Alexandre Albinet
email: alexandre.albinet@gmail.com

Eva Leoz-Garziandia

Hélène Budzinski

Eric Villenave

Eric Viilenave

Polycyclic Aromatic Hydrocarbons (PAHs), nitrated PAHs and oxygenated PAHs in ambient air of the Marseilles area (South of France): concentrations and sources

Keywords: Polycyclic aromatic hydrocarbons, Nitrated polycyclic aromatic hydrocarbons, Oxygenated polycyclic aromatic hydrocarbons, Atmospheric pollution

Polycyclic aromatic hydrocarbons (PAHs), nitrated

PAHs and oxygenated PAHs in ambient air of the Marseilles area (South of France)

Introduction

Polycyclic Aromatic Hydrocarbons (PAHs) are ubiquitous environmental contaminants. In the atmosphere, they are released as by-products from the incomplete combustion or pyrolysis of organic matter and fossil fuel. They have been intensively these last 30 years studied because of their potential carcinogenic and/or mutagenic properties [START_REF] Iarc | Overall evaluations of carcinogenicity an updating of IARC monographs[END_REF].

PAH derivatives containing nitro or oxygen groups [i.e. nitrated PAHs (NPAHs) and oxygenated PAHs (OPAHs), respectively] have been recognized as direct acting mutagens and carcinogens [START_REF] Durant | Human cell mutagenicity of oxygenated, nitrated and unsubstituted polycyclic aromatic hydrocarbons associated with urban aerosols[END_REF][START_REF] Durant | Human lymphoblast mutagens in urban airborne particles[END_REF][START_REF] Hannigan | Bioassay-Directed Chemical Analysis of Los Angeles Airborne Particulate Matter Using a Human Cell Mutagenicity Assay[END_REF][START_REF] Lewtas | Bioassay directed fractionation of the organic extract of SRM 1649 urban air particulate and ambient matter[END_REF][START_REF] Pedersen | Human-cell mutagens in respirable airborne particles in the northeastern United States. 1. Mutagenicity of fractionated samples[END_REF][START_REF] Pedersen | Human cell mutagens in respirable airborne particles from the Northeastern United States. 2. Quantification of mutagens and other organic compounds[END_REF][START_REF] Schuetzle | Sampling of vehicle emissions for chemical analysis and biological testing[END_REF]. As PAHs they are also formed during combustion processes or, in the atmosphere, by both gas and heterogeneous reactions of PAHs induced by atmospheric oxidants (OH, NO 3 , O 3 ) [START_REF] Arey | The formation of nitro-PAH from the gas-phase reactions of fluoranthene and pyrene with the OH radical in the presence of NO x[END_REF][START_REF] Atkinson | Formation of ring-retaining products from the OH radical-initiated reactions of benzene and toluene[END_REF][START_REF] Atkinson | Kinetics and nitro-products of gas-phase OH and NO 3 radical-initiated reactions of naphthalene, fluoranthene and pyrene[END_REF][START_REF] Atkinson | Atmospheric chemistry of gas-phase polycyclic aromatic hydrocarbons: formation of atmospheric mutagens[END_REF][START_REF] Barbas | Photochemical oxidation of phenanthrene sorbed on silica gel[END_REF]Environmental Health Criteria (EHC) 229., 2003;[START_REF] Helmig | OH radical-initiated gas-phase reaction products of phenanthrene[END_REF][START_REF] Perraudin | Identification and quantification of primary ozonation products of phenanthrene and anthracene adsorbed on silica particles[END_REF][START_REF] Sasaki | Products of the Gas-Phase OH and NO 3 Radical-Initiated Reactions of Naphthalene[END_REF].

However, atmospheric sources (primary or secondary) and concentrations of these PAHs derivatives are not yet well documented.

Several monitoring campaigns in various regions, such as Roma, Milano, Napoli (Italy), São Paulo (Brazil) [START_REF] Ciccioli | The ubiquitous occurence of nitro-PAH of photochemical origin in airborne particles[END_REF][START_REF] Ciccioli | Formation and transport of 2-nitrofluoranthene and 2-nitropyrene of photochemical origin in the troposphere[END_REF], Birmingham (England) [START_REF] Dimashki | Measurements of nitro-PAH in the atmospheres of two cities[END_REF], København (Denmark) (Feilberg et al., 2001;Feilberg and Nielsen, 2001), Athína (Greece) [START_REF] Marino | Nitro-PAH in ambient particulate matter in the atmosphere of Athens[END_REF], Barcelona (Spain) [START_REF] Bayona | Sources and seasonal variability of mutagenic agents in the Barcelona city aerosol[END_REF], Kanazawa (Japan) [START_REF] Hayakawa | Comparison of polycyclic aromatic hydrocarbons and nitropolycyclic aromatic hydrocarbons in airborne particulates collected in downtown and suburban Kanazawa, Japan[END_REF], Baltimore (Bamford and Baker, 2003), Los Angeles [START_REF] Reisen | Atmospheric reactions influence seasonal PAH and nitro-PAH concentrations in the Los Angeles basin[END_REF], Houston [START_REF] Wilson | Concentrations and phase distributions of nitrated and oxygenated polycyclic aromatic hydrocarbons in ambient air[END_REF] (USA) have already demonstrated the existence of NPAHs in the atmosphere and have proposed different mechanisms explaining their formation. Furthermore, most of the studies concerning NPAHs focus on a limited number of compounds (generally nitrofluoranthene and nitropyrene isomers) and solely in the particulate phase. To the best of our knowledge, only Bamford and Baker (2003) have performed an extensive study on NPAH gas/particle partitioning considering a large variety of compounds (molecular weights between 173 and 292 g mol -1 ).

The number of studies concerning OPAHs is much more limited. Several OPAHs have been identified in ambient air samples but they still have not been quantified [START_REF] Alastair | Online coupled LC-GC-ITD/MS for the identification of alkylated, oxygenated, and nitrated polycyclic aromatic compounds in urban air particulate extracts[END_REF][START_REF] Bodzek | Identification of oxygen derivatives of polycyclic aromatic hydrocarbons in airborne particulate matter of upper Silesia (Poland)[END_REF][START_REF] König | Determination of oxygenated polycyclic aromatic hydrocarbons in airborne particulate matter by capillary gas chromatography and gas chromatography / mass spectrometry[END_REF][START_REF] Oda | Identification of polycyclic aromatic hydrocarbons of the particulate accumulated in the tunnel duct of freeway and generation of their oxygenated derivatives[END_REF][START_REF] Ramdhal | Polycyclic aromatic ketones in environmental samples[END_REF]. The only quantified and identified OPAHs are all very different, from ketones to dione [START_REF] Allen | Measurement of oxygenated polycyclic aromatic hydrocarbons associated with a size-segregated urban aerosol[END_REF][START_REF] Chung | Aerosol-borne quinones and reactive oxygen species generation by particulate matter extracts[END_REF]Feilberg et al., 2001;[START_REF] Fraser | Air Quality Model Evaluation Data for Organics. 5. C6-C22 Nonpolar and Semipolar Aromatic Compounds[END_REF][START_REF] Kishikawa | Concentration and trend of 9,10phenanthrenequinone in airborne particulates collected in Nagasaki city, Japan[END_REF][START_REF] Leoz-Garziandia | Sampling and analysis of polycyclic aromatic hydrocarbons (PAH) and oxygenated PAH in diesel exhaust and ambient air[END_REF][START_REF] Ligocki | Measurement of gas/particle distributions of atmospheric organic compounds[END_REF][START_REF] Liu | Oxidant denuder sampling for analysis of polycyclic aromatic hydrocarbons and their oxygenated derivates in ambient aerosol: Evaluation of sampling artefact[END_REF][START_REF] Del Rosario Sienra | Oxygenated polycyclic aromatic hydrocarbons in urban air particulate matter[END_REF][START_REF] Niederer | Determination of polycyclic aromatic hydrocarbons and substitutes (nitro-, oxy-PAHs) in urban soil and airborne particulate by GC-MS and NCI-MS/MS[END_REF][START_REF] Nielsen | The variation of street air levels of PAH and other mutagenic PAC in relation to regulations of traffic emissions and the impact of atmospheric processes[END_REF][START_REF] Schnelle-Kreis | Occurrence of particleassociated polycyclic aromatic compounds in ambient air of the city of Munich[END_REF][START_REF] Tsapakis | The composition and sources of PM 2.5 organic aerosol in two urban areas of Chile[END_REF][START_REF] Yassaa | Organic aerosols in urban and waste landfill of Algiers metropolitan area: Occurrence and sources[END_REF]. OPAHs source studies in real atmosphere are quasi non existent in the literature [START_REF] Chung | Aerosol-borne quinones and reactive oxygen species generation by particulate matter extracts[END_REF]Feilberg et al., 2001;[START_REF] Kishikawa | Concentration and trend of 9,10phenanthrenequinone in airborne particulates collected in Nagasaki city, Japan[END_REF][START_REF] Liu | Oxidant denuder sampling for analysis of polycyclic aromatic hydrocarbons and their oxygenated derivates in ambient aerosol: Evaluation of sampling artefact[END_REF][START_REF] Nielsen | The variation of street air levels of PAH and other mutagenic PAC in relation to regulations of traffic emissions and the impact of atmospheric processes[END_REF][START_REF] Schnelle-Kreis | Occurrence of particleassociated polycyclic aromatic compounds in ambient air of the city of Munich[END_REF]. On the other hand, their formation pathways have already been studied in laboratory [START_REF] Barbas | Photochemical oxidation of phenanthrene sorbed on silica gel[END_REF][START_REF] Bunce | Reaction of naphthalene and its derivatives with hydroxyl radicals in the gas phase[END_REF][START_REF] Esteve | Heterogeneous reactivity of OH radicals with phenanthrene[END_REF][START_REF] Perraudin | Identification and quantification of primary ozonation products of phenanthrene and anthracene adsorbed on silica particles[END_REF][START_REF] Wang | Formation of 9,10-phenanthrenequinone by atmospheric gasphase reactions of phenanthrene[END_REF].

