S1
SUPPLEMENTAL DATA
INORGANIC MERCURY ACCUMULATION IN RICE (Oryza
sativa L.)
BO MENG,† XINBIN FENG,*† GUANGLE QIU,† DINGYONG WANG,‡ PENG
LIANG,‡ PING LI,† LIHAI SHANG†
†State Key Laboratory of Environmental Geochemistry, Institute of Geochemistry,
Chinese Academy of Sciences, Guiyang, P.R. China
‡College of Resources Environment, Southwest University, Chongqing, P.R. China
15 pages in total, 4 Tables, 1 Figure
1
S2
Sites Description. The Hg mining centre of Gouxi, located in Tongren district,
eastern Guizhou province, is one of the largest artisanal Hg mining centers in China
(Fig. S1). Hg mining activities at Tongren area can be dated back to the Qin Dynasty
(221 B.C.). Large-scale mining activities were officially closed in 2001. However,
small-scale artisanal retorting activities were on-going in Gouxi area during the
rice-growing season of 2007. The briefly process of artisanal Hg mining is that the
cinnabar is crushed and roasted at 700-800 °C to produce Hg vapor. Then, the Hg
vapor is condensed in a wooden barrel and cooled by water. It has been demonstrated
that the abandoned mine wastes and artisanal Hg smelting activities have resulted in
serious Hg contamination of the ambient air, stream water, soil, and organisms in the
areas of interest (Li et al., 2008; Li et al., 2009; Qiu et al., 2005; Qiu et al., 2008;
Meng et al., 2010; Horvat et al., 2003). Meng et al. (2010) reported elevated
concentrations of Hg0 vapor in Gouxi ambient air reaching up to approximately 1650
ng m-3. The concentration of total Hg (THg) in surface soil samples (upper 0~20 cm),
moss samples, and stream water samples in Gouxi area can reach up to 20 mg kg-1, 44
mg kg-1, and 2300 ng L-1, respectively (Li et al., 2008; Qiu et al., 2008).
The control site of Huaxi, located southwest of Guiyang city (about 30 km
distance from the city) (Fig. S1), has no direct point sources of Hg contamination. The
corresponding measured concentrations of THg in ambient air (2.12-12.8 ng m-3),
surface soil (upper 0~20 cm, 0.25-0.30 mg kg-1), and drinking water (5.4 ± 1.9 ng L-1)
are significantly lower than those found in Gouxi (Li et al., 2008; Li et al., 2009;
Meng et al., 2010; Qiu et al., 2005; Zheng, 2007).
Experimental Design. In order to investigate the dynamic process of IHg
accumulation in rice plant, four experimental plots of rice-plantation were carefully
designed (Table S1).
2
S3
The first plot was a control site-paddy (CS-P) consisted of a rice paddy field
located at control-site Huaxi receiving a comparatively low Hg input by atmospheric
deposition (27 ± 17 ng L-1). This field with low soil Hg concentration (0.29±0.023 mg
kg-1) was irrigated with local drink water containing low levels of Hg (5.4±1.9 ng L-1).
The second plot was a control site-box (CS-B) (dimension 1.5×1.5×0.4 m) filled
up with soil (depth 30 cm) obtained from the Hg-contaminated paddy field (top soil
0-10 cm; 30±1.1 mg kg-1) in Gouxi and received water from the same irrigation plant
as CS-P. CS-B was mounted on the roof about 2.5 m above the surrounding ground in
order to avoid any contamination from soil particles by splashing during heavy
rainfall, and was situated beside CS-P in Huaxi. Therefore, it received a low Hg input
by atmospheric deposition (27 ± 17 ng L-1).
The main difference between CS-P and CS-B was the concentration of THg in
soil at CS-B; it was significantly higher than those at CS-P. The CS-P was assumed to
represent the regional background level where the rice plant was exposed to low Hg
deposition flux/air and low Hg soil. In contrast, the objective of CS-B was to
understand the influence of Hg contaminated soil on the mechanisms and process
controlling IHg accumulation in rice plant when the plant was exposed to the low Hg
deposition flux from the air pathway. Hence, both CS-P and CS-B were irrigated with
low Hg, local drinking water.
Analogous to CS-P, the third plot was an artisanal site-paddy (AS-P) consisting
of a rice paddy field located at Gouxi receiving an elevated Hg input by atmospheric
deposition (2900 ± 1400 ng L-1). This rice paddy with an elevated Hg soil
concentration was irrigated with stream water polluted with Hg (4200±4900 ng L-1).
