Dr Paco Bustamante

Jean-Louis Teyssié

Scott W Fowler

Olivier Cotret

Bruno Danis

Pierre Miramand

Michel Warnau

BIOKINETICS OF CADMIUM AND ZINC ACCUMULATION AND DEPURATION AT DIFFERENT STAGES IN THE LIFE CYCLE OF THE CUTTLEFISH SEPIA OFFICINALIS

Keywords: Cadmium, Zinc, Bioaccumulation, Depuration, Cephalopods, Sepia officinalis, Radiotracers

Bioaccumulation of 109 Cd and 65 Zn by the cuttlefish Sepia officinalis was studied at different stages of its life cycle, i.e. in embryos, juveniles and adults, following exposures via sea water, sediments and food. Cuttlefish eggs efficiently accumulated both elements from sea water with bioconcentration factors reaching 46 for 109 Cd and 79 for 65 Zn after 11 days of exposure. Most of the radiotracers was found in the capsule membrane of the eggs, demonstrating that the capsule acts as a shield to protect embryos against metals. Juveniles and adults efficiently bioconcentrated both radiotracers from sea water, with the muscular tissues containing 62% of the total 109 Cd and 84% of the total 65 Zn. Loss kinetics followed a single exponential function for 65 Zn while for 109 Cd, loss was best described by a double exponential model. Biological half-lives for elimination were ca. 2 months for both elements. After 29 days of depuration in uncontaminated sea water, 76 to 87% of the radiotracers were found in the digestive gland. For both elements the dissolved phase can be considered as a significant source of accumulation. In an experiment with radiolabelled sediments, transfer factors were very low, even after 29 days of exposure. Food chain transfer experiments demonstrated that both juveniles and adults assimilated 109 Cd and 65 Zn very efficiently. Moreover, loss of ingested radiotracers was much slower than elimination of 109 Cd and 65 Zn taken up from sea water, indicating a very strong retention of dietary Cd and Zn by juvenile as well as by adult cuttlefish. As with direct uptake from sea water, ingested radiotracers were mainly found in the digestive gland with fractions reaching 82% for 65 Zn and 97% for 109 Cd after 29 days of depuration. These tracer experiments indicate that (1) food is likely the primary pathway for Cd and Zn bioaccumulation in Sepia officinalis and (2) the digestive gland plays a major role in the subsequent storage and presumed detoxification of these elements regardless of the uptake pathway.

INTRODUCTION

Cephalopods constitute a class of marine molluscs which are found in a great variety of habitats from coastal waters to very deep ocean environments. They have also been found to live under extreme conditions such as near hydrothermal vents (e.g. the hydrothermal octopus Vulcanoctopus hydrothermalis, [START_REF] Gonzalez | Vulcanoctopus hydrothermalis gen. et sp. nov. (Mollusca, Cephalopoda): an octopod from a deep-sea hydrothermal vent site[END_REF]. Cephalopods are benthic (e.g. octopus), nectobenthic (cuttlefish), neritic and pelagic (squids), and it follows that they are a primary food source for many marine predators such as marine mammals or seabirds [START_REF] Clarke | Cephalopods as prey. III. Cetaceans[END_REF][START_REF] Croxall | Cephalopods as prey. I. Seabirds[END_REF][START_REF] Smale | Cephalopods as prey. IV. Fishes[END_REF].

Whatever their morphological and evolutionary diversity and heterogeneity in their geographic distribution, cephalopods have in common the ability to concentrate Cd to extremely high levels [START_REF] Martin | High copper concentrations in squid livers in association with elevated levels of silver, cadmium, and zinc[END_REF][START_REF] Miramand | High concentrations of some heavy metals in tissues of the Mediterranean octopus[END_REF][START_REF] 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], Bustamante et al. 1998a, 2000). Cephalopods are therefore a potential threat for higher trophic levels. Indeed, their predators are well known to display high Cd concentrations as well (Bustamante et al. 1998b). The reason for such a high Cd bioaccumulation capacity is still poorly understood. Thus, the primary objective of the present work was to investigate biokinetics of Cd uptake and elimination in cephalopods in order to better characterize their bioaccumulation, tissue distribution and retention capacity.

The common cuttlefish Sepia officinalis was selected as a model to study Cd transfer in cephalopods via sediments, sea water and food. Furthermore, bioaccumulation was followed at different stages of the life cycle of S. officinalis, viz. in embryos, early juveniles and adults, to better define the physiological basis for enhanced Cd bioconcentration.

In addition to Cd, Zn was also considered in this study. This essential element has chemical properties similar to those of Cd that, in turn, could interfere with Zn metabolism [START_REF] Nieboer | The replacement of the nondescript term 'heavy metal' by a biologically and chemically significant classification of metal ions[END_REF]. Biokinetics were determined using carrier-free radiotracers in order to measure element fluxes in real time at environmentally realistic contaminant concentrations [START_REF] Fisher | Scavenging and retention of metals by zooplankton fecal pellets and marine snow[END_REF][START_REF] Warnau | Biokinetics of selected heavy metals and radionuclides in the common Mediterranean echinoid Paracentrotus lividus: sea water and food exposures[END_REF].

MATERIAL AND METHODS

Organisms

Eggs, newly hatched juveniles and adult common cuttlefish (Sepia officinalis L.) were used in the experiments. Cuttlefish eggs were obtained from the Marine Station of Banyuls sur Mer (France) and were maintained in an aquarium until hatching. Young cuttlefish were then kept in a separate aquarium (open circuit, 20 l h -1 flow rate, constant aeration, 38 p.s.u., 16.5 ± 0.5 °C, 12h/12h light/dark cycle) and fed with brine shrimp (Artemia salina) for several days before the experiments.

In addition, two different groups of adults were investigated. The first group was reared in the Musée Océnaographique (Principality of Monaco) from hatching up to one year old organisms, and conditioned to feed on dead fish and mussels. The second group consisted of adults collected by net fishing off Monaco. They were maintained individually in aquaria for acclimation to laboratory conditions for two weeks prior to investigations. During acclimation, the wild cuttlefish were also conditioned on a diet of dead fish and mussels.

In order to maintain animals in good living conditions and to prevent cannibalism, food was provided ad libitum for both size classes before and during the experiments. Different food items were given to young and adult cuttlefish because of their specific biological characteristics: diet of young individuals is restricted to small, relatively soft and fleshy crustaceans while adults feed on larger crustaceans including crabs, molluscs (including cephalopods), and fish (Boucher-Rodoni et al. 1987).

Prior to any experimentation, adults were anaesthetised in sea water containing 2% ethanol in order to determine their sex, weight and mantle length. At the same time, a numbered plastic marker was inserted into the mantle fin to identify each animal.

Radiotracers and radioanalyses.

Carrier-free radiotracers, 65 Zn (T 1/2 = 243.9 d) and 109 Cd (T 1/2 =462.6 d), were purchased from Amersham, UK. Stock solutions of 65 Zn and 109 Cd were prepared in 0.1 N HCl.

Activities of the radiotracers were measured using a high-resolution γ-spectrometry system consisting of coaxial Ge N or P type detectors Intertechnique) connected to a multichannel analyser and a PC employing spectral analysis software (Interwinner, Intertechnique). The absolute activities of the samples were determined by comparison with known standards of appropriate geometry and were corrected for background and physical decay of the radiotracers.

Counting times were adapted to obtain relative propagated errors less than 5%. However, in a few cases, this counting precision could not be obtained even after 48 h of counting, due to the very low activity in some minute organs. Counting times ranged from 10 min to 1 h for whole cuttlefish, mussel and brine shrimp radioanalyses, and from 10 min to 48 h for the dissected tissues.

Experimental procedures.

Contamination of Sepia officinalis from sediments.

A group of nine newly hatched cuttlefish (mean wet weight ± SD: 0.327 ± 0.063 g) were placed for 29 days in a 20 l plastic aquarium (open circuit, 20 l h -1 flow rate, constant aeration, 38 p.s.u., 16.5 ± 0.5 °C) containing ca. 3 l of natural sea water running over a 4 cm layer of spiked sediments containing 120 ± 9 KBq 65 Zn g -1 and 49 ± 8 KBq 109 Cd g -1 wet wt over the experiment time. The height of sea water was maintained low in order to minimise the movements required for feeding and to maximise the time of contact with sediments. All cuttlefish were fed twice daily with Artemia salina and were periodically γ-counted to follow the radiotracer uptake kinetics. At the end of the experiment, 3 individuals were dissected to determine the distribution of the radiotracers among digestive gland, cuttlebone and remainder (rest of the organs).

