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Journal of Hazardous Materials 264 (2014) 98– 106

Contents lists available at ScienceDirect

Journal of Hazardous Materials

jou rn al hom epage: www.elsev ier .com/ locate / jhazmat

oliar exposure of the crop Lactuca sativa to silver nanoparticles:vidence for internalization and changes in Ag speciation

amille Laruea, Hiram Castillo-Michelb, Sophie Sobanskac, Lauric Cécillona,arah Bureaua, Véronique Barthèsd, Laurent Ouerdanee, Marie Carrière f,éraldine Sarreta,∗

ISTerre, Université Grenoble 1, CNRS, F-38041 Grenoble, FranceESRF, Beamline ID21, Grenoble, FranceLASIR, UMR CNRS 8516, Université Lille 1, Bât C5, 59655 Villeneuve d’Ascq Cedex, FranceCEA/LITEN/DTNM/L2T, CEA Grenoble, 38054 Grenoble Cedex 9, FranceLCABIE/IPREM-UMR 5254, Université de Pau et des Pays de l‘Adour, 64053 Pau Cedex 9, FranceUMR E3 CEA-UJF/LAN, 38054 Grenoble Cedex 9, France

i g h l i g h t s

Ag-NPs are internalized inside lettuceleaves after foliar exposure to a sus-pension of Ag-NPs.A classical washing process is inef-ficient at decreasing significantly Agcontent.Ag-NPs in plants undergo oxidation,and resulting ionic Ag are complexedwith organic compounds includingthiol-containing molecules.Foliar exposure to Ag-NPs doesnot lead to detectable phytotoxicitysymptoms.

g r a p h i c a l a b s t r a c t

r t i c l e i n f o

rticle history:eceived 7 August 2013eceived in revised form 17 October 2013ccepted 25 October 2013vailable online 1 November 2013

eywords:cotoxicology

a b s t r a c t

The impact of engineered nanomaterials on plants, which act as a major point of entry of contaminantsinto trophic chains, is little documented. The foliar pathway is even less known than the soil-root pathway.However, significant inputs of nanoparticles (NPs) on plant foliage may be expected due to depositionof atmospheric particles or application of NP-containing pesticides. The uptake of Ag-NPs in the cropspecies Lactuca sativa after foliar exposure and their possible biotransformation and phytotoxic effectswere studied. In addition to chemical analyses and ecotoxicological tests, micro X-ray fluorescence, microX-ray absorption spectroscopy, time of flight secondary ion mass spectrometry and electron microscopy

lantsg nanoparticlesicro-XAS

oF-SIMSiodistribution

were used to localize and determine the speciation of Ag at sub-micrometer resolution. Although nosign of phytotoxicity was observed, Ag was effectively trapped on lettuce leaves and a thorough washingdid not decrease Ag content significantly. We provide first evidence for the entrapment of Ag-NPs bythe cuticle and penetration in the leaf tissue through stomata, for the diffusion of Ag in leaf tissues, andoxidation of Ag-NPs and compcrucial for better assessing the

∗ Corresponding author. Tel.: +33 0 4 76 63 51 99.E-mail addresses: camille.larue@rub.de (C. Larue), castillo@esrf.fr (H. Castillo-Michel),

L. Cécillon), sarah.bureau@ujf-grenoble.fr (S. Bureau), veronique.barthes@cea.fr (V. Barthèeraldine.sarret@ujf-grenoble.fr (G. Sarret).

304-3894/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jhazmat.2013.10.053

+

lexation of Ag by thiol-containing molecules. Such type of information is risk associated to Ag-NP containing products.

© 2013 Elsevier B.V. All rights reserved.

sophie.sobanska@univ-lille1.fr (S. Sobanska), lauric.cecillon@irstea.frs), laurent.ouerdane@univ-pau.fr (L. Ouerdane), marie.carriere@cea.fr (M. Carrière),

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C. Larue et al. / Journal of Haza

. Introduction

In the last decade, nanotechnology has emerged as a majoreld of research and investments for industries. Thanks to theirnique properties, NPs are included in more and more consumerroducts. 313 consumer products containing Ag-NPs have been

isted over the 1317 products of the database in 2011 [1]. Ag-Ps are mainly used as an anti-bacterial agent. Major products

nclude cosmetics, paints, fabrics, food containers, medical equip-ent and pesticides. The production of Ag-NPs is estimated at 500

ons per year worldwide [2]. Eventually, most NPs will end upn the environment. Current knowledge on the fate of Ag-NPs inhe environment has been recently reviewed [2] and their poten-ial toxicological and ecotoxicological impact raises much concern3,4].

