G Uzu

S Sobanska
email: sophie.sobanska@univ-lille1.fr

Y Aliouane

P Pradere
email: p.pradere@stc-metaux.com

Camille Dumat
email: camille.dumat@ensat.fr.

Study of lead phytoavailability for atmospheric industrial micronic and submicronic particles in relation with lead speciation

Keywords: lead, PM 2.5 and PM 10, soil-lettuce transfer, phytoavailability

The soil-lettuce lead transfer from atmospheric industrial sub-micronic and micronic particles depends on particle size.

Introduction

Due to its numerous past and present uses and high persistence, lead is a major environmental contaminant [START_REF] Wang | Transfer characteristics of cadmium and lead from soil to the edible parts of six vegetable species in southeastern China[END_REF]. Potentially toxic for living organisms even at low concentrations, lead constitutes a risk for humans who can absorb it in various ways [START_REF] Canfield | Intellectual impairment in children with blood lead concentrations below 10 µg per deciliter[END_REF]. In the context of contaminated gardens, elevated lead intake by humans can be due to the consumption of crop plants grown on soils with relatively high plant-available metal concentrations, ingestion of contaminated soil, either accidentally or intentionally (pica), inhalation of soil particles and drinking water with high soluble concentrations of metals [START_REF] Alexander | Genotypic variations in the accumulation of Cd, Cu, Pb and Zn exhibited by six commonly grown vegetables[END_REF]. The total quantities of lead emitted in the environment by industries have decreased sharply in recent decades (Glorennec et al., 2007) and are strictly controlled in Europe nowadays. Lead was recently classified as a substance of very high concern in the European REACH law (Regulation EC 1907/2006 of the European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals). However, particles enriched with lead are still generated especially by lead-recycling plants [START_REF] Batonneau | Speciation of PM 10 sources of airborne nonferrous metals within the 3-km zone of lead/zinc smelters[END_REF][START_REF] Ohmsen | Characterization of fugitive material within a primary lead smelter[END_REF] and constitue the main source of lead pollution for soils [START_REF] Miquel | Les effets des métaux lourds sur l'environnement et la santé[END_REF][START_REF] Donisa | Heavy Metal Pollution by Atmospheric Transport in Natural Soils from the Northern Part of Eastern Carpathians[END_REF].

According to [START_REF] Zhang | Comparison of microand nano-size particle depositions in a human upper airway model[END_REF], emitted particles present a large granulometric spectrum in the atmosphere, but during the last decade the proportion of fine particle matter (PM) increased with the use of more effective filters in industry. Indeed PM 10 are target species of the World Heath Organization (WHO, 2001) and the European Union Framework Directive on ambient air quality assessment (EU, 1999), due to their adverse effects on the environment and human health. While micrometric and submicrometric fractions contribute very little to ambient particle mass, they may occur in substantial number concentrations. Most of the studies dealing with the characterization of metal-enriched particles in the ambient air provide information on quantitative measurements for PM 10 fractions (EU directives 96/62 and 99/30) and very few on the sub-micronic fraction [START_REF] Lazaridis | Long-range transport of aerosol particles and precursors -determining the relationship between emissions and observed concentration levels[END_REF]. The lack of knowledge regarding metal speciation in the industrial particles results mainly from a lack of analytical tools, both sensitive and specific to the size of the particles.

These fine particles are hihgly reactive due to their high specific area and can be transported over long distances in the troposphere [START_REF] Barrie | Scavenging Ratios: Black Magic or Useful Scientific Tool[END_REF]. They could therefore present a greater impact on the biosphere than coarse particles (Fernandez Espinosa, 2005). [START_REF] Ruby | Lead Bioavailability: Dissolution Kinetics under Simulated Gastric Conditions[END_REF] concluded that the bioaccessibility of lead rises strongly in particles under 2.4 µm size. But, the phytoavailability of lead in industrial particles as a function of their size and speciation have not been studied yet. In comparison with zinc, lead generally shows a relatively low mobility in soils (Dumat et al., 2006). It can however migrate through the soil with dissolved organic matter [START_REF] Cecchi | Multi-metal contamination of a calcic cambisol by fallout from a lead-recycling plant[END_REF] or be mobilized by certain plants [START_REF] Arshad | A field study of lead phytoextraction by various scented Pelargonium cultivars[END_REF]. Moreover, carried from the air to the soils as fine particles, lead could be released more easily in soil solution [START_REF] Komarnicki | Lead and cadmium in indoor air and the urban environment[END_REF].

We therefore focused our study on the links between soil-plant transfer of lead, size and speciation of particles emitted by a lead recycling plant, currently the main source of atmospheric emissions [START_REF] Cecchi | Multi-metal contamination of a calcic cambisol by fallout from a lead-recycling plant[END_REF]. The objectives were the following: (i) the elemental and molecular characterisation of micrometer and nanometer sized lead-rich particles and (ii) to study the influence of particle characteristics on lead soil-plant transfer.

The physico-chemical characterization of industrial PM 10 and PM 2.5 particles collected in the plant was investigated using both bulk and micro analysis techniques:

(i) MEB-EDS to determine the morphology and chemistry on the scale of a particle [START_REF] Laskin | Analysis of individual environmental particles using modern methods of electron microscopy and X-ray microanalysis[END_REF][START_REF] Choel | quantitative determination of low-Z elements in single atmospheric particles on boron substrates by automated scanning electron microscopy-energy-dispersive X-ray Spectrometry[END_REF]; (ii) Raman microspectrometry to study particle speciation [START_REF] Batonneau | Speciation of PM 10 sources of airborne nonferrous metals within the 3-km zone of lead/zinc smelters[END_REF]2006;[START_REF] Sobanska | Chemistry at level of individual aerosol particle using multivariate curve resolution of confocal Raman image[END_REF][START_REF] Sobanska | Chemistry at level of individual aerosol particle using multivariate curve resolution of confocal Raman image[END_REF].

The transfer of lead from particles to the lettuce Lactuca sativa a widely grown garden vegetable was investigated in the laboratory: two different uncontaminated calcareous soils were spiked with PM 10 and PM 2.5 for soil-plant experiments with a biotest device that enabled careful study of rhizosphere and roots in addition to the transfer to the shoots [START_REF] Chaignon | A biotest for evaluating copper bioavailability to plants in a contaminated soil[END_REF]. The study was finally completed by CaCl 2 extraction experiments carried out according to [START_REF] Houba | State of the art and future developments in soil analysis for bioavailability assessment[END_REF] to estimate lead phyto-availability.

The hypothesis tested throughout all these experiments was that particle characteristics have a significant influence on lead soil-plant transfer and translocation.

Materials and methods

Particle sampling and size separation.

A secondary lead smelter which currently recycles batteries was chosen as a representative example of the smelter metal industry to develop a methodology aimed at the risk assessment of industrial lead particles. The plant of the Chemical Metal Treatment Company (STCM) is located in the urban area of Toulouse (43°38'12" N, 01°25'34" E). According to the French authorities (DRIRE, 2007), 328 kg of Total Suspended Particles (TSP) including 31 kg of lead were emitted by this factory in 2007.

Three sources of particles corresponding to the three work units involving different steps in the process are identified in the plant: (i) the battery grinding unit where battery components are separated under wet conditions (ii) the smelter where lead pastes are processed in rotary furnaces at 1200°C and finally (iii) the refinery where lead is purified from unwanted metals or enriched. The same process has been used for thirty years in the plant. This study focuses on channeled emissions only generated by the furnace unit, considering the transfer towards the biosphere. The particle collection, performed three months after complete cleaning of the three work units (March, 12, 2007), is therefore representative of that emission period. Our aim was to characterize the reactivity of the particles in relation with their previously determined speciation and size, not to follow one specific parameter over time.

1kg of particles was collected in polyethylene bags from the air-sleeve filters of the furnace, then sealed and transferred in sealed opaque containers for transport to the laboratory. Samples were passed through a 2-mm stainless steel AFNOR sieve. Process dust was stored in a cool (4°C) dark place with Merck desiccant.

Because 80 % of emitted particles were smaller than 10 µm, PM 2.5 and PM 10 were size-segregated by artificial resuspension in a Teflon bag [START_REF] Batonneau | Speciation of PM 10 sources of airborne nonferrous metals within the 3-km zone of lead/zinc smelters[END_REF][START_REF] Young | Resuspension of soil as a source of airborne lead near industrial facilities and highways[END_REF] and collected by impaction onto a PM 10/2.5 Dekati inertial impactor.

The cascade impactor consists of two successive stages with aerodynamic cut-off diameters of 10 and 2.5 µm when it operates at 10 L/min airflow. PM 10 present an aerodynamic diameter between 2.5 and 10 µm, whereas the PM 2.5 stage collects particles < 2.5µm.