The main objectives of this study were to characterize the NPAHs and OPAHs in both the gas and particulate phases in three different sites in the Marseilles area, and to identify the main primary and/or secondary sources of these contaminants. Marseilles (South of France) is an important city (around 1 million inhabitants) of the Mediterranean area characterised by an important solar irradiation, a heavy traffic and several industrial activities. Sampling locations were chosen according to their potentially important O 3 and NO x concentrations, in order to make possible secondary formation of such compounds. Others atmospheric constituents, such as O 3 , NO, NO 2 , PM 10 and PM 2.5 were recorded (not at all sampling sites) in order to investigate their correlations with PAHs, NPAHs and OPAHs concentrations.

Methods

Sampling sites and meteorological conditions

Measurements were carried out in the Marseilles area from Thursday 07/22/2004 to Thursday 07/29/2004. These 7 days were characterised by high temperatures (averaging 26 to 32°C) and intermediate O 3 concentrations (from 66 to 130 µg m -3 ). No precipitation occurred during the entire duration of the campaign. Three different types of sites (urban, sub-urban and rural) were instrumented for the sampling of PAHs and their derivatives (Fig. 1). The urban sampling site (43°18'20"N, 5°23'40"E, 64 m.a.s.l.) was installed downtown in a park named "Cinq Avenues", away from direct emissions of PAHs. Samplers were located on the roof of the Observatory of Marseilles. The downwind sub-urban site (43°17'17"N, 5°30'42"E, 72 m.a.s.l.) was located in the city of "La Penne sur Huveaune" (6 000 inhabitants), distant from direct vehicular sources but close (<1 km) to a Rilsan's factory (polyamide from castor oil, Atofina). Samplers were installed on the roof of the municipal stadium. The rural site (43°19'45"N, 5°43'4"E, 701 m.a.s.l.) was located in the massif of "La Sainte-Beaume" in the village of "Plan d'Aups" Sainte-Baume (800 inhabitants), about 30 km from the urban areas of Marseilles, Aix en Provence and Toulon. The sampling site was located in the upper part of the village, far from local sources. The surroundings of the station are covered by typical Mediterranean vegetation.

Sample collection

Both ambient air particulate (on quartz filter, Schleicher & Schuell,∅=150 mm) and gas (on PUF polyurethane foams, Tisch Environmental, 79 mm long) phases were sampled using modified high volume samplers equipped with PM 10 head (Megatec Digitel DA-80, 30 m 3 h -1 ) in order to determine parent PAHs and PAH derivatives concentrations. Prior to sampling, quartz fiber filters were calcined at 500°C during 12 h to remove any organic compounds that may be present on the filters.

PUF were pre-washed with dichloromethane using pressurised liquid extraction (PLE; Dionex trade name ASE for accelerated solvent extraction) with a Dionex ASE 300 system (90°C, 100 bars, heat time at 5 min, static time at 5 min, 3 cycles). Samplings were performed every 12 h (starting at 08:00 local time). After collection, filters were stored at -18°C in individual pre-washed glass Petri dishes.

PUF were also stored with their glass holder at -18°C. All samples were wrapped in aluminium foil and placed in opaque reclosable poly bags.

Overall, 40 total filters together with 40 PUFs were collected during this sampling campaign.

Field blanks (ie, exposed PUFs and filters without any air filtration) were collected bi-weekly for each sampling site. NO x (NO and NO 2 ), O 3 , PM 10 and PM 2.5 measurements were performed by AIRMARAIX, the local air quality network, with a 15 min time resolution (Environnement S.A. AC31M; Environnement S.A. 0341M; R&P TEOM 1400A, PM 10 and PM 2.5 head; respectively). All measurements were performed at the urban site. PM 10 and PM 2.5 were not available at the sub-urban site and only O 3 was measured at the rural site.

Sample extraction and analysis

The complete PAH, NPAH and OPAH analytical procedures are presented in detail elsewhere [START_REF] Albinet | Simultaneous analysis of oxygenated and nitrated polycyclic aromatic hydrocarbons on standard reference material 1649a (urban dust) and on natural ambient air samples by gas chromatography-mass spectrometry with negative ion chemical ionisation[END_REF]. Briefly, filters and PUF were extracted with dichloromethane using Dionex ASE 200 and ASE 300 systems, respectively. Extracts were divided into two fractions. The first one, being used for the determination of PAH concentrations and the second one for the NPAH and OPAH quantification. Both fractions were evaporated under a nitrogen stream (Zymark Turbovap II) down to a volume of 500 µl and adjusted to 1 ml with acetonitrile (PAHs) or dichloromethane (NPAHs and OPAHs). 15 PAHs (see Table1) were quantified using HPLC with fluorescence/UV detection with a ternary elution gradient (methanol, water, acetonitrile). Two analytical systems were used for the determination of PAH concentrations. The first one was used for high concentration levels (Varian 9012 LC pump, Varian 9075 fluorescence detector, Varian 310 UV detector) and the second one, for very low concentration levels (Dionex P580 pump, Dionex RF200 fluorescence detector, Dionex UVD1705 UV detector). Two reversed-phase columns (Supelco Supercosil LC-PAH C18, 250 mm × 4.6 mm × 5 µm and Alltech Alltima C8, 250 mm × 4.6 mm × 5 µm) connected in series were used in order to have a better separation of PAHs. NPAH and OPAH concentrations were determined simultaneously using gas chromatography-mass spectrometry with negative ion chemical ionisation (GC/NICI-MS) and selective ion monitoring mode (SIM). Analyses were performed using a Perkin-Elmer Clarus 500 gas chromatograph coupled to a Perkin-Elmer Turbomass Gold mass spectrometer. The column used was a 5% phenyl-substituted methylpolysiloxane (DB-5MS, 30 m × 0.25 mm I.D., 0.25 µm film thickness, J&W Scientific, USA). The method included a liquid chromatography purification procedure on solid phase extraction (SPE) cartridges (alumina and silica). A total of 17 NPAHs and 9 OPAHs were quantified (see Table1).

Quality assurance

PAH analytical procedure was validated during a (french) national inter-comparison campaign [START_REF] Leoz-Garziandia | Essais interlaboratoires sur les Hydrocarbures Aromatiques Polycycliques (HAP)[END_REF]. Three kinds of samples were analysed in order to identify the critical steps of the analytical protocols (low and high concentrations of prepared standard solutions, extracts of natural ambient air samples and diesel Standard Reference Material 2975). For each sample, four replicate analyses were carried out by each laboratory and mean values were calculated.

Application of the OPAH and NPAH analytical procedure has been performed on standard reference material (SRM 1649a: urban dust), giving repeatable results with a mean coefficient of variation (standard deviation/mean) about 0.2 and in good agreement with the few data available in the literature [START_REF] Albinet | Simultaneous analysis of oxygenated and nitrated polycyclic aromatic hydrocarbons on standard reference material 1649a (urban dust) and on natural ambient air samples by gas chromatography-mass spectrometry with negative ion chemical ionisation[END_REF].

Analytical limits of detection defined as the lowest concentration of the compound than can be detected (S/N=3) were used to calculate the methodology limits of detection for each individual compound. Individual NPAH detection limits in a typical air sample of 360 m 3 ranged between 0.01 and 0.07 pg m -3 . OPAH detection limits for an air sample of 360 m 3 ranged between 0.01 and 2.6 pg m -3 . PAH detection limits ranged between 16.7 and 111.1 pg m -3 for HPLC Varian system and between 0.08 and 1.11 pg m -3 for HPLC Dionex system.

The formation of NPAHs during sampling by heterogeneous reaction with nitrogen oxides was investigated in previous studies [START_REF] Arey | Formation of nitroarenes during high-volume sampling[END_REF]Bamford et al., 2003;[START_REF] Dimashki | Measurements of nitro-PAH in the atmospheres of two cities[END_REF].

