Determination of Geographical Origin of Wuchang Rice with the Geographical Indicator by Multielement Analysis

e study aims to investigate whether the multielement analysis result can be used as a ngerprint to identify the geographical origin of Wuchang rice. e element contents of rice and soil samples from three regions in China (Wuchang, Qiqihar, and Jiamusi) were analyzed. e concentrations of 16 elements (Na, Mg, Al, K, Ca, V, Mn, Fe, Co, Cu, Zn, As, Rb, Sr, Cd, and Pb) in 194 rice samples and 112 soil samples from the harvest season in 2013 and 2014 were determined. e analysis of variance and linear discriminant analysis were performed to analyze the variation among regions and rice genotypes and classify the geographical origins of rice. Only the element of Cu showed signicant dierences among dierent genotypes. In the discriminant analysis, the overall correct identication rates of the rice samples obtained in 2013 and 2014 were, respectively, 96.6% and 89.6% and the overall correct identication rate for Wuchang rice reached 100%.


Introduction
Rice (Oryza sativa L.) is a staple food for nearly half of the world population.According to FAO statistics, the rice production of China is 144,850,000 metric tons and ranks rst in the world.It is necessary to discriminate geographical origins of rice in order to prevent mislabeling and adulteration problems [1].
Wuchang is a city located in Heilongjiang Province of China, and its rice cultivation history is more than 200 years.Based on its good quality and taste, Wuchang rice has gained many titles, such as "Green Food," "Organic Food," "Chinese Famous Brand," "Certi ed Products by American Food Nutrition Association," and "Chinese Protected Designation of Origin".Because of its limited production, Wuchang rice is frequently subjected to fraud practices, such as partial or total substitution by lower quality varieties [2].
e elements in plants are mainly from the surroundings (water and soil).Due to the di erences in hydrological characteristics and geological background, di erent plants have di erent element pro les, which provide the possibility of the geographical traceability for foodstu including both plants and animals [19]. is technique had been applied to identify the geographical origins of cereals including wheat [16] and rice [1,15,20,21].e special quality and flavor of Wuchang rice come from its unique origin and climate.It is necessary to figure out the characteristics of element fingerprints of Wuchang rice in order to protect this famous brand.
In this study, we collected rice and soil samples from Wuchang and other two adjacent regions (Qiqihar and Jiamusi) in 2013 and 2014.Different genotypes of rice were also collected in order to investigate whether the differences in multielements among genotypes existed.Furthermore, the discrimination model was established and assessed by the selected elements displaying significant differences among three regions.1).At the same time, 112 soil samples (0-20 cm and 20-40 cm) were also collected after harvesting the rice.

Sample Pretreatment.
Collected rice grains were washed by deionized water thoroughly and dried in an oven (DHG-9123A, Jinghong, China) at 38 °C until the weight was unchanged.All the rice kernel samples were ground with a sample miller (LM-3100, Perten, Sweden) to obtain fine powder.Meanwhile, soil samples were air-dried, finely ground with a ball mill (QM-3SP2, Planetary Ball Mill, Nanjing, Nanjing Nanda Instrument Plant), and sieved (the sieve pore diameter of 0.075 mm).

