Vol 2. December, 2013 ISSN 2306-8256

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CONTENTS

Spatial Analysis of Sulfur dioxide (SO2) concentration in Karachi Megapolis, Pakistan

Syed Nawaz-Ul-Huda, Farkhunda Burke, Erma Anwar,Imtiaz Ahmed, Muhammad Miandad ,

Muhammad Azam

1

Integrated geological and geophysical research on Lava effusion in Ziarat, Balochistan, Pakistan Asif Nazeer Rana, Muhammad Saeed, Mehtab Ur Rahman, , Syed Ali Abbas

17

Effect of multiple harvests on chemical composition of important nutrients of Alfalfa (Medicago

sativa L.) grown in Uthal, Lasbela District, Balochistan, Pakistan.

Saeed Ahmed, Abdul Hameed Baloch, Imtiaz Ahmed

30

Genetic differentiation of two Chrysichthys species using mitochondrial DNA sequencing

Nwafili S.A, Eminue B.O , Jamabo. N

36

Preliminary observation on Baseodiscus hemprichii (ribbon worm)

Faiz Muhammad, Muhammad Shafi, Muhammad Aslam

44

Identification and phylogenetic analysis of halophilic fungus isolated from a man-made solar

saltern in Thailand

Imran Ali, Sudip K. Rakshit, Napa Siwarungson, Hunsa Punnapayak, Pongtharin Lotrakul, Sehanat

Prasongsuk, Ali Akber, Zia ur Rehman

47

In vitro antibacterial activity of Sorghum halepense

Rooh-Ul-Amin, Muhammad Adil, Kashif Hayat, Arbab Sikandar, Farmanullah, Saeed Khan, Hazrat Nabi

53

Antagonistic potential of marine isolate DK6-SH8 against fish pathogens Muhammad Naseem Khan, Meng Li, Zulfiqar Ali Mirani, Jingxue Wang And Hong Lin

61

Physico-chemical properties of goat, sheep and camel milk of Balochistan Haseena Sajid, Shafia Muzafar, Abida Peer Muhammad, Illahi Bakhsh Marghazani, Sajid Ali Khosa,

Nasrullah, Ahmed Nawaz Khosa

70

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 1

ENVIRONMENTAL SCIENCES

RESEARCH ARTICLE

Spatial analysis of sulfur dioxide (SO2) concentration in Karachi

Megapolis, Pakistan

Syed Nawaz-ul-Huda1, Farkhunda Burke

1, Erma Anwar

2,Imtiaz Ahmed

3, Muhammad

Miandad1and Muhammad Azam

2

1Department of Geography, University of Karachi, Karachi, Pakistan.

2Department of Geography, Federal Urdu University of Arts, Sciences and Technology,

Gulshan-e-Iqbal Campus, Karachi, Pakistan. 3Faculty of Water Resources Management, Lasbela University of Agriculture, Water and

Marine Sciences, Uthal, Balochistan, Pakistan.

ABSTRACT

Rapid growth of motor vehicles in cities of Pakistan has brought in its wake a range of serious

socio-economic, environmental, health and welfare impacts. Of these impacts, those resulting

from urban air pollution, due to emissions from motor vehicles among other sources, have been

the focus of considerable public concern and policy attention. Vehicular smoke, burning of

garbage and low greenery have a predominant role in Karachi’s air pollution which subsequently

are causes of serious environmental degradation and lung diseases among the population. The

present study focuses on high traffic volume locations of Karachi for the study of SO2

concentration based on Minimum Curvature Interpolation technique. The study also focuses on

24 hours ambient data in selected places and identification of zones of SO2 concentration in

Karachi megapolis.

Keywords: Karachi, Pakistan, SO2, Minimum Curvature Interpolation, Burns Road

_____________________________________________________________________________ Correspondence: Syed Nawaz-ul-Huda Address: Department of Geography, University of Karachi, Karachi, Pakistan.

Email: nawaz_huda@hotmail.com Phone: +92-333-3177399 Received : 02 Feb, 2013 Revised : 25 Jun, 2013 Accepted: 26 Jun, 2013 Copyright: ©2013 Huda et al. This is an open-access article distributed under the terms of the Creative

Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any

medium, provided the original author and source are credited Competing Interests: The authors have declared that no competing interests exist. ______________________________________________________________________________

INTRODUCTION

Clean air is an essential component of life

but air pollution plays a prominent role in its

spoilage and urban atmosphere. It is affected

by industrial development and high volume

of growing traffic. Air pollution is a severe

problem in most cities of the developing

world as compared to cities of the developed

world (Sivaramasundaram and

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 2

Muthusubramanian, 2010; Viana et al, 2006;

Miranda, 2012 & Bell, 2007). The greatest

human and economic impacts of air

pollution are increased incidence of illness

and premature death which result from

human exposure to elevated levels of

harmful pollutants. The most common air

pollutants are sulfur dioxide (SO2), oxides of

nitrogen (NOx), carbon monoxide (CO),

particulate matter (PM) and volatile organic

compounds (VOCs) in urban areas, which

come from a wide variety of sources. The

single most important source generally

being fossil fuels (Gorham 2002 & Tiwari et

al, 2010).

Rapid growth in the number of motor

vehicles in cities of Pakistan has brought in

its wake a range of serious socio-economic,

environmental, health and welfare impacts.

Of these impacts, those resulting from urban

air pollution, due to emissions from motor

vehicles among other sources, have been the

focus of considerable public concern and

policy attention (Ilyas, 2007; Aziz and

Bajwa,2007; Aziz and Bajwa,2008; Ghouri

et al, 2007; Alam, et al., 2011;Majid, et al.,

2012b & Ali and Athar, 2010).

Sulfur oxides are one of the most abundant

pollutants (Dwivedi and Shashi, 2012).

Sulfur dioxide (SO2) is one of the major

oxides of sulfur. It is a heavy, pungent,

colorless gas. It forms from the combination

of sulfur from emissions of coal burning

industries and atmospheric oxygen. Sulfur

dioxide is highly reactive and hence is not

cumulative. The maximum residence time is

probably 10 days. Much of the compound

combines with atmospheric water to form

sulfuric acid. Atmospheric sulfuric acid

causes the leaves of plants to turn yellow. It

dissolves limestone and marble, and is

highly corrosive of iron and steel.

SO2 reduces atmospheric visibility and

blocks out sunlight (Yang, et al, 2009). It is

also responsible for decreased wind speed

and temperature in winter due to its

increased concentration (Luvsan, et al.,

2012) thus being contributory factor of

serious repository ailments in urban

environments especially among children

(Smargiassi, et al., 2009; Dockery et al.,

1996). It is a major irritant to the eyes and

respiratory system and is lethal at a few

parts per million. SO2, which is emitted in

direct proportion to the amount of sulfur in

fuel, causes changes in lung function in

persons with asthma and exacerbates

respiratory symptoms in sensitive

individuals (Gasana, 2012; Thriel, et al.

2010 & Koenig, 1999).

Karachi is the capital city of the province of

Sindh, and the largest and thickly populated

(16 millions) city of Pakistan. Located

strategically between 24.750 to 25.656 N

and 66.653 to 67.574 E on the coast of the

Arabian Sea, north-west of the Indus River

delta, it covers an area of 3,600 km² (Fig.1).

High volume of ground traffic in the urban

areas, mostly heavily populated cities acts as

one of the major factors in climate change

and cities including Karachi have observed

an increasing trend in temperature (Alam

and Rabbani 2007; Edmilson et al. 2007; Liu

et al, 2007; Yin et al. 2007; Chung et al.

2004 & Sajjad et al, 2009). Vehicular

smoke, burning of garbage and low greenery

(Azam, et al., 2012) are significant

contributors to the air pollution of Karachi

and one of the prime causes of serious

environmental degradation and henceforth

lung diseases among the population. SO2

concentration has become one of the

essential factors (Naddafiet al.2012) in

accelerating weathering of monuments,

buildings, and other stone and metal

structures (Plate.1).

The current study focuses on the hypothesis

that high traffic volume in Karachi is a

major cause of high SO2 pollution in various

parts of the city. In this scenario, the

objectives of this study are:

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 3

Analysis of 24 hours SO2 data from

selected sample sites of the city.

Identification ofSO2 concentration zones

in Karachi Megapolis.

Demarcation of probable expansion of

SO2 through Minimum Curvature

method.

S h a

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– P a

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N o r t h e r n

M a n g h o p i r R

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Landhi

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Gulshan

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New

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Jauhar

Malir

Cantonment

Quaidabad

S e a V i e w

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inhas Rd

Gulshan-e-

Maymar

Industrial Area Rd

Hub R

iver Rd (R

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PAF

Masroor

Korangi

Creek

– F

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Karachi Port

0 5

kilometers

Mauripur

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M a l i r R i v e r

Arabian

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Arabian Sea

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N o

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12 15

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1719 18

21

1. SUPARCO 2. Karimabad 3. Liaquatabad 10 4. Tin Hatti 5. Guru Mandir 6. Old Numaish 7. Garden Road 8. Tibet Center 9.

Maulvi Musafir Khana Road 10. Merewether Tower 11. Shaheen Complex 12. Burns Road 13. Preedy Street 14. Empress

Market 15. Metropole Hotel 16. FTC 17. Teen Talwar 18. Sunset Boulevard & Gizri Road 19. Gizri Road & Punjab Colony

20. Drigh Road 21. KPT 22. North Nazimabad 23. Nazimabad 24. Mauripur Road 25. Sohrab Goth 26. Gulshan Chowrangi

27. Gulbai 28. Maritime Museum

11

Fig 1: Study Area and Sample Sites

SO2 Affected Part Renovated portion

Plate 1: SO2 affected limestone buildings in the study area.

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 4

Due to reported news regarding rapid

growth of chronic diseases and speedy

weathering of limestone constructed

buildings and monuments; the present study

has focused only on SO2 analysis.

MATERIAL AND METHOD

For the present study 28 locations of varied

local activities including traffic density

based on previous study of SUPARCO have

been selected. Air quality samples were

collected during the period of March 2007 to

October 2007 with the help of Sulfur

Dioxide Monitor Z-1300XP equipment.

Various techniques for the scholarly study of

air pollutants have been applied

(Hadjimitsis, 2009; Wald, et al, 2009).

In present study, surface gridding analysis

has been designed, based on Minimum

Curvature method through Mapinfo

Professional 11/ Encom Discover 12. This

technique is widely used for analysis in the

Earth Sciences (Huda et al., 2011; Briggs,

1974; Kurtzman and Kadmon 1999).

Selection of this method is based on its

smoothness of possible surface within the

area of grid analysis. For the purposes of

spatial analysis, this method is fast, effective

and suitable over a wide range of smoothly

varying regional data.

RESULTS AND DISCUSSION

Spatial analysis, one of the basic tenets of

Geography, is a convenient method of

providing an insight into the measurement of

atmospheric pollution of any area. Under

this technique, ambient expansion can be

observed through visual contacts (Fig.2 to

25). Highest concentration of SO2 has been

observed at various places, among which

Guru Mandir, Mauripur, Metropole and

Sohrab Goth have emerged as the most

prominent locations (Fig.2). These areas are

the high traffic volume belts in Karachi

megapolis. Mauripur, located near Karachi

port is the biggest Trailer Truck stand where

hundreds of Trailers are parked and loaded

round the clock. Sohrab Goth, which is one

of the intercity bus terminuses, is another

busiest traffic zone in the study area located

at the urban periphery. Guru Mandir, which

is a junction of heavy and light traffic, is

located in the center of the city. The Hotel

Metropole junction is another crossroad of

light traffic especially for VIP movement in

the megapolis.

Fig.3 shows almost the same result as that of

the previous hour, however, SO2

concentration movement is diverted to other

directions. The area of concentration has

spread out markedly towards the eastern

part. KPT Interchange, gateway of Landhi-

Korangi Industrial area witnesses heavy

traffic after the mid night since the city

traffic law allows heavy traffic only after

that time. Figs.4 and 5 depict similar

concentration zones. In Figs.6 and 7, Preedy

Street and Drigh Road have emerged as new

zones of SO2 concentration. Fig. 8 depicts an

increase in traffic flow in the city and by

7am the coverage of low SO2 concentration

zones shows a marked decrease (Fig.9).

High pollution zones can be observed from

8am to 11am in the southern part of the city

where business offices, trade centers and

other centers of occupational activities are

concentrated, which lead to an increase in

traffic volume (Figs.10 to 13). Figs 14 and

15 reveal that FTC area appears as being a

high SO2 concentration zone, while

concentration in this area further increases at

14:00 and 15:00 hours (Figs. 16 to 17).

In the study area during the late afternoon

hours, except for some locations, high level

of SO2 can be observed in the northern,

eastern, southeastern and western parts. At

16:00 and 17:00 hours, the central part

shows the same pattern of SO2 concentration

(Figs. 18 &19). The level of pollution

concentration shows an increase that extends

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 5

from Empress Market (14) to FTC (16)

areas at this time. Fig. 20 shows the level of

spread of this area further towards

Merewether Tower (10), engulfing Gizri

Road and Punjab Colony areas as well. By

19:00 hours, depicted in Fig. 21, the SO2

zone moves from FTC towards KPT. At this

time Gulshan Chowrangi also emerges as

another high level SO2 zone. By 20:00 hours

(Fig. 22) SO2 high concentration zone

further spreads from Gulshan Chowrangi to

Sohrab Goth by 21:00 hours towards Drigh

Road area, while increase in concentration at

FTC and its neighboring locations are also

recorded (Fig. 23). In the study area, traffic

volume decreases during the night hours

especially between 20:00 and 23:00 hours.

Except for some critical locations, this

pattern is visible in most of the worst traffic

congested areas where level of SO2 falls

during the nighttime. Daily analysis reveals

that SUPARCO and Maritime Museum

locations are zones of least concentration of

SO2, while high concentration zones have

been recorded at Mauripur Road, Guru

Mandir, Sohrab Goth, Merewether Tower,

Empress Market, Drigh Road, FTC, Gulshan

Chowrangi and KPT areas (Figs. 24 &

25).Hourly variation in concentration can be

observed at SUPARCO location from 13:00

to 20:00 hours when volume of vehicle

movement is considerably higher than

during the nighttime to early morning hours

(Fig.26). Maritime museum is another

location of low concentration of SO2, where

hourly observations reveal negligible

variations because the area lies in the

jurisdiction of cantonment administration

and generally public vehicles do not halt

here for a long time; most of them flowing

in a stream (Fig. 27). Preedy Street reveals

marked variation in terms of SO2

concentration round the clock. Being the

busiest trade center of the city, the volume

of traffic is quite high. The building

structures are multistoried, mostly of stone.

Low concentration of SO2 has been recorded

during the late night to early morning hours

(Fig. 28). Tin Hatti has recorded very little

difference round the clock, with consistently

high readings except for a few hours during

late night. The area is mainly residential,

consisting of single story houses. Buses and

cars in thousands ply through this area (Fig.

29).

Concentration of SO2 at Teen Talwar area is

highly varied. Peak hours are 9am to 18:00

hours but from 19:00 to 21:00 hours the

level shows a decrease and retains high

concentration from 22:00 to 00:00 hours.

This area is a high class residential area of

the megapolis, yet the peaks of SO2 during

late night hours are much higher as

compared to that of working hours (Fig. 30).

The peak hours of SO2 concentration at

Gulshan Chowrangi area is between 18:00 to

00:00 hours (Fig. 31) because of heavy

traffic due to the presence of marriage halls.

Social functions in the megapolis are

arranged mainly during the nighttime, after

working hour’s business activities in that

area then for extend even till late hours i.e.,

22:00 hours. At Maulvi Musafir Khana SO2

concentration can be observed at a low level

during midnight to early morning (Fig. 32).

Empress Market is a purely trading area

where high level concentration of SO2 has

been recorded during 17:00 to 23:00 hours

during which worst traffic congestion is a

common sight (Fig. 33). Ghizri Road and

Punjab Colony’s location portray almost

same picture as that of Empress Market (Fig.

34). High level concentration hours at Tibet

Center location are 18:00 to 00:00 hours

(Fig. 35). Old Nomaish is another location

where concentration level increases

gradually from 12:00 to 20:00 hours and

then rapidly between 21:00 to 00:00 hours

(Fig. 36). SO2 concentration reveals

remarkable variation at Merewether Tower

with reference to 24 hours data. Peak hours

can be in the study area during the late

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 6

afternoon hours. Except for some locations,

high level of SO2 can be observed between

13:00 and 14:00 hours and between 18:00

and 20:00 hours, the peak being recorded at

19:00 hours. Subsequently it drops between

21:00 and 23:00 hours (Fig. 37). Gulbai

peak hours started from 11am and gradually

increased till 17:00 hours while the peak has

been recorded at 18:00 hours. Decreasing

trend is visible from 19:00 hours till 8:00 am

(Fig. 38).

Shaheen Complex shows great variation

between morning and night hours in terms

of SO2 concentration. Lowest volume has

been recorded at 6am, which subsequently

shows gradual increase, dropping one again

at 13:00 hours. Peak hours are 20:00 to

21:00 hours and decreasing trend

commences from 21:00 hours, while

concentration reveals sinking trend till early

morning (Fig. 39). Garden area is a highly

congested area with reference to traffic

volume, and SO2 concentration level

increases between 20:00 to 00:00 hours.

14:00 hour records peak SO2 concentration

probably because of traffic rush due to

plying of school vans, which plays a

significant role in traffic congestion (Fig.

40). In Burns road area, level of

concentration trend has been observed to be

a little different from other locations because

high SO2 concentration has been recorded

even during the fore noon and afternoon

times (Fig. 41). Boulevard and Gizri area

depicts very interesting variation regarding

SO2 concentration where decreasing trends

start from midnight to early morning.

Subsequently, increasing trend commences

from 7:00 to 14:00 hours. Another decreased

trend can be observed from 15:00 to 17:00

hours, while a repeated increase from 20:00

to 00:00 hours is visible. This increased

phenomenon is a real picture of traffic

trends of this location (Fig. 42).

SO2concentration is constant but at a lower

level at Karimabad. Increased peaks can be

observed between 12:00 and 14:00 hours

and between 17:00 to 22:00 hours (Fig.

43).Hourly variation in concentration can be

observed at SUPARCO location from 13:00

to 20:00 hours when volume of vehicle

movement is considerably higher than

during the nighttime to early morning hours

(Fig.26). Maritime museum is another

location of low concentration of SO2, where

hourly observations reveal negligible

variations because the area lies in the

jurisdiction of cantonment administration

and generally public vehicles do not halt

here for a long time; most of them flowing

in a stream (Fig. 27). Preedy Street reveals

marked variation in terms of SO2

concentration round the clock. Being the

busiest trade center of the city, the volume

of traffic is quite high. The building

structures are multistoried, mostly of stone.

Low concentration of SO2 has been recorded

during the late night to early morning hours

(Fig. 28). Tin Hatti has recorded very little

difference round the clock, with consistently

high readings except for a few hours during

late night. The area is mainly residential,

consisting of single story houses. Buses and

cars in thousands ply through this area (Fig.

29).

Concentration of SO2 at Teen Talwar area is

highly varied. Peak hours are 9am to 18:00

hours but from 19:00 to 21:00 hours the

level shows a decrease and retains high

concentration from 22:00 to 00:00 hours.

This area is a high class residential area of

the megapolis, yet the peaks of SO2 during

late night hours are much higher as

compared to that of working hours (Fig.

30).The peak hours of SO2 concentration at

Gulshan Chowrangi area is between 18:00 to

00:00 hours (Fig. 31) because of heavy

traffic due to the presence of marriage halls.

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 7

NNN

Fig.2, 00:00 hours Fig.3, 1:00AM

Fig.4, 02:00 hours Fig.5, 03:00 hours

Fig.6, 04:00 hours Fig.7, 05:00 hours

Fig.8, 06:00 hours Fig.9, 07:00 hours

1. SUPARCO 2. Karimabad 3. Liaquatabad 10 4. Tin Hatti 5. Guru Mandir 6. Old Numaish 7. Garden Road 8.

Tibet Center 9. Maulvi Musafir Khana Road 10. Merewether Tower 11. Shaheen Complex 12. Burns Road 13.

Preedy Street 14. Empress Market 15. Metropole Hotel 16. FTC 17. Teen Talwar 18. Sunset Boulevard & Gizri

Road 19. Gizri Road & Punjab Colony 20. Drigh Road 21. KPT 22. North Nazimabad 23. Nazimabad 24. Mauripur

Road 25. Sohrab Goth 26. Gulshan Chowrangi 27. Gulbai 28. Maritime Museum

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

Sulphur Dioxide Concentration at different hours of the day-Karachi

Social functions in the megapolis are

arranged mainly during the nighttime, after

working hour’s business activities in that

areas then for extend even till late hours i.e.,

22:00 hours. At Maulvi Musafir Khana SO2

concentration can be observed at a low level

during midnight to early morning (Fig. 32).

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 8

Empress Market is a purely trading area

where high level concentration of SO2 has

been recorded during 17:00 to 23:00 hours

during which worst traffic congestion is a

common sight (Fig. 33). Ghizri Road and

Punjab Colony’s location portray almost

same picture as that of Empress Market (Fig.

34). High level concentration hours at Tibet

Center location are 18:00 to 00:00 hours

(Fig. 35). Old Nomaish is another location

where concentration level increases

gradually from 12:00 to 20:00 hours and

then rapidly between 21:00 to 00:00 hours

(Fig. 36). SO2 concentration reveals

remarkable variation at Merewether Tower

with reference to 24 hours data. Peak hours

can be in the study area during the late

afternoon hours. Except for some locations,

high level of SO2 can be observed between

13:00 and 14:00 hours and between 18:00

and 20:00 hours, the peak being recorded at

19:00 hours. Subsequently it drops between

21:00 and 23:00 hours (Fig. 37). Gulbai

peak hours started from 11am and gradually

increased till 17:00 hours while the peak has

been recorded at 18:00 hours. Decreasing

trend is visible from 19:00 hours till 8:00 am

(Fig. 38).

Shaheen Complex shows great variation

between morning and night hours in terms

of SO2 concentration. Lowest volume has

been recorded at 6am, which subsequently

shows gradual increase, dropping one again

at 13:00 hours. Peak hours are 20:00 to

21:00 hours and decreasing trend

commences from 21:00 hours, while

concentration reveals sinking trend till early

morning (Fig. 39). Garden area is a highly

congested area with reference to traffic

volume, and SO2 concentration level

increases between 20:00 to 00:00 hours.

14:00 hour records peak SO2 concentration

probably because of traffic rush due to

plying of school vans, which plays a

significant role in traffic congestion (Fig.

40). In Burns road area, level of

concentration trend has been observed to be

a little different from other locations because

high SO2 concentration has been recorded

even during the fore noon and afternoon

times (Fig. 41). Boulevard and Gizri area

depicts very interesting variation regarding

SO2 concentration where decreasing trends

start from midnight to early morning.

Subsequently, increasing trend commences

from 7:00 to 14:00 hours. Another decreased

trend can be observed from 15:00 to 17:00

hours, while a repeated increase from 20:00

to 00:00 hours is visible. This increased

phenomenon is a real picture of traffic

trends of this location (Fig. 42). SO2

concentration is constant but at a lower level

at Karimabad. Increased peaks can be

observed between 12:00 and 14:00 hours

and between 17:00 to 22:00 hours (Fig.

43).North Nazimabad shows smooth traffic

flow between 1am to 10am. The SO2 level

increases at 19:00 hours and subsequently

decreases between19:00 hours till midnight

(Fig. 44). KPT Interchange is another

location of excessive traffic in the

megapolis. Throughout the day, SO2

concentration level at this location can be

observed as being exceedingly high. KPT

being the gateway to the highly populated

areas of Landhi and Korangi, including

Landhi-Korangi Industrial Zone, is the

junction of both light and heavy vehicular

traffic (Fig. 45).