During a winter high-NOx episode, the maximum percentage of artefact formation of the observed NPAHs was less than 2-3% [START_REF] Arey | Formation of nitroarenes during high-volume sampling[END_REF]. Results from a study conducted in a tunnel showed that the formation artefact represented less than 0.1% of the studied NPAHs [START_REF] Dimashki | Measurements of nitro-PAH in the atmospheres of two cities[END_REF].

Authors concluded that filter artefacts were not significant and were not problematic for NPAH ambient air samplings.

Concerning OPAHs, the study of [START_REF] Liu | Oxidant denuder sampling for analysis of polycyclic aromatic hydrocarbons and their oxygenated derivates in ambient aerosol: Evaluation of sampling artefact[END_REF], carried out with ozone scrubbers dealing with the identification of secondary compounds, does not allow to conclude about the formation of OPAHs during sampling.

Results and discussion

OPAH and NPAH concentrations

Table 1 presents all the OPAH and NPAH (filter + PUF) concentrations measured at the three instrumented sites. This table also presents, when available, the average temperature, and the NO, OPAH concentration levels are of the same order of magnitude as PAHs while NPAH concentrations are one to two orders of magnitude lower. Among all the 17 NPAHs and 9 OPAHs measured, only 1,4-anthraquinone was not detected in all samples either in gas or in the particulate phases. 9-Fluorenone and 9,10-anthraquinone were the most abundant OPAHs quantitatively measured at the three sites and accounting respectively for about 60% and 20% of the total OPAH concentration. The lightest NPAHs, namely 1-and 2-nitronaphthalene, were the most abundant NPAHs. They account respectively for about 30-50% and 15-30% of the total studied NPAH concentration.

The OPAH concentrations measured at the urban and the sub-urban sites were similar to those already reported in previous studies for comparable sampling seasons (summer or spring) and site characteristics (urban) [START_REF] Allen | Measurement of oxygenated polycyclic aromatic hydrocarbons associated with a size-segregated urban aerosol[END_REF][START_REF] Chung | Aerosol-borne quinones and reactive oxygen species generation by particulate matter extracts[END_REF][START_REF] Fraser | Air Quality Model Evaluation Data for Organics. 5. C6-C22 Nonpolar and Semipolar Aromatic Compounds[END_REF][START_REF] Leoz-Garziandia | Sampling and analysis of polycyclic aromatic hydrocarbons (PAH) and oxygenated PAH in diesel exhaust and ambient air[END_REF][START_REF] Del Rosario Sienra | Oxygenated polycyclic aromatic hydrocarbons in urban air particulate matter[END_REF][START_REF] Tsapakis | The composition and sources of PM 2.5 organic aerosol in two urban areas of Chile[END_REF][START_REF] Wilson | Concentrations and phase distributions of nitrated and oxygenated polycyclic aromatic hydrocarbons in ambient air[END_REF][START_REF] Yassaa | Organic aerosols in urban and waste landfill of Algiers metropolitan area: Occurrence and sources[END_REF]. However, OPAH concentrations determined by Allen et al. [START_REF] Allen | Measurement of oxygenated polycyclic aromatic hydrocarbons associated with a size-segregated urban aerosol[END_REF] at Boston in particulate phase, are 10 times higher than those reported here (benzanthrone, benzo[a]fluorenone and benzo[b]fluorenone). In all previous studies with samplings realised in summer season, 9fluorenone and 9,10-anthraquinone are also the major measured OPAHs.

Atmospheric concentrations of NPAHs such as, 1-, 2-nitronaphthalene, 2+3-nitrofluoranthene, 1and 2-nitropyrene for the urban and sub-urban sites were similar to those reported in Roma [START_REF] Ciccioli | The ubiquitous occurence of nitro-PAH of photochemical origin in airborne particles[END_REF][START_REF] Ciccioli | Formation and transport of 2-nitrofluoranthene and 2-nitropyrene of photochemical origin in the troposphere[END_REF], Athína [START_REF] Marino | Nitro-PAH in ambient particulate matter in the atmosphere of Athens[END_REF], Barcelona [START_REF] Bayona | Sources and seasonal variability of mutagenic agents in the Barcelona city aerosol[END_REF] and Los Angeles [START_REF] Reisen | Atmospheric reactions influence seasonal PAH and nitro-PAH concentrations in the Los Angeles basin[END_REF] at similar sampling seasons. Nevertheless, except for 9nitroanthracene and 2+3-nitrofluoranthene, individual total NPAH concentrations are 2 to 30 times higher (for 3-nitrophenanthrene and 1-nitronaphthalene, respectively) than those reported by Bamford and Baker (Bamford and Baker, 2003) for comparable sampling conditions in Baltimore (Summer; urban and sub-urban sites).

NPAH and OPAH gas/particle partitioning

In general, the fraction of OPAHs and NPAHs associated with the particulate phase is strongly depending on the molecular weight (Table 1). As in previous reported studies [START_REF] Atkinson | Atmospheric chemistry of gas-phase polycyclic aromatic hydrocarbons: formation of atmospheric mutagens[END_REF]Bamford and Baker, 2003;[START_REF] Fraser | Air Quality Model Evaluation Data for Organics. 5. C6-C22 Nonpolar and Semipolar Aromatic Compounds[END_REF][START_REF] Leoz-Garziandia | Sampling and analysis of polycyclic aromatic hydrocarbons (PAH) and oxygenated PAH in diesel exhaust and ambient air[END_REF][START_REF] Liu | Oxidant denuder sampling for analysis of polycyclic aromatic hydrocarbons and their oxygenated derivates in ambient aerosol: Evaluation of sampling artefact[END_REF][START_REF] Reisen | Atmospheric reactions influence seasonal PAH and nitro-PAH concentrations in the Los Angeles basin[END_REF][START_REF] Wilson | Concentrations and phase distributions of nitrated and oxygenated polycyclic aromatic hydrocarbons in ambient air[END_REF], the lightest compounds (1-napthtaldehyde to 9phenanthrenecarboxaldehyde for OPAHs; 1-and 2-nitronaphtalene for NPAHs) were detected mainly in the gas phase (>50%). More than 90% of the compounds with a number of aromatics cycles ≥4 were detected in the particulate phase in all samples. Despite similar ambient temperatures (26 to 32 °C), the partitioning of the nitrophenanthrene isomers (3 aromatics cycles) measured in the particulate phase presents strong variations between the sampling sites. The variability of the different sources associated with each individual NPAHs could explain these partitioning variations.

The same hypothesis may explain the partitioning dissimilarities of benzo[a]fluorenone according to the sampling locations. Finally, it is important to note that a light compound as 2-nitrofluorene (MW=211 g mol -1 ) was essentially associated to the particulate phase (>80%) at any sampling site.

At the rural site, only 9-phenanthrenecarboxaldehyde presented a significant variation of gas/particle partitioning from day to night-time samples with higher parts on the filters systematically observed during the night. Same changes were observed at the sub-urban site for this OPAH and for three NPAHs namely, 9-nitroanthracene, 3-and 9-nitrophenanthrene. During the day, these gaseous OPAHs and NPAHs are fastly degraded by photolysis. Lower photolysis during night-time period allows a longer life time for these compounds emitted and/or formed in gaseous phase. Such a longer lifetime allows a larger adsorption to ambient particles.

NPAHs: gas phase formation vs. direct sources

Some source specific ratios of NPAHs were investigated in order to assess the relative importance of primary sources versus secondary sources arising from PAH atmospheric degradation for the three sampling sites. The ratio of 2-nitrofluoranthene on 1-nitropyrene (2-NF/1-NP) concentrations is generally used to evaluate the relative contribution of both types of sources (Bamford and Baker, 2003;[START_REF] Ciccioli | Evaluation of nitrated polycyclic aromatic hydrocarbons in anthropogenic emission and air samples[END_REF][START_REF] Ciccioli | Formation and transport of 2-nitrofluoranthene and 2-nitropyrene of photochemical origin in the troposphere[END_REF]Feilberg and Nielsen, 2001;[START_REF] Marino | Nitro-PAH in ambient particulate matter in the atmosphere of Athens[END_REF]Zielinska et al., 1989a). 2-NF is only produced from the gas phase reaction between fluoranthene and NO2 initiated by OH during the day and initiated by NO 3 during the night [START_REF] Arey | The formation of nitro-PAH from the gas-phase reactions of fluoranthene and pyrene with the OH radical in the presence of NO x[END_REF][START_REF] Atkinson | Evidence for the transformation of polycyclic organic matter in the atmosphere[END_REF]. 1-NP is strictly arising from direct emissions (it has never been observed to result from any know gas phase reaction) [START_REF] Arey | Atmospheric reactions of PAHs including formation of nitroarenes[END_REF][START_REF] Nielson | Reactivity of polycyclic aromatic hydrocarbons toward nitrating species[END_REF][START_REF] Paputa-Peck | Determination of nitrated polynuclear aromatic hydrocarbons in particulate extracts by capillary column gas chromatography with nitrogen selective detection[END_REF]. Assuming reasonably for 2-NF and 1-NP the same removal rates and the same photolysis rates (Fan et al., 1996b;[START_REF] Feilberg | Effect of aerosol chemical composition on the photodegradation of nitro-polycyclic aromatic hydrocarbons[END_REF][START_REF] Kamens | A methodology for modeling the formation and decay of nitro-PAH in the atmosphere[END_REF], a value for the 2NF/1NP ratio less than five shows the relative extent of primary emission sources whereas a ratio value larger than five will highlight the importance of the secondary formation of NPAHs [START_REF] Ciccioli | Formation and transport of 2-nitrofluoranthene and 2-nitropyrene of photochemical origin in the troposphere[END_REF]. Fig. 2 presents the observed 2+3-NF/1-NP ratios at the three instrumented sites. It should be noted that the separation of both isomers of nitrofluoranthene (2-and 3-nitrofluoranthene) could not be achieved on the 5% phenyl-substituted methylpolysiloxane phase (DB5-MS) used in this study. Considering the relative low proportion of 3-nitrofluoranthene reported in other previous studies [< 1% in (Bamford and Baker, 2003;Zielinska et al., 1989b) and <50% in (Feilberg et al., 2001)], it was finally decided to us to use the 2+3-NF/1-NP ratio with care by fixing here the upper limit at 10 instead of 5. Indeed, the urban and sub-urban sites are clearly influenced by the primary NPAH sources (ratio <10) whereas "Plan d'Aups" (the rural site) shows an evidence for the production of secondary NPAHs by gas phase reactions (ratio >10).

