Biochar impact on physiological and biochemical attributes of spinach

Global J. Environ. Sci. Manage., 1(3): 245-254, Summer 2015
*Corresponding Author Email: atariq@cdfa.ca.gov
Tel.: +1 916-262-0855; Fax: +1 916-262-0855
Note. This manuscript was submitted on December 16, 2014;
approved on February 24, 2015; published online on June 1,
2015. Discussion period open until October 1, 2015; discussion
can be performed online on the Website “Show article” section
for this article. This paper is part of the Global Journal of
Environmental Science and Management (GJESM).
Biochar impact on physiological and biochemical attributes of spinach
Spinacia oleracea (L.) in nickel contaminated soil
1
U. Younis;2*
M. Athar; 1
S. A. Malik;1
M.H. Raza Shah; 1
S. Mahmood
1
Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan
2
California Department of Food and Agriculture, 3288 Meadowview Road, Sacramento, California, USA
ABSTRACT: Disastrous effect of nickel on spinach was discussed by number of authors but the effect of amendments
like biochar with nickel on Spinacea oleracea L. is not still discussed by any author of the world because biochar was
used as soil amendments which play a vital role in reducing mobilization and uptake of nickel by spinach plants. As
nickel contaminated plants are very harmful for the consumption by living organisms. Nickel can be gathered in
agronomic soils by anthropogenic actions such as Ni-Cd batteries. In this study, the growth, physiological, photosynthetic
and biochemical responses of Spinacia oleracea grown in Ni-spiked soil (0, 25, 50 and 100 mg Ni/Kg soil) at three levels
of cotton-sticks-derived biochar (0, 3 and 5 %) were evaluated. The results exposed significant decrease in growth,
photosynthetic, physiological, and biochemical traits of S. oleracea when grown in Ni-polluted soil. However, this
decrease was less pronounced in cotton-sticks-derived biochar amended soil. A steady rise in the MDA (0.66 µg/g to
2.08 µg /g), ascorbic acid (1.24 mg/g to 1.57 mg/g)and sugar concentrations (1.73 mg/g to 2.16 mg/g)was observed with
increased concentration of Ni. The increasing percentages of cotton-sticks-derived biochar from 3 % to 5 % decreased
Ni concentrations in root and shoot of experimental plant. Higher production of chlorophyll, amino acids and protein
with cotton-sticks-derived biochar amendment looked like alleviation in Ni toxicity. Therefore, it is concluded that, Ni
toxicity and availability to the plants can be reduced by cotton-sticks-derived biochar amendments.
Keywords: Charcoal, cotton-sticks-derived biochar (CSB), Nickel toxicity, Pyrolysis, Spinacea oleracea
ISSN 2383 - 3572
Received 16 December 2014; revised 21 February 2015; accepted 24 February 2015; available online 1 June 2015
INTRODUCTION
Spinach (Spinacia oleracea) is the important leafy
vegetable that belongs to the family Amaranthaceae.
Due to easy, pre-cleaned and pre-packaged units the
demand of spinach among the peoples of world is
continuously increasing (Lucier and Plummer, 2004).
Having rich source beta carotene, folate, vitamin C,
calcium, iron and highest ORAC (Oxygen Radical
Absorbance Capacity) spinach is highly consumed all
overtheworld(Dicoteau,2000). Theage related macular
degeneration is also reduced due to the consumption
of spinach as it is a rich source of lutein i.e. carotenoid
(Morelock and Correll, 2008). The growth of spinach is
observed best in sewage irrigated soil with broad
leaves. As a result the quantity improved but the quality
of spinach is destroyed due to hyper accumulation of
heavy metals in its shoot and leaves (Wagner and
Kaupenjohann, 2014).
