Plant and Soil 249: 373–382, 2003.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.
373
Effects of arsenate and phosphate on their accumulation by an
arsenic-hyperaccumulator Pteris vittata L.
Cong Tu1 & Lena Q. Ma2
Soil and Water Science Department, University of Florida, Gainesville, FL 32611-0290, USA 1 Present address:
Department of Plant Pathology, North Carolina State University, Raleigh, NC 27695-7616, USA. 2 Corresponding
author∗
Received 8 April 2002. Accepted in revised form 4 October 2002
Key words: accumulation, arsenate, hyperaccumulator, interaction, phosphate, Pteris vittata L.
Abstract
Arsenate and phosphate interactions are important for better understanding their uptake and accumulation by plant
due to their similarities in chemical behaviors. The present study examined the effects of arsenate and phosphate on
plant biomass and uptake of arsenate and phosphate by Chinese brake (Pteris vittata L.), a newly-discovered arsenic
hyperaccumulator. The plants were grown for 20 weeks in a soil, which received the combinations of 670, 2670,
or 5340 µmol kg−1 arsenate and 800, 1600, or 3200 µmol kg−1 phosphate, respectively. Interactions between
arsenate and phosphate influenced their availability in the soil, and thus plant growth and uptake of arsenate and
phosphate. At low and medium arsenate levels (670 and 2670 µmol kg−1 ), phosphate had slight effects on arsenate
uptake by and growth of Chinese brake. However, phosphate substantially increased plant biomass and arsenate
accumulation by alleviating arsenate phytotoxicity at high arsenate levels (5340 µmol kg−1 ). Moderate doses of
arsenate increased plant phosphate uptake, but decreased phosphate concentrations at high doses because of its
phytotoxicity. Based on our results, the minimum P/As molar ratios should be at least 1.2 in soil solution or 1.0 in
fern fronds for the growth of Chinese brake. Our findings suggest that phosphate application may be an important
strategy for efficient use of Chinese brake to phytoremediate arsenic contaminated soils. Further study is needed
on the mechanisms of interactive effects of arsenate and phosphate on Chinese brake in hydroponic systems.
Introduction
Arsenic (As) is toxic whereas phosphorus (P) is essential for plants. They are both Group VA elements and
thus have similar electron configurations and chemical properties. In soil, therefore, arsenate and phosphate will compete with each other for soil sorption
sites, resulting in a reduction in their sorption by
soil and an increase in solution concentrations (Livesey and Huang, 1981; Manning and Goldberg, 1996;
Smith et al., 2002). For example, Livesey and Huang
(1981) found that phosphate significantly suppressed
the sorption of arsenate by soils. Gao and Mucci
(2001) have recently reported similar competitive effects between arsenate and phosphate. They observed
∗ FAX No: +1-352-392-3902. E-mail: [email protected]
that increasing arsenate in solution results in the enhanced competition between phosphate and arsenate
for sorption sites and a subsequent decrease in the
amount of phosphate sorbed.
Similarly, it may be difficult for plants to distinguish between arsenate and phosphate. Thus uptake
of arsenate and phosphate by plants is very likely to
be competitive. Furthermore, after entering a plant, arsenate may replace phosphate in ATP synthesis, and/or
in various phosphorolysis reactions, thus interfering
with phosphate metabolisms and causing toxicity to
a plant (Dixon, 1997). In contrast, phosphate may
be able to alleviate arsenate toxicity by improving
phosphate nutrition (Sneller et al., 1999).
