Speciation Matters: Bioavailability of Silver and Silver Sulfide

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Speciation Matters: Bioavailability of Silver and Silver Sulfide Nanoparticles to Alfalfa (Medicago sativa) John P Stegemeier, Fabienne Schwab, Benjamin P. Colman, Samuel M. Webb, Matthew Newville, Antonio Lanzirotti, Christopher Winkler, Mark R. Wiesner, and Gregory Victor Lowry Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b01147 • Publication Date (Web): 24 Jun 2015 Downloaded from http://pubs.acs.org on June 29, 2015

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Environmental Science & Technology

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Speciation Matters: Bioavailability of Silver and Silver

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Sulfide Nanoparticles to Alfalfa (Medicago sativa)

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John P. Stegemeier,1,2† Fabienne Schwab,1,3† Benjamin P. Colman,1,4 Sam Webb,5 Matt Newville,6

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Antonio Lanzirotti,6 Christopher Winkler,7 Mark R. Wiesner,1,3 and Gregory V. Lowry1,2*

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1 Center for the Environmental Implications of NanoTechnology (CEINT)

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

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3 Civil & Environmental Engineering, Duke University, Durham, NC 27708, United States

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4 Department of Biology, Duke University, Durham, NC 27708, United States

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5 Stanford Synchrotron Radiation Lightsource, Stanford University, Menlo Park, CA 94025, United States

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6 Advanced Photon Source, Argonne National Lab, Lemont, IL 60439, United States

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7 ICTAS Nanoscale Characterization and Fabrication Laboratory, Virginia Tech, Blacksburg, VA 24061, USA

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†equal contribution

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*corresponding author: Gregory V. Lowry, [email protected], +1 (412) 268-2948

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Keywords: heavy metals, plant uptake, particle uptake, fate, environmental nanotechnology,

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nanoparticle bioaccumulation, defense mechanisms, colloidal silver, colloids

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AgNO3

Ag(0) - NPs

Ag2S - NPs Ag+ ion Ag-NPs Ag2S-NPs SixOy-NPs

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Abstract

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Terrestrial crops are directly exposed to silver nanoparticles (Ag-NPs) and their

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environmentally-transformed analog silver sulfide nanoparticles (Ag2S-NPs) when wastewater

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treatment biosolids are applied as fertilizer to agricultural soils. This leads to a need to

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understand their bioavailability to plants. In the present study, the mechanisms of uptake and

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distribution of silver in alfalfa (Medicago sativa) were quantified and visualized upon

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hydroponic exposure to Ag-NPs, Ag2S-NPs, and AgNO3 at 3 mg total Ag/L. Total silver uptake

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was measured in dried roots and shoots, and the spatial distribution of silver was investigated

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using transmission electron microscopy (TEM) and synchrotron-based X-ray imaging

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techniques. Despite large differences in release of Ag+ ions from the particles, Ag-NPs, Ag2S-

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NPs, and Ag+ became associated with plant roots to a similar degree, and exhibited similarly

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limited (S). The regions associated with high Ag:S in the root exposed to AgNO3

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shows discrete silver accumulations near bumps in the root resembling emerging lateral roots

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(red dots in map in Figure 3 top, right column; Ag:S correlation plots, Figure S2). The lowest

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Ag:S ratios were located at the root tips suggesting silver associated with thiols, which may have

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been generated in response to the Ag.

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The root exposed to Ag-NPs showed high Ag:S ratios near the root cap cells of the root tip,

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pointing to accumulation of Ag in or on these cells, which is in agreement with the results of the

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high energy X-ray maps (Figure 2). High Ag:S ratios were found at the exterior of the root

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suggesting nanoparticles attached to the root surface before uptake. Low Ag:S ratios were found

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in the interior of the root. This distribution of Ag in the Ag(0)-NP exposed roots suggests that

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Ag NPs on the root exterior are dissolving and entering the root as dissolved ions. This

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mechanism was proposed by Leclerc and Wilkinson (2014) for uptake of Ag by algae exposed to

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Ag(0)-NPs.60 Uptake of Ag-NPs via partial Ag dissolution is also supported by the TEM results

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discussed in the sections below, by the relatively high solubility of Ag-NPs, and the similar,

