Ecotoxicology and Environmental Safety 122 (2015) 528536

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Ecotoxicology and Environmental Safety

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

Zinccadmium interactions: Impact on wheat physiology and mineral

acquisition
Nadeem Sarwar a,b, Wajid Ishaq a, Ghulam Farid c, Muhammad Rashid Shaheen d,
Muhammad Imran e,f,n, Mingjian Geng g, Saddam Hussain g,nn
a

Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad 38000, Pakistan
Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad 38040, Pakistan
c
Department of Agriculture Extension, Gujranwala, Punjab, Pakistan
d
Institute of Horticultural Sciences, University of Agriculture, Faisalabad 38040, Pakistan
e
Department of Soil Science, Bahauddin Zakariya University, Multan 60800, Pakistan
f
Department of Soil Science, Muhammad Nawaz Shareef University of Agriculture, Multan 61000, Pakistan
g
College of Resources and Environment, Huazhong Agricultural University, Wuhan, Hubei 430070, China
b

art ic l e i nf o

a b s t r a c t

Article history:
Received 17 April 2015
Received in revised form
3 September 2015
Accepted 4 September 2015

Increasing concentration of Cd in soil is of great concern due to risk of its entry into food chain. Zinc (Zn)
being antagonist to Cd is an important micronutrient to ameliorate its toxic effects on plants and to limit
its entry into food chain. A pot experiment was conducted using Cd contaminated soil (30 mg Cd kg
1
soil as 3CdSO4  8H2O) to investigate the effect of soil and foliar applied Zn on physiological response and
Cd concentration in wheat. In soil, Zn was applied at 15 and 30 mg Zn kg
1 soil as zinc sulfate
(ZnSO4  7H2O). For foliar applications, 3 and 6 g L
1 ZnSO4 solution was sprayed on completing eight
weeks of growth. Results indicated that Zn application could effectively improve physiological performance and mineral content of wheat grown on Cd contaminated soils. Among different Zn fertilization
treatments, foliar application of 3 g L
1 ZnSO4 solution recorded the maximum soluble proteins and the
minimum grain-Cd concentration. Soil application of ZnSO4 or foliar application at 6 g L
1 did not affect
Cd concentration in grains. Zinc application through both the methods signicantly increased phosphorus (P), potassium (K) and Zn concentrations in shoots. Concentration of P and K in grains showed
positive relationship with that of Zn. In crux, present study suggests that foliar application of Zn at
booting stage in a suitable concentration (3 g L
1 ZnSO4 solution) can effectively ameliorate the adverse
effects of Cd and decrease grain-Cd of wheat grown in Cd contaminated soil.
& 2015 Elsevier Inc. All rights reserved.

Keywords:
Cadmium toxicity
Nutrient acquisition
Soil contaminants
Wheat physiology
Zn application

1. Introduction
Cadmium (Cd) is considered highly toxic heavy metal for both
the plants and animals. The risk of Cd toxicity increases when its
concentration in agricultural soils increases due to various anthropogenic activities including application of phosphate fertilizers and sewage sludge and deposition of that emitted from metal
industries, power stations, cement industries and urban trafc
Abbreviations: ANOVA, analysis of variance; Cd, cadmium; EC, electrical conductivity; Gs, stomatal conductance; K, potassium; PBM, plant biomass; P, phosphorus; Pn, photosynthetic rate; RDW, root dry weight; RFW, root fresh weight;
ROS, reactive oxygen species; SDW, shoot dry weight; SFW, shoot fresh weight; Tr,
transpiration rate; Zn, zinc
n
Corresponding author at: Department of Soil Science, Muhammad Nawaz
Shareef University of Agriculture, Multan 61000, Pakistan.
nn
Corresponding author.
E-mail addresses: m.imran@mnsuam.edu.pk (M. Imran),
shussain@webmail.hzau.edu.cn (S. Hussain).
http://dx.doi.org/10.1016/j.ecoenv.2015.09.011
0147-6513/& 2015 Elsevier Inc. All rights reserved.

(Astol et al., 2012; Fahad et al., 2015a, 2015b). Once Cd enters into
plant roots, it can adopt either apoplastic or/and symplastic
pathways to reach xylem cells (Salt and Rauser, 1995; Liu et al.,
2014). Cadmium bound to negatively charged aerosols and dust
particles may also penetrate in plant leaves via cuticle (Kvesitadze
et al., 2006). Its toxicity inhibits plant growth and yield by decreasing carbon xation due to its adverse effects on chlorophyll
content and photosynthetic rate (Hassan et al., 2005; Anjum et al.,
2015). In plants, it induces water stress with the symptoms of
decrease in stomatal conductance, transpiration rate and leaf relative water content (Chen and Huerta, 1997; Astol et al., 2012).
This is the result of physiological damages like decrease in size and
number of xylem vessels (Gong et al., 2005), intracellular spaces
and amount of chloroplast, and cell enlargement (Sandalio et al.,
2001). In plant cells, Cd might displace Zn from Zn-binding molecules, or attach to Ca-binding sites and thereby disturb intracellular signaling processes (Meda et al., 2007). Cd efciently
competes with Fe, Mn, Zn, and Ca transport and commonly

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529

Table 1
Properties of soil used in the experiment.
Soil
characteristic

Textural
class

pH

ECe

Unit
Value

Loam

(mmol L
1)1/2 cmol kg
1 %

%
mg kg
1
mg kg
1
mg kg
1

dS m
1
7.75 7 0.18 1.75 7 0.03 1.69 70.06
4.63 7 0.15 1.63 70.04 0.65 7 0.01 2.02 7 0.09 1347 7.19 0.147 0.01

