International Journal of Phytoremediation, 11:313–328, 2009
C Taylor & Francis Group, LLC
Copyright ISSN: 1522-6514 print / 1549-7879 online
DOI: 10.1080/15226510802564918
EFFECTS OF NITROGEN AND PHOSPHORUS LEVELS,
AND FROND-HARVESTING ON ABSORPTION,
TRANSLOCATION AND ACCUMULATION OF ARSENIC BY
CHINESE BRAKE FERN (PTERIS VITTATA L.)
Seenivasan Natarajan,1 Robert H. Stamps,1 Uttam K. Saha,2
and Lena Q. Ma2
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1
University of Florida, Institute of Food and Agricultural Science (IFAS),
Mid-Florida Research and Education Center (MREC), Department of
Environmental Horticulture, Apopka, FL, USA
2
University of Florida, Soil and Water Science Department, Gainesville, FL, USA
This hydroponic experiment was conducted to determine the effects of nitrogen (N)
and phosphorus (P) levels and frond-harvesting on the effectiveness of arsenic (As)hyperaccumulator Chinese brake fern (Pteris vittata L.) to remove As from contaminated
groundwater collected from south Florida. Three-month old ferns were grown in 38-L
plastic tanks (two ferns per tank) containing 30-L of As-contaminated water (130 µg·L−1
As), which was amended with modified 0.25 strength Hoagland’s solution #2. Two N (26 or
52 mg·L−1) and two P levels (1.2 and 2.4 mg·L−1) were tested in one experiment, whereas
the effect of frond-harvesting was tested in a separate experiment. Initially, N had little
effect on plant As removal whereas low P treatment was more effective than high P and
As was reduced to <5 µg·L−1 in 28 d compared to 35 d. For well-established ferns, N and
P levels had little effect. Reused fern, with or without harvesting the As-rich fronds, took
up arsenic more rapidly so the As concentration in the groundwater declined faster (130
to ∼10 µg·L−1 in 8 h). Regardless of the treatments, most As (85–93%) was located in
the aboveground tissue (rhizomes and fronds). Frond As concentrations were higher for
non-harvested ferns than for ferns where fronds were partially harvested prior to treatment.
Conversely, rhizomes accumulated more arsenic in ferns where fronds had been partially
harvested. Low-P treatment coupled with reuse of more established ferns with or without
harvesting fronds can be used to effectively remove arsenic from contaminated water using
P. vittata
KEY WORDS: Pteris vittata, phytofiltration, arsenic, nitrogen, phosphorus, frond harvesting,
groundwater, kinetic parameters, translocation factor, bioconcentration factor
INTRODUCTION
Arsenic (As) is ubiquitous in the environment. Its adverse effects on human health
include cancer, diabetes, and cardiovascular diseases (Abernathy et al., 2003; Guo, 2004;
Pontius et al., 1994). To adequately protect human health from detrimental effects of As, the
Address correspondence to Robert H. Stamps, Department of Environmental Horticulture, University of
Florida, Institute of Food and Agricultural Science, Mid-Florida Research and education Center, Apopka, FL
32703, USA. E-mail: rstamps@ufl.edu
313
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S. NATARAJAN ET AL.
United States Environmental Protection Agency (USEPA) reduced the maximum contamination limit of drinking water from 50 to 10 µg·L−1, effective in January 2006 (USEPA,
2001a). Among 74,000 community water systems in the US, USEPA estimated that approximately 4100 of these systems serving 13 million people must take measures to comply
with the new standards at total annual cost of approximately $181 million (USEPA, 2001b).
Various remediation technologies for As-contaminated groundwater are available
such as oxidation-reduction, precipitation and co-precipitation, solid/liquid separation, ion
exchange, physical exclusion, and biological methods (Garelick et al., 2005). However,
most of these technologies have significant limitations in terms of requiring technical
skills to operate, cost, and residual substrate handling (USEPA, 2000). Phytofiltration,
a biological method of using arsenic hyperaccumulating plants is an emerging “green
technology,” which is environment friendly and requires minimum technical knowledge to
operate (Elless et al., 2005; Gratao et al., 2005). Several studies using plants to remove
toxic substances from water demonstrated promising results. Aquatic plants such as Scirpus
lacustril and Phragmites karka successfully removed chromium from water (Chandra
et al., 1997). Hydroponic systems have been used to remove uranium, lead and cesium
from contaminated water using seedlings of sunflower (Helianthus annus) and Indian
mustard (Brassica juncea) (Dushenkov and Kapulnik, 2000; Dushenkov et al., 1997).
