Journal of Environmental Protection, 2011, 2, 932-939
doi:10.4236/jep.2011.27106 Published Online September2011 (http://www.SciRP.org/journal/jep)
Effects of EDTA and DTPA on Lead and Zinc
Accumulation of Ryegrass*
Hua-Yin Zhao1, Li-Jin Lin2, Qiao-Lun Yan1, Yuan-Xiang Yang1, Xue-Mei Zhu1#, Ji-Rong Shao3
1College of Resource and Environment, Sichuan Agricultural University, Chengdu, China; 2Ya’an Soil and Water Conservation
Monitoring Substation, Ya’an, China; 3College of Life Science, Sichuan Agricultural University, Ya’an, China.
Received May 24, 2011; revised July 2nd, 2011; accepted August 15th, 2011.
ABSTRACT
The tailing ponds of lead-zin c mine s are artificial environment pollution sources, and also important dangerous sources
of heavy metal contamination in lead-zinc mining areas. To study the effects of Ethylene Diamine Tetracetic Acid (ED-
TA) and Diethylene Triamine Penlaacetic Acid (DTPA) on phytoremediation of lead-zinc mining area soil, two chela-
tors (EDTA and DTPA) were used in enrichment plan t ryegrass to improve the uptake of Pb and Zn from soil. The re-
sults showed that when the doses of 0, 0.5, 1 and 2 mmol/kg ED TA and DTPA were used , the biomass of ryegrass (Lo-
lium multiflorum Lam.) and its nutrient (N, P, K, Ca and Mg) content increased, whereas EDTA and DTPA with a dose
of 4 mmol/kg decreased the biomass of ryegrass and its nutrient (N, P, K, Ca and Mg) content. EDTA and DTPA sig-
nificantly enhan ced the contents o f Zn and Pb in ryegrass as compar ed with the con trol. As for Pb, the content of Pb in
root and shoot reached a maximum of 2730.54 and 2484.42 mg/kg respectively when the dose of EDTA and DTPA was
2 mmol/kg. In the case of Zn, the content of Zn in root and shoot reached a maximum of 2428.37 and 2010.43 mg/kg
respectively. The total Pb and Zn accumulations and translocation ratio in ryegrass had also been enhanced. The re-
sults indicated that EDTA and DTPA had great potential to be used for ryegrass to remedy Pb and Zn contamination
soil of lead-zinc mining area, but sh o uld be used c a uti ousl y beca use of their environmental risks.
Keywords: EDTA, DTPA, Enri chment Plant, Ryegrass, Lead and Zinc
1. Introduction
With the development of lead-zinc mineral resources,
heavy metals were released and transferred into the eco-
logical environment [1], which caused serious environ-
mental problems. The contents of organic matter, nitro-
gen and phosphorus in the soil around the mine tailing
area were reduced to levels that were 20% - 30% of those
found in normal conditions [2,3]. As a result, it was ur-
gent to apply ecological remediation methods in the
lead-zinc mining area.
Recently, phytoremediation has appeared as an alter-
native reliable [4,5]. The use of hyperaccumulator spe-
cies in continuous phytoextraction processes is limited by
the low bioavailability of pollutants to the root uptake [6].
Some studies found that the application of chelating
agents showed positive effects on increasing the solubil-
ity of heavy metals in soil, thereby enhancing phytoex-
traction [7-9], and enhancing the amount of bioavailable
lead more than 100 times [10,11]. At present, most of the
reported hyperaccumulators or accumulators have not
had a very good effect on remedying heavy metals be-
cause of their small biomass and slow growth [12,13]. So,
it could be a reliable practice to increase metal bioavail-
ability, uptake and accumulation in the shoots of plants
by applying chelator [9,14-16].
Annual ryegrass (Lolium multiflorum Lam.) is one
kind of accumulator and graminaceous monocotyledon,
and it has a strong regeneration ability and resistance to
pests and diseases with its high biomass [17,18]. We
aimed to access the influence of EDTA and DTPA on
phytoremediation for contaminated soil of lead-zinc
mining areas using ryegrass, providing a case of the use
and promotion of chelator-induced phytoextraction in
contaminated soil.
2. Materials and Methods
*Project supported by Program of Science and Technology Bureau o
f
Sichuan Province, China (No. 2008FZ0180).
