Spatial Analysis of Sulfur Dioxide (SO2) Concentration in Karachi

Vol 2.
December, 2013
ISSN 2306-8256
Patron-in-Chief Dr. Abdul Hameed Bajoi Vice Chancellor LUAWMS Patron Dr. Gul Hasan Pro Vice-Chancellor LUAWMS Editor-in-Chief Dr. Muhammad Aslam Editors Dr. Abdul Raziq Abdul Qayoom Buzdar Imtiaz Ahmed
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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 Integrated geological and geophysical research on Lava effusion in Ziarat, Balochistan, Pakistan Asif Nazeer Rana, Muhammad Saeed, Mehtab Ur Rahman, , Syed Ali Abbas 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 Genetic differentiation of two Chrysichthys species using mitochondrial DNA sequencing Nwafili S.A, Eminue B.O , Jamabo. N Preliminary observation on Baseodiscus hemprichii (ribbon worm) Faiz Muhammad, Muhammad Shafi, Muhammad Aslam 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 In vitro antibacterial activity of Sorghum halepense Rooh-Ul-Amin, Muhammad Adil, Kashif Hayat, Arbab Sikandar, Farmanullah, Saeed Khan, Hazrat Nabi Antagonistic potential of marine isolate DK6-SH8 against fish pathogens Muhammad Naseem Khan, Meng Li, Zulfiqar Ali Mirani, Jingxue Wang And Hong Lin 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
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Lasbela University Journal of Science and Technology (2013) Vol-2
ISSN 2306-8256
ENVIRONMENTAL SCIENCES RESEARCH ARTICLE
Spatial analysis of sulfur dioxide (SO2) concentration in Karachi Megapolis, Pakistan

Syed Nawaz-ul-Huda1, Farkhunda Burke1, Erma Anwar2,Imtiaz Ahmed3, Muhammad Miandad1and Muhammad Azam2
1 2
Department of Geography, University of Karachi, Karachi, Pakistan. Department of Geography, Federal Urdu University of Arts, Sciences and Technology, Gulshan-e-Iqbal Campus, Karachi, Pakistan. 3 Faculty 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 Karachis 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
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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 km (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:
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Analysis of 24 hours SO2 data from selected sample sites of the city. Identification ofSO2 concentration zones in Karachi Megapolis.
M
Demarcation of probable expansion of SO2 through Minimum Curvature method.

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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
Fig 1: Study Area and Sample Sites

SO2 Affected Part Renovated portion
Plate 1: SO2 affected limestone buildings in the study area.
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Malir Cantonment
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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 LandhiKorangi 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
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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 hours 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 Colonys 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
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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.
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Sulphur Dioxide Concentration at different hours of the day-Karachi

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Social functions in the megapolis are arranged mainly during the nighttime, after working hours 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).
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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 Colonys 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).
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25 22 2 3 23 4 27 7 8 13 6 14 16 5 28 20 27 23 4 7 8 13 6 14 16 5 28 20 1 26 22 2 3 25 1 26
24 10
12
24 10
12
11
15 17 18 21
11
15 17 18 21
19
19
Fig.10, 08:00 hours

25 22 2 3 23 4 27 7 8 13 6 14 16 5 28 20 1 26
ppb
Fig.11, 09:00 hours

25 22 2 3 23 4 27 7 8 13 6 14 16 5 28 20 1 26
ppb
24 10
12
24 10
12
11
15 17 18 21
11
15 17 18 21
19
19
Fig.12, 10:00 hours

25 22 2 3 23 4 27 7 8 13 6 14 16 5 28 20 1 26
ppb
Fig.13, 11:00 hours

25 22 2 3 23 4 27 7 8 13 6 14 16 5 28 20 1 26
ppb
24 10
12
11
15 17 18 21
24 10
12
11
15 17 18 21
19
Fig.14, 12:00 hours

25 22 2 3 23 4 27 7 8 13 6 14 16 5 28 20 1 26
ppb Fig.15, 13:00 hours
19
ppb
25 1 26
22 2 3 23 4 27 7 8 13 6 14 16 5 28
20
24 10
12
24 10
12
11
15 17 18 21
11
15 17 18 21
19
19
Fig.16, 14:00 hours
ppb
Fig.17, 15:00 hours
ppb
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25 22 2 3 23 4 27 7 8 13 6 14 16 24 10 17 19 18 21 5 27 28 20 7 8 13 6 14 16 5 23 4 28 20 1 26 22 2 3 25 1 26
24 10
12
11
15
12
11
15 17 18 21
Fig.18, 16:00 hours

