Dr Paco Bustamante

R P Cosson

I Gallien

F Caurant

P Miramand

Cadmium detoxification processes in the digestive gland of cephalopods in relation to accumulated cadmium concentrations

Keywords: cephalopods, cadmium, metallothionein, detoxification

The high concentrations of cadmium recorded in the digestive gland of cephalopods from various temperate and subpolar waters suggest that these molluscs have developed efficient cadmium detoxification mechanisms. The subcellular distribution of cadmium in the digestive gland cells was investigated in seven cephalopod species from the Bay of Biscay (France) and the Faroe Islands. In most species, cadmium was mainly found in the cytosolic fraction of the digestive gland cells, reaching up to 86% of the total cadmium for the squid Loligo vulgaris from the Bay of Biscay. But species with the highest total level of cadmium showed a higher percentage of cadmium associated to insoluble compounds. The quantification of metallothioneins (MTs) by the polarographic method was performed in order to evaluate the involvement of these proteins in the detoxification of the high amounts of bioaccumulated cadmium. Metallothionein levels in cephalopods ranged form 742 ± 270 to 3478 ± 1572 µg/g wwt. No relationship could be established between total cadmium, cytosolic cadmium and MT levels suggesting the occurrence of other Cd-binding ligands. Although these proteins have not been characterised, as cadmium in the digestive gland of cephalopods is mainly associated with soluble ligands, a high potential transfer to predators can be predicted.

INTRODUCTION

Cephalopods are regarded as key species in many marine ecosystems [START_REF] Amaratunga | I.F. Caddy, Advances in assessment of world cephalopod resources[END_REF][START_REF] Rodhouse | Antarctic cephalopods -a living marine resource ?[END_REF]). They represent an essential link in marine trophic chains and are eaten by many marine top predators, fish, birds and mammals (see the reviews of [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][START_REF] Klages | Cephalopods as prey. II. Seals[END_REF]. Owing to their ecological importance, the few high background levels of cadmium reported in these molluscs [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] 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], Bustamante 1998, Bustamante et al. 1998ab) are of particular interest. Moreover, species from subpolar areas showed higher levels of cadmium than those living in temperate ones (Bustamante 1998, Bustamante et al. 1998ab) while exposure to soluble metals in natural seawater is extremely low in polar regions [START_REF] Mart | Comparative studies on the distribution of heavy metals in the oceans and coastal waters[END_REF][START_REF] Donat | Trace elements in the oceans[END_REF]. In both areas, cadmium is mainly accumulated in the digestive gland [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][START_REF] Bustamante | Etude des processus de bioaccumulation et de détoxication d'éléments traces (métaux lourds et terres rares) chez les mollusques céphalopodes et bivalves pectinidés. Implication de leur biodisponibilité pour le transfert vers les prédateurs[END_REF], Bustamante et al. 1998a), reaching up to 98% of the total body cadmium in some species.

Consequently, this organ appears to have a key function in the metabolism of cadmium in cephalopods. Very high levels of cadmium in the tissues of cephalopods would be expected to be toxic to the organisms unless efficient storage and detoxification mechanisms are available [START_REF] Simkiss | Metal detoxification and bioaccumulation in molluscs[END_REF][START_REF] Phillips | Strategies of metal sequestration in aquatic organisms[END_REF].

One well know detoxification strategy of marine invertebrates involves the binding of some trace metals to metallothioneins. These proteins which play a role in the homeostasis of the essential elements, copper and zinc [START_REF] Engle | Metallothionein and metallothionein-like proteins: physiological importance[END_REF][START_REF] Cosson | Metallothioneins and detoxification. Is the use of detoxification proteins for MTs a language abuse ?[END_REF][START_REF] George | Metallothioneins as indicators of trace metal pollution[END_REF], are induced by various metals (i.e. Cd, Cu, Zn, Ag and Hg). As a consequence, metallothioneins are considered to be involved in cadmium detoxification [START_REF] Dallinger | Strategies of metal detoxification in terrestrial invertebrates: Synopsis and perspectives[END_REF], 1996[START_REF] Roesijadi | Metallothionein in metal regulation and toxicity in aquatic animals[END_REF], 1995[START_REF] Viarengo | Mini-review. Mechanisms of heavy metal cation homeostasis in marine invertebrates[END_REF]. The occurrence of metallothioneins in cephalopods has not been demonstrated although the occurrence of proteins with a molecular weight similar to metallothioneins has been shown. These are associated mainly with copper, and, to lesser extent cadmium but little zinc [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].

Taking into account the very high levels of cadmium observed in several cephalopod species, the aim of our study was to investigate how they manage to tolerate such amounts of toxic metal. Our analyses provided us with information on the subcellular distribution of cadmium and with the involvement of metallothioneins in the detoxification processes. A comparison has been made between cephalopods from temperate and subpolar waters.

MATERIALS & METHODS

The level of metallothionein (MTs) and the subcellular distribution of metals were determined in the digestive gland of several species of cephalopods collected by bottom trawl in waters of the Bay of Biscay and Faroe Islands during 1997.

Description of the samples

Cephalopods were sampled 1) during a cruise on the Magnus Heinason in august 1997 on the Faroe eastern continental shelf, at several stations located between 60°30' and 62°50' N, and 3°40' and 7° 60' W; 2) during the RESSGASC cruise in February and May 1997 on the continental shelf of the Bay of Biscay (French Atlantic coast), at several stations located between 45° 14' and 46° 07' N, and 01° 24' and 01° 52' W.

Only living animals were selected for on board-dissection (see below). We investigated both pelagic (i.e. squids) and benthic (i.e. cuttlefishes and octopuses) cephalopod species commonly encountered in French nearshore waters, i.e. Loliginidae and Ommastrephidae squids (Loligo vulgaris, Illex coindetii), cuttlefish (Sepia officinalis, S. elegans, S. orbignyana) and some other species from the Faroe Islands, i.e. the Ommastrephidae squid Todarodes sagittatus and the octopus Eledone cirrhosa. Table 1 gives the origin, number of individuals, mantle length and weight for each species of cephalopod.

Preparation of the samples

The digestive gland of each animal was isolated on board and deep-frozen in liquid nitrogen.