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 9

Fig.10, 08:00 hours Fig.11, 09:00 hours

Fig.12, 10:00 hours Fig.13, 11:00 hours

Fig.14, 12:00 hours Fig.15, 13:00 hours

Fig.16, 14:00 hours Fig.17, 15:00 hours

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

ppb ppb

ppb ppb

ppb ppb

ppb ppb

1. SUPARCO 2. Karimabad 3. Liaquatabad 10 4. Tin Hatti 5. Guru Mandir 6. Old Numaish 7. Garden Road 8.

Tibet Center 9. Maulvi Musafir Khana Road 10. Merewether Tower 11. Shaheen Complex 12. Burns Road 13.

Preedy Street 14. Empress Market 15. Metropole Hotel 16. FTC 17. Teen Talwar 18. Sunset Boulevard & Gizri

Road 19. Gizri Road & Punjab Colony 20. Drigh Road 21. KPT 22. North Nazimabad 23. Nazimabad 24.

Mauripur Road 25. Sohrab Goth 26. Gulshan Chowrangi 27. Gulbai 28. Maritime Museum

Sulphur Dioxide Concentration at different hours of the day-Karachi

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 10

Fig.18, 16:00 hours Fig.19, 17:00 hours

Fig.20, 18:00 hours Fig.21, 19:00 hours

Fig.22, 20:00 hours Fig.23, 21:00 hours

Fig.24, 22:00 hours Fig.25, 23:00 hours

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

1

26

25

2028

22

2

3

23

4

5

6

14

7

138

211819

17

16

1511

129

10

24

27

ppb ppb

ppb ppb

ppb ppb

ppb ppb

1. SUPARCO 2. Karimabad 3. Liaquatabad 10 4. Tin Hatti 5. Guru Mandir 6. Old Numaish 7. Garden Road 8.

Tibet Center 9. Maulvi Musafir Khana Road 10. Merewether Tower 11. Shaheen Complex 12. Burns Road 13.

Preedy Street 14. Empress Market 15. Metropole Hotel 16. FTC 17. Teen Talwar 18. Sunset Boulevard & Gizri

Road 19. Gizri Road & Punjab Colony 20. Drigh Road 21. KPT 22. North Nazimabad 23. Nazimabad 24. Mauripur

Road 25. Sohrab Goth 26. Gulshan Chowrangi 27. Gulbai 28. Maritime Museum

Sulphur Dioxide Concentration at different hours of the day-Karachi

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 11

Fig. 27, Maritime Museum Fig. 28, Preedy Street Fig. 29 , Tin Hatti

1 23

4

5

6

7

8

9

10

11121314

15

16

17

18

19

20

21

22

2324

0

10

15

5

1 23

4

5

6

7

8

9

10

11121314

15

16

17

18

19

20

21

2223

24

0

5

10

15

20

Peak and Slack Levels of SO2 Round the Clock at different Samples Sites - Karachi

Fig. 26 , SUPARCO

4

1 23

4

5

6

7

8

9

10

11121314

15

16

17

18

19

20

21

22

232412

8

0

ppb 1 23

4

5

6

7

8

9

1011

12131415

16

17

18

19

20

21

2223

24

0

10

20

30

1 2

34

5

6

7

8

9

10

11121314

15

16

17

18

19

20

21

2223

24

0

10

20

30

40

Fig. 31, Gulshan Chowrangi

0

10

20

301

23

4

5

6

7

8

9

10

11121314

15

16

17

18

19

20

21

2223

24 1 23

4

5

6

7

8

9

10

11121314

15

16

17

18

19

20

21

2223

24

0

10

20

30

40

Fig. 33, Empress MarketFig. 32, M Musafir Khana

12

34

5

6

7

8

9

1011

12131415

16

17

18

19

20

21

2223

24

0

10

20

30

Fig. 30, Teen Talwar

Fig. 43, Karimabad Fig. 42,Boulevard & Gizri Fig. 44, North Nazimabad Fig. 45, KPT

0

5

10

15

20

251 2

34

5

6

7

8

9

1011

12131415

16

17

18

19

20

21

2223

24

0

10

20

30

401 2

34

5

6

7

8

9

1011

12131415

16

17

18

19

20

21

2223

24

0

10

20

30

401 2

34

5

6

7

8

9

1011

12131415

16

17

18

19

20

21

2223

24

0

10

20

301 2

34

5

6

7

8

9

1011

12131415

16

17

18

19

20

21

2223

24

Fig. 41, Burns Road

0

10

20

30

401 2

34

5

6

7

8

9

1011

12131415

16

17

18

19

20

21

2223

24

0

5

10

15

20

251 2

3

4

5

6

7

8

9

1011

12131415

16

17

18

19

20

21

2223

24

Fig. 40, Garden RoadFig.39, Shaheen Complex

0

10

20

30

401 2

3

4

5

6

7

8

9

1011

12131415

16

17

18

19

20

21

22

2324

0

10

20

30

401 2

3

4

5

6

7

8

9

1011

12131415

16

17

18

19

20

21

2223

24

Fig.38, Gulbai

.

Fig. 35, Tibat Center ColonyFig. 34, Ghizri Rd &Punjab Colony Fig. 37, Merewether Tower

1 23

4

5

6

7

8

9

1011

12131415

16

17

18

19

20

21

2223

24

0

10

20

30 1 23

4

5

6

7

8

9

10

11121314

1516

17

18

19

20

21

22

2324

0

10

20

30

40

Fig. 36, Nomaish

1 23

4

5

6

7

8

9

1011

12131415

16

17

18

19

20

21

22

2324

0

10

20

30

401

23

4

5

6

7

8

9

10

11121314

1516

17

18

19

20

21

22

2324

0

5

10

15

20

25

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 12

Fig.51, Metropole Hotel Fig.53, Guru Mandir

Fig. 47, Drigh Road Fig. 49, Nazimabad

0

10

20

30

401

23

4

5

6

7

8

9

1011

1213

1415

16

17

18

19

20

21

2223

24

0

10

20

30

401 2

34

5

6

7

8

9

1011

12131415

16

17

18

19

20

21

2223

24

0

10

20

30

401 2

34

5

6

7

8

9

1011

12131415

16

17

18

19

20

21

2223

24

0

5

10

15

20

25

301 2

34

5

6

7

8

9

1011

12131415

16

17

18

19

20

21

2223

24

Fig. 46, FTC Fig. 48, Liaquatabad 10

0

10

20

301 2

34

5

6

7

8

9

1011

12131415

16

17

18

19

20

21

2223

24

0

10

20

301 2

34

5

6

7

8

9

1011

12131415

16

17

18

19

20

21

2223

24

0

10

20

30

401

23

4

5

6

7

8

9

1011

12131415

16

17

18

19

20

21

2223

24

0

10

20

30

401

23

4

5

6

7

8

9

1011

12131415

16

17

18

19

20

21

2223

24

Fig. 50, Sohrab Goth Fig. 52, Mauripur Road

ppb

FTC shows high SO2 concentration

variations round the clock. During midnight

to early morning the level is considerably

decreased, while concentration increasing

trend can be observed between 7:00 and

13:00 hours. Peak hours are 13:00 and 19:00

hours till 20:00 hours, which is in

accordance the with traffic flow on this road

(Fig. 46). Sohrab Goth depicts more or less

constant values between 15 and 17ppb

during03:00 to 10:00 hours and highest peak

at noon from 11:00 hours and then gradually

increases to a peak of 27ppb around 12

noon. During the night hours especially at

21:00 hours, highest concentration level of

the day i.e. approximately 29 ppb has been

recorded (Fig. 50). Metropole Hotel, where

most of the traffic consists of new and old

cars, have recorded high level of

concentration during the working hours i.e.,

09:00 till 20:00 hours. Lowered SO2

concentration has been recorded between

early morning 03:00 and 07:00 hours (Fig.

51). Mauripur Road showed a marked drop

in concentration level of SO2 during late

night hours i.e., 00:00 hours and afternoon

time i.e.,16:00 to19:00 hours (Fig. 52).

Observations of SO2 concentration at Guru

Mandir round the clock, showed a notable

variation. Peak readings have been recorded

at 03:00, 12:00,16:00, 18:00 and 21:00

hours around 30ppb and highest at 21:00

hours, more than 30ppb (Fig.53).

According to WHO (2006) guidelines

regarding air quality on the basis of 24

hours mean data, except for SUPARCO, all

locations in the study area record high

concentration of SO2 (Fig. 54). Burns Road

has recorded highest concentration due to

high volume of traffic and congestion of

surrounding buildings. The area is also

known as Food Street, where hundreds of

people come for lunch and dinner. During

11:00 to 00:00 hours Burns Road is one of

the busiest locations in terms of people’s

activities in the study area. Guru Mandir,

Mauripur and FTC are areas with second

highest SO2 concentration, where traffic

keeps flowing round the clock.

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 13

Study Area 24 hours mean

Gu

ru M

an

dir

SU

PA

RC

O

Mari

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um

Pre

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treet

Tin

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Teen

Talw

ar

Gu

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an

Ch

ow

ran

gi

Mu

safi

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han

a R

oad

Em

pre

ss M

ark

et

Giz

ri R

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Tib

et

Cen

ter

Old

Nu

mais

h

Mere

weth

er

To

wer

Gu

lbai

Sh

ah

een

Co

mp

lex

Gard

en

Ro

ad

Bu

rns

Ro

ad

Su

nse

t B

ou

lev

ard

Kari

mab

ad

No

rth

Nazim

ab

ad

KP

T

FT

C

Dri

gh

Ro

ad

Lia

qu

ata

bad

10

Nazim

ab

ad

So

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op

ole

Ho

tel

Mau

rip

ur

Ro

ad

0

10

20

30

40

50

60

70

80(u

g/m

3)

SO

2C

on

cen

trati

on

WHO 24 hours mean

Fig 54: SO2 concentration in Study Area and WHO 24 hours mean

CONCLUSION

Karachi megapolis, aspiring to become a

World Class City, can least afford a polluted

environment. Institutions, both in the public

as well as private sectors, must be revamped

with resources and skills necessary to

control vehicular emissions. With reference

to third world countries, in view of financial

constraints, such measures must be cost

effective in order to ensure success. This

may be possible by extending attractive

incentives to both individual and firms, and

by promoting and adopting advance and

cleaner technologies and fuels. This will go

a long way in achieving a millennium goal,

i.e. improving environmental quality, an

inherent part of quality of life.

REFERENCES

Alam, K, Blaschke T, Madl P, Mukhtar A,

Hussain M., 2011, Aerosol size

distribution and mass concentration

measurements in various cities of

Pakistan : Journal of Environmental

Monitoring 13:1944-52.

Alam, M and Rabbani G.MD., 2007,

Vulnerabilities and responses to

climate change for Dhaka: Environ

Urban 19(1):81–97.

Ali, M., Athar, M., 2010, Impact of transport

and industrial emissions on the

ambient air quality of Lahore City,

Pakistan: Environmental Monitoring

Assessment. 171:353-63.

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 14

Azam, M., Safi, M and Burke, F.,

2012,Impact of open green spaces on

quality of life a case study of Kiamari

Town – Karachi: The Research Journal

of Sciences and Technology

3(1&2):35-46.

Aziz, A., Bajwa IU., 2007,Minimizing

human health effects of urban air

pollution through quantification and

control of motor vehicular carbon

monoxide (CO) in Lahore:

Environmental Monitoring

Assessment; 135:459-64.

Aziz, A, BajwaIU., 2008, Erroneous mass

transit system and its tended

relationship with motor vehicular air

pollution (an integrated approach for

reduction of urban air pollution in

Lahore: Environmental Monitoring

Assessment. 137:25-33.

Bell, M.L., Dominici F., Ebisu K., Zeger

S.L., Samet J.M., 2007, Spatial and

temporal variation in PM2.5 chemical

composition in the United States for

health effects studies: Environmental

Health Perspective 115(7):989–995.

Briggs, I. C. (1974), Machine Contouring

Using Minimum Curvature,

Geophysics, 39(1): 39-48.

Chung, Y-S, Yoon M-B, KimH-S (2004)On

climate variations and changes

observed in South Korea. Clim Change

66:151–161.

Dockery, D.W., Cunningham, J., Damokosh,

A.L., Neas, L.M., John D. Spengler,

J.D., Koutrakis, P., Ware, J.H.,

Raizenne, M and Speizer, F.E 1996,

Health Effects of Acid Aerosols on

North American Children: Respiratory

Symptoms: Environmental Health

Perspectives, 104(5): 500-505.

Dwivedi, A.K. andShashi 2012, Ambient air

sulphur dioxide and sulphate

accumulation in deciduous and

evergreen plants: Journal of

Environmental Biology, 33:1-3.

Edmilson, F.D., Christopher, R.M., William

C. R and Pedro S.L, 2007, Interactions

of an urban heat island and sea-breeze

circulations during winter over the

metropolitan area of São Paulo, Brazil:

Boundary-Layer Meteorol 122(1):43–

65.

Gasana , J., Dillikar, D., Mendy, A., Forno,

E., Vieira, E.R., 2012, Motor vehicle

air pollution and asthma in children: a

meta-analysis, Environmental

Research, 117: 36-45.

Ghauri, B, Lodhi, A., Mansha, M., 2008,

Development of baseline (air quality)

data in Pakistan: Environmental

Monitoring Assessment. 127:237-52.

Gorham, R., 2002,Air pollution from ground

transportation: an assessment of

causes, strategiesand tactics, and

proposed actions forthe international

community, The Global Initiative on

Transport Emissions A Partnership of

the United Nations and the World

Bank, Division for Sustainable

Development, Department of

Economic and Social Affairs, United

Nations.

Hadjimitsis, D.G., 2009, Aerosol optical

thickness (AOT) retrieval over land

using satellite image-based algorithm:

Air Quality Atmosphere and Health,

2:89–97.

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 15

Huda, S.N., Burke, F., Miandad , M. and

Rana, M.N., 2011, Analysis of

National Assembly 55-Rawalpindi

Franchise: An Electoral GIS

Perspective of Pakistan: The Research

Journal of Sciences and Technology,

2(1&2):35-48.

Ilyas, S.Z., 2007,A review of Transport and

Urban Air Pollution in Pakistan: J.

Appl. Sci. Environ. Manage.

11(2):113-121.

Koenig, J.Q., 1999, Air pollution and

asthma, Journal of Allergy and

Clinical Immunology, 104(4): 717-

722.

Kurtzman. D., and Kadmon, R.,

1999,Mapping of temperature

variables in Israel: a comparison of

different interpolation methods:

Climate Research, 13: 33-43.

Liu, W, Ji .C.,Zhong J., Jiang X., Zheng Z.,

2007, Temporal characteristics of the

Beijing urban heat island: Theor. Appl.

Climatol. 87:213–221.

Luvsan, M., Shie, R., Purevdorj, T.,

Badarch, L., Baldorj, B., Chan, C.,

2012.,The influence of emission

sources and meteorological conditions

on SO2 pollution in Mongolia:

Atmospheric Environment, 61:542-

549.

Majid, H., Alam, K., Madl, P & Hofmann,

W., 2012, Exposure assessment and

associated lung deposition calculations

for vehicular exhaust in four

metropolitan cities of Pakistan:

Environmental Monitoring

Assessment.

Miranda, R.G., Fatima, A. M., Fornaro, A.,

Astolfo, R., Andre, P.A. and Saldiva,

P, 2012, Urban air pollution: a

representative survey of PM2.5 mass

concentrations in six Brazilian cities:

Air Quality Atmosphere and Health,

5:63–77.

Naddafi, K., Hassanvand, S.H., Yunesian,

M., Momeniha, F., Nabizadeh, R.,

Faridi, S and Gholampour, A., 2012,

Health impact assessment of air

pollution in megacity of Tehran,

Iran.:Iranian Journal of Environmental

Health Sciences & Engineering, 9:28-

34.

Sajjad, S.H., Hussain, B., Khan, M. A.,

Raza, A., Zaman, B and Ahmed, I.,

2009, On rising temperature trends of

Karachi in Pakistan: Climatic Change,

96:539–547.

Sivaramasundaram, K., and

Muthusubramanian , P., 2010, A

preliminary assessment of PM10 and

TSP concentrations in Tuticorin, India,

Air Quality Atmosphere and Health,

3:95–102.

Smargiassi, A., Kosatsky, T., Hicks, J.,

Plante, C., Armstrong, B., Villeneuve,

P.J., Goudreau, S., 2009, Risk of

asthmatic episodes in children exposed

to sulfur dioxide stack emissions from

a refinery point source in Montreal,

Canada: Environ. Health. Prospect,

117(4):653-659.

Tiwari, V., Hanai, Y and Masunaga, S.,

2010, Ambient levels of volatile

organic compounds in the vicinity of

petrochemical industrial area of

Yokohama, Japan: Air Quality

Atmosphere and Health, 3:65–75.

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 16

Viana, M, Querol X, Alastuey A., 2006,

Chemical characterization of PM

episodes in North-Eastern Spain:

Chemosphere 62:947–956.

Wald, L, Basly L, Balleynaud, J.M., 1999,

Satellite data for the air pollution

mapping. Proceedings of the 18th

EARseL symposium on operational

sensing for sustainable development

(Enschede, Netherlands, 11–14 May

1998) edited by Nieeuwenhuis, G.J.A,

Vaugham, R.A., Molenaar, M.: 133–

139.

WHO-World Health Organization, 2006, Air

quality guidelines, global updates

2005: particulate matter, ozone,

nitrogen dioxide and sulphur dioxide.

Germany; chap. 10:217–305.

Yang, S., Yuesi, W., and Changchun, Z,

2009, Measurement of the vertical

profile of atmospheric SO2 during the

heating period in Beijing on days of

high air pollution Atmospheric

Environment, 43(2):468-472.

Yin, D, Zhiqing X, Yan Z, Yafeng S,

Jingang W., 2007, Impact of urban

expansion on regional temperature

change in the Yangtze River Delta: J

Geogr. Sci. 17(4):387–398.

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 17

GEOLOGICAL SCIENCES

RESEARCH ARTICLE

Integrated geological and geophysical research on Lava effusion in

Ziarat, Balochistan, Pakistan

Asif Nazeer Rana, Muhammad Saeed, Mehtab- ur- Rahman and Syed Ali Abbas

Geosciences Advance Research Labs, Geological Survey of Pakistan, Park Road, Shehzad

Town, Islamabad

__________________________________________________________________________

ABSTRACT

The geological and tectonic legacy of Balochistan has endowed it with massive mountain belts

and arcs, syntaxes as characterized by severe bending of the mountain belts from the ongoing

convergence of the Indo-Pakistan, Eurasian and Arabian plates. The Province is a seismically

active and tectonically unstable region. The eruptive/effusive vent activity on 27th

January

2010 at the Tor Zawar Mountain at Sari, Ziarat is a unique testimony, substantiating the earlier

risk/hazard findings of the area, as no previous post-Tertiary volcanic activity has ever been

reported earlier in the history of the Indo-Pak Subcontinent. Integrated geological and

geophysical surveys were undertaken during January-April 2010 to investigate the short lived

toothpaste lava to map, detect and delineate the changes resulting in the sub surface litho

logical and structural disposition at the vent site. A holistic approach is adopted for the

interpretation and analyses of the Total magnetic field intensity, Electrical resistivity and

Ground penetration radar surveys along with the geology, petrography and the geochemical

analyses of the molten material, which are presented along with a probable model. Keywords: Lava effusion, Ziarat, Balochistan, magnetic survey, Ground Penetrating Radar

Correspondence: Asif Nazir Rana

Address: Geosciences Advance Research Labs, Geological Survey of Pakistan, Park Road, Shehzad

Town, Islamabad

Email: asif_gsp@yahoo.com

Phone: +92-051- 9255137 Fax: +92-051- 9255136

Received: 25July 2013 Revised: 08 September 2013 Accepted : 08 September 2013

Copyright: ©2013 Rana et al. This is an open-access article distributed under the terms of the Creative

Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any

medium, provided the original author and source are credited

Competing Interests: The authors have declared that no competing interests exist.

____________________________________________________________________________

The synthesis of the magnetic, resistivity

soundings and profiling and ground

penetration radar survey indicate the presence

of highly magnetic dual lobe sources,

resistive and prominent reflectors from the

radar soundings in and around the vent site.

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 18

The resistivity pseudo sections delineate the

lateral and vertical molten flows which have

apparently solidified at shallow depth. The

GPR mapping due to ideal ground conditions

has optimum penetration with high definition

reflector topography, internal scatterers and

hyperbolas. The radar imaging explicitly

shows folding of the overlying fine grained

classics, whereas fracturing in the compact,

hard and brittle rock units of compact

gravels/limestone and volcanics due to the

pressure exerted by the intrusion.

The geological map of the study area

characterizes the presence of older volcanic

rocks which are remnants of past volcanic

episodes. Lava effusion appears as an

interactive play and involvement of the older

volcanics, ascending magma from depth and

dual tectonic-magmatism generating the

eruptive activity. The epicentral/focal locations

and migration of the past and present events in

the area strongly suggest the role of regional

tectonics and a positive connectivity of the

weaker Sibi Re - entrant, Quetta -Kalat fault

zone and the Quetta Transverse zone.

INTRODUCTION

An outpouring of molten material was

reported from Tor Zawar Mountain near Wam

which is about 90 km from Quetta and 36 km

from Ziarat on the main Quetta-Ziarat road

(Fig.A). Ziarat and Harnai areas have a known

seismic history. The name Wam (Wham)

locally means fear of the unknown and the

village was razed to the ground more than

once in the past. Wam was the worst affected

village in the doublet earthquake of 6.4M of

2008 which, was felt over a large area in

Balochistan.

Earthquake events as documented from 8th

to

27th

January 2010 indicate the epicentral

locations of the isolated events of January

2010, appear to be linked events resulting in

the main event of 27th

January, although the

counter clockwise focus have large spatial

separation probably caused by dual level

seismicity as substantiated by the focal depth

of the events varying from 10–60 Km. It is

reckoned that intense and major rock

deformation of sizeable dimension in the area

has taken place.

The present unique volcanic activity

probably enacted by nature after a span of

many millennia in this area provided a

surface thumb print warranting massive

research and follow up exploratory work in

continuity to allay the apprehensions,

concern and fear for the safety and security

of the people.

It is hoped that investigation at the vent site

would probably be a prelude for further

studies by the academic research and

professional public and private sector

institutes and other stakeholders.

Tectonic setting & general geology

The area lies between the active regional

Bibai and Gogai thrusts. The geological map

of the study area characterizes the presence

of older volcanic rocks which are remnants of

past volcanic episodes. The Urghargai fault

northwest of Ziarat is a right lateral wrench

fault which has horizontally displaced the

rock formations comprising the Bibai and

Gogai nappes by more than 2000 ft. This

particular lineament which runs for nearly 40

km in the NNW-SSE direction without any

trace of surface rupture may have been

reactivated in 2008 Gogai Earthquake.

However the origin of the magmatic/

hydrothermal solution from depth associated

with the regional concealed fault at the

moment is somewhat speculative.

Ziarat District is roughly a rectangular piece

of mountainous country comprising several

scenic valleys and is famous for its cool

climate and one of the world’s largest and

oldest Junipers forests. The altitude ranges

from 1, 800 to 3, 488 m.

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 19

Sedimentary rocks ranging in age from

Triassic to Quaternary are well exposed in

the Kach Ziarat area in the following

sequence (A. H Kazmi, 1979).

