Variations in Cadmium Concentrations in Rice And

ANALYTICAL SCIENCES MAY 2020, VOL. 36 531 2020 © The Japan Society for Analytical Chemistry

Original Papers Variations in Cadmium Concentrations in Rice and Oxidation- Reduction Potential at the Soil Surface with Supplementation of Fermented Botanical Waste-based Amendment in Large-scale Farmland

Nobuhiko WADA,*1,*2 Gao DI,*1 Hideyuki ITABASHI,*1 Masanobu MORI,*3† Yusuo LIN,*4 Shaopo DENG,*4 Weiwei XU,*5 Weiwei GUO,*6 Yuanheng LUO,*6 and Dianyu ZHU*7

*1 Graduate School of Science and Technology, Gunma University, 1-5-1, Tenjin-cho, Kiryu 376–8515, Japan *2 Shanghai Shenglong Environment Remediation Materials, Co. Ltd, Building 6, No. 936, Guoshun Road, Nanqiao Town, Fengxian District, Shanghai 201400, China *3 Faculty of Science and Technology, Kochi University, 2-5-1 Akebono-cho, Kochi 780–8520, Japan *4 Nanjing Institute of Environmental Science, MEP, P. R. China, Jiangwangmiao Road 8, Xuanwu District, Nanjing, Jiangsu Province 210042, China *5 Geological Survey of Jiangsu Province, 700 Zhujiang Road, Xuanwu District, Nanjing, Jiangsu Province 210018, China *6 Shanghai Shenglong Environment Remediation Technologies, Co. Ltd, Floor 13, Building No. 2, 100 Qinzhou Road, Xuhui District, Shanghai 200235, China *7 School of the Environment, Nanjing University, 163 Xianlin Road, Qixia District, Nanjing, Jiangsu Province 210023, China

We monitored the relationship between the cadmium (Cd) concentration uptake of rice and the oxidation-reduction potential (ORP) at the soil surface with the supplementation of fermented botanical waste-based amendment (FBWA), an organic fertilizer prepared from woody and food wastes. This study was carried out for 3 years in the western part of Jiangsu Province, China. It was found that the Cd concentration taken up by rice was correlated to a decreased the ORP of the cultivated soil. The yield of rice was ∼1.20 times higher than that of the control plot. The effects of reducing the Cd content in rice and increasing the rice yield remained for 2 years after FBWA application. Finally, Cd was immobilized in the soil with adsorption to FBWA or the decomposed products. The ORP measurement during rice cultivation might be a key index to predict the suppression effect of Cd uptake into the rice or limitation of the sustainable effect by the FBWA.

Keywords Cadmium, rice, reduction, farmland, amendment, soil analysis

(Received October 5, 2019; Accepted March 3, 2020; Advance Publication Released Online by J-STAGE March 13, 2020)

Land and Resources10 conducted the Geological Survey of Introduction China and measured pollutant levels in farmlands, forestlands, and unused land. Overall, 16.1% of the sampled locations had The cadmium (Cd) uptake by rice grown in Cd-contaminated contaminant levels exceeding thresholds established by the soils is a serious problem worldwide. In Thailand,1 Vietnam,2 Environmental Quality Standard for Soils (GB 15618-1995). Bangladesh,3 Pakistan,4 and China,5 researchers have investigated Therefore, cultivation methods for reducing the Cd contamination the potential health risks of ingesting rice harvested from in rice have mainly been developed in Japan and China. These Cd-contaminated soils. All studies emphasized the need for include autoclaved lightweight-concrete (ALC), which contains measures to mitigate Cd contamination. Excessive Cd causes calcium silicate;11 inorganic adsorbent added to farmland soil genetic instability in living organisms and inhibits plant growth.6 during irrigation;12 biochar soil amendment (BSA) composed of In agroecosystems, genotoxic compounds significantly decrease hay, wood chips, sawdust, or cereal straw waste;13–29 or a mixture the crop yield and quality.4,7,8 In Japan in 1955, illness was of cedar bark, rice bran, and soybean waste.30 reported in persons who consumed rice cultivated on soil The immobilization of Cd in soil might effectively suppress irrigated with Cd-contaminated river water.9 crop Cd uptake. However, spraying industrial or food wastes China is the largest producer and consumer of rice in the onto soils often decreases the crop yield because it causes world. From April 2005 to December 2013, the Ministry of irrigation water eutrophication, which inhibits plant growth. Therefore, a practical cultivation method is needed that both † To whom correspondence should be addressed. reduces rice Cd uptake and maintains high yields in Cd- E-mail: [email protected] contaminated farmlands. 532 ANALYTICAL SCIENCES MAY 2020, VOL. 36

