Journal of Cleaner Production 263 (2020) 121491

Contents lists available at ScienceDirect

Journal of Cleaner Production

journal homepage: www.elsevier.com/locate/jclepro

Phytoremediation of uranium and cadmium contaminated soils by

sunflower (Helianthus annuus L.) enhanced with biodegradable
chelating agents
Li Chen a, b, Jin-yan Yang b, Dan Wang a, *
a
College of Life Science and Engineering, Southwest University of Science and Technology, Mianyang, 621010, Sichuan, PR China
b
College of Architecture and Environment, Sichuan University, Chengdu, 610065, Sichuan, PR China

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

Article history: Applying biodegradable chelating agents to assist in phytoremediation is a promising method to increase
Received 9 January 2020 the remove efficiency of metal pollutants from contaminated soils. The effects of biodegradable chelating
Received in revised form agents on improving the phytoremediation capacity in uranium (U) and cadmium (Cd) contaminated soil
30 March 2020
was investigated using sunflowers, which were grown in pots containing soil with U and Cd added at
Accepted 31 March 2020
Available online 7 April 2020
15 mg/kg. After 2 months of growth, citric acid (CA), oxalic acid (OA) and ethylenediamine disuccinate
(EDDS) at various concentrations (0, 2.5, 5.0 and 7.5 mmol/kg) were applied. The results showed that
Handling Editor: Prof. Bing-Jie Ni plant biomass decreased by 12.12% for shoot and 15.74% for root under U and Cd combined stress.
Meanwhile, chelating agent treatments, especially with EDDS, enhanced U and Cd stress in plants by
Keywords: decreasing biomass, inhibiting photosynthesis, and increasing malondialdehyde and H2O2 levels. The U
Helianthus annuus L. uptake of plants after CA addition was significantly greater than that after OA and EDDS addition.
Biodegradable chelating agents Nevertheless, EDDS addition has better effects on Cd uptake than CA and OA addition. U and Cd remove
U and Cd contaminated soil efficiencies reached the maximum following the application of 5.0 mmol/kg CA and 5.0 mmol/kg EDDS,
Plant growth
which were 177.48% and 181.51% higher than that of the control, respectively. Furthermore, the
Stress tolerance
bioavailable U content in soils treated with CA were higher than that in soils treated with EDDS, whereas
Remove efficiency
bioavailable Cd content significantly increased due to EDDS addition. These results suggest that biode-
gradable chelating agents have significant effects on improving the U and Cd phytoremediation potential
of sunflowers.
© 2020 Elsevier Ltd. All rights reserved.

1. Introduction
Increasing attention has been given to Uranium (U), a special metal
element, due to the development of the nuclear industry (Hu et al.,
2019). Uranium is considered as a dangerous environmental
pollutant due to its chemotoxicity and radiotoxicity (Selvakumar
et al., 2018); moreover, mining and use of U resources also pollute
the soil environment, especially in soil surrounding U tailings (Li et al.,
2019). Previous studies have reported that the U content
(3.21e62.37 mg/kg) in the topsoil (total of 205 samples) around U
tailings (Heng yang, Hunan Province, China) significantly exceeded
the average value (3.03 mg/kg) of topsoil U content in China (CNEMC,
1990; Ma et al., 2017). Furthermore, soil around U tailings also contain
* Corresponding author.
E-mail address: wangdanxkd@163.com (D. Wang).
other heavy metals such as Cadmium (Cd), which is one of the most
hazardous pollutants in soil because of its widespread pollution, high
mobility and severe toxicity (He et al., 2017; Zhang and Liu, 2018).
Therefore, recent studies have focused on remediating contaminated
soils around U tailings especially in areas where heavy metal pollut-
ants occur in the topsoil combined with pollution.
To date, different remediation technologies including adsorp-
tion (Chen et al., 2020a), electrokinetic remediation (Guedes et al.,
2019) and soil washing (Pourfadakari et al., 2019) have been
developed to remove toxic metals from soils. Nevertheless, these
remediation methods have some disadvantages in large-scale
practice, including low efficiency, high expenditure and the pro-
duction of secondary pollution (Gurajala et al., 2019). In contrast,
phytoremediation, using plant to remove heavy metal pollutants
from the soil, has been considered to have great potential in
remediating heavy metal-contaminated soils in situ because of its
cost-efficiency and eco-friendliness (Gurajala et al., 2019; He et al.,

