Health Risk Assessment of Inorganic Mercury and Methylmercury via Rice Consumption in the Urban City of Guiyang, Southwest China

Rice consumption is the main methylmercury (MeHg) exposure route for residents in mercury (Hg) mining areas. However, there is limited studies on mercury in commercial rice, which has high liquidity and can be directly consumed by urban residents. This study measured the total Hg (THg) and MeHg concentrations in 146 rice samples purchased from the markets in Guiyang city, southwest China, and both the inorganic Hg (IHg) and MeHg estimated daily intakes (EDIs) and hazard quotients (HQs) were calculated according to rice consumption. The THg concentrations in all rice samples (range: 0.97 to 13.10 μg·kg−1; mean: 3.88 μg·kg−1) were lower than the Chinese national standard (20 μg·kg−1). The average MeHg concentration in rice was 1.16 μg·kg−1. The total HQs (THQs) ranged from 0.0106 to 0.1048, with a mean of 0.0462, which was far lower than 1. This result suggests that there were low Hg exposure levels through consumption of commercial rice in residents of Guiyang.


Introduction
Mercury (Hg), one of the most toxic heavy metal pollutants, has been a public concern since the recognition of Minamata disease in 1956. Both inorganic and organic Hg exist in the environment. Inorganic Hg (IHg) is much less toxic than methylmercury (MeHg) to humans [1], and the absorption rate of IHg in food by the human, which is about 8% [1][2][3][4], also much less than that of MeHg (95%) [5,6]. MeHg is the most toxic organic form of organic Hg; MeHg can cross the blood-brain barrier and through placenta in humans after entering the body, causing permanent damage to the central nervous system [1,7]. The Minamata Convention, formulated by the United Nations Environment Programme, came into effect on August 16, 2017, which aimed at controlling and reducing mercury emissions globally. Fish consumption is considered to be the primary route of human MeHg exposure [6,8]; however, recent studies have reported that rice consumption is the main MeHg exposure pathway for local residents in Hg mining regions due to elevated MeHg concentrations [2,5,9,10].
As a staple food, rice constitutes 20 percent of the total food energy intake of the world's population [11]. In Asia, the energy from rice and its by-products contributes to over 70% of residents' daily dietary intake [12]. Studies have found that rice that grown in Hg mining areas can accumulate high concentrations of MeHg in grain, and concentrations of MeHg as high as 174 µg·kg −1 were recorded in brown rice, approximately 2-3 orders of magnitude higher than that in the edible parts of other crops [9,13].
Within the last decade, studies of Hg rice exposure have mainly concentrated on Hg-polluted areas, particularly at abandoned Hg mining regions in Guizhou Province, southwest China. Horvat et al. [13] first confirmed the high contamination of rice with Hg (highest concentration: 569 µg·kg −1 for THg; 145 µg·kg −1 for MeHg) in the Wanshan Hg mine in Guihou Province. Feng et al. [5] first reported a significant correlation between the estimated rice MeHg intake and hair MeHg levels in the Wanshan Hg mining region, suggesting that rice consumption was the main pathway of MeHg exposure for local populations. Recently, Li et al. [14] reported that Hg exposure via rice consumption in the Wanshan region was comparable to that via a fish diet.
Therefore, Guiyang was selected as the study area, and market rice from Guiyang was collected. The purpose of this study was to assess the health risks of both inorganic Hg (IHg) and MeHg exposure in association with the consumption of commercial rice from markets in Guiyang City, the capital city of Guizhou Province. We aimed to examine and calculate (1) the concentrations of THg and MeHg in commercial rice; (2) the estimated values of the daily intakes (EDIs) of MeHg and inorganic mercury (IHg) via rice consumption; and (3) the hazard quotients (HQs) and total hazard quotients (THQs). The results will provide a better understanding of the MeHg and IHg exposure risks via consumption of commercial rice in Guiyang City, southwest China.

Study Area
Guiyang (106 • 07 E-107 • 17 E; 26 • 11 N-26 • 55 N) is the capital of Guizhou Province, southwest China, with an altitude of approximately 1100 meters. The annual mean temperature is 15.3 • C. The annual average relative humidity is 77%. The annual mean rainfall is 1129.5 mm. Guiyang City has a population of approximately 4.8 million, whose staple food is rice. In 2016, the output of rice in Guiyang was 17,810 tons, accounting for about 40% of the total output of cereal crops. And the per capita consumption of rice is 61.53 kg·year −1 (169 g·d −1 ), occupying approximately 58% of the total food consumption.

