Environ Sci Pollut Res

DOI 10.1007/s11356-015-4396-8

RESEARCH ARTICLE

Citric acid enhances the phytoextraction of chromium, plant

growth, and photosynthesis by alleviating the oxidative damages
in Brassica napus L.
Sehar Afshan 1 & Shafaqat Ali 1 & Saima Aslam Bharwana 1 & Muhammad Rizwan 1 &
Mujahid Farid 1 & Farhat Abbas 1 & Muhammad Ibrahim 1 &
Muhammad Aamer Mehmood 2 & Ghulam Hasan Abbasi 3

Received: 10 December 2014 / Accepted: 17 March 2015

# Springer-Verlag Berlin Heidelberg 2015

Abstract Chromium (Cr) toxicity is widespread in crops
grown on Cr-contaminated soils and has become a serious
environmental issue which requires affordable strategies for
the remediation of such soils. This study was performed to
assess the performance of citric acid (CA) through growing
Brassica napus in the phytoextraction of Cr from contaminat-
ed soil. Different Cr (0, 100, and 500 μM) and citric acid (0,
2.5, and 5.0 mM) treatments were applied alone and in com-
binations to 4-week-old seedlings of B. napus plants in soil
under wire house condition. Plants were harvested after
12 weeks of sowing, and the data was recorded regarding
growth characteristics, biomass, photosynthetic pigments,
malondialdehyde (MDA), electrolytic leakage (EL), antioxi-
dant enzymes, and Cr uptake and accumulation. The results
showed that the plant growth, biomass, chlorophyll contents,
and carotenoid as well as soluble protein concentrations sig-
nificantly decreased under Cr stress alone while these adverse
effects were alleviated by application of CA. Cr concentration
in roots, stem, and leaves of CA-supplied plant was signifi-
cantly reduced while total uptake of Cr increased in all plant
parts with CA application. Furthermore, in comparison with
Responsible editor: Elena Maestri
* Shafaqat Ali
shafaqataligill@yahoo.com
1
Department of Environmental Sciences and Engineering,
Government College University, Allama Iqbal Road,
Faisalabad 38000, Pakistan
2
Department of Bioinformatics and Biotechnology, Government
College University, Allama Iqbal Road, Faisalabad 38000, Pakistan
3
Department of Soil Science University College of Agriculture and
Environmental Sciences, The Islamia University of Bahawalpur,
Bahawalpur, Pakistan
Cr treatments alone, CA supply reduced the MDA and EL
values in both shoots and roots. Moreover, the activity of
superoxide dismutase (SOD), guaiacol peroxidase (POD), cat-
alase (CAT), and ascorbate peroxidase (APX) in shoots and
roots markedly increased by 100 μM Cr exposure, while de-
creased at 500 μM Cr stress. CA application enhanced the
activities of antioxidant enzymes compared to the same Cr
treatment alone. Thus, the data indicate that exogenous CA
application can increase Cr uptake and can minimize Cr stress
in plants and may be beneficial in accelerating the
phytoextraction of Cr through hyper-accumulating plants such
as B. napus.
Keywords Biomass . Brassica napus . Chromium . Citric
acid . Electrolyte leakage . Guaiacol peroxidase .
Phytoextractions
Introduction
The increasing concentration of heavy metals in agricultural
soil is an important environmental issue which can limit future
land-use options as the heavy metals are toxic and nonbiode-
gradable (Sharma and Pandey 2014; Habiba et al. 2015).
Among various heavy metals, chromium (Cr) is the seventh
most abundant element of the earth’s crust (Katz and Salem
1994). Anthropogenic activities such as tanning, mining, and
electroplating are the main sources of soil Cr contamination.
Cr is a nonessential element for plants and induces toxicity in
many plant species (Zeng et al. 2011; Singh et al. 2013; Gill
et al. 2015). Higher concentration of Cr in plants caused re-
duction in root growth and biomass, chlorosis, photosynthetic
impairment, and finally plant death (Das et al. 2014).
Recently, Singh et al. (2013) extensively reviewed the Cr toxic

