Bull Environ Contam Toxicol (2012) 89:658–663

DOI 10.1007/s00128-012-0725-5

Pesticide Residues in Tobacco Leaves from the Kushtia District

in Bangladesh
Mohammad Abdur Rahman • Alamgir Zaman Chowdhury •
Mohammed Moniruzzaman • Siew Hua Gan • Mohammad Nazrul Islam •

Zeenath Fardous • Mohammad Khorshed Alam

Received: 27 March 2012 / Accepted: 29 June 2012 / Published online: 11 July 2012
Ó Springer Science+Business Media, LLC 2012

Abstract In this study, tobacco leaf samples (n = 26) Bangladesh is predominantly an agricultural country, with
were collected from different areas in Kushtia to deter- over 70 % of its population dependent on farming and
mine the presence of residues of cypermethrin, diazinon, approximately 23 % of its gross domestic product derived
heptachlor, methoxychlor dichlorodiphenyltrichloroethane from this sector (Akhter 2011). Together with jute, cotton,
(DDT), dichlorodiphenyldichloroethan (DDD) and dichlor- sugarcane, tea and betel leaf, tobacco has been listed as one
odiphenyldichloroethylene (DDE). The analysis was con- of the six major cash crops in Bangladesh (Akhter 2011).
ducted by a High Performance Liquid Chromatography Tobacco (Nicotiana tabacum L.) from the family solana-
(HPLC) system that was equipped with a photodiode array ceae grows in drylands, is water demanding and consumes
detector. Both cypermethrin and diazinon were detected in large quantities of fertilizers and pesticides.
tobacco samples from six districts, namely, Mirzapur, Pesticide residues can remain on tobacco leaves after
Shahebnagar, Kodalipara, Pragpur, Farakpur and Taragunia. processing tobacco into its manufactured forms. Further-
The highest concentration of cypermethrin was found in more, and compared with other crops, tobacco leaves have a
Kodalipara (2.00 ppm) while the highest concentration of greater surface to weight ratio (Drossopoulos et al. 1998),
diazinon was detected in a sample from Pragpur (0.15 ppm). and with excessive use of pesticides, pesticide residues may
The pesticide DDT was only detected in the sample from accumulate in the leaf tissue. When tobacco is smoked, the
Pragpur at 4.00 ppm. This is the first study in Bangladesh residues are burnt together with the tobacco and both active
that reports pesticide residue concentrations in tobacco leaf and passive smokers inhale the mixture. A study to deter-
samples. mine the chlorinated pesticide residue levels in mainstream
smoke reported that the transfer rate of pesticides from
Keywords Cypermethrin
Diazinon
Pesticide
DDT
tobacco into smoke averaged approximately 12 % of that in
DDD
DDE
Tobacco
HPLC tobacco before combustion (Clapp et al. 1972). Another
study reported that dichlorodiphenyltrichloroethane (DDT)
and its derivatives transfer into mainstream smoke at up to
M. A. Rahman
A. Z. Chowdhury
M. Moniruzzaman
20 % of the residue in unsmoked tobacco (NHMRC 1982).
M. N. Islam
Z. Fardous
M. K. Alam
These findings indicate that pesticide residues in tobacco
Agrochemicals and Environmental Research Division, Institute
of Food and Radiation Biology, Atomic Energy Research are likely to increase the known adverse effects of inhaling
Establishment, Ganakbari, Savar, Dhaka 1349, Bangladesh tobacco smoke.
Tobacco is mainly cultivated in different locations of
M. Moniruzzaman
Khagrachari, Bandarban, Chittagong Hill tracks and
Department of Pharmacology, School of Medical Sciences,
Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Kushtia. A study conducted by the Policy Research for
Malaysia Development Alternatives (UBINIG) in three different
districts indicated that Kushtia has 53.33 % of the total farm
S. H. Gan (&)
holdings of tobacco, followed by Cox’s Bazar (36.04 %)
Human Genome Centre, School of Medical Sciences, Universiti
Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia and Bandarban (10.41 %) (Akhter 2011). Tobacco is prone
e-mail: [email protected] to many diseases and therefore requires chemical treatments