Arsenate competes with phosphate as a substrate
for the phosphate uptake system in many species, in-
374
cluding angiosperms (Asher and Reay, 1979; Jacobs
and Keeney, 1970), mosses (Wells and Richardson,
1985), lichens (Nieboer et al., 1984), fungi (Beever
and Burns, 1980) and bacteria (Silver and Misra,
1988). Many studies have shown that arsenate reduces
phosphate uptake by plants (Asher and Reay, 1979;
Jacobs and Keeney, 1970). Since the plant uptake system has a higher affinity for phosphate, only mild
inhibition of arsenate on phosphate plant uptake is
observed. Also, such competitive inhibition may be
insignificant as arsenate is toxic to plants at higher
levels. In recent years some studies have also shown
that at low levels arsenate can increase phosphate uptake (Burlo et al., 1999; Carbonell et al., 1998). These
authors assumed that such enhancement of plant phosphate uptake resulted from a physiological phosphorus
deficiency caused by low arsenate, since arsenate can
substitute for phosphate within the plants but is unable
to carry out phosphate’s role in energy transfer.
Phosphate have long been reported to suppress
plant uptake of arsenate (Asher and Reay, 1979; Khattak et al., 1991; Meharg and Macnair, 1991; Rumberg
et al., 1960; Woolson et al., 1973). In a hydroponic
solution containing 50 µM arsenate, sufficient phosphate will alleviate arsenate toxicity and improve plant
growth. Plant arsenate uptake rate is reduced by 75%
at 0.5 mM phosphate (Meharg and Macnair, 1991). A
molar P/As ratio of at least 12 is needed to protect
plants against arsenate toxicity (Walsh and Keeney,
1975). Nonetheless, plant arsenate uptake and toxicity
depends on both the P/As ratio and phosphate nutrition
levels. A hydroponic study has shown that at the same
P/As ratio arsenate is much less toxic at high phosphate levels since more arsenate is taken up by the
plants at low phosphate levels (Sneller et al., 1999).
In soil, however, the influence of phosphate on arsenate phytotoxicity varies. This is because soil properties
affect the availability of phosphate and arsenate. With
1100 µmol kg−1 arsenate supply, additions of up to
9700 µmol kg−1 phosphate do not influence arsenate toxicity on a silt loam soil (Jacobs and Keeney,
1970; Woolson et al., 1973). This can possibly be
attributed to the soil having a high phosphate fixation capacity and available phosphate probably does
not increase much after phosphate addition. However,
applying the same amount of phosphate actually enhances arsenate toxicity in a sandy soil, which is
due to the displacement of sorbed arsenate from the
soil by phosphate. When sufficient phosphate is added to maintain an available phosphate/arsenate molar
ratio of about 16, phosphate improves plant yields
(Woolson et al., 1973). At very high levels of added arsenate (13 mmol arsenate kg −1 ), however, phosphate
does not overcome arsenate toxicity even at a molar
phosphate/arsenate ratio of 24.
Chinese brake (Pteris vittata L.) is a newlydiscovered arsenic hyperaccumulator (Ma et al.,
2001). It accumulates 160–853 µmol As kg−1 in its
aboveground parts from uncontaminated soils containing 6.3–100 µmol As kg−1 , and takes up as much as
310 mmol As kg−1 when grown in soil spiked with
20 mmol kg−1 arsenate (Ma et al., 2001). Addition
of 670 µmol kg−1 arsenate to a sandy soil increases
the fern biomass by 107%. Moreover, this fern can
tolerate up to 6.7 mmol kg−1 arsenate in a sandy soil
and removed up to 26% of the added arsenate after
18 weeks (Tu and Ma, 2002). Unfortunately, there
is no information available about arsenate and phosphate interactions in Chinese brake. The objectives of
this study were to (1) examine the effects of arsenate
and phosphate interactions on fern biomass production
and uptake of arsenate and phosphate, and (2) determine the molar ratios of phosphate to arsenate in both
soil and plant for better fern growth. The results will
provide critical information for better understanding
arsenate hyperaccumulation by Chinese brake and optimizing soil conditions for arsenate phytoextraction.
Materials and methods
Soil characterization
The soil used in this study is a sandy soil (sandy,
siliceous, hyperthermic grossarenic paleudult) from
Gainesville, Florida. The soil pH was measured using a 1:1 soil to water ratio; cation exchange capacity (CEC) was determined by an ammonium acetate
method (Thomas, 1982); organic matter content was
measured by the Walkley Black method (Nelson and
Sommers, 1982); and particle size was measured by
the pipette method (Day, 1965). Selected physical and
chemical properties of the soil are presented in Table 1.