The average concentration of THg in soil of AS-P was 11 ± 3.4 mg kg-1, which was
significantly higher than the range of 0.01-0.5 mg kg-1, representative of
3
S4
uncontaminated soils globally (Senesi et al., 1999). The objective of AS-P was to
understand the source and process of IHg accumulation in rice plants when they were
exposed to the elevated Hg in the air and soil (the opposite to CS-P).
The fourth plot is an artisanal site-box (AS-B) with the same dimension as CS-B
filled up with soil to a depth of 30 cm obtained from the uncontaminated paddy field
in Huaxi (topsoil 0-10 cm). It had a low level of Hg concentration (0.44±0.028 mg
kg-1). This box was mounted on the roof about 2.5 m above the surrounding ground in
order to avoid any cross-contamination from native soil particles during heavy rainfall,
and was situated beside (approximately 300 m) AS-P in Gouxi. Therefore, it received
a high Hg deposition flux. Rice plants were irrigated with local drink water containing
low levels of Hg (14±2.7 ng L-1).
For the AS-B, we intended to understand whether the newly deposited Hg impact
the process and accumulation of IHg in rice plant. If we choose the serious
contaminated stream water (THg: 4200 ± 4900 ng L-1) as the irrigation water for
AS-B, the newly deposited Hg and irrigation water Hg (stream water) in soil will not
be distinguished. Owing to the highly elevated Hg concentration in precipitation
(2900 ± 1400 ng L-1) compared to that in the local drink water (14±2.7 ng L-1) at
Gouxi, the relative importance of Hg input from local drink water to the plot AS-B
could be negligible. Hence, the local drink water but not the stream water was chosen
as the irrigation water.
Description of Sample Collection. The experimental design and sampling campaigns
were same to our previous study (Meng et al., 2011). For each experiment plot, 2-6
rice plants, as well as the corresponding soil from the root zone (10-20 cm depth),
were collected during each sampling campaign randomly. In detail, each rice sample
comprised of a composite of at least five sub-samples of the rice plants in the same
4
S5
experimental plot, and the amount of each rice sample was recorded. All rice samples
were cleaned using drink water in-situ followed by de-ionized water rinses after being
brought back to the laboratory, and finally air dried and stored in polyethylene bags to
avoid cross-contamination.Soil samples were collected by hand with the disposable
polyethylene glove at the same site as the rice plant sample, and then were sealed,
double-bagged, stored in an ice-cooled container before being shipped to the
laboratory within 24 hours, and then stored in a refrigerator at -17 ºC prior to being
freeze-dried.
Rice plant tissues were ground to 150 mesh (IKA-A11 basic, IKA). Similarly,
freeze-dried soil samples were homogenized to 150 mesh with a mortar before
chemical analysis. Precautions were taken in order to avoid any cross contamination
during sample preparation. The grinder was thoroughly cleaned after processing each
sample. Powdered samples were transferred into an open plastic dish and separately
enclosed in polyethylene bags, then placed in a desiccator kept at +4 ºC in the dark.
Samples of irrigation water and precipitation were collected concurrently using
approved methodologies (Oslo, 1998; U.S. EPA, 1996). In this study just prior to a
precipitation event, a rain guage (measuring cylinder) was exposed to the air.
Immediately after the event, samples were poured into precleaned borosilicate glass
bottles. There was no collection of dry atmospheric deposition of Hg. All water
samples were collected by hand using ultra-clean handling protocols, transferred into
precleaned borosilicate glass bottles and promptly acidified to 0.5% (v/v) using
adequate volumes of concentrated hydrochloric acid. The bottled samples were then
sealed, double-bagged, and transported to the laboratory within 24 hours. Prior to Hg
analysis, samples were stored in a refrigerator at +4 ºC in the dark.
Description of Analytical Methods. For THg analysis, a rice plant sample was
5
S6
digested in a freshly prepared mixture of HNO3/H2SO4 (4:1, v/v) at 95°C (U.S. EPA,
2002; Horvat et al., 1991). Similarly, soil was digested using a fresh mixture of HCl
and HNO3 (1:3, v/v). THg was determined by cold vapor atomic fluorescence
spectrometry (CVAFS, Tekran 2500, Tekran Instruments) preceded by BrCl oxidation,
SnCl2 reduction, pre-concentration, and finally thermo-reduction to Hg0 (U.S. EPA,
2002). For MeHg analysis, rice plant samples were treated using a
KOH-methanol/solvent extraction technique (Liang et al., 1996). MeHg was
determined using aqueous ethylation, purge, trap, and GC-CVAFS detection (Brooks
Rand Model III, Brooks Rand Labs) following the U.S. Environmental Protection
Agency method 1630 (U.S. EPA, 2001; Liang et al., 1994).