Chinese brake fern (Pteris vittata), an arsenic hyperaccumulator discovered by
Ma et al., (2001), rapidly accumulated As in its fronds at concentrations as high as
22,000 mg·kg−1. Subsequently, several studies reported its possible use in phytoremediation
of As contaminated soil and water (Ma, et al., 2001; Meharg, 2003; Natarajan et al., 2008;
Zhao et al., 2002). However, a major cost for using P. vittata for As phytoremediation is
the plant material as the current wholesale price for 50 plugs is $28.00. Another major
concern of using phytoremediation technique is secondary contamination from nutrients
that are added to promote fern growth. Specifically, excess N and P in the water system
would result in accelerated eutrophication and algal growth, which have detrimental effects
on water quality (Carpenter et al., 1998; Fried, Mackie, and Nothwehr, 2003). The USEPA
recommendations for maximum nitrate and phosphate concentrations to help maintain
healthy water systems and minimize algal growth are 10 and 0.1 mg·L−1, respectively
(Kalkhoff et al., 2000).
Several studies examined factors regulating P. vittata growth and As-uptake. Tu and
Ma (2003) suggested that maximum plant biomass can be achieved by adjusting solution
pH based on As-concentration, and maximum As accumulation occurs when solution P
concentration is low and pH ≤ 5.21. Stamps (2007) reported that hydroponic production of
P. vittata increased linearly with increasing Hoagland’s #1 solution strength and was greater
in aerated than non-aerated solutions. Frond color (greenness) and biomass production
increased linearly with increase in fertilizer application rates when the plants were grown
in a soilless medium (Stamps and Rock, 2004). There are no well-defined cost effective and
environment friendly cultural practices to grow these fern for maximum As removal from
contaminated water. Although it was suggested that frond harvesting at 15-cm improved
the fern re-growth (Stamps and Rock, 2003), there are no studies reported regarding effects
of harvesting on its arsenic removal capacity.
Therefore long- and short-term experiments were conducted to study the effects
of nutrient levels and frond-harvesting practices on As removal by growing P. vittata in
contaminated groundwater. In a previous paper (Natarajan et al., 2008) the effects of plant
density, fertilizer concentrations, and repeated harvesting on phytoremediation efficiency
and plant growth was presented. The specific objectives of this study were to determine 1)
DIFFERENTIAL AS-DISTRIBUTION IN PTERIS VITTATA WITH TIME OF EXPOSURE
315
the optimum N and P application rates for maximum As removal while sustaining healthy
fern growth, 2) the effect of frond-harvesting on As-uptake, kinetic parameters of arsenic
absorption, and As-distribution in P. vittata. The results from this study should shed light on
optimizing parameters for maximizing As removal from contaminated water using P. vittata.
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MATERIALS AND METHODS
Two separate experiments were conducted with the first one (long-term: 26 weeks)
focusing on the effects of N and P and the second one (short term: 48 h) on effects of
frond-harvesting on As removal from water by P. vittata. The ferns used in the N and P
experiment were harvested three times (3 cycles) whereas the frond-harvesting experiment
was repeated once. The As-contaminated groundwater (∼130 µg·L−1 As) which may have
been contaminated by past use of As-based herbicides, was collected from South Florida
(Natarajan et al., 2008). Nutrient content of groundwater analyzed before adding nutrients
was as follows: pH of 8, total As ∼130 µg·L−1, soluble salts 130 mg·L−1, Ca 18.3 mg·L−1,
Mg 1.4 mg·L−1, K 3.7 mg·L−1, Na 13.3 mg·L−1, NH4 -N 0 mg·L−1, NO3 -N 0.40 mg·L−1,
P 0 mg·L−1, SO4 -S 3.43 mg·L−1, micronutrients (B, Cu, Fe, Mn, and Zn) 0 mg·L−1.
N and P Experiment
Ferns used in this experiment were grown from 3-month old sporlings germinated on
rock wool. Fern plugs were suspended in 8-cm net pots on 5-cm thick Styrofoam floats (two
per float) in 38-L plastic tanks, which were filled with 30 L of As-contaminated groundwater
(Figure 1a). Hydroponic tanks containing two net pots and rock wool plugs per float, but
no ferns, were used as controls. The solutions were continuously aerated and water losses,
both through evaporation (tanks with no ferns) and evapotranspiration and plant growth
(tanks with ferns), were replaced every week with dilute 0.25 strength Hoagland’s solution
#2 (0.25 HS). Electrical conductivities (ECs) and pHs measured during the experiment
ranged between 540–590 µS cm−1 and 7.7–8.1 respectively.
To test the effects of N and P on As removal by P. vittata, the As-contaminated
groundwater was amended with 0.25 HS with further reduced N (26 or 52 mg·L−1) and P
(1.2 or 2.4 mg·L−1) levels. Water samples were collected weekly for 12 weeks in Cycle 1.
