Metallomics View Article Online
Published on 06 August 2014. Downloaded by York University on 23/10/2014 12:41:24.
PAPER
Cite this: Metallomics, 2014, 6, 1951
View Journal | View Issue
Selenium modulates mercury uptake and distribution in rice (Oryza sativa L.), in correlation with mercury species and exposure level† Jiating Zhao, Yufeng Li, Yunyun Li, Yuxi Gao,* Bai Li, Yi Hu, Yuliang Zhao and Zhifang Chai Rice cultured in Hg- and/or Se-contaminated fields is an important food source of human Hg/Se intake. There are elevated Hg and Se levels in the soil of the Wanshan District, Guizhou Province. Here we attempted to explore how a Hg antagonist, Se, modulates the absorption and accumulation of inorganic mercury (IHg) and methylmercury (MeHg) in rice. The effects of Se on the content and transportation of Hg in hydroponic and soil cultured rice plants were examined. The results show that IHg mainly accumulated in the rice roots, but some also accumulated in the rice grain. In comparison to IHg, MeHg can be concentrated in the rice grain, and the proportion of MeHg in the rice grain may account for above 40% of the total Hg. Se can protect against Hg phytotoxicity in rice and inhibit IHg accumulation
Received 27th June 2014, Accepted 6th August 2014
in rice tissues, but was not remarkable for MeHg at a low dosage exposure level in this study. These
DOI: 10.1039/c4mt00170b
rice. This study illustrates that Se plays an important role in modulating Hg uptake, transportation and accumulation in rice. Therefore, Se is considered to be a naturally existing element that effectively
www.rsc.org/metallomics
reduces Hg accumulation in rice, which may have significant implications for food safety.
discrepancies imply mechanistic differences between IHg and MeHg absorption and accumulation in
1. Introduction Mercury (Hg), considered to be a global contaminant, is one of the most hazardous toxic elements. Its organic form, such as methylmercury (MeHg), causes great concern due to its high toxicity towards human beings.1,2 Although the average concentration of Hg in the earth’s crust is only about 0.05 mg kg 1 and the concentration of Hg in most foodstuffs is generally below 0.02 mg kg 1 (mainly present in inorganic forms),3 its potential environmental and health risks are considerable because once Hg deposits in aquatic or terrestrial systems it can be transformed into MeHg and bio-accumulate in the food chain. Due to the high Hg mobility in paddy fields, Hg levels in rice generally exceed those found in other staple cereals by an order of magnitude.4,5 Furthermore, rice tissues accumulate high concentrations of Hg, which renders rice-eating people more prone to a high Hg intake.6 The problem is more serious in regions, such as the Guizhou Province of China, where some paddy fields are contaminated with Hg, and people are unintentionally using CAS Key Lab of Nuclear Radiation and Nuclear Energy Technology, CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics (IHEP), Chinese Academy of Sciences (CAS), Beijing 100049, China. E-mail: [email protected] † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c4mt00170b
This journal is © The Royal Society of Chemistry 2014
Hg-containing waters to irrigate crops for a long time.7,8 It has been reported that the average Hg concentration in rice from Wanshan (Guizhou province, China) is approximately 78 mg kg 1, which is much higher than the maximum value (20 mg kg 1) recommended for consumption by the Standardization Administration of People’s Republic of China,9,10 and the MeHg content in the rice grain may reach 44.3 mg kg 1.7 Therefore, rice consumption is considered to be the major MeHg exposure pathway for the population in the Hg-contaminated area of Guizhou Province, China. Selenium (Se) is either an essential or toxic element depending on the ingested dose, and the range between its beneficial and toxic dosage is quite narrow.11–13 As an essential element, Se is important for the activity of antioxidative enzymes, such as glutathione peroxidase and thioredoxin reductase. It is also well-known that Se shares a lot of chemical similarities with sulfur, and that selenols are even more reactive towards Hg than thiols, making them play important roles in counteracting Hg toxicity.14,15 To date, many studies have been conducted on the interactions between Hg and Se in some organisms, suggesting that a universal Hg–Se antagonism exists among these biological organisms.16,17 Although data continues to accumulate to support the antagonistic effect in animals, the antagonism between Hg and Se in plants remains less explored. Due to the high affinity of Se for Hg, Se usually coexists with Hg in polluted fields. As previously reported, except
Metallomics, 2014, 6, 1951--1957 | 1951
View Article Online
Published on 06 August 2014. Downloaded by York University on 23/10/2014 12:41:24.
Paper
for high Hg levels, Se concentrations in the soil samples from the Wanshan district may be up to 36.6 mg kg 1. About 10% of the cropland contains more than 3.0 mg kg 1 of Se, which is the limit for Se-excessive soil.18 Early studies have suggested that Se, as an Hg antagonist, may be able to form an insoluble complex with Hg in rhizosphere soil and thus reduce the absorption and translocation of Hg to aerial parts of the plants.19,20 In the present study, aware of the elevated Hg and Se status in Guizhou Province, we sought to examine how Se could modulate absorption, transportation, accumulation and distribution of IHg and MeHg in rice. As the results suggest, the possible Hg–Se antagonism may offer an alternative strategy for minimizing Hg accumulation and phytotoxicity in rice.
2. Materials and methods Based on our investigations in Guizhou Province, there were elevated doses of Hg and Se co-existing in some of the paddy fields. To explore the antagonistic effect between Hg and Se at controllable conditions, the hydroponic cultivation for the study of rice seed germination and distribution of Hg affected by Se in rice seedlings under model conditions was carried out in our lab. Meanwhile, paddy soil cultivation was carried out to investigate the effects of Se on IHg and MeHg accumulation in different tissues of rice plants under controllable conditions. 2.1
Seed germination and hydroponic cultivation
Full and undamaged rice seeds were selected and treated with 10% NaCl solution for 15 minutes, and then washed with running water for 6 hours. All the seeds were sowed in an artificial climatic chamber, which was adjusted to the optimal conditions for rice germination: controlled temperature 30 1C, humidity 60% and light 100% with 16 h in the day time; at night, temperature 26 1C, humidity 80%. A total of 24 groups of healthy seeds were sowed in the culture medium (25% Hoagland solution) containing 0, 0.01, 1, and 10 mg L 1 Hg (HgCl2) and 0, 0.1, 0.5, 1, 5, and 10 mg L 1 Se (Na2SeO3), proceeding the crossover experiment. Each group contained 100 rice seeds. The seed germination percentage was determined after 5 days and 10 days of cultivation, respectively. After thirty days of hydroponic cultivation, the rice seedlings were harvested and separated into roots and aerial parts, freeze dried and ground into fine powders. The Hg content in different tissues of the rice were determined using inductively coupled plasma-mass spectrometry (ICP-MS) to analyze the effects of Se on the absorption and accumulation of Hg in the rice plants at the seedling stage. 2.2
Rice cultivation in paddy soil
Hg-contaminated paddy soil samples (top soil of 0–20 cm depth) were collected from a region of Qingzhen (Guizhou Province). The soil samples were air-dried for 6 days and ground into a o2 mm homogenized fraction. These paddy soils were used for the rice cultivation. Four sets of the soils were placed in plastic boxes. The control group was irrigated
1952 | Metallomics, 2014, 6, 1951--1957
Metallomics
with water. The Se treated soils (0.5, 1, and 5 mg kg 1 Se in each group, respectively) were obtained by irrigating with a series of Se-containing solutions. The soils were left in the plastic boxes for one month to allow for adsorption and equilibration of Se. The homogeneous seedlings after one month of hydroponic cultivation from the control group were transplanted into Hg-contaminated paddy soil. Throughout the experiment, watering and fertilization were done manually as required. After 3 months of rice growth, all the rice plants were harvested and washed thoroughly with deionized water. Each plant was separated into root, stalk and leaf, and rice grain, and then air dried. Parts of the plants were lyophilized at 50 1C and then ground into fine powders and stored at 20 1C before ICP-MS analysis. 2.3
Total mercury analysis in the rice seedling
To explore the effects of Se exposure on the accumulation of Hg in rice seedlings, the contents of Hg in the tissues of hydroponic cultivated rice were investigated. A triplicate of approximately 20 mg of the dried powder from each group was digested with 4 mL HNO3 (BV-III grade reagent) and 0.5 mL H2O2 (MOS grade reagent) in a digestion vessel. The mixture was left standing overnight at room temperature, and then digested at 160 1C for 3 hours in sealed pots in a drying oven. The paddy soil samples were digested with HNO3, HF and HClO4 (5 : 5 : 2, v/v/v) in uncovered PTFE jars on an electric hot plate. The resulting solutions were volatilized at a temperature below 90 1C, to a volume of 0.5–1 mL, and then diluted with 2% HNO3 containing 0.1% b-mercaptoethanol up to 10 mL for Hg determination. The Hg content in rice and soil samples were measured with ICP-MS (Thermo Elemental X7, USA). The detailed operating conditions of ICP-MS for Hg analysis are shown in Table S1 (ESI†). Optimization was carried out every time with a normal tuning solution (In 10 ppb, Bi 10 ppb). The calibrating standards of Hg were prepared by diluting the Hg standard stock solution with 2% (v/v) HNO3 containing 0.1% (v/v) b-mercaptoethanol, and then measured in the same manner with the sample solutions.21 The certified reference materials, poplar leaf CRM (GSV-3, China) and certified reference soil (GBW 07405), were used to validate the method, respectively. 2.4 Speciation analysis of IHg and MeHg in paddy soil cultured rice plant The rice tissues (roots, stalk and leaves, seeds) from Hg and Se exposed rice plants and the control group (without Se treatment) were rinsed with Milli-Q water, air-dried for 1 week, then freeze-dried and ground into a fine powder. A triplicate of 0.1 g powder of each sample was weighed and mixed with 5 mL of 6 mol L 1 HCl, shaken overnight at 28 1C, then ultrasonically extracted for 1 h. After 3 extractions, all the extractions were collected, adjusted to pH 6.7 and filtered using a 0.22 mm microporous membrane prior to analysis. The Hg content in the residues after extraction was analyzed using ICP-MS after digestion with HNO3 and H2O2.
