Florence Mouchet
email: florence.mouchet@ensat.fr

Christian Gancet

Emmanuel Flahaut

Eric Pinelli

Jeanàcharles Boutonnet

Laury Gauthier

International standardized procedures for in vivo evaluation of multi-walled carbon nanotube toxicity in water

Keywords: Multi-walled carbon nanotubes, ecotoxicity, standardized bioassays, bacteria, algae, fish, amphibians

The classical approach in ecotoxicological evaluation of chemical substances consists of conducting standardized bioassays on organism models. In this work, the potential impact of industrial multiwalled carbon nanotubes was investigated by ecotoxicological standardized procedures using aquatic organisms of different trophic levels, namely bacteria, green algae, invertebrates, fish, and amphibians. The results indicated (1) inhibition of growth in amphibians at 50 mg L ¡1 and higher, and (2) no effects on daphnia and fish up to 100 mg L ¡1 . With the exception of algae (for which Fe deficiency is measured), it seems that the observed toxicity may be due to physiological effects in relation to the ingestion of carbon nanotubes not necessarily related to their intrinsic effects.

Introduction

The publication of [START_REF] Iijima | Helical Microtubules of Graphitic Carbon[END_REF] generated unprecedented interest in the world of carbon nanostructures and led to an exponential growth in research on carbon nanotechnology. Carbon nanotubes (CNTs) can be described as graphene sheets rolled up to form cylinders that are closed at both ends. There are two main types, i.e. single-walled CNTs (SWNTs) and multi-walled CNTs (MWNTs). They have remarkable physical, i.e. mechanical, electric, and thermal and chemical properties (inertness, stability), making them a material of choice for polymer composites, electromagnetic shields, super capacitors, gas including hydrogen storage devices, batteries, structural composites, or medical applications [START_REF] Eklund | International Assessment of Research and Development of Carbon Nanotube Manufacturing and Applications[END_REF], especially of MWNTs for biomedical engineering, used in biosensors, as vehicles for drug delivery, and in gene therapy [START_REF] Kostarelos | Promises, Facts and Challenges for Carbon Nanotubes in Imaging and Therapeutics[END_REF].

Most likely, during production and use, some quantities will get into the environment, especially the aquatic compartment. Even if toxicological data are available, obtained most often with in vitro systems [START_REF] Guadagnini | Toxicity Screenings of Nanomaterials: Challenges Due to Interference with Assay Processes and Components of Classic In Vitro Tests[END_REF]) and with animal models [START_REF] Van Der Zande | Carbon Nanotubes in Animal Models: A Systematic Review on Toxic Potential[END_REF], nevertheless ecotoxicological exposure and effect data are necessary for understanding the potential hazards these new carbon-based materials may pose for the environment. As new substances, CNTs require registration under the Toxic Substances Control Act in the USA and in the European Union according to REACh (Registration, Evaluation, Authorization and Restriction of Chemicals) regulations (EU 2008). Some (eco)toxicological and environmental properties of SWNTs and MWNTs are listed in the report ENV/JM/MONO 13/REV ( 2008), but more is required for the proper evaluation of the potential ecotoxicity of CNTs. Aquatic ecotoxicity assessment of CNTs is a challenge since tests have been developed for water-soluble chemical compounds. Nevertheless, standard environmental hazard assessment is generally appropriate for nanoecotoxicological research [START_REF] Crane | Ecotoxicity Test Methods and Environmental Hazard Assessment for Engineered Nanoparticles[END_REF], especially using the test battery concept [START_REF] Kahru | Biotests and Biosensors for Ecotoxicology of Metal Oxide Nanoparticles: A Mini Review[END_REF][START_REF] Blaise | Ecotoxicity of Selected Nano-materials to Aquatic Organisms[END_REF]) in order to accumulate knowledge about their ecotoxicity [START_REF] Kahru | From Ecotoxicology to Nanoecotoxicology[END_REF] toward a wider range of biological species providing valuable insight into likely exposure scenarios [START_REF] Zhao | Recent Progress and Perspectives on the Toxicity of Carbon Nanotubes at Organism, Organ, Cell, and Biomacromolecule Levels[END_REF].

The aim of the present work is to contribute to the ecotoxicological assessment of the potential impact of MWNTs as an example of industrial CNTs in aquatic organisms belonging to different trophic levels by carrying out ecotoxicological standardized procedures. The selected species were decomposers (bacteria), primary producers (photosynthetic green algae, Pseudokirchneriella subcapitata), primary consumers (invertebrates Daphnia magna), and secondary consumers (vertebrate fish and amphibians, Danio rerio and Xenopus laevis).

Materials and methods

MWNTs preparation of suspensions

MWNT (Graphistrength® C100, Arkema, Colombes, France) suspensions in ultrapure water were prepared by sonication for 10 min at 45 kHz, 80 W (USC 300T, VWR, Fontenay sous Bois, France) just before each bioassay. The physical characteristics and transmission electron microscopic observations of the MWNTs were previously described by [START_REF] Mouchet | CNTs Ecotoxicity in Amphibians: Assessment of Multi-walled Carbon Nanotubes (MWNT) and Comparison with Double-Walled Carbon Nanotubes (DWNT)[END_REF]. Figure 1 displays scanning electron micrographs of MWNTs.

Biological bioassays

Activated sludge respiration inhibition test, OECD (1984) Guideline 209

The inoculum was activated sludge of a small biological domestic wastewater treatment plant (Abidos, France). MWNTs were studied at 500 and 5000 mg L ¡1 . Dissolved oxygen concentrations were determined with an oxygen electrode (Stirrox G, WTW, Weilheim, Germany) and meter (OXI 538, WTW). The inhibitory effect was expressed as percentage of the mean respiration rate of two controls, calculated from the recorder trace as mg O 2 L ¡1 h ¡1 over a period of 10 min. The inhibition was expressed as percentage relative to the mean of the respiration rates in two controls: % inhibition D [1 ¡ (2R s /(R c1 CR c2 ))] £ 100, where R s is the oxygen consumption rate at the tested concentration of test substance, and R c1 and R c2 are the oxygen consumption rates for controls 1 and 2. The sensitivity of the test system and the method were evaluated with 3,5-dichlorophenol.