In the present study, we ameliorated Cd-contaminated soil by The pH of soil suspensions and water samples were determined applying fermented botanical waste-based amendment (FBWA) using a pH meter (pH scan20, Bante Instruments, Shanghai, consisting of locally sourced decomposing bark sawdust, China; PB-21, Sartorius, Göttingen, Germany; and D-72, hardwood stems, tofu waste sludge, and rice bran. These Horiba, Kyoto, Japan). The ORP of these samples was obtained materials were broken down with white rot fungus. Lignin, using an ORP tester (ORPscan20, Bante Instruments, Shanghai, which constitutes 20 – 35% of the cell walls of coniferous China; and D-72, Horiba, Kyoto, Japan). woody bark and sawdust (the main raw materials of FBWA), is The cation-exchange capacity (CEC) in the FBWA was known to adsorb heavy metals, such as Cd.31–34 However, even measured by using the barium acetate method (Measurement if applying woody bark alone to the soil, the decomposition is Act #51 1992). The phosphorus (P) and potassium (K) were slow and takes a long time to assimilate into the soil. Therefore, analyzed in the oxidant by using energy dispersion X-ray we mixed rice bran (an agricultural residue) and okara (the fluorescence (ED-XRF) at JBCA (Japan Bark Compost strained lees of tofu) with bark at equal weight. They play the Association). The pH and the ORP of the paddy soil and water role as a forcing bed for white-rot fungi added to decompose the were measured both before and after spraying with FBWA. bark. The fungi actively decomposes bark by consuming any Aqueous soil suspensions were collected in 180-mL plastic cups waste products. and left to stand 1 min before any pH and ORP determinations. Several studies have reported the suppression of Cd The pH meter was calibrated at pH 4, 7, and 9. The ORP meter uptake by rice using amendments derived from industrial was calibrated using a standard solution manufactured by wastes.14,16,20,23,27,29,30 The applications of such amendments are Horiba (Kyoto, Japan). mostly conducted in Japan and China because rice is a staple food there. Table S1 in Supporting Information (SI) lists earlier Preparation of FBWA studies on the amendments used to reduce Cd in rice cultivated FBWA consisting of shredded bark sawdust and hardwood in Japan and China. However, most of the experiments listed stems, were obtained from moldy wood. Bean curd was are “pot studies” or small field-scale experiments. obtained from tofu manufacturing. Rice bran was obtained The present study was conducted concerning large-scale crop from rice polishing. All raw materials were purchased from cultivation on a 9636 m2 farmland continuously for 3 years. In industrial production facilities in China (undisclosed). Equal particular, we focused on whether the variation of the oxidation- quantities of bark, bean curd, and rice bran were mixed with reduction potential (ORP) related to that of the Cd concentration white rot fungus (consisting mainly of Phanerochaete in harvested brown rice, and clarify whether there was a positive chrysosporium) cultivated at Sasutera Co. Ltd. (Niigata, Japan). effect of FBWA addition. In our previous study, the Cd The mixture was fermented at 60 – 80°C for 7 d using only concertation in brown rice harvested in an organic amendment- white rot fungus. The final moisture content was 10%. supplied soil that provided a decrease of ORP in surface soil during rice cultivation was largely reduced compared with that Location of farmlands in