Ahmed Tlili
email: .ahmed.tlili@cemagref.fr

Marjorie Marechal
email: marjorie.marechal@cemagref.fr

Bernard Montuelle
email: bernard.montuelle@cemagref.fr

Bernadette Volat
email: bernadette.volat@cemagref.fr

Ursula Dorigo
email: ursula.dorigo@thonon.inra.fr

Annette Bérard
email: annette.berard@avignon.inra.fr

Use of the MicroResp TM method to assess pollution-induced community tolerance to metals for lotic biofilms

Keywords: river biofilm, pollution-induced community tolerance, substrate induced respiration, heavy metals, MicroResp TM

Understanding the ecological status of aquatic ecosystems and the impact of anthropogenic contamination requires correlating exposure to toxicants with impact on biological communities. Several tools exist for assessing the ecotoxicity of substances, but there is still a

I. Introduction

In a setting of agricultural land use, small lotic ecosystems (Strahler order below 3) are very sensitive to pollution because of generally low dilution and contiguous contamination sources (spraying of pesticides, run off…) (Dorigo et al., 2003). In these ecosystems, biofilms (attached microbial communities of autotrophic and heterotrophic, eukaryotic and prokaryotic populations) play a fundamental role in the aquatic trophic web and geochemical cycles [START_REF] Battin | Contributions of microbial biofilms to ecosystem processes in stream mesocosms[END_REF]. In agricultural watershed rivers, biofilms are exposed directly to toxicants that run off from fields, and their structure and function can be affected. The vast diversity of microbial species and the multitude of biological and physico-chemical processes induce a large structural and functional complexity into the matrix biofilm. Also, species composing the biofilm may have ranging sensitivities and responses towards various anthropogenic pressures [START_REF] Barranguet | Copper-induced modifications of the trophic relations in riverine algalbacterial biofilms[END_REF]. During exposure to toxic agents such as heavy metals, the most sensitive organisms may be overtaken by more resistant or more tolerant ones. Thus the entire community may be restructured, present physiological alterations and come finally to display an overall increase in tolerance to the toxicant. This process underlies the concept of pollution-induced community tolerance (PICT), developed by [START_REF] Blanck | Pollution-Induced Community Tolerance -A new ecotoxicological tool[END_REF] as an ecotoxicological tool to assess xenobiotic impact at the community level.

Diverse approaches to examining the tolerance or resistance of a bacterial community to heavy metals have been developed. For example, Díaz-Raviña et al. (2007) investigated vineyard soil copper contamination inducing bacterial tolerance with growth measurements using thymidine (Tdr) and leucine (Leu) incorporation techniques. In aquatic systems Tdr or Leu incorporation techniques have also been used to assess the tolerance of bacterial communities in biofilm and sediments (Paulsson et al., 2000;Lesley and Grant, 2007).

Despite good results, these techniques remain difficult to implement and are not possible in all laboratories as they use radio-labelled elements. Biolog TM is a conventional approach applied first to obtain catabolic fingerprints of the bacterial communities or a community-Comment citer ce document : Tlili, A., Maréchal, M., Montuelle, B., Volat, B., Dorigo, U., Berard, A. (2011). Use of the MicroResp (TM) method to assess pollution-induced community tolerance to metals for lotic biofilms. Environmental Pollution, 159 (1), 18-24. DOI : 10.1016/j.envpol.2010.09.033

E

Version définitive du manuscrit publié dans / Final version of the manuscript published in : Environmental Pollution, 2011, Vol.159, no.1, 18-24, DOI: 10.1016/j.envpol.2010.09.033 Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 4 level physiological profile (CLPP), and more recently to assess tolerance to metals of the bacterial communities in soil and aquatic biofilms (Boivin et al., 2006;Stefanowicz et al., 2009). However, this microplate technique, based on the analysis of a bacterial community's use of several carbon sources, has major drawbacks: incomplete assessment of the whole microbial community, a long incubation time inducing bacterial selection and growth (Preston-Mafham et al., 2002) and the use of artificial culture media with buffer that may have artificial significant effects on the speciation and in consequence on the bioavailability and the toxicity of toxicants [START_REF] Barranguet | Copper-induced modifications of the trophic relations in riverine algalbacterial biofilms[END_REF]. A respirometric technique based on the analysis of the substrate-induced respiration (SIR) response (using gas chromatography analysis) was also investigated with a multiple carbon-source substrate for CLPP (Garland and Mills, 1991), and in a few studies, to test microbial tolerance to heavy metals at the community level in soils (Witter et al., 2000;Rajapaksha et al., 2004). More recently this approach was used to assess induced tolerance to copper of bacterial communities in river biofilms (Tlili et al., 2010). However, the method is still tedious, especially when assaying many microbial samples simultaneously in a multitude of bottles that have to be processed separately to measure the amount of released CO 2 (Chapman et al., 2007). Miniaturized systems offer the considerable advantage of allowing sample and replicate multiplication. In soil studies, Campbell et al. (2003) developed the MicroResp TM technique, which is an alternative method that combines the advantages of the Biolog TM technique, using the microplate system, and of the SIR approach with ability to measure CO 2 production during short-term incubation from a whole soil microbial community.

We hypothesized that the use of this new microrespirometric technique could be modified and applied to perform ecotoxicological bioassays on river biofilms for metal tolerance comparison purposes. The objectives of our study were thus (i) to test the MicroResp TM technique for a metal-contamination assessment in an aquatic ecosystem, using biofilms as a biological model in a PICT approach, (ii) to take a catabolic fingerprint of the heterotrophic

II. Materials and methods

In-situ biofilm

The biofilm used in our study was taken from the River Morcille, located in the Beaujolais vineyard area. The Morcille is a small first-order stream (7 km long) under strong agricultural pressure, essentially from vineyards, which occupy about 80% of its 8.5 km 2 catchment area.

The Morcille watershed is part of the Long-Term Ecological Research Rhône Basin (ZABR).

Regular physico-chemical surveys (hydrology, nutrients, pesticides and metals) and biological monitoring have been conducted on this site (Dorigo et al., 2007;Rabiet et al., 2008;Morin et al., 2010;Montuelle et al., 2010). Three sampling sites were selected along a gradient of increasing percentage contribution of vineyards to the catchment area: Saint-Joseph (vineyard percentage cover 11%), Les Versauds (53%) and Saint-Ennemond (65%) (see Table . 1 for physico-chemical details). Several annual surveys were performed to characterize metal contamination. In 2009 (n = 43 samples), Saint-Joseph was the least polluted site with a mean 0.51 ± 0.02 µg.L -1 of Cu, 1.13 ± 0.21 µg.L -1 of Zn and 1.94 ± 0.46 µg.L -1 of As. By contrast, the two other sites studied were considered polluted: Les Versauds was characterized by a mean 2.05 ± 0.25 µg.L -1 of Cu, 0.83 ± 0.42 µg.L -1 of Zn and 11.5 ± 0.46 µg.L -1 of As, and Saint-Ennemond by a mean 3.93 ± 0.77 µg.L -1 of Cu, 7.05 ± 3.56 µg.L -1 of Zn and 9.26 ± 4.99 µg.L -1 of As. Other metals including Cd, Ni, Fe or Al were also detected at low levels (means near limits of quantification) and there was no gradient of contamination from upstream to downstream. milli-Q water before use) in laboratory microcosms under controlled conditions. Fifteen-litter aquariums were filled with river water from the reference site (Saint-Joseph), which had been filtered through a 50 µm mesh to remove most of the grazers (water was renewed once a week). The water was kept at a temperature of 18 ± 2 °C, and exposed to a light intensity of 260 µmol m -2 s -1 , with a light/dark regime of 18:6 h. Three-week-old biofilms were used for the MicroResp TM assays (Tlili et al., 2008). method. Briefly, biofilms were scraped from stones (500 cm² per sample) collected from the three sampling sites described previously (St. Joseph (J), Les Versauds (V) and Saint-Ennemond (E)), and homogenized in 150 mL of 0.22 µm-filtered water from the reference site (J). Biofilm suspensions were placed directly in the deep well (500 µL) and 50 µL of each tested metal (Cu, Cd or Ag) was added separately. After pre-incubation 30 µL of glucose was added to each of the deep wells, and the MicroResp TM system was assembled as described previously and incubated in the dark at room temperature (23 ± 1 °C). After incubation, the absorbance of the detection microplate was measured at 590 nm (Biotek Synergy HT spectrophotometer), the amounts of released CO 2 were calculated and the results were expressed in µg CO 2 .mg -1 AFDW.h -1 . Dose-response curves were plotted using CO 2 values produced by the biofilm at each metal concentration as a percentage of the CO 2 produced by control biofilms (without toxicant). The EC 50 values were calculated for each metal and for each site.

The MicroResp

Catabolic diversity fingerprint

With a view to establishing a CLPP of the heterotrophic biofilm compartment, a multiple carbon-source substrate-induced respiration approach (using the MicroResp CO 2 .mg -1 AFDW.h -1 . In addition to the SIR measurements, the basal respiration (without substrate addition) was measured for each biofilm.

Biofilm DNA extraction, amplification and automated ribosomal intergenic spacer analysis (ARISA)

To characterize the diversity of the river biofilm bacterial communities, we used the ARISA molecular fingerprint technique. Three replicates of 2 mL of each biofilm suspension were centrifuged at 14,000g for 30 minutes, and the supernatant was discarded and pellet stored at -80 °C before extraction. Nucleic acid extraction was performed on biofilm pellets using the FAST DNA kit (QBIOgene, Illkirch, France) according to the manufacturer's instructions.

The PCR conditions and the PCR template preparation for the ARISA conditions were those described by Ranjard et al. (2003). The primers used were S-D-Bact-1522-b-S-20 (3′end of 16S genes) and L-D-Bact-132-a-A-18 (5′end of 23S genes) (Ranajard et al., 2003).

Data treatment

version

). The significant level was set to 5% for all the statistical tests with four replicates for each parameter. Bacterial ARISA profiles were compared with regards to the presence or absence of bands, using the Pearson similarity index. Matrices were used to perform Ward's method of hierarchical cluster analysis (HCA) using the XLSTAT Software Package (2009 version).

