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Effects of organic amendments on Cd, Zn and Cu bioavailability in
soil with repeated phytoremediation by Sedum plumbizincicola
Article in International Journal of Phytoremediation · December 2012
DOI: 10.1080/15226514.2011.649436 · Source: PubMed
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EFFECTS OF ORGANIC AMENDMENTS ON
CD, ZN AND CU BIOAVAILABILITY IN SOIL
WITH REPEATED PHYTOREMEDIATION BY
SEDUM PLUMBIZINCICOLA
a
a
b
a
Longhua Wu , Zhu Li , Ikuko Akahane , Ling Liu , Cunliang Han
a
b
a
, Tomoyuki Makino , Yongming Luo & Peter Christie
c
a
Key Laboratory of Soil Environment and Pollution Remediation,
Institute of Soil Science, Chinese Academy of Sciences, Nanjing,
China
b
National Institute for Agro-Environmental Sciences, Ibaraki, Japan
c
Agri-Environment Branch, Agri-Food and Biosciences Institute,
Belfast, United Kingdom
Available online: 24 Apr 2012
To cite this article: Longhua Wu, Zhu Li, Ikuko Akahane, Ling Liu, Cunliang Han, Tomoyuki Makino,
Yongming Luo & Peter Christie (2012): EFFECTS OF ORGANIC AMENDMENTS ON CD, ZN AND CU
BIOAVAILABILITY IN SOIL WITH REPEATED PHYTOREMEDIATION BY SEDUM PLUMBIZINCICOLA ,
International Journal of Phytoremediation, 14:10, 1024-1038
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International Journal of Phytoremediation, 14:1024–1038, 2012
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Copyright ISSN: 1522-6514 print / 1549-7879 online
DOI: 10.1080/15226514.2011.649436
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EFFECTS OF ORGANIC AMENDMENTS ON CD, ZN AND
CU BIOAVAILABILITY IN SOIL WITH REPEATED
PHYTOREMEDIATION BY SEDUM PLUMBIZINCICOLA
Longhua Wu,1 Zhu Li,1 Ikuko Akahane,2 Ling Liu,1 Cunliang
Han,1 Tomoyuki Makino,2 Yongming Luo,1 and Peter Christie3
1
Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil
Science, Chinese Academy of Sciences, Nanjing, China
2
National Institute for Agro-Environmental Sciences, Ibaraki, Japan
3
Agri-Environment Branch, Agri-Food and Biosciences Institute, Belfast, United
Kingdom
Organic materials with different functional groups can be used to enhance metal bioavailability. Traditional organic materials (rice straw and clover) and ethylenediamine disuccinic
acid (EDDS) were applied to enhance metal uptake from polluted soil by Sedum plumbizincicola after repeated phytoextraction. Changes in pH, dissolved organic carbon (DOC) and
metal concentrations were determined in the soil solution after EDDS application. Amendment of the soil with ground rice straw or ground clove resulted in higher concentrations
of Cd only (by factors of 1.92 and 1.71 respectively) in S. plumbizincicola compared to
control soil. Treatment with 3 mmol kg−1 EDDS increased all the metals studied by factors
of 60.4, 1.67, and 0.27 for Cu, Cd, and Zn, respectively. EDDS significantly increased soil
solution DOC and pH and increased soil plant-available metals above the amounts that the
plants could take up, resulting in high soil concentrations of soluble metals and high risk of
ground water contamination. After repeated phytoremediation of metal contaminated soils
the efficiency of metal removal declines as the concentrations of bioavailable metal fractions
decline. Traditional organic materials can therefore be much more effective and environmentally friendly amendments than EDDS in enhancing phytoremediation efficiency of Cd
contaminated soil.
KEY WORDS: organic amendments, heavy metals, bioavailability, repeated phytoremediation, Sedum plumbizincicola
INTRODUCTION
Heavy metal pollution of soils is widespread in many parts of the world and the cleanup of these soils is a difficult task. Various in-situ and ex-situ remediation techniques have
been developed such as solidification, stabilization, soil washing and phytoremediation
(Tandy et al. 2004). In recent years the use of repeated phytoremediation (a technique
based on the utilization of metal hyperaccumulators that have the capacity to accumulate,
Address correspondence to Longhua Wu, Key Laboratory of Soil Environment and Pollution Remediation,
Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China. E-mail: lhwu@issas.ac.cn
1024
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EFFECTS OF ORGANIC AMENDMENTS ON CD, ZN AND CU
1025
translocate and tolerate high concentrations of metals over their complete growth cycle)
in managing contaminated sites has attracted considerable interest (Kumar et al. 1995;
Epelde et al. 2008). Sedum plumbizincicola has a remarkable capacity to extract zinc and
cadmium from polluted soils in south and east China and field experiments have indicated
the potential of S. plumbizincicola for Cd and Zn phytoremediation (Wu et al. 2006,
2008). However, after repeated phytoextraction, the efficiency of plant heavy metal uptake
decreases slightly and there is less metal accumulation in the aboveground parts of the
plants. Bioavailable forms of metals cannot be replenished from non-available forms as
quickly as plants assimilate them, leading to a decrease in the fractions of soil bioavailable
metals. The availability of metal in the soil for plant uptake is one important limitation for
successful phytoremediation (Blaylock et al. 1997). The application of organic materials
to soil has been proposed to achieve larger fractions of bioavailable metals and this may
help to maintain higher phytoremediation efficiency.