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albeit spatially less uniform, uptake profile of silver in plants exposed to Ag-NPs and AgNO3 in

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Figure 3. 14 ACS Paragon Plus Environment


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The silver in the root exposed to Ag2S-NPs was highly correlated with the signal of sulfur

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(Figure 3). Regions exhibiting a high Ag:S ratio were non-existent, demonstrating that silver

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remained co-located with sulfur. This means that Ag2S-NPs either was almost exclusively taken

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up in nanoparticulate form or the plants strongly responded to the NP exposure by generation of

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thiols, such as glutathione, for subsequent biotransformation. Glutathione is known to play an

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important role in the detoxification of heavy metals by plants61, 62. Dimkpa et. al. has shown

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elevated levels of the oxidized form of glutathione in the roots of wheat grown in the presence of

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silver ions and NPs63. Accumulation of intact Ag2S-NPs in the root tip was minimal given the

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absence of an appreciable Ag signal. Silver from the Ag2S-NPs exposed samples was primarily

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located at the elongation zone of the root, highlighting a clear difference in the uptake

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mechanisms (but not the mobility) of silver between the pristine and sulfidized silver

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nanoparticles. Overall, the silver from the Ag2S-NPs was less uniformly internalized and less

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prevalent in the root tip than for Ag-NPs or AgNO3, likely a result of two factors: the lower

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silver ion concentration associated with Ag2S-NPs; and the sorption of Ag2S-NP with cells at the

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root tip that are readily sloughed off by the root (Figure 2).

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3.4 Microscopic analysis of NPs in plant tissues

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Two types of particles were exclusively found on or in the NP-exposed roots: Large

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particles with low electron density adsorbed to border cells, and small electron dense particles in

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the cell walls and channels. There were thousands of very low electron density particles adhered

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to the border cells of the Ag-NP and Ag2S-NP treatments with diameters of 7414 nm and 8434

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nm, respectively (Figure 4 N+S, Figure 5, Table S2). Some of these particles were also found in

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the intracellular spaces (data not shown). The smaller particles were found in the intercellular

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spaces and interfibrillar channels of the cell wall and had diameters of 2.52±0.83 and 2.54±0.77


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nm in the Ag-NPs and Ag2S-NPs treatments, respectively (inserts in Figure 4 M+R), which is

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consistent with previous observations of uptake pathways.16, 64 Although alfalfa has been shown

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to generate silver NPs of similar size following exposure to silver nitrate in agar,65 no such NPs

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were present in the control cell wall and the cell walls of plants exposed to AgNO3 ( Figure 4C

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and H). The spatial resolution and sensitivity of TEM/EDX was insufficient to identify the

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elemental composition of these few nm-sized particles within the cell wall matrix. Nevertheless,

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the location of the particles in the cell wall and interfibrillar channels (Figure 4 M+R, and the

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lack of these particles in the control and the AgNO3 treatment (Figure 4 C+H) suggests that Ag-

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NPs and Ag2S-NPs at least partially dissolved in the acidic environment of the root border cells,

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and then diffused into the intercellular spaces and subsequently translocated along the apoplast.

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Analysis of many transmission electron microscope images revealed a variety of

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electron-dense particles of different sizes and shapes in and on the alfalfa roots (Figure 5, Figures

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S6 and S7). The types, sizes, and shapes of these particles (Figure 5 and Table S2) depended on

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the form of silver used to expose the plants, providing useful insights into how the plant

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responded physiologically to each form of silver at a microscopic (cellular) level.

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treatments (control, AgNO3, Ag-NPs, and Ag2S-NPs), the cytoplasm in the cells of the root

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cortex contained abundant small particles (Figure 4 B, G, L, in the insert of Q, and Figure S6). In

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control roots these cytoplasmic particles (average diameter 7.9-9.9 nm) were evenly distributed

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or loosely grouped (Figure 4 B, Figure S6). In the AgNO3 and Ag-NP roots there were also

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larger aggregates of these cytoplasmic particles of 3318 nm and 5517 nm in diameter,

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respectively (average±standard deviation; Figure 4 G, and insert in L). In contrast to the control,

