Johnny Gasperi
email: gasperi@univ-paris12.fr

Vincent Rocher

Solène Gilbert

Sam Azimi

OCCURRENCE AND REMOVAL OF PRIORITY POLLUTANTS BY LAMELLA CLARIFICATION AND BIOFILTRATION

Keywords: Lamella clarifier, biofiltration, priority pollutants, Water Framework Directive

This study investigates the occurrence of all priority substances (n = 41) listed in the Water Framework Directive and additional substances (n = 47) in raw sewage, as well as the removal performance of lamella clarification and biofiltration techniques. Once the efficiency of both types of techniques has been assessed for typical wastewater parameters, the differences in each technique's ability to remove pollutants becomes obvious; nevertheless, pollutant removal in quantitative terms still depends on the physico-chemical properties of the compounds used and operating conditions within the selected facility. For lamella clarification, the removal of organic chemicals was found to be primarily correlated with their sorption potential and, hence, strongly dependent upon log K ow of the compound under study.

Compounds with a strong hydrophobic character (log K ow > 4.5) are removed to a significant extent (approx. 85%), while hydrophilic compounds (log K ow < 3.5) are poorly removed (< 20%). For biofiltration, the removal of chemicals appears to be compound-dependent, although this outcome involves several mechanisms, namely: i) physical filtration of total suspended solids, ii) volatilisation, iii) adsorption on biomass, and iv) biotransformation of substances. Even if the complex processes within a biofilter system do not yield an accurate prediction of pollutant removal, two groups of chemicals can still be clearly identified: i) hydrophobic or volatile compounds, for which moderate to high removal rates are observed (from 50% to over 80%); and ii) hydrophilic, non-volatile and refractory compounds for which a low removal rate would be expected (< 20%).

Introduction

The European Community's strategy to combat surface water pollution by means of adopting a control policy was set forth in the European Water Framework Directive 2000/60/EC (WFD, Decision No. 2455/2001/EC). This directive offered the first list of 33 substances or groups of substances to be identified as action priorities at the Community level and required EU Member States to ensure a "satisfactory chemical and biological status for surface waters" by 2015. This proposal has therefore mandated a gradual reduction in emissions, losses and discharges of all priority substances, along with a phase-out or cessation of emissions, losses and discharges of priority hazardous substances over a 20-year period.

In pursuit of the WFD objectives, several steps have been taken to considerably reduce stormwater pollution (storage tanks, stormwater treatment facilities, etc.) and improve the efficiency of wastewater treatment technologies. On the one hand, these improvements have led to a significant decrease of carbonaceous and nutriment pollution in receiving waters, while on the other these improvements have played an important role in minimising the release of xenobiotics into the aquatic environment. Regarding this point, among the various wastewater technologies available, conventional activated sludge wastewater treatment plants (AS-WWTPs) are the most well-documented. Numerous studies have already been conducted on the fate of certain priority substances or emerging pollutants in AS-WWTPs [START_REF] Fauser | Phthalates, nonylphenols and LAS in an alternately operated wastewater treatment plant--fate modelling based on measured concentrations in wastewater and sludge[END_REF][START_REF] Jacobsen | Combined chemical analyses and biomonitoring at Avedoere wastewater treatment plant in 2002[END_REF][START_REF] Katsoyiannis | Persistent organic pollutants (POPS) in the sewage treatment plant of Thessaloniki, northern Greece: occurrence and removal[END_REF][START_REF] Vogelsang | Occurrence and removal of selected organic micropollutants at mechanical, chemical and advanced wastewater treatment plants in Norway[END_REF][START_REF] Clara | Removal of selected pharmaceuticals, fragrances and endocrine disrupting compounds in a membrane bioreactor and conventional wastewater treatment plants[END_REF]. Despite the contribution of this experimental work, data are currently lacking on the removal of WFD priority pollutants using more compact wastewater treatment technologies, which have now become widely implemented in modern WWTPs. Among such technologies, lamella clarification and biofiltration (aerated or non-aerated biological filters) are particularly attractive.

Lamella clarification uses a chemical additive followed by flocculation and a lamellar clarifier. A coagulant (causing destabilisation of colloidal particles) and polymer (promoting floc formation) are then injected into the influent wastewater prior to entering the flocculation zone. Since this technique is operated more compactly and contains less exposed surface area, lamella clarification is considered an essential process in wastewater treatment and is widely applied to provide advanced primary treatment, in addition to producing a highly-clarified effluent [START_REF] Tchobanoglous | Wastewater Engineering. Treatment and Reuse[END_REF]. This type of method can also serve to treat wet weather flows or combined sewer overflows.

The biofiltration technique has been derived from drinking water production filters and combines physical and biological purification processes using an immersed filtration material (aerated or not, depending on the desired treatment), onto which the bacteria populations ready for pollution abatement will settle. The benefits of these immersed biological systems lie in their compactness (small site encumbrance), modularity (treatment procedure can be adapted to match the wastewater flow the plant is able to accommodate) and intensiveness (short hydraulic retention time). As a result of these advantages, biofilters have become an alternative to activated sludge tanks and are perfectly suited for treatment plants built in large urban areas, where real estate development pressures make available land scarce.

Though both types of techniques are implemented in modern WWTPs and despite them being highly recommended and increasingly popular for stormwater management, only limited data are presently available on the removal of all WFD priority substances through application of such techniques. Most studies in the literature focus on a group of substances or on performance in removing carbonaceous and nutriment pollutants [START_REF] Mendoza-Espinosa | A review of biological aerated filters (BAFs) for wastewater treatment[END_REF][START_REF] Chang | Pre-denitrification and post-nitrification in an integrated anaerobic/aerobic filter system for advanced treatment of municipal wastewater[END_REF][START_REF] Imasuen | High-rate clarification of municipal wastewaters: a brief appraisal[END_REF][START_REF] Rocher | Le traitement des eaux usées par biofiltration : cas de la station Seine Centre (SIAAP)[END_REF]2008).

This study was therefore launched as part of the OPUR (Observatory of Urban Pollutants in Paris) research programme, with the objective of examining the removal of all priority pollutants and additional compounds by means of lamella clarification and biofiltration. For this purpose, the Seine Centre WWTP, which is localized in the Parisian suburban and combines both technologies, was selected during a total of three sampling campaigns. A total of 88 substances (ranked into 13 groups of compounds) were determined in raw sewage as well as at particular points within the WWTP. The distribution of pollutants between the dissolved and sorbed phases of wastewater was also investigated.

Materials and methods

Wastewater treatment plant description

The Seine Centre plant receives some 240,000 m 3 .d -1 of wastewater. The treatment process comprises: screening, grit removal, primary sedimentation using coagulant and flocculant, followed by biofiltration units (Fig. 1). Raw sewage is initially pre-treated; this stage includes screening and grit/oil removal, thus allowing the removal of coarse floating solids, sand and some grease components. Following this pre-treatment, the wastewater undergoes physical and chemical treatment, which traps a large amount of particles and phosphorus. This step is performed by lamellar settling (use of 9 settling tanks -Densadeg®), with the addition of both a coagulant (ferric chloride) and flocculant (anionic polymer).

After primary treatment, a biological treatment takes place over 3 stages of biofilters.

The first stage (24 Biofor®-type filters with biolite as the medium) is aerated and mainly serves to remove carbonaceous pollution. The second stage (29 Biostyr®-type filters with biostyrene as the medium) is also aerated and allows for the nitrification of ammoniacal pollution. Lastly, the denitrification step occurs during stage 3 (12 Biofor®-type filters), which is not aerated. Denitrification requires adding methanol, which acts as an exogenous carbonaceous substrate. Once this biological treatment has been completed, effluents are discharged into the Seine River.

Experimental procedure

During this study, 3 sampling points, corresponding to raw sewage (RS), decanted effluents (DE) and final effluents (FE), were considered (Fig. 1). In 2008, three sampling campaigns were carried out (March, September and December). At each site, 24-h composite samples were collected using automatic refrigerated samplers (at 4°C). To avoid problems associated with sample contamination and/or pollutant adsorption during sampling, the samplers were equipped with glass bottles and Teflon® pipes. In accordance with the constraint to analyse 88 substances on the particulate material (i.e. from 0.2 to 2.0 g of particles were required), large volumes had to be collected (10 l for RS, 30 l for DE and FE), and the sampling campaigns had to be performed on three consecutive days. Each day, 3 or 4 groups of compounds were analysed. To avoid any modification of pollutant distribution between the dissolved and particulate phases, samples were filtered as soon as possible on a 0.45-µm filter. After filtration, the dissolved phase was quickly sent to the laboratory for analysis. As for the particulate material, filters were first lyophilised and then sent to the laboratory.

Based on the WFD list, 41 individual substances were initially considered. Depending on the chemical group, 1 to 13 additional substances were also included (Table 1), thereby accounting for 88 compounds. Except for the metals, halogenated volatile organic compounds (HVOCs) and BTEX (benzene, toluene, ethylbenzene and xylenes), which were analysed on the total fraction, the dissolved and particulate fractions were assessed for each individual compound. Analysis work was performed at the IPL-Bretagne laboratory, certified by France's Environment Ministry (via the COFRAC accreditation committee).

Results and discussion

Priority pollutants in raw sewage

Of the 88 molecules targeted, 51 (including 18 substances listed in the WFD) were detected in RS. All non-detected compounds along with their limits of detection (LOD) have been listed in the Supplementary Material section (Table 1). Among the pollutants detected, 12 substances (7 pesticides, mono-and di-butyl tin, polychlorobiphenyls 183 and 153, and ethylbenzene) were highlighted since they could all be detected once during the three campaigns performed, with concentrations for these elements lying at or close to the LOD.

In all, 39 substances were thus selected to study chemical removal using the lamella clarification method. Total concentrations, as well as their respective LOD, are reported in Fig. 2. The array of molecules detected and corresponding concentration ranges are typically representative of data found in the literature on wastewater (Gasperi et al., 2008a in France, or Rule et al., 2006a andb in the United Kingdom). As regards contaminant levels, three groups of chemicals can be distinguished.

