.

Figure 4 :

 4 Figure 4: Three-dimensional representation of a block of lamellar ice crystals (left) and corresponding particles entrapped (right). Block dimension: 360x360x360 microns 3 , 1.3 microns particles. White arrows indicate the protruding dendrites, which can eventually turns into lamellar crystals, black arrows indicate twin dendrites, probably resulting from tip splitting.

Figure 5 :

 5 Figure 5: Interface velocity (fit, dashed line) and particle fraction (continuous line) vs. position of the interface, steady state, 1.3 microns particles. The z=0 position corresponds to the bottom of the picture in Fig. 1a. No significant variations of the particles fraction are observed.

Fig. 6 :

 6 Fig. 6: Structural wavelength () and interface velocity (fit)(▲) vs. position, as measured on reconstructed cross-sections. 1.3 microns particles.

Figure 7 :

 7 Figure 7: Solidification sequence in the steady state, high resolution radiography, 0.4 microns particles. Time picture was taken: a=t0, b= t0+4s, c= t0+9.7s, d= t0+16s, e= t0+24.7s. Scale bar: 150 microns.

Figure 8 :

 8 Figure 8: Detail of the solidification sequence in the steady state (Figure 7), high resolution radiography, 0.4 microns particles. Details of the dendrites tip. No visible concentration of particles ahead of the dendrites tip is observed. Interface velocity: 20 microns/s. Scale bar: 50 microns. Arrows indicate the dendrites tip. The solidification front is moving vertically, bottom to top.

Figure 9 :

 9 Figure 9: Solidification sequence in the steady state, high resolution, 3.4 microns particles. Time picture was taken: a=t0, b= t0+4s, c= t0+9.7s, d= t0+16s, e= t0+24.7s. Scale bar: 150 microns.

Figure 10 :

 10 Figure 10: interface position and velocity from high resolution observations, for 0.4 microns (▲) and 3.4 microns () particles.

Figure 11 :

 11 Figure 11: Three-dimensional representation of an isolated lamellar crystal, 1.3 microns particles. Perspective (a), side (b) and top view (c). Crystal height represented: 360 microns. The surface of the crystal is not faceted.

Figure 12 :

 12 Figure 12: Growth termination of dendrites, three-dimensional representation. Black arrows indicate the origin of the dendrites and white arrows their termination. The dendrites can either stop in between the crystals (a), or reach the next adjacent crystal (b).

Figure 13 :

 13 Figure13: Growth velocities and crystal orientation. Ice, under these conditions of temperature and pressure, is hexagonal, with strong anisotropies of interface kinetics between the a and c axis, resulting in platelets-like crystals.

Figure 14 :

 14 Figure 14: Solidification sequence, in situ tomography, 0.4 microns particles. Evidences of constitutional supercooling. The solidification front is moving vertically, bottom to top. Scale bar: 500 microns. Legend: 1: unfrozen suspension, 2: concentrated particles ahead of the interface, 3: ice crystals, 4: concentrated particles surrounded by ice 5: polymer mold, 6: outside background, 7: bubble. Approximate time: a: t0, b: t0+30s, c: t0+60s.

Figure 15 :

 15 Figure 15: Evidence of local instabilities in the frozen structure (a), leading to large crack-like defects in the final structure (b). Scale bars: 200 microns. a: tomography picture, b: SEM picture after sublimation and sintering.

Table 1 :

 1 Characteristics of powders used in this study

	Manufacturer Product name D50 (microns) SSA (m²/g)
	Taimei	TM DAR	0.2	14.5
	Ceralox	SPA 0.5	0.4	8
	Almatis	CT1200SG	1.3	3.7
	Almatis	CT800SG	3.4	1

Acknowledgements

We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation beam time and we would like to thank Elodie Boller and Jean-Paul Valade for their irreplaceable assistance in using beamline ID19. Financial support was provided by the National Research Agency (ANR), project NACRE in the non-thematic BLANC programme.


Etienne Munch

Eduardo Saiz

Antoni P Tomsia

Sylvain Deville
email: sylvain.deville@saint-gobain.com

Architectural control of freeze-cast ceramics through additives and templating

Keywords: porous ceramics, freeze-casting, ice, hierarchical materials, additives, templating

The freezing of concentrated colloidal suspensions is a complex physical process involving a large number of parameters. These parameters provide unique tools to manipulate the architecture of freeze-cast materials at multiple length scales in a single processing step. However, we are still far from developing predictive models to describe the growth of ice crystals in concentrated particle slurries. In order to exert reliable control over the microstructural formation of freeze-cast materials, it is necessary to reach a deeper understanding of the basic relationships between the experimental conditions and the microstructure of the growing solid. In this work, we explore the role of several processing variables (e.g., composition of the suspension, freezing rate, and patterning of the freezing surface) that could affect the formulation strategies for the architectural manipulation of freeze-cast materials. We also demonstrate, using freeze-cast lamellar structures, how reducing the lamellar thickness by less than half increases compressive strength by more than one order of magnitude.

I. Introduction

Emerging technologies in energy storage, catalysis or tissue engineering demand new materials with highly specialized and often seemingly incompatible functional properties such as high surface area, low density, and highly specific strength or toughness. Today, a number of researchers are working on the development of new processing routes capable of delivering materials to fulfill these complex requirements. Organized hierarchical structures, often inspired by natural structures and biological materials [START_REF] Sanchez | Biomimetism and bioinspiration as tools for the design of innovative materials and systems[END_REF] , are rapidly becoming attractive solutions to these challenges. Nonetheless, the complex architecture of natural materials continues to be extremely difficult to reproduce in a controlled manner. As a consequence the vast majority of current processing techniques yield materials with functional properties that are disappointing when compared to those of natural biomaterials.

Freeze-casting is a versatile processing technique that solidifies a colloidal ceramic suspension under conditions in which the freezing dispersant interacts with suspended ceramic particles, pushing them away from the advancing solid front. [START_REF] Deville | Freeze-casting of porous ceramics: A review of current achievements and issues[END_REF][START_REF] Zhang | Aligned twoand three-dimensional structures by directional freezing of polymers and nanoparticles[END_REF][START_REF] Gutierrez | A biocompatible bottom-up route for the preparation of hierarchical biohybrid materials[END_REF][START_REF] Araki | Porous ceramic bodies with interconnected pore channels by a novel freeze casting technique[END_REF] After the frozen liquid has been removed by sublimation, a replica of the solidified solvent is formed. Our group has recently demonstrated how freeze-casting can be used to fabricate porous lamellar ceramics with complex structures which resemble the inorganic component of nacre. [START_REF] Deville | Freezing as a path to build complex composites[END_REF] The freezing of concentrated colloidal suspensions is a complex physical process involving a large number of parameters [START_REF] Peppin | Solidification of colloidal suspensions[END_REF][START_REF] Shanti | Particle redistribution during dendritic solidification of particle suspensions[END_REF][START_REF] Deville | Ice-templated porous alumina structures[END_REF][START_REF] Peppin | Experimental verification of morphological instability in freezing aqueous colloidal suspensions[END_REF] .

