Environmental Pollution 157 (2009) 2550–2557

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Environmental Pollution
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The relationship of root porosity and radial oxygen loss on arsenic tolerance and
uptake in rice grains and straw
X.Q. Mei a, Z.H. Ye a, *, M.H. Wong b, **
a
State Key Laboratory for Bio-control, and School of Life Sciences, Sun Yat-sen (Zhongshan) University, 135 Xin Gang West Road, Guangzhou 510275, PR China
b
Croucher Institute for Environmental Sciences, and Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, PR China
Rice with high radial oxygen loss and porosity of root accumulates low As in grains.

a r t i c l e i n f o a b s t r a c t

Article history: The correlations among arsenic (As) accumulation in grains and straw, rates of radial oxygen loss (ROL),
Received 18 September 2008 and porosity of roots using 25 rice cultivars were investigated based on two pot experiments: (1) soil
Received in revised form with addition of 100 mg As kg1 for analysis of As in grains and straw, and (2) deoxygenated solution for
24 February 2009
analyzing rates of ROL and porosity of roots. The results showed that there were great differences in grain
Accepted 28 February 2009
As (0.71–1.72 mg kg1) and straw As (15.6–31.7 mg kg1), rates of ROL (7.40–13.24 mmol O2 kg1 root
d.w. h1), and porosity (20.91–33.08%) among the cultivars. There were significant negative correlations
Keywords:
between As in grains or straw and ROL and porosity, and significant positive correlations between rates
Oryza sativa L.
Accumulation of ROL and porosities, respectively. Rice cultivars with high porosities tended to possess higher rates of
Aerenchyma ROL, and had higher capacities for limiting the transfer of As to aboveground tissues.
Arsenic Ó 2009 Elsevier Ltd. All rights reserved.
Radial oxygen loss (ROL)
Root porosity

1. Introduction Some rice genotypes accumulated significantly lower As levels

Arsenic (As) has imposed adverse environmental problems that
have received worldwide attention, especially As contamination of
paddy soils throughout southeast Asia (Meharg, 2004; Juhasz et al.,
2006; Stone, 2008). Paddy rice (Oryza sativa L.) grown on As-
contaminated soils usually possesses high As levels in shoot
(including grains) (Xie and Huang, 1998; Abedin et al., 2002; Zhu
et al., 2008a). Rice grains collected in As-contaminated districts of
Bangladesh had concentrations that were 10-fold higher than the
‘normal’ level of about 0.2 mg g1 As (Meharg and Rahman, 2003).
Besides As contamination, grain yield is also decreased when rice is
grown in paddy soils contaminated by As (Akter et al., 2005). Since
it is not cost-effective to remediate these paddy soils, and rice is
particularly susceptible to As accumulation compared to other
cereals as it is generally grown under flooded conditions where As
mobility is high (Zhu et al., 2008b), the only option is to select and
breed rice cultivars with low accumulation and high tolerance of As
(Meharg, 2004), in order to ensure safe crop production, even
under on As-contaminated sites.
* Corresponding author. Tel.: þ86 20 39332858.
** Corresponding author. Tel.: þ86 852 34117746; fax: þ86 852 34117743.
E-mail addresses: lssyzhh@mail.sysu.edu.cn (Z.H. Ye), mhwong@hkbu.edu.hk
(M.H. Wong).
in their grains than others although they grew in the same As-
contaminated paddy soil (Williams et al., 2005; Liu et al., 2006).
Currently, the mechanisms involved in As accumulation by rice are
still poorly understood, henceforth it is crucial to understand the
internal/external factors of rice that may play important roles in As
uptake and tolerance in order to select/breed the rice varieties with
high tolerance and low accumulation of As in grains.
Wetland plants commonly possess aerenchyma tissues con-
taining enlarged gas spaces (Evans, 2003). Porosity (% gas space per
unit tissue volume) in plant tissues results from the intercellular
gas-filled spaces which form a constitutive part of the develop-
ment. It has been reported that the porosity of root differs markedly
among rice varieties, and can be further enhanced by the formation
of aerenchyma, flooding or deoxygenated nutrient solution
(Colmer, 2003a; Colmer et al., 2006). In roots, O2 is required for
respiration to provide sufficient energy for growth, maintenance
and nutrient uptake. However, significant amounts of O2 supplied
via aerenchyma to roots in anaerobic substrates may diffuse into
the rhizosphere, a process termed radial oxygen loss (ROL) (Arm-
strong, 1979).
ROL from root to the rhizosphere is considered to be essential for
detoxification of phytotoxins such as Fe2þ, Mn2þ, H2S, S2, HS and
organic acids by direct oxidation or by oxidizing aerobic microor-
ganisms maintained in the rhizosphere region (Armstrong and