The first group includes metals and di(2-ethylhexyl) phthalate (DEHP); it displays the highest concentrations (10-300 µg.l -1 ). Metal concentrations lie between 180 and 260 µg.l -1 for Zn, between 70 and 90 µg.l -1 for Cu, and 13-15 µg.l -1 for Pb. Like for DEHP, the concentration is bound within the 10-25 µg.l -1 range. The presence of these substances stems from their extensive use both throughout industry and in household products. Metals are currently used as chemical additives in a wide variety of consumer products or input into a number of metal finishing processes (Rule et al., 2006a and b). Similarly, DEHP is widely used as an additive in plastics [START_REF] Wams | Diethylhexylphthalate as an environmental contaminant -a review[END_REF].

The second group (6 volatile compounds, nonylphenol -NP, and amino methyl phosphonic acid -AMPA) contains total concentrations in the 1.0-6.0 µg.l -1 range. Due to their excellent degreasing properties, the HVOCs and BTEX presence can certainly be related to their widespread use as a degreasing agent and solvent. For example, tetrachloroethylene is introduced primarily for dry cleaning and metal degreasing purposes yet can also be found in numerous household products (CEPA, 1993). The presence of NP (1.0-1.7 µg.l -1 ) results mainly from the degradation of alkylphenol polyethoxylates, which are extensively used as non-ionic surfactants in many industrial, commercial and laboratory detergents and industrial processes [START_REF] Ying | Environmental fate of alkylphenols and alkylphenol ethoxylates--a review[END_REF]; this presence might also be correlated with the direct application of alkylphenols as plasticisers in plastics. Lastly, AMPA has been observed in RS (< 0.03-1.5 µg.l -1 ). This compound, generally considered to be a primary degradation product of glyphosate (Rueppel et al., 1977), was more frequently detected in RS and with higher concentrations than glyphosate (< 0.03-0.08 µg.l -1 ). According to [START_REF] Skark | The occurrence of glyphosate in surface water[END_REF], AMPA may also be formed by the degradation of phosphonic acids in detergents.

The final group includes 26 substances and yields the lowest total concentrations (between 0.005 and 0.5 µg.l -1 ). Polycyclic aromatic hydrocarbons (PAHs), a number of pesticides (diuron and, to a lesser extent, chlorpyrifos, isoproturon, metaldehyde and atrazine) and tributyl tin (TBT) have been observed. These substances are frequently detected in wastewater, as previously cited by [START_REF] Gasperi | Review on the hydrocarbon fate within combined sewers: Case of the "Le Marais" urban catchment (1994-2005)[END_REF]2008a) for Paris wastewater or by Thomaidis et al. (2006) for TBT in Greek wastewater. Of the 14 pesticides investigated, only diuron was actually detected in all samples, with concentrations ranging from 0.03 to 0.16 µg.l -1 . This marked presence in Paris wastewater is attributed to the city's extensive use, given that this pesticide accounts for a significant proportion of the total contribution from public works agencies [START_REF] Blanchoud | Contribution by urban and agricultural pesticide uses to water contamination at the scale of the Marne watershed[END_REF]. Moreover, the presence of TBT can likely be explained by its use as one of the main active ingredients in biocides for controlling a broad spectrum of organisms (wood treatment, antifungal action in textiles, and industrial water systems).

Since this section only discusses total concentration values, more detailed information on the partitioning of chemicals is available in the Supplementary Material section (Table 2).

Pollutant removal during wastewater treatment

This study is aimed at examining the removal of priority pollutants by means of clarification and biofiltration. Prior to assessing pollutant removal levels, some precise knowledge is required for removing conventional pollutants. Consequently, the efficiency of both techniques was examined as an initial step for typical wastewater parameters, such as total suspended solids (TSS), chemical and biochemical oxygen demand (COD and BOD), ammonium (NH 4 + ), total Kjeldahl nitrogen (TKN), total phosphorus (P tot ) and orthophosphates (PO 4

3-

). Data obtained in 2008 at the Seine Centre plant were processed; all analyses were conducted on 24-h composite samples at similar points in the plant.

Lamella clarifier

Conventional wastewater parameters

The removal rates (in %) of conventional wastewater parameters (TSS, total and dissolved COD -CODtot and CODs, BOD, TKN, P tot ) with clarification are illustrated in Fig. 3 (the box plot illustration indicates the median, d25 and d75 percentile removal rates). In addition, median concentrations ± standard deviations (SD) are shown for RS and DE.

First of all, Fig. 3 displays the high efficiency of lamella clarifier for TSS and total COD pollution. For these elements, median removal rates were evaluated at 86% for TSS and 63% for CODtot, giving rise to DE concentrations of 34 ± 10 mg.l -1 and 166 ± 31 mg.l -1 , respectively. No seasonal variations or temperature influence was remarked, and the decanted effluent concentrations appeared not to be influenced by the quality of raw sewage entering the facility. This trend has resulted primarily from adapting clarification operating conditions in accordance with the quality of influent, by adjusting the ferric chloride (25 and 40 g.m -3 ) and polymer doses (0.3 and 0.45 g.m -3 ). BOD was also removed to a great extent by clarification (63% removal rate, with a concentration in DE at 69 ± 15 mg.l -1 ). This high removal rate is correlated with the elimination of particles and colloids. The dissolved organic pollution removal is rather slight, yet nevertheless able to reach a 20% median (Fig. 3).

For nitrogen, only a small portion is removed by clarification (40 mg N.l -1 in RS vs. 35 mg N.l -1 in DE). This limited elimination rate is related to the removal of organic nitrogen (mainly associated with particles), whereas NH 4 + is predominant in wastewater (approx. 80% of total nitrogen) and principally in dissolved form (Boari et al., 1997).

Furthermore, phosphorus pollution removal is considerable, as demonstrated by the removal rates for PO 4 3-and P tot (81% and 75%, respectively). DE concentrations equal roughly 1.4 ± 0.3 mg P.l -1 for P tot and 0.5 ± 0.2 mg P.l -1 for PO 4 3-

. The high phosphorus pollution elimination rate is tied to particle sedimentation and, to a greater extent, to PO 4 3precipitation with the ferric ion (FePO 4 ).

During the three sampling campaigns, median influent flow was about 240,000 m 3 .d -1 .

The removal rates for conventional wastewater parameters with lamella separator were close to those obtained using conventional and typical operating modes (87% ± 2% for TSS, 63% ± 4% for CODtot, 64% ± 3% for BOD, 75% ± 6% for P tot , and 81% ± 10% for PO 4

3-

).

Priority substances

Table 2 summarises, for all molecules detected, the observed removal rates with lamella separator. According to these results, the pollutants studied can be classified as: i)

"poorly removed" compounds, when the elimination rate is below 20%; ii) "weakly removed" compounds, when this rate ranges between 20% and 50%; iii) "moderately removed", with an elimination rate of 50% to 80%; and lastly iv) "efficiently removed", i.e. a rate above 80%.

• Experimental results

According to the previously defined criteria, metals are moderately removed by clarification (> 33% for Pb and between 50% and 80% for Zn and Cu). Total metal concentrations decreased markedly between RW and DE, thus confirming the strong impact of lamella clarifier and suggesting that the removal of metals is likely to be strongly correlated with the affinity of these metals for particles (Buzier et al., 2006). As reported by [START_REF] Gasperi | Wastewater quality and pollutant loads in combined sewers during dry weather periods[END_REF] for Paris wastewater, 40%-80% of Zn and 75%-95% of Pb and Cu are associated with particles.

Depending on the physico-chemical properties of the pollutant, removal rates for organic pollutants vary significantly. HVOCs and BTEX are only weakly removed by clarification (< 20%), while heavy molecular weight PAHs (4-6 rings, HMW PAHs) are efficiently removed (> 80%). The differences observed are in fact tied to the hydrophobicity of the compounds, which may be reflected by the octanol -water coefficient (K ow ). As suggested by [START_REF] Rogers | Sources, behaviour and fate of organic contaminants during sewage treatment and in sewage sludges[END_REF] and [START_REF] Byrns | The fate of xenobiotic organic compounds in wastewater treatment plants[END_REF], hydrophobic compounds (log K ow > 4) are associated mainly with particles and hence efficiently removed by means of decantation. On the other hand, hydrophilic compounds (log K ow < 4) are weakly removed since they preferentially occur during the dissolved phase. This trend is clearly highlighted in Fig. 4. For hydrophilic compounds (log K ow < 4), removal rates vary considerably yet still lie below 20%. In this study, 18 substances with a strong hydrophilic character are involved (6 pesticides, 6 HVOCs and BTEX, 3 light molecular weight PAHs (LMW PAHs), and 3 individual substances). For hydrophobic compounds (log K ow > 4), three groups are distinguishable: i) Group 1 (4.0 < log K ow < 4.5), with removal rates between 20% and 50%;

Group 2 (4.9 < log K ow < 7.2), with removal rates of 50% to 80%; and iii) Group 3 (5.6 < log K ow < 6.7), with removal rates exceeding 80%.

• Theoretical approach

Three main mechanisms require special attention in order to predict the removal rates of pollutants during wastewater treatment, i.e.: i) sorption, ii) volatilisation, and iii) biodegradation [START_REF] Byrns | The fate of xenobiotic organic compounds in wastewater treatment plants[END_REF]. For high-rate clarification, it can reasonably be assumed that sorption prevails while both volatilisation (no stripping) and biodegradation (short hydraulic retention time and no high biomass concentration) processes exert only a minor influence.

The removal of chemicals as part of the clarification technique is therefore predominantly linked to their sorption potential on primary settled sludge. According to [START_REF] Karickhoff | Organic pollutant sorption in aquatic systems[END_REF], the adsorption of hydrophobic non-polar organic compounds is commonly described as a linear function of both the K ow of the solute and the organic carbon content of the adsorbent (f oc in g C.g -1 ); moreover, the tendency to accumulate in sewage sludge solids can be assessed using both parameters. The phase partitioning of a given pollutant between dissolved and sorbed phases can thus be computed using the eq. 1 adapted from [START_REF] Byrns | The fate of xenobiotic organic compounds in wastewater treatment plants[END_REF].