https://doi.org/10.1016/j.jclepro.2020.121491
0959-6526/© 2020 Elsevier Ltd. All rights reserved.
2 L. Chen et al. / Journal of Cleaner Production 263 (2020) 121491
2019). However, its remediation capacity is still questionable due to
low metal uptake in the tested plant; thus, this method requires
further improvements. Some techniques including intercropping
with various tested plants (De Conti et al., 2019), adding plant
growth regulators (Chen et al., 2020b), adding endophytic bacteria
(Wang et al., 2019a), and adding chelating agents such as ethyl-
enediaminetetraacetic acid (EDTA), ethylenediamine disuccinate
(EDDS), citric acid (CA) and oxalic acid (OA) (Wang et al., 2018; Hu
et al., 2019), have been developed. In particular, the use of EDTA can
significantly promote heavy metal accumulation in plants growing
in contaminated soils; however, EDTA addition can increase the risk
of leaching of heavy metal pollutants from soil into groundwater
due to its low biodegradability (Han et al., 2019). Thus, biode-
gradable chelating agents with no adverse effects are currently
desirable. CA, OA and EDDS have been favoured by researchers due
to their high biodegradability and due to their ability to stimulate
metal uptake in plants (Moslehi et al., 2019; Wang et al., 2019b).
Nevertheless, chelating agents also have adverse effects on plant
growth and development as they can cause etiolation, withering
and even death when present in excess amounts (Hasan et al.,
2019). Sidhu et al. (2018) previously reported that 2 mmol/kg
EDDS significantly decreased shoot and root biomass of the tested
plant (Coronopus didymus (L.) Sm.), with significant increases in the
H2O2 and malondialdehyde (MDA) contents of the plant leaves.
Sunflower (Helianthus annuus L.) has a higher metal bio-
concentration factor (including U and Cd), translocation factor and
strong stress tolerance; this has been confirmed by various studies
(Alsabbagh and Abuqudaira, 2017; Zehra et al., 2020). Meanwhile,
sunflower also has other advantages including a short growth cycle,
easy management and high biomass. Therefore, sunflower can be
considered as a promising candidate for removing heavy metal
pollutants from contaminated soils. We therefore hypothesized
that CA, OA and EDDS are potential biodegradable chelating agents
for improving the remediation of U- and Cd-contaminated soils.
This experiment aimed to (1) explore the influence of CA, OA and
EDDS addition in soil on plant growth and stress; (2) evaluate the
effects of the three chelating agents on enhancing U and Cd uptake,
translocation and remediation efficiency in the soil; and (3) analyse
the mechanisms by which chelating agents improve the capacity of
U and Cd phytoremediation using sunflower.
2. Materials and methods
2.1. Materials
Sunflower (Helianthus annuus L.) seeds were purchased from
Sichuan Xike Seed Industry Co., Ltd. Three biodegradable chelating
agents (CA, OA and EDDS) were obtained from Mianyang Rong-
sheng Chemical Reagent Co., Ltd. Soil samples (silty clay) with pH
7.08 were collected at depths of 0e25 cm from a field in the agri-
cultural experimental station of the Southwest University of Sci-
ence and Technology, China. The collected soil was not
contaminated by heavy metal pollutants according to the soil
environmental quality risk control standard (SEQ, 2018). Back-
ground U and Cd contents in the soil sample were 3.48 mg/kg and
0.29 mg/kg, respectively. The basic properties of the soil are as
follows: organic matter content 19.03 g/kg, available phosphorus
40.12 mg/kg, available potassium 59.15 mg/kg, available alkali
hydrolysable nitrogen 158.93 g/kg, cation exchange capacity
105.72 mmol/kg, clay 36.5%, silt 45.2% and sand 18.3%.
2.2. Experimental design and plant cultivation
All pot experiments were performed in a greenhouse at the
State Defense Key Laboratory of Nuclear Waste and Environmental
Security (Mianyang, Sichuan Province). The soil sample was air-
dried, crushed and then mixed. The gravels and weeds in the soil
sample were also removed. Identical plastic pots (20 cm
diameter
18 cm height) were each filled with 3.0 kg of the pre-
pared soil sample; a tray was placed under the pot to prevent U and
Cd leaching from the hole on the plastic pot. Soil sample in the
plastic pot was then spiked with UO2(CH3CO2)2$2H2O and
CdCl2$2.5H2O, corresponding to 15 mg U kg1 and 15 mg Cd kg1
of; soil without U and Cd treatment was used as control (CKK).
Meanwhile, the soil sample was fertilised with 225 mg N kg1,
65 mg P kg1 and 227 mg K kg1 using (NH4)2SO4, KH2PO4 and
K2SO4. In addition, soil aging was conducted in these plastic pots for
4 weeks. All treatments were arranged according to a completely
randomized design and was performed in triplicate. Subsequently,
two uniform seedlings of sunflower were transplanted into each
plastic pot (Fig. 1). The soil was regularly irrigated with deionized
water, and the soil moisture of each treatment was maintained at
approximately 60% of field water capacity.
To compare the roles of the additives, the three chelating agents
(CA, OA and EDDS) with concentrations of 0 (blank control, CK), 2.5,
5.0 and 7.5 mmol/kg were used in this experiment. Meanwhile,
chelating agents were formulated as a 100 mL solution with a pH
level of 6.5, and then slowly added to the rhizosphere soil via irri-
gation in three applications at each 3 day interval (in the first and
second applications, 33 mL additive was applied to each treatment;
in the third application 34 mL was added). To avoid the death of the
sunflower seedlings, the prepared CA, OA and EDDS were applied
based on the above-mentioned experimental design 2 months after
transplantation. Subsequently, the sunflowers in the pots were
regularly irrigated and were harvested 1 week after the third
application.
2.3. Measurement methods
2.3.1. Plant biomass, chlorophyll, MDA and H2O2 contents
At harvesting, the sunflowers were removed from the pots and
then separated into shoots (aboveground parts) and roots. The
shoots and roots were washed carefully with distilled water, dried
at 75  C, and after 30 min dried at 105  C until the weight remained
Fig. 1. The seedling stage (a) and flowering stage (b and c) of sunflowers in this
experiment (completely randomized design).
 L. Chen et al. / Journal of Cleaner Production 263 (2020) 121491
unchanged. After the dry weights (DW) of the shoots and roots
were measured, the single plant dry weight (dry weight per plant)
was calculated as the sum of the shoot and root dry weights. In this
study, chlorophyll, MDA and H2O2 contents were measured using
the fresh leaves of the tested plant. After extracting 0.25 g of fresh
