How Rice (Oryza sativa L.) Responds to Elevated As under Different

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How rice (Oryza sativa L.) responds to elevated As under different Si-rich soil amendments William A Teasley, Matthew Alan Limmer, and Angelia L. Seyfferth Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b01740 • Publication Date (Web): 10 Aug 2017 Downloaded from http://pubs.acs.org on August 11, 2017

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Environmental Science & Technology

Title: How rice (Oryza sativa L.) responds to elevated As under different Si-rich soil amendments

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Authorship: William A. Teasley, Matthew A. Limmer, Angelia L. Seyfferth*

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Affiliations:

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Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716, USA.

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*Corresponding author: Angelia L. Seyfferth, phone: (302) 831-4865, fax: (302) 831-4865,

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email: [email protected]

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ABSTRACT

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Several strategies exist to mitigate As impacts on rice and each has its set of tradeoffs

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with respect to yield, inorganic As content in grain, and CH4 emissions. The addition of Si to

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paddy soil can decrease As uptake by rice but how rice will respond to elevated As when soil is

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amended with Si-rich materials is unresolved. Here, we evaluated yield impacts and grain As

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content and speciation in rice exposed to elevated As in response to different Si-rich soil

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amendments including rice husk, rice husk ash, and CaSiO3 in a pot study. We found that As-

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induced yield losses were alleviated by Husk amendment, partially alleviated by Ash

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amendment, and not affected by CaSiO3 amendment. Furthermore, Husk was the only tested Si-

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amendment to significantly decrease grain As concentrations. Husk amendment was likely

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effective at decreasing grain As and improving yield because it provided more plant-available Si,

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particularly during the reproductive and ripening phases. Both Husk and Ash provided K, which

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also played a role in yield improvement. This study demonstrates that while Si-rich amendments

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can affect rice uptake of As, the kinetics of Si dissolution and nutrient availability can also affect

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As uptake and toxicity in rice.


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INTRODUCTION Arsenic (As) is a ubiquitous contaminant in the world’s rice supply and threatens human

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health and grain yield, particularly for rice grown under elevated As conditions.1-3 Paddy

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cultivation mobilizes soil As through the reductive dissolution of Fe oxide, hydroxide, and

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oxyhydroxide minerals (hereafter referred to as Fe (oxyhydr)oxides)) and the subsequent

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reduction of inorganic arsenate (Asi(V)) to the more mobile, arsenite (Asi(III)), which is readily

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taken up by rice.4 Rice contamination is further exacerbated in locales (e.g., Bangladesh) where

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groundwater contaminated with inorganic As (Asi) is used to irrigate rice.5, 6 In addition to Asi,

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the organic pentavalent As species (Aso) dimethylarsinic acid (DMA) and monomethylarsonic

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acid (MMA) can be found in rice paddy porewaters due to microbial methylation and may

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accumulate in rice grain.7 While Asi is a known human carcinogen, both Asi and Aso can

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decrease grain yield, as both are suspected agents of Straighthead disorder in which panicles

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remain erect due to poor grain filling.7, 8 Several strategies are being developed to mitigate As

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impacts on rice and each has its set of tradeoffs with respect to grain yield, As content in grain,

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and CH4 emissions. Increasing plant-available Si can effectively alleviate the negative impacts of As on rice,9-

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plant components that dictate the overall impact of Si addition on rice’s response to As.

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Increasing Si can alleviate As uptake because a) the dominant form of As in flooded paddies is

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Asi(III)4 which is chemically similar to Si (H3AsO3, pKa = 9.2; H4SiO4, pKa = 9.8), b) Asi(III) is

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taken up by Si transporters in rice,16 c) sufficient Si can downregulate Si transporter expression17

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and thus minimize Asi(III) uptake,10, 18, 19 d) increasing Si favors ferrihydrite stabilization over

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higher ordered phases (e.g. goethite), thus providing more surface area for As adsorption onto Fe

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phases,9, 20, 21 and e) excess Si may prevent re-release of As by subsequent Si polymerization

but Si addition to soil causes a shift in the dynamic interactions between porewater, soil, and