soil without the amendment.30 Moreover, the rice yield in soil A schematic representation of the farmland in the present with FBWA supplementation was also observed in the current study is shown in Fig. 1. A field trial was conducted in the study because it is of practical importance to consider the effect western part of Jiangsu Province, China at ∼5 m altitude. Both of supplementation on plant growth. Furthermore, we rice and wheat were cultivated there annually. The area of the investigated the mass transfer of Cd from soil to brown rice experimental farmland was 9635.4 m2 (90.9 m E–W and using the concentration factor and the Cd uptake rate. 106.0 m N–S). The farmland was situated both east and west of a 20-m-wide canal. Several pottery and chemical factories are located to the south of the farmland (Fig. 1). Experimental The experimental farmland consisted of six plots (A–F) and an embankment passage 30–40 cm wide. For each plot, the soil Reagents, materials, and instruments and the rice plant samples were collected within the 1 × 1 m of All reagents were obtained from Wako Pure Chemical area of three equal parts (1 – 3) per each plot (A – F) as shown Industries, Ltd. (Osaka, Japan) or from Suzhou Crystal Chemical in Fig. 1. In detail, the rice plants were collected from the Co. Ltd. (Suzhou, China). Purified water was obtained from center and four corners in the area, and the soil samples were Merck Serono S.A.S. (Geneva, Switzerland) and from EUG- collected using a cylindrical stainless-steel soil sampler (35 mm CY2-20 (Nanjing YouGen Environmental Protection Technology diameter) at 15 cm depth from the center. Co. Ltd., Nanjing, China). Before evaluating the effect of FBWA spraying on the Cd Soil, water, and rice samples were analyzed at the Geological uptake in rice, the heavy metal concentration was measured in Survey of Jiangsu Province and at the School of the Environment, irrigation water pumped from canals adjacent to the farmland. Nanjing University, China. Prior to the experiment, the The average Cd concentration, determined by ICP-MS, was elemental compositions of soil samples were determined by <0.20 μg L–1 (Table S3 in SI), which was well below the energy dispersive X-ray fluorescence (ED-XRF; Mesa-50, environmental quality threshold for farmland used for edible Horiba, Kyoto, Japan). agricultural product cultivation in China (5.0 μg L–1; HJ/T332- Heavy metals were evaluated by inductively coupled plasma 2006). The concentrations of other heavy metals were lower mass spectrometry (ICP-MS; Thermo Elemental X7 Quadrupole than the basic and selection control values established for ICP-MS, Thermo Fisher Scientific, Waltham, MA, USA). The irrigation water quality (Table S3 in SI). However, the Cd experimental conditions of ICP-MS are summarized in Table S2 concentrations in the farmland soil were 7 – 10 times higher in SI. In this approach, a mixture of germanium and indium than the environmental quality standard (0.30 mg kg–1; GB (0.5 mg L–1 each) was used as an internal standard to determine 15618 – 1995; Table S4 in SI) and also exceeded the averages the 52Cr, 58Ni, 63Cu, 66Zn, 77Se, 105Pd, and 111Cd concentrations. for farmlands in Hunan Province (0.73 mg kg–1), Guangxi The quantification limits of targeted elements estimated from Province (0.70 mg kg–1), and Sichuan Province (0.46 mg kg–1), the absolute calibration curves (1.0 – 5.0 μg L–1) were 0.02 – as reported by Liu et al.35 0.10 μg L–1. ANALYTICAL SCIENCES MAY 2020, VOL. 36 533