Annual ryegrass (L. multiflorum Lam.), ethylene diamine
tetracetic acid (EDTA) and diethylene triamine penlaace-

Effects of EDTA and DTPA on Lead and Zinc Accumulation of Ryegrass933
tic acid (DTPA) were selected as experimental plant and
chelators. Soil was selected from the lead-zinc mining
area of Tangjia Mountain which located in Hanyuan
county, Sichuan province, China. The most relevant
characteristics of the soil were (mean values) as follows,
pH 6.83, organic matter 6.82 g/kg, total N 0.756 g/kg,
total P 0.658 g/kg, total K 17.951 g/kg, available N
93.156 mg/kg, available P 3.426 mg/kg, available K
75.221 mg/kg CEC 5.368 cmol/kg, total Pb 2921.32
mg/kg, total Zn 1841.5 mg/kg, available Pb 378.79
mg/kg, available Zn 287.37 mg/kg.
The experiment was arranged in a randomized block
design with two chelators of 5 levels and four replicates
per treatment. Five concentrations of DTPA were 0
mmol/kg (A0), 0.5 mmol/kg (A1), 1 mmol/kg (A2), 2
mmol/kg (A3) and 4 mmol/kg (A4), and EDTA were 0
mmol/kg (B0), 0.5 mmol/kg (B1), 1 mmol/kg (B2), 2
mmol/kg (B3) and 4 mmol/kg (B4). After sieving (< 5
mm), 1.5 kilograms of dried soil were stored in plastic
pots (15 cm×15 cm), and soil samples were mixed with
fertilizers containing 100 mg/kg N in the form of urea, 80
mg/kg P as KH2PO4, and 100 mg/kg K as KCl. In all
treatments, ryegrass was grown and placed in the artifi-
cial climate chamber with the environmental conditions,
temperature 26˚C in day and 22˚C at night, humidity
75%, light intensity 20000 LX and illumination 12 hour
per day. Each pot contained 3 seedlings. When the rye-
grass had been growing for 2 months, two mixed chelator
solutions were added to the soil. Plants were harvested
after 14 days of adding chelator solutions and deactivated
enzymes in 105˚C, and baked in the oven in 75˚C until
constant weight.
Plant samples were nitrated by HCl—HNO3—HClO4,
and the contents of Pb, Zn, P, K, Mg and Ca were deter-
mined by ICP—AES, and the content of N was deter-
mined by Kjeldahl nitrogen determination. The data was
treated and analyzed by Microsoft Excel 2003 and SPSS
13.0.
3. Results and Analysis
3.1. Biomass and Root-shoot Ratio of Ryegrass
The results showed that, with a single chelator, the bio-
mass of roots and shoots of ryegrass increased at first,
and then decreased with the increasing concentrations of
the chelator (Table 1), and the application of EDTA at
the dose of 2 mmol/kg produced the maximum biomass
of roots and shoots (1.49 and 1.66 g/plant respectively).
When the dose of DTPA was 2 mmol/kg, the maximum
biomass of root and shoot were 1.56 and 1.51 g/plant
respectively. With the combined application of EDTA
and DTPA, the trends in change of the biomass were
similar to the application of a single chelator . For 2
mmol/kg dose of EDTA and DTPA, the maximum bio-
mass of roots and shoots were 2.01 and 1.89 g/plant re-
spectively. The root-shoot ratio of ryegrass showed the
reverse law of the biomass. Whatever two chelators were
used, they both showed significant effects (P < 0.01) on
Table 1. The effects of EDTA and DTPA on biomass and root-shoot ratio of ryegrass.
Biomass
Treatments Root (g/plant) Shoot (g/plant) Root/Shoot
A0B0 1.10M 1.34P 0.82
A0B1 1.37I 1.55I 0.88
A0B2 1.21K 1.46K 0.83
A0B3 1.49G 1.66H 0.90
A0B4 1.10M 1.36N 0.81
A1B0 1.33I 1.43L 0.93
A1B1 1.62E 1.68G 0.96
A1B2 1.80C 1.81C 0.99
A1B3 1.71D 1.77E 0.97
A1B4 1.06N 1.35O 0.79
A2B0 1.40H 1.46K 0.96
A2B1 1.70D 1.71F 0.99
A2B2 1.81C 1.81C 1.00
A2B3 1.00O 1.28R 0.78
A2B4 1.90B 1.87B 1.02
A3B0 1.56F 1.51J 1.03
A3B1 1.99A 1.87B 1.06
A3B2 1.81C 1.78D 1.02
A3B3 2.01A 1.89A 1.06
A3B4 0.96P 1.26S 0.76
A4B0 1.38H 1.38M 1.00
A4B1 1.26J 1.31Q 0.96
A4B2 1.18L 1.26S 0.94
A4B3 1.07N 1.18T 0.91
A4B4 0.82Q 1.09U 0.75
Note: The data followed by uppercase letters indicate the difference at 1% level, the same as following tables.

