2.1 Soil collection
Nineteen paddy soils were sampled from the main rice producing areas in China. The soil samples were collected from the surface soil layer (0-20 cm topsoil) of uncontaminated farmland. The soils were air-dried at room temperature, and then passed through a 2 mm sieve. The soil pH (1:2.5 soil to water ratio), organic matter (OM, K2CrO4:H2SO4 Oil-Bath-Heating), clay content (pipette method), cation exchange capacity (CEC, 1 M ammonium acetate leaching method at pH 7.0), sulfate content (1:10 soil to water ratio, measured by ion chromatography) and free FeOx/ MnOx/ AlOx (dithionite-citrate-bicarbonate: DCB method) are measured according to Lu (2000). The results are displayed in Table S1.
2.2 Experimental design
The soil samples (6 kg) of each soil type were placed in separate plastic pots, and IHg (inorganic Hg, indicating HgCl2 solution in this study) was added at four levels (0, 1, 2, 4 mg/kg). These treatments were named as CK, Hg1, Hg2 and Hg3. Each treatment had three replications. After a 60-day aging process under flooding conditions, the soils of all pots were sampled for the determination of THg and MeHg in the soil. These soil samples were stored in a -20℃ refrigerator. The soil samples were freeze-dried and passed through a 0.149mm sieve for THg and MeHg analysis.
The rice cultivar in this study is Huiliangyou 996 (hybrid indica), which is widely planted in China. The rice seeds were pre-grown in a seeding tray for 20 days. Next, the rice seedlings were transplanted into the prepared pots after the soil aging process. The flooding conditions lasted during the whole rice growth period. Seeds from mature rice plants were harvested, washed with deionized water, and then freeze-dried at -50℃. After that, rice grains were shelled, and the resulting brown rice was milled into powder for THg and MeHg determination.
The determination of THg in soil and brown rice was performed according to the following method: The soil samples were digested with (1+1) aqua regia in 100℃ water bath heating (Meng et al. 2010), while brown rice was digested with HNO3: H2O2 (4:3) by using the high pressure vessels according to Determination of total mercury and organic mercury in foods (GB5009.17-2014) (Ren et al. 2014; Ding et al. 2014). The digestion solutions were then measured by cold vapor atomic fluorescence spectrometer (CVAFS, AF-610D2, Beijing Rayleigh Analytical Instrument, China).
For MeHg determination, soil and brown rice samples were digested by 25% KOH/methanol (w/w) and kept in an incubation shaker at 60°C for 4 h (Bloom. 1989; Liang et al. 1994), and measured by cold vapor atomic fluorescence spectrometry (Brooks Rand, USA) according to the USEPA method 1630.
For QA and QC, blanks were excluded and standard materials (GBW07407 for total Hg in soil, GBW10049 for total Hg in brown rice, ERM-cc580 for MeHg in soil and TORT-2 for MeHg in brown rice) were included in the analysis. Analysis of these materials yielded total Hg concentrations of 780±66 µg/kg, 12.8±2.49 µg/kg, 71.2±4.07 µg/kg and 322±53 µg/kg, respectively, which were similar with the certified values of 740±110 µg/kg, 12.0±2.3 µg/kg, 75.5±4.0 µg/kg and 355±56 µg/kg. The LOD (limit of detection) and LOQ (limit of quantitation) of CVAFS for total Hg determination and GC-CVAFS for MeHg determination were 2 µg/kg and 0.2 pg respectively.
2.3 Data analysis
The maximum allowable limit of THg in brown rice is 20 µg/kg according to the National Food Safety Standard of China (GB 2762-2017). The maximum allowable limit of MeHg in rice grain has not been set by any countries or international organizations. The United States Environmental Protection Agency (USEPA) recommended 0.1 µg/kg/d as the probable daily intake (PDI) of MeHg from rice consumption. Equation 1 shows the correlation between the PDI and MeHg concentration in rice (Li el at. 2015):
where the PDI is given in µg/kg/day, C is the MeHg concentration in rice (µg/kg), IR is the daily intake rate of rice (kg/d), and BW is the average weight of the exposed population (kg). The maximum allowable limit of MeHg in brown rice can be calculated by Equation 2:
The BW and IR of adult (Duan et al. 2013), children aged 6-17 years old (Zhao et al. 2016), and children aged 0-5 years old (Wang et al. 2016a) were collected, and the maximum allowable limits of MeHg in brown rice were calculated. Accordingly, the strictest dose limit of MeHg in rice was 16.3 µg/kg (Table S2).
The data in this study were collected and calculated in Excel, and the figures were drawn by SigmaPlot 10.0. The prediction models in this study were built by linear stepwise regression using SPSS 19.0. In the model building process, more parameters could increase the accuracy and likelihood function of the models, which would result overfitting phenomenon. Therefore, the Akaike information criterion (AIC) and Bayesian information criterion (BIC) were used in this study. The calculated equations applied for calculations are shown in Equations 3 and 4:
where k is the number of parameters, and L is the likelihood function. The model with the minimum AIC was chosen as the most suitable one.
where k is the number of parameters, L is likelihood function. The model with minimum BIC was chosen as the most precise model. The BIC penalized more model parameters than the AIC, which led the BIC to prefer to choose simple models with fewer parameters. The column charts and linear regression equations were generated using SigmaPlot 10.0 and SPSS 19.0 respectively.