Multielement Analysis of Samples.
e digestion methods of each rice powder sample and the certified reference material of rice flour (GBW10011) were described as follows.Firstly, 0.25 g homogenized sample was treated in 6 mL of concentrated HNO 3 (Beijing Institute of Chemical Reagents, Beijing, China) for 2 h in the Teflon digestion vessel.en, 2 mL of BV-III grade of H 2 O 2 (Beijing Institute of Chemical Reagents, Beijing, China) was added into each vessel.After 30 min digestion for releasing nitrogen oxides, the vessels were transferred into microwave digestion instrument (CEM MARS Xpress, CEM, Matthews, USA) and the temperature was gradually increased to 180 °C within 40 min for digestion.As for soil samples, approximately 0.10 g of the homogenized sample was treated with 8 mL of concentrated HNO 3 (Beijing Institute of Chemical Reagents, Beijing, China) and then 2 mL HF (Beijing Institute of Chemical Reagents, Beijing, China) into each vessel to digest the soil sample by gradually increasing the temperature to 185 °C within 50 min.After digestion, all the liquid was cooled and diluted into a plastic vase with 18.2 MΩ•cm ultrapure water (Milli-Q, Millipore, USA) until the total solution weight was approximately 120 g.To avoid crosscontamination, Teflon digestion vessels were cleaned in a bath of 10% (v/v) nitric solution for 48 h in advance.
e concentrations of 16 isotopes ( 23 Na, 24 Mg, 27 Al, 39 K, 43 Ca, 51 V, 55 Mn, 56 Fe, 59 Co, 60 Cu, 63 Zn, 75 As, 85 Rb, 88 Sr, 111 Cd, and 208 Pb) in rice were determined with a highresolution inductively coupled plasma mass spectrometer (HR-ICP-MS) (Agilent 7700 Series, Agilent, Santa Clara, USA).An online internal standard solution of 72 Ge, 115 In, and 209 Bi was used to correct matrix effects and compensate for possible deviations in the instrument performance.e operation conditions for ICP-MS were provided as follows: radio frequency power, 1280 W; the temperature of the atomizing chamber, 2 °C; the sampling depth, 8 mm.e flow rates of cooling gas, carrier gas, and auxiliary gas were, respectively, 1.47 L•min −1 , 1 L•min −1 , and 1 L•min −1 .
e CRM of wheat flour (GBW10011) was digested and determined according to the above method to verify the determination procedure.e recoveries of all the elements of CRM ranged from 80% to 120%, and the linear range, LOD, and LOQ are listed in Table 2.
e determination procedure of all the samples was carried out in three replicates.If the relative standard deviation of internal standard concentration was higher than 5%, the sample was remeasured.
e measured data were corrected with the water content measured before digestion to obtain the element concentrations based on dry matters.

Statistical Analysis.
One-way analysis of variance (oneway ANOVA) was carried out to access the statistically significant differences in the element contents of rice and soil among different regions and different genotypes.Linear discriminant analysis was performed to establish a discriminant model for identifying the geographical origin of rice samples with SPSS for Windows version 18.0 (SPSS Inc., Chicago, IL, USA).

Elements in Rice from Different Regions.
e elements of Fe, Cu, As, Cd, or Pb were not significantly different among the samples obtained in 2013.e elements of Ca, Fe, Sr, or Pb were not significantly different among the samples obtained in 2014.e samples from Wuchang had the lowest contents of Mg, K, Fe, Co, and Zn, and the concentrations of Na, Mg, K, Ca, Co, Zn, and As in the samples from Jiamusi are the highest among three regions.e concentration of Fe in the rice samples from Qiqihar was the highest among the rice samples from three regions (Table 3).

Elements in Soil from Different Regions.
e concentrations of elements were expressed as mean ± standard deviation (SD) for soil samples from the depths of 0-20 cm and 20-40 cm (Table 4).Soil in Qiqihar was rich in Mg, Al, K, Ca, Fe, Co, Cu, Zn, As, Sr, and Cd. e soil samples from Jiamusi showed the lowest concentrations of Mg, Ca, Fe, Zn, and Cd, and Wuchang soil had the lowest contents of Na, Al, K, V, Mn, Co, As, Rb, and Pb.

Elements in Rice among Different Genotypes in One
Region.
e concentrations of elements in rice samples of the three genotypes (Longjing 31, Longjing 39, and Kongyu 131) in the region of Jiamusi from the year of 2014 were 2 Journal of Food Quality expressed as the mean and standard deviation (SD) for each of the tested categories (Table 5).ere are 11 samples of Longjing 31, 5 samples of Longjing 39, and 5 samples of Kongyu 131, respectively.Significant differences in Cu were found between Longjing 39 and Kongyu 131, but no significant difference in other elements was observed among different genotypes.

