Impact of Surface Charge on Cerium Oxide Nanoparticle Uptake and

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Impact of surface charge on cerium oxide nanoparticle uptake and translocation by wheat (Triticum aestivum) Eleanor Spielman-Sun, Enzo Lombi, Erica Donner, Daryl L Howard, Jason M Unrine, and Gregory V. Lowry Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 02 Jun 2017 Downloaded from http://pubs.acs.org on June 2, 2017

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Environmental Science & Technology

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Impact of surface charge on cerium oxide nanoparticle uptake and

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translocation by wheat (Triticum aestivum)

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Eleanor Spielman-Sun§, Enzo Lombi‡, Erica Donner‡, Daryl Howard⊥,

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Jason M. Unrine˟, Gregory V. Lowry*§

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§

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Civil & Environmental Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, 15213, United States

‡

Future Industries Institute, University of South Australia, Mawson Lakes, South Australia, 5095, Australia

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⊥Australian

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˟ University of Kentucky, Department of Soil and Plant Science, Lexington, Kentucky 40546,

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Synchrotron, Clayton, Victoria, 3168 Australia

United States

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*Corresponding author:

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Address: Carnegie Mellon University, Pittsburgh, PA 15213

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Phone: (412) 268-2948

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Fax: (412) 268-7813

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Email: [email protected]

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ACS Paragon Plus Environment

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TOC ART

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ABSTRACT

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Nanoparticle (NP) physiochemical properties, including surface charge, affect cellular uptake,

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translocation, and tissue localization. To evaluate the influence of surface charge on NP uptake

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by plants, wheat seedlings were hydroponically exposed to 20 mg/L of ~4nm CeO2 NPs

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functionalized with positively charged, negatively charged, and neutral dextran coatings. Fresh,

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hydrated roots and leaves were analyzed at various time points over 34h using fluorescence X-

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ray absorption near-edge spectroscopy to provide laterally resolved spatial distribution and

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speciation of Ce. A 15-20% reduction from Ce(IV) to Ce(III) was observed in both roots and

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leaves, independent of NP surface charge. Due to its higher affinity with negatively charged cell

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walls, CeO2(+) NPs adhered to the plant roots the strongest. After 34h, CeO2(-) and CeO2(0) NP

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exposed plants had higher Ce leaf concentrations than the plants exposed to CeO2(+) NPs. While

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Ce was found mostly in the leaf veins of the CeO2(-) NP exposed plant, Ce was found in clusters

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in the non-vascular leaf tissue of the CeO2(0) NP exposed plant. These results provide important

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information for understanding mechanisms responsible for plant uptake, transformation, and

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translocation of NPs, and suggest that NP coatings can be designed to target NPs to specific parts

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of plants.


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INTRODUCTION

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Nanotechnology has the potential to play a critical role in global food production.1–5 The

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unique properties of nanoparticles (NPs) can be harnessed for developing formulations that

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contain nanoscale active ingredients designed for direct use on agricultural soils or crops. These

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products have the capacity to reduce the energy and water inputs for food production by

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providing a more targeted and efficient delivery system for fertilizers and pesticides. However,

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properly utilizing the characteristics of these nanoparticles, such as particle size, coating,

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chemical composition, and shape, requires an understanding of the factors influencing the

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fundamental interactions between plant tissues and engineered NPs.

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Several hydroponic studies have investigated the accumulation and translocation of bare

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cerium dioxide nanoparticles (CeO2 NPs) in plants.6–19 Most of these studies demonstrated a

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dose-dependent increase of cerium (Ce) in the roots of various plant species and limited

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translocation of Ce to the shoots. There are a few studies that have specifically documented the

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speciation and transformation of CeO2 NPs in plant tissue. Hernandez-Viezcas et al. grew

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soybean in soil amended with 1000 mg/L CeO2 NPs and used Micro X-Ray Fluorescence (μ-

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XRF) and X-Ray Absorption Near Edge Structure (XANES) spectroscopy to determine that

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while most of the Ce(IV) remained untransformed in the plant, there was a small percentage of

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Ce(III) in the pod.20 Zhang et al. used XANES and Scanning Transmission X-ray Microscopy

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(STXM) analysis and found accumulation in cucumber of both CeO2 NPs and Ce(III) as cerium

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phosphate in roots and as cerium carboxylates in shoots.14 Similar dissolution and transformation

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of CeO2 NPs has been observed in hydroponically grown wheat, pumpkins and sunflowers8 as

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well as in soil cultivated corn.21


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Surface charge is a key property that can control NP transport in environmental and

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biological systems. Zhu et al. compared the uptake of functionalized gold nanoparticles (AuNPs)

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with well-defined surface charges into various terrestrial plants, including radish, ryegrass, rice,

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and pumpkin, and confirmed that while positively-charged NPs more readily attach to plant

