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Toxic effects of heavy metals (Cd, Cr and Pb) on seed germination and growth and DPPH-scavenging
activity in Brassica rapa var. turnip
Maryam Mehmood Siddiqui, Bilal Haider Abbasi, Nisar Ahmad, Mohammad Ali and Tariq Mahmood
Toxicol Ind Health 2014 30: 238 originally published online 7 August 2012
DOI: 10.1177/0748233712452605
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Article

Toxic effects of heavy metals


(Cd, Cr and Pb) on seed germination
and growth and DPPH-scavenging
activity in Brassica rapa var. turnip

Toxicology and Industrial Health


2014, Vol. 30(3) 238249
The Author(s) 2012
Reprints and permissions:
sagepub.co.uk/journalsPermissions.nav
DOI: 10.1177/0748233712452605
tih.sagepub.com

Maryam Mehmood Siddiqui1, Bilal Haider Abbasi1,


Nisar Ahmad1, Mohammad Ali1 and Tariq Mahmood2

Abstract
Toxicity of heavy metal is a wide spread environmental problem affecting all life forms including plants. In the
present study the toxic effects of heavy metals, cadmium (Cd), chromium (Cr) and lead (Pb) on seed germination
rate (%), germination index (G-index) and growth (mm) of Brassica rapa var. turnip have been investigated. The
seeds were soaked either in distilled water (control) or in aqueous solutions of Cd, Cr and Pb (1 g/l, 2.5 g/l and 5 g/l)
at 4 C in dark for 24 hours. Prior to inoculation onto MS0 medium, the soaked seeds were either washed with
sterile distilled water or inoculated without washing on solidified MS0 medium at 25 + 2 C with 16/8-hour
photoperiod in a growth chamber to germinate in vitro. Such stress conditions revealed that by increasing the
concentration of heavy metals, the germination rate (%), G-index value and growth (mm) decreased significantly,
suggesting their toxic effect on B. rapa var. turnip. This study further revealed that experiment with seed washing
resulted in less toxicity of selected heavy metals on germination and growth of B. rapa var. turnip, as compared to
experiment without washing. However, the resulting toxicity order of the selected heavy metals remained the
same (Cd > Cr > Pb). Significant decrease has been observed in seed viability and germination potential and
finally heavy metals completely ceased further growth and development of plants. The 2, 2-diphenyl-1picrylhydrazyl (DPPH)-scavenging activity revealed that significantly higher activity was observed in control
plants without heavy metals treatment. Furthermore, the Cd-treated plants showed decreased antioxidant
activity. Cr and Pb were less toxic as compared to Cd (control > Pb > Cr > Cd). This study revealed that
selected heavy metals not only affected plant development but also disturbed plant metabolic pathways.
Keywords
Brassica rapa var. turnip, heavy metals, toxicity, seed germination, DPPH

Introduction
In todays world of fast pacing technology and industrialization, heavy metal toxicity has become a global
threat to all life forms: plants, animals and ultimately
humans. The undesirable accumulation of toxic heavy
metals, particularly due to various anthropogenic
activities is not only causing worldwide devastation
of agricultural soils and products but also posing serious food safety issues, health risks and disruption of
ecosystems. As a result, unexpected consequences
and ecological crisis are anticipated with heavy metal
pollution (Amico et al., 2008; Heidari and Sarani,
2011; Kachout et al., 2009; Leon et al., 2005; Malik
et al., 2010; Yadav et al., 2009).

Heavy metals such as arsenic, cadmium, chromium,


mercury and lead are potentially toxic and have no
known biological function in plants (Peralta-Videa et
al., 2009). Usually an unwanted exposure of heavy
metal/metals produces detrimental effects on plants,
1

Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan


2
Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan
Corresponding author:
Bilal Haider Abbasi, Department of Biotechnology, Quaid-i-Azam
University, Islamabad 45320, Pakistan.
Email: bhabbasi@qau.edu.pk