In the laboratory, the samples were kept at -80°C until used. Each sample was weighed (wet weight) and homogenised with a mortar and pestle on ice with 10 volumes of a 0.02 M TRIS-HCl, 0.25 M sucrose buffer [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], containing 1 mM PMSF (phenylmethylsulfonylfluoride) as protease inhibitor and 5 mM DTT (dithiothreitol) as reducing agent, at pH 8,6. The homogenates were centrifuged at 100 000g for 1 h at 4°C in a Berkman LE-70 ultracentrifuge. Particle-free supernatants (cytosols) were separated from the pellet containing cellular membranes, nucleus, lysosomal and mitochondrial material. Two aliquots (1 ml each) were removed from the supernatant for protein and metallothionein quantification. Aliquots of the homogenates, cytosols and pellets were analysed for cadmium.

Cadmium analyses

The content of cadmium in the different fractions was determined by atomic absorption spectrophotometry (AAS) after acid digestion with 4 ml of 65% HNO 3 and 1 ml of 70% HClO 4 for 24 hours at 80°C. The acid was removed from the samples by evaporation and the residues were diluted in 10 ml of 1 N nitric acid. Cadmium was determined by flame and flameless AAS using a using a Varian spectrophotometer Vectra 250 Plus with deuterium background correction. Blanks and reference materials (dogfish liver DOLT-2, NRCC) were taken through the procedure in the same way as the sample. Our results (21.2 ± 1.5 µg.g -1 dwt, n=3) were in good agreement with certified values (20.8 ± 0.5 µg.g -1 dwt). Levels of cadmium are given relative to the fresh weight of tissue.

Quantification of metallothioneins

The amounts of MTs were determined using the polarographic method [START_REF] Olafson | An electrochemical approach to quantification and characterisation of metallothioneins[END_REF][START_REF] Thompson | An improved electrochemical method for the quantification of metallothioneins in marine organisms[END_REF][START_REF] Olafson | Electrochemical detection of metallothionein[END_REF] in supernatants obtained after heat denaturation (70°C, 15 min.) and centrifugation (15 000g, 10 min.). The levels of MTs were expressed relative to the fresh weight of tissues using rabbit liver metallothionein (Sigma) for the standard addition calibration. All the reagents used did not give a polarographic signal during the dosage of metallothioneins [START_REF] Erk | Advantages and disadvantages of voltametric method in studying cadmium-metallothionein interactions[END_REF].

Gel-filtration chromatography

Aliquots of heat-treated cytosol from the digestive gland of each species of cephalopods were chromatographed on a Waters Protein-Pack 125 (7.8x300 mm) column and eluted with 0.1 M TRIS-HCl, 0.25 M sucrose buffer, pH 8.6. The column was maintained at 4°C and the samples were collected as 1.5 ml fractions. The ultraviolet absorbency of the eluate was measured at λ=254 nm and λ=280 nm. Differential pulse polarography was used to determine the thiolic content of each chromatographic fraction as explained above. The column was calibrated for molecular weight estimations with Bovine Serum Albumin (66 kDa), Rabbit Liver Metallothionein (12.5 kDa) and Aprotinine (6.5 kDa) as standard markers.

RESULTS

Subcellular distribution

The partitioning of Cd between the pellet and the soluble fractions as a function of the total Cd content in the digestive gland is shown in Figure 1. A significant negative correlation exists between these parameters (df=6, r=0.68, P<0.05). The distribution of cadmium between the pellet and the supernatant was fairly stable in each species. Two-way ANOVA did not show any significant differences among size and sex for the species with numerous individuals (i.e. Todarodes sagittatus and Loligo vulgaris) (Figure 2).

Partitioning of cadmium between the pellet and the supernatant was highly variable between species (Figure 1). Nevertheless, more than 50% of cadmium was present in the soluble fraction in most of the species, reaching up to 86 ± 12% for the squid Loligo vulgaris from the Bay of Biscay; the only exception was the octopus Eledone cirrhosa from the Faroe Islands where only 42 ± 17% of the total cadmium was found associated to the soluble fraction.

Species with the lowest cadmium levels displayed the highest percentage of soluble cadmium (e.g. L. vulgaris).

The subcellular distributions of cadmium in the digestive gland of the squids Loligo vulgaris from the Bay of Biscay and Todarodes sagittatus from the Faroe Islands are shown in Figure 3. The amount of cadmium in the cytosolic fraction increased significantly as a function of total cadmium level in the digestive gland of both species (df=16, r=0.991, P<0.001 for L. vulgaris and df=20, r=0.887, P<0.001 for T. sagittatus) whilst the amount of cadmium in the insoluble fraction increased significantly with the total cadmium level in the digestive gland only for T. sagittatus (df=20, r=0.906, P<0.001).

Metallothioneins

Typical chromatographic elution profiles of the absorbance (λ=254, 280 nm) in heat treated cytosol from the digestive gland of cephalopods are shown in Figure 4. Except for the squid Loligo vulgaris, the principal cadmium-binding component has an apparent molecular weight of approximately 6.5 kDa, eluting between fractions 6-7. However, all the cephalopods chromatographic profiles displayed absorbency peak around 12.5 kDa that corresponds to the metallothionein pool. Thus, 75% of the polarographic activity was detected in these fractions showing unequivocally that thiolic response in the heat-treated cytosol corresponds to metallothionein fractions. Sepia officinalis was the only exception with 44% of the polarographic activity at 12.5 kDa and 40% for molecular weight around 3 to 4 kDa. For L. vulgaris, another peak at 254 nm was detected in the fraction 2, corresponding to proteins having a molecular weight around 80 kDa.

The levels of metallothioneins in the heat-stable soluble fraction of the digestive gland of cephalopods and the levels of total cadmium in this tissue are shown in Figure 5.

Metallothionein levels were fairly stable within species but were highly variable between species exhibiting a significant decrease (df=6, r=0.81, P<0.001) when the Cd concentrations increased in the digestive gland. The concentrations ranged from 700 ± 300 µg.g -1 wwt in the octopus Eledone cirrhosa from the Faroe Islands up to 3500 ± 600 µg.g -1 wwt in the squid Loligo vulgaris from the Bay of Biscay and corresponded respectively to the highest and the lowest total cadmium levels in the digestive gland (i.e. 4 ± 2 µg.g -1 wwt and 397 ± 126 µg.g -1 wwt).