leaves using 80% aqueous acetone, the absorbance of the filtrates
was recorded with an ultraviolet spectrophotometer (UV-1900,
Shimadzu (China) Co., Ltd, Shanghai, China) at 663, 652 and 645 nm
to determine the chlorophyll concentration according to the
method of Lichtenthaler and Buschmann (2001). This experiment
firstly investigated MDA and H2O2 responses in sunflowers under U
and Cd, under chelating agents, and under their combined stress.
Using fresh leaves, MDA and H2O2 content were detected with the
thiobarbituric acid (TBA) reaction and with titanium tetrachloride
(TiCl4) using the methods of Mohammadi et al. (2018) and
Hawrylak-Nowak et al. (2015), respectively.
2.3.2. Heavy metals analyses
U and Cd concentrations in the shoots and roots of sunflowers
were measured using the method described by Chen et al., 2020b.
The bioavailability of heavy metals (U and Cd) in soils was examined
with the Mehlich III extractant (0.013 N HNO3, 0.015 N NH4F, 0.2 N
CH3COOH, 0.25 N NH4NO3, and 0.001 N EDTA adjusted to pH 2.5)
according to the method described by Elrashidi et al. (2015). Briefly,
1.0000 g of dried soil sample around the roots was mixed with 50 mL
Mehlich III extractant in a 50-mL centrifuge tube and oscillated for
30 min (in triplicate). After filtration, the supernatant was taken for
U and Cd analysis. U and Cd concentrations in the shoots and roots of
sunflowers and in the supernatant were determined by ICP-MS
(Agilent Technologies 7500 Series, Santa Clara, USA). The detection
limit is 0.03 ng/L for U and 0.4 ng/L for Cd, respectively. In addition,
the standard soil samples (GSS14) obtained from the National
Institute of Metrology (Beijing, China) were used as the standard
reference material for quality assurance and quality control.
2.4. Computational method
To assess the effects of CA, OA and EDDS on U and Cd phytor-
emediation by the tested plants, the bioconcentration factor (BCF),
translocation factor (TF) and remove efficiency (RE) were calculated
using the following equations (Wang et al., 2019b; Chen et al.,
2020b).
Csh orr
BCF ¼
Cs
Csh
TF ¼
Cr
ðCsh
Msh þ Cr
Mr Þ
RE ¼
ðCs
Ms Þ
where Csh and Cr represent the metal concentration (mg/kg) in the
shoots and roots, respectively. Cs is the initial soil metal concen-
tration (mg/kg). Msh, Mr and Ms represent the mass (g) of the shoot,
root and soil, respectively.
2.5. Statistical analysis
All data presented in this study were analysed by Excel 2007,
SPSS 22.0 and Origin 2018b. Meanwhile, all data are shown as
mean ± standard error (triplicate). Duncan’s multiple comparison
test was used to evaluate significant differences between different
treatments, and the significance level used was p < 0.01.
3
3. Results and discussion
3.1. Effects of biodegradable chelating agents on plant biomass and
total chlorophyll content
The plant biomass (DW) and total chlorophyll content of sun-
flower after the different treatments are shown in Fig. 2. The
biomass of shoots and roots decreased by 12.12% and 15.74% under
U and Cd combined stress, respectively, compared to the biomass
observed in the treatments without U and Cd (CKK) (Fig. 2a and b).
The application of chelating agents, especially EDDS, further
decreased sunflower shoot and root biomass. The minimum dry
weight of the shoot and root was observed in the 7.5 mmol/kg EDDS
setup. EDDS treatments decreased shoot biomass by 3.28e30.31%
and the root biomass by 0.73e23.81% compared to those of the
control groups (CK); the observed reduction was more evident with
increased EDDS concentrations. These results suggest that the
tested plant is more sensitive to EDDS treatment phytotoxicity than
to CA and OA treatments. Liang et al. (2019) also found that the
application of 2.5 mmol/kg EDDS significantly decreased rhizoid
and frond biomass of Pteris vittata L. in metal-contaminated (As, Cd
and Pb) soil, which resulted in the 27.7% and 39.3% decrease of
rhizoid and frond biomass, respectively, compared to control
groups (without EDDS addition). These results may be attributed to
the competition between metals (U and Cd) and other vital
micronutrients like Mg, Zn and Fe in the soil due to the improve-
ment of U and Cd bioavailability by EDDS addition in the soil-plant
system (Ferraz et al., 2012). Furthermore, EDDS-metal complexes
could impair pigment generation and increase oxidative damage of
plant cells, thus leading to plant growth inhibition (Yin et al., 2015;
Ye et al., 2015). These were also observed in our studies; however,
no obvious decreases in the shoot and root biomasses of OA and CA
treatments were observed compared to the control groups (CK).
These results indicate that the effects of chelating agents on plant
growth are mainly related to the chelating agent type, dose and
environmental factors such as soil fertility levels, soil moisture
content and soil structure (Bian et al., 2018; Wang et al., 2019b).
Furthermore, chelating agents can also decrease the chlorophyll
content in plant cells due to their phytotoxicity (Yin et al., 2015). In
this study, EDDS addition significantly (p < 0.01) decreased the total
chlorophyll content of sunflower leaves (Fig. 2d). The minimum
total chlorophyll content in sunflower leaves was observed in the
7.5 mmol/kg EDDS addition, which was also in accordance with the
plant biomass. There are two potential reasons for these results:
(1) first is that EDDS-metal complexes can inhibit chlorophyll syn-
thesis via the suppression of chlorophyll-synthesizing enzyme ac-
tivity (v-amino levulinic acid dehydratase) (Bhattacharya et al.,
(2) 2010), second is due to the substitution of central Mg2þ ions in
chlorophyll molecules by Cd2þ, and EDDS addition significantly
increased the bioavailable Cd content in the soil and further pro-
moted sunflower Cd uptake (Pandey and Bhattacharya. 2018).
(3)
Chlorophyll synthesis inhibition by EDDS-metal complexes has
been reported in several studies: for instance, the results of Ye et al.
(2015) indicated that 8 mmol/kg EDDS markedly reduced the
chlorophyll content of soybean in multi-metal contaminated soils
compared to that of control (without EDDS addition).
3.2. Effects of biodegradable chelating agents on lipid peroxidation
and active oxygen
The overproduction of reactive oxygen species (ROS) is generally
considered as the primary response of plants to abiotic stress,
especially against heavy metals (Berni et al., 2019). U and Cd are
well-known as inducers of overproduction of ROS including H2O2,
superoxide (O2) and singlet oxygen (1O2) (Dabral et al., 2019). In
4 L. Chen et al. / Journal of Cleaner Production 263 (2020) 121491