Algal growth inhibition test (P. subcapitata), OECD (2006) Guideline 201 P. subcapitata (CCAP 278/4 stock) was obtained from the Culture Centre of Algae and Protozoa (Ambleside, UK). The cell density (measured fluorescence, Cytofluor 2350, Millipore, Molsheim, France) for the preliminary test was 1.21 £ 10 6 cells mL ¡1 , and for the definitive test, 1.09 £ 10 6 cells mL ¡1 . Algae were exposed under static conditions over a time period of 72 h to MWNTs dispersed in water (EN ISO 8692, 2004) at (1) 100, 50, 10, 5, 1, and 0 mg L ¡1 in the preliminary test, and (2) 1000,500,230,105,48,22,10, and 0 mg L ¡1 for the definitive test. The MWNT concentrations resulting in 0% and 100% of the uninhibited cell growth rate, and growth rate inhibition causing a 50% reduction in biomass (E b C 50 ) within 72 h (E b C 50 À72 h) and in growth rate (E r C 50 À72 h) were estimated. The sensitivity of the test system and the method were evaluated by performing an algal growth inhibition test on K 2 Cr 2 O 7 (Sigma, Lyon, France). The growth inhibition data were analyzed using an Excel sheet to calculate the effective concentration (EC 50 value) and the 95% confidence interval. Probit analysis was used to calculate the 24-, 48-, and 72-h EC 50 values. The no-observed effect concentration (NOEC), the highest tested concentration at which no significant inhibition of growth is observed relative to the control, was estimated by Dunnett's test. Values of pH (345 pH meter, Mettler Toledo, Viroflay, France) and dissolved O 2 (OXI 538 oxymeter, WTW) were measured.

Analytic complementary experiments have been carried out to study the ecotoxicological response of algae in relation to a potential deficiency of ionic metallic species, with the well-known property of CNTs to adsorb ionic species [START_REF] Li | Ferric Ion Adsorption and Electrode Sorption by Carbon Nanotubes and Nanofibres Films[END_REF][START_REF] Stafiej | Adsorption of Heavy Metal Ions with Carbon Nanotubes[END_REF]. B, Mn, Fe, Co, Ni, Cu, Zn, and Mo were measured in algal media (water dilution) with and without MWNTs after filtration (0.45 mm to remove most of MWNTs) by ICPÀMS (ICPÀMS 7500, Agilent, Les Ulis, France) at the end of the experiment. Detection limits were 1 mg L ¡1 . Metal traces were measured in water dilution alone, with and without Fe. The algal growth inhibition test was carried out with and without Fe to check the effect of iron deficiency. D. magna acute immobilization test, OECD (2004) Guideline 202 D. magna Straus (Cladocera, Crustacea), clone 5 and clone A, were from stock breeding in the laboratory reared in Volvic® water added of 0.1 mL L ¡1 B 12 solution (1 mg L ¡1 , Alfa Aesar, Kandel, Germany), 0.1 mL L ¡1 Na 2 SeO 3 ¢5H 2 O solution (6.7 mg L ¡1 , Sigma), 1 mL L ¡1 solution of Ca(NO 3 ) 2 ¢4H 2 O (208 g L ¡1 , Sigma) and MgCl 2 ¢6H 2 O (28 g L ¡1 , Sigma), and unicellular green freshwater algae (P. subcapitata and Chlorella vulgaris). A stock suspension at 100 mg L ¡1 was used to realize dilutions of 100, 50, 10, 5, 1, and 0.1 mg L ¡1 of MWNTs in water (EN ISO 6341, 1996) for the preliminary test. In the definitive test, based on the results of the preliminary test, a limit test was performed at 100 mg L ¡1 . Five D. magna aged from 6 to 24 h were added to each test flask. Two preliminary and four definitive test replicates were prepared for each concentration. As controls, two preliminary tests and four definitive test flasks without MWNTs were prepared under the same conditions. After 24-h incubation (definitive test), mobile D. magna were counted and flasks were placed back for continued incubation. At 48 h, mobile D. magna were counted again (preliminary and definitive tests). The sensitivity of the test system and the method were evaluated every month by performing an inhibition test with K 2 Cr 2 O 7 (Sigma). At 24 h and at the end of the 48-h test period, the actual concentrations inhibiting the mobility of daphnids by 50%, i.e. EC 50 À24 h and EC 50 À48 h, were estimated. The NOEC was estimated when possible. Dissolved O 2 (OXI 538 oxymeter, WTW) and pH (345 pH meter, Mettler Toledo) were measured at the highest concentration and in the control at the beginning and at all concentrations, and in the control at the end of the test.

D. magna reproduction test, OECD (2008) Guideline 211 Daphnia were exposed to MWNTs in a semi-static test from 5 to 100 mg L ¡1 . Exposure water was prepared with Volvic® water complemented as follows: 0.1 mL L ¡1 B 12 solution (1 mg L ¡1 , Alfa Aesar, Kandel, Germany), 0.1 mL L ¡1 Na 2 SeO 3 ¢5H 2 O solution (6.7 mg L ¡1 , Sigma), 1 mL L ¡1 solution of Ca(NO 3 ) 2 ¢4H 2 O (208 g L ¡1 , Sigma), and MgCl 2 ¢6H 2 O (28 g L ¡1 , Sigma). The test was performed with one Daphnia per vessel and with 10 replicates for each concentration. Ten control flasks without MWNTs were prepared under the same conditions. The positive control was with K 2 Cr 2 O 7 (Sigma). For each exposure concentration, the percentage of inhibition of reproduction was recorded after 21 days. The results of the acute toxicity test were used to define the concentration range for the reproduction test. The MWNT concentrations resulting in 0% and 100% inhibition of reproduction were determined by observation, and EC 50 was estimated by calculation using the HILL model (an Excel® macro REGTOX http://www.normalesup.org/ »vindimian/fr_download.html). The lowest observable effect concentration (LOEC) and NOEC were determined using Dunnett's test.

Fish acute toxicity test (D. rerio), OECD (1992) Guideline 203

The organisms used for the test were D. rerio (Teleostei, Cyprinidae), batch n 10/Br/01/1 supplied by Aquatrade (Saint Forgeux, France). The sensitivity of the biological reagent was checked at least once for each new batch of fish by determining the lethal concentration at 24 h (LC 50 À24 h) of K 2 Cr 2 O 7 . Fish were exposed under static conditions to 1, 35, 50, and 100 mg L ¡1 of MWNTs dispersed in water (EN ISO 7346, 1998) for the preliminary test and to 100 mg L ¡1 for the definitive test. Two replicate test chambers were maintained for each treatment and each control group. For the range-finding test, MWNT suspensions were directly prepared for each concentration, and for the definitive test by weighing the respective amounts of MWNTs into 100 mL water and under adjustment to the 5 L in the test tanks. In both tests, the fish were considered dead if no reaction was observed when no respiratory movement was observed upon stimulation of their caudal peduncle. Visible anomalies were noted, as were any sublethal effects such as loss of balance, altered pigmentation, changes in swimming behavior, or respiratory malfunction. The dead fish were counted and removed from the aquaria. At 24, 48, and 72 h and at the end of the 96-h test period, the concentrations killing 50% of the fish, i.e. LC 50 À24, À48, À72 and À96 h, were estimated. Dissolved O 2 (OXI 538 oxymeter, WTW) and pH (345 pH meter, Mettler Toledo) were monitored.