The 2-NF/2-NP concentration ratio (2-nitrofluoranthene/2-nitropyrene) is usually used to evaluate the occurrence and the importance of the gas phase production of NPAHs from OH and/or NO 3 initiated oxidation pathways [START_REF] Arey | Nitroarene products from the gas-phase reactions of volatile polycyclic aromatic hydrocarbons with the OH radical and N 2 O 5[END_REF][START_REF] Atkinson | Atmospheric chemistry of gas-phase polycyclic aromatic hydrocarbons: formation of atmospheric mutagens[END_REF]Zielinska et al., 1989a). 2-NF is the major NPAH product of the gas phase reaction of fluoranthene with OH (Arey et al., 1986) and the only nitrofluoranthene formed from the NO 3 initiated reaction [START_REF] Zielinska | Reaction of dinitrogen pentoxide with fluoranthene[END_REF]. 2-nitropyrene is the only nitropyrene formed from the reaction with OH but is not formed from the gas phase reaction of pyrene with NO 3 [START_REF] Arey | The formation of nitro-PAH from the gas-phase reactions of fluoranthene and pyrene with the OH radical in the presence of NO x[END_REF][START_REF] Atkinson | Kinetics and nitro-products of gas-phase OH and NO 3 radical-initiated reactions of naphthalene, fluoranthene and pyrene[END_REF]Zielinska et al., 1986). Ratios close to 10 indicate day-time OH reactions, and ratios > 100 rather suggest nighttime NO 3 formation pathways. At "Plan d'Aups", the 2-NF/2-NP ratio value [average ratio (±1σ) = 3.7 (±4.4)] is consistent with the day-time OH reaction as the dominant gas phase formation pathway compared to the NO 3 initiated reaction.

Focusing on the study of the 2+3-NF and 1-NP concentrations at the same site, an important coevolution of these compounds during the sampling week (r=0.83, P<0.05) can be observed. The same phenomenon, but lightly shifted is observed between the variations of the 2+3-NF/1-NP ratio and the 1-NP concentrations (Fig. 3). These observations suggest an external contribution secondary NPAHs at this site (higher values of the 2+3-NF/1-NP ratio and lower 1-NP concentrations) with the simultaneous existence of local NPAH sources ([2+3-NF] and [1-NP] correlation). A thorough study of the highest 2+3-NF/1-NP ratio values (>10) compared with the wind directions observed during the campaign period, shows that the external contribution of the secondary NPAHs would come from the urban areas of Marseilles (SW) and Aix en Provence (NW) (Fig. 1 and3). This result is consistent with the observations already made by Marinoni et al. at [START_REF] Marinoni | Physicochemical properties of fine aerosols at Plan d'Aups during ESCOMPTE[END_REF]. By contrast, at the urban and sub-urban sites, the 2+3-NF/1-NP ratio is fairly constant and below 5, and the 1-NP and 2+3-NF concentration evolutions are well correlated (r=0.93 and 0.76, P<0.05), confirming the primary origin of the measured NPAHs by the proximity of the emissions sources.

Source apportionment of NPAHs

To assess NPAH source distribution, relationships between ambient NPAH concentrations and NO, NO x , O 3 , PM 10 and PM 2.5 were investigated. The correlation between some NPAHs and more specially those considered as source tracers such as 1-NP (direct emission), 2-NP and 2+3-NF (secondary compounds), were also studied.

At the urban site of "5 avenues", except for a few compounds such as 1-and 2-nitronaphthalene (1-NN and 2-NN), 9-nitroanthracene (9-NA), 7-nitrobenz[a]anthracene (7-NB[a]A) and 3nitrophenanthrene (3-NPh), no significant correlation was observed between individual NPAH and NO, NO x , O 3 , PM 10 and PM 2.5 concentrations (Table 2). The lack of significant positive correlations between direct emission tracers (1-NP, predominantly diesel engines) and NO and NO x may be due to the large contribution of gasoline emissions to the NO and NO x levels measured at this site compared to diesel sources. Indeed, the gasoline influence is confirmed by the strong correlation between NO (r=0.72), NO x (r=0.79) and the ratio {(B[ghi]P+Cor)/Σ PAHp} 1 representative of gasoline emissions and previously used by [START_REF] Marchand | Polycyclic aromatic hydrocarbons (PAHs) in the atmospheres of two French alpine valleys: sources and temporal patterns[END_REF] for its capacity to discriminate gasoline to diesel influences.

Therefore, the variations of the NPAH concentrations seem to be linked to changes in the gasoline source contribution (r=0.80, P<0.05), with a constant contribution from diesel sources represented by a fairly constant (B[b]F+B[k]F)/Σ PAHp) ratio.

1 Σ PAHp: sum of filter concentrations of 10 PAHs mainly present in the particulate phase: from B[a]A to Cor Previous studies have already shown the primary diesel sources of 1-NN, 2-NN, 9-NA,7-NB[a]A [START_REF] Bezabeh | Determination of nitrated polycyclic aromatic hydrocarbons in diesel particulate-related standard reference materials by using gas chromatography/mass spectrometry with negative ion chemical ionization[END_REF][START_REF] Chiu | An improved method for nitro-PAH analysis[END_REF]Feilberg et al., 2001;[START_REF] Paputa-Peck | Determination of nitrated polynuclear aromatic hydrocarbons in particulate extracts by capillary column gas chromatography with nitrogen selective detection[END_REF] whereas such compounds seem to be also emitted by gasoline engines (Table 2). The strong correlation between 3-NPh and NO and the B[g,h,i]P+Cor)/Σ PAHp ratio lets suppose a gasoline origin of this compound at this site.

At the sub-urban site of "Penne sur Huveaune", except for 2-nitrofluorene (2-NFluo) and 6nitrochysene (6-NChr), all NPAHs (and OPAHs) were significantly correlated with NO and NO x and interrelated. NPAHs and OPAHs were also correlated with PAHs. On the other hand, no correlation was observed with PAH ratios used to discriminate source influences {i.e. (B[ghi]P+Cor)/Σ PAHp for gasoline and (B[b]F+B[k]F)/Σ PAHp) for diesel} [START_REF] Marchand | Polycyclic aromatic hydrocarbons (PAHs) in the atmospheres of two French alpine valleys: sources and temporal patterns[END_REF]. This could be explained by the very close presence of emission sources to the sampling point, emissions, which are not necessarily traffic oriented. The proximity with the Atofina plant may disturb the PAH, NPAH and OPAH chemical signatures and influence the gas phase distribution, making very difficult the interpretation of the data with respect to the sources.

Despite of an evident production of secondary NPAHs at the rural site of "Plan d'Aups", no correlation was observed between any NPAH and secondary NPAH tracers (2-NP and 2+3-NF).

At the urban site of "5 avenues" 1-NP, 2-nitrofluorene (2-NFluo) and 6-nitrochrysene (6-NChr) are strongly correlated (r=0.99 and 1.00, respectively). This strong correlation is also observed at the rural site of "Plan d'Aups" (0.98 and 0.97, respectively). Usually, 1NP is the predominant component in diesel particulate matter. However, it is not always the dominating substance.

Especially in heavy-duty diesel, 2-NFluo concentration may exceed that of 1-NP by a factor of 1.8-15. When 1-NP is the dominant substance, a 2-NFluo concentration equal to 15% of that of 1-NP is commonly observed (Environmental Health Criteria (EHC) 229., 2003). Strong correlations observed in our study and the previous literature data demonstrated that 1-NP, 2-NFluo and 6-NChr are primarily emitted by diesel vehicles.

