Distribution and Speciation of Copper and Arsenic in Rice Plants

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Distribution and Speciation of Copper and Arsenic in Rice Plants (O.sativa japonica ’Koshihikari’) Treated with Copper Oxide Nanoparticles and Arsenic during a Life Cycle Jing Liu, Kyle Wolfe, Phillip M. Potter, and George P. Cobb Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b00234 • Publication Date (Web): 28 Mar 2019 Downloaded from http://pubs.acs.org on March 28, 2019

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Distribution and Speciation of Copper and Arsenic in Rice Plants (O.sativa japonica ’Koshihikari’) Treated with Copper Oxide Nanoparticles and Arsenic during a Life Cycle

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Jing Liu a*, Kyle Wolfe a, Phillip M. Potter b, George P Cobb a

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a

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Waco, Texas, 76798-7266, USA.

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b

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USA.

Department of Environmental Science, Baylor University, One Bear Place #97266,

Oak Ridge Institute for Science and Education (ORISE), Oak Ridge, TN 37831-0117,

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Emails:

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*

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Kyle Wolfe: [email protected]

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Phillip M. Potter: [email protected]

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George P Cobb: [email protected]

Jing Liu (Corresponding author): [email protected]

1 ACS Paragon Plus Environment

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Abstract

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A 6×2 factorial study was conducted to investigate the effects of copper oxide

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nanoparticles (nCuO, 0.1–100 mg/L), arsenic (As, 0–10 mg/kg) and their interaction on

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uptake, distribution and speciation of Cu and As in rice plants (Oryza sativa

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japonica ’Koshihikari’). Arsenic (in As-addition treatments) and Cu in seedling roots

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(SRs) were 1.45 and 1.58 times of those in soil, respectively. Arsenic and Cu

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concentrations further increased in mature plant roots (MRs), which were 2.06 and

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2.35times of those in soil, respectively. Arsenic and Cu concentrations in seedling shoots

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(SSs) were 79% and 54% lower than in SRs, respectively. The mature stems, however,

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contained only 3% and 44% of As and Cu in SSs. Copper in flag leaves did not vary

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much compared with stems, while As was 14.5 times of that in stems. Species

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transformations of Cu and As were observed in rice including reductions of Cu (II) to Cu

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(I) and As (V) to As (III). Arsenic in dehusked-grains was negatively correlated with Cu

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and was lowered by nCuO below the WHO (World Health Organization) maximum safe

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concentration for white rice (200 ng/g). This may alleviate As adverse effects on humans

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from rice consumption.

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Keywords: distribution, speciation, copper oxide nanoparticles, arsenic, rice, life cycle

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Introduction

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High arsenic (As) concentrations in the environment pose direct and indirect adverse

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effects on humans. Rice (Oryza sativa) plant growth throughout the life cycle has been

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adversely affected by As 1-4. Seed germination and biomass of rice plants has been shown

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to be decreased by As, and the nutritional quality of rice grains have been lowered due to

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As accumulation 1-4. Rice grains containing high As concentrations provide a chronic As 2 ACS Paragon Plus Environment


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exposure pathway for humans. Therefore, regulations or guidelines have been developed

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for acceptable As concentrations in rice. The WHO (World Health Organization)

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proposed maximum safe concentrations of inorganic As at 0.2 mg/kg for white rice and

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0.35 mg/kg for brown rice 5, 6. The US Food and Drug Administration (FDA) proposed an

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action level (or limit) for inorganic As in infant rice cereals at 0.1 mg/kg 7. Studies have

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been conducted to reduce As accumulation in rice grains by adding soil amendments,

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water management (e.g., irrigation and drainage), and genetic manipulation 8-12.