DISCUSSION

In our study, except in Eledone cirrhosa from the Faroe Islands, cadmium was mainly located in the soluble fraction of the digestive gland cells, probably bound to cytosolic proteins. Very few studies have investigated the subcellular distribution of trace elements in the digestive gland of cephalopods and results are quite different between authors [START_REF] Rocca | Copper distribution in Octopus vulgaris Lam[END_REF][START_REF] Decleir | Determination of proteinbound copper and zinc in some organs of the cuttlefish Sepia officinalis L[END_REF][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]. Copper has been investigated in all these studies, but the subcellular distribution of cadmium has only been studied by [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] and [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] 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] found that most of the cadmium was bound to the organelles of the digestive gland cells of the squid Todarodes pacificus, with only 26 ± 3 % of the metal in the cytosolic fraction. On the contrary, most of this metal, i.e. 70 ± 10 %, was bound to cytosolic proteins in the squid Nototodarus gouldi [START_REF] Finger | Molecular association of Cu, Zn, Cd and 210 Po in the digestive gland of the squid Nototodarus gouldi[END_REF]. Despite very different subcellular distributions between T. pacificus and N. gouldi, total cadmium levels in the digestive gland were in the same range within both species, ranging from 40 to 100 µg.g -1 dwt for T. pacificus and from 11 to 88 µg.g -1 dwt for N. gouldi [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]. Our results concerning seven cephalopod species are in good agreement with those of [START_REF] Finger | Molecular association of Cu, Zn, Cd and 210 Po in the digestive gland of the squid Nototodarus gouldi[END_REF] but not with the results of [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] (Figure 1).

The subcellular distribution of cadmium seems to vary according to the levels of total cadmium in the tissue (Figure 1). Thus, comparing species, as total cadmium levels increase in the tissue, the metal is proportionately more abundant in the insoluble fraction, bound to organelles and/or membranes. Trace element precipitation into metal-rich granules is an efficient metal detoxification mechanisms reported for a great number of invertebrate species [START_REF] Coombs | Mechanisms of immobilisation and detoxification of metals in marine organisms[END_REF][START_REF] Brown | The form and function of metal-containing 'granules' in invertebrate tissues[END_REF][START_REF] Taylor | Inorganic deposits in invertebrates tissues[END_REF]. These granules function in the storage and excretion of essential and non-essential metals, and their production is common in all major phyla [START_REF] Brown | The form and function of metal-containing 'granules' in invertebrate tissues[END_REF][START_REF] George | Subcellular accumulation and detoxification of metals in aquatic animals[END_REF]. Thus, the observed increase of total cadmium levels in the digestive gland of cephalopods, resulting in the storage of cadmium in an insoluble form rather than via binding to cytosolic proteins, could be considered as more efficient to counteract the toxicity of the metal. Ultrastructural observations failed to show such insoluble compounds in several cephalopod species [START_REF] Mangold | L'appareil digestif et la digestion[END_REF]. Similar observations were made in the digestive gland cells of the squid Todarodes sagittatus from the Faroe Islands, a species which exhibited relatively high cadmium levels [START_REF] Bustamante | Etude des processus de bioaccumulation et de détoxication d'éléments traces (métaux lourds et terres rares) chez les mollusques céphalopodes et bivalves pectinidés. Implication de leur biodisponibilité pour le transfert vers les prédateurs[END_REF]): thus, microanalytical investigations of the digestive gland cells of this Faroese squid did not show any cadmium accumulations likely to explain the high cadmium levels of the digestive gland [START_REF] Bustamante | Etude des processus de bioaccumulation et de détoxication d'éléments traces (métaux lourds et terres rares) chez les mollusques céphalopodes et bivalves pectinidés. Implication de leur biodisponibilité pour le transfert vers les prédateurs[END_REF]. The absence of granule-like structures has been noted for the digestive gland cells of the loliginid squids Loligo forbesi and L. vulgaris, and in the cuttlefish Sepia officinalis [START_REF] Boucaud-Camou | Feeding and digestion in Cephalopods[END_REF][START_REF] Boucher-Rodoni | Fine structure and absorption of ferritin in the digestive organs of Loligo vulgaris and L. forbesi (Cephalopoda, Teuthoidea)[END_REF]. Nevertheless, metal-rich spherules, mostly containing copper, have been described in S. officinalis [START_REF] Martoja | Localisation cytologique du cuivre et de quelques autres métaux dans la glande digestive de la seiche, Sepia officinalis L. (Mollusque Céphalopode)[END_REF]. These authors suggested that the metals would be complexed by metallothioneins in these spherules. However, the presence of such structures remains controversial, as their existence has not been confirmed by other studies (Renata Boucher-Rodoni, pers. comm.). Among organelles, lysosomes would be expected to play an important role in cadmium detoxification as has been reported for bivalves [START_REF] Moore | Lysosomal cytochemistry in marine environmental monitoring[END_REF][START_REF] Taylor | Mechanisms of metal immobilisation and transport in cells[END_REF][START_REF] Marigomez | Morphological patterns of cell and tissue systems involved in metal handling and metabolism[END_REF]. In invertebrate organisms, lysosomes are known to accumulate both essential and toxic metals from the cytosol of the digestive gland cells. Lysosomes contain several hydrolytic enzymes and function as a digestive system in the cells. Finally, at their last stage of maturity, they accumulate cellular waste products which cannot be degraded [START_REF] Dallinger | Strategies of metal detoxification in terrestrial invertebrates: Synopsis and perspectives[END_REF]. It is therefore not surprising that metals are found in lysosomal residual bodies [START_REF] Dallinger | Mechanisms of metal incorporation into cells[END_REF].

In cephalopods, the digestive gland is involved in digestive and absorptive functions [START_REF] Boucaud-Camou | Localisation d'activités enzymatiques impliquées dans la digestion chez Sepia officinalis L[END_REF][START_REF] Boucaud-Camou | Fine structure and function of the digestive cell of Sepia officinalis (Mollusca : Cephalopoda)[END_REF][START_REF] Boucher-Rodoni | Fine structure and absorption of ferritin in the digestive organs of Loligo vulgaris and L. forbesi (Cephalopoda, Teuthoidea)[END_REF]. The lysosomal system is highly developed with several typical structures, i.e. heterolysosomes and heterophagosomes, "boules", residual bodies and brown bodies.

Although microanalytical investigations have not shown any accumulation of cadmium [START_REF] Bustamante | Etude des processus de bioaccumulation et de détoxication d'éléments traces (métaux lourds et terres rares) chez les mollusques céphalopodes et bivalves pectinidés. Implication de leur biodisponibilité pour le transfert vers les prédateurs[END_REF], such lysosomal structures are probably involved in the compartmentalisation of metals in the digestive gland cells of cephalopods as has been reported for other mollusc species [START_REF] Coombs | Mechanisms of immobilisation and detoxification of metals in marine organisms[END_REF][START_REF] George | A model for heavy metal homeostasis and detoxification in mussels[END_REF][START_REF] Viarengo | Mini-review. Mechanisms of heavy metal cation homeostasis in marine invertebrates[END_REF][START_REF] Marigomez | Morphological patterns of cell and tissue systems involved in metal handling and metabolism[END_REF]. Moreover, lysosomes can contain degradation products of metallothionein indicating that they may serve as a final storage site of degraded metallothioneins and possibly, of other metal-binding proteins [START_REF] Dallinger | Mechanisms of metal incorporation into cells[END_REF].

