Gabriela A Vázquez-Rodríguez

Rosa Icela Beltrán-Hernández

Claudia Coronel-Olivares

Jean-Luc Rols

R I Beltrán-Hernández

J.-L Rols

J.-L Rols

Standardization of activated sludge for biodegradation tests

Keywords: Bioassays, Bioanalytical methods, Sludge, Reference materials

Activated sludges are an inoculum source commonly used in biodegradation studies, as wastewater treatment facilities constitute an entry point to the environment for many chemicals. In this paper, the main issues relating to the use of activated sludge in biodegradability tests are presented. Special attention is also devoted to discussing the factors affecting both the activity of the microbial communities and the test results. After a short survey of the state of the art of microbiology of activated sludge, the paper focuses on the methods used to reduce the variations in the diversity, quality and quantity of these communities. Finally, use of surrogates as reference materials in biodegradability tests is discussed.

Introduction

Biodegradability can be defined as the intrinsic capacity of a substance to be structurally simplified by the action of microorganisms. It is one of the properties of the substances that determine their potential environmental hazard, and a desirable feature of chemicals used and then released to aquatic and terrestrial environments in large quantities. In the European Union (EU), the REACH (Registration, evaluation and authorization of chemical substances) system is new substance-oriented legislation based on the precautionary principle, whereby chemicals are considered to be unacceptably hazardous until proven otherwise [START_REF] Combes | An overall strategy for the testing of chemicals for human hazard and risk assessment under the EU REACH system[END_REF]. According to REACH, data on biodegradation properties are required for classification, labelling, and packaging of a substance, and for registration of any chemical produced or imported into the EU in quantities higher than 10 tons per year and per manufacturer or importer [START_REF] Foth | Concept of REACH and impact on evaluation of chemicals[END_REF].

The OECD methodology offers a tiered approach for measuring biodegradability [START_REF]Revised introduction to the OECD guidelines for testing of chemicals, Section 3. Organization for the Economic Cooperation and Development[END_REF]. The first-tier tests (i.e., the 301 series of ready biodegradability tests, RBT) are simple screening assays providing limited time (typically 28 days) and opportunity for biodegradation to occur, because the test substance is provided as the sole source of carbon and energy and small amounts of unadapted inoculum are used [4]. The second tier consists of inherent biodegradability tests, which involve a prolonged exposure of the chemical to the microorganisms and make the conditions more favourable for biodegradation, as in the 302 series of OECD tests. The highest tier refers to simulation tests in well-defined environments (i.e., aerobic sewage treatment facilities in the OECD 303 series) to assess degradation kinetics, half-life times, and degradation products. Under current REACH legislation, positive results in an RBT or in an inherent test allow the assumption that the chemical will not persist in the environment [5]. The lack of biodegradation (<20%) in an inherent test provides sufficient evidence to expect persistence, and hence the chemical is regarded as a preliminary "P/vP" (persistent/very persistent) substance in a PBT/vPvB (persistent, bioaccumulative and toxic/very persistent and very bioaccumulative substance) assessment [5].

It is widely recognized that mixed cultures have a higher biodegradation potential than axenic ones, as a microbial community is able to use a wider variety of substrates because of the collective action of individual species [START_REF] Painter | Detailed review paper on biodegradability testing[END_REF][START_REF] Van Heerden | Biolog for the determination of microbial diversity in activated sludge systems[END_REF]. Consequently, biodegradability tests are inoculated with natural populations of microorganisms to ensure the presence of a multiplicity of species and to mimic, to some extent, actual ecosystems or man-made environments. Activated sludge is regarded as a suitable and diverse source of microorganisms, and thus is the most common inoculum for biodegradation tests. Assays inoculated with activated sludge give an impression of a microbial community present in a wastewater treatment plant (WWTP), and their results are environmentally relevant as a preliminary assessment of the fate of chemicals during sewage treatment.

Although a complex and undefined inoculum leads to more representative results in biodegradability studies, its use has several drawbacks. Ideally, activated sludges from different WWTPs would be similar enough to lead to comparable and reliable results, depending only on the molecular structure of the test compound. Nevertheless, geographical and temporal differences in the structure and function of activated sludge communities are broadly reported [START_REF] Hantula | Rapid classification of bacterial strains by SDS-polyacrylamide gel electrophoresis: population dynamics of the dominant dispersed phase bacteria of activated sludge[END_REF][START_REF] Forney | Structure of microbial communities in activated sludge: potential implications for assessing the biodegradability of chemicals[END_REF]. Because any microbial population can to adapt to any assimilable carbon source, variations in the inoculum affect mainly the reproducibility of tests on not readily biodegradable compounds. When the biodegradability of these substances is tested, the results depend both on their chemical structure and the composition of the inocula (i.e., the specific fraction of the viable biomass able to grow on the chemical).

This article overviews the factors affecting both the characteristics of activated sludge and the results of biodegradation tests. The methods used to reduce the variations associated with the inoculation of activated sludge are also presented and discussed. Special attention is devoted to the use of surrogates in biodegradability testing.

Microbiology of the activated sludge process

Activated sludge is a specialized, aerobically-grown culture containing flocculated and non-flocculated organisms. The heterotrophic bacteria aggregated in the flocs and freely dispersed in the liquor constitute the most abundant microbial group. Based on DAPI staining (4′,6-diamidino-2-phenylindole; imparting fluorescence to DNA), the number of bacteria has been estimated to be in the range from 1×10 12 to 10×10 12 per gram of VSS (VSS: volatile suspended solids) [START_REF] Seviour | Microbial ecology of activated sludge[END_REF]. The percentage of metabolically active cells is typically around 80% of this total count [START_REF] Seviour | Microbial ecology of activated sludge[END_REF]. Autotrophic nitrifying bacteria and fungi may be present, but algae are normally absent. Protozoa (classified into flagellates, sarcodines, and ciliates) are virtually ubiquitous in activated sludge, frequently reaching a large proportion (around 5% of the dry weight of solids [START_REF] Seviour | Microbial ecology of activated sludge[END_REF]). Some metazoa (classified into rotifers and nematodes) may also occur. In this man-made ecosystem, protozoa act as predators of bacteria and fungi, serving also as food source of metazoa. In addition to improving effluent quality, protozoan grazing of dispersed bacteria is regarded as a major factor shaping the bacterial community in activated sludge [START_REF] Seviour | Microbial ecology of activated sludge[END_REF].

Conventional studies with culturable isolates have shown that microorganisms of the Proteobacteria phylum predominate in activated sludge. In fact, this bacterial group can be regarded as the main group responsible for pollutant removal [11]. In activated sludge from a municipal WWTP, the most common culturable isolates are Gram-negative, non-fermentative, and oxidase-positive bacteria [START_REF] Pike | Microbial aspects of pollution[END_REF]. The usual genera detected are Zoogloea, Pseudomonas, Achromobacter, Alcaligenes, Comamonas, and Flavobacterium. These culture-dependent techniques have also confirmed that bacterial diversity in activated sludge is different from that present in the sewage entering the plant, where Escherichia coli, Streptococcus faecalis, Salmonella typhi, and P. aeruginosa are commonly found [START_REF] Pike | Microbial aspects of pollution[END_REF].

Microbial community analysis based on fluorescent in situ hybridization (FISH) and clone library data using 16S rRNA sequence comparisons indicate that culturedependent methods are selective and then they do not provide an actual activated sludge structure [START_REF] Wagner | Probing activated sludge with oligonucleotides specific for proteobacteria: inadequacy of culture-dependent methods for describing microbial community structure[END_REF]. For instance, culture on nutrient-rich agar plates resulted in Acinetobacter (γ class of Proteobacteria) being responsible for 30-60% of total colony, forming units (CFU), whilst its abundance was estimated between 3-9% when a cultureindependent technique was employed [START_REF] Snaidr | Phylogenetic analysis and in situ identification of bacteria in activated sludge[END_REF]. By using group and genus specific rRNA oligonucleotide probes, the relative abundance of Proteobacteria was confirmed, as its α, β, and γ classes accounted for approximately 80% of all active bacteria found in activated sludge samples [START_REF] Wagner | Probing activated sludge with oligonucleotides specific for proteobacteria: inadequacy of culture-dependent methods for describing microbial community structure[END_REF]. However, the predominance of organisms belonging to the Betaproteobacteria class has also been established [START_REF] Wagner | Probing activated sludge with oligonucleotides specific for proteobacteria: inadequacy of culture-dependent methods for describing microbial community structure[END_REF][START_REF] Snaidr | Phylogenetic analysis and in situ identification of bacteria in activated sludge[END_REF]. The genera Hydrogenophaga, Comamonas and Acidovorax, which are well known for utilization of a wide range of carbon sources and seem therefore to be well-suited to wastewater treatment, are also included in this group and are now regarded as among the dominant bacteria in activated sludge [START_REF] Snaidr | Phylogenetic analysis and in situ identification of bacteria in activated sludge[END_REF].

Culture-independent methods have enabled the detection of large numbers of previously undescribed species of the Actinobacteria, Chloroflexi, Bacteroidetes, and Firmicutes phyla, along with the unusual Planctomycetes [START_REF] Seviour | Microbial ecology of activated sludge[END_REF][START_REF] Wagner | Probing activated sludge with oligonucleotides specific for proteobacteria: inadequacy of culture-dependent methods for describing microbial community structure[END_REF][START_REF] Snaidr | Phylogenetic analysis and in situ identification of bacteria in activated sludge[END_REF][START_REF] Wagner | Microbial community composition and function in wastewater treatment plants[END_REF]. Chloroflexi includes representatives of bulking filamentous bacteria. Nostocoidia limicola, another filamentous bacterium, belongs to the Actinobacteria phylum. Methanogenic Archaea (Euryarchaeota phylum) may also occur in activated sludge, where they account for <1% of the total cells [START_REF] Seviour | Microbial ecology of activated sludge[END_REF].