25 22 2 3 23 4 27 7 8 13 6 14 16 5 28 20 1 26
ppb
19
Fig.19, 17:00 hours

25 22 2 3 23 4 27 7 8 13 6 14 16 5 28 20 1 26
ppb
24 10
12
24 10
12
11
15 17 18 21
11
15 17 18 21
19
19
Fig.20, 18:00 hours

25 22 2 3 23 4 27 7 8 13 6 14 16 5 28 20 1 26
ppb
Fig.21, 19:00 hours

25 22 2 3 23 4 27 7 8 13 6 14 16 5 28 20 1 26
ppb
24 10
12
24 10
12
11
15 17 18 21
11
15 17 18 21
19
19
Fig.22, 20:00 hours

25 22 2 3 23 4 27 7 8 13 6 14 16 5 28 20 1 26
ppb
Fig.23, 21:00 hours

25 22 2 3 23 4 27 7 8 13 6 14 16 5 28 20 1 26
ppb
24 10
12
11
15 17 18 21
24 10
12
11
15 17 18 21
19
19
Fig.24, 22:00 hours
ppb
Fig.25, 23:00 hours
ppb
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Peak and Slack Levels of SO2 Round the Clock at different Samples Sites - Karachi
23 22 21 20 19 18 17 16 13 12 Fig. 26 , SUPARCO 14 23 22 21 20 19 18 17 16 15 11 14 13 12 10 9 24 30 20 10 0 1 2 15 11 10 9 2412 1 8 4 0 2
ppb
3 4 5 21 6 20 7 19 8 18 17 16 15 22 23
24 15 1 10 5 0
3 4 5 6 21 20 7 19 18 8 17 16 22
23
24 301 20 10 0
3 4 5 6 7 8 9 10 21 20 19 18 17 16 22
23
24 20 1 15 10 5 0
3 4 5 6 7 8 9 10
9 10 14 13 12 11
15
14 13
12
11
15
14
13
12
11
Fig. 27, Maritime Museum 3 4 5 21 6 20 7 19 8 18 17 16 15 14 13 12 11 10 9 23 22 24 40 1 30 20 10 0 2 3 4 5
Fig. 28 , Preedy Street 24 30 1 2 23 22 21 6 20 7 19 8 18 17 16 15 14 13 12 20 10 0
Fig. 29 , Tin Hatti 3 4 5 6 21 20 22 23 24 40 1 30 20 10 0 9 16 15 14 13 12 11 10 2 3 4 5 6 7 8
7 19 8 9 10 11 18 17
Fig. 30 , Teen Talwar 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 24 40 30 20 10 0 1 2 3 4 5 6
Fig. 31 , Gulshan Chowrangi 23 22 21 20 24 25 1 20 15 10 5 0 2 3 4 5 6
Fig. 32, M Musafir Khana . 22 21 20 24 30 1 23 20 10 0 9 16 15 14 13 12 11 10 2 3 4 5
Fig. 33 , Empress Market 23 22 21 6 20 7 19 8 18 17 16 15 14 13 12 11 10 9 24 40 1 30 20 10 0 2 3 4 5 6 7 8
7 19 8 18 17 16 15 14
7 19 8 9 10 11 18 17
13
12
Fig. 34,Ghizri Rd & Punjab Colony 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 24 40 1 30 20 10 0 2 3 4 5
Fig. 35 , Tibat Center Colony 23 22 21 24 40 1 30 20 10 0 9 16 15 14 13 12 11 10 2 3 4 5 6
Fig. 36 , Nomaish 24 25 1 20 15 10 5 0 2 3 4 5 6
Fig. 37 , Merewether Tower 23 22 21 20 24 40 1 30 20 10 0 9 16 15 14 13 12 11 10 2
23 22 21 20
3 4 5 6 7 8
6 20 7 19 8 18 17
7 19 8 18 17 16 15
7 19 8 9 10 18 17
14
13
12
11
Fig.38 , Gulbai
Fig.39, Shaheen Complex
Fig. 40, Garden Road
Fig. 41, Burns Road
23 22 21 20 19 18 17 16
2425 1 20 15 10 5 0
3 4 5 21 6 20 7 19 8 18 9 10 17 16 22
23
2440 1 30 20 10 0
3 4 5 21 6 20 7 19 8 18 9 10 17 16 22
23
2440 1 30 20 10 0
3 4 5 21 6 20 7 19 8 18 9 10 17 16 22
23
2430 1 20 10 0
3 4 5 6 7 8 9 10
12 13 Fig. 42,Boulevard & Gizri
15
14
11
15
14
13
12
11
15
14
13
12
11
15
14
13
12
11
Fig. 43, Karimabad
Fig. 44, North Nazimabad
Fig. 45, KPT
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23 22 21 20 19 18 17 16 15
2440 1 30 20 10 0
3 4 5 6 21 20 7 19 8 9 10 18 17 16 22
23
2440 1 30 20 10 0
ppb
4 5 21 6 20 7 19 8 18 9 10 17 16 22
23
2430 1 25 20 15 10 5 0
3 4 5 21 6 20 7 19 8 18 9 10 17 16 22
23
2440 1 30 20 10 0
3 4 5 6 7 8 9 10
14
13
12
11
15
14
13
12
11
15
14
13
12
11
15
14
13
12
11
Fig. 46, FTC 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 10 0 2430 1 20 2 3 4 5
Fig. 47, Drigh Road 23 22 21 6 20 7 19 8 18 17 16 15 14 13 12 11 10 9 10 0 2430 1 20 2 3 4 5
Fig. 48, Liaquatabad 10 23 22 21 6 20 7 19 8 18 17 16 15 14 13 12 11 10 9 2440 1 30 20 10 0 2 3 4 5
Fig. 49, Nazimabad 23 22 21 6 20 7 19 8 18 17 16 15 14 13 12 11 10 9 2440 1 30 20 10 0 2 3 4 5 6 7 8
Fig. 50, Sohrab Goth
Fig.51, Metropole Hotel
Fig. 52, Mauripur Road
Fig.53, Guru Mandir
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 peoples activities in the study area. Guru Mandir, Mauripur and FTC are areas with second highest SO2 concentration, where traffic keeps flowing round the clock.
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80 70 60
SO2 Concentration (ug/m3)
50 40 30
WHO 24 hours mean