Investigations with differing techniques have established that ethyl mercury
species are not present in detectable amounts in provisions, including rice flour, wheat
flour, and dogfish liver (Lin et al., 2008; Shi et al., 2005) in one of the studies, in such
a way that the sum of IHg and MeHg concentration compared favorably with that of
THg (Lin et al., 2008). Therefore, the concentration of IHg in rice plant tissues was
calculated here as the difference between the concentration of THg and MeHg in the
sample (Meng et al., 2010).
THg in water samples was quantified using dual amalgamation CVAFS (Tekran
2500, Tekran Inc., Toronto, Ontario, Canada) within 28 days of sampling following
the approved methodology (U.S. EPA, 2002; Bloom et al., 1988).
Detailed Information Concerning the QC/QA. Quantification for THg and MeHg
in rice tissues sample was conducted using daily calibration curves with the
coefficient of variation (r2) ≥ 0.99. Quality control measures consisted of method
blanks, triplicates, matrix spikes, and several certified reference materials. Field
blanks of water samples were 0.13 ng L-1 for THg. The method detection limits (3)
6
S7
were 0.01 μg kg-1 for THg and 0.002 μg kg-1 for MeHg in rice tissues. The precision
and bias for triplicate samples were less than 7.2% for THg and MeHg analysis. The
recoveries for matrix spikes ranged from 96 to 110% for THg analysis, and from 86 to
105% for MeHg. The following certified reference materials were employed: rice
(GBW08508 and GBW10010, National Research Centre for Certified Reference
Materials), poplar leaf (GBW07604, National Research Centre for Certified Reference
Materials), and shellfish (TORT-2, National Research Council of Canada). The results
of the certified reference material analysis are given in Table S2.
Statistical analysis was performed using SPSS 13.0 software (SPSS). The IHg
measurements in samples are generally described by giving the mean ± standard
deviation (SD). Relationships between covariant sets of data were subjected to
regression analysis. Correlation coefficients (r) and significance probabilities (p) were
computed for the linear regression fits. Differences are declared as significant in case
that p < 0.05. Moreover, the data was analyzed by principal component analysis.
7
S8
Table S1. Summarization of the experimental design.
Experimental
Code
Soil Source
Water Source
plots
Huaxi,
Control plot
CS-P
Uncontaminated
uncontaminated
Control
Gouxi,
CS-B
Uncontaminated
transplant
contaminated
Gouxi,
Treatment plot AS-P
Contaminated.
contaminated
Treatment
Huaxi,
AS-B
Uncontaminated
transplant
uncontaminated
8
Air Source
Huaxi,
uncontaminated
Huaxi,
uncontaminated
Gouxi,
contaminated
Gouxi,
contaminated
S9
Table S2. List of certified reference materials used in the present study and
corresponding analytical result.
Producer
CRM
n
Hg speciation Obtained value Certified value
NRC CRM* GBW07604 10 THg (mg kg-1) 0.027±0.0013
0.026±0.003
-1
NRC CRM* GBW10010 10 THg (mg kg ) 0.0053±0.0001 0.0053±0.0005
NRC CRM* GBW08508 10 THg (mg kg-1)
0.038±0.001
0.038±0.003
NRCC***
TORT-2
10 THg (mg kg-1)
0.27±0.017
0.27±0.06
-1
NRCC***
TORT-2
10 MeHg (μg kg ) 0.15±0.0041
0.15±0.013
*NRC CRM: National Research Centre for Certified Reference Materials.
