Uptake and Transformation of Silver Nanoparticles and Ions by Rice

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Environmental Processes

Uptake and Transformation of Silver Nanoparticles and Ions by Rice Plants Revealed by Dual Stable Isotope Tracing Qingqing Yang, Wanyu Shan, Ligang Hu, Yao Zhao, Yinzhu Hou, Yongguang Yin, Yong Liang, Fuyi Wang, Yong Cai, Jing-fu Liu, and Guibin Jiang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02471 • Publication Date (Web): 10 Dec 2018 Downloaded from http://pubs.acs.org on December 10, 2018

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Environmental Science & Technology

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Uptake and Transformation of Silver Nanoparticles and Ions by Rice

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Plants Revealed by Dual Stable Isotope Tracing

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Qingqing Yang

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Yin *, ‡, †, Yong Liang †, Fuyi Wang §, Yong Cai *, †, , Jingfu Liu ‡, and Guibin Jiang ‡

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†Institute

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‡State

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Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China

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§Beijing

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in Beijing; CAS Key Laboratory of Analytical Chemistry for Living Biosystems, CAS

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Research/Education Center for Excellence in Molecular Sciences; Institute of Chemistry,

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Chinese Academy of Sciences, Beijing 100190, China

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Department

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33199 USA

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* Corresponding author.

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Tel.: 86-10-62844175 (Y. Yin), 001-305-348-6210 (Y. Cai)

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E-mail: [email protected] (Y. Yin), [email protected] (Y. Cai)

†, ‡,

Wanyu Shan †, Ligang Hu

‡, †,

Yao Zhao §, Yinzhu Hou §, Yongguang

of Environment and Health, Jianghan University, Wuhan 430056, China

Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for

National Laboratory for Molecular Sciences; National Centre for Mass Spectrometry

of Chemistry and Biochemistry, Florida International University, Miami, FL

1

ACS Paragon Plus Environment


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TOC/Abstract Art

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ABSTRACT

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Knowledge on the uptake and transformation of silver nanoparticles (AgNPs) and Ag+

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ions by organisms is critical for understanding their toxicity. Herein, the differential

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uptake, transformation, and translocation of AgNPs and Ag+ ions in hydroponic rice

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(Oryza sativa L.) is assessed in modified Hewitt (with Cl– ions, HS(Cl)) and Hogland

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solutions (without Cl– ions, HS) using dual stable isotope tracing (107AgNO3 and

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109AgNPs).

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both) for 14 days, a stimulatory effect was observed on root elongation (increased by

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68.8 and 71.9% for HS(Cl) and HS, respectively). Most of the Ag+ ions (from 107Ag+

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ions and 109AgNPs) were retained on the root surface, while the occurrence of AgNPs

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(from

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uptake of AgNPs and/or reduction of Ag+ ions. Higher fractions of Ag+ ions in the

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shoot suggest an in vivo oxidation of AgNPs. These results demonstrated the

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inter-transformation between Ag+ ions and AgNPs and the role of AgNPs as carriers

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and sources of Ag+ ions in organisms, which is helpful for understanding the fate and

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toxicology

After co-exposure to

109AgNPs

and

107Ag+

107Ag+

ions and

109AgNPs

at 50 μg L-1 (as Ag for

ions) was observed in the root, suggesting the direct

of

Ag.

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■ INTRODUCTION

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Silver (Ag) is widely used in imaging, electrical and electronics equipment,

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catalyst, jewelry, and coin.1 The annual global demand for Ag surpassed 27,551 tons

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in 2016.2 It was estimated that global silver discards, including tailings and waste,

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account for approximately 57% of the silver mined.3 Additionally, in recent years, the

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antibacterial action of Ag salts and Ag nanoparticles (AgNPs) has been employed in

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numerous consumer products and medical devices.2 Due to its inevitable release, the

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concentrations of Ag in the effluents of wastewater treatment plants ranged from 0.06

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to 2.6 g L-1, several orders of magnitude higher than typical ‘‘background’’ surface

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water levels.4 Although the environmental concentrations of AgNPs in surface water

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were estimated at the level of ng L-1, they are expected to continue increasing in the

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near future.5 The AgNPs released directly from consumer products (e.g., washing

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machines, outdoor coatings, and textiles) in the leaches can be up to tens, even

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hundreds, of g L-1.6-8 In addition, AgNPs are also used as fungicides against various

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plant pathogenic fungi in vitro or in the field.9, 10 The accumulation of Ag+ ions and

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AgNPs is widely observed in crop plants.11 More importantly, recent studies revealed