0269-7491/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2009.02.037
 X.Q. Mei et al. / Environmental Pollution 157 (2009) 2550–2557
Armstrong, 2005). A number of wetland plants, including rice, are
known to form iron (Fe) plaque on their roots by oxidizing Fe2þ to
Fe3þ, resulting from the oxidizing activity of plant roots and asso-
ciated microorganisms (Crowder and St-Cyr, 1991; Ye et al., 1997).
Recently, it has been discovered that Fe plaque formed on root
surface has an important effect on As accumulation by rice (Chen
et al., 2005). The oxidizing capacity and ROL of plant root are
considered the most important biotic factor controlling Fe plaque
formation (Mendellsohn et al., 1995). It was reported that flood
tolerance was positively related with ROL or aerenchyma and
porosity of plants grown in waterlogged soil (Armstrong, 1979;
Chabbi et al., 2000). However, so far the effects of ROL on As
accumulation and tolerance by rice are uncertain. The objectives of
this study were therefore to investigate the relationships between
the rate of ROL, porosity of roots, levels of As accumulation in
aboveground parts (grains and straw), and As tolerance of rice plant
among different cultivars.
2. Materials and methods
2.1. Pot trial under waterlogged condition
Seeds of 25 rice (O. sativa L.) cultivars [Basmati 370, C039, Fengaizhan, Fen-
ghuazhan, Ganxiangnuo, Guangluai, IR 56, Jingxian 89, Khazar, Molixinzhan,
Qiguizao, Qihuangzhan, Qishanzhan, Sanerai, Sanhuangzhan, Sanluzhan 7, Shengtai
1, Suyunuo, Texianzhan 13, Zhenguiai, Zhong 4188, Zhonghua 11, Zhongerruanzhan,
Yuefengzhan, Yuexiangzhan] were obtained from the Rice Research Institute in
Guangdong Province and Professor Chuanguo Li (Principal Breeder), and Professor
Guiquan Zhang, Academy of Agriculture, South China Agricultural University. All
cultivars belonged to Indica, except for Khazar, Suyunuo and Zhonghua 11 which
belonged to Japonica.
The control soil (average As concentration: 9.10 mg kg1) collected from a paddy
field (0–20 cm depth) at the campus of South China Agricultural University was
amended with As (0 and 100 mg As kg1 as Na2HAsO4 $ 7H2O, As(V)), thoroughly
mixed and allowed to equilibrate for 3 weeks, and then placed in plastic pots (7.5 cm
diameter and 14 cm high, 1 kg soil pot1). The soil used for the pot experiment
contained 11.2% organic matter, 1.30 g kg1 total N and 1.14 g kg1 total P, respec-
tively, with a pH value of 5.98. The pots were painted black and had no drainage
holes.
Seeds were disinfected in 30% H2O2 (w/v) solution for 15 min, followed by
thorough washing with deionized water, and then germinated in moist perlites.
After 3 weeks, uniform seedlings were selected and transplanted to PVC pots (same
size as described above, one plant per pot). There were four replicates for each
treatment/cultivar. The plant pots were placed in a greenhouse and arranged in
a randomized completed block design during the rice-growing season (from early-
March to mid-July). To ensure normal growth and development of rice plants,
potassium chloride (KCl) solution (in distilled water) was applied after trans-
plantation of the seedlings to give 28.6 mg K kg1 soil, and nitrogen was supplied as
a solution of urea [CO(NH2)2] (in distilled water) in four equal splits to give a total of
76.3 mg N kg1 soil during the growth period. The soil was maintained under
flooded conditions (with 2 cm of water above soil surface) during the whole growth
period of 95–135 days, which varied among different cultivars.
After measuring plant height (panicle top to level of soil in the pot) and tiller
numbers, the aboveground portion of each rice plant was harvested at maturity by
cutting at 4 cm above the soil, and separated into straw and grain. The samples were
washed thoroughly with tap water and deionized water, and then oven-dried at
55  C to constant weight.
Oven-dried straw and grain samples were ground separately using a Retsch-
grinder (Type: 2 mm, Made in Germany), and digested with HNO3/H2O2 below
110  C (Cai et al., 2000). Arsenic concentrations both in straw and grain digests were
determined using inductively coupled plasma mass spectrometry (ICP-MS). Blanks
and tea standard material (GBW-08303) (China Standard Materials Research Center,
Beijing, PR China) were used for quality control. The As recovery rates were 90  10%.
2.2. Pot trial under deoxygenated nutrient condition
2.2.1. Rice cultivation
The same seeds used in the soil pot trials were used in the deoxygenated
nutrient experiment. Five days after germination, seedlings were subsequently
exposed to 0.25 and 0.5 strength Hoagland nutrient solutions (Hoagland and Amon,
1950) for 7 days and 14 days, respectively, and then uniform seedlings were selected
and transplanted to PVC pots (7.5 cm diameter and 14 cm high, one plant per pot)
filled with deoxygenated nutrient solution containing 0.1% (w/v) agar. Wiengweera
et al. (1997) showed that dilute agar prevents convective movements in the solution
so that it may mimic the changes in gas composition found in waterlogged soil (e.g.
2551
decreased O2, increased ethylene) better than other methods used to impose root-
zone O2 deficiency in solution culture. The pots were placed in a greenhouse and
arranged in a completely randomized design, and the solutions were renewed once
every 6 days. After 30 days, the plants were used for measurements of ROL and rate
of ROL, and root porosity.
2.2.2. Preparation of titanium(III) citrate solutions
Stock solutions of the reduced titanium(III) citrate buffer were prepared using
commercially available titanium(III) chloride in HCl solution (Aldrich Chemical Co.,
Milwaukee, WI, USA), as described by Zehnder and Wuhrmann (1976) and Kludze
et al. (1993). Deoxygenated water (300 ml) was added to 17.65 g of sodium citrate to
give 0.2 M sodium citrate solution. Titanium chloride (1.16 M, 30 ml) was then added
to the sodium citrate solution, with pH adjusted to 5.6 by adding saturated sodium
carbonate. All preparations were done under N2 atmosphere.
2.2.3. Measurement of radial oxygen loss (ROL)
ROL of rice seedlings was determined according to the method described by
Zehnder and Wuhrmann (1976) and Kludze et al. (1993). Nutrient solution (40 ml)
was poured into each 50 ml test tube and the solution was purged with Ar gas for
1200 s to remove dissolved O2. Rice seedlings, previously carefully washed of any
foreign matter, with the base of the plants coated with paraffin, were inserted into
the tubes (one per test tube). The roots were completely immersed into the nutrient
solution. A 5 ml aliquot of Ti3þ-citrate solution was then injected into each test tube
with a plastic syringe, followed immediately by layering the solution surface with
20 mm of paraffin oil to inhibit contamination by atmospheric O2. Control treat-
ments did not contain any plants. All the test tubes were kept at 25  C. After 6 h, the
test tubes were gently shaken and solution samples were collected with a syringe
through a rubber tubing that had been introduced into the solution alongside the
roots.
Absorbance of the partly oxidized Ti3þ-citrate solution was measured at 527 nm
using a Perkin–Elmer 3 UV/VIS spectrophotometer. Released O2 on the whole-plant
root system was determined by extrapolation of the measured absorbance to
a standard curve. Net rate of ROL was calculated (Kludze et al., 1993) as:
ROL ¼ cðy  zÞ
where ROL ¼ radial oxygen loss, mmol O2 plant1 day1; c ¼ initial volume of Ti3þ-
citrate added to each test tube, L; y ¼ concentration of Ti3þ in solution control
(without plant), mmol Ti3þ L1; and z ¼ concentration of Ti3þ in solution after 6 h
treatment with plants, mmol Ti3þ in solution plant1 L1.
cðy  zÞ
Rate of ROL ¼
G
where Rate of ROL ¼ rate of radial oxygen loss, mmol O2 kg1 root d.w. h1; G ¼ root
dry weight, kg.
2.2.4. Measurement of root porosity
Root porosity (% gas volume/root volume, POR) was measured for adventitious
roots of rice by pycnometer method (Jensen et al., 1969; Kludze et al., 1993). About
0.6 g of fresh roots (2–2.5 cm from the root apex) were used for porosity determi-
nation. Porosity was calculated using the following formula:
ðp and vrÞ  ðp and rÞ
Porosity ¼  100
ðp þ rÞ  ðp and rÞ
r ¼ mass of fresh roots, in g; p ¼ mass of water filled pycnometer, in g; p and r ¼ mass
of pycnometer with fresh roots and water, in g; and p and vr ¼ mass of pycnometer
with vacuumed roots and water, in g.
2.3. Statistical analysis
All results are presented as arithmetic means with standard errors. A statistical
comparison of means of plant data was examined with one-way ANOVA followed by
the Tukey-HSD test as available in the SPSS statistical package. Correlation coeffi-
cient analyses were conducted using Origin 6.0.
3. Results
3.1. Growth and biomass of rice
A number of visible toxic symptoms, including growth retar-
dation, reduction in tiller, and grain or straw dry weight, associated
with As treatment were observed, especially in some varieties
(such as: C039, Sanerai, Shengtai 1). Grain and straw biomass (% of
control) of 25 rice cultivars grown in soil with the addition of
100 mg As kg1 (As(V)) soil are presented in Fig. 1. Compared with
rice grown in control soil, grain and straw biomass of all tested rice
2552 X.Q. Mei et al. / Environmental Pollution 157 (2009) 2550–2557