Bacteriophages are another important group in activated sludge, able to change the microbial diversity of the ecosystem by driving bacterial lysis [START_REF] Ewert | Enumeration of bacteriophages and host bacteria in sewage and activated-sludge treatment process[END_REF][START_REF] Khan | Bacteriophages isolated from activated sludge processes and their polyvalency[END_REF]. The presence of bacteriophages in activated sludge is a result of their abundance in sewage (as E. coli phages) and of their own multiplication in the system [START_REF] Khan | Bacteriophages isolated from activated sludge processes and their polyvalency[END_REF]. Some phages, for example those hosted by enteric bacteria, seem to decline in numbers, even by ten orders of magnitude, during the activated sludge process [START_REF] Dias | Microbial ecology of activated sludge -II. Bacteriophages, Bdellovibrio, coliforms, and other organisms[END_REF]. By contrast, 3.5 to 18.3-fold augmentations of the amount of phage-like particles through the treatment have also been reported [START_REF] Ewert | Enumeration of bacteriophages and host bacteria in sewage and activated-sludge treatment process[END_REF]. Temporal dynamics of both phages infecting specific bacterial strains and their hosts have been demonstrated by several studies [START_REF] Shapiro | Bacteriophage predation regulates microbial abundance and diversity in a fullscale bioreactor treating industrial wastewater[END_REF][START_REF] Otawa | Abundance, diversity, and dynamics of viruses on microorganisms in activated sludge processes[END_REF]. This correlation between host abundance and phage counts is consistent with the "killing the winner" hypothesis, establishing that a rise in abundance of a bacterial strain leads to a rise in the counts of its specific phage [START_REF] Shapiro | Bacteriophage predation regulates microbial abundance and diversity in a fullscale bioreactor treating industrial wastewater[END_REF]. Several methodological problems with phage quantification and detection are responsible for hindering the assessment of their actual abundance and diversity in activated sludge. Consequently, the role of bacteriophages in the performance of the activated sludge process remains unclear [START_REF] Seviour | Microbial ecology of activated sludge[END_REF].

Effect of selective pressures on activated sludge communities

It is commonly assumed that activated sludge plants of similar configurations and treating wastewater of similar composition will have roughly similar microbial community structure, regardless of their location [START_REF] Seviour | Microbial ecology of activated sludge[END_REF]. However, the microbial populations found in activated sludge are responding continuously to changes in environmental conditions. In addition to the influent wastewater composition, the presence of toxic compounds, excess or deficiency of specific nutrients, pH, and the concentration of dissolved oxygen in the aeration tank can exert selective pressures on both the microbial structure and function of the sludge. A minor change in the environmental conditions may lead to restructuring of the community, even if the change affects only a limited number of microbial species [START_REF] Forney | Structure of microbial communities in activated sludge: potential implications for assessing the biodegradability of chemicals[END_REF]. So, activated sludge structure is now regarded as being in dynamic equilibrium rather than under "steady-state" conditions. This dynamic equilibrium arises also from the natural process of competition between microorganisms for essential resources, which is controlled mainly by the sludge retention time (SRT) or sludge age [START_REF] Saikaly | Bacterial competition in activated sludge: theoretical analysis of varying solids retention times on diversity[END_REF]. SRT is the mean residence time of cells in the plant, and its value may yield oscillations in the abundances of species. For instance, abundances of protozoa and metazoa are positively related to higher SRT [START_REF] Chipasa | The influence of soluble microbial products on microbial community composition: hypothesis of microbial community succession[END_REF][START_REF] Gray | Activated sludgetheory and practice[END_REF] and presumably to lower cell densities in the dispersed phase. It has been also reported that bacterial communities in younger sludges (i.e., cultivated at low SRT) have a higher diversity of genotypes than bacterial groups in older sludges [START_REF] Chipasa | The influence of soluble microbial products on microbial community composition: hypothesis of microbial community succession[END_REF]. As diversity is positively related to ecosystem stability, SRT is a key condition driving resistance to disturbances and resilience (rate of recovery after a disturbance) in activated sludge systems [START_REF] Saikaly | Bacterial competition in activated sludge: theoretical analysis of varying solids retention times on diversity[END_REF].

The mass loading rate (MLR), commonly referred to as the food-to-microorganism ratio, is the daily organic matter load per biomass quantity. The MLR is controlled by sludge wasting rates; if these are high, sludge age decreases and MLR increases. The MLR determines the rate of biological oxidation and the volume of new biomass by maintaining microbial growth in the log, declining, or endogenous growth phases [START_REF] Gray | Activated sludgetheory and practice[END_REF]. In a high-MLR (and consequently with a low sludge age) process, the microbial community has a high proportion of organisms in the stationary and even log growth phases, as there is excess substrate available per unit of biomass. Under these conditions, the rate of metabolism is enhanced and the microbial activity is higher. In contrast, in a low-MLR system (i.e., in an extended aeration plant) the quantity of organic substrate is low in relation to sludge mass, and the microorganisms become nutrient-starved. In this case, a larger proportion of dead cells exists, and most of the living microorganisms are in an endogenous phase.

The characteristics of activated sludges collected in municipal WWTP operating at low, mean, and high MLR (0.1, 0.5, and 0.9 kg BOD 5 kg VSS -1 d -1 , corresponding to SRT of 30, 5, and 3 days, respectively) were compared in a previous study [START_REF] Vázquez-Rodríguez | Activated sludge as inoculum for ready biodegradability testing of chemicals: effect of source[END_REF]. Total cell counts were quite homogeneous in these sludges (in the range 1.1×10 12 to 1.7×10 12 cells g TSS -1 ) (TSS: total suspended solids). However, the cultivable cell contents were higher in the sludge from the process operated at the highest MLR. The higher level of activity of this sludge was confirmed by its specific oxygen uptake rate and by its dehydrogenase activity (DHA), which assesses the bulk potential of sludge to oxidize carbonaceous substrates. Three inocula were prepared by settling of the sludges and recovery of the supernatants, which were then analysed. The densest inoculum was the high-MLR supernatant, as indicated by its higher active and cultivable cell contents. This supports the idea that a low SRT favours the growth of dispersed bacteria (probably by limiting protozoan growth and grazing), as mentioned above. High-MLR supernatant also had higher respiratory activity and a wider range and magnitude of hydrolytic activity determined by the API-ZYM system. These characteristics suggest that the high-MLR sludge has a biodegradation potential higher than that originating from the low-MLR treatment plant, which may be also attributed to the cell starvation induced by these last processes [START_REF] Gray | Activated sludgetheory and practice[END_REF].

Effect of cell density on biodegradability tests

For some OECD methods of the first tier (301 A, B and F), the inoculation level is specified only in terms of volume of sample per volume of test medium, with a maximum density threshold (30 mg TSS L -1 ) of unadapted biomass established as sole control [4]. As a consequence, biodegradation of a given chemical is less likely to occur in those RBT than in an inherent test, for example the Zahn-Wellens assay, which is inoculated with 0.2-1 g TSS L -1 of activated sludge [25]. With a decrease in the cell density, the number of organisms able to degrade a specific chemical in the test vessel also decreases [26]. It may be also expected that the fraction of biomass able to grow on a xenobiotic chemical is lower than the fraction growing on substances commonly found in sewage. Previous ring studies have shown that the percentage of positive results in biodegradability tests is approximately proportional to the logarithm of the total viable cell density at the beginning of the test [START_REF] Blok | Probability of biodegradation, a novel concept for improving chemical classification and risk assessment[END_REF]. In this way, Zahn-Wellens test gave 79-98% positive results, whereas in RBT this value varied around 40%.

The most stringent RBT of the 301 OECD series are the tests 301 D and E, because of their low cell density. In these methods, a more dilute inoculum without sludge flocs is needed and hence raw sewage or a secondary effluent from a domestic WWTP are preferred (one drop L -1 and 0.5 mL L -1 , respectively). After biodegradation of 29 chemicals in several OECD tests, it was found that the 301 D and E assays gave 14 and 38% of positive results whereas the 301 B test provided 54% [START_REF] Painter | Detailed review paper on biodegradability testing[END_REF]. During a study of the biodegradability of 16 chemicals [START_REF] Waggy | Comparison of 20-d BOD and OECD closed-bottle biodegradation tests[END_REF], the 301 D test was found to be as stringent as the 20-day BOD test (BOD: biochemical oxygen demand), irrespective of the inoculum used (i.e., filtered sewage, secondary effluent, or Polyseed, a commercial BOD seed). It was concluded that both tests can be interchangeably used, which also suggests that these sources have equivalent biodegradation potential toward readily biodegradable substances.

Effect of different activated sludge sources in biodegradability tests

The MLR (via the SRT) of a treatment plant exerts a selective pressure on activated sludge, and modifies its diversity, physiological state, and biodegradation potential. So, the choice of a given activated sludge source affects both the rate of biodegradation and the biodegradability test results. This could explain the results of other authors [START_REF] Van Ginkel | Influence of the size and source of the inoculum on biodegradation curves in closed-bottle tests[END_REF], who tested the biodegradation of dodecyl benzene sulfonate (DBS, a borderline case of ready biodegradability) in closed-bottle assays and obtained positive results using a sludge from a municipal WWTP with an SRT of 6 days (mean MLR), while the sludge derived from a WWTP with an SRT of 30 days (low MLR) yielded negative results.