Contamination of Sepia officinalis from sea water.

Eggs. The eggs were placed in a 75 l glass aquarium containing natural sea water spiked with 65 Zn (6 KBq l -1 ) and 109 Cd (13 KBq l -1 ) for 11 days. Spiked activities corresponded to very low additions of stable metals in the sea water, viz. 0.3 pM Zn and 1.16 pM Cd; this allowed experimentation under simulated conditions that were environmentally realistic.

The spiked sea water was changed daily to maintain radiotracer concentrations relatively constant and to avoid any build-up of exometabolites. During the experiment, activities of the eggs were measured at days 1, 2, 3, 6, 9, and 11. At each time, 3 eggs were dissected to determine the distribution of the tracers among the capsule membrane, peri-embryonar liquid and embryo.

Juveniles. Eight newly hatched cuttlefish (mean weight ± SD: 0.460 ± 0.060 g) were placed for 36 h in a 20 l plastic aquarium containing sea water spiked with 65 Zn (6 KBq l -1 ) and 109 Cd (13 KBq l -1 ). After that time, radiolabelled juvenile cuttlefish were held for 29 d in clean flowing sea water (75 l glass aquarium, open circuit, 20 l h -1 flow rate, constant aeration, 38 p.s.u., 16.5 ± 0.5 °C). In order to facilitate the retrieval and counting of each individual during the loss experiment, juveniles were held individually in the aquaria in separate circular plastic containers (10 cm diameter, 5 cm height) covered with a plastic netting. Cuttlefish were fed twice daily with Artemia salina and were periodically γ-counted to follow the radiotracer loss kinetics. After 29 d of loss, four individuals were dissected to determine the distribution of the tracers among digestive gland, cuttlebone and remainder.

Adults.

A group of five sexually mature male cuttlefish (mean weight ± SD: 253 ± 97 g) was held for 8 h in a 75 l glass aquarium containing sea water spiked with 20 KBq 65 Zn l -1 and 13 KBq 109 Cd l -1 . In terms of stable metal addition, these activities corresponded to concentrations of 1 pM Zn and 1.16 pM Cd. Afterwards, the cuttlefish were radioanalyzed and transferred to a flowing sea water aquarium (3000 l, open circuit, 600 l h -1 flow rate, constant aeration, 38 p.s.u., 16.5 ± 0.5 °C) for 6 d. Three adults were dissected after 24 h and the remaining ones were dissected after 6 days of depuration. For each individual, the branchial heart appendages, branchial hearts, gills, digestive tract (after removal of the gut contents), genital tract, ink sack, digestive gland, kidneys, mantle skin, mantle muscle, head and cuttlebone were separated, weighed, and their radiotracer content measured.

Contamination of Sepia officinalis through the food.

In order to better reflect the different feeding habits of juvenile and adult cuttlefish (Boucher-Rodoni et al. 1987), two different prey were selected for the feeding experiments. Mussels (Mytilus galloprovincialis) and brine shrimp (Artemia salina) were exposed for 7 d in a plastic aquarium containing 4 l of natural sea water spiked with 6 KBq 65 Zn l -1 and 13 KBq 109 Cd l -1 .

Radiolabelled sea water was changed daily and the organisms were subsequently used as food for cuttlefish.

Juveniles. Eight newly hatched cuttlefish (mean weight ± SD: 0.380 ± 0.030 g) were placed in individual plastic containers (10 cm diameter, 5 cm height), and held in a 75 l glass aquarium under the same conditions as in the previous experiments. These individuals were then fed for one hour with the previously radiolabelled A. salina. At the end of the feeding period, each individual was immediately γ-counted. From that time on, cuttlefish were fed twice a day with uncontaminated A. salina and regularly γ-counted to determine radiotracer loss kinetics and assimilation efficiency. Throughout the depuration period, faeces were removed three times per day to reduce direct contamination by recycled radiotracers leaching from the faeces. After 29 d, five juveniles were dissected to determine body distribution of the radiotracer.

Adults. Eighteen sexually mature cuttlefish (11 females and 7 males; mean weight ± SD: 164 ± 34 g and 107 ± 23 g, respectively) were placed in a 3000 l aquarium under conditions described above and fed for two hours with the soft parts of the previously labelled mussels. Following ingestion, each individual was γ-counted and the same procedure followed as for the juveniles. In addition, three adult cuttlefish were dissected after each radioanalysis to determine the radiotracer content of their different tissues.

Data analyses.

Uptake of the radiotracers from sea water and sediments was expressed as change in concentration factors (CF) and transfer factors (TF) (CF and TF = Bq g -1 wet organism divided by the time-integrated Bq g -1 in sea water or sediments, respectively) over time. Uptake kinetics in the eggs were described using a single-component first-order kinetic model

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

Radiotracer elimination was expressed in terms of percentage of remaining radioactivity, i.e. radioactivity at time t divided by initial radioactivity measured in the organisms at the beginning of the depuration period. When radiotracer loss plotted against time displayed an exponential shape, the kinetics were described either by single-component exponential model

A t = A 0 e -λt
where A t and A 0 are remaining activities (%) at time t (d) and 0, respectively, and λ is the depuration rate constant (d -1 ) which allows the calculation of the radiotracer biological half-life (T b1/2 = ln 2/λ), or by a 2-component exponential model

A t = A 0s e -λst + A 0l e -λlt
where the 's' subscript refers to a short-lived component (s component) and the 'l' subscript refers to a long-lived component (l component) [START_REF] Hubbell | Radiotracer, gravimetric and calorimetric studies of ingestion and assimilation rates of an isopod[END_REF][START_REF] Whicker | Radioecology: nuclear energy and the environment[END_REF]. The exponential model showing the best fit (decision based on calculation of the determination coefficients, R², and examination of the residuals) was then selected.

Constants of the models and their statistics were estimated by iterative adjustment of the model and Hessian matrix computation, respectively, using the non-linear curve-fitting routines in the Systat 5.2.1 Software [START_REF] Wilkinson | Systat: the system for statistics[END_REF]. Changes in radiotracer distribution among cuttlefish tissues were tested for significance by the G procedure (adapted from the log-likelihood ratio test) for 2 x k contingency tables [START_REF] Zar | Biostatistical Analysis[END_REF]. Changes in % of radioactivity in a single tissue during the depuration period were tested by one-way ANOVA (after arcsin transformation of data) followed by the Tukey's multiple comparison test. The significance level for statistical analyses was always set at α =0.05.

RESULTS

Contamination from the sediments

Although cuttlefish spend most of their time on the bottom sediments, very low activities were recorded in the individuals exposed to spiked sediments, even after an exposure as long as 29 d. The transfer factors between sediment and cuttlefish remained less than 0.10 for both 65 Zn and 109 Cd (Figure 1). The heterogeneity of the results was substantial and no realistic accumulation kinetics could be calculated for this experiment.

After 29 d, the dissection of 3 individuals showed that the digestive gland contained most of the two metals studied, i.e. 75 ± 8% for 65 Zn and 68 ± 7% for 109 Cd while the remainder and cuttlebone contained respectively 23 ± 8 and 2.5 ± 0.1% for 65 Zn and 20 ± 6 and 11 ± 1% for 109 Cd.

Contamination from sea water

Eggs. The experiment was carried out for 11 d at the end of which elevated concentration factors were reached in the eggs for both 65 Zn (79 ± 5, m ± SD) and 109 Cd (46 ± 2, m ± SD). The uptake of both tracers displayed saturation kinetics (Figure 2). Most of the radiotracers (96-99% for 65 Zn and 99% for 109 Cd) was always found associated with the capsule membrane of the eggs (Figure 3).

Juveniles and adults.

Due to obvious safety problems resulting from maintenance of cuttlefish in aquaria containing spiked sea water, the exposure time was shortened as much as possible. Preliminary experiments indicated that a 36 h exposure was necessary to obtain juveniles with activity levels suitable for efficient detection; a 8 h exposure was found to be sufficient for adults.

Activities (Bq g -1 wet wt) recorded in the different cuttlefish tissues after a 8 h exposure are given in Table 1. In the adults, 65 Zn exhibited the highest activities in the skin and gills (121 and 119 Bq g -1 , respectively) while the branchial hearts contained the highest activities of 109 Cd (185 Bq g -1 ). 65 Zn appears to be more bioavailable to cuttlefish than 109 Cd since the whole body CF was 3 times higher for Zn than for Cd.