Plants play a critical role in the fate of NPs in the envi-onment through their uptake, bioaccumulation and transfer torophic chains [5]. Crop plants are more likely to be exposed toPs than wild plants due to the application of sewage sludgen agricultural soils and use of Ag-NPs in plant protection prod-cts. After root exposure, uptake of Ag-NPs by roots was shown6–8], as well as phytotoxic effects: reduced seed germination, per-urbed root and shoot growth, and decreased evapotranspiration6,7,9–12]. Genotoxic effects were evidenced as well [7,13–15].he foliar exposure pathway has been much less investigated,lthough such information is necessary for a comprehensive riskssessment of nanomaterials. Indeed, NPs have been used inlant protection products for several years so far [16,17]. 110roducts containing Ag-NPs were registered as pesticides byhe US-EPA in 2010 [16], and several patents for Ag-NP fungi-ides were deposited [18]. The use of such products is expectedo expand in the future [18–21]. Moreover, new applicationsf NPs such as controlled release of agrochemicals for nutri-ion and protection against pests and pathogens, delivery ofenetic material and sensitive detection of plant disease are cur-ently under investigation [19,20]. Although these developmentsay be beneficial for the environment (decreased inputs), there

s an urgent need of better evaluating the risks associated tohese products [20,22]. To the best of our knowledge, publishedtudies on foliar exposure to NPs concerned TiO2-NPs [23], CeO2-Ps [24] and polystyrene-NPs [25], but none was realized ong-NPs.

The purpose of this study was to investigate the impact of a foliarxposure to Ag-NPs on a leafy crop plant in terms of phytotoxicity,o determine the proportion of Ag retained on/in the leaves afterhorough washing, and also to get insights on the mechanisms ofoliar uptake and possible transformations of Ag-NPs.

Lettuce (Lactuca sativa) was chosen as model species becausef its widespread occurrence in kitchen gardens or farmlandsnd large foliar surface making it an ideal model to studyhe foliar transfer of atmospheric contaminants [26,27]. Plantsere exposed to 1–100 �g Ag-NPs per g fresh weight (FW)

nd to Ag+ ions. This latter treatment aimed at evaluatinghe ionic contribution resulting from the dissolution of Ag-Ps. Total Ag accumulation in shoots was measured before andfter rinsing with slightly acidic water (as performed classi-ally by consumers) [26,27]. Impact of Ag-NP exposure on plantiomass, chlorophyll, protein, total glutathione and phytochelatinPC) contents and oxidative stress were evaluated. We com-ined scanning electron microscopy observation of whole leavesnd micro X-ray fluorescence (�XRF) mapping of leaf cryo-ections to localize Ag-NPs and their weathering products in

eaf tissues. Changes in Ag-NP speciation were evaluated usingg LIII-edge X-ray absorption near edge structure spectroscopy

XANES) and time of flight-secondary ion mass spectrometryToF-SIMS).

Materials 264 (2014) 98– 106 99

2. Material and methods

2.1. Nanoparticle characterization and dispersion

Uncoated Ag-NPs were provided by the paint company PPG(Pittsburgh, Pennsylvania, USA). These NPs were fully character-ized: shape, nominal diameter (transmission electron microscopyTEM – TECNAI OSIRIS) and specific surface area (BET). Before eachexposure, stock suspensions were prepared with NPs dispersed inultrapure water (10, 100, 1000 mg L−1) and homogenized 3 min inan ultrasonic bath. NP hydrodynamic diameter was assessed bydynamic light scattering using a NanoZS (Malvern) and is expressedin number. The zeta potential in ultrapure water and the pointof zero charge were measured with a NanoZS coupled to a MPT2titrator (Malvern). Finally, Ag-NP dissolution was measured in asuspension of 1000 mg Ag-NPs L−1 with an ion specific silver elec-trode (Thermo Scientific) calibrated with a series of AgNO3 standardsolutions.

2.2. Plant culture and exposure

Experimental details on plant growth and exposure conditionsare provided in supporting information. Briefly, young lettuceplantlets were exposed to 1, 10 or 100 �g Ag-NPs g−1 FW. Theseconcentrations are comparable to those chosen by Kim et al. [28]to study the effect of a nanosilver based pesticide against the rosemildew. The contribution of Ag+ coming from Ag-NP dissolutionwas accounted by exposing plants to 0.5 �g AgNO3 g−1 FW. Con-trol plants sprayed with ultrapure water were grown alongside.After a 7-day exposure, plants were harvested.

