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Pakistan Journal of Scientific and Industrial Research
Vol. 53, No. 1

Contents

January - February 2010

Physical Sciences
Effect of Thermal Shocking and Quenching on the Degradation Behaviour of a
Thin PZT Disc
Riffat Asim Pasha and Muhammad Zubair Khan

Comparison of Ion Chromatography with Ion Selective Electrodes for the
Determination of Inorganic Anions in Drinking Water Samples
Muhammad Hakim, Farhat Waqar, Saida Jan, Bashir Mohammad, Wasim Yawar and
Shah Alam Khan

Physical and Chemical Evaluation of Oils of Two Varieties of Carthamus tinctorius
Grown in Pakistan
Razia Sultana, Rubina Saleem and Ambrat

Analysis of Caffeine and Heavy Metal Contents in Branded and Unbranded Tea
Available in Pakistan
Asma Inayat, Shahid Rehman Khan, Muhammad Nawaz Chowdhry and Amran Waheed

Measurement of Atmospheric Concentrations of CO, SO2, NO and NOx in Urban Areas
of Karachi City, Pakistan
Durdana Rais Hashmi, Farooq Ahmad Khan, Akhtar Shareef, Farhan Aziz Abbasi,
Ghulam Hussain Sheikh and Alia Bano Munshi

Seasonal and Year Wise Variations of Water Quality Parameters in the
Dhanmondi Lake, Dhaka, Bangladesh
Shamshad Begum Qureshi

Biological Sciences
Salt Tolerance Evaluation of Rice (Oryza sativa L.) Genotypes Based on Physiological
Characters Contributing to Salinity Resistance
Jalal-ud-Din, Samiullah Khan and Ali Raza Gurmani

Parasitic Contamination in the Table Vegetables Planted in Shiraz Plain, Iran
Meraj Madadi

Microbiological Quality of Drinking Water and Beverages in Karachi, Pakistan
Anila Siddiqui, Korish Hasnain Sahir and Seema Ismat Khan

Short Communication
Feeding Inter-Relationship of Caranx hippos (Linneaus), Chrysichthys nigrodigitatus
(Lacepede), Ethmalosa fimbriata (Bowdich) and Mugil cephalus (Linneaus) in
Lagos Lagoon, Nigeria
Adebiyi Adenike Fatimat

Technology
Production and Characterization of Chitosan from Shrimp (Penaeus semisulcatus)
Shell Waste of UAE
Fazilatun Nessa, Saeed Ahmed Khan and Farah Mohammad Anas Al-Khatib

Physical Sciences
Pak. J. Sci. Ind. Res. 2010 53 (1) 1-5

Effect of Thermal Shocking and Quenching on the
Degradation Behaviour of a Thin PZT Disc
Riffat Asim Pasha* and Muhammad Zubair Khan
Department of Mechanical and Aeronautical Engineering, University of Engineering and Technology, Taxila, Pakistan
(received October 22, 2008; revised November 16, 2009; accepted November 21, 2009)

Abstract. Thin lead zirconate titanate discs were subjected to thirty five thermal shocks from two different temperatures
in deionized water and their relative dielectric constant, coupling factor and impedance values were measured with a view
to investigating the behaviour of thin piezoelectric (PZT) discs at frequency of maximum and minimum impedance.
Noticeable differences were observed in the electrical properties of the material, probably due to the change in dipole
lengths and their orientations during thermal shocking. The results can be useful in modeling and designing of smart
components for predicting their behaviour during such expected shocking conditions prior to fabrication.
Keywords: piezoelectric material, thermal shock, deionized water, dielectric constant, impedance, PZT

Introduction

Lead zirconate titanate ceramics show decrease in the dielectric constant and the resonance frequency when subjected
to thermal shocks. Importance of temperature stability for
dielectric constants and resonance frequencies have been
discussed by Lee and Kim (2005). Earlier thermal shock resistance of the materials was evaluated by water quenching.
Degradation of various properties of the piezoelectric devices
in the presence of water and AC voltage was investigated by
Xiang et al. (2007). They concluded that water is an important
cause of degradation of piezoelectric (PZT) ceramics.
However, limited work has been published on the effect of
thermal shocking, quenching and on the degradation behaviour of thin piezoelectric ceramic discs. In this study, the
degradation phenomenon of thin PZT ceramic disc have
been investigated when exposed to repeated heating and
quenching cycles below its curie temperature.

Piezoelectric materials are used in various electromechanical
applications where they are influenced by various cyclic
loadings. Thermal cycling or thermal fatigue in most electronics materials may cause degradation in their internal characteristics. Thermal fatigue test methods include quench
method and repeated heating method for thermal shocks
which have been earlier discussed (Lamon and Pherson, 1991;
Lamon, 1981). Influence of temperature on the electromechanical and fatigue behaviour of piezoelectric ceramics has
been studied by Wang et al. (1998). Temperature gradient is
developed due to sudden change in temperature in the
ceramic materials and therefore, thermal stress is generated.
Effect of thermal shocks has been studied by developing
newly designed equipment. There are various popular thermal
shock methods available in ascending and descending orders.
Some of them popular for ascending thermal shocks, include
hot jet gas method, high power radiation, melt immersion test,
ribbon test method and high power laser heating method.
Similarly, various test methods for descending thermal shocks
are quenching in water, fluidized bed or a cold air jet impinging
on hot discs; quenching in contact with huge brass rods and
indentation method have been mentioned by Panda et al.
(2002). Earlier, thermal shocks in a plate of finite thickness had
been attempted. Thermal shock and thermal fatigue of ferroelectric thin films were investigated by Zheng et al. (2005). In
all of the above methods, the water quenching method is
mostly used for thermal shock tests in which samples are
heated to a particular temperature and then quenched in
water bath. Fatigue studies show that material degradation
of PZT ceramics are strongly influenced by temperature.

Materials and Methods
Lead zirconate titanate piezoelectric discs, nickel electroded
on major faces, 0.191 mm thick and 12.7 mm in diameter, were
used for the experimentation. The thin piezoelectric ceramic
discs were heated at the heating rate of 9 °C/sec up to 100 °C,
and 150 °C, using a thermal chamber and then quenched in
deionized water at a temperature of about 20 °C. For all
thermal cycling and quenching experiments, 2 PZT test
samples were used and subjected to identical conditions.
The temperature of the PZT samples was recorded using a
spring loaded thermocouple and data acquisition system
attached directly to the samples. In order to observe degradation phenomenon of the PZT ceramic, the capacitance, dissipation factor and impedance were measured at a frequency of
1 kHz at the start and after every five heating and quenching

*Author for correspondence; E-mail: asimpasha@uettaxila.edu.pk

Riffat Asim Pasha and Muhammad Zubair Khan

Results and Discussion

cycles. Data was collected for a total of thirty-five thermal
shocks and their relative frequencies of maximum and
minimum impedance were observed between 100 kHz and
200 kHz. The capacitance and impedance at these frequencies
were recorded using impedance analyzer and dielectric test
fixture (model 1645 B). The fixture was attached to an LCR
meter and impedance analyzer 4294 A which uses a 4 pair
terminal measurement configuration. The values of capacitance measured with impedance analyzer were used to
calculate dielectric constant (KT3 and effective coupling
factor (Keff) by using the following equations from IEEE
Standard 177 (IEEE Standard, 1976) and Moulson and
Herbert (2005):

The changes in dielectric constant and coupling factor were
measured as a function of frequency of maximum and minimum impedance (fm and fn, respectively). Increase in the value
of the capacitance of the as-received PZT ceramic was
observed to be 5.8 × 104 pF which gradually decreased with
increasing thermal cycling (100 °C - 20 °C) to 1.72 × 104 pF. A
corresponding change in the fm was observed with a value of
160 kHz for the PZT sample at the start and then the value
decreased to 116.5 kHz. This represented a 28% decrease in
the fm after the ceramic was thermal cycled. A similar change
was observed for the fn which decreased from 165.5 kHz for
the as-received to 153.3 kHz after 35 thermal cycles. For the
thermal cycling (150 °C - 20 °C) change in fm was observed
from 160.2 kHz to 141 kHz and from 165.175 kHz to 157.5 kHz
in fn. Change in dielectric constant and coupling factor for
thirty five shocks in deionized water has been tabulated in
Table 1.

ta × Cp
T
K3 = ————
A × ∈0
Effective and transverse coupling factors (Keff) were determined by using the following relationships:
Keff = SQRT (fn2 - fm2) / fn2

Figure 1 indicates the value of capacitance directly measured
by impedance analyzer for the unshocked discs at frequency
of maximum impedance. Discs were shocked in deionized
water from 100 °C to 20 °C for thirty five shocks when their
capacitance value decreased from 58.041 nF to 17.237 nF
(Fig. 1 and 2). Interestingly, PZT discs shocked from 150 °C
to 20 °C showed a less decrease in capacitance value after
having thirty five shocks. In this case capacitance value at
frequency of maximum impedance decreased to 24.189 nF
(Fig. 3).

K31 = SQRT (Ψ/(1+Ψ),
where Ψ = π/2 (fn/fm) × tan | π/2 × (fn - fm) / fm |
Abbreviations used are as follows:
fm = frequency of maximum impedance
fn = frequency of minimum impedance
Cp = equivalent parallel capacitance
ta = average thickness of testing material
A = area of guarded electrode
K 3T = dielectric constant
Keff = effective coupling factor
K31 = coupling factor with transverse excitation
∈0 = permittivity at free space (8.854 × 10-12)
Ψ = phase angle

[Hz]
[Hz]
[F]
[m]
[m2]

A comparison of the graphical output for dielectric constant
for the PZT samples before thermal cycling and then after
exposing the ceramic to thirty five heating and quenching
shocks is shown in Fig. 4. Dielectric constant remains independent when measured at 1kHz. The dielectric constant is an

Table 1. Change in dielectric constant and coupling factor for two different thermal shocking conditions
Shocking from 100 °C to 20 °C
T
3

Shocking from 150 °C to 20 °C

T
3

Shocks #

K
at 1kHz

K
at fm

K eff

0
5
10
15
20
25
30
35

1853
1891
1911
1930
1918
1922
1928
1928

9888
9481
7726
6392
5291
4790
3450
2935

0.255
0.313
0.324
0.306
0.364
0.427
0.621
0.651

K 31

K
at 1kHz

K3T
at fm

Keff

K 31

0.279
0.34
0.352
0.333
0.393
0.457
0.644
0.671

1869
1915
1923
1932
1954
1976
1976
1976

10462
8532
7005
4366
3926
3504
3912
4121

0.23
0.27
0.3
0.32
0.34
0.36
0.41
0.44

0.26
0.29
0.32
0.35
0.37
0.38
0.41
0.47

Thermal Shocking and Quenching Effect on PZT Disc

A: Cp SCALE
B: D SCALE

20 nF/div REF
100 mU/div REF

0 F
500 mU
0

58.0411 nF
854.265 mU
160 kHz
Cp l

A: Cp SCALE
B: D SCALE

5 nF/div REF
100 mU/div REF

10 nF
500 mU

EX1

24.1897 nF
624.817 mU
141 kHz
Cp l

160 kHz
Cp l

START 100 kHz

OSC 500 mVolt

STOP 200 kHz

Frequency

2 nF/div REF
100 mU/div REF

10 F
500 mU

17.2376 nF
472.24 mU
116.5 kHz
Cp l

0
116.5 kHz
Cp l

START 100 kHz

OSC 500 mVolt

STOP 200 kHz

Frequency

Fig. 1. Value of capacitance for un-shocked disc w.r.t.
frequency (100 kHz-200 kHz); capacitance in nF at
frequency of maximum impedance.

A: Cp SCALE
B: D SCALE

141 kHz
Cp l

OSC 500 mVolt

STOP 200 kHz

Frequency

Fig. 2. Value of capacitance after thirty five shocks w.r.t.
frequency (100 kHz-200 kHz); capacitance in nF at
frequency of maximum impedance when shocked
from 100 °C - 20 °C.

intrinsic property of the ceramic material and the results
show that this value decreased with increasing thermal
cycles at fm and vice versa. The relative difference in the
frequencies of the maximum and minimum impedance values
depends on the material coupling factor and the resonator
geometry (i.e., dimensions of the ceramic PZT sample). For

Fig. 3. Value of capacitance after thirty five shocked w.r.t.
frequency (100 kHz-200 kHz); capacitance in nF at
frequency of maximum impedance when shocked
from 150 °C - 20 °C.

this reason, quantities known as the effective coupling
factor (Keff) and the transverse excitation factor (K31) were
calculated and compared as a function of the number of
thermal shocks (Fig. 5). It was found that both the values of
K31 and Keff increased with increasing thermal cycles to
which the PZT ceramic was exposed.
The change in modulus of impedance for two different
shocking conditions were evaluated (Fig. 6). It can be seen
that the modulus of impedance for both fm and fn, when
shocked from 100 °C to 20 °C, increased whereas for the other
conditions, it started decreasing after twenty five shocks.
Another interesting result is that when impedance at fm and
fn increased, the difference became larger at later shocks.
Decrease in dielectric constant in thermal shocking is the
expected normal behaviour. Various coupling factors were
close to each other but a noticeable change was observed
due to shocking and quenching effect. All changes may have
occurred due to change in dipole moments and their expected
random orientations. This reorientation may change the
length of dipoles, due to which specimen undergoes a change
in its piezoelectric properties.
The change in fm and fn causes the change of mechanical
quality factor. This response of the material can be utilized in
designing of oscillators. It is observed that the difference in
these two stated frequencies (fm and fn) is small as compared
to their impedance peaks during thermal shocking. The

Riffat Asim Pasha and Muhammad Zubair Khan

15000

Shocked from

10000

100 C -20 C at 1kHz
100 C -20 C at fm

5000

100 C -20 C at fn

-5000

150 C -20 C at 1kHz
O

150 C -20 C at fm

-10000

150 C -20 C at fn

-15000

Number of shocks

Fig. 4. Change in dielectric constant against number of shocks, at frequency 1kHz and frequencies of maximum and minimimum
impedance.

0.8
Shocked from

0.6

K eff, 100 C - 20 C
0.4

K 31, 100 C - 20 C

0.2

K eff, 150 C - 20 C
K 31, 150 C - 20 C

0
0

Number of shocks

Fig. 5. Change in coupling factor (K31, Keff) against number of shocks from 100 °C - 20 °C and from 150 °C - 20 °C in deionized
water.

160
140
120
100
80
60
40
20
0

Shocked from
O

100 C -20 C at fm
100 C -20 C at fn
150 C -20 C at fm
0

150 C -20 C at fn

Number of cycles

Fig. 6. Change in modulus of impedance (|Z|) against number of shocks from 100 °C - 20 °C and from 150 °C - 20 °C in deionized
water.

results suggest that the PZT ceramics suffer a noticeable
change in polarization when exposed to repeated heating
and quenching cycles, well below the curie temperature
(350 °C) for the PZT ceramic. It is thought that significant
depolarization of the PZT ceramic occurs due to the disorien-

tation of the ferroelectric domains and this reorientation is
affecting the critical piezoelectric properties by thermal
shocking and quenching. The behaviour is normal but the
number of peaks increases due to expected change in length
and reorientation of dipoles. Development of dipole moments

Pak. J. Sci. Ind. Res. 2010 53 (1) 6-13

Comparison of Ion Chromatography with Ion Selective Electrodes for
the Determination of Inorganic Anions in Drinking Water Samples
Muhammad Hakima*, Farhat Waqar a, Saida Jana, Bashir Mohammad a, Wasim Yawar a
and Shah Alam Khanb
a

Central Analytical Facility Division, PINSTECH, P. O. Nilore, Islamabad, Pakistan
b
PCSIR Laboratories Complex, Jamrud Road, Peshawar - 25120, Pakistan
(received July 8, 2009; revised December 15, 2009; accepted December 19, 2009)

Abstract. Fluoride, chloride and nitrate anions were determined in drinking water samples using techniques of ion selective
electrodes (ISE) and non-suppressed/suppressed ion chromatography (IC). Detection limit, percentage recovery and run
time were evaluated for the two methods. Detection limits for ISE [0.02, 0.20 and 1.7 ppm (μg/mL) for fluoride, chloride
and nitrate, respectively], were better than those for non suppressed IC (2.0, 1.0 and 2.0 ppm for fluoride, chloride and
nitrate, respectively). Suppressed IC was used to measure fluoride. Statistical analysis of the data revealed no evidence of
systematic difference between ISE and non suppressed IC for chloride and nitrate. Fluoride concentrations in all water
samples were lower, while chloride and nitrate concentrations in some samples were higher than the maximum contaminant
levels established by the United States Environmental Protection Agency.
Keywords: drinking water, nitrate, chloride, fluoride, ion selective electrode, ion chromatography

effluents, different food additives and as a result of leaching
or runoff from agricultural land (WHO, 2004).

Introduction
Due to increase in population, urbanization and continued
industrial growth, per capita water availability in Pakistan
has decreased from 5000 m3/annum in 1951 to 1100 m3/annum
in 2007 (WWF, 2007). The increasing gap between water
demand and supply has led to severe water shortage in
almost all sectors and has adversely affected the quality of
drinking water; consequently, water pollution has become
a serious problem in the country and most of the reported
health problems are directly or indirectly related to water
(PCRWR, 2008).

Various analytical methods have been proposed for the determination of fluoride in aqueous solutions, such as colorimetric, conductometric, complexometric and potentiometric
methods (APHA, 1985). Some methods are rapid, sensitive,
precise and relatively free of interferences. Traditional methods
used for determination of fluoride, chloride and nitrate anions
are based on colorimetric method, which due to interference
by various ions, require special treatment of the samples like
distillation or reduction and special analytical skills. Ion
chromatography is becoming more popular for the analysis
of water samples and is also recognized by the US Environmental Protection Agency (US EPA, 2009) as a method of choice
for the determination of anions in water samples (Bosch et al.,
1995; Cheam, 1992; Pereira, 1992; Frankenberger et al., 1990).
Potentiometry has been widely used for quite some time due
to its simplicity and prompt results. However, the selectivity
is rather limited, especially if chemically similar ions are
present in the sample. Recent developments in separation
techniques have led to an improvement especially in the
determination of fluoride in terms of selectivity and sensitivity (Weiss et al., 1995; Vasconcelos et al., 1994). Results of
determination of bromide, chloride, fluoride, nitrate and
sulphate using ion chromatography (IC) had been compared
with those obtained by colorimetry for rainfall, cloud water
and stream waters. According to that, there was no significant
difference in chloride and nitrate measurements between the

There are various sources of contaminants in drinking water
which, when exceeding certain levels, are harmful to man.
These contaminants are microorganisms, inorganic and
organic chemicals and certain radioisotopes. Inorganic
anions may affect the quality of water. Fluoride, chloride
and nitrate have considerable importance in the quality of
drinking water. Specially, the excess of nitrate and fluoride
in drinking water has intense effects on human health
(Meenakshi and Maheshwari, 2006; Fraser and Chilvers, 1981).
Excess nitrate in drinking water could cause serious illness
in infants below the age of six months. Fluoride might be the
reason for different bone diseases and tenderness of bones
in children (US EPA, 2009).
Fluoride, chloride and nitrate in groundwater and surface
water originate from natural sources, sewage, industrial
*Author for correspondence; E-mail: hakimsiwag@hotmail.com

Inorganic Ion Detection by ISE and IC

two methods. For fluoride, the IC method gave lower values
than the colorimetry, especially for the stream waters. Since,
the colorimetric method determines total fluorine, differences
in the values might be expected, for example fluoride forms
complexes with the available aluminium, especially in the
stream water (Neal et al., 2007). Statistical analysis of
fluoride concentrations in rain water samples as obtained
by capillary electrophoresis (CE), IC and ISE indicated that
there were no systematic differences between CE and ISE,
but the fluoride concentrations obtained by IC were significantly higher. The observed differences are most likely
due to presence of aluminium cations (Van den Hoop et al.,
1996). A fully validated dual ion chromatographic method,
complying with ISO 17025, has been developed at the
chemical laboratory of the Athens Water Supply and
Sewerage Company (EYDAP SA) for the concurrent determination of ten ions (F-, Cl-, NO3-, Br-, PO43-, SO42-, Na+, K+,
Ca2+ and Mg2+) in surface, ground and potable water samples
(Miskaki et al., 2007).

7
were same as that of the standard. This solution was stirred
at constant rate. The electrode tip was dipped in solution
while the meter was in mV mode. Increments of 10 ppm
standard solution were made after 90 second intervals to
get 0.01, 0.02, 0.04, 0.06, 0.10, 0.29, 0.48 and 1.10 ppm concentrations. For nitrate and chloride, 1, 10, 50, 100, 500 ppm
standard solutions were used and 2 mL of ISA (ion strength
adjuster) was added to 100 mL of standard solution. Same
amount of ISA was added to 100 mL of water samples. Rest of
the procedure was same. Calibration curve was obtained by
plotting a graph between electrode potential and concentrations from which the unknown concentrations of F-, Cl- and
NO3- in water samples were calculated.

Reagents. High purity distilled deionized water was used
throughout the work. Anion standards solutions were
prepared using sodium salt of fluoride (Merck, Germany),
chloride (Merck, Germany) and nitrate (BDH Chemical,
England). Other chemicals were analytically pure reagents
from RDH Chemicals, Germany.

Ion chromatography. Ion chromatograph consisted of
Kanauer HPLC quaternary pump Model K-1001 (Germany)
with maximum operating pressure of 400 bars and flow range
of 0.001-9.999 ml/min. HAMILTON PRP-X-100 polymer base
reverse phase No. R-79439 (USA) anion exchange column
PRP X-100 (150 mm × 4.1 mm) having 10 μm particle size with
comparative guard column was used. A comparative guard
column was also used. Alltech model 650 conductivity
detector (USA) was used as detector. Alltech model 640
suppressor (USA) and Metrosep A supp 3 (Metrohm,
Switzerland) anion exchange column (250 mm × 4.6 mm)
having particle size 9 μm, packed with polystyrene/divinylbenzene copolymer were used with comparative guard
column in suppressed ion chromatography. The volume of
sample loop used for injection was 20 μL. 4 mM solution of
p-hydroxy benzoic acid was used as mobile phase for non
suppressed ion chromatography while 1.8 mM Na2CO3/
1.7 mM NaHCO3 solution was used as mobile phase for
suppressed ion chromatography.

Ion selective electrode. Cole-Parmer ion selective electrode
chloride model EW-27502-13 (USA), Cole-Parmer combination
ion selective electrode fluoride model EW-27504-14 (USA) and
Cole-Parmer combination ion selective electrode nitrate model
EW-27504-22 (USA) were used. The response was in mV given
by OAKTON pH/mV/Ion Meter (pH 2100 series USA). Glacial
acetic acid and sodium chloride were used as low level total
ionic strength adjuster buffer (TISAB-2) for low level fluoride
measurement by ion selective electrodes. Sodium nitrate and
ammonium sulphate were used as ionic strength adjuster for
chloride and nitrate, respectively.

Optimal mobile phase and its flow rate were used for separation of F-, Cl- and NO3-, using the standard solutions.
Standard solutions of varying concentrations of fluoride,
chloride and nitrate were prepared from standard stock
solutions. These solutions were injected into ion chromatograph. Peak areas and heights of all these solutions were
measured and calibration curves for fluoride, chloride and
nitrate were obtained. All water samples were filtered through
0.45 μm pore diameter membrane syringe filters and injected.
The concentration of these anions in samples was determined
using these calibration curves.

By serial dilution, 10 ppm fluoride standard was prepared by
diluting 1000 ppm standard solution. 50 mL low level TISAB-2
was added to 50 mL of the above standard solution. In a 150 mL
beaker, 50 mL of distilled water and 50 mL low level TISAB-2
were added. The volume of real water samples and TISAB-2

ICP-OES instrument. The ICP-OES instrument used in the
present work is ARL 3580 model, made by Applied Research
Laboratories, Switzerland. The instrument is equipped with
a monochromator, a polychromator and a spark excitation
source besides ICP source. Both the monochromator and the

The aim of the present study was to optimize a simple, selective and efficient method for simultaneous determination of
chloride, fluoride and nitrate ions in drinking water samples
collected from various sources by using ion chromatography
and ion selective electrodes.