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239

affecting their growth, development and/or other physiological processes (Fargasova, 2001; Heiss et al.,
2003; Kupper et al., 1998; Peralta et al., 2001). In contrast to this, some plant species bear a remarkable potential to tolerate and grow in heavy metal contaminated
sites. Such plants can do so through metal exclusion,
accumulation or indication depending upon the plant
species and type of metal (Peralta-Videa et al., 2009;
Xiong and Wang, 2005). Because of long residence
time, toxicity, nonbiodegradability, irreversible nature
of contamination and accumulation of heavy metals in
food chain, the existing technologies are not very useful
to decontaminate heavy metal polluted sites (Malik et
al., 2010; Munir et al., 2010). So far, phytoremediation
that is a new emerging technology that uses green plants
to extract, sequester or detoxify heavy metal pollutants
seems promising to do away heavy metals from the
environment in a more effective, less expensive and
environmental friendly manner than conventional technologies (Bah et al., 2010; Kachout et al., 2009).
Effect of heavy metals on early growth stages of a
plant is a widely studied subject by the researchers,
because plants early growth stages such as seed
germination serve as an important indicator in determining the toxicity effects of heavy metals on plants
(Kuriakose and Prasad, 2008; Salvatore et al.,
2008). Besides this, such studies seem helpful in the
identification of new and better heavy metaltolerant
plant species as we are living in an age where it has
become inevitable to phytoremediate toxic heavy
metals from the polluted sites.
Therefore, the current study has been done to
assess the effects of Cd, Cr and Pb on the seed germination and growth of Brassica rapa var. turnip.
Turnip is a winter season food crop of worldwide economic significance, belonging to a well-known plant
family called Brassicaceae (or mustard family;
Abbasi et al., 2011a). Many plant species from this
family are known to have remarkable heavy metal
stress tolerance such as B. napus, mustard, etc.
(Amico et al., 2008; Meng et al., 2009; Xiong and
Wang, 2005). The inhibitory effects of these and other
heavy metals on different plant species have been
immensely reported in previous studies (Peralta
et al., 2001). Similarly, the inhibitory effects of some
heavy metals (Be, Ni, T1, and V) has already been
reported on seed germination of six plant species
including turnip (Carlson et al., 1991). However, this
study is particularly aimed at assessing the effect of
Cd, Cr and Pb (as they are heavy metals of international concern) on a local turnip variety (Bah et al.,

2010). Thus, this study supposed to be useful in determining heavy metal stress tolerance to grow on Cd, Cr
and/or Pb polluted sites or its subsequent phytoremediation potential to clean up the contaminated sites by
cultivating B. rapa.

Materials and methods


Seeds and seed soaking solutions of selected
heavy metals
Seeds of B. rapa var. turnip were obtained from
National Agricultural Research Council (NARC),
Pakistan. These were stored in a clean plastic bag and
kept in dark at room temperature (15-16  C) until
further use. To assess the effects of selected heavy
metals (Cd, Cr and Pb) on the seed germination and
subsequent seedlings growth of B. rapa var. turnip,
their respective salts that is cadmium chloride
(CdCl2; MW 183.32 g/mol), chromium chloride
(CrCl3.6H2O; MW 266.47 g/mol) and lead acetate
(Pb (CH3COO)2.3H2O; MW 379.369 g/mol), respectively, were selected (UNI-CHEM1). Then the seed
soaking solutions of three different concentrations
(1, 2.5, 5 g/l) of these tested heavy metals were prepared in autoclaved distilled water.

Sterilization of seeds
Seeds were surface sterilized according to the method
of Abbasi et al. (2011b), with some modifications.
Briefly, seeds were immersed in 0.1% HgCl2 for 1
min. Subsequently, they were washed three times
with sterile distilled water to avoid any fungal/ bacterial contamination and then air-dried on dry filter
papers prior to their inoculation on medium.

Seed germination experiments


According to previous reports, direct exposure
method that is placing seeds on filter paper, previously
moistened with heavy metal solution, is one the most
frequently used method to study the effects of heavy
metals on the early growth stages in plants (Kuriakose
and Prasad, 2008). Recently, some scientists found
that it was more appropriate to perform such toxicity
tests on agar (solidified medium) than on filter paper
(Salvatore et al., 2008). Thus, based on previous
reports and our lab experience, a novel approach has
been devised to understand the effect of different concentrations of selected heavy metals on seed viability,
its germination potential and growth, over a specific
time period (i.e. 24 hours seed soaking in respective

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Toxicology and Industrial Health 30(3)