effects in different plant species at morphological, physiolog-
ical, biochemical, and molecular levels. Thus, remediation of
Cr-contaminated soil has become an important environmental
issue worldwide (Ali et al. 2012).
Phytoremediation is an emerging technology which uses
the green plants to remediate or sequester heavy metals from
contaminated soils (Raziuddin et al. 2011). It is widely accept-
ed and is mainly considered for cleanup of polluted sites due
to its visual advantages, extensive applicability, and cost-
effectiveness (Su and Wong 2004; Boonyapookana et al.
2005). Phytoextraction is a promising treatment method,
which uses high biomass-producing plants to hyper-
accumulate metals from soils and water into plant harvestable
tissues. However, the degree of metal translocation from root
to aerial plant parts depends upon plant species, metals, and
environmental conditions (Farooq et al. 2013; Anwaar et al.
2015). Moreover, the efficiency of concerned metal uptake by
plants is associated with the bioavailability of metals from soil
for root uptake.
Although most of heavy metals have low bioavailability in
soils, there is a need to meet stringent cleanup targets. To
overcome the limitations of natural phytoextraction has led
to several studies on different chelates that increase the bio-
availability of heavy metals (Evangelou et al. 2007). For this
purpose, different synthetic and natural chelators are used to
enhance the bioavailability of metals in contaminated medi-
um. Among synthetic chelating agents, ethylenediaminetetra-
acetic acid (EDTA) and diethylene triamine pentaacetic acid
(DTPA) are commonly used as they are efficient in
complexing metals and increasing their concentration in the
upper plant parts (Kanwal et al. 2014). However, they are
nonbiodegradable and can cause ground water contamination
due to uncontrolled leaching in the soil (Meers et al. 2005;
Anwer et al. 2012; Bareen 2012). Organic acids could be an
interesting alternative to the persistent synthetic chelating
agents described above. Organic chelating agents are low-
molecular-weight organic acids such as citric acid (CA) and
can form complexes with heavy metals and have higher de-
gree of biodegradability and less leaching hazard as compared
to synthetic chelating agents (Meers et al. 2005; Bareen 2012).
So synthetic chelating agents should be replaced by natural
organic acids for the mobilization and availability of metals
from the contaminated medium. Recently, it has been reported
that citric acid (CA) significantly enhances metal solubility
and uptake by plants (Yeh and Pan 2012; Freitas et al.
2013). To date, many studies have reported the effects of or-
ganic chelating agents on the extraction of heavy metals from
the solution cultures (Gunawardana et al. 2011; Das et al.
2014; Ehsan et al. 2014); there are few reports on the role of
these chelating agents during phytoextraction of heavy metal
from contaminated soils (Chigbo and Batty 2013). Thus, we
need longer term (as compared to hydroponic cultures) and
more realistic soil-based studies to better understand the
Environ Sci Pollut Res
practical implications of CA-mediated phytoextraction and
tolerance of metals so that successful field experiments can
be conducted.
The cultivation of high-biomass-producing crops along
with solubilization through natural chelating agents may be
attractive for the remediation of contaminated soils. Recently,
however, biofuel plants are being used as phytoextractors of
heavy metals as the biomass produced can be converted to
biofuel, and it may pose an economic return as well (Melo
et al. 2012). Brassica napus L. was chosen as the plant can-
didate due to its inherent capability to hyper-accumulate
metals and high biomass production (Vamerali et al. 2010;
Szczygłowska et al. 2011; Bareen 2012). It has also been
reported that B. napus can be grown on contaminated soils
for remediation purposes, and the oil obtained from this plant
can be used as a source of biodiesel (Park et al. 2008) because
this plant contains 40–44 % oil in its seeds (Laaniste et al.
2004).
The present study was conducted in a wire house experi-
ment to investigate the effect of CA on (1) Cr bioaccumula-
tion; (2) growth and biomass and photosynthetic pigments;
and (3) electrolyte leakage and malondialdehyde (MDA) and
activities of the antioxidant enzymes: superoxide dismutase
(SOD), catalase (CAT), guaiacol peroxidase (POD), and
ascorbate peroxidase (APX) in B. napus grown in Cr-
contaminated soil.
Materials and methods
Soil treatments and B. napus plant
A clay loam (25 % sand, 12 % silt, 51 % clay) soil was used in
this study and was collected at 0–15 cm depth from botanical
garden of the Government College University Faisalabad,
Pakistan. Before pot experiment, the soil was air-dried and
passed through a 2-mm diameter sieve to remove stones and
crop residues. The soil was characterized chemically and
physically (Table 1). Mature seeds of B. napus L. genotype
(Faisal Canola (RBN-03060) developed from a cross KS-75 x