Effects of EDTA and DTPA on Lead and Zinc Accumulation of Ryegrass 934
the biomass of root and shoot of ryegrass.
3.2. Pb Content in Ryegrass Plant
With the increased EDTA dose, Pb content in the roots of
ryegrass increased before a dose of 2 mmol/kg, and de-
creased when a dose of 4 mmol/kg was used (Figure 1).
DTPA had approximately the same effects on Pb content
in roots. When the dose of EDTA and DTPA was 2
mmol/kg, Pb content in roots reached the maximum of
2730.54 mg/kg, and it was 3.15 times higher than the
control. The results also showed that EDTA increased Pb
content more than DTPA did with the same dose. Both
chelators showed significant effects (P < 0.01) on Pb
content in roots.
EDTA and DTPA had approximately the same effect
on the Pb content in shoots of ryegrass (Figure 2). Pb
content in shoots reached the maximum of 2484.42
mg/kg, which was 2.15 times greater than the control,
when the dose of EDTA and DTPA was 2 mmol/kg.
EDTA enhanced Pb content in shoots more than DTPA
did with the same dose. Both chelators showed signifi-
cant effects (P < 0.01) on Pb content in shoots.
3.3. Zn Content in Ryegrass Plant
Zn content in the roots of ryegrass had the same variation
with the doses of EDTA and DTPA increasing (Figure
3). When the dose of EDTA and DTPA was 4 mmol/kg,
Zn content in roots reached the maximum of 2428.37
mg/kg, and it was 9.82 times greater than the control.
EDTA enhanced Zn content in shoots more than DTPA
at the same dose. Both chelators showed significant ef-
fects (P < 0.01) on Zn content in root.
EDTA and DTPA had approximately the same effect
on the Zn content in shoots of ryegrass (Figure 4). When
the dose of EDTA and DTPA was 4 mmol/kg, Zn content
in shoots reached the maximum of 2010.43 mg/kg, and it
was 5.14 times higher than the control. With the same
dose, EDTA enhanced Zn content in shoots more than
DTPA did. Both chelators showed significant effects (P
< 0.01) on Zn content in shoot.
3.4. The Correlation between DTPA/EDTA and
Pb and Zn Content in Ryegrass
The results of partial-correlation analysis showed that
DTPA and EDTA didn’t significantly correlate with Pb
content in roots but did significantly correlate with Pb
content in shoots (Table 2). The correlation between
DTPA/EDTA and Zn content in roots and shoots were
significant. The results indicated that two chelators had
collaborative effects on Zn content in ryegrass plants.
Figure 1. The effects of DTPA and EDTA on Pb content in the roots.
Figure 2. The effects of DTPA and EDTA on Pb content in the shoots.

Effects of EDTA and DTPA on Lead and Zinc Accumulation of Ryegrass935
Figure 3. The effects of DTPA and EDTA on Zn content in the roots.
Figure 4. The effects of DTPA and EDTA on Zn content in the shoots.
Table 2. The partial-correlation coefficients of chelators
and Pb/Zn content in ryegrass plant.
Pb Zn
Chelators Root Shoot Root Shoot
EDTA 0.501 0.781** 0.648* 0.814**
DTPA 0.421 0.706* 0.801** 0.775**
Note: n = 9, r0.05 = 0.632, r0.01 = 0.765, * and ** showed the significant
levels of 5% and 1%, respectively.
3.5. Total Pb and Zn Accumulation in Ryegrass
Plants
DTPA and EDTA enhanced total Pb and Zn accumula-
tion in roots and shoots compared with the control (Ta-
ble 3). With the increase of one chelator dose, the total
Pb and Zn accumulation in root and shoot increased at
first and decreased at last. The maximum of total Pb and
Zn accumulation in roots were 4.99 and 3.77 mg/plant
respectively, and the maximum of Pb and Zn accumula-
tion in shoots were 5.16 and 4.38 mg/plant respectively.
The results showed that EDTA and DTPA had signifi-
cant effects (P < 0.01) on the total Pb and Zn accumula-
tion in ryegrass plants. Compared with the control, most
of the Pb translocation ratios were enhanced, and all of
the Zn translocation ratios were enhanced. This showed
that DTPA and EDTA could promote transfer of Pb and
Zn from roots to shoots.
3.6. Nutrients Contents in Ryegrass Plants
DTPA and EDTA enhanced the nutrient (N, P, K, Ca and
Mg) contents in ryegrass plants compared with the con-
trol (Table 4). The nutrient contents increased at first and
decreased at last when the dose of one chelator increased.