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 06 August 2014. Downloaded by York University on 23/10/2014 12:41:24.
Metallomics
The IHg and MeHg content in different rice tissues were analyzed using a HPLC-ICP-MS hyphened technique. A high performance liquid chromatography (HPLC) system consisting of a liquid chromatography pump (WAT055028 metal-free 626 pump, Waters, Milford, USA), a 5 mm Symmetryshield RP18 column (150 3.9 mm, Waters, Milford, USA) and a 5 mm Symmetryshield RP18 guard column (20 3.9 mm, Waters, Milford, USA), was used for the separation of IHg and MeHg. A 5% (v/v) methanol water solution containing 0.06 M ammonium acetate and 0.2% (v/v) 2-mercaptoethanol was used as the mobile phase of HPLC (pH = 6.7). The mobile phase was filtered using a 0.22 mm microporous membrane. The flow rate was 1 mL min 1 and the sample injection volume was 100 mL. The column was connected directly to the nebulizer of the ICP-MS system (Thermo Elemental X7, Thermo Electron Co., US) with polyether ether ketone (PEEK) tubing. The methylmercury chloride stock standard solution was prepared as a 1000 mg Hg L 1 solution by dissolving methylmercury chloride (Alfa Aesar) into 2% HNO3. The mercury chloride stock standard solution was obtained as a 1000 mg Hg L 1 solution (GBW 08617, National Research Centre for CRMs, China). The working standard solutions were prepared in Milli-Q water by dilution of the stock solutions as required daily and stored in the dark. All reagents were of analytical reagent grade. 2.5
Elemental image of Hg and Se in rice tissues with SRl-XRF
Hg and Se localization in different rice tissues were surveyed with synchrotron radiation micro X-ray fluorescence (SRm-XRF). The roots of the rice cultivated in Hg-contaminated paddy soils with 5 mg kg 1 Se treatment, and the control group (without Se treatment), were frozen quickly and then cut into 40 mm-thick slices with a freezing microtome (CM1850, Germany). The leaves of the corresponding rice plants were clamped between two layers of cellophane to keep them flat and dried at room temperature. The longitudinal sections containing the embryo of the rice grains were prepared. All of the slices were fixed onto 3 mm-thick Mylar films (polycarbonate), and then dried at 20 1C until analyzed by SRXRF.22
Paper
Elemental distribution was imaged using SRXRF at BL15U in Shanghai Synchrotron Radiation Facility (SSRF, Shanghai, China). The storage ring ran at an energy of 3.5 GeV with a current intensity of 200–300 mA. An excitation energy of 13 keV was chosen to excite the elements. The SR beamline was monochromatized with a Si(111) double-crystal monochromator and focused to 5 5 mm2 with a K–B system for analysis of the root slices (80 80 mm2 for the leaves and the seed slices analysis). The samples were fixed on a moving platform and moved along the horizontal vertical direction using stepped motors and the pixel step size was set to 5 mm for root slices scanning and 80 mm for leaves and seed slices scanning, with a dwell time of 2 seconds. The fluorescence intensities of the elements were recorded and analyzed using a 7 element Si (Li) detector combined with a multiple channel analyzer (e2v, UK). The counts of the elements of Hg and Se were normalized to that of the I0 to correct the effect of the SR beam flux variation on the signal intensity, and then imaged using Igor Pro. 5.01 software.
3. Results and discussion 3.1 Effect of Se on seed germination and growth of rice exposed to Hg The effects of Se and Hg on rice seed-germination are shown in Fig. 1. It can be seen from Fig. 1 that the seed germination percentage of the control group (only irrigated with Hoagland solution) exceeded 60% after five days of cultivation. Interestingly, Hg exposure at lower levels (less than 0.1 mg L 1) was found to promote rice seed-germination, indicating obvious hormesis of the heavy metal as previously reported.23,24 However, a significant inhibitive effect (about 50% of the control group) of Hg on seed germination was observed when Hg concentration in the culture medium was more than 1 mg L 1. For Se treated groups, the seed germination percentage was elevated when total Se concentration was less than 1 mg L 1 in the culture medium. In particular, the seed germination percentage even exceeded 20% compared to the control group when Se treatment was at 0.5 mg L 1 level. When the Se treatment level
Fig. 1 The seed-germination of rice under different dosages of Hg or/and Se exposure. (a) Seed-germination after 5 days of Hg or/and Se exposure; (b) seed-germination after 10 days of Hg or/and Se exposure. * significant difference at p o 0.05 from the control group.
This journal is © The Royal Society of Chemistry 2014
Metallomics, 2014, 6, 1951--1957 | 1953
View Article Online
Published on 06 August 2014. Downloaded by York University on 23/10/2014 12:41:24.
Paper
was 5 mg L 1 or 10 mg L 1, seed-germination was inhibited. However, the inhibitive effect of Se was much lower than that of Hg at the same levels. For the rice seed co-exposed to Hg and Se, seed-germination was significantly promoted when both Hg and Se exposure levels were at lower concentrations (red line of Fig. 1). On the 5th day, the seed germination percentage of the lower Hg and Se exposed groups reached nearly 100%, which was much higher than that of Hg or Se exposed alone. Conversely, the inhibitive effects were profoundly intensified and the seedgermination percentage decreased when both Se and Hg exposure were at higher levels (Z5 mg L 1). On the 10th day, the seed-germination percentage of most groups exceeded 90%, except for those exposed to high levels of Hg and Se. The protective effect of Se against Hg phytotoxicity was evident only at lower levels (less than 1 mg L 1) of Se. With further increasing of the Se level in the growth medium, the protective effect of Se on Hg phytotoxicity diminished, but its toxic effect emerged. This indicates that the effect of Se on Hg phytotoxicity in rice depends on their dosage. 3.2
Effect of Se on Hg accumulation in rice seedlings
Based on our investigations of the Hg-contaminated paddy fields in Qingzhen (Guizhou Province, China), the Hg concentration may reach up to 116.2 mg L 1 in irrigated water and 29.6 mg kg 1 in the soils, while Se concentration is at a relatively lower level (12.7 mg L 1 in the surface water and 189.6 mg kg 1 in the soil). In the present study, the Hg content in the roots, stalks and leaves of the rice seedlings was determined with ICP-MS to elucidate the influence of Se on Hg uptake and transportation in hydroponic cultured rice under such a range of Hg and Se exposure conditions (0.1–10 mg L 1). The results are shown in Fig. 2. In Fig. 2, Hg content in rice roots in the lower Hg exposed group (0 and 0.1 mg L 1 Hg exposure groups) gradually increased with increasing Se level (from 0 to 10 mg L 1 Se) in
Metallomics
the culture medium. Trace amounts of Hg in the control group were possibly caused by volatilization from the neighboring environment. However, the Hg content in the roots in the high Hg exposure groups (1 mg L 1 and 10 mg L 1) was significantly decreased as Se exposure level increased, indicating that Se can inhibit Hg uptake in rice grown in Hg-contaminated environments, and the inhibitive effect can be promoted at higher Se exposure levels. On the other hand, the ratio of Hg content in the stalks and leaves to that in the roots was about 1 : 20 when there was no Se addition, indicating a low transportation efficiency of Hg from the rice roots to the aerial parts. Hg accumulation in the stalks and leaves of the higher Hg exposure groups (1 mg L 1 and 10 mg L 1 Hg exposure) was profoundly inhibited by Se, and the transportation efficiency of Hg could be prominently decreased with Se addition, as shown in Fig. 2. The ratio of Hg concentration in the stalks and leaves to that in the roots may even reach 1 : 40 when 5 mg L 1 or 10 mg L 1 Se was added. These results imply that Se treatment at appropriate dosages can significantly inhibit Hg uptake and transportation from the root to the aerial parts of rice grown in a condition with a high Hg level. This may be ascribed to the formation of a Hg–Se complex which is insoluble and unavailable for plants in a Hg/Se co-existing environment, as previously reported in other plants.25–27 Otherwise, the retention of Hg–Se compounds can also prevent Hg leaching or mobilization in the paddy field ecosystem, reducing the spread of Hg contamination to water and the surrounding environment. 3.3
Effect of Se on IHg and MeHg accumulation in rice tissues
The IHg and MeHg compounds in different tissues of the soil cultured rice were extracted with 6 M HCl. The extractability of total Hg was above 80% with the HCl method in this study. Two major Hg species in the leachate, i.e. IHg and MeHg, were separated and analyzed using HPLC-ICP-MS.28 In the HPLCICP-MS study, recovery experiments were carried out to verify
Fig. 2 The concentrations of Hg in hydroponic cultured rice seedlings under different dosages of Hg and Se exposure. * significant difference at p o 0.05 from the control group.