Characterization of particles

Elemental total contents of the two size fractions were determined by ICP-OES (IRIS Intrepid II XDL) after heated digestion with standard acid (HNO 3, HCl and HF, Suprapur, Merk) in a PTFE vessel. Levels of C, H, S and O were determined after burning in an elemental analyser with coulometric-catharometric detection and IR.

X-ray diffraction patterns of PM 10 and PM 2.5 were recorded on an INEL diffractometer equipped with a curved detector CPS 120 and Co (Kα) radiation allowing a 120°/2θ detection. Crystallized compounds were identified by comparison with the diffraction patterns of the JCPDS database. The relative abundance of each crystallized phase detected in samples was qualitatively estimated by using the relative intensity of the strongest X-ray pattern peaks of each considered phase. It should be noticed that only crystallized phases with an abundance superior to 5 % in weight can be detected by XRD.

Particles were characterized using complementary microscopy and imaging techniques to determine elementary and molecular composition, size, morphology and heterogeneity of the individual lead-rich particles.

X-ray elemental mapping and secondary images were obtained using an Environmental Scanning Electron Microscope (ESEM) working in high-vacuum mode.

The Quanta 200 FEI instrument was equipped with an energy-dispersive X-ray (EDX) detector. ESEM was operated at 10-25 kV. Particles were dispersed on carbon substrates and analysed without further preparation. Because of the ESEM configuration, light element detection (C, N, and O) was ambiguous.

Raman microspectrometric measurements were carried out with a LabRAM confocal spectrometer (Jobin Yvon, Horiba Gr, France). The spot size of the laser focused by a 100×objective (numerical aperture (NA) = 0.90) was estimated to be 1 μm 2 in size. A liquid nitrogen-cooled CCD (Jobin-Yvon, 2048 pixels × 512 pixels) was used for detection. The Raman backscattering was excited at 632.8 nm provided by an internal, air-cooled, linearly polarized helium-neon laser. The laser power delivered to the sample was 8 mW. The microscope stage was XY-motorized and computercontrolled for point by point scanning with 0.1 μm resolution, 1 μm reproducibility and 90 mm × 60 mm spatial range. The glass plate with the impacted particles was mounted on the microscope stage without any further preparation. Data acquisition consisted in recording many spectra in point-by-point scanning mode with 1 μm as a minimum step, one accumulation and 30 s spectrum acquisition time. For identification of Raman spectra the experimental spectra were compared to reference spectra using Spectral Library Search ID 301 software (Thermo Galactic).

Measure of soil-plant lead transfer performing microculture experiments

Lettuce was chosen because it is a common vegetable widely grown for human consumption and has recently been used by several authors for metal transfer studies (Khan et al., 2007;[START_REF] Waisberg | The effect of pH, time and dietary source of cadmium on the bioaccessibility and adsorption of cadmium to/from lettuce (Lactuca sativa L. cv. Ostinata)[END_REF][START_REF] Alexander | Genotypic variations in the accumulation of Cd, Cu, Pb and Zn exhibited by six commonly grown vegetables[END_REF]. Moreover, lettuce is often used around plants to estimate atmospheric pollution.

The biotest presented on figure 1 was first described by Niebes et al. (1993) and then adapted by [START_REF] Chaignon | A biotest for evaluating copper bioavailability to plants in a contaminated soil[END_REF]. A small PVC cylinder (25 mm inner diameter) was closed by a polyamide net (900 µm mesh) inserted into a larger cylinder, itself closed by a finer polyamide mesh (30 µm, Fyltis/Nytel, Sefar filtration). A space of 3 mm was left between the net and the finer mesh, where the roots could develop as a mat.

Commercial lettuce seeds, "Batavia blonde dorée" cultivar, were surface sterilized with 0.9% CaClO for 15 min and rinsed with deionised water. Three seeds were sown in the container on the surface of the coarser mesh.

Lettuces were first grown hydroponically for 21 days to obtain a large flat mass of roots that fully covered the fine mesh.

The devices were placed on top of troughs containing an aerated complete nutrient solution with the macroelements: 5 mM KNO 3 , 5 mM Ca(NO 3 ) 2 , 2 mM KH 2 PO 4 and 1.5 mM MgSO 4 and oligoelements: 9.11 µM MnSO 4 , 1.53 µM ZnSO 4 , 0.235 µM CuSO 4 , 24.05 µM H 3 BO 3 , 0.1 µM Na 2 MoO 4 and 268.6 µM Fe/EDTA. The height of the nutrient solution was adjusted daily to keep the fine mesh wet during the whole preculture period. The experiment was conducted in a growth chamber (temperature 24 ± 0.5 °C/18 ± 0.5 °C day/night cycles; photoperiod 16 h under daylight fluorescent lamps providing 400 µmol.m -2 .s -1 [Philips 600W, Eindhoven, Netherlands] and 8 h darkness; relative humidity 70%).

Then, the second step involved an eight-day soil-plant contact period: five replicate plants for each treatment were transferred onto the various soil samples.

Top soils of two calcic cambisol profiles (FAO, 1998) produced by quaternary alluvial deposits, were sampled. These two calcareous soils (noted soil-1 and soil-2) with various physicochemical properties (table 1) were chosen because that kind of soil happens to be observed around several battery recycling plants. Moreover, if the lettuce species could favor lead transfer, the choice of calcareous soils could reduce it. The soils differ by texture, soil cultivation, organic matter (OM) and CEC: soil-1 was uncultivated loamy-sandy clay with 2% of OM and soil-2 was sandy-clayey loam under sunflowers with 1% of OM and a higher CEC.

These two soils were spiked with PM 2.5 and PM 10 up to [Pb] = 1650 ± 20 mg.kg -1 . 60 mg of particles were added per box containing 10 g of soil and were agitated for a day. Soils were turned over every fifteen days for four months. This long period was chosen to allow time for natural equilibration of the various sorption mechanisms in the soil [START_REF] Alexander | Genotypic variations in the accumulation of Cd, Cu, Pb and Zn exhibited by six commonly grown vegetables[END_REF]. The relatively high total lead soil concentration was chosen to be representative of a real soil pollution situation observed for atmospheric fallout from an STCM plant [START_REF] Cecchi | Multi-metal contamination of a calcic cambisol by fallout from a lead-recycling plant[END_REF] and we wanted to be sure that enough lead was available for the plant in soil solution. Surveys of garden soils in several countries have shown wide ranges of concentrations of heavy metals. For example, [START_REF] Culbard | Metal contamination in British suburban dusts and soils[END_REF] found concentrations of up to 14,100 mg Pb kg -1 . Some soils were left without crops, to determine the pH possible variation due to soil solution influence.

During the growth, pH was measured daily and nutrient solution level was maintained constant. pH of nutrient solution was 5.5±0.3 during the hydroponic and soil contact phases.

This biotest device presents two main advantages: (i) the roots are physically separated from the soil which enables total recovery of the shoots, roots and soil; (ii) the thickness of the soil layer used enables it to be considered it as rhizosphere soil and provides enough rhizosphere material to evaluate root-induced changes in metal speciation.

Roots and aerial parts were collected and analysed separately after the soil plant contact period. Biomass was determined before oven-drying at 80°C for 48H. Lead bound to the outer root cell walls, called [Pb] adsorbed , was determined according to the method of acidic desorption as described by Ferrand et al. (2006): roots were shaken end over end with 40mL of 0.001M HCl for 3 min, and then 360 µL of 1 M HCl was added to yield a final concentration of 0.01 M HCl. After shaking for another 5 min, the suspension was filtered through ashless paper. Then, plant roots and aerial parts were mineralized separately in a 1:1 mixture of HNO 3 and H 2 O 2 at 80°C for 4h. After filtration, the major and trace element concentrations were determined with an IRIS Intrepid II XDL ICP-OES. The concentration in roots after mineralization is considered as uptaken lead ([Pb] roots ). The accuracy of the acidic digestion and analytical procedures was verified using the reference material Virginia tobacco leaves, CTA-VTL-2, ICHTJ.

Estimation of lead availability by CaCl 2 extraction experiments

For the determination of the phytoavailable fraction, according to [START_REF] Menzies | Evaluation of extractants for estimation of the phytoavailable trace metals in soils[END_REF] neutral salts extractants provide the most useful indication. The 0.01 M CaCl 2 extraction procedure gives a good indication of lead phytoavailability [START_REF] Meers | Phytoavailability assessment of heavy metals in soils by single extractions and accumulation by Phaseolus vulgaris[END_REF]. The CaCl 2 procedure, first described by [START_REF] Houba | State of the art and future developments in soil analysis for bioavailability assessment[END_REF], was performed on the two contaminated soils and particle samples (PM 10 and PM 2.5 ). 30 mL of 0.01 M CaCl 2 solution was mixed with 3 g of soil (1:10 solid solution ratio) in 50ml polypropylene centrifugation tubes placed on a shaker table (Heidolph promax 1020) at 50 oscillations/min for 2h, 20°C. After extraction, the tubes were centrifuged at 10,000 g for 30 min (Avanti 30 centrifuge Beckman). The supernatant liquid was then filtered (0.22µm), acidified to 2% with distilled HNO 3 (15M, suprapur 99.9%) and stored at 4°C before analysis. That CaCl 2 extraction procedure was performed on particles, reference soils, and the two spiked soils before and after culture. Extracted metal concentrations were determined by an IRIS Intrepid II XDL/ Thermo Electron Corporation model ICP-AES Calibration used reference materials (ion 915 from National Water Research Institute, Canada).