These parameters provide unique tools to manipulate the architecture of freeze-cast materials at multiple length scales from the nano to the macro levels in a single processing step. However, in order to exert reliable microstructural control, it is necessary to obtain a deeper understanding of the basic relationships between experimental conditions (e.g., freezing rates, composition of the dispersion) and the microstructure of the growing solid. In this work, we explore the role of several processing variables (e.g., composition of the suspension, freezing rate, and patterning of the freezing surface) that could be used in the formulation of effective strategies for multiscale manipulation of freeze-cast materials.

While we are not attempting to fully quantify the precise influence of each given parameter, we have illustrated how they can provide a new toolbox for fabricating novel freeze-cast structures.

As an example, we will show how reducing the thickness of freeze-cast lamellae by less than half results in increased compressive strength by more than one order of magnitude. As demonstrated by our experiments, our technique not only promises to guide the design and fabrication of new hierarchical and porous materials, but it can also benefit any scientific field of research, including physics, chemistry, biology, or geology, where the solidification of concentrated colloidal suspensions plays a critical role.

II. Experimental procedure

Slurries were prepared by mixing distilled water with a small amount of ammonium polymethacrylate anionic dispersant (1 wt% of powder) (Darvan C, R. T. Vanderbilt Co., Norwalk, CT), an organic binder (polyvinyl-alcohol), alumina powder (Ceralox SPA05, Ceralox Div., Condea Vista Co., Tucson, USA), and different types of additives in various proportions (see the results and discussion section for the precise amounts). The following additives have been used: sucrose, minimum 99.5% (Sigma, St. Louis, MO, USA); trehalose, D(+)-trehalose dihydrate from corn starch (Sigma, St. Louis, MO, USA); glycerol (Mallinckrodt, Inc., St Louis, MO, USA); ethanol, ethanol reagent denatured HPLC grade (Sigma, St. Louis, MO, USA); sodium chloride, Morton iodized salt (Morton, Philadelphia, PA, USA); gelatine, Gelatine Knox Original, unflavored (Knox, Kraft Food, USA); citric acid, monohydrate 99.5+% (Alfa Aesar, Ward Hill, MA, USA).

Slurries were ball milled for 20 hours with alumina balls and de-aired by stirring in a vacuum desiccator until air bubbles were completely removed (typically 30 minutes). The powder used in the study has a specific area of 8.1 m 2 /g and an average grain size of 400 nm (data provided by manufacturer).

Slurries were frozen by pouring them into a Teflon ® mold (18 mm diameter, 30 mm length) placed at the top of a cold finger (copper rod), and cooled by liquid nitrogen (Fig. 1). Freezing kinetics were controlled by a heater placed on the metallic rods, and a thermocouple placed on the side of the mold. An additional vibration was applied to homogenize the cooling suspension.

Samples were freeze-dried (Freeze Dryer 8, Labconco, Kansas City, MI) for 24 hours. The green bodies thus produced were sintered in air for 2 hours at 1500°C, with heating and cooling rates of 5°C/minute (1216BL, CM Furnaces Inc., Bloomfield, NJ). The microstructure of the samples was analyzed by optical and environmental scanning electron microscopy (ESEM, S-4300SE/N, Hitachi, Pleasanton, CA). The wavelength was measured in parts of the sample where a constant thickness of the ceramic lamellae was established.

Compression tests have been performed on cubes (444 mm 3 ) on an MTS 810 material test system, at a constant displacement rate of 10 m/s -1 . Five specimens were used for each experimental condition.

The samples processed in this study have dimensions typically in the centimeter range (Fig. 2).

The planar dimensions are only limited by the experimental setup, and no scaling up issues should be expected. The achievable thickness is limited by the ability to maintain a homogeneous structure along the thickness. An improved experimental setup [START_REF] Waschkies | Control of Lamellae Spacing During Freeze Casting of Ceramics Using Double-Side Cooling as a Novel Processing Route[END_REF] allowed 40 mm thick freeze-cast samples to be processed, exhibiting a homogeneous structure along the thickness.

III. Results and discussion

III.1 Control of lamellae orientation

Among the common difficulties encountered in the pursuit of hierarchical materials is the control of the number of dimensions in which the structures are ordered. Typical freeze-cast materials exhibit only a one-dimensional ordering of the structure: the porosity is continuous along the propagation direction of the solid-liquid interface, but the pores are randomly oriented in a plane perpendicular to the ice-front propagation. A second dimension can be added for polymeric materials by performing several freezing steps [START_REF] Zhang | Aligned twoand three-dimensional structures by directional freezing of polymers and nanoparticles[END_REF] , although this approach cannot be used with particle suspensions. Obtaining such an improved control in a single-step requires a different strategy. The initial instance of directional freezing is usually characterized by the formation of a planar ice front, which nucleates and grows on the cold finger [START_REF] Deville | Ice-templated porous alumina structures[END_REF] . This front moves very fast, trapping the ceramic particles. Subsequently, a planar-to-cellular-to-lamellar transition sequence occurs, and a steady state is achieved in which lamellar ice grows while expelling the ceramic particles that accumulated in the layers between ice crystals [START_REF] Deville | Ice-templated porous alumina structures[END_REF] . When a well-polished cold surface is used, the planar-tolamellar transition results in the formation of randomly oriented lamellar domains of sizes on the order of 200-500 m (Fig. 3a). This is very similar to what happens when fast nucleation is induced by pouring the suspension of a surface at temperatures far below the freezing point of the suspension. Using simple, unidirectional patterns on the cooling surface, it is possible to manipulate the direction of the ice lamellae arising from the planar to lamellar transition and to obtain longrange ordering with well-oriented structures on the length scale of the whole sample (Fig. 3b), suggesting the ordering is controlled by nucleation.

The lamellae orient perpendicularly to the linear patterns, introducing a second dimension to the ordering of the structure. The crystals' perpendicular orientation to the pattern may be explained by a preferred nucleation at sharp-angles of the pattern. Further investigations will be necessary to clarify these aspects.

Patterns require the right dimension to achieve lamellae alignment. The thickness of the planar ice layer, the particle size, and the speed of the ice front determine the wavelength of the lamellar structure. Pattern sizes vary between 40 to 100 µm for 10 to 50 µm lamellae, respectively.