1954 | Metallomics, 2014, 6, 1951--1957
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 06 August 2014. Downloaded by York University on 23/10/2014 12:41:24.
Metallomics
Paper
Fig. 3 The contents of IHg (left image) and MeHg (right image) in tissues of the paddy soil cultured rice with different dosages of Se treatment. * significant difference at p o 0.05 from the group only exposed to Hg.
the viability of this method. The recovery of IHg and MeHg using the HPLC-ICP-MS method reached about 90%, which was satisfactory. The content of IHg and MeHg in the rice roots, stalks and leaves, and grains are shown in Fig. 3. The IHg levels in rice tissues followed the trend: roots 4 stalks and leaves 4 rice grains, while the MeHg levels were in the order of roots 4 rice grains Z stalks and leaves. This distribution tendency of IHg and MeHg is in agreement with a previous study.29 It is notable that the Hg species in the roots, stalks and leaves is mainly IHg, which accounts for more than 95% of the total Hg, while less than 5% of the total Hg in these parts of rice is in the form of MeHg (Fig. 4). However, MeHg might represent above 40% of the total Hg in the rice grains (Fig. 4). The proportion of MeHg distribution in different rice tissues is in the order of rice grains c stalks and leaves 4 roots. This result further confirmed a previous report in which the rice grains were considered an intensive bio-accumulator of MeHg, and rice intake can be an important pathway of MeHg exposure in human beings living in Hg-contaminated areas.6 The content of IHg in rice roots, stalks and leaves was substantially decreased with Se supplement, especially when
Fig. 4 The ratio (%) of IHg and MeHg in total Hg in tissues of the paddy soil cultured rice with different dosages of Se treatment.
This journal is © The Royal Society of Chemistry 2014
Se treatment was higher than 1 mg L 1. However, there was no significant decrease of IHg in rice grains resulting from Se addition (Fig. 3). The MeHg level in the rice roots was somewhat decreased with increasing Se levels. Nevertheless, there was no significant decrease of MeHg content in the leaves and rice grains with Se treatment (Fig. 3). It is notable that, the proportions of IHg in the rice roots, stalks and leaves, and rice grains were substantially decreased with Se addition, suggesting that the decrease of total Hg in rice might be primarily attributed to the inhibitive effect of Se on IHg uptake and transportation in the rice root and shoot, rather than a direct effect of Se on MeHg in rice (the transfer factors of IHg and MeHg are shown in Fig. S1, ESI†). The obvious discrepancy between IHg and MeHg accumulation in different rice tissues, along with the difference in effects of Se on the accumulation of IHg and MeHg in rice tissues indicate a mechanistic difference underlying IHg or MeHg uptake and accumulation in rice. As previously reported by Schwesig and Krebs, organic Hg was more prone to transportation to the ground vegetation than IHg.30 As reported in another study, naturally existing phytochelatins in plants could sequester a divalent Hg ion rather than MeHg to mitigate the phytotoxicity of Hg.31 In a recent study by Hua Zhang et al.,18 according to the investigations of Hg and Se content in rice plants collected from several Hg-contaminated sites in Guizhou Province, China, the authors concluded that Se content was positively correlated with the contents of IHg in the paddy soil samples, and the elevated Se in the soil samples could inhibit the absorption of IHg in the rice root, but not that of MeHg. In the present study, at a controllable condition, we obtained a similar result in the rice plants under different doses of Se treatment conditions by eliminating the possible influence factors from different sampling sites. According to our study, it can be speculated that the effects of Se on the accumulation and translocation of IHg and MeHg in rice plants might be quite variable. The detailed molecular mechanisms and the influence factors of Se/IHg (MeHg) antagonism in rice plant still requires further study.
Metallomics, 2014, 6, 1951--1957 | 1955
View Article Online
Paper
Published on 06 August 2014. Downloaded by York University on 23/10/2014 12:41:24.
3.4 The effect of Se on micro-zone distribution of Hg in rice tissues The SRXRF technique is a powerful tool for non-destructive elemental analysis with exceptional sensitivity.32 To reveal the influence of Se treatment on Hg micro-zone distribution in rice grown up in Hg-contaminated soil with or without Se treatment, 2D elemental distribution of the roots, leaves and rice grains were imaged using micro-SRXRF. The normalized X-ray fluorescence intensities are scaled from blue (minimum) to red (maximum). These images visually demonstrate the distributions and accumulations of Hg and Se in rice. The results are shown in Fig. 5. Fig. 5a1 shows that Hg was mainly localized in the epidermis and the pericycle of the rice root. Due to the high affinity of Hg for the sulfhydryl groups in the surface of the root,33,34 Hg can be enriched to quite a high concentration in the epidermis of the rice roots. Moreover, Hg can also be accumulated in the vessel of the roots, implying that Hg is able to penetrate the root surface into the vascular cylinder and then transport upwards. For Se/Hg co-exposed rice, one can see that the distribution of Se correlates well with that of Hg in the rice roots (Fig. 5b1 and b2), and both of them principally concentrate in the epidermis and pericycle of the rice root. Comparing Fig. 5b1 with 5a1, a substantial decrease in Hg accumulation in the epidermis and stele of the rice root can be found. This is consistent with the ICP-MS results shown in Fig. 2. The correlation between Hg and Se distribution in rice roots, combined with the strong affinity of Se to Hg, suggests that Hg and Se may form a Hg–Se complex in the rice root. This may explain how Se addition could inhibit Hg uptake and
Fig. 5 The distribution of Hg and Se in different tissues of the paddy soil cultured rice measured by m-SRXRF. (a) The cross section of the root tip from rice under Hg exposure (a1, Hg XRF image); (b) the cross section of the root tip from rice under Hg and Se exposure (b1, Hg XRF image; b2, Se XRF image); (c) the leaf from Hg exposed rice; (d) the leaf from Hg and Se co-exposed rice; (e) the rice grain from Hg exposed rice; (f) the rice grain from Hg and Se co-exposed rice.