The efficiency of phytoremediation can be enhanced by the addition of chelating
agents such as EDTA to the soil to increase solubility and plant uptake of metals (Blaylock
et al. 1997). Several years ago, the easily biodegradable chelating agent EDDS was proposed
as a safe and environmentally benign replacement for EDTA for chelant-enhanced phytoremediation (Grčman et al. 2001). EDDS is (S, S)-EDDS-Na3 , N, N -ethylenedi-(L-aspartic
acid) trisodium salt, a powerful, readily biodegradable chelating agent. EDDS-enhanced
phytoremediation has been proposed as an effective tool for the phytoextraction of heavy
metals from soils by non-hyperaccumulating plants (Luo et al. 2005). Tandy (2004) suggested that EDDS is a better metal extractant for Zn and Cu than EDTA at pH values above
6. However, there is still concern about the persistence of soluble chelate-metal complexes
in the soil with respect to their toxicity and possible leaching to groundwater (Sun et al.
2001; Romkens et al. 2002; Bandiera et al. 2010).
Rice straw is an organic material that may increase the short-term solubility of heavy
metals and could perhaps be a more environmentally-friendly aid to phytoremediation
than synthetic chelating agents. Straw contains substantial amounts of organic compounds
which may be able to re-distribute heavy metal fractions in soil (McGrath and Cegarra
1992; Narwal and Singh 1998; Shuman 1999; Walker et al. 2003). It has been reported
that more than six hundred million tonnes of straw are produced each year in China, more
than 50% of which is burned for immediate land clearing and rapid waste disposal (Yang
and Sheng 2003; Zhong et al. 2003). Rice straw may therefore serve as an effective and
cheap organic amendment for the promotion of phytoremediation of soils contaminated
with heavy metals.
Clover (Trifolium sp.) is a genus of about 300 species of leguminous plants. Legumes
are ecologically important in that they can fix atmospheric nitrogen in their root nodules
and have therefore been a valuable nutrient resource throughout farming history.
In the current study the effects of two organic amendments, ground clover (Trifolium repens, Haifa) and ground rice straw (both dried), and EDDS on Cd, Zn, and Cu
bioavailability were investigated in soil with repeated phytoremediation. The aims of the
study were to find a low cost and high efficiency organic amendment for enhancing the
plant availability of heavy metals to enhance their phyto-extraction efficiency and speed
up remediation, to compare the metal mobilizing efficiencies and rhizosphere effects of
the two traditional organic amendments, and to examine the limitations of EDDS in soil
heavy metal mobilization and the concomitant environmental risk of metal leaching in the
soil.
1026
L. WU ET AL.
MATERIALS AND METHODS
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Sample Collection and Preceding Phytoremediation Pot Experiment
The soil was collected from the arable layer (top 15 cm) of an agricultural field
adjacent to a copper smelter near Hangzhou City, Zhejiang Province, East China. The soil
is a Typic Agri-Udic Ferrosol (Gong et al. 1999). The adjacent copper smelter was operated
from 1989 to 2000 when large quantities of metals were emitted to the atmosphere in the
form of fly ash and then deposited on soils near the smelter because the unregulated factory
had no safe working practices or equipment for dust removal.
Selected properties of the soil were analysed by standard methods (Sparks et al.
1996). Soil pH (in H2 O) was 7.24, the organic carbon content was 29.1 g kg−1, and the
soil cation exchange capacity (CEC) was 11.8 cmol (+) kg−1. Total N, P, and K were 2.21,
0.22 and 22.9 g kg−1, respectively. Available N was 105 mg kg−1, Olsen-P was 6.70 mg
kg−1, and available K was 160 mg kg−1. Soil pH was measured with a glass electrode at a
soil:water ratio of 1:2.5 (Lu et al. 2000). Available phosphorus was extracted with 0.5 M
NaHCO3 by the Olsen method. Total P was determined by H2 SO4 /HClO4 digestion and
analysed by the molybdenum blue method. Total and available nitrogen were determined
by Kjeldahl digestion and distillation. Available K was determined by flame photometry
(Model 6400-A, Shanghai Analytical Instrument Factory, Shanghai) after extraction with
1 M NH4 OAc. Total K was determined using flame photometry after extraction following
aqua-regia digestion. The concentrations of Cu, Cd and Zn extracted by aqua-regia were
564, 15.9, and 1205 mg kg−1 respectively.