More complex lamellae orientations that can be dictated by functional requirements are difficult to control by simple cold-finger patterning. We have developed a simple epitaxy technique that places a mold filled with a pure solvent (water, in our case) on a cold finger. A second mold filled with the freeze-casting suspension is subsequently placed on top. When the cold-finger temperature reaches the solidification point, pure ice crystals grow in the patterned mold and extend onto the ceramic suspension, resulting in a macroscopic ice pattern directed by the mold surface features. This way, it was possible to replicate (Fig. 3c) the circular macrostructure of natural lamellar materials such as the osteons in bone [START_REF] Black | Harvesiaon osteons -size, distribution, internal structure, and orientation[END_REF] or the Euplectella sponge spicules [START_REF] Aizenberg | Skeleton of Euplectella sp.: Structural Hierarchy from the Nanoscale to the Macroscale[END_REF] , adding another level of structural control at macroscopic dimensions that can promote additional mechanisms to manipulate the mechanical response.

III.2 Using additives to control the structure

Controlling the structure at the micron and submicron levels requires the manipulation of the thickness and roughness of the growing ice crystals. Again, comparison with natural materials points out the importance of several microstructural features: the thickness and separations between the ceramic layers (the structural wavelength), the roughness of the ceramic walls at the microscopic and submicroscopic levels, and the presence of inorganic ceramic bridges between lamellae [START_REF] Meyers | Biological materials: Structure and mechanical properties[END_REF][START_REF] Barthelat | An experimental investigation of deformation and fracture of nacre-mother of pearl[END_REF] . These features play a crucial role in the mechanical response of natural layered materials such as nacre. A processing technique able to provide structural control from the macroto the nano-levels will make it possible to fabricate truly biomimetic structures.

It has already been shown how the solid content of the solution and the ice-front speed can be manipulated to control the wavelength of the lamellar structures resulting from freeze-casting [START_REF] Deville | Ice-templated porous alumina structures[END_REF] . The strategy explored in this work is the use of chemical additives that will affect the growth kinetics and microstructure of ice as well as the topology of the ice-water interface. Additives influence several physical parameters controlling the microstructure of the growing ice crystals; for example, additives can change the phase diagram of the solvent, the value and anisotropy of the solid/liquid and particle/liquid interfacial energies, the degree of under-cooling ahead of the liquid front, the viscosity of the solvent, and the forces between ceramic particles in the suspension. Some of these effects are interrelated, but as a result, the morphology of the ice crystals and their roughness can be manipulated down to submicron levels.

For our work, we selected additives commonly used in fields as diverse as cryopreservation, ice cream processing, and marine glaciology. Some additives are well-known antifreezers, including sodium chloride, carbohydrates such as sucrose and trehalose, glycerol, and ethanol.

Others such as gelatin were used to affect the viscosity of the suspension. Finally, we reduced pH by adding citric acid to manipulate inter-particle forces [START_REF] Lewis | Colloidal processing of ceramics[END_REF] .

Several of the additives used in this work (e.g., NaCl) have a eutectic phase diagram with water.

As a consequence, the solid and liquid regions are separated by a zone in which solid and liquid can coexist (Fig. 4) during ice growth in a temperature gradient. The height of this zone depends on the temperature gradient and on the particular phase diagram (the slope of the liquidus line). As the freezing front advances, a second phase will precipitate. Like the ice, this is a fugitive phase that will disappear during drying or sintering, but its morphology will have a strong influence on the microstructure of the porous scaffold. Through their influence on the interfacial energies and the solidification path, additives will dictate whether the materials' final architecture can be switched (Fig. 5a-d) from lamellar (e.g. no additive, trehalose, or sucrose) to cellular (e.g. gelatin, glycerol, or combining sucrose with a reduction of the pH using citric acid), or to a lamellar structure with a bimodal pore width distribution (ethanol), where pores with a very large cross section (a few hundred microns) are observed in addition to the basic lamellar structure previously shown. Such a structure exhibits interesting similarities with natural structures such as wood [START_REF] Gibson | The Mechanical-Properties of Natural Materials .2. Microstructures for Mechanical Efficiency[END_REF][START_REF] Wegst | The mechanical efficiency of natural materials[END_REF] , which provides an additional degree of hierarchy to the structure, and could prove to be useful in applications (catalysis, filtration) where a compromise should be found between specific surface area and the accessibility of fluids through the pore channels.

Besides the overall architecture, additives can also be used to modify the roughness of the ceramic walls (Fig. 5e-h). For example, the water-NaCl diagram (Fig. 4) has a eutectic point relatively close to the melting point of ice (-21 o C). During cooling, crystalline NaCl•2H2O will precipitate. This second phase will also expel the ceramic particles during solidification and subsequently disappear during firing at high temperature, resulting in a sharp, faceted microstructure (Fig. 5-g). On the other hand, sucrose and trehalose water diagrams [START_REF] Roos | Amorphous state and delayed ice formation in sucrose solutions[END_REF][START_REF] Chen | Literature Review: Supplemented Phase Diagram of the Trehalose-Water Binary Mixture[END_REF] are very similar, and for the structures used in this work, result in a second glassy phase when the temperature reaches -40 to -50ºC. Correspondingly, samples prepared with these two additives exhibited rounded, smooth features. In addition, the additives modified the interfacial energies and the degree of supercooling ahead of the freezing front, affecting the overall morphology of the growing ice crystals. Sucrose and trehalose promote the evenly distributed growth of homogeneous and regular dendrites over the surface of the lamellae (Fig. 5-h). The relative dimensions (width and height) of the dendrites can be modified by increasing the concentration of an additive in the suspension; for example, the dendrites become thinner at higher concentrations. In contrast, ethanol leads to very different topography, with pocket-like dendrites covering the surface of the lamellae (Fig. 5-f). The variations in surface roughness are also reflected in the specific surface area of the material. Additions of sucrose, ethanol, and trehalose lead to different values of specific surface areas according to their different roughnesses (sucrose 0.47 m 2 .g -1 , ethanol 0.36 m 2 .g -1 , and trehalose 0.41 m 2 .g -1 ).

It must be pointed out that, despite the obvious microstructural differences, the relationship between the structural wavelength and the average speed of the ice front seems almost independent of the additive used (Fig. 6). The only exception is ethanol; the underlying reasons for this behavior

are not yet understood, but it could possibly be related to the coexistence of a liquid and a solid (crystal) phase at relatively low temperatures (e.g., -72°C) and the complexity of crystal growth from the ethanol-water solution.

III.3 Using additives to control lamellar bridges

Another critical microstructural feature is the presence of ceramic bridges between adjacent lamellae. The bridges play a crucial role in the mechanical response, preventing Euler buckling during compression, and hindering crack propagation in the direction parallel to the lamellae. The velocity of the ice front during the freeze-casting process is below the critical velocity for particle trapping [START_REF] Deville | Ice-templated porous alumina structures[END_REF] . During the freezing of highly concentrated suspensions, the splitting and healing ice crystal tips can result in the encapsulation of ceramic particles and the formation of bridges between lamellae. Each additive effects interfacial tension and interparticle forces leading to a particular bridge structure. For example, trehalose promotes a high density (<> = 27.3 cm -1 ) of thin bridges (<w>=1.6 m), whereas sucrose leads to a low density (<> = 16.1 cm -1 ) of wider bridges (<w>=2.2 m).