100
95

Grain biomass (% of control)

90
85
80
75
70
65
60
55
50
45
40
IR 56
Yuexiangzhan
Basmati 370
Suyunuo
Zhonghua 11
Sanluzhan 7

Fenghuazhan
Zhongerruanzhan
Qiguizao
Jingxian 89
Qishanzhan
Qihuangzhan
Fengaizhan
Zhenguiai
Guangluai
C 039
Molixinzhan
Ganxiangnuo
Yuefengzhan
Texianzhan 13
Zhong 4188
Sanerai
Sanhuangzhan
Shengtai 1
Khazar

100
95
Straw biomass (% of control)

90
85
80 Indica
75 Japonica
70
65
60
55
50
45
40
Zhongerruanzhan
Suyunuo

Qihuangzhan
Zhonghua 11
Basmati 370
Fengaizhan
Molixinzhan
Qishanzhan
Zhong 4188
Qiguizao
IR 56
Yuexiangzhan
Zhenguiai
Texianzhan 13
Fenghuazhan
Sanluzhan 7
Jingxian 89
Ganxiangnuo
Yuefengzhan
Sanhuangzhan
Guangluai
Sanerai
C 039
Shengtai 1
Khazar

Cultivars
Fig. 1. Grain and straw biomass (% of control) of rice grown in soil with addition of 100 mg As kg1 (as Na2HAsO4 $ 7H2O) (mean  SE, n ¼ 4).