(d) Cd-5 g/l

(a) dH2O/Cd 0 g/l (Control)

(b) Cd-1 g/l

(e) dH2O/Cr 0 g/l (Control)

(f) Cr-1 g/l

(g) Cr-2.5 g/l

(h) Cr-5 g/l

(i) dH2O/Pb 0 g/l (Control)

(j) Pb-1 g/l

(k) Pb-2.5 g/l

(l) Pb-5 g/l

(c) Cd-2.5 g/l

Figure 1. Experiment without washing demonstrating a 7-day-old experiment on inhibitory effect of Cd, Cr and Pb on
seed germination and growth of Brassica rapa var. turnip ((a) to (l)), upon increase in concentration of these heavy metals in
their respective seed soaking solutions.

salt solutions of Cd, Cr and Pb). In this study, the basic


idea for the experiments has been adapted from the
work of Wei et al. (2009). In short, surface sterilized
seeds were first soaked in test solutions at 4 C in dark
for 24 hours to get heavy metal treatment followed by
their inoculation on MS0 medium (Murashige and
Skoog, 1962). In this study, seeds soaked in autoclaved
distilled water were used as controls.
Germination of heavy metals-treated seeds without
washing prior to inoculation. This experiment was performed in triplicate for seven days. Out of the seeds
soaked in respective concentrations (1.0, 2.5, 5.0
g/L) of Cd, Cr or Pb against control (i.e. seeds soaked
in distilled water), five seeds were inoculated in a
100-ml Erlenmeyer flask containing 30 ml of solidified MS medium (pH 5.5) in each flask without
washing with distilled water prior to inoculation.

After inoculation, flasks were tightly plugged and


incubated in growth chamber to allow seed germination at 25+2 C with 16/8-hour photoperiod. The germinated seeds were counted for seven days after seed
inoculation and scores were recorded. Seeds were
scored as germinated when the breakage of seed coat
was visible.
Germination of heavy metals-treated seeds with washing
prior to inoculation. This experiment was performed in
the same way as the above mentioned experiment
(section on Germination of heavy metals-treated
seeds without washing prior to inoculation) with the
only difference that the soaked seeds were washed
with distilled water prior to their inoculation on to the
medium.
At the end of each experiment, average germination rate (%), germination index (G-index; according

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241

Figure 2. Seed soaked germination experiment without washing prior to inoculation: germination rates attained over
7 days of seed incubation in Brassica rapa var. turnip upon exposure to different concentrations of Cd. The data shown
here are an average of three replicates with standard error.
Table 1. Experiment without washing: effect of various concentrations of Cd, Cr and Pb on G-index of Brassica rapa var.
turnip.

Cd
Cr
Pb

Control (dH2O)

1.0 g/l

2.5 g/l

5.0 g/l

4.93 + 0.09
4.94 + 0.08
4.89 + 0.17

2.89 + 1.65
4.58 + 0.39
4.56 + 0.51

1.47 + 1.07
2.05 + 0.60
3.18 + 1.78

0.28 + 0.49
0.71 + 0.62
3.13 + 1.78

Cd: cadmium; Cr: chromium; Pb: lead.

Table 2. Experiment without washing: effect of various concentrations of Cd, Cr and Pb on seedling growth (average) of
Brassica rapa var. turnip.

Cd
Cr
Pb

Control (dH2O)

1.0 g/l

2.5 g/l

5.0 g/l

8.04 + 0.36
8.14 + 0.21
8.22 + 0.30

1.1 + 1.08
6.7 + 0.67
8.08 + 0.79

0.00 + 0.00
1.4 + 0.96
4.7 + 0.84

0.00 + 0.00
0.6 + 0.42
1.32 + 0.73

Cd: cadmium; Cr: chromium; Pb: lead.

to the method of Akinci and Akinci, 2010) and


seedling growth (mm) were determined at the given
concentrations of Cd, Cr and Pb.