Figure 1 a) An illustration of hydroponic tank with two ferns; and b) an experimental set up of Pteris vittata in
hydroponic tanks, arrows indicate partially harvested fern at 15-cm height from rhizome.
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S. NATARAJAN ET AL.
At the end of 12-weeks, As-rich fronds (6-month old) were harvested at a 15-cm height
above the rhizome and the same ferns were used for Cycles 2 and 3. After first harvest and
refilling with fresh As-water, water samples were first collected two days after initiation
with subsequent sampling every week for five weeks in Cycle 2. In well-established ferns
(7.3-month old) after second harvest, water samples were collected at 2 h intervals for 8 h in
Cycle 3. All treatments were replicated three times (2 N concentrations × 2 P concentrations
× 3 replications = 12 tanks).
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Harvesting Experiment
The same well-established ferns used in Cycle 3, which had by this time developed
extensive root and frond systems, were used in this experiment to determine the effect
of frond-harvesting on As removal by P. vittata. Before initiating this experiment, all
fronds except for the croziers (immature, unfurling fronds) were harvested close to the
rhizome to minimize the residual As from the previous experiment. Harvested ferns were
pre-cultured for 3 weeks in As-free nutrient solution amended with 0.25 HS with low N
(26 mg·L−1) and P (1.2 mg·L−1) to promote frond biomass growth. The nutrient solution
was continuously aerated and water lost through evapotrapiration was replaced weekly as
described previously.
Ferns with three to four mature fronds after 3 weeks of preculture were selected.
To determine the effect of frond-harvesting, half of the ferns (6 tanks) were partially
harvested at 15-cm above the rhizome (Figure 1b). Nutrient solution was then replaced
with As-contaminated groundwater (120 µg·L−1) with no nutrients added for both partially
harvested and non-harvested tanks. All the tanks were arranged in a completely randomized
design. Water samples (10 mL) were collected at 2-hour intervals for 8 h. Half of the fern
plants were harvested at the end of 8 h and the remainder at the end of 48 h. Ferns were
separated into roots, rhizomes and fronds after harvest. Separated plant parts were washed
twice with tap water and once with distilled deionized water, drained, bagged individually
and oven dried at 55◦ C for five days.
Arsenic Analysis
Dried plant samples were ground to fine powder (20 mesh) and digested with
concentrated HNO3 and deionized H2 O (1:1, v/v), followed by 30% H2 O2 for As
determination (USEPA, 1983). The As-concentration of water samples and tissue extract
was determined using a graphite furnace atomic absorption spectrophotometer (GFAAS)
(SIMMA 6000; PerkinElmer, Wellesley, MA), blanks and internal standards were included
for quality assurance.
Short-Term Arsenic Uptake and Kinetic Parameters
A rapid depletion of arsenic in the groundwater reflects the net arsenic uptake
by P. vittata roots. Hence, kinetics of arsenic influx was determined using the solution
depletion technique as described by Claassen and Barber (1974). A parabolic spline or
Michaelis-Menten equation can be used to describe the net influx rate of arsenic.
I=
I max (C − C min)
,
Km + (C − C min)
(1)
DIFFERENTIAL AS-DISTRIBUTION IN PTERIS VITTATA WITH TIME OF EXPOSURE
317
where I is the net influx rate, expressed as µmol g−1 root fresh weight (fwt) h−1; I max
is the maximum net influx rate; and Km is the ion concentration when I = 1/2 I max. Km
indicates the plant root affinity for the ion and C min is the lowest ion concentration at
which plant roots can extract a particular ion from solution. These kinetic parameters can be
calculated using several procedures. In this study, a parabolic equation (square polynomial)
computed by least square regression as described by Tu et al. (2004) was used to obtain the
kinetic parameters.
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Statistical Analysis
Values are expressed as mean ± standard error. The data were analyzed for significant
differences (P ≤ 0.05) using PROC GLM and ANOVA procedures of SAS (SAS Institute,
2001). For kinetic parameters, a linear regression and variance analysis were carried out
using PROC REG and ANOVA. Tukey’s least significant differences (LSD) test was used
to compare the means.
RESULTS AND DISCUSSION
There are three major differences between this study and previous ones (Wang et al.,
2002; Tu and Ma, 2002; Huang et al., 2004). First, in this study, we used As-contaminated
groundwater containing relatively high-As concentrations (∼130 µg L−1). Second, we
used large volume of water per plant (15 L per plant) compared to 0.6–0.8 L per plant
in Tu and Ma (2002) and Huang et al. (2004). Third, this study compared the effect of
frond harvesting practice on fern’s As uptake capacity and its distribution pattern within
the plant. In short, this experiment is more applicable to real world since the data were
based on As-contaminated groundwater, realistic water to plant ratio and frond harvesting
practice. Since the groundwater macro and micro nutrient content was very negligible, a
weak nutrient solution was used to sustain the healthy growth of the ferns.