Amphibian (X. laevis) bioassays

Eggs were obtained from the Ecolab laboratory. The procedure for rearing of X. laevis and breeding until they reached the development stage appropriate for experimentation À stage 50 of the development table of Nieuwkoop and Faber [START_REF] Nieuwkoop | Freshwater Alga and Cyanobacteria, Growth Inhibition Test[END_REF]) À is described by [START_REF] Mouchet | Characterisation and In Vivo Ecotoxicity Evaluation of Double-Wall Carbon Nanotubes in Larvae of the Amphibian Xenopus laevis[END_REF]. MWNT dilutions were made in 20 mL of ultra-pure water in glass tubes, and then sonicated (Bioblock 89863, Fisher Scientific, Illkirch, France) for 5 min before their transfer to the exposure media. Exposure was in reconstituted water (RW), i.e. distilled tap water to which nutritive salts were added as described in ISO 21427À1 [START_REF] Iso | ISO International Standard. Water Quality À Evaluation of Genotoxicity by Measurement of the Induction of Micronuclei À Part 1: Evaluation of Genotoxicity Using Amphibian Larvae[END_REF]. The negative control condition (NC) was RW alone.

The first exposure was under static conditions for 96 h. Larvae were exposed in triplicate groups of 10 animals per flask containing either RW or test media at 10, 50, 100, and 500 mg L ¡1 MWNTs in RW. Each day, the number of dead larvae was counted and the lethal concentration at which mortality occurred for 50% of the animals (LC 50 ) was calculated. The sensitivity of the test system and the method were evaluated using CdCl 2 (Sigma).

The second type of exposure was performed for 12 d according to ISO 21427À1 [START_REF] Iso | ISO International Standard. Water Quality À Evaluation of Genotoxicity by Measurement of the Induction of Micronuclei À Part 1: Evaluation of Genotoxicity Using Amphibian Larvae[END_REF] for the amphibian micronucleus test (MNT) with a daily renewal of the exposure medium. For the positive control (PC), cyclophosphamide (Sigma) in RW at 20 mg L ¡1 was used. Xenopus larvae were exposed to 0.05, 0.1, 0.5, 1, 5, 10, 25, and 50 mg L ¡1 of MWNTs in RW. Larvae were exposed in groups of 20 animals in dishes containing either control media (NC and PC) or test media (0.1, 1, 10, and 50 mg L ¡1 of raw MWNTs in RW). Acute toxicity (mortality) of larvae exposed to MWNTs was examined for 12 d by visual inspection and counting. Chronic toxicity, i.e. growth inhibition, was evaluated by measuring the size of each surviving larva (n D 20) at the beginning of exposure (t 0 ) and at the end of the exposure at day 12 (t 12 ). The measurements and statistical analyses were performed according to [START_REF] Mouchet | Use of the International Amphibian Micronucleus Standardized Procedure (ISO 21427À1) for In Vivo Evaluation of Double-Walled Carbon Nanotubes Toxicity and Genotoxicity in Water[END_REF] using a Krus-kallÀWallis test followed by Dunn's test to isolate the group(s) that differ(s) from others, using a multiple comparison procedure with unpaired data versus the NC group (a < 0.05). Graphic representations are proposed based on the growth rate calculated as mentioned in [START_REF] Mouchet | Use of the International Amphibian Micronucleus Standardized Procedure (ISO 21427À1) for In Vivo Evaluation of Double-Walled Carbon Nanotubes Toxicity and Genotoxicity in Water[END_REF].

The MNT was performed according to ISO 21427À1 [START_REF] Iso | ISO International Standard. Water Quality À Evaluation of Genotoxicity by Measurement of the Induction of Micronuclei À Part 1: Evaluation of Genotoxicity Using Amphibian Larvae[END_REF]. At the end of 12 d of exposure À stage 54 [START_REF] Nieuwkoop | Freshwater Alga and Cyanobacteria, Growth Inhibition Test[END_REF]) À larvae are anaesthetized by immersion in a MS222 solution (0.2 g L ¡1 , Sigma) and a blood sample was obtained from each larva by cardiac puncture. The number of erythrocytes containing one micronucleus (MN) or more (micronucleated erythrocytes) was determined under a microscope in a total of 1000 erythrocytes per larva. The statistical method was described in [START_REF] Mouchet | Characterisation and In Vivo Ecotoxicity Evaluation of Double-Wall Carbon Nanotubes in Larvae of the Amphibian Xenopus laevis[END_REF]. Values of pH (345 pH meter, Mettler Toledo) were measured three times during the 12 days of exposure just before the renewal of the exposure medium (pH 24 h ) and just after (pH 0 h ). Al, Fe, and Mo were measured in RW with and without MWNTs after filtration (0.45 mm to remove most of MWNTs) by ICPÀMS (ICPÀMS 7500, Agilent).

Results

Activated sludge respiration inhibition test

The method was applied with respect to the following criteria: (1) the difference in respiration rates between the two controls was below 15% (Table 1) and (2) EC 50 of the control test with the reference 3,5-dichlorophenol was between the validity specified range of 5 and 30 mg L ¡1 (14 mg L ¡1 ). MWNTs did not affect the respiration rate of activated sludge in the conditions of the test up to a concentration of 500 mg L ¡1 at 54 and 28 mg O 2 mg L ¡1 h ¡1 , respectively, for 500 and 5000 mg L ¡1 of MWNTs. Inhibition percentage was 0% and 42.6% for 500 and 5000 mg L ¡1 , respectively. EC 50 (3 h) was, therefore, higher than 5000 mg L ¡1 .

Algal growth inhibition test (P. subcapitata)

The study was performed in compliance with the following quality criteria: (1) biomass in the control cultures increased exponentially by a factor of 102 higher than 16 within the 72-hour test period which corresponds to a specific growth rate of 0.92 d ¡1 ; (2) the mean coefficient of variation for section-by-section specific growth rates (days 0À1, 1À2, and 2À3, for 72-h tests) in the control cultures did not exceed 35%; and (3) the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7%.

In both tests, inhibition percentage of cell growth and growth rate increase with the increase in MWNT concentrations (Table 2). Total inhibition was observed to 500 mg L ¡1 of MWNTs for the cell growth and to 1000 mg L ¡1 for the growth rate. The MWNTs concentration causing a 50% reduction in cell growth (E b C 50 ) was estimated at 34 (23À47) mg L ¡1 , and the growth rate (E r C 50 ) was estimated at 120 (87À160) mg L ¡1 . The NOEC was also estimated at 10 mg L ¡1 for the growth rate inhibition and less than 10 mg L ¡1 for the cell growth. It has to be emphasized that the endpoint used for regulatory purposes is the growth rate and not the cell growth (biomass increase). It was observed that the majority of MWNT particles did not remain in suspension between the beginning and the end of the tests but gathered at the lower part of each flask. Microscopic observations confirmed that the algae appeared normal at the end of the test: The normal shape of P. subcapitata algae is a crescent-shaped cell with an average length of 5À10 mm. An increase in the pH was globally observed in both tests for a given concentration between the beginning and the end of the exposure in accordance with classical measures with algae (Table 3). This may be associated with consumption of the dissolved CO 2 due to the growth of algae. Above 230 mg L ¡1 of MWNTs, pH becomes stable during 72 h. Evolution of dissolved O 2 concentration during 72 h is not significant. Table 4 highlights the decrease of Fe concentrations under MWNT exposure. 11.65 mg kg ¡1 of Fe was measured in the presence of Fe and the absence of MWNTs, whereas no Fe was measured in the presence of both Fe and MWNTs. Zn concentrations also decreased in less proportion from 3.4 mg L ¡1 in the presence of Fe and the absence of MWNTs. Other elements were not affected by the treatment. The results of cell growth and growth rate inhibition without Fe check the effect of iron deficiency and demonstrate iron absorption by MWNTs. Indeed, both inhibition rates lead to 88.88% and to 57.22% in Fe-deprivation medium, whereas there is no inhibition of growth in presence of Fe.