Cadmium sequestration seems to depend on levels of Cd contamination in the digestive gland. While the cytosolic content of cadmium increased significantly as a function of cadmium levels in the tissue in both Loligo vulgaris from the Bay of Biscay and Todarodes sagittatus from the Faroe Islands (Figure 3), the accumulation in the insoluble fraction was correlated with cadmium levels only for the Faroese squids. This difference in cadmium partitioning could be explained by the following hypothesis. When a threshold level, located between the highest Loligo vulgaris and the lowest Todarodes sagittatus total cadmium level in the digestive gland (i.e. 8 and 12 µg.g -1 wwt, respectively) is reached, the detoxification processes are modified and the storage of cadmium in organelles starts. Cd-binding proteins would be efficient enough to detoxify cadmium in loliginid squids from the Bay of Biscay while in ommastrephid ones, this system would be overloaded and lead to an alternative process assuring an efficient detoxification of cadmium. On an ultrastructural point of view, the digestive gland cells of loliginid squids are different from those of other cephalopods.

They do not have the "boules" structures characteristic of most cephalopod species [START_REF] Boucher-Rodoni | Fine structure and absorption of ferritin in the digestive organs of Loligo vulgaris and L. forbesi (Cephalopoda, Teuthoidea)[END_REF]. In the cuttlefish Sepia officinalis, some of these "boules" are considered as heterolysosomes and heterophagosomes involved in intracellular digestion [START_REF] Boucaud-Camou | Structure et fonction de la glande digestive ("foie") de la Seiche, Sepia officinalis L. (Mollusque, Céphalopode)[END_REF][START_REF] Boucaud-Camou | Fine structure and function of the digestive cell of Sepia officinalis (Mollusca : Cephalopoda)[END_REF]. Although brown bodies which contain residues of the digestion, account for the digestion ability of endogenous or exogenous compounds by loliginid digestive cells, the lack of "boules" in these cells might mean that particle capture and intracellular digestion do not occur widely in their digestive glands [START_REF] Boucher-Rodoni | Fine structure and absorption of ferritin in the digestive organs of Loligo vulgaris and L. forbesi (Cephalopoda, Teuthoidea)[END_REF]. Generally, in the Loliginidae family, the lysosomal system is less developed than in other cephalopod species. Consequently, loliginid squids could be physiologically limited to detoxify cadmium via binding to insoluble compounds. Alternatively, these squids could have developed mechanisms favouring the excretion of cadmium.

The occurrence of metallothioneins was suspected when considering the chromatograms of metalloproteins from the digestive gland of the squids Nototodarus gouldi, Todarodes pacificus and Ommastrephes bartrami, eluting between 10 kDa and 18 kDa [START_REF] Tanaka | Subcellular distribution and binding of heavy metals in the untreated liver of the squid ; comparison with data from the livers of cadmium and silver exposed rats[END_REF][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]. Since then, our investigations are among the first to attempt to determine the importance of these proteins in the detoxification processes of cadmium in cephalopods.

The quantification of metallothioneins showed up high specific differences, with the highest metallothionein levels corresponding to the lowest total cadmium levels in the digestive gland (i.e. Loligo vulgaris from the Bay of Biscay) and on the contrary, the lowest levels of metallothioneins corresponding to the highest total cadmium levels (i.e. Eledone cirrhosa from the Faroe Islands) (Figure 5). These observations are in accordance with the variation of the percentage of soluble cadmium (see above).

Metallothionein levels measured in cephalopods from this study (i.e from 0.7 ± 0.3 to 3.5 ± 0.6 mg.g -1 wwt) were of the same order of magnitude than those reported for other mollusc species. Thus, in the digestive gland of the mussel Mytilus galloprovincialis and the gastropod Littorina littorea, reported levels range from 0.6 to 2.1 mg.g -1 wwt [START_REF] Pavicic | Quantitative determination of metallothionein-like proteins in mussels. Methodological approach and field evaluation[END_REF][START_REF] Bebianno | Metallothionein induction in Littorina littorea (Mollusca: Prosobranchia) on exposure to cadmium[END_REF]. Cadmium levels in cephalopod digestive glands however were one or two orders of magnitude higher than those of these molluscs. Cadmium is largely considered as an inducer of the synthesis of metallothioneins in several phyla, and owing to the high cadmium levels in cephalopods, high metallothionein levels could be expected. It was not the case. On the contrary, considering the different cephalopod species in our study, metallothionein levels were negatively correlated with total cadmium levels in the digestive gland (Figure 5). In this mollusc class, metallothioneins do not represent the major response to the accumulation of cadmium in the tissue and cannot be considered as a biomarker of exposure to this metal. Cadmium detoxification could occur with other metalloproteins than metallothioneins. This hypothesis is strengthened by results found by other authors working on cephalopods. [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] and [START_REF] Castillo | Evidence for the presence of heavy metal binding proteins in the squid, Onychoteuthis borealijaponica[END_REF] have shown that most of the cytosolic cadmium in the digestive gland of the squids Todarodes pacificus and Onychoteuthis borealojaponica was bound to proteins weighing more than 70 kDa. Only a small part of the soluble cadmium was bound to low molecular weight proteins (<3 kDa) or to proteins of size similar to metallothionein (10 kDa to 16 kDa). Moreover, [START_REF] Finger | Molecular association of Cu, Zn, Cd and 210 Po in the digestive gland of the squid Nototodarus gouldi[END_REF] have reported the occurrence of Cd-binding proteins with a high molecular weight (> to 70 kDa) in the digestive gland of the squid Nototodarus gouldi.

CONCLUSIONS

The high levels of cadmium reported for cephalopod species from different areas of the world suggest that these organisms have developed efficient detoxification mechanisms. The subcellular distribution of cadmium in the digestive gland between cytosol and organelles suggests that the major part of this metal is associated with cytosolic proteins, except in Eledone cirrhosa from the Faroe Islands. The quantification of metallothioneins suggests a limited participation of these proteins in the detoxification of cadmium in cephalopods. An alternative mode of sequestration (lysosomal) could be activated when the level of cadmium in the digestive gland reaches a threshold value. Thus, further studies are needed to characterise the proteins involved in cadmium detoxification in cephalopods.