20 10 0
Preedy Street Tin Hatti Maritime Museum Metropole Hotel Empress Market Gulshan Chowrangi North Nazimabad Garden Road Sunset Boulevard Musafir Khana Road Shaheen Complex Liaquatabad 10 Merewether Tower Mauripur Road Old Numaish Sohrab Goth Tibet Center Teen Talwar SUPARCO Guru Mandir Burns Road Gizri Road Gulbai Drigh Road Nazimabad Karimabad KPT FTC
Study Area 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.
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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):989995. 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 66:151161. 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
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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): 717722. 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. 87:213221. 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, 61:542549. 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.
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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.
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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.
separation probably caused by dual level seismicity as substantiated by the focal depth of the events varying from 1060 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 worlds largest and oldest Junipers forests. The altitude ranges from 1, 800 to 3, 488 m.
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
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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
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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 frotscoria, pea size globules-lapilli, bunches of finely spun glassy hair- peles 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
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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.
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.
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.
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
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crustal assimilation and re-melting of preexisting 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
18 15
N a 2O + K 2 O
Phonolite
12 9
P-N P-T B+T Benmorite
Trachyte
Mugearite Hawaiite B-A Trachyandesite Dacite Andesite
Rhyolite
6 3 0 35
Nephelin
Basalt
45
55
65
75
SiO2
Fig 8: Total Alkali vs Silica Diagram