III. Results and discussion

MicroResp TM protocol applied to ecotoxicological bioassays on microbial biofilms

In toxicological bioassays, adding a mineralizable carbon source overcomes a problem that arises when measuring basal respiration, namely mineralization of unknown native organic production was approximately linear between 8 h and 15 h incubation time, with respiration rates of 160.0 ± 14.9 and 160.6 ± 12.1 µg CO2.mg -1 AFDW.h -1 for 8 h and 15 h incubation time respectively. In addition, analysis of the bacterial community profiles (ARISA) of each of the four incubation times tested showed no change in the diversity of this community (data not shown). Incubation for 8 h would have sufficed, but for practical reasons we opted to conduct our bioassays with an incubation time of 15 h (overnight).

During preliminary bioassays with Cu, no dose-response curves were obtained (data not shown). The quantities of measured CO 2 corresponding to the well containing the most concentrated Cu solutions were high. The pH measurement of these biofilm solutions showed that the addition of large amounts of Cu to the biofilm suspensions induced a very rapid and marked pH decrease, linked to an abundant release of CO 2 , which could not be of biological origin but was probably due to chemical reactions. Oren and Steinberger (2008) suggested that dissolution of CaCO 3 (contained in the biological samples, in their case soil) may follow pH decline, and be accompanied by abiotic CO 2 evolution that may be mistakenly taken for respired CO 2 . Fig. 1 shows that 3 h of copper pre-incubation is needed to obtain a doseresponse curve and a percentage SIR inhibition below 20% (19.01 ± 0.1%) with the maximum copper concentration (17.2 × 10 2 µM). This dose-response curve did not change with biofilms pre-incubated for longer times (4 h and 6 h). These results suggest that this chemical artefact has ceased after 3 h. We therefore opted for this time of 3 h pre-incubation with copper. Finally, the protocol used for short-term bioassays can be summarized as follows: the biofilm suspension was distributed in deep wells (500 μL per well), to which we added 50 µL of the metal solution (increasing nominal concentrations). The microplate was then pre-incubated in the dark at room temperature for 3 h, 30 µL of the glucose solution (120 mg.mL -1 ) was added Cu EC 50 values were estimated to evaluate tolerance to this pollutant of the biofilm communities sampled at three sites along the River Morcille. EC 50 values increased upstream to downstream, ranging from 48.5 to 179.4 µM copper (Table .3, Fig. 2). The most Cusensitive communities were from the upstream site "Saint-Joseph" (J), while the most Cutolerant communities were downstream at "Saint-Ennemond" (E). The measured sensitivity at the intermediate site "Les Versauds" (V) was closer to E than to J. Cu concentrations in the water from the three sampling sites, collected in the same period as our study, and the EC 50 values measured were closely correlated (n = 4, Pearson R² = 0.72, p = 0.0004). Also, diversity analysis by ARISA of bacterial communities showed a differentiation of J, E and V.

Bacterial diversity from the intermediate site V was more closely similar to the most contaminated site E than the pristine area J (Fig. 3). These results agree with previous studies (Boivin et al., 2006;Dorigo et al., 2007) and are congruent with the predictions of the PICT concept and validation (Blanck, 2002), suggesting that Cu constitutes a selective pressure, at least on the bacterial and heterotrophic portion of the Morcille biofilms, producing changes in species composition, catabolic structure (see paragraph 2.3) and Cu substrate-induced respiration tolerance. However, given the presence of gradient contaminations with other metals (As and Zn) from the Morcille upstream to downstream, these differences in bacterial structure cannot be attributed to Cu alone.

On the other hand, environmental factors such as light, current velocity, temperature or nutrient content could affect toxicant bioavailibility and the biofilm tolerance to these toxicants. During long-term exposure, for example, Guasch et al. ( 2004) and Tlili et al. (2010) showed that phosphorus supply caused an increased induced tolerance of biofilms to copper.

Also, Guasch et al. ( 2002) evaluated the effects of chronic copper exposure on natural periphyton in a non-polluted calcareous river, and they concluded that the water pH in the river was important in affecting the bioavailability of the metals and therefore their toxicity towards the biological community. It is therefore necessary to have not only a reference site that is globally comparable to the study site, but also a sampling strategy (spatial and temporal) that minimizes the interference of environmental factors in the detection of PICT (Dorigo et al., 2009). Care must also be taken in a PICT approach to ascertain that bioassays (2002) stipulated that bioavailability may interfere in the PICT detection as a confounding factor, for example when the waters used in the short-term tests are different. This could then affect the EC X values obtained and finally the PICT signal may represent a bioavailability gradient. However, this remark concerns a regional survey [START_REF] Blanck | Variability in zinc tolerance, measured as incorporation of radio-labeled carbon dioxide and thymidine, in periphyton communities sampled from 15 European river stretches[END_REF]) that compared very different sampling sites (from different rivers and countries) in the context of bioassays that used the filtered natural water from each sampling area. This was not the case in our study, where we used the same filtered water of the upstream sampling area J (pH values in the range 7.2-8) for periphytic suspensions and toxic solutions of all bioassays and in the context of a small river basin. In addition, in a previous study we measured pH in the biofilmriver suspension (using water from site J), glucose and copper added at the beginning and end of the short-term incubation, and observed no pH changes during the bioassays despite demonstrating an induced tolerance to copper based on the substrate-induced respiration as a functional parameter (measured by gas chromatography, data not shown). Rusk et al. (2004) investigated tolerance of soil biological nitrification to metals using sterilized, metal-treated soils from the reference area reinoculated with a similar soil containing the microbial communities to be tested. Our approach using filtered water from a reference area (upstream)

to perform the PICT bioassays, in which we suspended periphyton scraped from the different investigated sites, is comparable to the approach of Rusk et al. ( 2004), who wanted to exclude the potentially confounding effects of variations in metal bioavailability in their tolerance bioassays. We can conclude that the bioassays conducted during our study were performed under homogeneous standardized conditions and minimized variations in metal bioavailability among the microbial samples tested. et al., 2000). These studies demonstrated that microbial tolerance to metals increased as soil metal pollutant concentrations increased.

However, there are few studies on the toxicity assessment of contaminants in aquatic ecosystems using the SIR to assess the induced tolerance to metals of heterotrophic biofilm communities. Dorigo et al. ( 2010), investigated the effects of Cu on heterotrophic biofilm communities collected from the same sampling sites (J; V and E), using the SIR measured by gas chromatography and the 16S rRNA gene DGGE method to highlight induced tolerance to Cu and bacterial diversity respectively. Like ours, her results strongly suggested Cu contamination-driven changes in biofilm community structure and in the tolerance to Cu of the bacterial community, confirming the PICT hypothesis.

This first biofilm ecotoxicological bioassays using MicroResp TM applied in a river contamination assessment context, highlights the efficiency of this technique for measuring the induced tolerance to metals of the heterotrophic biofilm community based on SIR.

Co-tolerance assessment

Co-tolerance may occur when communities that have been exposed to one toxicant, but not to another, become tolerant to both toxicants. Occurrence of co-tolerance depends on the means of conferring tolerance and on the tolerance mechanisms [START_REF] Blanck | Pollution-Induced Community Tolerance -A new ecotoxicological tool[END_REF]. Tolerance to Ag and Cd was measured for biofilms from the three sampling sites (J, V and E) (Table .3). In contrast to results for copper, no significant changes in tolerance to Ag or Cd were observed from upstream to downstream sites. These results are congruent with Ag and Cd concentrations measured in water from J, V and E sites, which were constantly below the limits of quantification. Therefore, these SIR measurements with MicroResp TM did not reveal any co-tolerance between Cu and Ag or Cd. Some studies have reported a co-tolerance of biofilm communities between copper and some metals such as Zn or Ni (Gustavson and , 1995;Ivorra et al., 1999). Soldo and Behra (2000) have shown a strong cotolerance to silver in autotrophic biofilm communities exposed to 5µM of copper. In contrast to our study, all these co-tolerance measurements were based on photosynthetic activity, and therefore especially targeted the phototrophic biofilm compartment. Detection of co-tolerance implies difficulty attributing a tolerance shift to the presence of a particular toxicant in the environment. Our results and those obtained in the above-cited studies show that depending on the kind of targeted activities and associated communities (phototrophic or heterotrophic), co-tolerance linked to specific modes of action of toxicants and detoxification processes (Soldo and Behra, 2000;Knauer et al., 2010) can be variable. MicroResp TM use, in addition to other tolerance measurements based on other activities in the context of co-tolerance studies, could thus offer a complementary approach to investigating mechanisms of tolerance.

Community-level physiological profiles

Principal component analysis (PCA) was applied to the data set obtained with the various carbon sources, normalized by the basal respiration values for each site (Fig. 4). The first two axes of the PCA accounted for more than 80% of the variability. Pristine area J and downstream area E were separated from the intermediate area V on the first axis F1, while the second axis F2 separated area J from area E (left panel). Biofilms from V were characterized by a higher mineralization of the glutamic acid and citric acid substrate and biofilms from J and E were characterized by a higher mineralization rate of glucose substrate (right panel).

Thus multivariate analysis showed a clear discrimination between the biofilms from the different sampling sites. Several soil studies investigated functional diversity with CLPP measurements and showed the utility of the MicroResp TM method to discriminate different soils or soils submitted to different pressures (Campbell et al., 2003(Campbell et al., , 2008;;Oren and Stenberger, 2008). Applied to an aquatic environment, MicroResp TM seems also to be a useful tool for assessing changes in the functional diversity of the microbial community (CLPPs) in biofilms.