At the three investigated sites, NPAHs mostly present negative correlation coefficients with O 3 (very noticeable at the sub-urban site of "la Penne sur Huveaune"). Such negative correlations have been already observed in previous works (Bamford and Baker, 2003;Feilberg et al., 2001). Studies of reactions of NPAHs with O 3 carried out in a smog chamber concluded that such degradation may take place. While photolysis remains the main degradation pathway for NPAHs, the reaction with O 3 could be an important night-time sink (Fan et al., 1996a;Fan et al., 1996b).

Source apportionment of OPAHs

A principal component analysis (PCA) was performed as a first approach. OPAH concentrations, some NPAH tracer concentrations and (B[ghi]P+Cor)/Σ PAHp ratio were compared for all samples (Fig. 4 and Table 3). Prior to the PCA, each compound concentration was normalized to the total OPAH or NPAH concentration. Due to the poor quantification of the 1-naphthaldehyde (low recovery factor), it was not taken into account in the PCA.

The first and the second principal components described 41% and 24% of the variability, respectively (Fig. 4). Four OPAH clusters are observed. The cluster formation as well as its position on the graph is closely related to its homogeneity with respect to potential sources or physicochemical properties.

The 9-fluorenone and to 2-nitronaphthalene cluster (cluster 1) can be explained by their largest presence in the gas phase. In addition, no significant correlations are observed with NPAH tracers and PAH source ratios, nor between 9-fluorenone concentration evolution and PM, NO and NO x concentrations. Finally, no information could be drawn about the primary and/or secondary origin of this compound.

A second OPAH cluster was constituted by 9-phenanthrenecarboxaldehyde, benzo[a]fluorenone, benzo[b]fluorenone and benzanthrone. Strong correlations between their concentrations and that of NO suggest a primary origin of these compounds (Table 4). Moreover, the absence of any correlation with the 1-NP and a significant correlation between the 9-phenanthrencarboxladehyde and the PAH gasoline ratio (Tables 3 and4) let suppose a probable gasoline vehicle origin of these OPAHs. In a previous study, Fielberg at al. (Feilberg et al., 2001) showed that the dominant source of benzanthrone in København center, was diesel vehicles; however it has not been clearly established in our case. The results presented in Tables 3 and4 show a possible contribution of the gasoline vehicles to its emissions.

The proximity between the benz[a]anthracene-7,12-dione (cluster 3) and the second OPAH cluster (see Fig. 4) demonstrates a good correlation between all these compounds suggesting a primary origin. The correlation with 1-NP as well as the absence of any correlation with the PAH gasoline ratio, indicates a potential diesel origin of this compound. This hypothesis is confirmed by the strong correlation observed with 1-NP, 2-Fluo and 6-NChr (r=0.79, 0.75 and 0.77, P<0.05) at the urban site.

The 9,10-anthraquinone is part of the fourth cluster (Fig. 4) and seems to have a double-origin. It is correlated with a primary diesel compound like 1-NP and with secondary compounds such as 2+3-NF and 2-NP (Table 3). Diesel origin is confirmed by the strong correlation observed with the 1-NP, 2-NFluo and 6-NChr at the urban site of "5 avenues " (r= 0.62, 0.67 and 0.66, P<0.05). The 9,10anthraquinone is the only OPAH positively correlated with O 3 at the urban site (even though the relation is not statistically significant) (Table 4). This observation suggests the gas phase formation of 9,10-anthraquinone by the ozonation of parent PAHs. The study of [START_REF] Perraudin | Identification and quantification of primary ozonation products of phenanthrene and anthracene adsorbed on silica particles[END_REF] in a simulated atmosphere showed that 9,10-anthraquinone and anthrone were the two major primary ozonation products of particle-bound anthracene. The formation of 9,10anthraquinone by the ozonation of anthracene was studied at the rural site of "Plan d'Aups". Fig. 5 shows a good correlation between the 9,10-anthraquinone and the O 3 concentrations from 07/23 night to 07/26 night. During the others days of the sampling week, low 9,10-anthraquinone levels are in agreement with high levels of Σ PAHg+p 2 . The absence or the low concentration levels of anthracene in ambient air can explain the low levels of 9,10-anthraquinone whereas high levels of O 3 and of Σ PAHg+p are observed (07/23, 27 and 28 days).

2 Σ PAHg+p: sum of total concentration (filter + PUF) of 5 PAHs mainly in the gas phase or partitioned between the gas and the particulate phases, from fluorene to pyrene.

The formation of 9,10-anthraquinone from the anthracene ozonation was observed at this site.

However, the next step will be the identification of when, before or during the sampling, this reaction takes place. Only one study has been carried out with ozone scrubbers dealing with the identification of secondary compounds [START_REF] Liu | Oxidant denuder sampling for analysis of polycyclic aromatic hydrocarbons and their oxygenated derivates in ambient aerosol: Evaluation of sampling artefact[END_REF]; it does not allow to conclude about the origin of the reaction. Further investigations on NPAH and OPAH formations in ambient atmosphere will be clearly necessary because of their potential toxicity and the lack of data available. Black triangles represent the average ratios. (ng m -3 ) at the rural site of "Plan d'Aups". * Σ PAHg+p: sum of total concentration (filter + PUF) of 5 PAHs mainly in the gas phase or partitioned between the gas and the particulate phases, from fluorene to pyrene.

  NO x , O 3 , PM 10 , PM 2.5 and each individual PAH concentrations. The arithmetic average of the concentrations of the field blanks was taken into account for the calculation of atmospheric concentrations. As it can be noted, NPAH and OPAH concentrations are consistent with the characteristics of the sampling sites (urban, sub-urban and rural) with the highest concentrations measured for the sampling stations close to car engine emissions. Concentrations of individual compounds show large daily basis variations related to their sources and sinks.

Fig. 1 .

 1 Fig. 1. Sampling sites location.

Fig. 3 .

 3 Fig. 3. A: Weekly variations of 1-NP and 2+3-NF concentrations and the 2+3-nitrofluoranthene/1-

Fig. 4 .

 4 Fig. 4. Correlation circle of normalized single OPAH concentrations (filter + PUF) with normalized

Fig. 5 .

 5 Fig. 5. Weekly evolution of 9,10-anthraquinone (pg m -3 ), O 3 (µg m -3 ), Σ PAHg+p * and anthracene

Table 2

 2 Pearson correlations coefficients (n=12) of total (filter + PUF) concentrations of individual NPAHs with PM 10 , PM 2.5 , NO, NO x , O 3 concentrations and with (B[ghi]P+Cor)/Σ PAHp * and (B[b]F+B[k]F)/Σ PAHp ratios at the urban site of "5 avenues"

		PM 2.5	PM 10	NO	NO x	O 3	(B[ghi]P+Cor)/Σ PAHp	(B[b]F+B[k]F)/Σ PAHp
	1-Nitronaphthalene	0.34	-0.04	0.50	0.79	-0.78	0.81	-0.41
	2-Nitronaphthalene	0.33	-0.06	0.36	0.71	-0.73	0.72	-0.34
	2-Nitrofluorene	0.10	0.00	-0.16	-0.25	0.35	0.12	0.07
	9-Nitroanthracene	0.44	0.22	0.66	0.53	-0.67	0.71	-0.17
	9-Nitrophenanthrene	0.13	-0.23	0.03	0.45	0.07	0.11	-0.25
	3-Nitrophenanthrene	0.70	0.62	0.81	0.53	-0.48	0.50	-0.32
	2+3-Nitrofluoranthene	0.30	0.10	-0.15	-0.05	0.16	0.23	-0.05
	4-Nitropyrene	0.25	0.35	0.56	0.13	-0.53	0.45	0.12
	1-Nitropyrene	0.17	0.07	-0.11	-0.19	0.31	0.15	0.01
	2-Nitropyrene	0.03	0.12	0.58	0.10	-0.59	0.21	0.31
	7-Nitrobenz[a]anthracene	0.20	-0.10	0.42	0.68	-0.83	0.65	-0.23
	6-Nitrochrysene	0.11	0.03	-0.15	-0.27	0.36	0.09	0.08
	6-Nitrobenzo[a]pyrene	0.39	0.19	0.07	0.26	-0.55	-0.36	0.53
	(B[ghi]P+Cor)/Σ PAHp	0.48	0.10	0.72	0.79	-0.60
	(B[b]F+B[k]F)/Σ PAHp	-0.27	-0.42	-0.47	-0.63	-0.27

In bold, correlation significant at the P<0.05 level (2 tailed)

In bold, underlined: no correlation was taken into account because the compound is essentially present in the gas phase (see Table

1

) * Σ PAHp: sum of filter concentrations 10 PAHs mainly present in the particulate phase: from B[a]A to Cor