For all analyses, except synchrotron analyses (see below), a threestep washing procedure was performed: leaves were first rinsedthoroughly with deionized water, then soaked for 10 min in 0.01 Macetic acid mimicking a washing with water and few droplets ofvinegar [29], and finally rinsed again with deionized water.

Fresh foliar biomass was determined; leaves were separated inaliquots for further experiments and kept frozen until analyses.

2.3. Phytotoxicity tests

Phytotoxicity tests were carried out on 5 replicates. Content inphotosynthetic pigments was determined according to Moran [30].Protein content was measured following the Bradford method [31].Lipid peroxidation was assessed by the quantification of thiobarbi-turic acid reactive species (TBARS) as described in Dazy et al. [32].Glutathione (GSH + GSSG) and PC (PC2, PC3, PC4, cysPC2, cysPC3and cysPC4) contents were determined by ultra-performance liq-uid chromatography–tandem mass spectrometry (UPLC–MS/MS)(Waters UPLC Acquity system coupled to a Waters quadrupoleMS/MS Xevo TQD) as previously described [33]. This protocolimplies a first step which reduces the oxidized species (GSSG andoxidized PCs), so measured values represent total concentrationsof glutathione (GSH + GSSG) and PCs. Experimental details for phy-totoxicity tests are provided in supporting information.

2.4. Measurement of total Ag content

Ag content in plant samples was quantified using inductivelycoupled plasma mass spectrometer (ICP-MS – Agilent 7500ce).About 500 mg of fresh plant sample was introduced in a teflon

beaker with 3 mL of 14 N ultrapure nitric acid (distilled once) andheated at 120 ◦C. After 72 h, samples were evaporated. 3 mL of HNO37 N were then added and the sample was evaporated again. Finallysamples were diluted in HNO3 2% for ICP-MS analysis and spiked

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00 C. Larue et al. / Journal of Haza

ith indium (20 ppb) to correct from a drift during measurements.g was quantified on four replicates for each condition.

.5. NP localization and Ag speciation1

.5.1. Analytical scanning electron microscopy (SEM)NP distribution on leaf surface and influence of the washing

ere investigated by SEM (Quanta 200, FEI) equipped with energyispersive X-ray spectroscopy (EDS; X flash 3001 Brucker). Let-uce leaves were oven-dried at 45 ◦C for 2 days and then mountedn the sample holder without further preparation. SEM was oper-ted at 15 kV in low vacuum (133 Pa) and in backscattered electronode. EDS analyses were performed either on spots of interest or

n scanning mode.

.5.2. Time of flight secondary ion mass spectroscopyToF-SIMS analyses were performed on the surface of lettuce

eaves to map Ag-containing molecular fragments using the samerocedure as described elsewhere [27] and reported in details in theupporting information. Briefly, the surface was sputtered using O2eam source operating at 0.5 kV. Analyses were performed with ai3+ source. The sputtering time was fixed to 60 s, correspondingo a depth analysis of about 100 nm in the present experimentalonditions.

.5.3. Synchrotron radiation based �XRF and �XANES�XRF and Ag LIII-edge �XANES measurements were performed

n the scanning transmission X-ray microscope on ID21 beamlinet the ESRF (European Synchrotron Radiation Facility, France) inryo-conditions using a vibration-free cryo-stage, passively cooledy a liquid nitrogen dewar.

Fresh leaves were thoroughly rinsed with deionized water,ently soaked with a tissue, and a small portion of leaf was flash-rozen, embedded in O.C.T® resin and then cut in thin sections20 �m) using a cryomicrotome. �XRF maps of leaf cross-sectionsnd whole leaves were recorded with various step sizes (from.3 �m × 0.3 �m to 3 �m × 3 �m) with incident energy of 3.42 keV,nd dwell time of 300–1000 ms. Elemental maps presented in thisaper were obtained after peak deconvolution using PyMCA soft-are [34]. Ag LIII-edge �XANES spectra were recorded in regions of

nterest of the maps. In addition, bulk XANES spectra were recordedn reference compounds in unfocused mode (200 �m pinhole).ore details are provided in supporting information.

.6. Statistical analysis

Statistical analysis was performed using R software with oneay ANOVA and Tukey tests. Significant differences compared to

he control were obtained for p ≤ 0.05.

. Results

.1. Nanoparticle characterization

Ag-NP physico-chemical characteristics are summarized in Fig-re S1 (supporting information). Briefly, Ag-NPs were mainlyound shaped (68%) with an average nominal diameter of 38.6 nm.nother fraction (32%) of these NPs was elongated with dimen-ions of 38.2 nm × 57.8 nm (Figure S1A and B). The average specificurface area measured by BET was 4.1 m2 g−1.