Experimental design
Our previous study showed that Chinese brake produced the highest biomass when 670 µmol kg−1 arsenate was added (Tu and Ma, 2002). Thus arsenate
concentrations were chosen at 1×, 4×, and 8× that
concentration, i.e., 670, 2670, and 5340 µmol arsenate
kg−1 , and referred to as low (AsL ), medium (AsM ) and
375
Table 1. Selected properties of the soil used in
this study
Property
pH (1:1 soil/water ratio)
Organic matter content (g kg−1 )
CECa (cmol(+) kg−1 )
Total P (mmol kg −1 )
Water-soluble P (µmol kg −1 )
Total As (µmol kg −1 )
Water-soluble As (µmol kg −1 )
Sand (g kg−1 )
Silt (g kg−1 )
Clay (g kg−1 )
This soil
7.3
11.0
4.4
20.8
96.8
9.2
0.27
882
91
27
a Cation exchange capacity of soil.
high (AsH ) levels, respectively. In loamy soils, phosphate was found to have no further benefit for plants at
greater than 3200 µmol kg−1 phosphate (Jacobs and
Keeney, 1970; Woolson et al., 1973). Therefore, phosphate concentrations were selected as 0.25, 0.5, and
1× the maximum phosphate concentration, i.e., 800,
1600 and 3200 µmol phosphate kg−1 , referred to as
low (PL ), medium (PM ), and high phosphate (PH ). One
control (As0 P0 ) without adding arsenate or phosphate
was also included for reference purposes.
for chemical analysis. Soil samples were collected
from each pot at transplanting the ferns for measuring
water-soluble arsenate and phosphate.
Chemical analysis
Plant (∼0.1–0.5 g) and soil (0.5–1.0 g) samples were
weighed into a 120-mL Teflon pressure digestion
vessel, mixed with 10 mL of nitric acid (concentrated trace-metals grade), and digested using USEPA
Method 3051 with a CEM MDS-2000 microwave
sample preparation system (CEM, Matthews, NC).
After cooling, the solution was filtered and diluted to
a volume of 100 mL. Water-soluble phosphate and arsenate were extracted with deionized water at 2:20 soil
to solution ratio by shaking for 2 h at 25 ± 1 ◦ C. Phosphorus concentrations in solution were measured on
a ICP-MS unit (Perkin-Elmer ELAN 6000, Norwalk,
CT). Arsenic was determined using a graphite furnace
atomic absorption spectrophotometer (Perkin-Elmer
SIMMA 6000).
Statistical analysis
The data (excluding As0 P0 ) were statistically evaluated with a two-way analysis of variance using SAS
programming (SAS Institute Inc., 1996). Means were
separated using the Duncan multiple range test at a 5%
level of probability.