The source of heavy metals are anthropogenic
activitieslikeminingand industrializationwhichdisrupted
the natural ecosystem (Shah and Nongkynrih, 2007;
Younis et al., 2015). Among these metalswhich are toxic
forhumansincludePb,As,Cd,Niand mercury(Ahluwalia
and Dinesh, 2007). Nickel is the 24th most plentiful
element of the earth crust (Cempel and Nikel, 2006).
Sewage sludge in high amounts adds large quantities of
Ni insoils that are near to the industries (Saltand Krämer,
2000). In soil the concentration of Ni accumulation can

Global J. Environ. Sci. Manage., 1(3): 245-254, Summer 2015U. Younis et al.
be varied from 3-100 ppm (Scott-Fordsmand, 1997). Ni
exists in soil in several forms such as crystalline
minerals (inorganic), on cations exchange surfaces
which are inorganic, cations surfaces that are organic,
as a free ion, water soluble and as a chelated compound
(Scott-Fordsmand, 1997). Nickel is toxic to human
health and causes cancer and other diseases on
consumption of contaminated food.
The edible part of many vegetables is the potential
sink of Ni where it accumulates and becomes toxic for
all the consumers (Satarug et al., 2012). In plants the
toxicity effects caused by Ni vary according to the
concentration of Ni in the soil solution (Mizuno, 1968).
The chlorosis and necrosis are usually observed in
those plants that are suffering from high toxicity of Ni.
Also it disturbs the natural mechanism of Iron (Fe)
uptake and mitotic activity in the plants which adversely
affect their growth (Madhava Rao and Sresty, 2000).
Seed germination is also reduced in many plants when
they are cultivated on sites contaminated with Ni
(Madhava Rao and Sresty, 2000). In case of cereals
especially wheat plants, the shoot growth becomes
retarded due to toxicity of Ni which causes reduction
in yield (Gajewska et al., 2006;Tripathi and Guar, 2006).
Therefore, there is a need to reclaim Ni in soil to reduce
its hazardous impacts on plants and animals.
For the reclamation of heavy metals through process
of immobilization in the soil mostly organic nature
amendments are suggested. In these organic nature
amendments biochar can prove very effective and can
significantly enhance the metals immobilization in the
soils through which metals bioavailability can be
reduced (Verheijen et al., 2010; Downie et al., 2009).
Recent studies show that the application of biochar in
the soil enhances the soil’s agronomic values as well
as reclaims the organic and inorganic contamination
through its high sorption capability (Asai et al., 2009;
Hossain et al., 2010; Uchimiya et al., 2012). The active
sites on the biochar due to presence of its functional
groups also make it an effective amendment for the
reclamation of heavy metals in the soil. Heavy metals
bind themselves with the active absorption sites of
biochar which reduce their mobility in the soil (Machida
et al., 2005).
There is still great uncertainty among the scientists
regarding the influence of the biochar on productivity
of agricultural commodities (Jha et al., 2010). In the
present study, mobility and bioavailability of Ni by
application of biochar and its subsequent effect on
the growth of Spinach have been studied. It is also
necessary to check the maximum rate of biochar
application in the soil which would not make it a harmful
degrading agent. Spinach is selected as an experimental
plant due to its extensive consumption as a nutrient,
capacity to uptake and store Ni ions.
MATERIALS AND METHODS
A pot experiment was conducted at the Botanical
Garden of Bahauddin Zakariya University Multan,
using a completely randomized factorial design
involving spinach (Spinacia oleracea) and two
treatment factors: biochar (0, 3, and 5 % w/w) and nickel
additions (0, 25, 50 and 100 mg Ni/kg soil using NiSO4
).
Each treatment was replicated four times. The source
of cotton-sticks-derived biochar (CSB) (Table 1) was
cotton-sticks treated at 450 °C in an especially designed
pyrolysis reactor. The plants were grown in clay pots
each filled with a mixture of 5 kg soil (Table 2) and the
calculated amounts of respective treatment factors (CSB
and Ni). Seeds of Spinacia oleracea were purchased
from the local market and sowing was done in all the
pots with their holes closed to prevent washing and
leaching of Ni. At the seedling stage, 10 plants per pot
were allowed to grow. The plants were irrigated on
regular basis to maintain 50 % ofwater holding capacity.