Materials and Methods

Muhammad Hakim et al.

polychromator are 1 meter focal length Paschen-Runge spectrometers having 1080 groves/mm concave grating mounted
in Rowland circles. The operating conditions of the ICP-OES
used are given in Table 1. Metal ions were investigated in
some water samples and their emission wavelengths were as
follows: Al (309.271 nm), Ca (393.366 nm), Fe (261.187 nm),
K (766.490 nm), Mg (279.553 nm), Mn (257.610 nm), Na
(588.995 nm), Ni (221.647 nm), Pb (220.353 nm), Si (251.611 nm),
Sr (407.771 nm) and Zn (213.856 nm).
Table 1. Operating conditions of ICP-OES
Generator frequency
Incident power
Out gas flow
Intermediate gas flow
Carrier gas flow
Observation height
Sample uptake

27MHz
1.25 kW
12 L/min.
0.8 L/min
1 L/min
16 mm above the coil
1-3 mL/min

injected at 1.0 mL/min flow rate at various pH of mobile phase.
The retention was decreased by increasing the pH of the
mobile phase. Variation of pH of the mobile phase led to shift
in the dissociation equilibrium and thus to change the
retention time. The peak height and peak area decreased, by
increasing the pH of mobile phase. This might be due to the
decrease in retention time. There was a shift in the base line
showing incomplete separation of anions. So optimum pH
was determined which was based on good resolution. The
clearer picture is given in Fig. 1. Consequently optimal pH
necessary for complete and in-time separation was 8.5.
60
pH 8.3
50

pH 8.5

pH 8.7

30
20

Samples. Surface water and groundwater are the main water
sources available to the residents of Islamabad. Drinking
water samples were collected from water sources in the month
of October 2008, from different sectors and nearby villages of
Islamabad. The water was allowed to flow from the source for
about 1-2 min in order to stabilize different parameters i.e.,
conductivity and pH. The collected samples were stored in
pre-cleaned, sterilized polyethylene bottles of one litre
capacity. The samples were cooled to 4 °C in clean and dust
free environment.
Statistical analysis. A paired t-test was performed to check
the validity of two methods (Miller and Miller, 1997). The
formula of paired t-test is:
_ _
xd √ n
_____
tcal =
Sd
where:
S
_ d = standard deviation
xd = mean of group one minus group two
n = the number of values
If tcal is less than ttab at a specific confidence limit then there is
no significant difference between the two methods.

Results and Discussion
Optimization of mobile phase for non-suppressed IC. In
order to obtain optimal separation, pH and flow rate of mobile
phase was optimized. Standards containing 10 ppm, 20 ppm
and 40 ppm of fluoride, chloride and nitrate, respectively, were

10
0
-10
-20
10

Time (min)
-

Fig. 1. Chromatogram of F- 10 ppm, Cl- 20 ppm and NO3
40 ppm at different pH at flow rate of 1.0 mL/min by
non-suppressed IC.
To see the effect of flow rate on retention time, a single
standard containing 10 ppm, 20 ppm and 40 ppm of fluoride,
chloride and nitrate, respectively, was injected by using
mobile phase of optimal pH 8.5. The effect of flow rate was
studied in the range of 0.8-1.2 mL/min. The results are shown
in Fig. 2. By increasing the flow rate, the retention time
decreased. The peak height and area also decreased with
retention time. This was due to faster separation of anions
resulting in incomplete separation of ions. The optimum
flow rate was 1 mL/min.
Performance characteristics. Performance characteristics in
terms of detection limit, percent recovery and total run time of
the analytical response were calculated from reproducibility
experiments which are shown in Table 2. The detection limits
for ISE were estimated based on three times standard deviation of response plus mean response from determination of

Inorganic Ion Detection by ISE and IC

Table 2. Performance characteristics of the applied techniques
Anions

Ion selective electrode
Detection
Run
Recovery
limit
time
(%)
(ppm)
(min)

Non-suppressed IC
Detection
Run
Recovery
limit
time
(%)
(ppm)
(min)

Detection
limit
(ppm)

Fluoride
Chloride
Nitrate

0.02
0.2
1.7

2
1
2

0.05
0.05
0.1

3
3
3

98.6
101.5
109.4

20
20
20

six blank samples. The detection limit is thus the corresponding concentration of the response from calibration curve
of each anion (Skoog et al., 2005). The detection limit for IC is
three times signal-to-noise ratio. Hence the detection limits
were found by using the standard whose response was three
times signal to noise. In order to evaluate the accuracy of
method, percentage recovery was calculated by adding
known amount of fluoride, chloride and nitrate to drinking
water samples according to the following equation:
spiked conc. _ actual conc.
Recovery (%) = ( ______________________ ) × 100
conc. of standard added
The total run time includes sample introduction, purging/
washing time and run time, whereas, the time needed for
pretreatment of the sample and to calculate the corresponding concentration were not taken into account.
Analysis of water samples. Measurement of pH. pH of all the
collected samples was measured which was in the range of
6.85-8.65 (Table 3). pH of most samples was in good agree-

102.7
103.2
98.5

Suppressed IC
Run
Recovery
time
(%)
(min)
25
25
25

99.3
102.6
106.5

ment with US EPA which is 6.5-8.5 except that of sample no.11
which was slightly higher. This sample was from Malal
stream in periphery of the village of Islamabad. People living
nearby this stream wash their clothes in the stream so pH may
be higher due to mixing of soapy water.
Determination of anions. Determination of concentrations
of fluoride, chloride and nitrate was carried out using ISE and
suppressed/non-suppressed ion chromatography. The results
are given in Table 3.
For the analysis of water samples, optimized non-suppressed
IC conditions were used. The chromatograms obtained by
injecting the samples were compared to standard chromatogram; peaks of these chromatograms were quite sharp and
resolution was also very good. Some of the chromatograms of
a standard and a sample are shown in Fig. 3 and 4, respectively.
Samples were analyzed by non suppressed/suppressed
IC as described above. Results obtained for chloride and
nitrate concentration by non suppressed IC are shown in
Table 3.

0.8 ml/min

1.0 ml/min

Chloride

Fluoride

Nitrate

1.2 ml/min
30

10
0

-10

-10
10

Time (min)

Time (min)
-

Fig. 2. Chromatogram of F- 10 ppm, Cl- 20 ppm and NO3
40 ppm at different flow rates at pH 8.5 by nonsuppressed IC.

Fig. 3. Chromatogram of F- 10 ppm, Cl- 20 ppm and NO3
40 ppm by non-suppressed IC at flow rate of
1.0 mL/min.

Muhammad Hakim et al.

Table 3. Results obtained by ion selective electrodes/pH electrode and non-suppressed ion chromatography (suppressed ion
chromatography for fluoride only)
Sample

Ion selective electrodes/pH electrode
Fluoride
Chloride
Nitrate
(ppm)
(ppm)
(ppm)

Non-suppressed ion chromatography
Fluoride
Nitrate
Chloride
(ppm)
(ppm)
(ppm)

Suppressed ion chromatography
Fluoride
(ppm)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24

7.45
7.45
7.30
7.20
7.30
6.85
7.50
7.35
7.30
7.35
8.65
7.05
7.85
8.00
7.80
7.25
7.30
7.55
8.10
7.55
8.00
7.65
7.05
7.15

0.34
0.77
0.15
0.43
0.64
0.33
0.13
0.79
0.96
0.59
0.38
0.23
0.19
0.23
0.89
1.08
0.25
0.21
0.20
0.07
0.08
0.08
0.25
0.08

nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd

0.31
0.73
0.15
0.41
0.60
0.41
0.10
0.81
0.92
0.62
0.36
0.22
0.20
0.24
0.90
1.12
0.24
0.26
0.22
0.05
0.06
0.09
0.27
0.07

92.11
114.10
53.93
67.10
128.09
1062.07
39.79
96.14
67.96
30.13
29.11
13.02
13.53
12.79
19.97
55.81
10.92
8.79
9.63
2.92
3.20
251.34
80.24
12.34

90.72
287.69
42.31
42.73
226.11
1321.69
19.05
70.59
39.24
38.27
6.01
16.39
15.90
19.15
22.48
31.94
23.75
28.03
16.88
6.82
6.91
379.91
2.88
23.52

80.61
222.70
38.20
39.10
205.23
1080.60
15.50
63.54
35.42
23.45
5.24
11.34
10.59
14.69
16.22
26.45
18.65
23.57
13.82
4.56
4.89
251.32
2.13
21.52

90.10
118.25
50.23
65.53
122.50
855.50
37.90
95.69
65.21
20.28
22.50
9.52
10.15
9.24
19.12
18.20
9.10
6.24
8.13
2.25
3.10
240.53
78.20
10.88

nd = not detected.
60
50

Chloride

100

40
Nitrate

Chloride
Fluoride

20
10

Nitrate

0
-10
-20
0

-30
5

10
Time (min)

Fig. 4. Chromatogram of sample # 2 by non-suppressed
IC.
Fluoride was not measured by non-suppressed ion chromatography due to its low concentration in the samples. So,
suppressed IC was used. Seven anion standards were injected
at the flow rate of 1 mL/min. The chromatogram is shown in
Fig. 5. Quantitative determination of fluoride in the water
samples was made by comparison of peak areas in the
chromatograms of the samples and that of the standard;
chromatogram of a sample is shown in Fig. 6. The results for

10
Time (min)

Fig. 5. Chromatogram of F - 3 ppm, Cl - 3 ppm, NO2
4 ppm, Br - 4 ppm, NO3 4 ppm, PO43 8 ppm and
2SO4 8 ppm at flow rate of 1.0 mL/min by suppressed IC.
fluoride analysis in water samples by suppressed IC are
given in Table 3.
The concentration of fluoride in all the water samples was
within the limits established by USEPA (4.0 ppm). The
chloride level was also within the permissible range i.e.,

Inorganic Ion Detection by ISE and IC

linearity in the results of the two methods from low concentration to the higher concentration.

105

100

Chloride
Nitrate

50
Fluoride
0
5

10
Time (min)

Fig. 6. Chromatogram of sample # 10 by suppressed IC at
flow rate of 1.0 mL/min.

A paired t-test was also performed to check the validity of
two methods. According to this test, if tcal is less than ttab
at a specific confidence limit then there is no significant
difference between the two methods. The results are given
in Table 5. The results of statistical analysis, according to
student’s t-test, shows that there is no significant difference
between the results obtained with non-suppressed ion
chromatography and ion selective electrodes for chloride
and nitrate determination in water samples.

1.2

y = 0.9905x + 0.0033
r = 0.9951

Table 4. Concentration of cations by ICP-OES
Cations

Sample 6
(ppm)

Sample 7
(ppm)

Sample 16
(ppm)

Al
Ca
Fe
K
Mg
Mn
Na
Ni
Pb
Si
Sr
Zn

nd
176.39
nd
5.50
328.42
nd
481.06
nd
nd
6.30
7.40
nd

nd
32.77
nd
8.94
17.79
nd
61.51
nd
nd
5.58
0.22
nd

nd
30.64
nd
1.70
37.41
nd
185.66
nd
nd
4.86
1.20
nd

nd = not detected.

250 ppm in all the samples except sample no. 6. In most of the
samples, nitrate level was higher than US EPA standard for
safe drinking water i.e. 10 ppm. Sample # 6 has the maximum
level of chloride and nitrate exceeding 1000 ppm. This is the
water obtained from house pump installed by boring in Nilore
colony situated in the surrounding area of Islamabad. The
underground water in these areas is in the narrow channels
rather than in large reservoirs. So the water may be in contact
with some rocks containing salts of nitrates and chlorides.
Thus, metal ion analysis especially of samples 6, 7 and 17
were performed using Inductive Coupled Plasma Optical
Emission Spectroscopy by conditions given in the experimental section. The results are given in the Table 4. It is clear from
the results that sample 6 contained a high concentration of
sodium, magnesium and calcium ions. So most probably the
nitrate and chloride of these cations may exist.
The results obtained by the two methods are compared in
Fig. 7-9. The correlation coefficient in each case shows good

0.9

0.6

0.3

0
0

0.3

0.6

0.9

1.2

IC (ppm)

Fig. 7. Comparison of the results for the determination of
fluoride in drinking water samples (n = 23) using IC
(suppressed) and ISE.
10000
y = 0.137+0.955 log(x)
1000

r = 0.9941

100

IC (ppm)

Fig. 8. Comparison of the results for the determination of
chloride in drinking water samples (n = 23) using IC
(non-suppressed) and ISE.

Muhammad Hakim et al.

10000

References

y = 0.0.155+0.968 log(x)
r = 0.9969

1000

100

IC (ppm)

Fig. 9. Comparison of the results for the determination of
nitrate in drinking water samples (n = 23) using IC
(non-suppressed) and ISE.

Table 5. Statistical analysis of the correlation between
chloride and nitrate concentrations by suppressed/nonsuppressed IC and ISE
Anions

Intercept

Slope

t cal

t tab

Fluoride

0.0033
±0.0005

0.9905
±0.01201

0.99022

- 0.70200 2.074

Chloride

0.16042
±0.02570

0.9637
±0.01636

0.99398

-1.46362

2.074

Nitrate

0.15521
±0.02604

0.9682
±0.0166

0.99386

-2.06386

2.074

Confidence limits = ±95; two tail; n=23.

Conclusion
Both ion chromatography and ion selective electrode were
employed for the determination of three anions (fluoride,
chloride and nitrate) in drinking water samples. ISE is a
preferred technique due to shorter analysis time and less
operational cost of the equipment. Ion chromatography is
sophisticated and reliable for simultaneous determination of
anions in routine water analysis. This technique can be used
for comparison and validation of methods.

Acknowledgement
Authors gratefully acknowledge the efforts of Central
Analytical Facility Division, PINSTECH, Islamabad in the
analysis of the water samples.

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Inorganic Ion Detection by ISE and IC

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13
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Pak. J. Sci. Ind. Res. 2010 53 (1) 14-19

Physical and Chemical Evaluation of Oils of Two Varieties of
Carthamus tinctorius Grown in Pakistan
Razia Sultana*, Rubina Saleem and Ambrat
Applied Chemistry Research Center, PCSIR Laboratories Complex,
Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-75280, Pakistan
(received August 25, 2009; revised December 8, 2009; accepted December 12, 2009)

Abstract. On evaluation of oils of two spineless varieties of Carthamus tinctorius, Thori-78 and Pawari-95 growing in
Sindh, Pakistan, the quality of the oil was found to be similar, only the oil content differed. The hexane-extracted oil
content of Thori-78 and Pawari-95 was 28.33±1.15 and 33.07±1.12, respectively. The oils contained 90.97% and
89.55% unsaturated fatty acids and 8.44% and 9.69%, saturated fatty acids, respectively. Linoleic acid was 75.42±0.59%
and 76.40±1.0% and oleic acid was 15.55±0.30% and 13.15±0.49% by weight, respectively, and were the predominant
fatty acids present in the oil.
Keywords: safflower oil, Thori-78, Pawari-95, linoleic acid, oleic acid

Introduction

in mono-unsaturated fatty acids) and high linoleic (high in
polyunsaturated fatty acids). Gas chromatography has been
an indispensable analytical technique ever since its first use
in the fatty acid determination of plant seed oil (Echard
et al., 2007; Peris-Vicente et al., 2006; Seppänen-Laakso
et al., 2002). High performance liquid chromatography
(HPLC) with ultraviolet and fluorescence detectors are the
alternative methods for separation of volatile short chain and
long chain fatty acids (Peris-Vicente et al., 2005; 2004; Chen
and Chuang, 2002).

Safflower (Carthamus tinctorius) is an annual herb belonging to the family Compositae. It is widely distributed throughout the world such as in Pakistan, India, Bangladesh, Afghanistan, Middle East, Thailand, China, Japan, Ethiopia, Sudan,
Tanzania, Keneya, Tunisia, Europe, Argintina, USA, Canada
and Australia (Knights et al., 2001). C. tinctorius flowers, seeds
and oil have wide range of medicinal uses in different countries. Flowers are used for the preparation of dyes and drugs
which are used for treating a number of disorders such as for
dilation of arteries, reduction of hypertension, increasing blood
flow, decreasing blood cholesterol, in treatment of rheumotoid
arthritis, menstrual problems, skin diseases, urinary problems
and jaundice etc. (Kaffka et al., 2001; Sastri, 1950). Seed
decoction is used as laxative in Pakistan. The oil is used in
Iran to treat liver and heart ailments and in charred state, used
in India in treatment of sores and rheumatism. In Northern
America, it is cultivated for using as bird feed, animal meal
and for industrial applications (Oyen et al., 2007; Mündel
et al., 2004; Oplinger et al., 1992).

Safflower oil can be used in cosmetics, foods, nutritional
supplements, personal care products, soaps and shampoos.
Cold press oil is golden yellow and is used for culinary
purposes. The oil obtained by dry hot distillation is dark and
sticky and is used only for greasing ropes and leather goods
which are exposed to water. Developed countries have
created the most significant market for safflower oil for use as
salad oil and cooking oil and in making margarine; being nonallergenic, it is considered to be one of the healthiest oils for
human consumption because it has a high ratio of polyunsaturated/saturated fatty acids.

Safflower is used as a substitute for saffron; its flowers are
commonly mixed with rice, pickles and other foods to give
an attractive colour (Sastri, 1950). America, India and
Africa are the main producers of safflower oil. Its seeds are
edible and are eaten after roasting. The seed oil content varies
from 24 to 36%, depending on the variety of safflower, soil
texture, climate and other conditions (Pritchard, 1991; Swern,
1964a). There are two types of safflower oil: high oleic (high

Safflower was introduced as oilseed crop in Pakistan in 1960.
It is mainly cultivated in Sindh and Baluchistan provinces.
Being a drought-tolerant crop, it is recommended for planting
in rainfed areas. In Sindh it is cultivated after the rice crop on
residual moisture. Due to the increasing interest in the
safflower oil for edible purposes based on its high content of
linoleic acid, our studies are mainly focused on the content
and physical and chemical evaluation of the oils of two spineless varieties of safflower, Thori-78 and Pawari-95, grown in

*Author for correspondence; E-mail: razias@hotmail.com

Carthamus tinctorius Oil Quality

Sindh, Pakistan. Proximate analysis of oils were carried out
for the content, glycerides composition and physical and
chemical parameters such as free fatty acid, acid value, peroxide value, iodine value, refractive index, saponification
value, unsaponifiable matter, specific gravity and colour; the
fatty acid composition of both the varieties of oils were investigated as methyl esters by gas chromatography.

Materials and Methods
Plant material. Two varieties of Carthamus tictorius seeds,
Thori-78 and Pawari-95, were collected from Tandojam,
Sindh, Pakistan. The fruit is an achene (dry, one seeded with a
thin hull) and resembles sunflower seed but is smaller in size
and creamish in colour. It is irregularly pear-shaped, smooth
and shiny up to 10 mm long.
Reagents. Solvents and chemicals such as n-hexane (95%),
n-heptane (99%), ethanol (95%), carbon tetrachloride (95.5%),
chloroform (99.5%), methanol (98.8%), sulphuric acid
(95.98%), hydrochloric acid (37%), acetic acid (100%),
glacial acetic acid (99.5%); sodium hydroxide (98%), potassium hydroxide (98%), sodium thiosulphate pentahydrate
(R.G), oxalic acid (extra pure), potassium dichromate (extra
pure), potassium iodide (extra pure), iodine monochloride
(R.G) and anhydrous sodium sulphate were purchased from
E. Merck (Damstadt, Germany) and Labscan (Bankok,
Thialand). Standards of fatty acid methyl esters were purchased
from Supelco (Bellefonte, PA, USA) and Sigma Aldrich Co.
(St. Louis, MO, USA).
Apparatus. The apparatus used included gas chromatograph
with flame ionization detector, model Clarus 500, from Perkin
Elmer Instruments LLC, (Shelton, CT, USA), capillary
column Rtx-2330 (60 × 0.25 mm × 0.20 ìm, film thickness)
from Supelco (Bellefonte, PA, USA), Lovibond model E
tintometer (Salisbury, UK), Abbé refractometer model 2W
(Shijiazhuang, China), Gallen Kamp air oven (West Mildlands,
UK) and vacuum oven (Melrose Park, IL, USA).
Oil extraction. Safflower, Thori-78 and Pawari-95, seeds
(500 g each) were crushed and finely ground to flour and then
subjected to extraction with n-hexane (0.5 litre) in a one litre
Soxhlet extractor for 8 h (AOCS, 2004). The fat was recovered using a rotary evaporator. The extracted fat was placed
in an oven at 60 °C for 1 h, transferred to a capped reagent
bottle and stored at 4 °C until required.
Quantitative separation of tri-, di- and mono-acylglycerols
of oil. The lipid class composition, comprising of TAGs,
DAGs, and MAGs mixture in C. tinctorius seed oil was
determined by solid-liquid adsorption chromatography
(SLAC), using silica gel as the adsorbent and eluted with

different solvent systems by following the AOCS method
(AOCS, 2004) with little modification. The effectiveness of
separation was verified by thin layer chromatography, using
solvent system (petroleum-ether and acetone: 9:1).
Fatty acid composition. Methyl esters of fatty acids were
prepared according to standard IUPAC method 2.301 (IUPAC,
1987). The chemical composition of fatty acid methyl esters
was accomplished with a Perkin Elmer gas chromatograph
model Clarus 500 fitted with a polar capillary column Rtx2330 (60×0.25 mm×0.20 μm, film thickness) and a flame
ionization detector. Nitrogen was used as carrier gas at a flow
rate of 3 mL/min. Other conditions were as follows: initial
oven temperature, 70 °C was maintained for 5 min then ramped
at 10 °C/min to 180 °C, followed by 3 °C/min to final temperature of 220 °C, where it was held for 15 mins; injector
temperature and detector temperature was 270 °C. A sample
volume of 0.3 μL was injected (splitless). Fatty acid methyl
esters were identified by comparing their relative and absolute retention times to those of authentic standards of fatty
acid methyl esters purchased from Supelco Sigma-Aldrich Co.
Quantification was done by a built-in data-handling
programme, provided by the manufacturer of the gas chromatograph. Analyses were performed in triplicate.
Physical and chemical analysis of the extracted oils. The
following tests for refractive index, specific gravity, colour,
free fatty acid, acid value, peroxide value, iodine value,
saponification value and unsaponifiable matter of the extracted
oils were performed by the standard methods of AOCS (2004).
Colour of the oils was determined by a Lovi bond tintometer
(Tintometer Ltd., Salisbury, UK) using a one inch cell.

Results and Discussion
Hexane extracted oil content of the two varieties of
C. tinctorius, Thori-78 and Pawari-95, seeds was found to be
28.33±1.15 and 33.07±1.12%, respectively; the high percentage of oil gives these varieties distinct potential for the oil
industry, because the average oil content of the seeds exceeds
those of conventional oil seeds i.e., cotton (15.0-24.0%),
canola (17-21%), soyabean (17-21%), olive (20-25%) which
are grown in the USA, Brazil and Asia (Pritchard, 1991) but
oil content is slightly lower than that of sunflower (25-35%).
Physical and chemical parameters of the oils are depicted in
Table 1. At room temperature, both varieties of seed oil were
present in a liquid state. The refractive index and specific
gravity of Thori-78 and Pawari-95 oils were determined at
40 °C, which were concordant with the reported value and
comparable with other vegetable oils (Rossell, 1991a; Swern,
1964a; 1964b). The values determined for free fatty acids as

Razia Sultana et al.