Greenhouse experiment
Soil (1.5 kg) was mixed with the designated amounts
of arsenate and phosphate, 200 mg N kg−1 and 100 mg
K kg−1 , and placed in a 2.5-L plastic pot. Arsenate and
phosphate were added as Na2 HAsO4 and NaH2 PO4 ,
respectively. Potassium was provided as KNO3 and N
as both KNO3 and NH4 NO3 . There were four replicates for each treatment using a complete randomized
experimental design. After 2 weeks of equilibrium,
one healthy fern plant with five to six fronds was transplanted into each pot. Plants were allowed to grow
for 20 weeks in the greenhouse (temperature ranged
14–30 ◦ C; average photosynthetically active radiation
was 825 µmol m−2 s−1 ) and were watered daily or as
needed. At the end of the experiment, the ferns were
harvested. Each individual fern was further separated
into roots (including rhizomes) and young, mature and
old fronds based on their ages. They were all washed
thoroughly with tap water, and then rinsed quickly
with 0.1 M HCl solution followed by several rinses
with deionized distilled water. All samples were ovendried for 3 days at 65 ◦ C, and ground to a fine powder
Results and discussion
Soil water-soluble arsenate and phosphate
It is well known that water-soluble fractions of soil
arsenic and phosphorus are readily available for plant
uptake. In the present experiment, arsenic speciation
did not show the existence of other arsenic species
in solution (unpublished data). Thus, the changes in
their water-soluble fractions are important for understanding interactive effects of arsenate and phosphate
on plant. Water-soluble arsenate and phosphate in the
soil were first determined (Figure 1). After two-weeks
of incubation following arsenate and phosphate applications, arsenate and phosphate interactions in the
soil significantly affected their water-soluble fractions
(Figure 1 and Table 2). Soluble arsenate was significantly slightly increased only by higher phosphate
application compared to the low phosphate levels
(Table 2 and Figure 1a, c). However, water-soluble
phosphate was greatly enhanced by arsenate additions
376
Table 2. Results of the two-way ANOVA and Duncan tests for the effects of arsenate and phosphate on their contents
in soil and Chinese brake and the dry biomass of the plant
Source of variation
ANOVA F values
As rate
P rate
As rate × P rate
Dry biomass
(g plant−1 )
390.0∗∗∗a
44.9∗∗∗
37.6∗∗∗
Duncan multiple range test
As rate
10.0 bb
AsL c
AsM
10.6 a
4.8 c
AsH
P rate
PL
7.2 b
8.9 a
PM
PH
9.2 a
Soluble As
Soluble P
—–(µmol kg−1 soil)—–
2198.9∗∗∗
9.2∗∗
5.0∗∗
78.2∗∗∗
37.0∗∗∗
2.1NS
Root As
Frond As Root P
Frond P
————-(mmol kg−1 d.w.)————216.1∗∗∗
2.0NS
5.1∗∗
124.2∗∗∗
1.5NS
2.8NS
4.9∗
2.4NS
5.8∗∗
8.3∗∗
6.7∗∗
3.6∗
100.5 c
589.3 b
1339.0 a
580.7 c
1091.4 b
1510.8 a
7.7 c
23.4 b
34.4 a
38.3 c
118.5 b
159.4 a
168.4 a
159.1 ab
145.8 b
112.4 a
117.4 a
104.3 b
646.8 b
659.7 b
722.3 a
731.1 c
1080.7 b
1371.0 a
23.0 a
20.5 a
21.9 a
113.0 a
100.0 a
103.1 a
160.1 a
164.3 a
148.9 a
106.9 b
108.9 b
118.2 a
a NS – not significant F ratio (P < 0.05), ∗ , ∗∗ , and ∗∗∗ significant at P < 0.05, 0.01, and 0.001, respectively.
b Treatment means from the ANOVA test. Values followed by the same letter, within the same source of variation, are
not significantly different (P < 0.05), Duncan multiple range test.
c As , As and As = 670, 2670 and 5340 µmol kg−1 arsenate, P , P , and P = 800, 1600 and 3200 µmol kg−1
L
M
H
L M
H
phosphate, respectively.
at all three phosphate levels (Table 2 and Figure 1b,
d). Within experimental arsenate levels, there was a
positive correlation between arsenate rates and soluble
phosphate (coefficient = 0.992–1.00). Compared to
low arsenate treatments, water-soluble phosphate increased by 70–106% and 164–197% at medium and
high arsenate levels, respectively.
A closer examination of arsenate and phosphate
availability in soil showed that 22–123% of the applied phosphate was water-soluble while only 13–26%
of the applied arsenate was water-soluble (Table 3),
indicating adsorption of more arsenate than phosphate
by the soil. This is also supported by increased watersoluble phosphate/arsenate molar ratios of 0.8–7.2 (P
molar concentrations in the soil divided by As molar
concentrations in the soil) compared to the added
phosphate/arsenate molar ratios of 0.1–4.8 (Table 3).