20 18 16
N a 2O + K 2 O
14 12 10 8 6
Alkaline
Fe2O3 is total iron; Major trace elements are analyzed on WD-XRF at GSPs 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.
4 2 0 35 40 45 50 55 60 65 70 75 80 85
Subalkaline
SiO2
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.
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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 Griffins 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.
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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
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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
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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 seismotectonic 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
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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,
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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. 10271036 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.
Ocean Tectonics, Geological Society of America Bulletin. Geological map No 26,1958, Reconnaissance geology of part of West Pakistan, Colombo plan cooperative 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.
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
Rana, A.N., and Akhtar, S.S., 2010, Effusion of Molten Material in Tor Zawar Mountain, Sari, Ziarat, Balochistan
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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 intracaldera 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 Baloch1 and Imtiaz Ahmed2
1 2
Faculty of Agriculture, LUAWMS, Uthal, Lasbela, Balochistan, Pakistan. Faculty 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
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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 stemleaf 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 6512'11"-6725'39" East longitudes and 2453'2"-2639'20" 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 6512'11"-6725'39" East longitudes and 2453'2"-2639'20" 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
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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 100C. 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 169C to 189C). 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 (Lpez 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 airdry 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
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pattern over the study period is gradually increasing (Table 1).
with maturity in alfalfa. However alfalfa fibre contains a high proportion of lignin compared with forage grasses resulting in low digestibility relative to grasses. 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 CP 25.4 20.9 17.5 14.4 14.9 CF 17.2 26.7 30.7 30.9 28.9 Percentage of dry matter Ash EE NFE 16.7 15.2 10.7 8.1 8.7 2.7 3.2 3.6 2.9 4.3 41.0 37.1 41.1 45.9 46.0 Ca 1.96 2.25 1.90 1.75 1.55 P 0.42 0.35 0.24 0.17 0.25
19.9 21.1
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 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
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 alfalfas low fibre content and the rapid rates of digestion and passage of that fibre. (Martin and Mertens, 2005).
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
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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 alfalfanutrient 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. Lpez, C.V.G., Garca, M.C.C., Fernndez, F.G.A., Bustos, C.S., Chisti, Y. and Sevilla, J.M.F., 2010, Protein measurements of microalgal and cyanobacterial biomass. Bioresource Tech. 101: 75877591 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
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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. 7446:23-37. Shahriari, M.H., G.R. SavaghebiFiroozabadi, 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. 4651.
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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. ______________________________________________________________________________
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
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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, sequencing amplification, and
l volumes containing 1.25 units of Taq polymerase, 200 nmolL-1 forward and reverse primers, 200 molL-1 each of dNTPs, 10 mMolL-1Tris (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 () 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 0.042450.002 while the total haplotype diversity was
The Muscles of the samples were preserved in 95% ethanol. Total genomic DNA was isolated using the standard PhenolChloroform-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
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0.9310.033. This indicates that both species are genetically diverse. Also the haplotype and nucleotide diversities for C. nigrodigitatus were (0.8460.085, 0.010210.002) and the haplotype and nucleotide diversities for C. walkeri were (0.8730.089, 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.
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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
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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)
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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 & AlvesGomes, 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. 14381443. Aurelle, D., and Berrebi, P., 2001, Genetic structure of brown trout (Salmo trutta, L.) populations from southwestern France: data from mitochondrial control region variability: Molecular Ecology, v. 10(6), p. 15511561. 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. 613627. 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. 19671971. Brown, G.G., Gadaleta, G., Pepe, G., and Saccone, C., 1986, Structural conservation and variation in the Dloop containing region of vertebrate mitochondrial DNA: Journal of Molecular Biology, v. 192, p. 503511.
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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 gentica da piramutaba Brachyplatystoma vaillantii (Valenciennes, 1840) (Siluriformes: Pimelodidae) no sistema EsturioAmazonas-Solimes: 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. 121128. 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. 19071919. 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:
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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.
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MARINE SCIENCES SHORT COMMUNICATION
Preliminary observation on Baseodiscus hemprichii (ribbon worm)