Fig A. Index Map of Pakistan Showing Study Area, Fig B. Geological Map of the Area

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 20

Geological investigations

The viscous lava flow was observed to

advance down slope for 8 meters reaching near

the foundation of an electric pylon and its

earth wire line. The high tension electricity

wires and earth support were found damaged

and cut off. Ruling out any possibility of

electric short circuiting from the nearby pylon

or thunder bolt/lightening striking at the pole

and absorbed into the subsurface, eruption of

lava was investigated in detail.

The erupted molten material (lava, scoria and

volcanic glass) was found to be cold and

solidified on the surface of the concentric

layers but underneath the viscous sheet of

molten material, the ground was still hot and

burning. Highly porous and crusty lava flow

showing vesicular structure had formed due to

escaping gases. Lava tubes and chambers were

formed when the surface of a basalt flow

highly charged with gas crystallized and still

molten lava within continued to move up.

Walking on cooled deposits of this lava was

similar to walking on crusted snow as the

viscous basaltic lava flow had developed ropy

surfaces like Pahoehoe-Hawaiin type lava

(Swanson, D.A., 1973). The glassy frot–scoria,

pea size globules-lapilli, bunches of finely

spun glassy hair- pele’s hair like structure were

observed at the vent site.

The volcanic activity may be indicative of

surface discharge of magmatic materials from

one central pipe and four satellite feeder

fissures that communicate with the heated

depths.

Fig3: Two larger fissures of 50.8 cm x133.2

cm and 71 cm x 33 cm brought lava to the

surface

Apparently the volcanic activity lived for

matter of a few hours and died out instantly.

The dimension of this lava structure was 1.9

m x 8.2 m in length and 15 cm to 0.6 m thick.

The solidified material on the surface was 2.9

m long and 1.5 m wide and formed

scoriaceous blocks with dangerously knife

sharp edges.

Fig4: The 35.5 cm cone shaped vent was

plugged by the solidification of the material

on the orifice.

Two larger fissures which brought lava to the

surface were of 50.8 cm x133.2 cm and 71 cm

x 33 cm. The two smaller ones were of 16.5

cm and 33 cm and 13 cm and all were found to

be still emitting heat. Solidified sheet of lava

formed after a thin skin of cool lava shoved

into folds by hot, more fluid lava just below

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

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the surface, was removed from the surface

after documenting the event. Samples of

volcanic material of different nature, including

volcanic glass, obsidian, scoria, pumice and

lava were collected for geochemical analyses

and petrographic studies.

The ejection of molten material and scoria

cone was excavated by digging a ditch of ~2 m

along the fissures to find the opening of the

vent and tracing of the source channel of the

extruded material. The complete structure of

the scoria cone and pipe was preserved for

display in the GSP Museum of Earth Sciences

for further research. The vent pipe of the main

cone was found to be 0.9 m deep from the

surface. The orifice was found to be widening

and inclined below the surface. The chamber

was found hollow up-to 50 cm blocked with

the solidification of material.

Fig5: Viscous lava had formed short stubby

flow down the mountain slope for 8 meters

The temperature of the chamber walls was

found still burning hot and when dry bushes

were put on the mouth of these chambers, they

caught fire.

Another smaller feeder which originated from

the central cone led towards SE direction. It

was measured to be about 4.75 m from the

main chamber.

Fig 6: After the upper surface had solidified,

the last of the molten lava drained away,

leaving an empty tunnel, with black icicles of

glass which adorned its side walls.

No fresh extrusion of volcanic material was

observed during field investigation.

However, heat was still coming out for ten

consecutive days.

Fig7: A chamber of 1m length and 5 cm

diameter lead vertically down to a funnel

shaped structure

Geochemical analysis

Cox et al (1979) diagram was used for fresh

lava specimens, nomenclature. Samples

overlap and fall in vacant domain below the

Hawaiite and above the basaltic field (Fig.2).

On SiO2 versus total alkali diagram, these

rocks are alkaline (Fig.3).The major elements

data classify the rocks as basaltic in

composition and alkaline. The geologists

inferred that it may be formed due to the

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

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crustal assimilation and re-melting of pre-

existing rocks of Bibai Volcanics under very

high pressure condition at depth as the data

corresponds to Bibai Formation (Bibai

volcanics of Kazmi, 1984; Siddiqui et al.,

1996). If these represent lava flow coming up

directly from the upper mantle, then the trace

elements data must have been depleted in Nb,

TiO2, Na2O and K2O.

Table 1: Geochemistry

Fe2O3 is total iron; Major trace elements are

analyzed on WD-XRF at GSP’s Geolab.

GEOPHYSICAL INVESTIGATIONS

The integrated techniques were adopted as the

effusive vent produced after an earthquake

resulted due to magmatic/lava activity which

was likely to have made major changes in the

sub surface detectable by the variation caused

in the magnetic properties of the rocks (host

sedimentary rocks enclosing magmatic

intrusive) , changed electrical rock

characteristics like resistivity in environments

of intrusions and shallow GPR scanning for

detecting near surface structural changes or

variations in the electrical properties of the

rocks.

35 45 55 65 750

3

6

9

12

15

18

Nephel in

P-N

B+T

P-T

Phonoli te

Benmorite

Mugearite

Hawaiite

Basalt

B-A Andesite

Dacite

Trachyandesite

Rhyolite

Trachyte

SiO2

Na

2O

+K

2O

Fig 8: Total Alkali vs Silica Diagram

35 40 45 50 55 60 65 70 75 80 850

2

4

6

8

10

12

14

16

18

20

Alkaline

Subalkaline

SiO2

Na

2O

+K

2O

Fig 9: Cox Diagram

Magnetic Survey

The total magnetic field intensity anomaly

map (Fig.10) shows the magnetic variation of

subsurface rocks in and around the

perforation vent. A circular rim of high

magnetic values is clearly discernable. This

high magnetic zone is bounded by the

continuous sharp gradient from all sides and

is comprised of about eight high magnetic

anomalies distributed within the circular

zone. The amplitude of the positive

anomalous poles vary from 700 to 1100 nT.

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 23

An interesting feature in the area is the

positive poles are inside the semi ring zone

and the negative poles relatable to these

positive poles are located outside the rim.

The anomaly rim is separated by a sharp

gradient in between them. The general trend

of this ring pattern shows positive values

with the negative values encountered in the

rest of the area, which indicates that all these

positive values are related to the same

causative dipolar body at depth. The four

positive magnetic peaks bound the vent site

with contour values ranging from 700 nT to

1100 nT. The magnetic anomalies indicate a

dipolar nature and the sharp gradient shows

shallow depth. The maximum depth to the

top of these bodies is about 11 meters. A

north-south trending sharp gradient is

observed near the profile W3 of the grid,

which probably indicates a concealed fault or

the sharp contact between sedimentary rocks

and volcanic intrusive. Maximum positive

anomaly signature is produced near the vent.

These anomalies are probably related to the

older Bibai volcanics or present activity of

fusion of the older rocks with the

hydrothermal/ magmatic intrusion ascending

and mixing with the older shallow volcanics

which could not reach the surface except at

the vent site. It is difficult to conclude,

whether this susceptibility contrast

corresponds to the already existing Bibai

volcanics and sedimentary rocks or

recent/new intrusion of different composition

showing the contrast. The composition of the

vent molten material is similar to Bibai

volcanics, dominantly basaltic (Mafic) which

are less viscous than the silicific lavas and

tends to be lighter than felsic lavas (Silica >

63 %).

The residual total magnetic field intensity

anomaly maps at an average ring radius of

24, 36, 48, 60, 72 and 84 meters are also

prepared by Griffin’s method for enhancing

the shallow anomalous features in and around

the vent.

Fig10: Total Field Magnetic Intensity

Anomaly

These maps also show the continuity of the

causative source, i.e the intrusion with depth.

The vent site is located at the Zero profile

and the residual total field magnetic intensity

anomaly map at average ring radius of 84

meters show that the main causative body lies

under E5 profile. This shows that the

causative intrusion likely moved from the

East.

Electrical resistivity survey

3 VES and 2 Dipole-Dipole profiles were

made at the vent site.

Vertical Electrical Sounding-1

The sounding depth attained at the vent site is

200 meters. The resistivity ranges from 1.91

Ohm-m to 341 Ohm-m. Alternating layers

of gravels/boulders with varying proportion

of clays are present down to the explored

depth. Anomalous very low resistivity 1.91

Ohm-m is attained by the bottom layer down

to the 200 meters.

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

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Vertical Electrical Sounding-2

The Vertical Electrical Sounding site is about

100 meters southeast of the vent site. The

resistivity of modeled four layers ranges from

73.6 Ohm-m to 417 Ohm-m. The resistivity

of the third and fourth layer decreases

gradually down to the explored depth of 300

meters, showing increasing conductivity with

depth.

Vertical Electrical Sounding-3

This investigated site is about 200 meters

southeast of the vent site. The resistivity

ranges from 45 Ohm-m to 288 Ohm-m. The

thickness of alluvium is 1.93 meters with

resistivity of 106 Ohm-m. The same trend of

decreasing resistivity at depth is observed as

that of site VES-2.

Dipole-Dipole Profiling Along E0 Profile

(South-North)

Dipole-Dipole survey along E0 profile of the

grid (Fig.6) with 30 meters dipole spacing.

The depth scanned is 90 meters. The pseudo

section appears laterally divided into two

sections. In the upper half of the section

alternate high and low resistivity zones are

observed along the survey line. This might be

due to the presence of boulders (high

resistivity) at shallow depth. A dome type

apparent resistivity pattern is observed

between G and H with peak contour value of

180 Ohm-m. This is deduced to corresponds

to the magmatic intrusive. The resistivity of

this zone is on the higher side, indicating that

the magma is already solidified. This

prominent zone of high resistivity is bounded

by decreasing resistivity at outer side,

showing the contact between sedimentary

units, and volcanics.

Fig 11 A) Vertical Electrical Sounding (VES-1) at the Vent Site B) Vertical Electrical Sounding

(VES-2) south of the Vent Site C) Vertical Electrical Sounding (VES-3) south of VES-2, Vent

Site

Fig 12A) Apparent Resistivity Pseudo Section along Zero Profile at the Vent Site B) Apparent Resistivity

Pseudo Section along E3 Profile at the Vent Site

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

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Dipole-Dipole Profiling along E3 Profile

(South-North)

The observed profile is 30 meters east of E0

profile. A distinct anomaly (Vertical to

Inclined) is indicated in the center of the

profile with a decreasing resistivity on the

outer side. The apparent resistivity of this

body is high with peak value of 190 Ohm-m

and bounded by decreasing resistivity. There

is a sharp resistivity gradient along this pattern

which probably shows the contact between

sedimentary and volcanic intrusive. Another

very high resistivity zone is observed in the

southern part of the section but its downward

extension is limited to only 60 meters depth.

This may correlates with a lateral flow (Sill)

of intrusion and is not exposed at the surface.

Ground Penetration Radar (GPR) Survey

Ground Penetrating Radar survey with 25

MHz unshielded with parallel antenna

configuration was observed from NW to SE

direction along a track that passes over the

vent in the study area. The depth of

penetration attained is above 50 meters.

Velocity calculated is 95 m/nsec by the

“hyperbola fitting” to an arch like pattern

along 90 meters profile length and at 900

nsec. The GPR echo shows the subsurface

geological section along the profile. At the

top an eight meters smooth pattern is

observed with some variations at some

places. This parallel and smooth signature is

produced because of the resolution of the

antenna. The resolution of the 25 MHz

antenna is about 1 meter. The distortion in

the sub surface structure at the vent site as

evidenced by the GPR section is probably

the intrusive activity that had taken place. A

prominent reflector is observed with top at

40 meters at the profile end. The north

western portion of the section appears more

disturbed than the rest.

Another very smooth bedding signature is

produced at 36 meters depth with some

disturbances at places, along 10-25 meters

profile length, a clear fracture at 50 meters, a

disturbed signal from 14 to 32 meters and

below 42 meters depth. A convex feature at

18 meters depth, look likes to be a resultant

fracture in the layer by the upward

stress/strain developed during intrusion of

magma. Along the profile the vent site is

located at 90 meters profile distance from

NW. A very good GPR signature at this

point at 42 meters depth as a

prominent/hyperbola is produced. This

strong signal directly under the vent site

leads to the presence of some hard and

compact source hosted within the

sedimentary surroundings. It is deduced that

this is from the solidified magma present in

the subsurface. The other features like

folding, fracturing and arc like structures are

the resulting signs of the volcanic intrusion

that took place causing structural change in

the subsurface under the vent site.

Fig13: Interpreted GPR Section at the Vent Site

The 27th

January 2010 earthquake was

resulted due to an effusion of molten

material. The vent site was investigated by

integrated geophysical surveys employing

magnetic, resistivity and GPR techniques.

The magnetic survey in the investigated area

shows a presence of a circular pattern of high

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 26

magnetic anomalies in the background of

low magnetic values in the area. The vent

site is located within the high anomalous

magnetic rim bounded by four positive

magnetic anomalous closures. The positive

and circular shaped corresponding negative

lobes of anomalies are separated by a

continuous sharp gradient from all sides. The

residual total magnetic field intensity

anomaly maps at different planes indicates

that the different magnetic anomalies

reflected in the total field magnetic intensity

anomaly map as different bodies are actually

the manifestation of a single causative source

at depth. The residual maps clearly show that

the causative source of these apparently

discrete anomalies at shallow depth is a

distinct single source which apparently shifts

towards east/southeast with increasing ring

radius/depth. The body center lies at E5

profile, 50 meters east of the vent in the

residual map at the average ring radius of 84

meters. VES-2 and VES-3 are located about

100 meters and 200 meters SE from the vent.

VES-2 and VES-3 show similar resistivity

response for the subsurface, but VES-1

encountered a relatively low resistivity layer

at 14.7 meters depth. The apparent resistivity

pseudo section along E0 profile shows a very

disturbed resistivity behavior. The upper and

lower sections along the profile are separated

laterally by a relatively low resistivity zone.

In both the upper and lower section,

alternating high and low resistivity values

are observed. These indicate

vertical/inclined/ segregated high and low

resistivity bodies. The comparison of

magnetic responses with apparent resistivity

pseudo section along E0 profile shows that a

high resistivity closure at depth of about 50

meters corresponds to high magnetic value.

This is also quite evident from the E3 profile

that high magnetic signature corresponds to

high resistivity. The GPR section also shows

a prominent reflector under the vent site at

42 meters depth. The GPR echo sounding

also indicates the folding and fracturing in

the subsurface structural litho configuration.

Visible folds are discernable in the low

resistivity layer (clays/shale) under the

gravels. Compact and brittle rock units,

however, have been fractured as shown in

the section. These anomalous magnetic,

resistivity signatures with the GPR echo

sounding results all interpreted in

conjunction leads to a deduction of an

intrusive characterized by high resistivity,

high magnetic and prominent reflector. The

solidified causative intrusive in the form of a

dyke/sill is a hard/compact body resulting

out from the hydrothermal /magmatic source

ascending from deeper zones at the vent site

and its surroundings. This intrusion has

caused significant structural changes and

fractures and folding in the subsurface.

Comparative total magnetic intensity

anomaly profile and GPR section shows an

arc like structure at the vent site located at 42

meters depth, corresponds to the high

magnetic response. The source of causative

body is shifted towards south east, also

corroborated by the apparent resistivity

pseudo sections along E0 and E3. A high

resistivity dome like structure an intrusive is

well correlated with the high magnetic along

E3 profile.

CONCLUSIONS

The event may be a hybrid between a

volcano-tectonic seismic event and seismo-

tectonic volcanic event. Apparently this

outpouring seems to be related to deep

seated basement geo-fracture which may

have been vertically active through much of

the geologic time.

The close proximity of the epicenter of 2008

and effusion of 2010 shows that the event,

though at different times, but being proximal

in the same zone appears related. The event of

the 2008 near Gogai had already rendered the

epicentral area as a weak ruptured zone which

was further weakened and become unstable

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

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with the tectonic stress from the 2010 event

resulting in an eruption at the Tor Zawar

Mountain.

The main event followed by the aftershocks

with scattered epicenters over a large area

shows the ruptures and rock fractures in the

subsurface occurring in a large area probably

along concealed regional fault or unstable

weakened tectonic/structural fronts much

deeper at the 10-50 km depth in the crust and

upper mantle.

The anomalies identified on the basis of

magnetic, resistivity, and GPR at the vent site

and surroundings indicate the presence of

intrusion with both vertical and lateral flows.

The alkaline basaltic lava flow at the vent site

appears to originate from the east/southeast of

a regional fracture, east of the vent site,

probably activated in the 2010 event which

resulted in the sluggish eruption.

The effusive lava is characterized by high

resistivity, high magnetic and prominent

reflector formed due to the crustal assimilation

and re-melting of pre-existing rocks of Bibai

Volcanics under very high pressure condition

at depth as the data corresponds to Bibai

Formation.

The odds are not zero that an earthquake

accompanied by an eruption will occur in

certain area during a certain period of time

and estimating eruption possibilities is an

interesting research field and one in which

more questions would be raised than

answered.

The present data caution us for an extremely

violent earthquake in this region, which may

or may not be accompanied with volcanic

effusions. The magnitude and scale of such

an eruption, even if a remote possibility

warrants that if we fail to plan, we plan to

fail.

ACKNOWLEDGEMENTS

The authors acknowledge the generous

support, encouragement and guidance of Dr.

Imran Khan, Director General, Geological

Survey of Pakistan in all stages of the

investigations.

REFERENCES

Armbruster, J., Seeber, L . Quittmeyer, R.,

and Farah, A.,1979, Seismic network

data from Quetta Pakistan, GSP

Record Vol.49

Bardintzeff, J.M., and McBirney., 2000,

Volcanology second Edition, Jones

and A., Bartlett Publishers,

Massachusetts, USA.

Bowen, R, 1979, Geothermal Resources,

Applied Science, Publishers Ltd.

New York, USA.

Carret, B, A., 2006, Geophysical hazards and

hazard awareness On Nisyros

Volcano Greece, M.SC Dissertation,

University College London.

Cox, K.G., Bell, J.D., and Pankhurst, R.J.,

1979,The interpretation of igneous

rocks, George Allen and Unwin,

Boston, 450 pp.

Farah, A., and Dejong, Kees A.,1979,

Geodynamics of Pakistan, GSP

Quetta.

Farah, A., Lawrence, R, D., And Dejong, K,

A., 1984, An overview of the

Tectonics of Pakistan, printed in

marine geology and oceanography of

Arabian sea and coastal Pakistan,

Bilal U Haq and John D Milliman

edition.

Farah, A., Abbas, G., De jong ., 1984,

Evolution of the lithosphere in

Pakistan.Hunting Survey Corp.Ltd.

(HSC), 1960, Reconnaissance geology,

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 28

Part of West Pakistan, publ.under

Columbo Plan Coop.Project, Govt. of

Canada, Toronto, Canada.

Kakar, D.M., Szeliga, W., and Bilham, R.,

2010, Seismic Potential of the

Pishin/Mach Shear Zone in Northern

Baluchistan, Pakistan, Seismological

Research Letters, v. 81, no. 2, pp.

324.

Kerr, A.C., Khan, M., and McDonald, I.,

2010, Eruption of basaltic magma at

Tor Zawar, Balochistan, Pakistan on

27 January 2010: geochemical and

petrological constraints on

petrogenesis, Mineralogical

Magazine, v. 74, no. 6, pp. 1027-

1036

Kazmi, A.H., 1979; The Bibai and Gogai

nappes in the Kach Ziarat areas of

north-eastern Balochistan,

Geodynamics of Pakistan. Ed: Farah

& Dejong.

Kazmi, A.H., 1984; Petrology of Bibai

Volcanics in Baluchistan, Geol

Bull.of Univ. of Peshawar, 7.Kazmi,

A.H & Jan, M. Qasim, 1997;

Geology and Tectonics of Pakistan.

Mona, Lisa and Jan, M.Q., 2010,

Geoseismological study of the Ziarat

(Balochistan) earthquake (doublet?)

Of 28 October 2008, Current

Science, v. 98, no. 1, p. 50-57.

Gawad, M, Abdel., 1971, Wrench

movements in the Balochistan Arc

and relation to Himalayan-Indian

Ocean Tectonics, Geological Society

of America Bulletin, Wrench

movements in the Balochistan Arc

and relation to Himalayan-Indian

Ocean Tectonics, Geological Society

of America Bulletin.

Geological map No 26,1958,

Reconnaissance geology of part of

West Pakistan, Colombo plan co-

operative Project.

IPI2Win-IPI Win, Resistivity Sounding

Interpretation software, (1990-2003),

Version: 3.0.1.a 7.01.03

Kazmi, A, H.,1979, Preliminary seismo

tectonic map of Pakistan, GSP, Map

series First Edition.

Kazmi, A, H.,1979, The Bibai and Gogai

Nappes in the Kach-Ziarat area of

north eastern Balochistan,

Geodynamics of Pakistan.

Lawrence, R,D., Khan, S,H., Dejong, K, A.,

Farah, A., and Yeats, R,S., 1981,

Thrust and strike slip faults along the

Chaman Transform zone Pakistan,

Thrust and Nappe tectonics. The

Geological Society of London.

Nakata, T., Otsuki, K., and Khan, S.H.,

1990, Active faults, stress field and

plate motion along the Indo-Eurasian

plate boundary, Tectonophysics,

Elsevier Science Publishers, B.V.

Amsterdam.

Rana, A.N., Akhtar, S.S. and Qadir,

G.T.,2008, Seismotectonic

investigations of October 29, 2008

earthquake of Gogai Ziarat,

Balochistan, Information Release No.

874, Geological Survey of Pakistan,

Islamabad.

Rana, A.N., and Akhtar, S.S., 2010, Effusion

of Molten Material in Tor Zawar

Mountain, Sari, Ziarat, Balochistan

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 29

on January 27, 2010, Information

Release No. 891, Geological Survey

of Pakistan, Islamabad, 24 pp.

(Summary in GSP News. V. 17, No.

1-12, p. 12.)

.

Rittman, A., and Vincent, E.A., 1960,

Volcanoes and their Activity ,

Interscience Publishers, A division of

John Wiley and Sons Inc New York

US.

Siddiqui, R. H, Khan I.H and Aziz A; Dec

1996, Geology and Petrogenesis of

Hot spot related magmatism on the

Northwestern Margin of Indian

Continent; Proceedings of

Geoscience Colloquim, GSP Vol. 16.

Siddiqui, R. H, Dec 1999, Petrogenetic

Study of Hotspot related magmatism

on the Northwestern Margin of

Indian Continent and its implications

for Paleosedimentary.

Shah, S.H.A, 1965; The structure of the area

between Ziarat and Wani, Sibi and

Loralai districts, W.Pakistan,

Rec.Vol. XIV Part II, Geological

Survey of Pakistan, Islamabad.

Swanson, D.A., 1973, Pahoehoe flows from

the 1969-1971 Mauna Ulu Eruption

Kilauea volcano, Hawaii, Geological

Society of America Bulletin, v. 84, p.

615-626.

Telford , W,M., Geldart,L,P., 1993, Applied

Geophysics, Second Edition

Cambridge Sheriff, R,E. university

Press , reprinted 1993 USA.

Verma,R,K., and Sekhar, CH, C., 1986.

Focal mechanism solutions and

nature of plate movements in

Pakistan, Journal of Geodynamics.

Welch, B, C., Dwyer, K, Helgen,M.,

Waythomas, C, F., Jacobel, Mt.

R,W., Geophysicalsurvey of intra-

caldera ice field of Veniamin,

Alaska.