Comment citer ce document : Tlili, A., Maréchal, M., Montuelle, B., Volat, B., Dorigo, U., Berard, A. (2011)

VI. Conclusion and perspectives

In the global context of assessing the impact of pollutants on the diversity and functioning of microbial communities in aquatic ecosystems, a range of diversified indicators for diversified functions and communities is needed (Clements and Rohr, 2009). Among such indicators, the PICT approach, which offers a good tool for assessing an ecosystem's history of exposure to pollution at the community level, and the establishment of catabolic fingerprint profiles of microbial communities, are now applied in diverse ecosystems [START_REF] Bérard | Application of the Pollution-Induced Community Tolerance (PICT) method to algal communities: its values as a diagnostic tool for ecotoxicological risk assessment in the aquatic environment[END_REF]Boivin et al., 2006;Kaufmann et al., 2006). However, this study is the first to investigate micro-SIR in a contaminated aquatic system as a tool for measuring induced tolerance in a microbial community and establishing catabolic physiological profiles on the whole heterotrophic biofilm. The purpose of this methodological study was not to make a direct translation of the EC 50 obtained in MicroResp TM plates to the situation of the biofilms exposed in the river, but to propose an easy short-term bioassay based on catabolism to detect relative differences in metal tolerance between long-term exposed communities that were sampled (complementary to investigations of their taxonomic composition for PICT methodology). Our results show that the MicroResp TM technique offers a convenient, rapid and sensitive method for assessing metal contamination of aquatic ecosystems. Like for soil microbial micro-SIR measurements, for results to be fully transportable between studies, this method would require interlaboratory calibration (Creamer et al., 2009). A modified MicroResp TM system could be developed, using microplates with larger wells than those currently used. The method could thus be applied with an in-place biofilm (grown on individual artificial substrates) to minimize disturbances of the microbial communities and be suitable for similar samples of periphyton, and for other measurements of additional activities such as photosynthesis (Dorigo and Leboulanger, 2001).

Catabolic diversity fingerprint

To establish a CLPP of the heterotrophic biofilm compartment, using a multiple carbon-source substrate.

Molecular biology analysis

To characterize the diversity of the river biofilm bacterial communities, using the ARISA molecular fingerprint technique.

Collected from the three sampling sites in the Morcille River. Vol.159, no.1, 18-24, DOI: 10.1016/j.envpol.2010.09.033 Fig.

3

F1 (57 06 %)

-1 -1 -0,75 -0,5 -0,25 0 0,25 0,5 0,75 1 F1 (57,06 %) F1 (57,06 %)

E

  TM technique) was used. The different carbon substrates used were: D-glucose (Prolabo), D-fructose (Sigma chemical Co.), D-sucrose (Fluka biochemica), D-ribose (Prolabo), D-galactose (Prolabo), Dmaltose (Prolabo), L-arginine (Sigma Chimie Fluka), glycine (Sigma Aldrich Chemie), Llysine hydrochloride (Sigma Chimie, Fluka), L-glutamic acid (Sigma Aldrich Chemie) and citric acid anhydre (Sigma Aldrich Chemie). Stock solutions containing 120 mg.mL -1 of each substrate were prepared in 0.22 µm-filtered Milli Q water and stored at 4 °C, until used. The pH of each solution was adjusted to river pH (7) to prevent any substrate-pH effects on microbial communities and minimize chemical artefacts due to carbonate-derived CO 2 . Final concentration was 6.2 mg of C per mL in each deep well. The results were expressed in µg Comment citer ce document : Tlili, A., Maréchal, M., Montuelle, B., Volat, B., Dorigo, U., Berard, A. (2011). Use of the MicroResp (TM) method to assess pollution-induced community tolerance to metals for lotic biofilms. Environmental Pollution, 159 (1), 18-24. DOI : 10.1016/j.envpol.2010.09.033 Version définitive du manuscrit publié dans / Final version of the manuscript published in : Environmental Pollution, 2011, Vol.159, no.1, 18-24, DOI: 10.1016/j.envpol.2010.09.033 Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 10

  eric.vindimian.9online.fr/). The CLPP data sets (normalized by the basal respiration values) were subjected to principal component analysis (PCA) (XLSTAT Software Package.

Fig

  Fig.1.

Fig. 2 .

 2 Fig. 2. Copper tolerance of heterotrophic communities at three sampling areas along a copper

Fig 3 .

 3 Fig 3. Cluster analysis (hierarchical ascendant classification based on Pearson correlation

Fig 4 .EFigEFigE

 4 Fig 4. Principal component analysis (PCA) of community-level physiological profiles (CLPP) of biofilms measured with the MicroResp TM method and normalized to basal respiration, from

EFig

  Fig.4

  Version définitive du manuscrit publié dans / Final version of the manuscript published in : EnvironmentalPollution, 2011, Vol.159, no.1, 18-24, DOI: 10.1016/j.envpol.2010.09.033

	biofilm community, and (iii) to implement an in-situ PICT application of MicroResp TM in a
	copper contamination context.
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Comment citer ce document : Tlili, A., Maréchal, M., Montuelle, B., Volat, B., Dorigo, U., Berard, A. (2011). Use of the MicroResp (TM) method to assess pollution-induced community tolerance to metals for lotic biofilms. Environmental Pollution, 159 (1), 18-24. DOI : 10.1016/j.envpol.2010.09.033 E

Table .

 . Version définitive du manuscrit publié dans / Final version of the manuscript published in : EnvironmentalPollution, 2011, Vol.159, no.1, 18-24, DOI: 10.1016/j.envpol.2010.09.033 During the different experiments with the MicroResp TM , a biofilm was grown on artificial substrates (microscope slides previously cleaned in a dilute nitric acid bath and rinsed with

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1 2. Biofilm microcosm Comment citer ce document : Tlili, A., Maréchal, M., Montuelle, B., Volat, B., Dorigo, U., Berard, A. (2011). Use of the MicroResp (TM) method to assess pollution-induced community tolerance to metals for lotic biofilms. Environmental Pollution, 159 (1), 18-24. DOI : 10.1016/j.envpol.2010.09.033 E

. Development of the MicroResp TM protocol

		E E
		Version définitive du manuscrit publié dans / Final version of the manuscript published in : Environmental Pollution, 2011, Version définitive du manuscrit publié dans / Final version of the manuscript published in : Environmental Pollution, 2011,
		Vol.159, no.1, 18-24, DOI: 10.1016/j.envpol.2010.09.033 Vol.159, no.1, 18-24, DOI: 10.1016/j.envpol.2010.09.033
		release) at room temperature. CO 2 -trap absorbance was measured at 590 nm (Biotek Synergy To enable the toxicant to penetrate into the cells and to cope with artefacts causing a chemical
		HT spectrophotometer) immediately before sealing to the deep-well plate (coefficient of release of CO 2 (Lindsay, 1979), a metal pre-exposure period was necessary before adding the
	Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript	variation below 5%), and after incubation. Quantities of CO 2 produced by the microbial samples were calculated using a calibration curve of absorbance values versus CO 2 quantity measured by gas chromatography (MTI 200 microcatharometer). Results were then expressed as µg CO 2 .mg -1 of sample.h -1 . In our biofilm study, the sample was considered as organic matter (ash-free dry weight, AFDW) of the biofilm suspension tested, and measured using the protocol described by Tlili et al. (2008). Briefly, three aliquots of each homogenized biofilm suspension (2 mL) were filtered through individual 25 mm CF/C Whatman glass fibre filters carbon substrate (glucose) and starting the incubation. The different tested times of pre-exposure to Cu were 0, 0.25, 0.5, 1, 2, 3, 4 and 6 h. 4.3. Short-term bioassays To validate the utility of the MicroResp TM method for short-term bioassays, using biofilms, a wide range of heavy metals was tested: CuSO 4 (Merck KgaA; purity≥99.0%;), ZnSO 4 (Sigma-Aldrich GmbH), CdN 2 O 6 .7H 2 O (Fluka Sigma-Aldrich; purity≥99.0%), AgNO 3 (CARLO ERBA reagenti; purity≥99.8%), N 2 NiO 6 .6H 2 O (Fluka Sigma-Aldrich Chemie (1.2 µm pore size). Total dry matter was measured by weighing the filters after drying at GmbH; purity≥97.0%), FeSO 4 .7H 2 O (Sigma Chemical co.; purity≥99.0%), CoN 2 O 6 .6H 2 O
		105°C for 24 h. The filters were then combusted in an oven at 480°C (Nabertherm P320) for (Fluka Sigma-Aldrich Chemie GmbH; purity≥98.0%), AlN 3 O 9 .9H 2 O (Merck KgaA;
	Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript	1 h, and reweighed to calculate mineral matter. AFDW was calculated by subtracting mineral matter from total dry matter. 4Table.2 4.1. Incubation time Campbell et al. (2003) propose an incubation time of 6 h with soil samples. However, in our case, we have to take into account the rate of CO 2 diffusion at the air-water interface, which purity≥98.5%) and Na 2 HAsO 4 .7H 2 O (Sigma-Aldrich GmbH; purity≥98.0%). Briefly, stock solutions containing 200 mM of each metal were prepared in water and stored at 4 °C, before dilution in the test wells. A semi-logarithmic series of concentrations was freshly prepared by serial dilution of the stock solutions in 0.2 µm-filtered river water. Final nominal test concentrations in the deep wells ranged from 0.5 µM to 17.2 mM (4 blanks and 4 replicates for each of the 9 concentrations). The carbon substrate used for all the SIR toxicological bioassays was D-glucose. Quantities of released CO 2 were measured for each bioassay as
		may be low. To ensure a relevant measure in the well airspace of the CO 2 released from the described in Section 3. Dose-response curves were plotted using CO 2 values produced by
	Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript	sample (the biofilm), we tested a range of incubation times: 4, 6, 8, and 15 h. Individual MicroResp TM microplates were used for each incubation time. Eight deep wells of each microplate were filled with 500 µL of biofilm suspension with 50 µL of milli-Q water and 30 µL of D-glucose (Prolabo) (the remaining wells of the microplate were filled with 580 µL of milli-Q water). Quantities of released CO 2 were measured at each incubation period, as described in Section 3. 4.2. Pre-incubation test with toxicant biofilm at each metal concentration as a percentage of CO 2 produced by a control biofilm (without toxicant). 5.
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		, Volat, B., Dorigo, U., Berard, A. (2011). Use of the Tlili, A., Maréchal, M., Montuelle, B., Volat, B., Dorigo, U., Berard, A. (2011). Use of the
		MicroResp (TM) method to assess pollution-induced community tolerance to metals for lotic MicroResp (TM) method to assess pollution-induced community tolerance to metals for lotic
		biofilms. Environmental Pollution, 159 (1), 18-24. DOI : 10.1016/j.envpol.2010.09.033 biofilms. Environmental Pollution, 159 (1), 18-24. DOI : 10.1016/j.envpol.2010.09.033

TM procedure

MicroResp TM system has been described in detail by

Campbell et al. (2003)

, but only a few ecotoxicological applications of the MicroResp TM technique have been investigated on soils

(Kaufmann et al. 2006

) and none on aquatic microbial communities. Here we present an adaptation of the procedure of

Campbell et al. (2003)

to aquatic biofilm suspensions and toxicological bioassays. MicroResp TM is a colorimetric method based on the colour change of a pH indicator dye caused by the release of CO 2 by heterotrophic communities. The system consists of two microplates (96 wells) placed face to face. One of these is a deep-well microplate (1.2 mL capacity, 96-deep-well microplate, NUNC) in which each well contains the microbiological sample (we used a 500 µL biofilm suspension obtained by scraping biofilm off glass substrata using a polypropylene spatula and suspending it in 0.2 µm of Nuclepore-filtered water from the reference site) with the carbon source (30 µL per well, 6.2 mg of C per well). To this deep-well microplate we added the toxicant (50 µL per well). The second microplate contained the detection gel (cresol red dye (12.5 ppm), potassium chloride (150 mM) and sodium bicarbonate (2.5 mM) set in a 1% gel of noble agar (final volume 150 µL per well)). The two microplates were sealed together with a silicone seal, with interconnecting holes between the corresponding wells. The assembly was clamped together and the system was incubated in the dark (to avoid any photosynthesis interference with CO 2

Comment citer ce document : Tlili, A., Maréchal, M., Montuelle, B.

