Distribution of nutrient and toxic elements in brown and polished rice

Accepted Manuscript Distribution of Nutrient and Toxic Elements in Brown and Polished Rice Gyuhan Jo, Todor I. Todorov PII: DOI: Reference:

S0308-8146(19)30517-5 https://doi.org/10.1016/j.foodchem.2019.03.040 FOCH 24487

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

2 November 2018 5 March 2019 9 March 2019

Please cite this article as: Jo, G., Todorov, T.I., Distribution of Nutrient and Toxic Elements in Brown and Polished Rice, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.03.040

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Distribution of Nutrient and Toxic Elements in Brown and Polished Rice

Gyuhan Jo1, Todor I. Todorov*

Office of Regulatory Science, Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, MD 20740

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Current address: Department of Biotechnology, College of Life Science and Biotechnology, Korea University, Anam-dong, Seongbuk-gu, Seoul, 02841, Republic of Korea *Corresponding Author: Todor I. Todorov, Office of Regulatory Science, Center for Food Safety and Applied Nutrition, Food and Drug Administration, 5001 Campus Dr, College Park, MD 20740, email: [email protected] Keywords: rice, polishing, ICP-MS, LA-ICP-MS, arsenic, nutrient elements, toxic elements

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Abstract Rice is a staple food in many countries around the world and it is a source of not only the nutrients, but also toxic elements. In this study, we evaluated four degrees of polishing and determined the elemental content (P, S, K, Mn, Fe, Ni, Cu, Zn, As, Se, Mo, Cd, Hg, Pb) in brown rice, rice bran and the resulting white rice using microwave assisted decomposition followed by inductively coupled plasma mass spectrometry (ICP-MS) detection. Additionally, individual rice grains at every polishing step were analyzed by laser ablation ICP-MS to generate elemental distribution maps. While P, K, Mn and Fe were predominantly located in bran layer, S, Cu, Zn, As, Se, Mo, Cd, and Hg were present in both the bran and endosperm. As the elemental distribution in the grain varies, polishing to produce white rice results in removal of different amounts of nutrient and toxic elements.

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1.Introduction Rice is one of the most consumed grains and is a large part of the diet in many countries. Currently, over 750 million tons are produced annually worldwide (FAO, 2018). Rice is usually grown under flooded conditions and the plant absorbs nutrients and other minerals from the water. Thus, minerals mobilized from the soil can be accumulated in the grain (Xu, McGrath, Meharg, & Zhao, 2008). The rice grain is composed from several main parts: hull/husk, bran, endosperm, and embryo (Juliano, 1972). The husk protects the grain and it is composed of fibrous tissue rich in cellulose and silica. Underneath the husk is the bran, which is composed of the pericarp (high in cellulose), seed coat and aleurone layers (high in oil, protein, vitamins and minerals). These layers are very low in starch. The embryo is located in the bottom part of the grain close to the stem and it contains mostly protein and minerals. The central and largest part of grain is the endosperm, containing mostly of carbohydrates and small amounts of little protein, fat, or minerals (Juliano, 1972). Depending on the end product, rice processing steps include (1) drying of the grain, (2) removal of the husk, and (3) milling to remove the bran layer for white rice production (Bond, 2004). During the milling process, the vitamin and mineral contents of rice are reduced. In the case of parboiled rice, an extra step is added in which the grain is first soaked in hot water and steamed before the drying stage (Bhattacharyya, 2004). This forces the migration of nutrients from the bran layer to the endosperm. The knowledge of the elemental composition of rice is important from both nutritional and toxicological perspectives. Multiple reports have investigated the levels of arsenic and other trace elements in the rice grain (Adedire, et al., 2015; Hensawang & Chanpiwat, 2017; Joy, et al., 2017; Kumarathilaka, Seneweera, Meharg, & Bundschuh, 2018; Lee, et al., 2018; Lombi, et al.,

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2009; Londonio, Morzan, & Smichowski, 2019; Ma, Wang, Jia, & Yang, 2016; Mataveli, et al., 2016; Meharg, Lombi, et al., 2008; Meharg, Sun, et al., 2008; Narukawa, Matsumoto, Nishimura, & Hioki, 2014; Runge, Heringer, Ribeiro, & Biazati, 2019; Segura, et al., 2016; Tattibayeva, et al., 2016; Yim, Park, Lee, Chung, & Shim, 2017; Zavala & Duxbury, 2008; Zhu, et al., 2008). Arsenic is a type one carcinogen that is readily taken up by the rice plant and the levels of arsenic are higher compared to other grains (ATSDR, 2007). The US Food Drug Administration has proposed an action level in infant rice cereals of 0.1 mg/kg inorganic arsenic (As3+ and As5+) and the European Commission has set a limit of 0.1 mg/kg inorganic arsenic for rice destined for the production of food for infants and young children (EC, 2015; USFDA, 2016). Because of the elevated As content, rice grains have been a subject of many studies. These include effects of polishing of the grain, washing before cooking, and cooking with various amounts of water (Gray, Conklin, Todorov, & Kasko, 2016; Hansen, et al., 2012; Naito, Matsumoto, Shindoh, & Nishimura, 2015). Narukawa et al, investigated the levels of 16 elements in polished rice and found that 70% of several elements, including As, were present in the outer 30% of the grain (Narukawa, Matsumoto, Nishimura, & Hioki, 2014). Naito et al, found that As levels in white rice were reduced to 29 – 34 % after 90 % degree of polishing compared from the starting brown rice (Naito, Matsumoto, Shindoh, & Nishimura, 2015). Choi et al, reported a 34 % decrease of As levels at the highest degree of polishing in their study (Choi, et al., 2014; Jeon, et al., 2014). Sun et al, found an order of magnitude difference between the As content in the rice bran and the bulk rice (Sun, et al., 2008). In addition to investigations of the rice elemental content as a function of polishing, more detailed information has been obtained using imaging techniques such as laser ablation inductively coupled plasma-mass