When in suspension, Ag-NPs were well dispersed with aydrodynamic diameter close to nominal diameter: 47.9 ± 29.2 nm

Figure S1A and C). Ag-NP suspensions were stable with a high neg-tive zeta potential of −29.5 mV. They had a negative surface chargen the pH range tested (2–8), and never reached their point of zeroharge (Figure S1D).

Materials 264 (2014) 98– 106

The release of Ag+ ions from a stock suspension at 1000 mgAg-NPs L−1 in ultrapure water was monitored. Concentrationsranging from 2.3 to 6.5 mg L−1 (i.e., between 2‰ and 7‰ oftotal Ag) were measured with a mean value of 5.3 mg L−1. Thisvalue was unchanged after one month, which suggests a limitedoxidation and dissolution of Ag-NPs. Based on these values, asolution containing 5 mg Ag+ L−1 (as AgNO3) was applied onplant leaves in order to test the effect of this dissolved pool ofAg.

3.2. Localization and speciation of Ag in the leaf tissue

The internalization of Ag-NPs in lettuce leaves was studied by�XRF on thin sections and portions of leaves (Figs. 1 and S2). Thecontrol leaf provides an estimate of the background signal for Ag(maximum counts of 50; Figure S2A–C). The XRF spectrum for thewhole map shows peaks related to plant matrix (Na, Mg, Si, P,S, Cl), and no peak at the Ag L�-edge energy (2.984 keV) (FigureS3A). Leaves exposed to Ag+ evidence an accumulation of Ag inthe exposed epidermis (Fig. 1A). The overall signal of the cross-section shows that Ag content is close to background level, but thespectrum extracted from all hot spots shows a clearly defined Agpeak (Figure S3B). This observation demonstrates that Ag+ pene-trated through the epidermis inside the leaf tissue. For the leavesexposed to Ag-NPs, Ag agglomerates (mean diameter of 2–3 �m)were detected all over the surface of leaves (Fig. 1B), includingstomata (Fig. 1C). These results are consistent with SEM results(Fig. 4G and H). Ag was also detected inside the leaf tissues: in theparenchyma, Ag hot spots of about 2 �m in diameter were observedeither inside cells or associated to the cell walls (Fig. 1D–F). Someof these hot spots presented a concentrated part surrounded by amore diffuse corona (Fig. 1F). This pattern suggests some dissolu-tion of Ag-NPs. Ag agglomerates were also observed between theguard cells and in the sub-stomatal chamber (Figs. 1G and S2D–F).The aperture size of the stomata was 16.0 ± 1.1 �m × 6.5 ± 0.7 �m,as determined by SEM observations. Finally, Ag-NPs were alsoobserved in the main vein, inside the bundles and in the cell wallthickenings (Fig. 1D). Thus, Ag spots were observed in all leaf tis-sues, both in the intra and extracellular compartments (FigureS3C). The distribution of Ag was not correlated with another ele-ment.

LIII-edge �XANES spectra were recorded on the Ag-rich spotsidentified by �XRF, and Ag speciation was determined by linearcombination fits (LCF) [35]. Fig. 2A shows the XANES spectra of Agreference compounds used for LCF. The Ag-NPs exposed to light andhumidity conditions of the growth chamber for one week (Fig. 2Band Table 1) showed a limited oxidation: 16% of Ag0 was convertedinto Ag+. In leaves exposed to Ag-NPs, a variety of speciation wasobserved, from 81% Ag-NPs to 100% Ag-GSH. In the majority ofthe analyzed regions, a mixture of Ag-NPs and secondary speciesincluding Ag-GSH and/or other Ag+ species was found (Table 1). Thenature of these later species is unclear because various fits of equiv-alent quality were obtained with different compounds (namely,Ag-chlorine, Ag-nitrate, Ag-phosphate, Ag-behenate, and ionic Agand Ag-malate in solution). However, AgCl is a likely (but possiblynot unique) species because it gave the best fit for 7 among 8 spec-tra. No difference in Ag speciation was observed with respect to thetissue. Surprisingly, leaves exposed to Ag+ contained the same threeAg species as those exposed to Ag-NPs, and some spots contained100% Ag-NPs (Table 1).