SAR

CEC

CaCO3

OM

Total Cd

Total Zn

AB-DTPA extractable Cd

AB-DTPA extractable Zn
mg kg
1
0.86 7 0.03

ECe: electrical conductivity of saturation extract, SAR: sodium absorption ratio, CEC: cation exchange capacity, CaCO3: lime content, OM: organic matter, values along with
means are the 7 S.E. of three replicates.

decreases the uptake and accumulation of these micronutrients

(Meda et al., 2007; Astol et al., 2012). Cadmium toxicity induces
production of reactive oxygen species (ROS) which may result in
cell membrane disintegration and destruction of cellular organelles and biomolecules (Ekmeki et al., 2008; Anjum et al., 2015).
Such damages not only decrease plant biomass but can also enhance translocation of Cd to aerial parts. Lack of sufcient energy
to sequester Cd in roots may also be responsible for higher
translocation of Cd from roots to aerial parts (Ekmeki et al., 2008).
Zinc is an important micronutrient which could help to minimize Cd accumulation in plants (Hassan et al., 2005, Saifullah
et al., 2014). It plays an important role in maintaining plasma
membrane integrity and is a cofactor of many essential antioxidant
enzymes (Zhao et al., 2005; Saifullah et al., 2014). Generally, Zn is
applied to plants through soil and foliar spray (Imran et al., 2015;
Fahad et al., 2015a, 2015b). Owing to its chemical similarity with
Cd, it might act as competitive ion for Cd absorption from soil by
roots and as well as interact with Cd within the transport system
of plants (Meda et al., 2007; Fahad et al., 2015a, 2015b). It is also an
integral part of various antioxidant enzymes of plants and under
optimum supply Cd cannot replace it in such enzymes to produce
oxidative stress. Studies reveal that the effects of Zn on Cd accumulation are not consistent and may differ with plant species and
Zn and Cd concentration in soil. The usefulness of soil applied Zn
in mitigating Cd problem is well documented (Adiloglu, 2002;
Fahad et al., 2015a, 2015b) but there is lack of evidences about the
role of foliar application of Zn to minimize Cd toxicity and its accumulation in wheat. Moreover, in calcareous soils with alkaline
pH, Zn availability to plants is generally low (Jahiruddin and Islam,
1999). Therefore, present study was conducted to evaluate and
compare the effectiveness of both soil and foliar applied Zn in
minimizing Cd toxicity and accumulation and its effect on physiology and mineral nutrients acquisition by wheat.

2. Materials and methods

2.1. Experimentation
A wire house pot experiment was conducted over approximately 4 months at the Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad (31.25N, 73.09E,
184 m above sea level). Due to high evapotranspiration, Faisalabad
features a semi-arid climate with mean annual rainfall of about
200 mm. The wire house had glass roof over it to ensure complete
sunlight availability and prevention from rain effects. The treatments comprised of; (1) control (uncontaminated soil), (2) Cd
contaminated soil (30 mg Cd kg
1 soil), (3) 15 mg Zn kg
1 soil
application to Cd contaminated soil (Cd Zn15-Soil), (4) 30 mg
Zn kg
1 soil application to Cd contaminated soil (Cd Zn30-Soil),
(5) foliar application of 3 g L
1 Zinc sulfate (ZnSO4) solution to Cdstressed plants (Cd Zn3-FA), and (6) 6 g L
1 Zinc sulfate (ZnSO4)
solution to Cd-stressed plants (Cd Zn6-FA). All the treatments
were arranged in completely randomized design replicated thrice.

2.2. Preparation of soil for pot experiment

Uncontaminated surface soil (015 cm depth; originally containing 2.02 ppm of total Cd) in bulk was collected from the research farm at University of Agriculture, Faisalabad. This soil has
been developed by deposited alluvium derived from Himalayas by
River Indus and its tributaries during late Pleistocene era (Riaz-ulAmin, 1986). Soil is relatively young having illite type clay mineral
(Ranjha et al., 1993). Representative soil sample was air dried,
ground with wooden pestle and mortar to pass through 2 mm
sieve and thoroughly mixed. Soil was analyzed for electrical conductivity of saturation extract (ECe), pH (pHs), lime content
(CaCO3), organic matter, cation exchange capacity (CEC) and soil
texture following standard methods described by Richards (1954)
and Page et al. (1982). The concentration of AB-DTPA extractable
Zn and Cd (Soltanpour, 1985) was determined using Atomic Absorption Spectrophotometer (Solar S-100, Thermo Electron Cooperation, USA). The properties of soil are given in Table 1.
Soil was spiked with Cd at 30 mg kg
1 by saturating with
3CdSO4  8H2O solution and was kept at about eld capacity for 60
days to equilibrate in plastic lined metallic containers. Soil was
again ground and mixed properly to make completely homogeneous. Soil of each treatment was lled in pots (10 kg pot
1) in
triplicate. Zinc was applied in soil at designed rates before pot
lling as described for Cd.
2.3. Plant material and growth conditions
Wheat (Triticum aestivum L.) variety Faisalabad-2008 was used
as test crop in the experiment. The seeds of the variety were obtained from Ayub Agriculture Research Institute (AARI), Faisalabad.
Recommended doses of NPK fertilizers (i.e., N 60 mg kg
1 soil,
P2O5 mg kg
1 soil and K2O60 mg kg
1 soil) were applied as
urea (46% N), single super phosphate (SSP, 18% P2O5) and potassium chloride (KCl, 60% K2O), respectively. The whole of P, K and
half dose of N were applied at seed sowing while remaining half of
N was applied in two equal splits after 30 days and 45 days of
germination. In each pot, 10 seeds were initially sown and nally
ve plants per pot were maintained. Uprooted plants were crushed and mixed into soil of same pot 20 days after germination.
Foliar application of Zn was done 55 days after germination at
designated rates, after covering the soil with plastic sheets. Spray
material was applied twice in two consecutive evenings. After 70
days of germination, one set of three replicates was harvested for
recording plant biomass and analyses of root, shoot and ag
leaves, while the 2nd set of three replicates was established till
maturity for grains analyses. Both the sets were planted at same
time keeping similar growth conditions.
2.4. Measurements of physiological characteristics
Chlorophyll content was measured at 60 days after germination
with the help of chlorophyll meter (Model SPAD-502, Minolta,
Japan) in three randomly selected ag leaves. Photosynthetic rate
(Pn; mM m
2 s
1), transpiration rate (Tr; mM m
2 s
1) and

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N. Sarwar et al. / Ecotoxicology and Environmental Safety 122 (2015) 528536

stomatal conductance (Gs; M m
2 s
1) were measured on two

consecutive days from two randomly selected ag leaves from
each replication using Infrared Gas Analyzer (IRGA) (LCi-SD, ADC
Bioscientic Ltd., England) at the same stage as chlorophyll
content.

