Demethylation of Methylmercury in Growing Rice Plants: an Evidence of Self-Detoxiﬁcation

Environmental Pollution 210 (2016) 113e120

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Demethylation of methylmercury in growing rice plants: An evidence of self-detoxiﬁcation

Xiaohan Xu a, b, 1, Jiating Zhao a, 1, Yunyun Li a, b, Yuqin Fan c, Nali Zhu d, Yuxi Gao a, Bai Li a, * Hanyu Liu e, Yu-Feng Li a, a State Environmental Protection Engineering Center for Mercury Pollution Prevention and Control, and Laboratory of Metallomics and Nanometallomics, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China b University of Chinese Academy of Sciences, Beijing 100049, China c Shandong Maternity and Child Care Hospital, Jinan 250001, China d Laboratory of Proteomics, Protein Science Core Facility Center, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China e Beijing National Day School, Beijing 100049, China article info abstract

Article history: Mercury (Hg) is a global pollutant that poses a serious threat to human and the environment. Rice was Received 17 July 2015 found as an important source for human exposure to Hg in some areas. In this study, the transportation Received in revised form and transformation of IHg and MeHg in rice plants exposed to IHg or MeHg were investigated. The IHg 8 December 2015 and MeHg concentrations in rice roots and shoots collected every five days were analyzed by HPLC-ICP- Accepted 8 December 2015 MS and SR-XANES. When exposed to MeHg, the percent of IHg in rice roots and shoots increased while Available online 18 December 2015 MeHg decreased significantly, suggesting prominent demethylation of MeHg occurred. However no Handling editor: Eddy Y. Zeng notable MeHg was found in both roots and shoots of rice plant when exposed to IHg. SR-XANES analysis further confirmed the demethylation of MeHg with rice. This study provides a new finding that deme- Keywords: thylation of MeHg could occur in growing rice, which may be a self-defense process of rice plant. Methylmercury © 2015 Elsevier Ltd. All rights reserved. Inorganic mercury Demethylation Rice SR-XANES

1. Introduction magnify through the food chain. For humans, the primary source of MeHg exposure is the consumption of contaminated foodstuff Mercury (Hg) is a global pollutant that poses a serious threat to (Gilmour et al., 1992; Celo et al., 2006; Harris et al., 2003; human health and the environment. Due to its bioaccumulation, Guimaraes et al., 2000). Once absorbed, MeHg can easily cause long-range transportation, strong neurotoxicity and persistence in permanent damage to central nervous system, especially for the the environment, Hg and its compounds have been regarded as developing fetuses and infants (Clarkson, 1997). priority-controlled pollutants by many agencies (Mastrine et al., Wetlands and the sediment-water interfaces are predominant 1999). As the largest producer, user and emitter of Hg in the sites in the environment where the methylation of Hg usually world, China is faced with great pressure of Hg pollution (Cheng happens (Guimaraes et al., 2000; Driscoll et al., 1998). Microbial and Hu, 2012; Zhang and Wong, 2007). activity is an important way of Hg methylation. Iron-reducing The toxicity of Hg is strongly dependent on its chemical species, bacteria, sulfate reducing bacteria and methanogens are three among which methylmercury (MeHg) is the most poisonous form main MeHg producers (Siciliano and Lean, 2002; King et al., 2001). and more prone to bio-accumulation in organisms. MeHg in the Recent studies found that methylation can also occur in the paddy environment can be generated by methylation of the inorganic ﬁeld ecosystem and the produced MeHg can be absorbed and mercury (IHg) through biological and chemical processes and can accumulated in rice grain (Galloway and Branﬁreun, 2004). As is reported in Qingzhen and Wanshan District, Guizhou Province, where acetic acid plants and largest mercury mines exist, the * Corresponding author. concentrations of mercury in the local soils can reach 83.9 mg/kg E-mail address: [email protected] (Y.-F. Li). and 496 mg/kg, respectively. Also in these areas, the total mercury 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.envpol.2015.12.013 0269-7491/© 2015 Elsevier Ltd. All rights reserved. 114 X. Xu et al. / Environmental Pollution 210 (2016) 113e120