Ready biodegradability of DBS was assessed in other work [START_REF] Vázquez-Rodríguez | Activated sludge as inoculum for ready biodegradability testing of chemicals: effect of source[END_REF] by using supernatants withdrawn from activated sludges of municipal WWTP operating at high, mean, and low MLR. Inoculation on a volume basis with the high-MLR supernatant led to a faster biodegradation pattern and to the shortest lag phase (time necessary to achieve 10% biodegradation; Table 1). These inocula were also used for testing the biodegradability of nitrilotriacetic acid (NTA) and pentaerythritol (PE) [START_REF] Vázquez-Rodríguez | Étude des possibilités de standardisation des inocula des tests de biodégradabilité facile: cas des boues activées de station d'épuration[END_REF], chemicals with erratic behaviour in ring biodegradability studies. As for DBS, the fastest kinetics were obtained with the high-MLR supernatant, and the same trend was observed when more biodegradable compounds, for example aniline and sodium acetate (SA), were evaluated in standard RBT. In this way, the test outcome is affected by the choice of a sludge source, because an active, high density inoculum is more likely to be obtained when activated sludge is sampled from a high-MLR treatment plant. Although a positive RBT result seems more probable with such an inoculum, more studies with non-readily biodegradable chemicals are needed to confirm this.

Use of preconditioned inocula

The OECD guidelines suggest preconditioning the inocula obtained from activated sludge or from secondary effluents by means of aeration for 5-7 days at the test temperature [START_REF]Revised introduction to the OECD guidelines for testing of chemicals, Section 3. Organization for the Economic Cooperation and Development[END_REF]. Preconditioning is intended to overcome the stress of transfer into the test medium and to reduce the endogenous respiration rates of sludges produced in high-MLR plants, because during this treatment the partially-degraded substrates present in the sludges can be oxidized. This aeration period is described as an optional alternative during the inoculum preparation, and so it is implicitly considered not to affect the spectrum or intensity of metabolic activity. In an earlier study [START_REF] Painter | Detailed review paper on biodegradability testing[END_REF], the biodegradation of PE was followed in flasks inoculated with fresh activated sludge and with the same sludge one week after shaking at the test temperature. By preconditioning, the lag period was reduced from 14.3± 2.8 to 11.9 ± 3.3 days, but there was no significant difference in dissolved organic carbon (DOC) removal neither was there variability in the assay between the two types of inocula (94±1% and 97±2% for the preconditioned and the fresh sludges, respectively).

In previous research [31], the possibility of homogenizing the characteristics of various activated sludges by means of preconditioning was explored. As mentioned earlier, the sludges cultivated in WWTP operating at different MLR (high, mean, and low MLR) were markedly heterogeneous in terms of cultivable cell density and dehydrogenase activity (measured as the reduction of 2-(4-iodophenyl)-3-(4 nitrophenyl)-5-phenyltetrazolium chloride, INT, to the corresponding formazan salt, INT-F). After a 7-day aeration period in a mineral medium and under agitation, these properties diminished significantly (Fig. 1). However, the measured characteristics of the supernatants were homogenized, because their cultivable cell contents (varying initially between 1.1 × 10 8 and 1.7 × 10 9 CFU L -1 ) were similar after preconditioning, with values between 1 and 2.4×10 7 CFU L -1 .

The raw and preconditioned supernatants were used afterwards for testing the ready biodegradability of SA and aniline. The tests inoculated with non-preconditioned supernatants had variable lag times (6.7-13.6 h and 36.9-60.1 h for SA and aniline tests, respectively), depending on the source of the sludge. In both cases, the high-MLR supernatant led to the shortest lag phase. When preconditioned supernatants were MLR (kg BOD 5 kg VSS -1 day -1 ) High (0.9) Mean (0.5) Low (0. used, SA biodegradation kinetics were relatively homogeneous and the lag times varied between 11.3 and 12.6 h. These lag times were higher than those obtained with nonpreconditioned inocula, contrary to the results reported for PE by Painter [START_REF] Painter | Detailed review paper on biodegradability testing[END_REF]. In the aniline tests, however, use of preconditioned inocula did not enable homogenization of the biodegradation kinetics, because only the mean-MLR supernatant could mineralize this chemical during a 90 hobservation period.

As evidenced by these results, a deprivation period leads to a decrease in the activity and viability of the microorganisms, and in the biodegradation potential. In axenic cultures of Ralstonia eutropha, phenol-oxygenating activity was completely lost after a 7-day starvation period [START_REF] Watanabe | Starvation improves survival of bacteria introduced into activated sludge[END_REF]. The starvation of microbial populations could cause the death and lysis of some species, whereas opportunistic ones could grow on the cellular material released depending on their ability to adapt to this substrate depletion. Because xenobiotic degradation is accomplished mainly by specialized bacterial groups, the ability to degrade a synthetic chemical such as aniline can be diminished or even lost because of the starvation-induced disappearance of a microbial species. On the other hand, the biodegradation of easily assimilable chemicals such as SA is carried out by more abundant, generalist bacterial populations and so its oxidation is more likely to occur, even after a starvation period. False-negative results can be expected in RBT inoculated with sludges subjected to a deprivation period, and consequently preconditioning is not well suited to homogenizing the quality of the inocula in these assays.

Use of precultured inocula

Preculture of the inoculum in a medium not provided with the test chemical has been suggested for improving the reproducibility of biodegradability assays. In the OECD testing scheme [START_REF]Revised introduction to the OECD guidelines for testing of chemicals, Section 3. Organization for the Economic Cooperation and Development[END_REF], this treatment is considered only in the MITI tests (301 C and 302 C of ready and inherent biodegradability evaluation, respectively). Initially, MITI inoculum is made up of at least ten natural sources, i.e. activated sludges, soils, surface or sea waters. The mixture of these samples is settled and the supernatant is grown on a glucose and peptone-based medium in semi-continuous mode. After one month of operation, the sludge may be used as an inoculum.

Although multiple sources are likely to increase the microbial diversity, this may be weakened or lost during the long preculture process of the MITI tests. Analysis of biomass levels, substrate utilization patterns and phospholipid fatty acid (PLFA) profiles revealed differences among freshly collected activated sludge samples and the same sludges after a standard MITI preculture [START_REF] Forney | Structure of microbial communities in activated sludge: potential implications for assessing the biodegradability of chemicals[END_REF]. In addition, precultured inocula contained greater numbers of Grampositive bacteria, with large depletion of heterotrophic eukaryotes. As significant differences in the substrate utilization patterns and in the PLFA profiles were detected during the preculture, a continuous shift in the dominant species is likely to occur. Changes in the inoculum structure induced by the long MITI preculture have been used to explain some loss of biodegradation potential of the sludges [START_REF] Nyholm | The European system of standardized legal tests for assessing the biodegradability of chemicals[END_REF][START_REF] Gerike | A correlation study of biodegradability determinations with various chemicals in various tests[END_REF].

In other work [START_REF] Thouand | The use of precultured inocula for biodegradability tests[END_REF], eight samples of activated sludge and eight samples of river water were precultured in a chemostat-type reactor with sodium acetate as the only source of carbon. The preculture enabled reduction of the variability of the inocula in terms of total cell count and endogenous dehydrogenase activity. However, when the biodegradation of PE and p-nitrophenol was tested, precultured inocula did not lead to an improved reproducibility of the assays. Resource competition theory predicts that competitive exclusion among species would occur during Short-term batch preculture on a complex substrate (yeast extract) was tested in an attempt to homogenize the characteristics of activated sludges from WWTP operating at different MLR (high, mean, and low). In Fig. 2a the counts of total, active and cultivable cells measured in the inocula are shown. Inocula were the sludge supernatants obtained after settling for 30 min. The total cells were measured by DAPI staining, the active cells were counted by the INT method, and the cultivable cell density by plating in solid non-selective medium; these methods are fully described elsewhere [START_REF] Vázquez-Rodríguez | Activated sludge as inoculum for ready biodegradability testing of chemicals: effect of source[END_REF]. The inocula had similar total (1.46-4.2×10 12 ) and active (1.02-1.8×10 12 ) cell densities; the main difference being the cultivable cell count of the high-MLR supernatant (1.49×10 9 ), which was one order of magnitude higher than those found in the other supernatants (1.2-1.84×10 8 ). After 14 h of preculture, the total (8.68×10 13 -1.42×10 14 ), active (3.16-4.42×10 12 ), and cultivable (5.0-7.6×10 10 ) cell contents of the inocula were augmented to similar levels (Fig. 2b). These inocula were used to test the biodegradability of two easily assimilable chemicals (SA and aniline) and two xenobiotic compounds (NTA and PE) showing erratic behaviour in biodegradation studies [START_REF] Painter | Detailed review paper on biodegradability testing[END_REF]. The biodegradability test used was the OECD 301 A test, based on the consumption of DOC. Inoculation was at 1% (v/v). The results, in terms of half-life times, are shown in Table 2.

In all the tests inoculated with non-precultured sludges the chemicals could be biodegraded. As mentioned above, the kinetics of biodegradation depend strongly on the inoculum source. High-MLR inoculum had the lowest half-life times, probably because of its higher cell density. Preculture of supernatants enabled the inoculation of tests with very similar levels of total, active, and cultivable cells.

In the case of easily assimilable compounds (aniline and SA), this also enabled their kinetics of biodegradation to be homogenized. However, for the less biodegradable compounds NTA and PE, inocula had a lower biodegradation potential after preculture. Apparently, even a short preculture imposes distinct selective pressures on activated sludges and causes a new community structure to appear, maybe not fairly simulating the original structure.