The soil was air-dried, sieved through a 2-mm nylon mesh sieve, and homogenized.
1.5 kg of soil was placed in each plastic pot. Six seedlings were transplanted into each
pot. S. plumbizincicola then grew for more than 13 months (from November 1, 2008 to
December 9, 2009) in a double-wall glass multi-span greenhouse (common energy-saving
type) located in Nanjing. During the experiment the minimum temperature was 15◦ C and
the maximum was 35◦ C with an average range of 19–31◦ C. The plant shoots at the first,
second, and third harvests were collected on March 26, June 11, and December 9, 2009.
Rhizosphere Pot Experiment
The soil for the rhizosphere pot experiment was selected on the basis of the above
preceding phytoextraction experiment. After the previous experiment ended on December 9,
2009, soil that had been phytoremediated in the preceding phytoremediation pot experiment
and non-remediated control soil were selected for the rhizosphere pot experiment in January
2010. S. plumbizincicola is an herbaceous species with very fine roots and small root systems
hence, it was difficult to recover the clean roots quantitatively from soil. After air-drying
at room temperature and removal of visible plant materials, the soil samples were sieved
through a 0.5-mm nylon mesh. Concentrations of aqua-regia extractable Cd, Zn, and Cu
were decreased to 542, 11.5, and 1059 mg kg−1, respectively. The organic amendments
(clover and rice straw) were air-dried, ground and analyzed before they were added to the
soils. The total concentrations of Cd, Zn and Cu were 0.54, 39.4, 18.5 mg kg−1 for clover,
and 0.49, 32.0, 16.1 mg kg−1 for rice straw.
Five treatments were established: (1) NP: soil without previous phytoremediation;
(2) P: soil with previous phytoremediation; (3) PC: soil with previous phytoremediation and
amended with clover; (4) PR: soil with previous phytoremediation and amended with rice
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EFFECTS OF ORGANIC AMENDMENTS ON CD, ZN AND CU
1027
straw; and (5) PE: soil with previous phytoremediation and amended with EDDS. Ground
clover and rice straw were added to the soils at a rate of 1% (3.6 g pot−1). There were four
replicates of all treatments except PE which had 8 replicates, half of which were used to
observe the changes in EDDS over time after addition to the soil.
In treatments PC and PR the soil in each pot was mixed prior to transplanting with
3.6 g of dry organic amendment and then watered to 70% of soil water holding capacity
for 3 weeks by weight and then air-dried.
360 g of soil (sieved through a 0.5-mm nylon mesh) was placed in a plastic rhizo-pot
(90 mm long, 70 mm wide and 70 mm high). The amounts of soil used per pot were 40 g
in the rhizobag as rhizosphere soil and 320 g outside as bulk soil. A plastic frame (10 mm
long, 70 mm wide, and 70 mm high) covered with 400 mesh nylon cloth was used to
separate the rhizosphere soil from the bulk soil in each rhizo-pot and was positioned to
one side of the rhizo-pot. Two porous soil moisture samplers (Rhizon SMS, Rhizosphere
Research Products, Wageningen, the Netherlands) were installed both in the rhizobag and
in the bulk soil 5 cm away from the rhizobag to allow sampling of the soil solution. Four
healthy seedlings of S. plumbizincicola were transplanted into the rhizobag soil on January
14, 2010, 3 weeks after mixing treatments three and four with organic material.
The pots were arranged randomly on a bench inside a glasshouse (day/night temperature 25/20◦ C, light period 14 h with photosynthetically active radiation flux 60 w m−2).
De-ionised water was added every day to maintain soil water content at about 70% of water
holding capacity by weight.
EDDS was applied (as ∼30% Na3 EDDS in 10 ml H2 O with pH 9.70; Sigma-Aldrich
reagent) to 8 pots of treatment PE when the plants were 57 days old (March 12) and 60 days
old (March 15) to give a final concentration equivalent to 3 mmol kg−1 dry soil. Plants were
harvested at 64 days old (March 19). The EDDS application was timed to increase heavy
metal solubility in the soil when shoot biomass was at maximum in order to maximize
heavy metal accumulation in the shoots.
In all treatments, soil solutions were collected on March 15 before the second application of EDDS, and there were eight collections of soil solution from four of the replicate
pots of treatment PE 1, 3, 6, 11, 21, 31, 41, and 51 d after EDDS application.
At harvest, the plants were cut just above the soil surface, washed with de-ionised
water, freeze-dried (Labconco 12 Port Chamber 7522800, Kansas City, MO, USA) and
weighed for subsequent analysis.