Fig. 2. Effects of three chelating agents on shoot biomass (a), root biomass (b), single plant biomass (c) and total chlorophyll content (d) of Helianthus annuus L. under U and Cd
treatments.
Note: data marked by the different lowercase letters represent statistically significant differences at p < 0.01 based on LSD test. Y line represents the control value (CK). CKK, soil
without U, Cd and chelating agent; CK, U and Cd contaminated soil without chelating agent; CA, citric acid addition; OA, oxalic acid addition; EDDS, ethylenediamine disuccinate
addition.

Fig. 3. Levels of MDA (a) and H2O2 (b) in Helianthus annuus L. with different treatments.
Note: data marked by the different letters represent statistically significant differences at p < 0.01 based on LSD test. Y line represents the value of the control (CK). CKK, soil without
U, Cd and chelating agent treatment; CK, U and Cd contaminated soil without chelating agent; CA, citric acid addition; OA, oxalic acid addition; EDDS, ethylenediamine disuccinate
addition.
 L. Chen et al. / Journal of Cleaner Production 263 (2020) 121491 5

Fig. 4. Effects of three chelating agents on U (a and b) and Cd (c and d) concentrations of Helianthus annuus L. in the soil.
Note: data marked by the different lowercase letters represent statistically significant differences at p < 0.01 based on LSD test. Y line represents the value of the control (CK). CK, U
and Cd contaminated soil without chelating agent; CA, citric acid addition; OA, oxalic acid addition; EDDS, ethylenediamine disuccinate addition.

addition, MDA, a final product of lipid peroxidation, is used to
indirectly determine the oxidative damage on plant cell mem-
branes, indicating that it could be a quantitative biomarker of plant
response against heavy metal stress (Shahid et al., 2017).
The impact of different treatments on MDA and H2O2 is shown
in Fig. 3. Under U and Cd combined stress, MDA and H2O2 contents
of sunflowers increased by 34.15% and 12.92%, respectively,
compared to those in the soil without U and Cd addition, suggesting
that MDA and H2O2 are the obvious responses of sunflowers against
U and Cd combined stress. Jiang et al. (2020) also reported that
MDA and H2O2 concentration in the leaves of rice (Oryza sativa L.)
significantly increased following 80 mmol/L Cd2SO4 treatment in
liquid medium compared to control (without Cd2SO4 addition). In
the current study, MDA and H2O2 contents of sunflower leaves were
further increased to varying degrees following the addition of three
chelating agents, with EDDS having a more prominent role than OA
and CA. The maximum MDA and H2O2 concentrations in sunflowers
under U and Cd treatments was observed in the set-up with
7.5 mmol/kg EDDS addition. EDDS addition induced more oxidative
damage to sunflowers under U and Cd combined stress, which can
be attributed to ROS overproduction, promotion of lipid peroxida-
tion, and loss of cell membrane integrity (Sidhu et al., 2018). This
might be due to the increased U and Cd accumulation in sunflower
tissues when treated with EDDS. Similar phenomena were
observed in our studies. Sidhu et al. (2018) also demonstrated that
MDA and H2O2 concentrations were significantly increased when
2 mmol/kg EDDS is added to nickel-stressed Coronopus didymus (L.)
Sm. Another possible reason for this is that EDDS is also toxic to
plant growth, and it can induce plant oxidative stress in uncon-
taminated soils (Li et al., 2018). Wang et al. (2018) found that
5 mmol/kg EDDS significantly decreased Commelina communis L.
shoot dry weight in uncontaminated soil and increased plant MDA
content. Furthermore, this study also showed slightly enhanced
MDA and H2O2 content after the application of 7.5 mmol/kg CA.
However, there are no significant differences between all OA ad-
ditions and control (CK), which is possibly due to the fact that OA
has a lower capacity to promote sunflower U and Cd uptake
compared with other chelating agents (Fig. 4).
3.3. Effects of biodegradable chelating agents on U and Cd
concentration
The addition of different chelating agents caused significant dif-
ferences in the U and Cd concentrations of the shoots and roots of
sunflowers, as shown in Fig. 4aed. In this study, the application of CA
and EDDS caused significant increases in the shoot and root U con-
centrations (Fig. 4a and 4b). The highest shoot (1.3 mg/kg) and root
(44.84 mg/kg) U concentrations was observed in the 5.0 and
7.5 mmol/kg CA additions, which were 5.87-fold and 2.79-fold higher
than those of the shoot (0.21 mg/kg) and root (16.05 mg/kg) of the
6 L. Chen et al. / Journal of Cleaner Production 263 (2020) 121491

Table 1
Influence of biodegradable chelating agents on the U and Cd phytoremediation efficiency by sunflower.

Chelating agent additions Bioconcentration factor Bioconcentration factor Translocation factor Remove efficiency (%)
of shoots of roots

U Cd U Cd U Cd U Cd
e e c d d c d
CK 0.014 1.20 1.07 3.83 0.013 0.32 0.111 1.46d
CA 2.5 mmol/kg 0.041c 1.48e 1.52c 4.18cd 0.027ab 0.36c 0.199bc 1.84cd
5.0 mmol/kg 0.082a 2.25cd 2.46ab 5.02bcd 0.033a 0.45bc 0.308a 2.46bc
7.5 mmol/kg 0.069ab 2.80c 2.99a 7.05a 0.023bc 0.40c 0.307a 2.86b
OA 2.5 mmol/kg 0.013e 1.43e 1.26c 4.31cd 0.011d 0.33c 0.121d 1.66d
5.0 mmol/kg 0.018e 1.56e 1.13c 4.64cd 0.016cd 0.35c 0.103d 1.76d
7.5 mmol/kg 0.021de 1.82de 1.36c 5.37bc 0.015cd 0.34c 0.120d 1.96cd
EDDS 2.5 mmol/kg 0.034cd 2.46cd 1.45c 5.50bc 0.024bc 0.46bc 0.161cd 2.68b
5.0 mmol/kg 0.059b 4.69a 2.40b 7.22a 0.025ab 0.65a 0.234b 4.11a
7.5 mmol/kg 0.067ab 3.74b 2.21b 6.29ab 0.031ab 0.60ab 0.198bc 2.85b

Notes: data marked by the different lowercase letters represent statistically significant differences at p < 0.01 based on LSD test.

Fig. 5. Influence of three chelating agents on bioavailable U (a) and Cd (b) contents in the soil.
Note: data marked by different lowercase letters represent statistically significant differences at p < 0.01 based on the LSD test. Y line represents the value of the control (CK). CK, U
and Cd contaminated soil without chelating agent, EDDS, ethylenediamine disuccinate addition; OA, oxalic acid addition; CA, citric acid addition.

Table 2
Pearson correlation coefficients among plant biomass (DW), oxidative damage, U uptake, U translocation, bioavailable U content and remove efficiency in the contaminated soil
(n ¼ 10). Meanwhile, * and ** represent significant correlations at level p < 0.05 and p < 0.01, respectively.