123
Bull Environ Contam Toxicol (2012) 89:658–663 659

with up to 16 applications of pesticides, including those that and then blended using an electric blender (Model Classic
are banned for use, such as aldrin, dieldrin and DDT (Khan 5 Speed, Crofion, China). The blended leaves were then
2007). As a result, farmers have increased pressure to use weighed using an analytical balance (Crystal-200, GI-
different types of pesticides to produce in large quantities. BERTINI, Italy). Five grams of each blended tobacco leaf
Farmers who may not be aware of the health effects and samples were transferred into a 250 mL Erlenmeyer flask
proper safety protocols for handling pesticides can be and then 100 mL of residue grade acetone was added using
exposed to their hazardous effects. a multi-Shaker (Max-4000, Guyson Corporation, USA)
Pesticides and fertilizers usually end up in soil and are over 12 h at 150 rpm. The extracts were then concentrated
thus transferred to waterways and food sources. Early to 5 mL on a rotary vacuum evaporator (Rotavapor-R 215,
exposures to pesticides may increase a person’s lifelong Buchi, Switzerland).
risk for cancer and harm the nervous and immune systems. The samples were cleaned up by following the method
Many of these compounds can cause moderate to severe described by (Fardous et al. 2007). Briefly, the acetone
respiratory and neurological damage or may act as geno- extract was subjected to clean up using florisil column
toxic and carcinogenic agents (Hayat et al. 2011). Acute chromatography. The florisil (60–100 mesh) was activated
exposure to high levels of DDT by any route is reported to at 200°C for 6 h and was subsequently deactivated with
lead to neurological effects including excitability, head- 2 % distilled water. The top 1.5 cm of the florisil column
ache, nausea, vomiting and dizziness (ATSDR 2002). The was packed with anhydrous sodium sulfate. Elution was
pesticide DDT is toxic not only to the nervous system but performed with a solvent mixture of double distilled hex-
also to the liver (ATSDR 2002). Both DDT and its ane (65 %) and dichloromethane (35 %) at 5 mL/min. The
metabolites dichlorodiphenyldichloroethane (DDD) and elute was concentrated using a rotary vacuum evaporator
dichlorodiphenyldichloroethylene (DDE) are xenoestro- and was transferred to a vial. Solvents were completely
gens, which influence both normal and neoplastic estrogen- removed under a gentle flow of fresh nitrogen. The evap-
responsive tissues (Muñoz-de-Toro et al. 2006). Accord- orated sample was dissolved in acetonitrile, and a 1 mL
ingly, it has been hypothesized that OCC contribute to the volume was used for the HPLC analysis. The procedure
risk of breast cancer. was repeated for all of the 26 extracted tobacco leaf
To date, no data are available pertaining to the levels of samples.
pesticide residues in tobacco leaf samples that are culti- After the sample clean up, aliquots of the final volume
vated throughout Bangladesh. These data could provide were quantified with a HPLC system (SHIMADZU LC-
information about the status of the tobacco leaves used and 10 Avp-Series Automated with LC Solution Software
the possible detrimental effects to human health should LabSolutions (LC solution Release 1.11SP1) that was
pesticides be present. In this study, we aim to determine the equipped with a SPD-M 10 Avp outfitted with a photodi-
presence and quantity of some selected organochlorine ode array (PDA) detector. A C18 Reverse Phase Alltech
pesticide residues (DDT and its metabolites DDD and analytical column (5 lm, 250 9 4.6 mm) was used and
DDE), organophosphate (diazinon) and pyrethroid (cyper- maintained at 30°C in a column oven. The mobile phase,
methrin) in tobacco leaf samples. which was a combination of 70 % ACN and 30 % water,
was filtered using a cellulose filter (0.45 lm) prior to use
and was allowed to run at 1.2 mL/min. Prior to the HPLC
analysis, the samples were passed through 0.45 lm nylon
Materials and Methods (Alltech Assoc) syringe filters and were manually injected
(20 ll) into the HPLC system each time. The identification
Reference grade standards for cypermethrin (99.0 %), of the suspected pesticide was performed by comparing
diazinon (99.0 %), dichlorodiphenyltrichloroethane (DDT) peak retention times in samples to those of peaks in the
(99.0 %), dichlorodiphenyldichloroethane (DDD) (99.5 %), pure analytical standard. Quantification was performed
dichlorodiphenyldichloroethylene (DDE) (98.5 %), hepta- using the method described by (Chowdhury et al. 2012).
chlor (99.50 %) and methoxychlor (99.5 %) were purchased A typical chromatogram from the analysis is shown in
from GmbH (D-86199 Augsburg, Germany). The organic Fig. 1. The calibration curves for cypermethrin, diazinon
solvents were n-hexane (Merck, Germany) and dichloro- and DDT were prepared between 0.1 and 5.0 ppm in
methane (BDH, England) and were of analytical grade while triplicates.
acetonitrile (Scharlau, EU) was of HPLC grade. The percentage recoveries were calculated using the
Tobacco leaf samples (n = 26) were collected from the following equation:
local area of Kushtia, Bangladesh between 2010 and 2011. Percentage of recovery = [CE/CM 9 100], where CE is
The leaves were labeled prior to storage at 4°C and were the experimental concentration determined from the cali-
extracted within 10 days. The tobacco leaves were dried bration curve and CM is the spiked concentration.