1956 | Metallomics, 2014, 6, 1951--1957
Metallomics
translocation in rice. Shanker et al. have reported the existence of a Hg–Se compound in the rhizosphere of radish, which can block the absorption of Hg through the root.19 Caruso et al. have confirmed the existence of a Hg–Se compound in the roots of Brassica juncea and soybean.20,26 Fig. 5c1, d1 and d2 show the effect of Se on Hg distribution in rice leaves. It can be seen that Hg is dispersed over the leaves, and some is specially located in the leaf vein (Fig. 5c1). Furthermore, the Se distribution pattern correlates well with that of Hg in the rice leaves (Fig. 5d2). The content of Hg in the leaves collected from the Hg/Se co-exposed rice plant (Fig. 5d1) is much less than that of the Hg exposed group (Fig. 5c1), indicating the inhibitive effect of Se on Hg transportation from the roots to the leaves through the vascular cylinder. The effects of Se on the distribution and accumulation of Hg in the rice grain are illustrated in Fig. 5e1, f1 and f2. From Fig. 5e1, it is shown that Hg is principally concentrated on the surface of the rice grain (the aleurone layer), especially along the growth site of the embryo. It is notable that a considerable amount of Hg can accumulate in the embryo part (Fig. 5e1), while less Hg is located in the rice endosperm. Fig. 5f1 and f2 show the distributions of Hg and Se in rice grain collected from Hg/Se co-exposed rice. Except for the location in the aleurone layer like Hg, a considerable proportion of Se can penetrate into the endosperm of the rice grain. Moreover, Se distribution in the embryo is more extensive than that of Hg. A similar Se distribution pattern was previously reported,35 in which much Se accumulation in the outer regions of the rice grain was observed, and an amount of Se was concentrated in the chalazal zone that shares a close proximity to the ocular vascular trace. Comparing Fig. 5f1 with Fig. 5e1, the concentration of Hg in the embryo part of the rice grain from Se and Hg co-exposed rice is much lower than that of the rice exposed to Hg alone. This indicates that Se can interfere with Hg accumulation in the rice grain, and suggests that Se treatment may mitigate Hg toxicity towards the growth and development of rice seeds, as the embryo is the budding point of the rice seed. In addition, the essential elements (Fe, Cu, Zn, K, Ca etc.) are mainly concentrated in the embryo of the rice grain (Fig. S2, ESI†). This suggests that the necessary nutrient elements, rather than the toxic elements, can be selectively accumulated in the rice grain. Furthermore, it implies the existence of a preventive mechanism for toxic element accumulation in rice. In conclusion, Se treatment can inhibit Hg uptake and transportation from the rice root to the aerial part, which finally results in lower Hg accumulation in the rice grain, especially in the aleurone layer and embryo. The effect of Se on MeHg is less remarkable than IHg, along with the discrepancy of IHg and MeHg accumulation in different rice tissues indicating mechanistic differences in the absorption and transportation of IHg and MeHg in rice. The present study may provide valuable information for sequestering Hg and novel insights for addressing food safety in Hg/Se polluted environments. More researches on the positions for the methylation of IHg in vitro or in vivo and the phyto-biological behaviors of IHg and MeHg in rice will be needed to further understand the ecological toxicology of this toxic element.
This journal is © The Royal Society of Chemistry 2014
View Article Online
Metallomics
Acknowledgements
Published on 06 August 2014. Downloaded by York University on 23/10/2014 12:41:24.
This work was financially supported by the National Natural Science Foundation of China (Grant No. 21377129, 11205168, 11375213), and the special project on treatment and control of water pollution (No. 2012ZX07203-006-06). SRXRF analysis was carried out at beamline BL15U in Shanghai Synchrotron Radiation Facility (SSRF). We thank Xiaohan Yu, Aiguo Li, Ke Yang et al. in SSRF, and Dongliang Chen and Wei Xu in Beijing Synchrotron Radiation Facility (BSRF) for their assistance during the SRXRF measurement and the data processing.
References 1 O. Lindqvist, K. Johansson and M. Aastrup, Water, Air, Soil Pollut., 1991, 55, xi-261. 2 T. W. Clarkson, Environ. Health Perspect., 1993, 100, 31–38. 3 WHO, International programme on chemical safety: environmental health criteria 118-inorganic mercury, World Health Organization, Geneva, 1991. 4 J. Burger, M. Gochfeld, C. Jeitner, S. Burke, T. Stamm, R. Snigaroff, D. Snigaroff, R. Patrick and J. Weston, Sci. Total Environ., 2007, 384, 93–105. 5 B. Meng, X. Feng, G. Qiu, Y. Cai, D. Wang, P. Li, L. Shang and J. Sommar, J. Agric. Food Chem., 2010, 58, 4951–4958. 6 X. Feng, P. Li, G. Qiu, S. Wang, G. Li, L. Shang, B. Meng, H. Jiang, W. Bai, Z. Li and X. Fu, Environ. Sci. technol., 2008, 42, 326–332. 7 H. Zhang, X. Feng, T. Larssen, L. Shang and P. Li, Environ. Sci. technol., 2010, 44, 4499–4504. 8 B. Meng, X. Feng, P. Li, C. Chen and L. Shang, Environ. Sci. technol., 2011, 45, 2711–2717. 9 M. Horvat, N. Nolde, V. Fajon, V. Jereb, M. Logar, S. Lojen, R. Jacimovic, I. Falnoga, L. Y. Qu, J. Faganeli and D. Drobne, Sci. Total Environ., 2003, 304, 231–256. 10 Maximum levels of contaminants in foods (in Chinese); GB2762-2005; Standardization Administration of People’s Republic of China: Beijing, 2005 pp. 171–173. 11 M. Stadlober, M. Sager and K. J. Irgolic, Food Chem., 2001, 73, 357–366. 12 A. D. Lemly, Ecotoxicol. Environ. Saf., 2004, 59, 44–56. 13 A. M. H. Debruyn and P. M. Chapman, Environ. Sci. Technol., 2007, 41, 1766–1770. 14 D. Tran, A. John Moody, A. S. Fisher, M. E. Foulkes and A. N. Jha, Aquat. Toxicol., 2007, 84, 11–18. 15 T. W. Clarkson, Crit. Rev. Clin. Lab. Sci., 1997, 34, 369–403. 16 D. Y. Yang, Y. W. Chen, J. M Gunn and N. Belzile, Environ. Rev., 2008, 16, 71–92.
This journal is © The Royal Society of Chemistry 2014
Paper
17 H. Y. T. Truong, Y. W. Chen, M. Saleh, S. Nehzati, G. N. George, I. J. Pickering and N. Belzile, Metallomics, 2014, 6, 465–475. 18 H. Zhang, X. Feng, J. Zhu, A. Sapkota, B. Meng, H. Yao, H. Qin and T. Larssen, Environ. Sci. Technol., 2012, 46, 10040–10046. 19 K. Shanker, S. Mishra, S. Srivastava, R. Srivastava, S. Dass, S. Prakash and M. M. Srivastava, Food Chem. Toxicol., 1996, 34, 883–886. 20 S. Mounicou, M. Shah, J. Meija, J. A. Caruso, A. P. Vonderheide and J. Shann, J. Anal. At. Spectrom., 2006, 21, 404–412. 21 J. Zhao, Y. Gao, Y. Li, Y. Hu, X. Peng, Y. Dong, B. Li, C. Chen and Z. Chai, Environ. Res., 2013, 125, 75–81. 22 J. Zhao, Y. Hu, Y. Gao, Y. Li, B. Li, Y. Dong and Z. Chai, Metallomics, 2013, 5, 896–903. 23 D. Cargnelutti, L. A. Tabaldi, R. M. Spanevello, G. de Oliveira Jucoski, V. Battisti, M. Redin, C. E. B. Linares, V. L. Dressler, ´. M. de Moraes Flores and F. T. Nicoloso, Chemosphere, E 2006, 65, 999–1006. 24 A. Stebbing, Sci. Total Environ., 1982, 22, 213–234. 25 K. Shanker, S. Mishra, S. Srivastava, R. Srivastava, S. Daas, S. Prakash and M. M. Srivastava, Plant Soil, 1996, 183, 233–238. 26 S. K. Yathavakilla and J. A. Caruso, Anal. Bioanal. Chem., 2007, 389, 715–723. 27 D. H. McNear, S. E. Afton and J. A. Caruso, Metallomics, 2012, 4, 267–276. 28 Y. Li, C. Chen, B. Li, Q. Wang, J. Wang, Y. Gao, Y. Zhao and Z. Chai, J. Anal. At. Spectrom., 2007, 22, 925–930. 29 M. Meng, B. Li, J. J. Shao, T. Wang, B. He, J. B. Shi, Z. H. Ye and G. B. Jiang, Environ. Pollut., 2013, 184, 179–186. 30 D. Schwesig and O. Krebs, Plant Soil, 2003, 253, 445–455. 31 E. M. Krupp, A. Mestrot, J. Wielgus, A. A. Meharg and J. Feldmann, Chem. Commun., 2009, 4257–4259. 32 Y. Gao, N. Liu, C. Chen, Y. Luo, Y. Li, Z. Zhang, Y. Zhao, B. Zhao, A. Iida and Z. Chai, J. Anal. At. Spectrom., 2008, 23, 1121–1124. 33 B. V. Cheesman, A. P. Arnold and D. L. Rabenstein, J. Am. Chem. Soc., 1988, 110, 6359–6364. 34 S. Carrasco-Gil, A. Alvarez-Fernandez, J. Sobrino-Plata, R. Millan, R. O. Carpena-Ruiz, D. L. Leduc, J. C. Andrews, J. Abadia and L. E. Hernandez, Plant, Cell Environ., 2011, 34, 778–791. 35 P. N. Williams, E. Lombi, G. X. Sun, K. Scheckel, Y. G. Zhu, X. B. Feng, J. M. Zhu, A. M. Carey, E. Adomako, Y. Lawgali, C. Deacon and A. A. Meharg, Environ. Sci. Technol., 2009, 43, 6024–6030.