The distributions of the radiotracers in body compartments of adult and juvenile cuttlefish are presented in Tables 1 (6 d of depuration) and 2 (29 d of depuration), respectively. Following exposure, the muscles of adults (i.e. mantle muscle and skin, and the head) contained the highest fraction of both radiotracers (84% for 65 Zn and 62% for109 Cd). The digestive gland contained the second highest proportion of 109 Cd (i.e. 25%). After 6 d of depuration, the global distribution of radiotracers changed since the fraction of 65 Zn increased significantly in the digestive gland (p=0.001), and 109 Cd in branchial hearts (p=0.004) and appendages (p=0.005).

In contrast, the tissues in direct contact with sea water generally exhibited a lower percentage of radioactivity after a 6 d depuration. This is particularly obvious in the case for 65 Zn in gills (p<0.001)and head (p=0.007) (see Table 1).

Loss of 65 Zn in juvenile cuttlefish followed a single-component exponential model (Figure 4A, Table 3) with a T b1/2 of approx. 2 months. In contrast, loss of 109 Cd was best described by a two-component model (Figure 4B, Table 3). The short term component was characterised by a very short T b1/2 (ca. 14 h) while the long-lived loss component which represented a large fraction of the 109 Cd content (A 0l = 71%) turned over with a T b1/2 of ca. 65 days.

After 29 d in uncontaminated sea water, both tracers were found in highest proportion in the digestive gland of the young cuttlefish (from 76 to 87%) (Table 2) while their lowest proportion was found in the cuttlebone (around 1% of the total activity).

Contamination via food

Eighteen adult cuttlefish ingested 123 radiolabelled mussels in 2 hours and 8 juveniles were fed radiolabelled brine shrimp ad libitum for one hour. The cuttlefish and their prey were immediately γ-counted for their tracer content (Table 4).

Loss kinetics of 65 Zn and 109 Cd ingested with food displayed a 2-component exponential model for both juveniles (Figures 4C &4D, Table 3) and adults (Figures 4E &4F, Table 3).

Regarding adults, the short-lived component involved about half the total activity in whole cuttlefish (59% for Zn and 47% for Cd) and both elements displayed very short T b1/2s (ca. 10 h) (Table 3). The short-lived loss component for juveniles represented a lesser fraction of radiotracer (38% for both elements) than that of adults (Table 3). The long-lived component of T b1/2l in both juvenile and adult cuttlefish (> 250 d). In contrast, in the young cephalopods, slow depuration rates characterising the long-lived component were noted for 65 Zn (T b1/2 =173 d) while in adults, depuration rates were much faster (T b1/2 =38 d). Young individuals incorporated metals more efficiently from brine shrimp (assimilation efficiency -AE-: 63% for Zn and 62% for Cd) than did adults ingesting mussels (AE = 59% for Zn and 47% for Cd) (Table 4).

The distribution of radioactivity in cuttlefish tissues was determined on several occasions in adults (Table 5) and only at the end of the loss period for juveniles (Table 2). At the end of the depuration period, the highest proportion of radiotracer was found in the digestive gland (i.e. 79 and 97% in adults and 82 and 91 % in juveniles for 65 Zn and 109 Cd, respectively). In adults, tissue distribution of 65 Zn varied significantly during the experiment (G test, p<0.05) (Table 5).

The proportion of 65 Zn activity decreased in the muscular tissues (mantle muscle and cephalic parts) and increased in the digestive gland (Tukey test, p=0.021) suggesting a lower depuration rate in digestive gland than in other organs (Table 5). For 109 Cd, a G test did not indicate any significant difference among radioactivity distributions during the experiment.

DISCUSSION

The use of radiotracers in very low concentrations allows the investigation of trace metal metabolism at realistic environmentally conditions. Furthermore, these radiotracer experiments permit simultaneously investigating several elements without inter-element interferences.

During embryonic development, Zn and Cd were efficiently taken up from sea water by the eggs, with CF of 79 for 65 Zn and 46 for 109 Cd after only 11 d of exposure. However, most of the radiotracers was associated with the capsule membrane of the egg, which thus would act as an efficient shield protecting the embryo against metal direct exposure. This observation is surprising for Zn since it is an essential element for the development of the embryos. That may in fact explain the large amounts of this metal occurring in ovaries, such as in the squid Illex argentinus in which the female gonad may account for 13.6% of the total Zn body burden [START_REF] Gerpe | Cadmium, zinc and copper accumulation in the squid Illex argentinus from the Southwest Atlantic Ocean[END_REF]. This suggests that eggs contain enough Zn for the needs of the embryo, and that additional metal could become toxic for the embryo. In fact Zn incorporation may also be blocked to inhibit Cd from crossing the capsule membrane, since the latter element has similar chemical properties as Zn and follows the same uptake pathways. Nevertheless, the exact mechanism causing the specific blockage of both metals in the capsule membrane of the eggs remains unknown.

Although the experimental contamination via sea water was only carried out for a short period of time, activities recorded in the whole cuttlefish suggest that they would efficiently accumulate these two elements directly from water. In other invertebrates such as echinoderms or bivalves, the major fraction of the radiotracers was found in the calcified parts, i.e. body wall of sea urchins [START_REF] Warnau | Biokinetics of selected heavy metals and radionuclides in the common Mediterranean echinoid Paracentrotus lividus: sea water and food exposures[END_REF]) and shell of mussels [START_REF] Guary | Recherches sur les transferts et la fixation du plutonium, de l'américium et du neptunium dans le milieu marin[END_REF], Fisher et al. 1996).

Although the cuttlefish Sepia officinalis has a calcareous compartment -the cuttlebonerepresenting around ca. 5% of the total body weight, it contained less than 1% of the total radioactivity of 65 Zn and 109 Cd after sea water exposure. This may be due to the internal location of the cuttlebone which has no direct contact with ambient sea water. Thus, trace elements are distributed more homogeneously among cuttlefish tissues than in invertebrates with calcareous compartments directly in contact with sea water (bivalves) or protected by a thin epidermis (sea urchins). In S. officinalis, most of the tracers taken up, i.e. 84% for 65 Zn and 62% for 109 Cd, was located in the tissues in direct contact with sea water, i.e. mantle, skin of the mantle, and head.

If direct sorption of metals on the cuttlebone is not possible (it is entirely surrounded by muscular tissue), transfer and redistribution of Cd to this compartment would be expected since the cuttlebone is mainly composed of calcium carbonate. Indeed, Cd mimics Ca because of their similar ionic radius [START_REF] Huheey | Inorganic chemistry[END_REF]. Therefore, due to its mineral composition, the cuttlebone of S. officinalis could play a storage role for some elements, including Cd. However, results presented here showed that transfer of radiotracers to the cuttlebone were very limited, even after a long depuration period, following either sea water or food exposure (Tables 2 and5).

After 6 d in uncontaminated sea water, the two elements were found to be distributed differently among body compartments (Table 1). This allowed the determination of three distinct groups of tissues: (1) those whose percentage of total radioactivity had decreased (muscular parts and gills); (2) those in which there was an increase in the percentage of total radioactivity (i.e. digestive gland, branchial hearts and appendages); and (3) those in which the percentage of the radioactivity remained similar (cuttlebone, kidney, ink sack, genital tract and gut). The tissues of the first group might have rapid depuration rates while those of the second group -which are also known or supposed detoxification organs [START_REF] Mangold | Appareils excréteurs et excrétion[END_REF])-would have either a longer retention capacity or would be the targets of preferential translocation of the elements from other organs. A similar distinction for the tissues has been made for the squid Sepioteuthis lessoniana which was exposed to stable Cd in sea water [START_REF] Koyama | Bioaccumulation of waterborne and dietary cadmium by oval squid Sepioteuthis lessoniana, and its distribution among organs[END_REF]; in this case the concentration of Cd in the digestive gland was still increasing several days after the beginning of depuration while loss was immediate in the gills and mantle. Koyama et al. suggested a stronger retention of the metal in the digestive gland than in other organs. Nevertheless, our results clearly demonstrated (1) the translocation of Zn to the digestive gland where the activity was more than 10 times higher after 6d of depuration, and

(2) the translocation of Cd to the branchial hearts and appendages in which activities increased by a factor 5 and 4, respectively, over the same period of time (see Table 1).

After bioaccumulation from sea water, Zn and Cd were released from juvenile cuttlefish following a single component ( 65 Zn) or a 2-component ( 109 Cd) exponential loss kinetics.