123
660 Bull Environ Contam Toxicol (2012) 89:658–663

mA (x100)
0.030 254nm,4nm
0.028
0.025
0.022
0.020
0.018
0.015 4.977
Cypermethrin
0.013
0.010
0.007
0.005
0.003
0.000
-0.003
-0.005
0. 1. 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 mi

Fig. 1 A typical Chromatogram showing cypermethrin standard peak (RT = 4.97)

Table 1 Percentage recoveries of the analyzed pesticides

Analyte Stock solution Spiking Spiking amount in 5 g of leaf Single spiking level Mean amount Mean
(ppm) volume (ll) sample (lg) (ppm) (lg)a recovery (%)
Spike Measure
d d

Cypermethrin 200 25 5 5 5 4.25 85.0

Diazinon 200 25 5 5 5 4.60 92.0
DDT 200 25 5 5 5 4.50 90.0
DDD 200 25 5 5 5 4.35 87.0
DDE 200 25 5 5 5 4.21 84.2
Heptachlor 200 25 5 5 5 4.43 88.6
Methoxychlor 200 25 5 5 5 4.53 91.0
RT retention time
a
mean values of triplicates

The mean percentage recoveries of cypermethrin, diaz- concentration of diazinon was detected in a sample from
inon, DDT, DDD, DDE, heptachlor and methoxychlor in Pragpur. The amount of pesticide residue that is detected in
the spiked positive controls of tobacco leaf samples after the tobacco leaves is an indication of the amount of pesticide
florisil clean up were greater than 80 %. This value com- applied, the time elapsed since the application and the
plies with the FDA guideline (FDA 2001) (Tables 1, 2), speed with which the pesticides dissipated into the envi-
indicating that the method is suitable for the analysis. The ronment. Cypermethrin and diazinon were detected in six
LOD and LOQs for the pesticides ranged from 0.01 to tobacco samples from Mirzapur, Shahebnagar, Kodalipara,
0.30 ppm and 0.03 to 0.10 ppm, respectively. Pragpur, Farakpur and Taragunia (Table 2). The highest
concentration of diazinon was from Pragpur, at 2.00 ppm
(Fig. 2), while the highest concentration of cypermethrin
Results and Discussion was from Kodalipara, at 0.12 ppm (Table 2). The pesticide
DDT was only detected in one sample from Pragpur, at
This is the first study to report the presence and levels of 4.00 ppm (Fig. 2).
pesticide residues in tobacco leaf samples from Bangla- The maximum levels of cypermethrin residues in
desh. Cypermethrin and diazinon were found in six tobacco tobacco leaves are neither available nor reported in other
samples from Mirzapur, Shahebnagar, Kodalipara, Prag- countries for comparison. In Italy and Spain, pesticide
pur, Farakpur and Taragunia. The highest concentration of residues are generally stipulated not to exceed the limit of
cypermethrin was found in Kodalipara while the highest detection, which is generally between 0.01 and 0.05 ppm

123
Bull Environ Contam Toxicol (2012) 89:658–663 661

Table 2 Levels of cypermethrin, diazinon, DDT, DDD, DDE, heptachlor and methoxychlor in tobacco leaf samples collected from different
locations of Kushtia district, Bangladesh
Sample Code Collection Area Mean valuea (ppm)
Cypermethrin Diazinon DDT DDD DDE Hepta-chlor Methoxy-chlor