Arsenate and phosphate interactions in soil have
previously been shown to increase their concentrations in soil solution by competing for sorption sites
(Gao and Mucci, 2001; Smith et al., 2002). For
example, application of phosphate fertilizer to arseniccontaminated soils has resulted in the displacement of
about 77% of the total arsenic in the soil and redistribution of arsenic to lower depths in the soil profile
(Woolson et al., 1973). Similarly, phosphate sorption
by soil is decreased by arsenate as demonstrated by
Table 3. Percentages of water-soluble arsenate and phosphate concentrations in total addition and the phosphate/arsenate molar ratios
in soil
Treatment
% of water-soluble/
total added
P
As
As0 P0
AsL /PL c
AsL /PM
AsL /PH
AsM /PL
AsM /PM
AsM /PH
AsH /PL
AsH /PM
AsH /PH
NAb
34
26
22
69
62
43
123
99
63
NA
13
15
17
19
23
24
25
25
26
P/As molar ratioa
Water-soluble
320
4.3
5.3
7.2
1.3
1.8
2.3
0.8
1.2
1.5
Total added
NA
1.2
2.4
4.8
0.3
0.6
1.2
0.1
0.3
0.6
a P molar concentrations in the soil divided by As molar concentra-
tions in the soil.
b Not applicable.
c As , As and As = 670, 2670 and 5340 µmol kg−1 arsenate,
L
M
H
PL , PM , and PH = 800, 1600 and 3200 µmol kg−1 phosphate,
respectively.
Gao and Mucci (2001) who reported that phosphate
sorption decreased from 57 to 48% and 57 to 42%
following the addition of 8.7 and 22 µM arsenate, respectively. In our experiment, when 5,430 µmol kg−1
377
Figure 1. Effects of different arsenate and phosphate levels on water-soluble arsenate and phosphate concentrations in a soil in the greenhouse
experiment. The treatment As0 P0 (). The levels of added phosphate were 800 (), 1600 (♦), and 3200 (×) µmol kg−1 . The levels of added
arsenate were 670 (), 2670 (), and 5340 () µmol kg−1 . Bars represent standard deviations of four replicates.
arsenate and 800–1600 µmol kg−1 phosphate were
concurrently added to the soil, all the phosphate was
essentially water-soluble (Table 3), i.e., no phosphate
was adsorbed by the soil.
Biomass of Chinese brake fern
For a hyperaccumulator, biomass is a key factor for
phytoremediation practices. Moreover, it is also an
overall measurement of plant health. Our previous experiment showed that the biomass of Chinese brake
was greatly enhanced by arsenate up to 1330 µmol
kg−1 and it survived even at 6700 µmol kg−1 (Tu
and Ma, 2002). The enhancement of fern biomass
by arsenate was also observed in this study up to
2670 µmol kg−1 arsenate (Figure 2 and Table 2). This
arsenate rate was two times greater than previously
reported (Tu and Ma, 2002). Addition of medium arsenate levels increased fern biomass by 10–24% as
compared to that in low arsenate. However, phosphate
only increased fern biomass at the high arsenate level.
At 5340 µmol kg−1 arsenate, the fern biomass was
greatly increased by 1600 µmol kg−1 phosphate compared to that at 800 µmol phosphate kg−1 addition,
though it was still lower than that of As0 P0 . However, no further biomass enhancement was observed
at 3200 µmol kg−1 phosphate. These findings indicate that phosphate has an alleviating effect on arsenate
phytotoxicity to Chinese brake only at high arsenate
levels (Figure 2). Interestingly, the molar ratios of
water-soluble phosphate to arsenate in the soil were
0.8, 1.2 and 1.5 for 5340 µmol arsenate kg−1 treatments with 800, 1600 and 3200 µmol phosphate kg−1 ,
respectively (Table 3). It seems that phosphate plays
a minor role in inhibiting arsenate phytotoxicity at a
soluble P/As molar ratio of greater than 1.2. This value
was much lower than the results obtained from nonhyperaccumulators (Hurd-Karrer, 1939; Woolson et
al., 1973).