(THg) concentrations in rice grains can be up to 8.8e569 mg/kg, in days for four times, respectively. Nutrient solutions with different which the concentration of MeHg is 1.20e170 mg/kg (Li et al., 2010a; dosages of IHg or MeHg were collected and renewed every time Qiu et al., 2008; Horvat et al., 2003). after sampling. The culture media was updated every five days with As a staple food for many people around the world, especially in new prepared Hg solutions. East and Southeast Asia, rice has attracted constant attention over the last several decades due to the increasing soil pollution (Horvat et al., 2003; Zhang et al., 2010a). For example, in some Hg mine 2.2. Total Hg (THg) analysis in IHg or MeHg exposed rice plants areas in China, the concentration of Hg in rice is much higher than the national limit of Hg in food, which has restricted the develop- Tissues separated from rice seedlings under Hg exposure were ment of agriculture and caused great damage to residents' health lyophilized at 50C and ground into fine powders after rinsed (Wang et al., 2011; Meng et al., 2014). Our previous work, whatever with deionized water completely. A triplicate of 20e30 mg each for the lab or field studies, indicated that the ratio of MeHg to IHg powdered sample was digested with a freshly prepared mixture of increased gradually from the roots to the grains in rice plants. MeHg 5 mL HNO3 (BV-III) and 0.5 mL H2O2 (MOS) in a digestion tank. The even account for over 50% of the total mercury in the rice grains mixtures were left overnight at room temperature and digested at (Zhao et al., 2014; Li et al., 2015). 160C in a series of sealed pots for 5 h (Zhao et al., 2013a). Subse- Studying the processes of uptake, transportation, trans- quently, the mixtures were volatilized to the volume of approxi- formation and accumulation of different forms of Hg in rice plant is mately 1 mL at the temperature of 90 C and diluted to 4 mL with helpful to understand the mechanism of Hg accumulation in rice 2% (v/v) HNO3 containing 0.1% (v/v) b-mercaptoethanol for Hg grain. Hg demethylation and methylation have ever been found in measurement. lower aquatic plants like unicellular diatom (Deng et al., 2013) and The concentration of total Hg in rice tissues were measured by some kinds of fish (Wang et al., 2013). However, it is still unclear if using ICP-MS (Thermo Elemental X7, USA) in a normal mode. The demethylation and methylation of Hg could also occur in higher running conditions of ICP-MS were listed as following: nebulizer plants like rice plant. Besides, demethylation of MeHg might be a (glass concentric), plasma gas flow (13.0 L/min), auxiliary gas flow self-defense process for organisms when facing Hg stress (Marie (0.70 L/min), nebulizer gas flow (0.72 L/min), collision gas (7.28% (v/ et al., 1995; Collin et al., 2009). v) H2 in He), dwell time (100 ms). A normal tuning solution (Bi In this study, rice plants were exposed to IHg or MeHg under 10 mg/L) was used during the process of optimization every time. hydroponic conditions. The concentrations of both IHg and MeHg Transportation factor (TF), the ratio of THg in rice shoots to that in roots and shoots were investigated using HPLC-ICP-MS in com- in rice roots, is used to express the Hg transportation ability from bination with synchrotron radiation X-ray absorption near-edge roots to aerial parts of rice plants (Meharg and Hartley-Whitaker, structure (SR-XANES) techniques to see if methylation or deme- 2002). thylation could occur or not in growing rice.