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Nanotechnology has revolutionized agriculture in the last decade to increase crop yield

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and improve food quality 13. Nanomaterials (NMs) can be introduced in plant growth

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systems as nanofertilizers, nanopesticides, and nanobiosensors 13. Copper oxide

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nanoparticles (nCuO) may serve as nanofertilizers by providing important micronutrients

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for plants, as well as nanopesticides due to antimicrobial properties 14. Moreover, nCuO

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has been studied to remove As from water systems 15, 16. All of these functions allow

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nCuO to improve rice plant growth, reduce As uptake into rice plants, and diminish As

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accumulation in the grains. In addition to the intentional applications, the unintentional

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discharge of nCuO is likely to increase due to emerging applications of its unique

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properties and high sustainability (wide-range industrial suitability, 100% recyclability,

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abundant availability, and effective antimicrobial properties) 14. Although some studies

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have evaluated effects of NMs or As on the life cycle of rice plants, no studies have been

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done that describe the interaction of nCuO and As during the life cycle growth of rice

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plants 17,18-20. The uptake, transport, and speciation of NMs in plants are also dependent

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on species and growth medium conditions.


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We conducted a factorial experiment to study effects of As (0 and 10 mg/kg soil) and

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nCuO (0–100 mg/L) individually and in combination on rice plants during the life cycle

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(from seed germination to seed maturation). Several hypotheses were investigated: nCuO

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and As interact to influence the uptake of As and Cu by rice plants during the life cycle;

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nCuO and As interact to affect the distribution and speciation of As and Cu in different

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parts of rice plants during the life cycle; and the accumulation of Cu and As in rice grains

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are influenced by nCuO and As interaction in the growth media.

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Methods

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Life Cycle Exposure in the Greenhouse

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Arsenic in the form of Na2HAsO4·7H2O 21, 22 (Sigma-Aldrich, lot # BCBM0939V) was

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added at 0 and 10 mg/kg (near average As concentration in Texas, US 23) to an artificial

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soil mixture (60% Grainger clay soil, Catalog # 2258, 40% Lowe’s topsoil, # 235384).

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nCuO (Nano-Arc®, 97.5%, 23–37 nm, APS powder, Alfa Aesar, MA, USA) was

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prepared at six concentrations (0, 0.1, 1.0, 10, 50, 100 mg/L) in 20% Hoagland’s solution.

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Twelve treatments (2×6 combinations of the nCuO and As) were studied including one

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control (without either As or nCuO), one As-only treatment, and 20 replicate growth

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containers (Berry Plastics ID: T60785CP, 2.5 L) were prepared for each treatment. Ten

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seeds (Oryza sativa japonica ’Koshihikari’, Kitazawa Seed Company, CA, USA) were

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wet-sown in each container after adding nCuO-containing solutions. Seedlings were

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thinned on day 18 and two well-established seedlings were kept for a 131-d life cycle in

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the greenhouse. Materials and procedural details are extensively presented in the

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Supporting Information (SI), and were published in our previous article 24.



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Uptake, Distribution and Speciation of Copper Oxide Nanoparticles and Arsenic

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Early seedlings shoots (SSs) and roots (SRs), mature plant roots (MRs), stems, flag

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leaves and their paired grains (husk and dehusked-grains) (Figure S4) were collected and

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digested before being analyzed for total As and Cu with inductively coupled plasma mass

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spectrometry (ICP-MS, Agilent 7900) (SI).

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Micro-XRF maps and X-Ray Absorption Near Edge Structure (XANES) with spectral

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acquisition near the Cu K-edge (8979 eV) and the As K-edge (11867 eV) were collected

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for samples (seedlings and grains) and reference standards at Beamline 13-ID-E in

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Argonne National Laboratory. Copper reference materials were: nCuO, CuO,

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CuSO4•5H2O, Cu3(PO4)2, cupric acetate, cuprous acetate, cupric oxalate, and synthesized

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cupric arsenate. Arsenic reference materials were: As2O3, As2S3, AsS, As2O5,

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schneiderhonite (Fe4As5O13), scorodite (FeAsO4•2H2O), dimethylarsonic acid (DMA),

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monomethylarsinic acid (MMA), and synthesized cupric arsenate. Details about sample

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preparation and data acquisition are included in the SI 25.