Phosphate effect on arsenate uptake and
accumulation in Chinese brake
Chinese brake is very efficient in absorbing arsenic
from soil and translocating it from roots to shoots (Ma
et al., 2001; Tu and Ma, 2002). Arsenic concentrations
in the plant are generally much higher in the fronds
than in the roots, with concentrations increasing with
378
Figure 2. Effects of different arsenate and phosphate levels on the
biomass of Chinese brake grown in a soil in the greenhouse experiment. The levels of added phosphate were 0 (), 800 (), 1600
(×), and 3200 (♦) µmol phosphate kg−1 . Bars represent standard
deviations of four replicates.
soil arsenate. These findings were also observed in
the present study (Figure 3), with the highest arsenate
concentrations found in the fern growing in the soil
receiving high arsenate (5340 µmol arsenate kg−1 ).
At low and medium arsenate levels (≤2670 µmol
kg−1 arsenate and water soluble P/As = 1.3–7.2),
phosphate slightly but not significantly increased arsenic concentrations in the roots and fronds of the
fern (Table 2 and Figure 3c, d). Interactive effects
of phosphate and arsenate was observed on root arsenic (Table 2). However, at high arsenate levels
(5340 µmol kg−1 ), phosphate at 1600 µmol kg−1 decreased arsenic concentrations by 23–25% in the roots
and fronds (Figure 3c, d). This appears to be due to
the dilution effects from greater biomass production,
which resulted from alleviation of arsenate phytotoxicity by phosphate (Figure 2). Previous studies showed
that the effect of phosphate on plant arsenate uptake depends on plant growing conditions (Asher and
Reay, 1979; Jacobs and Keeney, 1970; Khattak et
al., 1991; Meharg and Macnair, 1991; Pickering et
al., 2000; Woolson et al., 1973). In a hydroponic
system, phosphate at 500 µM reduced arsenate uptake by 75% in both tolerant and non-tolerant plant
genotypes of soft grass (Holcus lanatus L.) grown in
50 µM arsenate solution (Meharg and Macnair, 1991).
Alfalfa (Medicago sativa L.) shoot arsenate concentrations were also decreased by phosphate (Khattak et
al., 1991). Even for Indian mustard (Brassica juncea
L.), a hyperaccumulator, grown in 500 µM arsenate hydroponic solution with phosphate addition at
1000 µM, a reduction of arsenate uptake by 55–72%
over the control was reported (Pickering et al., 2000).
Since arsenate and phosphate are generally transported by the same uptake system, which has a much
greater affinity for phosphate than arsenate (Asher
and Reay, 1979; Meharg and Macnair, 1990; Meharg et al., 1994), phosphate can effectively reduce
the arsenate uptake and toxicity of plants. In the soil
systems, however, phosphate may either reduce arsenic concentrations in the plant through competition
and/or enhanced plant growth by alleviating arsenate
phytotoxicity, or increase arsenate uptake by the plant
and hence phytotoxicity, depending on soil conditions
and/or relative phosphate/arsenate levels (Creger and
Peryea, 1994; Jacobs and Keeney, 1970; Woolson et
al., 1973). This is because addition of phosphate usually increases arsenate concentrations in soil solution
by replacing arsenate sorbed by soil particles (Smith
et al., 2002).
Plant arsenic accumulation takes both arsenate uptake and plant biomass into consideration, providing
a better indication of phosphate effects on arsenate
phytoextraction (Table 4). Compared to low phosphate
level treatments, applying more phosphate fertilizer
enhanced total arsenate accumulation by the fern from
the soil, especially at high arsenate levels (5340 µmol
kg−1 ), where adding 1600–3200 µmol kg−1 phosphate resulted in increases up to 286% in arsenate
accumulation with biomass contributing more than
arsenate concentration. The highest arsenate accumulation (1042 µmol arsenate plant−1 ) by the fern
was recorded in AsM PH treatment (water soluble P/As
molar ratio = 2.3), accounting for 26% of initial total
soil arsenic. These findings have great implications for
optimizing phytoextraction of soil arsenic.