2. Materials and methods 2.3. IHg and MeHg analysis in IHg or MeHg exposed rice plants

According to previous studies, in typical Hg polluted areas such Forty mg fine powders of roots and shoots were transferred into as Wanshan and Qingzhen Districts, Guizhou province of South- 5 mL centrifuge tubes with 2.5 mL 6 M HCl added to extract IHg and western China, Hg polluted water from abandoned Hg mining and MeHg in the samples. Following 45 min of ultrasonic oscillation, the discharged by chemical factories were used as irrigation sources for mixtures were shaken at 28 C overnight by using an orbital shaker. farmlands and paddy fields (Li et al., 2015; Matsuyama et al., 2009). Subsequently, after 2 extractions, the upper extracts were trans- The Hg concentration in Hg polluted water and soil in the sample ferred into 15 mL centrifuge tubes and pH was adjusted to the value sites of Wanshan District is about 116.2 mg/L and 29.6 mg/kg, of 6.7 by adding ammonium water. 1 mL of freshly adjusted solu- respectively (Zhao et al., 2014). In this study, the exposure dosage of tions were extracted and diluted to 4 mL with previously prepared Hg is set to be 0.5 mg/L and 1 mg/L to roughly simulate the growth mobile phase and filtered by a 0.22 mm microporous membrane conditions of rice in Hg polluted areas. before analysis. The IHg and MeHg concentrations in the nutrient solutions collected were also analyzed following the aforemen- 2.1. Rice cultivation and sample preparation tioned procedure. The IHg stock standard solutions (HgCl2,GBW 08617, National Research Centre for CRMs, China) and MeHg stock Mature and undamaged rice seeds (Oryza sativa L.) selected in standard solutions (CH3HgCl, GBW 08675, National Research advance were sterilized with 10%(V/V) sodium chloride (NaCl) so- Centre for CRMs, China) were diluted with mobile phase to obtain lution for 15 min and washed with running water for 6 h. Subse- standard solutions used in the analysis. The well conducted stan- quently, after thoroughly rinsed with Milli-Q water (18.2 MU cm), dard curve and determination limit of MeHg and IHg (LOD, 0.2 mg/L þ þ all seeds were germinated on filter papers in an incubator at 28 C for MeHg and 0.3 mg/L for Hg2 ) analyzed by HPLC-ICP-MS hy- in darkness for one week. The homogeneous seedlings were phenated technique are shown in Fig. SI-1 and Table SI-1. selected and transplanted in 25% Hoagland solution for three weeks The concentrations of IHg and MeHg in samples were analyzed in an artificial climatic chamber with optimal conditions for seed- with HPLC-ICP-MS (Zhao et al., 2014; Li et al., 2007). Different Hg lings growth: temperature 30 C in daytime (14 h, humidity 60%, species were separated by a high performance liquid chromatog- light intensity 100%); about 26 C at night (10 h, humidity 80%). raphy system which includes two primary parts: a liquid chroma- Hoagland solution was replenished twice a week. tography pump (WAT055028 metal-free 626 pump, Waters, After three weeks of growth, the seedlings were cultivated in Milford, USA) and a 5 mm Symmetryshield RP18 column newly prepared Hoagland solutions and each group includes 40 (150 3.9 mm, Waters, Milford, USA). The flow rate and sample seedlings. The IHg or MeHg concentration in the culture media was injection volume during analysis were 1 mL/min and 100 mL, 0.5 or 1 mg/L with pH 6.5, respectively. As culture time went on, respectively. The mobile phase (pH ¼ 6.7) used in the detection total mercury (THg) concentration in culture media decreased and contained 60 mmol/L ammonium acetate, 5% (V/V) methyl alcohol, the pH value changed a little (6.0e6.5). Rice roots and shoots were 0.3% (V/V) b-mercaptoethanol and were filtered by a 0.22 mm harvested and washed with deionized water thoroughly every five microporous membrane prior to use. X. Xu et al. / Environmental Pollution 210 (2016) 113e120 115