Determination of 2, 2-diphenyl-1-picrylhydrazyl
(DPPH)-scavenging antioxidant activity
The DPPH-scavenging activity was determined
according to the method of Ahmad et al. (2010b) and
Abbasi et al. (2011b) with slight modification. DPPH

powder, 1.25 mg. was dissolved in 40 ml ( 4) of


ethanol to prepare stock solution. With 0.5 ml of
sample solution, 1 ml of DPPH solution was added
separately. These solution mixtures were kept at room
temperature in dark for 30 min (incubation period). Its
absorbance was measured at 517 nm. Lower absorbance of the reaction mixture indicated higher free
radical scavenging activity using the equation:
%DPPH  scavengingactivity 100  1  AE=AD

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Toxicology and Industrial Health 30(3)

Figure 3. Seeds soaked germination experiment without washing prior to inoculation: germination rates attained over
7 days of seed incubation in Brassica rapa var. turnip upon exposure to different concentrations of Cr. The data shown here
are average of three replicates with standard error.

AE is absorbance of the solution when extract has


been added at a particular level, and AD is the absorbance of the DPPH solution with nothing added
(blank, without extract). Whereas the percentage of
free radical scavenging activity (% FRSA) in ethanol
extract of these plant materials was recorded after the
incubation period of 30 min.

Data analyses
The average and standard error of three replicates for
each treatment were calculated using Microsoft Excel
software for seed germination rate (%), germination
index (G-index) and growth (mm). For statistical
analysis, three replicates were conducted for DPPHscavenging activity and the experiments were
repeated twice. Analysis of variance (ANOVA) and
Duncans Multiple Range Test (DMRT) was used for
comparison among treatment means.

Results and discussion


Germination of heavy metals-treated seeds
without washing prior to inoculation
This experiment clearly demonstrates that seed soaking in the given concentrations (1, 2.5 and 5 g/l) of
selected heavy metals (Cd, Cr and Pb) had deteriorating effects on B. rapa var. turnip seeds which either

resulted in a marked reduction or inhibition of seed


germination (about 793% of control) and subsequent
seedlings growth (Figure 1). According to results,
*100% seed germination took place in seeds soaked
in dH2O (control) after 1 day of seed incubation. However, either subsequent delay or inhibition to seed germination and growth occurred with increase in heavy
metal concentration, depending upon the heavy metal
used over the period of 7 days of study (Figures 2 to
4). In addition to this, inhibitory effect of these heavy
metals was more pronounced on seedling growth than
on seed germination. These results were found in great
agreement with a number of previously reported
results. Peralta et al. (2001) have also reported reduction in seed germination and growth of alfalfa as the
metal concentration in the growing media was
increased. They reported that exposure to 40 ppm Cd
and Cr (IV) had significantly inhibited seed germination (45 and 55%, respectively) and shoot growth as
compared to other heavy metals used. Similarly,
Fargasova (1994) and Li et al. (2005) have also
reported in their studies that seedling growth is more
sensitive to heavy metals (Hg2, Pb2, Cu2 and Zn2)
than seed germination, whereas inhibition was more
pronounced for both, germination and growth, in the
presence of Cd2.
Likewise, according to the results, Cd displayed the
highest inhibition in the seeds soaked in different Cd

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243

Figure 4. Seed soaked germination experiment without washing prior to inoculation: germination rates attained over
7 days of seed incubation in Brassica rapa var. turnip upon exposure to different concentrations of Pb. The data shown
here are average of three replicates with standard error.

concentrations (1, 2.5 and 5 g/l) and seed germination


rates were reduced from 100% (control) to 60, 33 and
7%, respectively (Figure 2). Like germination rate,
G-index values and seedlings growth (mm) also
depicted strong inhibition to seed germination and
growth of B. rapa var. turnip at all of the given Cd
concentrations and were significantly smaller than
control (Tables 1 and 2).
In a number of previous studies on Cd toxicity in
plants, Cd has been reported as the one determining
the highest toxic effects on different species
(Li et al., 2005; Peralta et al., 2001; Salvatore et al.,
2008). Similarly, Kiran and Sahin (2006) have
demonstrated inhibitory effect of Cd on seed germination, growth and increase in mitotic abnormalities,
with the increase in CdCl2 concentration (0.0625,
0.125, 0.250, 0.500 and 1.00 mM). Response to toxic
Cd is quite variable among different plant species as
has been reported by Salvatore et al. (2008) that lettuce was the plant that suffered more among the tested
vegetable plants. Similarly, Jun-Yu et al. (2008) have
reported Xiushui 110 (a rice variety) has a higher Cd
tolerance than Xiushui 11, a rice variety with low Cd
tolerance. In most of the studies, decrease in germination, growth or enzymes activity can be seen with
increase in Cd concentrations but an increase in the
total chlorophyll content has been reported in tomato