2.5. Determination of metals, phosphorus and soluble protein

content
Plant samples were subjected to Cd and Zn determination
following the method described by AOAC (1990). One gram of
plant sample (0.5 g for ag leaves samples) was taken in a conical
ask and kept overnight after adding 12 mL mixture of HNO3 and
HClO4 (3:1 ratio). Samples were digested using hot plate till clear
solution was obtained. Then samples were cooled at room temperature, diluted to 25 mL with distilled water and stored in airtight plastic bottles. Metals were determined using Atomic Absorption Spectrophotometer (Solar S-100, Thermo Electron Cooperation, USA). Sodium and potassium were measured using
ame photometer (Jenway PFP-7, Keison International Ltd., UK).
Phosphorus concentration in root and shoot digests was determined using UVvisible spectrophotometer (Chapmann and
Pratt, 1961).
Protein determination was performed on ag leaves collected
at 60th day of germination. The samples were stored in a refrigerator at 4 C. Leaves (0.3 g) were homogenized in a pestle and
mortar with 2 mL of 50 mM phosphate buffer (pH 7.8) containing
0.2 mM EDTA and centrifuged it at 13,000 rpm for 25 min. Supernatant was used for the determination of soluble proteins according to Bradford (1976).

2.6. Statistical analysis

All the data collected were analyzed to conrm its variability
following analysis of variance (one-way ANOVA) technique using
Statistix 8.1 version computer software (Statistix 8.1, Tallahassee,
Florida, USA). The differences between treatments were separated
using Least Signicance Difference (LSD) test at 0.05 probability
level. The relationships between different variables were evaluated by using linear regression analysis.

3. Results
3.1. Plant biomass
The data regarding plant biomass (PBM) is given in Fig. 1A.
Exposure to Cd (30 mg kg
1) did not signicantly affect PBM of
wheat. However, Cd Zn15-Soil signicantly increased PBM as
compared with Cd Zn30-Soil. Foliar applied Zn (Cd Zn3-FA and
Cd Zn6-FA) could not signicantly increase PBM compared with
control or Cd. Moreover, no visual symptoms of Zn and/or Cd
toxicity/deciency were observed on leaves, stem and roots.
3.2. Soluble proteins
Plants grown under 30 mg Cd kg
1 soil did not differ from Cd
untreated plants (control) regarding soluble proteins (Fig. 1B). Soil
application of 15 mg Zn kg
1 soil (Cd Zn15-Soil) or 3 g L
1 foliar
ZnSO4 spray (Cd Zn3-FA) signicantly decreased the soluble
proteins compared with control or Cd. However, 30 mg Zn kg
1
soil (Cd Zn30-Soil) or 6 g L
1 foliar ZnSO4 application (Cd Zn6FA) could not affect soluble protein contents (Fig. 1B).
3.3. Chlorophyll content and gas exchange attributes
The maximum chlorophyll content (SPAD-value) was observed
for control plants which decreased signicantly under Cd stress
(Fig. 2A). Application of Zn either through soil or foliar spray did
not improve chlorophyll content. Although Pn of Cd treated plants
also decreased compared to control, but change (15%) remained
non-signicant (Fig. 2B). Application of Zn at 30 mg kg
1
(Cd Zn30-Soil) signicantly improved Pn compared with Cd.
However, soil application of Zn at 15 mg kg
1 (Cd Zn15-Soil) and
foliar application at both the rates (Cd Zn3-FA and Cd Zn6-FA)
had non-signicant effect on Pn of wheat plants. The Tr of wheat
remained unaffected under Cd stress and also by both of soil and
foliar applied Zn treatments (Fig. 2C). The Gs signicantly decreased following exposure of plants to Cd stress (30 mg kg
1)
(Fig. 2D). Cadmium stress did not affect Gs and only soil Zn application at 30 mg kg
1 soil (Cd Zn30-Soil) signicantly increased Gs compared with control.
3.4. Zinc concentrations in plant parts
Treatments affected root-Zn signicantly (Fig. 3A), being the

Fig. 1. Effect of Zn application on (A) plant biomass (PBM), and (B) soluble proteins content of wheat grown on Cd contaminated soil. Error bars above means are 7S.E.
Treatments bars not sharing a letter in common differ signicantly at 5% probability level by LSD test. Treatments are; control uncontaminated soil, no Zn application,
Cd contaminated soil with 30 mg Cd kg
1soil, Cd Zn15-soil soil application of ZnSO4 at 15 mg kg
1 under Cd stress, Cd Zn30-soil soil application of ZnSO4 at
30 mg kg
1 under Cd stress, Cd Zn3-FA foliar application of 3 g L
1 ZnSO4 solution under Cd stress, Cd Zn6-FA foliar application at 6 g L
1 ZnSO4 solution under Cd
stress.