Metallomics, 2014, 6, 1951--1957 | 1957
Published on 06 August 2014. Downloaded by York University on 23/10/2014 12:41:24.
PAPER
Cite this: Metallomics, 2014, 6, 1951
View Journal | View Issue
Selenium modulates mercury uptake and distribution in rice (Oryza sativa L.), in correlation with mercury species and exposure level† Jiating Zhao, Yufeng Li, Yunyun Li, Yuxi Gao,* Bai Li, Yi Hu, Yuliang Zhao and Zhifang Chai Rice cultured in Hg- and/or Se-contaminated fields is an important food source of human Hg/Se intake. There are elevated Hg and Se levels in the soil of the Wanshan District, Guizhou Province. Here we attempted to explore how a Hg antagonist, Se, modulates the absorption and accumulation of inorganic mercury (IHg) and methylmercury (MeHg) in rice. The effects of Se on the content and transportation of Hg in hydroponic and soil cultured rice plants were examined. The results show that IHg mainly accumulated in the rice roots, but some also accumulated in the rice grain. In comparison to IHg, MeHg can be concentrated in the rice grain, and the proportion of MeHg in the rice grain may account for above 40% of the total Hg. Se can protect against Hg phytotoxicity in rice and inhibit IHg accumulation
Received 27th June 2014, Accepted 6th August 2014
in rice tissues, but was not remarkable for MeHg at a low dosage exposure level in this study. These
DOI: 10.1039/c4mt00170b
rice. This study illustrates that Se plays an important role in modulating Hg uptake, transportation and accumulation in rice. Therefore, Se is considered to be a naturally existing element that effectively
www.rsc.org/metallomics
reduces Hg accumulation in rice, which may have significant implications for food safety.
discrepancies imply mechanistic differences between IHg and MeHg absorption and accumulation in
1. Introduction Mercury (Hg), considered to be a global contaminant, is one of the most hazardous toxic elements. Its organic form, such as methylmercury (MeHg), causes great concern due to its high toxicity towards human beings.1,2 Although the average concentration of Hg in the earth’s crust is only about 0.05 mg kg 1 and the concentration of Hg in most foodstuffs is generally below 0.02 mg kg 1 (mainly present in inorganic forms),3 its potential environmental and health risks are considerable because once Hg deposits in aquatic or terrestrial systems it can be transformed into MeHg and bio-accumulate in the food chain. Due to the high Hg mobility in paddy fields, Hg levels in rice generally exceed those found in other staple cereals by an order of magnitude.4,5 Furthermore, rice tissues accumulate high concentrations of Hg, which renders rice-eating people more prone to a high Hg intake.6 The problem is more serious in regions, such as the Guizhou Province of China, where some paddy fields are contaminated with Hg, and people are unintentionally using CAS Key Lab of Nuclear Radiation and Nuclear Energy Technology, CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics (IHEP), Chinese Academy of Sciences (CAS), Beijing 100049, China. E-mail: [email protected] † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c4mt00170b
This journal is © The Royal Society of Chemistry 2014
Hg-containing waters to irrigate crops for a long time.7,8 It has been reported that the average Hg concentration in rice from Wanshan (Guizhou province, China) is approximately 78 mg kg 1, which is much higher than the maximum value (20 mg kg 1) recommended for consumption by the Standardization Administration of People’s Republic of China,9,10 and the MeHg content in the rice grain may reach 44.3 mg kg 1.7 Therefore, rice consumption is considered to be the major MeHg exposure pathway for the population in the Hg-contaminated area of Guizhou Province, China. Selenium (Se) is either an essential or toxic element depending on the ingested dose, and the range between its beneficial and toxic dosage is quite narrow.11–13 As an essential element, Se is important for the activity of antioxidative enzymes, such as glutathione peroxidase and thioredoxin reductase. It is also well-known that Se shares a lot of chemical similarities with sulfur, and that selenols are even more reactive towards Hg than thiols, making them play important roles in counteracting Hg toxicity.14,15 To date, many studies have been conducted on the interactions between Hg and Se in some organisms, suggesting that a universal Hg–Se antagonism exists among these biological organisms.16,17 Although data continues to accumulate to support the antagonistic effect in animals, the antagonism between Hg and Se in plants remains less explored. Due to the high affinity of Se for Hg, Se usually coexists with Hg in polluted fields. As previously reported, except
Metallomics, 2014, 6, 1951--1957 | 1951
View Article Online
Published on 06 August 2014. Downloaded by York University on 23/10/2014 12:41:24.
Paper
for high Hg levels, Se concentrations in the soil samples from the Wanshan district may be up to 36.6 mg kg 1. About 10% of the cropland contains more than 3.0 mg kg 1 of Se, which is the limit for Se-excessive soil.18 Early studies have suggested that Se, as an Hg antagonist, may be able to form an insoluble complex with Hg in rhizosphere soil and thus reduce the absorption and translocation of Hg to aerial parts of the plants.19,20 In the present study, aware of the elevated Hg and Se status in Guizhou Province, we sought to examine how Se could modulate absorption, transportation, accumulation and distribution of IHg and MeHg in rice. As the results suggest, the possible Hg–Se antagonism may offer an alternative strategy for minimizing Hg accumulation and phytotoxicity in rice.
2. Materials and methods Based on our investigations in Guizhou Province, there were elevated doses of Hg and Se co-existing in some of the paddy fields. To explore the antagonistic effect between Hg and Se at controllable conditions, the hydroponic cultivation for the study of rice seed germination and distribution of Hg affected by Se in rice seedlings under model conditions was carried out in our lab. Meanwhile, paddy soil cultivation was carried out to investigate the effects of Se on IHg and MeHg accumulation in different tissues of rice plants under controllable conditions. 2.1
Seed germination and hydroponic cultivation
Full and undamaged rice seeds were selected and treated with 10% NaCl solution for 15 minutes, and then washed with running water for 6 hours. All the seeds were sowed in an artificial climatic chamber, which was adjusted to the optimal conditions for rice germination: controlled temperature 30 1C, humidity 60% and light 100% with 16 h in the day time; at night, temperature 26 1C, humidity 80%. A total of 24 groups of healthy seeds were sowed in the culture medium (25% Hoagland solution) containing 0, 0.01, 1, and 10 mg L 1 Hg (HgCl2) and 0, 0.1, 0.5, 1, 5, and 10 mg L 1 Se (Na2SeO3), proceeding the crossover experiment. Each group contained 100 rice seeds. The seed germination percentage was determined after 5 days and 10 days of cultivation, respectively. After thirty days of hydroponic cultivation, the rice seedlings were harvested and separated into roots and aerial parts, freeze dried and ground into fine powders. The Hg content in different tissues of the rice were determined using inductively coupled plasma-mass spectrometry (ICP-MS) to analyze the effects of Se on the absorption and accumulation of Hg in the rice plants at the seedling stage. 2.2
Rice cultivation in paddy soil
Hg-contaminated paddy soil samples (top soil of 0–20 cm depth) were collected from a region of Qingzhen (Guizhou Province). The soil samples were air-dried for 6 days and ground into a o2 mm homogenized fraction. These paddy soils were used for the rice cultivation. Four sets of the soils were placed in plastic boxes. The control group was irrigated
1952 | Metallomics, 2014, 6, 1951--1957
Metallomics
with water. The Se treated soils (0.5, 1, and 5 mg kg 1 Se in each group, respectively) were obtained by irrigating with a series of Se-containing solutions. The soils were left in the plastic boxes for one month to allow for adsorption and equilibration of Se. The homogeneous seedlings after one month of hydroponic cultivation from the control group were transplanted into Hg-contaminated paddy soil. Throughout the experiment, watering and fertilization were done manually as required. After 3 months of rice growth, all the rice plants were harvested and washed thoroughly with deionized water. Each plant was separated into root, stalk and leaf, and rice grain, and then air dried. Parts of the plants were lyophilized at 50 1C and then ground into fine powders and stored at 20 1C before ICP-MS analysis. 2.3
Total mercury analysis in the rice seedling
To explore the effects of Se exposure on the accumulation of Hg in rice seedlings, the contents of Hg in the tissues of hydroponic cultivated rice were investigated. A triplicate of approximately 20 mg of the dried powder from each group was digested with 4 mL HNO3 (BV-III grade reagent) and 0.5 mL H2O2 (MOS grade reagent) in a digestion vessel. The mixture was left standing overnight at room temperature, and then digested at 160 1C for 3 hours in sealed pots in a drying oven. The paddy soil samples were digested with HNO3, HF and HClO4 (5 : 5 : 2, v/v/v) in uncovered PTFE jars on an electric hot plate. The resulting solutions were volatilized at a temperature below 90 1C, to a volume of 0.5–1 mL, and then diluted with 2% HNO3 containing 0.1% b-mercaptoethanol up to 10 mL for Hg determination. The Hg content in rice and soil samples were measured with ICP-MS (Thermo Elemental X7, USA). The detailed operating conditions of ICP-MS for Hg analysis are shown in Table S1 (ESI†). Optimization was carried out every time with a normal tuning solution (In 10 ppb, Bi 10 ppb). The calibrating standards of Hg were prepared by diluting the Hg standard stock solution with 2% (v/v) HNO3 containing 0.1% (v/v) b-mercaptoethanol, and then measured in the same manner with the sample solutions.21 The certified reference materials, poplar leaf CRM (GSV-3, China) and certified reference soil (GBW 07405), were used to validate the method, respectively. 2.4 Speciation analysis of IHg and MeHg in paddy soil cultured rice plant The rice tissues (roots, stalk and leaves, seeds) from Hg and Se exposed rice plants and the control group (without Se treatment) were rinsed with Milli-Q water, air-dried for 1 week, then freeze-dried and ground into a fine powder. A triplicate of 0.1 g powder of each sample was weighed and mixed with 5 mL of 6 mol L 1 HCl, shaken overnight at 28 1C, then ultrasonically extracted for 1 h. After 3 extractions, all the extractions were collected, adjusted to pH 6.7 and filtered using a 0.22 mm microporous membrane prior to analysis. The Hg content in the residues after extraction was analyzed using ICP-MS after digestion with HNO3 and H2O2.