Statistical data treatment

The plant absorption and chemical extraction data obtained were subjected to analysis of variance (ANOVA) with one factor, using the software Statistica, Edition'98 (StatSoft Inc., Tulsa, OK, USA). For each bioassay, mean values with different letters represent a significant difference (p < 0.05) as measured by the LSD Fisher test. Letters are reported on the figures and tables.

Results

PM 10 and PM 2.5 characterization:

Elemental concentrations in particles are shown in table 2. All results are given as the mean of the three replicates for each sample (PM 10 and PM 2 . 5 ) and standard deviations never exceed 7%. No significant difference except for Fe in the total elemental concentrations was observed in relation with the size of the particle. Major elements found in the samples were, by mass: Pb (27%), O (15%) and S (7.5%) for both fractions. High levels of Na (3-4%) were due to the industrial recycling process where Na is used to lower the melting point of Pb. Several other metals: Cd (2.5%), Zn (0.5%), Fe (0.1-0.4%) and Sb (0.1%) are also present. The remaining elements to complete to 100% are expected to be chloride and other trace metals.

XRD patterns of PM 10 and PM 2.5 provide identification of major crystallized species in bulk samples. The results are presented in table 3. Particles are mainly composed of metallic sulfides, sulfates, oxides and perchlorates. A significant amount of Na 2 SO 4 was found in PM 10 samples. Regardless of the size, the major phases identified were the same and one more Fe species was detected for PM 2.5 .

The ESEM-EDX analysis provided the morphology and elemental composition of individual particles in the two size fractions. PM 10 samples exhibited both particles with a size range between 2 and 10 µm and large aggregates composed of many micron-sized particles without specific shapes (figure 2). The main elements detected by EDX in these aggregates were, by order of importance, Pb, S, Cl, Sn, Na, and Fe. The chemical complexity of aggregates did not allow the detection of minor elements. Elemental mapping recorded on PM 10 showed the chemical heterogeneity of aggregates. PM 2.5 samples are composed of fine aggregates of few submicronic particles exhibiting characteristic features i.e. needles (< 100 nm), nano crystals (< 500 nm), cubes (~300 nm) and balls (~500 nm). Elemental analysis of particles showed that needles mainly contain Pb and S, nano crystals are composed of Pb, S and Cl while Na and S are detected in cubic particles. Compared to the PM 10 sample, numerous Pb only-rich nanoballs were observed in PM 2.5 (figure 2).

Raman microspectrometry combines the analytical capability of Raman spectroscopy to distinguish in situ a wide range of chemical substances in aerosols with the spatial resolution of a confocal optical microscope (~1 µm 3 ) which enables investigation of individual particles. The scanning of a large area of sample (300 µm 2 ) by automated analyses enables the analysis of a huge number of particles. The data treatment of all the spectra recorded in this area leads to the major and minor component identification. xPbO.PbSO 4 (x = 1,2,3), PbSO 4 , -PbO, PbCO 3 , Na 2 SO 4 , and ZnSO 4 were identified in both PM 10 and PM 2 . 5 samples as major species. The laserdamaging effect generates complete and irreversible oxidation of PbS to PbSO 4 as described previously [START_REF] Batonneau | Microscopic and imaging Raman scattering study of PbS and its photo-oxidation products[END_REF]. The Raman mapping of species within particles exhibits a large heterogeneity of particle composition. A typical Raman image of PM 2.5 particles is shown in figure 3. A large number of particles are composed of Uzu et al., 2008 Revised manuscript for Environmental pollution, full paper 13 lead sulphate and are agglomerated with other compounds such as sodium sulphate or carbonates in both PM 10 and PM 2.5 size fractions. The results suggest that no significant differences in lead speciation are observed in accordance with the particle size.

However, for the minor lead compounds (like carbonates) not exactly quantified by XRD, differences in percentages between the two size fractions could exist.

Assessment of the available fraction with CaCl 2 extraction

Figure 4A and 4B shows respectively total lead concentrations extracted by CaCl 2 and percentages of extracted lead with CaCl 2 (relative, with respect to total lead concentrations in different subtracts) for various samples: the single particles, soil-1 and spiked soil-1 before and after an eight-day exposure of Lactuca sativa.

Due to high levels of lead in particles (330,000 mg.kg -1 ), maximum lead quantities were extracted from PM 10 and PM 2.5 . But, in comparison with total lead contents, lead in uncultivated spiked soils was 0.2% exchangeable, while PM 10-2.5 presented a 0.01% exchangeability. Reference soils were under ICP-OES limit detection. After culture, a significantly greater lead quantity was extracted by CaCl 2 for the soil spiked with the finest PM 2.5 fraction than in PM 10 . A 1 unit pH decrease was measured after plant-soil contact while the pH values of the control soils (left without crops but with nutrient solution influence) remained constant. Concerning CaCl 2 extraction, no significant differences were observed between the two particle sizes in the PM 10-2.5 and spiked soil-1 before culture; The same trends described above for soil-1 were also observed for soil-2.

Influence of lead exposure on plant growth.

Fresh biomass data are shown in figure 5. After eight days of soil-plant exposure, biomasses for aerial parts grown on spiked soils were slightly lower than in the respective unspiked soils. No significant biomass differences were observed among the two types of spiking (PM 10-2.5 ). In controls, fresh weights were approximately 18.5 ± 2.5 g for aerial parts and 3.7 ± 0.7 g for roots while in spiked soils, weights reached 12 ± 4 g and 2.5 ± 1 g respectively. This slight influence of lead on plant biomass could be due to lead toxicity [START_REF] Sharma | Lead toxicity in plants[END_REF] or water status [START_REF] Parys | The effect of lead on photosynthesis and respiration in detached leaves and in mesophyll protoplasts of Pisum sativum[END_REF].

Absorption and adsorption of lead in the lettuce

Figure 6 presents results of lead transfer from soil to the various compartments of the plant (roots and shoots) and the distribution between adsorption (noted [Pb] adsorbed ) and absorption for roots ([Pb] roots ) respectively described in the materials and method section as lead only adsorbed at the root surface and lead truly taken up by the plant.

Measurements on the reference samples (Tobacco leaf VTL-2) validated the assay: the concentration found was 21.4 ± 1.1 mg.kg -1 , for a certified value of 22.1 ± 1.2 mg.kg -1 . Several trends were observed dealing with the influence of the particle type, soil type and lead location. For both soils, a significant increase of lead adsorption on roots, absorption and translocation throughout the shoots was observed, when the finest particles were added in comparison with the PM 10 . Global uptakes ([Pb] adsorbed +

[Pb] roots , + [Pb] shoots ) were greater for soil 1 than for soil 2. The main lead fraction was adsorbed on root membranes where concentrations were up to 985 ± 147.kg -1 for PM 10 contaminated soils and up to 1281 ± 195 mg.kg -1 for PM 2.5 contaminated soils. The soils spiked with PM 10 allowed a lead uptake of 230 ± 24 mg.kg -1 by roots and a translocation to aerial parts of 7.7 ± 2.7 mg.kg -1 . While in the PM 2.5 spiked soils, root and shoot concentrations reached 275 ± 40 mg.kg -1 and 12.19 ± 3 mg.kg -1 respectively.

Discussion

(1) Influence of particle size on soil-plant transfer and lead translocation.

Whatever the soil, for a given total lead concentration (1650 mg Pb.kg -1 soil), higher lead soil-plant transfer and translocation were observed for the finest particles.

Roots exposed to PM 2.5 spiked soils allowed a 20% greater lead uptake and a 30% increase in adsorbed lead. Shoots presented a 60% increase in translocated lead in PM 10 spiked soils. For the first time in industrial particles, the transfer of lead to the soil solution and its translocation throughout the plant is reported to increase as the particle size decreases. Douay et al. (2006), measuring lead concentrations in lettuces in urban gardens (1572 mg Pb.kg -1 ) observed a shoot concentration of 5 mg Pb.kg -1 DW. The soil to plant metal transfer measured in this study (for one week of lead exposure) was relatively high in comparison with previously reported data in publications dealing with vegetables (BAPPET, 2007), probably because of particle size, but also due to numerous factors that influence the soil-plant transfer of metals [START_REF] Kabata-Pendias | Soil-plant transfer of trace elements an environmental issue[END_REF][START_REF] Dumat | Metal ion geochemistry in smelter impacted soils and soil solutions[END_REF]Dumat et al., & 2006;;[START_REF] Costa | Cadmium uptake by Lupinus albus (L.): cadmium excretion, a possible mechanism of cadmium tolerance[END_REF][START_REF] Oliver | The effects of zinc fertilization on cadmium concentration in wheat grain[END_REF].