cultivars were reduced under As treatment, and the reduction (% of 3.3. Radial oxygen loss and porosity of roots of rice grown in 0.1%
control) was significantly different (P < 0.01) among the 25 rice agar solution
cultivars, ranging from 12.6 (IR 56) to 55.5% (Shengtai 1) for grains,
and from 13.8 (Suyunuo) to 56.0% (Shengtai 1) for straw biomass. ROL and porosity of the same 25 rice cultivars were measured
There were significant (P < 0.01) correlations between grain As dry after being grown in 0.1% agar nutrient solutions for 30 days (Table
weight (% of control) and straw As dry weight (% of control) of the 1). The results showed that both the rate of ROL and the root
rice varieties tested (r ¼ 0.68, n ¼ 25, data not shown). porosity of rice were varied due to genetic differences among the
cultivars. Rates of oxygen loss ranged from 7.40 to 13.24 mmol
O2 kg1 root d.w. h1 (average 9.75 mmol O2 kg1 root d.w. h1),
3.2. Total arsenic in straw and grain
and porosities of rice roots ranged from 20.91 to 33.08% (average
26.73%).
The total As concentrations in husked grain and straw of all rice
cultivars under both the control soil and soil with addition of
100 mg As kg1 (As(V)) conditions are presented in Table 1. Under 3.4. Correlation between ROL, porosity, biomass (% of control) and
the control soil condition, husked grain As concentrations ranged As concentration in grain and straw
from 0.08 to 0.22 mg kg1, with an average of 0.15 mg kg1. Straw
As concentration ranged from 0.59 to 2.52 mg kg1, with an average There were positive significant relationships between grain or
of 1.53 mg kg1. While under 100 mg kg1 As exposure, husked straw biomass (% of control) and ROL and porosity of roots (Fig. 2).
grain As concentrations ranged from 0.71 to 2.72 mg kg1, with an Furthermore, significant negative correlations were found between
average of 1.72 mg kg1. Straw As concentration ranged from 15.60 porosity of root and grain total As concentration (P < 0.01) or straw
to 31.70 mg kg1, with an average of 22.27 mg kg1. total As concentration (P < 0.01). The negative correlations
 X.Q. Mei et al. / Environmental Pollution 157 (2009) 2550–2557 2553

Table 1
Total As concentrations (mg kg1 d.w.) in grain and straw in 25 rice cultivars grown in the control soils (contained 9.10 mg As kg1, CK) and soil with addition of 100 mg As kg1
(As(V)), and root porosity (POR) (%) and rate of ROL (mmo1 O2 kg1 root d.w. h1) of the rice cultivars exposed to 0.1% agar with 1/2 strength Hoagland solution for 30 days
(mean  SE, range, n ¼ 4).