Effects of N & P on As Removal by P. Vittata
During the initial establishment of young ferns (6 month old) in Cycle 1, it took 28
to 35 days for P. vittata to reduce As from 130 to <10 µg·L−1 (Figure 2a). For water
samples analyzed at 28 days, As concentrations in the low P (1.2 mg· L−1) treatment were
significantly lower (<5 µg·L−1) than high P (2.4 mg·L−1) treatment (P = 0.05). It took
an additional 7 days to drop to the same level in the high P treatments. These results
suggest that ferns tend to absorb more arsenic when P concentrations are low. Several
studies demonstrated that plants take up arsenate by the phosphate pathway due to their
chemical similarity (Meharg and Hartley-Whitaker, 2002; Meharg and Macnair, 1990,
1991a; Meharg, Naylor and Macnair, 1994). Wang et al., (2002) reported that the presence
of P in solution culture markedly decreased arsenate influx, whereas P starvation for 8 days
increased the maximum net influx by 2.5 fold.
In the present study, when established ferns (6 to 7.3 month old) were reused in
Cycles 2 and 3, P treatments showed no difference in As-depletion from contaminated
groundwater (P = 0.05) (Figure 2b and 2c). In Cycle 2 (after the fronds were harvested),
water As concentrations rapidly declined to less than 10 µg·L−1 in 2 days and continued to
maintain low concentrations during subsequent weeks compared to control with no ferns
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S. NATARAJAN ET AL.
-1
Arsenic concentration ( g·L )
(a)
160
Cycle 1
140
120
100
80
60
N-26 P-1.2
N-26 P-2.4
N-52 P-1.2
N-52 P-2.4
Control (no plants)
40
20
0
0
7
14
21
28
35
(b)
160
Cycle 2
-1
Arsenic concentration ( g L )
140
120
100
80
60
40
20
0
0
2
7
14
21
28
Time (days)
(c)
160
Arsenic concentration ( g·L -1)
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Time (days)
140
Cycle 3
120
100
80
60
40
20
0
0
2
4
6
8
Time (hours)
Figure 2 Effect of nitrogen (N) (26 or 52 mg·L−1) and phosphorus (P) (1.2 or 2.4 mg·L−1), which was supplied
using a modified 0.25 strength Hoagland’s solution #2, on As removal by Pteris vittata from contaminated
groundwater containing 130 µg As L−1. a) Three-month old sporlings, b) well-established six months old fern,
and c) well-established ferns 7.3 months old with extensive root and frond systems. Error bars represents means
± SE (n = 3).
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DIFFERENTIAL AS-DISTRIBUTION IN PTERIS VITTATA WITH TIME OF EXPOSURE
319
(P = 0.05) (Figure 2b). This indicates the fern’s major role in As uptake and that there
was no leaching of As from roots. With even more well-established ferns in Cycle 3,
As-depletion from water was extremely rapid (from 130 to ∼10 µg·L−1 in just 8 h) (Figure
2c). Arsenic uptake rate calculated based on initial and final arsenic concentration in water,
increased by more than 80 fold from Cycle 1 to Cycle 3 (Table 1) or from using 3-month
old ferns to 7.3 month old ferns. This characteristic is useful when using P. vittata ferns
repeatedly for phytofiltration of As-contaminated water.
Based on Cycle 3, the As removal rate was 0.2 mg As /h/plant (Table 1), which is the
highest reported rate for P. vittata (Tu and Ma, 2002; Wang et al., 2002; Huang et al., 2004).
The increase in plant As removal may be attributed to two factors: 1) more established ferns
with relatively large root and shoot structures, i.e. bigger ferns take up more As; and 2)
frond-harvest induced new growth promotes more As-uptake by P. vittata (discussed later).
Also, it is interesting to note that low P has no influence on increasing arsenic uptake when
well-established ferns are used.
In addition, our study was the first one to demonstrate the increased effectiveness of
As removal via reused P. vittata, which is in contrast to Tu and Ma (2002). This may be
partly because the duration of the earlier study was very short (3 days), younger ferns were
used (3–4 months old), and ferns were reused without frond harvesting. In contrast, in our
study, the ferns were re-used for a longer duration (26 weeks), and in each cycle ferns were
more established than they were in the previous cycle. The slower arsenic depletion in earlier
studies were also attributed to high arsenic status in the plants that might have inhibited
As-uptake (Tu and Ma, 2002), as also observed in barley whose phosphate uptake was
restricted under high nutrient status (Lee, 1982). In our study, periodic frond harvesting at
15-cm height during the initial stages of fern growth may have proved beneficial to the plant
by lowering arsenic load and helped to produce new growth for enhanced arsenic uptake.