Application of the microrespirometric method to a field situation

	5.1. PICT approach (short-term bioassays)
	The relevance of the MicroResp TM technique in a PICT approach was tested with biofilms
	sampled from the River Morcille. Induced tolerances to Cu, Cd and Ag of the heterotrophic
	biofilm communities were then tested by toxicological bioassay using the MicroResp

TM Comment citer ce document : Tlili, A., Maréchal, M., Montuelle, B., Volat, B., Dorigo, U., Berard, A. (2011). Use of the MicroResp (TM) method to assess pollution-induced community tolerance to metals for lotic biofilms. Environmental Pollution, 159 (1), 18-24. DOI : 10.1016/j.envpol.2010.09.033 E Version définitive du manuscrit publié dans / Final version of the manuscript published in : Environmental Pollution, 2011, Vol.159, no.1, 18-24, DOI: 10.1016/j.envpol.2010.09.033 Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 9

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	matter that may be different between sampling stations. Glucose mineralization activity was
	detected by our MicroResp TM modified design within 8 h incubation time, and net CO 2
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  Version définitive du manuscrit publié dans / Final version of the manuscript published in : EnvironmentalPollution, 2011, Vol.159, no.1, 18-24, DOI: 10.1016/j.envpol.2010.09.033 and the detection microplate was positioned. The system was sealed and incubated for 15 h in the dark (without photosynthetic activity, which could induce an increase of the pH and interfere with the CO 2 release) and at room temperature (23 ± 1 °C).CO 2 -trap absorbance was measured at 590 nm (or better at 570 nm,Rowell, 1995) immediately before sealing to the deep well plate, and after 15 h incubation.The MicroResp TM technique enabled us to plot dose-response curves with all the tested metals. The EC 50 obtained with the different tested metals in short-term bioassays are shown in Table.3. Compared with the other metals, silver seems to be the most toxic metal to the SIR (EC 50 = 3.96 µM). Aluminium (EC 50 = 2317.86 µM) and cobalt (EC 50 = 6170.63 µM) were the least toxic, while Zn, Cu and Cd had similar intermediate toxicities. However, in our study some of the metal salts used included a nitrogen source (e.g. AgNO 3; N 2 NiO 6 .6H 2 O or CoN 2 O 6 .6H 2 O) that could interfere with the respiration measurements and so bias the EC 50 Vibrio fischeri) are also comparable to our range of EC 50 values (e.g. EC 50 (5 mn) = 941 µM CdCl 2 and 168 µM CuCl 2 and EC 50 (15mn) = 421 µM CdCl 2 and 49 µM CuCl 2 , inMacken et al. 2009). Therefore, our dose-response curves and EC 50 obtained with the SIR, related to heterotrophic organisms, confirm that the MicroResp TM Version définitive du manuscrit publié dans / Final version of the manuscript published in : EnvironmentalPollution, 2011, Vol.159, no.1, 18-24, DOI: 10.1016/j.envpol.2010.09.033

	Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript to each well Manuscrit d'auteur / Author manuscript 2. Application of the microrespirometry method to a field situation 2.1. Copper-induced tolerance
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Comment citer ce document : Tlili, A., Maréchal, M., Montuelle, B., Volat, B., Dorigo, U., Berard, A. (2011). Use of the MicroResp (TM) method to assess pollution-induced community tolerance to metals for lotic biofilms. Environmental Pollution, 159 (1), 18-24. DOI : 10.1016/j.envpol.2010.09.033 E values. A second control using NaNO 3 in addition to glucose might be a good option to overcome this interference. Witter et al., (2000) obtained similar results when testing soil bacteria tolerance (based on SIR) to these three metals (e.g. EC 50 Zn value of about 250 ppm).

EC 50 values calculated for the percentage of 95 substrates used completely within 8 days of incubation in Cu (0-300 mM)-amended BIOLOG GN plates inoculated with a river biofilm bacteria consortium were in the range 10-30 µM

[START_REF] Barranguet | Copper-induced modifications of the trophic relations in riverine algalbacterial biofilms[END_REF]

). Sensitivities obtained with other bioassays such as the well-known Microtox® (based on bioluminescence reduction of the marine bacterium technique could be a useful method to classify heavy metals according to their toxicity toward river biofilms. Table.4. Comment citer ce document : Tlili, A., Maréchal, M., Montuelle, B., Volat, B., Dorigo, U., Berard, A. (2011). Use of the MicroResp (TM) method to assess pollution-induced community tolerance to metals for lotic biofilms. Environmental Pollution, 159 (1), 18-24. DOI : 10.1016/j.envpol.2010.09.033 E

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	are controlled and standardized, and that different physicochemical parameters are
	homogeneous and similar during short-term exposure of the tested communities. Blanck
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Table .

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		In previous studies, a respirometric technique based on the analysis of the substrate-induced
		respiration (SIR) response was developed to test the microbial tolerance to toxicants at the
	Manuscrit d'auteur / Author manuscript	community level in soil ecosystems (Witter
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4. Fig.2. Fig.3. Comment citer ce document : Tlili, A., Maréchal, M., Montuelle, B., Volat, B., Dorigo, U., Berard, A. (2011). Use of the MicroResp (TM) method to assess pollution-induced community tolerance to metals for lotic biofilms. Environmental Pollution, 159 (1), 18-24. DOI : 10.1016/j.envpol.2010.09.033 E

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	Wängberg
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	Fig.4.
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. Use of the MicroResp (TM) method to assess pollution-induced community tolerance to metals for lotic biofilms. Environmental Pollution, 159 (1), 18-24. DOI : 10.1016/j.envpol.2010.09.033 E

  Version définitive du manuscrit publié dans / Final version of the manuscript published in : EnvironmentalPollution, 2011, Vol.159, no.1, 18-24, DOI: 10.1016/j.envpol.2010.09.033 Version définitive du manuscrit publié dans / Final version of the manuscript published in : EnvironmentalPollution, 2011, Vol.159, no.1, 18-24, DOI: 10.1016/j.envpol.2010.09.033 .E.Y., Massieux, B., Breure, A.M., Greve, G.D., Rutgers, M., Admiraal, W., 2006. Biology and Fertility of Soils. 45, (6) 623-633. Diaz-Ravina, M., Calvo De Anta, R., Baath, E., 2007. Tolerance (PICT) of the Bacterial Communities to Copper in Vineyards Soils from Spain. J. Environ. Qual. 36, 1760-1764.Version définitive du manuscrit publié dans / Final version of the manuscript published in : EnvironmentalPollution, 2011, Vol.159, no.1, 18-24, DOI: 10.1016/j.envpol.2010.09.033 Bourrain, X., A., B., Leboulanger, C., 2003. Seasonal changes in river microalgae sensitivity to atrazine and isoproturon, along a contamination gradient. The Science of the Version définitive du manuscrit publié dans / Final version of the manuscript published in : Environmental Pollution, 2011, Vol.159, no.1, 18-24, DOI: 10.1016/j.envpol.2010.09.033 Comment citer ce document : Tlili, A., Maréchal, M., Montuelle, B., Volat, B., Dorigo, U., Berard, A. (2011). Use of the MicroResp (TM) method to assess pollution-induced community tolerance to metals for lotic biofilms. Environmental Pollution, 159 (1), 18-24. DOI : 10.1016/j.envpol.2010.09.033 E Version définitive du manuscrit publié dans / Final version of the manuscript published in : Environmental Pollution, 2011, Vol.159, no.1, 18-24, DOI: 10.1016/j.envpol.2010.09.033 Comment citer ce document : Tlili, A., Maréchal, M., Montuelle, B., Volat, B., Dorigo, U., Berard, A. (2011). Use of the MicroResp (TM) method to assess pollution-induced community tolerance to metals for lotic biofilms. Environmental Pollution, 159 (1), 18-24. DOI : 10.1016/j.envpol.2010.09.033 E Version définitive du manuscrit publié dans / Final version of the manuscript published in : Environmental Pollution, 2011, Vol.159, no.1, 18-24, DOI: 10.1016/j.envpol.2010.09.033 Version définitive du manuscrit publié dans / Final version of the manuscript published in : Environmental Pollution, 2011, Vol.159, no.1, 18-24, DOI: 10.1016/j.envpol.2010.09.033 Version définitive du manuscrit publié dans / Final version of the manuscript published in : Environmental Pollution, 2011,Vol.159, no.1, 18-24, DOI: 10.1016/j.envpol.2010.09.033

	Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript	Boivin, MFunctional recovery of biofilm bacterial communities after copper exposure. Environmental Pollution. 140, (2) 239-246. Campbell, C.D., Chapman, S.J., Cameron, C.M., Davidson, M.S., Potts, J.M., 2003. A rapid microtiter plate method to measure carbon dioxide evolved from carbon substrate amendments so as to determine the physiological profiles of soil microbial communities by using whole soil. Applied and Environmental Microbiology. 69, (6) 3593-3599. Campbell, C.D., Cameron, C.M., Bastias, B.A., Chen, C.R., Cairney, J.W.G., 2008. Long term repeated burning in a wet sclerophyll forest reduces fungal and bacterial biomass and responses to carbon substrates. Soil Biology & Biochemistry. 40, (9) 2246-2252. Kaufmann, K., Chapman, S.J., Campbell, C.D., Harms, H., Hohener, P., 2006. Miniaturized test system for soil respiration induced by volatile pollutants. Environmental Pollution. 140, Preston-Mafham, J., Boddy, L., Randerson, P.F., 2002. Analysis of microbial community functional diversity using sole-carbon-source utilisation profiles -a critique. Fems Tlili, A., Bérard, A., Roulier, J.L., Volat, B., Montuelle, B., 2010. PO 4 3-dependence of biofilm autotrophic and heterotrophic communities tolerance to copper and diuron. Aquatic Table.1. Means (± standard deviation, n = 43) of selected chemical parameters (pH; cond.: conductivity [µS.cm -1 ]; DOC [mg.L -1 ]; NH 4 + [mg.L -1 ]; NO 2 -[mg.L -1 ]; NO 3 -[mg.L -1 ] and Table. 2. Chronological summary of the different experiments and steps performed during the study Dorigo, U., Total Environment. 318, 101-114. Dorigo, U., Leboulanger, C., Bérard, A., Bouchez, A., Humbert, J.F., Montuelle, B., 2007. Lotic biofilm community structure and pesticide tolerance along a contamination gradient in a vineyard area. Aquatic Microbial Ecology. 50, (1) 91-102. Dorigo, U., Lefranc, M., Leboulanger, C., Montuelle, B., Humbert, JF., 2009, Influence of sampling strategy on the assessment of the impact of pesticides on periphytic microbial communities in a small river. FEMS Microbial Ecology, 67, 491-501. Dorigo, U., Berard, A., Rimet, F., Bouchez, A., Montuelle, B., 2010. In situ assessment of (2) 269-278. Knauer, K., Leimgruber, A., Hommen, U., Knauert, S., 2010. Co-tolerance of phytoplankton communities to photosynthesis II inhibitors. Aquatic Toxicology. 96, (4) 256-263. Lesley, A., Ogilvie, A.B., Alistair Grant, B., 2008. Linking pollution induced community tolerance (PICT) and microbial community structure in chronically metal polluted estuarine sediments. Marine Environmental Research. 65, 187-198. Lindsay, W.L., 1979. Carbonate equilibria, in: Lindsay, W.L. (Eds.), Chemical Equilibria in Soils. Wiley, M. J, New York, pp. 78-84. Microbiology Ecology. 42, (1) 1-14. Rabiet, M., Margoum, C., Gouy, V., Carluer, N., Coquery, M.. . 2008. Transfert des pesticides dans un petit bassin versant viticole. Influence des conditions hydrologiques sur le transport de ces contaminants. Ingénieuries EAT. Rajapaksha, R.M.C.P., Tobor-Kaplon, M.A., Baath, E., 2004. Metal toxicity affects fungal and bacterial activities in soil differently. Applied and Environmental Microbiology. 70, (5) 2966-2973. Ranjard, L., Lejon, D.P.H., Mougel, C., Schehrer, L., Merdinoglu, D., Chaussod, R., 2003. Toxicology. 98, (2) 165-177. Witter, E., Gong, P., Baath, E., Marstorp, H., 2000. A study of the structure and metal tolerance of the soil microbial community six years after cessation of sewage sludge applications. Environmental Toxicology and Chemistry. 19, (8) 1983-1991. PO 4 3-[mg.L -1 ]) obtained from the three sampling areas (J: Saint-Joseph; V: Les Versauds and E: Saint-Ennemond) over two years (2009 and 2010). Site pH cond DOC NH 4 + NO 2 -NO 3 -PO 4 3-J 7.27 ± 0.29 161.72 ± 17.65 2.67 ± 0.88 0.04 ± 0.03 0.02 ± 0.01 6.82 ± 1.28 0.07 ± 0.03 Step No. Biofilm used Experiment 1 Development of the protocol, applied to aquatic ecosystems and using river 1. Incubation time To ensure a relevant measure in the well airspace of the CO 2 released from 2. Pre-incubation test with toxicant To cope with artefacts causing a chemical release of CO 2 . Cultivated in laboratory microcosms under MicroResp TM the sample. controlled conditions
	Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript	periphyton recovery in a river contaminated by pesticides. Aquatic Toxicology. 98, (4) 396-406. Garland, J.L., Mills, A.L., 1991. Classification and Characterization of Heterotrophic Microbial Communities on the Basis of Patterns of Community-Level Sole-Carbon-Source Utilization. Applied and Environmental Microbiology. 57, (8) 2351-2359. Guasch, H., Paulsson, M., Sabater, S., 2002. Effect of copper on algal communities from oligotrophic calcareous streams. Journal of Phycology. 38, (2) 241-248. Guasch, H., Navarro, E., Serra, A., Sabater, S., 2004. Phosphate limitation influences the Macken, A., Giltrap, M., Ryall, K., Foley, B., Mcgovern, E., Mchugh, B., Davoren, M., 2009. A test battery approach to the ecotoxicological evaluation of cadmium and copper employing a battery of marine bioassays. Ecotoxicology. 18, (4) 470-480. Montuelle, B., Dorigo, U., Bérard, A., Bouchez, A., Tlili, A., Gouy, V., Pesce, S., 2010. The periphyton as a multimetric bioindicator to assess the impact of land use on river: an overview on the Ardières-Morcille experimental watershed (France). Hydribiologia. inpress, Morin, S., Pesce, S., Tlili, A., Coste, M., Montuelle, B., 2010. Recovery potential of periphytic communities in a river impacted by a vineyard watershed. Ecological Indicators. Sampling strategy in molecular microbial ecology: influence of soil sample size on DNA fingerprinting analysis of fungal and bacterial communities. Environmental Microbiology. 5, (11) 1111-1120. Rusk, J.A., Hamon, R.E., Stevens, D.P., Mclaughlin, M.J., 2004. Adaptation of soil biological nitrification to heavy metals. Environmental Science & Technology. 38, (11) 3092-3097. Soldo, D., Behra, R., 2000. Long-term effects of copper on the structure of freshwater periphyton communities and their tolerance to copper, zinc, nickel and silver. Aquatic Toxicology. 47, (3-4) 181-189. 7.41 201.33 3.28 0.05 0.03 7.58 0.23 biofilms. 3. Short-term bioassays V ± 0.27 ± 18.57 ± 0.91 ± 0.04 ± 0.01 ± 3.03 ± 0.13 E 7.51 ± 0.24 233.61 ± 33.52 4.52 ± 1.82 0.08 ± 0.04 0.06 ± 0.06 7.33 ± 2.69 To validate the utility of the 0.28 ± 0.17 MicroResp TM method for short-term bioassays with different metals. Experiment 2 Application of the microrespirometric method to a field 1. PICT approach (short-term bioassays) To validate the relevance of the MicroResp TM technique in a PICT approach, by testing copper, silver and situation cadmium.
		sensitivity to copper in periphytic algae. Freshwater Biology. 49, (4) 463-473. 10, (2) 419-426. Stefanowicz, A.M., Niklinska, M., Laskowski, R., 2009. Pollution-induced tolerance of soil
	Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript	Dorigo, U., Leboulanger, C., 2001. A PAM fluorescence-based method for assessing the effects of photosystem II herbicides on freshwater periphyton. Journal of Applied Phycology. 13, (6) 509-515. Gustavson, K., Wängberg, S.-Å. 1995. Tolerance induction and succession in microalgae communities exposed to copper and atrazine. Aquatic Toxicology. 32, 283-302. Ivorra, N., Hettelaar, J., Tubbing, G.M.J., Kraak, M.H.S., abater, S., Admiraal, W., 1999. Translocation of microbenthic algal assemblages used for in-situ analysis of metal pollution in rivers. Arch. Environ. Contam. Toxicol. 37, 19-28.Kaufmann, K., Chapman, S.J., Oren, A., Steinberger, Y., 2008. Coping with artifacts induced by CaCO3-CO2-H2O equilibria in substrate utilization profiling of calcareous soils. Soil Biology & Biochemistry. 40, (10) 2569-2577. Paulsson, M., Nystrom, B., Blanck, H., 2000. Long-term toxicity of zinc to bacteria and algae in periphyton communities from the river Gota Alv, based on a microcosm study. Aquatic Toxicology. 47, (3-4) 243-257. bacterial communities in meadow and forest ecosystems polluted with heavy metals. European Journal of Soil Biology. 45, (4) 363-369. Tlili, A., Dorigo, U., Montuelle, B., Margoum, C., Carluer, N., Gouy, V., Bouchez, A.,Bérard, A., 2008. Responses of chronically contaminated biofilms to short pulses of diuron -An experimental study simulating flooding events in a small river. Aquatic Toxicology. 87, (4) 252-263.
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Berard, A. (2011)

. Use of the MicroResp (TM) method to assess pollution-induced community tolerance to metals for lotic biofilms. Environmental Pollution, 159 (1), 18-24. DOI : 10.1016/j.envpol.2010.09.033 E Comment citer ce document : Tlili, A., Maréchal, M., Montuelle, B., Volat, B., Dorigo, U., Berard, A. (2011). Use of the MicroResp (TM) method to assess pollution-induced community tolerance to metals for lotic biofilms. Environmental Pollution, 159 (1), 18-24. DOI : 10.1016/j.envpol.2010.09.033 E Chapman, S.J., Campbell, C.D., Artz, R.R.E., 2007. Assessing CLPPs using MicroResp TM -A comparison with biolog and multi-SIR. Journal of Soils and Sediments. 7, (6) 406-410. Clements, W.H., Rohr, J.R., 2009. Community Responses to Contaminants: Using Basic Ecological Principles to Predict Ecotoxicological Effects. Environmental Toxicology and Chemistry. 28, (9) 1789-1800. Creamer, R.E., Bellamy, P., Black, H.I.J., Cameron, C.M., Campbell, C.D., Chamberlain, P., Harris, J., Parekh, N., Pawlett, M., Poskitt, J., Stone, D., Ritz, K., 2009. An inter-laboratory comparison of multi-enzyme and multiple substrate-induced respiration assays to assess method consistency in soil monitoring. Comment citer ce document : Tlili, A., Maréchal, M., Montuelle, B., Volat, B., Dorigo, U., Berard, A. (2011). Use of the MicroResp (TM) method to assess pollution-induced community tolerance to metals for lotic biofilms. Environmental Pollution, 159 (1), 18-24. DOI : 10.1016/j.envpol.2010.09.033 E Comment citer ce document : Tlili, A., Maréchal, M., Montuelle, B., Volat, B., Dorigo, U., Berard, A. (2011). Use of the MicroResp (TM) method to assess pollution-induced community tolerance to metals for lotic biofilms. Environmental Pollution, 159 (1), 18-24. DOI : 10.1016/j.envpol.2010.09.033 E Comment citer ce document : Tlili, A., Maréchal, M., Montuelle, B., Volat, B., Dorigo, U., Berard, A. (2011). Use of the MicroResp (TM) method to assess pollution-induced community tolerance to metals for lotic biofilms. Environmental Pollution, 159 (1), 18-24. DOI : 10.1016/j.envpol.2010.09.033 E Comment citer ce document : Tlili, A., Maréchal, M., Montuelle, B., Volat, B., Dorigo, U., Berard, A. (2011). Use of the MicroResp (TM) method to assess pollution-induced community tolerance to metals for lotic biofilms. Environmental Pollution, 159 (1), 18-24. DOI : 10.1016/j.envpol.2010.09.033 E