Elements in Rice between Different
Years. e concentrations of elements in rice samples of two years including three regions were expressed as the mean and standard deviation (SD) for each of the tested categories (Table 6).Except the elements of V, Cu, Cd, and Pb, other elements found significant difference between the harvest year of 2013 and 2014 (P < 0.05).

Linear Discriminant Analysis.
According to the element determination results of the samples in 2013 and 2014, we established the geographical origin discrimination model with the elements (Na, Mg, Al, K, V, Mn, Co, Zn, Rb, and Cd) with significant differences among various geographical origins for both years.e overall correct identification rates of the rice samples from three regions obtained in 2013 and 2014 were, respectively, 96.6% and 89.6%.Although the cross-validation rates decreased slightly, all the samples in Wuchang were significantly isolated from rice samples in the other two geographical origins (Table 7).According to the scatter plots, all the rice samples in Wuchang can be separated clearly from the rice samples from other regions based on multielement analysis results (Figure 1).

Discussion
According to previous studies, the element profiles in grain from three regions had their own characteristics and were closely related to the geologic background, soil type, and climate [22,23].Wuchang city belongs to temperate    e concentrations of elements in soil from Qiqihar and Jiamusi were consistent with previous results.Cao et al. [25] reported that the concentration ranges of As, Cu, Pb, and Zn in rice soil of Sanjiang Plain were, respectively, 1.36-80.98,1.97-665.62,0.03-27.86,and 4.61-125.50mg/kg.e mean values obtained in this study falls within these ranges.In addition, the soil types of Qiqihar and Jiamusi are, respectively, black soil and planosol.e contents of As and Zn  Journal of Food Quality in black soil are higher than those in planosol [25].Our study also gave the consistent results.
In our study, only the element of Cu showed significant differences among the samples of different genotypes.e similar results had been reported in wheat genotypes [26,27].ey found that, compared with the environment or region, the genotype contributed most to the variation of Cu in rice, indicating that the genotype of rice also affected the accumulation of Cu in grains.When discriminating the geographical origin of rice by the element of Cu, it would be better to consider only one genotype in case of variety interference.
Apart from the factor of the genotype, the concentrations of most elements (Na, Mg, Al, K, Ca, Mn, Fe, Co, Zn, As, Rb, and Sr) were found to be significantly different among two years.In previous reports, the contents of Mg, Al, Ca, Fe, Zn, and As in wheat were most affected by the harvest year comparing with the region or genotype according to the result of multiway variance analysis [19], and the harvest year was the main source of the variations of the concentrations of Fe and Zn in maize [28]; these results are consistent with ours.e significant differences for some element contents could be explained by the changing weather conditions such as the annual temperature, precipitation, and sunshine.
According to the result of linear discriminant analysis, the overall correct identification rate of the rice samples from three regions was higher than 89.6% for single year samples.Satisfactory results were obtained for distinguishing Wuchang rice from rice produced from other regions (correct identification rate: 100%) based on mineral profiles in two years, and there was no significant concentration difference in the elements (except Cu) among the three genotypes in the same region.It is promising and reliable to identify rice according to their geographic origins by using multielement analysis in combination with statistical analysis.
ere are also some other methods for rice geographical traceability, such as stable isotopic ratios, near-infrared technique, and volatile component.Among them, the multielement method is time-saving because all the elements can be determined once for each sample.In addition, the concentration of element in samples is very stable compared with organic composition since some organic composition in food may change with the temperature, sunshine, and storage time.As a result, multielement analysis is a promising method for Wuchang rice geographical traceability and is more suitable for application in marketing.

Table 2 :
Quality control for determining the elements of rice.

Table 3 :
Elements for all rice samples harvested in the year of 2013 and 2014 among three different regions (µg•g −1 ).

Table 1 :
e information of rice samples from three regions.
Different letters in rows are statistically significantly different at P < 0.05.

Table 5 :
Elements for all rice samples among three different genotypes at Jiamusi from the year of 2014 (µg•g −1 ).
Different letters in rows are statistically significantly different at P < 0.05.

Table 6 :
Elements for all rice samples between two different years (µg•g −1 ).

Table 7 :
Linear discriminant analysis of elements in rice from three regions.