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that Ag+ ions and AgNPs via oral exposure could be accumulated and retained in the

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brain of rats and breast-fed offspring mice.12,

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inevitable environmental release, the environmental and health impact of Ag,

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especially from AgNPs, are of increasing concern.8, 14

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13

Given the increasing use and

Nevertheless, the mechanisms through which AgNPs exert toxicity to organisms 4


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have been debated for nearly a decade, as it remains unclear whether the toxicity

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comes from the nanoparticle itself or through the Ag+ ions.15 Although the

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extracellular oxidative release of Ag+ ions has been demonstrated as the main cause of

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AgNPs toxicity in certain cells and organisms,15-19 it still does not fully explain the

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mechanism of AgNPs toxicity. Compared with Ag+ ions, AgNPs resulted in

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different20-24 or higher25-28 toxicity in various cell or organism models, suggesting that

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there is a nanoparticle-specific effect. Such an effect can be at least partially explained

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by the specific uptake, delivery, and intracellular dissolution of AgNPs in the target

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organs or cells.21 Therefore, knowledge of the uptake and translocation of AgNPs in

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biological organisms is critical for a better understanding of the nanoparticle-specific

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effect of AgNPs.29

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The different uptake, translocation, and accumulation of AgNPs than Ag+ ions in

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algae and plant need to be further clarified. Although a study doubts that AgNPs

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could be directly internalized in the nanoparticle form by a freshwater algal species

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Raphidocelis subcapitata,30 most studies suggest and have reported that AgNPs could

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be taken up and internalized in algae and plants, with their bioavailability comparable

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to,31 lower than32-34 or even higher than35-37 that of Ag+ ions depending on

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experimental conditions. These experiments, however, have conventionally been

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conducted by exposing individual plants to AgNPs or Ag+ ions separately. This

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practice could not rule out the bias associated with the variations in the uptake and

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accumulation of Ag in different individual plants. One possible solution is to use the 5



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same individual plant for simultaneous exposure of both AgNPs and Ag+ ions.

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However, due to the coexistence of Ag+ ions and AgNPs in an experimental setting,

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an effective distinction between differential uptake and translocation of AgNPs and

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Ag+ ions in a single plant sample is required. Stable isotope tracing is a powerful

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technique to probe the environmental and biological fates of metal-containing

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engineered nanoparticles.38-40 Silver has two stable isotopes (107Ag and

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allowing the respective labeling of AgNPs and Ag+ ions and, therefore, the use of dual

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stable isotope tracing to monitor their differential environmental fate and

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bioaccumulation.41

109Ag),

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In the present study, rice (Oryza sativa L.), an important food crop and monocot

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model species, was used as a plant model to assess the uptake and translocation of

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Ag+ ions and AgNPs (both at 50 g L-1) as well as their effect on plant growth. Dual

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stable isotope (107Ag+ and

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inter-transformation of Ag+ ions and AgNPs and to distinguish the different uptake

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and translocation of Ag+ ions and AgNPs in the rice plant. This study is helpful to

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better understand the interactions of plants with Ag+ ions, AgNPs, or other soluble

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nanoparticles.

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■ MATERIALS AND METHODS

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Chemicals and Reagents.

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109Ag-enriched

99

International (Ontario, Canada). Polyvinylpyrolidone (PVP, molecular weight 10,000)

109AgNPs)

tracing was used to probe the possible

107Ag-enriched

silver foil (isotope purity >99%) and

AgNO3 (isotope purity >99.8%) were purchased from Trace Sciences

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was obtained from Sigma-Aldrich (St. Louis, MO, USA). AgNO3 standard (1000 mg

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L-1) was purchased from the National Institute of Metrology (Beijing, China). All

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other reagents used in this study were of analytical grade or above. Ultrapure water

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(18.3 MΩ cm-1) from a Milli-Q gradient system (Millipore, Bedford, MA, USA) was

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used throughout the experiments.

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Preparation and Characterization of

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obtained by dissolving the

107

agent to sterically stabilize AgNPs,42 was used to prepare

108

PVP-coated

109

reported method with slight modifications.43 Briefly, 0.375 g PVP was dissolved in 70

110

mL ultrapure water. After addition of 2.25 mL of 0.1 mol L-1

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was stirred in an ice-bath for 5 min, followed by adding 2.75 mL of NaBH4 (0.08 mol

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L-1). The mixture was further stirred in an ice-bath for 30 min. Residual

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and PVP were removed by centrifugal ultrafiltration (Amicon Ultra-15 100 kDa,

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Millipore, MA), and the obtained

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times, after which the stock suspension was stored at 4 °C in the dark for later use.