Chemical Analysis
Analysis of Total Cd, Zn, and Cu in Soil and Plant Shoots. Soil total heavy
metal concentrations were determined using a Series X7 ICP-MS (Thermo Electron Corp.,
Madison, WI, USA) after digestion of ∼0.25 g samples with 14 ml of HCl:HNO3 :HClO4
(4:2:1, v/v). For quality assurance, replicate samples, blanks, and a certified reference material (GBW07401, provided by the Institute of Geophysical and Geochemical Exploration,
Langfang, Hebei Province, China) were included in all analyses.
Dry plant samples (∼0.5 g) were digested using a mixture of 6 ml HNO3 and
4 ml HClO4 , and concentrations of Cd, Zn, and Cu were determined using AAS (Varian
SpectrAA 220 FS). A certified reference material (GBW07603, provided by the Institute
of Geophysical and Geochemical Exploration, Langfang, Hebei Province, China) was used
for quality control. The data obtained by the methods above were within the certified ranges
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1028
L. WU ET AL.
of Cu, Zn, and Cd (data not shown), indicating that the analysis of the metal concentrations
was reliable.
Determination of Cd, Zn, and Cu Speciation. In the rhizosphere pot experiment, soil sampled from the rhizobag was defined as rhizosphere soil and soil sampled at a
distance of 5 cm from the rhizobag was considered to be bulk soil. In the pot experiment,
concentrations of Cd, Zn, and Cu in rhizosphere or bulk soil solution were evaluated using
samples obtained directly from the SMS suction samplers.
Soil available metals were extracted by shaking at 25◦ C for 16 h with 1.0 M NH4 OAc
(soil:extractant ratio, 1: 5), then centrifuging (15 min at 4500 rpm) and filtering. Cu, Cd,
and Zn concentrations were then determined with a Thermo X7 ICP-MS.
Determination of EDDS in Soil Solution. Details of the EDDS method were
described previously (Sun et al. 2006; Wu et al. 2007). The soil solution samples (1 ml)
were derived by CuSO4 (1 ml) to a total volume of 2 ml. CuSO4 was used to convert
all metal–EDDS complexes to Cu-EDDS for efficient detection at 254 nm. The samples
were then filtered through a 0.45-µm nylon membrane filter (Nalgene, syringe filter) prior
to analysis. The samples were analyzed using high performance liquid chromatography
(Agilent Model 1100, Agilent, Santa Clara, CA, USA) equipped with a UV detector at
254 nm, reverse phase Agilent-Zorbax SB-C18 (5 µm, 4.6 × 150 mm). The mobile phase
consisted of a 0.03 -M acetate buffer at pH 4.0 with 20% tetrabutyl ammonium hydroxide
(40 ml L−1) as a counter ion and was filtered through a 0.45-µm nylon membrane.
Determination of DOC in Soil Solution. The DOC fractions in soil solution
were analysed directly with a Multi N/C 2100 TOC analyzer (Analytik Jena AG, Jena,
Germany).
Statistical Analysis
Data were analysed by one-way analysis of variance using SPSS version 13.0 for
Windows. Differences in mean shoot biomass or heavy metal concentrations between
treatments were tested by Duncan’s multiple range test at the 5% level. Data are presented
as mean of four replicates ± standard error of the mean (SEM).
RESULTS
Changes in Soil Solution Chemical Properties: pH, DOC,
and Heavy Metals
Soil Solution pH. The rhizosphere solution pH in soil without previous phytoremediation was significantly lower than in soil not previously remediated but there was
no difference among treatments in bulk soil solution pH. Compared to treatment P (soil
previously remediated), addition of ground clover or rice straw or EDDS had no effect
on soil solution pH in either rhizosphere or bulk soil 3 days after EDDS addition (Figure
1A). In the EDDS treatment soil solution pH showed complex patterns of change in both
rhizosphere and bulk soils, which did not appear to differ significantly 31 days after EDDS
application. Overall, EDDS application produced a marked increase in soil solution pH
(Figure 1F).
Soil Solution DOC and EDDS. Analysis of DOC in the soil solutions revealed
no marked difference between treatments with the exception of EDDS addition (Figure 1B)
which increased soil solution DOC concentrations. Compared to the control (treatment P),
1029
7.8
8.0
0
100
200
300
400
500
600
700
800
900
7.0
7.2
7.4
7.6
b
NP
1
a
3
Rhzisphere
Bulk
a
6
PC
11
(A)
21
Treatments
a
a
PR
31
a
(D)
Day after EDDS application
P
a
a
41
PE
a
51
Rhizosphere
Bullk
a
0
200
400
600
800
1000
0
30
60
200
90
300
400
500
NP
1
b
b
3
6
P
b
b
11
21
(B)
PR
b
31
Treatments
PC
b
(E)
Day after EDDS application
b
Rhizosphere
Bulk
41
b
PE
a
51
Rhizosphere
Bullk
a
7.4
7.6
7.8
8.0
8.2
7.2
7.5
7.8
8.1
b
1
NP
a
3
P
6
a
PC
11
21
(C)
Treatments
b
a
PR
31
b
a
Day after EDDS application
(F)
b
Rhizosphere
Bulk
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-1
-1
41
PE
a
51
Rhizosphere
Bulk
a
Figure 1 Physicochemical properties and changes in PE treatment after EDDS application. (A) Soil solution pH and (B) DOC observed 3 days after EDDS addition, and (C)
soil pH at the end of the experiment, and changes in (D) DOC, (E) EDDS and (F) pH after EDDS application. Days denote time after the first addition of EDDS; the second
EDDS application was made three days after the collection of the soil solution. Values are the means of four replicates. NP, P, PC, PR, and PE mean the treatment soils without
phytoremediation, with phytoremediation, with phytoremediation applied with clover powder, with phytoremediation added with rice straw powder, and with phytoremediation
added with EDDS respectively. Different letters in the same colour column mean significantly different at p < 0.05.