BD 01 Bharamara ND ND ND ND ND ND ND
BD 02 Dharampur ND ND ND ND ND ND ND
BD 03 Amlasadarpr ND ND ND ND ND ND ND
BD 04 Sholodag ND ND ND ND ND ND ND
BD 05 Kholishakuni ND ND ND ND ND ND ND
BD 06 Rifiatpur ND ND ND ND ND ND ND
BD 07 Boholbaria ND ND ND ND ND ND ND
BD 08 Bittipara ND ND ND ND ND ND ND
BD 09 Bahadurpur ND ND ND ND ND ND ND
BD 10 Mirzapur 1.72 0.11 ND ND ND ND ND
BD 11 Shahebnagar 0.62 0.10 ND ND ND ND ND
BD 12 Kodalipara 2.0 0.12 ND ND ND ND ND
BD 13 Daulatpur ND ND ND ND ND ND ND
BD 14 Mothurapur ND ND ND ND ND ND ND
BD 15 Pragpur 1.45 0.15 4.0 ND ND ND ND
BD 16 Hosenabad ND ND ND ND ND ND ND
BD 17 Boiragirchar ND ND ND ND ND ND ND
BD 18 Philipnagarchar ND ND ND ND ND ND ND
BD 19 Farakpur 1.41 0.09 ND ND ND ND ND
BD 20 Allardarga ND ND ND ND ND ND ND
BD 21 Taragunia 0.84 0.06 ND ND ND ND ND
BD 22 Majdiarchar ND ND ND ND ND ND ND
BD 23 Gongarampur ND ND ND ND ND ND ND
BD 24 Kiamari ND ND ND ND ND ND ND
BD 25 Kazipara ND ND ND ND ND ND ND
BD 26 Dharmodah ND ND ND ND ND ND ND
All samples are measured in triplicate
BD tobacco leaf sample, ND not detected
a
Mean value of the triplicate measurements

Fig. 2 A chromatogram for a 0020

mAU (x1000)
254nm 4nm(100)
tobacco leaf sample from
Pragpur showing the presence 0018
of cypermethrin (RT = 4.94), 7.16
diazinon (RT = 7.16) and DDT 0015 Diazinon
(RT = 16.29)
0013 16.29
DDT
0010
4.94
Cypermethrin
0007