Fig. 1 Location of the farmland in Jiangsu Province, China. P: Pumping facilities. WI-1 and WI-2: water intake positions. The numbers in the experimental farmland represent the sampling points divided in three equal parts per plot.

Soil sample analysis Table 1 Area, soil quantity, and percent of fermented botanical The pH and ORP were measured after shaking 10 g of a soil waste-based amendment (FBWA) in each farmland plot sample in 25 mL of pure water (liquid-to-solid ratio 2.5) for Plot A B C D E F 10 min. The soil surface water level and temperature during rice cultivation were regularly measured using a ruler and a Area/m2 1823 1728 1823 1071 1961 1230 thermometer, respectively. To determine the Cd concentrations, Soil quantity/ton 273 259 273 161 294 184 the collected soil samples were dried, and pulverized in ceramic FBWA, % — 0.1 0.5 1.0 0.5 0.1 and agate mortars to particle sizes of <0.01 mm, and then analyzed in a portable ED-XRF (Mesa-50, Horiba, Japan) according to a method of Imanishi et al.36 Soil samples taken from the soil surrounding the rice plants were collected using a used a different variety, used in 2016 and 2018, because we cylindrical stainless-steel soil sampler (35 mm diameter) at could not secure sufficient quantities of “#2845” to spray on 15 cm depth within the centers (1 m diameter) of three equal large farms. parts per plot. The samples were air-dried at room temperature. In 2017 and 2018, rice was planted again following the wheat Each 0.05-g soil sample was placed in a 50-mL polytetra- harvest. To determine the persistence of the effects of FBWA fluoroethylene (PTFE; Teflon) crucible and dissolved in a from 2016, no FBWA was added in 2017 and 2018. The soil –1 –1 mixture of 3 mL 13 mol L HNO3, 7 mL 27 mol L HF, and conditioning and rice sampling area were unified from 2016 to –1 0.25 mL 18 mol L H2SO4 according to standard methods used 2018. The cultivation schedules are presented in Table S5 in SI. in China (NJTC/DM07-D28). The crucible was heated on a hotplate at 220°C for 3 h and 2 – 3 drops of H2O2 were added to Pretreatment of harvested rice remove organic matter. The inner wall of the crucible was Rice sample pretreatments, Cd determinations, and yield –1 rinsed with pure water, and then 10 mL of 3.5 mol L HNO3 estimates are summarized in the flowchart of Fig. S1 in SI. The was added. The sample solution was brought to a final volume Cd concentrations in the harvested rice samples were determined of 25 mL after cooling, and was filtered using a syringe filter by extraction, as described in the “Identification standards for (0.45-μm membrane). The Cd concentrations were then hazardous wastes-identification for extraction toxicity” (GB determined by ICP-MS. 5085.3-2007, HJ/T 299-2007, and GB/T 15441-1995). The Cd concentrations in the extracts were determined by ICP-MS. Cultivation conditions in the farmland The effects of the FBWA application rate on soil were assessed Theoretical yields, concentration factor, and statistical analysis in terms of the rice Cd uptake and yield. On August 11, 2016, Rice stocks were sown at rates of 25 – 30 m–2. The rice yield FBWA was sprayed about two weeks before the rice was per unit area differed among plots. In addition, the rice yields headed. The quantities of FBWA sprayed were determined in conditioned fields were compared using theoretical yields from the soil weight calculated using depth 15 cm of a rice root (Yt), which were estimated from Eq. (1), and were based on the and the area in each plot, based on the soil specific gravity number of panicles (Np) to unit of surface area, the number of –3 (1.2 g cm ). As summarized in Table 1, 0.1% FBWA was rice grains (Nr), and the 1000-grain weight of ripe rice (G1000) applied in plots B and F, 0.5% FBWA was applied in plots C (GB/T 5519-2008): and E, and 1.0% FBWA was applied in plot D. FBWA was not –2 6 sprayed onto plot A (control plot). The rice varieties sown were Yt(kg) = Np(m ) × Nr × G1000(g)/10 × 0.85, (1) “#2845” in 2016 and 2018 and “#5055” in 2017, which are commonly cultivated in the experimental area. In 2017, we where 0.85 is the weight loss ratio of the samples after drying. 534 ANALYTICAL SCIENCES MAY 2020, VOL. 36

Table 2 Characterization of the raw material mixtures before adding white rot fungus (Comparison of FBWA with bark compost obtained from JBCAa)

Mixture of raw Standard level of Parameter FBWA waste materials bark compost

pHb — 7.8 ± 0.0 5.5 – 8.0 Hydrogen 5.8% 5.6 ± 0.0% — Carbon (C) 42.3% 43.5 ± 3.0% — Nitrogen (N) 1.9% 2.5 ± 0.2% >1.2% Phosphorus (P)c — 0.8 ± 0.1% >0.5% Potassium (K)c — 1.3 ± 0.1% >0.3% C/N ratio 22.3 17.7 ± 0.3 <35 CEC cmol kg–1 d — 68.0 ± 6.2 >70 a. Samples were dried at 100°C before analysis. JBCA: Japan Bark Compost Association. b. pH was determined for suspensions made by adding 1 g sample to 10 mL pure water. c. P and K were measured in oxide form by ED-XRF. d. CEC: Cation-exchange capacity.