plants, treated with Cd (10, 20, 30 and 40 mg) whereas


growth, plant biomass, leaf number and leaf area were
found to be negatively correlated with the concentration of Cd (Rehman et al., 2011). Thus, these contrasting findings suggest that Cd is highly toxic to affect
different vital processes in plants, but its toxicity
range varies in different plant species, depending
upon their ability to tolerate Cd.
These results also show that Cr was less toxic than Cd
to affect seed germination and growth in turnip. In case
of Cr, significantly less difference was found between
control and seeds soaked in low Cr concentration
(Cr 1 g/l). However, considerable inhibitory effect was
seen at its medium (2.5 g/l) and high concentration value
(5 g/l; Figures 1 and 3). Hence, the resulting germination
rates found at applied Cr concentrations were 100, 73
and 20%, respectively (Figure 3). Similarly, G-index
values and growth (mm) for Cr concentrations were less
than the control but larger than those found at the corresponding Cd concentrations (Tables 1 and 2).
In nature, Cr exists either in Cr (III) and Cr (IV)
oxidation states as the most stable forms of Cr such
that Cr (IV) is more toxic than Cr (III) (PeraltaVidea et al., 2009; Zayed and Terry, 2003). Effects
of both Cr (III) and Cr (IV) on plants have been
widely reviewed, depicting detrimental effects of
Cr on plants growth, development and other

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Toxicology and Industrial Health 30(3)

Figure 5. Seed soaked germination experiment with washing prior to inoculation: germination rates attained over 7 days
of seed incubation in Brassica rapa var. turnip upon exposure to different concentrations of Cd. The data shown here are
average of three replicates with standard error.

Figure 6. Seed soaked germination experiment with washing prior to inoculation: germination rates attained over 7 days
of seed incubation in Brassica rapa var. turnip upon exposure to different concentrations of Cr. The data shown here are
average of three replicates with standard error.

physiological processes (Peralta-Videa et al., 2009;


Shanker et al., 2005; Zayed and Terry, 2003). Generally, plants exposure to low Cr levels is known to
have stimulating effects on plant growth and yield,
whereas its synergistic effect is possible upon

increase in Cr concentration (Akinci and Akinci,


2010; Peralta-Videa et al., 2009; Zayed and Terry,
2003). Similar findings have also been reported by
Zou et al. (2006) which showed that Cr (VI) had a stimulating effect on root growth of Amaranthus viridis

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245

Figure 7. Seeds soaked germination experiment with washing prior to inoculation: germination rates attained over 7 days
of seed incubation in Brassica rapa var. turnip upon exposure to different concentrations of Pb. The data shown here are
average of three replicates with standard error.

exposed to 105 M Cr (IV), but it had inhibitory effect


at 104 M and 103 M Cr (IV). On the other hand,
some scientists have conclusively argued the nonessentiality of Cr to plants by showing that if Cr is required
for normal plant growth, the required concentrations in
plant tissues ought to be much lower than the levels of
any known essential nutrient (Zayed and Terry, 2003).
This experiment also reveals that the least inhibitory effect was shown by Pb as compared to Cd and
Cr and both germination and growth were quite similar to control. For Pb, the germination rates obtained
at the given Pb concentrations were 93, 80 and 73%,
respectively, which shows that seed germination rates
were almost similar to control (100%) at the given Pb
concentrations (Figure 4). However, at low Pb concentration (1 g/l), the corresponding G-index values
and growth lengths (mm) were also very similar to
control but larger than Cd and Cr, respectively; but
with increase in Pb concentration, decrease in corresponding values was found, particularly in case of
growth (Tables 1 and 2). This shows that in case of
Pb, seedling growth was more sensitive to various
Pb concentrations as compared to seed germination.
Several reports already exist on Pb phytotoxic
effects showing that excess Pb is toxic and leads to
a number of toxicity symptoms in plants for example
inhibition to seed germination, stunted growth,
chlorosis, blackening of root system, changes in