OA and acid values are comparable with the reported values
of the crude oil (Dhellot et al., 2006; El-Adawy and Taha,
2001). Very low value of free fatty acid and acid value in the
present analysis is an indication of the good quality of crude
oil. Peroxide values (Table 1), indicating the presence of hydroperoxides in oils, were high, thus showing low resistance
to oxidation (Onyeike and Acheru et al., 2002); thus oils could
be used after slight refining. The analyzed crude oils were
high in colour index 2.13R + 45.4Y + 0.71N (Thori-78) and
2.48R + 46.16Y + 0.81N (Pawari-95). Intense colour of
vegetable oils depend mainly on the presence of various
colouring pigments of plants such as carotenoids, chlorophyll
etc., which are effectively removed during refining and bleaching steps of oil processing. Vegetable oils with minimum
values of colour index are good for edible purpose.

Table 1. Proximate and physicochemical characteristics of
C. tinctorius oils
Parameters

Thori-78

Pawari-95

Oil content

28.33±1.15
(27.11-29.88)

33.07±1.12
(31.5-34.0)

Free fatty acid
(% as OA)

0.52±0.012
(0.51-0.54)

0.53±0.004
(0.53-0.54)

Acid value (mg/kg)

1.12±0.15
(0.99-1.34)

1.06±0.02
(1.04-1.09)

Peroxide value
(Meq/kg)

17.2±0.25
(16.94-17.54)

20.75±0.38
(20.2-21.1)

Iodine value
(g of I/100 g of oil)

134.82±0.68
(133.99-135.67)

136.16±0.96
(134.89-137.21)

Saponification value 187.56±2.34
(mg of KOH/ g of oil) (184.89-190.60)

188.96±2.18
(186.5-191.8)

Unsaponifiable
matter (%)

0.41±0.06
(0.32-0.46)

0.57±0.05
(0.50-0.62)

Refractive index
at 40 oC

1.4734±0.0005
(1.4730-1.4742)

1.4679±0.0007
(1.4672-1.4689)

Specific gravity
at 40 oC

0.9064±0.002
(0.9031-0.9085)

0.9240±0.0004
(0.9234-0.9245)

Colour (Red unit)

2.13±0.04
(2.1-2.2)

2.48±0.08
(2.4-2.6)

(Yellow unit)

45.4±0.29
(45.0-45.7)

46.16±0.62
(45.5-47.0)

(Blue unit)

(Neutral unit)

0.71±0.06
(0.65-0.80)

0.81±0.01
(0.79-0.83)

Iodine values were comparatively high due to the presence of
high content of unsaturated fatty acids and are comparable
with the values of poppy, soybean and sunflower oils (Rossell,
1991a). High iodine value shows that both the varieties of
seed oils have good qualities of oils, required for edible and
drying purposes (Eromosele et al., 1994). Values of saponification and unsaponifiable matter of Thori-78 and Pawari-95
oils are concordant with the values of sunflower, poppy seed
and soybean oils (Rossell, 1991a; Swern, 1964a; 1964b), indicating them to be good source of industrial oil which can be
used in the manufacture of soap and liquid soap.
Table 2 shows glyceride composition of oils of both the
varieties of C. tinctorius. The content of triacylglycerides is
over 83%. Comparison of free fatty acid value, acid value,
saponification value, unsaponifiable matter, iodine value, refractive index, specific gravity and colour of the studied oils
with those of the known edible oils reveal that the quality of
both the varieties of oil have great potential for edible usage.
Table 2: Glyceride composition of C. tinctorius seed oils
(wt. %)
C. tinctorius
variety

Monoglyceride

Diglyceride

Triglyceride

Thori-78
Pawari-95

5.70±0.55
5.77±0.50

7.62±0.87
8.72±0.53

85.61±0.61
83.93±0.88

Values are means ± SD, analyzed in triplicate.

Fatty acid composition of the oils of the two varieties was
determined using gas chromatography (Fig. 1 and 2 and
Table 3). The principal fatty acid components in Thori-78 and
Pawari-95 were palmitic (C16:0), stearic (C18:0), oleic (C18:1) and
linoleic (C18:2) acids. Linoleic acid is predominantly present
in both the varieties as compared to other varieties, U.S.-10,
S.-208 and V. F.-stp (53-1) grown in Pakistan (Table 3) (Raie,
2008). Fatty acid composition was more or less similar to that
of sunflower, soybean, corn, and cotton seed oils and safflower
oil originating from different geographic regions (Cosge
et al., 2007; Rossell, 1991b). These results suggest that these
varieties of C. tinctorius can serve as potential dietary sources
of mono unsaturated fatty acid (MUFA) and poly unsaturated
fatty acid (PUFA).
Present studies revealed that seed oils of C. tinctorius varieties, Thori-78 and Pawari-95, indigenous to Pakistan have very
good potential for edible and industrial usage and also for use
in developing nutritionally balanced formulations blended with
other high stearic or high oleic oils. These Oils are used in the
food and pharmaceutical industries to produce cooking oils,

Carthamus tinctorius Oil Quality

C18:2

C18:1
C18:1

C16:0

C16:0
C18:0

C18:0

Retention time (min)

Fig. 1: Gas chromatogram of fatty acids of safflower
(Thori-78) seed oil; major components are
labelled.

Fig. 2: Gas chromatogram of fatty acids of safflower
(Pawari-95) seed oil; major components are
labelled.

food supplements and skin care products. The good drying
property and high content of linoleic acid and absence of
linolenic acid and wax and low content of free fatty acid, colour
and unsaponifiable compounds make them suitable for use in
the production of high quality paints, alkyd resins, coatings,
varnishes and linoleum. They can also be used in the production of biodiesel.

Pakistan imports huge amount of palm oil and soybean from
foreign countries to fulfil the increasing demand of oil in
the country. Moreover, Pakistan has suitable atmosphere for
cultivating all the conventional and non-conventional oilseed
crops. Cultivation of safflower varieties at larger scale could
fulfil the requirements of the country and save enormous
amount of foreign exchange spent otherwise.

Table 3. Fatty acid composition of high linoleic C. tinctorius varieties grown in Pakistan (wt. %)
Fatty acids

Thori-78

Pawari-95

US-10

S.-208

V.F.-stp
(53-1)

Myristic Acid
(C14:0)
Palmitic Acid
(C16:0)
Stearic Acid
(C18:0)
Oleic Acid
(C18:1)
Linoleic Acid
(C18:2)
Others

3.1

0.9

2.8

6.45±0.57
(5.66-7.02)
1.99±0.09
(1.89-2.12)
15.55±0.30
(15.31-15.98)
75.42±0.59
(74.65-76.11)
0.59±0.04
(0.54-0.64)

6.92±0.37
(6.41-7.28)
2.77±0.49
(2.24-3.42)
13.15±0.49
(12.67-13.81)
76.40±1.0
(75.01-77.32)
0.76±0.06
(0.68-0.83)

10.2

9.4

12.0

5.5

2.3

3.6

14.4

14.0

15.7

66.8

73.4

65.9

8.44

9.69

18.8

12.6

18.4

90.97

89.55

81.2

87.4

81.6

Total saturated
fatty acids
Total unsaturated
fatty acids

Values are mean ± SD, analyzed in triplicate.

Conclusion
These studies were focussed on the yield and physical and
chemical evaluation of seed oils of C. tinctorius varieties,
Thori-78 and Pawari-95, cultivated in the region of Sindh,
Pakistan. It was revealed that oils of both the varieties have
very good potential for developing nutritionally blended
formulations balanced with other high saturated fatty oils, as
well as for different industrial usage due to the presence of
high percentage of polyunsaturated fatty acids. These can also
be used in the production of biodiesel and thus in different
industries in Pakistan.

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Pak. J. Sci. Ind. Res. 2010 53 (1) 20-24

Analysis of Caffeine and Heavy Metal Contents in Branded and
Unbranded Tea Available in Pakistan
Asma Inayat a*, Shahid Rehman Khan a, Muhammad Nawaz Chowdhry b and Amran Waheed b
a

Applied Chemistry Research Centre, PCSIR Laboratories Complex Lahore - 54600, Pakistan
b
College of Earth and Environmental Science, University of Punbjab, Lahore, Pakistan
(received November 10, 2008; revised December 2, 2009; accepted December 10, 2009)

Abstract. In the investigation of caffeine and heavy metal contents in four branded and six unbranded tea samples
collected from local markets of Lahore, Faisalabad and Peshawar, the amount of caffeine and heavy metals in all the
branded tea samples were in agreement with the international standards. In unbranded tea samples, though the amount
of caffeine was within standard limits but two of the samples collected from Peshawar had high concentrations of lead
being, 13.69 and 15.78 mg/kg, consumption of which can lead to serious problems.
Keywords: tea, caffeine, heavy metals

Introduction

metals and essential trace elements that can otherwise
accumulate in bone, hair and soft tissues such as liver,
kidney, brain or lungs (Tautkus et al., 2004).

Tea (Camellia sinensis) is one of the most popular beverages
all over the world. According to an estimate, 2.5 million metric
tonnes of dried tea is manufactured annually, 75% of which is
processed as black tea and consumed in different countries.
In UK, on an average, one litre of tea is consumed per person
per day (Al-Oud, 2003). Different brands of tea are manufactured to meet the increasing demands of consumers worldwide. Positive and negative effects of tea on the health have
been investigated by many researchers, recently (Yao et al.,
2006a).

Materials and Methods
Caffeine. Preparation of tea solution. Two grams of tea were
added to boiling water, (200 mL) in a 250 mL conical flask placed
on a hot plate at 90 °C while stirring for 10 min by a magnetic
bar. Then the tea solution was filtered through cotton wool
and the residue was washed thrice with distilled water
(10 mL). The tea solution was cooled to room temperature and
washings were diluted to 250 mL with distilled water. The
sample was analyzed in duplicate.

Caffeine ascribes quality characteristics to tea, such as
briskness, taste etc., and has been considered an important
quality parameter in the evaluation of tea quality (Yao et al.,
2006b). Caffeine is a pharmacologically active substance and,
depending on the dose, can be a mild nervous system
stimulant. Caffeine does not accumulate in the body and is
normally excreted within several hours of consumption
(Mumin et al., 2006; Obanda et al., 1999). Human body
requires both metallic and non-metallic elements for healthy
growth and development within certain permissible limits.
The optimum concentration needed for this purpose varies
widely from one element to another, from infant to childhood
to adult and from male to female (Atta, 1995). Determination
of these elements in beverages, water, food, plant and soil is
thus of utmost important. Tremendous research has been
rendered on finding tolerance limits for daily intake of nearly
all essential elements needed for healthy growth and sound
physiological changes in human body. There is a fairly
narrow gap between the essential and the toxic levels of

Measurement. To 10 mL of tea solution, 5 mL HCl (0.9 mL of
36% HCl was diluted to 1000 mL with distilled water) and 1 mL
lead acetate solution (100 g of lead acetate was dissolved in
small quantity of water and diluted to 200 mL with distilled
water) were added and diluted upto 100 mL with distilled
water. The solution was then filtered through Whatman filter
paper # 42. Filtrate 25 mL and 0.3 mL sulphuric acid (167 mL of
98% H2SO4 was diluted to 1000 mL with distilled water) were
placed in a volumetric flask and diluted to 50 mL with distilled
water. The solution was filtered using the same type of filter
paper. Absorbance of the filtrate was measured using spectrophotometer (Spectronic Unicam) at 274 nm. Readings were
taken in duplicate.
Standard curve. Caffeine stock solution (20 mg caffeine/10 mL,
w/v distilled water) was diluted to 200 mL with distilled water.
Next, 0, 10, 20, 30, 40 and 50 mL of the diluted caffeine solution
were separately mixed, each with 4 mL HCl in a volumetric
flask and diluted to 100 mL with distilled water. Thereafter, the

*Author for correspondence; E-mail: asimainayat@hotmail.com

Caffeine and Heavy Metals in Tea

measuring steps were repeated as described above. Readings
of the absorption of the standard solution against its concentration were used to prepare the standard curve. Following
formula was used to calculate the caffeine contents:
Caffeine (%) = (E/1000) × Vo × (100 × V1) × (50/25) / W
or
= 0.2 EVo /V1 /W
where:
E is caffeine (mg) from the standard curve against the reading of the spectrophotometer
E/1000 is used to convert ‘mg’ into ‘g’,
Vo is the total volume of tea solution (250 mL),
V1 is the volume used for the measurement (10 mL),

solutions. For the determination of metals in tea, 3 g of the
oven dried (105 °C) samples were preheated for 30 min at
250 °C and burned for one hour at 800-850 °C. The resulting
ash was wetted with double distilled water and mixed with 10
mL of diluted HCl (1:1). The mixture was mildly boiled, cooled
to room temperature, filtered (if necessary), transferred to
100 mL volumetric flask and diluted with double distilled
water.
A Varian (AA 240) flame atomic absorption spectrophotometer equipped with hollow cathode lamps was used for the
analysis. The instrumental setup was adjusted according to
the manufacturer’s instructions.

Results and Discussion

100/V1 indicates 10 mL tea solution that were diluted to
1000 mL,
50/25 shows another dilution from 25 mL filtrate made to
50 mL in the measurement and
W is the dry weight of the tea sample.
Heavy metals. Double distilled water and analytical grade
reagents were used in all the experiments. Standard solution
(10 ppm) of heavy metals i.e., Zn, Cu, Cd, Co, Pb, Mn and Ni
were prepared by taking 1 mL of each heavy metal stock
solution (1000 ppm) in a 100 mL volumetric flask and diluted
upto the mark with double distilled water. Heavy metal
working standard solutions i.e., 0.5, 1.0, 1.5 and 2.0 ppm were
prepared by diluting 5, 10, 15 and 20 mL, respectively, standard
solution (10 ppm) of each metal made upto 100 mL with double
distilled water.
The calibration curve used for the determination of heavy
metals in tea samples by flame atomic absorption spectrometry (FAAS) was established using the working standard

The spectrophotometric method is the most common
method used for the determination of caffeine. Flame atomic
absorption spectrometry (FAAS) is one of the techniques
most extensively used for determining various elements with
a significant precision and accuracy. This analytical technique
is remarkable for its selectivity, speed and fairly low operation
cost. However, in some cases it is rather difficult to determine
traces of heavy metals in environmental samples due to unsufficient sensitivity or matrix interferences. Thus, a pre-concentration or/and separation step is necessary.
Caffeine. Caffeine content in different tea samples ranged from
3.41 to 3.74% with a mean of 3.56% of the dry mass (Table 1).
Earlier studies showed that caffeine content in black tea was
affected by clone of plant, season and stage of plucking of
leaves (Obanda and Owuor, 1995). Caffeine content can vary
in tea leaves from 24% to 40% depending on the maturity i.e.,
the young leaves may contain more caffeine than the older tea
leaves (Owuor and Orchard, 1992; Owuor et al., 1987). The
results (Table 1) of the current study showed that the mean
caffeine content in tea varieties marketed in Pakistan was

Table 1. Concentrations of caffeine and heavy metals in branded and unbranded tea samples
Tea sample
(code)

Caffeine
(%)

Branded (S 1)
Branded (S 2)
Branded (S 3)
Branded (S 4)
Unbranded (LHR 1) (S 5)
Unbranded (LHR 2) (S 6)
Unbranded (FSD 1) (S 7)
Unbranded (FSD 2) (S 8)
Unbranded (PWR 1) (S 9)
Unbranded (PWR 2) (S 10)

3.41
3.45
3.42
3.46
3.64
3.72
3.53
3.49
3.74
3.73

1.20
1.05
0.75
0.75
0.50
1.0
0.50
0.75
1.45
0.75

nd
nd
nd
nd
0.25
0.28
0.25
0.50
0.34
0.25

2.75
40.50
0.25
39.75
19.50
15.70
16.10
25.75
25.50
16.50

nd = not detected.

Heavy metals (mg/kg)
Co
Ni
0.69
0.60
0.65
0.61
0.10
1.29
1.20
1.14
1.02
0.89

4.58
4.58
4.59
4.58
9.71
9.27
9.98
8.66
10.01
9.69

nd
nd
0.25
1.00
nd
0.76
0.84
0.25
nd
0.75

4.64
3.28
4.75
4.28
4.64
4.59
4.20
3.75
13.69
15.78

Asma Inayat et al.

22
similar to the values reported in literature, 3.32 to 31.81%, and
also that the tea varieties originating from different sources,
with differences in clones, seasons and maturity, differ in
caffein content (Gulati et al., 1999).

Cadmium. Cd is another toxic metal not required by any
living being. In the four branded tea samples (1, 2, 3 and 4)
Cd was not detected while in other samples its value ranged
from 0.20 to 0.50 mg/kg (Table 1). It may have been introduced
during packaging or the tea plants might have absorbed this
pollutant from the surrounding air and the soil. Thus consumption of such tea for long period of time is dangerous due
to Cd contamination. Cd damages nerve cells; it inhibits the
release of acetylcholine and activates cholinesterase enzyme,
resulting in hyper activity of the nervous system. By altering
calcium and phosphorus metabolism, toxic level of Cd can
contribute to arthritis, osteoporosis and neuromuscular
diseases. In cardio-vascular system, Cd replaces Zn in the
arteries making them brittle and inflexible. Cd accumulates
in the kidneys and once accumulated, it stays there and is not
removed through excretion, resulting in high blood pressure
and kidney diseases.

Heavy metals. Results of the study show that different tea
samples contain the elements Cu, Cd, Mn, Co, Ni, Zn and Pb
in various proportions (Table 1 and Fig. 1). Variations in
elemental contents from sample to sample can be attributed to
the differences in botanical profile as well as the mineral composition of the soil in which plants are cultivated. Other
factors responsible for variation in elemental contents may be
pre-ferential absorbability of the plant, fertilizers, irrigation
water, climatic conditions and, most of all, differences in the
methods of processing and packing of tea leaves. Deposition
of various metals from the vehicular emission and other
sources on the open tea leaves could contribute to differences in analytical results.

Copper. In all the tea samples, high Cu concentration was
found. In sample-9, concentration of Cu was 1.45 mg/kg
(Table 1), while in all other samples, its concentration varied
from 0.50 to 1.20 mg/kg. Although Cu is an essential enzymatic
element for normal plant growth and development but at
excessive levels, it can be toxic. Phytotoxicity can occur if its
concentration in plants is higher than 20 mg/kg. Copper
accumulation in body can result in a tendency for hyperactivity in autistic children. An excess of Cu can cause
stuttering, insomnia and hypertension as well as oily skin,
loss of skin tone (due to blocking of vitamin C absorption)
and dark pigmentation of the skin, usually around the face. Cu
can also make nails brittle and thin and may contribute to hair
loss especially in women.

Lead. Lead was found in variable amounts in all tea samples
(Table 1). For example high concentration of lead i.e., 13.69
and 15.78 mg/kg were found in two tea samples (S-9 and 10,
respectively), while in other samples the amount of Pb varied
from 3.28 to 4.75 mg/kg which is lower than the prescribed
Caffine limit of WHO (1998) for Pb content in herbs i.e. 10 mg/kg. It is
believed that 95% of Pb present in plants is due to foliar
Cu
uptake from the surroundings. As majority of the tea varieties
Cd
Mn
are imported from other countries, so source plants with
Co
different origins have different air, water and soil environNi
ment. The high concentration of Pb in plants growing above
Zn
the ground is due to air borne Pb and the surrounding
Pb
polluted environment.

50
1

Caffeine

Conc. (mg/kg)

3
4

5
6

7
8

1 2 3 4 5 67 8

S-1

S-2

1 2 3 4 5 67 8

S-3

1 2 3 4 5 67 8

S-4

1 2 3 4 5 67 8

S-5

1 2 3 4 5 67 8

S-6

Sample number

Fig. 1. Concentration of caffeine and heavy metals in different tea samples.

1 2 3 4 5 67 8

S-7

1 2 3 4 5 67 8

S-8

1 2 3 4 5 67 8

S-9

1 2 3 4 5 67 8

S-10

Caffeine and Heavy Metals in Tea

Zinc. Zn is an important metal required by both plants and
animals. The highest concentrations of Zn (1.0 mg/kg) was
found in one branded sample (sample-4), while in other
samples, its amount varied from 0.25 to 0.84 mg/kg (Table 1).
In few tea samples, its concentration was very low. Zn is very
important for plant and human life. In the blood about 85%
of Zn combines with proteins for the transport of the latter
after its absorption and its turnover is rapid in the pancreas.
Deficiency of Zn causes diabetic hyposmia, hypogensia or
coma.
Nickel. In all the studied unbranded tea samples, higher
amount of Ni was recorded: 10.01 mg/kg in sample-9, followed
by 8.66-9.98 mg/kg in five samples (samples 5, 6, 7, 8 and 10),
while equal amounts of Ni (4.58 mg/kg) was found in all the
branded samples. Ni plays an important role in the production
of insulin in the pancreas. Its deficiency results in disorder of
the liver. However, higher concentration, of Ni have adverse
effects on health. For example Ni tends to accumulate in
kidneys causing kidney damage. Being a common ingredient
in fashion jewellery, Ni can cause allergic reactions in some
wearers; eczema and even asthma attacks may develop. A
steady exposure ot Ni can cause cancer of lungs and nasal
sinus.
Cobalt. As shown in Table 1, the highest concentration of Co
was found in one sample (sample 6, 1.29 mg/kg), followed
by 1.20 mg/kg in sample-7. Other tea samples had a concentration of 0.60 mg/kg to 1.14 mg/kg. Thus as a whole,
considerable variations were recorded in all the tea samples
which may be due to the differences in the air, soil and water
environment of the source plants as well as local activities
in their nearby surroundings. Human body needs Co in trace
amounts and it is toxic in higher concentrations. Co in the
form of vitamin B12 is in its physiologically active form.
Manganese. Mn was the most abundant metal found in tea
samples in variable amounts (Table 1). For example high Mn
concentration was found in two branded samples No.2 and 4,
being 40.50 and 39.75 mg/kg, respectively, and 25.75 and 25.50
mg/kg in two unbranded samples, samples 8 and 9, respectively. The least amount of Mn was found in one branded
sample (sample 3) being 0.25 mg/kg. The presence of Mn in
tea samples may be due to its use as a colouring material for
tea leaves.
Daily intake of heavy metals. Due to lack of information about
the maximum allowable levels of heavy metals in tea leaves
the discussion will be extended to the acceptable daily intake
that can be taken into account through foods and drinking
water. For intance, the expected calculated intake of Mn in the
present study was 121.38 mg/day for tea (Table 2). This value

23
is much higher than the intake through food (0.008) and at the
same time nearly equal to 3.8 mg/day through drinking water
according to the standards of US Environmental Protection
Agency (MAFF, 1997). The calculated overall mean intake of
Ni through tea was 49.39 mg/day. This value is in agreement
with the human requirements (50 mg/day). However, this value
is higher than the average intake in the UK (0.13 mg/day)
recorded for the total diet studied (MAFF, 1999). According
to MAFF (1998) tea beverage has considerable amount of Ni
that could significantly contribute to daily intake of metals.
Calculated intake of Cu through tea was 5.22 μg daily or
0.005 mg daily. This value was much lower than that of 1.2 mg
daily set by UK and WHO (1998).

Table 2. Daily intake of different heavy metals
Heavy metal

Total (ppm)

Intake (mg/day)

Cu
Cd
Mn
Co
Ni
Zn
Pb

0.87
0.18
20.23
0.91
7.56
0.38
6.36

5.22
1.12
121.38
5.46
49.39
2.31
38.16

The mentioned values were calculated based on the assumption that the average consumption of tea beverage for a single
person is three cups a day with one packet of 2 g in a cup, i.e.,
6 g of tea particles/day.
The results show (Table 2) that only small part of heavy metal
content through tea leaves may be introduced into beverage
preparation. All branded and unbranded tea samples were
found to contain heavey metal contents within safe levels
except for only two unbranded samples collected from
Peshawar, which had high concentrations of Pb.