) ] [ 10 3 . 6 1 ( 1 100 7 RS OW OC TSS K F FDW × × × × + × = FDW FSS - = 100
Eq 1 adapted from [START_REF] Byrns | The fate of xenobiotic organic compounds in wastewater treatment plants[END_REF] with: FDW = Fraction of chemical dissolved in water FSS = Fraction of chemical sorbed on solids F OC = 0.7 g C.g -1 [START_REF] Tchobanoglous | Wastewater Engineering. Treatment and Reuse[END_REF] [TSS] RS = Total suspended solid concentration in raw sewage

The fraction of chemical dissolved in water (FDW) is shown in Fig. 5 as a function of log K ow (black squares). Experimental partitioning, when available, is also indicated on this figure (white squares). Once the fraction of pollutant sorbed on the solid (FSS) had been evaluated, the theoretical removal was calculated according to the TSS removal rate. Fig. 5 then compares experimental (median value ± SD) with theoretical removal rates.

For compounds offering the possibility of comparison, a good correlation between prediction, experimental FDW and removal rates can be observed. Below a log K ow of around 2, FDW is predominant and, consequently, the compounds are weakly removed (< 20%). In contrast, the compounds with a log K ow of above 5 are preferentially associated with particles and hence efficiently removed (80% ± 10%), except for DEHP which displays a slightly lower removal rate (70% vs. 86%). For compounds with a log K ow between 4 and 5, FDW ranges from 12% to 51%, meaning that removal rates can vary significantly (20%-80%).

The good correlation between experimental and theoretical results justifies the empirical predictive approach for easily determining the evolution of pollutants during primary treatment. The above procedure provides a suitable description of sorption behaviour and therefore furnishes important information on the evolution of a wide array of non-polar organic compounds during lamella clarification. Theoretical approaches are critical to wastewater management since the set-up and execution of experimental campaigns is complex and expensive. Model generalities for screening purposes can be extended to polar organics and trace metals through the use of alternative formulations to describe the relevant partitioning phenomena. Although a number of empirical methods can be implemented to predict the extent of partitioning, the Kd (distribution coefficient) for polar or charged compounds in aquatic systems can be evaluated using laboratory tests, as performed by [START_REF] Ternes | A rapid method to measure the solid-water distribution coefficient (K-d) for pharmaceuticals and musk fragrances in sewage sludge[END_REF] for pharmaceuticals and musk fragrances.

Biofiltration

Conventional wastewater parameters

The removal rates for typical wastewater parameters with biofiltration are displayed in Fig. 6; in addition, median concentrations ± SD have been indicated for DE and FE.

Results reveal that a significant portion of TSS are treated when settled effluents flow through the biofiltration units (89% ± 5%). TSS concentrations in the final effluents were typically found in the 3-7 mg.l -1 range. According to [START_REF] Rocher | Le traitement des eaux usées par biofiltration : cas de la station Seine Centre (SIAAP)[END_REF], the first stage performs the physical filtration of particles, while the subsequent biofiltration units play a minor role in particle filtration.

By combining the physical and biological purification processes, biofiltration can effectively treat carbonaceous, nitrogenous and phosphorous pollution. For carbonaceous pollution, the 3-stage biofilter system removes over 92% and 83% of BOD and COD loads, respectively. Like for TSS, BOD is mainly consumed during the first stage [START_REF] Rocher | Le traitement des eaux usées par biofiltration : cas de la station Seine Centre (SIAAP)[END_REF]: concentrations do not exceed 10 mg O 2 .l -1 (with the median being 5.9 mg O 2 .l -1 ). In spite of the significant removal of COD loads, a residual organic pollution, mainly in dissolved form, is commonly observed in discharged effluents at a level of about 30 mg O 2 .l -1 .

For the nitrogenous pollution, removal rates of roughly 99% are observed for NH ), leading to FE concentrations of 0.4 and 0.2 mg P.l -1 , respectively. The high level of phosphorous abatement is related to the assimilation of phosphorus by bacteria.

During the three campaigns conducted, the biofiltration units operated at nominal flow, and the removal rates of typical wastewater parameters were close to those obtained with conventional operating procedures (87% ± 7% for TSS, 84% ± 3% for CODt, 92% ± 3%

for BOD, 96% ± 1% for TKN, and 99% ± 1% for NH 4

+

).

Priority substances

Table 3 lists the removal rates of priority substances using biofiltration. The removals for 23 substances remaining in decanted effluents are described. Detailed information on dissolved and sorbed chemicals in DE is available in the Supplementary Material section (Table 3). As previously stated, the pollutants can be classified as: i) poorly removed (< 20%), ii) weakly removed (20-50%), iii) moderately removed (50-80%), and iv) efficiently removed (> 80%).

Depending on the compound, strong differences appeared regarding removal efficiency with biofiltration. On the whole, all pesticides (diuron, atrazine, isoproturon, metaldehyde and desethylatrazine) were poorly removed (< 20%), while all volatile compounds (1 HVOC, 4 BTEX and 3 LMW PAHs) were at least half removed. For other pollutants, removal rates varied from 20% to 80% (Table 3). Observed differences are due to differing processes occurring in the biofilter system. In theory, four mechanisms are involved herein: i) physical filtration, ii) volatilisation, iii) adsorption, and iv) biotransformation of substances. Although the exact apportioning of each mechanism cannot be assessed, it nevertheless remains possible for each chemical to be identified with a predominant mechanism.

i) Physical filtration. As previously mentioned, biofiltration units act as a physical filter, removing roughly 89% ± 5% of TSS. This retention rate leads to a reduction in particulate pollutants. High removal rates are also observed for most of the hydrophobic compounds; this finding appears to be positively correlated with TSS removal. The highest particulate fraction removal rates were observed for NP (90% ± 4%), followed by fluoranthene (85% ± 2%), phenanthrene (83% ± 2%) and pyrene (82% ± 1%).

ii) Volatilisation. During both the 1 st and 2 nd biofilter system stages, the physical forces due to injecting diffused air induce atmospheric transfer (called air stripping). Given the use of air injection, volatile organic compounds can be volatilised. According to [START_REF] Rogers | Sources, behaviour and fate of organic contaminants during sewage treatment and in sewage sludges[END_REF], compounds with a Henry's constant (K H ) above 10 -4 atm.m -3 .mol -1 and a K H / log K OW ratio exceeding 10 -9 exhibit a high potential to volatilise. In this study, 8 compounds (toluene, xylenes, dichloromethane, chloroform, tri-and tetra-chloroethylene, naphthalene and phenanthrene) feature K H > 10 -4 atm.m -3 .mol -1 . All these compounds are either moderately (50-80%) or efficiently (> 80%) removed. As regards BTEX and HVOCs, tetrachloroethylene has the highest removal rate (82% ± 4%), followed by trichloroethylene (76% ± 8%), toluene (71% ± 4%) and dichloromethane (70% ± 14%), while the lowest rate for volatile compounds was recorded for chloroform (39% ± 20%). The fact that tri-and tetra-chloroethylene are more widely removed can be correlated with their higher K H value (1.81 and 1.04 x 10 -2 atm.m -3 .mol -1 ). For naphthalene and phenanthrene, the removal rates were also substantial, as

shown by the major reduction in dissolved concentrations (89% ± 6% and 82% ± 6%, respectively).

iii) Adsorption on biomass. Adsorption can also occur within the biofilter system. The adsorption process corresponds to electrostatic interactions of positively-charged groups of chemicals with the negatively-charged surface of micro-organisms and, consequently, concerns ionic species. In this study, the adsorption process has been clearly highlighted for Zn and TBT, whose losses amount to about 30% and 20%, respectively. iv) Biotransformation of substances. Like for dissolved organic matter (with a removal rate of 79%), chemicals can be degraded by biomass during biofiltration treatment. According to [START_REF] Byrns | The fate of xenobiotic organic compounds in wastewater treatment plants[END_REF], the complete mineralisation of xenobiotic compounds in treatment systems is rare, and the term biotransformation would more accurately describe potential changes to the composition and molecular structure of such a compound. Depending on their biodegradability, the three following pollutant types are distinguished: i) easily biodegradable, ii) moderately biodegradable, and iii) resistant to biodegradation or refractory.

Given the short hydraulic retention time in the biofilter system (approx. 90 min for all three stages), it can reasonably be assumed that only the easily or moderately biodegradable compounds can be biodegraded.

For example, biodegradation process can explain the differences of removal between volatile compounds exhibiting a quite similar volatilization potential (2.7 x 10 -4 < K H < 6.3 x 10 -3 atm.m -3 .mol -1 ). Indeed, a more important removal is observed for readily biodegradable compounds such as naphthalene and phenanthrene in comparison to moderately degradable compounds (dichloromethane and chloroform). This observation is in accordance with study led by [START_REF] Mcnally | Biodegradation of three-and four-ring polycyclic aromatic hydrocarbons under aerobic and denitrifying conditions[END_REF], who demonstrated that LMW PAHs prove to be readily biodegradable under both aerobic and denitrifying conditions.

Similarly, biodegradation process can be responsible of a decrease of dissolved concentration for no volatile compounds (K H < 10 -4 atm.m -3 .mol -1 ). For example, a reduction of dissolved concentrations was observed for DEHP (from 20 to 60 % depending on the sampling campaign) and nonylphenols (10 -60 %) which are recognized as easily biodegradable compounds [START_REF] Ying | Environmental fate of alkylphenols and alkylphenol ethoxylates--a review[END_REF][START_REF] Fauser | Phthalates, nonylphenols and LAS in an alternately operated wastewater treatment plant--fate modelling based on measured concentrations in wastewater and sludge[END_REF]. A slighter losses have even been observed for fluoranthene (0 -30 %) and fluorene (55 -83 %).