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The concentration of 109AgNPs was measured by an inductively coupled plasma-mass

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spectrometer (ICP-MS) (7700ce, Agilent, Santa Clara, CA). The morphology of

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109AgNPs

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JEOL, Japan). TEM samples were prepared by dropping 10 μL aliquots of the

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aqueous sample onto an ultrathin carbon-coated copper grid and drying at room

109AgNPs

107Ag+

107Ag-enriched

109AgNPs. 107Ag+

ions were

foil in HNO3. PVP, as a usual capping

were synthesized from

109AgNPs

and

109AgNO

3

109Ag-enriched

AgNPs.

following a previously

109AgNO

3,

the solution

109Ag+

ions

were washed with ultrapure water three

was characterized by transmission electron microscopy (TEM, JEM-2100,

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109AgNPs

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temperature in a vacuum box. The size distribution of

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the TEM images using Nano Measurer 1.2 software and Gaussian fitting. The

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hydrodynamic diameter and Zeta potential of

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Nano (ZEN3600, Malvern Instruments, Worcestershire, UK) at 25 °C according to

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our previous study.44 The UV-vis spectra of

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Shimadzu UV-3600 spectrometer (Kyoto, Japan). The stability of AgNPs (0.2 or 1 mg

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L-1) in hydroponic solution alone and hydroponic solutions supplemented with NaCl

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(4 mmol L-1), CaCl2 (2 mmol L-1), or Ca(NO3)2 (2 mmol L-1) was investigated by

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UV-vis spectroscopy.

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Rice Plant Growth and Treatment. The Indica rice seeds (genotype Zhuliangyou

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No. 1) were sterilized in 30% H2O2 for 10 min, and washed with de-ionized water

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8-10 times. Then, the seeds were germinated at 27 °C in Petri dishes under the dark.

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After 7 days (2-3 leaf stage), the seedlings were transferred into pots (four rice

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seedlings in each pot) within 1/4 strength Hewitt hydroponic solution (pH 5.5 ± 0.3).

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The seedlings were grown in greenhouse with 14/10 h of day/night (light intensity,

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250-360 μmol m-2 s-1 Osram lamp). The temperature was kept at 28-30 °C during the

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daytime and 22-25 °C during the night. The hydroponic solution was renewed every 3

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days. Two common hydroponic solutions, namely modified Hewitt solution45 (with

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Cl– ions, abbreviated as HS(Cl)) and Hogland solution35 (without Cl– ions,

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abbreviated as HS), were used in the exposure experiments to assess the role of Cl–

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ions in the uptake of Ag+ ions and AgNPs. Detailed formulae of the hydroponic

109AgNPs

was estimated from

were measured by Zetasizer

109AgNPs

were recorded using a

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solutions are shown in Table S1.

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At the 6-7 leaf stage (25 days after germination), 109AgNPs and 107AgNO3 (50 μg L-1,

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as Ag for both) were spiked into the hydroponic solutions of the exposure groups (five

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pots with four plants in each pot). The Ag-spiked hydroponic solution was renewed

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every 2 days for a total of 14 days. Control experiments without the addition of Ag+

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ions and AgNPs were also performed in each trial.

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Analysis of Rice Plant Growth. After exposure, the rice plants were firstly washed

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with flowing tap water and then rinsed with ultrapure water three times. The root and

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shoot were separated, dried in a lyophilizer at -40 °C, and weighed. The root

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morphology of rice plants was analyzed on a desktop scanner (AGFA SNAP SCAN

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1236) with WinRHIZO Pro v.2003B software (Regent Instrument Inc., Montreal,

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Canada).

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Detection of AgNPs on/in the Root by Scanning Electron Microscopy (SEM)/

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TEM. Freeze-dried roots (1 cm) were treated by carbon spraying (~5 nm) with Gatan

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682 PECSTM, and then observed with ZEISS Merlin SEM under the voltage of 10 or 5

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kV. To detect whether AgNPs exist in the Ag exposed rice root, fresh roots were

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prepared for TEM following a standard procedure.35 The roots were washed with

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de-ionized water, prefixed in 2% glutaraldehyde for 24 h, rinsed in 0.1 mol L-1

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phosphate buffer (pH 7.2), and post-fixed in 1% osmium tetroxide for 2 h. After

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rinsing again in 0.1 mol L-1 phosphate buffer (pH 7.2), the roots were dehydrated in

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ethanol, ethanol-acetone mixture, and acetone. Specimens were sequentially 9



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infiltrated and embedded in acetone-Quetol 812 epoxy resin (2:1) for 4 h,

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acetone-Quetol 812 epoxy resin (1:2) for 8 h, and Quetol 812 epoxy resin for 24 h.