2.4. Hg speciation analysis using SR-XANES 0.5 mg/L MeHg exposed rice showed a rising trend in general as the cultivation time went on, albeit the reduction from day 5 to day 10. The roots of rice plants under 1 mg/L IHg and MeHg exposure The phenomenon was found to occur more remarkably in 1 mg/L during the whole cultivation period were ground into uniform fine IHg or MeHg exposed groups. As illustrated in Fig. 1f, while being powders after lyophilized at 50 C. Then the powders prepared exposed to 1 mg/L IHg solution, the TF of THg in rice plants reduced were pressed to 1 mm thick tablets and smeared on Scotch tapes much more sharply than that exposed to 0.5 mg/L IHg. Additionally, before detection. TF of THg in rice plant exposed to MeHg solution increased grad- The Hg L3-edge XANES of rice roots were collected at 14 W ually from day 5 to day 20, and was much higher than that in rice beamline (3.5 GeV, 250 mA) in Shanghai Synchrotron Radiation exposed to IHg solution after 15 days cultivation, which is consis- Facility (SSRF, China). A Si (111) double-crystal monochromator was tent with the report that MeHg is more liable to be transported to applied in the beamline 14 W equipment to minimize the harmonic the overground part than IHg in plants (Schwesig and Krebs, 2003). content of the beam. The XANES data of all samples were recorded The differences between THg accumulation in rice roots and shoots in fluorescence mode (using a 19-elemental Ge solid detector) at under different Hg species exposure, along with the discrepancy in room temperature. By using ATHENA software, data of rice roots TF of THg in rice imply the different mechanism of absorption and were fit to standard compounds after background subtraction and translocation for IHg and MeHg in rice plants. normalization. Components that have the least proportion of composition were removed and the rest were fit repeatedly until an 3.2. IHg and MeHg in rice plants optimal fit was achieved (Zhao et al., 2013b; Ressler et al., 2000; Li et al., 2010b). The discrepancy between IHg and MeHg in rice roots and shoots under different concentrations of IHg or MeHg exposure are dis- 2.5. Quality control played in Fig. 2.InFig. 2a, IHg concentration in roots and shoots of 0.5 mg/L IHg exposed rice increased gradually with the increase of The quality assurance/control for THg and MeHg analysis was cultivation time while no MeHg could be detected in roots during conducted using certified reference material (CRM), replicates, the whole cultivation period. However, it is worth mentioning that extraction and recovery efficiency and the method blank. Details of a small amount of MeHg was detected in rice shoots sampled on the QA/QC were described in the supporting information (see SI file, day 20, but it is not clear if the MeHg comes from the roots or Table SI-2 and Table SI-3). methylated in the shoots. For the metabolism of heavy metals in plant, it has been found that there is arsenic methylation in rice 3. Results and discussions plants (Arao et al., 2011; Zhao et al., 2009), however, the evidence regarding the in vivo methylation of Hg at the present stage is very 3.1. THg accumulation and translocation in rice plants few. Whether or not the Hg methylation could occur in rice plants still remains unclear. As displayed in Fig. 2b, there was IHg detected As a control group, THg, MeHg and IHg in rice plant without Hg in roots and shoots of rice under 0.5 mg/L MeHg exposure and the exposure was much lower than the limit of detection (LOD) of concentration kept growing evidently as the cultivation time went HPLC-/ICP-MS. As shown in Fig. 1, THg concentration of 0.5 mg/L on. Conversely, the MeHg concentration in rice roots decreased IHg exposed rice ranged between 80 and 240 mg/kg in roots gradually. Combining with the stably increasing trend of THg in rice (Fig. 1a) and from 3 to 5 mg/kg in aboveground parts (Fig. 1b). THg roots under 0.5 mg/L MeHg exposure (shown in Fig. 1a), the concentration in both roots and shoots increased as the cultivation decrease of MeHg displayed that with the increase of cultivation time went on, which can also be observed in the 0.5 mg/L MeHg time, MeHg was increasingly transformed into IHg. So far, there is exposed group (110e260 mg/kg in roots and 7e20 mg/kg in shoots, almost no study on in vivo MeHg demethylation in higher plants. In as shown in Fig. 1a and b). This trend was also true in 1 mg/L IHg or the present study, at least, the results can hint the close correlation MeHg exposed group (as shown in Fig. 1c and d). The THg con- of MeHg demethylation with rice plant growth. Similar to the centration of rice roots is remarkably higher than that of rice shoots growing trend of IHg concentration in rice shoots, the MeHg con- in both exposure groups, which implies that the root surface acts as centration increased as the time went on. This suggests that both a potential Hg barrier and the translocation of Hg through the root IHg and MeHg can accumulate in rice shoots but we cannot figure system to the overground parts can be reduced (Meng et al., 2014). out whether IHg found in rice shoots was derived from in vivo In Fig. 1, compared to the 0.5 mg/L IHg or MeHg exposed group, the transformation or due to the transportation and accumulation of THg in rice roots of 1 mg/L IHg or MeHg exposed group increased IHg from rice roots. Under 1 mg/L IHg exposure (Fig. 2c), the con- more sharply as the cultivate time increased, suggesting that when centration of IHg in rice roots and shoots followed the same pattern exposed to higher dosages of Hg, more Hg were absorbed and as the 0.5 mg/L IHg exposed group, but there were some exceptions accumulated in rice roots. The significant differences between Hg on MeHg concentrations in rice tissues. Contrary to the situation in concentrations in rice roots and in shoots under IHg or MeHg the 0.5 mg/L IHg exposed rice group, the MeHg was detected in rice exposure demonstrates the low transportation efficiency of Hg roots after 15 days cultivation. Just as Fig. 2c showed, rice roots from root to the above-ground parts of rice plant. Besides, when sampled day 15 and day 20 had a small amount of MeHg while a exposed to MeHg, the THg in rice roots and shoots are prominently little MeHg in rice shoots was detected during the whole cultivation higher than that of rice roots and shoots exposed to the same period. We imply that the discrepancy between situations exposed dosage of IHg, especially in 1 mg/L IHgor MeHg exposed group. In a to two different doses of IHg may be attributed to the relatively low recent study by Zhang et al. (Zhang et al., 2010b), it was found that MeHg concentration in the samples. That is, MeHg in rice roots of the bioaccumulation factors of MeHg were on average more than 0.5 mg/L IHg exposed group might be lower than the detection limit 800 times higher than those of IHg in rice. In the present study at a of HPLC-ICP-MS and could not be detected. Under 1 mg/L MeHg controlled condition and the same IHg or MeHg exposure levels, it exposure (Fig. 2d), the changes of IHg and MeHg concentrations in also confirms that MeHg might be much more prone to accumu- rice roots and shoots are similar as the situation of rice plants under lating in rice plant than IHg can do. 0.5 mg/L MeHg exposure, with the distinction that MeHg concen- In Fig. 1e, TF of THg for 0.5 mg/L IHg exposed rice reduced tration in rice shoots remains barely unchanged in the rice culture gradually and notably and tended to be stable finally. TF of THg for process. 116 X. Xu et al. / Environmental Pollution 210 (2016) 113e120