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531

Fig. 2. Effect of Zn application on (A) chlorophyll content (SPAD value), (B) photosynthetic rate (Pn; mM m
2 s
1), (C) transpiration rate (Tr; mM m
2 s
1), and (D) stomatal
conductance (Gs; M m
2 s
1) of wheat grown in Cd contaminated soil. Error bars above means are 7 S.E. Treatments bars not sharing a letter in common differ signicantly
at 5% probability level by LSD test. Treatments are; control uncontaminated soil, no Zn application, Cd contaminated soil with 30 mg Cd kg
1soil, Cd Zn15-soil soil
application of ZnSO4 at 15 mg kg
1 under Cd stress, Cd Zn30-soil soil application of ZnSO4 at 30 mg kg
1 under Cd stress, Cd Zn3-FA foliar application of 3 g L
1 ZnSO4
solution under Cd stress, Cd Zn6-FA foliar application at 6 g L
1 ZnSO4 solution under Cd stress.

maximum (97 mg kg
1) with Cd Zn15-Soil, and the minimum in

control (53 mg kg
1). Exposure to Cd non-signicantly increased
Zn concentration in roots (6%) compared to control. Soil-applied
Zn treatments (Cd Zn15-Soil and Cd Zn30-Soil) signicantly
increased root Zn concentration compared to no Zn application
treatments (control and Cd). While foliar applied Zn (Cd Zn3-FA
and Cd Zn6-FA) did not increase the root Zn concentration
compared to control and Cd. Increase in root Zn concentration by
Cd Zn15-Soil, Cd Zn30-Soil, Cd Zn3-FA and Cd Zn6-FA was
52.02%, 70.88%, 6.13% and 8.71% respectively over that of Cd.
Treatments also affected the ag leaves-Zn signicantly
(Fig. 3C), being the maximum (374 mg kg
1) with Cd Zn6-FA.
The treatments viz., Cd, Cd Zn15-Soil and Cd Zn30-Soil were
statistically similar with control for ag leaves-Zn. However, foliar
applied-Zn treatments (Cd Zn3-FA and Cd Zn6-FA) signicantly
increased ag leaves-Zn compared with all other treatments. Increase in ag leaves Zn concentration by Cd Zn15-Soil,
Cd Zn30-Soil, Cd Zn3-FA and Cd Zn6-FA was 6.46%, 28.37%,
455.39% and 536.83% respectively over that of Cd.
The maximum (83 mg kg
1) grain-Zn was recorded for
Cd Zn6-FA, followed by 75 mg kg
1, 67 mg kg
1, 64 mg kg
1,
59 mg kg
1 and 56 mg kg
1 for Cd Zn30-Soil, Cd, Cd Zn15-Soil,
Cd Zn3-FA and control, respectively (Fig. 3E). Exposure to 30 mg
Cd kg
1 soil (Cd) did not signicantly inuence grain-Zn content
compared with untreated plants (control). The treatments
Cd Zn15-Soil and Cd Zn30-Soil were statistically similar with
Cd regarding grain-Zn concentration. However, Zn concentration

in Cd Zn30-Soil was signicantly higher compared to that of

control. Foliar application of 6 g L
1 ZnSO4 (Cd Zn6-FA) signicantly increased grain-Zn concentration compared with Cd and
control (Fig. 3E) but Cd Zn3-FA (3 g L
1 ZnSO4 solution) was not
effective for grain-Zn concentration. Treatments Cd Zn30-Soil
and Cd Zn6-FA increased grain-Zn concentration by 33.24% and
47.63% compared with control while 12.51% and 24.74% compared
with Cd, respectively.
3.5. Cadmium concentrations in plant parts
Treatments affected root-Cd signicantly (Fig. 3B), being the
maximum (173 mg kg
1) with Cd Zn30-Soil, followed by
169 mg kg
1, 159 mg kg
1, 149 mg kg
1, 143 mg kg
1 and
7.59 mg kg
1 with Cd Zn6-FA, CdZn3-FA, Cd, Cd Zn15-Soil
and control, respectively. Exposure to 30 mg kg
1 Cd (Cd) signicantly increased root-Cd as compared with control. Application
of Zn either through soil or foliar spray did not affect the Cd
concentration in roots of wheat grown in Cd contaminated soil.
However, root Cd concentration was lower in plants fertilized with
15 mg kg
1 Zn (Cd Zn15-Soil) compared to those treated with
30 mg Zn kg
1 soil Zn (Cd Zn30-Soil) and 6 g L
1 ZnSO4 solution
(Cd Zn6-FA).
Cadmium concentration in ag leaves was also different among
applied treatments (Fig. 3D), being the maximum (15.78 mg kg
1)
with Cd Zn15-Soil and the minimum with control
(0.14 mg kg
1). Exposure to Cd signicantly increased ag leaves

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N. Sarwar et al. / Ecotoxicology and Environmental Safety 122 (2015) 528536

Fig. 3. Effect of Zn application on (A) root Zn concentration, (B) root Cd concentration, (C) ag leaf Zn concentration, (D) ag leaf Cd concentration, (E) grain Zn concentration, and (F) grain Cd concentration of wheat grown in Cd contaminated soil. Error bars above means are 7 S.E. Treatments bars not sharing a letter in common differ
signicantly at 5% probability level by LSD test. Treatments are; controluncontaminated soil, no Zn application, Cd contaminated soil with 30 mg Cd kg
1soil, Cd Zn15soil soil application of ZnSO4 at 15 mg kg
1 under Cd stress, Cd Zn30-soil soil application of ZnSO4 at 30 mg kg
1 under Cd stress, Cd Zn3-FA foliar application of
3 g L
1 ZnSO4 solution under Cd stress, Cd Zn6-FA foliar application at 6 g L
1 ZnSO4 solution under Cd stress.