Based on the results ferns can be grown in As contaminated groundwater with minimum
nutrient addition (0.25 HS) with further reduced N (26 mg L−1) and P (1.2 mg L−1)
and the same plants can be re-used for effective phytofiltration of arsenic.
Effects of Frond-Harvesting on Plant As Removal
This is the first study to report the effects of frond-harvesting practice on As-removal
capacity of fern. These results are very useful in practical application of these ferns in
Table 1 Effects of nitrogen and phosphorus levels in 0.25-strength Hoagland’s solution #2 on arsenic (As) uptake
rate (µg·h−1) from contaminated groundwater by Pteris vittata during three sequential remediation cycles using
the same plants. Values are means ± SE (n = 3)
Arsenic uptake rate (µg·h−1)z
26 mg·L−1
52 mg·L−1
Nitrogen Phosphorus
1.2 mg·L−1
2.4 mg·L−1
1.2 mg·L−1
Cycle-1
Cycle-2
Cycle-3
4.60 ± 0.03
67.0 ± 0.50
382 ± 6.30
4.00 ± 0.60
69.0 ± 0.04
343 ± 1.90
4.60 ± 0.01
68.5 ± 0.30
389 ± 4.10
ZArsenic uptake rate
=
vol. of water in liters×(initial As conc.−final As conc.)
time in hours
2.4 mg·L−1
4.60 ± 0.02
68.0 ± 0.30
399 ± 1.30
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S. NATARAJAN ET AL.
As-phytoremediation where As-rich fronds were harvested for disposal and same ferns
may be reused for long-term cleanup process. Results show that both partially harvested
and non-harvested ferns effectively removed As from water, i.e., a rapid decline in As
concentrations was observed during the first four hours (Figure 3a) and after eight hours, As
concentrations were reduced to about 11.6 µg·L−1, similar to data from Cycle 3. Cumulative
As uptake calculated based on root fresh weight exhibited a hyperbolic saturation curve:
an almost linear increase in As-uptake in the first four hours followed by a slower increase
or leveling off with time, in both partially harvested (R2 = 0.92) and non-harvested ferns
(R2 = 0.88) (Figure 3b). These results suggest that when the ferns are fully established,
frequent frond harvesting may not be required and the same plants can be reused for
remediation purpose. However, delaying frond-harvest for longer period may result in
potential problem of arsenic leaching from decaying dead and dry fronds (Kertulis-Tartar,
2006). Therefore to optimize the frequency of frond harvesting, and to reduce the labor and
biomass disposal costs, it may be best to only harvest mature and senescing fronds. Pteris
vittata can accumulate arsenic up to 22,000 mg·kg−1 dry weight, with a threshold value of
approximately 10,000 mg·kg−1 dry weight for phytotoxic symptoms to appear (Tu and Ma,
2002; Wang et al., 2002). The threshold tolerance for phytotoxicity of P. vittata is much
greater than non-hyperaccumulating plants (5 to 100 mg·kg−1 dry weight) (Kabata-Pendias
and Pendias, 1992).
Based on our results and from earlier studies, it is possible to adjust the frond
harvesting intervals in P. vittata depending on the As contamination levels at the site and
initial appearance of phytotoxic symptoms. Also, the results suggest that during this short
term exposure of ferns to As-contaminated groundwater for phytofiltration, absence of
nutrients did not reduce its arsenic uptake capacity. Hence, a batch of mature ferns (two
ferns/30 L) may be maintained in weak nutrient solution (0.25 HS) as stock plants and
can be transferred to As-contaminated groundwater with no added nutrients to remove
As. A regular rotation of ferns between As-free nutrient-solution and As-contaminated
groundwater (no added nutrients) may be adopted for continuous As-phytofiltration. Elless,
et al., (2005) suggested a similar regime where 10 hydroponic tanks (eight ferns/45 L)
were connected in series and continuously filled with As-contaminated water (14 µg·L−1).
Further studies are needed to determine the number of cycles that these ferns can be
effectively used to remove As from contaminated groundwater without added nutrients.
Also, nutrient regime of groundwater differs from region to region, hence a complete water
analysis may be useful in determining the nutrient requirement for the ferns that are used
in phytofiltration.
Kinetics of Arsenic Uptake
Kinetic parameters of ion absorption can be used to describe the plant arsenic
uptake pattern. According to Michaelis-Menten equation the uptake pattern under different
substrate concentration indicates that, at low substrate concentration ion uptake operates
via high affinity system (HAS), and at high substrate concentration via low affinity system
(LAS) (Epstein, 1976). Arsenic concentrations of ≤ 100 µM correspond to a HAS and 100
µM –10,000 µM to a LAS (Meharg and MacNair, 1994). Since the initial As-concentration
in this experiment was 1.7 µM (130 µg·L−1), the arsenic uptake here probably controlled
by a HAS.