III.4 Structure/properties relationships

By controlling the solid content, the cooling rate and the use of diverse additives, freeze-casting of water-based suspensions allows the design of inorganic scaffolds with a wide range of structures ranging from lamellar to cellular, and from low-to high-density bridging between lamellae, and having various levels of roughness at a local scale (Fig. 5). To illustrate the importance of the materials' structure, the comparison between the compressive strengths of a highly uniform lamellar alumina prepared by adding 4 wt % sucrose in water and a cellular structure formed using 10 wt % sucrose in water under acid conditions (pH=2.5) is shown in Fig. 7. Both structures have the same relative porosity and average size of the ceramic wall. However, the cellular structure exhibits a bimodal distribution of wall thickness (Fig. 7a) while the lamellar structure has a very narrow distribution (Fig. 7b). The microscopic roughness of the lamellae is not expected to influence the mechanical behavior of the highly porous materials. As expected, the cellular structure has a relatively more isotropic response, and for the same average lamellae thickness is stronger (Fig. 7c) . However, the thickness of the lamellae has a tremendous impact on the strength of the lamellar material, and decreasing the thickness by less than half increases the strength by almost an order of magnitude (Fig. 7d). This increase may not be fully surprising if we consider the refinement of the structure, the increasing presence of bridges for refined lamellae and the smaller defect size in thinner layers. A systematic assessment of the failure modes in these complex lamellar structures can guide the design of optimized microstrucures.

III.5 Multiscale control of the structure

Processing techniques for cellular ceramics usually have little control over a material's characteristics, yielding very specific porous microstructures of controlled pore size and shape.

Hence, a processing route must be chosen based on the desired structure. Freeze-casting is a very versatile technique, streamlined control of several important structural features, in a wide range of length scales, each of which can be tailored with some degree of freedom, by different mechanisms. The different features of a freeze-cast ceramic structure and their corresponding controls are summarized in Table 1. Some of these controls, such as the influence of the solidification interface velocity on the lamellae's thickness, or the use of solvents other than water, have been demonstrated in previous papers. Additional strategies to control the structure have been developed with the freeze-casting of polymers, such as consecutive solidification stages using different materials to process biaxially aligned structures [START_REF] Zhang | Aligned twoand three-dimensional structures by directional freezing of polymers and nanoparticles[END_REF] ; however, it is not always possible to transfer these methods to ceramic freeze-casting.

We have introduced two new powerful strategies: templating the cooling surface and the use of common additives. The templating is completely independent of the other strategies to control the structure of the materials. The use of additives offers a number of advantages over modifying a solvent; for example, additives broaden the number of possible pore-shape morphologies, and the same equipment can be used to process different materials. However, much more work is needed to assess the precise influence of additives, and to determine the degree of a control's interdependence or independence.

IV. Conclusions

Processing techniques for porous materials in general and cellular ceramics in particular yield very specific porous microstructures with controlled pore size and shape. However, the manipulation of pore orientation, morphology, and surface topography is typically very limited.

Freeze-casting appears to be a very versatile technique, allowing the use of different mechanisms to control several important structural features, in a wide range of scales, all of which can be tailored independently. The various physicochemical processes controlling the solidification of the colloidal suspensions provide a set of tools to control various features of the structure in a single processing step. This versatile, streamlined process allows the development of truly biomimetic, hierarchical structures. The right combination of experimental freezing conditions and different suspension formulations makes it possible to adjust the orientation of lamellae, and also provides a new means to manipulate the order, the size and shape of pores, the wavelength of the structure, the roughness of lamellae surface, and the density and thickness of bridges between lamellae. This also makes it possible to refine the porous structure, and to consequently manipulate a material's mechanical response.

Figures

Figure 1 :

 1 Figure 1: Experimental setup

Figure 2 :Figure 3 :

 23 Figure 2: Typical sample sizes and geometries

Figure 5 :

 5 Figure 5: Effect of additives on a material's architecture. The graph shows how different additives

Figure 6 :

 6 Figure 6: Wavelength vs. time to freeze for 2 cm tall samples. Error bars represent the standard

Figure 7 :

 7 Figure 7: Link between structure and mechanical properties. (a-b) Normalized distribution of

Table 1 :

 1 Structural features of freeze-cast materials and their controls

Acknowledgments

This work was supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences and Engineering Division, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231


P Bustamante

F Caurant

S W Fowler

P Miramand

Cephalopods as a key of the transfer of cadmium to top marine predators

Keywords: Cephalopods. Sub-Arctic. Heavy metals. Cadmium. Transfer. Top predators. Seabirds. Marine mammals. Dose Cadmium, like other heavy metals, is well

Cephalopods of 11 species differing in their geographic distribution (French , Irish and faroese waters) and feeding habitats, were analysed for their cadmium contents.

When comparing the cadmium levels of these cephalopods, it show that the squid Todarodes sagittatus and the octopus Eledone cirrhosa coming from the Faroe Islands contain respectively 17 and 18 times more than the animals from the French Atlantic coast. High cadmium levels in cephalopods from sub-polar areas were related with high cadmium concentrations in the tissues of top predators in these areas. Weekly doses of cadmium were estimated for the Faroese pilot whales and the Adélie Penguins and were compared to the "Provisional Tolerable Weekly Intake" defined by the World Health Organisation for humans. Our results show that cephalopods constitute an important source of cadmium for cephalopods predators.