The removal of chemicals in a biofiltration system is compound-dependent and dues to several mechanisms. The multiplicity and complexity of processes involved in chemical removal do not allow for a simple generalisation of results obtained. Though an accurate prediction of pollutant removal is not possible from their physico-chemical properties alone, two groups of chemicals with differing biofiltration-induced removal rates can still be clearly identified: i) hydrophobic or volatile compounds, for which moderate to high removal rates are observed; and ii) hydrophilic compounds, which are neither volatile nor easily biodegradable and hence expected to be poorly removed. High removal rates were indeed recorded for hydrophobic compounds (mainly in the particulate fraction) or easily volatilised compounds following air injection. Total removal ranged from 50% to over 80% for compounds with K H > 1 x 10 -4 atm.m -3 .mol -1 and between 50% and 80% for hydrophobic compounds (log K ow > 4) due to TSS filtration. On the other hand, poor removal was observed for pesticides. This low rate (< 20%) with biofiltration is tied to the low hydrophobicity (log K ow < 2.6), combined with both the low volatilisation potential (1.53 x 10 -9 < K H < 3.46 x 10 -5 atm.m -3 .mol -1 ) and low biodegradability of such compounds.

Concentrations in final effluents

To provide a complete overview of biofiltration performance, the quality of discharged effluents was also investigated. Out of the 39 pollutants initially detected in RS, 20 compounds including 10 substances listed in the WFD exhibited concentrations above the discharged effluent detection limit (Fig. 7). More detailed information on the dissolved and particulate concentrations is available in the Supplementary Material section (Table 4). These higher concentrations were found for Zn (40-50 µg.l -1 ) and DEHP (2.1-5.8 µg.l -1 ), followed by chloroform (1.2 µg.l -1 ), tetrachloroethylene (0.4-1.2 µg.l -1 ) and NP (0.1-0.3 µg.l -1 ). High levels of DEHP and, to a lesser extent of NP can pose an environmental threat since these compounds are recognised as endocrine disrupters and have been found to persist as pollutants in the environment, in addition to being blamed for causing developmental disorders and/or fertility problems [START_REF] Jobling | Altered sexual maturation and gamete production in wild roach (Rutilus rutilus) living in rivers that receive treated sewage effluents[END_REF][START_REF] Ying | Environmental fate of alkylphenols and alkylphenol ethoxylates--a review[END_REF]. For the other compounds, concentrations lie within the ng.l -1 range and typically vary from 0.005 to 0.3 µg.l -1 . In final effluents, 8 pesticides were present with concentrations generally ranging between 0.03 and 1.23 µg.l -1 . Given their poor removal during clarification and biofiltration treatments, the effluents revealed concentration ranges quite similar to those observed in raw sewage, except for AMPA. Of the three campaigns carried out, AMPA was detected twice with higher concentrations in final effluents than in RS or DE. This increase could reflect local AMPA production relative to the degradation of glyphosate, but more likely to the degradation of some detergent components (Koplin et al., 2006;[START_REF] Skark | The occurrence of glyphosate in surface water[END_REF].

Conclusion

This study has investigated the occurrence of priority substances in raw sewage as well as the removal of a wide range of contaminants by means of both lamella separator and biofiltration techniques. 88 substances, ranked into 13 groups of compounds, were determined in raw sewage and at particular points in the WWTP.

This paper first confirmed that a broad array of contaminants is present in raw sewage.

Of the 88 molecules investigated, 39 substances (18 of which appeared on the WFD list) were detected. On the whole, metal concentrations ranged from 15 to 260 µg.l -1 , while other organic pollutants were found to lie in the µg.l -1 range, with the exception of DEHP (13.5-24.7 µg.l -1 ) and volatile organic compounds (0.8-4.8 µg.l -1 ). The presence of most of these chemicals has resulted from their extensive use in a wide variety of consumer products and household appliances.

The performance of both techniques, as regards the removal of conventional pollutants and priority substances, was then assessed. The differing capability of each technique to remove pollutants was obvious; nonetheless, pollutant removal was still quantitatively dependent upon the physico-chemical properties of the chemicals and operating conditions within the facility.

By operating at low coagulant and flocculant doses (25-40 g FeCl 3 .m -3 and 0.30-0.45 g polymer.m -3 ), lamella clarification can effectively clarify wastewater, thus achieving total solid and organic load reductions of 86% and 65% respectively for COD. The adjustment of coagulant and polymer doses, according to RS quality entering the facility, enables maintaining this performance level. As for the chemicals, lamella clarifier can also effectively retain most of those tracked. Backed by the theoretical approach, the removal of organic chemicals is highly dependent on the log K ow of the compound under consideration.

Compounds with a strong hydrophobic character are removed to a significant extent (similar to TSS). This removal mechanism operates through TSS sedimentation and through sorption to sludge particles with subsequent transfer to sludge processing systems. Conversely, hydrophilic compounds (18 compounds with log K ow < 4 out of the 39 detected in raw sewage) were poorly removed (< 20%).

By combining physical and biological purification processes, biofiltration can effectively treat both carbonaceous and nitrogenous pollution. Concerning the removals of priority pollutants, this study has also demonstrated that chemical removal in biofiltration system is compound-dependent and derived from several mechanisms, including: i) physical filtration of suspended solids, ii) volatilisation or air stripping, iii) adsorption on biomass, and iv) biotransformation of substances. Of the 23 substances detected in decanted effluents, 12

were removed at over 50% and 3 over 80%, while 5 pesticides were poorly removed (< 20%).

While the combination of complex processes within biofilter systems does not allow an accurate prediction of pollutant evolution, two groups of chemicals are nonetheless clearly distinguished for their removal rates with biofiltration: i) hydrophobic or volatile compounds, for which moderate to high rates are observed; and ii) hydrophilic compounds, which are neither volatile nor easily biodegradable and hence expected to be poorly removed.

By examining the occurrence and removal of a broad set of pollutants by lamella clarification and biofiltration, this study has provided relevant information on both wastewater treatment technologies and their ability to remove contaminants. Considering that both techniques are currently implemented in WWTPs, the data generated for a predetermined number of hazardous substances may be used in the future to identify other techniques of potential significance and/or deserving of implementation within the Water Framework 4,7 ± 2 29 ± 6 24 ± 6 5,9 ± 3 1,8 ± 0,6 0,4 ± 0,1 0,2 ± 0,1 DE FE Unity mg.l -1 mg O 2 .l -1 mg O 2 .l -1 mg O 2 .l -1 mg N.l -1 mg P.l -1 mg P. (2) The substance in brackets is listed in the WFD.

Figure caption

  N.l -1 ). Water transit through the biofiltration units is accompanied by the biological oxidation of ammonia-containing pollutants. The nitrification process intensifies during the second stage, when over 95% of ammoniacal pollution is oxidised (0.d -1 , with an aeration of approx. 100 Nm 3 /applied kg of NH 4 + ). Within the stage 3, nitric nitrogen formed upon the oxidation of ammonia-containing pollutants is reduced to atmospheric nitrogen. The efficiency of nitrification and denitrification stages minimizes the discharges of nitrogen inputs (NH 4 + / NO 3 -) in aquatic systems. Moreover, biofiltration allows for the efficient removal of phosphorous pollution (73% for P tot and

Fig. 1 -Fig. 3 -Fig. 4 -Fig. 5 -Fig. 6 -Fig. 2 -

 134562 Fig. 1 -Wastewater treatment plant studied .......................................................................................... 25 Fig. 2 -RS concentrations (µg.l -1 ) and limits of detection .................................................................... 26 Fig. 3 -Removal of carbonaceous, nitrogenous and phosphorous pollutions by lamella separator ...... 27 Fig. 4 -Removals of pollutants with lamellar clarifier, according to log K OW ...................................... 28 Fig. 5 -Comparison between experimental and theoretical removals by lamellar clarifier .................. 29 Fig. 6 -Removal of carbonaceous, nitrogenous and phosphorous pollutions by biofiltration .............. 30 Fig. 7 -FE concentrations (µg.l -1 ) and limits of detection .................................................................... 31

Fig. 4 -

 4 Fig. 4 -Removals of pollutants with lamellar clarifier, according to log K OW

Fig. 7 -

 7 Fig. 6 -Removal of carbonaceous, nitrogenous and phosphorous pollutions by biofiltration

( 3 )

 3 Analytical methods: ICP = inductively coupled plasma, AAS = atomic absorption spectrometry, GC = gas chromatography, GC-ECD = GC with electron capture detector, GC-MS = GC with mass spectrometer, GC-MSMS = GC gas chromatography with tandem mass spectrometer, HPLC-fluo = High pressure liquid chromatography with fluorescent detector, UPLC-MSMS = ultra performance liquid chromatography with tandem mass spectrometer. (4) Phase considered with D = dissolved, P = particulate, T = Total

Table 1 -

 1 Priority pollutants and analytical methods

	Groups 1	Total 2	Standards	Methods 3	Phase 4
	Alkylphenols	5 (2)	ISO 18857-1	GC-MSMS	P + D
	BTEX	5 (1)	NF EN ISO 11423-1	GC-MS	T
	Chloroalkanes	1 (1)	Internal method	CG-ECD	P + D
	Chlorobenzenes 5 (3)	EN ISO 6468	GC-MS	P + D
	Chlorophenols	1 (1)	NF EN 12673 et ISO 6468	GC-MSMS	P + D
	HVOCs	7 (4)	NF EN ISO 10301 + 6468	GC-MS	T
	PAHs	16 (8)	ISO 17993	HPLC-Fluo	P + D
	Metals	8 (4)	NF EN ISO 11885 + 1483	ICP and AAS T + D
	Organotins	3 (3)	NF EN ISO 17353	GC-MS	P + D
	PBDEs	3 (1)	ISO 22032	CG-ECD	P + D
	PCBs	8	NF EN ISO 6468	GC-MS-MS	P + D
	Pesticides	25 (12)	NF EN ISO 11369+ Internal method	GC-MS UPLC-MSMS	P + D
	Phthalates	1 (1)	Internal method	GC-MS	P + D

(1) Compound groups: BTEX = benzene, toluene, ethylbenzene and xylenes, HVOC = halogenated volatile organic compounds, PAHs = polycyclic aromatic hydrocarbons, PBDE = polybromodiphenylethers, PCB = polychlorobiphenyls.