Recently, a study has pointed out that reactor operation (semi-continuous vs. continuous), nature of the carbon source (simple vs. complex), loading rate, reactor configuration, and addition of exogenous bacteria can modify the microbial diversity in bioreactors [START_REF] Pholchan | Systematic study of the effect of operating variables on reactor performance and microbial diversity in laboratory-scale activated sludge reactors[END_REF]. However, microbial diversity could not to be systematically increased in that study. To avoid the selection of specific populations with high affinity for the substrates used, and to maintain high microbial diversity, continuous changing of the frequency, composition, and amount of substrate feed has been suggested [START_REF] Forney | Structure of microbial communities in activated sludge: potential implications for assessing the biodegradability of chemicals[END_REF]; this undoubtedly increases the complexity of the preculture method.

Batch or continuous, on complex or defined media, preculture has a strong effect on the microbial quality and quantity of activated sludge. This leads to a diminution in the biodegradation potential of inocula, especially for xenobiotic compounds, and could give more false negative results in RBT. Because preculture does not seem to increase the reproducibility of biodegradation tests, it should be avoided even in its standardized form (i.e. MITI test procedure) for screening purposes.

Use of inocula preadapted to the test chemical

Preadaptation has been defined as the preincubation of an inoculum in the presence of the test substance with the purpose of enhancing the ability of the inoculum to degrade Fig. 2 Total, active, and cultivable cell densities of three inocula obtained from different WWTP (a) before and (b) after preculture on complex substrate it [START_REF] Painter | Detailed review paper on biodegradability testing[END_REF]. For a readily biodegradable compound, for example p-nitrophenol, it has been found that a total concentration of 5×10 10 cells L -1 is needed to ensure 99.9% success in a 28day biodegradability test [26]. However, such a high cell concentration in an indigenous inoculum leads to important organic contamination in the assay. As preadaptation of the inoculum to the test compound enables the number of specific biodegraders to be increased without introducing excess exogenous carbon, it has been suggested for augmenting the reproducibility of RBT. In fact, a similar test response can be obtained when an adapted inoculum contains at least 1.2×10 5 specific biodegraders per litre [26]. Use of preadapted inocula also enables diminution of lag times; for p-nitrophenol, lag periods were between 2.8 and 52 h when adapted populations were used, whereas lag times for non-adapted inocula can reach several weeks [26].

Preadaptation is affected by the pre-exposure period to the test chemical, among other factors. The ability of sludges to degrade 11 chemicals was studied by varying the length of exposure in semi-continuous units over a 67-day period [START_REF] Painter | Detailed review paper on biodegradability testing[END_REF]. The sludges were taken at various times [START_REF] Seviour | Microbial ecology of activated sludge[END_REF][START_REF] Dias | Microbial ecology of activated sludge -II. Bacteriophages, Bdellovibrio, coliforms, and other organisms[END_REF]26, and 67 days), and then used to inoculate an ISO test based on DOC consumption. Two chemicals, N-methylaniline and t-butanol, were only biodegraded when preadapted inocula were used. For some chemicals (PE, 3-chlorobenzoic acid, and 2-nitrobenzoic acid), the time required to achieve the test pass level (DOC removal >70%) diminished as the exposure period of the inoculum was augmented. For sulfanilic acid and diethylene glycol, adaptation of the inocula led to erratic results, and benzene 1,3-sulfonic acid, and 2-chlorobenzoic acid were not degraded by any of the adapted sludges or by the original inoculum.

Although adaptation is a process of crucial importance in the real environment (e.g., in natural media exposed intermittently to a chemical), regulatory authorities have traditionally ruled out preadaptation in RBT to avoid falsepositive results. If a chemical compound does not reach the pass level in an RBT, the OECD recommends performing an inherent biodegradability test [START_REF]Revised introduction to the OECD guidelines for testing of chemicals, Section 3. Organization for the Economic Cooperation and Development[END_REF]. REACH has recently introduced several enhancements of the RBT to assist in the prioritization of PBT/vPvB assessments by reducing the need for higher-tier testing [5]. These enhancements include an increase in the duration of the test to 60 days (if biodegradation is still occurring on day 28) and the possibility of adapting the biomass to the test compound. So, semi-continuous biodegradability tests and low-level preadaptation (consisting in the use of a sample from a completed RBT to seed a subsequent RBT) are now allowed. In this way, for PBT/vPvB assessment, a substance reaching the pass level in an enhanced biodegradability test can be regarded as "not P" [5]. Enhanced RBT methods have still to be standardized and validated [START_REF]Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures[END_REF].

Use of surrogate microbial communities

Several workers have pointed out the need for a certified biological material with the same metabolic potential as an activated sludge that can be used for quality control and to provide traceability [START_REF] Paixão | Evaluation of two commercial microbial inocula as seed in a 5-day biochemical oxygen demand test[END_REF][START_REF] Paixão | Alternative inocula as activated sludge surrogate culture for a toxicity test[END_REF][START_REF] Paixão | Biodegradability testing using standardized microbial communities as inoculum[END_REF][START_REF] Paixão | Assessing microbial communities for a metabolic profile similar to activated sludge[END_REF]. So, the use of surrogate cultures has been proposed to fulfil the need of an activated sludge with constant characteristics. The reliability of two commercial inocula (Bi-Chem and Biolen) has been studied in standard BOD 5 tests [START_REF] Paixão | Evaluation of two commercial microbial inocula as seed in a 5-day biochemical oxygen demand test[END_REF]. For both inocula, the assay reproducibility was adequate when a standard solution of glucose and glutamic acid was tested because the variation around the mean BOD 5 value was between 4.52 and 9.37%. These commercial inocula and activated sludge were used to test the BOD 5 of several wastewater samples, and the results were found to be significantly correlated (95% confidence). This correlation was explained by the similarity of microbial strains found in the three inocula by means of the API identification system: Gram-positive, aerobic, spore-forming bacteria of the genus Bacillus and Gramnegative bacteria belonging to the genera Enterobacter and Pseudomonas. In another study [START_REF] Paixão | Alternative inocula as activated sludge surrogate culture for a toxicity test[END_REF], the substrate utilization patterns of Bi-Chem and Biolen were studied and compared with that of activated sludge by using Biolog plates. Activated sludge was positive for all the 31 carbon sources tested, whereas Bi-Chem and Biolen were considered positive for 30 and 22 substrates, respectively. From the authors' point of view, the overall similarity of catabolic profiles observed between activated sludge and Bi-Chem supports its use as surrogate inoculum. Use of commercial inocula (i.e., BOD seed, Bi-Chem and Polyseed) was also studied for testing biodegradable dissolved organic carbon (BDOC) [START_REF] Khan | Application of commercial biochemical oxygen demand inocula for biodegradable dissolved organic carbon determination[END_REF]. The performance of the commercial inocula was compared with that of activated sludge and indigenous inocula for a standard solution of sodium tartrate, surface waters, and secondary effluents. Commercial seeds had similar BDOC exertion levels with analogous kinetics in all tests, but they tended to underestimate the actual BDOC values of the samples. The activated sludge inoculum provided higher and more accurate BDOC values, and faster exertion kinetics than the commercial and indigenous inocula. This fact was explained by the wider species diversity and the greater number of acclimated cells of the activated sludge samples. Because activated sludge also resulted in the lowest coefficients of variation, it can be regarded as the most suitable inoculum for the BDOC test.

The potential of Bi-Chem and Biolen to be used as alternative inocula to activated sludge was evaluated by assessing the biodegradability of diethylene glycol in the Zahn-Wellens assay [START_REF] Paixão | Biodegradability testing using standardized microbial communities as inoculum[END_REF]. Whereas complete biodegradation was achieved in 10-12 days with activated sludge, none of the commercial seeds reached the test validity criterion (biodegradation >70% on the 14th day, [25]). Tabka et al. [START_REF] Tabka | Inoculum standardization for biodegradability tests[END_REF] also studied the biodegradability of several chemicals in the Zahn-Wellens assay using activated sludge and Actizym, a commercial seed. Both inocula degraded ethylene glycol with almost identical kinetic patterns; however, activated sludge had a higher biodegradation potential, because 1,4-nitrophenol was degraded only when this inoculum was used. To the best of our knowledge, no commercial surrogate has been tested in the RBT, and so their usefulness for improving the reproducibility of these assays has not been demonstrated.

Another approach consists in using a representative model composed of a reduced number of bacterial species. Well-defined mixtures containing 4 to 8 selected bacterial strains in equal proportions were compared with an activated sludge in terms of substrate utilization patterns measured by Biolog plates [START_REF] Paixão | Assessing microbial communities for a metabolic profile similar to activated sludge[END_REF]. The strains studied were Acinetobacter sp., Enterobacter sp., Xanthomonas sp., Staphylococcus capitis, Pseudomonas sp., P. putida, Bacillus subtilis, and B. cereus, which are nonpathogenic species commonly found in water and soil. Because four of these mixtures (composed of four to six strains) had repeatable functional diversity similar to that of an activated sludge, they were suggested as potential biological reference material in activated sludge-based bioassays. In a study already mentioned [START_REF] Paixão | Biodegradability testing using standardized microbial communities as inoculum[END_REF], a defined consortium composed of equal parts of six bacterial strains (i.e., Acinetobacter sp., Enterobacter sp., S. capitis, P. putida, B. subtilis and B. cereus) was compared with activated sludge samples using diethylene glycol as reference chemical in the Zahn-Wellens assay. Among the surrogates, the defined consortium behaviour was the most similar to that of activated sludge, although not as fast.

Although surrogates have some similarities to activated sludge (i.e., catabolic profile) and equivalent behaviour with regard to easily assimilable compounds, they do not seem to provide more reproducible results with regard to the ability to degrade either xenobiotic chemicals or persistent samples (as organic carbon in secondary effluents). The performance of the surrogates has been only studied in the Zahn-Wellens test, which uses a high concentration of biomass. Therefore, it is unlikely that they enable RBT reliability to be increased.