D. magna acute immobilization test

The study was performed in compliance with the following quality criteria: (1) the immobilization in the control did not exceed 10% at the end of the test, (2) daphnids in the control were not trapped at the surface of the water, and (3) the dissolved oxygen concentration remained above 3 mg L ¡1 over the test period. No immobilization effect is observed, regardless of the MWNT concentration in both tests, at 24 and 48 h. After the 24-h and 48-h test periods, the actual concentrations inhibiting the mobility of daphnids, i.e. EC 50 À24 h and EC 50 À48 h, were estimated to be both higher than 100 mg L ¡1 . Neither pH nor concentrations of dissolved O 2 were impacted in the presence of 100 mg L ¡1 of MWNTs during 48 h (Table 5). The appearance of the test suspensions was visually checked at the beginning and at the end of the test: as flasks were continuously maintained under axial rotation by use of a cylindrical roller device, MWNTs remained in suspension.

D. magna reproduction test

The study was performed in compliance with the quality criteria: (1) the mortality in the controls (parent females) did not exceed 20% at the end of the test, and (2) the average cumulative number of living young produced per surviving parent female was higher than 60 in the controls at the end of the test. The percentage of inhibition of the reproduction was dose dependent (Table 6). Inhibition percentage ranged from 0.30 (to 10 mg L ¡1 of MWNTs) to 21.06% (at 100 mg L ¡1 of MWNTs). The EC 50 value was 317.75 mg L ¡1 , and LOEC and NOEC were 100 and 47 mg L ¡1 , respectively. After filling and between each renewal, there was sedimentation of the MWNTs at the bottom of the flasks.

Fish acute toxicity test (D. rerio)

The study was performed in compliance with the following quality criteria: (1) the mortality in the control did not exceed 10% at the end of the test;

(2) the concentration of dissolved oxygen in the test vessels remained above 60% of the air saturation value at the end of the test;

(3) the pH did not vary by more than 1 unit. The results indicated no mortality effect regardless of the MWNTs concentration in both tests and irrespective of exposure time, 24, 48, 72 or 96 h (Table 7). LC 50 were then higher than 100 mg L ¡1 at each time. pH (Table 7) and saturation in oxygen (Table 8) were stable both in the preliminary and definitive tests. Thanks to the stirring device, it was observed that many of the MWNT particles remained in suspension within each tank.

Amphibian (X. laevis) bioassays

No mortality was observed until 72 h of exposure whatever the MWNT concentration. Very low mortality was observed from 50 mg L ¡1 at 96 h of exposure and was not significant compared to the negative control (0 mg L ¡1 ). EC 50 was then estimated to be higher than 500 mg L ¡1 . Results of Xenopus exposure for 12 days in semi-static conditions indicated 20% mortality at 50 mg L ¡1 of MWNTs. No mortality was observed at lower concentrations. Growth inhibition results (Figure 2) were significantly evidenced in larvae exposed to 25 (no growth of larvae) and 50 mg L ¡1 of MWNTs (four times less). MN induction in Xenopus larvae after 12 days of exposure to the referent genotoxic CP (Figure 3) was significant compared to the NC. This result validates the bioassay. The results of MN induction in larvae exposed to MWNTs indicated no genotoxicity compared to the NC group. Median values were distributed without MWNTs' doseÀeffect relation. pH values (Table 9) were slightly lower after 24 h of exposure (pH 24 h ) compared to just after the renewal of the exposure medium (pH 0 h ). It may be in relation to the excretion process of larvae for 24 h and the acidification of the exposure media. There was no pH modification in relation to MWNT concentrations. Metals were dosed in water exposure in the presence or absence of 50 mg L ¡1 of MWNTs, without larvae, after 24 h of contact (Table 10). 12.8 § 0.6 mg L ¡1 of Mo and 131.5 § 5.4 mg L ¡1 of Al were measured in medium in the presence of MWNTs, whereas concentration of Fe was under the quantification limit (<10 mg L ¡1 ).