Rainbow) were taken from Oilseeds Research Institute,
AARI, Faisalabad, Pakistan (Mahmood et al. 2012). The
seeds were washed with distilled water thoroughly, and they
were sown in earthen pots each containing 5 kg soil (dry
weight) under wire house conditions. In each pot, ten seeds
were sown thinned to five individuals per pot after 15 days of
germination, and the pulled-up plants were crushed carefully
into the same pot. Each soil pot was fertilized with a 500-mL
solution containing 2.19 g L−1 N (as (NH2)2CO), 0.5 g L−1 P
(as (NH4)2HPO4), and 2.14 g L−1 K (as K2SO4). Half of the
fertilizer solution was applied after 15 days of germination and
remaining half after 30 days of germination. All plastic and
Environ Sci Pollut Res
Table 1 Properties of soil used for the pot experiment
Physicochemical properties
Texture Clay loam
Sand (%)
Silt (%)
Clay (%)
pH (1/2.5 soil to water ratio)
ECe (dS m−1)
SAR (mmol−1)1/2
Organic matter (%)
Available P (mg kg−1)
HCO3 (mmol L−1)
Cl− (mmol L−1)
SO42− (mmol L−1)
Ca2+ +Mg2+ (mmol L−1)
Na2+ (mmol L−1)
K+ (mmol L−1)
Available Cu2+ (mg kg−1)
Available Zn2+ (mg kg−1)
glasswares were rinsed with 10 % HNO3 and then washed
with distilled water till neutral pH.
Treatments
After 4 weeks of germination, uniform plants were treated
with increasing concentrations of Cr (0, 100, and 500 μM)
along with increasing concentration of CA (0, 2.5, and
5.0 mM). In total, there were nine, T1: Cr (0 μM) + CA
(0 mM); T 2 : Cr (100 μM); T 3 : Cr (500 μM); T 4 : CA
(2.5 mM); T 5 : CA (5 mM); T 6 : Cr (100 μM) + CA
(2.5 mM); T 7 : Cr (100 μM) + CA (5 mM); T 8 : Cr
(500 μM)+CA (5 mM), T9: Cr (500 μM)+CA (5 mM), treat-
ments. Each treatment was triplicated, and the experimental
pots were rotated randomly in the wire house and weeds were
removed when present. Each treatment was applied in the soil
in solution form prepared in distilled water to the pots after
4 days interval for next 8 weeks.
Plant sampling and analysis
Plants were harvested after 8 weeks of treatments. Roots were
carefully separated from the soil. All the samples were thor-
oughly washed with deionized water, and excess water was
removed with the help of tissue paper. All samples were sep-
arated into shoots, roots, and leaves. Then, growth variables
including root and shoot length, no. of leaves per plant, area of
leaves, and root and shoot fresh weights were measured. After
this, half of each of the sample was oven-dried at 70 °C for
about 3 days until constant weight, and the other half was used
for the determination of antioxidant enzymes. After weighing,
dried plant samples were grounded and were used for further
analysis. The plants were safely disposed off after the comple-
tion of the experiment.
27
20 Pigment content assay
53
6.7 After 8 weeks of treatments, the uppermost fully extended
2.9 fresh leaves of B. napus plants were used for the determination
6.5 of chlorophyll (a and b) and carotenoid contents. The pigment
0.31 contents were extracted in the dark with 85 % (v/v, Sigma)
2.17 aqueous acetone solution at 4 °C by continuous shaking until
3.55 color had completely disappeared from the leaves. Then, the
2.34 assay mixture was centrifuged at 4000 rpm for 10 min at 4 °C,
6.67 and supernatant was taken. Light absorbance at 663, 644, and
3.5 452.5 nm was determined by spectrophotometer (Halo DB-
3.7 20/ DB-20S, Dynamica Company, London, UK) (Metzner
0.06 et al. 1965). The concentrations of chlorophylls and caroten-
0.35 oids were calculated by using the adjusted extinction coeffi-
0.85 cients and equations (Lichtenthaler 1987).
Chromium content analysis
For Cr content determination, plant sample (0.5 g) was taken
in a 100-mL flask, and then 15 mL of concentrated HNO3
(70 %, Sigma) was added. Then, the flasks were put on a
hot plate which temperature was gradually increased up to
275 °C, and dense yellow fumes appeared from the flask.
When the quantity of dense yellow fumes became low, then
hydrogen peroxide was added until fumes disappeared. When
samples became colorless, the extracts were diluted to 25 mL
with distilled water and were analyzed for Cr contents by
means of flame atomic absorption spectrometry (AAS) made
by novA A400, Analytik Jena, Germany, followed by Ehsan
et al. (2013).
Determination of electrolyte leakage and MDA
Electrolyte leakage (EL) was measured according to the meth-
od of Dionisio-Sese and Tobita (1998). After 8 weeks of treat-
ments, the uppermost fully expanded leaves were cut into
small parts of about 5 mm length and positioned in test tubes
containing 8 mL of distilled water. Then, the tubes were incu-
bated in water bath at 32 °C for 2 h, and then electrical con-
ductivity of the initial medium (EC1) was measured. After
this, all samples were again placed in autoclave at 121 °C
for 20 min so that all electrolytes could release, then these
samples were cooled to 25 °C, and then again electrical con-
ductivity (EC2) was measured by using pH/conductivity me-
ter (model 720, INCO-LAB Company, Kuwait) and computed
by using the following formula.
EL ¼ ðEC1=EC2Þ  100:

The level of lipid peroxidation in leaf tissue was
measured in terms of malondialdehyde (MDA; a product
of lipid peroxidation) content determined by the thiobar-
bituric acid (TBA) reaction using the method of Heath
and Packer (1968), with minor modifications as de-
scribed by Dhindsa and others (1981) and Zhang and
Kirham (1994). A 0.25-g leaf sample was homogenized
in 5 mL of 0.1 % trichloroacetic acid (TCA). The ho-
mogenate was centrifuged at 10,000×g for 5 min. To
1 mL aliquot of the supernatant, 4 mL of 20 % TCA
containing 0.5 % TBA was added. The mixture was
heated at 95 °C for 30 min and then quickly cooled
in an ice bath. After centrifugation at 10,000×g for
10 min, the absorbance of the supernatant at 532 nm
was measured and the value for the nonspecific absorp-
tion at 600 nm was subtracted. The MDA content was
calculated by using an extinction coefficient of
155 mM−1 cm−1.
Evaluation of antioxidant enzymes and protein content
Antioxidant enzymes including superoxide dismutases
(SOD), guaiacol peroxidase (POD), catalase (CAT), ascorbate
peroxidase (APX), and soluble protein in roots and leaves
were evaluated spectrophotometrically. After 8 weeks of treat-
ment, fully expanded leaves of plants and roots samples were
taken for enzymatic analysis. The leaves and roots samples
(1.0 g) were quickly frozen in liquid nitrogen and grounded
with precooled pestle and mortar. This pattern was stan-
dardized in 0.05 M phosphate buffer (pH 7.8) and fil-
tered through four layers of muslin cloth and centri-
fuged at 12,000×g, at 4 °C for 10 min. The final su-
pernatant was for the estimation of SOD and POD ac-
tivities according to Zhang (1992). The soluble protein
content in the supernatant was measured according to
(Bradford 1976) using coomassie brilliant blue G-250
as dye and albumin as a standard.
Catalase (CAT; EC 1.11.1.6) activity was determined ac-
cording to (Aebi 1984). The assay mixture (3.0 mL) consisted
of 100 μL enzyme extract, 100 μL H2O2 (300 mM), and
2.8 mL 50 mM phosphate buffer with 2 mM CA (pH 7.0).
The CAT activity was assayed by measuring the decrease in
absorbance at 240 nm as consequence of H2O2 disappearance
(ε=39.4 mM−1 cm−1).
Ascorbate peroxidase (APX; EC 1.11.1.11) activity was
assayed according to the method of Nakano and Asada
1981. The reaction mixture consisted of 100 μL enzyme ex-
tract, 100 μL ascorbate (7.5 mM), 100 μL H2O2 (300 mM),
and 2.7 mL 25 mM potassium phosphate buffer with 2 mM
CA (pH 7.0). The oxidation activity of ascorbate was deter-
mined by measuring the change in wavelength at 290 nm (ε=
2.8 mM−1 cm−1).
Environ Sci Pollut Res
Statistical analysis
All values described in this study are mean of three replicates.
Analysis of variance (ANOVA) was done by using a statistical
package, SPSS version 16.0 (SPSS, Chicago, IL), followed by
Tukey’s post hoc test between the means of treatments to
determine the significant difference.
Results
Effect of chromium and citric acid on growth and biomass
Plant growth, estimated as plant height, root length, number of
leaves per plant, and leaf area, significantly decreased at both
Cr levels (100 and 500 μM) alone as compared to control
(Fig. 1). The maximum decrease in growth parameters was
observed with highest Cr (500 μM) treatment alone. The ad-
verse effects of Cr stress on the B. napus plants were alleviated
when CA was applied along with Cr stress. CA application
markedly increased the plant height, root length, number of
leaves per plant, and leaf area under Cr stress. The highest
increase in growth parameters was observed with highest
CA (5 mM) treatment without Cr stress. Fresh and dry weights
of root, stem, and leaves significantly decreased at both levels
of Cr stress alone as compared to those of control (Table 2).
When compared with control, this decrease in root, stem, and
leaf dry biomass was 43, 44, and 39 % at 100 μM Cr treatment
and was 72, 73, and 70 % at 500 μM Cr treatment, respec-
tively. Application of CA, at both levels, significantly in-
creased the fresh and dry weights of plants as compared to
the same treatment without CA application. Degree of in-
crease in biomass with CA application was dose dependent
Cr concentration and uptake
Chromium contents in roots stem and leaves are presented in
Table 3. Cr concentrations in all three plant parts of B. napus
seedlings were dramatically increased at both levels of Cr
(100 and 500 μM) relative to control plant (Table 3). Cr con-
centration was higher in roots irrespective of Cr levels follow-
ed by stem and leaves. In the presence of 2.5 mM CA, how-
ever, Cr concentrations in roots were significantly decreased
by 13.2 and 14.3 % compared with the 100 and 500 μM Cr
treatment alone, respectively, while in the presence of 5.0 mM
CA, this decrease Cr in roots was 20.8 and 23.6 % compared
with the 100 and 500 μM Cr treatment alone, respectively.
Similarly, the application of 2.5 mM CA decreased 13.1 and
29.9 % Cr compared with 100 and 500 μM Cr treatment
alone, respectively, while 5.0 mM CA under Cr stress, this
decrease in Cr concentration was 23.9 and 42.7 % than those
of 100 and 500 μM Cr treatment alone, respectively.
Moreover, 2.5 mM CA decreased 8.3 and 23.4 % Cr in leaves
Environ Sci Pollut Res