Shoot biomass Root biomass MDA H2O2 Shoot U contents Root U contents Bioavailable U contents

Shoot biomass 1
Root biomass 0.724* 1
MDA 0.943** 0.652* 1
H2O2 0.933** 0.485 0.924** 1
Shoot U contents 0.585 0.005 0.486 0.705* 1
Root U contents 0.608 0.112 0.439 0.683* 0.924** 1
Translocation factor 0.438 0.116 0.442 0.595 0.897** 0.686* 0.582
Bioavailable U content 0.243 0.145 0.070 0.356 0.815** 0.883** 1
Remove efficiency 0.352 0.212 0.211 0.501 0.937** 0.939** 0.930*

control, respectively. Similarly, CA and EDDS treatments also showed
significant increases in sunflower Cd uptake (p < 0.01) compared to
the control (Fig. 4c and 4d). Cd concentration changed from 19.02% to
290.35% for the shoots and 12.66%e88.56% for the roots due to the
addition of chelating agents. The maximum Cd concentration was
70.38 mg/kg for the shoot and 108.27 mg/kg for the root following
5.0 mmol/kg EDDS addition, which was significantly greater (p < 0.01)
than that of the shoot (18.03 mg/kg) and root (57.42 mg/kg) of control.
This may be attributed to the fact that chelating agents can form
chelant-Cd complexes with Cd2þ due to their negatively charged
hydroxyl or carboxyl groups, which are conducive to the uptake of Cd
by sunflowers, thus leading to increases in Cd concentration of the
shoots and the roots (Bian et al., 2018; Wang et al., 2019b). Moreover,
there are two possible mechanisms behind the plant uptake of
chelant-metal complexes. The first possibility is that chelant-metal
complexes are directly absorbed by plants. Second, the complexes
are dissociated prior to their uptake in soil-plant systems (Hasegawa
et al., 2019). The specific processes still require further investigation.
 L. Chen et al. / Journal of Cleaner Production 263 (2020) 121491
In this study, we observed that CA addition has a better capacity to
in promoting sunflower U uptake compared to OA and EDDS addition,
whereas EDDS addition had a better effect on Cd uptake compared to
CA and OA addition. These results suggest that the chelated abilities of
CA and EDDS to change the form of metals (U and Cd) are different
because they have different physicochemical properties (Lestan et al.,
2008; Bian et al., 2018). Hu et al. (2019) also showed that CA had good
potential to activate immobilized U in soils and significantly increased
the Macleaya cordata shoots and roots U concentration compared to
EDDS treatments. Wang et al. (2019b) tested chelating agents-assisted
Cd phytoremediation by Amaranthus hypochondriacus L. and found
that EDDS treatments had a stronger ability to increase Cd concen-
tration of the shoots and roots compared to CA treatments. However,
U and Cd concentrations of the shoots and roots had no significant
differences following OA addition compared to the control (p < 0.01).
The possible reason for this result is that OA has less potential to
mobilize U and Cd in the soil compared to CA and EDDS treatments
(Hu et al., 2019; Liang et al., 2019).
3.4. Effects of biodegradable chelating agents on the translocation
and remove efficiency of heavy metals
Chelating agents have the capacity to increase the translocation
and remove efficiency of heavy metals by the tested plants. The
bioconcentration factor and translocation factor of U and Cd in
sunflowers are presented in Table 1. All bioconcentration factors of
U were extremely low compared with the bioconcentration factor
of Cd, suggesting that the absorption of U by sunflowers from the
soil was more difficult than Cd absorption. Application of CA and
EDDS had a significantly positive effect on U and Cd translocation
from soil to shoots/roots (p < 0.01), and the shoot bioconcentration
factor under 5.0 mmol/kg CA and EDDS addition reached a
maximum of 0.082 (U) and 4.69 (Cd), corresponding to an increase
of 485.71% (U) and 290.83% (Cd), respectively. Meanwhile, the
application of CA and EDDS significantly (p < 0.01) increased the
translocation factor of U and Cd, suggesting that CA and EDDS
favoured the translocation of U and Cd from roots to shoots. The
translocation factor of U reached a maximum (0.033) following the
5.0 mmol/kg CA treatment, which was 2.53-fold that of the control,
indicating that CA is better at increasing the translocation factor of
U than EDDS and OA treatment. A possible reason is that CA
addition hindered the sorption of U by the soil particles, thereby
enhancing U bioavailability in the soil-plant system, and improved
the ability of sunflowers to transfer U from the roots to the shoots
(Hu et al., 2019). EDDS treatment significantly (p < 0.01) increased
the translocation factor of Cd in the tested plants compared with CA
and OA treatments. A possible reason for this is the mutual selec-
tivity of the plants, type of chelating agents and type of heavy metal
ions involved; as such, various chelating agents may have different
effects on heavy metal translocation from shoots to roots (Bian
et al., 2018; Mai et al., 2019). The translocation factor of Cd
Table 3
Pearson correlation coefficients among plant biomass (DW), oxidative damage, Cd uptake, Cd translocation, bioavailable Cd content and remove efficiency in the contaminated
soil (n ¼ 10). Meanwhile, * and ** represent significant correlations at level p < 0.05 and p < 0.01, respectively.
Shoot biomass Root biomass MDA
Shoot biomass 1
Root biomass 0.724* 1
MDA 0.943** 0.652* 1
H2O2 0.933** 0.485 0.924**
Shoot Cd contents 0.838** 0.472 0.707*
Root Cd contents 0.771* 0.525 0.580
Translocation factor 0.806** 0.387 0.733*
Bioavailable Cd content 0.944** 0.636* 0.992**
Remove efficiency 0.688* 0.318 0.515
7
reached a maximum (0.65) under 5.0 mmol/kg EDDS treatment,
which was 2.03-fold that of the control (0.32).
The remove efficiencies of U and Cd in CA, OA and EDDS treated
soils are also shown in Table 1. The U remove efficiency was
significantly lower than the Cd remove efficiency in all treatments