concentrations of these metals in cephalopods, one of the essential links in marine trophic chains (Amaratunga 1983[START_REF] Rodhouse | Antarctic cephalopods -a living marine resource ?[END_REF][START_REF] Rodhouse | Cephalopods occupy the ecological niche of epipelagic fish in the Antarctic Polar Front Zone[END_REF], have rarely been documented. The studies have mostly concerned essential elements [START_REF] Ghiretti-Magaldi | Pathways of terminal respiration in marine invertebrates. I. The respiratory system in cephalopods[END_REF][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] Martin | High copper concentrations in squid livers in association with elevated levels of silver, cadmium, and zinc[END_REF][START_REF] Schipp | Distribution of copper and iron in some central organs of Sepia officinalis (Cephalopoda). A comparative study by flamless atomic absorption and electron microscopy[END_REF][START_REF] Miramand | High concentrations of some heavy metals in tissues of the Mediterranean octopus[END_REF][START_REF] Smith | Concentrations of the elements Ag, Al, Ca, Cd, Cu, Fe, Mg, Pb and Zn, and the radionuclides 210 Pb and 210 Po in the digestive gland of the squid Nototodarus gouldi[END_REF][START_REF] Finger | Molecular association of Cu, Zn, Cd and 210 Po in the digestive gland of the squid Nototodarus gouldi[END_REF], Miramand & Bentley 1992, Bustamante et al. in press) and reports of levels of toxic metals such as cadmium are few [START_REF] Renzoni | Mercury concentration in the water, sediments and fauna of an area of the Tyrrhenian coast[END_REF][START_REF] Martin | High copper concentrations in squid livers in association with elevated levels of silver, cadmium, and zinc[END_REF][START_REF] Ueda | Amounts of trace elements in marine cephalopods[END_REF][START_REF] Miramand | High concentrations of some heavy metals in tissues of the Mediterranean octopus[END_REF][START_REF] Smith | Concentrations of the elements Ag, Al, Ca, Cd, Cu, Fe, Mg, Pb and Zn, and the radionuclides 210 Pb and 210 Po in the digestive gland of the squid Nototodarus gouldi[END_REF][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] 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], Yamada et al. 1997, Bustamante et al. in press). The studies addressing cadmium levels in cephalopods have shown varied results with high values often present in tissues [START_REF] Miramand | High concentrations of some heavy metals in tissues of the Mediterranean octopus[END_REF][START_REF] Smith | Concentrations of the elements Ag, Al, Ca, Cd, Cu, Fe, Mg, Pb and Zn, and the radionuclides 210 Pb and 210 Po in the digestive gland of the squid Nototodarus gouldi[END_REF][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] 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 very high concentrations in certain cases is remarkable (Martin & Flegal 1975, Bustamante et al. in press). All of these studies have shown the cephalopod ability to concentrate cadmium in the digestive gland.

In this paper, we hypothesise that cephalopods may be a significant source of cadmium, one of the most toxic metals in the marine environment, to their predators which in some areas exhibit high levels of this metal in liver and kidney. Indeed, a large number of top predators regularly include cephalopods in their diets, or at least opportunistically.

Cephalopods are eaten by many oceanic animals, such as marine mammals [START_REF] Clarke | Cephalopods as prey. III. Cetaceans[END_REF][START_REF] Klages | Cephalopods as prey. II. Seals[END_REF], seabirds [START_REF] Croxall | Cephalopods as prey. I. Seabirds[END_REF] and fish [START_REF] Smale | Cephalopods as prey. IV. Fishes[END_REF]. Over 80 % of odontocete species and two baleen whales species regularly include cephalopods in their diets [START_REF] Clarke | Cephalopods as prey. III. Cetaceans[END_REF]. In 28 odontocetes, cephalopods comprise the main food with the Ziphiidae and Physeteridae being the principal consumers. Nevertheless, cephalopods are an important part of the diets of all the other odontocetes species (Monodontidae, Phocaenidae, Delphinidae, Stenidae). Among the various cephalopods groups, the Ommastrephidae are the most common cephalopod family in the diets of odontocetes in oceanic waters, and Loliginidae are the main family in neritic waters [START_REF] Clarke | Cephalopods as prey. III. Cetaceans[END_REF]. For 31 of the 33 existing species of pinnipeds, it is either known or suspected that cephalopods are a primary food source in their diets [START_REF] Klages | Cephalopods as prey. II. Seals[END_REF]. Altough, data are few, the Ommastrephidae, Loliginidae, Onychoteuthidae, Gonatidae and Octopodidae are the most common cephalopods families recorded in the diet of seals. In the same way, a great number of seabirds species, like penguins, procellariforms, pelecaniforms and alcids, are known to feed on cephalopods, mainly squid [START_REF] Croxall | Cephalopods as prey. I. Seabirds[END_REF]. Numerous seabirds species prey up on cephalopods which are only as important as fish or crustaceans for some albatrosses and petrel species [START_REF] Croxall | Cephalopods as prey. I. Seabirds[END_REF]. Penguins, auks and terns also eat significant quantities of squid during certain seasons. Squid are generally the most common items for seabirds, and the Ommastrephidae, Onychoteuthidae, Histioteuthidae and Gonatidae families probably represent the greatest fraction [START_REF] Croxall | Cephalopods as prey. I. Seabirds[END_REF].

To estimate the intake of cadmium for cephalopod consumers, cadmium concentrations in cephalopods originating from French, Irish and Faroe Island waters were determined and compared. Then, the weekly intake of cadmium in predators due to ingestion of cephalopods is estimated and compared with the "Provisional Tolerable Weekly Dose" (PTWI) recommended by the World Health Organisation.

MATERIALS AND METHODS

A large sampling of several species of cephalopods, caught by trawl between 1992 to 1997 in Bay of Biscay, English Channel, Atlantic Irish coast and Faroe Islands waters have been used in the analysis. Each individual has been weighted and measured (mantle length, total length), and the sex determined. The origin, sampling date, number of individuals, mantle length and body weight for each species of cephalopods are given in Table 1. Digestive gland was totally removed from the dissected individuals and treated separately from the remains of the animals. Cadmium concentrations in the whole animals were calculated from remains and digestive gland concentrations. Tissue samples were dried for several days at 80°C to constant weight. Two aliquots of approximately 300 mg of each homogenized dried sample were digested with 4 ml of 65 % HNO 3 and 1 ml of 70 % HClO 4 during 24 hours at 80°C. After evaporation, the residues were dissolved in 0.3 N nitric acid. Blanks were carried through the procedure in the same way as the sample. Cd was determined by flame atomic absorption spectrophotometry (AAS) for the digestive gland and by graphite furnace for the remaining tissues using a Varian spectrophotometer Vectra 250 Plus with Deuterium background correction.

All glassware and plastic was cleaned with HNO 3 / HCl 1N and rinsed with deionized water.

Reference materials, dogfish liver DOLT-2 and MA-A-2 fish-flesh standard (IAEA), were treated and analyzed in the same way. The results for standards were in good agreement with certified values. All the results are given in micrograms of metal per gram of the wet weight tissue (µg/g w wt). The detection limits for Cd were 0.05 µg/g by flame AAS and XXXX by flameless AAS.

Results

Our sampling includes most of the species commonly encountered in French near shore waters and some other species from the Irish and Faroe Island waters. It represents males and females over a large range of size and weight. Cd concentrations (µg/g wet wt) in these whole cephalopods are reported in Table 2 along with other values from the literature. In our analysis, the octopus Eledone cirrhosa from the Faroe Islands exhibited the highest Cd concentrations, 9.06 ± 3.38 µg/g, while the lowest were encountered in the Loliginidae squid, Alloteuthis subulata, Loligo forbesi and L. vulgaris, from European waters with values around 0.10 µg/g w wt.