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spectometry (LA-ICP-MS), micro-x-ray fluorescence (micro-XRF) and synchrotron-x-ray fluorescence (synchrotron-XRF) (Basnet, Amarasiriwardena, Wu, Fu, & Zhang, 2014; Carey, et al., 2012; Choi, et al., 2014; Jeon, et al., 2014; Lombi, et al., 2009; Meharg, Lombi, et al., 2008; Promchan, Gunther, Siripinyanond, & Shiowatana, 2016). Meharg et al, and Lombi et al, used synchrotron based XRF, particle induced X-Ray emission (PIXE) and LA-ICP-MS to investigate the distribution of As and other elements in the rice grain (Lombi, et al., 2009; Meharg, Lombi, et al., 2008). For brown rice they observed higher levels of As, Zn, Cu and Fe in the pericarp and aleurone layers of the grain. For white rice the distribution for these elements was more even throughout the grain. Hansen et al, showed that while Cu, Zn, Fe and Mn were located in outer layer of the grain, Cu and Zn were removed at a slower rate during polishing compared to Fe and Mn (Hansen, et al., 2012). Using LA-ICP-MS, Choi observed that the concentration of As was significantly higher near the surface of the grain (Choi, et al., 2014). This study aims to investigate the contents of 14 elements in brown rice that was subsequently polished stepwise to produce white rice. The concentrations of the elements were measured at each step in both the resulting rice and the removed rice bran. Spatially resolved LA-ICP-MS analysis of the grains at each polishing step was performed. The elemental maps provided detailed information on the effect of the milling process on essential and toxic element distributions in rice. The LA-ICP-MS results correlate with measurements of the digested fractions obtained during the milling polishing process.

2.Materials and Methods 2.1.Instrumentation

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Rice samples were polished using a consumer rice polisher (Twinbird E-751, Tsubame, Japan) and ground using a tube mill (IKA, Wilmington, NC, USA). Samples were decomposed using an accelerated microwave digestion system (CEM Mars Express, Matthews, NC, USA). Elemental analysis was carried out using an ICP-MS instrument (Thermo Electron ICAPq, West Palm Beach, FL, USA), equipped with a peltier-cooled spray chamber, perfluoroalkoxy nebulizer (PFA-ST, Elemental Scientific Inc, Omaha, NE, USA) and a Cetac ASX-560 autosampler (Omaha, NE, USA). The sample was transferred to the ICP-MS using a 0.381 mm i.d. peristaltic pump tubing (Meinhard Inc, Golden, CO, USA) at 20 rpm resulting in a 0.12 mL/min sample uptake rate. The analysis was performed in kinetic energy discrimination (KED) mode using 4.6 mL/min He as collision gas to minimize interferences. Rice grains were sectioned using a Thermo Electron Finesse microtome (Waltham, MA, USA). For elemental imaging studies, the ICP-MS was connected to an Electro Scientific Industries NWR 193e laser ablation system equipped with a 100 mm x 100 mm two volume sample cell and a 193 nm excimer laser (Portland, Oregon, USA). Helium at 0.7 L/min was used as a sweep gas and was mixed with 5 mL/min nitrogen and 0.6-0.65 L/min argon (tuned daily) inside the ICP-MS torch similar to the design by Douglas et at. (Douglas, Managh, Reid, & Sharp, 2015). The same size tubing diameter was used from the sample cell cup to the ICP plasma in order to minimize aerosol dispersion. This resulted in shorter than 100 ms washout time for the analyzed elements. 2.2.Chemicals Double distilled nitric and hydrochloric acids (Optima grade), and high purity hydrogen peroxide (Optima grade) were obtained from ThermoFisher Scientific (Waltham, MA, USA). High purity 2-propanol (electronic grade, 99.9999%) was obtained from Sigma Aldrich (St.