Concluding remarks

Activated sludges are, with secondary effluents, the most utilized sources of inocula for biodegradation tests. This is mainly because of their availability, and specific general features (for example their continuous exposure to sewage and their aerobic character) that make them more consistent than other natural sources. These general features are not sufficient to ensure a limited variability range of inoculum quantity and quality for testing ready biodegradability with standard methods. Differences in the structure and activity of activated sludge communities could arise from dissimilarities in wastewater composition, but largely from WWTP operation (i.e., MLR or SRT).

However, activated sludges are a relevant inoculum because their use in biodegradation studies enables a preliminary approach to the fate of chemicals during wastewater treatment. Furthermore, activated sludges seem to have substantial biodegradation potential, higher than that of other natural sources. The metabolic potential of activated sludge toward xenobiotic compounds can hardly be found in commercial surrogates or in other well-defined microbial consortia. It is our view that any manipulation of the inocula incurring in a diminution of their biodegradation potential, for example preconditioning or preculture, does not constitute an option for standardization.

Biodegradation experiments inoculated with fresh activated sludge simulate in a more realistic way the complexity of microbial processes occurring in aquatic environments. Such complex microbial assemblages are necessary for first-tier testing of chemicals, requiring maximum microbial diversity, even at the expense of reproducibility. For not readily biodegradable chemicals, for which there is a small number of specific degraders in environmental samples, this problem could be overcome by using inocula from different origins. Additionally, recent enhancements promoted by REACH (allowing low-level adaptation of inocula in RBT) have led to promising improvement of the reliability of ready biodegradation tests. Both alternatives have still to be standardized and validated for a wide set of chemicals.

Fig. 1

 1 Fig. 1 (a) Cultivable cell densities and (b) dehydrogenase activity of three inocula obtained from different WWTP before and after preconditioning

Table 2

 2 Half-life times (h) obtained in OECD 301 A tests inoculated with non-precultured and precultured sludges from different WWTP

		Tests with non-precultured inocula			Tests with precultured inocula
	Inoculum	SA	Aniline	NTA	PE	SA	Aniline	NTA	PE
	High-MLR	8.5	43.5	115	190	5.2	35.3	NB	NB
	Mean-MLR	-	50.8	-	-	-	35.3	-	-
	Low-MLR	12.5	59.6	165	248	5.2	36.3	NB	NB
	SA, sodium acetate; NTA, nitrilotriacetic acid; PE, pentaerythritol
	NB, not biodegraded in the observation period (288 h)

Acknowledgements The authors sincerely appreciate the help of Mr Randall Lewis in improving the English of the manuscript.


Hassen Fourati

Noureddine Manamanni

Lissan Afilal

Yves Handrich
email: yves-jean-handrich@c-strasbourg.fr

an application in Bio-logging

Rigid body motions capturing by means of wearable inertial and magnetic MEMS sensors assembly: from the reconstitution of the posture toward the dead reckoning

INTRODUCTION

The rigid body attitude and orientation estimation problems are highly motivated from various applications. For example, in rehabilitation and biomedical engineering [START_REF] Zhou | Applications of wearable inertial sensors in estimation of upper limb movements[END_REF], the attitude is used in stroke rehabilitation exercises to record patient's movements in order to provide adequate feedback for the therapist. In human motion tracking and biomechanics [START_REF] O'donovan | A inertial and magnetic sensor based technique for joint angle measurement[END_REF], the attitude serves as a tool for physicians to perform long-term monitoring of the patients and to study human movements during everyday activities. Moreover, the attitude estimation is extensively used in tracking of handheld microsurgical instrument [START_REF] Ang | Kalman filtering for real-time orientation tracking of handheld microsurgical instrument[END_REF]. In aerial and marine vehicles [START_REF] Mahony | Nonlinear complementary filters on the special orthogonal group[END_REF], the attitude is used to achieve a stable controller.

Recently, the problem of attitude and orientation tracking has been treated in another scientific field: The Bio-logging. This latter stands in the intersection of animal behavior and bioengineering and aims at obtaining new information from the natural world and providing new insights into the hidden lives of animal's species [START_REF] Rutz | New frontiers in biologging science[END_REF], [START_REF] Ropert-Coudert | Diving into the world of biologging[END_REF]. Bio-logging generally involves a freeranging animal-attached electronic device (called also bio-logger) that records aspects of the animal's biology (behavior, movement, physiology) [START_REF] Halsey | Fine-scale analyses of diving energetics in king penguins Aptenodytes patagonicus: how behaviour affect costs of a foraging dive[END_REF], [START_REF] Bost | Change in dive profiles as an indicator of feeding success in king and Adélie penguins[END_REF] and its environment. Thirty years ago, several tagging technologies such as satellite tracking (the Argos system) [START_REF] Le Boeuf | Foraging ecology of northern elephant seals[END_REF] and Time-Depth-Recorders (TDRs) [START_REF] Kooyman | Genesis and evolution of bio-logging devices: 1963-2002[END_REF] have been used to provide a basic knowledge on the function of free-ranging organisms. The recent advances in electronic miniaturization, sensors and digital information processing allowed researchers studying animal's biology with a high level of detail and across the full range of ecological scales.

Many marine and terrestrial animals are studied during their daily activities. The posture and orientation tracking of these free-ranging animals represents one of the recent biology aspects studied in Bio-logging. Indeed, some scientific researches started to focus on this topic using low-cost sensors based on Micro Electro-Mechanical System (MEMS) technology as a 3-axis accelerometer and a 3-axis magnetometer. The obvious advantage of this new approach is the gain access to the third dimension space, which is the key to a good understanding of the diving strategies observed in these predators [START_REF] Elkaim | Marine Mammal Marker (MAMMARK) dead reckoning sensor for In-Situ environmental monitoring[END_REF]. The main question to answer is how it is possible to extract the gravity components of the body animal [START_REF] Johnson | A digital acoustic recording tag for measuring the response of wild marine mammals to sound[END_REF][START_REF] Watanabe | A new technique for monitoring the behaviour of terrestrial animals; a case study with the domestic cat[END_REF][START_REF] Wilson | Prying into the intimate details of animal lives: use of a daily diary on animals[END_REF]? This information is exploited after to deduce the corresponding posture (attitude) and consequently the Dynamic Body Acceleration.

In this chapter, we propose the addition of 3-axis gyroscope measurements to the sensors already used (3-axis accelerometer and 3-axis magnetometer) in Bio-logging. The use of gyroscope with accelerometer and magnetometer, mounted in triad configuration, in Bio-logging has never been done before in our knowledge. In our opinion, it can improve the estimation precision of the attitude especially during dynamic situation of the animal motion [START_REF] Mahony | Nonlinear complementary filters on the special orthogonal group[END_REF], [START_REF] Fourati | A rigid body attitude estimation for Bio-logging application: A quaternion-based nonlinear filter approach[END_REF], [START_REF] Fourati | A nonlinear filtering approach for the attitude and Dynamic Body Acceleration estimation based on inertial and magnetic sensors: Bio-logging application[END_REF]. The main idea of the algorithm is to use a complementary filter coupled with a Levenberg Marquardt Algorithm (LMA) to process the measurements from a 3-axis gyroscope, a 3-axis magnetometer and a 3axis accelerometer. The proposed approach combines a strap-down system, based on the time integral of the angular velocity, with the LMA that uses the Earth's magnetic field and the gravity vector to compensate the attitude predicted by the gyroscope. It is important to note that the resulting structure is complementary: high bandwidth rate gyro measurements are combined with low bandwidth vector observations (gravity and Earth's magnetic field) to provide an accurate attitude estimate. Thanks to the knowledge of the estimated attitude, it is now possible to reconstitute the Dynamic Body Acceleration of the animal in order to evaluate its daily diary [START_REF] Wilson | Prying into the intimate details of animal lives: use of a daily diary on animals[END_REF] (sleeping, walking/flying, running, and hunting) and provide important insights into some of the stresses faced by free-ranging animals especially the king penguin and badger.

In the second part of the chapter, the problem of 3D position estimation in the case of pedestrian locomotion is addressed. The final goal would be to apply this work in Bio-logging to reconstruct the trajectory of animal. Previous works in this domain have focused only on marine animals using a 3-axis accelerometer and speed sensor to estimate the 3D position [START_REF] Wilson | Prying into the intimate details of animal lives: use of a daily diary on animals[END_REF]. In this chapter, we are interested to the case of terrestrial animal. Based only on measurements provided by a 3-axis accelerometer, a 3-axis magnetometer and a 3-axis gyroscope (Inertial Measurement Unit, a position estimation approach is proposed. A dead reckoning approach is used where the Dynamic Body Acceleration is integrated to estimate at first the linear velocity and then the position. A complementary filter approach is used to estimate the body attitude that is necessary to reconstruct the Dynamic Body Acceleration. An experimental evaluation of the proposed approach is carried out in the case of human locomotion and where an Inertial Measurement Unit is attached to the foot of the subject.

This chapter is organized as follows: section 2 describes the problem statement and our motivations for motion estimation in the case of the attitude and position in Bio-logging. Section 3 details the attitude parameterization and the sensor measurement models used in this work.

Section 4 details the structure of the proposed complementary filter for the attitude estimation.

Section 5 is devoted to experimental results to illustrate the effectiveness of the proposed algorithm. Section 6 introduces some concepts about 3D position estimation approach in the case of pedestrian locomotion with our first experimental results. Finally, section 7 summarizes the main conclusions of the chapter.