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roots, negatively-charged NPs are most efficiently translocated into the plant roots.22 The same

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trend has been observed for AuNPs coated with variously charged organic ligands on rice and

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tomato23 and for cationic or anionic coated CdSe/ CdSnS quantum dots on poplar trees.24 There

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are a few studies focused on surface charge dependent CeO2 NP plant uptake, including Zhao et

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al. who exposed corn to alginate coated CeO2 NPs21 and Barrios et al. who exposed tomato to

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citric acid coated CeO2 NPs.25 However these studies focus more on the effects of uptake with

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and without coating, and less on the translocation and spatial distribution of Ce in the plant. Here

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we studied NP surface charge in a well-controlled hydroponic system to gain a better

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understanding of the influence of surface charge on NP uptake, transformation, and translocation

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to leaves, and tissue localization in wheat plants.

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In situ imaging of metal distribution in hydrated biological systems can provide mechanistic

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details of plant uptake and translocation, but is challenging because target metal concentrations

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are low (mg/kg dry plant tissue) and element speciation and distribution can be altered during

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sample preparation (including drying, embedding, and staining) as well as by the analytical

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process itself (e.g. X-ray beam damage).26,27 Furthermore, in the case of most biological samples,

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the highly hydrated nature of the specimens is not compatible with most major analytical

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techniques operating under vacuum such as TEM and non-environmental SEM. However,

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advances in synchrotron-based techniques now allow the in situ examination of metals and

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metalloids in hydrated and fresh plant tissues at low metal concentrations.28–30 In this


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experiment, we obtained spatially resolved 2D maps of metal speciation in plant samples by

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XANES mapping, i.e. scanning the same area repeatedly with decreasing energies across an

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absorption edge, thus collecting a XANES spectra for each pixel of an entire image.31,32 This

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approach differs from other studies where a μ-XRF map is obtained and μ-XANES spectra are

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then collected from specific regions of interest within that map, or where XANES is performed

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on the bulk, freeze-dried tissue.26,28–30,33 XANES mapping has the advantage of providing more

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detailed information on speciation distribution for the entire sample. This technique has

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previously been performed on plant tissues by Wang et al. to compare selenate and selenite

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exposed wheat and rice plants32, Kopittke et al. to compare arsenate and arsenite exposed wheat

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and rice plants, 34 and De Brier et al. to study iron speciation in mature wheat grains.35

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The main goals of the present study were to evaluate the influence of surface charge on

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CeO2 NP uptake, distribution, and speciation in a model plant, specifically wheat (Triticum

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aestivum), and elucidate details about the reduction of Ce(IV) to Ce(III) in the different plant

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compartments. CeO2 NPs were chosen as a model NP because they are easy to synthesize in a

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narrow size distribution, to coat with various charges, and to disperse in aqueous solution;

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cerium also has a low background level in plants. We used fluorescence-XANES imaging to

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investigate the in situ laterally resolved speciation of Ce within fresh wheat roots and leaves with

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the overarching goal of gaining a better understanding of how NPs can be engineered to increase

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translocation and to target specific parts of plants through functionalization.

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MATERIALS AND METHODS

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CeO2 Nanoparticles. Cerium dioxide NPs with three different charges were synthesized as

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reported previously in Collin et al.36 Briefly, uncharged dextran coated CeO2 NPs (CeO2 NP (0))

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with a nominal 4 nm primary particle diameter were produced via alkaline precipitation from a

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solution of cerium chloride salt and dextran. This coating was then further functionalized, either

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with diethylaminoethyl groups to create a net positive charge (CeO2 (+)) or with carboxymethyl

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groups to create a net negative charge (CeO2 (-)). The particles were diluted to 20 mg/L as Ce in

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the exposure medium (¼ strength Hoagland’s) and probe sonicated (550 Sonic Dismembrator,

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Fisher Scientific) for 1 min at 10 s intervals to ensure dispersion. The hydrodynamic diameter

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and electrophoretic mobility of the NPs in the exposure medium (20 mg/L as Ce in ¼ strength

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Hoagland’s) were measured using a Nano Zetasizer (Malvern Instruments, Malvern). The initial

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pH and ionic strength of the exposure medium were 5.6 and 5.2 mM, respectively, and these

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were not further adjusted. The apparent zeta potential was calculated from the electrophoretic

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mobility using the Hückel approximation. NP dosing concentrations were selected to avoid acute

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toxicity while ensuring adequate signal for µ-XRF imaging.

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Plant Growth: For µ-XRF, wheat (Triticum aestivum cv. shield) seeds were surface sterilized

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with 10% w/v bleach (VWR Analytical) for 10 min and then thoroughly rinsed with DI water.

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The sterilized seeds were germinated on deionized water-moistened filter paper in a Petri dish.