Table . 4

 . Version définitive du manuscrit publié dans / Final version of the manuscript published in : EnvironmentalPollution, 2011, Vol.159, no.1, 18-24, DOI: 10.1016/j.envpol.2010.09.033 Version définitive du manuscrit publié dans / Final version of the manuscript published in : EnvironmentalPollution, 2011, Vol.159, no.1, 18-24, DOI: 10.1016/j.envpol.2010.09.033 . EC 50 values (in µM of copper, silver or cadmium) for the three sampling sites (J: Saint-Joseph; V: Les Versauds; E: Saint-Ennemond). C.I. confidence interval (n = 4; α = 0.5).Version définitive du manuscrit publié dans / Final version of the manuscript published in : EnvironmentalPollution, 2011, Vol.159, no.1, 18-24, DOI: 10.1016/j.envpol.2010.09.033 Respiration values (expressed as a percentage of control) after short-term bioassay with different pre-incubation periods ( 0; 0.25; 0.5; 1; 2; 3 ; 4 and 6 h) before addition of the carbon substrate (glucose). Error bars represent standard deviations (n = 4).

		Table. 3. Sensitivity (expressed as EC 50 in µM) of the heterotrophic biofilm community to Fig. 1.
		different tested metals in short-term bioassays using MicroResp TM . C.I. confidence interval
	Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript	(n = 4; α = 0.5)	Tested metal EC 50 µM Silver 3.96 Zinc 82.41 Copper 156.45 Cadmium 194.95 Arsenic 244.46 Nickel 403.53 Iron 772.88 Tested metal Site EC 50 µM Copper J 48.52 V 172.37 133.50 -227.91 C.I. 2.60 -6.27 15.77 -323.18 122.27 -196.44 110.99 -336.18 178.52 -348.43 87.14 -1184.89 392.85 -1532.95 C.I. 41.02 -59.13 E 179.42 117.11 -266.18 Silver J 31.99 25.99 -40.67 V 22.39 17.25 -31.37 E 25.52 19.22 -36.01 Cadmium J 82.48 65.59 -103.61 V 111.15 91.04 -142.36 E 82.48 65.59 -103.61
			Aluminium	2317.86 1515.83 -3731.99
			Cobalt	6170.63 4270.30 -8519.66
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Comment citer ce document : Tlili, A., Maréchal, M., Montuelle, B., Volat, B., Dorigo, U., Berard, A. (2011). Use of the MicroResp (TM) method to assess pollution-induced community tolerance to metals for lotic biofilms. Environmental Pollution, 159 (1), 18-24. DOI : 10.1016/j.envpol.2010.09.033 E Comment citer ce document : Tlili, A., Maréchal, M., Montuelle, B., Volat, B., Dorigo, U., Berard, A. (2011). Use of the MicroResp (TM) method to assess pollution-induced community tolerance to metals for lotic biofilms. Environmental Pollution, 159 (1), 18-24. DOI : 10.1016/j.envpol.2010.09.033 E Comment citer ce document : Tlili, A., Maréchal, M., Montuelle, B., Volat, B., Dorigo, U., Berard, A. (2011). Use of the MicroResp (TM) method to assess pollution-induced community tolerance to metals for lotic biofilms. Environmental Pollution, 159 (1), 18-24. DOI : 10.1016/j.envpol.2010.09.033 E

Acknowledgments

The authors thank G. Sevenier (UMR EMMAH, INRA-UAPV, France) for technical advice on the MicroResp TM technique, M. Coquery, J. Gahou for metal analysis, B. Motte (UR MAEP, CEMAGREF-Lyon, France) for field sampling and ATT for editing the Englishlanguage version of the manuscript. We thank Onema (French National Agency for Water and Aquatic Ecosystems) for financial support (Program 26 "Assessment and Remediation of Pesticide Effects").


Fabrice Theoleyre

Eryk Schiller

Andrzej Duda

Efficient Greedy Geographical Non-Planar Routing with Reactive Deflection

Keywords: greedy geographical routing, detection of voids, packets deflection, wireless mesh networks

We present a novel geographical routing scheme for spontaneous wireless mesh networks. Greedy geographical routing has many advantages, but suffers from packet losses occurring at the border of voids. In this paper, we propose a flexible greedy routing scheme that can be adapted to any variant of geographical routing and works for any connectivity graph, not necessarily Unit Disk Graphs. The idea is to reactively detect voids, backtrack packets, and propagate information on blocked sectors to reduce packet loss. We also propose an extrapolating algorithm to reduce the latency of void discovery and to limit route stretch. Performance evaluation via simulation shows that our modified greedy routing avoids most of packet losses.

I. INTRODUCTION

We consider wireless mesh networks composed of a large number of wireless routers providing connectivity to mobile nodes. They begin to emerge in some regions to provide cheap network connectivity to a community of end users. Usually they grow in a spontaneous way when users or operators add more routers to increase capacity and coverage.

We assume that mesh routers benefit from abundant resources, may only move, quit, or join occasionally, so that the topology of a typical mesh network stays fairly stable. The organization of mesh networks needs to be autonomic, because unlike the current Internet, they cannot rely on highly skilled personnel for configuring, connecting, and running mesh routers. Spontaneous growth of such networks may result in a dense and unplanned topology with some uncovered areas.

Unlike traditional approaches, geographical routing presents interesting properties for spontaneous wireless mesh networks: it does not require any information on the global topology since a node choses the next hop among its neighboring routers on the basis of the destination location. Consequently, the routing scheme is scalable, because it only involves local decisions. Geographical routing is simple, because it does not require routing tables so that there is no overhead for their creation and maintenance. Joining the network is also simple, because a new mesh router only needs an address based on its geographical position. Such addresses can be obtained from a dedicated device (e.g. GPS) or with methods for deriving This work was partially supported by the European Commission project WIP under contract 2740, the French Ministry of Research project AIRNET under contract ANR-05-RNRT-012-01 and ARESA under contract ANR-05-RNRT-01703. consistent location addresses [START_REF] Capkun | Gps-free positioning in mobile ad hoc networks[END_REF], [START_REF] Rao | Geographic routing without location information[END_REF]. The most familiar variant of geographical routing is greedy forwarding in which a node forwards a packet to the neighbor closest to the destination [START_REF] Bose | Routing with guaranteed delivery in ad hoc wireless networks[END_REF], [START_REF] Karp | Greedy perimeter stateless routing for wireless networks[END_REF]. Greedy forwarding guarantees loop-free operation, but packets may be dropped at blocked nodes that have only neighbors in the backward direction. Blocked nodes appear at some places near uncovered areas (voids) or close to obstacles.

Our main contribution is to propose a new greedy routing that correctly deals with voids. First, we define a new mechanism to reactively detect voids and surround them, which significantly reduces packet loss. The information of detected voids propagates backwards so that subsequent packets to the same direction benefit from this reactive detection. Second, we propose a mechanism in which voids deviate packets and shorten the length of a route compared to classical approaches. Our routing scheme works in any network topology independently of whether it corresponds to a planar graph or not.

We start by describing the related work on geographical routing in Section II. Section III presents the details of the proposed new greedy routing protocol. Then, we evaluate its performance via simulation in Section IV and conclude. The reader can read [START_REF] Theoleyre | Efficient greedy geographical non-planar routing with reactive deflection[END_REF] for a more complete version of this article.

II. RELATED WORK

Geographic information can largely reduce complexity of routing in spontaneous mesh networks. The most simple and widely used protocol is greedy geographic routing [START_REF] Bose | Routing with guaranteed delivery in ad hoc wireless networks[END_REF], [START_REF] Carlsson | Non-euclidian geographic routing in wireless networks[END_REF], [START_REF] He | A novel geographic routing algorithm for ad hoc networks based on localized delaunay triangulation[END_REF], [START_REF] Casari | Efficient non planar routing around dead-ends in sparse topologies using random forwarding[END_REF]: when a node receives a packet, it uses the following forwarding rule:

"forward the packet to the node with the best improvement". Improvement is usually defined with respect to the distance towards the destination. Since improvement is not negative, there is no routing loop and the algorithm is scalable because the decision is local.

We assume that the nodes either have a dedicated localization device (e.g. GPS) or are able to estimate their positions distributively (e.g. [START_REF] Capkun | Gps-free positioning in mobile ad hoc networks[END_REF], [START_REF] Niculescu | Ad hoc positioning system (APS) using AOA[END_REF] that use respectively triangulation and angle of arrival). A pragmatic approach to this problem is to assume that a subset of mesh routers know their exact positions via GPS devices and other nodes can compute their positions with respect to its neighbors [START_REF] Bulusu | Adaptive beacon placement[END_REF].