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XRF mapping data were processed with Larch software 26. XANES data were processed

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with Athena (Demeter 0.9.26) 27 and analyzed with linear combination fitting method. R

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factor and reduced χ2 were criteria for determining the best fit.

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Statistical Analyses

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Generalized linear models (GLMs) were used to analyze the main and interactive effects

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of nCuO and As in growth media on their uptake into rice. Data distributions were

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defined in the model. Models were validated and accepted with low residual

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heterogeneity, followed by the analysis of variance (ANOVA) to study the individual

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main effects of nCuO and As and their interaction. Data were expressed as means ±


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S.E.M (standard error of measurement). Means were considered different when p < 0.05.

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All p values were adjusted with Tukey method. With the exception of spectral

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deconvolution, all statistical analyses were performed in R (version 3.3.2) 28.

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Results and Discussion

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Primary effects of nCuO and As and their interaction on major morphological and

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physiological parameters of rice plants during a life cycle growth has been discussed

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previously 24. Arsenic mainly inhibited seedling growth, and nCuO generally mitigated

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As adverse effects. Arsenic decelerated panicle heading process, while nCuO accelerated

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this process. Both As and nCuO increased grain yield in the studied concentration range.

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Total Cu and As uptake, transport and speciation inside plants were also shown to be

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dependent on treatments.

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Uptake and Distribution of Copper and Arsenic in Rice Plants

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18-d Seedlings

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Copper was highly concentrated in rice SRs and was influenced by nCuO concentration,

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As addition, and the interaction of As and nCuO (p < 0.05) (Figure 1, Figure S7, Table

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S5). Copper concentration in SRs exponentially increased with that in soil in nCuO alone

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treatment and tended to reach a plateau (Figure 1), while Cu in SRs linearly increased

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with that in soil in As+nCuO treatments. In contrast, Cu in SSs increased in an

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exponential manner with Cu concentration in soil and plateaued in both treatment groups.

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Compared with control, Cu concentrations in SRs decreased at nCuO 10 mg/L but

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increased at nCuO 50 and 100 mg/L (Figure S7, Table S5). Arsenic-only treatment

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decreased Cu in SRs compared with control. Arsenic addition to nCuO at 50 mg/L also

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decreased Cu in SRs compared with nCuO alone treatment at 50 mg/L (p < 0.05). 6 ACS Paragon Plus Environment


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Arsenic addition increased Cu in SRs at a medium and a high nCuO concentration (10

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and 100 mg/L) (p < 0.05). Copper in SSs was much lower than in SRs and soils (p
stem ~ husk.

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Arsenic concentrations generally followed the order:

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a) nCuO treatment: root > flag leaf > stem > husk > dehusked-grain;

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b) As+nCuO treatment: root > flag leaf > husk > dehusked-grain > stem.

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Uptake Mechanisms of Copper and Arsenic by Rice Plants

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Being an essential micronutrient, Cu was taken into the plant from roots via specific Cu

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transporters (e.g., COPT) 29. Adequate Cu uptake in rice plants was within the range of

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15–26 mg/kg found by other researchers 30. This explains the rapid Cu uptake (16–21.2

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mg/kg) in SRs at nCuO < 10 mg/L in our study (Table S5). When excess Cu is provided,

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plants employ complex homeostatic regulations to address the dual nature of Cu as being


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essential and toxic by assuring Cu delivery to cuproproteins and inhibiting excess Cu

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uptake (e.g., by sequestering excess metal with polypeptides) 29, 31. Research by other

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groups showed that the TCP16 from TCP (TEOSINTE BRANCHED 1, CYCLOIDEA

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and PROLIFERATING CELL FACTOR 1) family of transcription factors significantly

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down-regulated the expression of Cu intracellular transporter (COPT3) in A. thaliana

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with hydroponic exposure to Cu2+ (635 µg/L) 32. This concentration is lower than the

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highest Cu concentration in solutions from in our study (Table S3). Therefore, total Cu

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uptake in SRs in treatments without As addition tended to reach relatively high maximum

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concentrations (Figure 1). Meanwhile, As addition to the growth media disrupted normal

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homeostatic Cu regulation 22. Therefore, in treatments with As addition, Cu in SRs

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increased linearly with Cu in soil. However, Cu uptake in MRs followed different

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patterns compared with 18-d SRs in our study. This was probably because Cu and As

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were transported to other parts when the plant grew and the biomass increased 33, 34.