Fig. 1. THg in rice roots and shoots under different forms and doses of Hg exposure (aed), and ratio of THg in rice shoots to that in rice roots (TF, e and f). (a) THg in roots of rice under 0.5 mg/L IHg or MeHg exposure. (b) THg in shoots of rice under 0.5 mg/L IHg or MeHg exposure. (c) THg in roots of rice under 1 mg/L IHg or MeHg exposure. (d) THg in shoots of rice under 1 mg/L IHg or MeHg exposure. (e) TF of THg in rice plants exposed to 0.5 mg/L IHg or MeHg solution. (f) TF of THg in rice plants exposed to 1 mg/L IHg or MeHg solution.

Fig. 3 showed the typical changes of IHg and MeHg species with rice growth. By combining chemical forms of Hg in pure MeHg between the control solutions and the rice tissues. As displayed in solution and tissues of MeHg exposed rice, as well as the remark- Fig. 3a, only IHg signal was detected in the IHg solution without rice ably decreased ratio of MeHg to IHg in rice plant under 5 days and culture, which strongly showed that after putting aside for 5 days, 20 days of 0.5 mg/L MeHg exposure (see Table SI-4), it can be the chemical form of Hg in IHg solution remains unchanged, that is deduced that rice plant itself, to some extent, is responsible for the no methylation occurred in the IHg solution. Meanwhile, in rice demethylation of MeHg. There were many studies which found that roots and shoots under IHg exposure (Fig. 3b) after cultivating for 5 the demethylation of MeHg occurred in animals (Collin et al., 2009; days, only IHg signal existed. Combining the situations in IHg so- Henny et al., 2002). Nevertheless, in vivo demethylation of MeHg in lution and tissues of IHg exposed rice, it can be stated that no higher plants has seldom been reported. Our experiments dis- methylation occurred in rice seedlings during its early cultivation played that demethylation of MeHg occurred with rice plant period. In Fig. 3c, only MeHg signal was detected in the MeHg so- growth. However, the mechanisms of demethylation of MeHg lution without rice culture, which was completely contrary to the discovered in rice plant and the possible impact factors still remain situation in rice tissues under MeHg exposure (Fig. 3d). In Fig. 3d, unknown and require further studies. both IHg and MeHg signals were detected in tissues of MeHg exposed rice and the ratio of IHg to MeHg in rice shoots was much 3.3. Binding forms of Hg in rice roots analyzed by m-XANES higher than that in rice roots.