Sample Collection and Preparation
A total of 146 white rice samples (84 brands) were purchased from markets in Guiyang from July 22 to August 22 in 2017. The samples covered both Chinese domestic (n = 137) and imported rice (n = 9, from Cambodia, Vietnam and Thailand). According to the package information, rice samples were briefly categorized into two main subspecies-Japonica and Indica. All of the rice samples collected from the markets were transported to the laboratory, rinsed with ultrapure water, freeze-dried, ground with a grinder (IKA-A11, IKA, Staufen, Germany), and stored in plastic bags for analysis.

Analytical Methods
For THg analysis, approximately 0.5 g dry rice samples were weighed and were then digested at 95 • C in a water bath with a fresh mixture of HNO 3 and H 2 SO 4 (4:1, v/v). The THg concentrations in rice samples were determined by BrCl oxidation, SnCl 2 reduction, purging, gold amalgamation, and cold vapour atomic fluorescence spectrometry (CVAFS) detection following Method 1631e [20].
For MeHg analysis, approximately 0.5 g of dry rice samples were weighed for digestion using 25% KOH-methanol/solvent extraction at 80 • C in a water bath. During extraction, acidification was needed after digestion of the rice sample. Then, MeHg in rice samples was extracted with dichloromethane and back-extracted into water. Finally, the concentration of MeHg was measured by gas chromatography (GC)-CVAFS according to Method 1630 [21]. IHg was obtained by the difference between THg and MeHg.

Quality Assurance and Quality Control
The standard reference materials (GBW(E)100359 for THg; TORT-2 for MeHg), duplicates, and method blanks were used for data quality control. The rice samples were measured in three replicates. The recoveries of THg and MeHg in the certified reference material ranged from 93% to 110% and from 90% to 115%, respectively. The detection limits for THg and MeHg were 0.013 µg·kg −1 and 0.003 µg·kg −1 , respectively.
All acids, so as HNO 3 , H 2 SO 4 and HCl (Sinopharm, Shanghai, China) used in the analysis were ultra-pure grade, and all other reagents, like KOH, were of an analytically pure grade. The CH 2 Cl 2 (Tedia, Fairfield, OH, USA) was Chromatographic pure grade. The Hg concentrations was mensurated by the Tekran 2500 (Tekran Inc., Toronto, ON, Canada). The Brooks Rand Model III (Brooks Rand, Seattle, WA, USA) was used to measure the MeHg concentrations. The water used in the analysis was double-deionized water (DDW). All of the glassware was soaked in nitric acid for more than 24 h and washed with DDW.

Assessment of Human Health Risk
The estimated daily intakes (EDI) of MeHg and IHg were calculated based on the concentrations of MeHg and IHg in milled rice and rice consumed per capita according to Equation (1). The non-cancer health hazard was estimated by employing hazard quotient (HQ) Equations (2) and (3). The health risk of Hg exposure through rice consumption was estimated according to the total hazard quotient (THQ), which was calculated by Equation (4): EDI is given in micrograms per kilogram of body weight per day (µg·kg −1 ·d −1 ); bw (body weight) is the average weight of the adult population. E F is the exposure frequency (365 days/year); E D is the exposure duration (70 years) and equal to the average lifetime; T A is the average exposure time for non-carcinogens (365 days/year × number of exposure years, assuming 70 years in this study) [16,22]; C are the IHg and MeHg concentrations in milled rice (µg·kg −1 ); IR is the daily intake rate of rice; HQ is the hazard quotient; RfD is the reference dose for the substance; PTWI is the provisional tolerable weekly intake of inorganic mercury; and THQ is the total hazard quotient. In this study, bw is 60 kg [9]; IR is 169 g·d −1 [23]. RfD is 0.1 µg·kg −1 ·d −1 [1], indicating the maximum dose of the compound (µg·kg −1 ·d −1 ) below which there is no known hazard of health effects. The PTWI is 0.57 µg·kg −1 ·d −1 (equal to 4 µg·kg −1 ·week −1 ) [24,25]. HQ and THQ were used to estimate the non-cancer health hazard. When the value is >1, it indicates a potential health risk due to Hg exposure from rice consumption. If the value is <1, it is assumed to be safe according to the risk of non-carcinogenic effects. If the HQ exceeds one, there is a chance that non-carcinogenic effects may occur, with a probability which tends to increase as the value of HQ increases [26].