Root transfer factors (TF-roots) and shoot transfer factors (TF-shoots) were calculated as ratios between [Pb] roots , [Pb] shoots with [Pb] soil . TFr ranged from 0.10 to 0.17 and TFs were between 0.005 and 0.007 (table 4). These relatively low values illustrate the known low mobility and availability of lead and are comparable to Khan et al.'s results (2008) reporting 0.15 for TFr and 0.07 for TFs. As transfer factors for vegetables decrease with increasing levels of lead in the soil [START_REF] Zheng | Health risk of Hg, Pb, Cd, Zn, and Cu to the inhabitants around Huludao Zinc Plant in China via consumption of vegetables[END_REF][START_REF] Wang | Transfer characteristics of cadmium and lead from soil to the edible parts of six vegetable species in southeastern China[END_REF], we therefore expected a greater influence of particle size on transfer with a lower total lead concentration in soil.

According to [START_REF] Sobanska | Microchemical investigations of dust emitted by a lead smelter[END_REF], lead smelter emissions contain PbS, PbSO 4 , PbSO 4 .PbO, Pb, α-PbO compounds and numerous nano balls were observed for PM 2.5 . [START_REF] Dumat | Metal ion geochemistry in smelter impacted soils and soil solutions[END_REF] and [START_REF] Cecchi | Multi-metal contamination of a calcic cambisol by fallout from a lead-recycling plant[END_REF] working on soils polluted by lead produced by industrial atmospheric fallout from smelters, concluded that lead chemical speciation strongly influences its bioavailability. The sequence of solubility constants from the CHESS data base for the lead is the following: PbCO 3 > PbSO 4 > PbO > Pb 0 . In water media and equilibrium conditions, lead carbonate and sulphate will be much more labile than PbO or Pb 0 species. However, according to [START_REF] Birkefeld | In situ investigation of dissolution of heavy metal containing mineral particles in an acidic forest soil[END_REF][START_REF] Birkefeld | In situ transformations of fine lead oxide particles indifferent soils[END_REF] which used an in-situ method to study dissolution and phase transformation of lead particles from a smelter in different soils, the sequence of solubility is strongly dependent on soil characteristics (like texture, pH, lime amount...). They observed that PbO was rapidly covered by lead-hydroxy carbonates (hydrocerussite) in some soils while in other it was relatively stable. Moreover, according to the general review of [START_REF] Ruby | Lead Bioavailability: Dissolution Kinetics under Simulated Gastric Conditions[END_REF], release of lead in the soil solution depends on particle size, speciation and soil geochemistry. In soil solution, ligands like fulvic acids or low weight organic acids excreted by roots can displace the equilibrium (Ferrand et al., 2006). Finally, as no significant difference among speciation forms was noticed between PM 10 and PM 2.5 , all changes observed in reactivity were attributed to size differences.

Whatever the particle size, greater lead absorption by lettuce was observed for soil-1 than for soil-2. In comparison with soil-2, soil-1 has more clay, its CEC is higher and its level of lime is lower (see Table 1). According to [START_REF] Twining | Soil-water distribution coefficients and plant transfer factors for 134 Cs, 85 Sr and 65 Zn under filed conditions in tropical Australia[END_REF], we could expect that lead transfer would be higher for soil-2. However, several hypotheses can explain the results observed: (i) the higher amount of carbonates present in the soil-2 could reduce lead absorption [START_REF] Birkefeld | In situ investigation of dissolution of heavy metal containing mineral particles in an acidic forest soil[END_REF][START_REF] Birkefeld | In situ transformations of fine lead oxide particles indifferent soils[END_REF]; (ii) the complex influence of soil organic matter on the transfer of metals [START_REF] Yin | The importance of organic matter distribution and exact soil : solution ratio on the desorption of heavy metals from soils[END_REF][START_REF] Inaba | Effects of dissolved organic matter on toxicity and bioavailability of copper for lettuce sprouts[END_REF]; (iii) for the total lead concentration studied, the relatively high quantity of lead could be available in the soil solution (due to high solubility of the fine particles) reducing the influence of soil characteristics.

Total lead in the roots ([Pb] roots + [Pb] adsorbed ) reached 1500 mgPb/ kg dry weight.

Lead in roots was predominantly adsorbed onto the cell walls, 80 % of the total lead in roots, and depends on the nature of the particles used for exposure: the uptake was greater for PM 2.5 than PM 10 . According to [START_REF] Seregin | Strontium transport, distribution, and toxic effects on maize seedling growth[END_REF], Pb 2+ binds to the carboxy groups at the root surface reducing the translocation rate of lead [START_REF] Pendergrass | Uptake of lead and arsenic in food plants grown in contaminated soil from Barber Orchard, NC[END_REF][START_REF] Pendergrass | Uptake of lead and arsenic in food plants grown in contaminated soil from Barber Orchard, NC[END_REF]. To illustrate this phenomenon, the translocation factors or shoot/root ratios calculated indicate the ability of plants to transport metals from the roots toward the aerial parts (Ferrand et al., 2006): they ranged between 0.03 and 0.04 (table 4). But considering the lower biomasses in roots than in shoots, we can also reason with lead quantities. Whatever the type of soil or spiking, the total lead quantity in shoots (Pb shoots dry weight) between 0.003 and 0.007 mg Pb translocated which represents up to 33% (PM 2.5 ) of the total lead uptake. This percentage is not negligible with respect to risks concerning consumption of vegetables.

(2) Behaviour of lead in the rhizosphere and assessment of lead availability Relatively high lead transfer was observed for the lettuce cultivated on the two alkaline spiked calcareous soils. PM 10 -2.5 can therefore release lead in particular in the rhizosphere of Lactuca sativa. The lead availability estimated by the CaCl 2 procedure was greater for soils spiked with the finest particles and for both soils it increased after soil-plant contact: [Pb] CaCl2 in spiked soil with PM 2.5 > [Pb] CaCl2 in spiked soil with PM 10 . Under the root activity influence, the CaCl 2 lead extracted from polluted soils increased and a one-unit pH decrease in soil was measured. [START_REF] Lin | Root-induced changes of lead availability in the rhizosphere of Oryza sativa L. Agriculture[END_REF] and [START_REF] Kidd | Metal extraction by Alyssum serpyllifolium ssp. lusitanicum on mine-spoil soils from Spain[END_REF] also observed that exchangeable lead was much higher in the rhizosphere than in the bulk soil. Producing exudates, plants can modify metal speciation and behaviour in the rhizosphere [START_REF] Lin | Root-induced changes of lead availability in the rhizosphere of Oryza sativa L. Agriculture[END_REF][START_REF] Laperche | Effect of apatite amendments on plant uptake of lead from contaminated soil[END_REF][START_REF] Welch | Micronutrient nutrition of plants[END_REF]. This phenomenon has been particularly observed for calcareous soil by [START_REF] Chaignon | A biotest for evaluating copper bioavailability to plants in a contaminated soil[END_REF]. As pH influences metal solubility and transfer [START_REF] Wang | Transfer characteristics of cadmium and lead from soil to the edible parts of six vegetable species in southeastern China[END_REF], the rhizosphere acidification could have displaced the equilibrium towards bicarbonates, which are less stable than carbonates [START_REF] Sauvé | Derivation of soil quality criteria using predicted chemical speciation Pb 2+ and Cu 2+[END_REF]. An effect of soil on particle solubility was also observed: particles present a CaCl 2 exchangeability ten times lower than in spiked soil. Quantities extracted for particles were only up to 1.5% of the solution extraction. Mixed with soil for four months, particle solubility could therefore have changed.

In order to estimate the transfer of lead from polluted soils towards lettuce, relationships were sought between: lead mobilised by CaCl 2 extraction performed on soils before culture and lead concentrations in lettuce (shoots and roots). Equations were obtained from 6 parameters (2 soils; uncontaminated, spiked with PM 10 or PM 2.5 ), and every condition was studied by 5 replicates finally involving 30 observations. Significant correlations were observed between lead concentrations in shoots (Equation 1 below, with r 2 = 0.8) or in roots (Equation 2 below, with r 2 = 0.9) and lead extracted by CaCl 2 . CaCl 2 extraction procedure seems to be a suitable method for the determination of phytoavailable lead. This test simplifies the matrix and could avoid in vivo tests.