Whole body loss was relatively slow for both 65 Zn and 109 Cd with mean biological half-lives of 53 and 65 d, respectively. Our results are quite similar to those obtained for Zn in the octopus Octopus vulgaris which displayed a mean half-life of 74 d [START_REF] Ueda | Accumulation of 65 Zn by Octopus Octopus vulgaris[END_REF]. Comparison for 109 Cd retention with other cephalopod species is not possible as there are apparently no data available in the literature. However, similar half-lives for 109 Cd have been reported for other mollusc species such as the mussels Mytilus edulis (67-78 d) and M. galloprovincialis (16-60 d) [START_REF] Fisher | Accumulation and retention of metals in mussels from food and water: a comparison under field and laboratory conditions[END_REF], Wang et al. 1996). For these bivalves, the dissolved phase is usually considered as a significant source of metal accumulation [START_REF] Nolan | Accumulation of metal radiotracers by Mytilus edulis[END_REF], Wang et al. 1996), since they have high filtration rates and can process large volumes of sea water. As cephalopods have lower respiration rates compared to the filtration rates of bivalves, the dissolved phase might be of lesser importance in metal uptake than it is for bivalves.

After 29 d of depuration, the residual radioactivity was mainly located in the digestive gland.

Thus, the digestive gland appears to be the major storage organ for both Zn and Cd. It is also noteworthy that 109 Cd activities in branchial hearts were relatively elevated following the exposure to contaminated sea water (Table 1). After 6 d of loss, branchial hearts and appendages exhibited a significantly higher percentage of total 109 Cd than at day 0. In fact, branchial heart of cephalopods generally contains very low Cd concentrations [START_REF] Miramand | High concentrations of some heavy metals in tissues of the Mediterranean octopus[END_REF][START_REF] Miramand | Concentration and distribution of heavy metals in tissues of two cephalopods, Eledone cirrhosa and Sepia officinalis, from the French coast of the English Channel[END_REF], even if they concentrate many elements such as copper, iron, cobalt, nickel or vanadium [START_REF] Ueda | Amounts of trace elements in marine cephalopods[END_REF], Nakahara et al. 1979[START_REF] Miramand | High concentrations of some heavy metals in tissues of the Mediterranean octopus[END_REF][START_REF] Miramand | Concentration and distribution of heavy metals in tissues of two cephalopods, Eledone cirrhosa and Sepia officinalis, from the French coast of the English Channel[END_REF]. Radiotracer investigations with Octopus vulgaris have also shown the ability of octopus to concentrate radionuclides such as americium and plutonium in the branchial hearts and appendages [START_REF] Guary | High concentrations of transuranic and natural radioactive elements in the branchial hearts of the cephalopods Octopus vulgaris[END_REF][START_REF] Guary | Experimental studies on the biokinetics of plutonium and americium in the cephalopod Octopus vulgaris[END_REF].

Furthermore, 241 Am is eliminated only very slowly from octopus tissues [START_REF] Guary | Experimental studies on the biokinetics of plutonium and americium in the cephalopod Octopus vulgaris[END_REF]. In contrast, Cd has a very short retention time in branchial hearts, which probably serve as excretory organs for this element. In fact, these organs have circulatory and excretory functions, since they are actively involved in the ultrafiltration of the haemolymph [START_REF] Mangold | Reproduction, croissance et durée de vie[END_REF]). Accordingly, Cd that is directly taken up in the cephalopod haemolymph through the gills could be excreted by these organs.

Following the dietary contamination of young and adult cuttlefish, loss kinetics were described by a 2-component exponential model. The T b1/2s of the elements were very short (≤ 1 d). In this case, the short-lived component would represent the unabsorbed fraction of the element which was directly eliminated with the faeces. Indeed, this loss rate corresponds quite well to the gutresidence time in both juvenile and adult Sepia officinalis for which digestion is typically completed in ca. 15 hrs at 20°C (Boucaud-Camou 1973). Thus, the remaining radioactivity can be considered as the absorbed fraction of the ingested dose. In adults, 41% of 65 Zn and 53% of 109 Cd from ingested mussels were readily incorporated into the tissues. These elements were absorbed to an even greater degree in juvenile cuttlefish with assimilation efficiencies (AE) of 63% for 65 Zn and 62% for 109 Cd from ingested brine shrimp. These differences between juveniles and adults could be due to differences in efficiency of digestion metabolism [START_REF] Mangold | Reproduction, croissance et durée de vie[END_REF]). However, since food type was different in experiments with juveniles (fed brine shrimp A. salina) and adults (fed mussels M. edulis), it is difficult to determine whether the differences in element AE are due to different energetic needs of juvenile and adult cuttlefish rather than to differences in element bioavailability. Indeed, different storage and/or detoxification processes occurring in mussels and brine shrimps could partially control metal bioavailability owing to the different physico-chemical speciation of the elements in the prey.

In general, metals located in the soluble subcellular fraction (i.e. cytosol) are more bioavailable to higher trophic levels whereas those bound to insoluble subcellular fraction have a lower potential for transfer to predators [START_REF] Reinfelder | The assimilation of elements ingested by marine copepods[END_REF][START_REF] Wallace | Bioavailability of biologically sequestered cadmium and the implications of metal detoxification[END_REF].

Likewise, it would be of interest to examine trace element assimilation efficiencies in cephalopods with respect to the subcellular location of the elements in each prey.

The biological half-life of 65 Zn was found to be longer in juveniles than in adults probably because the newly hatched cuttlefish incorporate large amounts of Zn in their cells to satisfy their metabolic needs. In this context, it was also expected that 109 Cd would have been retained more efficiently (T b1/2 = 257 d) than an essential element such as Zn. Cd was also strongly retained in the tissues of adults, with an estimated T b/2 tending towards infinity. These very elevated retention capacities concerned almost exclusively the digestive gland. Indeed, the digestive gland retained 91% and 97 % of the total body burden of 109 Cd in juvenile and adult cuttlefish, respectively. Thus, assimilated Cd that is contained in the digestive gland may be considered as actually stored. The occurrence of Cd T b1/2 of the same time-scale order as the cuttlefish lifespan (1-2 y in the Mediterranean) (Boletzky 1983) suggests that during their evolution, cephalopods have developed a detoxification strategy favouring the storage of this toxic element instead of its elimination.

The high degree of Cd retention might thus be a result of a very efficient detoxification system which allows storing this toxic metal as one or another detoxified form. Such a detoxification process could involve precipitation or co-precipitation of metals into metal-rich granules leading to a highly stable form. This sequestering process has been observed for heavy metals in several invertebrate species [START_REF] Coombs | Mechanisms of immobilisation and detoxification of metals in marine organisms[END_REF], Brown 1982[START_REF] Taylor | Inorganic deposits in invertebrates tissues[END_REF]. However, in cephalopods, subcellular investigations have shown that Cd is mainly associated with soluble (cytosolic) compounds, but this proportion decreases when concentration of total Cd increases in the organ (Bustamante et al. in press). This is mainly due to the transfer and sequestration of Cd in the lysosomal system which is very well developed in cephalopods. Nevertheless, neither ultrastructural nor microanalytical investigations have shown the presence of any granules in the digestive gland of squids or cuttlefish (Boucaud-Camou & Boucher-Rodoni 1983, Bustamante 1998).

Cd analyses of the digestive gland of adult cuttlefish which died during collection or acclimation period indicate Cd concentrations (18.6 ± 7.3 µg g -1 dry wt) which are similar to those measured in the same species from the Bay of Biscay (9.4 ± 4.1 µg g -1 dry wt; Bustamante 1998) or the English Channel (12.9 ± 0.3 µg g -1 dry wt; [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]. Given that 64% of the total Cd in the digestive gland of S. officinalis from the Bay of Biscay were located in the soluble fraction of this organ (Bustamante et al. in press), it is most likely that the accumulated 109 Cd was mainly bound to soluble compounds in the cuttlefish used in our experiments. Such an efficient detoxification system could involve cytosolic proteins such as metallothioneins as suggested by the chromatographic results obtained with different squid species [START_REF] Tanaka | Subcellular distribution and binding of heavy metals in the untreated liver of the squid ; comparison with data from the livers of cadmium and silver exposed rats[END_REF][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] Castillo | Isolation and partial characterisation of cadmium binding proteins from the oceanic squid, Ommastrephes bartrami[END_REF]. But proteins with a high molecular weight (> to 70 kDa) may play an important role in Cd detoxification allowing cephalopods to accumulate high Cd levels [START_REF] Tanaka | Subcellular distribution and binding of heavy metals in the untreated liver of the squid ; comparison with data from the livers of cadmium and silver exposed rats[END_REF][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] Castillo | Isolation and partial characterisation of cadmium binding proteins from the oceanic squid, Ommastrephes bartrami[END_REF]. The lower retention efficiency for Cd that was noted in juvenile cuttlefish (257 d vs infinity in adults) would result from the incomplete development of the digestive gland in early juveniles. Indeed the digestive gland is not yet fully grown (and

thus not yet fully physiologically active) in juveniles, since its two segments are still separated by the anterior lobe of the inner yolk sack (Boucher-Rodoni et al. 1987). Interestingly, the structure of the digestive gland in juveniles appears to be similar to that of adults 30 days after hatching, and it becomes functional as soon as the cuttlefish begins to feed (i.e. 1-2 days after hatching). Thus, it would be of particular interest to compare the detoxification processes of Cd by cytosolic proteins in both juvenile and adult cephalopods.