Discussion

The aim of the present work is not to compare the biological effects between different biological models but to contribute to a better understanding of the ecotoxicity of CNTs and their environmental exposure assessment to provide valuable insight into likely exposure scenarios at different levels of the trophic chain. Synthetic results of the biological effects in organisms after MWNT exposure are presented in Table 11. The results are as follows: no toxicity in activated sludge (bacteria) at 500 mg L ¡1 of MWNTs, no acute toxicity in fish and daphnia up to 100 mg L ¡1 of MWNTs, inhibition of growth in amphibian larvae at 25 mg L ¡1 of MWNTs, and a notable effect at high concentrations of MWNTs with the growth inhibition test in algae (EC 50 D 120 mg L ¡1 and NOEC D 10 mg L ¡1 ). Observed toxicity in this present work is in accordance with much of the data published in the literature relative to the potential toxicity of raw CNTs in aquatic organisms. An LOEC of 10 mg L ¡1 was observed in daphnia [START_REF] Roberts | In Vivo Biomodification of Lipid Coated Carbon Nanotubes by Daphnia Magna[END_REF], in marine copepod [START_REF] Templeton | Life-Cycle Effects of Single-Walled Carbon Nanotubes (SWNTs) on an Estuarine Meiobenthic Copepod[END_REF]) and in amphibians [START_REF] Mouchet | Characterisation and In Vivo Ecotoxicity Evaluation of Double-Wall Carbon Nanotubes in Larvae of the Amphibian Xenopus laevis[END_REF]). Zhu and collaborators [START_REF] Zhu | Acute Toxicities of Six Manufactured Nanomaterial Suspensions to Daphnia magna[END_REF]) calculated EC 50 to 8.72 and 1.30 mg L ¡1 for immobilization of daphnia exposed to raw SWNTs and MWNTs, respectively, and to 22.57 and 2.42 mg L ¡1 for mortality exposed to raw SWNTs and MWNTs, respectively. Kennedy and collaborators [START_REF] Kennedy | Factors Influencing the Partitioning and Toxicity of Nanotubes in the Aquatic Environment[END_REF]) calculated EC 50 for mortality after 48 h of raw MWNTs exposure in daphnia at 50.9 mg L ¡1 . No toxicity was evidenced for hydra and crustaceans up to 100 mg L ¡1 of raw SWNTs [START_REF] Blaise | Ecotoxicity of Selected Nano-materials to Aquatic Organisms[END_REF], whereas toxicity to algae exposed to SWNTs was observed at 10 mg L ¡1 . [START_REF] Cheng | Effect of Carbon Nanotubes on Developing Zebrafish (Danio rerio) Embryos[END_REF] observed hatching delay in fish eggs exposed to 120 mg L ¡1 of raw SWNTs and 240 mg L ¡1 of raw DWNTs. Neither mortality nor growth inhibition was observed in urodele amphibian larvae up to 1 g L ¡1 of raw DWNTs [START_REF] Mouchet | Assessment of the Potential In Vivo Ecotoxicity of Double-Walled Carbon Nanotubes (DWNTs) in Water, Using the Amphibian Ambystoma mexicanum[END_REF]), whereas mortality was observed at 50 mg L ¡1 and growth inhibition from 10 mg L ¡1 in anuran amphibian larvae exposed to raw DWNTs [START_REF] Mouchet | Characterisation and In Vivo Ecotoxicity Evaluation of Double-Wall Carbon Nanotubes in Larvae of the Amphibian Xenopus laevis[END_REF][START_REF] Mouchet | Use of the International Amphibian Micronucleus Standardized Procedure (ISO 21427À1) for In Vivo Evaluation of Double-Walled Carbon Nanotubes Toxicity and Genotoxicity in Water[END_REF] as well as growth inhibition at 50 mg L ¡1 for raw MWNTs exposure [START_REF] Mouchet | CNTs Ecotoxicity in Amphibians: Assessment of Multi-walled Carbon Nanotubes (MWNT) and Comparison with Double-Walled Carbon Nanotubes (DWNT)[END_REF]. [START_REF] Kahru | From Ecotoxicology to Nanoecotoxicology[END_REF] Toxicity obtained from algae in the present work appears to be in relation to the effect of Fe deficiency on algal growth. Indeed, algal growth experiments in the absence of iron indicated 88.8% of cell growth inhibition (biomass) and 57.2% of growth rate inhibition (cell multiplication) compared to the absence of inhibition when iron was present. Moreover, among the 8 micro-nutrients that are present in the algal culture medium (boron, manganese, iron, cobalt, nickel, copper, zinc, and molybdenum), iron is strongly removed by the MWNTs from the filtrated culture medium (<1 mg kg ¡1 ). When compared to the initial algal medium in which iron concentration is 11.65 mg kg ¡1 , this leads to suspect iron adsorption by MWNTs. Iron ion adsorption has already been demonstrated in chemical studies using different types of CNTs [START_REF] Li | Ferric Ion Adsorption and Electrode Sorption by Carbon Nanotubes and Nanofibres Films[END_REF]. In contrast to the present growth inhibition of P. subcapitata due to iron deprivation, [START_REF] Schwab | Are Carbon Nanotube Effects on Green Algae Caused by Shading and Agglomeration?[END_REF] indicated that observed inhibition of C. vulgaris and P. subcapitata is in relation to light masking by CNTs, which can adhere to algal surfaces and hence restrict light accessibility to the cells, resulting in the inhibition of growth. [START_REF] Long | Systematic and Quantitative Investigation of the Mechanism of Carbon Nanotubes' Toxicity Toward Algae[END_REF] indicated that their MWNTs significantly inhibited the algal growth of Chlorella sp. with a negligible contribution of metal catalyst residues in the MWNTs and nutrient elements adsorbed by MWNTs. These authors hypothesize that the toxicity of algae could mainly be explained by the combined effects of oxidative stress, agglomerations and physicals interactions, and shading effects, with the quantitative contributions from these mechanisms depending on the MWNT size and concentration. In any case, comparison of results between these different works must be limited because of the diversity of studied CNTs. Nevertheless, [START_REF] Verneuil | Multi-walled Carbon Nanotubes, Natural Organic Matter and the Benthic Diatom Nitzschia palea: "A Sticky Story[END_REF] indicated that only direct exposure to 50 mg L ¡1 of MWNTs (the same type of MWNTs as in the present study) led to growth inhibition of Nitzschia palea after 48 h and suggested that EPS (extracellular polymeric substances) provide considerable protection against MWNTs, without alteration of the photosynthesis.

Concerning the absence of genotoxicity in erythrocytes of Xenopus larvae, the present results are in agreement with the previous ones obtained on amphibian larvae in the same conditions of exposure to MWNTs [START_REF] Mouchet | CNTs Ecotoxicity in Amphibians: Assessment of Multi-walled Carbon Nanotubes (MWNT) and Comparison with Double-Walled Carbon Nanotubes (DWNT)[END_REF] and DWNTs [START_REF] Mouchet | Assessment of the Potential In Vivo Ecotoxicity of Double-Walled Carbon Nanotubes (DWNTs) in Water, Using the Amphibian Ambystoma mexicanum[END_REF][START_REF] Mouchet | Characterisation and In Vivo Ecotoxicity Evaluation of Double-Wall Carbon Nanotubes in Larvae of the Amphibian Xenopus laevis[END_REF]. The majority of the time, if acute and chronic toxicities are generally observed after CNTs exposure of different biological models, genotoxicity, especially via micronucleus induction mechanism, is not demonstrated. [START_REF] Kim | Aspect Ratio Has No Effect on Genotoxicity of Multi-wall Carbon Nanotubes[END_REF] indeed obtained no genotoxicity of raw MWNTs according to OECD test guidelines 471 (bacterial reverse mutation test), 473 (in vitro chromosome aberration test with and without S9), and 474 (in vivo micronuclei test). Di Sotto et al. ( 2009) [START_REF] Szendi | Lack of Genotoxicity of Carbon Nanotubes in a Pilot Study[END_REF] also reported that MWNTs had no mutagenic effect in bacteria systems. In the same way, [START_REF] Wirnitzer | Studies on the In Vitro Genotoxicity of Baytubes®, Agglomerates of Engineered Multi-walled Carbon Nanotubes (MWCNT). Toxicity of Engineered Nanomaterials[END_REF] indicated no genotoxicity of raw MWNTs testing for chromosome aberrations in V79 cells and for gene mutations in the Salmonella microsome test. Nevertheless, genotoxic effects may be produced either by direct interaction of particles with genetic material or by secondary damage from particle-induced reactive oxygen species. In this context, some authors demonstrated oxidative stress by MWNTs [START_REF] Reddy | Multi-wall Carbon Nanotubes Induce Oxidative Stress and Cytotoxicity in Human Embryonic Kidney (HEK293) Cells[END_REF][START_REF] Srivastava | Multi-walled Carbon Nanotubes Induce Oxidative Stress and Apoptosis in Human Lung Cancer Cell LineÀA549[END_REF]), and, for example, in Xenopus larvae after MWNT exposure [START_REF] Saria | Evaluation of Oxidative Stress and DNA Damage in Xenopus laevis Tadpoles Exposed to Multi-walled Carbon Nanotubes[END_REF].