MOTIVATIONS AND PROBLEM FORMULATION

Recent technological advances have revolutionized the approach of the animals in their environment, and have enabled researchers in biology and eco-physiology to leave their laboratories to study these adaptations on the animal models living freely in their natural environment. Bio-logging has been introduced as the science that studies the behavior, physiology, ecology and environment properties of free-living animals (bioclimatic, global change, etc…) that are often beyond the border of our visibility or experience. Bio-logging has found its origin in the marine environment [START_REF] Kooyman | Genesis and evolution of bio-logging devices: 1963-2002[END_REF] and has diversified into the study of flying and terrestrial species. This scientific area refers often to the study of free-ranging animals in their natural environment through miniaturized electronic devices (called also bio-loggers [START_REF] Naito | New steps in bio-logging science[END_REF]) usually attached to their bodies. These systems measure and record biological parameters or physico-chemical properties related to the individual and / or its environment using various types of sensors (luminosity, pressure, velocity, etc…). The loggers provide time tracking of physical and biological parameters over periods ranging from several hours to several months or sometimes a year and at sampling rates ranging from minutes to several times per second. The King penguin and badger are the major biological models studied in Strasbourg University thanks to the Bio-logging technology. Biologists are recently interested to reconstruct the motions of these animals (3D attitude and position) under several acceleration profiles, to be able to study their behaviour during long periods.

In this chapter, we are interested to:

• Firstly, propose a robust alternative approach to estimate the movement patterns (attitude or orientation) of rigid body, which represents the animal body, to be applied after in the case of penguin (see Fig. 1). To achieve this goal, we use a wearable inertial and magnetic MEMS sensors assembly based on a 3-axis accelerometer, a 3-axis magnetometer and a 3-axis gyroscope (Inertial Measurement Unit). Furthermore, the estimated attitude is used to calculate three components of Dynamic Body Acceleration (DBA) of the animal, which provides for biologists important information about the energy budgets of free-living animals.

• Secondly, we address the problem of 3D position estimation in the case of human pedestrian locomotion, based on attitude and DBA estimations. The goal is to obtain results that remain promising to reconstruct the animal position for an application in Bio-logging.

MATERIALS AND METHODS

RIGID BODY ATTITUDE AND COORDINATE SYSTEMS

A rigid body is considered as a solid formed from a finite set of material points with deformable volume [START_REF] Goldstein | Classical Mechanics. 2 nd Ed[END_REF]. Generally, the rigid body attitude represents the direction of its principal axes relative to a reference coordinate system and its dynamics expresses the change of object orientation. In the navigation field, the attitude estimation problem requires the transformation of measured and computed quantities between various frames. The rigid body attitude is based on measurements gained from sensors attached to this latter. Indeed, inertial sensors (accelerometer, gyroscope, etc…) are attached to the body-platform and provide inertial measurements expressed relative to the instrument axes. In most systems, the instrument axes are nominally aligned with the body-platform axes. Since the measurements are performed in the body frame, we describe in

MATHEMATICAL MODEL OF ATTITUDE REPRESENTATION

In this chapter, we use the quaternion algebra to describe the rigid body attitude. The unit quaternion, denoted by q , is expressed as:

0 0 1 2 3 1 vect q q q q q i q j q k H = + = + + + ∈ (1)
where 1 2 3 vect q q i q j q k = + + represents the imaginary vector, 0 q is the scalar element and H can be written such as:

[ ] { } 3 1 0 0 1 2 3 / 1, , , T T T T vect vect
H q q q q q q q q q q q

×   = = = ∈ℜ = ∈ℜ   (2)
The rotation matrix in term of quaternion can be written as:

( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 2 0 1 1 2 0 3 1 3 0 2 2 2 1 2 0 3 0 2 0 1 2 3 2 2 0 2 1 3 2 3 0 1 0 3 2 1 2 2 2 2 1 2 2 2 2 1
B N q q q q q q q q q q M q q q q q q q q q q q q q q q q q q q q q

  + - + -     = - + - +     + - + -   (3)
We invite the reader to refer to [START_REF] Kuipers | Quaternion and Rotation Sequences[END_REF] for a more details about quaternion algebra.

3-AXIS INERTIAL/MAGNETIC SENSORS PACKAGE MEASUREMENT

MODELS

The sensors configuration consists of a 3-axis accelerometer, a 3-axis magnetometer and a 3-axis gyroscope containing MEMS technologies. A detailed study of these sensors is given in [START_REF] Beeby | MEMS Mechanical Sensors[END_REF].

3-axis accelerometer

An accelerometer measures the acceleration of the object that it supports. If three accelerometers are mounted in orthogonal triad in a rigid body, such that their sensitive axes coincide with the principal axes of inertia of the moving body. The output of a 3-axis accelerometer in the bodyfixed frame ( ) B is given by the following measurement vector [START_REF] Guerrero-Castellanos | Estimation de l'attitude et commande borne en attitude d'un corps rigide: Application à un hélicoptère à quatre rotors[END_REF]:

( )( ) B N f f M q a G δ = -+ (4)
where

[ ] 0 0 T G g = and T x y z a a a a   = 
 represent, respectively, the gravity vector and the Dynamic Body Acceleration (DBA) of the rigid body, given in the Earth-fixed frame ( )

N . 3 f
δ ∈ℜ is a noise vector assumed to be independent, white and Gaussian.

( )

B N M q is the rotation matrix defined in [START_REF] Ang | Kalman filtering for real-time orientation tracking of handheld microsurgical instrument[END_REF] and reflecting the transition between the frames ( ) N and ( )

B .

3-axis magnetometer

A magnetometer, sometimes called magnetic compass is a device for measuring the direction and intensity of a magnetic field and especially the Earth's magnetic field. The output of a 3-axis magnetometer in the body-fixed frame ( )

B is given by the following measurement vector [START_REF] Guerrero-Castellanos | Estimation de l'attitude et commande borne en attitude d'un corps rigide: Application à un hélicoptère à quatre rotors[END_REF]:

( ) B N h h M q m δ = + ( 5
)
where m is the magnetic field expressed in the Earth-fixed frame ( )

N by: [ ] ( ) ( ) 0 cos 0 sin T T x z m m m m I m I = =     ( 6
)
h δ is a white Gaussian noise and ( )

B N
M q is the rotation matrix expressed in [START_REF] Ang | Kalman filtering for real-time orientation tracking of handheld microsurgical instrument[END_REF]. Currently, the parameters of the theoretical model of the geomagnetic field m closest to reality can be deduced from [START_REF] Astrosurf | [END_REF].

3-axis gyroscope

A gyroscope is an inertial sensor that measures the angular velocity of reference attached to the sensor compared to an absolute reference frame along one or more axes [START_REF] Titterton | Strapdown Inertial Navigation Technology. 2 nd Ed[END_REF]. The output of a 3axis gyroscope in the body-fixed frame ( )

B is given by the measurement vector [START_REF] Guerrero-Castellanos | Estimation de l'attitude et commande borne en attitude d'un corps rigide: Application à un hélicoptère à quatre rotors[END_REF]:

G G ω ω b δ = + + (7)
where

3
ω ∈ℜ is the real angular velocity, 3 b ∈ℜ is a slowly time varying function [START_REF] Beeby | MEMS Mechanical Sensors[END_REF] called also bias and G δ is a white Gaussian noise.

DESIGN APPROACH FOR ATTITUDE ESTIMATION: COMPLEMENTARY

FILTER

In this chapter, the objective is to design an attitude estimation algorithm based on inertial and magnetic MEMS sensors. The application in mind is related to a free-ranging animal case in Biologging [START_REF] Fourati | Posture and body acceleration tracking by inertial and magnetic sensing: Application in behavioural analysis of free-ranging animals[END_REF]. By considering the rigid body kinematic model, a complementary filter is proposed in order to take advantage from the good short-term precision given by rate gyros integration and the reliable long-term accuracy provided by accelerometer and magnetometer measurements.

This leads to better attitude estimates [START_REF] Mahony | Nonlinear complementary filters on the special orthogonal group[END_REF]. It is important to note that the resulting approach structure is complementary: high bandwidth rate gyro measurements are combined with low bandwidth vector observations to provide an accurate attitude estimate [START_REF] Brown | Introduction to Random Signal and Applied Kalman Filtering[END_REF].

RIGID BODY KINEMATIC MOTION EQUATION

The rigid body motion can be described by the attitude kinematic differential equation [START_REF] Shuster | A survey of attitude representations[END_REF],

which represents the time rate of attitude variation, expressed in a quaternion term q , as a result of the rigid body angular rates measured by the gyroscope:

1 2 T vect G vect q q ω I q q × ×   - =     +       ɺ (8)
where

• 0 T T vect q q q   = 
 is the unit quaternion that denotes the mathematical representation of the rigid body attitude between two frames: body-fixed frame ( ) B and Earth-fixed frame ( )

N . Note that [ ] 1 2 3
T vect q q q q = represents the vector part of q . It is customary to use quaternions instead of Euler angles since they provide a global parameterization of the body orientation, and are well-suited for calculations and computer simulations.

• G ω represents the angular velocity vector expressed in ( ) B and 3 3 I × is the identity matrix of dimension 3.

• vect q ×   
 represents the standard vector cross-product (the skew-symmetric matrix) which is defined such as:

1 3 2 2 3 1 3 2 1 0 0 0 vect q q q q q q q q q q × × -           = = -           -     (9)

THE DESIGN STATE MODEL

Let us consider the following system model ( )

1
S composed of (8) with the output y that represents the linear measurement model. The output 6 y ∈ℜ of this system is built by stacking the accelerometer and magnetometer measurements.