Table 3

 3 Pearson correlation coefficients (n=40) related to the Fig.4

		2-NN 2+3-NF 1-NP 2-NP (B[ghi]P+Cor)/Σ PAHp
	9-Fluorenone	0.47	-0.58	-0.64 -0.24	-0.13
	9-phenanthrenecarboxaldehyde -0.35	0.00	0.32 -0.26	0.46
	9,10-Anthraquinone	-0.36	0.62	0.59	0.33	0.00
	Benzo[a]fluorenone	-0.26	0.02	0.17 -0.10	0.12
	Benzo[b]fluorenone	-0.44	0.10	0.32 -0.07	0.29
	Benzanthrone	-0.37	-0.04	0.13 -0.09	0.47
	Benz[a]anthracene-7,12-dione	-0.48	0.33	0.54 -0.14	0.18
	In bold, correlation significant at the P<0.05 level (2 tailed)

Table 4

 4 Pearson correlation coefficients (n=12) of total concentrations (filter + PUF) of OPAHs with PM 10 , PM 2.5 , NO, NO x and O 3 concentrations and with (B[ghi]P+Cor)/Σ PAHp and (B[b]F+B[k]F)/Σ PAHp ratios at the urban site of "5 avenues"

		PM 2.5	PM 10	NO	NO x	O 3	(B[ghi]P+Cor)/ Σ PAHp	(B[b]F+B[k]F)/ Σ PAHp
	9-Fluorenone	0.40	0.10	0.34	0.20	-0.67	0.49	0.28
	9-phenanthrenecarboxaldehyde	0.51	0.32	0.86	0.64	-0.50	0.79	-0.41
	9,10-Anthraquinone	-0.45	0.01	-0.28	-0.54	0.42	-0.22	0.10
	Benzo[a]fluorenone	0.43	0.53	0.82	0.37	-0.48	0.38	-0.18
	Benzo[b]fluorenone	0.48	0.50	0.76	0.32	-0.59	0.40	-0.05
	Benzanthrone	0.48	0.43	0.87	0.57	-0.81	0.58	-0.18
	Benz[a]anthracene-7,12-dione	0.32	0.16	0.18	0.05	-0.21	0.29	0.11
	(B[ghi]P+Cor)/Σ PAHp	0.48	0.10	0.72	0.79	-0.60	-	-
	(B[b]F+B[k]F)/Σ PAHp	-0.27	-0.42	-0.47	-0.63	0.08	-	-

In bold, correlation significant at the P<0.05 level (2 tailed)

Acknowledgements

The funding of this research was provided by the French Ministry of Ecology and Sustainable Development (MEDD) and the ADEME agency. Albinet. A. and Leoz-Garziandia E. wish to thank AIRMARAIX for NO x , O 3 , PM 10 and PM 2.5 measurements and sampling site access, Yoann Fagault for his greeting in his laboratory, Nadine Guillaumet and Sébastien Fable for PAH analyses, Amandine Fievet and Cristina Alegre for PAH sampling.

 Zielinska, B.,Arey, J.,Atkinson, R. and McElroy, P.A. Formation of methylnitronaphthalenes from the gas-phase reactions of 1-and 2-methylnaphthalene with OH radicals and N 2 O 5 and their occurrence in ambient air. Environ Sci Technol 1989a;23: 723-729. Zielinska, B.,Arey, J.,Atkinson, R. and Wiener, A. M. The nitroarenes of molecular weight 247 in ambient particulate samples collected in southern California. Atmos Environ 1989b;23: 223-229.

Table 1 Total (filter + PUF) PAH, OPAH, NPAH concentrations (pg m -3 ) and associated particulate fraction (%), T (°C), NO, NO x , PM 10 , PM 2.5 and O 3 concentrations (µg m -3 ) for the three instrumented sites "5 avenues" (urban)


Françoise Rogerieux
email: francoise.rogerieux@ineris.fr

Modifications of Phleum pratense grass pollen allergens following artificial exposure to gaseous air pollutants (O 3 , NO 2 , SO 2 )

Keywords: traffic-related pollution, grass pollen, allergen, two-dimensional electrophoresis, ozone, nitrogen dioxide, sulphur dioxide

Background: Air pollution is frequently proposed as a potential cause for the increased incidence of allergy in industrialised countries. Our objective was to investigate the impact of the major gaseous air pollutants on grass pollen allergens.

Methods: Timothy grass pollen was exposed to O 3 , NO 2 , SO 2 alone or mixed.

Allergen contents were analysed by 2D-immunoblot using grass pollen-sensitive patient sera.

Results: For O 3 -treated pollen, immunoblotting showed an acidification of allergens Phl p 1b, 4, 5 and 6 and an IgE recognition decrease in Phl p 1, 2, 6, 13. NO 2exposure induced a decrease in Phl p 2, 5b and 6 recognition and SO2-treatment induced a decrease in Phl p 2, 6 and 13 recognition. Moreover, samples treated with a mix of NO 2 / O 3 or NO 2 / SO 2 showed a higher decrease in allergen content, compared to samples treated with only one pollutant. The O 3 acidification was also observed with the mix NO 2 /O 3 .

Conclusion:

Exposure of pollen to gaseous pollutants induced a decrease in allergen detection in pollen extracts. This decrease could be due to a mechanical loss of allergens from the altered pollen grains and/or. post-translational modifications affecting allergen recognition by IgE.

Introduction

Respiratory allergic diseases are increasing world-wide in both prevalence and severity, affecting in particular subjects living in urban areas [START_REF] Amato | Environmental urban factors (air pollution and allergens) and the rising trends in allergic respiratory diseases[END_REF][START_REF] Ring | Why are allergies increasing?[END_REF] . Although the role played by air pollution has yet to be clarified, a body of evidence suggests that urbanisation, with its high levels of transport emissions and a westernised lifestyle are linked to the rising frequency of these diseases observed in most of the industrialised countries [START_REF] Von Mutius | Increasing prevalence of hay fever and atopy among children in Leipzig, East Germany[END_REF][START_REF] Nicolai | Urban traffic and pollutant exposure related to respiratory outcomes and atopy in a large sample of children[END_REF][START_REF] Trasande | The role of air pollution in asthma and other pediatric morbidities[END_REF] . Atmospheric pollution is a complex mixture of gaseous and particulate compounds interacting with each other. The main classical gaseous air pollutants are nitrogen dioxide (NO 2 ), ozone (O 3 ) and sulphur dioxide (SO 2 ). Table I summarise for each one, the main sources and European norms for atmospheric concentrations compared to the doses used in our study.

Laboratory studies confirmed the epidemiological evidences that inhalation of some pollutants, either individually or in combination, adversely affects lung function in asthmatics [START_REF] Jenkins | The effect of exposure to ozone and nitrogen dioxide on the airway response of atopic asthmatics to inhaled allergen: dose-and time-dependent effects[END_REF][START_REF] Barck | Brief exposures to NO2 augment the allergic inflammation in asthmatics[END_REF][START_REF] Tunnicliffe | The effect of sulphurous air pollutant exposures on symptoms, lung function, exhaled nitric oxide, and nasal epithelial lining fluid antioxidant concentrations in normal and asthmatic adults[END_REF][START_REF] Vagaggini | Ozone exposure increases eosinophilic airway response induced by previous allergen challenge[END_REF] and increases asthma severity [START_REF] Van Zijverden | Adjuvant activity of particulate pollutants in different mouse models[END_REF][START_REF] Nygaard | Ambient air particles from four European cities increase the primary cellular response to allergen in the draining lymph node[END_REF][START_REF] Dybing | Respiratory allergy adjuvant and inflammatory effects of urban ambient particles[END_REF] . However, the exact nature and role of outdoor pollutants in allergic sensitisation is still not entirely clear [START_REF] Ring | Environmental pollution and allergy[END_REF] .

Alternatively, air pollution can directly affect pollen grains. As an example, exposure to pollutants (CO, NO 2 and SO 2 at 10,000 ppm each) of 3 tree species and 1 grass pollens, has been shown to alter the allergen content [START_REF] Ruffin | Effects of certain atmospheric pollutants (SO2, NO2 and CO) on the soluble amino acids, molecular weight and antigenicity of some airborne pollen grains[END_REF][START_REF] Ruffin | The effect of some environmental gaseous pollutants on pollen-wall proteins of certain airborne pollen grains[END_REF] . After exposure of P. pratense pollen to high concentrations of SO 2 (16.5 mg/m 3 =7 ppm), but not to NO 2 (1 mg/m 3 =1 ppm), Phl p 5 recognition using mAb Bo1 was significantly reduced depending on exposure duration [START_REF] Behrendt | Air pollution and allergy: experimental studies on modulation of allergen release from pollen by air pollutants[END_REF] . Pollen that had been collected at road-sides showed significantly reduced allergen release in comparison with pollen collected in rural meadows [START_REF] Behrendt | Localization, release and bioavailability of pollen allergens: the influence of environmental factors[END_REF] . Moreover morphological modifications were reported in pollen shape and tectum under polluted conditions [START_REF] Chehregani | Increasing allergy potency of Zinnia pollen grains in polluted areas[END_REF][START_REF] Senechal | Pollen grains and urban air pollution: a three year study (abstract)[END_REF] . Pollen grains collected from polluted areas were covered with large amounts of pollutants such as airborne particles, heavy metals, nitrate, sulphur [START_REF] Behrendt | Air pollution and allergy: experimental studies on modulation of allergen release from pollen by air pollutants[END_REF] . Furthermore, our laboratory experiments have shown that NO 2 (0.5 to 50 ppm) and O 3 (0.1 to 5 ppm) treatments can alter pollen tectum and facilitate intra-cytoplasmic granules release, in dose-dependent manner [START_REF] Motta | Traffic-related air pollutants induce the release of allergen-containing cytoplasmic granules from grass pollen[END_REF] . These granules, which contain most of the pollen major allergens, have been implicated in thunderstorm-associated asthma [START_REF] Suphioglu | Thunderstorm asthma due to grass pollen[END_REF][START_REF] Marks | Thunderstorm outflows preceding epidemics of asthma during spring and summer[END_REF] . They have also been shown to trigger primary and secondary allergic responses in pollen-sensitised Brown Norway rats [START_REF] Motta | Phleum pratense pollen starch granules induce humoral and cell-mediated immune responses in a rat model of allergy[END_REF][START_REF] Motta | Intratracheal Instillation of Cytoplasmic Granules from Phleum pratense Pollen Induces IgE-and Cell-Mediated Responses in the Brown Norway Rat[END_REF] .