At maturity, the plants were harvested. Fresh and dry
biomasses were determined. The concentrations of Ni
in the plant samples were determined using di-acid
digestion followed by using atomic absorption
spectrophotometer equipped with graphite furnace and
hydride/HydreA134 technology, NovAA400,Analytic
Jena (Rashid, 1986). Total soluble protein and amino
acids were determined following the procedures of
Bradford (1976). The concentration of ascorbic acid
Table 1: Physicochemical characteristics of biochar derived from cotton sticks
pH
9.5
EC (dS/m)
1.52
Volatile matter
(%)
26
Ash(%)
62
Fixed carbon
(%)
23
Nitrogen (%)
1.12
Phosphorus
(mg/kg)
0.47Biochar
Potassium
(mg/kg)
64
246

was determined using the formula of Keller and
Schwager, (1977). Plant malondialdihyde contents were
assayed as thiobarbituric acid (TBA) method and
soluble sugar was determined with the anthrone reagent
(Cakmak and Horst, 1991). Gas exchange attributes
(photosynthetic rate, transpiration rate (E) and sub-
stomatal CO2 concentration (Ci)) were determined using
an open system LCA-4 ADC portable infrared gas
analyzer (Analytical Development Company,
Hoddeson, England). The photosynthetic pigments
were determined following the method ofArnon (1949).
Statistical analysis
The data were analyzed for statistical differences
by performing analysis of variance (ANOVA) test using
statistical software package SPSS version 18.0. The
Tukey-HSD Test was used to find significant
differences between various treatments.
RESULTS AND DISCUSSION
Growth
Fig. 1 is plotted by taking nickel concentrations (0
mg Ni/Kg, 25 mg Ni/Kg, 50 mg Ni/Kg, 100 mg Ni/Kg
soil) on X-axis and fresh and dry biomass on Y-axis. It
represented from the figure that the fresh-biomass of
S. oleracea was greater at 3 % CSB application rate
than at 5 % CSB and control treatments. The increase
in Ni concentrations induced negative effect on fresh
weight which decreased from110.69 g(control) to 62.83
g (100 mg Ni /kg). However, the addition of CSB with
increasing levels (3% and 5%) in the Ni-spiked soil
significantly enhanced the fresh biomass and reduced
the negative impact of Ni (Fig. 1). The dry biomass of
experimental plants was also significantly (P < 0.01)
decreased by the application of Ni levels from 25 mg
Ni/kg to 100 mg Ni/kg (Fig. 1). However, the CSB
application revealed significant increase of dry biomass
at all levels of Ni. The dry biomass increased from 7.49
g (control + 100 mg Ni/kg) to 11.88 g with biochar
application (3% CSB + 100 mg Ni/kg).
Spinach is a source of many vitamins and minerals
like,A, B2, B6, C, E, K, folate, zinc, selenium, copper,
magnesium, calcium, iron and potassium. It gives good
supply of dietary fiber and omega-3 fatty acids.
Pollution of agricultural industries is a main source of
contamination in spinach. So, the foremost purpose of
this study was to assess the role of biochar on spinach
in Ni-contaminated soil. For this, Spinacia oleracea
was selected due to its hyper accumulating ability of
heavy metals. Similarly, biochar has the ability to adsorb
Ni and reduced its toxicity. The Results of the study
clearly indicated an increased in plant weight with
increase of biochar application in controlled and Ni
contaminated soil (Fig. 1). This increase was mainly
due to biochar improving the soil’s overall
physicochemical properties. Moreover, biochar
improves the ion uptake and water-holding capacity of
soils, leading to increased water usage ability by the
plants (Uzoma et al., 2011). In our case,CSB reduced Ni
stress, resulting in improved plant growth which is
reduced innickel stress. Madhava Rao and Sresty(2000),
observed a negative impact of Ni on the fresh and dry
biomass of roots and shoots of plants due to Ni storage
in the plants body parts (Pandey and Sharma, 2002).