Indeed, there is a great variability in tissue concentrations in a same area and between different areas for the same species. Cadmium concentrations in the cuttlefish Sepia elegans and the octopuses E. cirrhosa and Octopus vulgaris from the Bay of Biscay, were 5 to 9 times higher than those found in the squid A. subulata and L. vulgaris (Table 2). Likewise, the octopus E. cirrhosa from the Faroe Islands exhibits cadmium concentrations 18 times higher than the same species from the Bay of Biscay (Table 2).

According to the World Health Organisation, the limit of cadmium intake is 1µg/kg/day for humans, i.e. 7 µg/kg/week. The minimum weight of cephalopods necessary to reach these limits has been calculated for each species (in g/kg) and is called "weekly dose" (Table 2).

For the Ommastrephidae squid Todarodes sagittatus from Irish waters and the octopus E. cirrhosa from the Faroe Islands, the weekly dose was reached with less than one gram of cephalopod. The total cadmium content in cephalopods was calculated using means of fresh weight and cadmium concentrations (Table 2). Cadmium contents of cephalopods were very high with more than 22 mg for a single T. sagittatus from Irish waters and more than 4 mg for an E. cirrhosa from Faroe Islands (Table 2).

DISCUSSION

Cadmium concentrations show high variability in cephalopods with respect to the families, geographical origin and feeding habits (benthic or pelagic). For example, in cephalopods from the Bay of Biscay, the lowest concentrations are encountered in Loliginidae while the highest are found in Octopodidae (Table 2). It is possible to classify the cephalopods families based solely on cadmium concentration: Loliginidae < Ommastrephidae < Sepiidae ≤ Octopodidae with mean concentrations according to families of approximately 0.13 µg/g, 0.25 µg/g, 0.50 µg/g and 0.52 µg/g respectively. These differences be diet related: benthic cephalopods (octopuses and cuttlefish) feed mainly on bottom invertebrates (mostly crustaceans, bivalves and polychetes; [START_REF] Boyle | Prey handling and salivary secretions in octopuses[END_REF][START_REF] Mcquaid | Feeding behaviour and selection of bivalve prey by Octopus vulgaris Cuvier[END_REF] while neritic and pelagic cephalopods (mostly squid) prey mainly on fish and cephalopods [START_REF] Rocha | The diet of Loligo vulgaris and Loligo forbesi (Cephalopoda: Loliginidae) in the Galician waters (NW Spain)[END_REF], Pierce et al. 1993[START_REF] Collins | Size selectivity in the diet of Loligo forbesi (Cephalopoda: Loliginidae)[END_REF]. From a same area, fish exhibit lower cadmium concentrations than benthic invertebrates [START_REF] Cossa | Le cadmium en milieu marin. Biogéochimie et écotoxicologie[END_REF], thus diets of the benthic cephalopods should be more rich in cadmium than these based on fish.

In addition, cephalopods show large differences of cadmium concentrations according to their origin. For example, the Ommastrephidae squid Todarodes sagittatus from the Faroe Islands contains very high cadmium concentrations (about 3.5 µg/g) while the cadmium levels in the same species of similar sizes from the Bay of Biscay are 17 times lower (Table 2). The same species from Irish waters exhibit very high values (i.e. 8.4 µg/g, Table 2) perhaps because they are very large individuals. Likewise, cadmium concentrations in the octopus Eledone cirrhosa are very different according to their origin; the lowest levels were encountered in individuals coming from the Bay of Biscay (0.5 µg/g) with more elevated concentrations in individuals from English Channel (1.2 µ/g) and Faroe Islands (9.1 µg/g) (Table 2). Furthermore, with respect tothe large differences in cadmium concentrations in cephalopods, species from sub-Arctic and sub-Antarctic areas contain very high cadmium concentrations (Table 2). According to the data in Table 2, both of the sub-polar areas appear to be regions where there is a cadmium enrichment in the food web. Thus, top predators from polar and sub-polar areas which ingest cephalopods should exhibit high cadmium concentrations in their tissues, particularly in the liver and kidney, the target organs for this metal. Such differences in temperate areas are confirmed when concentrations in the tissues of marine mammals (Table 3) and seabirds (Table 4) are considered. For example, the beluga Delphinapterus leucas exhibit cadmium concentrations in kidney ranging from 10 to 22 µg/g in arctic area whereas in the same species from the estuary of Saint Lawrence, cadmium concentrations in this tissue are about 1.5 µg/g wet wt (Table 3). Likewise, cadmium concentrations in the liver of the harbor porpoise Phocoena phocoena from the Atlantic waters vary from less than 0.07 to 0.5 µg/g whereas some individuals from Greenland have levels as high as 11.9 µg/g in the liver and 72.5 µg/g wet wt in the kidney (Table 3). In French waters, stranded dolphins have been used to investigate heavy metal concentration. Cadmium concentrations recorded in the liver and kidney of Stenella coeruleoalba and Delphinus delphis are, respectively, 17 and 6, and 42 and 18 times lower than concentrations found in Faroe Island pilot whales (Table 3). Stenella coeruleoalba and Delphinus delphis are known to eat neritic squid, particularly Loliginidae, however fish comprise the main portion of their diet. Cadmium transfer from squid is not negligible, and the lowest cadmium concentrations recorded in neritic squid from French waters (i.e. Loliginidae and Ommastrephidae) may explain these low values.

In older individuals of the species from the Arctic and sub-Arctic areas (i.e. pilot whales, beluga, narwhal), the cadmium concentrations in kidney are close to or higher than critical values established for humans (Table 3). Values as high as 500 to 900 µg/g wet wt have been measured in some pilot whales from the Faroe Islands [START_REF] Caurant | Bioaccumulation de quelques éléments trace (As, Cd, Cu, Hg, Se, Zn) chez le globicéphale noir (Globicephala melas, Delphinidé) pêché au large des Iles Féroé[END_REF]. These critical values are about 200 µg/g wet wt in the renal cortex [START_REF] Lauwerys | Cadmium. In: Toxicologie industrielle et intoxication professionnelle[END_REF]). Such cadmium concentrations could lead to severe toxic effects which are nevertheless difficult to show in wild animals. Despite these high concentrations, there is no evidence of a decrease of these populations in these areas, an observation which suggests physiological adaptations in cadmium metabolism. Indeed, during over time, consumers of cephalopod have been submitted to high doses of cadmium, and they probably evolved to withstand cadmium toxicity with efficient detoxification processes.