-1
DOC in soil solution
(mg L )
EDDS in soil solution(mgL )
Soil solution pH
DOC in soi lsolution(mg L)
Soil pH
Soil solution pH
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1030
L. WU ET AL.
DOC concentrations in rhizosphere and bulk soil solution in treatment PE increased 8.44
and 9.86 fold (p < 0.05), respectively. However, the soil solution DOC in treatments with
application of clover and rice straw did not seem to be enhanced.
Six days after EDDS addition the DOC concentration in the soil solution declined
rapidly (Figure 1D). This may be due to degradation of the EDDS (Tandy et al. 2006),
which could be validated by the decrease in EDDS concentration in the soil solution with
time according to our experiment (Figure 1E), and carbon in the form of EDDS showed
a similar declining trend as DOC in the soil solution. Comparing Figures 1D and E, we
calculated that during the period between the 1st day and the 11th day after EDDS addition,
about 40.8–53.3% of the DOC in the rhizosphere soil solution was from EDDS and the
equivalent value in the bulk soil solution was 52.5–77.5%, indicating the induction of a
large percentage of DOC by the addition of EDDS. There was a sharp decrease in EDDS
in both rhizosphere and bulk soil solution from days 6 to 21, showing rapid degradation of
EDDS in the soil (Figure 1E).
Concentration of Cd, Cu, and Zn in the Soil Solution. Repeated phytoremediation (3 crops of S. plumbizincicola) decreased the Cd concentration in the soil solution
but had no marked effect on Cu and Zn concentrations. Compared to the control, the
metal concentrations in soil solution of the clover and rice straw application treatments
(PC and PR) showed no significant change, with the exception of the Cd concentration in
rhizosphere soil solution and the Cu concentration in bulk soil solution of PC treatment,
which were slightly higher than that in the control (Table 1). The Cu-solubilising effect of
EDDS can be seen clearly in the soil solution (Table 1). Three days after EDDS addition,
the Cu concentration of soil solution in the PE treatment increased dramatically, but slight
decreases were found for Cd in both rhizosphere and bulk soil, which may be contributed
to Cd absorption by the plants, and there was no significant effect on the Zn concentration
in the soil solution (Table 1).
The dynamics of Cd, Cu, and Zn concentrations in soil solution after EDDS application are presented in Table 2. Compared to the heavy metal concentrations in soil solution
of treatment P (Table 1), 1 day after the first EDDS addition there was no significant change
in Cd and there was slight increase in Zn, but there was a marked increase in Cu (Table 2).
After the second EDDS addition all three metals in the soil solution increased greatly and
then decreased markedly as time passed, but there were still high concentrations of Cu
and Zn in the soil solution at the end of the experiment (51 days after EDDS application).
Unexpectedly, the Cu and Zn concentrations in the soil solution on the 11th day were lower
than on the 6th and the 21st days, possibly related to soil pH which was relatively high
on that day (Table 2 and Figure 1F). Thus, although it is a biodegradable chelating agent,
EDDS may enhance heavy metal mobility substantially over an extended time period and
this could result in high environmental risk.
Soil pH and NH4 OAc-Extractable Cd, Cu, and Zn
A significant effect of EDDS addition on increasing soil pH was observed only in
rhizosphere soil and there was no effect on for bulk soil. None of the other treatments
exerted any significant effect on the pH of bulk or rhizosphere soil (Figure 1C).
Concentrations of NH4 OAc-extractable Cd and Zn may reflect their bioavailability
to S. plumbzincicola as demonstrated in a previous study (Liu et al. 2011). As expected,
repeated phytoextraction by S. plumbzincicola decreased the NH4 OAc-extractable fractions
of Cd and Zn compared with the control soil with no previous phytoextraction (Table 3).