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Louis, MO, USA). All elemental standards were prepared gravimetrically from 1000 mg/kg stock solutions (Inorganic Ventures, Christiansburg, VA, USA). All solutions were prepared using deionized water (MilliQ Element, Millipore, Billerica, MA, USA). Low viscosity Spurr type polymer containing cycloaliphatic epoxide resin (ERL 4221), flexible epoxy resin (DER 736), nonenyl succinic anhydride and 2-dimethylaminoethanol were used for embedding the rice grains for imaging analysis (RT 14300, Electron Microscopy sciences, Hatfield, PA, USA). Certified reference materials used were NIST 1568, NIST 1568b (NIST, Gaithersburg, MD, USA), and NMIJ 7503-a (NMIJ, Tsukuba, Ibaraki, Japan). 2.3.Rice polishing and preparation Brown long grain rice was purchased from a local grocery store. A 150 g portion of the rice was kept as a reference starting material. Four other 150 g portions of the rice were polished in duplicate at the different settings of the polisher (removal of 30%, 50%, 70% and 100% of the bran layer). The 100% bran removal setting resulted in the preparation of white rice. Both the rice and bran portions from each setting were ground using a tube mill and kept for analysis. A small portion of grains (approximately 20 g) from each polishing step was kept unhomogenized for the laser ablation ICP-MS imaging analysis. The embedding media was prepared using the directions from the manufacturer by gently mixing 10 g of ERL 4221 resin, 8 g of DER 736 resin and 25 g nonenyl succinic anhydride, after which 0.3 g of 2-dimethylaminoethanol were added. The reagents were thoroughly mixed and placed in an oven at 90˚C for 50 min at which point the rice grains were added to the embedding media. The polymerization was complete after 8 hours at 90˚C. The rice grains were then sectioned using a microtome to obtain a flat cross-section that was used in the LA-ICP-MS analysis.

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2.4.Rice sample analysis Ground rice and rice bran samples of approximately 0.5 g were decomposed in 11.4 g HNO3 and 1 g H2O2 using microwave digestion with a 25 min ramp to 200˚C followed by a 15 min hold at the same temperature. The digests were transferred to 100 mL tubes, 5.2 g of 10 % HCl was added to stabilize Hg and keep it in the solution and were diluted with deionized water for a final weight of 100 g. All samples were prepared and analyzed in triplicate. Blank samples were prepared with 0.5 g of deionized water undergoing the same procedure. Samples were transferred to autosampler tubes and quantified by ICP-MS using an external calibration with 2 ng/g Rh in 1% HNO3, 0.5% HCl and 4% 2-propanol as internal standard added inline through a Y-connector. Instrument parameters are summarized in Supplementary material Table S1. The laser ablation system was optimized using NIST CRM 612 glass (NIST, Gaithersburg, MS, USA) and fine-tuned using pressed cellulose powder fortified with 24 elements to maximize intensity and signal stability (<5% RSD over 30 second line scan) for Fe, As, and U, while maintaining a Th/U between 0.95 and 1.05 and oxides below 0.3% (measured as UO/U). The Th/U ratio in both the NIST CRM 612 glass and the spiked cellulose powder were 1.01 and 0.99, respectively. The laser ablation ICP-MS analysis parameters are summarized in Supplementary Material Table S2. Rice cross-sections were placed in the laser ablation system sample cell and were ablated at 2.1 J/cm2 using a 30 m spot size at a repetition rate of 100 Hz. The stage was moved in a line scan mode at 50 m/s and the distance between the lines was 35 m. The ablation with 2.1 J/cm2 fluence resulted in approximately 30 m deep laser line. Monitored elements included P (m/z=31), S (m/z=34), K (m/z=39), Mn (m/z=55), Fe (m/z=57), Cu (m/z=65), Zn (m/z=66), As (m/z=75), and Cd(m/z=111). In addition, carbon was monitored at m/z=13 and it showed uniform signal throughout the grain, indicating that the amount of

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sample ablated was uniform across the rice grain. Total acquisition time for all isotopes was 100 ms. Data processing included gas blank subtraction and quantification using NIST CRM 1568b rice powder for P, S, K, Mn, Fe, Cu, Zn, and As. Because the certified level of Cd in CRM 1568b is low, 0.022 mg/kg, NJIM CRM 7532-a was used for the Cd quantification of that element (0.429 mg/kg). The CRMs were pressed as pellets and provided matrix matching for elemental quantification of the rice grains. The raw data was processed using Microsoft Excel generated macro (Microsoft, Redmond, WA, USA) and the line scans were combined to generate elemental images using Surfer 12 (Golden Software, Golden, CO, USA). To simplify data processing, the ablated area for all rice grains was set to 3 mm by 2.7 mm and each elemental image was composed of 89 lines. This resulted in 86 min acquisition time for the individual grains and data processing under one hour. Five sections from separate grains were prepared and analyzed by LA-ICP-MS for the brown rice and each of the polishing steps.