minerals and water balance, upsets in hormonal status, and so on (Peralta-Videa, 2009; Sharma and
Dubey, 2005). Like Yang et al. (2010) showed a significant inhibition to seed germination and seedling
growth in a wheat variety, called Xihan 2, subjected
to high Pb (NO3)2 concentrations and exogenous
H2O2. According to the results, Pb showed minimum
inhibition to seed germination and growth in B. rapa
var. turnip, compared to Cd and Cr. Similarly, Mami
et al. (2010) have also reported the less toxic effect of
Pb on seed germination and growth indices of the
selected tomato varieties and was in the order:
Cu2 > Fe2 > Pb2 and Cu2 > Pb2 > Fe2, respectively, at five different doses (0, 0.001, 0.01, 0.1 and
1%) of the selected metals. So the least toxicity of
Pb in this study can be due to less solubility and mobilization to uptake by B. rapa var. turnip or may be a
high-tolerance potential of a plant to adapt to Pb
stress, probably due to its high chlorophyll stability
(Peralta-Videa, 2009; Sudhakar et al., 1992).

Germination of heavy metals-treated seeds with


washing prior to inoculation
This experiment demonstrates that prior washing of
seeds soaked in the given concentrations of Cd, Cr and
Pb, with distilled water, before their inoculation on
MS0 medium did not significantly reduce or attenuate

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Toxicology and Industrial Health 30(3)

(a) dH2O/Cd 0 g/l (Control)

(b) Cd-1 g/l

(c) Cd-2.5 g/l

(d) Cd-5 g/l

(e) dH2O/Cr 0 g/l (Control)

(f) Cr-1 g/l

(g) Cr-2.5 g/l

(h) Cr-5 g/l

(k) Pb-2.5 g/l

(l) Pb-5 g/l

(i) dH2O/Pb 0 g/l (Control)

(j) Pb-1 g/l

Figure 8. Experiment with washing demonstrating a 7-day-old experiment on inhibitory effect of Cd, Cr and Pb on seed
germination and growth of Brassica rapa var. turnip (A-L), upon increase in concentration of these heavy metals in their
respective seed soaking solutions.
Table 3. Experiment with washing: effect of various concentrations of Cd, Cr and Pb on G-index of Brassica rapa var.
turnip.

Cd
Cr
Pb

Control (dH2O)

1.0 g/l

2.5 g/l

5.0 g/l

4.93 + 0.09
4.94 + 0.08
4.89 + 0.17

2.85 + 1.13
4.84 + 0.15
4.89 + 0.08

1.04 + 0.92
3.37 + 1.35
4.04 + 0.43

0.33 + 0.57
1.75 + 0.64
3.09 + 0.44

Cd: cadmium; Cr: chromium; Pb: lead.

Table 4. Experiment with washing: effect of various concentrations of Cd, Cr and Pb on seedling growth (average) of
Brassica rapa var. turnip.

Cd
Cr
Pb

Control (dH2O)

1.0 g/l

2.5 g/l

5.0 g/l

7.9 + 0.42
8.14 + 0.21
8.22 + 0.30

4.6 + 1.56
7.76 + 0.51
8.04 + 0.29

2.3 + 2.17
7.68 + 0.97
7.98 + 0.32

0.00 + 0.00
0.48 + 0.35
8.0 + 0.35

Cd: cadmium; Cr: chromium; Pb: lead.

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Heavy metal

20

40

60

80

100
a

Control
Pb-100 M
Pb-300 M
Pb-500 M
Pb-0.5 mM
Pb-1 mM
Cr-100 M
Cr-300 M
Cr-500 M
Cr-0.5 mM
Cr-1mM
Cd-100 M
Cd-300 M
Cd-500M
Cd-0.5 mM
Cd-1mM

ab
ab
bc

c
bc
c
cd
d
e
c
c
cd
d
de
0

20

40

60

80

100

DPPH-Scavenging activity (%)

Figure 9. 2, 2-Diphenyl-1-picrylhydrazyl (DPPH)-scavenging antioxidant activity in cadmium-, chromium- and


lead-treated (different concentrations) plants in Brassica
rapa var. turnip.