Cd concentration compared to control. None of the applied treatment affected ag leaf-Cd, however, plant grown on Zn applied
soil at 15 mg kg
1soil (Cd Zn15-Soil) had higher ag leaf Cd than
those receiving Zn at 30 mg kg soil (Cd Zn30-Soil) or foliar applied Zn (Cd Zn3-FA and Cd Zn6-FA).
Cadmium contamination of soil increased its concentration in
grain from 0.04 to 10.9 mg kg
1 soil (Fig. 3F). Soil application of Zn
and foliar spray of 6 g L
1 ZnSO4 (Cd Zn6-FA) did not affect grain
Cd concentration. However, 3 g L
1 spray of ZnSO4 solution
(Cd Zn3-FA) signicantly decreased grain-Cd (Fig. 3F).

3.6. Phosphorus concentration in plant parts

Grain-P signicantly differed among treatments (Fig. 4A), being
maximum (3.3 g kg
1) with Cd Zn6-FA, followed by 3.0 g kg
1,
2.6 g kg
1, 2.4 g kg
1, 2.4 g kg
1 and 2.4 g kg
1 with Cd,
Cd Zn3-FA, control, Cd Zn15-Soil and CdZn30-Soil, respectively. Exposure to 30 mg kg
1 Cd signicantly increased grain P
concentration by 23.5% compared with control. Application of Zn
either through soil (Cd Zn15-Soil and Cd Zn30-Soil) or foliar
spray (Cd Zn3-FA and Cd Zn6-FA) did not signicantly inuence
grain-P concentration, while Cd Zn6-FA signicantly increased

N. Sarwar et al. / Ecotoxicology and Environmental Safety 122 (2015) 528536

533

Fig. 4. Effect of Zn application on (A) P-concentration in grain, (B) P-concentration in shoot, (C) Na-concentration in grain, (D) Na-concentration in shoot, (E) K-concentration
in grain, and (F) K-concentration in shoot of wheat grown in Cd contaminated soil. Error bars above means are 7S.E. Treatments bars not sharing a letter in common differ
signicantly at 5% probability level by LSD test. Treatments are; control uncontaminated soil, no Zn application, Cd contaminated soil with 30 mg Cd kg
1soil, Cd Zn15soil soil application of ZnSO4 at 15 mg kg
1 under Cd stress, Cd Zn30-soil soil application of ZnSO4 at 30 mg kg
1 under Cd stress, Cd Zn3-FA foliar application of
3 g L
1 ZnSO4 solution under Cd stress, Cd Zn6-FA foliar application at 6 g L
1 ZnSO4 solution under Cd stress.

grain P concentration compared with control, Cd Zn15-Soil,

Cd Zn30-Soil and Cd Zn3-FA. Treatments Cd and Cd Zn6-FA
were found statistically at par with each other.
Similarly, treatments affected shoot-P signicantly (Fig. 4B),
being maximum (1.7 g kg
1) with Cd Zn6-FA and control, followed by 1.4 g kg
1, 1.4 g kg
1, 1.3 g kg
1 and 1.3 g kg
1 with Cd,
Cd Zn30-Soil, Cd Zn3-FA and Cd Zn15-Soil, respectively. Exposure to 30 mg kg
1 Cd non-signicantly decreased (14.76%) P
concentration in roots compared with control. Shoot P concentration in Cd Zn6-FA was statistically at par with control and

Cd but varied signicantly compared

Cd Zn30-Soil and Cd Zn3-FA.

with

Cd Zn15-Soil,

3.7. Sodium concentration in plant parts

The treatments under investigation affected grain-Na signicantly (Fig. 4C), being maximum (1.8 g kg
1) with Cd Zn6-FA,
followed by 1.7 g kg
1, 1.4 g kg
1, 1.4 g kg
1, 1.3 g kg
1 and
1.3 g kg
1 with Cd Zn3-FA, Cd, Cd Zn30-Soil, control and
Cd Zn15-Soil, respectively. Exposure of wheat to 30 mg kg
1 Cd

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N. Sarwar et al. / Ecotoxicology and Environmental Safety 122 (2015) 528536

increased grain-Na by 8.97% compared with control, however,

these two treatments were statistically similar with each
other (Fig. 4C). The treatments Cd Zn15-Soil and Cd Zn30-Soil
decreased grain-Na by 9.62% and 3.76% compared with Cd, but
were statistically similar with each other. Plants where ZnSO4
was applied as foliar spray (Cd Zn3-FA and Cd Zn6-FA) showed
signicant increase in Na content in grain compared to all other
treatments. Increase in Na content in Cd Zn3-FA and Cd Zn6-FA
was 22.07% and 25.59% respectively over that of Cd. Similar
trend was observed in shoot-Na concentration where Cd Zn30Soil, Cd Zn3-FA and Cd Zn6-FA gave signicantly high concentration of Na as compared to control and Cd (Fig. 4D).
3.8. Potassium concentration in plant parts
Grain-K signicantly differed among treatments (Fig. 4E), being
the maximum (3.4 g kg
1) with Cd Zn6-FA, followed by
3.3 g kg
1, 3.1 g kg
1, 3.1 g kg
1, 3.0 g kg
1 and 2.1 g kg
1 with
Cd, Cd Zn30-Soil, Cd Zn3-FA, control and CdZn15-Soil, respectively. Exposure to 30 mg kg
1 Cd signicantly increased the
grains K concentration by 9.97% as compared with control. Application of 15 mg Zn kg
1 soil (Cd Zn15-Soil) remained similar
to control and decreased K by 9.57% over that of Cd. However, the
treatments Cd Zn30-Soil and Cd Zn3-FA were statistically similar with Cd.
Similarly, shoot-K signicantly differed among applied treatments (Fig. 4F), being maximum (43.1 g kg
1) with Cd Zn6-FA,
followed by 42.0 g kg
1, 41.3 g kg
1, 30.5 g kg
1, 24.9 g kg
1 and
23.9 g kg
1 with Cd Zn30-Soil, Cd Zn3-FA, Cd Zn15-Soil, Cd,
and control, respectively. Exposure to 30 mg kg
1 Cd signicantly
increased grain K concentration (3.9%) compared to that of control.
Application of Zn either through soil (Cd Zn15-Soil and
Cd Zn30-Soil) or foliar spray (Cd Zn3-FA and Cd Zn6-FA) also
signicantly increased shoot K concentration compared with
control and Cd.