Exposure media for the different organisms would play a role in the observed toxicity of the present work. Nevertheless, characterizations of the MWNT suspension in exposure medium do not appear essential to place it in relation with biological effects because they are observed at very high and unrealistic concentrations. The effects obtained in the present work are globally weakly marked, probably in relation with the MWNTs' limited bioavailability in the water column for organisms because of CNTs' sedimentation at the bottom of containers. As displayed in Figure 1, raw MWNTs appeared as large rather spherical agglomerates of bundles without free or isolated nanotubes. Observed effects in organisms may be in connection with exposure to these agglomerates inducing respiratory and/or intestinal clogging in relation with their absorption and not necessarily related to the intrinsic effects of CNTs [START_REF] Mouchet | CNTs Ecotoxicity in Amphibians: Assessment of Multi-walled Carbon Nanotubes (MWNT) and Comparison with Double-Walled Carbon Nanotubes (DWNT)[END_REF]2011;[START_REF] Petersen | Potential Release Pathways, Environmental Fate, and Ecological Risks of Carbon Nanotubes[END_REF]). This result is also in accordance with the observation of CNTs in the guts of aquatic organisms such as Lumbriculus variegatus [START_REF] Petersen | Ecological Uptake and Depuration of Carbon Nanotubes by Lumbriculus variegates[END_REF], Arenicola marina [START_REF] Galloway | Sublethal Toxicity of Nano-titanium Dioxide and Carbon Nanotubes in a Sediment Dwelling Marine Polychaete[END_REF], D. magna [START_REF] Zhu | Acute Toxicities of Six Manufactured Nanomaterial Suspensions to Daphnia magna[END_REF], Hyalella azteca, Leptocheirus plumulosus, and Ceriodaphnia dubia [START_REF] Kennedy | Influence of Nanotube Preparation in Aquatic Bioassays[END_REF]. [START_REF] Li | The Responses of Ceriodaphnia dubia Toward Multi-walled Carbon Nanotubes: Effect of PhysicalÀChemical Treatment[END_REF] describe the ingestion of CNTs in C. dubia followed by excretion in exposure media. Many of these studies tend to highlight that ingested CNTs by organisms may enter the ecological pyramid via their move up through the food chain. Moreover, excreted CNTs may contribute to maintaining a pressure contamination of CNTs in media.

Nevertheless, a few mg L ¡1 of Mo and Al (and no Fe) were measured in the water medium of amphibian exposure after 24 h containing the higher concentration of MWNTs (50 mg L ¡1 ). It would suggest that they may contribute, especially Al, to the toxicity observed in amphibians to high concentrations of MWNTs, although no genotoxicity was observed. This result encourages us to investigate the potential release of metal impurities at lower concentrations and in function of time in the different water exposures.

Conclusion

The present knowledge concerning the ecotoxic effects of CNTs is rather limited and deserves to be documented more extensively. First, the ecotoxicological hazard assessment needs approaches and measurement tools using standardized test methods. Then, adaptation of well-known protocols is necessary. This work is thus a contribution to the assessment of the potential ecotoxicity of CNTs within the aquatic compartment; it could be helpful for regulatory purposes. The results indicate that MWNT effects are weakly marked and expressed at unrealistic nominal concentrations (approximately 10 mg L ¡1 ), in relation with probable MWNT ingestion. Considering their increasing use in commercial products, this study emphasizes the need to further study their ecotoxicity and highlights that assessing the risks of CNTs requires a better understanding of their toxicity, bioavailability, and behavior in relation with their intrinsic physicochemical properties.

Figure 1 .

 1 Figure 1. Scanning electron microscopy observations of MWNTs, raw MWNTs from the same sample are observable in balls at different magnifications: (a) 100 X, (b) 3000 X and (c) 20,000 X.

Figure 2 .

 2 Figure 2. Growth inhibition measurement of Xenopus larvae after 12 days of semi-static exposure to MWNTs.Note: Ã indicates a significant lower length compared to the control (0 mg L ¡1 ). Growth rate is calculated as a percentage based on the length measurement of larvae at the beginning of the exposure and at the end.

Figure 3 .

 3 Figure 3. Micronucleus induction measurement (median § IC 95%) in erythrocytes of Xenopus larvae after 12 days of semi-static exposure according to the concentration of MWNTs. Note: Ã indicates a genotoxic condition compared to the negative control NC (0 mg L ¡1 of MWNTs).PC: Positive Control, Cyclophosphamide, genotoxic of reference to 20 mg L ¡1 .MNE /oo: Micronucleated erythrocytes.

Table 1 .

 1 Results of respiration and inhibition rate of microorganisms of activated sludge after 3 h in the presence of MWNTs.

				MWNTs (mg L ¡1 )
		C1	C2	500	5000
	Respiration rate (O 2 mg L ¡1 h ¡1 )	52.5	45.0	54.0	28.0
	% inhibition	À	À	0	42.6
	C1, C2: control 1, 2

Table 2 .

 2 Average percentage inhibition of cell growth (I Ai ) and growth rate (I mi ) of the freshwater algae Pseudokirchneriella subcapitata exposed to MWNTs for 72 h: (a) preliminary test; (b) definitive test.

		Nominal concentration of MWNTs (mg L ¡1 )	I Ai (%)	I mi (%)
	(a)	0	0	0
		1	0	0
		5	0	0
		10	0	1
		50	27	7
		100	54	13
	(b)	0	0	0
		10	13	2
		22	26	6
		48	54	14
		105	90	44
		230	99	73
		500	100	82
		1000	100

Table 3 .

 3 Measured pH and O 2 concentrations in the preliminary test (a) and in the definitive test (b) of the exposure of the freshwater algae Pseudokirchneriella subcapitata to MWNTs.

			pH		Dissolved O 2 (mg L ¡1 )
		Nominal Concentration
		of MWNTs mg L ¡1	T 0	T 72 h	T 0	T 72 h
	(a)	0	7.89	7.91	9.0	9.6
		1	7.92	9.42	9.1	9.7
		5	7.94	9.29	9.1	9.5
		10	7.94	9.40	9.0	9.7
		50	7.95	8.83	9.0	9.4
		100	7.96	8.62	9.0	9.3
	(b)	0	7.96	7.98	8.8	8.8
		10	7.90	8.90	9.0	8.9
		22	7.91	8.60	9.0	8.8
		48	7.89	8.30	9.0	8.7
		105	7.90	8.04	9.0	8.6
		230	7.91	7.97	9.0	8.5
		500	7.96	7.97	9.0	8.5
		1000	8.03	7.99	8.9	8.4

Table 4 .

 4 Measured metal species in mg kg ¡1 using ICPÀMS in alga medium in presence or absence of Fe and MWNTs.

				B	Mn	Fe	Co	Ni	Cu	Zn	Mo
	Alga medium	¡Fe	¡MWNTs	53.2	110.4	<1	<1	<1	<1	1.6	2.7
	Alga medium	CFe	¡MWNTs	39.5	122.8	11.6	<1	<1	<1	3.4	3.3
	Alga medium	CFe	CMWNTs	43.8	94.8	<1	<1	<1	<1	<1	3.2
	ICPÀMS: Inductively coupled plasmaÀmass spectrometry.