References
Al-Oud, S.S. 2003. Heavy metal contents in tea and herb
leaves. Pakistan Journal of Biological Sciences, 6:
208-212.
Atta, M.B. 1995. Aluminium content in dust black tea leaves
and its beverages. Menofiya. Journal of Agricultural
Research, 20: 137-150.
Gulati, A., Ravindranath, S.D. 1999. Seasonal variations in
quality of kangra tea Amellia sinensis (L.) O kuntze) in
Himachal Pradesh. Journal of the Science of Food and
Agriculture, 71: 231-236.

24
MAFF, 1999. Total diet study: Aluminium, arsenic, cadmium,
chromium, copper, lead, mercury, nickel, selenium, tin
and zinc. Food Surveillance Information Sheet No. 191.
Ministry of Agriculture Fisheries and Food (1997), UK.
MAFF 1998. Lead, Arsenic and other metals in food. Food
Surveillance Paper No.52. London, UK.
MAFF 1997. Total diet study : metals and other elements.
Food Surveillance Information Sheet No. 131, Ministry
of Agriculture Fisheries and Food, UK.
Mumin, M.A., Akhter, K.F., Abedin, M.Z., Hossain, M.Z. 2006.
Determination and characterization of caffeine in tea,
coffee and soft drinks by solid phase extraction and
high performance liquid chromatography (SPE-HPLC).
Malaysian Journal of Chemistry, 8: 45-51.
Obanda, M., Owuor P.O., Taylor, S.J. 1999. Flavanol composition and caffeine content of green leaf as quality
potential indicators of Kenyan black teas. Journal of
the Science of Food and Agriculture, 74: 209-215.
Obanda, M., Owuor, P.O. 1995. Changes in black tea quality
chemical parameter due to storage, duration and packaging method. Tea, 16: 34-40.

Asma Inayat et al.
Owuor, P.O., Orchard, J.E. 1992. Effects of storage time in a
two stage withering process on the quality of seedling
black tea. Food Chemistry, 45: 45-49.
Owuor, P.O., Obanda, A.M., Tsushida, T., Horita, H., Murai, T.
1987. Geographical variation of theaflavin, thearubigins
and caffeine in Kenyan clonal black teas. Food Chemistry,
26: 223-230.
Tautkus, S., Kazlauskas, R., Kareiva, A. 2004. Determination
of copper in tea leaves by flame atomic absorption
spectrometry. Chemistry, 15: 49-52.
WHO, 1998. Guidelines for Drinking Water Quality, 2nd
edition, Addendum to volume 2: Health Criteria and
other Supporting Information, pp. 46-61, World Health
Organization, Geneva, Switzerland.
Yao, L.H., Jiang, Y.M., Caffin, N., Arcy, B.D., Datta, N., Liu, X.,
Singanusong, R. Xu, Y. 2006a. Phenol compounds in tea
from Australian supermarkets. Food Chemistry, 96: 614-620.
Yao, L., Liu, X., Jiang, Y., Caffin, N., Arcy, B.D., Singanusong,
R., Datta, N., Xu, Y. 2006b. Compositional analysis of
teas from Australian supermarkets. Food Chemistry,
94: 115-122.

Pak. J. Sci. Ind. Res. 2010 53(1) 25-29

Measurement of Atmospheric Concentrations of CO, SO2, NO and NOx
in Urban Areas of Karachi City, Pakistan
Durdana Rais Hashmi*, Farooq Ahmad Khan, Akhtar Shareef, Farhan Aziz Abbasi,
Ghulam Hussain Sheikh and Alia Bano Munshi
Centre for Environmental Studies, PCSIR Laboratories Complex, Sharah-e-Dr. Salimuzzaman Siddiqui,
Karachi-75280, Pakistan
(received May 28, 2009; revised December 8, 2009; accepted December 12, 2009)

Abstract. In the assessment of variation trends in ambient air quality at five selected regions of Karachi city, four air
pollutants namely carbon monoxide, sulphur dioxide, nitrogen oxide and nitrogen dioxide were monitored, along with
metrological parameters, for eight consecutive days. The results suggested that all the pollutants were mainly due to
the emissions from motor vehicles and industries, owing to the absence of regulatory laws/standards about ambient air
quality in Pakistan. The results have been discussed with reference to recommendations of the World Health Organization for the same.
Keywords: air pollution, industrial emission, vehicular emission, atmosphere

rate base-line data on these localities by air pollution monitoring analyzers, to identify major sources of air pollution and
suggest their remedial measures. The data so generated may
assist in the formulation of the country air quality standards.
Information about the industries was obtained from different
civic agencies and the Department of Industries.

Introduction
Karachi is the largest metropolitan city of Pakistan having an
estimated population of above 10 million. Total amount and
complexity of toxic pollutants in the environment of Karachi are
increasing day by day with the rapid increase of population
and proportional increase of industries, vehicular traffic and open
air garbage burning. Rate of atmospheric pollution is 40 percent
higher in Karachi than the other cities of Pakistan (Qureshi, 1997).

Materials and Methods
The subtropical city of Karachi is located in a semiarid zone.
It is the largest industrial and commercial centre in Pakistan
and declared as one of the twenty mega cities of the world
(Mage et al., 1996). Growing urban population, industrialization and traffic congestion are the main causes of air pollution
in Karachi city. In order to assess the load of air pollutants in
the environments of the city, monitoring of different air
pollutants was carried out at five different locations (as shown
in location map) of the city, categorized as follows:

Typical major ambient air pollutants in the urban environment include CO, SO2, NO, NOx, HC and PM10. CO is
formed during combustion of carbon containing compounds.
It is a toxic gas and its prolonged exposure, even at very low
levels, may adversely affect central nervous system. When
inhaled, it reacts with the haemoglobin of the blood stream
to form carboxy-haemoglobin. CO attaches to haemoglobin
roughly about 210 times more than the oxygen (All
Refer.com, 2005). SO2 is also generated by the combustion
of high sulphur fuels. SO2 is toxic to human body especially
for persons having previous history of respiratory diseases,
such as emphysema; besides, it also causes pneumonia.
Nitrogen oxides are generated at high temperatures during
combustion. Their ultimate effect on human beings is still
not clearly understood, but they act as irritants to breathing
and create discomfort to eyes and also destroy the celia in
the respiratory system.

1- Region A: the site with urban background, moderately
populated, having low vehicular traffic density. It is
one km distant from the main super highway. The area
around the sampling site is sparsely populated.
2- Region B: a commercial site, densely populated, having
high vehicular traffic density. This site is the busiest
intersection of Karachi, surrounded by multistoried
commercial as well as residential buildings. The population around this site mostly belongs to high income group.
3- Region C: an industrial area in district South of Karachi,
with nearly 2000 different types of industries, approximately 60 percent comprising of textile mills, while

Present study was carried out in various industrial, residential/
commercial and down-town regions of Karachi city to gene*Author for correspondence; E-mail: drhpak@yahoo.com

Durdana Rais Hashmi et al.

Location map: sample collection points

other industries are related to pharmaceuticals, chemicals, detergents, iron and steel, sulphur refining, vegetable
oils, beverages and food products.
4- Region D: an industrial area in district East of Karachi
with approximately 2000 various types of industries
including tanneries (more than 100 units), pharmaceutical, textile and chemical units and refineries etc.
5- Region E: also an industrial area in district East of
Karachi with 300 industrial units of different categories including textile, food, chemical, pharmaceutical
and engineering units.
Measurement of major ambient air pollution components
such as CO, SO2, NO and NO2 was carried out in summer
season, for eight consecutive days at each of the five
stations. Average variations of the pollutants were
recorded for 11 hours at hourly intervals, at the selected
regions (A-E).
Air quality measurements were performed, using air analyzer, designed and fabricated by Environmental SA,
France. Average values of CO, SO2, NO, NO2 and NOx

concentrations for 15 min were used for determining daily
average hourly concentrations. The daily hourly average
concentration values were further averaged for determining values for 8 days and for time weighted average (TWA)
values for 1 h, 8 h and 24 h for each region.
UV fluorescent SO2 Analyzer Model AF21 M consisted of
zinc ray UV lamp with stabilized power supply, continuous
energy monitor and compensation for measurement at
constant energy level and integrated carbon kicker for
continuous removal of interfering hydrocarbons.
The chemiluminescent NO-NOx Analyzer Model AC 31M
was of two channel type coupled with serial R232 output
signal processing and continuous zero control by the microprocessor. The air sampled by a pump placed at the circuit
end, is carried, on the one hand via a converter oven towards
the NOx chamber and on the other hand, directly into NO
chamber. The radiation emitted in the NOx chamber is
proportional to NO+NO2 (reduced to NO).
Concentration of carbon monoxide was measured by Snift
CO Analyzer (Model 50). The meter was kept at about 1.2 m

Inorganic Ion Concentrations in Urban Karachi Atmosphere

above the ground level and readings were taken at intervals
of 15 minutes.

SO2 is considered to be the vehicular traffic, whereas, at
regions C, D, and E, the combustion of fuels in the nearby
industries.

Results and Discussion

The highest average concentrations of NO were found to be
17.2, 231.3, 193.5, 157.4 and 132.5 ppb (Fig. 3), and those of
NO2, 9.8, 127.3, 122.2, 103.2 and 79.2 ppb (Fig. 4), whereas
the highest average concentrations of NOx were found
to be 27.0, 358.6, 315.7, 260.6 and 211.7 ppb (Fig. 5) in the
selected regions A, B, C, D and E, respectively.

Hourly average variations of the pollutants, recorded at five
selected regions A, B, C, D and E are graphically presented in
Fig. 1 to 5. Table 1 gives the time weighted average (TWA)
values for 1, 8, and 24 h, along with permissible ambient air
quality limits, recommended by WHO.
Maximum average concentration of CO was found to be 4,
21, 11, 9, and 6 ppm in regions A, B, C, D and E, respectively
(Fig. 1). The main source of CO at regions A and B may be
motor vehicles plying on nearby main super highway and
University Road, where traffic density is high, whereas, at
regions C, D and E, the combustion of fuels in nearby industries and power generation plants.
Maximum hourly average concentrations of SO2 were 5.7,
349.1, 74.5, 42.2 and 21.2 ppb at the regions A, B, C, D, and
E, respectively, the highest concentration of SO2 being
349.1, recorded at region B (Fig. 2). The main cause of this
high concentration of SO2 at this region may be very high
traffic density due to narrow and congested roads, surrounded
by high rising buildings. At region A, the main source of

1000

100

8:00
A 3.4
B 175.2
C 29.1
D 41.3
E 10.2

Local time in hr

5.7 3.9
349.1 123.6
58.9 31.1
15.2 15.7
21.1 6.7

2.9 5.1
142.1 179.6
19.3 29.6
13.9 39.6
6.7 10.2

2.1 2.9 4.3 2.1
202.4 221.3 140.6 197.2
34.2 36.7 23.6 41.9
24.1 21.5 16.3 20.1
13.4 11.9 9.2 8.1

1.7 3.1 2.8
213.7 250.9 249.1
67.3 74.5 61.3
42.2 34.4 27.1
17.6 21.2 19.3

Fig. 2. Hourly average SO2 concentrations in regions A-E.

250

200

9:00 10:00 11:00 12:00 1:00 2:00 3:00 4:00 5:00 6:00 7:00

150

10
50
5
0 8:00 9:00 10:00 11:00 12:00 1:00 2:00 3:00 4:00 5:00 6:00 7:00
A 3
4
2
2
2
4
3
4
2
3
3
4
B 13 21 10 11 13 15 19 11 13 17 19 21
9
C 7
6
5
9 10 7
5
7
9 11 10
8
5
5
D 6
4
6
7
3
5
7
6
9
5
3
3
3
4
6
2
3
4
5
6
E 4

Local time in hr

Fig. 1. Hourly average CO concentrations in regions A-E.

8:00 9:00 10:00
A 13.5 10.1 9.6
B 167.5 152.9 135.3
C 182.2 193.5 112.3
D 131.4 112.2 98.6
E 105.6 91.3 80.5

Local time in hr

11:00 12:00 1:00 2:00
9 8.6 8.1 7.5
117.6 96.2 84.8 80.5
100.4 79.1 68.8 63.7
80.5 61.3 53.5 51.2
61.4 46.7 41.6 40.2

3:00 4:00 5:00 6:00 7:00
6.7
95.1
60.4
47.8
38.1

7.9 9.2 12.3 17.2
112.7 130.5 119.3 231.3
81.3 112.3 134.4 186.5
64.5 97.2 123.3 157.4
51.1 82.6 107.9 132.5

Fig. 3. Hourly average NO concentrations in regions A-E.

Table 1. Concentration of CO, SO2 and NO2 evaluated for 1, 8 and 24 h (TWA) and permissible limits of WHO.
Pollutants
Region A

Time weighted average (TWA) values
Region B
Region C
Region D
Region E

SO2

3
2.1
4

12.25
15.75
159.1

6.7
8.8
42.1

3.7
4.1
30.6

3.5
3.8
15.9

NO2

5.6

79.4

47.7

35.9

32.9

WHO

Unit

30
10
350
100-150
400
150

mg/m3
μg/m3
μg/m3

Averaging
time
1h
8h
1h
24 h
1h
24 h

Durdana Rais Hashmi et al.

140
120
100
80
60
40
20
0

Local time in hr

8:00 9:00 10:00 11:00 12:00 1:00 2:00 3:00 4:00 5:00 6:00 7:00

A 5.2 4.1
B 96.4 90.1
C 103.5 122.2
D 71.2 64.2
E 57.5 50.3

3.3
81.2
63.3
98.6
80.5

3.1
69.1
52.5
40.3
31.4

2.7
61.6
41.6
30.5
23.7

2.1
54.5
36.8
27.6
20.8

1.8
52.1
32.1
23.2
20.1

3.1
51.5
30.6
27.1
17.2

4.2
67.7
41.4
36.7
24.6

6.4
91.3
58.2
47.6
32.7

7.9
116.5
101.2
84.7
59.9

9.8
127.3
119.6
103.2
79.2

Fig. 4. Hourly average NO2 concentrations in regions A-E.
400
350
300
250
200
150
100
50
0

Local time in hr

A
B
C
D
E

8:00 9:00 10:00 11:00 12:00 1:00 2:00 3:00 4:00 5:00 6:00 7:00
18.7 14.2 12.9 12.1 11.3 10.2 9.3 9.8 12.1 15.6 20.2
263.9 243 186.7 186.7 157.8 139.3 132.6 146.6 180.4 221.8 307.8
285.7 315.7 152.9 152.9 120.7 105.6 95.8 91 122.7 170.5 235.6
202.6 176.4 120.8 120.8 91.8 81.1 74.4 74.9 101.2 144.8 208
163.1 141.6 122.7 92.8 70.4 62.4 60.3 55.3 75.7 115.3 167.8

27
358.6
306.1
103.2
79.2

Fig. 5. Hourly average NOx concentrations in regions A-E.
Carbon monoxide is not easily detected by olfactory senses.
High concentration of this pollutant in central parts of the
cities due to traffic jams may creat serious problems. (ALA,
2000; WDNR, 2000). Exposure to carbon monoxide may lead
to head ache, tiredness, dizziness, nausea, vomiting and
drowsiness and in very acute situations, to unconsciousness
and even death (Malakootian and Yaghmaeian, 2004). Exposure to elevated carbon monoxide level is associated
with impairment of visual perception, work capacity, manual
dexterity, learning ability and performance of complex tasks
(Aziz and Qureshi, 2003).
In region A, the highest hourly average concentration of CO
was recorded to be 4 ppm, from 7:00 to 9:00 a.m. and from
5:00 to 7:00 p.m. In the morning, the movement of traffic is
down town and is reverse in the evening. Variations in the
concentrations of carbon monoxide show that the concentration gradually increases till 9:00 a.m. and then comes down at
1:00 p.m. and again increases around 6:00 p.m. which are the
rush hours. In region A, the air pollution was generated by
vehicular traffic as the air currents were coming from main
Super Highway that has quite high traffic density.

In region B, the pollution generation is mainly due to vehicular traffic. This site is the busiest intersection on M.A. Jinnah
Road having high traffic density and traffic jams with highrising buildings on both sides of the road creating tunnel
effect. On the contrary, in regions C,D, and E, the
pollution generation may be due to the emissions from
nearby industries, power generation plants and boilers of the
industries.
Sulphur dioxide originates mostly from the combustion of
trace amounts of inorganic and organic sulphur, contained in
the fuel. The estimated background concentration of SO2 is
0.2 ppb and calculated atmospheric residence time is 4 days
(Bhatia, 2005). Short term high level of SO2 may enhance
respiratory diseases, lung function disturbance and even
mortality in adults and children (Nautiyal et al., 2007). The
maximum average concentrations of SO2 at regions A and B
were found to be 5.7 and 349.1 ppb, respectively, during 7.00
to 9.00 a.m. At these stations, the high concentration of SO2
may be due to the fuel combustion by vehicular traffic. At
regions C, D and E the highest values of SO2 were 74.5, 42.2
and 21.2 ppb, respectively, at 6:00 p.m. The main source of
SO2 at station C may be power plants and boilers of the industries and at stations D and E, a large oil refinery, all located in
SW directionof the areas. Relatively high concentration of
SO2 obtained at regions C, D, and E, during specified period
may be due to emissions from the nearby industrial units.
Nitrogen oxides are of great concern, being precursors in
ozone production in the presence of sun light. NO and NO2
are emitted together from combustion sources and exist in
equilibrium in the atmosphere; together, they are usually
referred to as NOx. The diurnal pattern of NO and NOx
has correlation with solar energy. A distinct photochemical
relation between NO, NOx and solar energy has been established and as the solar energy increases during the day time,
the level of NO, NOx decreases. The reaction of photochemical oxidants has a time scale of one to a few minutes (Clark,
1988). At region C, the highest concentrations of NO and
NOx were found to be 193.5 ppb and 306.1 ppb, respectively,
between 7.00 to 9.00 a.m. which may originate from the combustion of industries and power plants, about 45-60 meters
away. The reaction is therefore, even more rapid here, having
a time scale of only few seconds. The chemical reaction
between the two mixing species was not completed due to
time lag and shows high concentration of NO and NOx
during the day time. However, at regions A, B, D and E, the
highest values of hourly average concentration of NO and
NOx were found before the sunrise which started decreasing
as the ultra-violet radiations from the sun increased, and again
increasing with the decrease of ultra-violet radiations from
the sun. The main contributor of NO and NO2 at regions A
and B were the emissions from the vehicular traffic due to

Inorganic Ion Concentrations in Urban Karachi Atmosphere

combustion of fuel, whereas, at regions D and E, the combustion gases emitted by the industries.
A comparison of the time weighted average values of all
the measured pollutants at the selected stations, with those of
the WHO recommended air quality guidelines shows that
the concentrations of ambient air pollutants found at these
stations are well within the WHO limits.
Air pollution has become a world wide public health problem, particularly in large towns and cities of the developing
countries where people are commonly exposed daily to very
high levels of pollution for 3-7 hours for the last many years
(Engel et al., 1998). Effect of air pollution on human health
varies according to the intensity, duration of exposure and
health status of exposed population. Air pollution increases
the risk of chronic obstructive pulmonary diseases and acute
respiratory infections in childhood, lung and chest cancer,
tuberculosis, prenatal outcomes including low birth weight
and eye diseases. The worst affected age group had been
between 50-60 years, followed by the lower age group of
45-55 years (Maddission, 1997).

Conclusion
The baseline data generated for major ambient air pollutants
at different urban sites of Karachi show that the concentration
of ambient air pollutants such as CO, SO2, NO and NOx, are
all within WHO threshold limits. The values recorded
indicate that all the pollutants are emitted by the industries
and motor vehicles. It is expected that the generated data
will play a part in laying the foundation for developing appropriate ambient air quality standards for Pakistan and their
implementation.

References
AllRefer.com 2005. Hemoglobin derivatives. Available at:
http://health.allrefer.com/health/hemoglobinderativesinfo.html.Accessed April 6, 2005.
ALA, 2000. Carbon Monoxide and the Environment,

American Lung Association,USA, available at: http;//
www.lunguas.org/air/carbon.
Aziz, J.A., Qureshi, T.A. 2003. Measurement of ambient
particulate matter and carbon monoxide in Peshawar.
Science Technology and Development, 22: 1-4.
Bhatia, S.C. 2005. Environmental Pollution and Control in
Chemical Process Industry, Delhi, pp. 104, published by
Khanna Publishers, Nai Sarak, India.
Clark, .P.A. 1988. Mixing models for simulation of plume
interaction with ambient air. Atmospheric Environment,
22: 1097-1106.
Engel, P., Hortodo, E., Ruel, M. 1998. Smoke Exposure of women
and young children in highland Guatemala, predictions
recall Accuracy. Human Organization, 54: 408-417.
Mage, D., Ozolins, G., Peterson, P., Webster, A., Orthofer, R.,
Vandeweerd, V., Gwynne, M. 1996. Urban air pollution
in megacities of the world. Atmospheric Environment,
30: 681-686.
Malakootian, Yaghmaeian, K. 2004. Investigation of carbon
monoxide in heavy traffic intersections of Municipal districts. International Journal of Environmental Science
and Technology, 1: 227-231.
Maddission, D.A. 1997. Meta Analysis of Air Pollution Epidemiological Studies, London Centre for Social and Economic Research on the Global Environment, University
College London, UK.
Nautiyal, J., Garg, M.I., Kumar, M.S., Khan, A.A., Thakur,
J.S., Kumar, R. 2007. Air pollution and cardiovascular
health in Mandi Gobindgarh, Punjab, India, -A pilot study.
International Journal of Environmental Research
and Public Health, 4: 268-282. (doi10.3390/)
Qureshi, O.R. 1997. Atmospheric pollution in Karachi 40
percent higher than other cities. Frontier Post, June 07,
Karachi.
WDNR, 2000. Carbon monoxide sources and health
effects. Wisconsin Department of Natural Resources,
USA, (http://www.dnr.state.wi.us).

Pak. J. Sci. Ind. Res. 2010 53(1) 30-36

Seasonal and Year Wise Variations of Water Quality Parameters in the
Dhanmondi Lake, Dhaka, Bangladesh
Shamshad Begum Qureshi
Chemistry Division, Atomic Energy Centre, Dhaka-1000, Bangladesh
(received April 18, 2008; revised December 11, 2009; accepted December 18, 2009)

Abstract. The quality of the surface water through 16 physicochemical variables was monitored at three sites of
Dharmondi Lake of Dhaka, Bangladesh, over 5 years during 2002-2007. The concentration of heavy metals (Pb, Cd, Cr,
Co, Ni, Cu) was below detection limits with few exceptions. No clear seasonal variation trend for Fe, Mn, Zn, PO43-, SO42,
Cl- and F- was observed which differed from year to year. Slight increasing tendency in case of sulphate, phosphate,
chloride concentrations and electrical conductivity was observed but it was not clear in other parameters. The levels of
all parameters were found well below the standards for drinking water.
Keywords: lake water, seasonal variability, pollution trend, water quality, heavy metals

Introduction

Trace toxic metals and physicochemical parameters e.g., Pb,
Cd, Cr, Ni, Co, Cu, Fe, Mn, Zn, EC, pH, Cl, F, SO4, PO4, CN, NO3
concentrations were monitored three times at three different
locations over a period of five years between March 2002 and
September 2007; data for the year 2003 is not available.