4. Discussion
Decrease in growth and plant biomass accumulation is generally considered as initial symptom of Cd toxicity especially in
plants grown on highly contaminated sites (Gong et al., 2005;
Ekmeki et al., 2008). Cadmium toxicity symptoms may appear
during vegetative and/or reproductive growth stages. However,
many plants can accumulate signicant quantities of Cd in their
body without visual toxicity symptoms and yield loss (Anjum
et al., 2015). Likewise in the present investigation, 30 mg Cd kg
1
soil neither caused any toxicity symptoms nor decrease PBM
compared with control. However application of 15 mg Zn kg
1
(Cd Zn15-Soil) increased PBM signicantly over that of Cd which
is a clear indication of the positive effect of Zn on wheat growth.
Khan et al. (2006) also reported that ve wheat cultivars (BPW343,
HT2329, BPW373, UP2338 and WH542) grown at highly contaminated soil did not show any decrease in biomass production.
Also, Naeem et al. (2014) reported no yield loss due to Cd stress
despite grain and straw Cd content ranging between 1.62.9 and
4.36.0 mg kg
1, respectively.
In present study, exposure of wheat plants to Cd stress severely
hampered the leaf gas exchange attributes. The decrease in Pn
(Fig. 2B) might be due to decrease in chlorophyll content or destruction of PSII reaction center in the leaf (Khan et al., 2006; Ci
et al., 2010). Khan et al. (2006) reported that increasing Cd concentration decreased Pn and the maximum decrease in Pn was
observed with 100 mg kg
1. They also reported that light and dark
reactions of photosynthesis also decreased due to Cd toxicity. Zinc
application did not improve chlorophyll content but did so for Pn

and Gs (Fig. 2). However, Ebrahim and Aly (2004) reported increase in both chlorophyll content and Pn of Zn supplied wheat
plants. Zn-induced increase in Pn without concomitant increase in
chlorophyll content suggest that Zn might has enhanced activities
of enzymes involved in photosynthesis. Since Zn application improved Gs in Cd-stressed plants (Fig. 2D), this positive effect of Zn
on Gs may be related to enhanced Pn of plants and other physiological attributes.
Soil application of Zn at 15 mg kg
1 (CdZn15-Soil) decreased
root Cd concentration whereas increased that of ag leaf (Fig. 3).
This implies that Zn application at 15 mg kg
1 increased translocation from root to shoot. Actually, Cd induces the synthesis of
phytochelatins (PCs) in roots which have ability to sequester metal
ions into roots (Sarwar et al., 2010; Chaffai and Koyama, 2011).
Phytochelatins form CdPC complexes which are sequestrated in
vacuole of root cells resulting in decreased Cd translocation from
roots to shoots (Hassan et al., 2005). But formation of ZnPC complexes in the presence of Zn (Perrin and Watt, 1971) could increase
the concentration of free Cd and as a result of which Cd translocation from root to shoot may be enhanced. Net accumulation and
complexation of metal ions in ag leaf may control the translocation of metal ions to grains. The results of the present study are in
accordance with Zhu et al. (2003) who conrmed that Zn application up to 100 mg kg
1 soil did not decrease shoot Cd concentration. Contrarily, grain-Cd signicantly decreased with foliar application of 3 g L
1 (CdZn3-FA) ZnSO4 solution (Fig. 3F). All other Zn
treatments were ineffective in decreasing Cd concentration in
grains. This suggests that antagonistic effect of Zn on Cd concentration in grains is related to its application rate. Beyond some
specic level, it may show synergism to grain Cd. This comparison
shows that foliar application of Zn proved to be more effective as
compared to its soil application. One possible reason is that, foliar
application enhanced ag leaf-Zn status many folds compared to
soil application. This enhanced level of Zn might act as an antagonist to Cd (Akay and Koleli, 2007). This implies that enhanced Zn
pool in ag leaf (Cd Zn3-FA) to a certain concentration is physiologically important to antagonize Cd translocation to grains along
with photosynthates but when ag leaf Zn concentration exceeded
beyond 350 mg kg
1 (CdZn6-FA), it failed to do so as affectively
as in CdZn3-FA (Fig. 3F). Akay and Koleli (2007) reported that
increasing rate of Zn application from 0 to 3 kg ha
1 decreased ag
leaves Cd from 12.9 to 5.5 mg kg
1 and grain-Cd from 12.0 to
4.0 mg kg
1. While in present study, application of 3 g L
1 ZnSO4
solution (Cd Zn3-FA) decreased the grain Cd concentration from
10.88 to 8.90 mg kg
1. Soil application of Zn (both low and high Zn)
did not decreased grain Cd concentration signicantly which might
be due to lower ag leaf concentration of Zn in soil applied treatments compared with foliar application treatments. Another possible reason might be that foliar applied Zn have enhanced the
concentration of soluble proteins including antioxidant enzymes
which either scavenged ROS more effectively (Davies, 2003) or
complexed Cd with them. Damage to membranes otherwise may
increase the translocation of Cd from shoot to grain (Sarwar et al.,
2010). Root-Zn was signicantly higher in CdZn15-Soil and
Cd Zn30-Soil (soil-applied) compared to that of Cd and control
(without Zn). However, foliar application of Zn (Cd Zn3-FA and
Cd Zn6-FA) did not increase root Zn concentrations signicantly
compared to that of non-Zn treatments (Fig. 3A). Maximum ag
leaf-Zn was recorded in CdZn6-FA followed by Cd Zn3-FA
(Fig. 3C). While, ag leaf-Zn with soil-applied Zn treatments were
found signicantly less compared to that with foliar applied treatments which indicates that a considerable fraction of Zn absorbed
by plant roots from soil-applied Zn, was retained in roots. Grain Zn
concentration was maximum in Cd Zn6-FA followed by CdZn30Soil, while all the other treatments differed non-signicantly for
grain-Zn compared to that of Cd. These results are in line with