( )

1 2 3 0 0 3 2 1 3 0 1 2 3 3 0 1 1 2 0 3 1 1 2 2 : Gx Gy Gz T vect Gx Gy Gz G Gx Gy Gz vect Gy Gx Gz T x y z x y z
q ω q ω q ω q q q ω q ω q ω q ω q ω q ω q ω q I q q S q ω q ω q ω q y

f f f h h h × ×  - - -            - - +      = =      + -     +            - +            =    ɺ ɺ ɺ ɺ (10)
By considering the rigid body kinematic equation and the linear measurement model y , the proposed system ( ) 1 S can take advantage from the good short term precision given by the rate gyros integration and the reliable long term accuracy provided by accelerometers and magnetometers measurements fusion [START_REF] Brown | Introduction to Random Signal and Applied Kalman Filtering[END_REF], [START_REF] Fourati | A quaternion-based Complementary Sliding Mode Observer for attitude estimation: application in free-ranging animal motions[END_REF], which leads to improve the quaternion estimation.

ATTITUDE COMPLEMENTARY FILTER

The aim of this approach is to ensure a compromise between the accuracy provided by shortterm integration of the gyroscope data and the long-term measurements precision obtained by the accelerometer and the magnetometer. To compensate for the drifts on the estimated quaternion that are observed during the integration of the differential equation ( 8), a correction term T is introduced in this equation based on a quaternion product ⊗ . We propose the following complementary filter:

( )

0 1 2 3 0 3 2 1 3 0 1 2 1 2 0 3 ˆˆˆ1 : ˆˆ2 ˆˆˆx y z x y z x y z y x z q q ω q ω q ω q ω q ω q ω q F T q ω q ω q ω q q ω q ω q ω q   - - -       - +     = ⊗     + -     - +         ɺ ɺ ɺ ɺ (11)
where

[ ] 4 0 1 2 3
ˆˆˆˆˆT q q q q q = ∈ ℜ represents the estimated quaternion. The correction term T is calculated from a fusion approach of accelerometer and magnetometer data. The quaternion product introduced in (11) allows to merge the magnetic and inertial measurements.

Let us present the method for calculating the correction term T . We consider the modeling error ( ) ( ) ˆδ q y y = -. The estimated output is given by ŷ :

ˆˆˆˆˆˆT x y z x y z y f f f h h h   =   (12)
Measurements of the estimated accelerations ˆx f , ˆy f and ˆz f can be calculated by assuming that the Dynamic Body Acceleration a is low ( )

2 2 a G ≪ [29]
. Thus we obtain:

1 ˆˆˆˆˆ0 T x y z q f f f f q G q -   = = ⊗ ⊗   (13)
where Measurements of the estimated Earth's magnetic field ˆx h , ˆy h and ˆz h can be calculated such as:

1 ˆˆˆˆˆ0 T x y z q h h h h q m q -   = = ⊗ ⊗   (14)
where [ ]

0 0 T q x z m m m = : Quaternion representation of the Earth's magnetic field [ ] 0 T x z m m m = .
The minimization of the modeling error ( ) δ q is performed from a regression method that minimizes the scalar squared error criterion function ( ) ξ q related to ( )

δ q : ( ) ( ) ( ) ˆˆT ξ q δ q δ q = ( 15
)
In this chapter, the Levenberg Marquardt Algorithm is used to minimize the non-linear function ( ) ξ q . This choice reflects the robustness demonstrated by this algorithm compared to other methods such as Gauss-Newton or gradient [START_REF] Dennis | Numerical Methods for Unconstrained Optimization and Nonlinear Equations[END_REF].

The unique solution to this problem can be written in the following form [START_REF] Marquardt | An Algorithm for the Least-Squares Estimation of Nonlinear Parameters[END_REF]:

( ) ( )

ˆη q Kδ q = ( 16
)
where

1 3 3 T T K k X X λI X - ×   = +  
is the gain of the filter used to minimize the error ( ) δ q .

6 3 X × ∈ ℜ is the Jacobian matrix defined by:

0 0 2 2 0 0 0 0 T z y z y T z x z x y x y x f f h h X f h f f h h f f h h × ×   - -         = - = - - -           - -   (17)
The constant λ is chosen to ensure the non-singularity of the minimization problem. The constant k determines the crossover frequency of the latter. It is used to tune the balance between measurement noises suppression and response time of the filter. Generally, it combines low bandwidth accelerometer/magnetometer readings with high bandwidth gyroscope measurements. Notice that, the complementary filter has a better convergence when k is chosen somewhere between 0.1 and 1 [START_REF] Mahony | Nonlinear complementary filters on the special orthogonal group[END_REF]. ( ) η q represents a part of the correction term T . To achieve the quaternion product in [START_REF] Elkaim | Marine Mammal Marker (MAMMARK) dead reckoning sensor for In-Situ environmental monitoring[END_REF], the term T must be of dimension 4. So, T is constructed as follows:

( )

1 0 0 0 0 0 0 1 0 0 0 T δ q K         =           (18)
The scalar part of quaternion error is chosen to 1 to force the error quaternion to represent small angles of rotation [START_REF] Deutschmann | Quaternion normalization in spacecraft attitude determination[END_REF]. Finally, the complementary filter can be written as follows:

( ) ( ) ( ) ( ) ( ) ( ) 1 2 3 0 0 3 2 1 2 3 0 1 3 1 2 0 ˆˆˆ1 0 0 0 0 0 0 ˆˆˆ1 0 1 : 0 2 ˆˆˆˆ0 ˆˆˆx y z x y z x y z y x z
q ω q ω q ω q q ω q ω q ω q F δ q K q q ω q ω q ω q q ω q ω q ω

  - + +             - +         = ⊗         + -                 - +   ɺ ɺ ɺ ɺ (19)

EXPERIMENTAL VALIDATION

EXPERIMENTAL TOOLS FOR ATTITUDE ESTIMATION: INERTIAL MEASUREMENT UNIT (MTI-G)

In order to evaluate the efficiency of the proposed complementary filter in real world applications, an experimental setup was developed resorting to an inertial and magnetic sensor assembly. The goal is to obtain an estimation of the quaternion that represents the orientation of a rigid body and to investigate its accuracy under various conditions. For the experiments, an Inertial Measurement Unit (IMU) was employed. The MTi-G from Xsens Motion Technologies

[33] is used. This MEMS device is a miniature, lightweight, 3D calibrated digital output sensor (3D acceleration from accelerometer, 3D angular rate from gyroscope, and 3D magnetic field data from magnetometer), a GPS enhanced Attitude and Heading Reference System with built-in bias, sensitivity, and temperature compensation. The MTi-G outputs data at a rate of 100Hz and records them on a computer (see Fig. 3). In addition, this device is designed to track the body 3D attitude output in quaternion representation using an embedded Kalman filter algorithm. The calibration procedure to obtain the gain, offsets and non-orthogonality of the sensors was performed by the manufacturer of the sensor module.

It is important to note that the MTi-G device serves as tool for the evaluation of the complementary filter efficiency and cannot be suitable for use in Bio-logging field due to its dependence on an energy source as well as its heavy weight. In the following set of experiment, the calibrated data from the MTi-G are used as input to the complementary filter.

EVALUATION TEST AND ANALYSIS IN FREE MOVEMENT OF ANIMAL

In this set of experiments, the accuracy of the complementary filter is evaluated during the free motion of a domestic animal (a dog). The MTi-G is attached to the back of the animal with its xyz axes aligned with those of the dog. The path followed by the animal was carried out in a football stadium as shown in Fig. 4. Inertial/magnetic measurements and attitude (in quaternion representation) are recorded using the MTi-G during the motion of the dog (see Fig. 5) and transmitted to a computer via USB port. It should be noted that, based upon measurements recorded by the accelerometer, we note that the animal motion consists of two acceleration profiles, one corresponding to the low frequencies of motion (during walk) and the other rather to the high frequencies (during trot and canter). The acceleration profile varies between [-15, 15 m/s 2 ] for x f and y f and [-5, +25 m/s 2 ] for z f . The increase in the acceleration level between the natural gaits is due to the Dynamic Body Acceleration a of the dog that is more important during the trot and the canter.

The recorded inertial and magnetic measurements from the MTi-G are used to estimate the attitude using the proposed complementary filter. The calculated attitude from the MTi-G is considered as reference of the dog's motion. Fig. 6 plots the evolution of the difference between the calculated quaternion using the MTi-G and the one estimated by the proposed approach.

Although some parts of the motion are with high dynamics, we can remark that the errors on quaternion's components don't exceed 0.03 on 0 q , 1 q , 2 q and 0.05 on 3 q . For more clarity to the reader, we also represent the attitude estimation results of the same movement using the Euler angles (roll, pitch and yaw). Fig. 7 shows the evolution of the difference between the Euler angles estimated by the complementary filter and the MTI-G.

It is clear that this mismatch between the estimated attitude by our approach and the MTi-G is small. Then, one can conclude about the performance of the complementary filter in attitude estimation of the animal body even in dynamic situations. Although our approach didn't exploit a GPS data as done in MTi-G, it is able to reconstruct the orientation of the dog given by the MTi-G with a small error.

TOWARD THE 3D POSITION ESTIMATION IN PEDESTRIAN LOCOMOTION

In this chapter, we focus on the problem of 3D position estimation of a mobile with pedestrian locomotion (human walking) using a dead reckoning approach. Our motivation is to go toward the estimation of the animal's 3D position in Bio-logging. In this context, a position estimation approach is proposed based only on proprioceptive measurements provided by a 3-axis accelerometer, a 3-axis magnetometer and a 3-axis gyroscope, where the Dynamic Body Acceleration is integrated to estimate at first the linear velocity and then the position. The complementary filter approach, presented in the previous section, is used to estimate the body attitude that is necessary to reconstruct the Dynamic Body Acceleration. The obtained results are satisfactory and remain promising for terrestrial animal in Bio-logging application.