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around As-bearing ferrihydrite minerals.22 However, Si can also desorb Asi(III) from the solid

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phase23 and lead to an increase in As uptake by rice particularly if the Si-rich amendment

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provides relatively low Si levels.19 Si additions have also been shown to shift grain speciation

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from Asi to Aso.10 Moreover, Si helps rice plants form stronger aerenchyma, which promotes

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more gas exchange, e.g., oxygen diffusion, with consequent impacts on Fe oxidation in the

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rhizosphere. Because of rice’s unique demand for Si, Si addition alters both geochemical and

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plant physiological parameters that influence the impact of Si on alleviating As in rice.11

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Recent work has shown that Si-rich rice residues can provide the Si to alleviate the

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negative outcomes of As in rice.9-11. We showed that Asi levels in grain decreased by 25 to 50%

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for rice grown in rice husk-amended flooded soil with low/background As without affecting

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grain yield or dissolved CH4 concentrations.10 In contrast, rice straw amendment to soil has been

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shown to increase grain As24 and CH4 emissions.25-27 While shown to be promising for

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low/background As conditions, how rice responds to elevated As when soil is amended with rice

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husk or rice husk ash has not yet been evaluated. In areas such as Bangladesh where dry-season

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rice is irrigated with As-contaminated water, rice grows under elevated soil As, resulting in

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elevated grain As and significant grain yield loss.28-33 In addition, those soils may be depleted in

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plant-available Si due to extensive soil weathering and intensive rice cultivation whereby Si-rich

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residues (i.e, husk and straw) are removed,34 which may exacerbate the negative effects on grain

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yield and quality caused by elevated As. Moreover, CaSiO3 is used as a Si source in some rice

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growing regions,35 but rice’s response to elevated As when grown in CaSiO3-amended and Si-

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depleted soil and how it directly compares to Si-rich rice husk and rice husk ash amendments is

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unresolved. Lastly, while our previous work10, 11 indicated that plant-based Si amendments did

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not affect dissolved CH4, it is unknown how emissions are affected.


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Here, we evaluated rice’s response to elevated As under different Si-rich amendments

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including fresh rice husk “Husk”, rice husk ash “Ash”, and calcium silicate “CaSiO3”. We also

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monitored the dynamic geochemical shifts that occur upon Si addition that influence As, C, and

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nutrient cycling in rice. Our findings illustrate that the properties of the Si amendment dictate

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the overall effect of its addition to soil on rice’s response to elevated As.

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MATERIALS AND METHODS

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Soil Collection and Characterization A well-weathered soil depleted in plant-available Si was collected from the University of

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Delaware farm in Newark, Delaware. The site had been used as an orchard prior to 1950 and has

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been fallow for at least the past five years. The soil is classified by the U.S. taxonomic system as

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a fine-loamy, mixed, semiactive, mesic Typic Hapludult in the Elsinboro series or as an Acrisol

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according to the Food and Agriculture Organization system. After collection, soil was hand-

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mixed over several weeks to allow for air-drying, taking care to preserve the soil structure as

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much as possible. The soil had a pH (1:1) of 5.2, a total C of 1.4%, and total N of 0.13 %

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(Elementar CHNS Cube). Plant-available nutrients in soil were approximated using CaCl2,

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BaCl2 and acetic acid extractions and analyzed with ICP-OES and ICP-MS (Table S1)36. Acid

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ammonium oxalate (AAO) and citrate-bicarbonate-dithionite (CBD) extractions were conducted

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according to standard methods36 to quantify poorly-crystalline and crystalline fractions,

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respectively, of Fe, As, and Si (Table S2). In addition, soil underwent a sequential extraction

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procedure37 to quantify As-soil phase associations prior to the addition of As (i.e, -As-control),

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and in +As soil post-harvest.



