The chronic exposure to arsenic (As) is associated with an increased risk of carcinoma in humans. Increases in the As concentration in paddy rice are of great concern, especially in Asian countries where rice is consumed as a staple food [1
]. Paddy rice is flooded during growing, which leads to the development of anaerobic conditions, leading to microbial reduction of arsenate (As-5) to arsenite (As-3). Since the adsorption affinity of As-3 to soil is weaker than that of As-5 [2
], the As concentration in the soil solution is increased with the development of anaerobic conditions [3
]. In addition, iron (Fe) (hydr)oxide, which adsorbs As, undergoes reductive dissolution, and then the As-3 associated with Fe (hydr)oxides is released into the solution as the adsorption phase diminishes [6
]. In contrast, when rice is grown as an upland crop, soil conditions remain aerobic and the As concentration is significantly lower than in paddy rice [5
] because the dissolution of As in the soil solution is decreased. Therefore, growing rice without flooding is one of the strategies to reduce As concentrations in rice grains; however, soil flooding has many benefits for rice production, such as the control of weeds, the prevention of damage from continuous cultivation and cool weather [9
]. It may also not be feasible to abandon the flooding practice for practical or economic reasons in areas that experience annual monsoon flooding. In addition, aerobic conditions cause an increase in the Cd concentration in rice grains [5
]. To decrease the As uptake by rice while retaining the benefits of soil flooding, the As concentration in the soil solution must be reduced under anaerobic conditions. Several Fe-bearing materials have been shown to be effective in reducing the As concentration in soil solutions under anaerobic conditions and thereby reduce the As concentration in rice grains [10
As well as Fe-bearing materials applied to soil, Fe plaque is known to influence As dissolution and uptake by rice [16
] The Fe plaque is deposited around the roots when Fe2+
is supplied from the soil solution due to the reductive dissolution of Fe minerals in soil under anaerobic conditions. Even when the soil matrix is predominantly under anaerobic conditions, the rice rhizosphere is partially aerobic due to the radial oxygen loss (ROL) through the root aerenchyma; thus, Fe2+
is oxidized and precipitated as ferric (hydr)oxide [20
]. Fe plaque is dominantly composed of ferrihydrite, although crystalline minerals, such as goethite and lepidocrocite, are also present [20
]. Since these Fe minerals strongly adsorb As, Fe plaque sequesters As around the roots [20
]. Both As-3 and As-5 are present in Fe plaque [22
]. Liu et al. [21
] showed that As-5 is the dominant As species associated with the Fe plaque of roots in mature rice plants, whereas Yamaguchi et al. [23
] showed that As-3 is the dominant species when the soil matrix is under anaerobic conditions and the proportion of As-5 increases at 1 month after harvest. The source of As sequestered in Fe plaque is the As-3 dissolved in the soil solution [4
]. Therefore, the results of Yamaguchi et al. [23
] implied that the As-3 associated with Fe plaque is not immediately oxidized to As-5. After the flooded water is drained, the oxidation of As-3 in the Fe plaque is faster than that in the bulk soil [23
]. However, oxidation rate of As-3 associated with Fe plaque is not well understood so far.
The formation and distribution of Fe plaque is heterogeneous and influenced not only by the degree of ROL, which is associated with the growth stage [24
] and genotype [25
] of rice, but also by various soil-derived factors [23
]. When larger amounts of Fe2+
are supplied from the soil by reductive dissolution, larger amounts of Fe plaque are formed around roots. Ultra et al. [28
] showed that the addition of 0.1 wt % amorphous Fe (hydr)oxide results in an increase in Fe plaque formation and thereby a reduction in As uptake by rice. The addition of Fe-bearing materials to soil increases the source of Fe2+
dissolved in the soil solution, which possibly influences Fe plaque formation. Various types of Fe-bearing materials have been investigated to identify promising soil amendments for decreasing As uptake by rice [10
]. However, the influence of these materials on Fe plaque formation and As sequestration in Fe plaque is not well understood.