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AGRICULTURAL SCIENCES

RESEARCH ARTICLE

Effect of multiple harvests on chemical composition of

important nutrients of Alfalfa (Medicago sativa L.) grown in

Uthal, Lasbela District, Balochistan, Pakistan.

Saeed Ahmed1, Abdul Hameed Baloch

1 and Imtiaz Ahmed

2

1Faculty of Agriculture, LUAWMS, Uthal, Lasbela, Balochistan, Pakistan.

2Faculty of Water Resources Management, LUAWMS, Uthal, Lasbela, Balochistan,

Pakistan.

ABSTRACT

The purpose of this paper was to study the effect of different harvests on alfalfa

(Medicago sativa L.)biomass through the assessment of DM (Dry Matter contents), CP

(Crude Protein), EE (Ether Extract), CF (Crude Fibre) and NFE (Nitrogen free extract).

Alfalfa was collected from five cuts (three cuts from 1-3 months, first harvest and

second harvest). The results of present study revealed that feed quality of alfalfa

harvested as silage depends, to a great extent, on the maturity of the stand. With

increasing maturity, CP, and Ash quantity decreases and on the other hand crude fibre

(CF) and nitrogen free extract (NFE) fractions, were increased. These fibre fractions

represent the more indigestible parts of the plant. As a result, digestibility and energy of

alfalfa as a fodder crop decreased with maturity.

Keywords:Medicago sativa L, multiple harvests, biomass , Lasbela District

_____________________________________________________________________ Correspondence: Abdul Hameed Baloch Address: Faculty of Agriculture, LUAWMS, Pakistan. Email: hameedbaloch67@yahoo.ca Phone: +92-333-2218439 Received: 05 Jun 2013 Revised: 03 Aug 2013 Accepted : 05 Aug 2013 Copyright: ©2013 Ahmed et al. This is an open-access article distributed under the terms of

the Creative Commons Attribution License, which permits unrestricted use, distribution, and

reproduction in any medium, provided the original author and source are credited Competing Interests: The authors have declared that no competing interests exist. ______________________________________________________________________

INTRODUCTION

Alfalfa (Medicago sativa L.) often called

the “Queen of Forages,” belongs to family

Fabaceae, cultivated as an important forage

crop, which is grown in areas oflimited

rainfall, high temperature and high level of

salinity (Safarnejad et al, 1996). Alfalfa

offers the prospect of enhancing the

management of major crop weeds. Alfalfa

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

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enhances and protects the soil due to its

vigorous and perennial root system, fast

growing protecting canopy and capability

to fix atmospheric nitrogen (Shahriari et al,

2007). Its vigorous growth combined with

annual harvest during the growing period

provides excellent weed control. As a

fodder crop, Alfalfa is rich in protein,

minerals and vitamins. The relative feed

value has been extensively used in ranking

fodder for sale, inventorying and assigning

forage lots to animal groups, according to

their quality needs and determination when

to harvest. The nutrient contents of the

feeding value largely depend upon the stage

of growth at the time of utilization.Because

nutritive value falls with advancing

maturity and associated increase in stem-

leaf ratio. In later stages of growth cell wall

constituents (cellulose, hemicellulose,

lignin) increased by 0.l6% of dry matter per

day with advancing maturity, which

decreases forages digestibility (Keftassa

and Tuvesson, l993). Although cutting at

earlier stages of growth improves forage

digestibility and crude protein content it

decreases total yield (Brink and Marten,

l989; Hesterman et al, l993). Compared

with any forage grass at similar stages of

growth, alfalfa has lower cell wall and

digestible fibre contents but higher

digestible cell and crude protein contents

(Campling, l984). It was suggested that

frequent cutting deceases alfalfa yields

(Judd and Radcliffe, 1970), however, it has

been found that increasing cutting height,

reduced yield per unit area of protein and

carotene (Ogden and Kehr, 1968).

District Lasbela is the 7th

largest district in

Balochistan and has an area of 15,153

square kilometres, Lasbela District, lies

between 65°12'11"-67°25'39" East

longitudes and 24°53'2"-26°39'20" North

latitudes. The major Rabi crops of this

district are Wheat, Barley, Mutters pulse

and Fodder. However, alfalfa as a major

fodder crop receives much attention in this

district. Alfalfa production has been

increasing in the Lasbela District to support

a rapidly expanding dairy industry.

Lactating dairy animals fed physically

effective alfalfa fibre are healthy and very

productive. Therefore, a common variety of

alfalfa (Omani), which is successfully

grown in Kech district, was introduced for

the first time in Lasbela District.

The purpose of the present study was to

investigate at the effect of cuttings and the

influence of cutting frequency on the

productive and qualitative characteristics of

alfalfa cultivars widely grown in the Kech

region in Pakistan. Our objectives in this

study were: to understand whether or not

cutting frequency affects alfalfa

productivity (biomass) and quality (CP

etc.,) and; to determine if this variety

(Omani) had higher yield and quality with

increased cutting frequency in Lasbela

District.

MATERIALS AND METHODS

The experiments were conducted in

Agronomy field of Lasbela University of

Agriculture Water and Marine Sciences

campus, Tehsil Uthal. The site is located at

latitude between 65°12'11"-67°25'39" East

longitudes and 24°53'2"-26°39'20" North

latitudes, in arid climate in the centre of

Lasbela District, where the summer is dry

and hot, while the winter is cool. Before

seeding of alfalfa the soil was fertilized

with DFA. Seeds of alfalfa cultivars

‘Omani’ were planted @ 2.5 kg/acre, on

October 1, 2012. The field was rolled after

planting. Once seeding appeared and

reached at 2-3 leaves stages regular urea

fertilizer was used to enhance the growth of

fodder. At different stages of growth

traditional manure was also applied. Pest

and weed controls were carried out

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

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according to general local practices and

recommendations.

The first harvest was taken on first month

after seeding followed by second and third

harvest at the interval of one month

respectively. The first cutting was done at

the first week of November, 2013 and the

second was done at the first week of

December 2013. The whole-plant samples

were taken from experiment quadrates (one

square meter) and dried at 100ºC. All

samples were grinded in a regular mill

through a 1-mm screen. The Following

analysis was conducted by using standard

methods.

The Crude Protein (CP) is approximated by

multiplying the Kjeldahl nitrogen analysis

by the factor 6.25. The plant materials were

treated with sulfuric acid, which

decomposes the organic substance by

oxidation to liberate the reduced nitrogen as

ammonium sulfate. In this step potassium

sulfate is added to increase the boiling point

of the medium (from 169°C to 189°C). The

solution is then distilled with a small

quantity of sodium hydroxide, which

converts the ammonium salt to ammonia.

Total nitrogen derived from the analysis is

converted into protein by multiplying with

factor that takes into account the nitrogen

content of a known or average amino acid

composition (López et al, 2010).

Ether Extract (EE), the ground sample of

alfalfa, is extracted with diethyl ether which

dissolves fats, oils, pigments and other fat

soluble substances. The ether is then

evaporated from the fat solution. The

resulting residue is weighed and referred to

as ether extract or crude fat.

Total Ash is determined by igniting in a

furnace at 600oC to oxidize all organic

matter. Ash is determined by weighing the

resulting inorganic residue.

Crude Fibre (CF) refers to organic matter

insoluble in a hot diluted sulphuric acid and

diluted sodium hydroxide solution (Goering

et al, 1972). Nitrogen free extract (NFE)

was mathematically calculated, as

difference between organic matters values

and analytically assessed organic

compounds. Dry Matter (DM), is the

determination of dry matter on ground air-

dry or partially dried (85% dry matter)

forages. Moisture is evaporated from the

sample by oven drying. Dry matter is

determined gravimetrically as the residue

remaining after drying.

Calcium (Ca) and Phosphorus (P) were

determined with spectrophotometer model

DR3900 HACH, USA. All chemical

analyses were carried out in triplicate for

each harvest of alfalfa.

RESULTS

The variability of DM, CP, CF, Ash, EE, and NFE of alfalfa as influenced by stage of growth is presented in Table 1. Dry matter (DM) content gradually increased after first cutting from 19.9% to 21.1% at 4 weeks interval (table 1). In contrast, crude protein (CP) content and Ash contents decreased by 51% and 50% respectively. It is because the nutritional substances from cell content decreased, accompanied by an increase of cell walls content, therefore a diminution of organic matters digestibility. Alfalfa harvested during bud stage presented 25.4% CP proportion,

which decreased till 14.9% CP (full

flowering,2nd

harvest). There were no significant

differences among different harvest of

alfalfa in terms of Ether Extract (EE) when

compared with crude protein concentration

of Nitrogen Free Extract (NFE), exhibited

an inconsistent change pattern with

advancing maturity of alfalfa. From the

result, it is also clear that of the chemical nutrients’ concentration (Ca, P) change

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

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pattern over the study period is gradually increasing (Table 1). The percentage of crude fibre (CF) gradually increased from 17.2% at one months old plant to 28.9% at second harvest (after 5 months of seeding). This is because the total content of cell wall was influenced by plants age at harvesting. As plants turned old, cellulose and lignin proportion increased,

while hemicelluloses decrease

DISCUSSION

The qualityof alfalfa as a forage crop is

depending upon the palatability and

maximizing intake and production of dairy

cows. Alfalfa is low in fibre and high in

protein compared with other forages, which

makes it an excellent compliment in dairy

rations. The quantity of crude protein is

higher in immature alfalfa but the protein is

rapidly fermented in the rumen to ammonia

and not used efficiently (Broderick and

Satter, 1998). Like any other forages grass,

the percentage of Crude Fibre (CF) the

proportions of fibre and lignin increase

with maturity in alfalfa. However alfalfa

fibre contains a high proportion of lignin

compared with forage grasses resulting in

low digestibility relative to grasses.

It was suggested that 60 to 80% of grass

fibre is potentially digestible; the possible

level of digestion of alfalfa fibre is only

40% to 60% due to its high lignin content

(Martin and Mertens, 2005). However,

alfalfa has a great advantage over grasses

because the rate of digestion of its

important digestible fibre is 2 to 3 times

that of forage grasses. It is also appear that

the indigestible fibre in alfalfa disintegrates

into particles that rapidly pass out of the

rumen. The higher intake and digestibility

often observed with alfalfa-based diets

compared to those containing grass is not

due to greater digestibility of alfalfa fibre,

but due to alfalfa’s low fibre content and

the rapid rates of digestion and passage of

that fibre. (Martin and Mertens, 2005).

CONCLUSION

The replacement of traditional forage

grasses to alfalfa in Lasbela District is

obvious because of the nutrient contents and

higher digestibility. An ideal alfalfa as an

alternative fodder crop would contain a

better balance of protein and rapidly

fermentable carbohydrate. Similarly it

would be desirable to have about 18% crude

protein, less ash, and about 30% crude fibre

CF. It would also be beneficial to have a

better balance of amino acids in the protein

and with a slower rate of degradation in the

Table 1: Composition of different nutrient contents in dry biomass of Medicago sativa L.

Harvest

1 month

2 months

3 months

First cutting

Second cutting

DM Percentage of dry matter

CP CF Ash EE NFE Ca P

25.4 17.2 16.7 2.7 41.0 1.96 0.42

20.9 26.7 15.2 3.2 37.1 2.25 0.35

17.5 30.7 10.7 3.6 41.1 1.90 0.24

19.9 14.4 30.9 8.1 2.9 45.9 1.75 0.17

21.1 14.9 28.9 8.7 4.3 46.0 1.55 0.25

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

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rumen to minimize its losses as ammonia.

Although the rate of digestion and passage

of alfalfa fibre is outstanding, compared

with any other forage grass but it would be

excellent if low lignin containing verities

introduce in Lasbela District. Similarly the

yield of alfalfa should be enhanced with a

reduction in the number of cuttings needed

to produce dairy-quality alfalfa forage

ACKNOWLEDGEMENT

We are grateful to Dr. Gul Hasan Vice

Chancellor and Mr. Amanullah Ronjha

Registrar, LUAWMS for providing

transport facilities.

REFERENCES

Brink, G.E. and Marten, G.C., 1989,

Harvest management of alfalfa-

nutrient yield vs. forage quality and

relationship to persistence: Jour. of

Prod. Agri. (2): 32-26.

Broderick, G.A. and Satter, L.D.,1998,

Balancing forage proteins. In State

Forage and Feeding and

Management Conf: Wisconsin Dells,

WI: p. 19-34.

Campling, R.C., 1984, Lucerne, red clover

and other forage legumes: feeding

value and animal production.

Thomson, D.J. (ed.) Forage

Legumes. Occasional Symposium

No. 16: British Grassland Society,

Hurley. 140-146.

Collin, H. A., Bruce, K. D. and McNeilly,

T., 1996, Characterization of alfalfa

(Medicago sativa L.) following in

vitro selection for salt tolerance:

Euphytica. 92(1-2): 55-61.

Goering, H.K., Gordon, Hemken, C.H.

R.W., Waldo, D.R., Van Soest, P.J.

and Smith, L.W., 1972, Analytical

estimates of nitrogen digestibility in

heat damaged forages. Jour. of Dairy

Sci. 55(9): 1275-1280.

Hesterman, O.B., Kells, J.J. and Tiffin,

P.L., 1993, Interaction among

harvest frequency, fertilizer and

herbicide use with intensively

managed alfalfa in the north-central

USA. Baker, M.J. (ed.). Proceedings

of the XVII International Grassland

Congress, New Zealand and

Australia, Vol. I. New Zealand

Grassland Association: Palmerstone

North, pp. 885-887.

Judd, P. and Radcliffe, J.C., 1970, The

influence of cutting frequency on

yield, composition and composition

and persistence of irrigated. Jour. of

Exper.Agri. and Animal Husb. .10:

48-52.

Keftassa, D. and Tuvesson, M., 1993, The

nutritive value of lucerne (Medicago

sativa, L.) in different development

stages. Swedish Jour. of Agri. Res.

23: 153-159.

López, C.V.G., García, M.C.C., Fernández,

F.G.A., Bustos, C.S., Chisti, Y. and

Sevilla, J.M.F., 2010, Protein

measurements of microalgal and

cyanobacterial biomass. Bioresource

Tech. 101: 7587–7591

Martin, N.P. and Mertens D.R., 2005,

Reinventing alfalfa for dairy cattle

and novel uses. In: Proceedings,

California Alfalfa and Forage

Symposium, 12-14 December, 2005,

Visalia, CA, UC Cooperative

Extension, Agronomy Research and

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 35

Extension Center, Plant Sciences

Department, University of

California, Davis 95616.

Ogden, R.L. and Kehr, N.R., 1968, Field

management for dehydration and

hay production. Proc. 10th

Tech.

Alfalfa Conf. USDA., AR.S. 74-

46:23-37.

Shahriari, M.H., G.R. Savaghebi-

Firoozabadi, M. Azizi, F. Kalantari

and Minai-Tehrani, D., 2007, Study

of growth and germination of

Medicago sativa (Alfalfa) in light

crude oil-contaminated soil. Res. J.

Agric. Biol. Sci., 3: pp. 46–51.

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 36

INTRODUCTION

The catfish Chrysichthys (lacepede), a

siluroid belonging to the Claroteidae is

widely distributed in fresh and brackish

waters of West Africa, (Holden etal, 1991).

The Chrysichthys species are among the

dominant species of commercial catches.

The realization of aquaculture potentials of

Chrysichthys nigrodigitatus has increased

the demands for its seeds for stocking,

leading to increased fishing pressure.

Different species of Chrysicthys seeds are

harvested because of difficulty in

distinguishing them at fry stage. Moreover,

the present state of taxonomy using

MARINE SCIENCES

RESEARCH ARTICLE

Genetic differentiation of two Chrysichthys species using

mitochondrial DNA sequencing

Nwafili S.A, Eminue B.O And Jamabo. N

Department of Animal Science and Fisheries, University of Port-Harcourt, PMB 5523 Port

Harcourt, Nigeria

______________________________________________________________________________

ABSTRACT

Genetic variation of two Chrysichthys species, Chrysichthys nigrodigitatus and Chrysichthys

walkeri from the Lagos lagoon were investigated at mitochondrial DNA level. Mitochondrial

DNA control region (D-loop) fragment was amplified. Twenty four D-loop fragments were

randomly sequenced. Among 13 individuals of C. walkeri and 11 individuals of C. nigrodigitatus

the number of haplotypes were 7, respectively. The haplotype and nucleotide diversities were

respectively 0.846 and 0.010 C. nigrodigitatus and 0.873 and 0.006 for C. walkeri. The NJ tree

clearly distinguished the two species into two clades with a mean genetic distance of 0.898. High

diversities despite heavy fishing may indicate high recruitment of the species.

Keywords:Chrysichthys species, Genetic variation,, Mitochondrial DNA

_____________________________________________________________________________

Correspondence: Nwafili S.A

Address:Department of Animal Science and Fisheries, University of Port-Harcourt, PMB 5523

Port Harcourt, Nigeria

Email: sylvanus.nwafili@uniport.edu.ng.

Phone: +2347037964676 Fax: +2347037964676

Received: 08 April 2013 Revised: 12 July 2013 Accepted 14 July 2013

Copyright: ©2013 Nwafili et al. This is an open-access article distributed under the terms of the

Creative Commons Attribution License, which permits unrestricted use, distribution, and

reproduction in any medium, provided the original author and source are credited

Competing Interests: The authors have declared that no competing interests exist.

______________________________________________________________________________

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 37

morphological keys on Chrysichthysis not

adequate for proper identification of species.

The species C. walkeri is also endangered

(IUCN, 2012) and its genetic features need

documentation. Molecular markers are

effective methods for detecting genetic

differences (Whitehead 2003).

Mitochondrial DNA (mtDNA) has been

widely used in resolving identity issues

owing to its higher mutation rate and lower

effective population size than nuclear DNA

(Brown et al, 1979; Birky et al, 1989). This

is because mtDNA is inherited maternally

without recombination, making it a powerful

tool for detecting past mtDNA lineages in

populations. Tudela (2009) demonstrated the

use of the control region identification of

tuna species. …….((

In this work, we employ mitochondrial

DNA control region to differentiate and

characterize two species of Chrysichthys.

The mitochondrial control region is

noncoding and has used to detect genetic

variations of fishes for the studies of

population structure and phylogeny because

it varies greater than the other regions

(Aurelle & Berrebi 2001; Hoelzel et al.

1998; Nesbo et al. 1998).

MATERIALS AND METHODS

Samples: 11 and 13 individuals of C.

nigrodigitatus and C. walkeri were

respectively obtained from Lagos lagoon,

Nigeria for the study.

DNA extraction, amplification, and

sequencing

The Muscles of the samples were preserved

in 95% ethanol. Total genomic DNA was

isolated using the standard Phenol-

Chloroform-Isoamyl-alcohol after digestion

of tissue with proteinase K. This was

followed by PCR amplification to obtain a

portion of the control region in a total of 20

µl volumes containing 1.25 units of Taq

polymerase, 200 nmolL-1

forward and

reverse primers, 200 µmolL-1

each of

dNTPs, 10 mMolL-1

Tris (PH 8.3), 50

mMolL-1

KCl, 1.5 mMolL-1

MgCl2.Part of

the control region was amplified using the

set of primers cited in Watanabe and

Nishida (2003) undercycling conditions of 3

min initial denaturation at 94 oC and 40

cycles of 45 s at 94 oC for denaturation, 45 s

at 50 oC for annealing, 45 s at 72

oC for

extension, and a final extension of 10 min at

72 oC.

MtDNAanalysis

The sequences were viewed and edited

using Seqman as implemented in

DNASTAR software (DNASTAR Inc.). The

alignment was carried out using ClustalX2.

The number of variable sites (Ps), number of

haplotypes (Nhap), nucleotide (π) and

haplotype (Hd) diversities was calculated

using Dnasp 4.0 (Excoffer et al, 2005). A

genetic distance was generated to see how

the species are genetically separated using

MEGA 3.1. This distance is the proportion

(p) of nucleotide sites at which two

sequences being compared are different. It

was obtained by dividing the number of

nucleotide differences by the total number of

nucleotides compared.

RESULTS AND DISCUSSION

The length of the region examined varied

between 513 and 514 after cutting off the

primers from both ends prior to alignment.

After the alignment, a total of 517

nucleotides sites were obtained. Among the

24 sequences examined 47 sites or 9.09%

were variable, which resulted in 14

haplotypes (Fig. 1). It also resulted in 44

parsimony informative sites. The total

nucleotide diversity was 0.04245±0.002

while the total haplotype diversity was

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 38

0.931±0.033. This indicates that both

species are genetically diverse. Also the

haplotype and nucleotide diversities for C.

nigrodigitatus were (0.846±0.085,

0.01021±0.002) and the haplotype and

nucleotide diversities for C. walkeri were

(0.873±0.089, 0.00563± 0.0015). The

haplotype and nucleotide diversities were

quite high despite the fact that Chrysichthys

were generally are heavily fished. This

could be due to an exceptionally high initial

population and possible recruitment from

adjacent basins. The estimates of the genetic

parameters are shown in Table 1. The

characteristic of the control region of the

two species as revealed by the nucleotide

composition are shown in Table 2.

Fig 1: Alignments of the variable sites of mtDNA CR sequences from the two fish species.

Dashes indicate indels introduced for optimal alignment. CN1- CN7 represents the haplotypes

from 13 individuals of Chrysichthys nigrodigitatus and CW1- CW7 are representatives

haplotypes of Chrysichthys walkeri.

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 39

Table 1: Estimates of Genetic parameters for mitochondrial DNA Control region of Chrysichthys

nigrodiigitatus and Chrysichthys walker

Table 2: Nucleotide composition of two Chrysichthys species: CN1-7 reperesent base

characteristics of C. nigrodigitatus and CW1-7 for those C. walkeri haplotypes

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 40

There was a bias towards the purines, which

were consistently higher than the

pyrimidines. Thetotal composition of

purines varied between consistently about

47% and 49.2% in C. nigrodigitatus and

about 50% in C. walkeri. The nucleotide

compositions of C. nigrodigitatus and C.

walkeri revealed in this study are similar to

those of the control regions for other

mitochondrial genomes, with a

predominance of purine bases (Chiang et al,

2006). The region was also A + T rich in

both species than the G + C content among

the sequences examined, which was

consistent with previous studies (Brown et

al, 1986; Cheng et al, 2010).

The net mean genetic distance between

Chrysichthys nigrodigitatus and

Chrysichthys walkeri computed using

MEGA 3.1 was 0.067% or 6.7%. This level

has been reported for other teleosts

including Clarias. The mean genetic

distance of 0.898 between the walker and

nigro clade (Fig. 2) implies that this region

of MtDNA is effective in differentiating

between the Chrysichthys species. Within

each clade in the neighbour joining tree (Fig.

2) are minor clades, indicating high diversity

and divergence among individuals within

the two species. For example, the overall

mean genetic distance within Walkeri was

9.1% while it was 16.9% within nigro.

Among some tilapiine species, Wu and

Yang (2012) found within species genetic

distance ranging from 0.00-0.072 and

interspecies range of 0.012-0.210. All of the

sequences were successfully differentiated

to the two species by the phylogenetic tree

with high bootstrap values.

Fig 2. Neighbour joining tree showing genetic differentiation between C. nigrodigitatus and C.

walkeri using the control region. Two clades nigro for C. nigrodigitatus and Walkeri for C.

walkeri can be distinguished with bootstrap value of above 83% (a reliability index)

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 41

CONCLUSION

The major objective of this study was to

characterize and distinguish C.

nigrodigitatus from C. walkeri. Dependence

on morphological differences wereunreliable

in resolving taxonomic and systematic

ambiguities. The mitochondrial DNA has

previously been shown to be a very efficient

molecular marker for identifying fish

species (Nwafili and Gao, 2007; Ardura et

al, 2010, Wu and Yang, 2012). Use of the

control region successfully identified

tilapine species inferred the phylogeography

of the dourada, Brachyplatystoma

rousseauxii (Castelnau) (Batista & Alves-

Gomes, 2006), and also the panmixia of the

piramutaba, Brachyplatystoma vaillantii

(Valenciennes) (Formiga-Aquino, 2004),

and the tambaqui, Colossoma macropomum

(Cuvier) (Santos et al, 2007).