Characteristic
pHs
( saturated paste)
ECe
TSS
Water Soluble Carbonates
Water Soluble Bicarbonates
Water Soluble Chlorite
Water Soluble Sulphate
Water Soluble Calcium + Magnesium
Water Soluble Sodium
Organic matter
Nickel
Unit
-
dS/m
meq/L
meq/L
meq/L
meq/L
meq/L
meq/L
meq/L
%
mg/Kg
Value
7.91
0.86
8.6
0
4.77
2.08
1.75
4.62
3.98
0.42
1.09
Table 2: Physicochemical characteristics of experimental soil
247

Global J. Environ. Sci. Manage., 1(3): 245-254, Summer 2015Biochar and nickel impact on spinach
Physiological and photosynthetic
The physiological attributes of the plants were
significantly influenced by the application of CSB and
Ni (Fig. 2). The photosynthetic rate, transpiration rate
and sub-stomatal CO2
concentrations increased with
increasing CSB percentages from 3% to 5%. By
increasing the level of Ni (from 25 mg Ni/kg to 100 mg
Ni/kg), physiological attributes decreased rapidly.
However, this decrease was less pronounced in the
medium containing CSB.
Similarly, Ni in the growth medium significantly
reduced the chlorophyll a, b and total chlorophyll of S.
oleracea (Table 3) expected for chlorophyll a at 25 mg
Ni/kg (1.64 mg/g fresh weight) and for chlorophyll b
(0.49 mg/g fresh weight) at 50 mg Ni/kg. There was a
significant decrease in the concentrations of pigments
with increasing Ni levels. Highest drop in total
chlorophyll contents from 1.63 mg/g fresh weight
(control) to 1.25 mg/g fresh weight were observed at
100 mg Ni/kg. It was observed that the CSB additions
caused less decrease of these pigments. The
carotenoid contents were also affected in a similar way
like chlorophyll. Similarly, the concentrations of
anthocyanin and lycopene gave treatment specific
response at various levels of Ni and CSB.
The photosynthetic parameters serve as
physiological indicators for determining the effects of
Ni toxicity (Monni et al., 2001). The substomatal CO2
concentration, photosynthetic and transpiration rates
decreased with increasing Ni levels but with the use of
BC, this decrease was changed to an increase (Fig. 2).
Similar results regarding Ni-induced reductions in
photosynthetic and transpiration rates in various
plants have been reported (Seregin and Ivanov, 2001).
These decreases may be due to a decrease in
chlorophyll contents, chlorophyll deprivation, and/or
disintegration and destruction of Rubisco with stomata.
It was observed that when plants were cultivated in
the Ni toxic soils, the Ni treated plants showed the
highest symptoms of chlorosis. These symptoms may
have appeared due to the competition between the Ni
with Ca and Mg intake in the plants. Less intake of Mg
resulted in the deficiency of chlorophyll production in
the plants and ultimately photosynthesis is reduced
(Molas, 1997). Seregin and Ivanov (2001), noted that
when plants uptake large amount of Ni and store it in
the lamella regions, the PS II become disturbed due to
which reduction in the photosynthesis takes place. It
also changes the structure of electron carriers by
changing the plastoquinone QA
and Fe to
plastoquinone QB
(Krupa and Baszynski, 1995). An
increase in chlorophyll contents was also observed
with increased biochar percentages, as observed
previously with Ni stress.
The decrease in photosynthetic pigments is due to
the inhibition of the activities of enzyme that play roles
in the synthesis of these pigments, such as δ-
aminolevulinic acid dehydratase and proto-
Fig. 1: Fresh and dry biomass of S. oleracea under different levels of nickel (0, 25, 50 and 100 mg Ni/kg soil) and cotton
sticks biochar (CSB, 0 %, 3% and 5 %).