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 06 August 2014. Downloaded by York University on 23/10/2014 12:41:24.
Metallomics
The IHg and MeHg content in different rice tissues were analyzed using a HPLC-ICP-MS hyphened technique. A high performance liquid chromatography (HPLC) system consisting of a liquid chromatography pump (WAT055028 metal-free 626 pump, Waters, Milford, USA), a 5 mm Symmetryshield RP18 column (150 3.9 mm, Waters, Milford, USA) and a 5 mm Symmetryshield RP18 guard column (20 3.9 mm, Waters, Milford, USA), was used for the separation of IHg and MeHg. A 5% (v/v) methanol water solution containing 0.06 M ammonium acetate and 0.2% (v/v) 2-mercaptoethanol was used as the mobile phase of HPLC (pH = 6.7). The mobile phase was filtered using a 0.22 mm microporous membrane. The flow rate was 1 mL min 1 and the sample injection volume was 100 mL. The column was connected directly to the nebulizer of the ICP-MS system (Thermo Elemental X7, Thermo Electron Co., US) with polyether ether ketone (PEEK) tubing. The methylmercury chloride stock standard solution was prepared as a 1000 mg Hg L 1 solution by dissolving methylmercury chloride (Alfa Aesar) into 2% HNO3. The mercury chloride stock standard solution was obtained as a 1000 mg Hg L 1 solution (GBW 08617, National Research Centre for CRMs, China). The working standard solutions were prepared in Milli-Q water by dilution of the stock solutions as required daily and stored in the dark. All reagents were of analytical reagent grade. 2.5
Elemental image of Hg and Se in rice tissues with SRl-XRF
Hg and Se localization in different rice tissues were surveyed with synchrotron radiation micro X-ray fluorescence (SRm-XRF). The roots of the rice cultivated in Hg-contaminated paddy soils with 5 mg kg 1 Se treatment, and the control group (without Se treatment), were frozen quickly and then cut into 40 mm-thick slices with a freezing microtome (CM1850, Germany). The leaves of the corresponding rice plants were clamped between two layers of cellophane to keep them flat and dried at room temperature. The longitudinal sections containing the embryo of the rice grains were prepared. All of the slices were fixed onto 3 mm-thick Mylar films (polycarbonate), and then dried at 20 1C until analyzed by SRXRF.22
Paper
Elemental distribution was imaged using SRXRF at BL15U in Shanghai Synchrotron Radiation Facility (SSRF, Shanghai, China). The storage ring ran at an energy of 3.5 GeV with a current intensity of 200–300 mA. An excitation energy of 13 keV was chosen to excite the elements. The SR beamline was monochromatized with a Si(111) double-crystal monochromator and focused to 5 5 mm2 with a K–B system for analysis of the root slices (80 80 mm2 for the leaves and the seed slices analysis). The samples were fixed on a moving platform and moved along the horizontal vertical direction using stepped motors and the pixel step size was set to 5 mm for root slices scanning and 80 mm for leaves and seed slices scanning, with a dwell time of 2 seconds. The fluorescence intensities of the elements were recorded and analyzed using a 7 element Si (Li) detector combined with a multiple channel analyzer (e2v, UK). The counts of the elements of Hg and Se were normalized to that of the I0 to correct the effect of the SR beam flux variation on the signal intensity, and then imaged using Igor Pro. 5.01 software.
3. Results and discussion 3.1 Effect of Se on seed germination and growth of rice exposed to Hg The effects of Se and Hg on rice seed-germination are shown in Fig. 1. It can be seen from Fig. 1 that the seed germination percentage of the control group (only irrigated with Hoagland solution) exceeded 60% after five days of cultivation. Interestingly, Hg exposure at lower levels (less than 0.1 mg L 1) was found to promote rice seed-germination, indicating obvious hormesis of the heavy metal as previously reported.23,24 However, a significant inhibitive effect (about 50% of the control group) of Hg on seed germination was observed when Hg concentration in the culture medium was more than 1 mg L 1. For Se treated groups, the seed germination percentage was elevated when total Se concentration was less than 1 mg L 1 in the culture medium. In particular, the seed germination percentage even exceeded 20% compared to the control group when Se treatment was at 0.5 mg L 1 level. When the Se treatment level
Fig. 1 The seed-germination of rice under different dosages of Hg or/and Se exposure. (a) Seed-germination after 5 days of Hg or/and Se exposure; (b) seed-germination after 10 days of Hg or/and Se exposure. * significant difference at p o 0.05 from the control group.
This journal is © The Royal Society of Chemistry 2014
Metallomics, 2014, 6, 1951--1957 | 1953
View Article Online
Published on 06 August 2014. Downloaded by York University on 23/10/2014 12:41:24.
Paper
was 5 mg L 1 or 10 mg L 1, seed-germination was inhibited. However, the inhibitive effect of Se was much lower than that of Hg at the same levels. For the rice seed co-exposed to Hg and Se, seed-germination was significantly promoted when both Hg and Se exposure levels were at lower concentrations (red line of Fig. 1). On the 5th day, the seed germination percentage of the lower Hg and Se exposed groups reached nearly 100%, which was much higher than that of Hg or Se exposed alone. Conversely, the inhibitive effects were profoundly intensified and the seedgermination percentage decreased when both Se and Hg exposure were at higher levels (Z5 mg L 1). On the 10th day, the seed-germination percentage of most groups exceeded 90%, except for those exposed to high levels of Hg and Se. The protective effect of Se against Hg phytotoxicity was evident only at lower levels (less than 1 mg L 1) of Se. With further increasing of the Se level in the growth medium, the protective effect of Se on Hg phytotoxicity diminished, but its toxic effect emerged. This indicates that the effect of Se on Hg phytotoxicity in rice depends on their dosage. 3.2
Effect of Se on Hg accumulation in rice seedlings
Based on our investigations of the Hg-contaminated paddy fields in Qingzhen (Guizhou Province, China), the Hg concentration may reach up to 116.2 mg L 1 in irrigated water and 29.6 mg kg 1 in the soils, while Se concentration is at a relatively lower level (12.7 mg L 1 in the surface water and 189.6 mg kg 1 in the soil). In the present study, the Hg content in the roots, stalks and leaves of the rice seedlings was determined with ICP-MS to elucidate the influence of Se on Hg uptake and transportation in hydroponic cultured rice under such a range of Hg and Se exposure conditions (0.1–10 mg L 1). The results are shown in Fig. 2. In Fig. 2, Hg content in rice roots in the lower Hg exposed group (0 and 0.1 mg L 1 Hg exposure groups) gradually increased with increasing Se level (from 0 to 10 mg L 1 Se) in
Metallomics
the culture medium. Trace amounts of Hg in the control group were possibly caused by volatilization from the neighboring environment. However, the Hg content in the roots in the high Hg exposure groups (1 mg L 1 and 10 mg L 1) was significantly decreased as Se exposure level increased, indicating that Se can inhibit Hg uptake in rice grown in Hg-contaminated environments, and the inhibitive effect can be promoted at higher Se exposure levels. On the other hand, the ratio of Hg content in the stalks and leaves to that in the roots was about 1 : 20 when there was no Se addition, indicating a low transportation efficiency of Hg from the rice roots to the aerial parts. Hg accumulation in the stalks and leaves of the higher Hg exposure groups (1 mg L 1 and 10 mg L 1 Hg exposure) was profoundly inhibited by Se, and the transportation efficiency of Hg could be prominently decreased with Se addition, as shown in Fig. 2. The ratio of Hg concentration in the stalks and leaves to that in the roots may even reach 1 : 40 when 5 mg L 1 or 10 mg L 1 Se was added. These results imply that Se treatment at appropriate dosages can significantly inhibit Hg uptake and transportation from the root to the aerial parts of rice grown in a condition with a high Hg level. This may be ascribed to the formation of a Hg–Se complex which is insoluble and unavailable for plants in a Hg/Se co-existing environment, as previously reported in other plants.25–27 Otherwise, the retention of Hg–Se compounds can also prevent Hg leaching or mobilization in the paddy field ecosystem, reducing the spread of Hg contamination to water and the surrounding environment. 3.3
Effect of Se on IHg and MeHg accumulation in rice tissues
The IHg and MeHg compounds in different tissues of the soil cultured rice were extracted with 6 M HCl. The extractability of total Hg was above 80% with the HCl method in this study. Two major Hg species in the leachate, i.e. IHg and MeHg, were separated and analyzed using HPLC-ICP-MS.28 In the HPLCICP-MS study, recovery experiments were carried out to verify
Fig. 2 The concentrations of Hg in hydroponic cultured rice seedlings under different dosages of Hg and Se exposure. * significant difference at p o 0.05 from the control group.