**NIST: National Institute of Standards and Technology.
***NRCC: National Research Council Canada.
****IAEA: International Atomic Energy Agency.
9
S10
Table S3. Concentrations of IHg in tissues of rice plant (dry wt with standard deviations) during the rice growing season
Experimental
plots
CS-P
CS-B
AS-P
AS-B
Rice growing
season
30
45
60
75
90
30
45
60
75
90
30
45
60
75
90
30
45
60
75
90
n
4
2
4
2
5
2
2
2
2
5
2
2
2
2
5
2
2
2
3
6
Root, mean±SD,
(mg kg-1)
0.15±0.035
0.19±0.019
0.12±0.025
0.13±0.0089
0.15±0.012
4.0±0.14
3.9±0.86
3.2±1.3
4.9±0.94
2.6±0.53
3.2±1.1
3.0±0.81
3.2±0.12
2.5±0.20
2.8±0.25
1.3±0.44
1.5±0.60
2.4±0.61
2.1±0.21
1.9±0.24
Stalk, mean±SD,
(mg kg-1)
0.041±0.030
0.037±0.018
0.015±0.0047
0.019±0.0017
0.031±0.0066
0.24±0.055
0.17±0.031
0.13±0.012
0.13±0.036
0.0670.012±
0.64±0.015
0.63±0.038
0.62±0.13
1.0±0.0080
1.6±0.16
1.0±0.018
1.2±0.096
1.4±0.14
2.6±0.13
3.5±0.83
10
Leaf, mean±SD,
(mg kg-1)
0.15±0.035
0.058±0.0057
0.052±0.0072
0.073±0.028
0.13±0.010
0.14±0.011
0.14±0.0048
0.14±0.016
0.20±0.0095
0.27±0.057
2.7±0.15
5.2±1.6
6.1±0.25
12±1.3
13±0.95
3.9±0.15
5.7±0.42
7.3±1.8
13±0.79
16±2.6
Hull, mean±SD,
(mg kg-1)
Seed, mean±SD,
(mg kg-1)
n/a
n/a
n/a
n/a
n/a
n/a
0.0073±0.00028
0.0073±0.0025
0.0042±0.00016
0.0042±0.0013
n/a
n/a
n/a
n/a
n/a
n/a
0.020±0.0020
0.020±0.0039
0.020±0.00347
0.021±0.0086
n/a
n/a
n/a
n/a
n/a
n/a
1.0±0.17
1.1±0.37
0.43±0.023
0.43±0.087
n/a
n/a
n/a
n/a
n/a
n/a
1.5±0.013
1.5±0.17
0.48±0.011
0.52±0.16
S11
Table S4. IHg mass (ng plant-1) in tissues of rice plant (dry wt with standard deviations) during the rice growing season.
Experimental
plots
CS-P
CS-B
AS-P
AS-B
Rice growing
season
30
45
60
75
90
30
45
60
75
90
30
45
60
75
90
30
45
60
75
90
n
4
2
4
2
5
2
2
2
2
5
2
2
2
2
5
2
2
2
3
6
Root, mean±SD,
(µg plant-1)
0.16±0.044
0.39±0.029
0.30±0.053
0.33±0.050
0.23±0.032
4.6±0.45
4.5±1.3
6.8±1.7
7.7±0.21
7.2±1.1
7.3±3.3
5.4±0.43
8.3±0.60
5.2±1.3
4.3±0.38
2.6±0.70
3.45±1.5
5.2±0.87
5.7±0.14
5.1±0.90
Stalk, mean±SD,
(µg plant-1)
0.21±0.14
0.42±0.22
0.33±0.095
0.32±0.039
0.51±0.091
1.3±0.32
1.8±0.31
2.9±0.22
2.1±0.50
1.1±0.18
5.2±0.62
11±1.2
21±3.3
24±2.1
22±1.4
6.1±0.61
12±0.67
29±2.2
63±2.7
48±8.6
11
Leaf, mean±SD, Hull, mean±SD,
(µg plant-1)
(µg plant-1)
n/a
0.26±0.072
n/a
0.52±0.029
n/a
0.51±0.072
0.48±0.20
0.033±0.0013
0.79±0.048
0.039±0.014
n/a
0.71±0.037
n/a
1.3±0.069
n/a
1.3±0.20
1.3±0.024
0.087±0.0050
1.7±0.41
0.11±0.018
n/a
17±0.41
n/a
58±22
n/a
64±6.1
98±14
5.0±0.99
88±8.6
5.4±1.9
n/a
20±1.9
n/a
42±1.5
n/a
107±0.80
111±1.3
6.8±0.16
107±19
7.7±1.0
Seed, mean±SD,
(µg plant-1)
n/a
n/a
n/a
0.038±0.0035
0.089±0.033
n/a
n/a
n/a
0.18±0.037
0.42±0.14
n/a
n/a
n/a
5.3±0.10
8.8±2.2
n/a
n/a
n/a
4.4±0.031
8.4±2.59
S12
Figure S1. The locations of the study area and the sampling sites in Tongren Hg
mining area (Gouxi) and the control site (Huaxi), Guizhou Province, China.
12
S13
Literature cited
1. Bloom NS, Fitzgerald WF. 1988. Determination of volatile mercury species at the
picogram level by low temperature gas chromatography with cold-vapor atomic
fluorescence detection. Anal Chim Acta 208:151-161.