N. Sarwar et al. / Ecotoxicology and Environmental Safety 122 (2015) 528536

535

important than others (Ebrahim and Aly, 2004; Rehim et al., 2014).
Ebrahim and Aly (2004) investigated that application of Zn as foliar spray form 050 mg kg
1 progressively increased both the
shoot K and P concentration in shoot of wheat. In the present
study, similar results were obtained for shoot and grains K and P
concentration (Fig. 4). All the Zn treatments increased the concentration of K in shoot as compared to plants where no Zn was
applied (control and Cd). There was a positive relationship between shoot Zn and K concentrations (Fig. 5A). In grain, Cd Zn6FA signicantly increased K concentration compared with all other
treatments (Fig. 4). This indicates that foliar application of Zn
might be a more efcient method which enhanced the K inux
from shoot to grains. In present study, strong positive association
between grain Zn and K concentration (Fig. 5B) and grain Zn and P
concentration (Fig. 5C) was observed, which is in accordance with
the ndings of Ebrahim and Aly (2004).
For grain-Na, both of the foliar application treatments
(Cd Zn3-FA and Cd Zn6-FA) performed better compared to all
the other treatments. Application of 6 g L
1 ZnSO4 solution
(Cd Zn6-FA) recorded the maximum P in both in shoot and grains
(Fig. 4A and B), while there was no effect on root P concentration.

5. Conclusions
Present study demonstrated that the soil application of Zn at
15 mg Zn kg
1 soil (Cd Zn15-Soil) could effectively improve
physiological performance and mineral content of wheat grown on
Cd contaminated soils and ameliorates toxic effects of Cd on
plants. However, it is not effective to decrease grain Cd of wheat
grown in soil with neutral to alkaline pH. Whereas, foliar application at booting stage in a suitable concentration (3 g L
1 ZnSO4
solution) cannot only enhance shoot and grain mineral content (P,
K, Na and Zn), but also decreases grain Cd concentration possibly
by providing sufcient Zn pool in shoot (particularly in ag leaf).
Still more research is required to fully understand the mechanisms
and functions of Zn-pool construct as a result of foliar applied Zn
to decrease grain-Cd.

References

Fig. 5. Relationship between (A) concentration of Zn and K in shoots,

(B) concentration of Zn and K in grains, and (C) concentration of Zn and P in grain of
wheat grown in Cd contaminated soil.

Ebrahim and Aly (2004), who reported that foliar application increased grain-Zn of wheat. In the present study, CdZn6-FA increased the grain Zn concentration by 47.6% compared with control
suggesting that foliar method of Zn application can be effectively
used for Zn biofortication of cereal grains. Habib (2009) reported
3-fold increase in grain Zn concentration compared to that with Zn
decient plants.
Zinc interacts with other plant nutrients in soil as well as in
plant tissues. Among these interactions with K and P are more

Adiloglu, A., 2002. The effect of zinc (Zn) application on uptake of cadmium (Cd) in
some cereal species. Arch Acker P Boden 48, 553556.
Akay, A., Koleli, N., 2007. Interaction between cadmium and zinc in barley (Hordeum vulgare L.) grown under eld conditions. Bangladesh J. Bot. 36, 1319.
Anjum, S.A., Tanveer, M., Hussain, S., Bao, M., Wang, L., Khan, I., Ullah, E., Tung, S.A.,
Samad, R.A., Shahzad, B., 2015. Cadmium toxicity in Maize (Zea mays L.): consequences on antioxidative systems, reactive oxygen species and cadmium
accumulation. Environ. Sci. Pollut. Res., 19. http://dx.doi.org/10.1007/
s11356-015-4882-z.
AOAC, 1990. Ofcial Methods of Analysis of AOAC International. AOAC International,
Arlington, VA, USA.
Astol, S., Zuchi, S., Neumann, G., Cesco, S., Sanit di Toppi4, L., Pinton, R., 2012.
Response of barley plants to Fe deciency and Cd contamination as affected by
S starvation. J. Exp. Bot. 63, 12411250.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal.
Biochem 72, 248254.
Chaffai, R., Koyama, H., 2011. Heavy metal tolerance in Arabidopsis thaliana. Adv.
Bot. Res. 60, 149.
Chapmann, H.D., Pratt, P.F., 1961. Methods of Analysis for Soil Plant and Waters.
University of California, USA.
Chen, Y., Huerta, A.J., 1997. Effects of sulfur nutrition on photosynthesis in cadmium-treated barley seedlings. J. Plant Nutr. 20, 845856.
Ci, D., Jiang, D., Wollenweber, B., Dai, T., Jing, Q., Cao, W., 2010. Cadmium stress in
wheat seedlings: growth, cadmium accumulation and photosynthesis. Acta
Physiol. Plant. 32, 365373.
Davies, M.J., 2003. Singlet oxygen-mediated damage to protein and its consequences. Biochem. Biophys. Res. Commun. 305, 761770.
Ebrahim, M.K.H., Aly, M.M., 2004. Physiological response of wheat to foliar application of zinc and inoculation with some bacterial fertilizers. J. Plant Nutr. 27,
18591874.