Arsenate effect on phosphate accumulation by
Chinese brake
Phosphorus concentrations in plants normally range
from 96 to 160 mmol kg−1 of dry matter during the vegetative stage, with the highest in the young and lowest
in the old parts of the plants (Marschner, 1995). In the
present study, phosphorus concentrations in the fronds
were 71–175 mmol kg−1 , which are within the normal
ranges of plant phosphorus contents. The distribution
of phosphorus in the fern is also similar to that of nonhyperaccumulators, with greater concentrations in the
roots than in the aboveground biomass, and greater in
young fronds than in old fronds. Phosphorus concentrations in the fern were little influenced by phosphate
rates (Table 2 and Figure 4). These results suggest
379
Figure 3. Effects of different arsenate and phosphate levels on arsenate concentrations in roots and fronds of Chinese brake grown in a soil in
the greenhouse experiment. The treatment As0 P0 (). The levels of added phosphate were 800 (), 1600 (♦), and 3200 (×) µmol kg−1 . The
levels of added arsenate were 670 (), 2670 (), and 5340 () µmol kg−1 . Bars represent standard deviations of four replicates.
Table 4. Arsenic accumulation (µmol plant−1 ) in Chinese brake as
influenced by different arsenate and phosphate levels in soil
Treatments
Roots
Fronds
As0 P0
AsL /PL a
AsL /PM
AsL /PH
AsM /PL
AsM /PM
AsM /PH
AsH /PL
AsH /PM
AsH /PH
0.21±0.1b
21.2±6.9
22.4±7.1
30.6±3.7
70.9±11
66.3±5.5
67.2±9.8
17.8±0.6
71.5±1.4
78.2±1.5
0.76±0.1
267.9±9.9
283.1±3.7
285.7±10
856.7±50
870.5±9.5
974.5±63
165.4±16
582.9±33
629.4±44
Total
0.97
289.1
305.5
316.2
927.6
936.8
1041.7
183.2
654.5
707.6
% of
soil As
7.0
28.4
30.0
31.0
23.1
23.3
25.9
2.3
8.2
8.8
a As , As and As = 670, 2670 and 5340 µmol kg−1 arsenate,
L
M
H
PL , PM , and PH = 800, 1600 and 3200 µmol kg−1 phosphate,
respectively.
b Mean ± SE (standard error).
that Chinese brake fern is unable to hyperaccumulate
phosphate from soil. In contrast, arsenate rates and the
interaction of arsenate and phosphate significantly influenced phosphorus concentrations in both roots and
fronds of the fern (Table 2). At high rates, arsenate
decreased phosphate accumulation in the roots and
fronds, obviously due to phytotoxicity (Table 2 and
Figure 4a, b), but increased phosphorus concentrations
in young fronds at medium rates of arsenate (Data
not shown). This result may help us to explain the
enhancement of fern biomass by arsenate at medium
rates.
The molar ratio and bioaccumulation preference of
arsenic to phosphorus in Chinese brake
The above results clearly showed that phosphate and
arsenate interaction influenced the growth of and arsenate and phosphate uptake by Chinese brake. So,
P/As molar ratios, P molar concentrations in the plant
divided by As molar concentrations in the plant, can
be a good index for their relative abundance and roles
in the plants. Generally, the molar P/As ratios in the
fern were higher in the roots (3–29) than in the fronds
(0.5–3.8) (Table 5). Much higher results have previously been reported for non-hyperaccumulating plants
grown in elevated arsenate environments such as corn
(Zea mays L. subsp. mays) seedlings with 102–4400
380
Figure 4. Effects of different arsenate and phosphate levels on phosphate concentrations in roots and fronds of Chinese brake grown in a soil in
the greenhouse experiment. The treatment As0 P0 (). The levels of added phosphate were 800 (), 1600 (♦), and 3200 (×) µmol kg−1 . The
levels of added arsenate were 670 (), 2670 (), and 5340 () µmol kg−1 . Bars represent standard deviations of four replicates.