The results showed that EDTA and DTPA had signifi-
cant effects (P < 0.01) on the nutrient contents in rye-
grass plants. This indicated that DTPA and EDTA could
enhance the absorption of nutritional elements from soil
to promote the growth of ryegrass plants.
4. Discussion
Previous studies found that in a certain range of concen-
trations, EDTA strongly inhibited plant growth [19]. In
this paper, with increased EDTA and DTPA doses, the
biomass in roots and shoots of ryegrass increased at first
and decreased at last, and the nutrient (N, P, K, Ca and
Mg) contents of ryegrass plants increased compared with
the control, which could be due to the active effects of
chelators on soil nutrients [20,21]. Meanwhile, EDTA
may stimulate the heavy metal extraction from the soil
[22], and reduced biological toxicityof heavy metals [23].
When the dose of EDTA and DTPA was 4 mmol/kg, the
biomass in roots and shoots decreased. This could be due

Effects of EDTA and DTPA on Lead and Zinc Accumulation of Ryegrass 936
Table 3. The effects of DTPA and EDTA on total Pb and Zn accumulation in ryegrass plant.
Root Shoot Translocation ratio
Treatments Pb (mg/plant) Zn (mg/plant) Pb (mg/plant) Zn (mg/plant) Pb (%) Zn (%)
A0B0 1.27O 0.41J 1.16K 0.33K 47.72 44.86
A0B1 3.09GHIJ 1.64GH 2.96GHI 1.68J 48.93 50.58
A0B2 2.74IJKLM 1.77GH 2.96GHI 2.16I 51.95 55.06
A0B3 3.38FGH 2.80C 3.83D 3.27BCDE 53.09 53.89
A0B4 2.46KLM 2.14EF 3.05GH 2.92DEFG 55.27 57.74
A1B0 3.05GHIJK 1.20I 2.69IJ 1.26J 46.89 51.18
A1B1 3.74DEF 2.16EF 3.46EF 2.33HI 48.05 51.83
A1B2 4.17BCDE 2.84C 4.20C 3.05CDEFG 50.19 51.76
A1B3 3.99CDE 3.25B 4.23C 3.56B 51.42 52.27
A1B4 2.40LMN 2.13EF 3.12GH 3.07CDEF 56.5 59.04
A2B0 3.27FGHI 1.55H 2.80HI 1.55J 46.15 49.99
A2B1 4.01CDE 2.28EF 3.60DE 2.56GHI 47.3 52.85
A2B2 4.29BCD 2.72CD 4.29C 3.08DEF 49.98 53.14
A2B3 2.37LMN 1.71GH 3.11GH 2.67FGH 56.74 60.91
A2B4 4.47ABC 3.66A 4.38BC 4.38A 49.5 54.49
A3B0 3.65EFG 2.40DE 3.27FG 2.33HI 47.24 49.21
A3B1 4.77AB 3.23B 4.38BC 3.33BCD 47.88 50.77
A3B2 4.42ABC 3.25FB 4.61B 3.53BC 51.05 52.06
A3B3 4.99 A 3.77A 5.16A 4.13A 50.82 52.32
A3B4 2.28MN 1.92FG 3.07GH 2.98DEFG 57.4 60.79
A4B0 3.19FGHI 2.27EF 2.95HI 2.68FGH 48.01 54.1
A4B1 2.95HIJKL 2.35E 2.92HI 2.81EFGH 49.73 54.43
A4B2 2.80HIJKLM 2.32E 2.97GHI 2.87DEFG 51.44 55.29
A4B3 2.57JKLM 2.14EF 2.83HI 2.82EFGH 52.43 56.8
A4B4 1.84N 1.65GH 2.42J 2.65FGH 56.77 61.62
Note: Translocation ratio (%) = Pb (Zn) accumulation in shoot / Pb (Zn) accumulation in root × 100%.
Table 4. The effects of EDTA and DTPA on nutrients contents of ryegrass plant.