In order to probe more precisely the leaf cuticle, the leaf surfacewas analyzed by ToF-SIMS (Fig. 3). For the control leaves, no Ag

was detected in the depth profile (Fig. 3A). On the leaf exposed toAgNO3, the analysis of a necrotic area demonstrated the presence ofa diffuse signal for Ag+ on the surface, as shown by the appearanceof the peak of Ag+ prior to the C4H7

+ peak indicative of the cuticle

C. Larue et al. / Journal of Hazardous Materials 264 (2014) 98– 106 101

Fig. 1. Elemental distribution in lettuce leaves by �XRF. Analyses for plant cross-sections exposed to 0.5 �g g−1 AgNO3 (A) or to 100 �g g−1 Ag-NPs (D–G) or analyses of plantsurface exposed to 100 �g g−1 Ag-NPs (B and C). Arrows followed by legend indicate some of the hot spots where �XANES spectra were recorded (Fig. 2). ep. epidermis, nu.nucleus, p. parenchyma, st. stomata, v.b. vascular bundle, and w. wall.

Fig. 2. Speciation by Ag LIII-edge �XANES. Spectra recorded on reference compounds (A) and in Ag-rich regions of the leaves with linear combination fits (dotted lines, plusdashed lines in case several solutions were obtained) (B).

102 C. Larue et al. / Journal of Hazardous Materials 264 (2014) 98– 106

Fig. 3. ToF-SIMS analyses of the cuticle of lettuce leaves for control plants (A), from a necwith maps of total ionic fragments, C4H7

+, Ag+ and Ag2Cl+ fragments and corresponding d

Table 1Ag speciation as determined by linear combination fits of LIII-edge �XANES spectra.

Proportion of Ag species (in molar %) Sum NSS (%)b

Ag0-NPs Ag+-GSH Other Ag+ speciesa

Ag-NPs under light 88 16 105 0.49Ag-NPs in H2O 88 16 105 0.34Exposure: Ag-NP

EpidermisSpot 1 78 (5) 28 (5) 105 0.12Spot 2 85 24 109 0.66Spot 3 71 31 102 0.17

81 (1) 21 (0) 102 0.17Spot 4 39 (4) 70 (3) 110 0.19

29 (6) 32 (19) 51 (13) 111 0.20Parenchyma

Spot 1 100 100 0.20Exposure: AgNO3

EpidermisSpot 1 100 100 0.32Spot 2 77 27 104 0.07Spot 3 108 108 0.43Spot 4 44 28 21 93 0.14

ParenchymaSpot 1 82 (8) 32 (8) 113 0.60Spot 2 69 (1) 30 (3) 99 0.42

49 51 100 0.44

a Three types of Ag species were distinguished: Ag-NP, Ag-GSH, and other Ag+

species, fitted with Ag chlorine, Ag nitrate, Ag phosphate, Ag behenate and aqueousspecies (Ag+ and Ag malate in solution). When several fits of equivalent quality(increase of NSS < 10%) were obtained with different standards from the “other Ag+

species” pool, proportions are expressed as mean percentage (SD) calculated forthese fits.

b Residual between fit and experimental data:NSS =

∑(�experimental − �fit)2/

∑(�experimental)2 × 100, in the 3335–3417 eV

r

(dtstc

impact of Ag-NP foliar exposure on lettuces. After a 7-dayexposure, the fresh foliar biomass was unchanged (p = 0.369)

ange.

Fig. 3B). Finally, for leaves exposed to Ag-NPs (Fig. 3C), Ag wasetected as small agglomerates of few micrometers in diameter, inhe form of Ag+ and Ag2Cl+ fragments in the leaf. Depth profilinghowed that Ag-linked fragments appeared concomitantly with

he C4H7

+ fragments, demonstrating that Ag was associated to theuticle.

rotic part of plants exposed to 0.5 �g g−1 AgNO3 (B) or to 100 �g g−1 of Ag-NPs (C)epth profiles.

3.3. Effect of vegetable washing on total Ag concentration

The influence of rinsing lettuce leaves on their content in Ag wasinvestigated by SEM-EDS analysis of leaf surface and ICP-MS. A con-trol sample sprayed with ultrapure water was enclosed (Fig. 4A) forcomparison. After a 7-day exposure and prior to washing, agglom-erates of Ag were observed all over the surface (Fig. 4B) with sizeranging from hundreds nanometers up to 25 �m with a mean sizeof 7 �m (for observable particles). After the washing step, feweragglomerates were observed on the leaf surface (Fig. 4C) and theirmean size was 2 �m. EDS analyses (Fig. 4D–F and I) confirmedthat these agglomerates contained Ag. Ag agglomerates were alsofound near and inside stomatal openings (Fig. 4G and H). Surface ofleaves exposed to Ag+ were also analyzed but Ag signal was belowdetection limit (data not shown).