The concentration factor (CF) of Cd from soil to brown rice was estimated from Eq. (2).

CF = Crice/Csoil, (2) where Crice is the concentration of Cd in brown rice and Csoil is that in soil. Differences in the Cd concentration among rice samples harvested from soils with and without FBWA were evaluated using a Student’s t-test at a significance level of 0.05.37 Triplicate samples of each soil condition were used. Descriptive statistics were calculated in Microsoft Excel 2010.

Results and Discussion

Characterization of FBWA FBWA was analyzed in accordance with criteria stipulated by the Japan Bark Compost Association (JBCA). The standard levels of bark compost cited were those officially released by JBCA (http://www.bark-assoc.jp/products2.html). The pH of the FBWA suspension in pure water was 7.8. The standard pH range specified by JBCA was 5.5 – 8.0 (Table 2). The nitrogen Fig. 2 Variations in the oxidation–reduction potential (ORP) in each (N), P, and K contents and the CEC were also within the plot of the cultivated farmland as functions of the elapsed days after standard ranges established by JBCA. The C/N ratio of the spraying FBWA (August 11, 2016). Three replicates were analyzed FBWA was 17.7, whereas that for the unfermented raw materials per plot. The error bars represent standard deviation from the mean. was 22.3. The C/N for woody waste was 131 (C: 47.1%; N: 0.36%). The low C/N ratio of the FBWA was the result of the high N content in the bean curd and rice bran (C/N <5), but not the woody waste. Detailed C and N contents of the bean curd ORP in farmland soils sprayed with different concentrations of and rice bran were not furnished because the suppliers refused FBWA to disclose this information. The N, P, and K contents (NPK The reduction of ORPs in the farmland soils was evaluated by content) in the FBWA were 2.4, 0.8 and 1.4%, respectively. The varying the quantities of FBWA sprayed on the soil. The ORP total NPK content was <5.0%. FAO38 recommends a total NPK measurements were started 1 day before FBWA application content of ≥20% in the chemical fertilizers applied to rice and (August 10, 2016). Following the FBWA application (August wheat. Therefore, the FBWA used in the present study had 11, 2016), on August 12 – 16, August 29 (18 days), September lower NPK than rice fertilizer, but the same as that of organic 14 (34 days), and September 27 (47 days) in 2016, the ORP bark compost. The concentrations of chromium (CrVI), Cd, measurements were made in triplicate for each observation arsenic (As), mercury (Hg), and lead (Pb) in the FBWA were all point. On August 10 in 2016, one day before FBWA application, below the maximum threshold levels. These results indicate the ORP was highly positive (100 – 200 mV; Fig. 2). It had that FBWA is probably safe to use as a soil amendment, and been measured immediately after the semi-dry soil was irrigated fermenting raw waste improves its quality and safety as a until the water level reached the soil surface. The ORPs of plots fertilizer. B–F rapidly declined immediately after FBWA application. ANALYTICAL SCIENCES MAY 2020, VOL. 36 535

Fig. 3 Cadmium (Cd) content in rice harvested from the farmland. Farmland was divided into blocks according to the irrigation water inflow. Fermented botanical waste-based amendment (FBWA) was sprayed onto the paddy soil at the following rates: A, 0%; B and F, 0.1%; C and E, 0.5%; and D, 1.0%. The variety of rice seed sprayed on the experimental farmland in 2017 was different to those in 2016 and 2018. Asterisks indicate significant differences between the Cd levels in the rice harvested from amended soil and those in the rice harvested from unamended soil at P <0.05 (*). Triplicate samples were taken for each plot.