The mtDNA control region has shown

strong phylogenetic signals and

discriminating power as a genetic marker for

identification of Chrysichthys. From Fig. 2,

two clearly different clades were

distinguished. However, regions such as cyt

b and CO1 have been suggested as more

useful for tracing long evolutionary

relationships. We therefore recommend that

these more conserved regions of the mtDNA

be used to elucidate the phylogenetic

relationship among the species of

Chrysichthys in Nigerian waters. In this

regard CO1 is now internationally used in

barcoding of fish organisms and could be

most useful for Chrysichthys identification.

Sampling should cover major distribution

areas throughout the country for the results

to be more realistic.

REFERENCE

Ardura, A., Linde, A.R., Moreira, J.C., and

Garcia-Vazquez, E., 2010,DNA

barcoding for conservation and

management of Amazonian

commercial fish: Biological

Conservation, v. 143(6), p. 1438-

1443.

Aurelle, D., and Berrebi, P., 2001, Genetic

structure of brown trout (Salmo

trutta, L.) populations from south-

western France: data from

mitochondrial control region

variability: Molecular Ecology, v.

10(6), p. 1551–1561.

Batista, J.S., and Alves-gomes, J.A., 2006,

Phylogeography of

Brachyplatystoma rousseauxii

(Siluriformes: Pimelodidae) in the

Amazon Basin offer preliminary

Amazon migratory catfish: Genetics

and Molecular Research, V. 5(4), p.

723-740.

Birky, C.W Jr., Fuerst P., and Maruyama T.,

1989,Organelle gene diversity under

migration, mutation, and drift:

equilibrium expectations, approach

to equilibrium, effects of

heteroplasmic cells, and comparison

to nuclear genes: Genetics, V. 121, p.

613–627.

Brown, W.M., George, J.R. M., and Wilson,

A.C., 1979. Rapid evolution of

animal mitochondrial DNA:

Proceedings National Academy of

Sciences, USA, V. 7(4), p. 1967-

1971.

Brown, G.G., Gadaleta, G., Pepe, G., and

Saccone, C., 1986, Structural

conservation and variation in the D-

loop containing region of vertebrate

mitochondrial DNA: Journal of

Molecular Biology, v. 192, p. 503-

511.

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 42

Cheng, Y., Xu, T., Shi, G., and Wang, R.,

2010, Complete mitochondrial

genome of the miiuy croaker

Miichthys miiuy (Perciformes,

Sciaenidae) with phylogenetic

consideration: Marine Genomics, v.

3, p. 201-209.

Chiang, H.C., Hsu, C.C., Lin, H.D., Ma,

G.C., Chiang, T.Y., and Yang, H.Y.,

2005, Population structure of bigeye

tuna (Thunnus obesus) in South

China Sea, Phillipine Sea and

Western Pacific Ocean inferred from

mitochondrial DNA: Fisheries

Research, v. 79, p. 219-225.

Excoffier, G., Laval, G., and Schneider, S.,

2005, Arlequin Version 3.0: An

integrated software package for

population genetics data

analysis:Evolutionary Bioinformatics

Online, v.1, p. 4750.

Formiga-Aquino, K., 2004, Variabilidade

genética da piramutaba

Brachyplatystoma vaillantii

(Valenciennes, 1840) (Siluriformes:

Pimelodidae) no sistema Estuário-

Amazonas-Solimões: Master Thesis,

National Institute for Amazonian

Research, Manaus, Brasil, 77p.

Hoelzel, A.R., M. Dahlheim and Stern,

1998, Low genetic variation among

killer whales (Orcinus orca) in the

eastern north Pacific and genetic

differentiation between foraging

specialists: Journal of Heredity, v.

89(2), p. 121–128.

Holden, M., and Reed, W., 1991, West

African freshwater fish.Longman

publishers LTD Singapore. 68p.

IUCN, 2012.IUCN Red List of Threatened

Species.Version 2012.1.IUCN

2012.IUCN Red List of Threatened

Species.

Nesbo, C.L., Arab, M.O., and Jakobsen,

K.S., 1998, Heteroplasmy, length

and sequence variation in the

mtDNA control regions of three

percid fish species (Perca fluviatilis,

Acerina cernua, Stizostedion

lucioperca): Genetics, v. 148(4), p.

1907–1919.

Nwafili, S.A., and Gao, T.X., 2007, Is the

Dutch domesticated Clarias

gariepinus a hybrid?: African

Journal of Biotechnology V.6(8), p.

1072-1076.

Santos, M.C.F., Ruffino, M.L., and Farias,

I.P., 2007, High levels of genetic

variability and panmixia of the

tambaqui Colossoma macropomum

(Cuvier, 1816) in the main channel

of Amazon River: Journal of Fish

Biology, V. 71(Suppl. A), p.33-44.

Vin˜ as, J., and Tudela S., 2009, A Validated

Methodology for Genetic

Identification of Tuna Species

(Genus Thunnus): PLoS ONE 4(10):

e7606.doi:10.1371/journal.pone.000

7606.

Watanabe, K., and Nishida, M., 2003,

Genetic population structure of

Japanese bagrid catfishes:

Ichtyological Research 50:140-148.

Whitehead, A., Aderson, S.L., Kuivila,

K.M., Roach, J.L., and May, B.,

2003, Genetic variation among

interconnected populations of

catostomus occidentalis:

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 43

implications for distinguishing impacts of

contaminants from biogeographic

structuring: Molecular Ecology, v.12,

p.2817-2833.

Wu, L., and Yang, J., 2012, Identification of

captive and wild Tilapia species existing in

Hawaii

by mitochondrial DNA control region

sequence PLoSONE,v. 7(12),p.51731.

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 44

MARINE SCIENCES

SHORT COMMUNICATION

Preliminary observation on Baseodiscus hemprichii (ribbon worm)

Faiz Muhammad1,

and Muhammad Shafi 2

1Centre of Excellence in Marine Biology, University of Karachi, Pakistan

2Lasbela University of Agriculture, Water and Marine Sciences, Uthal, Pakistan

ABSTRACT

Baseodiscus hemprichii were sampled from tidal rock pools; (5" in depth) from Buleji.

Observations were made on abundance and morphology of these two nemerteans. Length

measured as 45 cm on death. The relative abundance of Baseodiscus hemprichiiis 0.5%.

Keywords: Baseodiscus hemprichii, Buleji, nemerteans

Correspondence: Faiz Muhammad

Address:Centre of Excellence in Marine Biology University of Karachi, Pakistan

Email: balouch_23@yahoo.com

Phone: +92-219261551 Fax: +92-21261398

Received: 5 May 2013 Revised: 8 Jun 2013 Accepted: 8 Jun 2013

Copyright: ©2013 Muhammad et al. This is an open-access article distributed under the terms

of the Creative Commons Attribution License, which permits unrestricted use, distribution, and

reproduction in any medium, provided the original author and source are credited

Competing Interests: The authors have declared that no competing interests exist.

______________________________________________________________________________

INTRODUCTION

Nemertean are commonly called as ribbon

worms, and are comprised two classes, 250

genera and 1150 species (Gibson 1995),

these animals are not found in abundance

(Thiel and Kruse 2001) and prey on

polychaetes and crustaceans (McDermott

and Roe, 1985) but some species are

scavengers (Heine et, al. 1991; Thiel 1998)

The size of nemertean varies ranging from

few millimeters to over 30 m such as Lineus

longissium. Scanty information is available

on this group of fauna because of difficulties

in their taxonomy.

In Pakistan insufficient studies were

conducted on Baseodiscus hemprichii.

Someauthors like (Puri1924) reported

Baseodiscus hemprichii while same species

was re-discovered (Kazmi and Gibson

1994). Present study is an attempt to give an

overview to this species.

MATERIALS AND METHODS

Observations were done at Buleji, rocky

shore (24o

51’N and 66o 48’E). At rocky

shore random tide pools were selected,

having mean depth of 5 inches, quadrate was

placed and animals were handpicked with

the help of forceps. Animal were narcotized

and preserved in 5% formalin for further

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 45

studies. Identification was done with key to

marine invertebrate (Wood hole). The

relative abundance was noted.

RESULTS AND DISCUSSION

aseodiscus hemprichii was found in tide

pools ( Fig 1A & B）,the average water

level of tide pool was five inches and it was

lying on the rocks and color of Baseodiscus

hemprichii was milky white ventrally while

single long straight red scarlet was present

dorsally on entire body. The head

hadtransversered mark and the length

measured as 45 cm on death.The relative

abundance of Baseodiscus hemprichiiwas

0.5%. Earlier from Pakistan, Puri (1924) and

Kazmi and Gibson (1994) recorded this

species from the Pakistani coast. However,

moredimensional studies are needed to

reveal the ecological role, its reproduction,

development and molecular identification

Figure 1 A and B showing Baseodiscus hemprichii in tide pool

REFERENCES

Gibson. R, 1995. Nemertean Genera and

species of the world: an annotated

checklist of original names and

description citations, synonyms,

current taxonomic status habitats and

recorded zoogeographic distribution.

J.Nat.Histo. Vol 29,pp 271-561.

Heine, J.N., J.B. McClintock, M.Slattery &

J.Weston, 1991.Energetic

compositin, biomass, and chemical

defense in the common Antarctic

nemertean Parborlaia corrugatus

McIntosh. J.exp.mar.Biol.Ecol.153:

15-25.

McDermott. J.J and Roe,P.,1985.Food,

feeding behavior and feeding

ecology of nemerteans. Am.Zool.25:

113-125.

Puri, I.M. 1924. Nemertine worms from

Karachi. Proceedings of the Lahore

Philosophical society 3:71-72

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 46

Kazmi Q.B and Gibson R. 1994.On the

rediscovery of Baseodiscus

hemprichii (Ehrenberg, 1831)

(Nemertea, Anopla, Baseodiscidae)

from Karachi waters.

Thiel, M., 1998.Nemertines as predators on

tidal flats- High Noon at low tide.

Hydrobiologia 265:241-250.

Thiel.M. and Kruse.I, 2001.Status of the

Nemertea as predators in marine

ecosystems. Hydrobiologia 456:21-

32.

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 47

MICROBIOLOGY

RESEARCH ARTICLE

Identification and phylogenetic analysis of halophilic fungus

isolated from a man-made solar saltern in Thailand

Imran Ali1,2,4

, Sudip K. Rakshit1, Napa Siwarungson

3, Hunsa Punnapayak

4, Pongtharin

Lotrakul4, Sehanat Prasongsuk

4, Ali Akber

1 and Zia Ur Rehman

1,2

1Food Engineering and Bioprocess Technology, School of Environment, Resources and

Development, Asian Institute of Technology, Klong Luang, Pathumthani 12120, Thailand. 2Institute of Biochemistry, University of Balochistan, Quetta, 83700, Pakistan.

3Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330,

Thailand. 4Department of Botany, Faculty of Science, Plant Biomass Utilization Research Unit,

Chulalongkorn University, Bangkok, 10330, Thailand.

ABSTRACT

The halophilic fungus was isolated from one of the man-made solar salterns located in Ban

Laem district of Phetchaburi province in Thailand. Harsh physical conditions were found in

hypersaline habitat. Morphological identification revealed that the fungus was having white

colored colonies, cotton like appearance, septate mycelia, pointed hyphae and medium sized

spores. The fungus was able to grow from 0-20% of NaCl concentration (w/v). The halophilic

fungus was identified as Engyodontium album with 100% similarity. Phylogenetic analysis

expressed the evolution pattern of isolated halophilic fungus.

Keywords:Halophilic fungus, solar saltern, Engyodontium album, Thailand

____________________________________________________________________________

Correspondence: Imran Ali

Address: Food Engineering and Bioprocess Technology, School of Environment, Resources

and Development, Asian Institute of Technology, Klong Luang, Pathumthani 12120, Thailand.

Email: imranalisheik@gmail.com

Phone: +66-2692-0583 Fax: +66-2524-6200

Received: 18 April 2013 Revised: 16 August 2013 Accepted: 20 August 2013

Copyright: ©2013 Ali et al. This is an open-access article distributed under the terms of the

Creative Commons Attribution License, which permits unrestricted use, distribution, and

reproduction in any medium, provided the original author and source are credited

Competing Interests: The authors have declared that no competing interests exist.

____________________________________________________________________________

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 48

INTRODUCTION

Extremophiles requiring salt concentrations

for their growth and reproduction are termed

as halophiles (Ali et al, 2012; Madigan et al,

1997; Reed, 1986). Larsen. (1962) classified

halophilic microorganisms into four classes

on the basis of their saline habitats: 1) Non

halophilic microorganism. This group

represents those microorganisms which can

survive and grow in salt concentrations

lower than 2% NaCl (w/v); 2) Slight

halophilic microorganisms: which can

survive and reproduce in NaCl concentration

of 2-5% (w/v); 3) Moderate halophilic

microorganisms: which can grow, survive

and reproduce in NaCl concentration of 5-

20% (w/v) and; 4) Extreme halophilic

microorganisms: which can grow, survive

and reproduce in NaCl concentration of 20-

30% (w/v).

Fungi that are halophilic microorganisms

have only been found in the last decade

(Gunde-Cimerman et al., 2000). According

to Gunde-Cimerman et al., (2009), the fungi

which can be isolated frequently across the

globe from natural hypersaline habitats

having upto 3 M concentration of sodium

chloride, can be categorized as halophiles.

Sporadic fungi which are capable of

tolerating in vitro concentration of 3 M

sodium chloride are termed as halotolerants

(Al-Abri, 2011). However, there is a

difference between halophilic fungi and

obligate halophilic fungi, as the later one

cannot grow and survive in NaCl free

medium.

The mycobiota of halophilic fungi in the

hypersaline environments around the world

comprise of different species of genus

Cladosporium, Alternaria, Scopulariopsis

and Wallemia (Butinar et al, 2005; Gunde-

Cimerman et al, 2000; Zalar et al, 2007) and

random species from the genus Aspergillus

and Penicillium (Ali et al, 2012; Ali et al,

2013, Cantrell et al, 2006).

Engyodontium album has been known

mostly as marine fungi and it has been

reported to be potential fungi for the

production of proteases (Chellappan et al,

2006; Chellappan et al, 2010). Interestingly

it has also been sampled from the

stratospheric air (Wainwright et al,

2003).However it has never been reported

from the hypersaline environment.

Our aim of this study is to report the

presence of Engyodontium album in

hypersaline environment (man-made solar

saltern) and to check its halotolerance.

METHODOLOGY

Site Description

The soil sample was collected from one of

the man-made solar salterns located at

Phetchaburi province of Thailand in the

month of April. Soil analysis confirmed the

site to be hypersaline habitat with NaCl

concentration of 13.11% and low levels of

total organic carbon, nitrogen and organic

matter were found (Ali et al., 2012)

.

Isolation of halophilic fungus

Fungus was isolated by serial dilution

method on potato dextrose agar (PDA)

having 15% of NaCl. The isolate was

separated from obligate halophilic fungi by

checking its growth in NaCl free medium

(Ali et al. 2012).

Morphological identification of fungus

Morphological study was aided by available

literatures (Barnett and Hunter, 1972;

Carmichael et al, 1980; Ellis, 1971; Ellis,

1976; Klich and Pitt, 1988; Subramanian,

1976). Microscopic studies were performed

by the help of stereomicroscope (Olympus

SZ30) and by fluorescent microscope

(Olympus BX60).

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 49

Molecular identification of halophilic fungus

DNA was isolated from the fungus by using

NucleoSpin® Plant II Kit (Macherey-Nagel,

Germany). Standard protocol for fungal

DNA isolation, provided with the kit was

used. Isolated DNA was sent for obtaining

internal transcribed spacer (ITS) 1-4

sequence to the mycology lab at National

Science and Technology Development

Agency (NSTDA) Pathumthani, Thailand.

Isolate was identified and sequence

similarities were studied by using Basic

Local Alignment Search Tool (BLAST)

from National Centre for Biotechnology

Information (NCBI) website. Phylogenetic

tree was reconstructed by neighbor joining

(NJ) method, using Editseq (DNASTAR

Lasergene), Clustal X version 1.81

(Thompson et al, 1997) and MEGA 4.0.2

(Tamura et al, 2007).

RESULTS AND DISCUSSION

Fungal isolation and morphological study

Morphological observations are summarized

in table 1.

Table1:Morphological observations of

Engyodontium album isolated from a man-

made solar saltern in Thailand

Parameter Result

Lab code SWAF

Colony color White

Colony appearance Cotton like

Spore size Medium

Mycelium Septate

Number of isolates 3

Halotolerance 0-20 % NaCl (w/v)

Colony color of cotton like SWAF (lab

code) was found white in color (Fig 1).

The isolated fungus was able to grow on

PDA supplemented with 15% NaCl (w/v),

which proves that this is halotolerant

fungus.Initial quantity of NaCl provided for

culture isolation was selected on the basis of

soil salinity which was found as 13.11% (Ali

et al, 2012).

Figure1: Colony of SWAF (Engyodontium

album) on PDA supplemented with 15 %

NaCl concentration (w/v).

The halotolerance of the fungus was found

ranging from 0-20 % of NaCl concentration

(w/v). The halotolerance test proved the

isolate to be halophilic fungus as it was

isolated from the hypersaline habitat and

was able to survive over 3 molar

concentration of NaCl. Total 3 numbers of

SWAF were found amongst 43 fungal

isolates, which makes approximately 7% of

its presence in total fungal population.

Microscopic observations revealed the

isolate having septated mycelia, pointed

fruiting hyphae and medium sized spores

(Fig 2).

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 50

Figure 2: Engyodontium album, under

stereoscopic microscope showing:

mycelium, reproductive hyphae and spores.

Molecular identification

SWAF was found to be Engyodontium

album strain NRRL 2312 when ITS 1-4

sequences obtained was compared using

BLAST tool analysis on NCBI. Accession

number was found as JF77960. The

similarity was found as 100% (Table 2).

Table 2: Information of species, strain,

similarity percentage and accession number

of Engyodontium album isolated from a

man-made solar saltern in Thailand.

Information Findings

Species Engyodontium album

Strain strain NRRL 2312

Similarity 100%

Accession number JF77960

Phylogenetic analysis

The phylogenetic tree for Engyodontium

album JF77960 is presented in figure 3.Tree

shows that Engyodontium album

DFFSCS022 is found to be the most closely

related species, while Engyodontium album

UTHSCSA and Engyodontium sp FSU9303

were found to be earlier evolved than

Engyodontium album JF77960.

Figure 3.Phylogenetic tree: The genus and

species are followed by their accession

number. Isolated fungus in the tree is given

in bold.

Engyodontium species and Lecanicillium

lecanii V56 were found to have a common

node at boot strap value of 96%.

Simplicillium wallacei CBS 101237 and

Cordyceps pseudomilitaris NBRC 101410

appeared to be far relatives of Engyodontium

album JF77960 in the phylogenetic tree.

CONCLUSION

This study reports the presence of

Engyodontium album from the hypersaline

habitat which is a man-made solar saltern

located in Phetchaburi province, Thailand.

To the best of our knowledge this is the first

time any strain of Engyodontium album

being reported as halophilic fungus. Our

findings will increase interests of scientific

community in Engyodontium album. In

depth, study of this strain will provide more

information about adaptations and responses

of Engyodontium album to its saline

environment. This halophilic fungus also

holds potential to be the sources of

important biological compounds and are

being studied further.

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 51

ACKNOWLEDGEMENT

We will like to thanks all lab staff of King

Mongkut’s University of Technology

Thonburi for their technical support and

guidance throughout this work.

REFERENCES

Al-Abri, S.K. 2011,Use of molecular

approaches to study the occurrence

of extremophiles and extremodures

in non-extreme environments:

Doctoral Thesis, University of

Sheffield, England, Sheffield:

University of Sheffield.

Ali, I. Kanhayuwa, L. Rachdawong, S.

Rakshit, S.K. 2012, Identification,

phylogenetic analysis and

characterization of obligate

halophilic fungi isolated from a man-

made solar saltern in Phetchaburi

province, Thailand: Annals of

Microbiology, doi: 10.1007/s13213-

012-0540-6

Ali, I. Siwarungson, N. Punnapayak, H.

Lotrakul, P. Prasongsuk, S.

Bankeeree, W. Rakshit, S.K.

2013,Screening of potential

biotechnological applications from

obligate halophilic fungi, isolated

from a man-made solar saltern

located in Phetchaburi province,

Thailand (in press) Pak. J. Bot.

Barnett, H.L. Hunter, B.B. 1972, Illustrated

Genera of Imperfect Fungi, 3rd edn:

Burgess Publishing Company.

Butinar, L. Zalar, P. Frisvad, J.C. Gunde-

Cimerman, N. 2005, The genus

Eurotium – members of indigenous

fungal community in hypersaline

waters of salterns: FEMS

Microbiology Ecology, v.51, p. 155–

166.

Cantrell, S.A. Casillas-Martinez, L. Molina,

M. 2006, Characterization of fungi

from hypersaline environments of

solar salterns using morphological

and molecular techniques:

Mycological research v.110, p. 962–

970.

Carmichael, J.W. Kendrick, W.B. Conners,

I.L. Sigler, L. 1980,Genera of

Hyphomycetes: University of

Alberta Press, Alberta, Canada.

Chellappan, S. Jasmin, C. Basheer, S.M.

Kishore, A. Elyas, K.K. Bhat, S.G.

Chandrasekaran, M. 2010,

Characterization of an extracellular

alkaline serine protease from marine

Engyodontium album BTMFS10:

Journal ofIndian Microbiology and

Biotechnology,v. 38, p. 743–752.

Chellappan, S. Jasmin, C. Basheer, S.M.

Elyas, K.K. Bhat, S.

Chandrasekaran, M. 2006,

Production, purification and partial

characterization of a novel protease

from marine Engyodontium album

BTMFS10 under solid state

fermentation: Process Biochemistry,

v. 41, p. 956–961.

Ellis, M.B. 1971,Dematiaceous

Hyphomycetes: Commonwealth

Mycological Institute, Kew England.

Ellis, M.B. 1976, More Dematiaceous

hyphomycetes: Commonwealth,

Mycological Institute, Kew,

England.

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 52

Klich, M.A. Pitt, J.I. 1988,A laboratory

guide to the common Aspergillus

species and their teleomorphs:

Commonwealth Scientific and

Industrial Research Organization,

Division of Food Processing in

North Ryde, N.S.W.

Larsen, H. 1962, Halophilism. Quoted in

R.H. Vreeland and L.I. Hochstein

(eds.). (1993). The Biology of

Halophilic Bacteria (p. 87). Boca

Raton: CRC Press

Madigan, M.T. Martinko, J.M. Parker, J.

1997,Brock: Biology of

Microorganisms: 8th ed. Prentice-

Hall, Inc., Upper Saddle River, New

Jersey.

Subramanian, C.V. 1976, Hyphomycetes:

Indian Council of Agricultural

Research, New Delhi.

Tamura, K. Dudley, J. Nei, M. Kumar, S.

2007, MEGA4: Molecular

Evolutionary Genetics Analysis

(MEGA) software version 4.0:

Molecular Biology and Evolution, v.

24, p. 1596-1599.

Thompson, J.D. Gibson, T.J. Plewniak, F.