To determine the efficiency of P. vittata to absorb arsenic from the contaminated
groundwater, kinetic parameters of As-uptake were calculated based on arsenic depletion
DIFFERENTIAL AS-DISTRIBUTION IN PTERIS VITTATA WITH TIME OF EXPOSURE
321
140
120
100
80
60
40
Partially harvested
20
Non-harvested
Control (no plants)
0
0
2
4
6
8
Time (h)
(b)
350
Partially harvested (– – –)
Non-harvested (-----)
250
200
Y = –2.2x2 + 43x – 9.6
R2 = 0.92
-1
(nmol·g root fwt)
300
Cumulative As uptake
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-1
Water As concentration ( g·L )
(a) 160
150
Y = –3.4x2 + 57x – 8.7
R2 = 0.88
100
50
0
0
2
4
6
8
Time (h)
Figure 3 Effect of frond-harvesting from rhizome on a) As-depletion by Pteris vittata from contaminated
groundwater containing 130 µg·L−1 As. Error bars represent ± SE (n = 6) and, b) cumulative As-uptake by P.
vittata based on root fresh weight. The quadratic parabolic curves were fitted to the scatter plot of the all data
(n = 30).
over a period of 8 h and root fresh weights. Arsenic uptake rates calculated at 8 h were
24.4 ± 0.7 and 26.9 ± 0.9 nmol·g−1 root fwt·h−1 for the harvested and non-harvested
treatment, respectively. Frond harvesting treatment did not show significant difference in
Km and C min, however, I max value of non-harvested ferns was greater than harvested
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S. NATARAJAN ET AL.
Table 2 Effect of frond-harvest on kinetic parameters of arsenic uptake by Pteris vittata over a period of 8 h
from contaminated groundwater containing 130 µg L−1 As, which was amended with 0.25-Strength Hoagland’s
solution #2. Values are means ± SE (n = 6)
Treatments
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Partial frond harvest
No frond harvest
I max (nmol g−1 root fwt h−1)
Km (µM)
48.0 ± 2.05
55.4 ± 1.81
0.55 ± 0.03
0.51 ± 0.01
C min (µM)
0.156 ± 0.03
0.124 ± 0.03
ferns (P = 0.05) (Table 2). Earlier studies indicated that uptake of metal ions in plants is via
transpirational mass flow (Ouyang, 2005). For conditions under which the transpiration rates
are high, the mass flow increases the supply of metal to the root interface, consequently
increasing the uptake (Lehto, et al., 2006). In the present study, the greater I max in
non-harvested ferns may be due to relatively higher transpiration rate from intact fronds
than partially harvested fronds. Hence, it may be beneficial if the fronds were left intact to
increase its I max and thus enhance arsenic removal from contaminated water.
C min, which is defined as the lowest concentration at which plants roots can extract
As, was 0.156 µM (11.7 µg·L−1) and 0.124 µM (9.3 µg·L−1) in harvested and nonharvested ferns, respectively. The Km calculated in this study were 0.51 and 0.55 µM,
and are similar to those reported in other studies (Tu et al., 2004 and Wang et al., 2002).
These values are much lower than reported in other non-hyperaccumulator plant species
(6–25 µM), such as Hordeum vulgare, Oryza sativa, Holcus lanatus, Agrostis capilaris and
Deschampsia cespitosa (Asher and Reay, 1979; Abedin et al., 2002; Meharg and Macnair,
1994, 1992, 1991b). Significantly, lower Km value indicates the enhanced arsenic uptake
efficiency of P. vittata, and its efficiency was not affected by frond harvesting at 15 cm height.
Arsenic Concentration and Distribution in Fronds, Rhizomes, and Roots
Results presented here are based on the assumptions that at the initiation of the
harvesting experiment the residual arsenic that may be still present in the ferns from
the N & P experiment, was negligible and equal in all the fern plants. Tissue analysis
in the present study indicated that fronds of non-harvested ferns (at the end of 8
and 48 h) and rhizome and fronds of partially harvested ferns at the end of 48 h
was relatively high in As compared to rest of the treatments (P = 0.05) (Figure 4).