One week after spraying 0.1 – 0.5% FBWA, the ORPs in plots amended with 0.5 – 1% FBWA had 66 – 80% less Cd than that B–F ranged from –150 to –100 mV. In contrast, the ORP in derived from control plots, while rice harvested from plots B unamended plot A gradually decreased relative to those for the and F amended with 0.1% FBWA had 24 and 44% less Cd, plots sprayed with FBWA. The ORP in plot D (1% FBWA) respectively, than that derived from control plots. remained stable at –100 mV from August 29 (18 days) to In 2017, the rice crop was sown after the wheat harvest and September 14 (34 days) in 2016. While the rice heading was soil conditioning. FBWA was not added to the cultivation soil August 25 (14 days), the rapid increases in ORP between the to determine the longevity of its initial application. Because the end of August and September might be because the water level rice variety seeded in that year was different to that in 2016, the above the soil surface was lowered from 10 – 15 cm depth to Cd contents taken up by brown rice were lower than those in 4 – 8 cm as a result of drought. A rapid decrease in the ORP 2016 but the trends in the Cd contents among plots were similar. was noted when FBWA was sprayed onto irrigated soil. Because the Cd concentration in rice also decreased in plot A, The ORPs of the soils sprayed with 0.5% FBWA and 1% the differences between the FBWA-amended plots and the FBWA in 2016 decreased even after the plots were irrigated on control plot in 2017 were smaller than those in 2016. August 10, 2017 (364 days since the FBWA application), even In 2018, the suppressive effect of FBWA on the Cd uptake by though FBWA was not applied in 2017. The ORPs of the soil rice was almost eliminated, that is, there were no significant surfaces in plots D and E were –43.7 ± 15.8 and –32.4 ± 13.8 mV differences between the amended plots and the control plot A. on August 9, 2017 (363 days), respectively, relative to that in In addition, the Cd concentration in rice harvested from plot C plot A (125.8 ± 24.0 mV). Furthermore, the ORP values amended with 0.5% FBWA was higher than that in plot A. decreased over time with the rice cultivation after water As mentioned above, we concluded that the longevity of the irrigation. Therefore, FBWA continued to reduce ORP in the suppression of Cd uptake by rice was 2 years from the initial year after its initial application. Even in 2018, negative ORP application of FBWA. values were obtained regardless of the plots. Rice yield Variations in rice Cd content after spraying FBWA onto Applying the technique evaluated in the present study to real contaminated soil farmland restoration requires not only decreases of the Cd Next, the variations in the Cd contents in brown rice were content in rice, but also validation of the rice yield. The compared between plots amended with FBWA and the control variation in the theoretical rice yield estimated from Eq. (1) for plot over a period of 3 years. each year is plotted in Fig. 4. In 2016, the Cd concentration of the rice harvested from When the yield in control plot A, which was a normal chemical control plot A was 0.66 mg kg–1, and those from plots B and F fertilizer only, was compared with those for plots B–F after the (where 0.1% FBWA was sprayed) were 0.50 and 0.37 mg kg–1 FBWA treatment in 2016, plots C and E (0.5% FBWA) in 2016 of the concentrations measured in plot A, respectively (Fig. 3). had the maximum yields of 13% higher rice yields than all other Those in rice harvested from plots C and E (where 0.5% FBWA plots (P <0.05). Therefore, FBWA-amended soils promoted was sprayed) were 0.19 and 0.14 mg kg–1, respectively, lower higher yields than unamended soil. than that of the rice harvested from plot A. Furthermore, the Cd The rice yield trend obtained in 2016 did not change in 2017, concentration in rice harvested from plot D (0.13 mg kg–1), though the species of rice seed in 2017 was different to that in where 1% FBWA was sprayed, was reduced by 80% compared 2016 and 2018. Because the cultivation situation, such as crop with that in plot A. Accordingly, rice harvested from plots rotation, soil type, and water flow, except for rice species, 536 ANALYTICAL SCIENCES MAY 2020, VOL. 36

Fig. 4 Rice yield in each fermented botanical waste-based amendment (FBWA)-sprayed farmland plot relative to that for the unamended control (plot A) in 2016 – 2018. FBWA was sprayed onto the paddy soil at the following rates: A, 0%; B and F, 0.1%; C and E, 0.5%; and D, 1.0%. Asterisks indicate significant differences between rice yields in soils amended with FBWA (B – F) and the unamended (A) at P <0.05 (*). Statistical analyses were conducted using three replicates per plot. In 2016, the rice yield was calculated by collecting all the rice harvested in each plot