Jeanmougin, F. Higgins, D.G. 1997,

The Clustal X windows interface:

flexible strategies for multiple

sequence alignment aided by quality

analysis tools: Nucleic Acids

Research, v. 24, p. 4876–4882.

Reed, R.H. 1986, Halotolerant and

halophilic microbes. In: Herbert RA,

Codd GA (eds), Microbes in

Extreme Environments. Academic

Press Inc, London, pp. 55-82.

Gunde-Cimerman, N. Ramos, J. Plemenitaš,

A. 2009, Halotolerant and halophilic

fungi: Mycological Research, v. 113,

p. 1231–1241.

Gunde-Cimerman, N. Zalar, P. de Hoog, S.

Plemenitaš, A. 2000, FEMS

Microbiol Ecology, v. 32, p. 235–

240.

Wainwright, M. Wickramasinghe, N.C.

Narlikar, J.V. Rajaratnam, P. 2003,

Microorganisms cultured from

stratospheric air samples obtained at

41 km: FEMS Microbiology Letters,

v. 218, p.161–165.

Zalar, P. de Hoog, G.S. Schroers, H-J.

Frank, J.M. Gunde-Cimerman, N.

2005, Taxonomy and phylogeny of

the xerophilic genus Wallemia

(Wallemiomycetes and Wallemiales,

cl. et ord. nov.): Antonie van

Leeuwenhoek, v. 87, p. 311–328.

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

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VETERINARY SCIENCES

RESEARCH ARTICLE

In vitro antibacterial activity of Sorghum halepense

Rooh-ul-Amin1, Muhammad Adil

2, Kashif Hayat

1, Arbab Sikandar

2, Farmanullah

3, Saeed

Khan4

and Hazrat Nabi4

1Department of Botany, Abdul Wali Khan University, Mardan, Pakistan

2Department of Basic sciences, College of Veterinary & Animal sciences, Jhang,

Pakistan 3Lasbela University of Agriculture, Water and Marine Sciences, Uthal, Pakistan

4Department of Livestock & Dairy Development, Khyber Pakhtunkhwa, Pakistan

ABSTRACT

Agar well diffusion assay was executed to evaluate the in vitro antibacterial activity of

chloroform, ethyl acetate and n-hexane extracts of Sorghum halepense against three

bacterial species i.e., Bacillus subtilis (ATCC-6633), Escherichia coli (ATCC-25922)

and Staphylococcus aureus (ATCC-25923). Results of the current study revealed that

chloroform, ethyl acetate and n-hexane extracts exhibited notable antibacterial action

against the tested gram positive bacteria, i.e., Bacillus subtilis and Staphylococcus

aureus. Whereas the sensitivity of gram negative bacteria (Escherichia coli) to

antibacterial action of plant extracts was considerably trivial. Moreover, chloroform

extract was superior to ethyl acetate and n-hexane extracts in terms of antibacterial

activity. By taking the overall results it was concluded that Sorghum halepense is a

potential source of natural antibacterial constituents. Additional research should be

undertaken to elaborate the mechanism of action and antimicrobial spectrum of Sorghum

halepense. Furthermore, screening of plant extracts for antibacterial efficacy against

resistant strains of Staphylococcus aureus, for instance methicillin-resistant

Staphylococcus aureus (MRSA) is also recommended.

Keywords:Sorghum hlepense, antibacterial, Escherichia coli, Staphylococcus aureus

_____________________________________________________________________ Correspondence: Muhammad Adil Address: Department of Basic sciences, College of Veterinary & Animal sciences, Jhang,

Pakistan Email: muhammad.adil@uvas.edu.pk Phone: +92-345-9358013 Received: 15 Jun 2013 Revised: 03 July 2013 Accepted: 10 July 2013 Copyright: ©2013 Adil et al. This is an open-access article distributed under the terms of the

Creative Commons Attribution License, which permits unrestricted use, distribution, and

reproduction in any medium, provided the original author and source are credited Competing Interests: The authors have declared that no competing interests exist. ________________________________________________________________________

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

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INTRODUCTION

Plants constitute the oldest and

Indispensablesource for the derivation

of vast number of drugs thus providing the

remedy for various human and animal

diseases.Severalforms of complementary

medicine practicedin developing countries

exclusively rely on the use of herbal

preparations to treat ailments. Growing

progress in the fields of medicinal

chemistry, pharmacognosy and ethno-

pharmacology has further extended the

scope and utility of herbal

medicines.Likewise the optimal application

of various drug discovery methods (such as

bio-prospecting and random screening) has

enabled scientists to exploit the medicinal

potential of more and more plant-

derivedsubstances. The antimicrobial

potential of medicinal plants has been

globally acknowledged (Valero and

Salmeron, 2003).

It has been reported that certain bacterial

isolates have acquired the capabilityfor

developing antibiotic resistance (Fluit et al.,

2000; Sahm et al., 2001; Schwaber et al.,

2006)that is regarded as a global challenge

for medical science. This emergent

antibiotic resistance is attributable to

indiscriminate use of antibiotics for treating

human and animal diseases. The problem is

further exacerbated by the inability of new

antibiotics to attack bacteria in disparate

ways therebyevading the resistant genes.

Therefore, finding antimicrobial agents

effective against resistant bacteria would be

an advantage.

Bacillus subtilis (B. subtilis), Escherichia

coli (E. coli)andStaphylococcus aureus (S.

aureus) have been implicated in causing

hospital-acquired infections (Fluit et al.,

2000; Karlowsky et al., 2004; Huang et al.,

2006). Regardless of being considered as

the normal inhabitants of humans, animals

and environment, these organisms manifest

an incredible tendency of acting as

opportunistic pathogens.

Sorghumhalepense(S. halepense) is a

globallydistributed perennial weed (Huang

et al., 2010; Loddo et al., 2012). Its

nutritional value is equivalent to that of

Sudangrassandalfalfa(Bennert,

1973).Conversely this plant can sometimes

instigate cyanide toxicity in livestock owing

to the presence of cyanogenic glycosides

(Findlay, 1975; Looker, 1981). The

allelopathic potential of S. halepense has

been well-documented due to the presence

of numerous phenolics and flavonoids in its

different parts (Huang et al., 2010; Liu et

al., 2011).S. halepense also possesses

ethnomedicinal value and is being used as

demulcent and diuretic (Naw Bahaar and

Bhat, 2012). This study was accomplished

to investigate the in vitro antibacterial

activity of chloroform, ethyl acetate and n-

hexaneextracts of S. halepense using agar

well diffusion method.

MTERIALS AND METHODS

Collection of plant and extraction

The plant material, specimen (Voucher

specimen # AWK/BOT/S.H/02) was

collected randomly from two areas i.e.,

Parmoli and Shewa Adda (district, Swabi,

Khyber Pakhtunkhwa, Pakistan). The

research work of this project was carried

out in Botany Laboratory, Department of

Botany, Abdul Wali Khan University,

Mardan, Pakistan. The plant material was

air dried by keeping it at room temperature

for three weeks in the dark conditions. The

dried plant material was milled to obtain a

fine powder. Two liters of methanol

solution was taken in an air tight glass jar

for soaking two hundred grams of the

minced plant material for 7 days at room

temperature. Filtration was performed

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

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using muslin cloth and filter papers to

extract the soaked material. Later on the

solvent was removed in a rotary evaporator

to yield crude methanolic extract that was

stored at 35°C. Next, the crude methanolic

extract was subjected to cold extraction

(also referred to as maceration) to attain

different fractions (Irshad et al., 2012).

Maceration was carried out using

chloroform, ethyl acetate and n-hexane.

For the preparation of each fraction, 150

ml of pertinent solvent was used to

dissolve 50 grams of crude methanolic

extract using a magnetic stirrer. After that,

the extracts were filtered and then

preserved for further processing.

Test micro-organisms

The pure cultures of B. subtilis (ATCC-

6633),E. coli(ATCC-25922) and S.

aureus(ATCC-25923)were collected from

Phytomedicine and Organic Biochemistry

Laboratory, Department of Chemistry,

University of Peshawar (Pakistan).

Determination of antibacterial activity

Agar well diffusion method (Bauer et al.,

1966) was employed to determine the

antibacterial activity associated with various

extracts of S. halepense. Mueller-Hinton

Agar medium was inoculated with tested

bacterial strains and six wells were made in

every plate using a sterile corkborer having

a diameter of 0.6 mm. Sterile micropipette

tips were used for loading150 μl of given

samples in their relevant wells.Same

amounts of dimethyl sulphoxide (DMSO)

were also loaded in their corresponding

wells designated as negative control wells.

Streptomycin-impregnated discs(10 μg/disc)

were placed in the centers of the

lawnsaspositive control.The culture plates

were incubated in bacterial incubators for

24 hours at 37 0C. Next day the results were

examined in terms of zone of inhibition

(measured in mm).

Statistical analysis

Data regarding the zone of inhibition were

analyzed through analysis of variance

(ANOVA) using completely randomized

block design. However the results of

negative control wells were not taken into

consideration, to elude the incompatibility

of data with mentioned statistical tool.

Significant difference between extracts and

positive control were further subjected to

Duncan’s multiple range test (Duncan,

1955), taking the level of significance at

0.01.

RESULTS

S. halepense was extracted with methanol

and then fractioned with n-hexane, ethyl

acetate and chloroform. Later on,these

fractions were screened for antibacterial

activity against gram-negative and gram-

positive bacteria. Data regarding the

antibacterial activity of various extracts of

S. halepensehas been presented in table-1.

It is obvious that all tested plant extracts

were capable to inhibit the growth of target

bacteria. S. aureus exhibited highest

susceptibility to antibacterial action of

chloroform extract followed byB.

subtilisandE. Colirespectively.

Table 1: In vitro antibacterial activity of

different extracts of S. halepense.

Bacterial

species

Zone of inhibition (mm)

Chloroform extract

Ethyl

acetate

extract

n-

hexane

extract

Streptomycin

S. aureus 14 12 10 21

E. coli 9 7 3 29

B. subtilis 12 10 8 24

Analogous pattern of bacterial sensitivity

was manifested against ethyl acetate and n-

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 56

hexane extracts. Significantly higher zones

of inhibition were observed in case of

positive control wells whereas negative

control wells did not demonstrate detectable

zones of inhibition.

Table 2: ANOVA-based comparison of

antibacterial activity of plant extracts and

control

Experimental

unit

Calculated

F-value

F-

table

value

Conclusion

Bacterial

species 0.42857 10.92 Non-

significant Plant

extracts 15.08 9.78 Significant

Both gram positive bacterial species (B.

subtilis and S. aureus)demonstrated a

comparable degree of growth inhibition in

response to the application of plant extracts.

Nevertheless gram negative bacteria (E.

coli) exhibited relatively less susceptibility

to the antibacterial activity of administered

plant extracts. Comparison of the bacterial

sensitivity to plant extracts was carried out

using analysis of variance (ANOVA). The

type of targeted bacteria did not induce

substantial modification in bacterial

sensitivityto respective plant extracts (as

signified in table: 2).

Table 3: Comparisons among the treatment

means

Plant extracts Mean values n-hexane extract 7ab

Ethyl acetate extract 9.66bc

Chloroform extract 11.66ac

Streptomycin 24.66

According to DMRT,values followed by

similar subscript are not significantly

different.

Remarkable discrepancy was evident in the

antibacterial activity of various plant

extracts. This phenomenal variation in the

antibacterial activity of different extracts

could be attributed to substantial polar

diversity of individual solvents thus

governing the composition and efficacy of

resultant extracts. Apparently chloroform

extract was found to yield highest

antibacterial activity against the tested

bacteria followed by ethyl acetate and n-

hexane extracts respectively (as illustrated

in table: 1). Therefore comparisons among

the treatment means were executed using

Duncan’s multiple range test (Duncan,

1955). However the comparison of

antibacterial potential among various

extracts reflected the lack of statistical

significance. Conversely, a statistically

significant variation was observed by

comparing the antibacterial activity of each

distinct plant extract with positive control

(as indicated in table: 3).

DISCUSSION

Results of the current study provided

evidence regarding theantibacterial activity

of chloroform, ethyl acetate and n-hexane

extracts of S. halepenseagainst the tested

bacteria. Our results reinforce the findings

of Nicollieret al., 1983, who reported that

methanolic extract of S. halepense inhibited

the growth of several bacteria. Besides,

many studies have reflected the

antimicrobial activity of S. halepense

against different pathogens (Bahraminejadet

al., 2011; Yanar et al.,2011 and Javaidet al.,

2012). The shoot portion of this plant

consists of many active phytotoxic

substances including ethyl p-

hydroxybenzoate and p-

hydroxybenzaldehyde which could be

expected to exert the consequent

antimicrobial activity (Javaidet al.,

2012).Appraisal of the biosynthesis of plant

phenolics stipulates that p-

hydroxybenzaldehyde is formed from

salicylic acid (Dicko et al., 2006) which is

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 57

known for having optimal antibacterial

activity (El-mougy, 2002). Furthermore, the

leaf and rhizome extracts of S. halepense

have been documented to contain many

flavonoids and phenolic compounds such as

dhurrin, taxiphyllin, sorgoleone, prunasin,

proanthocyanidins, p-coumaric acidand

chlorogenic acid (Nicollier et al., 1983;

Czarnota et al., 2003). Another probable

mechanism for the antimicrobial activity of

phenolic compounds could be the chelation

of iron with subsequent disruption of

microbial oxidative metabolic system (O,

Connell and Fox, 2001).

Crude phenolic, saponin and methanolic

extracts of Sorghum have been recorded to

exert inhibitory action on the growth of L.

monocytogenes, B. cereus, S. aureus and E.

coli (Khadambi, 2005; Soetan et al., 2006;

Mohamed et al., 2009). Several studies have

evinced the effectiveness of sorghum

proanthocyanidins against influenza virus,

herpes simplex virus (Lu et al., 2004;

Hamauzu et al., 2005) and human

melanoma (Gomez-Cordoves et al.,2001).

Proanthocyanidins exhibit their

antimicrobial action through the inhibition

of hydrolytic enzymes and inactivation of

microbial adhesions (Cowan, 1999).

The differential sensitivity of microbial

strains to administered extracts can be

elucidated on the basis of peculiarity in the

structure of bacterial cell wall. Gram

negative bacteria tend to be relatively

resistant to antibacterial substances by

virtue of outer membrane in their cell wall

and surrounding glycocalyx. Outer

membrane imparts hydrophilic nature to the

cell wall and acts as a strict permeability

barrier(Smith-Palmer et al., 1998). Another

integral element of gram negative cell wall

is the periplasmic space that contains

essential enzymes meant for the

neutralization of foreign substances (Duffy

and Power, 2001).

By taking the overall results it was

concluded thatS. halepenseis a potential

source of natural antibacterial constituents.

Nevertheless additional research should be

undertaken to elaborate the mechanism of

action and antimicrobial spectrum ofS.

halepense. Furthermore, screening of plant

extracts for antibacterial efficacy against

resistant strains of S. aureus, for example

methicillin-resistant S. aureus (MRSA) is

also recommended.

REFRENCES

Bahraminejad, S., Abbasi, S., and Fazlali,

M., 2011, In vitro antifungal activity

of 63 Iranian plant species against

three different plant pathogenic fungi,

African Journal of Biotechnology, v.

10, p. 16193-16201.

Bauer, A. W., Kirby, W. M., Sherris, J. C.,

and Turk, M., 1966,Antibiotic

susceptibility testing by a standardized

single disk method, American Journal

of Clinical Pathology, v. 4, p. 493-

496.

Bennett, H. W., 1973, Johnsongrass,

carpetgrass and other grasses for the

humid south, in Heath, M. E.,

Metcalfe, D. S., and Barnes, R. F.,

editors, Forages, Iowa State

University Press, Ames, Iowa, p. 286-

293.

Cowan, M. M., 1999, Plants products as

antimicrobial agents. Clinical

Microbiology Reviews, v. 12, p. 564-

582.

Czarnota, M. A., Rimando, A. M., and

Weston, L. A., 2003, Evaluation of

root exudates of seven sorghum

accessions,Journal of

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 58

ChemicalEcology, v. 29, p. 2073–

2083.

Dicko, M. H., Gruppen, H., Traoré, A. S.,

Voragen, A. G. J., and Van Berkel,

W. J. H., 2006, Phenolic compounds

and related enzymes as determinants

of sorghum for food use,

Biotechnology and Molecular Biology

Review, v.1, p. 21-38.

Duffy, C. F., and Powell, R. F., 2001,

Antioxidant and antimicrobial

properties of some Chinese plant

extracts, International Journal of

Antimicrobial Agents, v. 17, p. 527-

529.

Duncan, D. B., 1955, Multiple rangeand

multiple F test, Biometrics, v. 11, p.

1–24.

El-Mougy, N. S., 2002, In vitro studies on

antimicrobial activity of salicylic acid

and acetylsalicylic acid as pesticidal

alternatives against some soilborne

plant pathogens, Egyptian Journal of

Phytopathology, v. 30, p. 41-55.

Findlay, R. M., 1975, Potential menace of

Johnsongrass, New Zealand Journal of

Agriculture,v. 130, p. 40-41.

Fluit, A. C., Jones, M. E., Schmitz, F. J.,

Acar, J. G., and Verhoef, J., 2000,

Antimicrobial susceptibility and

frequency of occurrence of clinical

blood isolates in Europe from the

SENTRY antimicrobial surveillance

program, 1997-1998, Clinical

Infectious Diseases, v. 30, p. 454-60.

Gomez-Cordoves C., Bartolome B., Vieira

W., and Virador V. M., 2001, Effects

of wine phenolics and sorghum

tannins on tyrosinase activity and

growth of melanoma cells, Journal of

Agriculture and Food Chemistry, v.

49, p.1620-1624.

Hamauzu, Y., Yasui, H., Inno, T., Kume,

C., and Omanyuda, M., 2005,

Phenolic profile, antioxidant property,

and anti-influenza viral activity of

chinese quince (Pseudocydonia

sinensis Schneid.), quince (Cydonia

oblonga Mill.), and apple (Malus

domestica Mill.) fruits, Journal of

Agriculture and Food Chemistry, v.

53, p. 928-934.

Huang, Y. C., Chou, Y. H., Su, L. H., Lien,

R. I., Lin, and T. Y., 2006,

Methicillin-Resistant Staphylococcus

aureuscolonization and its association

with infection among infants

hospitalized in neonatal intensive care

units, Pediatrics, v. 118, p. 469-474.

Huang, H., Liu, Y., Meng, Q., Cui S. W.

H., and Zhang, C., 2010,

Flavonolignans and other phenolic

compounds from Sorghum halepense

(L.) Pers,Biochemical Systematics and

Ecology, v.38,p. 656-658.

Irshad, S., Mahmood, M., and Perveen, F.,

2012, In-vitro anti-bacterial activities

of three medicinal plants using agar

well diffusion method, Research

Journal of Biology, v. 2, p. 1-8.

Javaid, A., Naqvi, S. F., and Shoaib, A.,

2012, Antifungal activity of

methanolic extracts of Sorghum

halepense against Macrophomina

phaseolina, Journal of Medicinal

Plants Research, v. 6, p.4102-4106

Karlowsky, J. A., Jones, M. E., Draghi, D.

C., Thornsberry, C., Sahm, D. F., and

Volturo, G. A., 2004, Prevalence of

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 59

antimicrobial susceptibilities of

bacteria isolated from blood cultures

of hospitalized patients in the United

States in 2002,Annals of Clinical

Microbiology and Antimicrobials, v.

3, p. 7.

Khadambi, T. N., 2005, Antimicrobial

properties of phenolic compounds

from sorghum, M. Sc. (Agric.), thesis:

University of Pretoria, Pretoria, p. 46-

65.

Liu, Y., Zhang, C., Wei, S., Cui, H., and

Huang, H., 2011,Compounds from the

subterranean part of Johnsongrass and

their allelopathic potential, Weed

Biology and Management, v. 11, p.

160-166.

Loddo, D., Masin, R., Otto, S., and Zanin,

G., 2012,Estimation of base

temperature for Sorghum halepense

rhizome sprouting, Weed Research, v.

52, p. 42-49.

Looker, D., l98l, Johnsongrass has an

Achilles heel, New Farm,v. 3,p. 40-

47.

Lu, L., Liu, S., Jiang, S., and Wu, S.,

2004,Tannins inhibit HIV-1 entry by

targeting gp41, Acta Pharmacologica

Sinica, v.25, p. 213-218.

Mohamed, S. K., Ahmed, A. A. A., Yagi,

S. M., and Abd Alla, A. E. W. H.,

2009, Antioxidant and Antibacterial

Activities of Total Polyphenols

Isolated from Pigmented Sorghum

(Sorghum bicolor) Lines, Journal of

Genetic Engineering and

Biotechnology, v. 7, p. 51-58.

Naw Bahaar, S. W., and Bhat, G. A.,

2012,Diversity and distribution of

ethno-medicinal flora in the rice field

agro-ecosystems of Kashmir valley (J

and K) India, Journal of Science, v. 1,

p. 63-71.

Nicollier, G. F., Pope, D. F., and

Thompson, A. C., 1983, Biological

activity of dhurrin and other

compounds from Johnson grass

(Sorghum halepense), Journal of

Agriculture and Food Chemistry, v.

31, p. 744–748.

O, Connell, G. E., and Fox, P. F., 2001,

Significance and applications of

phenolic compounds in the production

and quality of milk and dairy

products: a review, International Dairy

Journal, v. 11, p. 103-120.

Sahm, D. F., Thornsberry, C., Mayfield, D.

C., Jones, M. E., and Karlowsky, J.

A., 2001, Multidrug-resistant urinary

tract isolates of E. coli: prevalence and

patient demographics in the United

States,Antimicrobial Agents and

Chemotherapy, v. 45, p. 1402-1406.

Schwaber, M. J., Navon-Venezia, S.,

Kaye, K. S., Ami, R. B., Schwartz, D.,

and Carmeli, Y., 2006, Clinical and

economic impact of bacteremia with

extended- spectrum-ß-lactamase-

producing

Enterobacteriaceae,Antimicrobial

Agents and Chemotherapy,v. 50, p.

1257-1262.

Smith-Palmar, A., Stewart, J., and Fyfe, L.,

1998, Antimicrobial properties of

plant essential oils and essences

against five important food-borne

pathogens, Letters in Applied

Microbiology, v. 26, p. 118-122.

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 60

Soetan, K. O., Oyekunle, M. A.,

Aiyelaagbe O. O., and Fafunso, M. A.,

2006, Evaluation of the antimicrobial

activity of saponins extract of

Sorghum bicolor L. Moench,African

Journal of Biotechnology, v. 5, p.

2405-2407.

Valero, M., Salmeron, and M. C., 2003,

Antibacterial activity of 11 essential

oilsagainst Bacillus cereus in

tyndallized carrot broth, International

Journal of FoodMicrobiology, v. 85,

p. 73-81.

Yanar, Y., Kadioglu, I. A., Gokce, I.,

Demirates, I., Goren, N., Cam, H., and

Whalon, M., 2011,In vitro antifungal

activities of 26 plant extracts on

mycelial growth of Phytophthora

infestans (Mont.) de Bary, African

Journal of

Biotechnology, v. 10, p. 2625-2629.