248

c c
c
c
b
b
b
b
a
a
a
a
0
2
4
6
8
10
12
14
16
18
20
0 25 50 100
P
h
o
to
syn
th
etic
rate
(µ
m
o
l
m
/sec) 0% CSB 3% CSB 5% CSB
c c
c
c
b b
b
b
a
a
a
a
0
0.5
1
1.5
2
2.5
3
3.5
4
0 25 50 100
T
ransp
iration
rate
(m
m
ol
m
/sec) 0% CSB 3% CSB 5% CSB
c
c
b
c
b b
a
b
a
a
c
a
0
5
10
15
20
25
0 25 50 100
S
u
b-sto
m
atal
C
O
2
C
on
cen
tration
s
(µ
m
ol/m
o
l)
Nickel application (mg Ni/Kg-soil)
0% CSB 3% CSB 5% CSB
Fig. 2: Photosynthetic rate, transpiration rate and sub-stomatal CO2
concentrations of
S. oleracea under different levels of nickel (0, 25, 50 and 100 mg Ni/kg soil) and
cotton sticks biochar (CSB, 0 %, 3% and 5 %) on. The different letters on bars
show significant differences (P < 0.01) between biochar application levels within
a nickel application.
Photosyntheticrate(µmolm/sec)Transpirationrate(mmolm/sec)
Sub-stomatalCO2Concentrations
(µmol/mol)
249

chlorophylli-dereductase. It controls the bio
manufacturing of pigments and Calvin cycle. Similarly,
Somashekaraiah et al., (1992) suggested that the
decrease in the chl.a and chl.b are resulted due to
damaging of chloroplast by Ni. The increase in
carotenoid contents in the present study is consistent
with the findings of Chaneva et al., (2009) who reported
increased carotenoid contents in maize under heavy
metal stress.
Biochemical attributes
The concentration of soluble proteins in the leaves
was significantly affected by the application of Ni and
CSB (Fig.3). The decrease inprotein concentration (15.37
mg/g to 6.46 mg/g) was noted with increasing levels of
Ni irrespective of CSB application. Similar results were
observed for total soluble amino-acids. Lowest
concentration (0.33 mg/g) of total soluble amino acids
was noted at 100 mg Ni/kg. The MDA contents were
also significantly (P < 0.01) influenced by Ni treatment
along with CSB. By increasing Ni concentrations the
MDA contents also increased from 0.67 µg/g (control)
to 2.07 µg/g (100 mg Ni/kg). However, by the addition of
CSB, this increase was changed into decrease at various
levels of Ni (2.07 µg/g to 1.14 µg/g at 100 mg Ni/Kg with
5% biochar).Asimilar pattern for the sugar and ascorbic
acid concentrations was observed at different levels of
Ni and CSB. However, sugar concentration increased
more rapidly at 100 mg Ni/kg as compared to MDA
and ascorbic acid.
Ni stress was also noticed in the form of decreased
soluble proteins in the plants. This decrease in
protein content might have been due to the reduction
of de novo synthesis of enzymes (Fig. 3). High amount
of Ni intake reduces the production of proteins in the
sunflower, maize as well as in soybean (Sharma and
Dhiman, 2013). In the forms of enzymes many cell
functions are performed by the proteins in the body
of plants. The increase in the concentrations of metal-
binding complexes (phytochelatins and
metallothioneins) with heavy metal pollution was also
demonstrated by Inouhe, (2005). The increase in
proteins with biochar application under Ni stress
might have resulted from the same reason. Amino
acids are critically important for metabolism of plants
as these are a connection between carbon and
nitrogen metabolism. The synthesis of amino acids
by plants is an environmentally controlled factor;
thus, plants produce high quantities of amino acids
under limited stress conditions (Pant et al., 2011).