0005

0003

0000

-0003

00 25 50 75 100 125 150 175 min

123
662 Bull Environ Contam Toxicol (2012) 89:658–663

(GAO 2003). The levels of cypermethrin detected in our compound DDT is the only pesticide that is currently
study, which were between 0.62 and 2.00 ppm, are higher produced in Bangladesh (Environment and Social Devel-
than these recommended values. In Bangladesh, the types opment Organization, ESDO; Bangladesh, November
of pesticides that are legally allowed for use on tobacco are 2005). Furthermore, Pragpur is located close to the border
unknown. The US General Accounting Office reported that area between India and Bangladesh, which may facilitate
17 out of the 73 pesticides that were commonly used on the illegal entry of DDT by retailers for disease control and
tobacco in the 1990s in US belong to three chemical classes crop production purposes. The illegal trans-boundary entry
(organochlorine, organophosphate and carbamates), and at of pesticides is a common phenomenon in Bangladesh
high doses, these pesticides are known to cause adverse (Environment and Social Development Organization
effects to human health, including death (GAO 2003). (ESDO) Bangladesh, November 2005). The study also
Diazinon is one of the pesticides that falls within this discovered some old stocks of DDT stored at different
category. Even though the EPA has concluded that most districts, namely, Rajshahi, Madaripur, Barisal, Pirojpur
of the 17 pesticides do not cause birth defects, the EPA and Noakhali, totaling to 6,868 kg.
has also concluded that five of these pesticides, and a The indiscriminate and overuse of pesticides creates
by-product of another, may cause cancer (GAO 2003). many problems. The use of pesticides has to be controlled
In the USA, diazinon is no longer approved for use on to avoid the contamination of food supplies and the envi-
tobacco. However, diazinon was detected on six of the ronment, but present measures in Bangladesh are inade-
tobacco samples in this study, indicating that it is still used quate and allow the sale of unregistered pesticides and
in Bangladesh. The highest level of diazinon was found in misuse of pesticides by farmers (Matin et al. 1998). There
the sample collected from Kodalipara, at 0.15 ppm. How- are also concerns about prevalent pesticide use leading to
ever, this level is below the residue limits at 1.00 ppm for the development of resistance in mosquitoes and flies,
Germany, Italy and Spain (GAO 2003). There are no making the control of diseases such as malaria more
reported residue levels for diazinon in the US because its difficult. For example, the excessive use of DDT and other
use has been discontinued. Even though the largest agri- pesticides has been responsible for the resurgence of
cultural use of DDT has been reported for cotton and fish malaria and possibly DDT resistant malaria in certain states
(Environment and Social Development Organization, (Chapin and Wasserstrom 1983). The DDT pesticide is
ESDO; Bangladesh, November 2005), DDT may also be highly toxic, persists in the environment and tends to
used in a tobacco leaf storehouses to control pests, and in accumulate in the body tissues of human and animals,
such a way, DDT may contaminate tobacco leaves. In the especially in fatty tissues.
1980s, high quantities of organochlorines were used in both In this study, we did not detect any DDD, DDE, hep-
agriculture and public health before organochlorines were tachlor or methoxychlor residue levels on the tobacco.
banned in 1993, exempting heptachlor (Matin et al. 1998). Since DDE and DDD are both metabolites of DDT, they
An estimated 100–300 tons of formulated organochlorines, may be below the detection limit of our machine and may
particularly DDT and lindane, were used annually between be present in amounts that are too small to be detected. No
1990 and 1993 in Bangladesh to control malaria (Matin study has assessed the presence of pesticides in human
et al. 1998). A report by the (Environment and Social milk samples in Bangladesh. However, the pesticides DDT,
Development Organization, ESDO; Bangladesh, Novem- chordane and heptachlor have been used for long time in
ber 2005) states that some of the persistent organic pollu- Bangladesh, and both rural and urban women have been
tants, such as heptachlor, dieldrin, DDT and chlordane, are exposed to each; thus, the milk samples of such exposed
still in use in different parts of Bangladesh, where they are women are very likely to contain residues of these pesti-
considered essential for ensuring agricultural production cides (Environment and Social Development Organization
and public health. (ESDO) Bangladesh, November 2005). The presence of
The USA Dairy and Tobacco Adjustment Act set max- pesticides in milk can be further studied.
imum allowable concentrations for 15 pesticide residues Even though the EPA regulates the specific pesticides
that are not approved for use in the USA. However, these that may be used in tobacco and the amounts to be applied
pesticides are believed to be used in other countries, as in some countries, the EPA does not regulate pesticide
allowed under the EPA’s guidelines (EPA 1989). For residues that are approved for the use on tobacco. Even
example, the summed maximum allowable limit for resi- though the residue levels on tobacco typically decline as the
due in tobacco for DDT, tetrachlorodiphenylethane (TDT) leaves are harvested, dried and processed or burned (GAO
and DDE (all of which are chemically related) is 0.4 ppm. 2003), varying residue levels may remain. A study reported
In our study, DDT was detected in one sample from that chlorinated pesticide residue such as DDT in Australian
Pragpur at 4.0 ppm. This concentration is ten times above cigarettes ranged between 18.8 and 53.2 ppm while that in
the stated combined limits for DDT. The chemical Finland ranged between 3.6 and 7.5 ppm (Morris 1972).