Moreover, taking into consideration that IHg was not detected in In this study, Hg L3-edge XANES is utilized to analyze the the MeHg exposure solution without rice plant while very limited binding forms of Hg in rice roots because of its unique advantages IHg was detected in the MeHg added solution with rice culture over other methods such as less or even no pretreatment proce- (Fig. SI-2), it may imply the occurrence of MeHg demethylation dure, and accurate reﬂection on the surrounding information of X. Xu et al. / Environmental Pollution 210 (2016) 113e120 117

Fig. 2. The concentration of IHg and MeHg with time in rice roots and shoots under different doses of IHg (a, c) or MeHg (b, d) exposure. (a) Under 0.5 mg/L IHg exposure; (b) Under 0.5 mg/L MeHg exposure; (c) Under 1 mg/L IHg exposure; (d) Under 1 mg/L MeHg exposure.

Fig. 3. Typical changes of chemical forms of Hg in solutions without rice culture (a, c) after putting aside for 5 days and in rice tissues (b, d) after cultivating for 5 days. (a) Chemical forms of Hg in solution of IHg exposed group without rice culture. (b) Chemical forms of Hg in rice tissues under IHg exposure. (c) Chemical forms of Hg in solution of MeHg exposed group without rice culture. (d) Chemical forms of Hg in rice tissues under MeHg exposure.

samples, including molecular structures and chemical bonds(Gao et al., 2008, 2007). The HgL3-edge XANES spectra of rice roots 118 X. Xu et al. / Environmental Pollution 210 (2016) 113e120

Fig. 4. Hg L3-XANES spectra of rice roots sampled from 5 days to 20 days of IHg or MeHg exposed rice plants and Hg standard compounds, HgCl2, MeHgCl, Hg(GS)2 (represent RS- Hg-SR bonding form) and MeHgCys (represent MeHg-SR bonding form). exposed to IHg or MeHg solution is illustrated in Fig. 4. like that of MeHg-SR, and the typical absorb peak near 12,290 eV As displayed in Fig. 4, there was no obvious variation in Hg va- was weakened gradually for the MeHg exposed rice roots cultivated lences of rice roots sampled from 5 days to 20 days under IHg or from 5 to 20 days. MeHg exposure because the spectra of all samples are broad and Table 1 demonstrates the quantitative values of Hg species in alike in general, which comes down to the structural feature that rice roots and standard Hg compounds that were transformed by the low-lying d orbitals in molecules of compounds are full of using the least-squares fit. In rice roots of IHg exposed group, only electrons and no strong dipole transitions exists. Additionally, the HgCl2 and RS-Hg-SR data could be well fitted to the sample data. No Hg spectra of rice roots collected from 5 days to 20 days under IHg methylated Hg such as MeHgCl and MeHg-SR forms could be fitted exposure (Fig. 4a) are more similar to the standard spectra of well to the sample spectra, which indicated no significant Hg(GS)2(represent RS-Hg-SR binding form), that is, the major methylation of Hg occurred. As obviously observed from Table 1, component of Hg in the roots of IHg exposed rice plant existed as while the proportion of RS-Hg-SR form increased as the cultivation the form of binding with sulfhydryl groups owing to the high af- time went on and finally reached up to 82.9% from 78.2% of the THg, þ finity of Hg to S in plants. The strong HgeS bond may also be related the proportion of Hg2 form decreased gradually from 21.8% to to some phytochelatins or proteins in plant(Clarkson, 1997). 17.1% of the THg. Previous reports have shown that in plants and Because the SR-XANES spectra of Hg(GS)2 and HgePCs are quite animal tissues, e.g. in fish(Harris et al., 2003), Hg ions are more similar and cannot be distinguished effectively by XANES, in this prone to bind with sulfhydryl groups in biothiols to form com- study we use RS-Hg-SR to represent the binding of Hg to sulfhydryl pounds rather than existing in the form of divalent cations (Krupp groups, such as Hg(GS)2,HgePCs and others. The Hg spectra of rice et al., 2008). Our detection is in line with those existed reports. roots collected from 5 days to 20 days under MeHg exposure In rice roots of MeHg exposed group during the whole cultiva- (Fig. 4b) were much similar to the standard spectra of Hg(GS)2 and tion time, Hg was mainly found to incorporate into MeHg-SR and MeHgCys (represent MeHg-SR binding form), while the other two RS-Hg-RS forms (account for above 83% of THg). Both Hg- and Hg standard substances, HgCl2 and MeHgCl, contains an obvious MeHg-containing compounds were ascertained, which illustrated shoulder peak, which may be attributed to the binding of Hg to Cl, that part of MeHg-containing compounds in rice roots were rather than S. The Hg L3-edge XANES spectra of MeHg exposed rice transformed into less toxic Hg-containing compounds and pro- roots cultivated for 5 days had an obvious absorb peak in 12,290 eV vided proof to verify the process of demethylation of