B: Boron À Mn: Manganese À Fe: Iron À Co: Cobalt À Ni: Nickel À Cu: Copper À Zn: Zinc À Mo: Molybdenum. Measured values correspond to mean value from two replicates.

Table 5 .

 5 Dissolved O 2 and pH measured at the beginning (T 0 ) and at the end (T 48 h ) of the exposure of Daphnia magna to MWNTs for the definitive test of immobilization.

		pH		Dissolved O 2 (mg L ¡1 )
	MWNT Concentrations
	mg L ¡1	T 0	T 48 h	T 0	T 48 h
	0	7.95	7.77	8.4	8.1
	100	7.95	7.73	8.5	8.3

Table 6 .

 6 Results of the Daphnia magna reproduction test. Percentage of inhibition of reproduction measurement after 21 days of exposure.

	MWNT concentrations (mg L ¡1 )	0	5	10	22	47	100
	Mean	193.70	197.67	193.11	186.40	169.70	152.90
	Standard deviation	21.78	64.40	61.74	15.60	27.08	46.73
	% Inhibition	À	2.05	0.30	3.77	12.39	21.06

Table 7 .

 7 Measured pH in (a) preliminary test at the beginning (0 h) of the experiment and at the end of exposure (96 h) and in (b) definitive test, each having a total exposure time of 24 h in fish experiment.

	pH

Table 8 .

 8 Measured O 2 concentrations (a) in the preliminary test at the beginning of the experiment (0 h) and at the end of exposure (96 h) and (b) in the definitive test.

			Dissolved O 2 (mg L ¡1 )
	(a)	MWNT concentrations (mg L ¡1 )	0 h	96 h
		100	8.0	9.2
		50	8.3	9.3
		35	8.3	9.2
		1	8.1	9.1
		0	8.2	9.1
				Dissolved O 2 (mg L ¡1 )
	(b)	MWNT concentrations (mg L ¡1 )	0 h	24 h	48 h	72 h	96 h
		100	95	99	96	97	98
		100	95	99	95	98	98
		0	95	98	93	98	98
		0	95	98	93	97

Table 9 .

 9 Measured pH values during the 12 days of exposure of Xenopus larvae to different concentrations of MWNTs. pH 0 h was measured just after the renewal of the exposure medium. pH 24 h was measured just before the renewal.

	MWNT concentrations (mg L ¡1 )	pH 0 h	pH 24 h
	0	8.00	7.31
	0.05	8.04	7.30
	0.1	8.04	7.26
	0.5	8.05	7.27
	1	8.14	7.22
	5	8.12	7.33
	10	8.27	7.19
	25	8.14	7.43
	50	8.09	7.29

Table 10 .

 10 Measured metal species (Al, Fe, Mo) in mg L ¡1 using ICPÀAES in amphibian mediums in the presence or absence of 50 mg L ¡1 of MWNT after 24 h of contact. Al: aluminum. Calculated values correspond to mean value from 4 replicates ( § standard error). Quantification limit (QL) is 10 mg L ¡1 for Fe and Mo, and 50 mg L ¡1 for Al.

		Fe	Mo	Al
	ÀMWNTs	<10 mg L ¡1	<10 mg L ¡1	<50 mg L ¡1
	CMWNTs	<10 mg L ¡1	12.80 § 0.58 mg L ¡1	131.50 § 5.36 mg L ¡1

Fe: Iron À Mo: Molybdenum À

Table 11 .

 11 Synthetic results of the biologic effects in organisms after exposure to MWNTs.

	LOEC mg L À1						100
	NOEC mg L À1		<10	10			47
	EC 50 mg L À1	>5000	34	120	>100	>100	317.75	>500
	Measure	0% at 500 mg L ¡1	42.6% at 5000 mg L ¡1						20% at 50 mg L ¡1	Significant inhibition from 25 mg L ¡1	No significant MN induction
	Biological endpoint Exposure time	Inhibition percentage 3 h	Cell growth À biomass 72 h	Growth rate À cell multiplication 72 h	Mobility 24 h	48 h	Reproduction 21 days	Mortality 96 h	Mortality 12 days	Growth inhibition 12 days	Micronuclei (MN) induction 12 days
	Bioassay Organism	OECD 209 Bacteria activated sludge	OECD 201 Fresh algae Pseudokirchneriella	subcapitata	OECD 202 Daphnid		OECD 211 Daphnia magna	ISO 21 427À1 Amphibian Xenopus laevis

  calculated on the basis of 34 median values a L(E)C 50 between 1 and 10 mg L ¡1 for SWNTs and MWNTs (L(E)C 50 derived from 77 individual values. The majority of the published results indicate that exposure to CNTs generally leads to biological disorder at different levels but usually above unrealistic concentrations of 10 mg L ¡1 . In surface water in Europe, for the simulation results of the predicted environmental concentrations,[START_REF] Gottschalk | Modeled Environmental Concentrations of Engineered Nanomaterials (TiO 2 , ZnO, Ag, CNT, Fullerenes) for Different Regions[END_REF] indicated lower CNTs concentrations, with 0.004 ng L ¡1 (most frequent value) and 0.0035 ng L ¡1 as the range of the lower quantile and 0.021 g L ¡1 as the upper quantile. Nevertheless, it could be hypothesized that CNT concentrations accumulate into the environment over time.

Acknowledgments

The authors acknowledge Annie Perrault and Floriane Bourdiol for their technical help. Thanks to the Groupement de Recherche de Lacq (ARKEMA) for MWNTs suspensions and technical help. We are also indebted to Michel Lagoin (Universit e Paul Sabatier, Toulouse, France) for reviewing the English version of the manuscript and to the language services of Elsevier.

Disclosure statement

Part of this research has been developed in the framework of the French collaborative laboratory NAUTILE (Nanotubes et Ecotoxicologie), which is the first public/private joint laboratory dedicated to the study of the ecotoxicological impact of CNTs in the aquatic environment. The framework agreement was signed by (1) Arkema France, (2) Centre National de la Recherche Scientifique (CNRS), (3) Institut National Polytechnique de Toulouse (INPT), and ( 4) Universit e Paul Sabatier (UPS) of Toulouse in September 2010. The authors declare no competing financial interests or conflict of interest.


One of the main fundamental problems in designing a sensor network is to detect and locate their positions. Hence, sensors can be utilized for data collection in order to detect and organize the objects of interest. In this paper, we present a novel mechanism for object localization and detection in protected areas and surveillance zones. The goal of the proposed approach is to enhance area supervision and localize objects efficiently in wireless sensor networks.