If we consider all existing data on cadmium concentrations in cephalopods, the range of these concentrations varies over 1 to 2 orders of magnitude between the lowest levels found in the French waters (i.e. Loliginidae with about 0.10 µg/g wet wt) and the highest in the Pacific Ocean (i.e. the Ommastrephidae Symplectoteuthis oualaniensis with about 13.71µg/g wet wt) (Table 2). Nevertheless, regardless of the origin of the cephalopods, we propose that cephalopods constitute an important source of cadmium for many oceanic top predators. We have estimated the weight of cephalopods necessary to reach the Provisional Tolerable Weekly Dose determined by World Health Organisation for humans which is relatively low (1µg Cd/ kg human/day). The highest weight estimated is about 70 g of cephalopod with Loliginidae squids from French waters and the lowest is about 1 g for several species (Table 2). This induces a high exposure to cadmium for marine predators which include cephalopods in their diets and could explain the high cadmium concentrations found in their target organs.

In the case of pilot whales whose diets have been well studied [START_REF] Desportes | La nutrition des odontocètes en Atlantique Nord-Est (côtes françaises et Iles Féroé)[END_REF], Desportes & Mouritsen 1993, Gannon et al. 1997ab), there is strong evidence for a large cadmium contribution from cephalopods for cadmium intake in these species. For example, very high values of cadmium have been recorded in the liver (63 µg/g) and kidney (78 µg/g) in the pilot whales from Faroe Islands (Caurant et al. 1994). Caurant et al. (1994) have estimated the total intake of cadmium from diet studies of the pilot whales from Faroe Islands which show a mean of 18.3 cephalopods per stomach with exceptional values as high as 380 cephalopod remains in one stomach [START_REF] Desportes | La nutrition des odontocètes en Atlantique Nord-Est (côtes françaises et Iles Féroé)[END_REF][START_REF] Desportes | Preliminary results on the diet of long-finned pilot whales of the Faeroe Islands[END_REF]. Our estimations with the cadmium concentrations found in the squid Todarodes sagittatus, the most common prey in the diet of the Faroe Island pilot whales [START_REF] Desportes | Preliminary results on the diet of long-finned pilot whales of the Faeroe Islands[END_REF], are about 765 µg of cadmium per cephalopod (Table 2). We have calculated a minimum weekly intake of 98 mg of cadmium per week. The maximum value was estimated to be about 2037 mg of cadmium.

With the mean weight for pilot whales of 690 kg, the total weekly intake is from 142 to 2952 µg/kg/week. This is much higher than values calculated by [START_REF] Caurant | Cadmium contamination in pilot whales Globicephala melas: source and potential hazard to the species[END_REF] using another squid species, Loligo forbesi; their values ranged from 25.2 to 516.6 µg/kg/week. All these dosages are much higher than values reported by [START_REF] Nogawa | (ed) Changing metal cycles and human health[END_REF] as limits beyond which metabolic disorders appear in human beings. This author has shown in Japanese exposed to cadmium by rice consumption that an increase of metabolic disorders was evident for a weekly intake of 1.41 to 2.03 mg of cadmium for people older than fifty years. For a mean weight of 70 kg, the weekly intake for humans is from 20.3 to 28.7 µg/kg/week. It is more difficult to calculate such values for pinnipeds, but on the 33 pinniped species, only two species, Baikal Seal and Caspian Seal, exclude cephalopods in their diets because they inhabit freshwater where cephalopods do not occur. [START_REF] Watanabe | Trace element accumulation in Baikal seal (Phoca sibirica) from the Lake Baïkal[END_REF] have recorded very low cadmium concentrations in both liver and kidney of Baikal Seal, Phoca sibirica, with mean values respectively of 0.28 ± 0.45 and 2.0 ± 1.1 µg/g wet wt, compared to levels in the marine species which include cephalopods in their diet (Table 3). These authors suggest that the low levels of cadmium are due to low concentrations in their fish prey.

Concerning seabirds, most of the species whose diets have been studied have only been investigated at one site and usually only during their chick-rearing period [START_REF] Croxall | Cephalopods as prey. I. Seabirds[END_REF]. Nevertheless, energy requirements have been calculated for seabirds and translated into food requirements, assuming an energetic value of 4.5 kJ per gram fresh weight food [START_REF] Franeker | Top predators as indicators for ecosystem events in the confluence zone and marginal ice zone of the Weddell and Scotia seas, Antarctica, November 1988 to January 1989 (EPOS Leg 2)[END_REF]. Such energy contents is approximately the value for krill whereas fish usually has higher and squids lower energy contents [START_REF] Brown | Energy requirements for growth of Salvin's Prions Pachyptila vittata salvini, Blue Petrel Halobaena caerulea and Great-winged Petrels Pterodroma macroptera[END_REF][START_REF] Croxall | Relationships between prey life-cycles and the extent, nature and timing of seal and seabird predation in the Scotia Sea[END_REF][START_REF] Jackson | Assimilation efficiencies of White-chinned Petrels (Procellaria aequinoctialis) fed different prey[END_REF]. For example, the Adélie penguin needs about 870 g of food per day. Estimate of squid occurrence in the diets of Adélie penguins from Weddell Sea and Adélie Land were 54 % [START_REF] Ridoux | The diets of five summer breeding seabirds in Adélie Land, Antarctica[END_REF] and3 % (Ainley et al. 1992) corresponding to a minimum of 26 and 470 g of squid by day, i.e. 182 and 3290 g per week, respectively. We lack cadmium concentrations in the squid species included in the diet of Adélie penguin (i.e. Psychroteuthis glacialis and Kondakovia longimana, [START_REF] Ridoux | The diets of five summer breeding seabirds in Adélie Land, Antarctica[END_REF], Ainley et al. 1992).

Nevertheless, taking the lowest cadmium concentration in squid from our study (e.g. 0.10 µg/g, Table 2), ingested cadmium by Adélie penguins would be about 18.2 µg in Weddell Sea and 3290 per week µg in Adélie Land. Assuming a mean body weight of 4.4 kg for an adult Adélie penguin, the doses estimated are 4.1 and 748 µg/kg/week, respectively. These dosages obtained with minimum values (e.g. energetic value for squid, lowest cadmium concentrations) are close to and much higher, to the Provisional Tolerable Weekly Dose of 7 µg/kg/week for humans. Thus, high cadmium concentrations such as 35.0 ± 17.6 µg/g wet wt found in the kidney of Adélie penguins from the Weddell Sea (Schneider et al. 1985, Table 4) are not surprising and can be explained by regular cephalopod consumption in this area.

CONCLUSION

Cadmium levels in top vertebrate predators can be very high in sub-polar areas remote from industrial sites (e.g. Faeroe Islands, Gough Islands, Kerguelen Islands). In contrast, in the shelf waters of industrial countries (e.g. French Atlantic coast), top predators regularly exhibit cadmium concentrations lower than those in similar species from sub-polar areas (Table 3).