Statistical Analysis
Statistical analysis of THg and MeHg in rice was performed using Excel 2016 (Microsoft, Redmond, WA, USA), Origin 8.0 (Originlab, Northampton, MA, USA) and SPSS 22.0 (IBM, Armonk, NY, USA). Pearson correlation was employed to study the correlation coefficients. Independent-sample t tests were performed to indicate the significance of the average values of THg and MeHg in rice.

Whole Market Samples
The THg concentrations in all rice samples ranged from 0.97 to 13.1 µg·kg −1 , with a mean of 3.88 µg·kg −1 . The THg concentrations in this study were lower than the maximum value of 20 µg·kg −1 recommended by the Ministry of Public Health of China [19].
Compared with the previously reported Hg values in rice from Chinese markets, the average value of 3.88 µg·kg −1 (n = 146) in rice from Guiyang market that we found was slightly lower than that reported data for THg in rice in China. In Zhoushan Island, the mean THg concentration was 9 µg·kg −1 (n = 6) [26]. Qian et al. [16] reported that the THg concentrations ranged from <0.02 to 31 µg·kg −1 , with a mean of 5.8 µg·kg −1 (n = 712), in rice from markets in 20 provinces. In Jiangsu province, the THg concentrations ranged from 1.0 to 13 µg·kg −1 , with a mean of 5.7 µg·kg −1 (n = 23) [27]. Li et al. [17] reported that the rice concentration from 7 provinces ranged from 0.86 to 47.2 µg·kg −1 , with a mean of 10.1 µg·kg −1 . Huang et al. [18] reported a mean THg concentration of 5 µg·kg −1 (n = 224) in rice from Zhejiang province. Interestingly, there is a decreasing trend of Hg in rice from markets.
Our results for both THg and MeHg were comparable or slightly lower than results reported from markets abroad (Figure 1). Batista et al. [29] reported that the THg concentrations of 44 rice samples in Brazilian market varied from 0.3 to 13.4 µg·kg −1 , representing most brands in the whole country. The THg and MeHg concentrations of rice samples collected from the supermarkets of Kampong city in Cambodia were in the range of 6.16-11.7 µg·kg −1 (a mean of 8.14 µg·kg −1 ) and 1.17-1.96 µg·kg −1 (a mean of 1.44 µg·kg −1 ) [30], respectively. Recently, Brombach et al. [31] reported that the THg and MeHg concentrations of 87 rice samples purchased in supermarkets in the United Kingdom, Germany and Switzerland were 3.04 ± 2.7 µg·kg −1 and 1.91 ± 1.07 µg·kg −1 , respectively.

Variations in Types
The average THg concentrations of Japonica and Indica rice were 3.96 ± 1.88 and 3.80 ± 1.90 µg·kg −1 , respectively. The MeHg concentrations of Japonica and Indica rice were 1.10 ± 0.48 and 1.22 ± 0.57 µg·kg −1 , respectively ( Table 1). The proportion of MeHg to THg in Japonica rice (31 ± 13%) was slightly lower than that in Indica rice (35 ± 15%) ( Figure 3). No significant difference of the THg (t test, p = 0.61) and MeHg (t test, p = 0.17) concentrations between Japonica rice and Indica rice were observed in the present study.

Variations in Types
The average THg concentrations of Japonica and Indica rice were 3.96 ± 1.88 and 3.80 ± 1.90 μg·kg −1 , respectively. The MeHg concentrations of Japonica and Indica rice were 1.10 ± 0.48 and 1.22 ± 0.57 μg·kg −1 , respectively ( Table 1). The proportion of MeHg to THg in Japonica rice (31 ± 13%) was slightly lower than that in Indica rice (35 ± 15%) ( Figure 3). No significant difference of the THg (t test, p = 0.61) and MeHg (t test, p = 0.17) concentrations between Japonica rice and Indica rice were observed in the present study.    The THg concentrations in Japonica rice were similar to those in Japonica rice (n = 24) (mean: 3.46 µg·kg −1 ; range: 0.73-10 µg·kg −1 ), as reported by Brombach et al. [31]; however, the MeHg concentrations of Japonica rice (1.63 µg·kg −1 ) were slightly higher than those found in the present study. Previous studies reported that different rice types may influence the MeHg concentrations in rice [27,33,34].