In contrast to most marine invertebrates which would accumulate Cd mainly from sea water [START_REF] Dahlgaard | Bioindicators for monitoring radioactive pollution of the marine environment-experiments on the feasibility of Mytilus as a bioindicator in estuarine environments with some comparisons to Fucus[END_REF][START_REF] Nolan | Accumulation of metal radiotracers by Mytilus edulis[END_REF], Fisher et al. 1996[START_REF] Warnau | Biokinetics of selected heavy metals and radionuclides in the common Mediterranean echinoid Paracentrotus lividus: sea water and food exposures[END_REF], cephalopods like S. officinalis would accumulate Cd principally from food. Once incorporated, the fate of this metal is mainly controlled by the digestive gland which acts as an efficient detoxification organ whatever the source of Cd is sea water, food, or sediment. This detoxification process occurs throughout the lifetime of the cephalopod except during embryonic development. Results for Zn were very similar to those for Cd in juveniles contaminated from sea water and from food. However, a lower retention Zn in adults could be a result of displacement of this element from the cytosolic proteins in the digestive gland by other metals such as Cd. Cd in whole juvenile cuttlefish previously exposed to radiolabelled sea water for 36 h (mean remaining activity ± SD, n =8 at day 0 and n=4 at day 29); C and D: Loss of 65 Zn and 109 Cd in whole juvenile cuttlefish previously fed with radiolabelled Artemia salina (mean remaining activity ± SD, n =8 at day 0 and n=5 at day 29); E and F: Loss of 65 Zn and 109 Cd in whole adult cuttlefish previously fed with radiolabelled mussels (mean remaining activity ± SD, n =18 at day 0 and n=3 at day 29). Parameters of the equations are given in Table 3.

Caption to figures

Figure 1 .

 1 Figure 1. Sepia officinalis. Transfer factors (TF) of 65 Zn and 109 Cd over time in whole cuttlefish exposed to spiked sediments (mean transfer factor ± SD, n=9 at day 0 to 22, and n=3 at day 29).

Figure 2 .

 2 Figure 2. Sepia officinalis. Uptake of 65 Zn and 109 Cd in whole eggs exposed for 11 d to radiotracer in sea water (mean concentration factor ± SD, n=3). Fitted model: CF t = CF equil (1e -kt ); CF t , CF equil : concentration factors at time t (d) and steady state, respectively; k: rate constant (d -1 ); R²: determination coefficient; p: probability of the model adjustment.

Figure 3 .

 3 Figure 3. Sepia officinalis. Radiotracer distribution (mean %) among the egg compartments at different times during the uptake phase.

Figure 4 .

 4 Figure 4. Sepia officinalis. A and B: Loss of65 Zn and 109 Cd in whole juvenile cuttlefish previously exposed to radiolabelled sea water for 36 h (mean remaining activity ± SD, n =8 at day 0 and n=4 at day 29); C and D: Loss of 65 Zn and 109 Cd in whole juvenile cuttlefish previously fed with radiolabelled Artemia salina (mean remaining activity ± SD, n =8 at day 0 and n=5 at day 29); E and F: Loss of 65 Zn and 109 Cd in whole adult cuttlefish previously fed with radiolabelled mussels (mean remaining activity ± SD, n =18 at day 0 and n=3 at day 29). Parameters of the equations are given in Table3.

  Figure 1

Table 1 .

 1 Sepia officinalis. Concentration factor (CF), radiotracer concentrations (mean Bq g -1 wet wt ± SD) and distribution (mean % ± SD) in the organs of adult cuttlefish after 8 h of exposure in sea water (n=3) and after 6 d of depuration (n=2).

	Tissue	% fresh	65 Zn
		weight

Table 2 .

 2 Sepia officinalis. Radiotracer distribution (mean % ± SD) among three body compartments of juvenile (J) and adult (A) cuttlefish exposed via sediments, sea water, and food followed by 29 d of depuration in uncontaminated sea water.

	Experiment	n	Compartment
		Digestive gland	Cuttlebone	Remainder
	Sediments (J)	3
	65 Zn	75 ± 8	3 ± 0	23 ± 8
	109 Cd	68 ± 7	11 ± 1	20 ± 6
	Sea water (J)	4
	65 Zn	87 ± 3	0.6 ± 0.1	13 ± 3
	109 Cd	76 ± 4	1.3 ± 0.5	23 ± 4
	Artemia (J)	5
	65 Zn	82 ± 2	1.3 ± 0.9	17 ± 2
	109 Cd	91 ± 3	1.2 ± 0.9	8 ± 3
	Mussels (A)	3
	65 Zn	79 ± 4	2 ± 1	19 ± 2
	109 Cd	97 ± 0	< 1	3 ± 0

Table 3 .

 3 Sepia officinalis. Parameters of the equations describing the loss kinetics of 65 Zn and 109 Cd in whole cuttlefish previously exposed through different pathways: (1) juveniles previously exposed for 36 h to radiotracers in sea water; (2) juveniles fed labelled brine shrimp Artemia salina; (3) adults which received a single ration of labelled mussels Mytilus galloprovincialis. O and T: one-and two-component loss model, respectively; ASE: asymptotic standard error; R²: determination coefficient; p: probability of the model adjustment. See text for other abbreviations.

	Pathway Model A 0s (ASE)	λs (ASE)	T b1/2s A 0l (ASE)	λl (ASE)	T b1/2l R²	p
	1. Sea water exposure
	65 Zn	O	92.5 (2.0) 0.013 (0.002)	53	-	-	-	0.98 < 0.001
	109 Cd	T	29.2 (6.7) 1.131 (0.530)	0.6	71.4 (4.2) 0.011 (0.005)	65	0.96 < 0.001
	2. Labelled brine shrimp exposure
	65 Zn	T	37.5 (3.9) 1.044 (0.232)	0.7	63.2 (2.3) 0.004 (0.003) 173.3 0.98 < 0.001
	109 Cd	T	37.8 (6.3) 0.653 (0.223)	1.1	62.3 (4.4) 0.003 (0.004) 256.7 0.96 < 0.001
	3. Labelled mussel exposure
	65 Zn	T	59.1 (4.2) 1.940 (0.545)	0.36	40.9 (3.0) 0.018 (0.007)	38	0.95 < 0.001
	109 Cd	T	46.7 (6.5) 1.509 (0.817)	0.46	53.1 (4.6) -0.010 (0.006)	∞	0.91 < 0.001

Table 4 .

 4 Radioactivity (Bq g -1 wet wt, mean ± SD) in Mytilus galloprovincialis soft parts (n=20) and in Artemia salina (n=10) used as radiolabelled food for adult and juvenile cuttlefish, respectively, and radioactivity (Bq, range) in both groups of Sepia officinalis fed these prey.