Table 2 -

 2 Removals of pollutants by lamellar clarifier

	n	< 20 %	20 -50 %	50 -80 %	> 80 %
	n = 3	tributyl tin toluene chloroform trichloroethylene tetrachloroethylene diuron metaldehyde naphthalene acenaphthylene acenaphthene	Pb* fluorene phenanthren e	Cu Zn chlorpyrifos DEHP nonylphenols para-tert-octylphenol anthracene dibenz(ah)anthracen e PCB 28 pyrene	benzo(a)pyrene benzo(b)fluoranthen e benzo(k)fluoranthen e benzo(ghi)perylene fluoranthene benzo(a)anthracene chrysene
		xylenes
		dichloromethane
		atrazine
	n = 2	desethylatrazine isoproturon	-	-	indeno(cd)pyrene
		AMPA
		chloromethylphenol
		butylphenol
	Total	18	3	10	8

* Given the Pb detection limit, the removal rate exceeds 30%.

Table 3 -

 3 Removals of pollutants by biofiltration

	1
	n	< 20 %	20 -50 %	50 -80 %	> 80 %
				toluene
				trichloroethylene	tetrachloroethylen
	n = 3	diuron	tributyl tin DEHP	nonylphenols fluoranthene	e naphthalene
				fluorene	phenanthrene
				pyrene
				chloroform
		atrazine		dichloromethane
	n = 2	isoproturon desethylatrazin metaldehyde	Zn	chloromethylpheno 4-ter-butyl phenol l	-
		e		benzo(a)pyrene
				xylenes
	Total	5	3	12	3


Diz

JF Feijoo

Limeres

Pedro Diz Dios
email: pedro.diz@usc.es

Keywords: Diagnostics, Genetics Oral Diseases - oral-facial-digital syndrome, cleft palate, aberrant frenula, lobulated tongue, molecular screening, mutation

Oral-facial-digital syndrome (OFDS) type 1 (OFD1) is an X-linked dominant condition associated with embryonic male lethality. It almost always affects the oral cavity, face, and digits. It is considered to be a ciliopathy caused by mutations in the OFD1 gene. A variety of mutations have been described, and a genotype-phenotype correlation has been suggested.

Objective and Methods

The proband was an 8-year-old Spanish girl with suspected OFD1. We extended the pedigree to three proband´s generations, performing a thorough physical examination and screening for OFD1 mutations in 9 individuals.

Results

The proband, her mother and her sister showed oral findings consistent with OFD1.

Ultrasound evaluation revealed the existence of renal cysts only in the proband's mother. The rest of the family (all male) had no relevant morphological abnormalities. A single-base deletion in exon 16 of OFD1 (c.2183delG) leading to a frameshift was detected in the proband, her mother, and her sister.

Conclusion

Since all three women had a similar oral phenotype, this new mutation might be involved in the development of the OFD1 oral manifestations. In cases of OFDS, physical examination (including the oral cavity and renal function), and genetic screening of the probands and their relatives are mandatory.

O

INTRODUCTION

The oral-facial-digital syndrome (OFDS) was first described by [START_REF] Mohr | A hereditary subletal syndrome in man[END_REF][START_REF] Mohr | A hereditary subletal syndrome in man[END_REF], when he reported a family with highly arched palate, lobate tongue with papilliform outgrowths, broad nasal root, hypertelorism, and digital anomalies. It is possible, however, that this condition was described previously, and in fact a report of apparent Mohr syndrome can be found 100 years earlier in the literature as 'Monstrorum humanum hexadactylum' [START_REF] Beckwith | Monstrorum humanum hexadactylum' (personal communication)[END_REF]. OFDS shows a striking phenotypic heterogeneity. To date, 13 types of OFDS have been described based on clinical manifestations and inheritance patterns [START_REF] Gurrieri | Oral-facial-digital syndromes: Review and diagnostic guidelines[END_REF].

OFDS type 1 (OFD1; OFDI; OMIM 311200) was described by [START_REF] Papillon-Leage | Hereditary abnormality of the buccal mucosa: abnormal bands and frenula[END_REF][START_REF] Papillon-Leage | Hereditary abnormality of the buccal mucosa: abnormal bands and frenula[END_REF][START_REF] Papillon-Leage | Hereditary abnormality of the buccal mucosa: abnormal bands and frenula[END_REF]. It is inherited as an X-linked dominant trait with embryonic male lethality [START_REF] Wettke-Schäfer | X-linked dominant inherited diseases with lethality in hemizygous males[END_REF]. Type 1 accounts for the majority of OFDS cases, and its estimated frequency is approximately 1 case per 50,000-250,000 live births [START_REF] Wahrman | The oral-facial-digital syndrome: a male-lethal condition in a boy with 47/xxy chromosomes[END_REF]. It has been suggested that OFDS reflects the pleiotropic effects of a morphogenetic impairment that almost invariably affects the oral cavity, face, and digits [START_REF] Gurrieri | Oral-facial-digital syndromes: Review and diagnostic guidelines[END_REF]. The main oral manifestations in OFD1 include pseudo-cleft of the upper lip, cleft palate, hyperplastic frenula, aberrant frenula, lobulated or cleft tongue (bifid or trifid), lingual hamartomas, ankyloglossia, hypodontia, supernumerary teeth, and hypoplastic mandible [START_REF] Lauterstein | Tooth anomalies in the oral-facial-digital syndrome[END_REF][START_REF] Melnick | Orofaciodigital syndrome, type I: a phenotypic and genetic analysis[END_REF][START_REF] Thauvin-Robinet | Clinical, molecular, and genotype-phenotype correlation studies from 25 cases of oral-facial-digital syndrome type 1: a French and Belgian collaborative study[END_REF]Hennekam et al., 2010a). The facies is remarkably characteristic, with frontal bossing, hypertelorism, dystopia cantorum, telecanthus, hypoplasia of alar cartilages, broad nasal root, low-set ears, flattened midfacial region, and microretrognathia [START_REF] Macca | The Molecular Basis of Oral-Facial-Digital Syndrome, Type1[END_REF]Hennekam et al., 2010). Digital findings include brachydactyly, syndactyly, clinodactyly, polydactyly, and irregular bone mineralization [START_REF] Al-Qattan | Classification of limb anomalies in oral-facialdigital syndromes[END_REF]Hennekam et al., 2010). Bilateral polycystic kidney disease is common in OFD1, though most cases are detected in adulthood [START_REF] Scolari | Oralfacial-digital syndrome type I: an unusual cause of hereditary cystic kidney disease[END_REF]. Central nervous system malformations include agenesis of the corpus callosum, arachnoid cysts, cerebellar abnormalities, hydrocephalus, and porencephaly. As many as 50% of cases have mild intellectual disability [START_REF] Holub | Central nervous system malformations in oral-facial-digital syndrome, type 1[END_REF][START_REF] Gurrieri | Oral-facial-digital syndromes: Review and diagnostic guidelines[END_REF].

OFD1 is caused by mutations in the Cxorf5 transcript, later named OFD1 (MIM# 300170) [START_REF] Ferrante | Identification of the gene for oral-facial-digital type I syndrome[END_REF], which comprises 23 exons encoding a 1011 amino acid protein. The gene encodes a centrosomal protein found in the primary cilia [START_REF] Romio | OFD1, the gene mutated in oral-facial-digital syndrome type 1, is expressed in the metanephros and in human embryonic renal mesenchymal cells[END_REF], and consequently OFD1 has been considered a ciliopathy [START_REF] Badano | The ciliopathies: an emerging class of human genetic disorders[END_REF]. It is widely expressed in metanephros, brain, tongue, and limb [START_REF] Romio | OFD1, the gene mutated in oral-facial-digital syndrome type 1, is expressed in the metanephros and in human embryonic renal mesenchymal cells[END_REF] mutations have been described, and some genotype-phenotype correlations have been suggested [START_REF] Thauvin-Robinet | Clinical, molecular, and genotype-phenotype correlation studies from 25 cases of oral-facial-digital syndrome type 1: a French and Belgian collaborative study[END_REF][START_REF] Prattichizzo | Mutational spectrum of the oralfacial-digital type I syndrome: a study on a large collection of patients[END_REF][START_REF] Macca | The Molecular Basis of Oral-Facial-Digital Syndrome, Type1[END_REF].

Diagnosis in a proband raises the level of suspicion for the proband's relatives. The objective of this study was to perform a thorough physical examination and screening for OFD1 mutations in a three-generation Spanish family with oral-facial-digital syndrome type 1, who had heterogeneous phenotypic findings.

MATERIALS & METHODS

Pedigree Construction

The proband was an 8-year-old girl who was referred by her pediatrician to the Special Needs Dentistry Unit at the University of Santiago de Compostela, Spain, with a diagnosis of multiple caries and abnormal dental eruption. She had a history of cleft palate, lobulated tongue, and syndactyly that had all been surgically corrected, along with agenesis of the corpus callosum and mild mental retardation. Physical examination also revealed hypertelorism, broad nasal root, sequelae arising from surgical closure of the cleft palate and bifid tongue, numerous thick fibrous bands in the lower mucobuccal fold (aberrant frenula), supernumerary maxillary deciduous canines, and absence of lower lateral incisors (Fig. 1). As OFD1 was suspected, we extended the pedigree to three generations, including 9 individuals (Fig. 2): 3 affected, 4

unaffected, and 2 deceased. All participants or their legal guardians (cases II6 and III1)

provided informed consent to inclusion in the study, which was approved by the Ethics Committee of the University of Santiago de Compostela.

Mutation Analysis

Genomic DNA was extracted from buccal epithelial cells using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The 23 exons of the OFD1 gene were amplified as described previously [START_REF] Ferrante | Identification of the gene for oral-facial-digital type I syndrome[END_REF]. PCR products were separated on polyacrylamide gels (T-9%, C-5%). After confirming the success of the amplification, the PCR product was purified with a Multiscreen PCR µ 96

Filter Plate (Millipore Corporation, Billerica, USA). Sequencing reactions were carried out on both strands of the PCR product using the ABI Prism BigDye Terminator v3.1

Cycle Sequencing Kit (Applied Biosystems, Foster City, USA) and the products were purified with the Montage®SEQ96 Sequencing Reaction Cleanup Kit (Millipore).

Electrophoresis was performed on an ABI PRISM 3730xl [START_REF] Wildeman | Improving sequence variant descriptions in mutation databases and literature using the Mutalyzer sequence variation nomenclature checker[END_REF], and the software was used to predict the affected protein from the variant coding region.

