Foliar exposure of the crop Lactuca sativa to silver nanoparticles: Evidence for internalization and changes in Ag speciation
Graphical abstract
Introduction
In the last decade, nanotechnology has emerged as a major field of research and investments for industries. Thanks to their unique properties, NPs are included in more and more consumer products. 313 consumer products containing Ag-NPs have been listed over the 1317 products of the database in 2011 [1]. Ag-NPs are mainly used as an anti-bacterial agent. Major products include cosmetics, paints, fabrics, food containers, medical equipment and pesticides. The production of Ag-NPs is estimated at 500 tons per year worldwide [2]. Eventually, most NPs will end up in the environment. Current knowledge on the fate of Ag-NPs in the environment has been recently reviewed [2] and their potential toxicological and ecotoxicological impact raises much concern [3], [4].
Plants play a critical role in the fate of NPs in the environment through their uptake, bioaccumulation and transfer to trophic chains [5]. Crop plants are more likely to be exposed to NPs than wild plants due to the application of sewage sludge on agricultural soils and use of Ag-NPs in plant protection products. After root exposure, uptake of Ag-NPs by roots was shown [6], [7], [8], as well as phytotoxic effects: reduced seed germination, perturbed root and shoot growth, and decreased evapotranspiration [6], [7], [9], [10], [11], [12]. Genotoxic effects were evidenced as well [7], [13], [14], [15]. The foliar exposure pathway has been much less investigated, although such information is necessary for a comprehensive risk assessment of nanomaterials. Indeed, NPs have been used in plant protection products for several years so far [16], [17]. 110 products containing Ag-NPs were registered as pesticides by the US-EPA in 2010 [16], and several patents for Ag-NP fungicides were deposited [18]. The use of such products is expected to expand in the future [18], [19], [20], [21]. Moreover, new applications of NPs such as controlled release of agrochemicals for nutrition and protection against pests and pathogens, delivery of genetic material and sensitive detection of plant disease are currently under investigation [19], [20]. Although these developments may be beneficial for the environment (decreased inputs), there is an urgent need of better evaluating the risks associated to these products [20], [22]. To the best of our knowledge, published studies on foliar exposure to NPs concerned TiO2-NPs [23], CeO2-NPs [24] and polystyrene-NPs [25], but none was realized on Ag-NPs.
The purpose of this study was to investigate the impact of a foliar exposure to Ag-NPs on a leafy crop plant in terms of phytotoxicity, to determine the proportion of Ag retained on/in the leaves after thorough washing, and also to get insights on the mechanisms of foliar uptake and possible transformations of Ag-NPs.
Lettuce (Lactuca sativa) was chosen as model species because of its widespread occurrence in kitchen gardens or farmlands and large foliar surface making it an ideal model to study the foliar transfer of atmospheric contaminants [26], [27]. Plants were exposed to 1–100 μg Ag-NPs per g fresh weight (FW) and to Ag+ ions. This latter treatment aimed at evaluating the ionic contribution resulting from the dissolution of Ag-NPs. Total Ag accumulation in shoots was measured before and after rinsing with slightly acidic water (as performed classically by consumers) [26], [27]. Impact of Ag-NP exposure on plant biomass, chlorophyll, protein, total glutathione and phytochelatin (PC) contents and oxidative stress were evaluated. We combined scanning electron microscopy observation of whole leaves and micro X-ray fluorescence (μXRF) mapping of leaf cryo-sections to localize Ag-NPs and their weathering products in leaf tissues. Changes in Ag-NP speciation were evaluated using Ag LIII-edge X-ray absorption near edge structure spectroscopy (XANES) and time of flight-secondary ion mass spectrometry (ToF-SIMS).
Section snippets
Nanoparticle characterization and dispersion
Uncoated Ag-NPs were provided by the paint company PPG (Pittsburgh, Pennsylvania, USA). These NPs were fully characterized: shape, nominal diameter (transmission electron microscopy TEM – TECNAI OSIRIS) and specific surface area (BET). Before each exposure, stock suspensions were prepared with NPs dispersed in ultrapure water (10, 100, 1000 mg L−1) and homogenized 3 min in an ultrasonic bath. NP hydrodynamic diameter was assessed by dynamic light scattering using a NanoZS (Malvern) and is
Nanoparticle characterization
Ag-NP physico-chemical characteristics are summarized in Figure S1 (supporting information). Briefly, Ag-NPs were mainly round shaped (68%) with an average nominal diameter of 38.6 nm. Another fraction (32%) of these NPs was elongated with dimensions of 38.2 nm × 57.8 nm (Figure S1A and B). The average specific surface area measured by BET was 4.1 m2 g−1.
When in suspension, Ag-NPs were well dispersed with a hydrodynamic diameter close to nominal diameter: 47.9 ± 29.2 nm (Figure S1A and C). Ag-NP
Discussion
If the internalization of Ag-NPs has been demonstrated after root [6], [7], [8], [36], [37] and fruit [38] exposure, nothing is known on their fate after foliar exposure. In the present study, Ag was found as micrometer-scale agglomerates on the surface of leaves, and in leaf tissues including epidermis, mesophyll and vascular tissues. Thus, Ag-NPs deposited on the surface were able to cross the epidermis and eventually to be transferred to the leaf tissue. The low solubility of Ag-NPs used in
Conclusion
This study gives first insights on the localization and speciation of Ag-NPs in a crop plant after foliar exposure. Results show that Ag-NPs were transferred in all types of tissues, and suggest that both stomatal and cuticular pathways were used. A washing procedure with slightly acidified water was not efficient at removing significant amount of contamination. Thus, Ag-NPs applied on crops may potentially be transferred to humans. Bioaccumulated Ag-NPs underwent dissolution and complexation
Acknowledgements
The authors would like to thank the FP7 of the European Union for the funding (Nanohouse project no. 247810) and French program LABEX Serenade (11-LABX-0064). TEM analyses were 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 operated by François Saint Antonin. Synchrotron experiments were performed on the ID21 beamline at the European Synchrotron Radiation Facility
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