I. INTRODUCTION

The scientific and industrial communities are lately showing a huge interest in wireless sensor network which consists of a set of smart sensors miniaturized with limited energy capacity [START_REF] Risteska Stojkoska | Application of wireless sensor networks for indoor temperature regulation[END_REF]. Rapid deployment, reduced cost and fault tolerance in sensor networks are key features that enable deployment of different sensor network applications in various fields. There is a growing requirement for each person, for example, to be able to locate or determine whereabouts of his/her family members and relatives, or locate a service (e.g., medical help, doctor, University cafeteria, etc.). Such requirements are becoming even more significant in the context of improving the quality of life in general. Firefighters working in hazardous environments require efficient tracking systems to avoid being ending up in danger in their interventions. In hospitals, wireless sensor networks can be exploited to locate the nearest doctor of a patient, in the case of an emergency [START_REF] Dasios | Hands-On Experiences in Deploying Cost-Effective Ambient-Assisted Living Systems[END_REF].

The main tasks for a surveillance system include detection and aggregation of wireless sensor network data. The network must be able to identify any object in its detection area. The most current and closest detection algorithm was able to integrate the energy values of the transmitted signals by sensors in a fixed time period [START_REF] Bahrepour | Having Use of wireless sensor networks for distributed event detection in disaster management applications[END_REF]. If those values exceed a threshold value, the sensors indicate the presence of the detected object. This simple method suffers significant drawbacks when no objects are detected mainly if the signal consists of noise, or when the setting of the threshold is very difficult.

In [START_REF] Ko | A grid-based distributed event detection scheme for wireless sensor networks[END_REF], Ko et al. propose a detection technique of an event at the sensor node that needs to be divided into clusters. Each cluster is defined by a cluster-head which supervise, contain and detect data events that happen between the sensor nodes. A cluster-head shares information collected with his clusterheads neighbors in order to define the main algorithm threshold value.

Work in [START_REF] Xiao | A Survey on Wireless Indoor Localization from the Device Perspective[END_REF] surveys different surveillance systems that can detect and track patients when they have problems. These systems are based on indoor location of small nodes. As a result, these sensors communicate with each other and are able to set up some environmental parameters measurements. In addition, they can estimate the patient's position as well using less energy. However, the accuracy of the position obtained by the sensor nodes is highly dependent on the technique used. The location algorithm of the above techniques is localization using an infrared-based system. This technique is not efficient because of the non-robust infrared signal which is disrupted by the presence of daylight.

Among the known data sensing and fusion methods, a cluster network structure for WSNs. This can process the original data in the sensor nodes and reduce unnecessary transmissions. Many clustering algorithms have been specifically designed for the wireless sensor network. Among them, K-means algorithms, it organizes the sensor nodes so that each group can communicate with the fusion center with relaunched manner. In addition, clustering can also reduce bandwidth communications and routing table size stored in individual nodes [START_REF] Luo | A kernel machine-based secure data sensing and fusion scheme in wireless sensor networks for the cyber-physical systems[END_REF] In this paper, we introduce a network-based monitoring system of wireless sensors depending on an algorithm which detects and localizes objects. Our objective is to improve the detection accuracy by integrating fuzzy logic in the aggregation phase of the detected data. In addition, we focus on determining the position of the detected object. Our contributions are subsequently threefold:

1-Integration of a fuzzy logic in order to optimize data processing which is detected by the multiple type of sensors.

2-A new clustering mechanism based on the k-means

algorithm which takes into account the distance between nodes for the cluster-head election process. 3-A new location mechanism based on the ultrasonic technique and finally, the implementation and operation of our system through the use of low cost hardware such as FPGAs and sensor Acoustic.

II. PROBLEMATIC AND OBJECTIVES

Monitoring of daily life activities of elderly people at home is a social problem which requires, on one hand, the need for an increased security for people living alone. On the other hand, we need to control health-related costs by delaying the entry into institution or hospitalization. The resident person must have tools that provide comfort and well-being by giving the user the ability to control his environment permanently, but also to alert in case of danger and prevent the possible risks loss. For this, solutions should be proposed to be able to monitor risky situations or possible danger (e.g., discomfort, fall, impaired balance and walking, malnutrition, poor adherence, hearing loss or vision, etc.) [START_REF] Piau | Aging society and gerontechnology: A solution for an independent living?[END_REF]. Some of these factors can have a significant impact with potentially serious consequences on the quality of life. It is therefore necessary to

A Novel Intelligent Mechanism for Monitoring in Wireless

Sensor Networks

Fekher Khelifi a,b , Abbas Bradai b ,Med Lassaad Kaddachi a , Priyanka Rawat c a Laboratory of Electronics and Microelectronics, University of Monastir, Monastir 5019, Tunisia b XLIM Institute, University of Poitiers, Poitiers 86130, France c University of Avignon, LIA-CERI, Avignon, France Fakher.Khelifi@ fsm.rnu.tn, abbas.bradai@univ-poitiers.fr, lassaad.Kaddachi@isigk.rnu.tn, priyanka.rawat@univ-avignon.fr develop open, flexible and low-cost solutions, which are easy to install for collecting relevant data representing the life activity of the resident in his living environment. The proposed solution is based on establishing a set of wireless sensors modules with low consumption of wires, configurable and easily interfaced. Our proposal is illustrated in Figure 1. During the detection phase, the acoustic sensors, infrared, ultrasonic are useful for detecting an object or a potential event. Each sensor detects its range during the operating period. When a sensor node detects a set of events, first, the node compares the detected value to the predefined threshold value and then triggers the transmitter to the cluster-head. Each cluster-head processes the values received by those nodes, using the fuzzy logic algorithm and sends the percentage of detection to the base station. If this percentage exceeds the threshold, the proposed system moves into the localization phase.

The localization phase of the object triggers, the moment it is detected by the sensor node. During this phase, the ultrasound sensors measure the distances using the propagation of the ultrasonic signal and time difference in radio frequency (RF). The centroid algorithm is used to calculate the location coordinates of the detected object. The cluster-head sends these location coordinates of the detected object to the base station. When the detected value by the sensor nodes is below the threshold, the system moves into the phase of new clusters configuration.

When the network is deployed, the clusters are formed using the k-means algorithm. Each cluster has a cluster-head which processes the data collected by its members, then the detection percentage are sent to the base station. If this percentage is below the threshold, the base station selects the nodes that have non-zero values in order to form a new cluster and to verify the existence of the object. This clustering technique reduces the communication in the network and subsequently minimizes the energy consumption and as a result reduces the amount of transmitted data.

IV. CONCLUSION

In this article, a novel monitoring mechanism is proposed for wireless sensor networks, in which we have proposed a technique for objects detection based on fuzzy logic. This technique improves the detection capability as well as increases the location accuracy using ultrasonic technology for data measurement and the centroid algorithm for data processing. To reduce the communication between nodes and the amount of data transmitted to the base station, we have proposed a novel clustering mechanism based on the k-means algorithm.

The performance analysis of our proposal has been carried out through the NS3 network simulator. Our system is realized using nexys2 FPGA which is responsible for data processing and is connected to the sensors which capture the data and send to the XBee PRO-OEM RF module for transmission V. REFERENCE

Figure 1 .

 1 Figure 1. Scenario for Data Collection

Figure 2 .

 2 Figure 2. Schematic diagram of the proposed Model