1954 | Metallomics, 2014, 6, 1951--1957
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 06 August 2014. Downloaded by York University on 23/10/2014 12:41:24.
Metallomics
Paper
Fig. 3 The contents of IHg (left image) and MeHg (right image) in tissues of the paddy soil cultured rice with different dosages of Se treatment. * significant difference at p o 0.05 from the group only exposed to Hg.
the viability of this method. The recovery of IHg and MeHg using the HPLC-ICP-MS method reached about 90%, which was satisfactory. The content of IHg and MeHg in the rice roots, stalks and leaves, and grains are shown in Fig. 3. The IHg levels in rice tissues followed the trend: roots 4 stalks and leaves 4 rice grains, while the MeHg levels were in the order of roots 4 rice grains Z stalks and leaves. This distribution tendency of IHg and MeHg is in agreement with a previous study.29 It is notable that the Hg species in the roots, stalks and leaves is mainly IHg, which accounts for more than 95% of the total Hg, while less than 5% of the total Hg in these parts of rice is in the form of MeHg (Fig. 4). However, MeHg might represent above 40% of the total Hg in the rice grains (Fig. 4). The proportion of MeHg distribution in different rice tissues is in the order of rice grains c stalks and leaves 4 roots. This result further confirmed a previous report in which the rice grains were considered an intensive bio-accumulator of MeHg, and rice intake can be an important pathway of MeHg exposure in human beings living in Hg-contaminated areas.6 The content of IHg in rice roots, stalks and leaves was substantially decreased with Se supplement, especially when
Fig. 4 The ratio (%) of IHg and MeHg in total Hg in tissues of the paddy soil cultured rice with different dosages of Se treatment.
This journal is © The Royal Society of Chemistry 2014
Se treatment was higher than 1 mg L 1. However, there was no significant decrease of IHg in rice grains resulting from Se addition (Fig. 3). The MeHg level in the rice roots was somewhat decreased with increasing Se levels. Nevertheless, there was no significant decrease of MeHg content in the leaves and rice grains with Se treatment (Fig. 3). It is notable that, the proportions of IHg in the rice roots, stalks and leaves, and rice grains were substantially decreased with Se addition, suggesting that the decrease of total Hg in rice might be primarily attributed to the inhibitive effect of Se on IHg uptake and transportation in the rice root and shoot, rather than a direct effect of Se on MeHg in rice (the transfer factors of IHg and MeHg are shown in Fig. S1, ESI†). The obvious discrepancy between IHg and MeHg accumulation in different rice tissues, along with the difference in effects of Se on the accumulation of IHg and MeHg in rice tissues indicate a mechanistic difference underlying IHg or MeHg uptake and accumulation in rice. As previously reported by Schwesig and Krebs, organic Hg was more prone to transportation to the ground vegetation than IHg.30 As reported in another study, naturally existing phytochelatins in plants could sequester a divalent Hg ion rather than MeHg to mitigate the phytotoxicity of Hg.31 In a recent study by Hua Zhang et al.,18 according to the investigations of Hg and Se content in rice plants collected from several Hg-contaminated sites in Guizhou Province, China, the authors concluded that Se content was positively correlated with the contents of IHg in the paddy soil samples, and the elevated Se in the soil samples could inhibit the absorption of IHg in the rice root, but not that of MeHg. In the present study, at a controllable condition, we obtained a similar result in the rice plants under different doses of Se treatment conditions by eliminating the possible influence factors from different sampling sites. According to our study, it can be speculated that the effects of Se on the accumulation and translocation of IHg and MeHg in rice plants might be quite variable. The detailed molecular mechanisms and the influence factors of Se/IHg (MeHg) antagonism in rice plant still requires further study.
Metallomics, 2014, 6, 1951--1957 | 1955
View Article Online
Paper
Published on 06 August 2014. Downloaded by York University on 23/10/2014 12:41:24.
3.4 The effect of Se on micro-zone distribution of Hg in rice tissues The SRXRF technique is a powerful tool for non-destructive elemental analysis with exceptional sensitivity.32 To reveal the influence of Se treatment on Hg micro-zone distribution in rice grown up in Hg-contaminated soil with or without Se treatment, 2D elemental distribution of the roots, leaves and rice grains were imaged using micro-SRXRF. The normalized X-ray fluorescence intensities are scaled from blue (minimum) to red (maximum). These images visually demonstrate the distributions and accumulations of Hg and Se in rice. The results are shown in Fig. 5. Fig. 5a1 shows that Hg was mainly localized in the epidermis and the pericycle of the rice root. Due to the high affinity of Hg for the sulfhydryl groups in the surface of the root,33,34 Hg can be enriched to quite a high concentration in the epidermis of the rice roots. Moreover, Hg can also be accumulated in the vessel of the roots, implying that Hg is able to penetrate the root surface into the vascular cylinder and then transport upwards. For Se/Hg co-exposed rice, one can see that the distribution of Se correlates well with that of Hg in the rice roots (Fig. 5b1 and b2), and both of them principally concentrate in the epidermis and pericycle of the rice root. Comparing Fig. 5b1 with 5a1, a substantial decrease in Hg accumulation in the epidermis and stele of the rice root can be found. This is consistent with the ICP-MS results shown in Fig. 2. The correlation between Hg and Se distribution in rice roots, combined with the strong affinity of Se to Hg, suggests that Hg and Se may form a Hg–Se complex in the rice root. This may explain how Se addition could inhibit Hg uptake and
Fig. 5 The distribution of Hg and Se in different tissues of the paddy soil cultured rice measured by m-SRXRF. (a) The cross section of the root tip from rice under Hg exposure (a1, Hg XRF image); (b) the cross section of the root tip from rice under Hg and Se exposure (b1, Hg XRF image; b2, Se XRF image); (c) the leaf from Hg exposed rice; (d) the leaf from Hg and Se co-exposed rice; (e) the rice grain from Hg exposed rice; (f) the rice grain from Hg and Se co-exposed rice.