Over the past 25 years, the quality of water bodies around
Dhaka city has deteriorated a lot due to unplanned
discharge of untreated effluents from factories and sewage.
Dhanmondi Lake is one of the biggest lake and a great recreation place for the people of Dhaka city. But, this Lake is being
contaminated due to the increase in human activities
during the last few years. Lot of construction work had also
been done during 1980 to 2006 along the valley of this Lake,
which had directly influenced the water quality of the Lake.
In addition, frequent floods during recent years have also
contributed in polluting the lake water. The number of tourists
has also increased in recent years, directly affecting the
quality of water. Fishing activities around the lake are another
source of contamination. Thus, a constant and systematic
monitoring is essential to study long term pollution in the
Lake environment especially when it is impacted by the
increasing tourist population which disturbs normal
activities in the area. Some short-term research work had been
carried out in the past on water quality parameters of the river,
and lake water in our laboratory (Quraishi et al., 2006; Azim
2005; Chowdhury et al., 2005; Hossain, 2005; Hadi et al., 1996;
1991; Maroof et al., 1985). But long-term monitoring is necessary to evaluate the pollution sources and to get a clear trend
of pollution. Therefore, a long-term monitoring program was
initiated in 2002 spread over a period of 5 years during 20022007, for a wide range of water quality parameters. The main
objectives of this work were (i) to establish background levels
for Mn, Fe, Zn, Cr, Ni, Co, Cu, Cd, and Pb in the lake water of
Dhaka City and (ii) to examine the seasonal and year-wise
variability of trace metals in lake water on seasonal basis.

Materials and Methods
Reagents. All chemicals were of analytical reagent grade.
HNO 3, HCl and H2SO4 were of analar grade from BDH
Laboratories. Certified reference material was obtained from
the National Institute of Standards and Technology, USA.
Commercially available 1000 mg/L (ICP grade) single element
standard solutions (Merck or SPEX Certiprep, Metuchen, NJ,
USA) were used in preparation of the working standards. Standard solutions were freshly prepared from 1000 ppm stock by
dilution with deionized water (DI ).
Sample collection, preparation and analysis. Water samples
were collected from three locations thrice in a year between
March 2002 and September 2007. Sampling was done in March
(pre-monsoon), July (monsoon) and September (post-monsoon). The locations of the three sampling sites are shown in
Fig. 1.
Each sample was divided into two portions, one for the
analysis of metals ions and another for that of anion. pH of
the portion for the analysis of metals was adjusted below 1 by
addition of nitric acid to prevent adsorption to the bottle and
the portion of anions was filtered, using Whatman filter #41 to
remove suspended matter and stored at 4 °C. Water sample
(250 mL) was quantitatively transferred to 250 mL beaker and
then heated on a hot plate with 2 mL of HNO3 until the total
volume was reduced to approximately 5 mL. The concentrate

E-mail: mumu3222@yahoo.com

Seasonal Quality Variations of Dhanmondi Lake Water

obtained was transferred to a 10 mL volumetric flask and made
up to volume.
Electrical Conductivity (EC) and pH were measured using
Jenway Conductivity Meter, model No. 4070 and WTW
Multiline P4 Universal Meter, respectively. Concentration of
anions (Cl-, F-, NO3-) were determined by ion selective electrode (ISE). For fluoride determination, 1:1 (sample: TISAB)
low level TISAB was used whereas for chloride and nitrate,
2% of 5M NaNO3 and 2% of 2M (NH4)2SO4 were used, respectively, as ionic strength adjuster (ISA) according to the users
manuals (Quraishi et al., 2006; Chowdhury et al., 2005). The
concentrations of chloride, fluoride and nitrate in samples
were measured by the ISE method based on direct calibration.
50 mL of each of the calibration standard solutions (0.01, 0.10,
1.0, 10.0, 100 ppm) were taken in a 100 mL beaker and the
required amount of ISA buffer was added to it. The electrode
potentials (mV) of the standards were measured using the
digital ion-selective electrode meter (Orion Ionalyzer/model
470 A). After measurement, a calibration curve was drawn by
plotting electrode potentials of the standards against their
respective concentrations. Then the target electrode was
connected to the meter for determination of target anion in the
real samples similarly treated as the standards. From the calibration curve constructed by the instrument as mentioned
above, the slope was found to be -57±3 mV/decad. The computer code in the instrument provided the concentration of
anion in the sample directly by carrying out the calculations
based on the calibration factor (slope: -57±3 mV/decad) and
the electrode potential value of the sample. SO42- and PO43concentration were measured by Shimadzu 1201 uv-visible
spectrophotometer. After digestion, CRM and samples were
analyzed for metal content using a three point calibration by
atomic absorption spectrophotometer (Perkin Elmer 3110
and 560, USA). Standard solutions were prepared from single
element standards in acid media matching with sample
solution. Calibration curve was constructed for all elements
using at least three different concentrations. Very good R
square value of 0.9995 was obtained for all the elements.
Quality assurance. Quality assurance measures included the
calculation of method detection limit, recovery and analysis
of standard reference materials. To determine the DL, a low
concentration standard solution was analyzed several times
and the standard deviation (δ) was calculated for the data.
The detection limits of the method were calculated using 10 δ,
recommended by IUPAC, including preconcentration factors
for the elements Pb, Cd, Cr, Ni, Co, Cu, Fe, Mn, Zn, as 10.0,
4.46, 5.07, 5.30, 1.50, 3.38, 3.0, 4.0 and 12.0 μg/L, respectively.
The accuracy of the method was checked by recovery assays
of known amounts of analyte added to the samples. The

E
N

S
W

DP3

DP1

DP2

Map of D............. lake

Fig. 1. Map of Dhanmondi lake
recovery values obtained for spiked samples were 95-102 % in
case of all elements. Procedural blanks were used throughout
the sample preparation and analysis to evaluate contamination from reagents, containers etc. and the procedures were
validated by triplicate analysis of water samples, reference
material and blanks.

Results and Discussion
The baseline-monitoring programme was initiated in 2001 to
monitor heavy metals in different lakes in Dhaka city by the
Table 1. Analytical results for analysis of NIST SRM 1643d
and 1640
Elements Method NIST certified
values for SRM
1643d, (μg/L)

Measured
values
(μg/L)

Fe
AAS
91.2 ± 3.9
91.5 ± 0.94
Mn
AAS
37.66 ± 0.83
35.55 ± 0.36
Zn
AAS
72.48 ± 0.65
69 ± 1.86
Cu
AAS
20.5 ± 3.8
21.77 ± 0.22
Cd
AAS
6.47 ± 0.37
7 ± 0.07
Cr
AAS
18.53 ± 0.20
18.75 ± 0.19
Ni
AAS
58.1 ± 2.7
54.54 ± 0.56
NIST SRM 1640, Trace elements in natural water (μg/L)
Cu
AAS
85.2 ± 1.2
80.28 ± 0.81
Mn
AAS
121.5 ± 1.1
129.12 ± 1.31

Trueness
(%)

100
94.4
95.2
106
108
101
93.9
94.2
106

Shamshad Begum Qureshi

Mar

0.8

Mar

July
Sept

SO4(mg/l)
SO (mg/L)

0.6
0.4

PO4
(mg/l)
PO
(mg/L)
4

0.2

July

Sept

30
20
10
0

0
2002

2004

2005 2006

2002

2007

2004

60
50
40
30
20
10
0

2007

Mar

July
Sept

July

0.3

Sept

0.2
0.1
0

2004

2005

2006

2002

2007

2004

Year

2006

2007

Year

Mar

July

Mn (ug/l)
(μg/L)
Mn

1.2
1
0.8
0.6

2005
Year

Year

FeFe
(mg/L)
(mg/l)

2006

0.4

Mar

2002

Sept

0.4
0.2
0

Sept

40
20
0

2002

2004

2005

2006

2002

2007

2004

2005

2006

2007

Year
Year

Mar
July

Sept

60
40
20
0

Mar

500
2
EC
uS/cm2)
EC( (μS/cm
)

100
Zn
(ug/l)
Zn (μg/L)

2005
Year
Year

(mg/L)
F F(mg/l)

Cl
Cl(mg/L)
(mg/L)

Year
Year

July

400

Sept

300
200
100
0

2002

2004

2005
Year
Year

2006

2007

2002

2004

2005

2006

Year
Year

Fig. 2. Seasonal variations of different parameters of the Lake water during five years (2002-2007).

2007

11
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
00

Annual
Annual Arithmetic
arithmetic
mean (mg/L)
Mean
(mg/l

Annual Arithmetic
Annual arithmetic
Mean
mean (mg/l
(mg/L)

Seasonal Quality Variations of Dhanmondi Lake Water

PO4
PO4

2002

2004

2005

SO4
SO4

40
40
30
30
20
20
10
10

2006 2007

2002 2004 Year
2005 2006 2007

2002

2006 2007
2002 2004
2004 2005
2005 2006
2007
Year
Year

2002

2004

2005

2006

2007

0.3
0.3
0.25
0.25
0.2
0.2
0.15
0.15
0.1
0.1
0.05
0.05
00
20 0 2

20 0 4

60
50
40
30
20
10
0

2004

2005

2006

30
20
10
0
2002

Annual
AnnualArithmetic
arithmetic
2
mean (μS/cm
)
Mean
(mg/l

0.4
0.4
0.2
0.2
00
2002
2004 2005
2005 2006
2006 2007
2007
2002 2004
Year
Year

2004

2005

2006

2007

YeYear
ar

0.6
0.6

2 0 07

2007

0.8
0.8

2 0 06

Ye a r

Ye a r Year

2 0 05
Year

Year
Year

2002

Annual
Arithmetic
Annual arithmetic
Mean
mean (mg/l
(mg/L)

2002 2004 2005 2006 2007

Annual
Arithmetic
Mean
Annual
arithmetic
mean
(μg/L)
(ug/l

Annual arithmetic
Mean
(ug/l
mean
(μg/L)

Annual Arithmetic Mean
Annual arithmetic
(mg/l)
mean
(mg/L)

40
40
38
38
36
36
34
34
32
32
30
30

Annual Arithmetic

Annual
Arithmetic
Annual
arithmetic
mean
(mg/L)
Mean
(mg/l

Year

Fe
EC

0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
00

2002 2004
2004 2005
2005 2006
2006 2007
2002
2007
Year
Year

Fig. 3. Year wise variations of different parameters of Lake water during five years (2002, 2004-2007).

DP 1

DP 2

DP 3

PO4
po4

So4
SO
4

Shamshad Begum Qureshi

20
10
0

July
2004

March Sept
2005 2005

DP 3

Mar Sept
2002 2002

July March Sept
2004 2005 2005

DP 2

0.3
0.3

DP 3

Sept Sept
2006 2007

DP 1

0.4
0.4

DP 2

DP 3

0.2
0.2
0.1
0.1

2002

July
July
2004

March Sept
Sept Sept
March
Sept Sept
Sept
2005
2005 2006 2007
2004 2005 2005 2006 2007

120

DP 1

100

DP 2

Sept
Sept
2002

2002

July
July
2004

2004

March
March
2005

2005

Sept
Sept
2005

2005

Sept
Sept
2006

2006

120

DP 1

100

DP 2

DP 3

Sept
Sept
2007

2007

20
0

0
Sept
Sep t
2002
2002

July
July
2004
2004

March
M
arch
2005
2005

Sept
Sep t
2005
2005

Sept
Sep t
2006
2006

Sept
Sep t
2007
2007

DP 1
DP 2

1.5

DP 3

Septt
Sep
2002
2002

July
Sept
Sept
July MMarch
arch Sep
t
Sep
t
2004
2005
2005
2006
2004 2005 2005 2006

500
500

DP1

400
400

DP2
DP3

300
300

Septt
Sep
2007
2007

EC
EC

Fe
Fe

DP 2

Sept
2007

DP 1

70
60
50
40
30
20
10
0
Sept
Sept
2002

Mn
Mn

Sept
2006

Sept
2002

DP 1

1.4
1.2
1
0.8
0.6
0.4
0.2
0

200
200

0.5

100
100

0
Sept
Sept
2002

2002

July
July
2004

March Sept
March
Sept
2005
2005
2004 2005 2005

Sept
Sept
2006

2006

Sept
Sept
2007

2007

0
0

Sept
Sept
2002

2002

July
July
2004

2004

March
M
arch
2005
2005

Sept
Sept
2005

2005

Sept
Sept
2006

2006

Sept
Sept
2007

2007

Fig. 4. Variations in different parameters of water between the sampling locations of the Lake (DP1, DP2 and DP3).

Seasonal Quality Variations of Dhanmondi Lake Water

Chemistry Division of Atomic Energy Centre. Analytical
results for analysis of NIST SRM 1643d and 1640 are summarized in Table 1. Good agreement was found between the
measured and the certified values. The data of heavy metals
and various physical parameter of water for 2002 and 20042007 (five years) are presented in Fig. 2-4. It was observed
that during the study period (2002 and 2004-2007), the levels
of various parameters including toxic metals were much lower
than the drinking water standard of Bangladesh. Annual mean
concentrations of phosphate in 2006 and 2007 were 0.45 and
0.81 mg/L, respectively, (Fig. 3) (MEF,1997) which were much
lower than the standard set as 6 mg/L. The mean fluoride
concentrations (0.20 mg/L) observed during the study period
was also much lower than the standard, which is set at 1.0 mg/L.
Mean sulphate concentration in Dhanmondi lake water
was 17.5 mg/L which was much lower than the drinking water
standard (400 mg/L). Concentrations of Pb, Cd, Cr, Co, Ni,
known as toxic metals, were found to be below the method of
detection limits. The levels of Fe, Mn and Zn, which are known
to be the essential elements, were found to be in the range of
60-1000, 11.6-60 and 3.2-89 μg/L, respectively, the levels of
Mn and Zn were much lower than the Bangladesh drinking
water standards (Mn: 100 μg/L and Zn: 5000 μg/L) but were
found much higher as compared to those of other lakes (Nojiri
et al., 1985).
Seasonal variations. Seasonal variations of phosphate,
sulphate, chloride, fluoride, electrical conductivity, iron,
manganese and zinc in the lake water are shown in Fig. 2
which were found to be inconsistent. Fluoride level in
September 2002, was markedly high as compared to those
in March and July. In the year 2004, a systematic decrease
of fluoride was noticed during March to September. In case
of phosphate, concentration was higher in March (premonsoon) than in the July (rainy season) in the year 2002.
But in the year 2005, just reverse trend was observed and
in the year 2006, the highest concentration was found in
March and the lowest in September. Therefore, it could be
concluded that seasonal variations of phosphate, sulphate,
chloride, fluoride, electrical conductivity, iron, manganese
and zinc were not consistent (Fig. 2). This inconsistency
indicated that there is no particular or permanent source of
contaminants but sudden contamination events appear
which cannot be related to the local pollution events or
other local anthropogenic origins but rather to the influence of tourism-related activities (Topalian et al., 1999).
Contamination by metals can be related to the metallic
objects used in fishing activities, metallic containers and/
or packing materials of food which are directly thrown
into the lake by the tourists and thus contamination can

increase with the increase in the number of tourists (Conde
and Garcia - Montelongo, 2004).
Year wise variations. Time course changes in the concentrations of phosphate, sulphate, chloride, fluoride, electrical conductivity, iron, manganese and zinc in the lake water are shown
in Fig. 3. Phosphate concentration decreased till 2005 and
increased again in 2006 and 2007. Cl level increased from
2002 to 2004, decreased in 2005 and again increased in 2006
and fell in 2007. Sulphate concentration significantly decreased
from 2002 to 2005 and again remarkably increased in 2006 and
2007. Fluoride concentration fell from 2002 to 2004 and
remained almost constant until 2007. Zinc concentration
increased from 2002 to 2005 and then sharply decreased in
2006 followed by 2007. On the other hand, iron concentration
showed a zigzag pattern as shown in Fig. 3. In case of
manganese, concentration increased till 2006 and then
suddenly decreased in 2007. An increasing tendency was
observed for EC from the year 2004 to 2007.
Variations between locations. The distribution of different
parameters was not uniform in surface water of Dhanmondi
Lake. Significant variations in iron, manganese, zinc and
phosphate concentrations were observed with regard to
sampling stations throughout the study period (Fig. 4).
Therefore, it was very difficult to find out a particular sampling station having the highest or the lowest contamination
point in a particular season during the monitoring period. It
also indicates that contamination sources are not fixed and
these differences can be related to tourist activities. It can be
also seen in this figure that no significant variations
between the sampling stations were observed in case of
sulphate, chloride, fluoride and electrical conductivity.

Conclusion
This study was focused on the evaluation of water quality of
Lake and consequently on the determination of the pollution
level of this aquatic environment. Seasonal variations did not
show any clear pattern and were difficult to explain. A possible
explanation might be made based on the fact that the lake is
acceptor of both regular and non-regular pollution pulses. An
increasing tendency was observed in case of electrical conductivity, phosphate, chloride and sulphate, whereas the level was
unchanged in case of fluoride during the last four years of the
study. Concentration of zinc, iron and manganese decreased in
2007 as compared to the previous year.

References
Azim, A. 2005. Study on Trace Elements and other Micronutrients in the Water Samples from the Rivers Buriganga and

Turga. M.Sc Thesis, 127 pp. University of Dhaka,
Bangladesh.
Chowdhury, M.N., Akhter, S., Khan, M.M.K., Quraishi, S.B.
2005. Studies on water quality parameters in flood
water. Journal of Bangladesh Academy of Sciences, 29:
195-200.
Conde, J.E., Garcia-Montelongo, F.J. 2004. Heavy metals in
the coastal environment of Las Galletas, Tenerife, Canary
Islands, Spain. Bulletin on Environmental Contamination and Toxicology, 73: 77-84.
Hadi, D.A., Tarafdar, S.A., Akhter, S. 1996. The level of
aluminium in drinking water supplies and river water.
Bangladesh Journal of Scientific and Industrial
Research, 31: 41-46.
Hadi, D.A., Tarafdar, S.A., Mia, Y. 1991. The study of nitrate,
nitrite and phosphate in some river water around Dhaka
City. Nuclear Science and Applications, 3: 69-75.
Hossain, Z. 2005. Study on Quality of Lake Water in Dhaka
City: Monitoring of Seasonal Variation of Water Quality
Parameters for Evaluation of Water Pollution. M.Sc.
Thesis, pp. 151, Gogonath College, Dhaka, Bangladesh.

Shamshad Begum Qureshi

Maroof, F.B.A., Hadi, D.A., Khan, A.H. 1985. Cadmium level
of surface water of the Dhaka City. Nuclear Science and
Applications, 16&17, Series B: 145-146.
MEF, 1997. Drinking water quality criteria. In: A Compilation
of Environmental Laws (Bangladesh Gazette Additional
28), M. E. Huq (ed.), pp. 52-54, Department of Environment, The Ministry of Environment and Forests,
Bangladesh.
Nojiri, Y., Kawai, T., Otsuki, A., Fuwa, K. 1985. Simultaneous
multi element determination of trace metals in lake waters
by ICP emission spectrometry with preconcentration and
their background levels in Japan. Water Research, 19:
503-509.
Quraishi, S.B., Chowdhury, M.N., Khan, M.M.K, Akhter, S. 2006.
Impact of flood on some water quality parameters of lake
waters in Dhaka City area. Nuclear Science and Applications, 15: 82-85.
Topalian, M.L, Rovedatti, M. G., Castane, P.M., Salibian, A.
1999. Pollution in a low land river system. A case study:
The Reconquista River ( Buenos Aires, Argentina).
Water, Air and Soil Pollution, 114: 287-302.

Biological Sciences
Pak. J. Sci. Ind. Res. 2010 53 (1) 37-41

Salt Tolerance Evaluation of Rice (Oryza sativa L.) Genotypes Based on
Physiological Characters Contributing to Salinity Resistance
Jalal-ud-Din*, Samiullah Khan and Ali Raza Gurmani
Plant Physiology Programme, Crop Sciences Institute, National Agricultural Research Centre,
Islamabad, Pakistan
(received May 22, 2009; revised December 9, 2009; accepted December 20, 2009)

Abstract. Seven newly developed rice cultivars i.e., KS-133, DR-83, DR-64, BR-601, Gomal, JP-5 and Gomal-6,
were evaluated for salinity tolerance in a glasshouse along with three varieties of known salinity tolerance i.e., KS-282
(tolerant), IR-6 (medium tolerant) and Basmati-385 (susceptible). Based on the survival percentage at 50 mol/m3
sodium chloride salinity imposed at seedling stage, rice cultivars KS-133, Gomal, and DR-83 showed high survival
comparable to that of salinity tolerant cultivars like KS-282, and were thus placed in tolerance range. Survival percentage of JP-5, Gomal-6 and DR-64 remained in medium tolerance range (35 to 38%) as that of IR-6. The rice cultivar
BR-601 showed only 13% survival and was found to be as sensitive towards salinity as Basmati-385. The results of
rice survival in saline medium showed good uniformity and the check varieties showed results corresponding to
those found elsewhere. Sodium (Na+) and potassium (K+) concentrations in the third leaf showed variations among
different rice cultivars under salinity. There was an inverse correlation between varietal leaf Na+ vs survival percentage
(r = -0.808) and Na+ vs leaf chlorophyll (r = -0.857). The correlation between K+ and final survival percentage was
direct (r = 0.744) and also leaf chlorophyll vs survival (r = 0.952). The shoot fresh and dry weights were greater in the
rice genotypes having higher final survival percentage under saline conditions. Therefore, in addition to final survival
percentage, the higher shoot fresh and dry weight under salinity could be also used as criterion for evaluation of
salinity tolerance of rice.
Keywords: salinity, rice, chlorophyll, salinity tolerance

Introduction

main factors contributing towards salinity resistance (Yeo
et al., 1990). Reduction in shoot growth under salinity limits
the volume of tissue for the uptake of newly arriving salt
and once it starts, the situation worsens. Accumulation of Na+
and Cl- in the leaves has been found to reduce photosynthetic
activity, with ultra-structural and metabolic damage (Flowers
et al., 1985). Salinity tolerance in rice can be enhanced by
reducing the influx of excessive amounts of sodium chloride
in the transpiration stream. The salt concentration in the shoot
can be reduced by lowering sodium transport to the shoot and/
or increasing plant vigour. Normally the more vigorous plants
under non-saline conditions show greater resistance to salts
(Yeo and Flowers, 1986). Some traditional cultivars and
landraces are more tolerant to various abiotic stresses than
elite cultivars. These cultivars are good source of tolerant traits;
however, they generally have poor agronomic traits.

Salinization of agricultural soils is one of the major abiotic
stresses reducing crop productivity worldwide. Over 6 % of
the global land area and over 20% of the irrigated land are
currently affected by salinity (Munns, 2005). As irrigated
system supplies roughly one-third of the world food supply,
therefore, addressing the problem of salinity is of great
concern especially with an increasing global population. Rice
is one of the most important food crops, but the yield of the
grain is very susceptible to salinity (Akbar et al., 1985). In
Pakistan out of 6.8 million hectares of salt-affected land, over
1.5 million hectares are under rice cultivation (Khan, 1998).
Thus, selection of rice cultivar having salt tolerant potential
that would grow over a range of soil salinity is a prerequisite
for generating income for rice farmers having sizable saltaffected lands.

Maximizing the salt tolerance of crop species mainly depends
on two factors: availability of genetic variation to tolerance
and exploitation of the genetic variation by screening and
selection of plants with superior performance under the
applied stress (Yamaguchi and Blumwald, 2005; Shannon
et al., 1994). Sensitivity of rice to salinity varies with the stage
of growth. Generally, it is very sensitive to salinity stress at

Salinity resistance in rice is a complex character and many
factors contribute to such resistance as occurs in species.
Physiological studies suggest that in rice, restriction of
sodium entry, higher potassium uptake, plant vigour, tissue
tolerance to absorbed ions and water-use efficiency are the
*Author for correspondence; E-mail: jalaludin10@yahoo.com.uk

Materials and Methods
Plant material and growth conditions. The experiment was
conducted in a glasshouse with temperature controlled between
25 ºC to 35±3 ºC. Seven newly developed rice cultivars, along
with three varieties of known salinity tolerance were used in
the study. The three rice varieties of known salinity tolerance
used as check were; KS-282 (salt resistant), IR-6 (moderate
resistant) and Basmati-385 (salinity sensitive). Sterilized rice
seeds were germinated and seedlings were grown in a sand
culture. Seven days old seedlings were transplanted into black
boxes filled with 5 L rice culture solution (Yoshida et al., 1976).
The solution was renewed once a week. Each cultivar was
replicated thrice in separate boxes having 30 plants per replicate. At day 11, the solution was salinized with NaCl at a
concentration of 50 mol/m3 (total electrolyte concentration
resulting in electrical conductivity of 6 ds/m). The concentration of 50 mol/m3 NaCl is an established and useful working
level for eliciting a wide range of varietal response (Yeo
et al., 1990).
Leaf sodium, potassium and chlorophyll concentrations.
Six days after salinization, third leaves of 10 plants of each
cultivar was analyzed for sodium (Na+), potassium (K+ )
and chlorophyll concentration as described by Din (1997).
Chlorophyll contents were extracted in 80% ethanol and
calculated according to Arnon (1949).

of dead plants from the total number of plants and expressed
as percentage.