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Soil As Spiking and Si Amendments. To control for the effect of As on grain yield and plant biomass, plants were grown in

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non-spiked (no added As, “-As-control”) soil and irrigated with deionized (DI) water with 4

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replicates. The remaining 16 pots were filled with As-spiked soil (“+As”), which was spiked

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according to the results of an adsorption isotherm (Fig. S1) with 0.125 µmol g-1 As (as Na salts)

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to achieve an initial equilibrium porewater concentration of 4 µM in a molar ratio of 80:20

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Asi(III):Asi(V).19 This concentration and ratio was chosen to resemble As speciation measured in

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an As-contaminated Bangladeshi paddy.5, 6 Arsenic solutions were mixed into the soil and

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allowed to equilibrate for 3 weeks, hand-mixing intermittently to ensure uniformity.

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Each +As treatment received one type of Si-rich amendment: fresh rice husk “Husk”, rice

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husk ash “Ash”, or calcium silicate “CaSiO3”. The +As-control group was spiked with As but

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received no Si amendment. Both Husk and Ash amendments were obtained from a rice milling

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factory in Battambang, Cambodia and applied at a rate of 1% (w/w) amendment/soil 10, 11, which

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previously demonstrated equivalent porewater concentrations of amendment-derived As and Si

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at that loading11. Amendment-derived As, which was 0.26 and 0.50 mg kg-1 for Husk and Ash,

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respectively, was negligible in comparison to the ~24 mg kg-1 As in spiked soil (Table S3 and

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Fig. S2). CaSiO3 was amended at 0.46% (w/w) to give a Si rate equivalent to that of the Husk

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amendment (Table S3); this rate equates to 5 Mg ha-1, an application rate previously shown to

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benefit rice plants38. After mixing each Si amendment thoroughly, the soil in each group was

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divided equally by weight into 4 L pots that each received 3.5 kg dry-weight equivalent of soil

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with 4 replicate pots per group.

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Plant Growth Rice seeds (Oryza sativa L., cv. M206) were sterilized in dilute bleach, rinsed, and

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germinated in non-spiked soil. Ten-day-old seedlings were transplanted into pots, three 6 ACS Paragon Plus Environment

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seedlings per pot, and each pot was fertilized with 120 mg N (as 0.4 M NH4NO3) and

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immediately flooded with deionized water to a height of 5 cm above the soil surface. For +As

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plants, DI water was replaced by 4 µM As solution (80:20 Asi(III)/ Asi(V)) after 1 week to

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simulate contaminated irrigation water.5, 19 Plants were kept flooded with this 4 µM As solution

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throughout growth. Plants were grown to maturity (120 d) in a climate controlled chamber (70%

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RH, 28˚/22˚C day/night cycle) using growth lights (Lumigrow, Novato, CA) set to a 16-h

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photoperiod.

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Porewater and Gas Flux Monitoring Porewater was drawn weekly into acid-washed, N2-flushed, crimp-sealed vials using

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Rhizon samplers and processed using previous methods10, 11, 19 for measurement of pH, Eh, and

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concentrations of Fe(II), As species, and total elements. Total element samples were acidified

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with trace metal grade (TMG) HNO3 and analyzed with ICP-OES. Speciation samples were

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frozen at each sampling. Eh was determined immediately using a combination Pt electrode with

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built-in temperature and Ag/AgCl reference electrodes and is reported relative to the standard

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hydrogen electrode. pH was determined using a meter and calibrated electrode. Fe(II) was

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determined using the colorimetric ferrozine method.39 Prior to As speciation analysis, select

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samples were thawed, acidified 1:1 with 2% TMG HNO3, filtered through a 0.2 µm nylon

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syringe filter, and analyzed within 24 hours. An Agilent 1200 HPLC and PRP-X100 column

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was used to separate As species10, 40 that were detected with an Agilent 7500cx Series ICP-MS

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operating in He collision mode. Only two samples per group per time point were analyzed for

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As species. Standard checks, blanks, and spike recoveries were included throughout each run.