1031
0.13 ± 0.01c
0.18 ± 0.05bc
0.24 ± 0.02b
0.22 ± 0.02b
73.0 ± 5.9a
16.3 ± 2.2a
6.08 ± 1.53b
13.0 ± 1.7a
7.69 ± 1.61ab
4.85 ± 0.34b
NP
P
PC
PR
PE
0.27 ± 0.05a
0.21 ± 0.07a
0.36 ± 0.04a
0.32 ± 0.08a
0.40 ± 0.08a
Zn (mg L−1)
19.7 ± 1.9a
7.48 ± 1.03b
9.26 ± 1.08b
7.42 ± 1.08b
4.56 ± 0.28c
Cd (µg L−1)
0.07 ± 0.01d
0.08 ± 0.02cd
0.18 ± 0.02b
0.12 ± 0.01c
66.2 ± 10.5a
Cu (mg L−1)
Bulk soil solution
0.25 ± 0.05a
0.38 ± 0.07a
0.31 ± 0.04a
0.25 ± 0.04a
0.23 ± 0.07a
Zn (mg L−1)
Values are the means of four replicates; different letters in the same column mean significantly different at P<0.05; NP, P, PC, PR, and PE mean the treatment soils without
phytoremediation, with phytoremediation, with phytoremediation applied with clover powder, with phytoremediation added with rice straw powder, and with phytoremediation
added with EDDS, respectively.
Cu (mg L−1)
Cd (µg L−1)
Treatment
Rhizosphere soil solution
Table 1 Soil solution Cd, Cu and Zn concentrations 3 days after the 1st EDDS addition
Downloaded by [Nanjing Institute of Soil Science], [L. H. Wu] at 05:41 12 June 2012
1032
183 ± 8
87.9 ± 7.8
281 ± 8
81.4 ± 18.8
18924 ± 2053
5179 ± 862
850 ± 72
1054 ± 130
10.5 ± 1.1
5.19 ± 0.49
80.6 ± 28.8
10.5 ± 0.95
5.62 ± 0.60
4.37 ± 0.50
2.80 ± 0.55
2.21 ± 0.37
1
3
6
11
21
31
41
51
4.18 ± 1.22
0.44 ± 0.08
62.7 ± 11.6
1.46 ± 0.41
100 ± 14
83.3 ± 14.1
61.0 ± 10.1
57.8 ± 6.0
Zn (mg L−1)
7.00 ± 0.21
4.52 ± 0.60
17.4 ± 3.0
10.1 ± 0.98
5.23 ± 1.05
3.78 ± 0.26
2.82 ± 0.27
2.00 ± 0.22
Cd (µg L−1)
72.6 ± 11.1
58.3 ± 10.9
184 ± 3
107 ± 12
2122 ± 240
679 ± 8
486 ± 29
371 ± 26
Cu (mg L−1)
Bulk soil solution
0.34 ± 0.10
0.15 ± 0.01
1.12 ± 0.38
0.22 ± 0.02
57.6 ± 17.2
61.1 ± 8.5
52.5 ± 7.4
42.1 ± 4.3
Zn (mg L−1)
NB: days are after the first addition of EDDS; the second EDDS application was made after 3 days the first addition but was carried out after the soil solution was collected on
this day. Values are the means of four replicates.
Cu (mg L−1)
Cd (µg L−1)
Days after EDDS addition
Rhizosphere soil solution
Table 2 Dynamics of soil solution Cd, Cu and Zn concentrations in the PE treatment
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Cu
12.4 ± 0.1b
10.9 ± 0.3b
11.5 ± 0.3b
11.3 ± 0.4b
191 ± 13a
Cd
1.84 ± 0.06a
1.21 ± 0.04b
1.43 ± 0.08b
1.38 ± 0.04b
0.81 ± 0.15c
7.81 ± 0.29b
5.95 ± 0.30c
6.57 ± 0.22c
6.21 ± 0.23c
39.7 ± 7.4a
Zn
2.39 ± 0.06a
1.60 ± 0.03b
1.58 ± 0.03b
1.64 ± 0.08b
1.42 ± 0.04c
Cd
12.1 ± 0.1b
10.6 ± 0.2b
11.3 ± 0.2b
11.3 ± 0.1b
123 ± 8a
Cu
Zn
9.18 ± 0.36a
7.13 ± 0.14b
7.46 ± 0.08b
7.64 ± 0.47ab
7.97 ± 0.55ab
Bulk soil (mg kg−1)
197 ± 4b
125 ± 18c
339 ± 45a
365 ± 36a
334 ± 49a
Cd
25.1 ± 2.4b
11.2 ± 1.6c
9.16 ± 1.13c
6.14 ± 0.64c
688 ± 101a
Cu
Zn
2527 ± 137b
2537 ± 130b
2464 ± 170b
2200 ± 115b
3227 ± 314a
Metals in plant (mg kg−1)
0.49 ± 0.08a
0.73 ± 0.08a
0.68 ± 0.06a
0.75 ± 0.06a
0.74 ± 0.06a
Shoot biomass
(g pot−1)
Values are the means of four replicates; different letters in the same column mean significantly different at P < 0.05; NP, P, PC, PR, and PE mean the treatment soils without
phytoremediation, with phytoremediation, with phytoremediation applied with clover powder, with phytoremediation added with rice straw powder, and with phytoremediation
added with EDDS, respectively.