	Radiotracer	M. galloprovincialis	S. officinalis	A. salina	S. officinalis
		(n=20)	(n=18 Adults)	Bq / ind.	(n= 8 Juveniles)
		(Bq g -1 wet wt)	(Bq)	(n=10)	(Bq)
	65 Zn	780 ± 129	249-4284	10.92 ± 0.32	30-98
	109 Cd	996 ± 162	117-4126	7.19 ± 0.37	19-68

Cd depuration had very low depuration rate constants (λ l ), which resulted in a very long

Acknowledgements. We thank Dr S. von Boletzky from the Marine Station of Banyuls (France) for providing us with part of the cuttlefish eggs, N. Tevenin and P. Gilles from the Musée Océanographique (Monaco) for supplying adult cuttlefish, and Prof. E. Boucaud-Camou for advice on rearing cuttlefish. MW is a Honorary Research Associate of the National Fund for Scientific Research (NFSR, Belgium). The Marine Environment Laboratory operates under a bipartite agreement between the International Atomic Energy Agency and the Government of the Principality of Monaco. LITERATURE CITED Boletzky S von (1983) Sepia officinalis. In : Boyle PR (ed) Cephalopod Life Cycles, Vol. 1. Species accounts. Academic Press, London, p 31-52 Boucaud-Camou E (1973) Etude de l'appareil digestif de Sepia officinalis L. (Mollusque Céphalopode). Essai d'analyse expérimentale des phénomènes digestifs. PhD Thesis, University of Caen, France, 208 p Boucaud-Camou E, Boucher-Rodoni R (1983) Feeding and digestion in Cephalopods. In: Saleuddin ASM, Wilbur KM (eds) The Mollusca, Vol. 5. Physiology, Part 2. Academic Press, New York, London, p 149-187 Boucher-Rodoni R, Boucaud-Camou E, Mangold K (1987) Feeding and digestion. In: Boyle PR (ed) Cephalopod life cycles. Vol II. Comparative Reviews. Academic Press, New York, London, p 85-108 Brown BE (1982) The form and function of metal-containing 'granules' in invertebrate tissues. Biol Rev Cam Philos Soc 57:621-667 Bustamante P (1998) Etude des processus de bioaccumulation et de détoxication d'éléments traces (métaux lourds et terres rares) chez les mollusques céphalopodes et bivalves pectinidés.


Véronique Langlais

Olivier Guillermet

David Martrou

André Gourdon

Sebastien Gauthier

Nc-AFM Imaging and Manipulation of a Triphenylene Derivative on KBr(001)

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

INTRODUCTION

Using single adsorbed molecules to perform elementary electronic functions or calculations is the main purpose of molecular electronics. But a precise control of a single molecule-based device requires to image the molecule. Imaging single molecules is now performed routinely by STM, which necessitates conducting surfaces (metals, semiconductors or ultrathin insulating layers on conducting substrates). This condition constitutes a severe limitation and in most cases using molecules adsorbed on bulk insulators would greatly simplify the device design and operation.

Due to the continuous development of Atomic Force Microscopy in non-contact mode (nc-AFM), the number of studies devoted to the adsorption of molecular layers or islands on bulk insulators is steadily increasing [1][2][3][4][5][6][7][8][9][10][11][12][13]. Nevertheless, only very few investigations of single molecules adsorbed on bulk insulators are reported until now, because, at room temperature, most molecules tend to diffuse on these substrates. No isolated molecules could be observed at room temperature so far except when trapped on step edges [3,4].

At low temperature, where diffusion is inhibited, the adsorption of Co-Salen on NaCl(001) [2] and NiO(001) [9] at 8K has been studied in detail by nc-AFM from constant ∆f images. Based on these experiments, the interest of using metallic tips on this type of systems was pointed out.

Here, we report on a study on individual isolated hexamethoxytriphenylene molecules (HMTP: Fig 1a) on the (001) surface of the bulk insulator KBr. This study is performed in UHV, at low temperature (5K). This system was already studied at room temperature [5].

In the following, after describing our experimental methods, we present nc-AFM frequency shift images of isolated HMTP obtained in the constant height mode. These images display submolecular contrast that we interpret on the basis of force-field-based numerical simulations. The same methods are then applied to understand why and in which conditions isolated HMTP molecules are manipulated (laterally or vertically) by the tip. This analysis is followed by a discussion and conclusions.

METHODS

Experimental

The KBr crystal was cleaved in air just before being introduced in the UHV system. It was annealed at 150°C for 1 hour in order to remove residual charges originating from the cleavage process. The sample was then transferred to the low temperature (5K) STM/nc-AFM (Scienta Omicron). HMTP molecules, in powder form, were then deposited on the cooled (T<10 K) surface from a heated crucible. The force sensor was a tuning fork of the qPlus sensor type with resonance frequency f0 and Q factor of the order of 25,000 Hz and 20,000 respectively. It was equipped with a glued electrochemically etched tungsten tip. The control electronics were provided by SPECS. All images, spectra, and manipulation experiments were performed at a temperature of 5K. All the displayed experimental images are oriented such that the vertical and horizontal directions correspond to <001> non polar crystallographic directions of the KBr surface.

Numerical

The COMPASS (Condensed-phase Optimized Molecular Potentials for Atomistic Simulation Studies) force-field [14] was used with Materials Studio [15] to optimize the conformation of an isolated molecule on KBr(001) in the presence of the tip. This force-field is well suited to calculate the adsorption of organic molecules on the surface of inorganic materials. It has been parameterized using condensed phase properties as well as ab initio and empirical data for isolated molecules [16,17]. Previous studies have shown that the interaction of organic molecules with ionic surfaces is dominated by van der Waals and electrostatic forces. In addition, these works indicate that the charge transfer between the substrate and the molecule is generally negligible for this type of surfaces [18], ensuring that force-field based calculations are sufficient for a realistic description of these systems. A constant ∆f image of HMTP molecules at low coverage on KBr(001) is displayed in Fig. 1b. Single molecules appear as circular, featureless objects, with a mean diameter of ≈ 1.3 nm and a height of ≈ 0.18 nm. These dimensions are compatible with the molecular model shown in Fig. 1a. The bigger object could originate from an aggregate of a few molecules. The image of the single molecules do not show any intra-molecular resolution. Trying from the imaging conditions of Fig. 1b to improve the resolution by optimizing either ∆f or the amplitude A led to molecular manipulation, where the molecules are laterally displaced by the scanning tip as illustrated in Fig. 1c.

RESULTS

NC-AFM imaging of HMTP molecules on the

b. Constant height images

The constant height mode, which produces ∆f images, is increasingly used since the pioneering work of Gross et al [19] on the high resolution imaging of pentacene/NaCl/Cu(111) at low temperature. It is also frequently used at room temperature (see for instance [13]). Its advantages are that one of the feedback loops of nc-AFM is disabled, simplifying the settings of the instrument and, more importantly, the tip can approach or go beyond the minimum of the ∆f(Z) curve without risking a tip crash, in contrast to the more conventional constant ∆f mode. While the constant height mode is generally restricted to very flat samples, it has also been used efficiently to image the top facet of clusters [20]. The evolution of the image of HMTP-A when Z decreases is characterized by the apparition of a nearly circular black depression, with a diameter that increases from 1 nm to 1.4 nm, on which submolecular clearer features emerge, reaching positive frequency shifts for the smaller Z. HMTP-B and C follow the same trends. This behavior is similar to the observations reported by Gross et al [19], where pentacene molecules where imaged at constant height with a CO-terminated tip. The black depression was associated with long range attractive electrostatic and van der Waals interactions, while the submolecular features were interpreted in terms of Pauli repulsive interactions. Despite these similarities, the situation is different here: the tip is not intentionally functionalized, and the size of the submolecular features does not go down to atomic resolution as in [19].

Beyond this general behavior, these images also share common features such as minima (2 for HMTP-A, 2 to 4 for HMTP-B, 2 to 3 for HMTP-C) with quite similar topologies. But they are not identical. Note that each of these series of images was obtained with the same qPlus sensor, but with different tip apices since the tip was often reconditioned by gentle contacts with the surface while measuring ∆f(Z) approach curves. In the following, we try to interpret the contrasts observed on the images of Fig. 2. More specifically, we address the following questions:

Which interactions are involved in the formation on the contrast? Why is the contrast different from one molecule to another? Can we extract information about the tip?

We have two objectives in mind. The first one is to get as much information as possible on the adsorption of HMTP on KBr(001). The second, more methodological, consists in identifying the experimental factors that limit the spatial resolution of nc-AFM on this type of system.

Modeling of HMTP on KBr(001)

The purpose of this modeling is not to reproduce precisely the experimental results. One of the main obstacles toward this objective is to determine the precise structure of the tip, a huge task which has been achieved in only a few cases (see for instance [21]). It requires to perform calculations with a large number of plausible tip structures until an acceptable fit with the experimental results is achieved.

Here, following the pioneering work reported in [22], we will consider that the tip is constituted of a KBr cluster linked to the W tip of the qPlus sensor. Indeed, it is generally accepted that when imaging the surface of ionic crystals, in particular KBr, the tip is rapidly covered by material that has been picked up on the sample surface [23]. The structure of the tip is kept as simple as possible, being modelled by a KBr cube of 8 atoms oriented with its diagonal perpendicular to the sample surface with either a Br ion or a K ion pointing toward the surface. It was checked that including more ions in the tip, for instance 36 atoms as shown in Supporting Information (SI) Figure S2, does not significantly change the conclusions of our study.