Recently, Zinia pollen exposed to polluted air has been shown to trigger higher allergic responses in guinea pig [START_REF] Chehregani | Increasing allergy potency of Zinnia pollen grains in polluted areas[END_REF] .

The aim of the present study was to characterise modifications of P. pratense pollen allergenic content following artificial exposure to the major gaseous air pollutants O 3 (0.1 ppm), NO 2 (2 ppm) and SO 2 (2 ppm). Based on our previous study [START_REF] Motta | Traffic-related air pollutants induce the release of allergen-containing cytoplasmic granules from grass pollen[END_REF] , the pollutant concentrations used were the ones inducing the lowest increase of damaged pollen and intra-cytoplasmic granules release compared to air-exposed pollen.

Compared to the ones used in most of the studies, these low concentrations were up to 10 times the European warning levels (Table I). Differing from other previous similar studies, an original fluidised bed exposure system was used in our study, in order to mimic environmental exposures of pollen and allergens were detected using sensitive patient sera after separation by 2D-PAGE electrophoresis.

Material and methods

Pollen and pollen extract

Pollen from Phleum pratense (Timothy grass) and Dactylis glomerata (Orchard grass) were purchased from Allergon (AB, Angelholm, Sweden) and stored at 4°C (nonexposed pollen). For pollen extracts, 50 mg of pollen were suspended in 500 microL of distilled water, incubated on a rotating drum for 1 hour at room temperature and then centrifuged for 10 min at 10,000 g 25 . The supernatant was stored at -20°C until use. Proteins concentrations were assessed as described by Bradford [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] and by Lowry et al. [START_REF] Lowry | Protein measurement with the Folin phenol reagent[END_REF] using bovine serum albumin as a standard.

Pollen exposure to pollutants in fluidized-bed

The exposure system consisted of a glass cylinder (250-mm height, 20-mm diameter) with a porous glass plate at the bottom. Two grams of P. pratense pollen were placed in the cylinder and formed a suspension of fine particles 25 to 30 mm thick from the porous glass plate. Dry air alone or with pollutants (0.1 ppm O 3 , 2 ppm NO 2 , 2 ppm SO 2 , the mix of 0.1 ppm O 3 and 2 ppm NO 2 or the mix of 2 ppm NO 2 and 2 ppm SO 2 ) were injected from the bottom at a constant rate of 1.2 L/min, for 4 hours. O 3 was generated by passing oxygen through an UV-ionizer (Sonimix 6023, LN Industries SA, Geneva). Ozone concentration was monitored inside the exposure system by an O 3 analyzer (O 3 41M, Environnement SA, France) and adjusted by modifying the UV lamp intensity. Concentrations of NO 2 and SO 2 were obtained from gas containers prepared and certified by the supplier (Air Liquide, France). Homogenous pollen exposure was obtained by a slight contact of the cylinder with a vortex [START_REF] Motta | Traffic-related air pollutants induce the release of allergen-containing cytoplasmic granules from grass pollen[END_REF] .

2D PAGE

Proteins from pollen extracts were separated by isoelectricfocusing (IEF) in a 4% polyacrylamide gel (CleanGel IEF, GE Healthcare, AB, Uppsala, Sweden) containing 2 % (w/v) Servalyt ® pH 2-11 (Serva, Heidelberg, Germany) in a Multiphor II electrophoresis chamber (AB). Two adjacent 4-mm-wide strips of a focused protein extract were cut and submitted side by side to a SDS-PAGE separation on a 8-18% gradient gel (ExcelGel, AB), given 2 quasi identical 2D separation. One of the 2-D gel separation was silver-stained according to Blum et al. [START_REF] Blum | Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels[END_REF] and the other used for an immunoblot.

Immunoblots

After 2-D gel electrophoresis, proteins were electroblotted onto a nitrocellulose (NC) sheet (Schleicher & Schull, Basel, Switzerland) with a semi-dry Novablot apparatus, according to the manufacturer's instructions (1h15, 1 mA/cm 2 ). All following incubations were performed using phosphate-buffered saline (PBS), containing 0.3% Tween 20 (PBS-Tw) at room temperature (about 22°C). NC sheets were blocked 1 hour with PBS-Tw and incubated (overnight) with either grass pollenallergic patient sera (1:10), or mouse monoclonal antibody (mAb) directed against Dactylis glomerata allergen 4 (Dac g 4), revealing also Phl p 4 29 (1:1 500) or rabbit polyclonal antibody directed against rDac g 3 30 (1:500). Phl p 3 can be easily identified by the polyclonal antibody directed against rDac g 3 due to their sequence homology in grass pollen family [START_REF] Andersson | Characteristics and immunobiology of grass pollen allergens[END_REF] . After extensive washing, the NC sheets were incubated 2 hours with either alkaline phosphatase (AP)-conjugated goat anti-human IgE (1:700, Sigma-Aldrich, St. Louis, USA), or AP-conjugated rabbit anti-mouse IgG (1:10 000, Sigma-Aldrich) or AP-conjugated mouse anti-rabbit IgG (1:5 000, Sigma-Aldrich). AP activity was detected using 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (Sigma-Aldrich) in 0.1 mole.L -1 Tris buffer, pH 9.5.

Patient sera

Sera number 10, 12 and 25 were selected from a group of 26 grass pollen allergic donors, for their ability to recognise a great number of grass pollen allergens after blotting [START_REF] Batard | Isotypic analysis of grass pollen-specific immunoglobulins in human plasma. 1. Specialization of certain classes and subclasses in the immune response[END_REF] .

Results: 1. Non-exposed and air-exposed pollen

To study the effect of gaseous pollutants on proteins and allergens, two-dimensional separations were performed on water-soluble extract from non-exposed and exposed P. pratense pollens. The 2D-gel silver staining of non-exposed protein extract revealed about 30-40 proteins with a large spectrum of relative molecular masses (Mr) and isoelectric points (pI) (fig 1a). Most of the proteins had a Mr comprised between 14 and 77 kD and a pI between 3.5 and 10.6.

The 2D immunoblot patterns of non-exposed timothy grass pollen extract obtained

Gaseous exposed pollen

Proteins from different pollen extracts

Timothy grass pollen grains were exposed to air, O 3 , NO 2 or SO 2 , alone or in combination. Using Bradford and Lowry methods, protein amounts in water soluble pollen extracts showed no significant difference between air-exposed and gaseous pollutant exposed pollens. By Bradford method, protein concentrations were 51.1 3.7, 51.9 3.6, 48.8 8.0, 51.9 4.0 µg of protein per mg of pollen, respectively for air-, O 3 -, NO 2 -and SO 2 -exposed pollen extract.

2D protein patterns

No significant differences in the 2D protein patterns were observed between non-

2D allergen pattern of O 3 exposed pollen

Acidic shift was also observed at the allergen level with the 3 sera. For example, with (pI 4.0-5.9) recognitions were strongly decreased when recognitions of major allergens Phl p 1 and 5 showed no significant difference. Whereas using serum #12, we observed a decrease in Phl p 1 and 5 recognition (data not shown).

2D allergen pattern of SO 2 exposed pollen

2D allergen pattern of gas mixture exposed-pollen

The allergen patterns obtained with serum #10 from combined gaseous pollutant exposed pollen extracts are shown on figure 3

Discussion:

When timothy grass pollen exposures were performed with realistic O 3 concentration or/and with NO 2 and SO 2 concentrations 10 times higher than the European warning level, we observed a decrease of some allergen recognition by IgE from three allergic patient sera. Acidification of several allergens was also observed following treatment using O 3 with or without NO 2 . These experimental gaseous exposures didn't induce any apparition of de novo allergens. The decrease of allergen recognition suggests either the allergen loss during the treatment (for example the release in atmosphere as free allergens) and/or their alteration.