Cephalopods are considered to be a vector for the transfer of cadmium to top marine predators [START_REF] Honda | Distribution of cadmium and zinc in tissues and organs, and their age-related changes in striped dolphin, Stenella coeruleoalba[END_REF][START_REF] Muirhead | Heavy metal concentrations in the tissues of seabirds from Gough Island, South Atlantic Ocean[END_REF][START_REF] Bustamante | Cephalopods as a vector for the transfer of cadmium to top marine predators in the north-east Atlantic Ocean[END_REF]. This is strengthened by our results which have demonstrated the subcellular localisation of cadmium (Table 2). Recent investigations using radiollabelled food with 109 Cd have showed that 109 Cd bound to cytosolic proteins was transferred with a high efficiency and 109 Cd bound to insoluble compounds was relatively unavailable [START_REF] Wallace | Bioavailability of biologically sequestered cadmium and the implications of metal detoxification[END_REF]. Thus, the high bioavailability of cadmium in the digestive gland cells indicates a high potential for the trophic transfer of the metal to their predators such as marine mammals and seabirds.

I. coindetii (BB)

S. elegans (BB) S. orbignyana (BB) Figure 1. Partitioning of cadmium (as % of soluble Cd) in the digestive gland of cephalopods as a function of total cadmium levels in this organ (expressed as µg Cd.g -1 wwt). Scale bars represent 1 standard deviation. BB : Bay of Biscay; FI: Faroe Islands. Figure 5. Metallothionein-like protein levels in the heat-stable soluble fraction of the digestive gland of several cephalopod species as a function of the level of total cadmium in this tissue. Each point represents the mean ± 1 standard deviation for a single species. BB : Bay of Biscay; FI: Faroe Islands.

Figure 4 .

 4 Figure 4. Chromatographic profiles of heat-denatured homogenates from the digestive gland of cephalopods : Waters Protein-Pack 125 (7.8 x 300 mm) column; 100mM Tris-HCl, pH 8.6; λ = 254 nm; a : Bovine Serum Albumine (66 kDa), b : Rabbit Liver Metallothionein (12.5 kDa), c : Aprotinine (6.5 kDa)

Table 1 .

 1 Characteristics of the cephalopod samples.

	Family	Number	Sex	Mantle length	Weight	Origin
	Species	of		(mm)	(g)
		individuals
	Sepiidae
	Sepia elegans	5	5 Ε	63 ± 6	33 ± 11	Bay of Biscay
	S. officinalis	8	2 Γ, 6 Ε	153 ± 63	507 ± 522	"
	S. orbignyana	7	3 Γ, 4 Ε	84 ± 20	153 ± 53	"
	Loliginidae
	vulgaris	27	15 Γ, 12 Ε	233 ± 88	434 ± 302	Bay of Biscay
	Ommastrephidae
	Illex coindetii	9	5 Γ, 4 Ε	206 ± 43	368 ± 109	Bay of Biscay
	Todarodes sagittatus	21	11 Γ, 10 Ε	228 ± 43	199 ± 46	Faroe Islands
	Octopodidae
	Eledone cirrhosa	4	4 Γ	101 ± 8	448 ± 85	Faroe Islands
	120				y = -6.1017 Log (x) + 82.35
					r = 0.680
	100
	80
	60
	40
	20
	0
	1	10		100		1000
			µg

Cd.g -1 wwt % Soluble Cd .

	L.vulgaris (BB)	T.sagittatus (FI)
	S. officinalis (BB)
		E.cirrhosa (FI)

Table 2 .

 2 Percentage of Cd, Cu and Zn in the soluble fraction of the cytosol from different cephalopod species.

	Family	Number	Metal percentage in the soluble fraction	Origin
	Species	of	Cd	Cu	Zn
		individuals
	Sepiidae
	Sepia elegans	5	59 ± 22	72 ± 17	60 ± 15	Bay of Biscay
	S. officinalis	8	64 ± 8	69 ± 9	60 ± 9	"
	S. orbignyana	7	70 ± 8	69 ± 6	72 ± 10	"
	Loliginidae
	Loligo vulgaris	27	86 ± 12	86 ± 15	82 ± 6	Bay of Biscay
	Ommastrephidae
	Illex coindetii	9	79 ± 15	84 ± 12	79 ± 17	Bay of Biscay
	Todarodes sagittatus	21	64 ± 10	70 ± 8	61 ± 9	Faroe Islands
	Octopodidae
	Eledone cirrhosa	4	42 ± 17	53 ± 14	50 ± 3	Faroe Islands

ACKNOWLEDGEMENTS

We thanks the captains and the crews of Magnus Heinason and Gwen-Drez RVs for their kind co-operation as well as the participating scientists, especially Rogvi Mouritsen on Magnus Heinason and Jacques Labastie on Gwen-Drez. This work was supported financially by the "Conseil Régional Poitou-Charentes" and Rhodia (Rhône-Poulenc).

Loligo vulgaris

Todarodes sagittatus


Louise Devigne

Franc ¸ois Pasteau

Marie Babel

Vishnu K Narayanan

Sylvain Guégan

Philippe Gallien

Design of a haptic guidance solution for assisted power wheelchair navigation

Smart powered wheelchairs can increase mobility and independence for people with disability by providing navigation support. This support can be supplied in the form of autonomous or semi-autonomous obstacle avoidance systems. However, for rehabilitation or learning purposes, it would be of great benefit for wheelchair users to have a better understanding of the surrounding environment while driving. Therefore, another way of providing navigation support is to communicate information through a dedicated and adapted feedback interface. We here propose a framework in which feedback is provided by sending forces through the wheelchair controller as the user steers the wheelchair. This solution is based on a low complex optimization framework able to perform smooth trajectory correction and to provide obstacle avoidance. The impact of the proposed haptic guidance solution on user driving performance was assessed during this pilot study for validation purposes through an experiment with 4 able-bodied participants. They were asked to drive a power wheelchair on an obstacle course with and without activation of the force feedback. Results of this pilot study showed that the number of collisions significantly decreased while force feedback was activated, thus validating the proposed framework.