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Unlike Cu, As is not required for plant health, but As species can enter plants as

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analogues of other essential elements. As(V) enters plant roots through phosphate

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transporters (e.g., OsPT4) 35. As(III) is taken up by rice roots through silicic acid

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transporters (Nodulin 26-like intrinsic membrane proteins, such as Lsi1) 20. Arsenic is

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more available for rice plants compared to other crop plants, because As bioavailability is

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relatively high in water than in other environmental compartments 36. Overall, As uptake

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was differentiated at various growth stages (Table S5-8). High phosphate concentrations (>

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6.2 mg/L phosphorus), which were observed in our study, may also have enhanced As

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desorption from soil mineral particles due to the competition for adsorption sites 16.

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However, a study by other groups demonstrated that As (V) and As(III) were both



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effectively adsorbed onto nCuO between pH 6–10, with little competition from other

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anions (e.g., phosphate, silica and sulfate) 15, 16. The post-treatment pH of soils in our

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study was covered in this range (7.4–7.9) 24. Our previous study also demonstrated that

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As concentration in the Hoagland solution decreased over time in the presence of nCuO

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37

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rhizosphere also interacted with As and nCuO, thus influencing their bioavailability and

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uptake to rice plants 38. Soil type also significantly influences Cu and As availability 39, 40.

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Our previous study with sand as the growth media showed that Cu concentrations in 18-d

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SRs and SSs were about 12 and 3 times greater, respectively, than those in our current

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study 37. While As concentrations in 18-d SRs and SSs grown in sand were 70 and 54

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times greater, respectively, than those in our current study.

. In addition, microbes in the growth media and root exudates (e,g., organic acid) to the


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Figure 2. Copper and arsenic concentrations in the mature rice plants from a greenhouse study of rice (O.sativa japonica ’Koshihikari’) with 131 d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution (n=5)



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Figure 3. Copper concentration in the mature rice plant roots from a greenhouse study of rice (O.sativa japonica ’Koshihikari’) with 131 d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution (n=5)


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Figure 4. Comparison of copper and arsenic concentrations in different parts of rice plants at two time points (Day 120 and Day 131, 2017) from a greenhouse study of rice (O.sativa japonica ’Koshihikari’) with 131-d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution (n=5). * means of the same treatment at the two time points are different (p < 0.05)

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a

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Figure 5. Relationships between copper and arsenic concentrations in the rice grain husk (a) and dehusked-grains (b) in treatments from a greenhouse study of rice (O.sativa japonica ’Koshihikari’) with 131-d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution (n=5)

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Transport and Speciation of Copper and Arsenic in Rice Plants

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The Cu XANES spectra of bulk CuO and nCuO were indistinguishable as the concentration we

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measured in plants 41. Thus we used “CuO” for the speciation in our samples. Spectral

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deconvolution of Cu XANES showed that Cu species in seedlings mainly included cupric acetate,

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cupric oxalate, CuO, and cuprous acetate (Figure 6, Table 1). Arsenic species in seedlings mainly

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included As2S3, As2O5, scorodite, dimethylarsonic acid, and methylarsinic acid (Figure 6, Table

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2). Copper oxide was observed in all samples, and the dominant species in SSs from nCuO 100

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mg/L treatment. Cupric oxalate was observed in control and As+nCuO100 mg/L treatment, and

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the percentage in SSs increased than in SRs from the same treatment. Cupric acetate was the

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dominant species observed in SRs from control and in SSs from As+nCuO100 mg/L treatment.

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Cuprous acetate was the main species observed in SRs from treatments receiving nCuO 100

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mg/L.