due to the poor U uptake capacity of sunflower. This result may be
attributed to the fact that U has higher molecular weight and lower
mobility in the soil compared to Cd (Malaviya and Singh. 2012).
Meanwhile, less U-related transporters in the tested plants may
also result in low absorption and translocation (Gavrilescu et al.,
2009; Mouser et al., 2009). CA addition induced the highest in-
crease in U remove efficiency, resulting to an efficiency 1.77-fold
greater than that of the control. Meanwhile, maximum Cd
remove efficiency of sunflowers was observed in the application of
5.0 mmol/kg EDDS. These results indicate that biodegradable
chelating agents have great potential in enhancing the phytor-
emediation of U and Cd by the tested plants. Wang et al. (2019b)
also showed that the application of EDDS caused higher Cd
remove efficiency by Amaranthus hypochondriacus L. compared to
CA treatment. In addition, plants having toxic heavy metal contents
can be disposed using various disposal technologies.
3.5. Effects of biodegradable chelating agents on bioavailable U and
Cd contents
The application of chelating agents can alter the chemical form
of heavy metals, further increasing the biological availability of
heavy metals in soil (Bian et al., 2018). The effects of CA, OA and
EDDS on the bioavailable U and Cd contents in the soil are shown in
Fig. 5a and 5b. CA, OA and EDDS addition increased the bioavailable
U and Cd contents in the soil (Fig. 5a and 5b). The maximum
bioavailable U and Cd content was 11.31 and 11.34 mg/kg in the
7.5 mmol/kg CA and EDDS addition, which was 102.69% (for CA)
and 69.01% (for EDDS) greater than those of the control. Never-
theless, the maximum U and Cd concentration of sunflowers
(shoots and roots) were observed at 5.0 mmol/kg CA and EDDS
addition. This is possibly due to the fact that sunflower physiologies
underwent severe stress following high levels (7.5 mmol/kg) of CA
and EDDS addition, thus leading to a decrease in U and Cd uptake
(Sidhu et al., 2018). Under OA addition, the bioavailable U and Cd
contents in the soil were not significantly different from those of
the control, indicating that OA had a poor capacity to transform
immobile U and Cd to a mobile form. This is mainly attributed to
structural and chemical properties of OA itself. Less carboxyl groups
in OA would go against the formation of chelant-metal complexes,
indirectly leading to metal sorption by the soil (Wang et al., 2019b).
CA addition had the greatest effect on mobilizing U from the soil,
whereas EDDS could more efficiently mobilize Cd from the soil. This
is likely due to the mutual selectivity of the metals and the various
chelating agents (Bian et al., 2018). Hu et al. (2019) also indicated
that specific chelating agents are suitable for particular metals in
H2O2 Shoot Cd contents Root Cd contents Bioavailable Cd contents
1
0.747* 1
0.671* 0.893** 1
0.735* 0.961** 0.748* 0.768*
0.920** 0.745** 0.628 1
0.598 0.966** 0.904** 0.570
8 L. Chen et al. / Journal of Cleaner Production 263 (2020) 121491
soil; for example, CA addition is better than OA and EDDS addition
for mobilizing U in soil.
3.6. Correlations among different parameters
Pearson correlation analysis was performed to determine the
correlations among plant growth, oxidative damage, metal uptake,
metal translocation, bioavailable metal contents and sunflower
remove efficiency (Tables 2 and 3). The shoot biomass of sunflower
showed significant (p < 0.01) negative correlations with oxidative
damage parameters (MDA and H2O2). Khan et al. (2019) also re-
ported that the decline in plant dry mass is because of the adverse
effects of the co-application of metals and chelating agents, which
significantly inhibits plant growth and increases MDA and H2O2
contents of Petunia hybrida L. Shoot and root U concentrations
exhibited significant positive correlations with the U remove effi-
ciency of sunflowers (p < 0.01), suggesting that U concentrations of
the shoot and root have more important roles in the phytor-
emediation process than plant biomass. Meanwhile, shoot Cd
concentration was significantly (p < 0.01) negatively correlated
with shoot biomass but was significantly (p < 0.05) positively
correlated with MDA and H2O2 contents. A possible reason is that
high level Cd could induce severe phytotoxicity, leading to a
decrease in shoot biomass and an increase in oxidative damage of
sunflower (Lian et al., 2020). Meanwhile, shoot and root Cd con-
centrations were also significantly positively correlated with Cd
remove efficiency (p < 0.01). Furthermore, bioavailable U and Cd
contents exhibited a significant positive correlation with shoot U
and Cd concentrations, respectively, suggesting that highly
bioavailable metal contents in the soil might cause an increase in
metal uptake in the tested plants.
4. Conclusions
This study was conducted to evaluate the effects of biodegradable
chelating agents on enhancing the phytoremediation of U and Cd
using sunflowers. The results indicated that chelating agents
enhanced the adverse effects of U and Cd combined stress to the
sunflower by reducing plant biomass and inhibiting photosynthesis.
Specifically, EDDS exhibited stronger inhibition effects on plant
growth and chlorophyll synthesis compared to CA and OA treat-
ments. Meanwhile, EDDS also significantly increased MDA and H2O2
contents, further elevating the oxidative damage of sunflowers. The
addition of chelating agents can effectively improve the capacity of
sunflowers to absorb and transport U and Cd to varying degrees. The
maximum remove efficiency of U and Cd occurred in the setups with
5 mmol/kg CA and EDDS addition which was 177.48% and 181.51%
greater than that of the control, respectively. CA application led to a
greater capacity in mobilizing U, whereas EDDS addition was more