70
Fig. 1 Effect of various Cr (0, A) CA (0 mM) 25
B)
100, and 500 μM) and citric acid a CA (2.5 mM) a
60 b
(0, 2.5, and 5 mM) treatments on c d CA (5 mM)

Root length (cm plant-1)

b

Plant height (cm plant-1)

20
plant height (a), root length (b), 50 c
e d
number of leaves per plant (c), f
and leaf (d) of Brassica napus 40 g 15 e
seedlings grown in soil. Values h f
are expressed as means of three 30 i g
10 h
replicates with standard
20 i
deviations. Different letters
5
indicate that values are significant 10
different at P<0.05
0 0

7
B)
140 D)
a
120 a a
6 b a
c
No. of leaves plant-1 e d 100

Leaf area (cm-1)

5 d
f
4 80 e
g f
3 h 60 g
i h
2 40 i

1 20

0 0
0 100 500 0 100 500
[Cr] in soil in µM [Cr] in soil in µM

compared with 100 and 500 μM Cr treatment alone, respec-
tively, and in the presence of 5.0 mM CA, this decrease in Cr
concentration was up to 19.5 and 35 % as compared to 100
and 500 μM Cr treatment alone, respectively.
Total Cr uptake significantly increased in roots, stem, and
leaves of B. napus plants at both levels of Cr (100 and
500 μM) relative to control plant (Table 3). Total Cr uptake
was highest in leaves followed by stem and roots. CA appli-
cation, at both levels, further enhanced the total Cr uptake in
all three plant parts as compared to respective Cr treatments
alone. These results depicted that CA application decreased Cr
concentrations in different plant parts while increased total Cr
uptake as compared to Cr treatments alone.
Photosynthetic pigments and protein contents
Chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophyll,
and carotenoid and soluble protein contents are presented in
Fig. 2. At both levels of Cr (100 and 500 μM), Chl a, Chl b,
total chlorophyll, and carotenoid concentrations in leaves of
B. napus significantly decreased as compared to those of
plants grown without Cr stress. CA application with and

Table 2 Effect of different concentration of chromium (Cr) (0, 100, and 500 μM) and citric acid (CA) (0, 2.5, and 5.0 mM) on biomass of B. napus
plants

Treatments Root weight (g plant−1) Stem weight (g plant−1) Leaf weight (g per plant−1)

Fresh Dry Fresh Dry Fresh Dry

Control 3.02±0.27a 0.72±0.04a 38.93±1.00a 2.52±0.21a 21.43±1.07a 12.95±0.56a

CA (2.5 mM) 4.08±0.21a 0.75±0.01a 40.64±1.29a 2.59±0.19a 22.58±0.80a 13.51±0.09b
CA (5.0 mM) 4.23±0.2a 0.78±0.03a 42.08±1.78a 2.67±0.24a 23.25±1.02a 14.86±0.58c
Cr (100 μM) 2.26±0.17b 0.41±0.02b 22.29±0.51b 1.41±0.10b 12.31±0.56b 7.87±0.38d
Cr100+2.5 CA 2.63±0.16c 0.48±0.03c 25.93±1.32c 1.64±0.10c 14.33±1.05c 9.16±0.58e
Cr100+5.0 CA 3.09±0.22d 0.56±0.04d 30.46±1.70d 1.93±0.11d 16.83±1.04d 10.76±0.36f
Cr (500 μM) 1.10±0.13e 0.20±0.03e 10.85±1.13e 0.67±0.03e 6.00±0.10 e 3.83±0.47g
Cr500+2.5 CA 1.50±0.20f 0.27±0.02f 14.82±1.54f 0.94±0.07f 8.19±0.71f 5.23±0.25h
Cr500+5.0 CA 1.90±0.16g 0.35±0.04g 18.70±1.68g 1.18±0.06g 10.33±0.58g 6.60±0.34i

Values are expressed as means of three replicates with standard deviations. Different letters indicate that values are significant different at P<0.05
 Environ Sci Pollut Res

Table 3 Effect of different concentration of chromium (Cr) (0, 100 and 500 μM) and citric acid (CA) (0, 2.5, and 5.0 mM) on Cr concentrations and
accumulation/uptake by B. napus plants

Treatments Cr concentrations (mg kg−1) Cr accumulation (mg plant−1)

Roots Stem Leaves Roots Stem Leaves

Control 0.00±0.00a 0.00±0.0a 0.0±0.0 a 0.0±0.0a 0.00±0.00a 0.00±0.00a

CA (2.5 mM) 0.47±0.36a 0.5±0.0a 0.0±0.0a 0.0±0.0a 0.001±0.00a 0.00±0.00a
CA (5.0 mM) 0.08±0.0a 0.7±0.0a 0.1±0.0a 0.0±0.0a 0.002±0.00a 0.00±0.00a
Cr (100 μM) 422.8±10.2b 175.6±13.1b 91.2±5.2b 0.173±0.002b 0.248±0.02b 0.718±0.012b
Cr100+2.5 CA 367.0±11.1c 152.6±8.0c 83.6±1.3c 0.176±0.003b 0.250±0.01b 0.766±0.018c
Cr100+5.0 CA 334.7±9.8d 133.6±4.8d 73.4±3.2d 0.187±0.004c 0.258±0.00b 0.790±0.006c
Cr (500 μM) 685.4±21.8e 378.4±24.7e 201.7±4.2e 0.137±0.004d 0.254±0.01b 0.772±0.020c
Cr500+2.5 CA 587.2±29.0f 265.3±13.1f 154.5±4.54f 0.159±0.006e 0.25±0.006b 0.808±0.010d
Cr500+5.0 CA 523.6±14.9g 216.7±11.8g 131.2±4.0g 0.183±0.009c 0.256±0.01b 0.866±0.024e