NP
P
PC
PR
PE
Treatment
Rhizosphere soil (mg kg−1)
Table 3 Soil NH4 OAc-extractable Cd, Cu and Zn concentrations, biomass and heavy metal concentrations in plant shoots after the experiment
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L. WU ET AL.
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The addition of clover and rice straw did not significantly affect the increase in NH4 OAcextractable metal fractions. However, EDDS addition decreased the NH4 OAc-Cd fraction
at plant harvest and NH4 OAc-extractable Cu and Zn increased compared with the other
treatments with the exception of Zn in the bulk soil (Table 3). Concentrations of NH4 OAcCu in the rhizosphere and bulk soil treated with EDDS (PE) increased by about 16.5- and
10.6-fold, respectively, while NH4 OAc-extractable Zn in the rhizosphere soil of treatment
PE exceeded that in the control (treatment P) by about 5.7-fold (Table 3).
Plant Biomass and Metal Concentrations in Plant Shoots
After EDDS was applied to the soil (3 mmol kg−1 in total) the plants lost their leaves
and plant growth was strongly inhibited. This inhibitory effect of EDDS on the plants may
have contributed to the enhancement of Cu uptake after EDDS application, together with
translocation from roots to shoots and resulting in Cu accumulation to toxic levels. Thus, a
significantly increase in the concentration of Cu in the plants may explain the phytotoxicity
effects exerted by EDDS (Table 3). However, shoot biomass did not differ significantly
among these treatments because the EDDS application was made just one week before
harvest.
At harvest the plant Cd concentrations followed the order PR > PC > PE > NP >
P. Shoot Cd concentrations were all markedly higher (p < 0.05) in treatments with organic
amendments (treatments PC, PR, and PE) than the controls (P and NP). EDDS application
also significantly increased the concentrations of Zn and Cu in the plants with respect to
control P.
DISCUSSION
Heavy metal concentrations in plants, a key factor in successful phytoextraction, will
be profoundly affected by the amount of plant available heavy metal in the soil. After
repeated extraction by the hyperaccumulator, the plant available metals in soil decreased
greatly and thus resulted in a low efficiency of subsequent phytoremediation. Numerous
previous studies had indicated that exogenous organic materials can increase soil dissolved
organic matter (DOM) concentrations and thereby significantly depress the sorption of
metals by soil (Xu et al. 1989; Temmninghoff et al. 1997; Antoniadis and Alloway 2002).
Organic materials may therefore be used to redistribute the metal fractions in soil and
enhance the mobility and bioavailability of the metals.
EDDS has been reported to be a promising and environmentally friendly metalmobilizing chemical chelating agent and can be applied to the soil to enhance plant heavy
metal uptake (Tandy et al. 2004; Evangelou et al. 2007). In the study by Komarek et al.
(2010), EDDS addition to soil increased Cu mobility up to 100-fold and enhanced plant
uptake of Cu up to 65-fold at the same time. Salati et al. (2010) also reported enhancing
effects of natural organic materials on metal phytoextraction. Similar results were also
observed in our study, with EDDS promoting soil solution Zn and Cu and soil NH4 OAcextractable Zn and Cu and thus enhancing their uptake by plants, and effects of rice straw
and clover on enhancement of plant Cd uptake were also found. Interestingly, previous
studies showed that the NH4 OAc-extractable and water-soluble fractions of metals have
positive relationships with metal uptake by S. plumbizincicola (Jiang et al. 2010; and Liu
et al. 2011). In the present study a large increase in Cd concentration in the plants was found
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EFFECTS OF ORGANIC AMENDMENTS ON CD, ZN AND CU
1035
(Table 3) in the PR, PC, and PE treatments but the NH4 OAc-extractable Cd and watersoluble Cd fractions in these treatments were not markedly changed and even decreased
compared to the control (P) with the exception of soil solution Cd in the PC treatment
rhizosphere soil (Tables 1 and 3). Cui et al. (2008) reported that the addition 6% rice
straw to soil decreased the concentration of soil solution Cd and Mohamed et al. (2010)
considered that the application of rice straw to soil can enhance the soil organic matter, pH
and CEC, and thus depress the soluble and exchangeable Cd concentrations. Nevertheless,
under submerged conditions the soluble Cd in soil would increase with the application of
rice straw (Shan et al. 2008) and the addition of rice straw ash to flooded rice paddy soils
also suppressed the release of Cu into the soil solution (Huang et al. 2011). One or several
of the explanations given below could clarify the disagreement between available Cd in the
soil and the Cd concentration in the plants in this research. First, in practice the soil available
Cd has been enhanced by the application of organic materials but S. plumbizincicola has a
great ability to absorb Cd from soil and this may have led to the larger amount of Cd taken
up than was released from the unavailable fractions by the organic materials. Second, the
experimental treatments did not induce any significant change in the amount of available
Cd, but may have formed a type of Cd fraction easily taken up by the plants, for example Cd
complexed with low molecular-weight organic material which might represent microbial
decomposition products of rice straw and clover, or root exudates induced by EDDS. Some
studies have indicated that plant roots can directly take up metals which were bound to
DOM or low molecular-weight organic acids (Hamon et al. 1995; Krishnamurti et al. 1997).