the toxic effects of these heavy metals on seed germination and growth of B. rapa var. turnip (Figures 5 to 8
and Tables 3 and 4). However, washing with water has
resulted in a slight increase in germination rates, Gindex values and growth, as compared to the results
obtained without washing of seeds (Figures 1 to 4;
Tables 1 and 2). Furthermore, despite the fact that
seeds washing with water resulted in less toxicity of
selected heavy metals, it can be clearly seen that negative effect of these heavy metals on seed germination
and growth was the common observation in both the
experiments, with increase in concentration of Cd, Cr
or Pb in soaking solutions as compared to control
seeds. Hence, this shows that even though the soaking
treatment has been over after a certain time period (24
hrs) and soaked seeds have also been washed with
water, heavy metals conglutinated on the surface
of seeds were still effective in inhibiting seed germination and subsequent seedling growth of B.
rapa var. turnip, particularly in the case of Cd as
shown in Figures 1 and 8 (Wei et al., 2009).
Thus, this study clearly shows that seed soaking in
Cd, Cr and Pb had deteriorated the germination potential of turnip seeds. This means that B. rapa seeds
were badly influenced by these heavy metals and lost
their viability and growing potential with increase in
heavy metal concentration in the soaking solutions.
This toxic (inhibitory) effect of these heavy metals
on turnip seed germination can be linked to various
metabolic adjustments, leading to modulation of different enzymes involving heavy metal-induced structural alterations, leading to cell death, interference

with the functions of metal ion cofactors, inhibition


to enzyme activity or their posttranslational processing,
growth retardation, and so on (Kuriakose and Prasad,
2008; Li et al., 2005; Meng et al., 2009). Similarly, a
number of reasons can be anticipated with the inhibition of growth in B. rapa var. turnip, induced by the
tested heavy metals: inhibition of mitosis, the reduced
synthesis of cell wall components, damage to the Golgi
apparatus, changes in the polysaccharide metabolism,
breakdown in photosynthesis (because of substitution
of Mg, the central atom of chlorophyll, by heavy
metals), decrease in the water potential (causing loss
of turgor and wilted growth), and so on (Heidari and
Sarani, 2011; Kupper et al., 1998).
Another noticeable finding of this study was the
great difference in the relative toxicity of tested
heavy metals, with the resulting toxicity order as
Cd > Cr > Pb. This difference in the relative toxicity
of Cd, Cr and Pb can be due to their variable selective permeability across the seed barriers, particularly, seed coat and embryonic tissues. This
permeability of heavy metals across the seed coat
is mainly governed by a relationship between metal
intake by the seed and media water status. Hence,
greater the concentration of heavy metal in the soaking solution, greater will be its influx into the plant
system and vice versa, leading to a multitude of
inhibitory reactions, manifested as reduced
germination and growth retardation (Akinci and
Akinci, 2010; Kuriakose and Prasad, 2008; Li
et al., 2005; Wierzbicka and Obidzinska, 1998).

Toxic effect of heavy metals on DPPH-scavenging


antioxidant activity
Plants produce complex secondary metabolites during
metabolism (Ahmad et al., 2010a, 2011a). Some of
these metabolites have potential to detoxify free radicals. But during stress conditions the scavenging
power of the metabolites decreases (Abbasi et al.,
2011b; Ahmad et al., 2011b, 2011c). In the present
investigation, heavy metals (Cd, Cr and Pb) are supplemented to MS-medium and the B. rapa seeds were
allowed to germinate. After germination, the in vitro
plantlets were collected for DPPH-scavenging activity. Analysis of the assay revealed that significantly
higher activity (87.058%) was observed in control
plants without heavy metals treatment (Figure 9). It
has been observed that cadmium was the most toxic
heavy metal on antioxidant activity (37.7451
55.372%). However, chromium was less toxic than

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248

Toxicology and Industrial Health 30(3)

cadmium (31.86259.627%). In comparison with Cd


and Cr, the Pb-treated plant exhibited higher antioxidant activity but lower than control (Control
87.058% > Pb 52.94175.549% > Cr 31.862
59.627% > Cd 37.745155.372%). From the current
observation, it has been concluded that these heavy
metals especially cadmium not only inhibit the plant
growth but can also affect the antioxidant activity.
Funding
This research received no specific grant from any funding
agency in the public, commercial, or not-for-profit sectors.

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