While there is no difference in As concentration of rhizome at the end of 8 h in both
partially harvested and non-harvested ferns, it was greater in partially harvested ferns than
non-harvested ferns at the end of 48 h (P = 0.05). Among partially harvested ferns As
concentration was equally distributed in fronds and rhizomes, whereas in non-harvested
ferns fronds accumulated greater As at the end of 8 and 48 h (P = 0.05). Root As
concentration in partially harvested fern was not different at the end of 8 and 48 h in,
however with increase in duration of exposure (8 to 48 h) concentration dropped to half
in non-harvested fern. Overall arsenic concentration in the roots were significantly lower
than rhizomes and fronds of ferns (P = 0.05). Results indicate that frond biomass may be
the driving force for greater As uptake, and with the duration of exposure accumulation of
As in the aboveground tissue increases. The influence of residual As that may be present
in the rhizomes and roots from the previous study may be negligible and equal among all
the ferns. Differential As-levels in different plant parts reported in the present study were
mainly due to duration of exposure and frond biomass levels.
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DIFFERENTIAL AS-DISTRIBUTION IN PTERIS VITTATA WITH TIME OF EXPOSURE
323
Figure 4 Tissue arsenic (As) concentration and relative distributions (percentages) of As in plant tissues in
partial frond-harvested and non-harvested Pteris vittata. The ferns were separated into rhizomes, roots and fronds
after exposure to 130 µg·L−1 As contaminated groundwater for 8 and 48 h.
For convenience, distribution of As among the different plant parts (frond, rhizome
and root) was expressed as percent of total absorbed As and corresponding values were
indicated in Figure 4. The highest percentage, i.e. 40 to 56% of absorbed As was quickly
transported to the fronds within 8 to 48 h in both partially harvested and non-harvested
fern. Rhizomes accumulated next highest percentage, 36 to 46%, and the least remained in
the roots, ranging from 7 to 15%. In other words, shoot tissue (both rhizome and fronds)
accumulated majority of the As (85 to 93%). This distribution is within the range reported
by Tu, et al. (2002), Zhang, et al. (2002) and Ouyang (2005).
In this study, rhizome acted both as sink and source for As-accumulation and
translocation within the plants. Singh and Ma, (2006) reported that, P. vittata exposed
to high arsenic had high As-accumulation in the rhizome and was described as primary
sink for As. Accumulation of arsenic in the rhizome was described as a “buffer storage”,
reported most likely as a plant’s adaptation to tolerate As-phytotoxicity, especially in
heavily contaminated sites (Liao et al., 2004). Similarly, in our study, rhizomes may
have served as “buffer storage” for As-accumulation both in the presence and absence
of sufficient frond biomass. Ouyang (2005) using a modified mathematical model showed
that, As-accumulation in the fronds may be as a result of transpirational mass flow from
roots to fronds through xylem. Based on this model it may be expected that with the
production of new fronds and time, in partially harvested and non-harvested ferns, As
accumulated in the rhizome will be translocated to the frond tissue via transpirational mass
flow and can be later harvested for disposal. Also, in the present study, the differential
As-accumulation in partially harvested vs. non-harvested ferns could be due to reduced
transpirational surface area in the former, having only 15-cm fronds (un-harvested portion).
This may have resulted in greater proportion of As being accumulated in rhizomes of
partially harvested ferns, compared to the fronds in non-harvested ferns during the 48 h
period.
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Arsenic Translocation Factor
For effective arsenic phytoremediation, rapid translocation of absorbed As to the
aboveground tissue is essential. Translocation factor (TF), which is defined as ratios of Asconcentration of the shoots to roots, was calculated to quantify the translocation efficiency of
P. vittata. Here TF was calculated separately for frond:root, frond:rhizome and rhizome:root,
to better understand the arsenic distribution in these tissues. Overall TF was greater than
1 in all the tissue of P. vittata, however, arsenic TF for frond:root and rhizome:root were
much higher, ranging from 2.5 to 8.5, than for frond:rhizome ranging from 1 to 1.5 (P =
0.05) (Figure 5a). Arsenic translocation from root to either frond or rhizome of P. vittata
increased with increase in duration of exposure, i.e., 8 to 48 h. Highest arsenic TF was
obtained in frond:root of non-harvested ferns at the end of 48 h (P = 0.05) indicating
that the As-translocation within fern is due to frond biomass and increased duration of
exposure. Lowest TF (close to 1) among the frond:rhizome of both partially harvested
and non-harvested ferns may be due to almost equal accumulation capacity of fronds and
rhizomes. Among both partially harvested and non-harvested ferns, TFs of rhizome:root
increased with an increase the duration of exposure (P = 0.05) and a similar trend was
observed for the TF of frond:root in non-harvested ferns.