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RESEARCH ARTICLE

Antagonistic potential of marine isolate DK6-SH8 against fish

pathogens

Muhammad Naseem Khan1, 2

, Meng Li1, Zulfiqar A Mirani

2, Jingxue Wang

1&Hong Lin

1

1 Food Safety Laboratory, College of Food Science and Engineering, Ocean University of

China, Qingdao, 266003, China

2 Microbiological Analytical Centre, PCSIR Labs. Complex Karachi, 75280, Pakistan

ABSTRACT

Oceanic hunt by marine organism enforces to produce contemporary and novel strategies to

compete, survive and reproduce their population. This fact facilitates to find new solutions to

control pathogenic bacteria. In this study Strain DK6-SH8 was identified via EzTaxon-e server,

and analyzed for its antagonistic potential against fish pathogens by organism-organism

interaction on agar plate. The results revealed that DK6-SH8 was 99.65% pairwise similar to

Vibrio kanaloae. Antagonistic test suggest that DK6-SH8 is active against Vibrio anguillarum,

Vibrio alginolyticus, Vibrio campbellii, Vibrio harveyi, Vibrio tubiashii and Vibrio vulnificus. It

has been concluded that Vibrio kanaloae DK6-SH8 could be potential candidate to control fish

pathogens.

Keywords:Vibrio kanaloae, fish pathogens, EzTaxon-e, antagonistic

______________________________________________________________________________ Correspondence:Jingxue Wang

Address: Food Safety Laboratory, College of Food Science and Engineering, Ocean University of China,

Qingdao, 266003, China

Email: snow@ouc.edu.cn Phone: +86-532-820-32203Fax:+86-532-820-32389

Received: 15 Aug 2013 Revised: 03 Sept 2013 Accepted: 15 Sept 2013

Copyright: ©2013 Wang et al. This is an open-access article distributed under the terms of the Creative

Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any

medium, provided the original author and source are credited

Competing Interests: The authors have declared that no competing interests exist.

______________________________________________________________________________

INTRODUCTION

Marine is the source of food and many

tremendous compounds.Aquatic

environment studies particularly microbial

interactions are the key for new solutions

for pathogenic microbial control. Diversity

and oceanic hunt among microorganism for

food and living space enforces to adapt

unique and contemporary physiological and

structure characteristic, this behavior is not

seen in soil organisms (Radajewski et al,

MARINE SCIENCES

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Online available at www.luawms.edu.pk pg. 62

2002). Additionally, these conditions

resulted in extraordinary unique compounds

production from marine microorganism as

compare to terrestrial microorganisms

(Wagner-Dobler et al., 2002) Many natural

compounds have been obtained from

various microbial sources, and are being

successfully used in different fields. In the

period of past five decades more than one

million compounds were isolated from

marine resources, and about 18% are from

microbial community (Bhatnagar and Kim,

2010).

Rosenfeld and Zobell (1947) first time

revealed that marine bacteria can produce

antimicrobial agents. After that many

studies for isolation and purification and

application of these antibiotics and bacteria

itself were reported. Application of bacteria

as probiotics in aquaculture is also on focus

in many research groups (Prado et al, 2009).

Synthetic and engineered antibiotics and

other chemical treatments are losing

acceptability among consumers. Green

technology to grow food and rearing

animals and aquaculture are gaining interest

over harmful effect of chemical treatments

(Yebra et al, 2004). Marine bacteria with

antibacterial potential could be a safe

andreliable option to control pathogens in

aquaculture.

The major fish pathogens among vibrios are

luminous vibrio, group of vibrio causes

luminous vibriosis in shrimp’s

cultures.These include V. parahaemolyticus,

V. alginolyticus, V. harveyi, V. damsel and

V. vulnificus. Among these V. harveyi has

been considered as very important pathogen

and predominantly involved in deterioration

of shrimp rearing industries and causes

huge economic loss (Leano et al, 1998,

Lightner and Redman, 1998).

Many members of marine Vibrios are

omnipresent in aquatic environment and

exhibit an unusually rapid growth rate,

which makes them predominant in

eutrophic environments (Aiyar et al, 2002,

Macian et al, 2000). To control these

pathogenic vibrios, bacteria which have

similar growth rate in aquatic environment

may have higher success rate. Considering

our previous report on isolation and

antibacterial activity of isolate DK6-SH8

(paper under review), we plan this study to

evaluate the antagonistic activity of isolate

DK6-SH8 against fish pathogens.

Moreover, EzTaxon-e database server was

compiled to identify the strain by

comparing 16S rRNA gene with valid,

identified and published bacterial strains.

The accuracy and validity of results from

ExTaxon-e was also discussed.

MATERIALS AND METHODS

BacterialStrain DK6-SH8 were selected

from 272 isolates (paper under review) on

the basis of initial screening of antibacterial

activity against four pathogenic indicator

strains including Staphylococcus aureus,

Escherichia coli, Listeria monocytogenes

and Vibrio cholrea. Strain DK6-SH8 was

isolated from surface attached marine

invertebrate samples obtained from coast of

Taiping bay in China (N 360 03’ 35.5”, E

120o 18’ 34.4”). Marine fish pathogenic

bacteria were obtained from College of

Marine Life Sciences, Ocean University of

China. All isolates were revived before

experiments from preserved culture in

Marine broth 2216E with 30% glycerol at -

80 oC.

Identification of marine isolates strain by

EzTaxon-e

Strain DK6-SH8 16S rDNA sequence

accession number (KC737551) was fetched

in ExTaxon-e server (http://eztaxon-

e.ezbiocloud.net/) and identify within

cultured database of systematically

identified and verified bacteria. The number

of percent pairwise similarity was obtained

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

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and isolationwas identified on the basis of

highest rank of type bacteria. The

phylogeny tree constructed via Mega 5.1

software by maximum likelihood method

and tree topology were reproduced by

bootstrap analysis of 1000 replicates (Hall,

2013) .

Antagonistic activity test

Antagonistic activity were tested by method

described by Jin et al, (2010) with

modification. Strain DK6-SH8 and putative

pathogenic strains were cultured for 24 h on

marine 2216E broth at 28 oC with 180 rev

min-1

. All test cultures were diluted 100

times with sterile physiologic saline (SPS).

Each putative pathogenic strain was spread

on a marine 2216E plate with sterile cotton

stick. After absorption, sterile oxford cups

were place on each test plate. After that 10

µL of 24 h old broth of strain DK6-SH8

was inoculated in oxford cups. All plates

were incubated for 24 h at 28 oC, and the

diameters of the inhibitory zones were

measured. Antagonistic activity of strain

DK6-SH8 cultured for 12, 24, 36, 48, 60

and 72 h also measured against Vibrio

anguillarum. Colonies were visualized

clearly after spraying aqueous solution of

MTT (2mg/mL) followed by incubation at

30 oC for 15 min.

Statistical calculations

Data were presented as mean of triplicate

experiments and statistical mean and

standard error of mean were analyzed by

statistical software SPSS (v 16.0).

RESULTS

Identification of marine isolates strain by

EzTaxon-e

The strain DK6-SH6 have shown highest

pairwise similarity to many strains of genus

vibrios, and have 99.65% similarity to

Vibrio kanaloae LMG 20539T(Table 1).

The phylogeny analysis also revealed that

strain DK6-SH8 cluster with Vibrio

kanaloae LMG 20539T (Figure 1). On the

basis of EzTaxon-e data base strain was

identified as Vibrio kanaloae DK6-SH8.

Table 1: Identification table of Strain DK6-SH8 by EzTaxon-e Database

Rank Name Strain Pairwise

Similarity (%) Diff/Total nt Accession Authors

1 Vibrio kanaloae LMG 20539(T) 99.65 5/1438 AJ316193 Thompson et al (2003a)

2 Vibrio pomeroyi LMG 20537(T) 98.75 18/1436 AJ491290 Thompson et al (2003a)

3 Vibrio splendidus ATCC33125(T) 98.70 18/1381 X74724 Baumann et al (1980)

4 Vibrio artabrorum Vb 11.8(T) 98.68 19/1441 EF599164 Dieguez et al (2011)

5 Vibrio gigantis CAIM 25(T) 98.54 21/1442 EF094888 Le Roux et al (2005)

6 Vibrio celticus Rd 8.15(T) 98.54 21/1438 EF599162 Beaz-Hidalgo et al (2010)

7 Vibrio atlanticus Vb 11.11(T) 98.34 24/1442 EF599163 Dieguez et al (2011)

8 Vibrio tasmaniensis LMG 21574(T) 98.33 24/1441 AJ514912 Thompson et al (2003b)

9 Vibrio crassostreae CAIM 1405(T) 98.33 24/1441 EF094887 Faury et al (2004)

10 Vibrio cyclitrophicus P-2P44(T) 97.98 29/1434 U57919 Hedlund and Staley (2001)

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

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Fig1: Phylogeny analysis of Marine

isolate.DK6-SH8 via MEGA 5.1 software by

Maximum likelihood method. Numbers at

the nodes indicated the bootstrap values of

1000 resembled data sets. Scale bar 0.001

represents sequence divergence

Antagonistic spectrum

Strain DK6-SH8 have shown good

antimicrobial activity against fish pathogens

including Vibrio anguillarum, Vibrio

alginolyticus, Vibrio campbellii, Vibrio

harveyi, Vibrio tubiashii and Vibrio

vulnificus,while there is no activity against

the strain Vibrio parahaemolyticus (Table

2). Antagonistic activity was observed after

12 h old broth of DK6-SH8 against Vibrio

anguillarum, and increase when more than

24 h old broth suspensions were used

(Figure 2). This shows that antagonistic

activity on plate culture was improved after

24 h of initial broth revival.

Table 2: Antagonistic activity of DK6-SH8 against fish pathogens

Microorganism Source Zone of inhibition in mm ± SEM*

Vibrio anguillarum LMG 4437T; cod, Norway 22 + 0.34

Vibrio alginolyticus LMG 4408T 19 + 0.58

Vibrio campbellii LMG 11216T; USA 13 + 0.34

Vibrio harveyi LMG 7890T; USA (1982) 14 + 0.89

Vibrio parahaemolyticus LMG 2850T; Japan IN

Vibrio tubiashii LMG 10936T; USA 23 + 0.34

Vibrio vulnificus LMG 13545T; USA 20 + 0.58

* Mean of triplicates + Standard Error of Mean IN : invisible

Fig 2: Zone on inhibition by strain DK6-SH8 against Vibrio anguillarum lawn on Marine agar

2216E after (1) 12 h old culture (2) 24 h (3) 36 h (4) 48 h ( 5) 60 h ( 6) 72 h ; ( A) without MTT

spray (B) after 2 mg/ mL MTT spray followed by incubation at 30 oC.

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DISCUSSION

Regardless of current development in

industrially validated identification options,

Identification of bacteria is still a difficult

assignment in a lot of microbiological

routine laboratories, particularly in

conditions where taxonomically new strains

are concerned. Genetic level identification

of strain for general and research purpose

has been widely used with well-curetted

gene database, such as EMBL, NCBI and

ExTaxon. Although, uncultured prokaryotic

species sequences and unpublished gene

sequences may leads to ambiguity and miss

calculation, if user are beginners. These

issued were overcome by new generation

database ExTaxon-e; this segmented and

separated uncultured and unpublished or

non-validated sequence when analyzing 16S

rDNA sequence. The results from this

database shows pairwise global sequence

alignment with basic local alignment search

tool (blast) of formally identified, validated,

up-to-date, nomenclature system. Isolate

DK6-SH8 was identified as Vibrio

kanaloae.Who was previously identified as

Vibrio sp. DK6-SH8 due to massive

comparison with cultured but unverified and

unpublished sequences (paper under

review). EzTaxon-e database seems to be a

very powerful tool for the taxonomic

research in right direction with accuracy.

Additionally, the sequenced strains relevant

information, real-time research updates and

meaningful data resource link for a

particular gene sequence or strains are added

advantages. (Kim et al, 2012).

TheVibrionaceae family, Gram-

negative Gammaproteobacteria omnipresent

in aquatic and salty environments

(Thompson et al, 2004), harbors strains with

antagonistic activity (Gram et al,

2010). Antimicrobials from Vibrio spp. can

decrease the quantity of additional microbial

population members and manipulate

microscale variations in challenging

bacterial populations (Long and Azam,

2001). Antibacterial actions have been

describedfrom V.parahaemolyticus (Radjasa

OK et al, 2007), V. alginolyticus (Austin et

al, 1995), V. anguillarum (Hjelm et al,

2004) ,and several unidentified Vibrio spp.

(Castro et al, 2002, Long et al, 2005). But,

the nature and frequency of antagonism

among vibrios is still mostly mysterious, and

only a little antibiotic Vibrio compounds

have been structure elucidated to date

(Kobayashi et al, 1994, Oclarit et al,

1994).Wietz et al (2010) also reported V.

coralliilyticus and V. neptunius with

antimicrobial compounds. These facts

suggest that, although, the vibrios are mainly

classified in pathogenic bacteria of fish and

human, but it could be a potential source to

control pathogens as well. Antagonistic

activity could be achieved by vibrios by

mean of antimicrobial compounds produced

and by application of vibrios itself as

probiotics in aquaculture.

So far conventional approaches such as use

of disinfectants and antimicrobial drugs to

control diseases have had limited success in

the prevention or cure of aquatic disease.

The massive use of antibiotics encourages

natural emergence of antibiotic resistant

bacteria, which can transfer their resistance

genes to other bacteria that have never been

exposed to the antibiotics (Davison, 1999).

This led to suggestions of suitable

alternative disease prevention methods,

which could be the use of non-pathogenic

bacteria as probiotic biocontrol agents

(Verschuere et al, 2000). From best of our

knowledge, all the previously identified

strains of Vibrio kanaloae were never being

reported as pathogens. The antibacterial

potential of isolate Vibrio kanaloae DK6-

SH8 against fish pathogens suggest that

stain Vibrio kanaloae DK6-SH8 could be a

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 66

potential source for control of pathogenic

strains.

CONCLUSIONS

We have successfully identified our isolate

DK6-SH8 as Vibrio kanaloae, which have

antagonistic activity against fish pathogens.

These results facilitate to investigate further

for purifying it antimicrobial compounds

and application in aquaculture.

ACKNOWLEDGEMENTS

Authors would like to thankful to Ocean

University of China (OUC) and Chinese

Scholarship Council (CSC) for providing

resources to complete this study.

REFRENCES

Aiyar, S. E., Gaal, T. & Gourse, r. L.2002,

rRNA promoter activity in the fast-

growing bacterium Vibrio

natriegens: Journal of Bacteriology,

v. 184, p. 1349-1358.

Austin, b., Stuckey, l. F., Robertson, P. A.

W., Effendi, I. & Griffith, D. R.

W.1995, a probiotic strain of Vibrio

alginolyticus effective in reducing

diseases caused by Aeromonas

salmonicida, Vibrio anguillarum and

Vibrio ordalii: Journal of Fish

Diseases, v. 18, p. 93-96.

Baumann, P., Baumann, l., Bang, S.

&Woolkalis, M.1980,Reevaluation

of the taxonomy ofVibrio, beneckea,

andPhotobacterium: Abolition of the

genusBeneckea: Current

Microbiology, v. 4, p. 127-132.

Beaz-Hidalgo, R., Dieguez, A. L.,

Cleenwerck, I., Balboa, S., Doce, A.,

De Vos, P. &Romalde, J.

L.2010,Vibrio celticus sp nov., a

new Vibrio species belonging to the

Splendidus clade with pathogenic

potential for clams: Systematic and

Applied Microbiology, v. 33, p. 311-

315.

Bhatnagar, I. &Kim, S. K.2010,Immense

Essence of Excellence: Marine

Microbial Bioactive Compounds:

Marine Drugs, v. 8, p. 2673-2701.

Castro, D., Pujalte, M.J., Lopez-Cortes, l.,

Garay, E. &Borrego, J. J.2002,

Vibrios isolated from the cultured

manila clam (Ruditapes

philippinarum): numerical taxonomy

and antibacterial activities: J Appl

Microbiol, v. 93, p. 438-47.

Davison, J.1999, Genetic Exchange between

Bacteria in the Environment:

Plasmid, v. 42, p. 73-91.

Dieguez, A. L., Beaz-hidalgo, R.,

Cleenwerck, I., Balboa, S., De vos,

P. &Romalde, j. L.2011, Vibrio

atlanticus sp nov and Vibrio

artabrorum sp nov., isolated from the

clams Ruditapes philippinarum and

Ruditapes decussatus: International

Journal of Systematic and

Evolutionary Microbiology, v. 61, p.

2406-2411.

Faury, N., Saulnier, D., Thompson, F. L.,

Gay, M., Swings, J. &Le Roux,

F.2004, Vibrio crassostreae sp nov.,

isolated from the haemolymph of

oysters (Crassostrea gigas):

International Journal of Systematic

and Evolutionary Microbiology, v.

54, p. 2137-2140.

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 67

Gram, L., Melchiorsen, J. &Bruhn, J.

B.2010, Antibacterial activity of

marine culturable bacteria collected

from a global sampling of ocean

surface waters and surface swabs of

marine organisms: Mar Biotechnol

(NY), v. 12, p. 439-51.

Hall, B. G.2013, Building phylogenetic trees

from molecular data with MEGA:

Mol Biol Evol, v. 30, p. 1229-35.

Hedlund, B. P. &Staley, J. T.2001, Vibrio

cyclotrophicus sp nov., a polycyclic

aromatic hydrocarbon (PAH)-

degrading marine bacterium:

International Journal of Systematic

and Evolutionary Microbiology, v.

51, p. 61-66.

Hjelm, M., Riaza, A., Formoso, F.,

Melchiorsen, J. &Gram,

L.2004,Seasonal incidence of

autochthonous antagonistic

Roseobacter spp. and Vibrionaceae

strains in a turbot larva

(Scophthalmus maximus) rearing

system: Appl Environ Microbiol, v.

70, p. 7288-94.

Jin, G., Wang, S., Yu, M., Yan, S. &Zhang,

X.-H.2010, Identification of a marine

antagonistic strain JG1 and

establishment of a polymerase chain

reaction detection technique based

on the gyrB gene: Aquaculture

Research, v. 41, p. 1867-1874.

Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H.,

Kim, M., Na, H., Park, S. C., Jeon,

Y. S., Lee, J. H., Yi, H., Won, s.

&Chun, J.2012, Introducing

EzTaxon-e: a prokaryotic 16S rRNA

gene sequence database with

phylotypes that represent uncultured

species: International Journal of

Systematic and Evolutionary

Microbiology, v. 62, p. 716-721.

Kobayashi, M., Aoki, S., Gato, K.,

Matsunami, K., Kurosu, M.

&Kitagawa, I.1994, Marine natural

products. XXXIV. Trisindoline, a

new antibiotic indole trimer,

produced by a bacterium of Vibrio

sp. separated from the marine sponge

Hyrtios altum: Chem Pharm Bull

(Tokyo), v. 42, p. 2449-51.

Le Roux, F., Goubet, A., Thompson, F. L.,

Faury, N., Gay, M., Swings, J.

&Saulnier, d.2005,Vibrio gigantis sp

nov., isolated from the haemolymph

of cultured oysters (Crassostrea

gigas): International Journal of

Systematic and Evolutionary

Microbiology, v. 55, p. 2251-2255.

Leano, E. M., Lavilla-Pitogo, C. R. &Paner,

M. G.1998, Bacterial flora in the

hepatopancreas of pond-reared

Penaeus monodon juveniles with

luminous vibriosis: Aquaculture, v.

164, p. 367-374.

Lightner, D. V. &Redman, R. M.1998,

Shrimp diseases and current

diagnostic methods: Aquaculture, v.

164, p. 201-220.

Long, R. A. &Azam, F.2001,Antagonistic

interactions among marine pelagic

bacteria: Appl Environ Microbiol, v.

67, p. 4975-83.

Long, R. A., Rowley, D. C., Zamora, e., Liu,

J., Bartlett, D. H. &Azam, f.2005,

Antagonistic interactions among

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 68

marine bacteria impede the

proliferation of Vibrio cholerae:

Appl Environ Microbiol, v. 71, p.

8531-6.

Macian, M. C., Garay, E., Gonzalez-

Candelas, F., pujalte, M. J. &Aznar,

R.2000, Ribotyping of Vibrio

populations associated with cultured

oysters (Ostrea edulis): Systematic

and Applied Microbiology, v. 23, p.

409-417.

Oclarit, J. M., Okada, H., Ohta, S.,

Kaminura, K., Yamaoka, Y., Iizuka,

T., Miyashiro, S.&Ikegami, S.1994,

Anti-bacillus substance in the marine

sponge, Hyatella species, produced

by an associated Vibrio species

bacterium: Microbios, v. 78, p. 7-16.

Prado, S., Montes, J., Romalde, J. L.

&Barja, J. L.2009, Inhibitory activity

of Phaeobacter strains against

aquaculture pathogenic bacteria: Int

Microbiol, v. 12, p. 107-14.

Radajewski, S., Webster, G., Reay, D. S.,

Morris, S. A., Ineson, P., Nedwell,

D. B., Prosser, J. I. &Murrell, J.

C.2002, Identification of active

methylotroph populations in an

acidic forest soil by stable-isotope

probing: Microbiology, v. 148, p.

2331-42.

Radjasa Ok, Sabdono A, Zocchi J&E.,

Z.2007, Richness of secondary

metabolite-producing marine

bacteria associated with the sponge

Haliclona sp: Int J Pharmacol, v. 3,

p. 275–279.

Rosenfeld, W. D. &Zobell, C. E.1947,

Antibiotic Production by Marine

Microorganisms: J Bacteriol, v. 54,

p. 393-8.

Thompson,F. L., Iida, T. & Swings, J.2004,

Biodiversity of vibrios: Microbiol

Mol Biol Rev, v. 68, p. 403-31, table

of contents.

Thompson, F. L., Thompson, C. C., Li, Y.,

Gomez-Gil, B., Vandenberghe, J.,

Hoste, B. &Swings, J.2003a, Vibrio

kanaloae sp nov., Vibrio pomeroyi

sp nov and Vibrio chagasii sp nov.,

from sea water and marine animals:

International Journal of Systematic

and Evolutionary Microbiology, v.

53, p. 753-759.

Thompson, f. L., thompson, c. C. & swings,

j.2003b, Vibrio tasmaniensis sp nov.,

isolated from Atlantic Salmon

(Salmo salar L.): Systematic and

Applied Microbiology, v. 26, p. 65-

69.

Verschuere, L., Rombaut, G., Sorgeloos, P.

&Verstraete, W.2000, Probiotic

bacteria as biological control agents

in aquaculture: Microbiology and

Molecular Biology Reviews, v. 64,

p. 655-+.

Wagner-Dobler, I., Beil, W., Lang, S.,

Meiners, M. &Laatsch, H.2002,

Integrated approach to explore the

potential of marine microorganisms

for the production of bioactive

metabolites: Adv Biochem Eng

Biotechnol, v. 74, p. 207-38.

Wietz, M., Mansson, M., Gotfredsen, C. H.,

Larsen, T. O. &Gram,

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 69

L.2010,Antibacterial Compounds

from Marine Vibrionaceae Isolated

on a Global Expedition: Marine

Drugs, v. 8, p. 2946-2960.

Yebra, D. M., Kiil, S. &Dam-Johansen,

K.2004, Antifouling technology -

past, present and future steps towards

efficient and environmentally

friendly antifouling coatings:

Progress in Organic Coatings, v. 50,

p. 75-104.

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VETERINARY SCIENCES

RESEARCH ARTICLE

Physico-chemical properties of goat, sheep and camel milk of

Balochistan

Haseena Sajid1, Shafia Muzafar

1, Abida Peer Muhammad

2, Illahi Bakhsh Marghazani

3, Sajid Ali

Khosa3, Nasrullah

3and Ahmed Nawaz Khosa

3

1 Sardar Bahadur Khan Women University, Quetta, Balochistan

2Health, Education, Agriculture, Livestock Upgradation Programs (HEAL UP)

Balochistan; 3

Faculty of Veterinary and Animal Sciences, Lasbela University of Agriculture, Water

and Marine Sciences (LUAWMS), Uthal, Balochistan

ABSTRACT

This Study was conducted to determine the physic-chemical properties of goat, sheep and camel milk

of Quetta district, Balochistan. Result showed comparatively highest (6.65) milk pH in sheep,

followed by goat (6.24) whilst lowest (5.47) in camel. The electrical conductivity of milk was

recorded maximum (7.80 ms) in goat, followed by sheep (7.20 ms) and minimum (3.18 ms) in camel.