Conclusions and perspectives

A significant size influence was found for soil-plant lead transfer and translocation throughout the lettuce when micronic and nanometric industrial particles were compared: roots exposed to PM 2.5 spiked soils allowed a 20% greater lead uptake and a 30% increase in adsorbed lead. Shoots presented a 60% increase in translocated lead in PM 10 spiked soils. Our results highlight that source characteristics strongly influence metal transfer: total metal soil concentration is insufficient to estimate the risk induced by soils polluted by metals. Lead speciation was quite similar in PM 10 and PM 2.5 particles i;e. PbS, PbO.PbSO 4 , PbO, PbCO 3 and Pb 0 , were predominant however differences could exist for minor lead components. Moreover, the high amount of Pbrich nano balls and nano crystals much more dispersed for PM 2.5 could influence the lead transfer in the rhizoshere acidified by plant root activity. Lead concentrations in the edible part of lettuces were very well estimated by 0.01M CaCl 2 extraction.

In order to check the generality of the size influence of industrial particles on soilplant transfer, further experiments could be performed on other vegetables and various soils testing aging effects. Moreover, the study of other metals and metalloids measured in the particles will be performed. Dumat, C., Quenea, K., Bermond, A., Toinen, S., Benedetti, M.F., 2006 Ferrand, E., Dumat, C., Leclerc-Cessac, E., Benedetti, M.F., 2006. Phytoavailability of zirconium in relation to its initial added form and soil characteristics. Plant and Soil 287, 313-325. Glorennec , P., Bemrah, N.,Tard,A., Robin, A., Le Bot, B., Bard, D., 2007. Probabilistic modeling of young children's overall lead exposure in France: Integrated approach for various exposure media. Environment International 33, 937-945.

  Equation 1: [Pb] shoots =2.11×[Pb] CaCl2 + 2.13 r 2 = 0.797 p<0.01 n = 30 Equation 2: [Pb] roots =35.02×[Pb] CaCl2 + 4.24 r 2 = 0.915 p<0.005 n = 30 Both for PM 10 and PM 2.5 , the CaCl 2 chemical extraction was therefore a good indicator of soil-plant transfer. As previously shown by Pueyo et al. (2004), the 0.01 mol l -1

  Figure legends

Figure 2 ESEM

 2 Figure2

Figure 3

 3 Figure 3

Figure 4

 4 Figure 4

Figure 5

 5 Figure 5

Figure 6

 6 Figure 6

  Figure 1

  . Study of the trace metal ion influence on the turnover of soil organic matter in cultivated contaminated soils. Environmental Pollution 142, 521-529. Falgayrac, G., Sobanska, S., Laureyns, J., Brémard, C., 2006. Heterogeneous chemistry between PbSO 4 and calcite microparticles using Raman microimaging. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 64, 1095-1101. FAO, 1998. World reference base for soil research. World Soil resources Report, v. 84. FAO, Rome. Fernandez Espinosa, A.J.,Rossini Oliva, S., 2006. The composition and relationships between trace element levels in inhalable atmospheric particles (PM 10 ) and in leaves of Nerium oleander L. and Lantana camara L. Chemosphere 62, 1665-1672.

	Ebert, M., Inerle-Hof, M. and Weinbruch, S., 2002. Environmental scanning
	electron microscopy as a new technique to determine the hygroscopic behaviour of
	individual aerosol particles. Atmospheric Environment 36, 5909-5916.
	European Commission, 96/62/CE directive (September 27, 1996) for the quality of
	ambient air; directive 22/04/99 setting maximum levels for lead in ambiant air; REACH
	directive Regulation (EC) No 1907/20061, Directive 67/548/EEC.

Table 1

 1 Physico-chemical characteristics of the two soils used for micro-culture experiments.

		unit	Soil-1	Soil-2
	pH		8.3	8.4
	CEC	cmol+/kg	19.9	8.9
	Clay		405	195
	Silt	(g.kg -1 )	261	390
	Sand		334	415
	C/N		9.15	8.9
	MO	(g.kg -1 )	18.8	12.5
	Total carbonates	(g.kg -1 )	36.8	98
	P 2 O 5 Joret Hebert	(g.kg -1 )	0.28	0.215
	Cd (HF extraction)		0.22	0.175
	Pb (HF)		35.5	21
		(mg.kg -1 )
	Zn (HF)		101	48
	Cu (HF)		28.5

Table 2

 2 Total elemental analysis of particles.

	Contents (mg.kg -1 ) ± 7%

Table 3

 3 Determination of main chemical speciation of lead in the PM 10 and PM 2.5 particles by X-Ray Powder Diffraction Analysis a

	Sample	Cristallized compound	Estimated % of phases
	PM 10	PbS,	55
		PbO.PbSO 4 , PbSO 4 , Pb(ClO 4 ) 2 , Pb,	25
		Na 2 SO 4	10
		CdS, Cd(ClO 4 ) 2 , ZnO, ZnSO 4	10
	PM 2,5	PbS,	55
		PbO.PbSO 4 , PbSO 4 , Pb(ClO 4 ) 2 , Pb,	25
		Na 2 SO 4	10
		CdS, Cd(ClO 4 ) 2 , ZnO, ZnSO 4, FeS 2	10
	a The major compounds are written in bold

Table 4

 4 Tranfer factors in roots (TFr) and shoots (TFs) and translocation ratios.

	Soil	TFs	TFr	Translocation
	S1 + PM 10	0.005	0.14	0.03
	S1 + PM 2.5	0.007	0.17	0.04
	S1	0.081	0.26	0.31
	S2 + PM 10	0.004	0.10	0.04
	S2 + PM 2.5	0.005	0.15	0.03
	S2	0.01	0.44	0.18

Acknowledgements

ADEME, the French Agency for Environment and Energy as well as the STCM, are gratefully acknowledged for their financial support and technical help. This research project was supported by the National CNRS CYTRIX-EC2CO program. We thank Dr J. Silvestre from ENSAT for cropping advice, P. Recourt from Laboratoire Geosystèmes, UMR CNRS 8157, University of Lille for ESEM-EDX measurements and


Gaëlle Uzu

Sophie Sobanska

Géraldine Sarret

Manuel Munoz

Camille Dumat
email: camille.dumat@ensat.fr

Manuel Muñoz

Foliar lead uptake by lettuce exposed to atmospheric fallouts

Metal uptake by plants occurs by soil-root transfer, but also by direct transfer of contaminants from the atmosphere to the shoots. This second pathway may be particularly important in kitchen gardens near industrial plants. The mechanisms of foliar uptake of lead by lettuce (lactuca sativa) exposed to the atmospheric fallouts of a lead-recycling plant were studied. After 43 days of exposure, the leaves thoroughly washed contained 335 ± 50 mg Pb.kg -1 DW. Micro X-ray fluorescence (µXRF) mappings evidenced Pb-rich spots of a few hundreds of micrometers in diameter located in necrotic zones. These spots were more abundant at the base of the central nervure. Environmental scanning electron microscopy coupled with energy dispersive X-ray microanalysis (ESEM-EDX) showed that smaller particles (few µm in diameter) were also present in other regions of the leaves, often located beneath the leaf surface. In addition, submicrometric particles were observed inside stomatal openings. Raman Microspectrometry (RMS) analyses of the leaves identified smelter-originated Pb minerals but also secondary phases likely resulting from the weathering of original particles. Based on these observations, several pathways for foliar lead uptake are discussed. A better understanding of these mechanisms may be of interest for risk assessment of population exposure to atmospheric metal contamination.

BRIEFS.

The mechanisms of foliar uptake of lead in lettuce exposed to atmospheric Pb-rich fallouts are studied.

INTRODUCTION

Particles emitted in the atmosphere present a large variety of sizes [START_REF] Zhang | Comparison of micro-and nano-size particle depositions in a human upper airway model[END_REF] and during the last decade, the proportion of fine particle matter (PM) increased with the use of more effective filters in industry [START_REF] Müller | Characterization of atmospheric aerosols with multiwavelength Raman lidar[END_REF].

PM 10 (PM whose aerodynamic diameter is less than 10 µm) are target species of the World Health Organization (3) and the European Union Framework Directive on ambient air quality assessment [START_REF][END_REF] due to their adverse effects on the environment and human health. While PM 2.5 , PM 1 and nanoparticles are minor components of total emitted particles, they are probably the most important in terms of environmental impact. Indeed, they can be transported over long distances in the troposphere [START_REF] Dordevic | Contribution of dust transport and resuspension to particulate matter levels in the Mediterranean atmosphere[END_REF][START_REF] Arimoto | Eolian dust and climate: relationships to sources, tropospheric chemistry, transport and deposition[END_REF] and due to their high specific area they can strongly impact the biosphere [START_REF] Uzu | Study of lead phytoavailability for atmospheric industrial micronic and sub-micronic particles in relation with lead speciation[END_REF][START_REF] Fernandez Espinosa | The composition and relationships between trace element levels in inhalable atmospheric particles (PM10) and in leaves of Nerium oleander L. and Lantana camara L[END_REF].

At the global scale, fallouts of atmospheric PM represent the main source of lead pollution in soils [START_REF] Donisa | Heavy Metal Pollution by Atmospheric Transport in Natural Soils from the Northern Part of Eastern Carpathians[END_REF].