Cultivara Total grain As Total straw As POR (%) Rate of ROL

CK Added As CK Added As
Basmati 3 0.16  0.008 (0.15–0.17) 1.33  0.02 (1.31–1.35) 1.25  0.09 (1.16–1.34) 15.60  0.15 (15.45–15.75) 28.97  0.43 (28.54–29.40) 11.64  0.36 (11.28–12.00)
C039 0.17  0.002 (0.17–0.18) 2.72  0.22 (2.50–2.94) 1.66  0.01 (1.65–1.67) 25.77  0.34 (25.43–26.11) 26.40  0.64 (25.76–27.04) 8.23  0.24 (7.99–8.47)
Fengaizh 0.16  0.004 (0.16–0.17) 2.33  0.09 (2.24–2.42) 1.89  0.06 (1.84–1.95) 23.37  0.49 (22.88–23.86) 25.77  0.29 (25.48–26.06) 8.05  0.26 (7.79–8.31)
Fenghuaz 0.19  0.009 (0.18–0.20) 1.78  0.12 (1.66–1.90) 1.97  0.09 (1.88–2.06) 29.20  0.38 (28.82–29.58) 23.33  0.91 (22.42–24.24) 8.05  0.63 (7.42–8.68)
Ganxiang 0.17  0.002 (0.17–0.18) 2.05  0.06 (1.99–2.11) 1.82  0.09 (1.73–1.90) 23.80  0.78 (23.02–24.58) 25.75  1.09 (24.66–26.84) 7.59  0.11 (7.48–7.70)
Guanglua 0.17  0.006 (0.15–0.19) 2.28  0.02 (2.26–2.30) 1.44  0.10 (1.34–1.53) 25.13  0.91 (24.22–26.04) 25.20  0.99 (24.21–26.19) 9.03  0.33 (8.7–9.36)
IR 56 0.14  0.008 (0.13–0.15) 1.05  0.07 (0.98–1.12) 0.78  0.06 (0.72–0.84) 16.20  0.22 (15.98–16.42) 25.50  0.53 (24.97–26.03) 10.39  0.59 (9.80–10.98)
Jingxian 0.15  0.005 (0.14–0.15) 2.22  0.15 (2.07–2.37) 1.35  0.11 (1.24–1.47) 21.60  0.99 (20.61–22.59) 25.39  0.78 (24.61–26.17) 9.52  0.24 (9.28–9.76)
Molixinz 0.15  0.008 (0.14–0.16) 0.71  0.02 (0.69–0.73) 1.54  0.05 (1.49–1.60) 18.80  0.39 (18.41–19.19) 29.15  0.69 (28.46–29.84) 10.00  0.10 (9.90–10.10)
Qiguizao 0.15  0.015 (0.13–0.17) 1.93  0.03 (1.90–1.96) 1.09  0.12 (0.97–1.21) 21.40  0.69 (20.71–22.09) 31.08  0.45 (30.63–31.53) 11.36  0.55 (10.81–11.91)
Qihuangz 0.13  0.008 (0.13–0.14) 1.87  0.15 (1.72–2.02) 1.96  0.13 (1.82–2.09) 24.17  0.53 (23.64–24.70) 24.30  0.16 (24.14–24.46) 9.31  0.18 (9.13–9.49)
Qishanzh 0.13  0.007 (0.12–0.14) 1.34  0.06 (1.28–1.40) 2.10  0.12 (1.98–2.22) 18.90  0.82 (18.08–19.72) 29.00  0.88 (28.12–29.88) 12.44  0.37 (12.07–12.81)
Sanerai 0.21  0.011 (0.20–0.22) 1.52  0.11 (1.41–1.63) 1.60  0.15 (1.45–1.75) 22.10  0.87 (21.23–22.97) 27.40  0.75 (26.65–28.15) 8.05  0.14 (7.91–8.19)
Sanhuang 0.19  0.008 (0.18–0.19) 2.65  0.11 (2.54–2.76) 2.42  0.10 (2.33–2.52) 25.40  0.85 (24.55–26.25) 21.43  0.57 (20.86–22.00) 8.05  0.25 (7.80–8.30)
Sanluzha 0.14  0.008 (0.13–0.14) 1.47  0.17 (1.30–1.64) 0.72  0.04 (0.67–0.76) 16.90  0.49 (16.41–17.39) 28.53  0.76 (27.77–29.29) 11.58  0.35 (11.23–11.93)
Shengtai 0.17  0.004 (0.16–0.17) 2.14  0.14 (2.00–2.28) 1.26  0.07 (1.19–1.33) 24.03  0.94 (23.09–24.97) 21.63  0.78 (20.85–22.41) 7.40  0.16 (7.24–7.56)
Texianzh 0.21  0.003 (0.21–0.21) 2.24  0.08 (2.16–2.32) 1.74  0.08 (1.66–1.82) 31.70  0.89 (30.81–32.59) 20.91  0.13 (20.78–21.04) 8.82  0.15 (8.67–8.97)
Zhenguia 0.20  0.001 (0.19–0.20) 2.26  0.06 (2.20–2.32) 1.94  0.09 (1.85–2.04) 28.30  0.84 (27.46–29.14) 24.85  0.46 (24.39–25.31) 8.23  0.17 (8.06–8.40)
Zhong 418 0.16  0.002 (0.16–0.16) 1.85  0.09 (1.76–1.94) 1.88  0.11 (1.78–1.99) 19.77  0.54 (19.23–20.31) 21.81  0.51 (21.30–22.32) 8.21  0.09 (8.12–8.30)
Zhongerr 0.11  0.010 (0.10–0.12) 1.07  0.07 (1.00–1.14) 1.53  0.08 (1.46–1.61) 23.35  0.66 (22.69–24.01) 29.48  1.06 (28.42–30.54) 11.57  0.37 (11.20–11.94)
Yuefengz 0.19  0.006 (0.18–0.19) 1.41  0.02 (1.39–1.43) 2.32  0.07 (2.24–2.39) 29.80  0.12 (29.68–29.92) 26.80  1.02 (25.78–27.82) 8.18  0.31 (7.87–8.49)
Yuexiang 0.13  0.001 (0.12–0.13) 0.96  0.02 (0.94–0.98) 1.67  0.03 (1.64–1.70) 21.07  0.75 (20.32–21.82) 29.92  0.87 (29.05–30.79) 11.99  0.27 (11.72–12.26)
Khazarb 0.08  0.002 (0.08–0.08) 1.73  0.23 (1.50–1.96) 0.64  0.04 (0.59–0.68) 15.97  0.16 (15.81–16.13) 30.40  0.52 (29.88–30.92) 10.47  0.13 (10.34–10.60)
Suyunuob 0.12  0.009 (0.11–0.13) 0.92  0.06 (0.90–0.94) 0.79  0.03 (0.76–0.82) 17.73  0.59 (17.14–18.32) 33.08  0.39 (32.69–33.47) 13.24  0.24 (13.00–13.48)
Zhonghuab 0.09  0.002 (0.09–0.10) 1.09  0.18 (0.91–1.27) 0.86  0.04 (0.82–0.90) 16.77  0.42 (16.35–17.19) 32.14  0.27 (31.87–32.41) 12.36  0.40 (11.96–12.76)
a
Only first eight letters will be used to name cultivars.
b
The cultivars are Japonica, and the others are Indica.