effective in mobilizing soil Cd. High amounts of chelating agent-
metal complexes may cause serious toxicity to plant growth and
physiological processes, which may even result in reduced metal
phytoremediation. In the future, large-scale studies under field
conditions should be conducted to examine the removal efficiency of
sunflowers under real-world conditions.
Declaration of competing interest
None.
CRediT authorship contribution statement
Li Chen: Investigation, Writing - original draft. Jin-yan Yang:
Writing - review & editing. Dan Wang: Funding acquisition, Project
administration.
Acknowledgements
This work was supported by the National Defense Foundation of
China (Grant No. 16ZG6101), and the Chengdu Science and Tech-
nology Project (Grant No. 2018-YF05-00760-SN).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.jclepro.2020.121491.
References
Alsabbagh, A.H., Abuqudaira, T.M., 2017. Phytoremediation of Jordanian uranium-
rich soil using sunflower. Water Air Soil Pollut. 228 (6), 219.
Berni, R., Luyckx, M., Xu, X., Legay, S., Sergeant, K., Hausman, J.F., Lutts, S., Cai, G.,
Guerriero, G., 2019. Reactive Oxygen Species and heavy metal stress in plants:
impact on the cell wall and secondary metabolism. Environ. Exp. Bot. 161,
98e106.
Bhattacharya, T., Chakraborty, S., Banerjee, D.K., 2010. Heavy metal uptake and its
effect on macronutrients, chlorophyll, protein, and peroxidase activity of Pas-
palum distichum grown on sludge-dosed soils. Environ. Monit. Assess. 169
(1e4), 15e26.
Bian, X.G., Cui, J., Tang, B.P., Yang, L., 2018. Chelant-induced phytoextraction of heavy
metals from contaminated soils: a review. Pol. J. Environ. Stud. 27, 2417e2424.
Chen, L., Zhu, Y., Luo, H., Yang, J., 2020a. Characteristic of adsorption, desorption,
and co-transport of vanadium on humic acid colloid. Ecotoxicol. Environ. Saf.
190, 110087.
Chen, L., Long, C., Wang, D., Yang, J., 2020b. Phytoremediation of cadmium (Cd) and
uranium (U) contaminated soils by Brassica juncea L. enhanced with exogenous
application of plant growth regulators. Chemosphere 242, 125112.
China National Environmental Monitoring Centre (CNEMC), 1990. Background
Values of Elements in Soils of China. China Environmental Science Press,
pp. 87e90.
Dabral, S., Yashaswee, Varma, A., Choudhary, D.K., Bahuguna, R.N., Nath, M., 2019.
Biopriming with Piriformospora indica ameliorates cadmium stress in rice by
lowering oxidative stress and cell death in root cells. Ecotoxicol. Environ. Saf.
186, 109741.
De Conti, L., Ceretta, C.A., Melo, G.W., Tiecher, T.L., Silva, L.O.S., Garlet, L.P.,
Mimmo, T., Cesco, S., Brunetto, G., 2019. Intercropping of young grapevines with
native grasses for phytoremediation of Cu-contaminated soils. Chemosphere
216, 147e156.
Elrashidi, M.A., Seybold, C.A., Wysocki, D.A., 2015. Effects of annual precipitation on
heavy metals in runoff from soils in the US great plains. Water Air Soil Pollut.
226 (12), 417.
Ferraz, P., Fidalgo, F., Almeida, A., Teixeira, J., 2012. Phytostabilization of nickel by
the zinc and cadmium hyperaccumulator Solanum nigrum L. Are metal loth-
ioneins involved? Plant Physiol. Biochem. 57, 254e260.
Gavrilescu, M., Pavel, L.V., Cretescu, I., 2009. Characterization and remediation of
soils contaminated with uranium. J. Hazard Mater. 163 (2e3), 475e510.
Guedes, P., Lopes, V., Couto, N., Mateus, E.P., Pereira, C.S., Ribeiro, A.B., 2019. Elec-
trokinetic remediation of contaminants of emergent concern in clay soil: effect
of operating parameters. Environ. Pollut. 253, 625e635.
Gurajala, H.K., Cao, X., Tang, L., Ramesh, T.M., Lu, M., Yang, X., 2019. Comparative
assessment of Indian mustard (Brassica juncea L.) genotypes for phytor-
emediation of Cd and Pb contaminated soils. Environ. Pollut. 254, 113085.
Han, R., Dai, H., Zhan, J., Wei, S., 2019. Clean extracts from accumulator efficiently
improved Solanum nigrum L. accumulating Cd and Pb in soil. J. Clean. Prod. 239,
118055.
Hasegawa, H., Mamun, M.A., Tsukagoshi, Y., Ishii, K., Sawai, H., Begum, Z.A.,
Asami, M.S., Maki, T., Rahman, I.M., 2019. Chelator-assisted washing for the
extraction of lead, copper, and zinc from contaminated soils: a remediation
approach. Appl. Geochem. 109, 104397.
Hawrylak-Nowak, B., Dresler, S., Matraszek, R., 2015. Exogenous malic and acetic
acids reduce cadmium phytotoxicity and enhance cadmium accumulation in
roots of sunflower plants. Plant Physiol. Biochem. 94, 225e234.
Hasan, M.M., Uddin, M.N., Ara-Sharmeen, I., Alharby, H., Alzahrani, Y., Hakeem, K.R.,
Zhang, L., 2019. Assisting phytoremediation of heavy metals using chemical
amendments. Plants 8 (9), 295.
He, J., Strezov, V., Kumar, R., Weldekidan, H., Jahan, S., Dastjerdi, B.H., Zhou, X.T.,
Kan, T., 2019. Pyrolysis of heavy metal contaminated Avicennia marina biomass
from phytoremediation: characterization of biomass and pyrolysis products.
J. Clean. Prod. 234, 1235e1245.
He, S., Yang, X., He, Z., Baligar, V.C., 2017. Morphological and physiological responses
of plants to cadmium toxicity: a review. Pedosphere 27 (3), 421e438.
Hu, N., Lang, T., Ding, D., Hu, J., Li, C., Zhang, H., Li, G., 2019. Enhancement of
repeated applications of chelates on phytoremediation of uranium contami-
nated soil by Macleaya cordata. J. Environ. Radioact. 199, 58e65.
Jiang, M., Jiang, J., Li, S., Li, M., Tan, Y., Song, S., Shu, Q., Huang, J., 2020. Glutamate
alleviates cadmium toxicity in rice via suppressing cadmium uptake and
translocation. J. Hazard Mater. 384, 121319.
 L. Chen et al. / Journal of Cleaner Production 263 (2020) 121491
Khan, A., Butt, T., Mirza, C.R., Yousaf, S., Nawaz, I., Iqbal, M., 2019. Combined
application of selected heavy metals and EDTA reduced the growth of Petunia
hybrida L. Sci. Rep. 9, 4138.
Lestan, D., Luo, C., Li, X., 2008. The use of chelating agents in the remediation of
metal-contaminated soils: a review. Environ. Pollut. 153 (1), 3e13.