Despite the strong decrease in industrial and vehicle lead emissions in recent decades [START_REF] Glorennec | Probabilistic modeling of young children's overall lead exposure in France: Integrated approach for various exposure media[END_REF], leadenriched PM are still emitted in the environment, especially by lead-recycling facilities (11-12-13). In a previous study on particles rejected by such a facility, a large proportion of submicrometric size particles was observed [START_REF] Uzu | Study of lead phytoavailability for atmospheric industrial micronic and sub-micronic particles in relation with lead speciation[END_REF]. Toxic for living organisms even at low concentrations [START_REF] Canfield | Intellectual Impairment in Children with Blood Lead Concentrations below 10 µg per Deciliter[END_REF], lead can be ingested as polluted soil and dust particles by children [START_REF] Alloway | Heavy metals in soils. 2 nd Edition[END_REF]. Additional routes of exposure, which are more important for adults, include drinking water contamination and consumption of locally produced vegetables grown in kitchen gardens [START_REF] Alexander | Genotypic variations in the accumulation of Cd, Cu, Pb and Zn exhibited by six commonly grown vegetables[END_REF].

Lead is known to be weakly mobile in soils [START_REF] Cecchi | Multi-metal contamination of a calcic cambisol by fallout from a lead-recycling plant[END_REF][START_REF] Dumat | Metal ion geochemistry in smelter impacted soils and soil solutions[END_REF], with a residence time estimated to several hundreds of years [START_REF] Klaminder | Flux rates of atmospheric lead pollution within soils of a small catchment in northern Sweden and their implications for future stream water quality[END_REF]. Lead concentrations in vegetables from kitchen gardens contaminated by atmospheric PM are not correlated with soil contents [START_REF] Douay | Assessment of a remediation technique using the replacement of contaminated soils in kitchen gardens nearby a former lead smelter in Northern France[END_REF]. The main reason is that plant uptake is correlated to the phytoavailable fraction of lead rather than to the total metal burden. However, this absence of correlation may be due to a direct metal contamination through the shoots. Airborne contamination of plants by lead and other metals (21-22-23) and by radionuclides [START_REF] Madoz-Escande | Foliar contamination of Phaseolus vulgaris with aerosols of 137Cs, 85Sr, 133Ba and 123mTe: influence of plant development stage upon contamination and rain[END_REF] was evidenced in previous studies. However, the mechanisms responsible for the foliar uptake are still unclear and the literature on this topic is very limited compared to soil-root transfer. Most studies on foliar transfer concern plant nutrients in the context of foliar fertilization (25-26-27-28) and to our knowledge there is no study on the mechanisms of foliar uptake of lead.

Terrestrial plants have kept from their aquatic ancestors the ability to absorb nutrients through the leaves [START_REF] Mengel | Alternative or complementary role of foliar supply in mineral nutrition[END_REF], and contaminants may follow the same pathway. Nutrients and contaminants have to cross several physical barriers before entering the cytosol of epidermal cells. This penetration is strongly dependant of weather conditions, plant species, physiological status and speciation of the element [START_REF] Schönherr | Cuticular penetration of potassium salts: Effects of humidity, anions, and temperature[END_REF].

There are two parallel routes for crossing the cuticle, the lipophilic and the hydrophilic pathway [START_REF] Schönherr | Calcium chloride penetrates plant cuticles via aqueous pores[END_REF][START_REF] Schonherr | Characterization of aqueous pores in plant cuticles and permeation of ionic solutes[END_REF].

The lipophilic pathway is not considered in this study because it concerns apolar and non-charged molecules, which cross the cuticle by diffusion in cutin and waxes. Ions and hydrophilic solutes follow the hydrophilic pathway via aqueous pores [START_REF] Schönherr | Calcium chloride penetrates plant cuticles via aqueous pores[END_REF]. This pathway requires dissolution of the compounds which depends mostly on the humidity, and on the hygroscopicity and solubility of the particles [START_REF] Schönherr | Cuticular penetration of potassium salts: Effects of humidity, anions, and temperature[END_REF].

When the humidity is above the point of deliquescence, the compound partly dissolves and penetration proceeds from this saturated solution. Aqueous pores are located over anticlinal walls and on cuticular ledges of stomata guard cells [START_REF] Schonherr | Characterization of aqueous pores in plant cuticles and permeation of ionic solutes[END_REF][START_REF] Eichert | Quantification of stomatal uptake of ionic solutes using a new model system[END_REF]. Once a contaminant has crossed the cuticle, it may remain in the apoplasm or be transported inside cells [START_REF] Chamel | Quantitative determination of water sorption by plant cuticles[END_REF]. Beside this hydrophilic pathway for solutes, a solid-state pathway also exists. Stomata enable the uptake of suspended nanoparticles and their diffusion in the apoplasm. The stomatal pathway is considered as highly capacitive because of its large size exclusion limit 10 nm-1µm and its high transport velocity [START_REF] Eichert | Size exclusion limits and lateral heterogeneity of the stomatal foliar uptake pathway for aqueous solutes and water-suspended nanoparticles[END_REF].

The first objective of the present study was to evaluate the transfer of lead from atmospheric contamination in lettuce, a widely cultivated vegetable already used as a model plant in metal transfer studies [START_REF] Alexander | Genotypic variations in the accumulation of Cd, Cu, Pb and Zn exhibited by six commonly grown vegetables[END_REF][START_REF] Waisberg | The effect of pH, time and dietary source of cadmium on the bioaccessibility and adsorption of cadmium to/from lettuce (Lactuca sativa L. cv. Ostinata)[END_REF][START_REF] Khan | Accumulation of polycyclic aromatic hydrocarbons and heavy metals in lettuce grown in the soils contaminated with long-term wastewater irrigation[END_REF]. Secondly, this work aimed at investigating the mechanisms of lead foliar uptake by monitoring Pb leaves content over a 43-day exposure time and determining its localization and speciation in the leaves. Such knowledge can be of high interest for risk assessment. Lettuces were first exposed to the fallouts of a plant recycling batteries emitting lead-rich particles (333,000 mg Pb/kg particles) have been previously characterized [START_REF] Uzu | Study of lead phytoavailability for atmospheric industrial micronic and sub-micronic particles in relation with lead speciation[END_REF]. The total content of lead in the leaves was determined after carefully washing. Micro X-ray fluorescence (µXRF) was used to study the distribution of lead and other elements on centimetric zones of leaves with a lateral resolution of 50 µm. Environmental scanning electron microscopy coupled with energy dispersive X-ray microanalysis (ESEM-EDX) was used to determine the morphology and elemental composition of lead deposits at a higher resolution, and Raman Microspectrometry (RMS) provided the molecular composition of lead-rich areas. The results obtained were then discussed and possible scenarii for lead foliar uptake were proposed. To our knowledge, this is the first study of foliar transfer of metals on vegetable samples originating from a field study using a combination of physical and chemical techniques.

EXPERIMENTAL SECTION

Lettuce exposure to atmospheric lead fallouts. Commercial lettuce seeds, ''Batavia blonde dorée'' cultivar, were surface sterilized with 0.9% CaClO for 15 min and rinsed with deionised water. Lettuces were first grown hydroponically for 10 days before transfer in pots in a greenhouse for 15 days to get plants of about 15 g (fresh biomass). After this period, 40 lettuces were placed in pots containing four kg of uncontaminated calcareous soil (total lead concentration: 25±2 mg.kg -1 ). Each pot contained one plant. A geotextile membrane was placed on the soil surface to protect it against atmospheric fallouts.

Plants were exposed for 43 days in the courtyard of a secondary lead smelter which recycles batteries [START_REF] Uzu | Study of lead phytoavailability for atmospheric industrial micronic and sub-micronic particles in relation with lead speciation[END_REF]. Every ten days, five replicates were harvested. In addition, five control plants placed in an urban area at 15 km from the smelter were harvested after 43 days. Lead concentration in the courtyard of the recycling-plant was 1 µg Pb.m -3 air and lead concentration in PM Emissions was 330 000 mg Pb.kg -1 .

According to the French authorities (36), 328 kg of Total Suspended Particles (TSP) including 31 kg of lead were emitted by this facility in 2007. The particles emitted by this smelter were characterized in a previous study by X-ray diffraction and Raman spectroscopy [START_REF] Uzu | Study of lead phytoavailability for atmospheric industrial micronic and sub-micronic particles in relation with lead speciation[END_REF]. Lead speciation was, in decreasing order of abundance, PbS, PbSO 4 , PbSO 4 • PbO, α-PbO and Pb 0 .

Chemical Analysis of soils and vegetables. After harvesting and removal of the roots, the shoot biomass was measured. Each lettuce was cut in four quarters and one was randomly picked for analyses.