Similar results were observed in our study. The
amount of amino acids increased at 25 mg Ni/Kg soil.
This increase was more visible at 5% biochar
application with Ni stress.
In many plants the oxidation of polyunsaturated
fatty acids named as lipids is also observed when
0.10 b
0.14 ab
0.16 a
0.12 b
0.13 a
0.12 b
0.09 b
0.11 a
0.09 b
0.08 a
0.08 a
0.07 b
0% CSB
3% CSB
5% CSB
0% CSB
3% CSB
5% CSB
0% CSB
3% CSB
5% CSB
0% CSB
3% CSB
5% CSB
1.63 c
2.23 b
2.61 a
1.64 c
2.05 a
1.80 b
1.51 c
1.63 b
2.20 a
1.25 c
1.49 b
1.82 a
0.47 c
0.74 b
1.08 a
0.44 c
0.76 b
0.91 a
0.49 b
0.63 a
0.42 c
0.31 b
0.48 a
0.34 b
2.10 c
2.98 b
3.68 a
2.09 c
2.81 a
2.71 b
2.00 c
2.27 b
2.63 a
1.57 c
1.97 b
2.17 a
0.55 c
0.68 b
0.71 a
0.46 c
0.68 a
0.65
0.50 a
0.50 a
0.51 a
0.41 a
0.38 b
0.34 b
0.10 c
0.11 b
0.13 a
0.09 c
0.11 b
0.12 a
0.09 b
0.08 b
0.10 a
0.07 b
0.09 a
0.09 a
Anthocyanin
(umol/ml)
Ni
Treatments
Chlorophyll
a
Biochar
application (%)
Chlorophyll
b
Total
Chlorophyll
Carotenoids Lycopene mg/g
Control
25
mg Ni/Kg
50
mg Ni/Kg
100
mg Ni/Kg
Table 3: Photosynthetic attributes of S. oleracea under different levels of nickel (0, 25, 50 and 100 mg Ni/Kg soil) and cotton
sticks biochar (CSB, 0 %, 3% and 5 %). The different letters within column show significant differences (P < 0.01)
between biochar application levels within a nickel application.
250

b a a
a
a a
a c
c
a
a
b
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 25 50 100
a
a b
a
b
b
a
b
b
c
c
c
0
0.5
1
1.5
2
2.5
0 25 50 100
a
b
b
a
b
a
a
b
c
c
c
c
0
0.5
1
1.5
2
2.5
0 25 50 100
a
b
c bc
c
b
c
b
a
a
a
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 25 50 100
Fig. 3: Biochemical attributes (Ascorbic acid, MDA, sugar, amino acids and protein) of S. oleracea under different levels of nickel
(0, 25, 50 and 100 mg Ni/kg soil) and cotton sticks biochar (CSB, 0 %, 3% and 5 %). The different letters on bars show
significant differences (P < 0.01) between biochar application levels within a nickel application.
Ascorbicacid(mg/g)
MDA(µg/g)
Sugar(mg/g)
Aminoacid(mg/g)
Protein(mg/g)
mg Ni/Kg soil
251

Global J. Environ. Sci. Manage., 1(3): 245-254, Summer 2015Biochar and nickel impact on spinach
they are cultivated in the Ni toxic soils. At the highest
levels of nickel application, the concentrations of
MDA, sugars, and ascorbic acid were increased (Fig.
3). The increases in these concentrations, especially
MDA and ascorbic acid, play protective roles against
Ni stress. The increase in MDA (polyunsaturated fatty
acids ) has also been observed in different plants with
Ni treatment (Demiral and Türkan, 2005). Similarly the
high concentration ofNi in the wheat plants also increase
the synthesis of MDA as found by many scientists
(Gonnelli et al., 2001). The increased in ascorbic acid
under Ni stress was also observed by Mishra and
Choudhuri (1999), who suggested the defensive role of
ascorbic acid in Ni stress. However, these increases were
much less in BC-treated soils, suggesting a role for BC
in reducing the toxicity of Ni by sorption it in soils.