Results and Discussion
Survival rate was used as an indicator of genotypic
performance to salinity. Based on the survival in saline
medium, the cultivars were divided into three tolerance ranks.
Survival of KS-133, Gomal and DR-83 was as high as that of
KS-282 and therefore, these three varieties were ranked as
tolerant. Survival percentage of JP-5, Gomal-6 and DR-64
was 35-36% as that of IR-6, and so these were ranked as
medium tolerant, while, BR-601 showed only 13% survival
as that of the sensitive, Basmati-385 (Table 1).
The concentrations of Na+, measured 5 days after salinization,
showed differences in various rice cultivars (Fig. 1). There
was an inverse correlation between leaf Na+ and survival
Table 1. Ranking of salinity tolerance of rice cultivars based
on the survival under NaCl salinity (50 mM)
Salinity rank

Rice genotypes

Survival (%)

Resistant
Resistant
Resistant
Resistant
Medium
Medium
Medium
Medium
Sensitive
Sensitive

KS-133
Gomal
DR-83
KS-282
JP-5
Gomal-6
IR-6
DR-64
Bas-385
BR-601

66
72
56
63
36
36
35
29
13
13

3.0

Leaf Na+ (mg/g d.wt.)

young seedling stage and less at reproductive stage (Lutts
et al., 1995). Hence, for selection a specific growth stage that
is more sensitive to salinity stress should be targeted. Rice is
considered to be generally salt sensitive; there is genetic
variation for salt tolerance at critical stages in the cultivated
gene pool (Moradi et al., 2003; Yeo and Flowers, 1983). Therefore, selection of highly salt tolerant rice cultivars could be
expected to provide useful material for breeding and for
experimental comparison with unselected lines, in order to
examine possible mechanism of salt tolerance. In the present
study, we have reported the screening of some newly developed rice cultivars for overall performance (survival) and
other physiological characters, such as leaf chlorophyll,
Na+ and K+ concentrations and biomass accumulation under
salt stress.

Jalal-ud-Din et al.

2.5
2.0
1.5
1.0
0.5

D
R
-6
4
Ba
s38
5
BR
-6
01

IR
-6

JP
-5
G
om
al
-6

D
R
-8
3
G
om
al

-1
33
KS

-2
82

0.0

Plant growth and survival tests. After salinization for
24 days, 30 plants (10 from each replicate) of each cultivar
were harvested and the shoot fresh and dry weights were
recorded. The number of dead plants was recorded every
day after the first plant had died and continued till more than
50% plants of the sensitive check variety, Basmati-385, were
dead; a plant was considered dead when it was totally bleached.
The final survival was calculated by subtracting the number

Rice cultivars
+

Fig. 1. Mean Na concentration (mg/g dry weight) of
third leaf of rice cultivars under NaCl (50 mol/
m3) salinity. Leaves were sampled 5 days after
salinization. Each bar represents standard error
of the mean.

Salt Tolerance in Rice Genotypes

3.0

IR
-6
D
R
Ba -64
s38
5
BR
-6
01

D
R
-8
3
G
om
al
JP
-5
G
om
al
-6

-1
33

-2
82
KS

Rice cultivars

Fig. 3. Chlorophyll concentration (mg/g dry weight) of
third leaf of rice cultivars under NaCl salinity
(50 mol/m3). Leaves were sampled 5 days after
salinization. Each bar represents standard error
of the mean.
0.08
0.07

Weight (g/plant)

In this study survival was used as a quantification of genotypic
performance to salinity. This assessment criterion is used in
the field for evaluation of salinity damage during mass
screening of rice cultivars (IRRI, 1996). Based on the survival
in saline medium, the cultivars were divided into three
tolerance classes (Table 1). Generally, survival or visual assessment of salt damage is the criterion for overall measurement
of plant performance. The characteristics would normally be
chosen on their better correlation with overall performance.
Yeo et al. (1990) concluded that survival under salinity strongly
correlates with the salinity resistance of rice. In the present
research study, the survival experiment was repeated three
times and the results showed good uniformity; the check

9
8
7
6
5
4
3
2
1
0

Chlorophyll (mg/g d.wt.)

(r = -0.808). Leaf K+ showed good correlation with the salinity
tolerance of rice cultivars (Fig. 2) where correlation with
survival under salinity was (r=0.744, Table 2). Total
chlorophyll contents measured in the third leaf after
salinization was greater in the tolerant as compared to the
sensitive (Fig. 3). The shoot fresh and dry weight showed
good correlation with the final survival under NaCl salinity
(Table 2). KS-133, Gomal and DR-83 had greater shoot fresh
and dry weights and DR-64 had the least (Fig. 4).

SFW
Fresh weight

SDW
Dry
weight

0.06
0.05
0.04
0.03
0.02

IR
-6
D
R
-6
4
Ba
s38
5
BR
-6
01

om
al
-6

JP
-5

-2
82

1.5

-1
33
D
R
-8
3
G
om
al

2.0

Leaf K+ (mg/g d.wt.)

0.01

2.5

Rice cultivars
1.0
0.5

D
R
-6
4
Ba
s38
5
BR
-6
01

IR
-6

JP
-5
om
al
-6
G

D
R
-8
3
G
om
al

-2
8
KS

-1
33

0.0

Rice cultivars
+

Fig. 2. Mean K concentration (mg/g dry weight) of third
leaf of rice cultivars under NaCl (50 mol/m3)
salinity. Leaves were sampled 5 days after
salinization. Each bar represents standard error
of the mean.

Fig. 4. Mean fresh and dry weights (g/plant) of shoots
of rice cultivars, when grown for 24 days in NaCl
(50 mol/m3) salinity. Each bar represents standard
error of the mean.
varieties, KS-282 and Basmati-385, showed corresponding
salinity tolerance as found earlier (Khan and Abdullah, 2003)
under field conditions.
In monocots, generally salinity tolerance is associated with
the ability of plant to exclude Na+ from shoot tissues (Tester

Table 2. Correlation coefficient (r) for relationship between overall performance (survival) and individual traits of the
available data

Survival
Chlorophyll

Shoot Na+

Chlorophyll

SFW

SDW

K+

0.808 ( - )*
(n = 30)
0.857 ( - )
(n = 30)

0.952 (+ )*
(n = 30)

0.762 (+ )
(n = 30)

0.886 (+ )
(n = 30)

0.744 (+ )
(n = 30)

* = signs + and – represent the positive and negative correlation, respectively.

and Davenpor, 2003). In the present study, there were
differences in the leaf Na+ concentration among the rice
cultivars; those having higher leaf sodium had poor survival
rate (Table 1). Potassium accumulation also showed good
correlation with the salinity tolerance of rice cultivars
(r = 0.744). In rice, genotypic variations in Na+ and K+ uptake
have already been reported; low concentration of Na+ and
higher K+ were correlated with the salinity tolerance under
salinity stress (Babu et al., 2007; Kader et al., 2006; Walia
et al., 2005). The chlorophyll contents measured in the third
leaf after salinization was greater in the tolerant as compared
to the sensitive varieties (Fig. 3). There was inverse
correlation between leaf chlorophyll and Na+ concentration
(r = -0.857). Yeo and Flowers (1983) established inverse
relationship between leaf chlorophyll and Na+ concentration.
The shoot fresh and dry weights were the greatest in the
tolerant genotypes followed by the medium tolerant varieties
and the least in sensitive rice cultivars (Fig. 4) and showed
high correlation with the final survival under saline conditions
(Table 2). Sankar et al. (2006) recorded the highest total
biomass and vigour index in salt tolerant cultivars. The
greater plant vigour (shoot fresh and dry weights) may
provide dilution of salt concentrations with growth and
tolerance within the tissues; these are the traits that may be
expected to be helpful in achieving greater salinity tolerance
(Yeo and Flowers, 1986). Richards (1983) concluded that
vigour is essential for plant survival and productivity under
saline environment. Akbar et al. (1985) identified more
vigorous accessions, which were non-dwarf land races, as
the most salt resistant in mass screening trials.

Conclusion
The data demonstrate that rice cultivars with low sodium and
high potassium uptake could lead to higher survival of rice
under saline conditions. The additional aspects of less
chlorophyll damage under such conditions are the potential
of resistant cultivars. Chlorophyll content could be used as an
index of salt tolerance for selection of rice tolerance against
salinity stress. It also demonstrates that amount of biomass of
rice seedlings under NaCl salinity could be used as a criterion
in ranking for salinity tolerance.

References
Akbar, M., Khush, G.S., Hellerislamber, D. 1985. Genetics
of salt tolerance in rice. In: Rice Genetics, Proceedings
of the International Rice Genetics Symposium IRRI, pp.
399-409, Manila, Phillipines.
Arnon, D.I. 1949. Copper enzymes in isolated chloroplasts:
polyphenol-oxidase in Beta vulgaris. Plant Physiology,

Jalal-ud-Din et al.

24: 1-15.
Babu, S., Sheeba, A., Yogameenakshi, P., Anbumalarmathi,
J., Rangasamy, P. 2007. Effect of salt stress in the selection
of salt tolerant hybrids in rice (Oryza sativa L.) under in
vitro and in vivo condition. Asian Journal of Plant
Sciences, 6: 137-142.
Din, J. 1997. Effect of Hormonal Pretreatments on the
Response of Rice (Oryza sativa L.) and Wheat (Triticum
aestivum L.) to Salinity. D. Phil. Thesis, pp. 25-26, University of Sussex, UK.
Flowers, T.J., Duque, E., Hajibagheri, M.A., McGonigle, T.P.,
Yeo, A.R. 1985. The effect of salinity on leaf ultra
structure and net photosynthesis of two varieties of rice:
Further evidence for cellular component of salt-resistance.
New Phytologist, 100: 37-43.
IRRI, 1996. Standard Evaluation System for Rice, The
International Rice Research Institute, Los Banos, Manila,
Philippines.
Kader, M.A., Seidel, T., Golldack, D., Lindberg, S. 2006.
Expressions of OsHKT1, OsHKT2, and OsVHA are
differentially regulated under NaCl stress in salt-sensitive
and salt-tolerant rice (Oryza sativa L.) cultivars. Journal
of Experimental Botany, 57: 4257-4268.
Khan, G.S. 1998. Soil Salinity/Sodicity Status in Pakistan. pp.
1-59, Soil Survey of Pakistan, Lahore, Pakistan.
Khan, M.A., Abdullah, Z. 2003. Salinity-sodicity induced
changes in the reproductive physiology of rice (Oryza
sativa L.) under dense soil conditions. Environmental and
Experimental Botany, 49: 145-157.
Lutts, S., Kinet, J.M., Bouharmont, J. 1995. Changes in plant
response to NaCl during development of rice (Oryza
sativa L.) varieties differing in salinity resistance. Journal
of Experimental Botany, 46: 1843-1852.
Moradi, F., Ismail, A.M., Gregorio, G. , Egdane, J. 2003.
Salinity tolerance of rice during reproductive development
and association with tolerance at seedling stage. Indian
Journal of Plant Physiology, 8: 105-116.
Munns, R. 2005. Genes and salt tolerance: bringing them
together. New Phytologist, 167: 645-663.
Richards, R.A. 1983. Should selection for yield in saline
regions be made on saline or non-saline soils? Euphytica,
32: 431-438.
Sankar, P.D., Subbaraman, N., Narayanan, S.L. 2006. Ranking
of salt tolerant rice lines based on germination and
seedling growth under salt stress conditions. Research
on Crops, 7: 798-803.
Shannon, M.C., Grieve, C.M., Francois, L.E. 1994. Whole
plant response to salinity. In: Plant-Environment
Interaction, R.E. Wilkinson (ed.), pp. 199-244, Marcel
Dekker Inc., New York, USA.

Salt Tolerance in Rice Genotypes

Tester, M., Davenpor, R. 2003. Na+ tolerance and Na+ transport
in higher plants. Annals of Botany, 91: 503-527.
Walia, H., Wilson, C., Condamine, P., Liu, X., Ismail, A.M., Zeng,
L., Wanamaker, S.I., Mandal, J., Xu, J., Cui, X., Close, T.J.
2005. Comparative transcriptional profiling of two
contrasting rice genotypes under salinity stress during the
vegetative growth stage. Plant Physiology, 139: 822-835.
Yamaguchi, T., Blumwald, E. 2005. Developing salt-tolerant
crop plants: challenges and opportunities. Trends in Plant
Sciences, 10: 615-620.
Yeo, A.R., Yeo, M.E., Flowers, S.A., Flowers, T.J. 1990.
Screening of rice (Oryza sativa L.) genotypes for
physiological characters contributing to salinity resistance,

and their relationship to overall performance. Theoretical
and Applied Genetics, 79: 377-384.
Yeo, A.R., Flowers, T.J. 1986. Salinity resistance in rice (Oryza
sativa L.) and a pyramidal approach to breeding varieties
for saline soils. Australian Journal of Plant Physiology,
13: 161-167.
Yeo, A.R., Flowers, T.J. 1983. Varietal differences in the
toxicity of sodium ions in rice leaves. Physiologia
Plantarum, 59: 189-195.
Yoshida, S., Forno, D.A., Cock, J.H., Gomez, K.A. 1976.
Laboratory Manual for Physical Studies of Rice,
rd
3 edition, International Rice Research Institute, Los
Banos, Laguna, Philippines.

Pak. J. Sci. Ind. Res. 2010 53 (1) 42-45

Parasitic Contamination in the Table Vegetables Planted
in Shiraz Plain, Iran
Meraj Madadi
Scientific Board of Islamic Azad University, Firoozabad, Iran
(received June 10, 2008; revised June 17, 2009; accepted September 2, 2009)

Abstract. Contamination with parasites of the vegetables grown in Shiraz plains and irrigated by urban and industrial
sewage-laden Shiraz Rookdhaneh Khoshk River and seasonal Soltanabad River was studied. It was found that 31.5% of the
farms irrigated by the river water directly, 30.9% of the farms irrigated by water of nearby located shallow wells and 33.7%
of the farms using water from the wells at a distance of one kilometer from the River were contaminated by Ascaris ova.
32.20% vegetables of farms irrigated by the wells located near Soltanabad river were contaminated with insects and larvae
and 24.5% with Ascaris worm. After Ascaris ova, the larvae of different insects, Strongyloides parasite, Sterocoralis and
Trichostrongylus were the contaminants most present.
Keywords: parasites, irrigation, vegetables, Shiraz rivers, Ascaris

Introduction

worms such as Trichostrongylus and hookworms, unicellular
creatures such as Antamoeba histolytica, Giardia lamblia,
Toxoplasma gondii which cause amoebiasis, giardiasis,
(lambliasis) and toxoplasmosis and other diseases (Fereydoun,
1987).

Table vegetables, particularly those irrigated with raw
sewage, play an effective role in the transfer of parasites
specially the soil parasites and thus in spreading contagious
and parasitic diseases to the consumers (Shariatpanahi, 2001;
Fereydoun, 1987). The vegetables irrigated with FiroozabadTehran creek in 1989 and 1990 and table vegetables used
in Yasooj in 1996, were found to be contaminated with
parasitic worm eggs (Sarkari, 1997; Vosooghi, 1990). In south
Louisiana in USA, where city and industrial sewage contaminated the agricultural lands, vegetables such as spinach,
parsley, onion, asparagus, spearmint, tomato, pea, carrot and
cabbage were found to be contaminated with heavy metals,
the latter being 1.60% more than the permissible limit of the
American National Health Society. Moreover 28.20% of the
farms were contaminated with the parasitic eggs found in
sewage (Ramelow et al., 1992).

Though irrigation method should be defined in relation
to the amount of water used and the type of plants being
irrigated, as each plant needs a different rate of water depending on the environment and geographical conditions such
as temperature, raining rate, latitude etc., it was observed that
these factors are ignored in Shiraz plain irrigation; here the
irrigation system is deep water which is traditional and uses
about 4,000-12,000 cubic meter water per hectare, yearly.
This system causes problems relating to drainage of water,
environmental contamination, agricultural damages and soil
errosion etc. (Rastegar, 1992).
The present study was undertaken to find the extent of
contamination of vegetables irrigated directly by Shiraz
Roodkhaneh Khoshk River and Soltanabad River and by
nearby lying well water. Taking into consideration the
diseases, such as diarrhoea, resulting from the above
mentioned practice, it is intended to propose safe means of
growing vegetables and prevent regional contamination of
vegetables with parasites and their transfer to the consumers.

In Japan, the researchers of Agriculture College, Tokyo
University, in 1962 conducted necessary tests for finding the
contamination of leek, parsley, sweet basil, spearmint and
green pepper growing in the fields which were irrigated with
city and industrial raw sewage and found these vegetables,
heavily contaminated with parasites whereas the content of
heavy metals was 2.13% more than the standards (Chino
et al., 1991).

Materials and Methods

The researches conducted in Iran show that the vegetables
may transfer eggs of worms such as Ascaris, Trichocephalus,
Hymenolepis nana, Taenia, Fasciolia hepatica, larvae of

City sewage is discharged into Shiraz Roodkhaneh Khoshk
River passing through the city centre. Some districts of the
city do not have proper sewage disposal means and the
factories beside the river also dump their waste products

E-mail: navid_madadi@yahoo.com

Parasitic Contamination of Vegetables in Iran

into it. Due to disposal of garbage into the river, some agricultural irrigation places have high BOD (the index indicating
sewage contamination) related to city sewage (WHO, 1989).

b) Radish, tomato, cucumber, eggplant, green pepper,
carrot, onion, cabbage and squash (eaten cooked except
carrot)

This study covers table vegetables planted in the farms
around Shiraz where the vegetables are consumed either raw
or cooked. The areas studied included Gheisar Aboonasr,
Mehraghan, Eghbal Abad, Torkan, Nasirabad, Kooshkak,
Mahfiroozan, Dasht Khezr, Noortaban, Khaljooy and
Sharifabad villages, mountainous region and Kaftarak
village, all of them located beside Roodkhaneh Khoshk River.

Parameters taken into consideration were area of the land
under cultivation, type and amount of harvest, fertilizer and
disinfectants, herbicides, fungicides and means of irrigation
including deep and semi-deep wells and river.

Samples of 58 shallow, semi deep and deep wells were
subjected to bacteriological tests. Results indicated that 98%
of the wells were contaminated with coliform, 100% of which
were soil coliform. Physical and chemical properties of water
did not conform to the standards, except that of 12 deep wells
lying at a distance of 1,500 m from the river.

The land for cultivation of table vegetables, domestic animals
provender, wheat, barley and potato was about 1,372 hectares
of which 128 hectares was under cultivation of the provender
including alfalfa, 168 hectares that of barley, wheat and maize
and 100 hectares were used for planting of potato. The land
used for cultivation of vegetables was 276 hectares of which
90 hectares were irrigated with river and 186 hectares irrigated
mostly by shallow wells.
Samples (140) of different vegetables cultivated in Kaftarak
and Soltanabad were selected on the basis of the factors
such as means of irrigation including shallow wells, wells
within one km radius of the river and the river itself; one type
of vegetable from each farm was sampled. 45 samples were
taken from farms irrigated with river and 95 samples, from farms
irrigated with deep, semi-deep and shallow wells.
The following vegetables, 250-500 g of each, were sampled:
a)

Sweet basil, leek, spearmint, cress, purslane, parsley
lettuce, tarragon, common dill and spinach (eaten raw).

Green bean, pea and okra (eaten cooked).

The samples were collected with gloved hands and put into
nylex bags and carried to the related laboratories (Gholami
and Mohammadi, 1998). Domestic animals provender was
sampled in the same way.

Results and Discussion

Findings show that the parasitic contamination of table
vegetables depends on location of the place/farm to be irrigated, source of irrigation and type of the parasites (Table 1).
Most of the farms around Roodkhaneh Khoshk river were
contaminated with “Ascaris” and larvae of different insects.
31.50% farms irrigated directly by river (Fig. 1), 31% farms
irrigated by the wells located around the Roodkhaneh
Khoshk river (Fig. 2) and 33.70% by the wells within one
kilometer radius of the river were contaminated with ascaris
eggs; mostly the fertilizer used was human faeces (Table 1);
bacteriological tests of shallow regional well water indicated
faecal coliform contamination, 3 to 45 MPN/100 mL, and total
coliform, 70 to 800 MPN/100 mL, whereas, total coliforms in
deep well water were 5 to 45 MPN/100 mL. Physical and
chemical specifications of the water used for irrigation of
Shiraz plains and Roodkhaneh Khoshk farms were not within
the standard limits necessary for agriculture with BOD more

Table 1: Parasitic contamination in the vegetables according to the irrigating source and the type of parasite
Parasite

Roodkhaneh
Khoshk water
No. of
%
parasites

Wells near
Roodkhaneh Khoshk
No. of
%
parasites

Well within radius
of 1 km from the river
No. of
%
parasites

Wells near
Soltanabad river
No. of
%
parasites

Trichostrongylus
Strongyloides stercoralis
Ascaris lumbricoides
Trichocephalus
Oxyure
Different insect
larvae and eggs

35
32
71
4
19

15.5
14.2
31.5
1.78
8.5

19
13
35
0
0

16.8
11.5
31
0.0
0.0

13
19
27
0
2

16.3
23.7
33.7
0.0
2.6

12
10
15
0
4

19.6
16.4
24.6
0.0
6.6

28.5

40.7

23.7

32.8

Total

225

100.0

113

100.0

Meraj Madadi

TricostrongyIus,
15.56%
Stercoralis,
14.23%

Oxyure,
14.23%
Trichocephalus,
1.78%

Ascaris,
31.55%, 30%

Fig. 1. Pollution (%) of vegetables irrigated via water of Khoshk
river with different parasites.

Other insects,
40.70%
TricostrongyIus,
16.82%
Ascaris,
31%

Tests showed that all the table vegetables cultivated in Shiraz
plains and irrigated with Roodkhaneh Khoshk river, and the
wells close to the river were contaminated with one or several
faecal parasites. The range of pollution in vegetables irrigated
directly by the river was (with the exception of 2% in cabbage)
from 6% (in gourd) to 35% (in green beans) (Fig. 3) and in
those irrigated by close-by lying wells was 4% (in gourd
cabbage and onion) to 10% (in green beans) (Fig. 4). Thus it
is clear that most of the contamination relates to the means
of irrigation and use of man faeces (US EPA, 1981).

Pollution (%)

Other insects,
28.45%

Stercoralis,
11.50%

40
35
30
25
25 21
17
20
14
13
15
10
10 9
10
5
0

18
7

9 10

12
7

6
2

Fig. 2. Pollution (%) of vegetables irrigated via wells close to
Khoshk river with different parasites.