The main drawback of greedy geographical routing is packet loss at blocked nodes near voids or obstacles. A node must drop a packet when the improvement associated with any of its neighbors is negative (cf. figure 1). In face routing the left-hand rule [START_REF] Karp | Greedy perimeter stateless routing for wireless networks[END_REF] tries to go around a void, but it requires the connectivity graph of nodes to be planar. Relative Neighborhood Graphs can yield planar graphs for Unit Disk Graphs (UDG) [START_REF] Cartigny | Localized LMST and RNG based minimum-energy broadcast protocols in ad hoc networks[END_REF], but in real wireless environments, the conditions for obtaining planar graphs are not satisfied due to asymmetric links and not circular radio coverage [START_REF] Kim | Lazy cross-link removal for geographic routing[END_REF]. To the best of our knowledge, there is no efficient and localized planarization algorithm proposed for a general connectivity graph. A possible solution to this problem is the following method: a border node initiates local flooding to find the next hop closer to the destination [START_REF] Stojmenovic | Loop-free hybrid single path flooding routing algorithms with guaranteed delivery for wireless networks[END_REF]. However, it results in long delays and significant overhead. Fotopoulou et al. [START_REF] Fotopoulou-Prigipa | GCRP: Geographic virtual circuit routing protocol for ad hoc networks[END_REF] propose to adapt this method to establish and maintain a virtual circuit. Funke et al. [START_REF] Funke | Topological hole detection in wireless sensor networks and its applications[END_REF] propose an algorithm inspired by topological geometry to discover the void limits, i.e. the border nodes by creating isosets. However, this method does not work for non UDG since isosets are not rings in general connectivity graphs.

We propose here a generic method to deal with voids by backtracking packets and discovering blocked areas near voids: deflection routing deviates packets outside them. Thus, the algorithm presented here is perfectly supplementary to existing methods: deflection improves greedy routing by trying to surround voids and any other technique presented above can be used when a void is reached.

III. REACTIVE DEFLECTION

Geographical routing is attractive for mesh networks, but suffers from two main drawbacks: blocked nodes can drop many packets and the route length may drastically increase when a surrounding mechanism tries to deviate a packet around a void (e.g. the left-hand rule in UDG). In this paper, we assume a general connectivity graph and propose to reactively detect blocked nodes and locally advertise blocked sectors to avoid packet losses. Such a technique is efficient in any type of networks and graphs since it does not assume any particular graph property.

Detection of blocked nodes can be done in a proactive way: locally flood information to detect voids. For example, we can discover the topology of the wireless mesh to detect elementary cycles in which no other node is located inside the ring. The location of nodes helps to surround voids. However, such an approach requires a complete knowledge of the mesh topology and is computationally intensive. In opposition to this approach, we have chosen a reactive method: a node becomes blocked with respect to a given destination when it cannot forward a packet to any neighbor closer to the destination. Hence, the part of the network not concerned by forwarding this packet does not generate any control traffic so that this approach is more scalable.

Let us first adopt the following notation: In our approach, a node chooses a neighbor closer to the destination and not blocked for this direction. If a node fails to forward a packet to a given destination, it will consider itself as blocked for this direction. It will advertise backwards a list of blocked directions so that its neighbors will not choose it as a next hop for these directions. If several non blocked neighbors exist, the forwarder chooses the neighbor closest to the destination, i.e. with the best improvement.

For advertising blocked directions, we propose to use the notion of blocked sectors: a node N advertises that it is blocked for any destination that falls in sector S (N, angle min , angle max , dist min ). Let us consider the topology illustrated in Figure 2. Node n 1 wants to forward a packet to destination d and it discovers that it is blocked for this destination since no neighbor exists in this direction. Thus, it backwards the packet to s with its two blocked sectors. Based on this information, s marks n 1 as blocked and forwards the packet to another neighbor closer to d (node n 2 in this case).

To limit the overhead, a node tries to merge all its blocked sectors before advertising them. It can only merge overlapping sectors having the same minimal distances (within some tolerance ∆ d ). Otherwise, the merged blocked sector may include nodes that are reachable-consider for instance the topology of figure 2: if node p merges sectors C and C , node p 1 may appear in the blocked sector. Thus, it would become unreachable from p 2 . Clearly, we must avoid such a merging. Only sectors with the same d min will be merged : tolerance ∆ d allows some merging of sectors with approximately equal minimal distances.

More formally, node N executes Algorithm 1. Procedure ReactiveDef lection() finds the next hop for forwarding a packet to destination D: the next hop must be closer to the destination and must be unblocked for D. If it does not return any node, it means that node N becomes blocked for destination D (variable BLOCKED(N,D) becomes true). Thus, node N updates its blocked sectors and sends the packet backwards to the previous hop with its list of blocked sectors piggybacked onto the packet. This scheme is loop-free: when a node sends a packet backwards, the receiver will update its blocked sectors and it cannot choose the same next hop for subsequent packets, because Algorithm 1 does not forward packets to blocked nodes. In networks with non UDG topologies, when a node does not have any neighbor closer to the destination, it is blocked. Such a border node has to discover a virtual next hop in a larger vicinity [START_REF] Bose | Routing with guaranteed delivery in ad hoc wireless networks[END_REF], [START_REF] Fotopoulou-Prigipa | GCRP: Geographic virtual circuit routing protocol for ad hoc networks[END_REF]. A border node can for example flood at a fixed distance to discover a node closer to the destination. However, the packets still keep on being forwarded until they reach these blocked nodes, even if the packets must after that turn back to surround a void.

Note that when we reduce packet loss with the previously described algorithm, we also reduce in the long term the route length. Indeed, the nodes around the void discover that they have blocked sectors. When they propagate the information about blocked sectors, nodes with all blocked neighbors also become blocked for this destination. Finally, each node discovers a blocked area and forwards packets outside this area. However, we need several useless packet transmissions and backtracking before the network converges, and blocked sectors are correctly constructed. We propose a mechanism to accelerate the convergence of this propagation process by extrapolating the location of a blocked area.

We propose to detect the border of a void based on only local neighborhood knowledge. We will show that even if a node has only local knowledge, i.e. about nodes at a limited distance, voids can be efficiently surrounded. When a node must transmit a packet backwards, it locally floods an hello packet containing the list of its neighbors and blocked sectors in a k hop scope.

To detect the border of a void, node N first searches for the blocked k-neighbor closest to the direction of the destination D, i.e. minimizing angle ∠((N, D), (N, BN )) for all blocked nodes BN . Then, N constructs the Maximum Connected Set of blocked nodes that contains BN : it adds BN to this set, and recursively adds all its blocked neighbors. Finally, N computes the forbidden sector that spans the maximum connected setit extrapolates the blocked area.

Figure 3 illustrates void detection with the knowledge of the 3-neighborhood topology. First, the source node detects if it knows a node with a blocked direction and takes the closest one to the direction of the destination. In the example the blocked node is B. Then, the source constructs the connected set of blocked nodes that includes node B: it obtains set {A, B, C, D}. Obviously, node F is not present in the set since it is not connected to A via other blocked nodes. In the same way, border node H is not in set {A, B, C, D}, because it is 2 hops away: it is border to another void. Finally, we obtain the forbidden sector for the destination. We can note that node E is not blocked since it can choose node F when A is blocked: E will never be blocked for the direction.

Algorithm 2 presents the modified protocol. Function ISIN-FORBIDDENSECTOR computes the forbidden sector and returns TRUE, if the node is located inside this sector. Function CLOSERTOSECTORLIMITS(P,Q) returns TRUE, if P is closer than Q to the forbidden sector limits.

In other words, if some next hops exist and do not lie in the computed forbidden sector, we choose the best one. Otherwise, if all possible next hops are in this forbidden sector, we choose the node closest to the limits of the forbidden sector. With this modified routing scheme, we forward packets outside the forbidden sector, because a void appears as something repellent to packets by creating forbidden sectors in a distributed manner while keeping routing loop-free.

IV. PERFORMANCE EVALUATION

We generated random meshes of 1000 nodes with two models: UDG [START_REF] Clark | Unit disks graphs[END_REF] and what we call a proxi-graph (a graph based on proximity). In a proxi-graph, each node chooses a radio range following a Gaussian distribution centered at 1 with standard deviation Std depending on the radio range (here, Std = 25% • (radio -range)). The proxi-graph owns a rectangular central void of size two fifth of the simulation disk radius. Besides, we discard disconnected topologies and use a disk simulation area to reduce border effects. Data traffic consists of 1,000 flows of 10 packets each from a random source to a random destination. A node advertises its neighborhood and blocked sectors 3 hops far since it achieves the best performances, as highlighted below. We evaluated the properties of routing itself: we assume ideal radio and MAC layers, i.e. packets do not experience any loss due to channel or MAC behavior to only test routing properties. We also simulated this protocol with ns2 to take into account more realistic radio conditions, but do not report here these results because of lack of space. The reader can refer to [START_REF] Theoleyre | Efficient greedy geographical non-planar routing with reactive deflection[END_REF] for a more exhaustive view of all the results.

We compare our routing algorithm with greedy geographic routing to quantify the reduction of packet loss: a node chooses the neighbor closest to the destination (other variants that minimize the angle difference, or the hop length do not exhibit better results). We measured packet losses (the proportion of packets sent by a source that never reach the destination), route length (the average route length in hops for the delivered packets), and stretch factor (the average ratio of the route length for a packet and the length of the shortest route for the associated source/destination pair). We plot the average values and the associated 95% confidence intervals.

A. Performance for Unit Disk Graphs

We first measure the route length obtained for deflection routing with different values of the k-neighborhood with density of 8 neighbors per node in Unit Disk Graphs. For densities of (1,2,3,4,5) neighbors, we obtain route lengths of respectively (33. 6, 33, 32.7, 33.2, 33.6) hops. Thus, we chose k = 3 since it provides an accurate route length and limits the overhead. Besides, for large k values, deflection routing tends to overestimate the size of voids and increase the route length.

Then, the following experiment shows (fig. 4(a)) that packet loss for greedy routing decreases with the increase of density: probability of having a large area without any node decreases so that voids are less probable to appear. However, more than 70% of packets are lost for low density. On the contrary, the proposed routing scheme lowers packet loss: almost no packet is lost (less than 4%) when density exceeds a small threshold (8 neighbors per node). Thus, nodes reroute less packets by means of reactive discovery so that the overhead is lower and delay improved. We can also notice that route length optimization has no impact on delivery ratio.