RESULTS

Clinical Diagnosis

The proband (II6) had morphological characteristics consistent with OFD1 [START_REF] Thauvin-Robinet | Clinical, molecular, and genotype-phenotype correlation studies from 25 cases of oral-facial-digital syndrome type 1: a French and Belgian collaborative study[END_REF][START_REF] Gurrieri | Oral-facial-digital syndromes: Review and diagnostic guidelines[END_REF]. Orofacial examination of the proband's mother (I2) revealed pseudocleft palate, bifid tongue, aberrant frenula, and absence of the lower lateral incisors (Fig. 1), as well as one nostril smaller than the other; she had a history of two spontaneous male miscarriages with severe malformations (II1 and II2) before conceiving the proband (21 and 17 years earlier). Examination of the proband's sister (II3) revealed a slight pseudocleft palate and bifid tongue, aberrant frenula, and absence of the lower lateral incisors (Fig. 1). Ultrasound evaluation of the kidneys in the three affected women (I2, II3 and II6) revealed the existence of small, bilateral renal cysts only in the proband's mother (I2). The rest of the family (all male) had no relevant morphological abnormalities.

Mutation Analysis

A single base deletion (c.2183delG) in exon 16 of the OFD1 gene leading to a frameshift was identified. Mutation nomenclature numbering is based on the GenBank accession number_NM003611, with +1 as the A of the ATG initiation codon. The variant c.2183delG was present in the proband (II6), her mother (I2), and her sister (II3) (Fig. 3). The predicted protein based on analysis with Mutalyzer 2.0 β-4 software is p.(Gly728Alafs*89). As expected, the other members of the family did not carry the mutation.

DISCUSSION

Clinical Findings

The diagnosis of OFD1 is usually established at birth on the basis of characteristic oral, facial, and digital abnormalities [START_REF] Toriello | Oral-Facial-Digital Syndrome Type I[END_REF]. In spite of the severe phenotypic findings observed in the proband, a suspected diagnosis OFDS had not been suggested until she was referred for oral evaluation at 8 years of age. In this case, the proband was the first affected family member. A pedigree was therefore constructed to rule out the possibility that other relatives might be undiagnosed due to incomplete penetrance or variable expressivity. It has been suggested that neither the oral nor the facial features of OFDS can be used to reliably distinguish X-linked from autosomal recessive variants [START_REF] Siebert | The oral-facial-digital syndromes[END_REF]. Oral abnormalities including pseudocleft palate, bifid tongue, aberrant frenula, and absence of the lower lateral incisors were the most relevant findings in both the proband's sister and her mother. In a worldwide cohort of 120 individuals clinically diagnosed with OFD1, cleft palate/high arched palate was present in 23.5%, tongue anomalies in 90.1%, aberrant frenula in 65.4%, and abnormal teeth in 42% [START_REF] Prattichizzo | Mutational spectrum of the oralfacial-digital type I syndrome: a study on a large collection of patients[END_REF]. None of these abnormalities is specific to OFDS and accessory frenula, for instance, may be more suggestive of Pallister-Hall syndrome (Hennekam et al., 2010b).

The proband's mother had renal cystic disease. Renal impairment can be present at birth but usually develops later on, occurring in 63% of patients older than 18 years [START_REF] Macca | The Molecular Basis of Oral-Facial-Digital Syndrome, Type1[END_REF]. Some reports describe renal disease as completely dominating the clinical course of the syndrome [START_REF] Feather | Oral-facial-digital syndrome type 1 is another dominant polycystic kidney disease: clinical, radiological and histopathological features of a new kindred[END_REF], indicating that morphological assessment and biochemical monitoring of renal function is necessary in OFD1 patients [START_REF] Prattichizzo | Mutational spectrum of the oralfacial-digital type I syndrome: a study on a large collection of patients[END_REF]. The proband's mother also had a history of two male miscarriages. Male lethality usually occurs in the first or second trimester of pregnancy [START_REF] Wettke-Schäfer | X-linked dominant inherited diseases with lethality in hemizygous males[END_REF], and only a few exceptional male cases of OFD1 have been reported to date [START_REF] Wahrman | The oral-facial-digital syndrome: a male-lethal condition in a boy with 47/xxy chromosomes[END_REF].

Efforts to classify OFDS into distinct types have met criticism based on reported individuals or family members with different phenotypic findings. As in the present case, affected family members may not exhibit defects of all three areas: oral region, face, and digits [START_REF] Siebert | The oral-facial-digital syndromes[END_REF]. Clinical variability is often seen in affected females even within the same family [START_REF] Toriello | Heterogeneity and variability in the oral-facial-digital syndromes[END_REF], possibly due to the different degrees of somatic mosaicism [START_REF] Thauvin-Robinet | Clinical, molecular, and genotype-phenotype correlation studies from 25 cases of oral-facial-digital syndrome type 1: a French and Belgian collaborative study[END_REF]. Although the findings described for OFD1 overlap with those observed in other OFDS types, it has been suggested that OFD1 can be distinguished because of its X-linked dominant inheritance with male lethality and the presence of cystic kidneys [START_REF] Gurrieri | Oral-facial-digital syndromes: Review and diagnostic guidelines[END_REF]. Consequently, the clinical findings in the proband, the pedigree analysis, and the molecular analysis of the OFD1 gene led to a diagnosis of OFD1.

Mutation Analysis

To date, 99 mutations have been described in the OFD1 gene, the majority of which have been identified in exons 3 (14%), 8 (14%), 9 (10%), 12 (6%), 13 (10%), and 16

(7%), which may represent mutational hotspots [START_REF] Macca | The Molecular Basis of Oral-Facial-Digital Syndrome, Type1[END_REF]. Intriguinly, no mutations or polymorphisms were identified beyond exon 16 [START_REF] Prattichizzo | Mutational spectrum of the oralfacial-digital type I syndrome: a study on a large collection of patients[END_REF].

The mutation in exon 16 identified in this study is the hundredth to be reported. This is a deletion mutation causing a frameshift. Previously, 8 frameshift mutations and 1 and Franco, 2009). As with many of the other mutations that have been described, it is predicted to cause premature truncation of the protein that would probably lead to loss of function [START_REF] Gurrieri | Oral-facial-digital syndromes: Review and diagnostic guidelines[END_REF][START_REF] Prattichizzo | Mutational spectrum of the oralfacial-digital type I syndrome: a study on a large collection of patients[END_REF].

In more than 20% of patients, bi-directional DNA sequencing of the exons and intronexon boundaries of the OFD1 gene remains negative, but in 23% of these cases a genomic deletion of the OFD1 transcript has been identified through a combination of quantitative multiplex PCR of short fluorescent fragments (QMPSF) and relative quantification by real-time PCR (qPCR) [START_REF] Thauvin-Robinet | Genomic deletions of OFD1 account for 23% of oral-facial-digital type 1 syndrome after negative DNA sequencing[END_REF]. However, only 85% of cases are explained by identifiable deletions and mutations, while the remaining cases are not identified using currently available techniques [START_REF] Macca | The Molecular Basis of Oral-Facial-Digital Syndrome, Type1[END_REF]. Recently, Thauvin-Robinet et al [START_REF] Thauvin-Robinet | Search for genomic imbalances in a cohort of 20 patients with oral-facial-digital syndromes negative for mutations and large rearrangements in the OFD1 gene[END_REF], in an effort to identify new regions containing candidate genes that could be implicated in OFDS, performed high-resolution array-CGH in a series of OFDS patients, but without success. Furthermore, genomic deletions of the OFD1 transcript have been identified

in cases in which direct sequencing of the exons was negative due to the presence of the wild-type allele [START_REF] Morisawa | Novel double-deletion mutations of the OFD1 gene creating multiple novel transcripts[END_REF][START_REF] Thauvin-Robinet | Search for genomic imbalances in a cohort of 20 patients with oral-facial-digital syndromes negative for mutations and large rearrangements in the OFD1 gene[END_REF].

When analysing genotype-phenotype correlations, mental retardation was more frequently associated with mutations in exons 3, 8, 9, 13 and 16 [START_REF] Thauvin-Robinet | Clinical, molecular, and genotype-phenotype correlation studies from 25 cases of oral-facial-digital syndrome type 1: a French and Belgian collaborative study[END_REF]. Polycystic kidney disease appears to be correlated with splice mutations [START_REF] Thauvin-Robinet | Clinical, molecular, and genotype-phenotype correlation studies from 25 cases of oral-facial-digital syndrome type 1: a French and Belgian collaborative study[END_REF], mainly located in exons 9 and 12 [START_REF] Prattichizzo | Mutational spectrum of the oralfacial-digital type I syndrome: a study on a large collection of patients[END_REF]. High-arched/cleft palate was found most frequently with missense and splice site mutations [START_REF] Prattichizzo | Mutational spectrum of the oralfacial-digital type I syndrome: a study on a large collection of patients[END_REF]. Cleft lip was more often associated with mutations in exon 3 [START_REF] Prattichizzo | Mutational spectrum of the oralfacial-digital type I syndrome: a study on a large collection of patients[END_REF]. Tongue anomalies were more often detected in patients showing a mutation in exon 12, and tooth abnormalities more frequently associated with mutations in coiled-coil domains [START_REF] Toriello | Oral-Facial-Digital Syndrome Type I[END_REF].

Extensive phenotypic variability has been detected even among female members of the same family, probably due to X inactivation [START_REF] Thauvin-Robinet | Clinical, molecular, and genotype-phenotype correlation studies from 25 cases of oral-facial-digital syndrome type 1: a French and Belgian collaborative study[END_REF][START_REF] Macca | The Molecular Basis of Oral-Facial-Digital Syndrome, Type1[END_REF]. Another possible explanation for the inter-and intrafamilial variability observed in OFD1 patients could be ascribed to modifier genes [START_REF] Prattichizzo | Mutational spectrum of the oralfacial-digital type I syndrome: a study on a large collection of patients[END_REF]. To our knowledge, in the literature no correlation has been found between OFD1 clinical features and frameshift compared with other mutations. Moreover, mutations located in exon 16 have not been related to any specific phenotype to date.