The KBr slab was four (001) atomic layers thick. The surface was constituted by 6x6 conventional unit cells, with a vacuum slab of 4 nm to avoid any coupling between neighboring supercells due to the periodic boundary conditions. The van der Waals and electrostatic energies were calculated using the Ewald summation method.

Adsorption geometry of HMTP on KBr(001)

The minimum energy conformation obtained by calculation is illustrated in Fig. 3. For this calculation the two upper layers of the slab as well as the molecule were allowed to relax. The adsorption energy is E0=0.87 eV. The molecule binds to the surface by positioning two of its oxygen atoms directly above the two K + ions colored in green in Figure 3, with a K + -O distance ≈ 0.3 nm, while the other four oxygen atoms are distant by more than ≈ 0.38 nm from any K+ ion. As can be seen on the side view in Figure 3b, this electrostatic interaction bends the central aromatic part of the molecule downwards. This constitutes another example of a adsorption stabilized by "charge matching" as already illustrated for instance for another triphenylene derivative [7] or for functionalized truxenes [3] on KBr(001).

Figure 4 displays the van der Waals surface of HMTP colored according to its molecular electrostatic potential. As expected, the oxygen atoms are surrounded by a negative potential while the methyl groups are surrounded by a positive potential. The charge matching derives from the nearly perfect equality between the distance separating the two binding oxygen atoms of the molecule (0.69 nm, Figure 4) and the distance between the two second nearest K + ions of the substrate (0.66 nm, Figure 3a) onto which the molecule is electrostatically bound.

Calculation of ∆f maps

In a first step, the total energy E of the system constituted by the molecule in its optimized conformation on the substrate in interaction with the tip was calculated, without any further relaxation. This calculation was performed for each position (x, y, z) of the tip. The tip-

surface force z E z y x F ∂ ∂ - = ) , , (
and its derivative relative to z were evaluated by numerical differentiation.

) , , ( z y x f ∆ was then calculated by numerical integration of the relation derived by Giessibl [24]:

[ ] ) 1 ( ) 2 sin( ) 2 sin( , , ( 2 ) , , ( 0 0
/ 1 0 2 0 0 t f A t f A D y x F dt A k f z y x f f π π π + - = ∆

∫

where D is the distance between the front tip ion and the substrate when A=0.

∆f images calculated for a Br -tip or a K + tip are displayed in Fig. 5 and6 respectively. Fig. 5c and 6c are obtained from the force derivative map according to the formula:

z F k f f A ∂ ∂ - = ∆ → 2 lim 0 0
giving the small amplitude limit of equation (1). As expected, these images are nearly identical to the images at A=10 pm. The images calculated with the experimental amplitude A=150 pm are very similar to the images calculated with A=10 pm. This is a first evidence that the image contrast formation arises from long range interaction, in contrast to the case of atomic resolution mentioned previously. In addition, the contrast obtained with the K + tip is qualitatively the opposite of the contrast obtained with the Br -tip. This observation clearly indicates that the image is dominated by the long range electrostatic interaction in the selected D range. It is immediately obvious that the calculation with the K + tip does not compare favorably with the experimental images of Figure 2. In contrast, a fair qualitative agreement between the Br - tip images and the experimental images is obtained, especially for HMTP-C, which displays, like in the calculation a unique maximum with two marked minima. Thus, we conclude that the tip used in the experiment was of the Br -type. This conclusion is also consistent with the electrostatic potential map of Fig. 4a: the negatively polarized tip is attracted by the positive (blue) regions, resulting in the two minima, while it is repelled by the central more negative (yellow) region. In addition, the electrostatic potential map shows that the appearance of the minima is closely related with the orientation of the O-CH3 bounds in the molecule. For instance, the potential is more positive when these bounds point toward the outside of each O pair, as indicated by the blue arrows in Figure 4a andc.

Note that this comparison also excludes a metallic tip, since in most cases the polarity of these tips is positive, i.e. their associated dipole is pointing outside [25,26].

The van der Waals and electrostatic contributions to the ∆f image of the minimum energy configuration of HMTP on KBr(001) are displayed in Figure 7. The van der Waals image only exhibits attractive interaction. In contrast both attractive and repulsive interactions contribute to the electrostatic image. The intramolecular contrast is therefore purely of electrostatic origin.

Figure 7: (a) van der Waals and (b) electrostatic contribution to (c) the ∆f image of the minimum energy configuration of HMTP on KBr(001).

Figure 8 shows, in addition to the minimum energy configuration already discussed, two other conformations of slightly lower adsorption energies that were obtained by optimizing the system after quenching a molecular dynamics simulation. They correspond to local relative minima of the potential energy. These conformations can be distinguished by the orientation of their methyl groups: conformation (b) is obtained from (a) by rotating around their CO bond the 2 upper methyl groups (surrounded by a blue ellipse in Figure 8a andb) while conformation (c) is obtained from (a) by rotating the lower right methyl group (marked by a red circle in Figure 8a andc). This last change induces a slight rotation of the whole molecule, which ends in a fully symmetric situation, aligned along a non-polar [100] direction of KBr(001).

Even if the images still share the common features mentioned previously, the location of the methyl groups has a noticeable influence: by changing the relative altitude of the central aromatic part, the oxygen atoms and the methyl groups, they alter the relative intensity of the maxima and minima of the images. The influence of the size of the tip and its orientation (tilt of the tip axis or rotation along an axis perpendicular to the surface) was also checked. The results, reported in Supporting Information Figure S2 andS3, only show a limited influence of these parameters.

To go further, the same kind of calculations were performed allowing the molecule and the first atomic layer of the KBr substrate to relax at each position of the tip. Three behaviors can be distinguished, depending on D. When the tip is far away from the surface, the relaxations of the molecule and the substrate induced by the tip are very small and do not affect the images. In the intermediate range, the molecule is reversibly displaced from its equilibrium position in the absence of the tip. These displacements can be quite large (~50 pm) and make the method for calculating the images previously described inadequate. A realistic modelling would require molecular dynamics simulation at each point, which is beyond the scope of this paper. For shorter D distances, irreversible displacements occur: the molecule is laterally or even vetically manipulated by the tip.

Manipulations

Reducing the tip-substrate distance from the stable imaging conditions used in Figure 2 led quite often to lateral molecular manipulation, as illustrated in the constant height ∆f image of Figure 9. The molecule is displaced along a polar [110] direction of the substrate, as quite often observed on alkali halide substrates. The ∆f profile along the manipulation path, displayed in Figure 9b shows that the molecule proceeds by jumps of 0.47 nm length, corresponding to the nearest neighbor distance along <110> directions. The shape of this profile suggests that the molecule is repelled by the tip: in the first part of the elementary manipulation step, the tip images the molecule until a sudden jump happens, corresponding to the translation of the molecule to the next equivalent site of the trajectory. This is typical of a "pushing" mode [27][28][29]. Note that it was necessary to lift the tip by 50 pm to release the molecule, as shown on the Z profile in Figure 9b. The tip always stays on the left of the molecule, meaning that the manipulation proceeds indeed by pushing. A careful examination of the manipulation sequence (SI) shows that one of the oxygen atoms of the molecule (O1 in Figure 10a) interacts strongly with the closest K + ions of the tip second layer (KTip in Figure 10a). This bond allows the tip to lift one of the branches of the molecule (Figure 10 c andd), facilitating the displacement toward the next site along the [100] substrate direction.

The length of this dO1-Ktip bond goes down to 0.27 nm. This corresponds to the equilibrium distance between an isolated K+ ion and an oxygen atom of the molecule, as given by the numerical simulation. This short distance indicates that the O1 is strongly bound to the tip that pushes on the molecule as suggested by the images of Figure 10. It means that, for manipulation, in contrast to imaging, the repulsive short-range part of the van der Waals interaction plays a significant role. Similar calculations with a K + tip show that in this case the molecule is manipulated at much larger tip-surface distance. The calculated sequence (V3 and V4 in SI) shows that even at large distance (closest approach = D-A = 0.98 nm) the molecule can be picked up by the tip.

The images of Figure 11 are extracted from this sequence.