I. INTRODUCTION

The use of a power wheelchair for individuals with mobility impairments has positive outcomes such as enhancing user autonomy and social inclusion [START_REF] Davies | Changes in the quality of life in severely disabled people following provision of powered indoor/outdoor chairs[END_REF] [START_REF] Evans | Older adults' use of, and satisfaction with, electric powered indoor/outdoor wheelchairs[END_REF]. However, heavy motor and/or perceptual impairments can lead to a variety of activity limitations [START_REF] Rhoda | Limitations in activity and participation experienced by stroke patients: A qualitative inquiry[END_REF]. In particular, perceptual impairments can impact the ability to independently operate a wheelchair safely [START_REF] Cawood | Impact of post-stroke impairments on activities and participation as experienced by stroke survivors in a western cape setting[END_REF]. In this context, several research teams developed autonomous or semi-autonomous wheelchair navigation solutions [START_REF] Li | Human cooperative wheelchair with brainmachine interaction based on shared control strategy[END_REF] [START_REF] Panzarella | Copilot: Autonomous doorway detection and traversal for electric powered wheelchairs[END_REF].

Although such systems assist the user while driving a wheelchair, providing feedback information (visual, auditory or haptic/tactile) could have additional benefits for learning and rehabilitation purposes [START_REF] Levin | Feedback and virtual environments for motor learning and rehabilitation[END_REF].

In the robotics field, feedback information is often communicated through haptic interfaces, especially for human robot interaction applications. Indeed, applying forces, vibrations, or motions to the user interface allows to communicate information between a user and a remote environment. Typically, this can be useful for robot teleoperation, interaction with a virtual environment or even remote surgery [START_REF] Stone | Haptic feedback: A brief history from telepresence to virtual reality[END_REF][9] [START_REF] Koehn | Surgeons and non-surgeons prefer haptic feedback of instrument vibrations during robotic surgery[END_REF]. This type of feedback is typically used for people with visual impairments [START_REF] Lahav | Haptic-feedback support for cognitive mapping of unknown spaces by people who are blind[END_REF] [START_REF] Jones | The efficacy of haptic simulations to teach students with visual impairments about temperature and pressure[END_REF], where haptic information compensates for the lack of visual information and thus allows the user to improve his/her interaction with the external environment. Some studies have also shown the benefits of using haptic feedback for motor rehabilitation process [START_REF] Broeren | Virtual reality in stroke rehabilitation with the assistance of haptics and telemedicine[END_REF] [START_REF] Choi | A rehabilitation method with visual and haptic guidance for children with upper extremity disability[END_REF].

It is then possible to create a communication channel between the user and the system he/she is using. This feedback provides additional information to users during their interaction such that their experience or their performance can be enhanced. Moreover, as such a feedback can improve user understanding while performing a given task, haptic feedback could be seen as an effective rehabilitation tool to train severely disabled people to drive a powered wheelchair [START_REF] Crespo | Haptic guidance can enhance motor learning of a steering task[END_REF].

Research works have been done on the use of haptic feedback for power wheelchair driving but are only focused on a specific line following task [START_REF] Marchal-Crespo | A robotic wheelchair trainer: design overview and a feasibility study[END_REF] [START_REF] Chen | Assisting versus repelling force-feedback for human learning of a line following task[END_REF]. Moreover, several teams investigated force feedback for power wheelchair driving. In [START_REF] Protho | An evaluation of an obstacle avoidance force feedback joystick[END_REF], the authors evaluated the impact of force feedback on user performance while driving a power wheelchair in a virtual environment. Results of trials with people with disability showed fewer collisions while the feedback was activated. Vander Poorten et al. [START_REF] Vander Poorten | Powered wheelchair navigation assistance through kinematically correct environmental haptic feedback[END_REF] also developed a haptic feedback algorithm for power wheelchair driving relying on the definition of a collision-free paths computed from a local map of the environment. Such systems typically use Potential Field Methods relying on the definition of a local map of the environment requiring information classically measured by expensive sensors such as laser range finder.

In this paper we present a novel approach for power wheelchair haptic navigation assistance which uses low-cost distance sensors and does not require to compute a local map of the environment. This solution relies on a sensor-based servoing approach we presented in [START_REF] Babel | HandiViz project: clinical validation of a driving assistance for electrical wheelchair[END_REF]. In this framework, the force feedback information is computed from the fusion of user manual control and sensor-based constraints without any a priori knowledge of the environment. A reactive force feedback is thus provided to the user while driving. This paper is organized as follows: section II describes the force feedback algorithm. Section III presents a simulation of the system in a virtual environment. Section IV determines first the experimental setup. Then we present the pilot study experiment we performed with able-bodied participants. Section VII presents the results of the experiment.

II. MODELING

The wheelchair is modeled as a 6 wheeled robot composed of 2 actuated wheels and 4 passive caster wheels. It is controlled with 2 variables, namely the linear velocity u and the rotational velocity ω. As shown on Fig. 1, we define the following notations

• let u = u ω
T be the velocity of the wheelchair with u and ω respectively the linear and angular velocities,

• let v si = u si ω si T be the velocity of the sensor s i with u si and ω si respectively the linear and angular velocities of the sensor s i , • let x i be the distance from the sensor s i to the obstacle,

• let θ i be the orientation of the sensor s i with respect to the robot frame z-axis,

• let e i be the difference between the measured distance

x i and its desired value x * i .

Fig. 1: Definition of the robot frame.

The translation between the sensor s i frame and the robot frame is given by r t si = a b 0

T with a and b depicted on Fig. 1. The rotation matrix relative to the sensor s i frame with respect to the robot frame si R r is given by

si R r =   cos θ i sin θ i 0 -sin θ i cos θ i 0 0 0 0   . (1)
From si R r and si t r , the velocity screw transformation matrix is given by

si W r = si R r [ si t r ] × si R r 0 3×3 si R r . (2)
with [ ] × the skew matrix expression. The velocity of the sensor s i in the sensor frame v si is derived from the robot velocity u in the robot frame using

v si = si T r u, (3)
with si T r = si W r r J r , (4)
where r J r is the robot jacobian expressed in the robot frame:

r J r = 1 0 0 0 0 0 0 0 0 0 0 1

T . (5)
III. METHOD In order to assist a user in controlling a wheelchair, we can communicate additional information by using haptic control devices which provide feedback forces to the user's hand. This way, the user benefits from a communication channel with the wheelchair controller.

A. Assistive algorithm computation

We can define an interaction matrix L xi for each sensor

s i such that ẋi = L xi v si . (6)
In order to modify a trajectory leading to a collision, we design a proportional corrector by constraining ẋi by a minimum value λ e si . We then get:

ẋi ≥ -λe si . ( 7
)
The combination of ( 6) and ( 7) results in

L xi si T r u ≥ -λe si (8)
In our case with N sensors, inequality ( 8) can be rewritten as

A u ≥ B ( 9
) with A =    A 0 . . . A N -1    and B =    B 0 . . . B N -1   .
For each sensor s i the following inequation

A i u ≥ B i ( 10
)
defines a half-plane in the wheelchair velocity domain. All the computed intersected half-planes define a convex area in the wheelchair velocity domain as shown on Fig. 3. This area is used in the haptic guidance framework which is presented below.