3.Results and Discussion 3.1.Analytical performance All data is reported on a dry weight basis. The analytical batches contained certified reference materials (NIST 1568, NIST 1568b, and NMIJ 7503-a), preparation of triplicate portions, and method blanks that underwent the entre sample preparation procedure. The rice analysis followed the performance criteria set in US FDA elemental analysis method 4.7 (Gray & Cunningham, 2018). The analytical performance of the microwave digestion ICP-MS and laser ablation ICP-MS methods is shown in Table 1. The instrument detection limits were calculated as 3 times the standard deviations of the measurement of 8 analytical blank solutions. The limit of quantification was calculated as 30 times the standard deviations of the

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measurement of 7 method blank solutions (blanks underwent the entire sample preparation and analysis procedure) from multiple digestions over a period of 1 week. The estimation of the limit of quantification includes error that is associated with sampling, sample preparation and the chemical analysis (USFDA, 2014). Accuracy of the analysis was measured using the 3 CRMs and resulted in 88-111% recoveries for all measured elements. The laser ablation ICP-MS analysis limit of detection was calculated as 3 times the standard deviation of the gas blank signal. The LODs for P, S, K, Mn, Fe, Cu, Zn, As, And Cd were 10, 100, 30, 1, 2, 0.4, 0.4, 0.08 and 0.2 g/g, respectively. These LODs were lower than the concentrations found in rice, except for Cd, where levels are an order of magnitude lower than the determined LOD. NMIJ 7532-a brown rice pressed pellets were used as quality control and resulted in recoveries of 96 -111% for Mn, 84 – 131% for Fe, 83 – 133% for Cu, 84 – 122% for Zn and 88 – 112 % for As. Recoveries were not reported for P, S, K , and Cd as they were not certified in NMIJ 7532-a and the Cd level in NIST 1568b was below the limit of detection. 3.2.Elemental content in brown and polished rice The content of 14 elements in brown rice, as well as the polished rice and rice bran resulting from each polishing step, are summarized in Table 2 (replicate analyses results are shown in the supplementary information Table S3). The levels determined in brown rice are consistent with previous studies (Choi, et al., 2014; Hansen, et al., 2012; Meharg, Lombi, et al., 2008; Naito, Matsumoto, Shindoh, & Nishimura, 2015; Narukawa, Matsumoto, Nishimura, & Hioki, 2014). The elemental concentrations are higher in brown rice compared to 100% polished rice (white rice) for all elements except for S and Se, consistent with the observations by Narukawa et al (Narukawa, Matsumoto, Nishimura, & Hioki, 2014). The decrease is most pronounced for P, K, Mn, and Fe whereas the levels of Cu, Zn, As, Mo, Cd and Hg were

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decreased to a lesser extent. Ni and Pb in polished rice were below the LOQ and therefore accurate assessment on the removed amount of these elements during the polishing process was not possible. Removal of 30% of the bran layer resulted in a weight decrease of 11%. Visual inspection revealed that the embryo was removed in over 99 % of the polished grains. Previous studies have reported the embryo to be 2 – 3 % of the brown rice grain weight (Champagne, 2004; Juliano, 1972). The content of P, K, Mn, and Fe was reduced by 31%, 33%, 43%, and 43%, respectively, when compared to the original brown rice. Higher degree of polishing (50%, 70%, and 100%) removed 14%, 15% and 18% of the grain weight. This resulted in reduction of Fe of 55%, 64%, and 66%. Figure 1 shows the content of the 14 elements, normalized to the content in brown rice. The first degree of polishing decreased the elemental content (with an exception of S, Cd and Pb) by the largest amount, but it also resulted in the largest amount of solid material that was removed from the grain. Further evaluation of the elemental content as a function of removed material during the polishing process showed that decrease was linear for P, K, Mn, Fe, Cu, Zn, As, Mo, and Hg with a correlation coefficient of 0.97 or higher. When comparing white rice to brown rice, for P, K, Mn and Fe, the decrease was 35 – 50% from the initial product, and for Cu, Zn, As, Mo, Cd, and Hg: 66 – 89 %. Elemental analysis of the polished fractions showed higher concentrations of all 14 elements compared to the brown and resulting white rice. The contents of the outer layers varied by element and were up to 13 times higher compared to the remaining inner endosperm portion of the grain. This is in agreement with previous studies that investigated As content and other elemental distributions in rice (Basnet, Amarasiriwardena, Wu, Fu, & Zhang, 2014; Carey, et al., 2012; Choi, et al., 2014; Lombi, et al., 2009; Meharg, Lombi, et al., 2008). Mass balance was