The molecule is at first partially lifted up by the interaction of the K + terminating tip ion with one then two of the oxygen atoms (O5 and O4) of the molecule (Fig. 11b andc). It is then rotated by 90° and translated along the scanning direction (Fig. 11d ande). After the inversion of the scanning direction, the molecule is pulled in the opposite direction and the two oxygen atoms interact now with a K + ion on the right side of the tip (Fig. 11f andg). Finally the molecule is picked up by the tip (Fig. 11h andi). Here also, as indicated in Figure 11c andi, the distances between O4 and O5 and the K+ ions of the tip approaches the O-K+ equilibrium distance of 0.27 nm, revealing the contribution of the repulsive short-range part of the van der Waals interaction. This finding could explain why most of the experimental images we obtained are of the "Br - tip" type: with K + tip the molecule is picked up before a clear contrast could be obtained.

Discussion and conclusions

The comparison of the images of Figure 5, calculated with a Br -tip and the experimental images of Figure 2 shows a fair qualitative agreement. They display a near central clear protrusion, reaching frequency shifts that are higher than on KBr, surrounded by two or more dark minima. Furthermore, the order of magnitude of ∆f is comparable for the D range chosen for the calculations. In addition, the comparison with the images of Figure 6, obtained with a K + tip, clearly excludes a positive polarity tip. These findings lead to the following conclusions:

-The tip used in the experiment has a negative polarity -The experimental tip surface distance is around D=1.1 nm. For much smaller values of D (below ~ 0.85 nm), the molecule is manipulated while for much higher values (above ~ 1.3 nm), the ∆f range becomes too small. In this range, the contrast is dominated by the electrostatic interaction as demonstrated in Figure 7.

These conclusions are very robust: they do not depend strongly on the size (Figure S2) and orientation of the tip (Figure S3). This robustness is obviously the consequence of the long range interactions that are involved in the image formation; the image is the result of a spatial average on a length scale given by the range of the interaction, i.e. nanometers in our case and is rather insensitive to atomic details. These findings are also consistent with the simulations of the manipulation process with the two kinds of tips shown in the preceding part. These simulations highlight the important role played by the bounds that form between the oxygen atoms of the molecule and some K+ ions of the tip, whatever the polarity of the tip. This is not surprising as the partial charge on the oxygen atom is by far the highest in the molecule (see Figure S1). The formation of these bounds means that in the manipulation process, the short-range repulsive contribution of the tip molecule potential plays an important role. The consequence is that, in contrast to the case of imaging, the atomic structure of the tip is expected to have a strong influence on the manipulation process.

The only way to improve the imaging resolution would be to reduce the tip-surface distance.

But, in this situation, the molecule is not stable enough on the surface to resist the force applied by the tip. This limitation depends in a complex way on the structure of the moleculesurface system. Even if the adsorption energy is not so small (E0=0.87 eV), the flexibility of the triphenylene core provides low force paths to displace or pick up the molecule, as shown in Figure 10 and11.

Nevertheless, the results of Figure 8 shows that the position of the methyl groups of the molecule have a noticeable influence. This result demonstrates that a precise adsorption structure determination should be possible from experimental images, even with a resolution limited by the long range nature of electrostatic forces. Video files V1.avi to V4.avi: Calculated manipulation sequences with a Br -tip and a K + tip.

Supporting Information Description

Table of Contents Graphics

Manipulation with a Br -tip

Video files: V1.avi and V2.avi Legend: Top view and side view along X of a calculated manipulation sequence with the Br - tip. D = 0.73 nm, A = 70 pm. The tip Br -ions is colored in blue. The surface K + ions on which the molecule is electrostatically bound at the beginning (end) of the sequence are colored in yellow (green). The Z oscillation of the tip is not displayed but only the frames where the tip is closest to the surface.

An inspection of V1 and V2 shows that the molecule interacts in a globally repulsive way with the two methoxy groups that include O1 and O2. The molecule "oscillate" (anticlockwise rotation on O1, clockwise rotation on O2) but stays in the same adsorption site until frame ~ 5650. The distances remain above 0.4 nm, i.e. in the electrostatic range (Fig. S4b).

From frame ~ 5650 to frame ~ 6300, the system is in a transitory state during which the molecule is laterally displaced (Fig. S4d) . Fig. S4c shows that before the manipulation, the molecule is bound to the surface by O1 on K1 and O2 on K2, as shown by the red and green curves (dO1K1 and dO2K2 < 0.35 nm). After the manipulation, the molecule binds to the surface by O2 on K3 and O1 on K5 (pink and dark yellow curves, dO2K3 and dO1K5 < 0.35 nm). ). It has moved by one nearest neighbor distance along <100>.

A jump occurs at frame 5684 (arrow 2 in Fig. S4 d ande). Fig. S4d shows that O1 gets closer to the tip (green and pink curves). Simultaneously O1 is lifted from the surface (Fig. S4e green curve). O1 is captured by the tip, lifting the extremity of the molecule from the surface (see also Fig. 10).

From frame ~ 5700 to frame ~ 5900, O3 and O4 get closer to K3 and K4 while O1 gets farther from K1. From frame ~ 5900 to ~ 6250, the molecule binds to the surface by O3 on K3 (blue curve in Fig. S4e ) and O4 on K4 (pink curve in Fig. S4e ).

A final jump occurs near frame 6300 (arrow 3 in Fig. S4 d ande). O2 binds to K5 and O1 binds to K3.

1 nc-

 1 AFM imaging and manipulation of a triphenylene derivative on KBr(001) V. Langlais a , O. Guillermet a,b , D. Martrou a , A. Gourdon a , S. Gauthier * a (a) NanoSciences Group, CEMES, CNRS UPR 8011, 29 rue J. Marvig, 31055 Toulouse (France) (b) Université de Toulouse, UPS, 29 rue J. Marvig, 31055 Toulouse (France) * Corresponding author: gauthier@cemes.fr

Figure 1 :

 1 Figure 1: (a) HMTP optimized in vacuum. Oxygen atoms are depicted in red, carbon in grey

Fig. 2

 2 Fig. 2 displays constant height ∆f images of 3 single HMTP molecules at different heights Z.

Figure 2 :

 2 Figure 2: Constant height ∆f images of 3 HMTP molecules at different height Z.

Figure 3 :

 3 Figure 3: (a) Top and (b) side view of HMTP in its minimum energy configuration on KBr(001). K + ions are depicted in violet, Br -ions in pale red. The two K + ions on which the

Figure 4 :

 4 Figure 4: Molecular van der Waals surface of HMTP colored according to its electrostatic

Figure 5 :

 5 Figure 5: ∆f images calculated with a Br -tip at D=11 Å. (a) A=150 pm, (b) A=10 pm, (c)

Figure 6 :

 6 Figure 6: ∆f image calculated with a K + tip at D=11 Å. (a) A=150 pm, (b) A=10 pm, (c)

Figure 8 :

 8 Figure 8: Side and top views and the corresponding images for the three lowest energy

Figure 9 :

 9 Figure 9: (a) Constant height ∆f image of a HMTP manipulation sequence. (b) Profile along

Figure 10 :

 10 Figure 10: Images extracted from V1 and V2 (SI). (b), (d) and (f): Views along +X. For the sake of simplicity, the tip is represented by two of its ions only. The Br -ion terminating the tip is colored in blue, its K + ion is colored in green. The yellow K + ions are the initial and final

Figure 11 :

 11 Figure 11: Images extracted from the K + tip manipulation sequence V4 (SI). Views along +X.

Figure S1 :Figure S2 :Figure S3 :Figure S4 :

 S1S2S3S4 Figure S1: Atomic charges of HMTP

Figure S4 :

 S4 Figure S4: Time evolution during the manipulation sequence of videos V1 and V2 of the distances between selected O atoms of the molecule and : -(b) and (d) the terminating Br-and one of the K+ of the tip -(c) and (e) K1 to K5 K+ ions of the surface (d) and (e) are enlargements of (b) and (c). See (a) for the labelling of the different species.

Acknowledgements: This work has been supported by the European Commission within

the PAMS project (Contract N° 610446).

Influence of the tip size

Influence of the tip orientation

Around frame ~ 6000, dO1-Ktip goes below 0.27 nm. This is less that the sum of the ionic radii of O -and K + . It indicates that the O1 is strongly bound to the tip that pushes on the molecule as suggested by the images of Figure 10.

Note that these two jumps are preceded by an unsuccessful event (arrow 1 in Fig. S4 d ande):

The molecule starts to move as immediately after jump 2 but goes back to its initial position (O1 on K1 and O2 on K2, red and green curves). It is only when X is larger by 0.1 nm, i.e. when the tip is closer to the molecule that the manipulation will be successful.

Manipulation with a K + tip

V3.avi: Top view of a calculated manipulation sequence with the K + tip.

V4.avi: Side view (seen along -X) of the same manipulation sequence as in V3.avi. A= 110 pm, D= 1.09 nm.