3D POSITION ESTIMATION APPROACH USING A DEAD RECKONING TECHNIQUE

The proposed approach is based on the dead reckoning technique [START_REF] Steinhoff | Dead reckoning from the pocket -An experimental study[END_REF]. It consists in deducing the position of a mobile from its last known position. Therefore, we used the physical relationship existing between the estimated Dynamic Body Acceleration of the mobile â and its estimated position p . It can be written using the following two equations:

( ) ( ) 2 1 ˆˆ. T T v t a t dt = ∫ (20) ( ) ( ) 2 1
ˆˆ.

T T p t v t dt = ∫ (21)
where v represents the estimated linear velocity of the mobile and [ ]

; T T denotes the sampling period.

The Dynamic Body Acceleration of the mobile â can be deduced from the equation ( 4):

( ) ( ) ˆB N a inv M q f G = - (22)
where ( )

B N
M q is the rotation matrix defined in (3). It is expressed in terms of the estimated quaternion from the complementary filter.

3 G ∈ ℜ is the gravity vector and 3 f ∈ℜ is the accelerometer measurements.

Based solely on equations ( 20) and ( 21), a significant and fast drift appears at the position estimation. This drift was predictable; it is mainly due to measurement noise in the accelerometer signal f and their numerical integration in ( 20) and (21). To overcome this problem, one introduces a correction step usually proposed for pedestrian navigation [START_REF] Ladetto | Capteurs et Algorithmes pour la localisation Autonome en Mode Pédestre[END_REF], [START_REF] Ojeda | Non-GPS Navigation for Security Personnel and First Responders[END_REF] in the case of human and terrestrial animal locomotions (see Fig. 8). The idea is to use an Inertial Measurement Unit attached to the human's foot. The accelerometer measurements f allow us to detect the moments when the acceleration norm 2 f equals gravity. At these moments, theoretically the Dynamic Body Acceleration â and the linear velocity v vanish since the foot reaches the ground (see Fig. 9). But in reality, they stay slightly different from zero at these moments. Therefore, a correction step is applied to reset the linear velocity to zero at these moments.

First, we calculate the squared norm of the measured acceleration 2 2 f by using the following formula:

2 2 2 2 2 x y z χ f f f f = = + + (23)
one then calculate the means mo of χ over a range of samples e :

χ mo e = (24)
Then, the variance of this squared norm is computed on a sliding window by using the following equation [START_REF]Personal Dead Reckoning System with Shoe Mounted Inertial Sensors[END_REF]:

( ) ( ) 2 1 1 1 i j e i j i j e V j χ mo e = = -+ = - -∑ (25)
where i χ is the squared norm of the measured acceleration f and j mo is the means of i χ over a chosen range of samples e . The variance of the squared norm is used to detect significant moments of movement of the subject such as where the foot touches the ground (Dynamic Body Acceleration theoretically equal to zero). The samples interval e must be carefully chosen so that the test of the variance is sufficiently sensitive to slow and rapid changes of the accelerometer signal.

To detect the moments when the estimated Dynamic Body Acceleration â vanishes, the following condition is imposed:

e V L < (26)
When condition ( 26) is performed, it is necessary to reset the components of velocity vector v to zero before using the equations ( 20) and [START_REF] Beeby | MEMS Mechanical Sensors[END_REF]. Otherwise, the integration procedure using these two equations is normally done without resetting the velocity.

EXPERIMENTAL RESULTS IN THE CASE OF HUMAN PEDESTRIAN

LOCOMOTION

To examine the accuracy of the position estimation by the proposed approach above, we perform experimental trials in the case of human walking. The Inertial Measurement Unit MTi To estimate the attitude of the human's foot during the 3D walking trajectory, we used the complementary filter approach proposed in the previous section. The estimated attitude q is then used to calculate the Dynamic Body Acceleration from [START_REF] Guerrero-Castellanos | Estimation de l'attitude et commande borne en attitude d'un corps rigide: Application à un hélicoptère à quatre rotors[END_REF]. Then we calculated the values of the squared norm of the measured acceleration i χ by using [START_REF] Astrosurf | [END_REF]. The means j mo for this acceleration is then calculated through [START_REF] Titterton | Strapdown Inertial Navigation Technology. 2 nd Ed[END_REF] with the range of samples 8 e = . Finally, the values of variance e V are derived using [START_REF] Fourati | Posture and body acceleration tracking by inertial and magnetic sensing: Application in behavioural analysis of free-ranging animals[END_REF].

Fig. 12 includes the squared norm of the measured acceleration i χ , the means j mo , the variance ( ) e V j and the moments where the foot touches the ground. Finally, we have applied equations [START_REF] Kuipers | Quaternion and Rotation Sequences[END_REF] and [START_REF] Beeby | MEMS Mechanical Sensors[END_REF] to extract the estimated position of the person along his trajectory. At each integration step, the condition ( 26) is verified and when it is checked the velocity vector v is initialized to zero. The limit L in this condition is experimentally set (depending on the nature of human walking) in order to adequately detect and differentiate the phases of walking and rest (the foot touches the ground). Figure 12 shows the validity of the proposed approach to locate the phase where the foot reaches the ground. Indeed, the detector shown in this figure is set to 1 when the foot touches the ground and changes to 0 during walk. The linear velocity estimation with and without the correction step is plotted in Fig. 13. The improvement provided by this step is obvious in all three axes (solid line versus dashed line). If this reset is not done, the observed differences are due to the integration of noise in the accelerometer measurements. We calculated in a first step the 3D position of the person using the usual integration procedure from equations ( 20) and ( 21) without correction step. Note that we have chosen to begin the estimation of position from the initial point ( ) 0, 0, 0 . We show in Fig. 14 the estimated 3D trajectory. A rapid and total divergence in estimation of position is observed in this figure . The error is about 300 meters on the X, Y and Z-axis. Fig. 15 illustrates the improvement in the estimation of 3D position after adding the correction step of the velocity using the condition [START_REF] Brown | Introduction to Random Signal and Applied Kalman Filtering[END_REF]. This procedure allows obtaining an estimation of position with an absolute error ranging between 5 and 10 meters on the X and Y-axes and 30 cm on the Z-axis.

CONCLUSION

This paper focused principally to the 3D motions estimation of rigid body. The application in mind concerns Bio-logging and aims to truck posture and orientation of free-ranging animals during their natural lives.

Firstly, a quaternion-based complementary filter for the rigid body attitude estimation has been designed. The proposed state estimation algorithm adds to the data from a 3-axis accelerometer and a 3-axis magnetometer the one provided by a 3-axis gyroscope. The main idea is to combine a strap-down system, based on the time integral of angular velocity, with the Levenberg Marqurdt Algorithm. The efficiency of the proposed approach is highlighted with a set of experiments on a domestic animal through sensor measurements provided by an Inertial Measurement Unit (MTi-G).

Secondly, the problem of 3D position estimation in the case of human pedestrian locomotion was addressed. The Dynamic Body Acceleration is integrated to estimate at first the linear velocity and then the position. The complementary filter approach, presented in the previous section, is used to estimate the body attitude that is necessary to reconstruct the Dynamic Body Acceleration. The obtained results are satisfactory and remain promising for terrestrial animal in Bio-logging application. T im e (s ) T im e ( s ) T im e (s ) T im e ( s )

FIGURE CAPTIONS

  Rigid body motions capturing by means of wearable inertial and magnetic MEMS sensors assembly: From the reconstitution of the posture toward the dead reckoning: an application in Bio-logging Hassen Fourati, Noureddine Manamanni, Lissan Afilal, Yves Handrich To cite this version: Hassen Fourati, Noureddine Manamanni, Lissan Afilal, Yves Handrich. Rigid body motions capturing by means of wearable inertial and magnetic MEMS sensors assembly: From the reconstitution of the posture toward the dead reckoning: an application in Bio-logging. MEMS: Fundamental Technology and Applications (Devices, Circuits, and Systems), CRC Press, pp.313-329, 2013, Devices, Circuits, and Systems (Book 15), 978-1466515819. hal-00909138

Fig. 2

 2 Fig. 2 the orientation of the body-fixed frame ( ) , , B B B B X Y Z with respect to the Earth-fixed

  [33] was used to collect magnetic and inertial measurements that are used firstly to estimate the attitude by the complementary filter. This device has the same characteristics as the Mti-G. The MTi is attached to the end of the human's foot as shown in Fig.10and used after to record data during an episode of walking during a few minutes. In this case, it is possible to obtain the norm of the Dynamic Body Acceleration â near to zero each time the foot touches the ground. To get an accurate reference of 3D walking trajectory, we chose to perform a known one along the corridors of our laboratory CReSTIC in Reims as shown in Figure11. This trajectory takes the form of a rectangle with a size ≈ 2*(22 meters*19 meters).

Fig. 1 Fig. 2 NFig. 3 Fig. 4 TheFig. 5 Fig. 6 Fig. 1 Fig. 4 The

 12345614 Fig.1 Schematic diagram of how an Inertial Measurement Unit is attached to a penguin Fig. 2 The coordinate system ( ) B of a rigid body represented in the Earth-fixed frame ( ) N

  c ific fo rc e s E a r th 's m a g n e tic fie ld s A n g u la r ra te s

Fig. 5 Fig. 8 FigFig. 12 Fig. 13 Fig. 14 Fig. 15

 5812131415 Fig. 5 Inertial and magnetic measurements recorded from the MTi-G

ACKNOWLEDGEMENT

The authors would like to thank both the Alsace and Champagne-Ardenne regions within the framework of the project (NaviMeles) for their financial support.

Difference on q 0

Difference on q 1

Difference on q 2

Difference on q 3