Values presented are means of three replicates with standard deviations. Different letters indicate that values are significant different at P<0.05

without Cr stress positively affected the photosynthetic pig- increased these pigment concentrations under Cr stress as
ment concentrations when compared with their corresponding compared to plants treated with Cr alone. The evaluation of
plants under Cr stress alone. CA application significantly protein contents also indicated a significant effect of CA

Fig. 2 Effect of various Cr (0, 30 A) CA (0 mM) 10 B)

100, and 500 μM) and citric acid a

[Chl b] in mg g-1 FW
[Chl a] in mg g-1 FW

a CA (2.5 mM)
(0, 2.5, and 5 mM) treatments on 25 a a 8 b
b CA (5 mM) c
chlorophyll a (a), chlorophyll b c
20
(b), total chlorophyll (c), d 6
carotenoids (d), and soluble e d
15 f e
proteins (e, f) of Brassica napus g 4 f
seedlings grown in soil. Values 10 g
h
are expressed as means of three i
5 2
replicates with standard
deviations. Different letters 0 0
indicate that values are significant
different at P<0.05 40 C) 10 D)
35 a a a
c b 8 a
[Chla + Chl b] in

30 b
[Carotenoids]
in mg g-1 FW

d c
mg g-1 FW

25 e 6 d
f e
20 g f
h 4 g
15 i
10
2
5
0 0

600 600
E) a F)
Protein content in root
Protein content in leaf

500 b 500
c
(mg g-1 FW)

400 400
(mg g-1 FW)

d
e a
300 f 300 c b
g d
200
h
200 f e g
i
i h
100 100

0 0
0 100 500 0 100 500
[Cr] in soil in µM [Cr] in soil in µM
Environ Sci Pollut Res

treatments on Cr stressed and unstressed plants (Fig. 2e, f).
Soluble protein concentrations significantly decreased in both
leaves and roots of plants under Cr stress alone relative to
control treatment. Both levels of CA under Cr stress signifi-
cantly increased the soluble protein contents than those of Cr
treatments alone. The protein contents also significantly in-
creased in CA-treated plants without Cr stress relative to con-
trol plants.
Effect of citric acid and Cr on electrolyte leakage
and MDA contents
Both levels of Cr significantly affected the activities of anti-
oxidant enzymes in roots and leaves. Addition of 100 μM Cr
treatment alone significantly enhanced the activities of antiox-
idant enzymes as compared to control and 500 μM Cr alone.
Addition of CA further enhanced the activities of antioxidant
enzymes and exhibited a synergetic effect. The combination
of CA with Cr treatments improved the activity of antioxidant
enzymes. The increase of the antioxidant enzyme activities in
Cr+CA treatments was significantly higher than that of the
respective Cr treatments alone.

Malondialdehyde and EL concentration were measured to Discussion

evaluate the oxidative damages in leaves and roots of
B. napus (Fig. 3). MDA and EL concentrations were not sta-
tistical different between the CA treatments alone and the
control. MDA and EL significantly increased with increasing
Cr concentrations in the soil relative to control plants. The
exogenous addition of both CA along with both Cr levels
significantly reduced MDA and EL contents in shoots and
roots as compared to Cr treatment alone suggesting that CA
application reduced oxidative stress induced by Cr in leaves
and roots.
Antioxidant enzyme activities in shoots and roots
Four key (SOD, POD, CAT, and APX) antioxidant enzyme
activities were evaluated to find out the effect of CA applica-
tion on the enzyme activities in Cr-stressed B. napus (Fig. 4).
Our results showed that plant growth attributes and biomass
were significantly reduced with increasing Cr supply in the
soil (Fig. 1 and Table 1). Toxic effects of Cr on plant growth
and biomass have already been observed in many plants
(Rodriguez et al. 2012; Ali et al. 2013a, b; Das et al. 2014).
This decrease in plant growth might be due to ultrastructural
changes in plant organs (Gill et al. 2015). In the present study,
however, decrease in plant growth may be due to reduction in
photosynthetic pigments (Fig. 2). CA application increased
plant growth and biomass under Cr stress as compared to
plants treated with Cr alone (Fig. 1 and Table 2). In the liter-
ature, conflicting results have been reported related to CA
effect on plant growth and biomass. Freitas et al. (2013) re-
ported that biomass of Sorghum bicolor decreased after the
application of 40 mmol kg−1 CA in lead (Pb)-contaminated