100 100
R SD N P
Straw biomass (% of control)

R SD N P
Grain biomass (% of control)

------------------------------- -------------------------------
90 0.76 8.22 25 < 0.001 90
0.66 8.93 25 < 0.01

80 80

70 70

60 60

50 50

40 40
7 8 9 10 11 12 13 14 7 8 9 10 11 12 13 14
Rate of ROL (mmol O2 kg-1 root d.w. h-1) Rate of ROL (mmol O2 kg-1 root d.w. h-1)

100 100
R SD N P R SD N P
Straw biomass (% of control)
Grain biomass (% of control)

-------------------------------- ------------------------------
90 0.62 9.87 25 < 0.001 90
0.55 9.96 25 < 0.01

80 80

70 70

60 60

50 50

40 40
20 22 24 26 28 30 32 34 20 22 24 26 28 30 32 34
POR (%) POR (%)

Fig. 2. Correlation between the grain or straw biomass (% of control) of rice grown in soils added with 100 mg As kg1 and the rates of radial oxygen loss (ROL) and root porosity
(POR) of rice grown in 0.1% agar solution.
2554 X.Q. Mei et al. / Environmental Pollution 157 (2009) 2550–2557

0.25
2.50

Grain total As conc. in CK (mg kg-1)

Straw total As conc. in CK (mg kg-1)

R SD N P
R SD N P
----------------------------------
-------------------------------------
-0.74 0.023 25 < 0.001
0.20 -0.56 0.43 25 < 0.01
2.00

0.15 1.50

0.10 1.00

0.05 0.50
7 8 9 10 11 12 13 14 7 8 9 10 11 12 13 14
Rate of ROL (mmol O2 kg-1 root d.w. h-1) Rate of ROL (mmol O2 kg-1 root d.w. h-1)

0.25 2.50

Straw total As conc. in CK ( mg kg-1)

Grain total As conc. in CK (mg kg-1)

R SD N P R SD N P
---------------------------------- ----------------------------------
0.20 -0.67 0.025 25 < 0.001 2.00 -0.54 0.43 25 < 0.01

0.15 1.50

0.10 1.00

0.05 0.50
20 22 24 26 28 30 32 34 20 22 24 26 28 30 32 34
POR (%) POR (%)

Fig. 3. Correlation between total As concentrations in grain or straw of rice grown in control soil (contained 9.10 mg As kg1) and rates of radial oxygen loss (ROL) and root porosity
(POR) of rice grown in 0.1% agar solution.

between rate of ROL and grain total As concentration (P < 0.01) or Williams et al., 2005, 2007; Xu et al., 2008). Concentrations of As in
straw total As concentration (P < 0.01) were also significant for the rice tissues usually follow the order of root > straw > grain
25 rice cultivars examined under both the control soil and soil with husk > grain (Abedin et al., 2002; Xu et al., 2008). Our results
addition of 100 mg As kg1 conditions (Figs. 3 and 4). showed that As concentrations in husked grains and straws ranged
from 0.08 to 0.22 mg kg1 and from 0.59 to 2.52 mg kg1 under the
4. Discussion control soil (contained 9.10 mg kg1 As) condition, and from 0.71 to
2.72 mg kg1 and from 15.6 to 31.7 mg kg1 under the soil with
4.1. Biomass (yield) and As levels in straw and husked grain addition of 100 mg kg1 (As(V)) condition, respectively (Table 1),
which were within the range reported by others (Abedin et al.,
Rice growth and yield were usually depressed when grown in 2002; Meharg and Rahman, 2003; Xu et al., 2008; Zhu et al., 2008a).
soils containing high levels of As under both greenhouse and field Under greenhouse conditions, Xu et al. (2008) reported that up to
conditions (Akter et al., 2005; Williams et al., 2005). Results from 2.5, 6.0 and 30 mg kg1 As were accumulated in rice grain
the present pot trial study clearly showed that the grain and straw (unpolished), rice husk and straw, respectively, when rice was
biomass of all rice cultivars were reduced when grown in soil with grown on soils with the addition of 10 mg As kg1. Another pot
the addition of 100 mg As kg1 compared to the control (Fig. 1). experiment conducted by Abedin et al. (2002) showed that straw
Similar results have been reported by Williams et al. (2005) when As concentration reached 91.8 mg kg1 when rice grown on soil
rice was grown in soil with the addition of 100 mg As kg1. Akter was continuously irrigated with As-contaminated water. Under
et al. (2005) showed that As in soil damaged the roots of rice, field conditions, some rice cultivated in areas irrigated with As-
resulting in inhibition of nutrient uptake; while Rahman et al. contaminated water in Bangladesh contained 1.23–2.05 mg kg1
(2007) revealed that the reduction of growth and yield of rice were total As in grain (Meharg and Rahman, 2003; Williams et al., 2005).
due to reduced chlorophyll content in rice leaf under As toxicity. Zhu et al. (2008a) reported 0.07–0.62 mg kg1 total As in rice grain
The present results also showed that toxic effects of As on rice samples from mining-impacted areas in Hunan, China.
biomass varied considerably among the rice cultivars studied. It has been noted that the uptake of As to consumable parts of
These indicated that there are great variations in As tolerance rice varied greatly with cultivars within a species (Williams et al.,
among different rice cultivars. 2005; Liu et al., 2006). Our results indicated that As levels in both
Accumulative evidence from pot trials and field samples clearly straw and husked grain varied considerably among different
showed that rice grown on As-contaminated land results in cultivars under both treatment conditions. Under the control soil
elevated As levels in rice straw and grains (Abedin et al., 2002; treatment, grain total As concentration of Sanerai and Texianzhan
 X.Q. Mei et al. / Environmental Pollution 157 (2009) 2550–2557 2555