1956 | Metallomics, 2014, 6, 1951--1957
Metallomics
translocation in rice. Shanker et al. have reported the existence of a Hg–Se compound in the rhizosphere of radish, which can block the absorption of Hg through the root.19 Caruso et al. have confirmed the existence of a Hg–Se compound in the roots of Brassica juncea and soybean.20,26 Fig. 5c1, d1 and d2 show the effect of Se on Hg distribution in rice leaves. It can be seen that Hg is dispersed over the leaves, and some is specially located in the leaf vein (Fig. 5c1). Furthermore, the Se distribution pattern correlates well with that of Hg in the rice leaves (Fig. 5d2). The content of Hg in the leaves collected from the Hg/Se co-exposed rice plant (Fig. 5d1) is much less than that of the Hg exposed group (Fig. 5c1), indicating the inhibitive effect of Se on Hg transportation from the roots to the leaves through the vascular cylinder. The effects of Se on the distribution and accumulation of Hg in the rice grain are illustrated in Fig. 5e1, f1 and f2. From Fig. 5e1, it is shown that Hg is principally concentrated on the surface of the rice grain (the aleurone layer), especially along the growth site of the embryo. It is notable that a considerable amount of Hg can accumulate in the embryo part (Fig. 5e1), while less Hg is located in the rice endosperm. Fig. 5f1 and f2 show the distributions of Hg and Se in rice grain collected from Hg/Se co-exposed rice. Except for the location in the aleurone layer like Hg, a considerable proportion of Se can penetrate into the endosperm of the rice grain. Moreover, Se distribution in the embryo is more extensive than that of Hg. A similar Se distribution pattern was previously reported,35 in which much Se accumulation in the outer regions of the rice grain was observed, and an amount of Se was concentrated in the chalazal zone that shares a close proximity to the ocular vascular trace. Comparing Fig. 5f1 with Fig. 5e1, the concentration of Hg in the embryo part of the rice grain from Se and Hg co-exposed rice is much lower than that of the rice exposed to Hg alone. This indicates that Se can interfere with Hg accumulation in the rice grain, and suggests that Se treatment may mitigate Hg toxicity towards the growth and development of rice seeds, as the embryo is the budding point of the rice seed. In addition, the essential elements (Fe, Cu, Zn, K, Ca etc.) are mainly concentrated in the embryo of the rice grain (Fig. S2, ESI†). This suggests that the necessary nutrient elements, rather than the toxic elements, can be selectively accumulated in the rice grain. Furthermore, it implies the existence of a preventive mechanism for toxic element accumulation in rice. In conclusion, Se treatment can inhibit Hg uptake and transportation from the rice root to the aerial part, which finally results in lower Hg accumulation in the rice grain, especially in the aleurone layer and embryo. The effect of Se on MeHg is less remarkable than IHg, along with the discrepancy of IHg and MeHg accumulation in different rice tissues indicating mechanistic differences in the absorption and transportation of IHg and MeHg in rice. The present study may provide valuable information for sequestering Hg and novel insights for addressing food safety in Hg/Se polluted environments. More researches on the positions for the methylation of IHg in vitro or in vivo and the phyto-biological behaviors of IHg and MeHg in rice will be needed to further understand the ecological toxicology of this toxic element.
This journal is © The Royal Society of Chemistry 2014
View Article Online
Metallomics
Acknowledgements
Published on 06 August 2014. Downloaded by York University on 23/10/2014 12:41:24.
This work was financially supported by the National Natural Science Foundation of China (Grant No. 21377129, 11205168, 11375213), and the special project on treatment and control of water pollution (No. 2012ZX07203-006-06). SRXRF analysis was carried out at beamline BL15U in Shanghai Synchrotron Radiation Facility (SSRF). We thank Xiaohan Yu, Aiguo Li, Ke Yang et al. in SSRF, and Dongliang Chen and Wei Xu in Beijing Synchrotron Radiation Facility (BSRF) for their assistance during the SRXRF measurement and the data processing.
References 1 O. Lindqvist, K. Johansson and M. Aastrup, Water, Air, Soil Pollut., 1991, 55, xi-261. 2 T. W. Clarkson, Environ. Health Perspect., 1993, 100, 31–38. 3 WHO, International programme on chemical safety: environmental health criteria 118-inorganic mercury, World Health Organization, Geneva, 1991. 4 J. Burger, M. Gochfeld, C. Jeitner, S. Burke, T. Stamm, R. Snigaroff, D. Snigaroff, R. Patrick and J. Weston, Sci. Total Environ., 2007, 384, 93–105. 5 B. Meng, X. Feng, G. Qiu, Y. Cai, D. Wang, P. Li, L. Shang and J. Sommar, J. Agric. Food Chem., 2010, 58, 4951–4958. 6 X. Feng, P. Li, G. Qiu, S. Wang, G. Li, L. Shang, B. Meng, H. Jiang, W. Bai, Z. Li and X. Fu, Environ. Sci. technol., 2008, 42, 326–332. 7 H. Zhang, X. Feng, T. Larssen, L. Shang and P. Li, Environ. Sci. technol., 2010, 44, 4499–4504. 8 B. Meng, X. Feng, P. Li, C. Chen and L. Shang, Environ. Sci. technol., 2011, 45, 2711–2717. 9 M. Horvat, N. Nolde, V. Fajon, V. Jereb, M. Logar, S. Lojen, R. Jacimovic, I. Falnoga, L. Y. Qu, J. Faganeli and D. Drobne, Sci. Total Environ., 2003, 304, 231–256. 10 Maximum levels of contaminants in foods (in Chinese); GB2762-2005; Standardization Administration of People’s Republic of China: Beijing, 2005 pp. 171–173. 11 M. Stadlober, M. Sager and K. J. Irgolic, Food Chem., 2001, 73, 357–366. 12 A. D. Lemly, Ecotoxicol. Environ. Saf., 2004, 59, 44–56. 13 A. M. H. Debruyn and P. M. Chapman, Environ. Sci. Technol., 2007, 41, 1766–1770. 14 D. Tran, A. John Moody, A. S. Fisher, M. E. Foulkes and A. N. Jha, Aquat. Toxicol., 2007, 84, 11–18. 15 T. W. Clarkson, Crit. Rev. Clin. Lab. Sci., 1997, 34, 369–403. 16 D. Y. Yang, Y. W. Chen, J. M Gunn and N. Belzile, Environ. Rev., 2008, 16, 71–92.
This journal is © The Royal Society of Chemistry 2014
Paper
17 H. Y. T. Truong, Y. W. Chen, M. Saleh, S. Nehzati, G. N. George, I. J. Pickering and N. Belzile, Metallomics, 2014, 6, 465–475. 18 H. Zhang, X. Feng, J. Zhu, A. Sapkota, B. Meng, H. Yao, H. Qin and T. Larssen, Environ. Sci. Technol., 2012, 46, 10040–10046. 19 K. Shanker, S. Mishra, S. Srivastava, R. Srivastava, S. Dass, S. Prakash and M. M. Srivastava, Food Chem. Toxicol., 1996, 34, 883–886. 20 S. Mounicou, M. Shah, J. Meija, J. A. Caruso, A. P. Vonderheide and J. Shann, J. Anal. At. Spectrom., 2006, 21, 404–412. 21 J. Zhao, Y. Gao, Y. Li, Y. Hu, X. Peng, Y. Dong, B. Li, C. Chen and Z. Chai, Environ. Res., 2013, 125, 75–81. 22 J. Zhao, Y. Hu, Y. Gao, Y. Li, B. Li, Y. Dong and Z. Chai, Metallomics, 2013, 5, 896–903. 23 D. Cargnelutti, L. A. Tabaldi, R. M. Spanevello, G. de Oliveira Jucoski, V. Battisti, M. Redin, C. E. B. Linares, V. L. Dressler, ´. M. de Moraes Flores and F. T. Nicoloso, Chemosphere, E 2006, 65, 999–1006. 24 A. Stebbing, Sci. Total Environ., 1982, 22, 213–234. 25 K. Shanker, S. Mishra, S. Srivastava, R. Srivastava, S. Daas, S. Prakash and M. M. Srivastava, Plant Soil, 1996, 183, 233–238. 26 S. K. Yathavakilla and J. A. Caruso, Anal. Bioanal. Chem., 2007, 389, 715–723. 27 D. H. McNear, S. E. Afton and J. A. Caruso, Metallomics, 2012, 4, 267–276. 28 Y. Li, C. Chen, B. Li, Q. Wang, J. Wang, Y. Gao, Y. Zhao and Z. Chai, J. Anal. At. Spectrom., 2007, 22, 925–930. 29 M. Meng, B. Li, J. J. Shao, T. Wang, B. He, J. B. Shi, Z. H. Ye and G. B. Jiang, Environ. Pollut., 2013, 184, 179–186. 30 D. Schwesig and O. Krebs, Plant Soil, 2003, 253, 445–455. 31 E. M. Krupp, A. Mestrot, J. Wielgus, A. A. Meharg and J. Feldmann, Chem. Commun., 2009, 4257–4259. 32 Y. Gao, N. Liu, C. Chen, Y. Luo, Y. Li, Z. Zhang, Y. Zhao, B. Zhao, A. Iida and Z. Chai, J. Anal. At. Spectrom., 2008, 23, 1121–1124. 33 B. V. Cheesman, A. P. Arnold and D. L. Rabenstein, J. Am. Chem. Soc., 1988, 110, 6359–6364. 34 S. Carrasco-Gil, A. Alvarez-Fernandez, J. Sobrino-Plata, R. Millan, R. O. Carpena-Ruiz, D. L. Leduc, J. C. Andrews, J. Abadia and L. E. Hernandez, Plant, Cell Environ., 2011, 34, 778–791. 35 P. N. Williams, E. Lombi, G. X. Sun, K. Scheckel, Y. G. Zhu, X. B. Feng, J. M. Zhu, A. M. Carey, E. Adomako, Y. Lawgali, C. Deacon and A. A. Meharg, Environ. Sci. Technol., 2009, 43, 6024–6030.
Metallomics, 2014, 6, 1951--1957 | 1957