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The polymerization was performed sequentially at 37 °C for 12 h, 45 °C for 12 h, and

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60 °C for 24 h. The above prepared specimens in epoxy resin were sectioned (~60 nm)

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using an U2 ultramicrotome (Reichert, OM) with a diamond knife. The ultra-thin

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sections were collected in 200-mesh copper grids. The grids were treated by carbon

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spraying (~5 nm) with Gatan 682 PECSTM, then viewed to examine subcellular

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localization of AgNPs in treated roots by using Hitachi SEM 5500 at TEM mode (30

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kV).

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Analysis of Silver Spatial Distribution in/on the Root by Laser Ablation

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(LA)-ICP-MS. The analysis of Ag distribution in/on the root cross sections by

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LA-ICP-MS was carried out using a NWR-213 (ESI, NWR Division, USA) laser

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ablation system coupled to an Agilent 7700x ICP-MS mass spectrometer. Line scans

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of 40 μm width across the surface area of the samples were performed using the

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following laser ablation parameters: energy output set to 100%, laser pulse frequency

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of 10 Hz, scan speed of 30 mm/s and a laser spot size of 40 × 40 μm. The line spacing

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was 0. Helium was used as the carrier gas at a flow rate of 600 mL min-1. The

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achieved spatial resolution was 15-40 m. The parameters for ICP-MS are as follows:

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radio frequency power 1550 W; registered isotopes,

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min-1; make up gas 0 L min-1; and plasma gas 15 L min-1.

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Analysis of Ag+ Ions and AgNPs in Rice Tissues by Liquid Chromatography

107Ag, 109Ag;

carrier gas l.07 L

10


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(LC)-ICP-MS. Tetramethylammonium hydroxide (TMAH) alkaline digestion46 was

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used to extract silver species in rice tissues. Previous studies have

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demonstrated that this alkaline digestion procedure can preserve the species of Ag

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in bilogical tissues.46 Shoot (~0.07 g)/root tissues (~0.02 g) were weighed into 15 mL

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centrifuge tubes, and to each tube 2 mL of 10% TMAH was added. The tissue

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samples were then solubilized by shaking at 300 r min-1 for 4 h at 25 ℃ (IS-RDD3

190

shaker, Crystal, TA). The alkaline digestion solutions were diluted 10 fold with

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deionized water before LC-ICP-MS analysis. The LC-ICP-MS system consists of LC

192

(UltiMate 3000, Dionex, Sunnyvale, CA) for chromatographic separation and

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ICP-MS (iCAP Q, Thermo Fisher, Waltham, MA) for element determination. The

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separation of AgNPs and Ag+ ions was performed on a Venusil XBP NH2 column (5

195

μm particle size, 1000 Å pore size, 4.6 × 250 mm, Bonna-Agela, Tianjin, China) at

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20 ℃ based on a size exclusion mechanism. The mobile phase consisted 1% (v/v)

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FL-70 and 10 mmol L-1 Na2S2O3 at a flow rate of 0.5 mL min-1. ICP-MS

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determination was performed with radio frequency power 1550 W, sampling depth 5

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mm and 0.022 sec integration time to monitor the 107Ag and 109Ag isotopes.

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Root samples were analyzed by LC-ICP-MS before and after removing adsorbed

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silver species. The root samples were washed with a mixture solution of 10 mmol L-1

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K3Fe(CN)6 and 10 mmol L-1 Na2S2O3, in which process the complexed Ag+ ions,

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adsorbed AgCl and AgNPs on the root surface can be effectively removed through

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chemical etching and dissolution.47, 48 The root samples were rinsed with deionized 11



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water and then digested for Ag analysis by LC-ICP-MS.

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Total Silver Concentration Measurements in Root and Shoot. To quantify Ag

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accumulation in various tissues, dried root and shoot samples were ground with a

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mortar and pestle. Tissue powders (~0.1 g) were digested with 4 mL HNO3 and 2 mL

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H2O2 under microwave irradiation. The concentrations of

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analyzed with ICP-MS (7700ce, Agilent, Santa Clara, CA) after dilution with

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deionized water. For the root samples, the total Ag concentration was also measured

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before and after K3Fe(CN)6-Na2S2O3 washing. Procedure blank, matrix spike, and

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precision check were included for QA/QC. The procedure blank was

Uptake and Transformation of Silver Nanoparticles and Ions by Rice

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