We hypothesized that the application of Fe-bearing materials to paddy soil enhance the deposition of Fe plaque around rice roots, and thereby influences As speciation and its dissolution to soil solution. Whether the Fe plaque acts as a sink or as a source of As for rice roots is still controversial. When As is associated with Fe plaque in the rhizosphere as As-5, it is barely dissolved in the soil solution. However, if As-3 is a major species associated with Fe plaque, it can be dissociated from Fe plaque and become a source of As for root uptake by rice or any crop that is subsequently cultivated after the harvest of rice. Since As uptake by rice is differs according to growth stage, the speciation of arsenic in the rhizosphere at different cultivation stages of rice must be clarified to understand the role of Fe plaque in the uptake of As from roots. The purpose of this study was to investigate the effects of Fe amendments on the speciation of As in the rice rhizosphere, including Fe plaque, when the soil shifts from anaerobic to aerobic conditions after drainage at different growth stages of rice. The deposition of Fe (hydr)oxide around roots and soil particles was observed by preparing resin-embedded thin-sections of soil. The speciation of As in the rhizosphere and bulk soil was determined by X-ray absorption near edge structure (XANES) spectroscopy. These results contribute to the understanding of how drainage and aeration associated with rice root aerenchyma influence the oxidation of As-3 in the presence of Fe amendments.
2. Materials and Methods
2.1. Pot Experiment
The soil for the pot experiments was collected from a plowed layer of paddy field and passed through an 8-mm mesh sieve. The soil was classified as Aeric Epiaquepts
according to US taxonomy, and the relevant soil properties, including the As concentration, are shown in Table S1
. Two types of commercially available Fe-bearing materials, ferrihydrite-base material (FB, Ishihara Sangyo Kaisha, Ltd., Osaka, Japan) and zero-valent iron-based material (ZVI, Kobe Steel, Ltd., Tokyo, Japan), were used. Chemical fertilizer (0.2 g of nitrogen, 0.087 g of phosphorus and 0.17 g of potassium) and Fe-bearing material (17.9 g of FB or 10.1 g of ZVI; corresponding to 500 kg Fe ha−1
) were mixed with 2.5 kg of oven-dried soil. Five sets of pots were prepared for each group: control soil (without Fe-bearing material), soil amended with FB and soil amended with ZVI, (15 pots in total).
A cylinder (80 mm in diameter and 200 mm in height) made of nylon mesh screen (NYTAL PA-25-63, Semitec, Osaka, Japan; mesh size: 63 µm) was filled with 0.6 kg of the soil and placed in the center of a pot (1/5000 a Wagner pot, 4000 cm3). The remaining soil was filled around the soil cylinder. The soil in each pot was mixed well with water to mimic the puddling of a paddy field before transplanting the rice seedlings. Three seedlings of Oryza sativa cv. Koshihikari were transplanted into the center of the soil cylinder to regulate the position of the rice root zone inside the compartment surrounded by the nylon mesh screen.
The soil surface was flooded continuously by tap water during rice cultivation. Two sets of pots for each of the three treatments (control, FB and ZVI) were used as samples of intermittent drainage. Fifty days after transplanting the rice seedlings, the flooding water was drained from the hole at the bottom of the pot, which mimics the intermittent drainage that occurs in paddy fields. The soil and root samples were collected 1 h and 7 days after the drainage as described in the following section. The remaining three sets of pots for each treatment were used as samples of drainage after harvest. Rice was grown until the grain maturing stage without intermittent drainage, and the flooding water was drained just before harvest (113 days after transplanting).
2.2. Eh, pH and Soil Solution Analyses
A platinum electrode (PRN-41, Fujiwara, Tokyo, Japan) for measuring the redox potential and a soil solution sampler were both buried at a depth of 5 cm from the soil surface inside the nylon mesh cylinder. The soil solution sampler was composed of a 10-cm porous part (OD 2.5 mm) and a connected polyethylene/polyvinyl chloride tube (5 Rhizon MOM 5 cm; Rhizosphere Research Products, Wageningen, The Netherlands); a polyvinyl chloride extension tube (PF; TOP Co., Ltd., Tokyo, Japan); and an evacuated plastic tube (Venoject II, TERUMO Co., Tokyo, Japan). The soil solution was collected six days before the intermittent drainage (44 days after transplanting) from five sets of pots for three treatments (control, FB and ZVI); 41 and 35 days before the drainage at harvest (9 and 15 days after heading; 72 and 78 days after transplanting, respectively) from three sets of pots for three treatments. The timing of soil solution sampling was within three weeks after heading when As uptake by rice is known to be high [5
]. The soil solution was immediately acidified by HNO3
to prevent precipitation of Fe (hydr)oxide. The soil redox potential and pH was recorded when the soil solution was collected. The As and Fe concentrations in the soil solution were determined with an inductively coupled plasma mass spectrometer (ICP-MS, Elan, PerkinElmer, Waltham, MA, USA, limit of quantification for As: 0.17 µg/L) and an inductively coupled plasma optical emission spectrometer (ICP-OES, VistaPro, Varian, Palo Alto, CA, USA, limit of quantification for Fe: 1 µg/L), respectively. Certified reference materials used were ERM-CA615 (Trace elements in groundwater: European Commission, JRC, IRMM, North-East Limburg, Geel, Belgium).