The leaves were cut at their base, and each one was washed, first in running tap water for 30 seconds and then in two baths of de-ionized water for one minute, in order to eliminate particles present on the leaf surface but not tightly bound. After draining the leaves with a salad-spin and drying at 50°C for 48h, leaf and soil samples were digested, in a 1:1 mixture of HNO 3 and H 2 O 2 at 80 °C for 4 h and in hot aqua regia, respectively. After filtration, lead concentration was measured by inductively coupled plasma -atomic emission spectrometry (ICP-AES) with an IRIS Intrepid II XDL. The accuracy of the acidic digestion and analytical procedures was checked using the reference material Virginia tobacco leaves, CTA-VTL-2, ICHTJ. Certified values for lead in Tobacco leaves were given for 22.1 ± 1.2 mgPb.kg -1 dry weight. Measured values for the three replicates were 22.0 ± 0.9, 22.4 ± 0.8 and 21.9 ± 0.8 mgPb.kg -1 dry weight. The leaves of the lettuces after 43 days of exposure were then studied by µXRF, ESEM-EDX and RMS.

Micro X-ray fluorescence (µXRF).

Elemental distributions in various regions of the leaves were determined by µXRF. Fresh washed leaves were freeze dried, flattened on the sample holder and placed under vacuum. µXRF spectra were collected with an EDAX Eagle III XRF spectrometer, equipped with a Rh anode and a poly-capillary that focuses the X-ray beam down to 30 µm full width at half maximum (FWHM). An EDX detector with 140 eV resolution was used to measure the X-ray fluorescence. The spectrometer was operated at 15 or 20 kV, and 300 to 450 µA. Centimetric sized X-ray maps were collected over 256 by 200 pixels with steps of 30 to 50 µm. The counting time was 600 to 2000 ms per pixel. Detection limits for such apparatus is around 100 ppm for Pb and other heavy elements.

Deconvolution of µXRF spectra were needed since the S K-and Pb M-lines overlap within the 140 eV resolution of the EDX detector (Fig. 1b). It was performed using the EDAX-integrated software Vision32. Despite the peak overlap, the statistics on Pb fluorescence signal was better using Pb M-line than Pb L-line at higher voltage (40 kV). Elemental maps were combined as RGB (red-green-blue) 3color maps using Matlab TM . In addition, the XRF spectra for the Pb-rich particles obtained by summing single-pixel spectra in the spot of interest were compared.

Environmental Scanning Electron Microscope (ESEM-EDX). Morphology and elemental

distribution were studied using an ESEM (Quanta 200 FEI) equipped with an EDX detector Quantax (Rontec). It was operated at 25 kV in the low vacuum mode. Semi-quantification measurements were obtained with ZAF correction and using real standards. Leaves were air dried and fixed on a carbon substrate before analysis. As for the µXRF analyses, Pb M-and S K-peaks were separated by deconvolution.

Raman microspectrometry (RMS).

Molecular identification of particles present on or beneath the leaf surface of lettuce leaves was performed using RMS. Leaf samples collected after 43 days of exposure were fixed on a glass plate and mounted on the microscope stage without further preparation. In most cases, particles (or leave areas) were analyzed by both ESEM-EDX and RMS after a careful relocalization, thus electronic and optical images as well as elemental and molecular compositions could be compared. RMS measurements were carried out using a Labram Confocal spectrometer and a Labram HR UV 800 (Jobin Yvon, Horiba Gr, France). The Raman backscattering was excited with 632.8 nm or 266 nm excitation wavelength supplied by a He-Ne and a solid MBD 266 system (Coherent -laser group), respectively. The beams were focused on the sample surface through an optical objective (visible Olympus objective, X100, 0.9 NA and UV Mitutoyo objective X80, 0.55 NA) with a lateral resolution (XY) less than 1 µm in both cases. The excitation using a UV laser (266 nm) was necessary to probe the surface of leaves since many biologically important molecules such as chlorophyll have an intense fluorescence emission when excited in the visible range (400 nm << 800 nm). Fluorescence cross sections are typically several orders of magnitude larger than Raman scattering cross-section and masks any underlying Raman spectra. The Raman spectra were analyzed based on the shifts in Raman peak values, changes in full width at half maximum (FWHM) ratios of Raman bands, and normalized intensity variations. For identification of chemical species, measured Raman spectra (band wave number and relative intensities) with flat base line were compared with spectra in established libraries using Spectral Library Search ID 301 software (Thermo Galactic). The hit quality index (HQI) represents the closeness of the match between the unknown and a particular library entry. Raman spectra libraries used are listed in the supporting information. In addition to these databases, Raman spectra of relevant Pb compounds including solid-state phases and aqueous solutions were recorded to check for possible differences due to instrumental functions. Further information about identification of Pb minerals using RMS can be found elsewhere [START_REF] Batonneau | Speciation of PM10 Sources of Airborne Nonferrous Metals within the 3-km Zone of Lead/Zinc Smelters[END_REF][START_REF] Sobanska | Microchemical investigations of dust emitted by a lead smelter[END_REF].

Statistical data treatment. Amount of lead in plant obtained were subjected to analysis of variance (ANOVA) with one factor, using the software Statistica, Edition'98 (StatSoft Inc., Tulsa, OK, USA).

Significant differences (p<0.05) were measured by LSD Fisher test.

RESULTS AND DISCUSSION

Foliar lead uptake. Pb average concentrations in washed leaves after 0, 13, 23, 34 and 43 days of atmospheric fallouts exposure were 0.22 ± 0.09, 69 ± 15, 139 ± 40, 217± 40, 335± 50 mg.kg -1 dry weight (DW), respectively. These values were all significantly different, and lead enrichment as a function of time followed a linear law, without plateau at 43 days of exposure:

[Pb] leaves = 6.98  (number of days of exposure), r 2 = 0.96, n=5.

No significant difference in leaf biomass was noticed between control and exposed plants (40 ± 5 g DW after 43 days), and plants stayed green and healthy despite some necrotic spots (see below). The comparison of the total lead concentrations in leaves between the exposed and control plants after 43 days (335 ± 50 and 5 ± 3 mg.kg -1 , DW, respectively) suggests a strong foliar lead uptake by exposed plants. Other lettuces cultivated at 250 and 400 meters from the smelter (not investigated in this study) contained 30 and 15 mg.kg -1 , DW, respectively. Thus, lead content strongly varies as a function of the distance to the smelter. A survey of reported metal contents in vegetables in potentially contaminated sites [START_REF]BAPPET-Database on ETM in vegetables[END_REF] showed a great variability as a function of sampling sites and environmental conditions.

According to EU requirements for consumption (4), lead concentration shouldn't exceed 0.3 mg Pb.kg -1 fresh biomass in leaf-vegetables corresponding to 3.26 mg Pb.kg -1 DW in our study (DW reached 10.85% in exposed lettuces). Thus, this threshold value were reached after 0.5 day of exposure in the plant courtyard. The relatively high lead content measured in control plants could result from soil-root uptake. However, this latter hypothesis is not likely because the control soil was chosen for its relatively high pH (8.4) and carbonates amount (98 g.kg -1 ) to minimize Pb availability [START_REF] Cecchi | Multi-metal contamination of a calcic cambisol by fallout from a lead-recycling plant[END_REF][START_REF] Birkefeld | In situ transformations of fine lead oxide particles in different soils[END_REF] and low lead content. Lead present in the control plant more likely results from diffuse Pb contamination of the urban environment.

After exposure, no variation of lead content in the soil contained in the pots was observed (< 30 mgPb.kg -1 ). However, this is not a proof that Pb root uptake did not occur. Supposing that Pb contained in the control plants only originates from the substrate (which likely overestimates the soil-root transfer), the amount of lead taken up by roots would represent only 1.5% of total Pb measured in the leaves after 43 days of exposure in the plant courtyard. So the soil-root uptake of Pb can be neglected relative to the foliar uptake. In conclusion, the strong Pb enrichment in exposed plants is due to a foliar contamination. Considering that vegetables were thoroughly washed, lead was at least strongly bound to the leaf surface or internalized.

Lead distribution in the leaves, spatially-resolved speciation and phyto-toxicity symptoms. The distribution of lead and other elements was investigated at the leaf-scale by µXRF on ten leaves on centimetric zones. Figure 1 shows the analysis of the basal part of a leaf. Four lead-rich zones of a few hundreds of micrometers corresponding to brownish necrotic zones were observed (spot 1 to 4 in Fig. 1c). The µXRF spectra for the four spots, obtained by summing single pixel spectra, are shown in The leaves were then studied at a higher resolution by ESEM-EDX and RMS. A variety of Pb-rich necroses were investiagted (Erreur ! Source du renvoi introuvable.a-c and Figure S3, SI). As shown by µXRF, EDX maps showed a heterogeneous distribution of Pb within a Ca-and Mn-containing phase (Erreur ! Source du renvoi introuvable.b). P and K elements were detected in all the necroses, suggesting the presence of an organic membrane covering the precipitates. This organic layer was clearly visible on the secondary electron images (e.g., Erreur ! Source du renvoi introuvable.b-c and Figure S3, SI) and on optical image performed under UV light (e.g., Figure S4, SI). The RMS measurements carried out using a laser beam with a wavelength in the visible region showed an intense fluorescence signal typical of organic substances (e.g. chlorophyll). Measurements using a laser beam at 266 nm allowed probing the compounds beneath this organic membrane. The Ca, Mn phase was identified by Raman as a mixed carbonate i.e (Ca 1-x Mn x )CO 3 thanks to a characteristic intense Raman band at 1087 cm -1 corresponding to the symmetric stretching ( 1 ) of carbonate group (symmetry D 3h ) in the calcite-like structure (space group ). Small particles of MnO 2 and PbSO 4 were found within this carbonate precipitate (Erreur ! Source du renvoi introuvable., SI). The Raman bands at ~1580 cm -1 and ~1300 cm -1 found on many spectra were assignated to carbonaceous material resulting from the degradation of the organic membrane by the laser beam.