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After four days, the seedlings were transferred to 100-mL plastic containers. Each container was

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filled with 80 mL of ¼ strength Hoagland’s medium37 and covered with a plastic lid with five

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holes. Five seedlings were transplanted to five of the holes with the roots suspended in a

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continuously aerated solution. Plants were grown at 25 °C under alternating conditions of 12 h of

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light and 12 h of dark. Nutrient solution was renewed every 3 days. After 7 days, the plants were


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transferred from the University of South Australia (Adelaide, Australia) to the Australian

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Synchrotron (Melbourne, Australia) where the plants were hydroponically exposed to 20 mg-

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Ce/L of CeO2 NPs. Plant tissue was removed at 1 h, 8 h, and 34 h to analyze by µ-XRF.

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Total Ce determination experiments were conducted at Carnegie Mellon University

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(Pittsburgh, PA). Plant growth conditions were replicated to match those used for the

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synchrotron experiment. At the various time points, plants were harvested in triplicate and roots

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and shoots separated for determination of fresh and dry mass. Briefly, dried plant tissue samples

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were digested overnight at room temperature in a 2:1 ratio of concentrated HNO3 and 30% H2O2,

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then heated to 100 °C for 30 minutes (protocol adapted from EPA Method 3050b38). Following

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digestion, the samples were diluted to 5% HNO3 using deionized water before analysis using

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inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7700x, Santa Clara, CA).

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Further details are provided in the supporting information.

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µ-XRF and Fluorescence XANES Imaging: Following exposure, roots were rinsed in a Ce free

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hydroponic solution and placed between two pieces of 4 µm-thick Ultralene, which formed a seal

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around the plant tissue to minimize dehydration. For each exposure solution, two replicate roots

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were examined, with all roots positioned vertically in the sample holder and scanned

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simultaneously. The tips and bases of the plant leaves were scanned separately due to the size of

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the sample mount. Samples were prepared and examined at the XFM beamline at the Australian

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Synchrotron, where an in-vacuum undulator is used to produce an X-ray beam. A Si(111)

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monochromator and Kirkpatrick-Baez (KB) mirror microprobe are used to obtain a

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monochromatic beam focused onto the specimen. The X-ray fluorescence emitted by the

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specimen was collected using the 384-element Maia detector (Rev C) placed in a backscatter


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geometry.39,40 For all scans, samples were analyzed continuously “on-the-fly” in the horizontal

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direction.

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An initial large area survey scan at 15.8 keV was conducted to identify the area of interest

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and obtain overall elemental distributions. Step sizes and scanning velocities were adjusted so

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each scan took around 2 h. Details are provided in the supporting information. Subsequently, a

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smaller area was chosen to conduct fluorescence XANES imaging with the XANES stack itself

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consisting of 108 individual maps at decreasing energies across the Ce LIII edge. Details

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regarding the energy steps are provided in the supporting information.

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The three nanoparticle types as standards were also analyzed as suspensions using

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fluorescence-XANES imaging. All standards were prepared to a final Ce concentration of 20

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mg/L. Further details about the preparation and imaging of these standards are provided in the

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supporting information. These XANES spectra and that of Ce(III) acetate, which was used as the

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model compound for Ce(III), are presented in the supporting information (Figure S1). The large

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peak at 5727 eV is a characteristic peak of the Ce(III) oxidation state; the two peaks at 5730 and

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5737 eV are characteristic of Ce(IV). These spectral differences are an important criterion for

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distinguishing Ce compounds of the two different oxidation states.

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µ-XRF spectra were analyzed using GeoPIXE.31 For the fluorescence-XANES stacks, the

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GeoPIXE “energy association” module was used to compare the ‘concentration’ ratios between

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two energies: 5,727 and 5,737 eV, which are the white lines for Ce(III) and Ce(IV) respectively.

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Because pixels were generally parallel to the 1:1 line, particularly at the highest Ce

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concentrations, selected pixel populations represented absolute changes in Ce(III) and Ce(IV)

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concentration. (See Figures S3, S5-6 in supporting information for more details). From the

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selected pixel populations, XANES spectra were extracted, background subtracted, and


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normalized using SIXPack (Version 1.4).41 Linear combination fitting (LCF) was performed

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using the collected CeO2 NP standards above and a Ce(III) acetate spectrum from Auffan et al.42

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Because the exact oxidation state of CeO2 NPs is difficult to characterize,43 we assume for the

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purposing of fitting that the starting materials are all Ce(IV) oxidation state.