Fig. 3 Effect of various Cr (0, 12 12

100, and 500 μM) and citric acid
A) CA (0 mM) B)
[MDA] in leaves µM g-1 FW

g
[MDA] in roots µM g-1 FW

10 CA (2.5 mM) 10
(0, 2.5, and 5 mM) treatments on f g
CA (5 mM) f
malondialdehyde (MDA) and e
electrolyte leakage (EL) in 8 8 e
d
leaves and roots of Brassica c d
napus seedlings grown in soil (a– 6 6 c
b
d). Values are expressed as means b
a
of three replicates with standard 4 a a 4 a
deviations. Different letters a a
indicate that values are significant 2 2
different at P<0.05
0 0

140 140
C) D) g
g
Root electrolyte leakage (%)
Leaf electrolyte leakage (%)

120 120 d
d e
100 f 100 e
e c
80 c 80 b
b
60 a a a 60
a
a a
40 40

20 20

0 0
0 100 500 0 100 500
[Cr] in soil in µM [Cr] in soil in µM
 Environ Sci Pollut Res

Fig. 4 Effect of different 240 240

concentrations of chromium (Cr)
A) CA (0 mM) B)
CA (2.5 mM) b

(Units g-1 FW)

200 200

SOD in roots
(0, 100, and 500 μM) and citric c

SOD in leaves
(Units g-1 FW)
CA (5 mM)
160 160 d
c b
acid (CA) (0, 2.5, and 5 mM) on e
superoxide dismutase (SOD) of d 120 f
120 e g
leaf (a) and root (b), guaiacol g f a a a
peroxidase (POD) of leaf (c) and 80 80
a a a
root (d), catalase (CAT) of leaf (e) 40
40
and root (f), and ascorbate
peroxidase (APX) of leaf (g) and 0 0
root (h) of B. napus plants grown
4000 1500
in soil. Values are expressed as C) b D)
POD in leaves

(Units g-1 FW)

(Units g-1 FW)
means of three replicates with

POD in roots
c b
standard deviations. Different 3000 c
letters indicate that values are d 1000
e d
significant different at P<0.05 f f e
2000 g g
a a a a a a
500
1000

0 0

5000 5000
E) b F) b
c
CAT in leaves
(Units g-1 FW)

(Units g-1 FW)

4000 c 4000 d

CAT in roots
d e
3000 e 3000 f
f
g g
2000 2000 a
a a a a a
1000 1000
0 0

700 b 700 b
G) c H)
600 600 c
(Units g-1 FW)

(Units g-1 FW)

APX in leaves

APX in roots

d d
500 e 500
400 f 400 e
g f
300 300
a a a g
200 200 a a a
100 100
0 0
0 100 500 0 100 500
[Cr] in soil in µM [Cr] in soil in µM