Liang, Y., Wang, X., Guo, Z., Xiao, X., Peng, C., Yang, J., Zhou, C., Zeng, P., 2019.
Chelator-assisted phytoextraction of arsenic, cadmium and lead by Pteris vittata
L. and soil microbial community structure response. Int. J. Phytoremediation 20
(10), 1032e1040.
Lian, J., Zhao, L., Wu, J., Xiong, H., Bao, Y., Zeb, A., Tang, J., Liu, W., 2020. Foliar spray
of TiO2 nanoparticles prevails over root application in reducing Cd accumula-
tion and mitigating Cd-induced phytotoxicity in maize (Zea mays L.). Chemo-
sphere 239, 124794.
Lichtenthaler, H.K., Buschmann, C., 2001. Chlorophylls and carotenoids: measure-
ment and characterization by UV-VIS spectroscopy. Curr. Protoc. Food Anal.
Chem. F4, 3.
Li, C., Wang, M., Luo, X., Liang, L., Han, X., Lin, X., 2019. Accumulation and effects of
uranium on aquatic macrophyte nymphaea tetragona georgi: potential appli-
cation to phytoremediation and environmental monitoring. J. Environ. Radioact.
198, 43e49.
Li, Z., Zhang, R., Zhang, H., 2018. Effects of plant growth regulators (DA-6 and 6-BA)
and EDDS chelator on phytoextraction and detoxification of cadmium by
Amaranthus hybridus Linn. Int. J. Phytoremediation 20 (11), 1121e1128.
Malaviya, P., Singh, A., 2012. Phytoremediation strategies for remediation of
uranium-contaminated environments: a review. Crit. Rev. Environ. Sci. Technol.
42 (24), 2575e2647.
Mai, X., Luo, D., Wei, L., Liu, Y., Huang, X., Wu, Q., Yao, G., Liu, G., Liu, L., 2019.
Evaluation method for the measuring comprehensive suitability of chelating
agents: a study of the temporal dynamics of heavy metal activation. Int. J.
Phytoremediation 21 (14), 1415e1422.
Ma, P.J., Wang, Z., Yi, F.C., Niu, S.J., Zhou, H.T., 2017. Spatial distribution and pollution
assessment of uranium in soil around uranium tailings. Atomic Energy Sci.
Technol. 51 (5), 956e960.
Mohammadi, H., Hatami, M., Feghezadeh, K., Ghorbanpour, M., 2018. Mitigating
effect of nano-zerovalent iron, iron sulfate and EDTA against oxidative stress
induced by chromium in Helianthus annuus L. Acta Physiol. Plant. 40 (4), 69.
Moslehi, A., Feizian, M., Higueras, P., Eisvand, H.R., 2019. Assessment of EDDS and
vermicompost for the phytoextraction of Cd and Pb by sunflower (Helianthus
annuus L.). Int. J. Phytoremediation 21 (3), 191e199.
Mouser, P.J., N’Guessan, A.L., Elifantz, H., Holmes, D.E., Williams, K.H., Wilkins, M.J.,
Long, P.E., Lovley, D.R., 2009. Influence of heterogeneous ammonium availability
on bacterial community structure and the expression of nitrogen fixation and
ammonium transporter genes during in situ bioremediation of uranium-
contaminated groundwater. Environ. Sci. Technol. 43 (12), 4386e4392.
Pandey, S.K., Bhattacharya, T., 2018. Effect of two biodegradable chelates on metals
9
uptake, translocation and biochemical changes of Lantana Camara growing in
fly ash amended soil. Int. J. Phytoremediation 20 (3), 214e224.
Pourfadakari, S., Ahmadi, M., Jaafarzadeh, N., Takdastan, A., Neisi, A.A., Ghafari, S.,
Jorfi, S., 2019. Remediation of PAHs contaminated soil using a sequence of soil
washing with biosurfactant produced by Pseudomonas aeruginosa strain PF2
and electrokinetic oxidation of desorbed solution, effect of electrode modifi-
cation with Fe3O4 nanoparticles. J. Hazard Mater. 379, 120839.
SEQ (Soil environmental quality), 2018. Soil Environmental Quality Risk Control
Standard for Soil Contamination of Agricultural Land. GB15618-2018. National
standard of China.
Selvakumar, R., Ramadoss, G., Menon, Mridula P., Rajendran, K., Thavamani, P.,
Naidu, Ravi, Megharaj, M., 2018. Challenges and complexities in remediation of
uranium contaminated soils: a review. J. Environ. Radioact. 192, 592e603.
Shahid, M., Shamshad, S., Rafiq, M., Khalid, S., Bibi, I., Niazi, N.K., Dumat, C.,
Rashid, M.I., 2017. Chromium speciation, bioavailability, uptake, toxicity and
detoxification in soil-plant system: a review. Chemosphere 178, 513e533.
Sidhu, G., Bali, A.S., Singh, H.P., Batish, D.R., Kohli, R.K., 2018. Ethylenediamine
disuccinic acid enhanced phytoextraction of nickel from contaminated soils
using Coronopus didymus (L.) Sm. Chemosphere 205, 234e243.
Wang, J., Zhu, Q., Shan, Y., Wang, Y., Song, X., Lei, X., 2018. A comparative study on
the efficiency of biodegradable EDDS and micro-electric field on the promotion
of the phytoextraction by Commelina communis L. in Cu-contaminated soils.
Geoderma 314, 1e7.
Wang, Q., Ma, L., Zhou, Q., Chen, B., Zhang, X., Wu, Y., Pan, F., Huang, L.K., Yang, X.E.,
Feng, Y., 2019a. Inoculation of plant growth promoting bacteria from hyper-
accumulator facilitated non-host root development and provided promising
agents for elevated phytoremediation efficiency. Chemosphere 234, 769e776.
Wang, K., Liu, Y., Song, Z., Wang, D., Qiu, W., 2019b. Chelator complexes enhanced
Amaranthus hypochondriacus L. phytoremediation efficiency in Cd-
contaminated soils. Chemosphere 237, 124480.
Ye, Z., Ge, M., Hu, H.X., Li, G., 2015. Effects of EDTA and EDDS on heavy metal
activation and accumulation of metals by soybean in alkaline soil. Soil Sediment
Contam. 24 (4), 353e367.
Yin, Y., Wang, Y., Liu, Y., Zeng, G., Hu, X., Hu, X., Zhou, L., Guo, Y., Li, J., 2015. Cadmium
accumulation and apoplastic and symplastic transport in Boehmeria nivea (L.)
Gaudich on cadmium-contaminated soil with the addition of EDTA or NTA. RSC
Adv. 5 (59), 47584e47591.
Zehra, A., Sahito, Z.A., Tong, W., Tang, L., Hamid, Y., Wang, Q., Cao, X., Khan, M.B.,
Hussain, B., Jatoi, S., He, Z., Yang, X.E., 2020. Identification of high cadmium-
accumulating oilseed sunflower (Helianthus annuus) cultivars for phytor-
emediation of an Oxisol and an Inceptisol. Ecotoxicol. Environ. Saf. 187, 109857.
Zhang, L., Liu, Z.R., 2018. Pollution characteristics and risk assessment of uranium
and heavy metals of agricultural soil around the uranium tailing reservoir in
Southern China. J. Radioanal. Nucl. Chem. 318 (2), 923e933.