The efficiency of the washing procedure was evaluated by ICP-MS analysis of the washing water of each step (Fig. 4J). The totalquantity of Ag-NPs deposited per lettuce is approximately 240 �g.The analysis of the first flushing water showed that 3.8 ± 1.8 �g ofAg were removed from the plant. The next two washing waterscontained 1.2 ± 0.1 �g Ag. Thus the washing permits to take off 2%of the total deposited particles. In parallel, lettuce leaves were ana-lyzed for their Ag content before and after washing (Fig. 4K). Theycontained 29.9 ± 6.6 and 18.9 ± 7.9 �g Ag g−1 FW, respectively. Thedecrease in total Ag content during the washing procedure wasnot significant. Nevertheless, there was a difference between theexpected concentration in leaves (100 �g g−1) and the measuredvalue. This discrepancy may originate from a dilution effect due toplant growth during the 7-day exposure. Another hypothesis wouldbe a leaking of NP suspension from the leaves after application ofthe treatment, or some inhomogeneity of the Ag-NPs suspension(although it was agitated manually before pipetting).

3.4. Phytotoxicity

Biological markers were followed to identify a potential

regardless of the concentration (Fig. 5A) with a mean biomassof 4092 ± 135 mg. Chlorophyll a, chlorophyll b, carotenoid and

C. Larue et al. / Journal of Hazardous Materials 264 (2014) 98– 106 103

Fig. 4. Effect of the washing: SEM-EDS analyses of the surface of lettuce leaves. Control plants exposed to ultrapure water (A) or to 100 �g g−1 Ag-NPs before (B) and after(C) washing. (D) Zoom of the insert in (C). (E and F) EDS analyses in scanning mode for Ag and K respectively of (D). SEM picture of stomata (G) with the correspondingE ght spl nt in

b

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rf

DS analysis in scanning mode for Ag (H). (I) Representative EDS analysis of the brieachates after each step of the washing process (J) and determination of Ag conteefore and after the washing process (K).

heophytin contents (Fig. 5B) were not affect with values of.141 ± 0.029 (p = 0.498), 0.235 ± 0.007 (p = 0.292), 0.401 ± 0.010p = 0.512) and 2.457 ± 0.064 (p = 0.493) mg pigments g−1 FW,espectively. Protein content in leaves was not significantly mod-fied neither (p = 0.88) ranging from 51.0 mg 100 mg−1 (FW) to7.0 mg 100 mg−1 FW (Fig. 5C). Similarly, TBARS content wasnchanged (mean value of 0.61 �g g−1 FW; p = 0.651) (Fig. 5D).

Concerning thiol-containing compounds, no significant differ-nces (p = 0.096) were observed between control (11.5 ± 2.7 �g g−1

W) and exposed lettuces (7.2 ± 2.3 �g g−1 FW) for total glu-athione content. Only phytochelatin PC2 could be detected anduantified in control plants (1.09 ± 0.11 �g g−1 FW), whereas nohytochelatin was detected in the exposed plants (<0.01 �g g−1

W).

. Discussion

If the internalization of Ag-NPs has been demonstrated afteroot [6–8,36,37] and fruit [38] exposure, nothing is known on theirate after foliar exposure. In the present study, Ag was found as

ots (electron-dense) in SEM pictures. ICP-MS determination of Ag content in waterlettuce leaves exposed to ultrapure water, 0.5 �g g−1 AgNO3 or 100 �g g−1 Ag-NPs

micrometer-scale agglomerates on the surface of leaves, and in leaftissues including epidermis, mesophyll and vascular tissues. Thus,Ag-NPs deposited on the surface were able to cross the epidermisand eventually to be transferred to the leaf tissue. The low solubilityof Ag-NPs used in this experiment suggested that ionic Ag speciesresulting from the dissolution of Ag-NPs contributed to a limitedextent to this transfer. These results are consistent with similarexperiments realized with other types of NPs. Au (10–20 nm), TiO2(14 nm) and CeO2 (37 nm) NPs were shown to accumulate in rape-seed and maize after foliar exposure [24,39,40].