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AgNO3, and Ag-NP exposed roots, the cytoplasm of Ag2S-NP exposed roots was mostly bare of

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the small cytoplasm particles as shown in Figure 4 Q. Instead, the cytoplasm contained large


In all


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9635 nm aggregates of smaller primary particles (17.27.8 nm in diameter, Figure 4 P and

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insert in Figure 4 Q, Figure S6and Figure S7A). These primary particles were significantly larger

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than the cytoplasm particles observed in the other treatments (ANOVA assuming unequal

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variances, Dunnett-C post-hoc test; Figure 5, Table S2). Hotspots of co-located Si and O in maps

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generated using S/TEM (Figure S7) suggest that the small cytoplasm particles and their

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aggregates mainly consisted of silicates. In the present hydroponic setup, the plants apparently

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formed SixOy particles in their cytoplasm. Potential sources for Si are dissolved Si species (i.e.

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orthosilicic acid) mobilized by the root exudates interacting with the glass marbles or the

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seedcoats, replacing natural silicate-containing rocks..

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Differences in the aggregation state of the naturally formed cytoplasmic silicate particles

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in plants exposed to different forms of silver suggest that they are part of the defense

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mechanisms against silver toxicity. In the control, the natural cytoplasmic silicate particles are

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not aggregated, while these particles are partially aggregated (AgNO3 and Ag(0)-NPs) or

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completely aggregated (nano-Ag2S) in plants exposed to silver. The degree of flocculation

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achieved (AgNO3Ag2S). This suggests that production of silicate aggregates was hampered

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under the condition of heavy stress by Ag+ exposure (AgNO3 and Ag(0)-NP), but was fully

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operational when exposed to lower levels of filterable silver (nano-Ag2S). More evidence for

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higher exposure to dissolved Ag+ are the stunted growth and structural damages of the root

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epidermis as observed by electron microscopy (data not shown) of plants exposed to AgNO3 and

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Ag-NPs compared to nano-Ag2S (Figure S1). We speculate that the naturally formed silicate

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aggregates in the roots of alfalfa may have served the plants to adsorb Ag+ or Ag2S-NPs and thus

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alleviated their toxicity and potential to translocate. This defense mechanism is common in


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plants against heavy metals or pests.38,

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sorptive naturally formed silicate aggregates (also called phytoliths) or silicate colloids in plant

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cells is already well known, e.g. for Cd in rice.38, 67, 68 In rice and other plants, silicate aggregates

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or colloids in roots can reduce translocation of heavy metals into shoots.

Co-precipitation of heavy metal ions on/in highly

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The XRF Ag element maps in Figure 2 confirm that in the Ag2S-NPs treatment little Ag

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was present in the root tip, the Ag:S ratio was low, and most Ag concentrated in the elongation

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zone above the root tip, indicating that the root tip itself containing the sensitive apical meristem

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was exposed to less toxic Ag+ ions than in other treatments. The question remains whether the

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individual cytoplasm particles or aggregates would show traces of Ag+ or even intact Ag2S-NPs

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at higher resolution. Such analysis is currently not feasible for NPs or this size in biological

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samples.

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4. Environmental Implications

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Overall, the present study suggests that nanoparticles of silver, even when transformed to

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low solubility Ag2S-NPs, strongly accumulate on and in the roots of alfalfa, an agriculturally

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important legume relevant for the human food chain (1,608-2,019 ppm dry weight,

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corresponding to a 536 to 673-fold increase from the nominal dose). Surprisingly different Ag

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distributions were present in the roots despite similar Ag uptake concentrations for different

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kinds of Ag NPs and ionic Ag+.

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This work suggests different uptake mechanisms for AgNO3, Ag-NPs, and Ag2S-NPs in

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alfalfa. Silver in the AgNO3 (i.e. Ag+ ion control) exposed sample was highly concentrated and

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uniformly distributed throughout the root tip cells. In contrast, the root tip exposed to Ag2S-NPs

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accumulated very little Ag inside the root tip cells. Instead, Ag aggregates adsorbed on the 18 ACS Paragon Plus Environment


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exterior of the root, and small particles

Speciation Matters: Bioavailability of Silver and Silver Sulfide

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