In the present paper, the three women with OFD1 carried the c.2183delG variant and had a similar oral phenotype, suggesting that the deletion could be implicated in the development of the oral manifestations of the syndrome. Further molecular studies will be required before adequate genetic counselling can be offered to all family members of affected individuals. Nevertheless, it is of critical importance to offer a thorough physical examination and molecular screening of OFD1

to the probands and their relatives, along with monitoring of renal function.

Figure caption

I2 (proband's mother, 43 years old) and II3 (proband's sister, 23 years old) had a pseudocleft palate, bifid tongue, aberrant frenula, and absence of the lower lateral incisors. II6 (proband, 8 years old) had a cleft palate and lobulated tongue that had been surgically corrected, along with aberrant frenula and absence of the lower lateral incisors.

Figure 2. Family pedigree

Figure caption

□ Unaffected male • Affected female ■ Deceased affected male Peripheral blood samples were not available for deceased members and those marked with an asterisk.

The proband is indicated with an arrow.

Figure 3 .

 3 Figure 3. Sequence analysis of PCR products of exon 16. a) Direct sequencing of the PCR product in an affected individual. b) Wild type.

  , which could explain the clinical expression of the syndrome. A variety of

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Oral Diseases -Manuscript CopyOral Diseases -Manuscript Copy

ACKNOWLEDGMENTS

We thank the family members for their willing cooperation, and Susana Rubido for her technical assistance.

COMPETING INTERESTS

The authors declare that they have no competing interests


C Yann

R Loison

R Gillard #

M Labeyrie

J-P Martinaud

Global technique analysis for reconfigurable reflectarray antennas

This paper focuses on the simulation of reconfigurable reflectarrays. A new method combining the 'surrounded-cell' approach and the compression method is presented. The method considers the real environment of the radiated elements and it only requires one lightweight electromagnetic simulation for the whole reflectarray.

I. INTRODUCTION

In recent years, reflectarray technology [START_REF] Berry | The reflectarray antenna[END_REF] is of growing interest in the field of telecommunications as it combines attractive features of reflector and array antennas. Reflectarrays have numerous advantages such as their ease of fabrication, low-profile, high-gain, lightweight, and electrically steerable capability. A microstrip reflectarray antenna consists of a reflecting surface fed by a horn antenna (Fig. 1).

This paper focuses on the simulation of reconfigurable reflectarrays. The concept of reconfigurable reflectarray is based on dynamic phase control of the scattered wave. Every cell is loaded with commutation elements such as diodes or MEMS [START_REF] Legay | A steerable reflectarray antenna with MEMS controls[END_REF]. The radiating elements have a similar physical structure and they differ only by active element states. This type of cell is able to adaptively change the phase of the radiated wave by electronically switching the active elements state. When all the actives elements are switched in optimal positions, it defines a configuration of the reflectarray. Consequently, by changing the configuration dynamically, the far-field pattern can be controlled. Reflectarray analysis is a major issue and due to the problem size, segmented methods have been developed.

In the case of passive reflectarrays, two commonly used methods are the Floquet approach [START_REF] Montgomery | Scattering by an infinite periodic array of microstrip elements[END_REF], and the 'surroundedcell' method [START_REF] Milon | Surroundedelement approach for the simulation of reflectarray radiating cells[END_REF]. The first one assumes that the unitary cell is extracted from an infinite periodic array of identical cells. However, it approximates mutual coupling given the fact that, in reality, the cells within the reflectarray are different. The 'surrounded cell' approach has been developed to take into account mutual coupling. This method is based on the analysis of the studied cell together with its neighbours.

For reconfigurable reflectarrays, the Floquet approach can be combined to the compression approach [START_REF] Kunisch | The compression approach : a new technique for the analysis of distributed circuits containing non linear elements, 21st EuMC Proceedings[END_REF], [START_REF] Dauguet | Extension of the compression approach to include the treatment of radiation pattern in the electromagnetic analysis of active planar antennas[END_REF] to simulate each unit-cell loaded by active elements. A unit cell extracted from an infinite array is electromagnetically simulated and a circuit simulation is realized to derive the cell response for all possible cell configurations [START_REF] Pereira | Four-state dual polarisation unit-cells for reflectarray applications[END_REF].

In this paper, a new method combining the 'surrounded-cell' approach and the compression method is presented.

II. PRINCIPLE

In order to compute the reflectarray radiation pattern, the proposed method consists in summing up the field radiated by each cell taking into account its actual environment. The radiation pattern of each surrounded element is computed through the simulation of sub-arrays composed of the considered cell and its neighbours. In order to compute the radiation pattern of the considered cell, the whole sub-array is illuminated and the field radiated by the considered cell is determined. The fact that cells only differ by active element states permits to determine all surrounded radiation patterns thanks to only one electromagnetic analysis and light postprocessing computations.

Each cell of the reflecting array surface is studied one after the other along with its neighbours (Fig. 2). For convenience, the unit cell is a dipole and only one active element, which is a diode, per cell is considered. Moreover, a sub-array of nine cells is considered but the method is expandable to a larger sub-array. The excitation surface includes the whole sub-array while the Huygens surface only surrounds the studied cell. This surface permits to determine the resulting far-field from the studied cell when all the cells of the sub-array are excited. Active elements, such as diodes in this example, have negligible size compared to wavelength so they can be considered as localized elements. The structure is decomposed into two parts. The first part includes localized elements and the second one, the distributed passive part, includes the subarray in which localized elements are replaced by ports (Fig. 3). The electromagnetic simulation of the distributed passive part is first carried out. Then, the compression method is applied in order to take into account on-and off-state equivalent impedances of diodes. A circuit simulation is realized (Fig. 4), that is to say a wave a 1 is sent on port 1 and the simulator computes the other coefficients a k and b k . The coefficient a 1 associated to the incident plane wave on port 1 depends on the desired amplitude and is equal to ŵ ŵ zzzzzzzzȎ Ŷ ô Ŵ with S the excitation surface, ŵ zzzzzzȎ the incident E-field on port 1 and " the vacuum impedance. The compute coefficients a i 2≤i≤10 corresponds to the combinations between the radiation patterns of the loaded structure previously exported.

Finally, the cell response is obtained by summing the contribution of each port. The E-field radiated by the cell is :

˗ zzzzzzzzzzzzzzȎ I #" (# zzzzȎ .
Note that the method is described for one polarization and one incidence angle of the incident field. The generalization is achieved simply by adding an excitation port for each polarization and incidence. The same procedure is repeated for all cells using the same electromagnetic simulation and individual responses are computed. In agreement with the superposition principle, the radiation pattern of the global antenna is the sum of all the unit-cell contributions.

III. METHOD ADVANTAGES

The major advantage of this method is that it allows actual mutual coupling effects to be accounted for. Unlike Floquet method, it considers the real environment of the radiated elements.

Moreover, it requires only one lightweight electromagnetic simulation for the whole reconfigurable reflectarray antenna which is time-saving.

Furthermore, the radiation pattern of any configuration of the reflectarray is determined promptly by circuit simulation. The method is independent of the number of cells in the array and it is expandable to analyze electrically large structures.

Lastly, any numerical simulation method, as for example MoM, FEM or FDTD, can be used for the electromagnetic simulation. A reconfigurable reflectarray composed of 64 cells is simulated using the electromagnetic HFSS ® commercial software and with different approaches at 8.7GHz.

Ŧ

Excitation surface Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Surface de Huygens Port 1 Ƌ Ƌ Ƌ Ƌ Ƌ Ƌ Ƌ Ƌ 2 3 4 5 6 7 8 Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ƌ 9 10 Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ Ŧ

Surface de Huygens

The unit cell consists of a printed circuit with 4 diodes inside a rectangular waveguide [START_REF] Delestre | New Challenges for Active ReflectArrays[END_REF] as shown on Fig. 5. The two pairs of diodes, located on the printed circuit, exhibit 4 phase values. Diodes have a forward "On" resistance of fractions of Ohms (Z On ) while their reverse blocking capacitance "Off" (Z Off ) is in the femtofarad range. I). By turning on or off the diodes, it permits to electronically vary the electromagnetic wave path and thus obtain the desired phase response. The cells are geometrically identical but the diodes are switched in different positions to control the far-field characteristics.

In this example, a small-sized array of 64 cells (Fig. 6) is studied so that the whole array can be simulated at one time with HFSS®, replacing diodes by their equivalent impedance, Z On or Z Off . This reference simulation allows to compare results from different approaches and to validate the proposed method. The array is illuminated by a normal-incident plane wave and the incident electric field is orthogonal to the slot.

As an example, the desired radiation pattern has a null in the θ = 0° direction and a main beam on each side of this null of radiation. To get these characteristics of the radiation pattern, the state of all diodes has to be fixed. The array is divided into two equal parts and the diodes are switched in such a way that there is 180° out of phase from each other. Then, destructive interference occurs, resulting in a null of amplitude at θ = 0°. As shown on Table I, approximately 180° out of phase is obtained between the On-On and the On-Off states of the cell. As a result, diodes on half part of the reflectarray are turned on while the other cells are in On-Off position (Fig. 6). The reconfigurable reflectarray is simulated with different approaches :

global simulation of the reflectarray replacing the diodes with their equivalent impedances, -Floquet approach combined with the compression technique, -proposed approach : surrounded cell method combined with the compression technique with 3x3 and 5x5 subarrays. The radiation patterns of the reflectarray with the different methods are overlaid on Fig. 7. The green trace shows the reference and corresponds to the global simulation. The proposed method with 5x5 sub-arrays is represented in red and the blue pattern is obtained with the same approach using 3x3 sub-arrays. First of all, the proposed approach gives better results than the Floquet approach, in cyan on Fig. 7. Secondly, the reconstruction from sub-arrays composed of 5x5 cells is more accurate than the method with 3x3 sub-arrays. As a consequence, the more neighbours are included in the simulation, the better the prediction is.

The statistical results set out in Table II compare the different approaches to the global simulation, which is the reference for this study. The ε central parameter estimates the amplitude difference between the studied approach and the reference in the θ = 0° direction. This central error is 6.5dB for Floquet and less than 1dB for the proposed method. The specified parameter σ is the standard deviation of the amplitude difference between the method under consideration and the reference for θ over the interval [-90, 90] degrees. With the proposed technique using 5x5 subarrays, σ is equal to 1.4dB which is low compared to the 10dB obtained with the Floquet method. The MAE parameter, which stands for "Mean Absolute Error", is an average of the absolute differences between the method and the reference over the same interval as the previous parameter. A distinct improvement is observed with the proposed method.