2.3. Soil and Root Sampling from Rice-Cultivation Pots
The nylon mesh limited root distribution and, hence, soil that was not directly influenced by the rice root was sampled from the compartment outside of the nylon mesh cylinder (MO) and soil that interacted with the rice roots (MI) and roots with root-attached materials including Fe plaque (RZ) was sampled from the area inside the nylon mesh cylinder. The soil and root samples were collected 1 h and 7 days after intermittent drainage and 1 h, 7 days and 27 days after drainage at harvest, as described in Section 2.2
. Each sample was collected from one pot per treatment. In this study, replicated measurements of bulk soil and root samples were sacrificed to allow for time-resolved sampling points. However, the mixing of samples from depth-intervals within the separated zones (MI, MO, and RZ), serves as an averaging of information and provides confidence that the measurements are representative.
Wet soil samples (MO and MI) were collected from a depth of 3–6 cm from the surface from inside and outside of the nylon mesh cylinder, respectively. The well-mixed portion of wet soil paste was packed in a plastic bag (c.a. 15 mm × 15 mm × 1 mm), sealed, and immediately frozen in liquid N2. The samples were stored at −30 °C until XANES analysis was carried out.
The soil and roots inside the nylon mesh cylinder at depths of 0–3 and 6–9 cm were frozen in liquid N2 and then freeze dried. Subsequently, the roots stained with the dark red color of Fe hydroxide (Fe plaque) were pulled out from the soil using tweezers. Visible soil particles attached to the roots were removed by hand or tweezers. The roots from depths of 0–3 cm and 6–9 cm obtained 1 h after intermittent drainage were mixed for the analyses because the quantity of roots obtained for the analyses was insufficient. The soil collected from outside of the cylinder was also freeze dried and then well mixed. These freeze-dried bulk soils (MO and MI) and RZ were used to determine the ammonium-oxalate extractable As and Fe concentrations (Aso and Feo) which is associated with poorly crystalline Fe hydroxide as described in the supporting information. The portions of freeze-dried roots were pulverized, and the powdered samples were pressed into pellets for the As speciation analysis by XANES spectroscopy.
2.4. Speciation of As by X-ray Absorption Near Edge Structure Spectroscopy
The arsenic K-edge XANES spectra were acquired using beamline BL01b1 at SPring-8 and beamline BL5S1 at the Aichi Synchrotron Radiation Center. A double-crystal Si(111) monochromator and two Rh-coated mirrors at a grazing incidence angle of 3.7 and 3.14 mrad were used at BL01b1 and BL5S1, respectively. The incoming beam was measured with an Ar 15% N2
85%-filled ion chamber. The XANES spectra of the soil paste (MO and MI) samples and RZ pellets were collected in the fluorescence detection mode using a 19-element Ge semiconductor detector, whereas those of the reference materials were collected in the transmission mode with an Ar 100%-filled ion chamber at ambient temperature. The frozen soil paste samples were thawed at an ambient temperature just before the analyses. During XANES analyses, the soil paste was kept sealed in a polyethylene bag because X-ray absorption by the polyethylene bag is negligible at the energy for As analyses. Our previous study showed that X-ray irradiation did not cause changes in As speciation for anaerobic soil paste during XANES analyses [4
]. The composition of As species was evaluated by a linear combination fitting (LCF) of the XANES spectra with reference compounds. The reference compounds included Na2
(rsenite, As-3 Wako pure chemical, Osaka, Japan), NaHAsO4
(arsenate, As-5 Wako pure chemical, Osaka, Japan), dimethylarsinic acid (DMA, Hayashi pure chemical, Osaka, Japan), orpiment (As2
Wako pure chemical, Osaka, Japan), and arsenopyrite (FeAsS, Francisco I. Madero Mine, Mexico, N’s Mineral, Niigata, Japan). The fitting range was ±10 eV from E0. The Athena in Demeter 0.9.25 program package was used to subtract the pre-edge background and normalize the spectra and LCFs. In the LCFs, the fraction of each reference compound was set as an adjustable parameter, and optimization was achieved by minimizing the residual of the fit, which was defined as the normalized root-square difference between the data and the fit (R-factor).