Translocation factor values presented in this study and earlier studies for P. vittata are
several fold greater than most non-hyperaccumulators, where roots accumulated highest
concentration of arsenic. For example arsenic TFs (shoot:root) were in the range of 0.02
to 0.1 in tomato (Lycopersicum esculentun.), Indian Mustard (Brassica juncea) and rice
(Oryza sativa) (Burlo, et al., 1999; Pickering, et al., 2000; Martin, et al., 1992). Higher
TF in frond:root or rhizome:root in our study indicates that, the arsenic concentrations in
the aboveground tissue (frond or rhizome) were much greater than in the roots. Arsenic-TF
reported by Luongo and Ma (2005) for P. vittata grown hydroponically in 1 mg·L−1 and
10 mg·L−1 arsenic solution were in the same range as reported in this study. Nevertheless,
arsenic TF as low as 0.17 to 3.98 and as high as 42 were reported in P. vittata growing on
soils with naturally elevated arsenic (50–261 mg·kg−1 As) near As-mines and artificially
spiked soils (98 mg·kg−1 As) respectively (Tu, et al., 2002; Wei and Chen, 2006).
Arsenic Bioconcentration Factor
Additionally, efficient As-phytoremediation also depends on the accumulation of
absorbed arsenic in the aboveground plant parts that can be harvested periodically to
remove As from a site. Bioconcentration factor (BCF), which is defined as the ratio of
As- concentration in plant tissue to the initial As-concentration in the growing media was
used to quantify the bioaccumulation. In our study, the overall arsenic-concentrations in the
aboveground tissues, both fronds and rhizome, were higher than those in the water indicating
a significant arsenic bioconcentration, depending upon the time of exposure (Figure 5b).
Shoot tissues (rhizome and frond) had higher BCF values than root tissue (P = 0.05).
Among the shoot tissues, BCF of fronds of both partially harvested and non-harvested
ferns and rhizomes of non-harvested ferns were greatest (P = 0.05). Almost equal BCF of
fronds in non-harvested ferns and rhizomes of partially harvested ferns at the end of 48 h
indicates that both these tissues have equal potential to bioaccumulate As.
Arsenic BCF in frond and rhizome varied depending upon the frond harvest treatment.
In partially harvested ferns BCF of both fronds (lower un-harvested portion) and rhizome
increased within 8 to 48 h, indicating rapid accumulation in shoot tissues (fronds or rhizome)
325
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DIFFERENTIAL AS-DISTRIBUTION IN PTERIS VITTATA WITH TIME OF EXPOSURE
Figure 5 Effect of frond-harvesting on a) translocation factor (TF) of frond/root, frond/rhizome and rhizome/root,
and b) bioconcentration factor (BCF) of fronds, rhizome and roots of Pteris vittata after exposed to contaminated
groundwater containing 130 µg·L−1 arsenic (As) for 8 and 48 h. Error bars represent ± SE (n = 3).
with time. However, BCF of root tissue in non-harvested ferns decreased within 8 to 48,
indicating rapid translocation from belowground tissue (roots). Apart from fronds of P.
vittata, as reported widely as a major arsenic accumulating tissue (Ma et al., 2001), it
was also reported that the rhizome of a fern as a significant source for nutrient elements
(Killingbeck et al., 2002; Singh and Ma, 2006; Liao, et al., 2004). Our results concur with
the earlier studies and it is clearly evident that both the shoot tissues (rhizome and roots)
are equally important As accumulating parts of P. vittata fern.
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CONCLUSION
In conclusion, the results from our study and others indicate that P. vittata had two
large As-storage locations, the rhizome and the fronds, and this differential As accumulation
under different conditions may also contribute to As-hyperaccumulation in P. vittata. Using
well established ferns, the arsenic depletion rate from contaminated water is very rapid (120
to ∼10 µg·L−1 in 8 h) and almost 85–93% of absorbed As was located in the shoots and only
7–15% remained in the roots within a short period (8–48 h). This may be because of larger
transpirational surface area (fronds), larger As-storage locations (fronds and rhizome) and
greater sorption (extensive root surface area). It may be recommended that in a long term
cleanup process where same ferns are repeatedly used, their established root system and
portion of shoots (rhizomes and croziers) may be left undisturbed, only arsenic-rich biomass
(mature fronds) may be harvested for disposal. Also the results show that, frond harvesting
had very little influence on its arsenic uptake capacity and hence, frond harvesting may
be delayed until several cycles of phytofiltration. However, further studies are warranted
to determine the most cost-effective and less labor intensive frond harvesting methods for
effective arsenic phytofiltration.
ACKNOWLEDGMENTS
This research was supported by Florida Power & Light Company and Florida
Agricultural Experiment Station. The authors gratefully acknowledge the assistance
provided by Diane Rock for fern propagation, Dr. Uttam Saha for GFAAS analysis,
and Loretta Satterthwaite for proofreading the manuscript. The valuable comments and
suggestions by two anonymous reviewers are also highly appreciated.
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