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An Ultraportable Greenhouse Gas Analyzer (LGR, Los Gatos Research, San Jose, CA,

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USA) was used with a dynamic dark flux chamber (Fig. S3) to monitor CO2 and CH4 flux 7 ACS Paragon Plus Environment


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weekly. Linear regressions were fit to data captured over 3 minute measurements using previous

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approaches41 and data were accepted only when the coefficient of fit (R2) was ≥ 0.95 with a p-

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value < 0.05. Using this approach, 95% of the measurements were acceptable. Cumulative

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emissions were calculated using linear interpolation to estimate flux between sampling points,

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and fluxes were converted to CO2-equivalence units using the 100-year GWP.42

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Plant Harvest and Analysis At grain maturity, plant organs were collected, dry weighed, and prepared for elemental

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analyses using previous methods.10 Root systems were treated per previous work to

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simultaneously quantify elements and characterize minerals in Fe-plaque.9 Briefly, roots were

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divided longitudinally to create two mirror images: one half underwent a dithionite-citrate-

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bicarbonate (DCB) extraction43 to remove Fe-plaque and quantify plaque-associated elements,

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and the other half was utilized for Fe mineral characterization (described next). Grain, husk,

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straw, and plaque-free roots were finely ground and microwave digested for elemental analysis

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using previous methods.10, 19 Undigested Si gels that formed after husk, straw, and root

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digestions were dissolved in 2M NaOH and quantified for Si using a colorimetric method.10, 44, 45

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Grain As species were determined with HPLC-ICP-MS after microwave-assisted extraction in

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2% TMG HNO3 at 100° C for 10 min40.

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Sample sets were accompanied by a National Institute of Standards and Technology

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standard reference material (SRM) rice flour (1568a), and recoveries were within 10% of their

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certified values (Table S4). While certified values for As species in SRM 1568a are not

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provided, the values we obtained for Asi, DMA, and MMA were 0.116, 0.195, and 0.005 mg/kg,

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respectively, and were within range of reported values40, 46-50 (Table S5). Species sums of grain

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samples from our study were within ±10% of total As values. 8 ACS Paragon Plus Environment

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S-XRD and Fe EXAFS and As XANES Analyses Root Fe-plaque minerals were concentrated onto nitrocellusoic membranes9 and

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characterized using synchrotron X-ray diffraction (S-XRD) and Fe extended X-ray absorption

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fine structure (EXAFS) spectroscopy at the Stanford Synchrotron Radiation Lightsource (SSRL).

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For XRD at beamline 11-3, an incident energy of 12700 eV (λ = 0.976 Å) and a MAR 345 Image

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Plate Detector were utilized. Resulting diffractograms were integrated in Q space between 0 and

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6 Å with wxdiff software and Match! software was used to find peak correlations. Several Fe

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mineral phases were thus identified to constrain fitting parameters for XAS analysis. Fe EXAFS was conducted on 11-2 and 4-3 at SSRL. The monochromator was calibrated

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with an Fe foil and the first inflection point was assigned to 7112.0 eV. Iron K-edge spectra

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were obtained with a Lytle detector from 200 eV below the absorption edge to k values of 15 Å-

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subtracted, normalized, and transformed to chi space (k-weight = 3) using Sixpack software.51

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Three replicates were analyzed per group. Linear combination fitting of k3 weighted EXAFS

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spectra in the range of 2-12 Å-1 was conducted utilizing results from XRD analysis including

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ferrihydrite, which is poorly resolved via XRD analysis due to its nanocrystalline structure.52

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Information regarding standard preparation can be found in Hansel et al.53

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. Three spectra were collected and averaged per sample. Averaged spectra were background-

Arsenic speciation in plaque samples was determined using X-ray absorption near edge

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structure (XANES) on beamline 11-2. An ACS certified sodium arsenate standard diluted in

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boron nitride was used to calibrate the incident energy of the beam and was assigned its first

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inflection point at 11874.0 eV. XANES spectra were obtained from 200 eV below the

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absorption edge to 350 eV above the edge in 8 minute scans and fluorescence signal intensity

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was monitored with a 100-element Germanium detector. Three repeated scans were averaged,

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background-subtracted, normalized and analyzed by linear combination fitting in the edge region 9 ACS Paragon Plus Environment


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11800-12000 eV using Sixpack.51 Standards used for linear combination fitting included Asi(III)

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as NaAsO2 and Asi(V) as Na3AsO4.

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Statistical Analyses SAS 9.4 was used to test for significant differences between groups using Proc GLM. If

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significant differences (P

How Rice (Oryza sativa L.) Responds to Elevated As under Different

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