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performed for the elemental analysis from the concentrations of the polished rice and bran portions. The sums of the concentrations of the resulting rice and removed layers were compared to brown rice and the combined recovery was 104 ± 8 % with a minimum of 84% and maximum of 120%. The concentrations for Ni and Pb were below the LOQ and were excluded from these calculations. Most of the elemental content was found in the outer layers of the grain. P, K, Mn, Fe were contained primarily in the outer most layer of the grain, Cu, Zn, As, Mo, and Cd, Hg more gradually spread towards inner layers, and S and Se were distributed more evenly in the grain. 3.3.Distribution of elemental content in the rice To further evaluate the elemental content in the grain, brown and polished rice sections were analyzed by LA-ICP-MS. The distribution of P, S, K, Mn, Fe, Cu, Zn, As, and Cd in brown rice is shown in Figure 2. Higher elemental concentrations were observed in the bran and outer layers of the endosperm, but the distributions varied between the different elements. P, K, Mn, and Fe were localized mainly in the pericarp and aleurone layers, with Mn also present in the endosperm. Previously, using PIXE and synchrotron X-ray fluorescence imaging, Lombi et al, observed higher levels of P, K, and Mn in in the aleurone/pericarp region (Lombi, et al., 2009). The P and K levels varied between 6000 and 20000 mg/kg in the bran layer and were distributed evenly around the grain perimeter. The endosperm contained much lower amounts of P and K and their levels were 400 – 500 mg/kg and 600 – 700 mg/kg, respectively. These values are in close agreement with our bulk grain analysis, where the grain contained 12000 mg/kg P and 8750 mg/kg K in the bran fraction and 1520 mg/kg P and 1230 mg/kg K in the resulting white rice fraction (Table 2). Based on image analysis, the average concentrations were calculated from all the points in the bran and endosperm parts of the image from cross-sections of 5 brown

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rice grains (summarized in Table 3, with individual rice grain results in the supplementary information Table S4). For P and K, the bran layer average concentration was 9590 mg/kg and 9510 mg/kg, respectively, and the endosperm areas showed 470 mg/kg and 620 mg/kg, respectively. The levels of these elements in the bran layer are in close agreement with the bulk bran analysis, 80% from the P in the bulk bran analysis and 109% from the K. The image calculated average for the endosperm shows a 2 – 3 times lower P and K concentration compared to the measurements in polished white rice. This difference is expected, as the average levels represent one cross-section of the grain vs the entire grain analysis. Additionally, there may be grain to grain variability in elemental composition and the average of 5 grains might not be sufficient to account for that variability. While Mn and Fe are also much higher in the bran layer compared to the endosperm (an order of magnitude difference), they are not distributed evenly around the grain perimeter. Mn and Fe were concentrated in the dorsal side of the bran layer with concentrations 5 times higher compared to the ventral side. The concentration ranges of Mn and Fe in the bran layer were 40 – 200 mg/kg with an average of 54 mg/kg and 30 – 150 mg/kg with an average of 49 mg/kg, respectively. The endosperm contained 8 – 12 mg/kg Mn (average 9 mg/kg) and 2 – 6 mg/kg Fe (average of 4 mg/kg) (Table 3). The image calculated averages for Fe agree well with the Fe measurements of bulk bran (103%) and white rice (122%). For Mn, the average concentration in endosperm from the imaging study also agrees with the measured values in the white rice (103% recovery), but the image calculated Mn in the bran is 44% from the bulk bran. This could be because in the imaging study only the bran is quantified, whereas in the bulk determination both the bran and the embryo are quantified together and higher levels of Mn in the embryo likely result in this difference.

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The S and Cu elemental images showed higher concentrations in the bran layer, but with a gradual decrease from the perimeter towards the center of the grain. Additionally, these elements were concentrated in the lateral sides of the bran, with lower levels in the ventral and dorsal parts of the grain. Previous studies in rice, wheat and rye grains have observed similar gradual decrease of S content from bran to center (Lombi, et al., 2009; Van Malderen, Laforce, Van Acker, Vincze, & Vanhaecke, 2017). The bran layer contained 2000 – 4500 mg/kg S (average 1830 mg/kg) and 4 – 20 mg/kg Cu (average 6.4 mg/kg). The endosperm S level were in the 500 – 2000 mg/kg range (average 920 mg/kg) and the Cu range was 0.7 – 5 mg/kg (average 1.8 mg/kg). Comparison with bulk rice measurement showed good agreement for S (107% of bulk bran, 88% of the white rice) and Cu (139% of the bulk bran and 95% of the white rice). Similarly, Lombi et al observed lower content of Cu in the center of the grain that gradually increases towards the bran layer (Lombi, et al., 2009). Zinc showed the most homogeneous distribution throughout the grain with 10 – 25 mg/kg in the endosperm (average 13 mg/kg) with only minor accumulations in the bran layer of 20 – 80 mg/kg (average 28 mg/kg). Similar to P and K, Zn was distributed homogeneously around the perimeter of the grain and was slightly elevated in the dorsal and ventral sides. The endosperm Zn concentration varied slightly and the concentration of the center of the grain was measured at 25 mg/kg. The image calculated averages for Zn agree well with the Zn measurements of bulk bran (64%) and white rice (94%). The lower concentration in the image analysis was because in this measurement study, only the bran was quantified, whereas in the bulk determination, both the bran and the embryo were quantified together, resulting in higher level in the bulk measurement technique.