Table 3 Cadmium (Cd) uptake rate into brown rice harvested in 2016 Plots Item A B C D E F

(i) Cd content in soil/mg kg–1 2.20 ± 0.34a 2.15 ± 0.43 3.44 ± 0.48 2.14 ± 0.06 2.85 ± 0.44 3.01 ± 0.44 (ii) Cd content in brown rice/mg kg–1 0.66 ± 0.15 0.50 ± 0.04 0.19 ± 0.13 0.13 ± 0.07 0.14 ± 0.12 0.37 ± 0.11 Concentration factor (CF)b 0.300 0.233 0.055 0.061 0.049 0.123 (iii) Theoretical rice yield/kgc 0.878 0.947 0.989 0.958 0.960 0.924 (iv) Cd in total soil/mgd 396.0 387.0 619.2 385.2 513.0 541.8 Cd uptake rate into brown rice, %e 0.147 0.122 0.031 0.031 0.026 0.063

a. The mean value and standard deviation summarizes the Cd content in soil and brown rice of three sampling points for each plot. b. The concentration factor was calculated by Eq. (2) in Theoretical yields, concentration factor, and statistical analysis. c. The theoretical rice yield was calculated by Eq. (1) in Theoretical yields, concentration factor, and statistical analysis. d. The total soil was calculated using item (i) in the table, total soil weight (Wsoil) collected with rice plant and specific gravity (1.2) of the soil for each plot per surface area: [Cd in total soil] = (i) × Wsoil × 1.2. e. [Cd uptake rate] = (ii) × (iii)/(iv) × 100 (%).

remained, the soil remediation with spraying FBWA would lead amount of FBWA sprayed on the farmland, through the masses to the acceleration of plant growth. In 2017, the rice yields in of Cd in the total soil of plots C, E, and F were 1.3 – 1.7 times the amended plots were increased by 5 to 16% over that of the higher than that in plot A. control plot A. The rice harvested from plots C–F had Cd In the Cd adsorption test using the batch method, the maximum concentrations lower than the standard level (<0.2 mg kg–1) in adsorption amount of Cd to FBWA showed higher or comparable China. However, in the third year, no significant differences in levels than those of the other amendments obtained from organic yields were observed. wastes in recent reports (Table S6 in SI).30,39–45 In fact, while the differences of ORPs between the amended Balance of Cd from soil to brown rice plots and the control plot in 2017 were smaller than those in Finally, using the analytical results from 2016, the balances of 2016, the Cd contents in rice of the amended plots were lower Cd from soil to brown rice for each plot were obtained from the than those of the control plot. Accordingly, the Cd in soil might Cd uptake rates in brown rice, estimated using (i) the Cd content be immobilized by organic matter generated through in soil before rice cultivation, (ii) that in harvested rice, (iii) the decomposition of the applied FBWA. theoretical yield of rice, and (iv) the amount of Cd per weight of Furthermore, as summarized in Table 3, the Cd uptake rates soil (∼150 kg) taken together when collecting the rice plant into brown rice were below 0.1% for plots C–F, and much (15 cm-depth) for each plot. As summarized in Table 3, 0.5 and smaller when compared with the amount of Cd in soil. Because 1% FBWA supplementation was demonstrated to effectively the straw and roots are mixed into the soil after harvest, suppress the Cd uptake into brown rice. In addition, through the most of the Cd in the soil is consequently retained in the soil. CF estimated from (i) and (ii) using Eq. (2), the Cd uptake was Considering the above results, FBWA is a useful amendment to effectively reduced by FBWA supplementation. While the rice suppress the movement of Cd into the plant, but is not suitable cultivated in control plot A absorbed 30% of Cd in soil, those in for phytoremediation, where the intention is to increase the soil with FBWA (plots B–F) absorbed 4.9 – 23% of Cd in soil. uptake of heavy metals. The Cd uptake rates in brown rice declined depending on the Throughout this study, for rice crops, the Cd concentrations ANALYTICAL SCIENCES MAY 2020, VOL. 36 537