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Copper oxide nanoparticles undergoes a series of transformations (e.g., aggregation,

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sedimentation, dissolution and speciation) in the environment. Ultimately, ionic Cu is the

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dominant species taken up by rice plants via Cu transporters, but nCuO could also enter plant

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roots via several pathways, and several barriers (e.g., mucilage, cuticle and cell wall, blockage of

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the pores of cell walls due to NP aggregation) hinder the process 42. Possible subcellular NP

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uptake pathways across the barriers were summarized in a review article, including passive

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diffusion through permeable region of cuticle and cell wall pores and facilitated transport by

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natural organic matter (e.g., humic acids and root exudates) and microorganisms (e.g., algae,

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bacteria, and fungi) 42. Once across the barriers, NPs penetrated cell membranes by binding to

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the receptor and subsequent fluid-phase endocytosis (FPE), by passive diffusion or by physically

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damaging the membrane 42. After entering the root, NPs could travel from root to xylem via

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apoplastic and symplastic routes, and then be translocated to other locations of the plants by

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xylem and phloem. This was verified by other research groups that nCuO was transported from

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roots to shoots via xylem and back translocated from shoots to roots via phloem in Maize (Zea

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mays L.) 43. Based on this research, the observed CuO in SRs and SSs in our study (Table 1)

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could be NPs 43. This previous research also observed that Cu(II) was reduced to Cu(I), which

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was consistent with our result that cuprous acetate content increased in SRs from nCuO

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treatment compared with control (Table 1). nCuO itself cannot be utilized by plants but may

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affect plant growth by interacting with other components (e.g., As). Although dissociated Cu

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ions from nCuO can be used as micronutrient, nCuO dissolution inside the plants has not been

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well understood. Moreover, during plant growth and development, different root exudates were

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secreted and influenced the behavior of nCuO in the rhizosphere and further uptake of Cu ion

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and nCuO 38. Concurrently, As and other components in the growth media also exerted dynamic

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influence on Cu speciation. 18 ACS Paragon Plus Environment


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Dominant As species in SRs and SSs varied among treatments (Figure 6, Table 2). Arsenic

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concentrations in SRs and SSs were relatively low, therefore there were more non-detectable

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species. DMA was dominant in SRs from control and SSs from As-only treatment. Scorodite was

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dominant in SRs from As and As+nCuO treatments, and SSs from As+nCuO treatment. As2S3

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was observed in all samples except in SSs from As-only treatment, while As2O5 were observed in

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both SSs and SRs from As-only treatment, and in SSs from As+nCuO treatment. Methylarsinic

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acid (MAA) was only observed in SSs from As-only treatment. for the literature suggests that the

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higher energy peak (11872 eV) in the spectra of SSs samples from As-only treatment is very

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similar to synthetic As(III)-(GLU)3 standard 44. This confirms to our understanding of the

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physiological mechanism of plants interacting with As 31.

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In our study, As(V) was the original test species added in the soil. However, As(V) could easily

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be reduced to As(III) under submerged rice cultivation in anaerobic or microaerobic soil and

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desorbed from the soil particles due to the lower sorption capacity of As(III) than As(V) 45.

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As(III) could also be oxidized to As(V) on the surface of nCuO 16. Once assimilated by the plant,

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As(V) could be relocated to the xylem and phloem by phosphate transporters such as OsPT1 and

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OsPT4 35, 46, while As(III) is transported from root towards stele through efflux transporter (Lis2)

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for silicic acid 20. Given the shared transport pathway in the xylem and phloem, As and nCuO

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could interact with each other inside the plants. Arsenic speciation inside the plants was

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dependent on the locations in the plant, which was also affected by nCuO in our study.

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a

b

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Figure 6. Copper (a) and arsenic (b) K-edge XANES spectra of reference standards and 18-d rice seedling root near seed and shoot near seed samples in control and treatments of nCuO100, and As+nCuO100 from a greenhouse study of rice (O.sativa japonica ’Koshihikari’) with 131-d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution . Solid lines are spectra for the samples, and dashed lines are linear combination fitting results for the sample.