Protein and allergen losses due to urban pollution exposure or to gaseous experimental exposure to high concentrations of pollutants were previously described 18, 19, 25 . However, in our experiments, we used lower pollutant concentrations than used by the former authors, inducing few damages on pollen grains and a weak intracytoplasmic granules release [START_REF] Motta | Traffic-related air pollutants induce the release of allergen-containing cytoplasmic granules from grass pollen[END_REF] . In these conditions, we have not observed any noticeable difference between air-and gaseous pollutant exposed pollen on protein concentration and on silver stained 2D gel profiles. The "allergen loss" hypothesis should be less suitable on the basis of our results but further work is needed. For example, pollen cytoplasmic granules and free allergens release could be measured following pollen exposures to gaseous pollutants.

Air pollutants could have an effect on the biochemical protein properties of pollen grains which might induce a decrease of the allergen recognition by IgEs. Goschnick et al. [START_REF] Goschnick | Surface and depth analysis of pollen treated with atmospheric trace gases (abstract)[END_REF] analysed the effect of nitrogen dioxide (1-100 ppm) or ozone (0.1 ppm) treatment on three species of pollen (timothy grass, mugwort and birch), showing incorporation of nitrogen as nitro-groups into the organic matrix of the pollen wall.

Recently, Franze et al. [START_REF] Franze | Protein nitration by polluted air[END_REF] reported pollen protein nitrations, in particular on birch allergen Bet v1, after exposition to a mixture of NO 2 (4.57 ppm) and O 3 (0.5 ppm).

After exposure to this gas mixture, the level of protein nitration depends on the concentration of O 3 and NO 2 and their highly reactive species (NO 3 or HNO 3 ) [START_REF] Franze | Protein nitration by polluted air[END_REF] .

Furthermore, post-translational modifications, such as hydroxylated proline residues, carbohydrate structures and disulfide formation [START_REF] Petersen | Post-translational modifications influence IgE reactivity to the major allergen Phl p 1 of timothy grass pollen[END_REF] or deglycosylation [START_REF] Batanero | IgE-binding and histamine-release capabilities of the main carbohydrate component isolated from the major allergen of olive tree pollen, Ole e 1[END_REF] , can affect the IgE reactivity. Also an alteration of the major phosphorylation site of an important allergenic epitope of casein affects the allergenicity of this protein [START_REF] Bernard | Post-translational phosphorylation affects the IgE binding capacity of caseins[END_REF] . In our experiments, we hypothesise that the observed decrease in IgE recognition induced by gaseous pollutant exposures could be due to a post-translational modification as previously described [START_REF] Goschnick | Surface and depth analysis of pollen treated with atmospheric trace gases (abstract)[END_REF][START_REF] Franze | Protein nitration by polluted air[END_REF][START_REF] Petersen | Post-translational modifications influence IgE reactivity to the major allergen Phl p 1 of timothy grass pollen[END_REF] . Identification of the allergen composition by mass spectrometry should allow the characterisation of chemical modifications that may be responsible of the decrease of allergen recognition. It could be interesting to investigate the role of these chemical modifications in the modified pollen allergenicity in vivo. For this purpose, we have studied the allergenicity of these polluted grass pollens in sensitised Brown Norway (BN) rats and until now we failed to observe any significant modification of the specific humoral (pollen-IgE) and cellular (lymphocyte proliferation in response to pollen) allergic responses (unpublished data).

In conclusion, the pollen exposure to gas pollutants induces a slight decrease in allergen detection in pollen extracts. This decrease could be due to post-translational modifications affecting allergen detection by IgE patient sera. Our preliminary results on BN rats emphasize the difficulty to evaluate nowadays the impact of these allergen

  using the three patient sera were similar. Patient serum #10 revealed about 30 allergen spots, named in www.allergome.com database, with a Mr range from 14 to 90 kD and a pI range from 3.5 to 10.6 (fig 1b). Allergens were located mainly around five different Mr ranges: 75-90, 65-70, 40-57, 25-26 and 17-24 kD, each of them showing a great pI variability. Three spots at 75-90 kD, pI 3.2-3.9 (unknown high molecular mass allergen) named [a] and 5 spots at 65-70 kD, pI 6.4-8.3 were revealed and could correspond to Phl p 13 isoforms. Using a mAb against Dac g 4 which reveals also Phl p 4, one spot (around 70 kD, pI 9.5-10.0) was recognised (data not shown). Two acidic allergen spots (pI 5.0-5.7) and 3 other acidic (4.8-5.7), 6 neutral ones (pI 5.8-8.0) and 1 basic one (pI 10.0) were strongly revealed with Mr ranges of 40-57 kD. They could correspond to Phl p 1, 5a, 5b and 1b respectively. Acidic allergen spots (from pI 4.0 to 5.9) were also detected at Mr 25-26 and 17-24 kD, probably corresponding to Phl p 6 and Phl p 2 respectively. Moreover, at Mr 17-24 kD, basic spots (from pI 7.0 to 9.0), named Phl p 3, visualised by the patient serum #10, were also recognised by the anti-Dac g 3 antibodies. This serum recognised weakly a very acidic allergen (pI around 3.0) with low Mr (14-20 kD) corresponding to profilin (Phl p 12). Compared to other tested sera, this one recognised the greatest allergen number and most of the known allergens. Serum #25 did not reveal Phl p 1b and 4 but recognised all others allergens revealed by serum #10 (data not shown). Serum #12 mainly recognised major allergens e.g. Phl p 1 and 5 (fig 1d). Allergen patterns of aqueous extract from non-exposed timothy grass pollen, revealed by IgE from patient sera #10 and 25, were similar to these of orchard grass pollen extract (data not shown). 2D-blots performed with extract from air-exposed pollen (Fig 1c) did not show any significant difference compared to non-exposed pollen extract (Fig 1b).

  exposed (fig 1a), air-exposed and NO 2 and/or SO 2 -exposed pollen extracts (data not shown) while O 3 treatment induced acidification of some proteins without visible change in silver staining intensity (fig 2a).

serum # 10 (

 10 fig 2b), allergen Phl p 4 and Phl p 1b switched from pI 10.0 to 8.3. Similarly, the two remaining acidic spots corresponding to Phl p 5a and 6 switched from pI 4.8-5.7 to 4.4-4.8 and from pI 4.0-5.9 to 4.3-4.8 respectively. In addition to this allergen acidification, a decrease in IgE recognition was observed with all sera. At Mr 75-90 kD, the patient serum #10 (fig 2b) revealed only an acidic spot around pI 3.2 corresponding to allergen [a]. Some of Phl p 13 spots in the pI 7-8 area have disappeared. Concerning Phl p 1 and Phl p 5a, IgE recognition decreased and the Phl p 1b strongly diminished compared to non-exposed pollen extract. Moreover Phl p 6 isoforms with pI 5.7-5.8 disappeared and in the Phl p 2 area (Mr 17-24 kD) a large decrease of isoform numbers was shown. 2.4. 2D allergen pattern of NO 2 exposed pollen Using the 3 sera, one of the Phl p 5b spots (pI 7.3-7.5) was not detected in the pattern from NO 2 -exposed pollen (fig 2c and data not shown). Using sera #10 and 25, two of the Phl p 6 spots (pI 5.7-5.8) have disappeared and one of the Phl p 2 spots (pI 5.7-5.8) was more weakly revealed than in control extract. No other differences were observed.

SO 2 -

 2 treatment induced a decrease of IgE recognition of several allergens using the 3 sera. With serum #10 (fig 2d), Phl p 13 (pI 6.4-8.3), Phl p 6 (pI 5.7-5.8) and Phl p 2

  . A strong reduction of IgE recognition was observed for the whole pattern from NO 2 /O 3 -exposed pollen extract (Fig 3a). In spite of similar allergen patterns, the reduction was slightly more pronounced on NO 2 /O 3 -exposed pollen (fig 3a) than on O 3 -exposed pollen (fig 2b). Allergen acidification shown with O 3 treatment was also observed in this case. The results previously described were also obtained with sera #12 and 25 (data not shown). In comparison with single pollutant exposed pollens (fig 2c and 2d), allergen pattern from NO 2 /SO 2 -exposed pollen (fig 3b) revealed a significant decrease of high Mr allergens: [a], Phl p 4 and 13. The recognition of the other allergens was similar to the one obtained after SO 2 treatment (fig 2b).

Figure 1 :

 1 Figure 1: 2D gel analysis of water-soluble extract from non-exposed and air-exposed

Figure 2 :

 2 Figure 2: 2D gel analysis of water-soluble extracts from O 3 -, NO 2 -and SO 2 -exposed

Figure 3 :

 3 Figure 3: 2D gel analysis of water-soluble extracts from NO 2 /O 3 -and NO 2 /SO 2 -

Acknowledgement

This work has been supported by the research project BCRD-AP2001-DRC05 provided by the French Ministry of the Ecology and Sustainable Development. We are very grateful to Christiane Mayer for laboratory assistance and we thanks Fréderic Bois for helpful discussion.

modifications on the allergenicity in vivo. Knowledge of this phenomenon would require further studies with different animal models of allergy.