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The distribution of As in the rice grain show similarities to both S and Cu, but also Zn. The bran layer contains higher As levels (0.6 – 2 mg/kg with an average of 0.86 mg/kg) compared to the endosperm (ranging from below the LOD of 0.08 to 0.3 mg/kg with an average of 0.17 mg/kg). The level of As in the endosperm gradually decreased starting at 0.4 mg/kg in the perimeter to the central part of the grain where it was below the limit of detection of the LAICP-MS determination of 0.08 mg/kg. These values agree well with the bulk bran (144%) and endosperm portion (85%) of the grain. The highest levels of As are observed in the dorsal and ventral sides of the bran layer, similar to the distribution for Zn. This is in agreement with previous studies for brown rice (Basnet, Amarasiriwardena, Wu, Fu, & Zhang, 2014; Choi, et al., 2014; Lombi, et al., 2009; Meharg, Lombi, et al., 2008). Table 3 shows a comparison of the LA-ICP-MS elemental image determined concentrations for the bran, endosperm, and entire slice with the concentrations determined by the bulk microwave digested brown rice. The LA-ICP-MS determinations are averages of 5 slices from 5 individual brown rice grains. Grain to grain variations from the LA-ICP-MS analysis are expressed as relative standard deviations from 5 different slices for the bran, endosperm, and entire slice. The calculated RSDs of the 5 grains, depending on the element, were 8 – 22%, with the lowest for the K at 8%, and highest for Cu at 22%. This indicates that the elemental content is relatively constant among individual grains and that the LA-ICP-MS is providing precise measurement among individual grains. Comparing the LA-ICP-MS average brown rice concentrations with the levels determined in the bulk brown rice produced recoveries in the range 58% to 122%, indicating good agreement between the two analyses.

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The detection limit for the elemental imaging analysis for Cd was 0.2 mg/kg. All points in the Cd generated images were below the LOD and the grain image was difficult to distinguish from the surrounding embedding polymer media (Figure 2). Elemental maps were generated for polished rice grains at each of the polishing steps and are shown in Figure 3 and in the supplementary information (Supplementary information Figures S1-S3). Each element is presented at the same concentration scale for brown and the 4 degrees of polishing, allowing comparison of the elemental content as a function of polishing. As the rice grains are polished and the bran layer is removed the concentrations in the rim of the grain decreases. The P concentration in the most outer layer of the brown rice grain was 6000 – 20000 mg/kg, for 30% polished rice it was 3000 – 20000 mg/kg, for 50% polished rice it was 2000 – 15000 mg/kg, for 70% polished rice it was 1000 – 12000 mg/kg and for 100% polished (white) rice it was in the 500 – 10000 mg/kg range. Similar to P, the K, Mn and Fe concentrations decreased as a function of polishing and the levels in 100% polished rice were 600 – 8000 mg/kg, 10 – 20 mg/kg and 6 – 60 mg/kg compared to 6000 – 20000 mg/kg, 40 – 200 mg/kg and 30 – 150 mg/kg in the brown rice, respectively. For S and Cu, removal of the bran layer did not result in a visual change in their elemental images, indicating that most of these elements were located in the inner aleurone layer and in the periphery of the endosperm. Slightly more of the Zn was located in the bran layer as higher levels were observed in brown rice (30 – 80 mg/kg) versus white rice (20 – 40 mg/kg). Polishing removed As in the bran layer as the lateral and dorsal As levels were decreased. In white rice, most As was still observed in the ventral part of the grain, but this may be due to variations of the polishing and may differ between grains. The accumulated As in the inner aleurone layers and the outer side of the endosperm remains unchanged, confirming the bulk

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digestion results that showed only 22% of the As content removed in the polishing from brown to white rice.

4.Conclusions Within this study, the effect of polishing of brown rice on the elemental content was investigated. Higher concentrations of all elements were measured in the bran layer compared to the remaining endosperm. This was especially pronounced for P, K, Mn and Fe, for which an order of magnitude difference was observed between the bran and the endosperm. For this reason, polishing of brown rice to white rice reduced the content of P, K, Mn and Fe by approximately a factor of two. The polishing reduced the remaining monitored elements by approximately 16-33% (Cu, Zn, As, Hg) and less than 10% (S, Se, Mo, Cd). Investigation of individual brown and polished rice grains by LA-ICP-MS revealed further elemental distribution detail. These results show good agreement with concentration of the measured fractions from the polishing process. The outer layer of the grain contained higher levels of P, K, Mn and Fe with much lower levels of these elements accumulated in the endosperm. While higher levels of S, Cu, Zn and As were observed in the bran layer, there was a gradual decrease of S, Cu and As in the endosperm towards the center of the grain, with Zn uniformly distributed in the endosperm. These elemental results explain the varied reduction of different nutrient and toxic elements during the process of polishing to produce white rice.

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Conflict of interest The authors declare that they have no conflict of interest

Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors. Informed consent: Not applicable.