Milk titratable acidity was highest in camel (0.14%) followed by sheep (0.11%) and goat (0.10%).

Milk specific gravity was highest in sheep (1.04), followed by camel (1.03) and goat (1.02). In milk

chemical properties, total solid, casein and ash contents were more in sheep (20.7%, 11.11%, 1.06%,

respectively), followed by camel (13.5%, 6.3%, 0.6%, respectively) and goat (11.6%, 4.4%, 0.4%,

respectively).

Keywords: Milk, goat, sheep, camel, physico-chemical properties

__________________________________________________________________________________ Correspondence: Illahi Bakhsh Marghazani

Address: Faculty of Veterinary and Animal Sciences, LUAWMS, Uthal, Balochistan, Pakistan.

Email: marghazani76@yahoo.com

Phone: +92-333-2218439

Received: 05 Jun 2013 Revised: 03 Aug 2013 Accepted: 15 Aug 2013

Copyright: ©2013 Ahmed et al. This is an open-access article distributed under the terms of the Creative

Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium,

provided the original author and source are credited

Competing Interests: The authors have declared that no competing interests exist.

__________________________________________________________________________________

INTRODUCTION

Milk is the secretion of the memory gland and

is the only food of the young mammals

during the first period of life. Milk from

various mammals such as cow, buffalo, goat,

sheep and camel is used for different

nutritional purpose e.g. feeding to young ones

and preparation of some nutritional products

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 71

such as milk, cream, butter, yogurt, ghee, sour

milk (Webb et al., 1974; Hassan, 2005). Goat

has been referred as the “poor man’s cow” due

to its great contribution to the health and

nutrition of the landless and rural areas

(Dresch, 1988). Goat milk differs from cow or

human milk in having better digestibility,

alkalinity and buffer capacity (Park, 1994).

Sheep milk is an excellent raw material for the

milk processing industry (Park et al., 2007).

Sheep milk has higher specific gravity,

viscosity, refractive index, titratable acidity and

lower freezing point than average cow milk

(Haenlein and Wendroff, 2006). Globally, there

are 19.5 million camels, Pakistan rank at 5th

position with 0.9 million heads of camels

(FAO, 1997). These animals are mainly used

for transportation and less for meat and milk.

Despite the large population of camel in

Pakistan, camel milk is not utilized to any

significant extent probably due to unawareness

of use and market value of camel milk or

because of saltish taste and high acidic nature

(Abu-Tarboush, 1996). Camel milk is highly

nutritious so that much generation of our

ancestors survived on his beverage alone. As

like other mammal’s milk camel milk is almost

a complete food consisting of proteins (mainly

casein) fat, salt and milk sugar (lactose) as well

as vitamins and minerals (Sawaya et al., 1984).

The major physic-chemical components of

milk include water, fat, proteins,

carbohydrates, minerals, organic acids,

enzymes and vitamins has been extensively

studied from various countries (Dobzanski et

al., 2005; Honda et al., 2003; Romonaityte,

2001), however in Balochistan limited work

has been carried out on physic-chemical

properties of milk of different species. Keeping

in view these facts, the present study planned

to determine the physico-chemical

characteristics of milk of goat, sheep and camel

species collected from district Quetta,

Balochistan.

MATERIALS AND METHOD

The study was consisted of two main phases

i.e., sample collection and laboratory work.

Phase -1: Sample collection

Three different areas for milk sampling

(approximately 250 ml each) in district Quetta

were selected. Total ninety milk samples of

goats (n=30), sheep (n=30) and camels (n=30)

were collected from those selected areas. All

milk samples were labeled for individual

species and locations and stored in plastic jars

at -20 0C till laboratory analyses.

Phase-2: Laboratory Analyses

Before laboratory analyses, the stored milk

samples were thawed properly. The physico-

chemical measurements i.e., pH, electrical

conductivity, specific gravity, titratable acidity,

casein, total solids and ash were performed

using Lactoscan-S Milk Analyser (50W,

Milkotronic Ltd., Bulgaria).

RESULTS AND DISCUSSION

Raw milk is a complete food for mammals. It is

a good source of protein, fats, lactose, minerals

and vitamins. Its composition is affected by

species, breeds, feeding, lactation stage and

other environmental factors (Enb et al., 2009).

The present work was carried out to compare

the physico-chemical parameters of milk

samples of goats, sheep and camels. In

chemistry, pH is the measure of the acidity or

basicity of a solution. The acidity of milk

sample is usually expressed as pH (Tasci,

2011). The mean values of pH of fresh milk

samples collected from goats, sheep and

camels were determined. The mean pH values

of milk samples of goat, sheep and camel were

6.24, 6.65 and 5.47, respectively (table 1). The

results showed that pH of sheep milk

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

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washigher than that of goat and camel milk. pH

value found in sheep milk were similar to the

reported findings (Asif and Sumaira, 2010;

Park et al., 2007; Haenlein and Wendorff,

2006 and Rashida Kanwal et al., 2004). The

pH value of goat milk recorded in our study

was slightly less than pH value than the

findings of Asif and Sumaira (2010). Likewise,

pH of camel milk determined in our study was

less than pHreported in earlier literature

(Zubeir and Ibrahim, 2007; Khaskheli, 2005).

Acidity in terms of lactic acid content is called

titratable acidity (Khaskheli et al., 2005).

Acidity of milk is due to the presence of lactic

acid, citric acid, and phosphoric acid (Bylund

1995). The titratable acidity is simple acid base

reaction. This test allows calculation of percent

acidity in milk (Rashida et al., 2004). Titratable

acidity (%) determined in goat, sheep and

camel milk was 0.10%, 0.11% and 0.14%,

respectively (table 1). The camel milk showed

maximum titratable acidity (0.14%) whilst goat

milk showed minimum titratable acidity

(0.10%). These findings are in agreement with

the reported findings (Park et al., 2007; Asif

and Sumaira, 2010; Heanlein and Wendorff,

2006, Rashida et al., 2004. The value of

titratable acidity in sheep milk is comparable

with the values reported by Abdalla and

Daffalla (2010) and Salwa et al., (2009).

Titratable acidity of camel milk recorded in

present study are in line with earlier workers

(Mint et al., 2011; Zubair and Ibrahim, 2007;

Khaskheli et al., 2005). The little difference in

mean values may be due to lactation stage of

species which had a great effect on titratable

acidity in milk (Bhosale et al., 2009; Zubair

and Marwa, 2007).

Electrical conductivity is dependent on the

concentration of anions, cations with Na+, K

+

and Cl- being most important (Mauropovinelli

et al., 2005). The conductivity range of milk

samples collected from goat, sheep and camel

milk were 7.80 ms, 7.20 ms and 3.18 ms

respectively (table 1). Minimum conductivity

was recorded for the camel milk followed by

the goat milk and highest was measured for

sheep milk. Conductance of the goat milk in

present study was observed to be within the

range whilst conductance of sheep and camel

milk was comparatively lower than the findings

of Park et al., (2007). Conductance of camel

milk was lower than findings of Janzekovic et

al., (2009). The variation in conductivity may

be due to the different level of electrolytes

present in milk samples (Imran et al., 2008).

As the content of chloride (Cl-) and sodium

(Na+) increases the content of lactose and

potassium (K+) decreases, which leads to the

higher electrical conductivity of milk (Billon et

al.,2007).

Specific gravity of milk samples of goat, sheep

and camel was 1.02, 1.04 and 1.03,

respectively (table 1). The specific gravity of

sheep milk was comparatively higher than

camel and goat. The specific gravity of sheep

milk determined in this research was slightly

higher than the findings of Asif and Sumaira

(2010) and Park et al., (2007) and lower than

value reported by Haenlian and Wendoff

(2006) and Rashida et al., (2004). The specific

gravity of the goat milk was less than that cited

by Bhosale et al., (2009), Imran et al. (2008)

and Rashida et al., (2004). The Specific gravity

of camel milk was comparable to that reported

by Mint et al., (2011) and Khaskheli et al.,

(2005). Sheep milk had highest value of

specific gravity due to its contents of solid not

fat (Mehmood and Sumaira, 2010). It was

observed that lactation age affects the specific

gravity of milk by increasing its value in milk

(Bhosale et al., 2009).

The milk protein has the high nutritional value.

The principal component of milk protein is

casein which constitutes 75% of all milk

protein (Hassan, 2005). Casein contents (table

2) were more in sheep (11.11), followed by

camel (6.3%) and goat (4.4%). Casein (%)

obtained from goat milk is in line with the

results obtained by Abdalla and Daffala (2010)

and Park et al., (2007). Sheep milk casein

%age found higher than the casein (%) value

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 73

(4.2%) reported by other workers (Mehmood

and Sumaira, 2010; Abdalla and Daffalla,

2010). Casein (%) determined for camel milk

was also higher than the casein (%) value

obtained by M. Khaskheli et al., (2005). It was

found that variation in casein level in goat,

sheep and camel milk was influenced by the

breed difference, health status and stage of

lactation (Mehmood and Sumaira, 2010 and

Bhosale et al., 2009). Casein composition in

goat and sheep milk is also influenced by

genetic polymorphism (Martin et al., 2003).

Milk samples collected from goat, sheep and

camel showed that total solid (TS) contents of

sheep was highest (20.7%) whilst lowest

(11.6%) in goat milk (table 2). The TS content

in camel milk were intermediate i.e., 13.5%.

The TS contents observed in present findings is

also fall in the reported literature (Mehmood

and Usman (2010); Bhosale et al., (2009),

Imran et al., (2008) and Rasheeda et al.,

(2004).Similarly, the present values obtained in

case of sheep milk (Talevski et al., 2009;

Roberta, 2002; Grevilla et al., 1997) and camel

milk (Zubair et al., 2007; Farag and Kebary,

1992 and Ahmed, 1990) are in agreement with

the earlier workers. One of the main reasons in

variation in total solid contents in different

species is partly due to inherited capabilities of

the animals or attribute due to various seasonal

and environmental factors (Khaskheli et al.,

2005).

The water of milk is removed by evaporation

and the dry residue is incinerated at a low red

heat, there after a white or nearly white ash is

left which contains inorganic residues like Ca ,

Mg, Na, K, P, Zn, Fe, phosphate, carbonates,

oxides (Gallego et al., 2006: Khaskheli et al.,

2005; Rasheeda et al., 2004). Ash contents %

in milk samples collected from goat sheep and

camel milk were 0.4%, 1.0% and 0.6%,

respectively (table 2). The result of this study

revealed that ash content % in goat milk was

lower than in sheep and camel milk.Ash

contents found in goat milk were slightly less

than that reported by Abdullah and Daffalla

(2010), Bhosale et al., (2009), andjandal

(1996)whilst obtained ash % value was higher

than findings of Rasheedaet al., (2004). Ash

contents found in sheep milk were nearly

comparable with that reported by Asif and

Sumaira (2010), Imran et al., (2009) and Park

et al., (2007). Ash contents found in camel

milk during this research was quite less than

reported by Mint et al. (2011) and Khaskheli et

al., (2005). Ash contents in camel milk vary

due to free grazing of camel on bushes or plant

growth at saline soil (Khaskheli et al., 2005).

Table 1: Physical parameters of goats, sheep and milk samples

Parameters Goat Sheep Camel

pH 6.24 6.65 5.47

Electrical conductivity

(ms)

7.80 7.20 3.18

Titratable acidity 0.10 0.11 0.14

Specific Gravity 1.02 1.04 1.03

Table 2: Chemical properties of goat, sheep and camel milk

Parameters Goats Sheep Camels

Total solids % 11.6 20.7 13.5

Casein % 4.4 11.11 6.3

Ash % 0.4 1.06 0.6

Lasbela University Journal of Science and Technology (2013) Vol-2 ISSN 2306-8256

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CONCLUSION

Based on the result of present study, it is

concluded that sheep milk has higher physico-

chemical characteristic than goat and camel

milk. The difference in physico-chemical

properties of goat, sheep and camel milk are

influenced by numerous factors such as genetic

(breed and genotype), physiological condition

(age, lambing ,body weight, number of lambing

stage and number of lactation), production

conditions (feeding and management condition

in the area) and individual characteristic of

particular animal are some of them. Physico-

chemical characteristics of milk are essential

for successful development of dairy industries

as well as for marketing the products. Therefore

regular survey of milk should be carried out by

the local authority for milk quality at various

critical control points. These findings may

helpful for the concerned lawmakers to monitor

the quality of milk products in Quetta

Baluchistan.

ACKNOWLEDGEMENT

The principal author is highly thankful to her

friends for their supporting company during

sample collection.

REFERENCES

Abdalla, M. O. M., and Daffalla, M. S., 2010,

Comparison of chemical and

microbiological parameters of charcoal

verses gas and solar energy treated milk:

Advance journal of food, science and

technology, v. 2(5), p. 286-290.

Abu-Tarboush, H. M., 1996, Comparison of

associative growth andproteolytic activity

of yogurt starters in whole milk from

camels and cows: J. Dairy Sci, v.79, p.

366-371.

Aganga, A. A., Amarteifio, J. O., and Nkile,

N., 2002, Effect of stage of lactation on

nutrient composition of Tswana sheep and

goat`s milk: Journal of Food Composition

and Analysis, v.15, p.533-543.

Ahmad, B., Qureshi, K. A, and Bajwa M.

A.,1990, Economic relationship of

cottonseed cake and wheat bran with milk

production: Pakistan Journal of

Agriculture and Science,v. 27(3), p. 203-

206.

Anonymous, 2009-10, Pakistan Economic

Survey 2008-09: Economic Advisory

Wing, Ministry of Finance, Govt. of

Pakistan, Islamabad.

Asif, M., and Usman, S., 2010, A comparative

study on the physico-chemical parameters

of milk smples collected from buffalo,

cow, goat and sheep of Gujrat, Pakistan:

Pakistan Journal of Nutrition, v. 9 (12), p.

1192-1197.

Bhosale, S. S., Kahate, P. A, Kamble, K.,

Thakare, V. M., and Gubbawar, S. G.,

2009, Effect of lactation on Physico-

Chemical properties of local goat milk:

Veterinary World, v. 2(1), p. 17-19.

Billon, P., Pledel, D., and Gaudin, V., 2007,

Effects of increasing the milk flow switch

point when using ACR’s on milking and

milk quality: Proc. Annual meeting of the

National Mastitis Council, p. 222-223.

Bylund, G., 1995, Dairy processing

handbook.Tetra Pak processing systems

AB. S-22186, Lund, Sweden.

Composition and nutritional quality of

camel milk: Journal of Food Science, v.

49, p.744.

Lasbela University Journal of Science and Technology (2013) Vol-2ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 75

Coni, E., Bocca, A., Ianni, D., and Caroli, S.,

1995, Preliminary evaluation of the

factors influencing the trace element

content of milk and dairy products: Food

Chemistry, v. 52(2), p. 123-130.

Dabeka, R. W., 1989, Survey of lead, cadmium,

cobalt and nickel in infant formulas and

evaporated milks and estimation of

dietary intakes of the elements by infants

0-12 months old: Science Total

Environment, v. 89(3), p. 279-289.

Dabeka, R. W., and McKenzie, A. D., 1987,

Lead, cadmium and fluoride levels in

market milk and infant formulas in

Canada: Journal of Association of

Analytical Chemistry, v. 70(4), p. 754-

757.

Dobrzañski, Z., Kolacz, R., Górecka, H.,

Chojnacka, K., and Bartkowiak A., 2005,

The content of microelements and trace

elements in raw milk from cows in the

Silesian region: Polish journal of

Environmental Study, v. 14(5), p. 685-

689.

Dresch, J., 1988, A plea for the goat:

Production-Pastorale-et-Societe OAE,

1982, v. 10, p. 81-83.

Enb, A., Abou donia, M. A., Abd-Rabou, N.S.,

Abou-Arab, A. A. K., and El-Senaity, M.

H., 2009, Chemical composition of raw

milk and heavy metals behavior during

processing of milk products: Global of

veterinary, v. 3, p. 268-275.

Farag, S. I., and Kebary, K. M. K., 1992,

Chemical composition and physical

properties of camels milk and milk fat:

Procd. 5th Egyptian Conference for Dairy

Science and Technology, p. 57–67.

Fuertes, J. A., Gonzalo, C., and Carriedo, J. A.,

and Primitive, F. S., 1998, Parameters of

test day milk yield and milk components

for dairy ewes: Journal of dairy science, v.

81, p. 1300-1307.

Fulya, T., 2011, Microbiological and chemical

properties of raw milk consumed in

burdur: Journal of Ani. and Vet. Adv., v.

10 (5), p. 635-641.

Gonzales, G. C., Caamo, D. L., and Schryvers,

A. B., 1990, Identification and

characterisation of a porcine-specific

transferring receptor in Actinobacillus

pleuropneumoniae: Molecular

Microbiology, v. 4, p. 1173–1179.

Haenlein, G. F. W., and Wendorff, W. L., 2006,

Sheep milk production and utilization of

sheep milk. In: Park, Y.W. and

G.F.W.Haenlein, (Eds.), Handbook of

Milk of Non-Bovine Mammals: Blackwell

Publishing Professional, Oxford, UK and

Ames, Iowa, USA, p. 137-194.

Hallen, I. P., Jorhem L., Lagerkvist, B. J., and

Oskarsson, A., 1995, Lead and cadmium

levels in human milk and blood: Science

Total Environment, v. 166 (1-3), p. 149-

155.

Hassan, S. S., 2005, Quality assurance of

various dairy products of Pakistan:

Department of Chemistry, University of

Peshawar.

Honda, R., Tawara, K., Nishijo, M.,

Nakagawa, H., Tanebe, K., and Saito, S.,

2003, Cadmium exposure and trace

elements in human breast milk:

Toxicology, v. 186 (3), p. 255-259.

Ikem, A., Nwankwoala, A., Odueyungbo, S.,

Nyavor, K., and Egiebor N., 2002, Levels

of 26 elements in infant formula from

Lasbela University Journal of Science and Technology (2013) Vol-2ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 76

USA, UK, and Nigeria by microwave

digestion and ICP-OES: Food Chemistry,

v. 77(4), p. 439-447.

Imran, M., Khan, H., Hassan, S. S., and Khan

R., 2008, Physico-chemical characteristics

of various milk samples available in

Pakistan: Journal of Zhejiang University

Science, v. 9(7), p. 546-551.

Iqbal, A., Gill, R. M., and Younas, M., 2001,

Milk composition of Pakistani camel

(Camelus dromedaries) kept under station/

farmer's condition: Emirate Journal of

Agricultural Science, v.(13), p. 7-10.

Janzekovic M., Brus, M., Mursec, B., Vinis, P.,

Stajnko, D., and Cus, F.,2009, Mastitis

detection based on electric conductivity of

milk: Journal of achievements in materials

and manufacturing engineering, v. 34 (1),

p. 39-46.

Javaid, S. B., Gadahi, J.A., Khaskheli, M.,

Bhutto, M. B., Kumbher, S., and Panhwar

A. H., 2009, Pakistan veterinary journal,

v. 29(1), p. 27-31.

Khaskheli, M., Arain, M. A. Chuadhary, S.,

Soomro, A. H., and Qureshi, T. A., 2005,

Physico-chemical quality of camel Milk:

Journal of Agriculture and Social

Sciences, v. 2, p. 164–166.

Krelowska-Kulas, M., 1990, Lead, cadmium,

iron, copper and zinc in fresh milk from

the selected areas of the Cracowregion:

Nahrung, v. 34(3), p. 13-217.

Martin, R., Langa, S., Reviriego, C., Jiminez,

E., Marin, M. L, Xaus, J., Fernandez, L.,

and Rodriguez, J. M., 2003, Human milk

is a source of lactic acid bacteria for the

infant gut: J. Pediatr., v. 143, p. 754-758.

Meiloudg, M., Bourayai, O., Samband, N., and

Houmeida, 2011, Composition of

Mauritanian camel Milk. Results of First

Study: International journal of agriculture

and biology, v. 13 (1), p. 145-147.

Park, Y. W., 1994, Hypo-allergenic and

therapeutic significance of goat milk:

Small Rum. Res., v. 14, p. 151-159.

Park,Y. W., Ju´arez, M., Ramosc, M., and

Haenlein, G. F. W., 2007, Physico-

chemical characteristics of goat and sheep

milk: Small Rum. Res., v. 68, p. 88–113.

Rajor, A., 2006,Experimental methods for

general and environmental chemistry (1st

edition): Standard publisher’s distributors,

v. 81, p. 8014-065.

Ramonaityte, D.T., 2001, Copper, zinc, tin and

lead in canned evaporated milk, produced

in Lithuania: the initial content and its

change at storage: Food Addition and

Contamination, v. 18 (1), p. 31-37.

Rashida, K., Toqeer, A., and Bushra, M., 2004,

Comparative analysis of quality of milk

collected from buffalo, cow, goat and

sheep of Rawalpindi/Islamabad region of

Pakistan: Asian journal of plant science,

v. 3 (3), p. 300-305.

Robert, B., 2002, Factors affecting the clotting

properties of sheep milk: Journal of the

science of food and agriculture, v. 82, p.

705-719.

Rodriguez, E. M., Sanz, A. M., and Diaz, R.C.,

2000, Concentration of iron, copper and

zinc in human milk and powdered infant

formula: International Journal of Food

Science and Nutrition, v. 51 (5), p. 373-

380.

Lasbela University Journal of Science and Technology (2013) Vol-2ISSN 2306-8256

Online available at www.luawms.edu.pk pg. 77

Salwa, B., Ali, S., Amira, A., and Hamadi, A.,

2009, Physicochemical characteristics and

renneting properties of camel milk: a

comparison with goat’s, ewe’s and cow’s

milk: International journal of dairy

technology, v. 62 (4), p. 505-513.

Simsek, O., Gultekin, R., Oksuz, O., and

Kurultay, S., 2000, The effect of

environmental pollution on the heavy

metal contents of raw milk: Nahrung, the

Nutrition Society, v. 44 (5), p. 360-363.

Talevski, G., Vasilevska, R., Csrbinovska, S.,

and Sireta, Z., 2009, Quality of the sheep

milk as a raw material in dairy industry of

Macedonia: Biotechnology of Animal

Husbandry, v. 25, p. 971-977.

Vander-Jagt, D. J., Okolo, S. N., Romero, L.,

Millson, M., and Glew, R. H., 2001, Lead

levels in the milk of Fulani women in

Nigeria: J. Natl. Med. Assoc., v. 93 (3),

p.104-108.

Vavilis, D., Bontis, J., Agorastos, T.,

Angelikakis, G., Zournatzi, V.,

Loufopoulos, A. Constantinou, A., and

Patsourou, A., 1997, Lead concentration

in early human milk of urban and rural

mothers: Clin. Exp. Obstet. Gynecol., v.

24 (4), p. 198-199.

Webb, B. H, Johnson, A. H., and Alford, J. A.,

1974,Fundamentals of dairy chemistry:

2nd

Ed. Westport, CT: AVI Publishing

Company.

Zubair, I. E. M., and El Ibrahium, M. I., 2009,

Effect of pasteurization of milk on the

keeping quality of fermented camel milk

(gariss) in Sudan: Traditional Dairy Food,

Karnal, India, v. 2, p. 1-9.