Quartacci et al. (2005) observed that application of 10 mmol kg−1 citric acid to soil had no
significant effect on the soluble Cd concentration but gave a 1.5 fold increase in Cd uptake
by Brassica juncea. Another study found that organic acids were much more important
than soil pH in influencing plant absorption of heavy metals (Huang et al. 1998). However,
this interesting observation cannot fully explain our results and further research is required
to elucidate the mechanisms involved.
Soil solution pH and DOC greatly influence heavy metal concentrations and speciation in soil (Gungor et al. 2010). However, few studies have considered the DOC and pH
in the soil solution and their dynamic changes with time after EDDS application (Wang
et al. 2007). In the present study when compared to the other treatments, EDDS application
markedly increased soil solution DOC (Figure 1B) but the incremental increase in DOC was
not solely from EDDS. We calculated that during the period from the 1st day to the 21st day
after EDDS application the DOC from EDDS amounted to only 40.8–53.3% (rhizosphere)
and 52.5–77.5% (bulk) of the total (Figures 1D and E). Therefore part of the DOC in the
soil solution must be derived from soil organic matter and/or root exudation which were
induced by EDDS. Yan and Lo (2011) considered that the soil structure could be disrupted
under excessive EDDS and this might lead to the dissolution of some soil organic matter.
Wang et al. (2007) reported that the ratio of concentration of Cu and EDDS in pore
water was close to 1:1 in 2.3 mmol kg−1 EDDS application treatments during the 3 to
24 day period after EDDS application but in the present this study the Cu concentration and
EDDS in soil solution showed no definite relationship and on day 21 there was a very high
Cu concentration but with a low concentration of EDDS in the soil solution (Table 2 and
Figure 1E). These results suggest that soil solution Cu was not all complexed with EDDS
and another factor may influence Cu speciation in the soil solution. This conclusion may be
supported by the relationship between Cu and pH in the soil solution. In the study by Wang
et al. (2007) Cu in solution was complexed with EDDS and the Cu-EDDS complex was not
readily affected by pH under the conditions of their study. However, in our study only part
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1036
L. WU ET AL.
of the Cu in the soil solution was complexed with EDDS so that the Cu concentration in the
soil solution was easily affected by soil solution pH. 11 days after EDDS application the
Cu concentration in the soil solution was significantly lower than between days 6 and 21,
and conversely, the solution pH on the 11th day was significantly higher than that between
days 6 and 21 (Table 2).
Numerous studies have indicated the EDDS is a promising biodegradable chelating
agent that can be applied for the remediation of heavy metal polluted soil (Luo et al.
2005; Meers et al. 2008). Recently some studies have highlighted heavy metal leaching and
phytotoxicity of EDDS as two important problems in the application of EDDS (Bandiera
et al. 2010; Komarek et al. 2010). Evangelou et al. (2007) conducted a review of the
literature and concluded that the increase in plant available heavy metals in soil resulting
from amendment with EDDS was much larger than the amount of heavy metals taken up
by plants, resulting in an excessive amount of mobile metals in soil and an increasing risk
of metal leaching, but the phytotoxicity of EDDS depended on the plant species studied
and the amount of EDDS used. This conclusion is supported by our study. After the first
application of 1.5 mmol kg−1 EDDS the plants showed no visible symptoms of toxicity,
but after the second application of 1.5 mmol kg−1 EDDS 3 days later all the plants in
the EDDS treatment were defoliated. In the soil solution the concentrations of metals
increased rapidly after EDDS amendment and the solution Cd concentration returned to
the control level 21 days after EDDS application, but Cu and Zn were maintained at very
high concentrations after 51 days (Table 2) and this would represent a very high risk of
groundwater contamination.
CONCLUSIONS
The results indicate that the most effective technique for maximising the phytoextraction of all the metals investigated is amendment with EDDS while the innovative organic
materials are effective for Cd only. Thus, further research might be useful to develop
different combinations of EDDS and the organic materials to achieve high rates of phytoremediation of all the metals together with minimal leaching risk of solubilised metals. For
example, a lower concentration of EDDS solution together with application of one or both
organic materials might provide the optimum strategy.
ACKNOWLEDGMENTS
This research was supported jointly by the Program of Innovative Engineering of the
National Natural Science Foundation of China (40921061, 40871155, and 40821140539)
and the Chinese Academy of Sciences (KSCX2-YW-G-053).
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