2.5. Analyses of Rice Grain
Rice grains were finely ground using a shaking mill (Shake Master; Bio Medical Science Inc., Tokyo, Japan) and 0.2 g of the powdered grain was digested at 105 °C using a hot block acid digestion system (DigiPREP MS; SCP Sciences, Inc., Quebec, QC, Canada). After cooling, 1 ml of 30% hydrogen peroxide solution was added, and the mixture was heated again for 1 h at 105 °C. The concentrations of As in the acid-digested solution were determined by ICPMS. Certified reference material used was NMIJ CRM7502-a (White rice flour: National Metrology Institute, AIST, Tsukuba, Japan).
2.6. Preparation of Soil Thin-Sections
The soil cylinder covered by the nylon mesh was pulled from the pot, and then the nylon mesh was removed. The soil cylinder was cut to a height of 3 cm. The soil inside the nylon mesh cylinder at a depth of 3–6 cm from the surface was immediately frozen using liquid nitrogen and then freeze dried. The freeze-dried soil block was embedded in unsaturated polyester resin (Sundhoma, DIC material, Tokyo, Japan) under vacuum. The resin-embedded soil block was kept in a draft chamber at ambient temperature for two weeks until the resin solidified. The solidified resin-embedded soil block was cut by bandsaw (Andosaw, TA300L, Aichi, Japan) and then polished using a grinder (PSG-52, Okamoto, Gunma, Japan) to a thickness of 30–50 µm. The method of soil thin-section preparation is described by Yamaguchi et al. [23
]. The thin-section image was obtained using an image scanner under a white color background (CanonScan LiDE 700F, Canon, Tokyo, Japan) and observed under a digital microscope (Keyence VHX-100, Osaka, Japan).
2.7. Statistic Analysis
Statistical analysis was conducted with an add-in program for MS Excel (BellCurve for Excel; Social Survey Research Information Co., Ltd., Tokyo, Japan). Before analysis of variance, normality of sample variances was assessed by Levene’s test for pH, As and Fe concentration of soil solutions, and Eh of soils during rice-pot cultivation. Two-way analysis of variance (two-way ANOVA) with sampling time and treatments as factors was used to analyze pH and Eh followed by the Tukey–Kramer multiple comparison test, whereas the Kruskal–Wallis test (nonparametric test) was applied for As and Fe followed by Steel–Dwass multiple comparison test.
The application of FB and ZVI to paddy soil resulted in a decrease in the dissolution of As into the soil solution under anaerobic conditions. At the time of intermittent drainage, formation of As sulfide was an important mechanism to restrict As dissolution in soil solution when soil was amended with ZVI. However, the fraction of As-S decreased in ZVI-amended soil after prolonged anaerobic conditions, possibly because sulfides were scavenged by increased Fe2+ dissolution. Despite the lower fraction of As-S than control, As concentration in soil solution of FB and ZVI amended soil was lower than the control. In addition to the sorption ability of As when FB and ZVI were applied, increased precipitation of Fe (hydr)oxide from dissolved Fe2+ provided sorption sites with As under anaerobic conditions. The decreased concentration of As in the soil solution limited supply of As to the rhizosphere, and therefore the sequestration of As with Fe (hydr)oxide in the rice rhizosphere was decreased by the application of FB and ZVI. Due to the ROL from the roots, the rice rhizosphere is known to be more oxic than bulk soil. Therefore, Fe2+ dissolved in the soil solution is deposited around roots as Fe plaque. The application of FB and ZVI caused increased deposition of Fe plaque as well as Fe (hydr)oxide deposition around soil particles while their appearances were heterogeneous. Despite the oxidation of Fe2+ followed by the deposition of Fe (hydr)oxide, our results showed that oxidation of As-3 was not sufficiently pronounced to generate higher proportions of As-5 in the rhizosphere than in the bulk soil, regardless of the application of Fe-bearing materials. It is likely that the partial aeration caused by ROL from roots was not enough to accelerate oxidation of As-3 to As-5. Therefore, intermittent drainage had little influence on oxidation of As-3 to As-5 both in rhizosphere and bulk soil. The fraction of DMA was higher in the rhizosphere than in bulk soil due to the possible influence from root-associated micro-organisms. After drainage, the oxidation process followed by Fe (hydr)oxide deposition occurred heterogeneously around soil particles and rice roots. Although heterogeneous, the application of FB and ZVI resulted in greater Fe (hydr)oxide deposition around the roots and soil particles. The deposited Fe (hydr)oxide-sorbed As-3 therefore contributed to the decreased concentration of As in the soil solution and rice grain. However, the influence of Fe amendments on the oxidation of As after drainage was not as pronounced as the heterogeneous oxidation process in the rice cultivated soil and rhizosphere with Fe plaque.