The mechanisms of foliar transfer of NPs and solutes arestill poorly understood. Two pathways have been proposed, forhydrophilic compounds via aqueous pores of the cuticle andstomata, and for lipophilic ones by diffusion through the cuticle.Although uncoated Ag-NPs are considered as hydrophilic, theirpossible coating by cuticular waxes might increase their lipophilic-

ity and favor their transfer through the cuticle [27]. These twopathways were described for small particles (few nm) [41]. Nev-ertheless, the uptake of 43 nm diameter polystyrene-NPs in thestomata of Vicia faba leaves was observed [25]. The presence of a

104 C. Larue et al. / Journal of Hazardous Materials 264 (2014) 98– 106

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ig. 5. Phytotoxicity tests after foliar exposure to 0.5 �g g−1 AgNO3 (ionic Ag) or toeaves. (D) Lipid peroxidation estimated by the TBARS content in leaves.

hin film of water on the leaf surface could build up a hydrauliconnection between the leaf surface and interior, and enhancehe transfer of solutes and particles [42]. In the present study,umidity conditions of the growth chamber could very likely gen-rate such an aqueous thin film. Some NPs were embedded inhe leaf cuticle and/or in cuticular waxes present on its surface,s shown by �XRF and ToF-SIMS, and NP accumulations werelso observed in the sub-stomatal chamber. Similar associationsith cuticular waxes and stomata were observed on lettuce leaves

fter a foliar contamination to Pb-rich nano- and microparticles26,27], but it is the first time that NPs are observed inside theub-stomatal chamber of this plant. CeO2-NPs were observed byEM close to stomata and also entrapped in surface waxes of maizeeaves [24]. The present study suggests that they might follow bothhe cuticular and stomatal pathways to penetrate inside lettuceeaves. The stomatal pathway may be easier since the cuticle of theub-stomatal chamber is generally thinner than the external one43].

Ag agglomerates of several �m in diameter were observedn various regions of the leaves. Isolated Ag-NPs also may beresent but not detected by �XRF at the Ag LIII-edge. Finally,g was detected inside cells as well as in the apoplasm. In atudy on Arabidopsis thaliana roots exposed to 20 and 40 nm Ag-Ps, authors also found Ag-NPs in the apoplasm and aggregatedlose to plasmodesmata [37] suggesting that both apoplasmicnd symplasmic pathways are active. An entry of Ag-NPs inhe cytoplasm of the alga Ochromonas danica accompanied byoxic effects was also evidenced [44,45]. The transfer of otherypes of NPs including TiO2 and carbon nanotubes inside cellsas observed in wheat and rapeseed [46,47]. The entry of NPs

nside cells may occur by damage of the cell membrane ory endocytosis, as observed by Liu et al. [48] for walled plant

ells exposed to carbon nanotubes. In our case TBARS assayid not evidence an increase in lipid peroxidation. However,

ocalized effects might be undetected by bulk analyses such asBARS.

Ps. (A) Foliar biomass. (B) Photosynthetic pigment content. (C) Protein content in

Ag speciation is of importance since it controls phytotoxicityand toxicity for consumers. Ag-NPs placed in the growth chamberunderwent very limited alteration. On the contrary, major changesin speciation were observed by �XANES for Ag-NPs present onthe leaf surface or internalized in the leaf tissue. These changesincluded partial or total oxidation of Ag-NPs, and formation ofsecondary Ag+ species. These dissolution phenomena were alsohighlighted for Lolium multiflorum under root exposure to Ag-NPs[7]. The predominant secondary species was Ag+ bound to GSH. GSHcan be considered as a proxy for thiol-containing ligands includ-ing cysteine, phytochelatins and metallothioneins. These moleculesare well-known for their implication in metal detoxification [49].Cysteine, the amino acid which bears the thiol termination inthese molecules, is an extremely strong binding ligand for theclass B metal ion Ag+ (log Ka = 12.3) [50]. Surprisingly, glutathioneand phytochelatin assays for exposed leaves did not reveal anincreased content in these molecules. The basal concentration ofthiol-containing ligands may be sufficient to bind Ag+ ions whichhave penetrated inside cells, and prevent perturbations of cellmetabolism. Another secondary species, AgCl, was evidenced by�XANES and ToF-SIMS. Cl is one of the main constituent of plantcells, the reaction between Ag and Cl is common in the environ-ment [2] and the complex AgCl has a very low solubility whichcould explain its presence. Chloride may come from the growthmedium which showed a relatively high content in extractablechloride (418 ± 1 mg Cl− kg−1), and also to a lesser extent to the tapwater used for watering the plants, which contained 9 ± 0.5 mg Cl−

L−1. Other likely species, although not identified firmly, includedAg+ bound to COOH and OH groups, which are proxy for cellwalls.