Treatments N (mg/g) P (mg/g) K (mg/g) Ca (mg/g) Mg (mg/g)
A0B0 13.2P 4.8N 20.9W 4.0M 1.0F
A0B1 19.8N 5.6K 28.7U 4.8J 1.3E
A0B2 24.7L 6.0I 36.1Q 5.0H 1.4D
A0B3 31.6ED 6.4F 41.4L 5.6D 1.5C
A0B4 32.7A 6.6D 45.7E 5.8B 1.7A
A1B0 17.4O 5.1M 26.6V 4.6L 1.3E
A1B1 21.6M 5.8J 30.8S 4.9I 1.4D
A1B2 26.5J 6.2H 38.7O 5.4E 1.5C
A1B3 32.0CD 6.5E 42.1K 5.7C 1.6B
A1B4 32.8A 6.8B 46.8B 5.9A 1.7A
A2B0 21.5M 5.4L 30.6T 4.7K 1.4D
A2B1 25.7K 6.0I 36.2P 5.3F 1.5C
A2B2 30.1G 6.4F 40.1M 5.6D 1.6B
A2B3 32.6AB 6.8B 44.7G 5.8B 1.7A
A2B4 32.8A 7.0A 47.0A 5.9A 1.7A
A3B0 27.4I 6.0I 34.2R 4.9I 1.5C
A3B1 29.1H 6.3G 38.7O 5.4E 1.6B
A3B2 31.1F 6.6D 42.6J 5.7C 1.7A
A3B3 32.8A 7.0A 46.4C 5.9A 1.7A
A3B4 32.7A 6.8B 46.0D 5.6D 1.6B
A4B0 31.2EF 6.2H 36.1Q 5.1G 1.6B
A4B1 31.8CD 6.4F 39.9N 5.6D 1.7A
A4B2 32.2BC 6.8B 43.2I 5.8B 1.6B
A4B3 32.6AB
6.7C 44.8F 5.6D 1.5C
A4B4 32.1C 6.5E 43.6H 5.4E 1.5C

Effects of EDTA and DTPA on Lead and Zinc Accumulation of Ryegrass 937
to the biological toxicity of free chelators as well as the
active effects of chelators on heavy metals [24,25]. Also,
the trend of the root-shoot ratio indicated that the eco-
logical adaptability of ryegrass plants had gradually in-
creased.
Some scholars found that 60% of lead in soil was ac-
tivated when 15 mmol/kg EDTA was added to acidic soil
(pH 5.5). As EDTA had oxygen atoms with four electron
pairs and nitrogen atoms with 2 electron pairs, it mainly
existed in soil in the form of H2[EDTA]2–, and in this
acidity range, heavy metal ions mainly existed in the
form of bivalent as well. So, EDTA and heavy metal ions
could form stable chelates [26,27]. DTPA and EDTA of
organic acid ligands had a strong ability of chelation with
the change of ligand and heavy metal elements, DTPA
played a more stable effect on chelating with lead and
zinc than that of EDTA [28]. Blaylock [29] found that
DTPA and EDTA could enhance Pb and Cd accumula-
tion in shoot of Brassiajuncea (1.6% and 1.0% respec-
tively). Xu [30] showed that EDTA enhanced Zn accu-
mulation in root and shoot of vetiver grass more than
7.3% and 37.4%. Combining with EDTA and DTPA, the
shoot of Thlaspi caerulescens accumulated more Zn than
the control [21]. Also, EDTA could improve the accu-
mulation of Pb and Zn in Chrysanthemum coronarium
and Cirsium japonicum [31,32], which had significant
correlation with its concentrations [33]. Meanwhile, this
study found that EDTA and DTPA enhanced the contents
of Pb/Zn in root and shoot of ryegrass plant, which was
consistent with the research of Wang [34], and increased
total Pb/Zn accumulation and translocation ratio in rye-
grass plant.
Bell [35] and Wenzel [36] reported that metal chelates
could enter the root from the endodermis cleft and trans-
fer into plants’ stem and leaves. In the marking test of
14C-EDTA-Pb, Blaylock [29] found that plants accumu-
lated Pb more easily with chelators. Salt [6] found that
chelation prevented precipitation and absorption of met-
als, thereby improving metals availability. But a proper
concentration of EDTA which induced the accumulation
of Pb was the key, and this may be due to the physio-
logical mechanism of roots which controlled the trans-
membrane transportation. In this paper, with the same
dose, EDTA enhanced total Pb and Zn accumulation in
shoots more than DTPA, which could be due to its weak
chelated ability and poor active ability.
5. Conclusions
EDTA and DTPA enhanced the biomass of ryegrass
when the dose less than 4 mmol/kg, and enhanced Zn and
Pb contents in roots and shoots of ryegrass. The total Pb
and Zn accumulations, translocation ratio and nutrient (N,
P, K, Ca and Mg) contents in ryegrass had also been en-
hanced. The partial correlation analysis showed that
DTPA and EDTA didn’t significantly correlate with Pb
accumulation in roots and did significantly correlate with
Pb accumulation in shoots of ryegrass, and the correla-
tion between DTPA/EDTA and Zn accumulation in roots
and shoots of ryegrass was significant. Therefore, EDTA
and DTPA have great potential to be used for ryegrass to
remedy Pb and Zn contaminated soil around lead-zinc
mining areas, but should be used cautiously because of
their environmental risks.
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