2. Chinese National Environment Protect Agency. 1995. Environmental quality
standard for soils. GB 15618-1995. Beijing, China. (in Chinese)
3. Horvat M, Lupsina V, Pihlar B. 1991. Determination of Total Mercury in Coal
Fly-Ash by Gold Amalgamation Cold Vapor Atomic-Absorption Spectrometry.
Anal Chim Acta 243:71-79.
4. Horvat M, Nolde N, Fajon V, Jereb V, Logar M, Lojen S, Jacimovic R, Falnoga I,
Qu LY, Faganeli J, Drobne D. 2003. Total mercury, methylmercury and selenium
in mercury polluted areas in the province Guizhou, China. Sci Total Environ
304:231-256.
5. Li P, Feng XB, Qiu GL, Shang LH, Wang SF, Meng B. 2009. Atmospheric
mercury emission from artisanal mercury mining in Guizhou Province,
Southwestern China. Atmos Environ 43:2247-2251.
6. Li P, Feng XB, Shang LH, Qiu GL, Meng B, Liang P, Zhang, H. 2008. Mercury
pollution from artisanal mercury mining in Tongren, Guizhou, China. Appl
Geochem 23:2055-2064.
7. Liang L, Horvat M, Bloom NS. 1994. An improved speciation method for
mercury by GC/CVAFS after aqueous phase ethylation and room temperature
precollection. Talanta 41:371-379.
8. Liang L, Horvat M, Cernichiari E, Gelein B, Balogh S. 1996. Simple solvent
extraction technique for elimination of matrix interferences in the determination
of methylmercury in environmental and biological samples by ethylation gas
13
S14
chromatography cold vapor atomic fluorescence spectrometry. Talanta
43:1883-1888.
9. Lin LY, Chang LF, Jiang SJ. 2008. Speciation Analysis of Mercury in Cereals by
Liquid Chromatography Chemical Vapor Generation Inductively Coupled
Plasma-Mass Spectrometry. J Agric Food Chem 56:6868-6872.
10. Meng B, Feng XB, Qiu GL, Cai Y, Wang DY, Li P, Shang LH, Sommar J. 2010.
Distribution patterns of inorganic mercury and methylmercury in tissues of rice
(Oryza sativa L.) plants and possible bioaccumulation pathways. J Agric Food
Chem 58:4951-4958.
11. Meng B, Feng XB, Qiu GL, Liang P, Li P, Chen CX, Shang LH. 2011. The
process of methylmercury accumulation in rice (Oryza sativa L.). Environ Sci
Technol 45:2711-2717.
12. Oslo, Commission P. 1998. JAMP guidelines for the sampling and analysis of
mercury in air and precipitation; Joint Assessment and Monitoring Programme,
pp 1-20.
13. Qiu GL, Feng XB, Li P, Wang SF, Li GH, Shang LH, Fu XW. 2008.
Methylmercury accumulation in rice (Oryza sativa L) grown at abandoned
mercury mines in Guizhou, China. J Agric Food Chem 56:2465-2468.
14. Qiu GL, Feng XB, Wang SF, Shang LH. 2005. Mercury and methylmercury in
riparian soil, sediments, mine-waste calcines, and moss from abandoned Hg
mines in east Guizhou province, southwestern China. Appl Geochem 20:627-638.
15. Senesi GS, Baldassarre G., Senesi N, Radina B. 1999. Trace element inputs into
soils by anthropogenic activities and implications for human health. Chemosphere
39:343-377.
16. Shi JB, Liang LN, Jiang GB. 2005. Simultaneous Determination of
14
S15
Methylmercury and Ethylmercury in Rice by Capillary Gas Chromatography
Coupled On-line with Atomic Fluorescence Spectrometry. J AOAC Int
88:665-669.
17. U.S. Environmental Protection Agency. 1996. Sampling Ambient Water for Trace
Metals at EPA water quality criteria levels. Office of Water, Washington, DC.
18. U.S. Environmental Protection Agency. 2001. Method 1630: Methylmercury in
Water by Distillation, Aqueous Ethylation, Purge and Trap, and CVAFS. Draft,
January. Office of Water, Washington, DC.
19. U.S. Environmental Protection Agency. 2002. Method 1631, Revision E: Mercury
in Water by Oxidation, Purge and Trap, and Cold Vapor Atomic Fluorescence
Spectrometry. August. Office of Water, Washington, DC.
20. Zheng, W. 2007. Mercury Species in the urban air of Guiyang. Ph.D. Dissertation.
The Graduate School of the Chinese Academy of Sciences (in Chinese with
English abstract), Beijing, China.
15