B. Haptic guidance and haptic feedback assistance

Two types of haptic navigation assistance for power wheelchair driving can be distinguished:

• haptic guidance: consists in assisting the user to safely control the wheelchair. To do so, a forces are communicated to the user through the wheelchair controller to provide additional information about the environment. In this case, the user has full control of the wheelchair and collisions can occur. • haptic feedback: the force feedback controller is used in conjunction with the obstacle avoidance framework. It aims at improving user understanding of the behavior of the system while correcting the trajectory. In this paper, we focus on the haptic guidance solution.

C. Haptic guidance algorithm

An appropriate information to be delivered to the user while driving would be the direction to which he/she should put the joystick in order to avoid collisions with obstacles. This information can be communicated to the user by sending appropriate forces through the wheelchair controller.

Let u op = (u op , ω op ) be the user desired velocity, and u cmd = (u cmd , ω cmd ) be the computed robot velocity. The novelty of the proposed haptic guidance framework is to directly compute the force magnitude and direction from the wheelchair velocity domain. Let k be the vector from u op to u cmd in the wheelchair velocity domain (Fig. 3). The force feedback vector F to be applied to the user interface will be computed from this information.

The computation of u cmd from u op under constraint ( 9) is an optimization problem using

u cmd = min u f (u op , u) A u ≥ B (11)
The function f is defined such as

f (u op , u) = α(u -u op ) 2 + β(ω -ω op ) 2 (12)
with α ≥ 0 and β ≥ 0.

As shown on Fig. 3, we define 2 sets P 0 and P 1 such as

P 0 = {u|Au ≥ B} P 1 = {u|Au > B} . ( 13
)
Hence a solution of the optimization problem (11) exists only if P 0 = ∅. Else, x * i is dynamically decreased to expand the size of P 0 . Fig. 2: Definition of P 0 and P 1 . Forbidden and allowed areas are represented respectively in red and green in the wheelchair velocity domain. Fig. 3: The algorithm output corresponds to the projection of user input within the allowed area. k represents the vector from the user input to the algorithm output.

There are two different cases: 1) if u op ∈ P 0 , then u cmd = u op , 2) otherwise, as the cost function is convex and its minimum is reached when u = u op , u cmd ∈ P 0 -P 1 . This means that it exists one i for which

A i u = B i .
Here the result u cmd of this optimization problem is shown in Fig. 3. It results from a search along P 0 -P 1 .

We define k(r k , θ k ) and F(r, θ) in polar coordinates with respectively r k and r their magnitudes and θ k and θ their directions. This representation leads to the following properties:

• force direction is the same as the direction of the vector k in the wheelchair velocity domain, leading to θ = θ k , • we define the force magnitude such that r = α r k with α > 0 a proportional gain which can be modified to adapt the force magnitude to the user strength, r k = k the euclidean distance between u op and u cmd in the wheelchair velocity domain so that

k = (u op -u cmd ) 2 + (ω op -ω cmd ) 2 . ( 14
)
This way, the force guides the user towards a joystick position for which the resulting user input is safe. Force magnitude is proportional to the distance between user input and its projection onto the allowed area.

To sum up, a set of constraints deduced from distance sensors is used to defined two areas in the wheelchair velocity domain. Then, if the user input is unsafe (i.e. within the forbidden area), a corresponding risk-free input is calculated by means of a projection within the allowed area. From this, we deduce the force to be sent to the user through the joystick. This force is directed from user input towards the corresponding risk-free input. Its magnitude is proportional to the euclidean distance between user input and the related projection within the allowed area. This way, the user is at the same time notified that there is a danger of collision and guided to a joystick position corresponding to safe input. It is to be emphasized that the user has full control of the wheelchair and can maintain his/her desired joystick position. The information communicated in the form of force feedback is only an indication of the risk-free joystick position the most compliant with his/her intention. Such a solution is then mainly meant to be used for learning or rehabilitation purposes.

IV. SIMULATIONS

In this section, we present the results of the very first step in the development of our haptic guidance solution. It consists in a simulation performed on a simulator for assisted power wheelchair driving we previously developed. This simulator is a flexible tool for development and testing of navigation assistance methods without having to equip a power wheelchair. In this framework, the user operates a force feedback jostick to drive a virtual robotized wheelchair in a virtual environment. The framework has been designed to be directly compliant with a monitor screen display, a Virtual Reality room or a Head Mounted Device and is presented in more details in [START_REF] Devigne | Design of an immersive simulator for assisted power wheelchair driving[END_REF].

A. Simulation conditions

The virtual environment is displayed on a computer screen as it is only used to demonstrate the properties of the proposed haptic guidance solution. The virtual environment used for this simulation is shown on Fig. 4. It consists of a 1.5 m wide and 10 m long corridor with 4 obstacles. The virtual wheelchair starts from the beginning of the corridor facing the top wall with an angle of 60 • . The wheelchair is here equipped with 22 virtual sensors positioned all around the virtual wheelchair according to Fig. 5b. The user is sitting in front of the computer screen and controls the virtual wheelchair (Fig. 5a) by operating a force feedback joystick (Fig. 5c). User view during the simulation is showed in Fig. 5d. The force feedback game controller is a commercially available joystick from Microsoft which we adapted to match wheelchair driving constraints by implementing automatic centering and reducing the number of degrees of freedom to 2.

In order to emphasize the properties of the proposed force feedback solution, the user voluntary tries to collide with the virtual obstacles while steering the wheelchair.

B. Simulation results

Simulation results are presented in Fig. 6. Fig. 6a shows the trajectory of the wheelchair during the simulation. Blue arrays represent the force vectors which are sent to the user through the force feedback joystick while driving. Fig. In Fig. 6, in part A, we observe that as long as the user is driving towards the wall on his left, he is being notified that there is a risk of collision by means of forces directly towards the opposite direction. Then, when the user modifies the angular velocity input, both the magnitude and direction of the force progressively decrease. When the wheelchair becomes parallel to the walls, as there is no risk of collision anymore (typically in D and H parts), the user does not receive any force feedback anymore. In B and E parts, the user drives towards the obstacle on his right, the algorithm then sends forces towards the left direction. We can observe the impact of this additional information on the angular velocity. Indeed the decreasing of the angular velocity input associated with a slight increase of the linear input shows that the user is adapting his joystick input while receiving force feedback information. We can observe similar results in C and F parts where the user drives toward the obstacle on his left.