In the case of the exposure to AgNO3, the formation of Ag-NPs was evidenced. Similar biosynthesis of Ag-NPs by plants was

observed in previous works [51,52]. Such reductive processes arestudied as green technologies for NP production [53]. Thus, onemay wonder whether Ag-NPs found in the leaf tissue under Ag-NP exposure result from direct transfer of deposited NPs, or from

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issolution/oxidation of Ag-NPs on the leaf surface, transfer of Ag+

ons in the leaf tissue and reduction into newly formed Ag0 NPs.t is difficult to answer to this question. A companion study onhe foliar transfer of TiO2-NPs in the same model plant and usinghe same protocol showed the presence of TiO2-NPs in the leafarenchyma (Larue et al., submitted for publication). Contrary tog-NPs, TiO2-NPs are very weakly soluble, so TiO2-NPs present in

he leaf parenchyma likely result from direct transfer. This com-anion study leads us to consider that both pathways are possible.

In the perspective of human exposure through the consumptionf lettuce exposed to atmospheric inputs of NPs, the efficiency of alassical washing was assessed. The washing process permitted toake off the bigger agglomerates from the surface but did not per-

it to decrease significantly the amount of Ag (19 �g Ag-NP g−1

W were detected). Likewise, Zhang et al. showed that Ag-NPsemained associated to pear epidermis even after washing, andere also transferred in the pulp [38]. In an experiment on maize

eaves exposed to CeO2-NPs which contained 50 �g CeO2 g−1 FW,n intensive washing led to a decrease in CeO2 content by a factorf five [24]. Leaf morphology, chemical composition and amountf wax and exudates present on leaves, density and morphologyf trichomes are probably key factors influencing the trapping ofarticles by leaf surfaces, and the stability of the binding. Micro-rganisms present on leaves and forming the phyllosphere maylso play a key role in the sorption and chemical transformationsf the particles. Finally, the nature and morphology of the particleshemselves may also influence these processes.

In order to better understand processes underlying the sorp-ion of particles on leaf surfaces on one hand, and their transfern the leaf tissue on the other hand, further studies comparinglant species with contrasted morphologies (cuticle thickness, tri-homes and stomatal densities) and biochemical characteristics ofhe phyllosphere (wax, exudates, microbial communities) woulde required.

The impact of the exposure of lettuce to Ag-NPs in termsf phytotoxicity was assessed. The various parameters testedbiomass, chlorophyll pigment content and protein content) wereot affected by the treatment. At the opposite, necroses werebserved on the same plant exposed to Ag+ and to Pb-rich nano-nd microparticles emitted by a smelter [54]. Ag-NP phytotoxicityas been demonstrated in several studies but exclusively after rootxposure to high concentrations of NPs [7,9–11]; thus not compara-le to our exposure scenario. In the perspective of plant productionnd gardening, the absence of visible sign of toxicity despite theccumulation of Ag-NPs is important to notice. In the present study,g+ released from NPs is mainly found associated to thiol groups orresent as AgCl, suggesting a detoxification process [2]. Still, toxicffects might be detectable on more sensitive endpoints such asNA damages or gene expression.

. Conclusion

This study gives first insights on the localization and specia-ion of Ag-NPs in a crop plant after foliar exposure. Results showhat Ag-NPs were transferred in all types of tissues, and suggesthat both stomatal and cuticular pathways were used. A washingrocedure with slightly acidified water was not efficient at remov-

ng significant amount of contamination. Thus, Ag-NPs appliedn crops may potentially be transferred to humans. Bioaccumu-ated Ag-NPs underwent dissolution and complexation of Ag+ byrganic molecules such as thiol-containing molecules, suggesting

detoxification process. Nevertheless, no toxicity symptom wasetectable by the tests used. This type of study shows the interestf combining micro-spectroscopic tools with sub-micron resolu-ion with chemical analyses and ecotoxicological tests to increase

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Materials 264 (2014) 98– 106 105

our understanding of the fate of nanomaterials in environmentaland biological systems.

Acknowledgements

The authors would like to thank the FP7 of the EuropeanUnion for the funding (Nanohouse project no. 247810) andFrench program LABEX Serenade (11-LABX-0064). TEM analyseswere obtained with the TEM OSIRIS, Plateform Nano-Safety, CEA-Grenoble funded by the Agence Nationale de la Recherche, program‘Investissements d’Avenir’, reference ANR-10-EQPX-39, and oper-ated by Franc ois Saint Antonin. Synchrotron experiments wereperformed on the ID21 beamline at the European SynchrotronRadiation Facility (ESRF), Grenoble, France. We thank Franc oisSaint-Antonin from LCSN/LITEN/CEA Grenoble for his help dur-ing TEM experiments and Nicolas Nuns from the Pôle Régionald’Analyse de Surface for ToF-SIMS measurements.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.jhazmat.2013.10.053.

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