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RESULTS AND DISCUSSION

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Characterization of CeO2 NPs: NPs have been previously characterized by TEM, FTIR and

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XRD by Collin et al.36 TEM indicated the CeO2 core of the particles was ~4 nm. Additional

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characterization of the particles at 20 mg/L as Ce in the nutrient solution was performed as

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follows. The number weighted average hydrodynamic diameter of the particles in the exposure

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medium were 12.0 ± 3.4, 19.4 ± 5.7, and 14.5 ± 3.3 nm, for the CeO2(+), CeO2(0), and CeO2(-)

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particles, respectively. The electrophoretic mobility of the particles in the nutrient solution were

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1.15 ± 0.30, -0.02 ± 0.21, and -1.59 ± 0.41 µm·cm·V-1·s-1 for the CeO2(+), CeO2(0), and CeO2(-)

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particles respectively. At pH of 5.6 and an ionic strength of 5.2 mM, this corresponds to an

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apparent zeta potential of +22.0 ± 6.1 mV, -0.5 ± 3.9 mV, and -30.3 ± 6.6 mV, for the CeO2(+),

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CeO2(0), and CeO2(-) particles respectively. The particles were stable in the exposure medium;

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less than 0.1 % dissolution was observed over 34 hours (Table S1); organic acid root exudates

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induced a small shift in the measured hydrodynamic diameter for all three particle types, but no

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significant difference in the aggregation behavior for the three particle types was observed

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(Figure S2).

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Total Ce Association/Uptake in Roots and Shoots: The surface charge of the CeO2 NPs had a

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substantial impact on the accumulation and translocation of Ce. The Ce concentrations

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associated with the wheat roots and shoots from the three different treatments are shown in

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Figure 1. These values include all Ce that was associated with the plant roots (internal and

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external) after collection and rinsing in NP-free Hoagland’s medium. As exposure time

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increased, total Ce uptake increased for all NP types in both the roots and shoots. The majority of

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the plant-associated Ce was sorbed on/in the roots rather than in the shoots, irrespective of the

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charge of the NPs (note the 1000-fold higher concentrations in the roots compared to the shoots

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in Figure 1). Du et al.44 and Rico et al.45 observed significantly less uptake of CeO2 NPs in their

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soil exposures with wheat, which is a common difference between hydroponic and soil

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exposures.46 Unlike our study, Schwabe et al. observed no translocation of CeO2 NPs in

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hydroponic wheat,8 though this is likely attributed to NP size differences. Numerous studies have

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demonstrated that smaller NPs are more likely to be internalized by plants than larger ones.47,48

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The smallest CeO2 NPs used by Schwabe et al. were 9 nm by TEM, which is more than twice the

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diameter of the particles used in this study. Thus we attribute the observed differences to a NP

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size effect.

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With regards to surface charge,, there was an overall greater mass of the positively charged

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NPs associated with the roots than the neutral or negatively charged NPs, which is consistent

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with other studies comparing the impact of surface charge on NP uptake by plants.22–24,49 This

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difference in association between the differently charged particles is likely due to electrostatic

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interactions between the charged particles and the root surface. It is well established that plant

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cell walls, including epidermal cells on the root surface, are negatively charged because of the

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abundance of polysaccharides containing galacturonic acid residues.50,51 Thus, CeO2(+) NPs


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accumulated on the root surface due to electrostatic attraction between the positively charged NP

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and the negatively charged root surface, while CeO2(-) NPs accumulated the least due to

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electrostatic repulsions. The uncharged particles had an intermediate degree of interaction

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(possibly limited by steric impediment). While the negatively charged and neutral particles had

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the lowest accumulation in/on the roots, these NPs were still able to overcome this electrostatic

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and/or electrosteric repulsion by forming strong hydrogen or covalent bonds with the root

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surface, through polyvalent cation bridging, or by diffusing into the mucilage.52 This distribution

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was confirmed with Ce maps on the wheat plant roots (Figure 2). Sufficient Ce was present on

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the roots exposed to negative and neutral NPs for 1h to allow elemental mapping; the neutral

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particles showed some regions of concentrated Ce, likely from particle adhesion (Figure 2).

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Overall, the leaves contained less than 1% of the total plant associated Ce, indicating that very

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little of the adhered Ce was translocated from the roots to the shoots. While there was no

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statistically significant difference in Ce accumulation in the leaves for each treatment after 8 h,

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by 34 h the CeO2(-) and CeO2(0) NP exposed plants had accumulated significantly more Ce in

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the leaves than the CeO2(+) NP exposed plants. This is particularly noteworthy since both had

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accumulated significantly less Ce on/in the roots than the CeO2(+) NP exposed plants.

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Figure 1. Total Ce concentrations in dried wheat plant roots (bottom) and leaves (top) after 1 h, 8 h, and 34 h. Note different scales for the roots and leaves. Total Ce uptake in the leaves after 1 h of exposure was not measured. Significant differences (based on ANOVA and Tukey HSD post hoc tests (p

Impact of Surface Charge on Cerium Oxide Nanoparticle Uptake and

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