123
Bull Environ Contam Toxicol (2012) 89:658–663 663

The maximum standard for DDT is 7.5 ppm (CEC 1976). organophosphorus and carbamate Pesticide residues in surface
Further research should be conducted to determine the water samples from the Rangpur district of Bangladesh. Bull
Environ Contam Toxicol 89:202–207
levels of potentially harmful pesticide residues in Bangla- Clapp WL, Shelar GR, Dobbins JT, Markunas PC (1972) The
desh cigarettes, as these findings will reflect the actual harm Determination of Chlorinated Pesticides in Mainstream Smoke
that both active and passive smokers are exposed to. The June 21. RJR on pesticides in mainstream smoke, Daily Doc
EPA recommends studies to be conducted even if the res- Drossopoulos J, Bouranis D, Kintzios S, Aivalakis G, Triposkoufi A
(1998) Distribution profiles of selected micronutrients in oriental
idues are below 0.1 ppm due to concerns about the toxicity field-grown tobacco plants as affected by nitrogen fertilization.
of pesticides. Furthermore, little is known directly about the J plant nutr 21:1391–1406
chronic effects of pesticides. The chronic effects of pesti- Environment and Social Development Organization (ESDO), Ban-
cides on human health are a broad area for further study. gladesh (November 2005) POPs Hotspots in Bangladesh.
Accessed on 3 Aug 2010
This is the first study to report the levels of pesticide EPA, U.S. Environmental Protection Agency (1989) Pesticide fact
residues in tobacco leaf samples in Bangladesh. Cyper- sheet number 199: Cypermethrin. Office of Pesticides and Toxic
methrin and diazinon were found in six tobacco samples substances, Washington, DC, pp 2–9
from Mirzapur, Shahebnagar, Kodalipara, Pragpur, Farak- Fardous Z, Islam MN, Hoque SM, Chowdhury MAZ, Rahman MA
(2007) Determination of some selected pesticide residues in
pur and Taragunia, indicating that these pesticides are still tomato from different locations of Bangladesh. Int J Sustain Agri
used on tobacco. The levels of cypermethrin are much Tech 3:4–7
higher than those reported in some countries, such as Italy FDA, U.S. Food and Drug Administration (2001) Guidance for
and Spain. However, the levels of diazinon are below the industry: bioanalytical method validation. CDER, Rockville
GAO (2003). Pesticides on tobacco. Federal activities to assess risks
maximum levels allowed in other countries. The pesticide and monitor residues Apr 24, GAO-03-485
DDT was detected in Pragpur at ten times the recom- Hayat K, Ashfaq M, Ashfaq U, Saleem MA (2011) Determination of
mended level. The Bangladesh government should monitor pesticide residues in blood samples of villagers involved in
the use of pesticides in tobacco fields. pesticide application at district Vehari (Punjab), Pakistan. Afr J
Environ Sci Tech 4:666–684
Khan MRA (2007) Tobacco Control, Social, Environmental and
Acknowledgments We would like to acknowledge the Universiti Economic Perspective Choices for Bangladesh. Department of
Sains Malaysia RU grant (1001/PPSP/815058). Agricultural Marketing, 1: Available at http://www.fairtradeto
bacco.org/wp-content/uploads/2011/07/Farida-Akhter_Tobacco-
to-Food-Production.pdf, Accessed 14 Jan 2012
References Matin M, Malek M, Amin M, Rahman S, Khatoon J, Rahman M,
Aminuddin M, Mian A (1998) Organochlorine insecticide
Akhter F (2011) Tobacco cultivation and its impact on food residues in surface and underground water from different regions
production in Bangladesh. 28 March, Dhaka. Available at http:// of Bangladesh. Agric Ecosyst Environ 69:11–15
www.fairtradetobacco.org/wp-content/uploads/2011/07/Farida- Morris P (1972) Chlorinated pesticide residues in Australian
Akhter_Tobacco-to-Food-Production.pdf. Accessed 13 Feb 2012 cigarettes. Bates No. 2000706359/6361, Available at http://
ATSDR (2002) Toxicological profile for aldrin and dieldrin. Atlanta, legacy.library.ucsf.edu/tid/rvj35e00, Accessed 18 Feb 2012
GA: US. Department of health and human services, Public health Muñoz-De-Toro M, Durando M, BeldomãÓNico PM, Bel-
service: 116 domãÓNico HR, Kass L, Garcãa SR, Luque EH (2006)
CEC, Commission of the European Communities (1976) Pesticide Estrogenic microenvironment generated by organochlorine res-
residues in tobacco and tobacco products. Information on idues in adipose mammary tissue modulates biomarker expres-
agriculture. Brussels, 1: 62 sion in ERalpha-positive breast carcinomas. Breast Can Res
Chapin G, Wasserstrom R (1983) Pesticide use and malaria 8:R47
resurgence in Central America and India. Soc Sci Med 17: NHMRC, Australian National Health and Medical Research Council
273–290 (1982) Report of the ninety-second session, Canberra. October
Chowdhury AZ, Jahan SA, Islam MN, Moniruzzaman M, Alam MK, 1981. Canberra: AGPS, 1
Zaman MA, Karim N, Gan SH (2012) Occurrence of

123