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USFDA. (2016). Inorganic Arsenic in Rice Cereals for Infants: Action Level, Guidance for Industry. Silver Spring, MD: Food and Drug Administration. Van Malderen, S. J. M., Laforce, B., Van Acker, T., Vincze, L., & Vanhaecke, F. (2017). Imaging the 3D trace metal and metalloid distribution in mature wheat and rye grains via laser ablation-ICPmass spectrometry and micro-X-ray fluorescence spectrometry. Journal of Analytical Atomic Spectrometry, 32(2), 289-298. Xu, X. Y., McGrath, S. P., Meharg, A. A., & Zhao, F. J. (2008). Growing rice aerobically markedly decreases arsenic accumulation. Environmental Science & Technology, 42(15), 5574-5579. Yim, S. R., Park, G. Y., Lee, K. W., Chung, M. S., & Shim, S. M. (2017). Determination of total arsenic content and arsenic speciation in different types of rice. Food Science and Biotechnology, 26(1), 293-298. Zavala, Y. J., & Duxbury, J. M. (2008). Arsenic in rice: I. Estimating normal levels of total arsenic in rice grain. Environmental Science & Technology, 42(10), 3856-3860. Zhu, Y. G., Sun, G. X., Lei, M., Teng, M., Liu, Y. X., Chen, N. C., Wang, L. H., Carey, A. M., Deacon, C., Raab, A., Meharg, A. A., & Williams, P. N. (2008). High percentage inorganic arsenic content of mining impacted and nonimpacted Chinese rice. Environmental Science & Technology, 42(13), 50085013.

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Figure Captions Figure 1. Elemental composition of brown and polished rice. The elemental content in polished rice has been normalized to the concentration of the starting brown rice.

Figure 2. LA-ICP-MS generated elemental images of P, S, K, Mn, Fe, Cu, Zn, As, and Cd of a cross-section of a brown rice grain. A microscopic white light image of the grain is also shown.

Figure 3. LA-ICP-MS generated elemental images of P, S, K, Mn, Fe, Cu, Zn, As, and Cd of a cross-section of a 100% polished rice grain (white rice). A microscopic white light image of the grain is also shown.

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Table 1. Analytical and method performance

Microwave digestion solution ICP-MS P S Sample size, g 0.5 0.5 Background, cps 69 697 Sensitivity, cps per 10 3 g/kg Instrument detection 0.002 0.2 limit, mg/kg (n=7) Limit of detection in 2 20 food samples, mg/kg (n=7)

K 0.5 2199 134

Mn 0.5 26 3300

Fe 0.5 37 162

Ni 0.5 152 667

Cu 0.5 138 6225

Zn 0.5 280 2033

As 0.5 9 3113

Se 0.5 2 268

Mo 0.5 60 15032

Cd 0.5 1 5337

Hg 0.5 50 28732

Pb 0.5 729 273929

0.002

0.000007

1

0.005

0.3

0.03

0.01

0.02

0.0003

0.004

0.002

0.00007

0.0004

0.0005

0.00004 0.00002 0.000003 0.00002 0.000003 0.00002 0.00002 0.0000004 0.000003 0.0000003

Limit of quantification in food samples, mg/kg (n=7)

13

160

7

0.04

2

0.2

0.06

0.2

0.002

0.03

0.02

0.0005

0.003

0.003

SRM recovery ranges

99100%

9599%

99101%

92-102%

93101%

101108%

90-100%

90101%

91-111%

90-93%

97100%

88-91%

94-103%

95-108%

Mn 30000 30000

Fe 500 500

Cu 100 4700

Zn 600 2800

As 800 5100

Cd 80 6400

1

2

0.4

0.4

0.08

0.2

96 -111%

84 131%

83 133%

84 122%

88 113%

Laser ablation ICPMS

Background, cps Sensitivity, cps per mg/kg Limit of Detection for LA-ICP-MS (mg/kg)

SRM recovery

P S K 24000 16000 300000 1050 52 11500 10

100

30

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Table 2. Elemental concentrations in brown and polished rice % weight P, S, K, Mn, removed mg/kg mg/kg mg/kg mg/kg brown rice 0 3030 ± 968 ± 9 2510 ± 28.9 ± 200 150 1.6

Fe, mg/kg 10.4 ± 1.1

Ni, mg/kg 0.22 ± 0.02

Cu, mg/kg 2.29 ± 0.03

Zn, mg/kg 18.3 ± 0.4

As, mg/kg 0.262 ± 0.006

Se, mg/kg 0.15 ± 0.004

Mo, mg/kg 0.74 ± 0.01

Cd, mg/kg 0.014 ± 0.001

Hg, mg/kg 0.0048 ± 0.0001

Pb, mg/kg 0.004 ± 0.001

30% polished rice 30% polished rice bran mass balance 50% polished rice 50% polished rice bran