Necroses associated to metal accumulations have already been observed for Cd in Brassica juncea L.

(41) and Thlaspi caerulescens [START_REF] Cosio | Distribution of cadmium in leaves of Thlaspi caerulescens[END_REF] in the case of metal root uptake, and for Pb in Arabidopsis thaliana in the case of foliar uptake [START_REF] Lummerzheim | Comparative microscopic and enzymatic characterization of the leaf necrosis induced in Arabidopsis thaliana by lead nitrate and by Xanthomonas campestris pv. campestris after foliar spray[END_REF]. Necroses are formed by dead cells and are symptoms of relatively acute toxicity. In the present study, metal-rich necroses suggest a toxic effect of metals (Pb, Zn, Fe, Cu, Mn depending on the spot, Pb being generally the most concentrated one). A likely scenario is the transfer of some metals in the intracellular compartment, leading to interferences with the cell metabolism.

Further microscopic investigations are required to confirm this hypothesis. In all leaves studied, the base of the central vein contained more particles than the other regions of the leaf. This phenomenon is likely due to rainfall and watering which flushed the particles towards the basal part of the leaves. It was already reported in the case of radionuclide-containing particles [START_REF] Madoz-Escande | Weather-dependent change of cesium, strontium, barium and tellurium contamination deposited as aerosols on various cultures[END_REF]. In order to reduce the risk induced by vegetables potentially exposed to atmospheric fallouts, a simple recommendation could consist in eliminating the basal part of leaves before consumption.

Particles were also present on the leaf surface (Figure S6, SI). Individual particles and aggregates were a few micrometers to a few tens of micrometers in diameter. The major elements detected by EDX in particles/aggregates were Pb, S, Cl, Fe, Ca, Si and Al. The RMS analysis of the particles allowed the identification of anglesite (PbSO 4 ), lanarkite (xPbO • PbSO 4 ), quartz (α-SiO 2 ), amorphous iron oxyhydroxyde (FeOOH), calcite (CaCO 3 ), hydrocerussite (Pb 3 (CO 3 ) 2 (OH) 2 ), cerussite (PbCO 3 ) and some fluorescent species attributed to clay minerals (Figure S7, SI). Clay may result from the deposition of non-contaminated dust. PbS was not detected because this mineral is almost silent in Raman (a weak and broad Raman band is observed at 451 cm -1 ), and this compound readily transform into lead sulphate products under the laser beam [START_REF] Batonneau | Microscopic and imaging Raman scattering study of PbS and its photo-oxidation products[END_REF].

PM were also frequently observed inside stomatal openings on adaxial and abaxial surfaces (Erreur ! Source du renvoi introuvable.d and Fig. S8, SI). These PM had a diameter comprised between 50 nm and 1 µm and minerals identified by RMS included PbSO 4 and PbO. The presence of these particles in stomata may have two consequences. Firstly, clogging ostioles disturbs leaf physiology by decreasing the stomatal conductance and gas exchanges and perturbing the water regime, transpiration and control of leaf temperature [START_REF] Hirano | Physical effects of dust on leaf physiology of cucumber and kidney bean plants[END_REF]. Secondly, toxic metals may penetrate inside leaves via stomata [START_REF] Eichert | Size exclusion limits and lateral heterogeneity of the stomatal foliar uptake pathway for aqueous solutes and water-suspended nanoparticles[END_REF].

Although some aggregates presented the same morphology as source particles characterized previously (7), 3-4% of needles crystallites were observed on leaf surface (ratio of needle-like particles to the total number of particles observed, Erreur ! Source du renvoi introuvable.e and Fig. S9, SI). These crystallites were not observed in source particles [START_REF] Uzu | Study of lead phytoavailability for atmospheric industrial micronic and sub-micronic particles in relation with lead speciation[END_REF]. EDX analyses showed Pb, K and Cl as major constituents of the needle structures whereas other elements such as Sn, Ca, K and Fe were found in non-spherical particles associated with the needles. These crystallites were not made of lead phosphate. RMS measurements showed that they were very beam sensitive and intense PbO and elemental carbon signals were observed after beam damage. These observations suggest that the needles are made of lead-organic species. This was confirmed by spectra recorded at very low laser power on which not very intense Raman bands at ~2800 cm -1 and ~ 1780 cm -1 characteristic of C-H and C-O bonds, respectively, were observed. These particles may correspond to secondary phases resulting from the dissolution of original particles on the leaf and precipitation with organic compounds present on the cuticle. These changes in particles' morphology and speciation may result from abiotic processes.

However, the surface of plant leaves is generally colonized by microorganisms which may participate to Our results suggest that various pathways may lead to foliar uptake of Pb by lettuce exposed to Pbrich PM. The fate of metal contaminants in the soil and their soil-plant transfer has to be evaluated in metal-contaminated soils, but the foliar transfer is another important issue, which is much less studied.

Indeed, in the context of agricultural activities established near metallurgic activities, plant metal uptake may occur predominantly via foliar contamination by micro-and nanoparticles. Microscopic and microspectroscopic investigations of the plants provided insights on the fate of PM deposited on lettuce leaves. Such knowledge may be of interest on a physiological point of view and in the perspective of risk assessment of atmospheric emissions in urban environments.

Figure 1b .

 1b Figure 1b. The spots contain the same elements but in varying proportions: Spot 1 is richer in S, Ca, Si

Figure 1 .

 1 Figure 1. (a) Optical image of the basal part of the lettuce leaf exposed to atmospheric fallouts.

Figure 2 .

 2 Figure 2 . ESEM-EDX analyses of Pb-rich regions on lettuce leaves exposed to atmospheric

Figure 3 .

 3 Figure 3. Tentative pathways for lead uptake after deposition of Pb-containing particles.

ACKNOWLEDGMENTS

ADEME, the French Agency for Environment and Energy as well as the STCM, are acknowledged for their financial support and technical help. This research project was supported by the National CNRS CYTRIX-EC2CO program. We thank M. Boidot for Figure 3, P. Recourt from Laboratoire Geosystèmes, UMR CNRS 8157, University of Lille for ESEM-EDX measurements, and M. Corazzi for his help during the µXRF measurements.

chemical processes taking place at this interface [START_REF] Andrews | The ecology and biogeography of microorganisms on plant surfaces[END_REF][START_REF] Lindow | Microbiology of the Phyllosphere[END_REF]. Further investigations are required to evaluate the bacterial colonization on the lettuce leaves and the possible participation of microorganisms to the alteration of the particles.

Potential mechanisms involved in foliar uptake

The results of this study clearly show that some lead is trapped both on the leaf surface and inside the leaf. Based on our observations and on the present knowledge on foliar uptake of elements, several mechanisms can be tentatively proposed for these processes (Figure 3). Firstly, particles deposited on the leaf surface may be trapped in the tiny folds of the leaf and remain as such. The original particles contain PbS which is highly reactive and subjected to oxidation and weathering processes, and secondary Pb-containing compounds may form on the leaf surface. The chemical processes of PbS oxidation under atmospheric conditions have been described previously [START_REF] Batonneau | Microscopic and imaging Raman scattering study of PbS and its photo-oxidation products[END_REF][START_REF] Choel | Fast evolution of tropospheric Pb-and Zn-rich particles in the vicinity of a lead smelter[END_REF], and lead to the formation of PbO, PbSO 4 , PbO • PbSO 4 and PbCO 3 . These transformations may occur in the atmosphere during the transport of particles, and after their deposition on plant leaves. In parallel, lead may penetrate inside the leaf by two pathways. Firstly, lead-containing nanoparticles observed in the stomata may penetrate in the apoplasm as solid compounds. In this study nanoparticles were observed only in stomatal openings. Further analyses, particularly the study of leaf cross sections, are necessary to test whether nanoparticles enter the leaves via stomata. In addition, particles were observed beneath an organic membrane, probably the cuticle. Again, further investigations are needed to determine their exact localization and the way they were transferred in this place. Secondly, lead resulting from the dissolution of source particles may diffuse through aqueous pores of the cuticle and of the stomata, following the hydrophilic pathway inducing the formation of necroses enriched with lead.