V. EXPERIMENT

As power wheelchair users can have weak upper limbs and/or tremors, it is mandatory to ask able-bodied participants to evaluate our haptic guidance solution before being able to obtain ethical approval regarding trials with people with disabilities. In this pilot study including 4 able-bodied participants, the impact of navigation assistance in the form of haptic guidance solution is assessed.

A. Experimental setup

We used the same wheelchair as in [START_REF] Devigne | Low complex sensor-based shared control for power wheelchair navigation[END_REF], a power wheelchair which we equipped with the same force feedback joystick as the one used for the simulation. Although it is the same wheelchair, sensor configuration has been upgraded: we empirically determined an optimal configuration composed of 22 sensors. Those sensors were installed on the wheelchair at the same location as in the simulation setup previously described (Fig. 5b).

The obstacle course we designed for this experiment (Fig. 7) is similar to the one used for the simulation (Fig. 4) but is more challenging as paths are narrower than the one used in simulation and it contains a turn around maneuver.

B. Experiment protocol

Participants were asked to drive a power wheelchair with the force feedback joystick (Fig. 8) on an obstacle course made with cardboard boxes (Fig. 7). Participants realized twice the course: once with haptic guidance and once without haptic guidance. The given instructions were

• driving along the obstacle course and go back,

• performing the obstacle course the faster they could without colliding with the obstacles. For each trial, the haptic guidance module can be activated or not. We switched the order of activation of the haptic guidance for each participant in order to avoid the learning effect.

C. Data Collection

1) Haptic guidance solution data: We use ROS to automatically record data during the trials. Here we recorded user input, the related risk-free input computed by the algorithm and force magnitude and direction. We also recorded the number of collisions as well as time completion.

2) User feedback: After completing the obstacle courses, participants were asked to answer the USE questionnaire [START_REF] Lund | Measuring usability with the use questionnaire12[END_REF] which consists in a 30 items Likert scale. The Questionnaire is composed of 4 sections: Usefulness (U), Ease of Use (EU), Ease of Learning (EL) and Satisfaction (S).

VI. EXPERIMENT RESULTS

This section presents the results of the experiment we performed. 4 able-bodied participants aged from 22 to 38 years old have volunteered to participate to this pilot study.

A. User performance

In order to evaluate the impact of the proposed haptic guidance solution on user performances while driving the power wheelchair, we recorded the number of collisions which occurred during the experiment with and without force feedback (Fig. 9a) as well as completion time.

As we can see, much fewer collisions occurred while haptic guidance was activated. With the haptic guidance activated, all participants collided the same obstacle. In fact, these collisions only occurred during a turn around maneuver which was really difficult as the space was very narrow compared to the length of the wheelchair. Moreover, there was no significant difference between time completion values with and without force feedback. Best score is for the ease of learning with 6.5 +/-0.41 and worst score is for the ease of use with 5.61 +/-0.42. Global questionnaire score is 5.99 +/-0.43.

Participants reported to be rather satisfied with the proposed haptic guidance solution. They felt the information given by the joystick was relevant and helpful for the navigation task. In particular, they reported having benefited from the haptic guidance while performing the turn around maneuver as it warned them while getting too close to obstacles and allowed them to better judge distances when obstacles were not easily visible as the force increased while approaching the obstacle with an unsafe joystick input. However, even if driving performances have been significantly increased, additional information provided by the force feedback joystick was not totally sufficient to avoid collisions during the turn around maneuver. Nevertheless, it globally incited participants to adapt their command and generally decreased the collision impact speed. Finally, participants did not report having felt any pain or discomfort while using the force feedback joystick but they reported to be unsatisfied with the haptic interface form factor itself: they found it difficult to manipulate because of its dimensions.

VII. DISCUSSION

High scores obtained with the USE questionnaire show that the participants were rather satisfied with the haptic guidance solution. Moreover, they found it helpful, particularly for the turn around maneuver. As we expected, they felt that the joystick was giving them additional information about the environment. While approaching an obstacle, they perceived the force as an indication of the risk of collision inducing them to reduce their speed and to modify joystick direction. While maneuvering the wheelchair, they perceived the feedback as an inducement to move the joystick in the right direction.

These preliminary results show the validation of our haptic guidance solution as a tool for learning or rehabilitation purpose as it incited the participants to adapt speed and direction to avoid collisions and allowed them to better understand the environment around the wheelchair.

VIII. CONCLUSION

In this paper, we presented a haptic guidance solution for power wheelchair assisted navigation. In order to validate our solution, we asked 4 able-bodied participants to perform an experiment consisting in driving a power wheelchair equipped with a force feedback controller on an obstacle course with and without haptic guidance activated. This pilot study is mandatory to obtain an approval from an ethical committee in order to perform trials with people with disability. While driving the wheelchair on the obstacle course, the number of collisions significantly decreased while haptic guidance was activated, thus validating the proposed framework. This pilot study allowed us to identify the remaining shortcomings of our system. Evidently, the game controller we used to validate our robotic solution is not not adapted to wheelchair control. Hence, we started working on the design of a dedicated force feedback joystick adapted to user needs and which dimensions are compliant with wheelchair standards. In addition, in future work we would like to assess and compare our 3 navigation assistance solutions (obstacle avoidance only, haptic guidance only and haptic feedback) with more healthy participants before running clinical trials with power wheelchair regular users once approved by an ethical committee.

Fig. 4 :Fig. 5 :

 45 Fig. 4: Top view of the map used for simulation.

  Fig. 6: Simulation results.

Fig. 7 :

 7 Fig. 7: Obstacle course, values correspond to distances in centimeters. Fig. 8: Participant driving on the obstacle course.

  (a) Number of collisions. (b) USE Questionnaire results. Mean and standard deviation for each category of the USE Questionnaire.

Fig. 9 :

 9 Fig. 9: Experiment results.

ACKNOWLEDGEMENT

The authors would like to thank Éric Bazin, Valentin Bureau and Nicolas Le Borgne from INSA Rennes who were of great help during the development process and experiments. Finally the authors would like to thank the people who participated to this study. This work has been funded by Inria associated team ISI4NAVE and by Institut des Neurosciences Cliniques de Rennes (INCR). This work is carried out as part of the IN-TERREG VA FMA ADAPT project " Assistive Devices for empowering disAbled People through robotic Technologies http://adapt-project.com/index.php.". The Interreg FCE Programme is a European Territorial Cooperation programme that aims to fund high quality cooperation projects in the Channel border region between France and England. The Programme is funded by the European Regional Development Fund (ERDF).