the suppression of Cd concentrations while increasing crop yields through applying an organic soil amendment (FBWA) to large-scale farmland. First, supplementation of the amendment with woody and food waste demonstrated suppression of Cd uptake by rice with a considerable reduction of ORP of the soil as the preliminary factor. In fact, the variations of the Cd concentrations in the rice were correlated to those of the ORP values. In particular, FBWA supplementation at 0.5% to Cd- contaminated soil provided suppression of the Cd uptake by rice with satisfactory rice yields without causing eutrophication of soil surface water for at least 2 years. Furthermore, the part of the fundamental data obtained in this study is adopted in the China Patent; CN107722987B, 2019-03-01.49 Accordingly, monitoring the ORP during rice cultivation would be a key index to previously estimate the suppression effect of Cd uptake into the rice by an organic amendment. In future studies, this rice cultivation method with supplementation Fig. 5 Relationship between the ORP of soil and Cd concentration of FBWA will be applied on rice farmlands outside of China in rice harvested from the tested farmland. The differences in the Cd and Japan. contents correlated with the average ORP (Eh) from Aug. 14 to Sep. 14. Tests were performed in triplicate samples per division. Sample number, 12; correlation coefficient (r), 0.694; and significant Acknowledgments difference, 0.002. This work was supported by a Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research Grant B were strongly correlated with the average ORP, depending on JP17H04482. We thank G. Chang (Shanghai Shenglong the quantity of FBWA applied (Figs. 2 and 3). In a previous Environment Remediation Materials, Co. Ltd.) for the study, when the depth of water above the soil surface was opportunity to examine the farmland and for coordinating the <1 cm, there was no suppression of Cd uptake by rice supplied collaborative investigation, T. Feng (Jiangsu Province Jingjiang with a fermented bark amendment.30 This means that the Cd City Agriculture Committee) for assisting with the measurement uptake suppression in rice is influenced by the bark amendment and evaluation of the harvested crops, Dr. Q. S. Luo (Yonker supplementation, but is also affected by the water conditions of Environmental Protection Co. Ltd.) for his valuable opinions the paddy soil. The positive correlations between the Cd content and discussions, K. Sagara and T. Akimoto (Gunma Univ.) for and ORP in 2016 and 2017 were provided across the whole their assistance in the measurements of the farmland soils, and experimental farmland (r = 0.694; Fig. 5). Accordingly, the M. Morita for support in producing FBWA from raw materials ORP value in the soil surface varied by supplementation of an in China. organic amendment would be an important index to evaluate the suppression of Cd uptake by harvested rice. FBWA is easily assimilated into the soil by pulverizing wood Supporting Information and food waste to 1 μm or less. Asakawa et al.46 reported that aerobic microorganisms consume oxygen in water on the surface Tables S1 – S6, and Figs. S1a and S2 are presented as supporting of the soil after supplementation with organic amendments. information. Table S1 summarizes the relevant examinations of Moreover, the concentration of insoluble Cd might increase. the restorative materials used for Cd reduction in rice in Japan Namely, Cd is immobilized when it binds to high-molecular- and China. Table S2 summarizes the operation conditions of weight organic matter, like humic acid or lignin, in woody ICP-MS used in this study. Table S3 summarizes the biomass.47,48 The changes in the solubility of Cd in farmland concentrations of heavy metals in irrigation water samples. soil will be investigated in future work to assist the discussion. Table S4 summarizes the pH and Cd concentration (mg kg–1) in The results indicate that FBWA at >0.5% is effective at soil samples from each division of the farmlands. Table S5 suppressing crop Cd absorption. However, supplementation summarizes the rice-wheat cultivation schedule from the 2016 with 1% FBWA resulted in a slightly lower rice yield than that to 2018. Table S6 summarizes the maximum adsorption obtained using 0.5% FBWA (Fig. 4). Higher FBWA capacity of Cd to FBWA alongside comparative adsorbent data concentrations might inhibit rice growth by causing oxygen from previous research. Figure S1 shows the procedures from deficiency at the soil surface. crop harvest to cadmium measurement in rice. 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