3.00 32
R SD N P R SD N P

Straw total As conc. (mg kg-1)

Grain total As conc. (mg kg-1)
30
-------------------------------------- -------------------------------------
2.50 -0.71 0.43 25 < 0.001 28 -0.67 3.39 25 < 0.001

26
2.00
24
22
1.50
20

1.00 18
16
0.50 14
7 8 9 10 11 12 13 14 7 8 9 10 11 12 13 14
Rate of ROL (mmol O2 kg-1 root d.w. h-1) Rate of ROL (mmol O2 kg-1 root d.w. h-1)

3.00 32
R SD N P R SD N P

Straw total As conc. (mg kg-1)

Grain total As conc. (mg kg-1)

------------------------------------ 30 -------------------------------------
2.50 -0.66 0.44 25 < 0.001 28 -0.62 3.67 25 < 0001
.

26
2.00
24

22
1.50
20

1.00 18

16

0.50 14
20 22 24 26 28 30 32 34 20 22 24 26 28 30 32 34
POR ( % ) POR ( % )

Fig. 4. Correlation between total As concentrations in grain or straw of rice grown in soils added with 100 mg As kg1 and rates of radial oxygen loss (ROL) and root porosity (POR)
of rice grown in 0.1% agar solution.

was 2.6-fold higher than that of Khazar, while straw total As constitutive porosity of their adventitious root when grown in
concentration of Sanhuangzhan was 3.8-fold high than that of aerated solutions (lowest was 16%, highest was 30%), and in stag-
Khazar. Similar results were also found when the cultivars grown in nant condition between 28 and 38% (Colmer et al., 1998). The
the soil with addition of 100 mg As kg1 (Table 1). Williams et al. porosities of adventitious roots in 25 selected rice genotypes
(2005) found that in all As-treated rice, shoot As increased investigated in the present study varied from 20.9 (Texianzhan 13)
compared to the control, but the magnitude of this increase varied to 33.1% (Suyunuo) when grown under stagnant conditions, which
considerably among cultivars, with shoot As increasing up to 50% further confirmed the great variation in porosity among rice
for some cultivars, and 400% for others. Similar results were found genotypes.
in the present study (Table 1). These results showed that different Similar to porosity of roots, substantial differences on rates of
genotypes have different capacities for assimilating/accumulating ROL of roots among wetland species (Chabbi et al., 2000; Jensen
As within the rice plant. et al., 2005; Van Bodegom et al., 2005) and rice genotypes have
It was subsequently observed that large variation existed in the been commonly observed (Kludze et al., 1993). Sorrell (1999)
shoot transport of As to rice grain, with the grain/shoot transfer reported that the rates of ROL of Juncus inflexu and Juncus effuse
factor (Grain/Shoot TF) ranging from 0.002 to 0.36 (Williams et al., were 8.1 and 14.57 mmol O2 kg1 root d.w. h1, respectively. Chabbi
2007). The variation of Grain/Shoot TF obtained in our study was et al. (2000) showed that Typha domingensis (1.8 mmol O2 kg1 root
smaller than the results of Williams et al. (2007), ranging from d.w. h1) and Cladium jamaicense (1.6 mmol O2 kg1 root d.w. h1)
0.038 in Molixinzhan to 0.11 in Khazar when exposed to 100 mg differed in rates of ROL under flooded conditions. The present
As kg1 soil. The present data also showed that the grain total As results also showed significant differences in rates of ROL among
concentrations were significant positively correlated with straw different rice cultivars, from the lowest 7.40 mmol O2 kg1 root d.w.
total As concentrations under both the control soil and the As h1 for Shengtai 1 to the highest 13.24 mmol O2 kg1 root d.w. h1
treatment soil conditions (P < 0.01) (Fig. 5), indicating that more As for Suyunuo.
was accumulated in straw, and eventually transported to grains.
4.3. Correlations between porosity or ROL and As tolerance
4.2. Porosity of root and rate of ROL and accumulation

Porosity resulting from constitutive intercellular gas spaces can
differ markedly among different species and genotypes, e.g. ranging
from 2 to 22% in selected non-wetland species and from 15 to 52%
in selected wetland species under O2-deficient conditions (Colmer,
2003b). Rice genotypes also differed significantly in the
Aerenchyma and/or porosity has long been regarded as an
essential feature for the survival of emergent macrophytes, such as
rice, in anaerobic (flooded) soils, and ROL from root to the rhizo-
sphere is essential for the detoxification of phytotoxins (e.g. Fe2þ,
Mn2þ, and H2S) (Armstrong and Armstrong, 2005). Previous studies
2556 X.Q. Mei et al. / Environmental Pollution 157 (2009) 2550–2557

A 0.25
R SD N P
A

Rate of ROL (mmol O2 kg-1 root d.w. h-1)

High grain total As conc. in CK (0.17-0.21 mg kg-1)
Grain total As conc. in CK(mg kg-1)

---------------------------------
0.60 0.027 25 < 0.01 Neutral grain total As conc. in CK (0.14-0.16 mg kg-1)
Low grain total As conc. in CK (0.08-0.13 mg kg-1)
0.20 14

13

12
0.15
11

10
0.10
9

8
0.05
0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 7
20 22 24 26 28 30 32 34
Straw total As conc. in CK (mg kg-1)
POR (%)