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Table 1. Copper speciation based on copper K edge XANES spectra of 18-d seedling root and shoot samples in control and treatments of nCuO and As+nCuO100 from a greenhouse study of rice (O.sativa japonica ’Koshihikari’) with 131-d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution. Fitting Criteria Samples Control_root near seed nCuO100_root near seed As+nCuO100_ root near seed Control_shoot near seed nCuO100_shoot near seed As+nCuO100_ shoot near seed

332 333 334 335

CuO

Cupric oxalate

Cupric acetate

Cuprous acetate

Fraction Sum

R factor

Reduced χ2

Fraction %

Fraction %

Fraction %

Fraction %

0.011

0.002

16.6

41.2

42.2

ND

100

0.005

0.001

39.7

ND

ND

60.3

100

0.003

0.001

22.25

23.1

26.05

28.6

100

0.007

0.002

28.3

71.7

ND

ND

100

0.007

0.002

58.05

ND

10.25

31.7

100

0.027

0.006

15.73

25.57

58.7

ND

100

Note: R factor, the residual sum of squares; ND=not detectable; Reduced χ2, a weighted sum of squared deviations. All standards were evaluated but some were omitted from the table for not present.



336 337 338 339

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Table 2. Arsenic speciation based on arsenic K edge XANES spectra of 18-d seedling root and shoot samples in control and treatments of nCuO and As+nCuO100 from a greenhouse study of rice (O.sativa japonica ’Koshihikari’) with 131-d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution.

Samples

Fitting Criteria Fitting Criteria R-factor

Control_root tip As_root near seed As+nCuO100_ root near seed As_shoot near seed As+nCuO100_ shoot near seed

0.05

As2S3

Reduced Fraction χ2 % 0.02 45.10

As2O5

Scorodite (FeAsO4•2H2O)

Dimethylarsonic Acid [DMA, As(III)]

Methylarsinic acid Fraction [MAA, As(V)] Sum Fraction %

Fraction %

Fraction %

Fraction %

8.90

46.00

ND

100

0.02

0.02

18.10

10.77

71.13

ND

ND

100

0.01

0.01

23.80

ND

76.20

ND

ND

100

0.05

0.02

ND

26.13

5.20

51.53

17.13

100

0.03

0.02

36.20

20.25

43.55

ND

ND

100

340 341 342

Note: R factor: the residual sum of squares; ND=not detectable; Reduced χ2, a weighted sum of squared deviations. All standards were evaluated but some were omitted from the table for not present.

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Agriculture and Human Health Implication

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Rice straw is an alternative of fertilizers used on-site in rice paddies, because nutrients in rice

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straw can be released to the field after straw decomposition for future rice cultivation 47.

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Meanwhile, As can also be remobilized from the straw residual and be available for rice growing

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in the next generation. In a previous study by other groups 47, the released As from incubated

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mixture of rice straw (5 g, 1.1 ± 0.34 mg As/kg dry weight) and soil (800 g, 9.49 mg As /kg dry

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weight) in 500 mL H2O was up to 1.3 % (~ 200 µg/L in pore water). Based on this percentage,

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As concentration from a mixture with the same relative ratio of straw, soil and water would be

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estimated up to 240 µg/L in our study, which is much lower than previously reported As

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concentration (500-8000 µg /L) in hydroponic studies that adversely affected rice seed

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germination and early seedling growth 21. Therefore, rice straw in Texas can be applied directly

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in the field as fertilizers.


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Copper and As concentrations in grains were related with those in flag leaves (Figure S11–12).