In transfer of parasites, three factors are involved: source of
infection, means of transfer and a sensible host, which
means the process depends on the way of distribution of the
parasite in a defined place at a defined time. The means by
which the parasite reaches from the source to the host are
clear (Neva and Brown, 2001). Some parasites reach the host
through direct contact whereas others have a more complicated life cycle and need to pass through some growth stages
such as free life or need an intermediate host to become infective. The transfer is accomplished through direct and indirect
contact, such as by means of food, water, soil and vertebrates
and arthropodes. Studies show that rate of contamination
with some worms in different parts of Iran is high; in Iran,
around 32 human parasites were found, some of which were
very common and frequent such as Ascaris (Mahvi, 1996).
The vegetables, contaminated with parasite eggs, transfer
them to the consumers (Monzavi, 1985). Raw sewage, human
faeces and contaminated water, used as fertilizer and for
irrigating farms, are dangerous sources of contamination for
the consumers of the final products because traditional
and deep water systems are still used here as the sources of
potable water.

Fig. 3. Parasitic pollution (%) of different vegetables irrigated
via water of Khoshk river.

Pollution (%)

than 100 mg/L. Also the level of some heavy metals was not
within the range applicable for agricultural use. Water EC
(electrical conductivity) of some well water was high, but all
the wells had coliform contamination.

12
10
8
6
4
2
0

7
5

9
7

6
5

10
8
6
4

Fig. 4. Parasitic pollution (%) of different vegetables irrigated
via wells close to Khoshk river.

The land measuring 276 hectares is used for cultivation of
table vegetables and is irrigated with Roodkhaneh Khoshk
river; these raw vegetables are distributed in Shiraz and other
adjoining cities. Also the wells located within a radius of one
km of the river, when tested bacteriologically, whenever less
deep and farther from the river, were more contaminated; it
shows that the wells were influenced by the river. The distant
well water had parasitic eggs found in human faeces (used
in the farm) and the differences were as follows:
a)

The parasitic contamination was more in the vegetables
of farms irrigated with the river water directly.

Parasitic Contamination of Vegetables in Iran

b) Contamination of the vegetables with parasite eggs was
less in the farms irrigated by hand and shallow wells
farther from the river than the farms irrigated directly by
the river water .

fertilizer, whose conditions of maintenance have been
observed, be used.

Chino, M., Moriyama, K., Saito, H., Mori, T. 1991. The amount
of heavy metals derived from domestic sources in Japan.
Water, Air and Soil Pollution, 57: 829-837.
Fereydoun, A. 1987. Medical Helminthology, vol. 1 & 2, 3rd
edition, Danesh Pajouh Publications, Tehran, Iran.
Gholami, M., Mohammadi, H. 1998. Water and Wastewater
Microbiology, Hayan Publications, Tehran, Iran.
Mahvi, A.H. 1996. Sewage Filtration in Torrid Zone, Jahad
Daneshgahi Publications Organization, Tehran, Iran.
Monzavi, M.T.I. 1985. Water Supply, 4th edition, Tehran
University Publications, Tehran, Iran.
Neva, F.A., Brown, H.W. 2001. Basic Clinical Parasitology
(Translated by A. Athari), Pezhman Publications, Tehran,
Iran.
Ramelow, G.J., Biven, S.L., Zhang, Y., Beck, J.N., Young, J.C.,
Callahan, J.D., Marcon, M.F. 1992. The identification
of point sources of heavy metals in an industrially
impacted water way by periphyton and surface sediment
monitoring. Water, Air and Soil Pollution, 65: 175-190.
Rastegar, M. 1992. Dry Farming, 1st edition, Behramand
Publications, Tehran, Iran.
Sarkari, B. 1997. Examining parasitic contamination of the
vegetables used in Yasuj city. Journal of Yasuj University of Medical Sciences, 3: 15-18.
Shariatpanahi, M. 2001. Principles of Quality and Treatment
of Water and Wastewater, 6th edition, Tehran University
Publications, Tehran, Iran.
US EPA, 1981. Process Design Manual for Land Treatment of
Municipal Wastewater, US Environmental Protection
Agency, Rept. 625 11-77-008, US. Govt. Printing Office,
Washington DC., USA.
Vosooghi, M. A. 1990. Examining the Worm Parasitic Eggs
Contamination of the Vegetables Irrigated by Firoozabad
River, MS. Thesis, Tehran Medical Sciences University,
Tehran, Iran.
WHO, 1989. Health Guidelines for the Use of Wastewater
in Agriculture and Aquaculture, WHO Technical
Report Series 778, World Health Organization, Geneva,
Switzerland.

Contamination in the farms, irrigated by deep wells more
than one km distant from the river, was less than the
above-mentioned two cases.

So, it is clear that the river water contaminated by sewage
played main role in contaminating the consumable raw
vegetables with parasite eggs.
It was found that green beans, sweet basil and tarragon were
more contaminated with the eggs and larvae of Ascaris and
Trichostrongylus than with the other insects. Also the farms
irrigated by the shallow wells and the wells, within a radius of
50 m of the river, had vegetables contaminated with parasite
eggs similar to the farms irrigated by the river water.
It was also found that the farms irrigated by the wells around
the seasonal river, Soltanabad, had eggs of strongylosis,
sterocoralis, ascaris and trichostrongylosis, more than other
parasite eggs.
The contamination of vegetables with parasite eggs of farms
irrigated by Roodkhaneh Khoshk river was found to be more
frequent as compared to those irrigated by the wells beside
the seasonal river, Soltanabad.

Conclusion
The results show that the vegetables cultivated in Shiraz
plain, irrigated/fertilized by sewage-containing water, play an
important role in transferring important parasites such as
ascaris, trichostrongylus, trichocephalus, S. stercoralis and
oxyure. Thus sewage and human fertilizers have been the
cause of contamination of farm vegetables irrigated by such
means. Irrigation of agricultural farms with such sources of
contamination is harmful from hygienic point of view of the
consumers of crops, grown in such farms.
It is therefore, proposed that the standards and regulations
relating to the irrigation water used specially for raw
vegetable crops should be observed. It is recommended that
unrefined as well as refined sewage may not be used for
irrigating vegetable crops and for fertilization; instead animal

References

Pak. J. Sci. Ind. Res. 2010 53 (1) 46-49

Microbiological Quality of Drinking Water and Beverages
in Karachi, Pakistan
Anila Siddiqui, Korish Hasnain Sahir and Seema Ismat Khan*
PCSIR Laboratories Complex, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-75280, Pakistan
(received February 20, 2009; revised October 21, 2009; accepted October 28, 2009)

Abstract. Microbiological assay of 780 water samples and 1220 beverage samples (412 branded and 808 unbranded),
collected from 490 different schools, both government (98 schools) and private (392 schools), situated in different areas
of the city of Karachi, was conducted for bacterial heterotrophic plate count, total coliforms, faecal coliforms, E. coli,
faecal streptococci, Pseudomonas and Salmonella species. The counts ranged from 0 to 2.5 × 105 cfu/mL and from 0 to
106 cfu/mL in water and beverage samples, respectively. About 36% of water samples and 48% of unbranded beverage
samples were contaminated with the indicator and the pathogenic bacteria; all the branded beverage samples were found
fit for human consumption from microbiological viewpoint.
Keywords: drinking water, beverages, microbiological quality

Introduction

safety and quality (Kohnen et al., 2005). However, several
studies have demonstrated that the total counts and number
of pathogens may get reduced due to the acidity and the
effect of CO2 during storage. (Mugochi et al., 1999; Simango
and Rukure, 1992; Sheth et al., 1988; Zschaler, 1979; King and
Nagel, 1975; 1967).

Raw water itself does not contain large number of microorganisms. Drinking water contains assimilable organic
compounds that allow a certain degree of bacterial growth
(Exner et al., 2005). Improperly installed hand pumps permit
infiltration of contaminated surface water, whereas unclean
storage devices and other factors contribute to disease cycle,
malnutrition and high mortality. Infectious diseases caused
by pathogenic bacteria are the most common and wide
spread health risk associated with drinking water. Some of
the pathogens, which are transmitted through contaminated
drinking water, lead to severe and sometimes life threatening
diseases particularly in children. The potential of drinking
water to transport microbial pathogens to large number of
population, causing subsequent illness, is well documented
in different countries.

In Pakistan people lack access to adequate supply of safe
water for daily use. The basic sanitary facilities are very poor
and are not available for half of the population. Sources of
microbial contamination of water and beverages include raw
materials, processing equipment or utensils, human activities,
sanitation practices, workers or handlers, waste materials,
animal and insect pests and microbial growth niches. Chemical
composition of foods and beverages and the environmental
factors, such as water activity, pH, temperature, etc., determine
the types of organisms that can grow there.

The most common and widespread risk associated with drinking water is its contamination by human or animal excreta. The
potential consequences of microbial contamination necessitates that its control be of paramount importance. Pathogens
in drinking water, presenting serious risk of diseases, include
Salmonella sp., pathogenic Escherichia coli, Pseudomonas,
Vibrio cholerae etc. Faecal specific bacteria such as coliforms,
faecal coliforms and E. coli are the parameters of importance
in monitoring faecal pollution.

The present study was undertaken keeping in view the
hazards of polluted or contaminated drinking water and the
effect of its use in beverages and ice lollies etc. The survey
provides base line data for authorities to set the guidelines
for microbiological quality of water and beverages within
the country.

Materials and Methods
Sample collection. A total of 780 water and 1220 beverage
samples were collected from 490 different schools/educational
institutes of Karachi, located in various areas including those
inhabited by low income, middle class and upper middle class
population, as well as rich and wealthy people. Both the
government and the private institutions were included in the

The survival and growth of microorganisms in processing
environments of foods, such as beverages, sherbets, ice
creams, ice-lollies, etc. may lead to contamination of the
finished products, resulting in reduction of microbiological
*Author for correspondence; E-mail: seema_ismat@hotmail.com

Microbiological Quality of Drinking Water

study, out of these 98 were government schools whereas 392
were private institutions. Insulated ice chest with ice packs
was used for collection and transportation of samples. The
collected samples were labelled with date and laboratory
code. Other necessary information about the samples, like
area or location of school, collection point etc. was recorded
on prescribed forms. Samples were collected in sterilized
screw-capped glass bottles.

300

Heterotrophic plate count (HPC), coliforms and faecal coliforms
were tested according to the Standard Methods for the Examination of Water and Wastewater (APHA, 1998; ISO 9308-1:
2000; 9308-2: 1990; 6222: 1988a; 8199: 1988b). E. coli 0157:H7
was tested by serological kit method (Pro-Lab Diagnostics)
according to Thompson et al. (1990) and faecal streptococci
were determined using ISO method 7899-1 (1984). Other
parameters were tested according to on-line Bacteriological
Analytical Manual of US FDA (2006; 2002; 2001). Salmonella
was confirmed using antisera (Pro-Lab Diagnostics).

Results and Discussion
Figures 1 and 2 present total heterotrophic plate count (HPC)
in water and beverage samples, respectively. The heterotrophic
plate count in water and beverage samples ranged from 0 to
2.5 × 105 cfu/ml and from 0 to <106 cfu/ml, respectively. HPC
may be used to assess the general bacterial content as well as
the efficiency of water treatment. The HPC standards for
drinking water vary a lot from country to country. According

343
350
300
250
200
150
100
50
0

207

177

48
5
<10

<100

<1000

<10000 <100000

HPC (cfu/mL)

Fig. 1. Heterotrophic plate counts of water samples.

272

250

Branded
Unbranded

221

200

164
121

150

100
50
0

Beverage samples were categorized as branded and unbranded. The unbranded beverages consisted of gola-gandas
(local name for an item made by crushed ice and additives),
sherbets (drinks) and carbonated and other drinks sold on
vending carts (thailas). All the water and beverage samples
were tested microbiologically for their heterotrophic plate
counts, total coliforms, fecal coliforms, E. coli 0157:H7, faecal
streptococci, Pseudomonas and Salmonella species.

318

350

0
<10

16 19
<100

<1000

<10000 <100000 <1000000

HPC (cfu/mL)

Fig. 2. Heterotrophic plate counts of beverage samples.
to WHO (1999) guidelines for drinking water quality, the limit
for HPC is 100 cfu/mL which has also been adopted by
Pakistan Standards and Quality Control Authority (PSQCA;
2004).
However, WHO guidelines for drinking water quality are
intended to be used as a basis for the development of national
standards in the context of local or national environmental,
socio-economical and cultural conditions (WHO, 1999). The
HPC standard for drinking water in Sri Lanka is 10,000 cfu/mL
(SLS, 2001; 1995), which is a pretty relaxed standard, compared
to the stringent standard of WHO. According to Canadian
standard, HPC is not considered as a parameter of drinking
water quality. A review study conducted by Allen et al. (2004)
reveals that there is no evidence to support health-based
regulations of HPC concentrations.
The national standards should be influenced by national
priorities and economic factors. Stringent standards could
limit the availability of water supplies, which is a significant
consideration in regions of water shortage. However, public
health must never be endangered. For this very reason, it is
suggested that the WHO standard for HPC must not be
adopted as the criterion for rejection of line water samples for
drinking purpose and the acceptance limit must be adopted
according to Sri Lankan standard or the Canadian guidelines.
The presence of indicator organisms and/or pathogens only
must be considered as the criterion for declaring a sample
unfit for human consumption. However, we do support
adoption of WHO guidelines for bottled water according to
which the samples must not contain any count at all as the
manufacturers/bottlers are claiming it to be pure and charging
extra money for that.
Figure 2 presents total bacterial counts in beverage samples.
According to the PSQCA (2002) standard PS: 1654-2002 R,
the freshly prepared carbonated drinks may contain <100
bacteria per mL of the sample whereas the count must

Seema Ismat Khan et al.

decrease to <30 within three days of storage. The counts in
the branded samples were mostly within the limit of PS
standard with only 19 of the 412 tested samples having counts
exceeding 100 mL.

organisms, only E. coli is specifically of faecal origin; other
thermotolerant coliforms may originate from organically
enriched waters such as industrial effluents or from decaying
plant materials and soils.

Coliform organisms are recognized as suitable microbial
indicators of drinking water quality. Coliforms include
heterogeneous lactose fermenting bacteria found in faeces
and environment. Detection of coliforms suggests inadequate treatment, post-treatment contamination or excessive
nutrients. Although coliforms may not always be related to
the presence of faecal contamination or pathogens, they are
useful for monitoring the microbial quality of public water
supplies. Presence of coliforms in the absence of faecal
coliforms suggests the use of secondary indicators like
faecal streptococci for confirmation of faecal contamination.

Out of 780 water samples, 185 (23.5%) and 156 (20%) (Fig. 3)
and out of 808 unbranded beverages, 549 (45%) and 463 (38%)
(Fig. 4) samples were found contaminated with faecal coliforms
and E. coli, respectively.

Figures 3 and 4 present the occurrence of organisms of public
health significance in water and beverage samples, respectively. The microbiological analyses revealed that a total of
36% water samples and 48% of unbranded beverages were
unfit for human consumption due to the presence of organisms of public health significance i.e. indicator organisms
and/or pathogenic bacteria. The branded samples, on the
other hand were all free from organisms of public health
significance.
In the present study, a total of 269 out of 780 water samples
(34.5%; Fig. 3) and 549 out of 808 unbranded beverages
samples (45%, Fig. 4) were found contaminated with coliform
bacteria (Noble et al., 2004, 2003; Reasoner and Galdreich,
1985).
Thermotolerant (faecal) coliform group comprises of
Escherichia, Klebsiella, Enterobacter and Citrobacter,
which are able to ferment lactose at 44-45 °C. Out of these

300
250
200

269
185
156

150
76

100
50

Fig. 3. Incidence of organisms of public health significance
in water samples.

The presence of Pseudomonas aeruginosa in potable water
also indicates serious deterioration in bacteriological quality
and is often associated with complaints about taste, odour
and turbidity linked to low rates of flow in distribution system
and a rise in water temperature.

600

549

549
463

500
400

Branded
Unbranded

300
200
100

0 0

0 12

0 0

Fig. 4. Incidence of organisms of public health significance
in beverage samples.

Conclusion
The present study has shown that the microbiological quality
of water and locally made beverages vended in schools is not
satisfactory and approximately 36% water samples and 48%
unbranded beverage samples were found to be unfit for
human consumption due to the presence of organisms of
public health significance i.e. indicator organisms and/or
pathogenic bacteria.
Water sources must be protected from contamination by the
human and animal excreta and other wastes to protect the
community from the risks of outbreaks of intestinal and other
infectious diseases. Moreover, effective treatment and
regular monitoring must be carried out in order to protect the
water reservoirs. Furthermore, strict sanitary and hygienic
regulations must be imposed on the local manufacturers of
beverages; the sanitary conditions of the vending carts and
the beverages being sold through them must also be effectively monitored.

Microbiological Quality of Drinking Water

References
Allen, M.J., Edberg, S.C., Reasoner, D.J. 2004. Heterotrophic
plate count bacteria - what is their significance in drinking water. Intternational Journal of Food and Microbiology, 92: 265-274.
APHA, 1998. Microbiological examination. In: Standard
Methods for the Examination of Water and Wastewater,
20 th edition, American Public Health Association,
Washington DC., USA.
Exner, M., Vacata, V., Gebel, J. 2005. Public health aspects of
the role of HPC - An introduction. In: Heterotrophic Plate
Counts and Drinking Water Safety, J. Bartram Cotruvo,
M. Exner and A. Glasmacher (eds.), AITBS Publishers,
New Delhi, India.
ISO 9308-1:2000(E). Detection and enumeration of
Escherichia coli and coliform bacteria. Part 1. Membrane
Filtration Method.
ISO 9308-2:1990(E). Water quality - detection and enumeration of coliform organisms, thermotolerant coliform
organisms, and presumptive Escherichia coli. Part 2:
Multiple Tube (Most Probable Number) Method.
ISO 6222:1988a. Enumeration of viable microorganisms.
Colony count by inoculation in or on nutrient culture
medium.
ISO 8199:1988b. General guide to enumeration of microorganisms by inoculation in or on culture medium.
ISO 7899-1:1984. Detection and enumeration of faecal streptococci-Part 1: Method by enrichment in a liquid medium.
King, Jr., A.D., Nagel, Ch. W. 1975. lnfluence of carbon dioxide
upon the metabolism of Pseudomonas aeruginosa.
Journal of Food Science, 40: 362-366.
King, Jr., A.D., Nagel, Ch. W. 1967. Growth inhibition of a
Pseudomonas by carbon dioxide. Journal of Food
Science, 32: 575-579.
Kohnen, W., Teske-Keiser, S., Meyer, H.G., Loos, A.H., Pietsch,
M., Jansen, B. 2005. Microbiological quality of carbonated drinking water produced with in-home carbonation
systems. International Journal of Hygiene and Environmental Health, 208: 415-423.
Mugochi, T., Parawira, W., Mpofu, A., Simango, C., Zvauya,
R. 1999. Survival of some species of Salmonella and
Shigella in mukumbi, a traditional Zimbabwean wine.
International Journal of Food Science and Nutrition,
50: 451-455.

49
Noble, R.T., Leecaster, M.K., McGee, C.D., Weisberg, S.B.,
Ritter, K. 2004. Comparison of bacterial indicator analysis
methods in stormwater-affected coastal waters. Water
Research, 38: 1183-1188.
Noble, R.T., Moore, D.F., Leecaster, M.K., McGee, C.D.,
Weisberg, S.B. 2003. Comparison of total coliform, fecal
coliform and enterococcus bacterial indicator response
for ocean recreational water quality testing. Water
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PSQCA, 2004. Pakistan Standard Specification for Drinking
Water. PS: 4639-2004 (R), Pakistan Standards & Quality
Control Authority, Standards Development Centre.
PSQCA, 2002. Pakistan Standard Specification for Carbonated Beverages. PS: 1654-2002 (R), Pakistan Standards
and Quality Control Authority, Standards Development
Centre.
Reasoner, D.J., Geldreich, E.E. 1985. A new medium for the
enumeration and subculture of bacteria from potable
water. Applied Environmental Microbiology, 49: 1-7.
SLS, 2001. Sri Lankan Standards 894, specification for packaged bottled drinking water.
SLS, 1995. Sri Lankan Standards 1038. Specification for bottled
natural mineral water.
Sheth, N.K., Wisniewski, T.R., Franson, T.R. 1988. Survival of
enteric pathogens in common beverages: an in vitro study.
American Journal of Gastroenterology, 83: 658-660.
Simango, C., Rukure, G. 1992. Survival of bacterial enteric
pathogens in traditional fermented foods. Journal of
Applied Bacteriology, 73: 37-40.
Thompson, J.S., Hodge, D.S., Borczyk, A.A. 1990. Rapid
biochemical test to identify verocytotoxin-positive strains
of Escherichia coli serotype O157. Journal of Clinical
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Online publication of Center for Food Safety & Applied
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Chapter 5 (2006).
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158-160.

Short Communication
Pak. J. Sci. Ind. Res. 2010 53 (1) 50-51

Feeding Inter-Relationship of Caranx hippos (Linneaus),
Chrysichthys nigrodigitatus (Lacepede), Ethmalosa fimbriata (Bowdich)
and Mugil cephalus (Linneaus) in Lagos Lagoon, Nigeria
Adebiyi Adenike Fatimat
Department of Marine Sciences, Faculty of Science, Univeristy of Lagos, Lagos, Nigeria
(received July 17, 2009; revised November 23, 2009; accepted December 5, 2009)

Abstract. Study of the feeding inter-relationship of Caranx hippos, Chrysichthys nigrodigitatus, Ethmalosa fimbriata
and Mugil cephalus in the Lagos Lagoon, Nigeria, revealed that algae and diatoms formed the main food items of the
four fish species; other food items were crustaceans, molluscs and detritus. Utilization of nearly identical food items
suggested inter-specific competition for food.
Keywords: feeding habits, Caranx hippos, Chrysichthys nigrodigitatus, Ethmalosa fimbriata, Mugil cephalus, Nigeria

Study of food and feeding habits of fish requires continuous
research, since successful fishery management, aquaculture and
capture fishery programmes are based on it (Oso et al., 2006).

Calanus finmarchicus, cladocera and crab appendages),
fish (fins, scales, eggs and bones), algae, plant material and
unidentifiable matter (Fig. 2).

Caranx hippos, Chrysichthys nigrodigitatus, Ethmalosa
fimbriata and Mugil cephalus are some of the fish species readily
available in the Lagos Lagoon, Nigeria in West Africa, and make
up an important part of artisanal fisheries. Several studies of
the food and feeding habits of these four fish species have been
made, some of which include the work of Oronsaye and
Nakpodia, (2005) and Blay (1995). However, information on
the feeding inter-relationship of these species is lacking. In
this paper, a report on the feeding inter-relationship among
the four fish species is presented.

Diatoms were the major food items of E. fimbriata as well.
Other food items were crustaceans (Calanus finmarchicus,
shrimp parts, isopods and cladocera), fish (bones, scales
and eggs), algae, plant materials and unidentifiable matter
(Fig. 3).
Major food items in the gut of M. cephalus were diatoms.
Other food items were crustaceans (Calanus finmarchicus and
shrimp parts), fish (scales and bones), algae, plant materials
and unidentifiable matter (Fig. 4).
Analysis of the food items in the gut of four fish species
revealed that C. hippos did not feed on fish in February and
April, molluscs in April and did not feed on plant material at
all. C. nigrodigitatus fed on plant material in Februaury and
fish in May. E. fimbriata did not feed on fish in February and
April, on plant material in April and did not feed on molluscs
at all throughout the months studied. M. cephalus did not feed
on fish in April and May and did not feed on molluscs at all.

Forty specimens of the above-mentioned four fish species were
caught in the Lagos lagoon each month during February to
May 2001. Body weights and lengths of fish were measured
and the stomach contents were studied. The organisms found
were identified to the species level and analyzed by numerical and frequency of occurrence method.
Analysis of the food of C. hippos revealed diatoms and algae
to form the major food items. Other food items were molluscs
(Aloidis trigona, bivalve shell and Tympanotonus fuscatus),
crustaceans (Calanus finmarchicus, shrimp and shrimp parts),
fish (eggs, bones, scales and flesh) and detritus and other
unidentifiable matter (Fig. 1).