We have also measured the route length stretch factor (fig. 4(b)). Greedy routing drops many packets when voids exist. Moreover, the packet drop probability is larger when the destination is farther. Since the route length is only measured for delivered packets, a poor delivery ratio creates mechanically a lower average route length. We can observe our optimized algorithm succeeds in slightly reducing the route length. More importantly, route length optimization results in deviating packets from voids and decreasing the load on the void's borders. The stretch factor is larger for low density since more voids are present and packets must be deflected and backtracked more often. The reader can note that greedy routing forwards one packet until it reaches a blocked node: this increases the load on these block nodes, even if they implement a void bypassing. Finally, the route length is reduced only for very low densities with the optimized version of deflection routing since blocked sectors interpolating is working only when voids are sufficiently large. Moreover, we could over-estimate the forbidden sector by adding a guarding angle around the blocked nodes: we would reduce the route length, but increase packet drops since we would over-estimate the presence of voids.

B. Performance for a proxi-graph with one rectangular void

We have evaluated packet loss rate in a proxi-graph with one central void (fig. 5(a)). We can see that packet loss increases compared to UDG, particularly for high density: dead-ends are more probable. Moreover, since the graph is not UDG, a node may choose a next hop in a greedy way although it does not have any neighbor in the direction of the destination. Thus, to increase density it is not sufficient to surround voids. We can remark that with our algorithms we significantly reduce packet loss ratio.

Finally, we have measured the route length (fig. 5(b)). We can remark the same trend as for UDG and for a proxi-graph with a void. Obviously, the route length is longer, because packets have to surround the rectangular void.

V. CONCLUSION

We have proposed a scheme for greedy geographical routing with reactive defect detection. The idea is to reactively detect blocked nodes and propagate the defect information by computing a set of blocked sectors. To reduce the route length and accelerate void detection in dense mesh networks, we have also proposed a method to extrapolate void location. Simulation results show packet loss as well as the route length decrease compared to greedy routing.

Fig. 1 .

 1 Fig. 1. Blocked nodes in greedy geographical routing.

Fig. 2 .

 2 Fig. 2. Examples of blocked nodes and blocked sectors.

•

  d(A, B): the Euclidean distance between the geographical coordinates of nodes A and B • ∠(AB, AC): the oriented angle between two coordinates of nodes (A, B) and (A, C) (by convention, we denote by ∠(AB) the normalized angle ∠( 1 0 , AB) ) • S (S, α, β, d min ): the sector of node S composed of all nodes N such that α ≤∠(SN )≤ β and such that d(S, N ) ≥ d min

Algorithm 1 7 :

 17 ReactiveDeflection(N→D) 1: next ← ∅ 2: for all n ∈ N eighbors do 3: if d(n, D) < d(N, D) and !BLOCKED(n,D) and d(n, D) < d(next, D) then if next = ∅ then 8: Blocked(N,D) ← true next ← previous hop 9: end if 10: return next

Fig. 3 .

 3 Fig. 3. Method for locally detecting the border of a void.

Algorithm 2 5 :← n 6 : 11 :

 25611 ModifiedReactiveDeflection(D,ForbiddenSector) 1: next ← ∅ 2: for all n ∈ N eighbors do 3: if d(n, D) < d(S, D) and !BLOCKED(n,D) then 4: if !ISINFORBIDDENSECTOR(n) and { ISINFORBID-DENSECTOR(next) or d(n, D) < d(next, D) } then next else if ISINFORBIDDENSECTOR(next) and ISIN-FORBIDDENSECTOR(n) and CLOSERTOSECTOR-LIMITS(n,next) then if next = ∅ then 12: Blocked(N,D) ← true next ← previous hop 13: end if 14: return next

Fig. 4 .

 4 Fig. 4. Performances under different routing schemes for UDG.

Fig. 5 .

 5 Fig. 5. Performances under different routing schemes for a proxi-graph with one central void.


Mohammad Nassiri
email: nassiri@imag.fr

Fabrice Theoleyre
email: theoleyr@imag.fr

Martin Heusse
email: heusse@imag.fr

Andrzej Duda
email: duda@imag.fr

Molecular Architecture for Autonomic Wireless Mesh Networks

INTRODUCTION

We consider wireless mesh networks that use 802.11 wireless LANs for interconnecting mesh routers. When mesh routers use only one network interface and a single radio channel, performance of packet forwarding quickly degrades with the number of hops due to channel contention and spatial problems such as hidden, exposed, masked, and blocked nodes. We observe that capacity of a wireless mesh network strongly depends on the ability of nearby mesh routers to communicate in parallel, which is only possible if the neighbor routers that may interfere use di erent channels.

The goal of this paper is to propose a mesh architecture for autonomic assignment and use of multiple channels to improve network capacity. We seek an autonomous and distributed way of operation-mesh routers organize themselves to form a desired mesh topology and take specific roles to achieve high performance. The architecture is coupled with an e⇤cient mechanism for packet forwarding over multiple wireless links that takes advantage of such a mesh network. We adopt a molecular analogy for defining our architecture.

MOLECULAR ARCHITECTURE

Our model assumes that each mesh router has only one wireless network interface that can switch channels. However we can easily generalize it to support mesh routers with multiple interfaces.

We adopt a molecular analogy for defining our architecture (cf. Figure 1). An atom is a basic entity from which we construct a mesh network. It is composed of a nucleus, a mesh router that uses a fixed channel for communicating with electrons, its immediate neighbors.

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CoNEXT' 07, December 10-13, 2007, New York, NY, U.S.A.

Copyright 2007 ACM 978-1-59593-770-4/ 07/ 0012 ...$5.00. Atoms bond together to form a molecule, which corresponds to a connected wireless mesh network. Electrons belong to adjacent atoms and communicate with their nuclei. There is no direct link between two electrons nor between two nuclei, e.g. mesh routers N and M in Figure 1 are nuclei of two atoms bonded by two mesh routers corresponding to electrons B and C.

There is a possible contention in an atom between entities that use the same channel (e.g. N, A, B, and C). Mesh routers can use any suitable MAC access method to control contention in an atom such as the standard IEEE DCF or any of its proposed optimizations.

To interconnect atoms and achieve high capacity, electrons switch between channels used by neighbor nuclei so that packets going through adjacent atoms benefit from parallel communications. During the construction of the mesh, a nucleus dynamically chooses its channel depending on the channels used in its neighborhood by other atoms. By choosing di erent channels, two neighbor atoms that may interfere when using the same channel limit interference problems, which contributes to achieving high capacity.

When neighbor nodes are tuned to possibly di erent channels, the problem of deafness arises: a node is trying to send its frame on one channel and its receiver is listening to another one. In our architecture, a nucleus node chooses its channel and lets it know to all electrons in its vicinity. The nucleus always stays on this channel: no deafness is possible. We define a molecular frame forwarding mechanism for communication between a nucleus and an electron that takes advantage of di erent channels used in adjacent atoms: an electron sends a CTS control frame to pull a frame-it tells a nucleus that it is ready to receive a frame on the nucleus channel. As it is up to an electron to pull a frame, an electron cannot be deaf, but the nucleus needs to notify its electrons that there are pending frames for them. To achieve this, the nucleus piggybacks the information on the destinations of pending frames onto data, ACK, and beacon frames. One important issue is to construct the mesh molecules. We want a mesh molecule composed of interconnected atoms with one nucleus per atom. Interconnection of adjacent atoms relies on one or more electrons common to two adjacent atoms with nuclei 2 hops apart. The molecular mesh structure may lead to the elimination of some links (e.g. to avoid a direct link between two electrons or two nuclei). At the same time, we want the resulting mesh molecule to remain connected. This kind of structure corresponds to the Independent Sets and more precisely to the Weakly Connected Dominating Set (WCDS) [START_REF] Dunbar | On Weakly Connected Domination in Graphs[END_REF]. We construct our mesh molecule in a distributed and localized manner.

The proper operation of a mesh molecule requires the knowledge of neighbor nodes: their state (nucleus or electron) and the channel to use for communication. As neighbor nodes may belong to di erent atoms and use di erent channels to transmit data, we define a multichannel neighbor discovery protocol.

Frame Forwarding Mechanism

Figure 2 illustrates the behavior of our mechanism for frame forwarding. When A (an electron) has a frame to send to N (a nucleus), it accesses the channel conforming to the channel access method and transmits the frame on the channel used by the nucleus.The transmission does not su er from deafness, because the nucleus always listens to the chosen channel. By piggybacking the information on the frame destinations present in its bu er (B, another electron) onto the data and ACK frames, N notifies B at instant t 2 that there is a pending frame to receive. Thus, B stays on the channel used by N, requests the frame from N, and receives it. Then, at instant t 3 , B forwards the packet contained in the frame to M using the channel chosen by M (channel 2).

When M is further forwarding the packet on channel 2, B finds out that it is not involved in this transmission: it may change channels at instant t 5 to receive notifications from its other nucleus at instant t 6 .

PRELIMINARY RESULTS

We provide some preliminary results on performance of our molecular mesh network. We can compare packet forwarding performance over a chain topology, which is a first test case for mesh networks. Figure 3 compares the expected performance of 802.11a DCF in a traditional multi-hop ad hoc wireless networks [START_REF] Chaudet | Performance Issues with IEEE 802.11 in Ad Hoc Networking[END_REF] with SSCH [START_REF] Bahl | SSCH: Slotted Seeded Channel Hopping For Capacity Improvement in IEEE 802.11 Ad Hoc Wireless Networks[END_REF] and our proposal. The throughput values for 802.11a and SSCH are extracted from the SSCH proposal [START_REF] Bahl | SSCH: Slotted Seeded Channel Hopping For Capacity Improvement in IEEE 802.11 Ad Hoc Wireless Networks[END_REF] that considers a chain of several nodes. Our results come from the following observation. In the chain configuration, packets go through several atoms interconnected by an electron. Packet forwarding benefits from parallel communication in adjacent atoms: in one atom, the nuclei and the electron share the same channel so the resulting throughput will be the half of the throughput of a single hop. In di erent atoms, frames use di erent channels so they are forwarded in parallel. There is also a slight overhead related to the use of the CTS control frame.

Figure 1 :

 1 Figure 1: Two atoms sharing two electrons

Figure 2 :

 2 Figure 2: Frame exchanges during packet forwarding in a chain.

Figure 3 :

 3 Figure 3: Throughput for di erent forwarding schemes.