B 3.00
B
R SD N P
High grain total As conc. (2.05-2.72 mg kg-1)

Rate of ROL (mmol O2 kg-1 root d.w. h-1)

----------------------------------
Neutral grain total As conc. (1.33-1.93 mg kg-1)
Grain total As conc. (mg kg-1)

2.50 0.57 0.48 25 < 0.01

Low grain total As conc. (0.71-1.09 mg kg-1)
14

2.00 13

12

1.50 11

10
1.00
9

0.50
14 16 18 20 22 24
Straw total As conc. (mg
Fig. 5. Correlation between grain total As concentration and straw total As concen-
tration grown in the control soil (A) and in soils added with 100 mg As kg1 (B).
showed that flood tolerance (Youssef and Saenger, 1996; Moog and
Bruggemann, 1998; Grimoldi et al., 2005; Mano et al., 2006) and
salinity tolerance (Rogers et al., 2008) of plants were correlated to
their degrees of aerenchyma/porosity and oxidizing ability.
However, little information is available about the relationship
between As tolerance or accumulation and degree of porosity and/
or ROL of rice, respectively.
The positive (significant) correlations between grain or straw
biomass (% of control) and porosity and ROL of rice (Fig. 2), and
negative (significant) correlations between grain As or straw As
concentrations and porosity and ROL of rice, respectively (Figs. 3
and 4). These results revealed that rice cultivars with higher
porosity and higher ROL tended to have higher As tolerance, and
the ability to inhibit As accumulation in grains and straw under
both the control and As-contaminated soil conditions (Fig. 6). The
results in Fig. 6 also showed that the correlation between rate of
ROL and porosity was positively significant (P < 0.01). These results
suggest that the rice cultivars with higher porosity had higher rates
of ROL, and also confirmed that higher root porosity will facilitate
more oxygen loss from roots to rhizosphere soil (Van Bodegom
et al., 2005).
Fig. 1 and Table 1 also showed that three Japonica cultivars of the
25 selected cultivars possessed higher porosities and rates of ROL,
8
26 28 30 32 34 7
kg-1) 20 22 24 26 28 30 32 34
POR (%)
Fig. 6. Correlation between grain total As concentration and rate of radial oxygen loss
(ROL) and root porosity (POR) of rice grown in the control soil (A) and in soils added
with 100 mg As kg1 (B). Symbols indicate Ward’s analysis clusters for grain As
concentrations.
higher grain and straw biomass (% of control) and lower As
concentrations in grain and straw than other Indica cultivars when
grown in both the control and As-contaminated soils. Further study
on this is needed.
Oxidizing capacity or ROL is considered the most important
biotic factor controlling Fe plaque formation (Mendellsohn et al.,
1995). Liu et al. (2006) reported that the amount (degree) of iron
plaque formed on the root surface of rice was significantly different
among the genotypes tested. The difference in iron plaque forma-
tion could reflect, at least partly, the genotypic difference in the
oxidation capacity of root during the growth period. Due to the high
adsorption capacity of iron (hydr)oxides, Fe plaque provides
a reactive substrate for As sequestration (Blute et al., 2004; Liu et al.,
2006). The formation of Fe plaque on root surface and in rhizo-
sphere resulted in an effective fixation and detoxification of As
(Doyle and Otte, 1997). As(III) is predominant species of As in
anaerobic soils (Zhao et al., 2009). The oxidative rhizosphere in
paddy soil is also likely to alter the speciation of As associated with
root surface. It is reported that most (70–80%) of the As in the Fe
plaque on the root of Phalaris arundinacea, Typha latifolia and rice
 X.Q. Mei et al. / Environmental Pollution 157 (2009) 2550–2557
was As(V), with the remaining (20–30%) As being As(III) (Hansel
et al., 2002; Blute et al., 2004; Liu et al., 2006). The presence of Fe
plaque enhances As(III) and decreases As(V) uptake by rice, but the
reasons are not clear (Chen et al., 2005). These all suggest that there
may be a strong relationship among porosity and ROL, degree of
iron plaque (formation) on root surface and in rhizosphere, and As
tolerance, accumulation in aboveground tissues of rice.
5. Conclusion
This is the first study revealing that rice cultivars with higher
porosity in their roots tend to result in more ROL from their roots,
leading to higher As tolerance and lower As levels in their grain and
straw. Porosity of root is considered a constitutive attribute of rice.
The present results are significant in screening rice cultivars with
high As tolerance and low As accumulation. The effects of ROL on
the changes of concentration and/or speciation of As, Fe and P, and
Fe plaque formation on roots and in rhizosphere soil, and the
correlations among the rates of ROL, degrees of Fe plaque, and As
levels and speciation in grains need further investigation.
Acknowledgements
We sincerely thank Guangdong Rice Research Institute and
South China Agricultural University for provision of rice seeds; and
Dr. H. Deng (South East Normal University) for technical assistance
in ROL measurement. We are also very grateful to National Natural
Science Foundation of China (30770417, 30570345), NSFC–Guang-
dong United Foundation (U0833004), Guangdong Natural Science
Group Foundation (06202438), and the Research Grant Council,
Hong Kong (HKBU 261407) for financial support.
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