11%

2± 0.01 4.84 ± 0.003 101% 1.93 ± 0.002 4.58 ± 0.01

14.8 ± 0.03 49.9 ± 1 102% 14.4 ± 0.1 45.5 ± 0.2

0.221 ± 0.008 0.598 ± 0.02 100% 0.212 ± 0.008 0.581 ± 0.005

0.141 ± 0.001 0.255 ± 0.004 103% 0.146 ± 0.007 0.277 ± 0.007

0.686 ± 0.009 1.14 ± 0.02 100% 0.656 ± 0.02 1.13 ± 0.01

0.014 ± 0.002 0.023 ± 0.002 106% 0.011 ± 0.0005 0.021 ± 0.002

0.0041 ± 0.0005 0.0083 ± 0.000003 94% 0.0035 ± 0.0004 0.0074 ± 0.0001

0.004 ± 0.001 0.017 ± 0.009 133% < 0.003

mass balance 70% polished rice 70% polished rice bran mass balance 100% polished rice (white rice)

14% 15%

101% 1.9 ± 0.09 5.3 ± 0.3 105% 1.91 ± 0.05

103% 14.1 ± 0.3 52.5 ± 4 109% 14.1 ± 0.2

101% 0.207 ± 0.01 0.695 ± 0.04 107% 0.205 ± 0.0008

110% 0.147 ± 0.01 0.299 ± 0.03 114% 0.144 ± 0.0002

98% 0.662 ± 0.02 1.2 ± 0.2 101% 0.66 ± 0.009

91% 0.012 ± 0.0007 0.025 ± 0.004 97% 0.012 ± 0.001

85% 0.0033 ± 0.00009 0.0082 ± 0.0009 84% 0.0032 ± 0.0003

N/A < 0.003

100% polished rice bran mass balance

2090 ± 23 11500 ± 580 104% 1840 ± 16 11200 ± 230

995 ± 22 1720 ± 36 111% 1040 ± 30 1710 ± 26

1690 ± 9 8950 ± 200 100% 1470 ± 4 8520 ± 79

16.5 ± 0.1 134 ± 5.1 103% 14.2 ± 0.13 122 ± 1.6

5.96 ± 0.79 52.8 ± 2 108% 4.71 ± 0.84 49.1 ± 2.9

< 0.2

105% 1630 ± 140 14500 ± 750 118% 1520 ± 45

117% 989 ± 61 1960 ± 210 117% 1040 ± 24

98% 1330 ± 91 10500 ± 470 108% 1230 ± 40

102% 13 ± 0.79 148 ± 5.9 116% 12.3 ± 0.36

105% 3.77 ± 0.55 57.9 ± 2.2 115% 3.6 ± 0.75

N/A < 0.2

18%

12000 ± 960

1710 ± 43

8750 ± 550

121 ± 7.3

47.6 ± 4.1

0.73 ± 0.06

4.61 ± 0.2

43.6 ± 2

0.599 ± 0.279 ± 1.13 ± 0.03 0.0004 0.02

0.022 ± 0.0071 ± 0.001 0

0.011 ± 0.002

18%

111%

120%

102%

109%

109%

N/A

105%

105%

105%

97%

N/A

11% 11% 14% 14%

15% 15% 18%

1.02 ± 0.43 N/A < 0.2 0.86 ± 0.31

0.86 ± 0.08 N/A < 0.2

24

112%

101%

81%

0.020 ± 0.009

0.012 ± 0.003 N/A < 0.003

Table 3. Elemental concentrations determined from the rice imaging analysis (n=5)

Bran layer Endosperm entire brown rice slice (LA-ICP-MS) brown rice microwave digestion ICP-MS

P, mg/kg 9590 ± 510 470 ± 30 1980 ± 220 3030 ± 200

S, mg/kg 1830 ± 140 920 ± 130 1070 ± 120 968 ± 9

K, mg/kg 9510 ± 750 620 ± 140 2080 ± 180 2510 ± 150

Mn, mg/kg 54 ± 5

Fe, mg/kg 49 ± 8

Cu, mg/kg 6.4 ± 1

9.5 ± 1.6 17 ± 3

4.4 ± 1.2 13 ± 2

28.9 ± 1.6

10.4 ± 1.1

1.8 ± 0.5 2.6 ± 0.6 2.29 ± 0.03

Highlights • • • • •

ICP-MS was used to determine the elemental content in brown and white rice The polishing of rice affects the levels of minor and trace elements LA-ICP-MS characterized elemental distribution in brown and white rice Highest concentrations of P, K, Mn, and Fe are found in the in the bran layer S, Cu, Zn and As are distributed more evenly throughout the rice grain

25

Zn, mg/kg 27.9 ± 3.7 13.2 ± 1.9 15.6 ± 1.8 18.3 ± 0.4

As, mg/kg 0.86 ± 0.08 0.17 ± 0.05 0.29 ± 0.06 0.262 ± 0.006

1.4

brown rice

11% polished

14% polished

15% polished

18% polished (white rice)

1.2

Elemental content normalized to brown rice

1

0.8

0.6

0.4

0.2

0 P

S

K

Mn

Fe

Ni

Cu

Zn

As

Se

Mo

Cd

Hg

Pb