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The flag leaf of a tiller emerges as the last leaf, and it contributes most to the grain filling process

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by transporting photosynthetic products into the spikelet 48. Relatively high concentrations of Cu

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and As in flag leaves as compared to stems also implied that flag leaves were an important

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pathway to transport As and Cu from the lower parts of plants to grains. Studies by other

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research groups showed that a phosphate transporter probably facilitated As mobilization from

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flag leaves to panicles, and a C-type ATP-binding cassette transporter (OsABCC1) was also

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shown to limit As transport to grains 35, 49. In our study, As in the dehusked-grain was lower than

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in husks, while Cu in the dehusked-grain was higher than in husks. Moreover, there was a

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negative correlation between Cu and As in dehusked-grains. These relationships implied an

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antiport process of Cu and As in the grain. However, it is unclear about the role of flag leaves

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during the process. Foliar application of NP suspension may be more efficient if Cu ion and

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nCuO can be absorbed and transported through flag leaves to grains and antiport As from grains

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simultaneously by interacting with the related transporters, which needs further study. In our

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study, As in dehusked-grains decreased at all nCuO+As treatments compared with As-only

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treatment (Table S8). The lowest As accumulation in dehusked-grains among all As-addition

371

treatments (128 ± 15.4 ng/g) was observed in As+nCuO 50 mg/L treatment. It was 36% lower

372

than the WHO maximum safe concentration of As in white rice (200 ng/g) for humans 50. Rice

373

containing 128 ng/g As converts to an exposure (for a 60-kg person consuming 200 g rice daily)

374

of 0.43 µg/kg/day, approximating the Minimal Risk Level for effects (other than cancer), which

375

was set at 0.4 µg/kg/day 51. Therefore, 128 µg/kg/day can reduce As exposure and avoid the

376

reported human effects by US Agency for Toxic Substances and Disease Registry (ATSDR) for

377

humans who consume at least 200 g rice per day 51.



Page 24 of 28

378

Arsenic contamination is a big issue globally and causes rice yield reduction and food safety

379

concerns. Thus, As remediation has become essential for healthy human life of the increasing

380

population worldwide. Although gene technology is efficient to lower As transport to the grains

381

52, 53

382

also be influenced consequently. Moreover, the safety of genetically modified food is still a big

383

controversy. Among a wide variety of NMs, Cu-based NMs exhibit great potential to remediate

384

As-contaminated environment, and simultaneously release the micronutrient and antimicrobial

385

agent Cu to improve plant growth. Therefore, nCuO provides an alternative to alleviate As

386

phytotoxicity in rice plants and reduce As accumulation in rice grains.

387

Acknowledgement

388

This work was supported by “C. Gus Glasscock, Jr. Endowed Fund for Excellence in Environmental

389

Sciences in the College of Arts and Sciences” at Baylor University. The authors thank Dr.Robert Doyle

390

for sharing the greenhouse space. The authors acknowledge Baylor University Mass Spectrometry Center

391

and Dr. Alejandro Ramirez for technical support. Acknowledgement to Dr. Bernd Zechmann in Center

392

for Microscopy and Imaging at Baylor University for helping with sample preparation for XANES spectra

393

collection, and Dr.Steve Dorwkin for facilitating soil particle size analysis. Thanks to Matt

394

Newville and Antonio Lanzirotti for helping with XANES data collection at Beamline 13-ID-E,

395

Argonne National Lab.

396

Declaration of Interest Statement

397

The authors report no conflicts of interest and are responsible for the publication.

398

Supporting Information

399

Additional text providing the materials and procedural details of the study; 12 figures showing

400

the environment condition in the greenhouse, the characterization of nCuO, a flag leaf paired

, rice yield may be decreased due to limited uptake of other nutrients. The nutrient value may


Page 25 of 28


401

with a panicle, portions of seedlings for XANES analysis, the elemental components of

402

synthesized cupric arsenate, Cu and As concentrations in 18-d seedlings, depicted Cu and As

403

distribution in rice seedlings, and relationships of Cu and As concentrations in different parts of

404

the plants; 7 tables demonstrating soil characteristics, concentrations of Cu and As in the growth

405

media and plants, and Cu speciation in dehusked rice grains.

406



Page 26 of 28

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Distribution and Speciation of Copper and Arsenic in Rice Plants

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