Moreover, for each parameter, the proposed method with 5x5 sub-arrays is more accurate than the same method using 3x3 sub-arrays. Thus, the more cells are included in the subarrays, the more accurate the results are. In addition, with 49 Go RAM and an Intel ® Xeon ® E5506 2.13GHz Quad Core, the simulation of the global antenna requires 407 minutes for each configuration of the reconfigurable reflectarray. In comparison, the new technique analysis requires 72 minutes for the first state and only 38 seconds for any configuration. In this paper, one specific configuration is studied but the method can easily analyze any configuration.

V. CONCLUSIONS In the present article, a fast and accurate technique to simulate a reconfigurable reflectarray is described and validated. This new method combines the 'surrounded-cell' and the 'compression' approaches and takes into account the actual coupling effects of neighbouring cells. The results show clearly that the proposed method is more accurate than the Floquet approach. Besides, the method requires only one electromagnetic simulation and light post-processing computations. Furthermore, this method is well-adapted for reconfigurable reflectarray no matter what the configuration is and how large the array is.

Fig. 1

 1 Fig. 1 reconfigurable reflectarray antenna in 2D

Fig. 2

 2 Fig.2 sub-array composed of 9 cells

Fig. 3

 3 Fig. 3 electromagnetic simulation of the 3x3 sub-array

Fig. 4

 4 Fig. 4 generalized S-matrix

Fig. 5

 5 Fig. 5 reflectarray phase shifting cell

Fig. 6

 6 Fig. 6 8x8 reflectarray

Fig. 7

 7 Fig.7comparison of the proposed method with 3x3, 5x5 sub-arrays and the Floquet approach for a 8x8 reflectarray

TABLE I PHASE

 I VALUES OF THE CELL WITH FLOQUET APPROACH AT NORMAL

	INCIDENCE
	State	Phase
	On-On	0°
	Off-Off	-93.1°
	On-Off	-176.5°
	Off-On	-224.6°

ACKNOWLEDGMENT

The authors thank Thales Systèmes Aéroportés, Elancourt, France for funding the study.


Emilie Bosc

Muriel Pressigout

Luce Morin

3D Video: new techniques and challenges

Three-dimensional television (3DTV) is meant to enhance conventional 2D television by the added feeling of depth. The introduction of 3DTV will be successful if the perceived image quality and the viewing comfort are at least comparable to conventional television. Indeed, 3DTV raises a number of challenges concerning various fields (technical and technological, evaluation protocols, etc.). The success of 3DTV at home is strongly related to the resolution of such questions. This paper discusses the 3DTV technology and the challenges to overcome.

I. INTRODUCTION

3D video has gained a growing interest for the last few years. Industrials have turned their attention to this technology and aim at providing users a brand new quality of experience that should offer more immersion. 3D video refers to tow main applications, namely 3D television (3DTV) and Free Viewpoint Video (FVV). 3DTV provides a depth feeling, and Free Viewpoint Video allows navigation inside the scene.

3DTV and FVV are thought as the logical evolution of conventional 2D television. Besides, 2D conventional techniques and protocols were naturally thought as appropriate candidates in view to an extension to 3D. However, it turns out not to be that simple: indeed, along the whole processing chain, from the acquisition to the display, choices should be studied in order to improve users' quality of experience.

This document identifies the new challenges brought out by 3DTV technology into different categories. Section II addresses the problem of data format and compression. Section III addresses the view synthesis question. Section IV addresses the evaluation question. Finally, Section V concludes the paper.

II. COMPRESSION METHODS CHALLENGE

Among the numerous data formats that were proposed as for 3D video [START_REF] Smolic | Coding algorithms for 3DTVa survey[END_REF], Multiview Video plus Depth (MVD) data is one of the most studied lately, but not exclusive (Layer depth Image (LDI) is also a candidate, derived from MVD). Indeed, most suited input data format is still questioned, regarding the targeted application.

MVD consists of a set of multiple sequences capturing the same scene at different viewpoints (namely texture data), with their associated per-pixel depth value (namely depth data) (Figure 1). Depth data provide information on scene geometry and help in virtual intermediate view generation.

Then, MVD data refer to a huge amount of data to be processed. Considering texture and depth information are required for view synthesis, efficient coding tools are needed.

Numerous methods have been proposed and an important part consists in improving the efficient H.264/AVC codec [START_REF]Advanced video coding for generic Audio-Visual services[END_REF], [START_REF] Wiegand | Overview of the h. 264/AVC video coding standard[END_REF] : H.264/MVC [START_REF]Text of ISO/IEC 14496-10:200X/FDAM 1 multiview video coding[END_REF] can be used as a compression method for both texture and depth videos of MVD. MVC stands for Multiview Video Coding [START_REF] Chen | The emerging MVC standard for 3D video services[END_REF].

However, most of the proposed methods are inspired from 2D codecs that are optimized for 2D perception. Yet, artifacts that may not be perceived in a 2D color image, may have drastic effects on depth data, as pointed out in [START_REF] Merkle | The effects of multiview depth video compression on multiview rendering[END_REF]. Indeed, the importance of depth data and texture data in view reconstruction is still discussed: because of the bandwidth constraints, it may be judicious to efficiently set this bit ratio between depth adn texture. Considering its capital role in the view synthesis processing, compression artifacts of depth data may lead to fatal synthesis errors when generating virtual views. For this reason, bit allocation between depth and texture data is still addressed [START_REF] Bosc | Bitrate allocation for multi-view video plus depth[END_REF].

III. VIEW SYNTHESIS ALGORITHMS

From depth data and texture data, new intermediate viewpoints can be synthesized with the help of depth-imagebased rendering techniques. The generated views can then be rendered on a conventional display, or a stereoscopic or an autosterescopic display. Generating a "virtual" view consists in synthesizing a novel view of the scene, from a viewpoint which differs from those captured by the cameras, relying on the available texture and depth data. The texture, that is the conventional 2D color sequences, gives the color information. The depth data are gray-scales images and are considered as a monochromatic signal. Each pixel of a depth map indicates the distance of the corresponding 3D-point from the camera. Based on projective geometry [START_REF] Hartley | Multiple view geometry in computer vision[END_REF], the 3D representation of a scene can be retrieved from a depth map.

Virtual view synthesis is essential because captured video sequences are generally not adapted to all 3D screen displays: to ensure depth feeling, synthesizing new viewpoint stereoscopic pairs is usually required. Depth Image Based Rendering algorithms (DIBR) algorithms are used but they are prone to projection errors, especially around depth discontinuities (Figure 2). Considerable effort ( [START_REF] Tanimoto | Reference softwares for depth estimation and view synthesis[END_REF]) has been made to overcome the challenge related to rendering. However, the proposed methods need to be validate through objective or subjective protocols that may differ from the 2D ones [START_REF] Chen | New requirements of subjective video quality assessment methodologies for 3DTV[END_REF].

IV. 3D PERCEPTION CHALLENGE

The compression question already consists of a serious challenge and in addition to this, is the problem of evaluating the proposed coding framework, or any new device. Those methods or devices need to be validated through robust measurements of particular features. The demand for highquality visual content makes the need for a reliable assessment protocol as essential. And indeed, the difficulty comes from the fact that 3D vision involves physiological mechanisms that are still not fully understood. In point of fact, thanks to recent studies that brought this complexity into focus, it is now more understandable. [START_REF] Seuntiens | Visual Experience of 3D TV[END_REF] reported that not only image quality should be taken into account for an assessment framework, but visual comfort and depth feeling as well. Concerning image quality, new distortion types have appeared, as discussed in [START_REF] Meesters | A survey of perceptual evaluations and requirements of three dimensional TV[END_REF], [START_REF] Cancellaro | Stereo video artifacts introduced by a distributed coding approach[END_REF]. Some of them are related to synthesis process, others are related to compression methods or display technologies. A few of them are: -keystone effect : the image look like a trapezoid.

ghosting effect: this is a shadow-like artifact.

cardboard effect: depth is perceived as unnatural, as discrete incoherent planes.

puppet-theater effect: characters in the scene appear miniaturized.

staircase effect: discontinuities appear between adjacent blocks. 3D video brings new types of artifacts that usual metrics may not be able to efficiently assess [START_REF] Bosc | Can 3D synthesized views be reliably assessed through usual subjective and objective evaluation protocols?[END_REF]. Especially, pixel-based metric PSNR (Peak Signal to Noise Ratio) revealed not to be sufficient for assessing synthesized views in which object translation may occur without affecting the perceived image quality. Furthermore, the problem of assessing 3D video refers first to the evaluated target. Assessment studies in 3D are not mature and there is no standardized assessment framework for 3D video. [START_REF] Chen | New requirements of subjective video quality assessment methodologies for 3DTV[END_REF] proposed new requirements for subjective video quality assessment. In particular, the authors point out the fact that the minimum of 15 observers, as recommended in ITU-BT.500, may not be sufficient because of the instability of viewers' assessment in 3D. As well, they suggest reconsidering the statistical analysis of viewers' opinion for appropriate rejection of incoherent responses. For 3DTV applications, the authors explained, above all, how test material (3D displays and content disparity) affects depth rendering. This invites to carefully analyze future experiment results.

V. CONCLUSION

There is a serious need to study and develop methods, tools, and requirements, in 3D video, that allow great 3D quality of experience. Task forces are required first to understand human visual system. That should be the key for future technical choices. This knowledge will help in defining more perceptual-based metrics and new assessments protocols. Then improvements are expected in the fields of compression, that should focus on 3D perception criteria, and view synthesis.

Fig. 1 .

 1 Fig. 1. MVD data consist of texture (left) and depth information (right).

Fig. 2 .

 2 Fig. 2. Rendering objects edges is a tough task for view synthesis algorithms.

ACKNOWLEDGMENT

This work is supported by ANR-PERSEE project n o ANR-09-BLAN-0170, ANR-CAIMAN project n o ANR-08-VERS-002.