Nickel concentration in plants
The concentration of Ni in root and shoot increased
significantly (P<0.01) with increasing Ni application
levels (Table 4), though, this increase was more
pronounced at rhizosphere level. Maximum value of Ni
concentration in root (6.652 ppm) was noted at 100 mg
Ni/kg followed by 50 mg Ni/kg (7.026 ppm), and the
Table 4: Effect of various application levels of nickel (0, 25, 50 and 100 mg Ni/kg soil) and cotton sticks biochar (CSB, 0 %,
3% and 5 %) on nickel concentration in S. oleracea. The different letters within column show significant differences
(P < 0.01) between biochar application levels within a nickel application.
minimum(0.159 ppm)was under 5% CSB. However the
addition ofCSB in Ni applied pots significantly reduced
the Ni concentrations in the roots and shoots of
respective plants.
CONCLUSION
The results of the present study clearly indicated
that different levels of nickel had negative effects on
growth, chlorophyll and physiological attributes of
spinach plants. Furthermore, the increased in
production of ascorbic acid, sugar and MDA indicate
the damage impose by nickel on plants. Though, the
cotton sticks biochar had revealed the capability to
sorb metal in the soil and delimited transfer of nickel
ions to the aerial tissue. Similarly, biochar application
also enhanced the growth and photosynthetic
attributes of spinach under nickel stress. Therefore,
biochar can be chose as an amendment in conditions
where irrigation water is impure with significant
quantity of nickel as well as for the operation of
unrestrained soils polluted with this metal.
ACKNOWLEDGEMENTS
This research constitutes a part of Ph.D. thesis of
the first author. The authors acknowledge the Higher
Education Commission Pakistan for financial support
252
0% CSB
3% CSB
5% CSB
0% CSB
3% CSB
5% CSB
0% CSB
3% CSB
5% CSB
0% CSB
3% CSB
5% CSB
0.199 a
0.166 b
0.159 c
6.723 a
6.716 a
6.360 b
7.026 a
6.054 b
5.032 c
7.652 a
7.492 b
7.459 c
0.099 a
0.096 b
0.093 c
5.254 a
4.662 b
4.629 b
6.060 a
5.115 b
5.065 c
7.546 a
5.364 b
5.298 b
Ni
Treatments
Root
Biochar % age
Shoot
Control
25
mg Ni/Kg
50
mg Ni/Kg
100
mg Ni/Kg
Nickel concentrations (mg Ni/Kg dry mass)

for this research work and Nouman Mirza of University
of Melbourne, Australia for critically reviewing the
manuscript.
CONFLICTOFINTEREST
The authors declare that there is no conflict of
interests regarding the publication of this manuscript.
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253

AUTHOR (S) BIOSKETCHES
Younis, U., Ph.D.; Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan.
Email: uzma.botany@hotmail.com
Athar, M., Ph.D, DSc., Professor; California Department of Food and Agriculture, 3288 Meadowview Road, Sacramento, California, USA.
Email: athar.tariq@cdfa.ca.gov
Malik, S.A., Ph.D., Professor; Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan.
Email; saeedbotany@hotmail.com
Raza Shah, M.H., MSc. Student; Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan.
Email: hrshah@hotmail.com
Mahmood, S., Ph.D., Professor and Director; Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan.
Email: drseemapk@gmail.com
How to cite this article: (Harvard style)
Younis, U.; Athar, M.; Malik, S.A.; Raza Shah, M.H.; Mahmood, S., (2015). Biochar impact on physiological and biochemical attributes
of spinach Spinacia oleracea (L.) in nickel contaminated soil, Global J. Environ. Sci. Manage., 1(3): 245-254.
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A.N.; Mahmood, S.; Malik, S.A., (2015). Growth, survival
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254

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