536

N. Sarwar et al. / Ecotoxicology and Environmental Safety 122 (2015) 528536

Ekmeki, Y., Tanyola, D., Ayhan, B., 2008. Effects of cadmium on antioxidant enzyme and photosynthetic activities in leaves of two maize cultivars. J. Plant
Physiol. 165, 600611.
Fahad, S., Hussain, S., Saud, S., Hassan, S., Chen, Y., et al., 2015a. Grain cadmium and
zinc concentrations in maize inuenced by genotypic variations and zinc fertilization. CLEAN Soil Air Water 42, 18.
Fahad, S., Hussain, S., Khan, F., Wu, C., Saud, S., et al., 2015b. Effects of tire rubber ash
and zinc sulfate on crop productivity and cadmium accumulation in ve rice
cultivars under eld conditions. Environ. Sci. Pollut. Res. 22, 1242412434.
http://dx.doi.org/10.1007/s11356-015-4518-3.
Gong, H.J., Zhu, X.Y., Chen, K.M., Wang, S.M., Zhang, C.L., 2005. Silicon alleviates
oxidative damage of wheat plants in pots under drought. Plant Sci. 169,
313321.
Habib, M., 2009. Effect of foliar application of Zn and Fe on wheat yield and quality.
Afr. J. Biotechnol. 8, 67956798.
Hassan, M.J., Zhang, G., Wu, F., Wei, K., Chen, Z., 2005. Zinc alleviates growth inhibition and oxidative stress caused by cadmium in rice. J. Plant Nutr. Soil Sci.
168, 255261.
Imran, M., Kanwal, S., Hussain, S., Aziz, T., Maqsood, M.A., 2015. Efcacy of zinc
application methods for concentration and estimated bioavailability of zinc in
grains of rice grown on a calcareous soil. Pak. J. Agric. Sci. 52, 169175.
Jahiruddin, M., Islam, M.R., 1999. Studies on Secondary and Micronutrients in Soils
and Crops of Some Major AEZs of Bangladesh. BARC, Farmgate, Dhaka, Annual
report 19981999.
Khan, N.A., Ahmad, I., Singh, S., Nazar, R., 2006. Variation in growth, photosynthesis, and yield of ve wheat cultivars exposed to cadmium stress. World J. Agric.
Sci. 2, 223226.
Kvesitadze, G., Khatisashvili, G., Sadunishvili, T., Ramsden, J.J., (Eds.), 2006. Biochemical Mechanisms of Detoxication in Higher Plants. Springer, Berlin.
Liu, H., Hussain, S., Peng, S., Huang, J., Cui, K., Nie, L., 2014. Potentially toxic elements concentration in milled rice differ among various planting patterns. Field
Crop Res. 168, 1926.
Meda, A.R., Scheuermann, E.B., Prechsl, U.E., Erenoglu, B., Schaaf, G., Hayen, H.,
Weber, G., von Wiren, N., 2007. Iron acquisition by phytosiderophores contributes to cadmium tolerance. Plant Physiol. 143, 17611773.
Naeem, A., Saifullah, Ghafoor, A., Farooq, M., 2014. Suppression of cadmium

concentration in wheat grains by silicon is related to its application rate and

cadmium accumulating abilities of cultivars. J. Sci. Food Agric. 12, 24672472.
http://dx.doi.org/10.1002/jsfa.6976.
Page, A.L., Miller, R.H., Keeney, D.R., 1982. Methods of soil analysis, part 2, chemical
and microbiological properties (2nd edition). Agronomy 9. ASA/SSSA, Madison,
WI.
Perrin, D.D., Watt, A.E., 1971. Complex formation in zinc and cadmium with glutathione. Biochim. Biophys. Acta 230, 96104.
Ranjha, A.M., Mehdi, S.M., Qureshi, R.H., 1993. Clay mineralogy of some selected
alluvial soils of Indus plains. Pak. J. Soil Sci. 8, 37.
Rehim, A., Hye, M.Z., Imran, M., Ali, M.A., Hussain, M., 2014. Phosphorus and zinc
application improves rice productivity. Pak. J. Sci. 66, 134139.
Riaz-ul-Amin, 1986. Tour Guide. Soil taxonomy and agro-technology transfer. In:
Proceedings of the XII International Forum. Soil Survey of Pakistan, October 9
23, Lahore, Pakistan, pp. 4963.
Richards, L.A., 1954. Diagnosis and Improvement of Saline and Alkali Soils. US
Department of Agriculture, USA, Agriculture Handbook No. 60.
Saifullah, Sarwar N., Bibi, S., Ahmad, M., Ok, Y.S., 2014. Effectiveness of zinc application to minimize cadmium toxicity and accumulation in wheat (Triticum
aestivum L.). Environ. Earth Sci. 71, 16631672.
Salt, D.E., Rauser, W.E., 1995. MgATP dependent transport of phytochelatins across
tonoplast of oat roots. Plant Physiol. 107, 12931301.
Sandalio, L.M., Dalruzo, H.C., Gomez, M., Romero-Puetras, M.C., del-Rio, L.A., 2001.
Cadmium-induced changes in the growth and oxidative metabolism of pea
plants. J. Exp. Bot. 52, 21152126.
Sarwar, N., Saifullah, Malhi, S.S., Zia, M.H., Naeem, A., Bibi, S., Farid, G., 2010. Role of
plant nutrients in minimizing cadmium accumulation by plant. J. Sci. Food
Agric. 90, 925937.
Soltanpour, P.N., 1985. Use of AB-DTPA soil test to evaluate elemental availability
and toxicity. Commun. Soil Sci. Plant Anal. 16, 323338.
Zhao, Z.Q., Zhu, Y.G., Kneer, R., Smith, S.E., 2005. Effect of zinc on cadmium toxicityinduced oxidative stress in winter wheat seedlings. J. Plant Nutr. 28, 19471959.
Zhu, Y.G., Zhao, Z.Q., Li, H.Y., Smith, S.E., Smith, F.A., 2003. Effect of zinccadmium
interactions on the uptake of zinc and cadmium by winter wheat (Triticum
aestivum) grown in pot culture. Bull. Environ. Contam. Toxicol. 71, 12891296.