(Woolson et al., 1973), tomato (Lycopersicon esculentum Mill.) with 500–4200 (Burlo et al., 1999), and
salt-water cordgrass (Spartina alterniflora L.) with
800–8000 (Carbonell et al., 1998). This further indicates that Chinese brake hyperaccumulates arsenate
in plant biomass. At high arsenate, the P/As ratios of
the fronds increased by applying more phosphate. By
looking at the fern biomass (Figure 2) and the corresponding P/As ratios in the fronds (Table 5), it can be
inferred that the P/As molar ratios of greater than 1.0
in the fronds seems to be necessary for normal growth
of the fern.
Bioaccumulation preference (BP) of arsenate to
phosphate by the plant measures the selectivity of the
fern in taking up arsenate from soil as compared to
phosphate, i.e., a higher number indicates a greater
preference of the fern for arsenate uptake. The BP was
calculated using the following equation:
Table 5. The molar ratio and bioaccumulation preference (BP)a of arsenate to phosphate in Chinese brake
grown in a soil with different arsenate and phosphate
levels
Treatment
As0 P0
AsL /PL c
AsL /PM
AsL /PH
AsM /PL
AsM /PM
AsM /PH
AsH /PL
AsH /PM
AsH /PH
P/As molar ratiob
Roots
Fronds
2272
28.8
24.5
16.7
7.9
5.8
6.0
3.2
5.8
4.1
763
3.8
2.7
2.7
1.0
1.0
1.0
0.5
0.7
0.8
Roots
0.1
0.1
0.2
0.4
0.2
0.3
0.4
0.3
0.2
0.4
BP
Fronds
0.4
1.1
1.9
2.7
1.3
1.8
2.3
1.7
1.7
1.8
a Bioaccumulation preference:
BP
BP
=
As concentrations in plant tissue/As contents in the soil
P concentrations in plant tissue/P contents in the soil
The BP values were found to be greater than 1 in the
fronds but less than 0.5 in the roots with an exception
shown in the control (Table 5). Therefore, arsenate
was preferentially accumulated in the aboveground
=
As concentrations in plant tissue/As contents in the soil
P concentrations in plant tissue/P contents in the soil
b P molar concentrations in the plant divided by As
molar concentrations in the plant.
c As , As and As = 670, 2670 and 5340 µmol kg−1 ,
L
M
H
PL , PM , and PH = 800, 1600 and 3200 µmol kg−1 ,
respectively.
381
parts over phosphate, especially in old fronds. However, in the control soil where arsenate was very low,
arsenic distribution in the aboveground biomass was
similar to that of phosphorus, i.e., both arsenic and
phosphorus were the highest in the young fronds and
lowest in the old fronds (data not shown). Based on
our data, it seems that phosphorus and arsenic distributions were similar in the plant at low soil arsenate
levels, but they were transported to different parts of
the plant at elevated concentrations.
In conclusion, synergistic interactions of arsenate and phosphate were observed in the soil. In the
plant, phosphate addition to soil increased arsenate
accumulation by Chinese brake, especially at high
soil arsenate concentrations by alleviating arsenate
phytotoxicity. Moderate addition of arsenate increased
phosphate uptake by the fern. To improve the fern
growth, the P/As molar ratios were required to be at
least 1.2 in soil solution or 1.0 in the fronds. The
present findings suggest that phosphate application
may serve as a feasible strategy for more efficient
phytoremediation of arsenic contaminated soils using
Chinese brake fern. The interactions of arsenate and
phosphate in the hyperaccumulator need further study
in hydroponic systems.
Acknowledgements
This research was partially supported by the National
Science Foundation (Grant BES-0086768 and BES0132114). The senior author gratefully thanks Ms.
Karen M. Parker at the Department of Plant Pathology, North Carolina State University, for improving
the English of the manuscript. The thoughtful comments by Prof. Dr. A. A. Meharg, the Editor, and two
anonymous reviewers are highly appreciated.
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Section editor: A.A. Meharg