Table 1

Relative proportion of mercury species in rice roots exposed to IHg or MeHg respectively as assessed by Hg LIII XANES least ﬁtting (%).

Groups Samples HgCl2 Hg(GSH)2 MeHgCl MeHgCys R-Factor IHg exposed group 5 days 21.8 78.2 ee 0.00067 10 days 20.2 79.8 ee 0.00095 15 days 18.3 81.7 ee 0.00083 20 days 17.1 82.9 ee 0.00061 MeHg exposed group 5 days 3.8 32.3 12.5 51.3 0.00039 10 days 3.2 41.9 8.3 45.6 0.00087 15 days 3.5 52.1 5.1 38.7 0.00056 20 days 3.3 56.9 3.6 36.2 0.00062 X. Xu et al. / Environmental Pollution 210 (2016) 113e120 119 methylmercury with rice growth. The demethylated Hg deter- high concentrations in environmental matrix yet low levels in fish on the e mined in the roots of MeHg exposed rice plant was mainly in RS- market. Environ. Sci. Technol. 46, 4695 4696. Clarkson, T.W., 1997. The toxicology of mercury. Crit. Rev. Clin. Lab. Sci. 34, 369e403. Hg-SR form like Hg(GSH)2, which accounted for above 32% of the Collin, A.E., Joshua, T.A., Julie, Y., Terrence, L.A., 2009. Mercury demethylation in THg. As the cultivation time went on, the proportion of RS-Hg-SR water bird livers: dose-response thresholds and differences among species. e form in rice roots increased gradually and finally reached up to Environ. Toxicol. Chem. 28, 568 577. Deng, G., Zhang, T., Yang, L., Wang, Q., 2013. Studies of biouptake and trans- 56.9% (shown in Table 1). The methyl-included Hg determined in formation of mercury by a typical unicellular diatom (Phaeodactylum tricornu- rice roots mainly presented as MeHg-SR form (similar to MeHgCys) tum). Sci. Bull. 58, 256e265. which accounted for above 51% of the THg, indicating the quick Driscoll, C.T., Holsapple, J., Schofield, C.L., Munson, R., 1998. The chemistry and þ transport of mercury in a small wetland in the Adirondack region of New York, combination of MeHg and thiols through the binding of Hg to S of e þ USA. Biogeochemistry 40, 137 146. eSH. As cultivation time went on, both the proportion of MeHg Galloway, M.E., Branfireun, B.A., 2004. Mercury dynamics of a temperate forested and MeHg-SR decreased obviously and eventually from 12.5% to wetland. Sci. Total Environ. 325, 239e254. Gao, Y., Chen, C., Chai, Z., 2007. Advanced nuclear analytical techniques for metal- 3.6% and from 51.3% to 36.2%, respectively. Meanwhile, the pro- loproteomics. J. Anal. At. Spectrom. 22, 856e866. portion of RS-Hg-SR increased gradually from 32.3% to 56.9% of the Gao, Y., Liu, N., Chen, C., Luo, Y., Li, Y., Zhang, Z., Zhao, Y., Zhao, B., Atsuo, L., Chai, Z., THg in MeHg exposed rice roots. MeHg in rice plant was reported to 2008. Mapping technique for biodistribution of elements in a model organism, be present almost exclusively as MeHg-SR form, a compound that Caenorhabditis elegans, after exposure to copper nanoparticles with microbeam synchrotron radiation X-ray fluorescence. J. Anal. At. Spectrom. 23, 1121e1124. was accountable for MeHg to transfer through the bloodebrain Gilmour, C.C., Henry, E.A., Mitchell, R., 1992. 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