This apparent natural accumulation of cadmium in marine mammals and seabirds can be explained by the contribution obtained from consuming cephalopods. The high variability of cadmium concentrations in cephalopods due to their geographical origin and their ecological behaviour may explain why top predators bioaccumulate cadmium in sub-polar areas remote from industrial inputs as opposed to the case in the shelf waters of industrial countries, where the same animals contain far lower cadmium concentrations in their tissues. Altough, in our study we have measured cadmium levels in 5 families of cephalopods including 11 different species, we still lack information about most of the important cephalopod families eaten by whales, pinnipeds and seabirds (i.e. Cranchidae, Gonatidae, Histioteuthidae, Onychoteuthidae, Octopoteuthidae). Future works focused on these families should further light on the transfer of cadmium to top predators in marine food chains.

Table 1 .

 1 Compilation of cadmium concentrations (µg/g wet wt) and cadmium content (µg) in whole cephalopods from different geographical locations in the Northeast Atlantic Ocean. The weekly dose is the cephalopod weight ingested necessary to reach the WHO 'Provisional Tolerable Weekly Intake' limit for humans of 7 µg Cd/kg/week. In most all cases values are Mean ± 1 SD.

	Origin	Species	Sample	Fresh	Water	Cd	Weekly	µg Cd per	Reference
			size	weight	content	concentration	dose	individual
				(g)	(%)	(µg/g wet wt)	(g/kg)
	Faroe Islands
	Loliginidae
	Loligo forbesi	21	123 ± 61	78 ± 1	0.46 ± 0.30	16.3	57 ± 46	This study
	Ommastrephidae
	Todarodes sagittatus	17	221 ± 125	75 ± 2	3.46 ± 1.49	2.0	765 ± 544	This study
	Octopodidae
	Eledone cirrhosa	4	448 ± 85	81 ± 1	9.06 ± 3.38	0.8	4059 ± 1699	This study
	West Irish shelf
	Loliginidae
	Loligo forbesi	18	91 ± 37	78 ± 1	0.11 ± 0.03	63.6	10 ± 5	This study
	Ommastrephidae
	Todarodes sagittatus	5	2692 ± 460 76 ± 3	8.41 ± 5.99	0.8	22640 ± 16583	This study
	Octopodidae
	Benthoctopus	1	1512	87	3.65	1.9	5519	This study
	ergasticus
	Opisthoteuthidae
	Opisthoteuthis sp.	1	1206	94	0.70	10.0	844	This study
	English Channel
	Loliginidae
	Alloteuthis subulata		-	78	0.31	-	-	Bryan (1976)
	Alloteuthis subulata	56	3.5 ± 2.0	78 ± 3	0.37 ± 0.17	18.9	1 ± 1	This study
	Loligo forbesi	9	1195 ± 585 78 ± 1	0.10 ± 0.04	70.0	120 ± 76	This study
	Loligo vulgaris	8	56 ± 49	75 ± 1	0.10 ± 0.03	70.0	6 ± 5	This study
	Octopodidae
	Eledone cirrhosa	15	494 ± 108	78	1.19 ± 0.24	5.9	588 ± 175	Miramand & Bentley
									(1992)
	Sepiidae
	Sepia officinalis	15	518 ± 74	76	0.33 ± 0.01	21.2	171 ± 25	Miramand & Bentley
									(1992)
	Bay of Biscay
	Loliginidae
	Alloteuthis subulata	92	8.4 ± 1.1	78 ± 2	0.10 ± 0.02	70.0	0.8 ± 0.2	This study
	Loligo vulgaris	21	118 ± 73	78 ± 3	0.14 ± 0.06	46.7	17 ± 12	This study
	Ommastrephidae
	Illex coïndetii	22	165 ± 191	79 ± 2	0.29 ± 0.09	25.0	48 ± 30	This study
	Todarodes sagittatus	5	93 ± 113	83 ± 4	0.21 ± 0.02	33.3	20 ± 24	This study
	Octopodidae
	Eledone cirrhosa	6	214 ± 146	81 ± 1	0.49 ± 0.10	14.3	105 ± 75	This study
	Octopus vulgaris	13	1486 ± 581 82 ± 1	0.54 ± 0.24	13.0	802 ± 475	This study
	Sepiidae
	Sepia elegans	14	28 ± 11	75 ± 2	0.90 ± 0.43	7.8	25 ± 16	This study
	Sepia officinalis	22	112 ± 85	77 ± 1	0.20 ± 0.06	28.0	22 ± 18	This study
	Sepia orbignyana	15	57 ± 24	76 ± 2	0.41 ± 0.28	17.9	23 ± 19	This study

Table 2 .

 2 Cadmium concentrations in whole fish from different locations in the North Atlantic and the Arctic Oceans. The weekly dose is the fish weight ingested necessary to reach the WHO 'Provisional Tolerable Weekly Intake' limit for humans of 7 µg Cd/kg/week. All values were converted from dry weight to wet weight concentrations, assuming a wet/dry weight ratio of 4.0.

	Origin	Species	Sample	Cd concentration	Weekly dose	Reference
			size	(µg/g wet wt)	(g/kg)
	North American Arctic
	Arctic cod (Boreogadus saida)	50	0.10 (0.05-0.19)	70 (37-133)	Macdonald & Sprague (1988)
	English Channel
	Trisopterus luscus	-	0.004-0.011	636-1750	Miramand, unpublished data)
	Potamoschistus minutus	10	0.013 ± 0.008	538 (250-1750)	Miramand et al. (1997)
				(0.004-0.028)
	Platichtys flesus	40	0.005 ± 0.002	1400	Miramand et al. in press
	Dicentrarchus labrax	20	0.006 ± 0.002	1167	Miramand et al. in press
	French Atlantic coast
	Potamoschistus minutus	-	0.002	3500	Amiard-Triquet et al. (1983)
	Platichtys flesus		0.003	2333	Amiard-Triquet et al. (1983)
	Calionymus lyra	-	0.009	778	Amiard-Triquet et al. (1983)
	Merlangius merlangius	-	0.005	1400	Amiard-Triquet et al. (1983)
	Pollachius pollachius	-	0.008	875	Amiard-Triquet et al. (1983)

Acknowledgements. We thank Estelle Gironnet for collecting cephalopods on the French trawler 'Rapsodie' in April 1997, IFREMER for use of the French trawler 'Gwen-Drez', and the 'Fiskirannsoknarstovan' for facilitating collections on the Faroese trawler 'Magnus Heinasson'. This work was supported financially by the "Conseil Régional Poitou-Charentes" and Rhône-Poulenc. LITERATURE CITED Amaratunga T (1983) The role of cephalopods in the marine ecosystem. In : IF Caddy (ed). Advances in assessment of world cephalopod resources. FAO Fish Tech Pap 231:379-415. Ainley DG, Ribic CA, Fraser WR (1992) Does prey preference affect habitat choice in Antarctic seabirds. Mar Ecol Prog Ser 90:207-221