The study reveals the food and feeding habits of the four fish
species. M. cephalus is a plankton feeder, feeding mainly on
algae and diatoms (Ramirez-Luna et al., 2008). In this study
the important food item of M. cephalus comprised of diatoms,
while other food items were algae, crustaceans, plant material
and detritus. The food items of C. nigrodigitatus included plant
materials, molluscs, crustaceans, fish and detritus. Dada and
Araoye (2008) also discovered similar food items in the
stomach of C. nigrodigitatus. Ajah et al. (2006) reported

Diatoms formed the major food items of C. nigrodigitatus.
Other food items were molluscs (Aloidis trigona, bivalve shell
and Tympanotonus fuscatus), crustaceans (shrimp parts,
E-mail: Adebiyi_fatima@yahoo.com

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Pak. J. Sci. Ind. Res. 2010 53 (1) 52-58

Technology

Production and Characterization of Chitosan from
Shrimp (Penaeus semisulcatus ) Shell Waste of UAE
Fazilatun Nessa*, Saeed Ahmed Khan and Farah Mohammad Anas Al Khatib
Dubai Pharmacy College, Al Muhaisanah 1, Al Mizhar, P.O.Box. 19099, Dubai - United Arab Emirates
(received June 11, 2009; revised December 1, 2009; accepted December 16, 2009)

Abstract. Chitosan was prepared from shrimp (Penaeus semisulcatus) shell waste by a chemical process involving
demineralization, deproteinization and deacetylation; conversion of chitin to chitosan (deacetylation) was achieved by
treatment with concentrated sodium hydroxide solution (55%) at room temperature (25 ºC). The present study was
undertaken to evaluate the influence of deacetylation process during chitosan production on the physicochemical and
functional properties of shrimp shell chitosan. Four experimental chitosan samples were prepared with deacetylation
for 40 h, for 50 h, with and without stirring as well as for 60 h and were subjected to physicochemical and functional
characteristic analysis. Change in duration of deacetylation process yielded some differences in each characteristic;
deacetylation for 40 h led to lower viscosity, solubility, water/fat binding capacity and degree of deacetylation and for
60 h resulted in increase in solubility but decrease in viscosity. Stirring during deacetylation process led to lower
viscosity, higher degree of deacetylation and higher fat binding capacity of the product. In contrast non-stirred sample
produced product with lower degree of deacetylation and higher viscosity. It was concluded that duration of
deacetylaylation process should be monitored constantly for optimal chitosan production depending on its intended
usages in food, pharmaceutical and biomedical industries.
Keywords: shrimp shell waste, deacetylation, chitosan, chitin

Introduction
Chitosan is a fiber-like substance derived from chitin, a
homopolymer of ß-(1→4)-linked N-acetyl-D-glucosamine.
Chitin is widely distributed in marine invertebrates, insects,
fungi, and yeast (Subasingle, 1995; Austin et al., 1981); however, it is not present in higher plants and higher animals.
Generally, the shells of selected crustaceans consist of 30-40%
protein, 30-50% calcium carbonate and calciumc phosphate
and 20-30% chitin (Acosta et al., 1993; Knorr, 1984). Chitin is
widely available from a variety of sources among which, the
principal source is shellfish waste such as that of shrimps, crabs
and crawfish (Rinaudo, 2006; Allan and Hadwiger, 1979). It
also exists naturally in a few species of fungi (Franco et al.,
2004; Andrade et al., 2000; Chung et al., 1994). Chitin and
chitosan have similar chemical structures (Fig. 1). Chitin is made
up of a linear chain of acetylglucosamine groups while chitosan
is obtained by removing enough acetyl groups (CH3-CO) from
the molecule so that it becomes soluble in most diluted acids.
This process is called deacetylation. The actual difference
between chitin and chitosan is the acetyl content of the polymer. Chitosan having a free amino group is the most useful
derivative of chitin (No and Meyers, 1992).

CH 2 OH

NHCOCH3
O

HO
CH 2 OH

NHCOCH3
O

O
NHCOCH3

CH 2 OH

Chitin
CH 2 OH

NH2

O
CH 2 OH

NH 2

O
O

CH 2 OH

Chitosan

Fig. 1. Structure of chitin and chitosan.
1997). Over the last several years, chitinous polymers, especially chitosan, have received increased attention as one of
the promising renewable polymeric materials for their extensive applications in the pharmaceutical and biomedical industries for enzyme immobilization and purification, in chemical
plants for wastewater treatment and in food industries for use
in food formulations as binding, gelling, thickening and stabilizing agent (Prashanth and Tharanathan, 2007; Franco et al.,
2004; Knorr, 1984).
Traditional isolation of chitosan from crustacean shell waste
consists of four basic steps: demineralization, deproteinization,
decolourization and deacetylation (Galed et al., 2008; No and
Meyers, 1995). Several procedures have been developed and
proposed by many researchers over the years for preparation
of chitosan from different crustacean shell wastes (Galed

Chitosan is a non toxic, biodegradable polymer of high
molecular weight (Zhang and Neau, 2001; Tomihata and Ikada,
*Author for correspondence; E-mail: nessa1995@yahoo.com

Production of Chitosan from Shrimp Shell Waste

et al., 2008; No and Meyers, 1995; No et al., 1989). Some of
these formed the basis of chemical processes for industrial
production of chitosan. But most of the reported processes
were carried out with 45% concentrated sodium hydroxide
solution at 100 °C or higher temperature with autoclaving
(Galed et al., 2008; Prashanth and Tharanathan, 2007; Domard
and Rinaudo, 1983; Horton and Lineback, 1965). Therefore,
the specific objectives of this work were to develop an
optimum shrimp shell chitosan production process at room
temperature (25 °C) with increased alkali strength without
decolourization step and to study the influence of deacetylation
process on the physicochemical and functional properties of
shrimp shell chitosan.

Materials and Methods
Shrimp shell chitosan production. Penaeus semisulcatus,
Metapenaeus mastersii and Penaeus latisulcatus are the
shrimp species found in UAE waters of which Penaeus
semisulcatus is the most common and commercially important species. Undersized shrimp shell waste of Penaeus
semisulcatus was obtained from a commercial shrimp shell
processor of Dubai, UAE. Upon receipt, shells (head, body
and tail) were washed under running warm tap water to
remove soluble organics, adherent proteins and other impurities. The shells were then dried in the oven (Mammert,
Germany) at 70 °C for a period of 24 h or longer until
completely dried shells were obtained. The moisture content
of dried shell was 0.48%. To obtain a uniform size product,
the dried shell was ground through a centrifugal grinding mill
and sifted with 20-mesh (0.841 mm) and 40-mesh (0.425 mm)
sieves. Dried ground shell powder was placed in opaque
plastic bottles and stored at room temperature until used. The
production of chitosan from shrimp shell waste was carried
out with a modified method of No et al. (1989). The dried
shrimp shell powder (5 kg) was demineralized with 8-10%
hydrochloric acid at ambient temperature with a solid to
solvent ratio of 1:15 (w/v), in an acid resistant vessel with
stirrer for 20-22 h until deminerali-zation was completed. The
demineralized shells were deproteinized with 8-10% sodium
hydroxide solution for 20-22 h at 65 °C with constant stirring
or without stirring at a solid to solvent ratio of 1:10 (w/v).
Samples were then washed with tap water and dried under
vacuum for 2-3 h until the powder was crispy. Removal of
acetyl groups from chitin was achieved by using concentrated
sodium hydroxide solution (55%) with a solid to solvent ratio
of 1:10 (w/v). Samples of four experimental shrimp shell
chitosans were prepared. The chemical reactions were carried out at room temperature (25 °C). Duration of deacetylation
process was 40 h for sample C40, 50 h for C50S (with magnetic

stirring), 50 h for C50WS (without stirring) and 60 h for C60.
The resulting chitosans were washed to neutrality in running
tap water, rinsed with distilled water, filtered and dried at
60 °C for 24 h in the oven. The obtained shrimp shell chitosan
was white to off white in colour and it was not necessary to
decolourize or bleach it.
Physicochemical and functional properties. Measurement
of nitrogen. Nitrogen of the crawfish chitosan was determined
using a microprocessor-based, software-controlled instrument
Model-TruSpec CN (Model # FP-428 Leco Corporation,
USA). There were three phases during an analysis cycle, i.e.,
purging, burning and analysis. The encapsulated sample was
purged of any atmospheric gases that had entered during
sample loading. During the burning phase, the sample was
dropped into a hot furnace (850 °C) and flushed with pure
oxygen for a very rapid combustion. Finally, in the analysis
phase, the remaining combustion product (nitrogen) was measured by the thermal conductivity cell. The final result was
displayed as percent nitrogen.
Ash. Ash of the crawfish chitosan was calculated according
to the standard method # 923.03 (AOAC, 1990). 2.0 g of
chitosan (triplicate) were placed into previously ignited,
cooled, and tarred crucible. The samples were heated in a
muffle furnace preheated to 600 °C for 6 h. The crucibles
were allowed to cool in the furnace to less than 200 ºC
and then placed in desiccator with a vented top. Crucibles
were cooled, weighed and ash content was recorded.
Degree of deacetylation. Chitosan samples prepared in the
form of KBr discs were studied for the degree of deacetylation
(DD) (Kassai, 2008; Khan et al., 2002). The prepared chitosan
KBr discs were kept in desiccators for 12 h and then placed in
sealed plates before scanning. The DD of chitosan was established using a FTIR (Fourier Transform Infrared Spectroscopy) instrument (Model # M2000, Midac Corp. USA) with
frequency of 4000-4/cm. The degree of deacetylation (DD)
of the chitosan was calculated using the baseline reported by
Khan et al. (2002). The computation equation for the baseline
is given below:
DD = 100 – [(A1655 / A3450) × 100 / 1.33]
where A1655 and A3450 are the absorbance at 1655 cm-1 of
the amide-I band as a measure of the N-acetyl group content and at 3450 cm-1 of the hydroxyl band as an internal
standard to correct for disc thickness. The factor ‘1.33’
denotes the value of the ratio of A1655/A3450 for fully
N-acetylated chitosan.
Viscosity. Viscosity of chitosan was determined with a
Brookfield viscometer (Model DV-II + Brookfield Engineering

Laboratories Inc., Stonghton, MA.). Chitosan solution was
prepared in 1% acetic acid at 1% concentration on dry
basis. Measurement was made in duplicate using a No. 27
spindle at 50 rpm on solutions at 25 °C with values reported
in centipoise (cP) unit.
Solubility. Crawfish chitosan sample (0.1 g in triplicate) was
placed in a centrifuge tube (known weight) then dissolved with
10 ml of 1% acetic acid for 30 min. The solution was then
centrifuged at 10,000 rpm for 10 min. The supernatant was
decanted. The undissolved particles were washed in
distilled water (25 ml) then centrifuged at 10,000 rpm. The
supernatant was removed and undissolved pellets were dried
at 60 °C for 24 h. Finally, the particles were weighed and the
percentage solubility was determined.
Water binding capacity (WBC). WBC of chitosan was
measured using a modified method of Knorr (1982). Initially
a centrifuge tube containing 0.5 g of sample was weighed,
10 ml of water was added and mixing was carried out on a
vortex mixer for one min to disperse the sample. The contents were left at ambient temperature for 30 min with intermittent shaking for 5 s every 10 min and then centrifuged
(Model # Z383K, HERMLE-National Labnet Company,
USA) at 3,500 rpm (6,000 × g) for 25 min. After the supernatant was decanted, the tube was weighed again. WBC was
calculated as follows:
WBC (%) = [water bound (g)/ initial sample weight (g)]× 100.
All experiments were carried out in triplicate.
Fat binding capacity (FBC). FBC of chitosan was measured
using a modified method of Knorr (1982). Initially a centrifuge tube containing 0.5 g of sample was weighed, 10 ml of
oil (three types of oil were used namely soybean, corn and
sunflower oils) were added and mixing was carried out on a
vortex mixer for 1 min to disperse the sample. The contents
were left at ambient temperature for 30 min with shaking
for 5 s every10 min and then centrifuged at 3,500 rpm
(6,000 × g) for 25 min. After the supernatant was decanted,
the tube was weighed again. FBC was calculated as follows:
FBC (%) = [fat bound (g)/ initial sample weight (g)] × 100.
Experiments were performed in triplicate.
Statistical analysis. All experiments were carried out in
triplicate, Average values (means) and standard deviations
were reported. Mean separations were analyzed using the
ANOVA and Tukey’s student range tests at á = 0.05.

Results and Discussion
Yield. Yield was calculated as the dry weight of chitin
obtained from 5 kg of dried shrimp shell powder. The yield of

Fazilatun Nessa et al.

chitin was 20% and that of chitosan ranged from 16-19%.
The highest yields were obtained from sample C40 (19%),
followed by C50WS (18%), C60 (17%), and C50S (16%). Results
are shown in Table 1. Brzeski (1982) reported about 14% yield
of chitosan from krill and 18.6% from prawn waste (Alimuniar
and Zainuddin, 1992). The yield of chitosan obtained (1518%) is lower than that (approximately 23%) of chitin
reported in the literature (No and Meyers, 1989). This may be
due to loss of sample mass/weight during deacetylation process as we used here 55% concentrated sodium hydroxide
solution, whereas in other methods 45% sodium hydroxide
solution was used. The moisture content of the shrimp shell
chitosan, determined by the gravimetric method (Black, 1965),
was in the range of 0.3% to 0.4% (Table 1).
Table 1. Proximate analysis of shrimp shell and commercial
chitosans (dry weight basis)
Sample

Yield Moisture
(%)

C40
19
C50S
16
C50WS
18
C60
17
Sigma 91**

Nitrogen

(%)

Ash

(%)
a

0.4 (0.25) *
a
0.3 (0.20)
a
0.4 (0.25)
a
0.4 (0.22)
b
2.5 (0.11)

(%)
a

8.33 (0.02) *
a
8.19 (0.01)
a
8.11 (0.05)
a
7.91 (0.05)
a
8.23 (0.09)

0.29 (0.07) *
a
0.3 (0.99)
a
0.3 (0.23)
a
0.3 (0.98)
a
1.5 (0.25)

* = numbers in parentheses are standard deviations; means with
different letters in each column are significantly different (P < 0.05);
** Sigma 91 is a commercial crab chitosan.

Nitrogen content. Nitrogen content of the shrimp shell
chitosan samples varied between 7.91% and 8.33% on a dry
basis, showing no significant differences (P >0.05) in nitrogen content, but the values were slightly higher than that
(7.06% to 7.97%) reported by No and Meyers (1995), for
chitosan from crab and shrimp shell on a dry basis. This is
probably due to the presence of protein residues as mentioned
by Rutherford and Austin (1978). Protein is bound by covalent bonds forming stable complex with chitin and chitosan.
Thus, it is very difficult to achieve 100% deproteinization.
Even with complete deprotinization, nitrogen was still present
since chitosan has the amino (-NH2) group.
Ash. Table 1 shows the ash content of shrimp shell chitosan
in the range of 0.29-0.3%. Ash measurement is an indicator of
the effectiveness of the demineralization step for removal of
calcium carbonate. Elimination of demineralization step
results in products having 31-36% ash (Bough et al., 1978).
Some residual ash of chitosans may affect their solubility,
consequently contributing to lower viscosity, or can affect other
more important characteristics of the final product. A high

Production of Chitosan from Shrimp Shell Waste

quality grade of chitosan should have less than 1% of ash
content (No and Meyers, 1995). An ash content of less than
1% from crab chitosans has been reported by No and Meyers
(1995). The results presented in Table 1 indicate that the
resultant chitosan sample was completely demineralized and
contained less than 1% ash.
Degree of deacetylation. The degree of deacetylation
(DD) of the studied shrimp shell chitosan samples ranged
from 55% to 76% (Table 2). According to No and Meyers
(1995), DD of chitosan ranges from 56% to 99% with an
average of 80%. Sample C50S (76%) had the highest DD,
followed by C 50WS, C 60 and C 40 (75%, 74%, and 55%,
respectively).
As in the Table 2, C40 had a very low solubility and viscosity
which may be due to the lower DD value. Therefore,
comparison among samples C50S, C50WS and C60, sample C50S
gave lower viscosity (136.6 cP) and higher DD (76%) value
which are very important characteristics of chitosan. The
medical and pharmaceutical applications of chitosan as
antitumor, hemostatic, hypocholesterolemic, antimicrobial
and antioxidant depends mostly upon DD and solubility (Jian
et al., 2008; Muzzarelli and Muzzarelli, 2005). However, we
expected that samples C50S, C50WS and C60 would have higher
DD with higher solubility but the values obtained were lower
than the expected ones. According to Kassai (2008) and Khan
et al. (2002) , the IR spectroscopic method is commonly used
for the estimation of chitosan DD values for its advantages: it
is relatively fast and does not require dissolution of the chitosan
sample in an aqueous solvent. DD values are not only highly
dependent on the source and method of purification (No et
al., 1989) but also on the type of analytical methods employed,
sample preparation and type of instrument used; other conditions may also influence the analysis of DD (Kassai 2008;
Khan et al., 2002).
Viscosity. The viscosity of chitosan solutions, reported
in the literature, generally ranges from 60 to 780 cP
(Alimuniar and Zainuddin, 1992). This range of viscosity
was also observed by Cho et al. (1998) for five commercially available chitosans. The results of viscosity, solubility
and degree of deacetylation of our shrimp shell chitosans
are shown in Table 2.
Bough et al. (1978) stated that viscosity of chitosans varied
considerably from 60 to 5,110 cP depending on the species.
Our shrimp shell samples had viscosity ranging from 90.7 to
170.2 cP. C40 had the lowest viscosity (90.7 cP) comparable
to that of other samples as of lower solubility may be due
to incomplete deacetylation of the sample. Whereas C50WS had
a very high viscosity (170.2 cP) (Table 2). Some factors

affect viscosity during the production of chitosan such as the
degree of deacetylation, molecular weight, concentration, ionic
strength, pH and temperature, etc. Moorjani et al. (1975)
reported that viscosity of chitosan decreased with increasing
time of demineralization. The viscosity of chitosan in acetic
acid tends to increase with decreasing pH but decrease with
decreasing pH in HCl. Intrinsic viscosity of chitosan is a
function of the degree of ionization as well as ion strength
(Bough et al., 1978). Deproteinization with 3% NaOH and
elimination of the demineralization step in chitin preparation,
decreased the viscosities of the final chitosan samples (Bough
et al., 1978). Moorjani et al. (1975) stated that it is not
desirable to bleach the material at any stage since bleaching
considerably reduces the viscosity of the final chitosan product. Our product, prepared without bleaching step, gave lower
viscosity which was desirable for preservation of foods against
microbial deterioration, formation of biodegradable films and
medical applications (Liu et al., 2008; Zeng et al., 2008).
Solubility. Three shrimp shell chitosan samples demonstrated
excellent solubility ranging from 98.01 to 99% with no
significant difference (Table 2), except sample C40, which
showed comparatively lower solubility (60.3%); it may be due
to lower degree of deacetylation. Brine and Austin (1981)
noted that lower solubility values suggested incomplete
removal of protein and acetyl group. Since solubility of
chitosan depends on the removal of acetyl group from chitin
therefore lower DD value and the presence of protein
contaminants remaining in the sample during the analysis
process could adversely interfere with the results.
Water binding capacity (WBC). Water binding capacity of
shrimp shell and commercial chitosans are shown in Table 3.
WBC differed among crawfish chitosan samples, ranging from
299.6 % to 745.4%. There were no significant differences in

Table 2. Viscosity, solubility and degree of deacetylation of
shrimp shell and commercial chitosans
Sample

Viscosity
(cP)

Solubility
(%)

Degree of
deacetylation
(%)

C40
C50S
C50WS
C60
Sigma 91**

90.7 (5.07)*a
136.6 (2.09)b
170.2 (3.66)c
154.29 (2.69)d
380.15 (3.44)e

60.3 (0.61)a
98.2 (0.66)b
98.01 (0.45)b
99.00 (0.56)b
89.88 (0.42)c

55
76
75
74
74

* = numbers in parentheses indicate standard deviation; means with
different letters in each column are significantly different (P < 0.05);
** Sigma 91 is a commercial crab chitosan.

Fazilatun Nessa et al.

Table 3. Water binding capacity and fat binding capacity of shrimp shell and commercial chitosans
Sample
C40
C50S
C50WS
C60
Sigma 91**

WBC (%)
299.6 (9.97)*a
738.8 (5.6)b
745.4 (4.9)b
732.2 (4.04)b
538.5 (4.99)c

Soybean oil

Fat binding capacity (%)
Corn oil

258.7(8.9)a
587.3 (5.3)b
571.5 (7.9)b
575.8 (6.5)b
379.7 (5.9)c

245.5 (4.8)a
599.2 (8.5)b
583.6 (7.3)b
577.5 (6.7)b
444.3 (5.3)c

Sunflower oil
255.7 (5.3)a
586.8 (9.9)b
579.4 (5.6)b
566.9 (7.6)b
398.6 (6.6)c

* = numbers in parentheses indicate standard deviation; means with different letters in each column are significantly different (P < 0.05);
** = Sigma 91 is a commercial crab chitosan.

WBC between C50S, C50WS and C60. These values were in agreement, except C40, with those reported by Cho et al. (1998)
where WBC for chitosans ranged from 458% to 805% for
five commercial chitosans from shrimp and crab shell. Sample
C40 had a lower WBC (299.6 %) than that of other samples; it
may be due to lower DA value.
Fat binding capacity (FBC). Fat binding capacity (FBC) of
four shrimp shell chitosans was measured using three types of
oils including soybean, corn, and sunflower oil. The results
are shown in Table 3. FBC differed among chitosan products,
ranging from 245.5% to 599.2%. Among our crawfish chitosan
samples, C50S showed the highest FBC values: 587.3% with
soybean oil, 599.2% with corn oil and 586.8% with sunflower
oil, although C50S had low viscosity (136.6 cP); C50S, C50WS
and C60 showed no significant difference in FBC.
The sample C40 showed the lowest FBC (245.5%-258.7%) as
it was not properly deacetylated; it seems higher deacetylation
facilitates oil binding capacity of chitosan. Several workers
suggested that the DD of chitosan is an important factor which
influences fat binding capacity of chitosan (Shahidi et al.,
2002). They suggested that increased DD causes increased
electrostatic force between chitosan and fatty and bile acid
and increased FBC. Moorjani et al. (1975) advocated that
changing the sequence of steps, when demineralization is
conducted prior to deproteinization and finally deacetylation,
results in an increase in FBC than when deproteinization is
conducted prior to demineralization and finally deacetylation.
Amongst the three types of oil used, soyabean oil generally
demonstrated more FBC with shrimp shell chitosan samples,
whereas sunflower oil showed the least FBC. Regardless of
the type of vegetable oils, the four prepared shrimp shell
chitosan samples showed desirable FBC ranging from 566.9%
(with sunflower) to 599.2% (with corn) which is in agreement
with those (314 to 535% with an average of 417%) reported
by No et al. (2000). Sample C40 showed lower value than the
reported value. It seemed degree of deacetylation influenced
the fat binding capacity of chitosan.

Conclusion
Throughout the literature on chitosan, the main emphasis is
on its quality and physicochemical properties which vary
widely with the crustacean species and the preparation
methods. Most of the reported preparation methods used high
temperature with 45% concentrated alkali and sometimes used
autoclave. Based on the reported practice this research study
attempted to present a process for the production of shrimp
shell chitosan at room temperature (25°C) with increased
alkali strength (55%); it could help to develop small industry
without wasting energy. This study also demonstrated that the
duration of deacetylation process affects the quality of the
products. In view of the foregoing, it is our recommendation
that for the purpose of achieving uniformity and proper
product quality control for particular usage of chitosan, the
relationship between the process protocols/conditions and the
resulting specific characteristics of chitosan products must be
monitored constantly and properly.

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