soil for 7 days before harvesting. Similarly, Araújo and
Nascimento (2010) also observed decrease in biomass of corn
plants under Pb stress after the application of 30 mmol kg−1 of
CA in soil for 9 days before harvesting. Lesage et al. (2005)
also reported that growth of sunflower severally decreased by
the use of CA in calcareous soil moderately contaminated with
different (Cu, Pb, Zn, and Cd) metals. They suggested that this
reduction in biomass might be due to the chelant-induced
metal mobility, chelate toxicity itself, and the chelant-metal
complexes. However, it has been reported by several re-
searchers that CA application increased plant growth and bio-
mass under metal stress in Juncus effusus (Najeeb et al. 2009)
and in Zea mays (Anwer et al. 2012). This increase in plant
growth and biomass might be due to enhanced nutrient uptake
by plants (Najeeb et al. 2011) or due to synthesis of
phytochelation (PCs) in plants (Muhammad et al. 2009). It
has been reported that sulfur (S) uptake decreased in plants
due to increase in Cr uptake by plants (Schiavon et al. 2008
and 2012), so the increase in biomass might also be due to
efficient S uptake and assimilation by plants in the presence of
CA. Overall, increase or decrease in plant growth and biomass
with CA application in different plants may be due to variation
in plant species and genotypes, time of CA applications, ex-
perimental conditions, and the duration of stress exposed.
Excess Cr decreased plant photosynthetic pigment concen-
trations of B. napus (Fig. 2). These results are in line with
previous findings that excess Cr decreased photosynthetic pig-
ment contents in many plant species (Singh et al. 2013; Gill
et al. 2015). Contrarily, pigment contents and carotenoids sig-
nificantly increased in Pisum sativum in response to Cr stress
Environ Sci Pollut Res
(Rodriguez et al. 2012). Inhibition in photosynthetic pigments
was also observed in B. napus under other metal stress such as
Cd (Ali et al. 2013a, b). This decrease in photosynthetic pig-
ment content may be due to marked distortion of chloroplast
ultrastructure under metal stress (Najeeb et al. 2011). In the
present study, reduction in biomass, chlorophyll, and caroten-
oid contents may be due to oxidative stress as observed by the
increased production of MDA and electrolyte leakage (Fig. 4).
Photosynthetic pigment contents increased in the treatment of
Cr+CA in comparison with the Cr exposure alone (Figs. 2 and
3). In the present study, however, increase in plant growth and
biomass and photosynthetic pigments of B. napus with CA
application under Cr stress may be due to enhanced antioxi-
dant enzyme activity (Fig. 4) which decreased the production
of MDA and electrolyte leakage (Fig. 3).
Chromium application induced oxidative stress in plants as
shown by increased production of MDA and electrolyte leak-
age (Fig. 4). Similar results have already been reported in
many studies (Singh et al. 2013). By contrast, CA+Cd addi-
tion significantly reduced the MDA and electrolyte leakage
compared with the Cr treatment alone, suggesting a protective
role of CA in preventing oxidative stress in B. napus. Plants
have developed a self-defense mechanism for the reduction of
oxidative stress which is the production of antioxidant en-
zymes (Mittler et al. 2004). Our results showed that activities
of key antioxidants such as SOD, CAT, POD, and APX in
both roots and leaves initially enhanced at 100 μM of Cr
concentration and then reduced at 500 μM of Cr concentration
(Fig. 4). This overexpression of antioxidant enzymes at lower
Cr (100 μM) stress in plants might be a powerful tool for the
survival of plants with the highest metal accumulation capac-
ity (Haouari et al. 2012; Muhammad et al. 2009). Decrease in
antioxidant enzyme activity at larger Cr stress might be due to
severe oxidative stress in plants (Mishra et al. 2006). Cr+CA
application significantly increased the activities of these en-
zymes in shoots and roots as compared to Cr treatment alone
(Fig. 4). It has been reported that CA helped plants to over-
come oxidative stress by enhancing their antioxidant enzyme
activities under metal stress (Meng et al. 2009; Najeeb et al.
2009, 2011). These results suggest that CA application scav-
enges oxidative stress, increase in electrolyte leakage and
MDA accumulation, in B. napus seedlings by enhancing the
activity of antioxidant enzymes.
Chromium application in the soil significantly increased Cr
uptake and accumulation in different parts of B. napus
(Table 3). Similar results have already been reported in differ-
ent B. napus cultivars under Cr stress (Gill et al. 2015). CA
treatments decreased Cr concentrations in different plant parts
as compared to the same Cr treatments alone (Table 3). These
results do not corroborate by the study of Freitas et al. (2013)
who reported increase in Pb concentration in maize and veti-
ver plants with CA (40 mmol kg−1) in Pb-contaminated soil.
In the present study, decrease in Cr concentration with CA
may also be due to ameliorative effect of CA on root structure
and shape (Najeeb et al. 2011). Meers et al. (2005) reported
that mobility of heavy metals (Cu, Zn, Cd, and Pb) in the soil
increased with CA application for some time and then de-
creased which might be due to degradability of CA. In the
present study, the decrease Cr concentration in different plant
parts was concomitant with an increase in biomass (Table 2)
indicating a dilution effect (the same uptake but larger bio-
mass) and thus increase in plant growth characteristics
(Fig. 1). Interestingly, application of CA+Cr increased Cr
uptake (concentration×biomass) by the plants relative to Cr
treatment alone (Table 3). Similar results have been reported
in different species under metal stress other than Cr (Najeeb
et al. 2009, 2011; Ehsan et al. 2014). Our results do not cor-
roborate by the study of Lesage et al. (2005) who reported that
CA was not effective as an amendment to enhance the extrac-
tion of Cu, Cd, and Zn by sunflower from a calcareous soil.
Increase in total uptake of Cr by B. napus is directly related to
increase in biomass in the presence of citric acid. Data regard-
ing CA effect on Cr uptake and phytoextraction is scarce
(Diwan et al. 2008; Tsetimi and Okieimen 2011). The data
that is provided in this article highlighted that B. napus plants
accumulated higher Cr in roots and leaves which is due to high
biomass production in the presence of CA which might justify
potential applications of CA in Cr phytoremediation strategies
using B. napus plants.
Moreover, half-life of heavy metal mobilization by CA
varied between 1.5 and 5.7 days while metal mobilization
remained constant in time for soils treated with EDTA
(Meers et al. 2005). Therefore, environmental risks associated
with chelate-assisted mobilization and leaching of metals can
also be minimized by the use of degradable CA as opposed to
permanent mobilization by using persistent amendments such
as EDTA.
Conclusion
In conclusion, the results demonstrate that plant growth,
biomass, and photosynthetic pigments decreased under
Cr stress while Cr concentration increased in different
plant parts. CA application significantly increased plant
growth, biomass, and chlorophyll content by enhancing
antioxidant enzyme activity, and it also increased metal
uptake in B. napus. Our results highlighted the role of
CA in enhancing Cr accumulation and phytoremediation
of Cr-contaminated soils.
Acknowledgments Authors thank the Higher Education Commission
of Pakistan for the financial support. The results presented in this paper
are a part of MPhil studies of Sehar Afshan.

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