Risk Assessment via Rice Consumption
The EDIs of IHg via rice consumption in the present study ranged from 0.0012 to 0.0326 µg·kg −1 ·d −1 , with a mean of 0.0076 µg·kg −1 ·d −1 . Compared to the Wanshan mining area, these values were 1-2 orders of magnitude lower than those reported by Li et al. [2], in which the EDI IHg values through rice consumption in two sites were 0.3191 and 0.1632 µg·kg −1 ·d −1 , respectively.

Conclusions
The study illustrated the THg and MeHg concentrations in the commercial rice samples from Guiyang. Results showed that THg concentrations were lower than the national tolerance limit of 20 μg·kg −1 in rice, suggesting a safe level of Hg. There was no significant difference in the THg and MeHg concentrations of the different types of rice. Low THQs (<1), based on the EDIs values, indicate the low Hg exposure of rice consumers in Guiyang. Though the generally low concentrations of THg and MeHg in rice from Guiyang markets, we suggest that the residents should consider different brands to avoid relatively high Hg exposure. The HQs of IHg exposure via rice consumption in the present study ranged from 0.0021 to 0.0570, with a mean value of 0.0134, which were one order of magnitude lower than those reported by Li et al. [2]. Considering the different brands, the HQ IHg values ranged from 0.0029 to 0.0554, and the HQ IHg of Japonica and Indica rice were 0.0047-0.0570 (mean: 0.0141) and 0.0021-0.0554 (mean: 0.0127), respectively. The HQs of IHg exposure via rice consumption in this study were less than 1, which indicated there was no health risk of IHg exposure via rice consumption for residents in Guiyang.
The EDIs of MeHg exposure through rice consumption in the present study were in the range of 0.0002-0.0076 µg·kg −1 ·d −1 . The top ten highest and lowest EDI MeHg values from different brands are showed in Figure 4. These EDI MeHg values in different brands ranged from 0.0002 to 0.0074 µg·kg −1 ·d −1 , with a mean of 0.0033 µg·kg −1 ·d −1 . The highest EDI MeHg value of 0.0074 µg·kg −1 ·d −1 was found for HBDM, which was approximately 35 times higher than that of the lowest EDI MeHg value for YYP. Considering the types of rice, the EDI MeHg values via consumption of Japonica and Indica rice ranged from 0.0002 to 0.0076 µg·kg −1 ·d −1 , with a mean of 0.0031 µg·kg −1 ·d −1 , and 0.0002-0.0075 µg·kg −1 ·d −1 , with a mean of 0.0034 µg·kg −1 ·d −1 , respectively.
Based on EDI MeHg and RfD, the calculated HQ MeHg values in the present study were between 0.0020 and 0.0756. Approximately 90% of the HQ MeHg values from the different brands were distributed between 0.0100 and 0.0600 (Table 2). Table 1 showed that the mean HQ MeHg values of Japonica rice and Indica rice were 0.0311 (0.0020-0.0756) and 0.0345 (0.0023-0.0750), respectively. In comparison, the EDIs and HQs of MeHg from Japonica rice and Indica rice were within the same range, exhibiting no significant differences. Compared to the HQ MeHg values reported in the literature, our data were comparable or lower than those reported from non-contaminated areas as well as markets. The HQ MeHg values found in the present study were lower than the HQ MeHg values found in the background site (a mean of 0.13 ± 0.052), as reported by Rothenberg et al. [33]. Li et al. [17] reported the HQ values of MeHg through rice consumption, ranging from 0.04 to 0.08, in 7 provinces in China. The highest HQ MeHg value of 0.0756 in this study was far below the highest HQ MeHg value of 0.39 via rice consumption of rice purchased from European supermarkets [31]. Moreover, the HQ MeHg values for residents in Guiyang were also lower than the HQ MeHg values reported for residents of Hg mine areas in China [9,35].
In the present study, the THQ values ranged from 0.0106 to 0.1048, with a mean of 0.0462, which was far below 1. This result might suggest low Hg exposure through the consumption of rice purchased from markets in Guiyang for the residents. Our data suggest that residents should regularly change the brands they consume.

Conclusions
The study illustrated the THg and MeHg concentrations in the commercial rice samples from Guiyang. Results showed that THg concentrations were lower than the national tolerance limit of 20 µg·kg −1 in rice, suggesting a safe level of Hg. There was no significant difference in the THg and MeHg concentrations of the different types of rice. Low THQs (<1), based on the EDIs values, indicate the low Hg exposure of rice consumers in Guiyang. Though the generally low concentrations of THg and MeHg in rice from Guiyang markets, we suggest that the residents should consider different brands to avoid relatively high Hg exposure.