Faiz Muhammad1, and Muhammad Shafi 2
1 2
Centre of Excellence in Marine Biology, University of Karachi, Pakistan Lasbela 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 51N and 66o 48E). 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
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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
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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 456:2132.
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Lasbela University Journal of Science and Technology (2013) Vol-2 MICROBIOLOGY RESEARCH ARTICLE
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Identification and phylogenetic analysis of halophilic fungus isolated from a man-made solar saltern in Thailand
Imran Ali1,2,4, Sudip K. Rakshit1, Napa Siwarungson3, Hunsa Punnapayak4, Pongtharin Lotrakul4, Sehanat Prasongsuk4, Ali Akber1 and Zia Ur Rehman1,2
1
Food Engineering and Bioprocess Technology, School of Environment, Resources and Development, Asian Institute of Technology, Klong Luang, Pathumthani 12120, Thailand. 2 Institute of Biochemistry, University of Balochistan, Quetta, 83700, Pakistan. 3 Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. 4 Department 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. ____________________________________________________________________________
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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 520% (w/v) and; 4) Extreme halophilic microorganisms: which can grow, survive and reproduce in NaCl concentration of 2030% (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; GundeCimerman 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).
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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 manmade solar saltern in Thailand
Parameter Lab code Colony color Colony appearance Spore size Mycelium Number of isolates Halotolerance Result SWAF White Cotton like Medium Septate 3 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).
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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 Species Strain Similarity Accession number Findings Engyodontium album strain NRRL 2312 100% 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.
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.
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ACKNOWLEDGEMENT We will like to thanks all lab staff of King Mongkuts 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.
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. 743752. 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. 956961. Ellis, M.B. 1971,Dematiaceous Hyphomycetes: Commonwealth Mycological Institute, Kew England.
Ali, I. Kanhayuwa, L. Rachdawong, S. Rakshit, S.K. 2012, Identification, phylogenetic analysis and characterization of obligate halophilic fungi isolated from a manmade solar saltern in Phetchaburi province, Thailand: Annals of Microbiology, doi: 10.1007/s13213012-0540-6 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. GundeCimerman, N. 2005, The genus Eurotium members of indigenous fungal community in hypersaline
Ellis, M.B. 1976, More Dematiaceous hyphomycetes: Commonwealth, Mycological Institute, Kew, England.
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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. PrenticeHall, 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. 48764882. 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. Plemenita, A. 2009, Halotolerant and halophilic fungi: Mycological Research, v. 113, p. 12311241. Gunde-Cimerman, N. Zalar, P. de Hoog, S. Plemenita, 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.161165. 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. 311328.
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VETERINARY SCIENCES RESEARCH ARTICLE
In vitro antibacterial activity of Sorghum halepense

Rooh-ul-Amin1, Muhammad Adil2, Kashif Hayat1, Arbab Sikandar2, Farmanullah3, Saeed Khan4 and Hazrat Nabi4
1 2
Department of Botany, Abdul Wali Khan University, Mardan, Pakistan Department of Basic sciences, College of Veterinary & Animal sciences, Jhang, Pakistan 3 Lasbela University of Agriculture, Water and Marine Sciences, Uthal, Pakistan 4 Department 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.
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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 ethnopharmacology 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 plantderivedsubstances. 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 nhexaneextracts 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
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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 35C. 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 (ATCC6633),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 l 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 g/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 Duncans 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 grampositive 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.
Zone of inhibition (mm) Ethyl nChloroform acetate hexane extract extract extract 14 9 12 12 7 10 10 3 8
Bacterial species S. aureus E. coli B. subtilis
Streptomycin 21 29 24
Analogous pattern of bacterial sensitivity was manifested against ethyl acetate and n-
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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 Ftable value Conclusion
Bacterial species Plant extracts
0.42857 15.08
10.92 Nonsignificant 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 n-hexane extract Ethyl acetate extract Chloroform extract Streptomycin Mean values 7ab 9.66bc 11.66ac 24.66
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 nhexane extracts respectively (as illustrated in table: 1). Therefore comparisons among the treatment means were executed using Duncans 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 phydroxybenzoate and phydroxybenzaldehyde which could be expected to exert the consequent antimicrobial activity (Javaidet al., 2012).Appraisal of the biosynthesis of plant phenolics stipulates that phydroxybenzaldehyde is formed from salicylic acid (Dicko et al., 2006) which is
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
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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. 493496. 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. 286293. Cowan, M. M., 1999, Plants products as antimicrobial agents. Clinical Microbiology Reviews, v. 12, p. 564582. Czarnota, M. A., Rimando, A. M., and Weston, L. A., 2003, Evaluation of root exudates of seven sorghum accessions,Journal of
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ChemicalEcology, v. 29, p. 2073 2083. Dicko, M. H., Gruppen, H., Traor, 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. 527529. Duncan, D. B., 1955, Multiple rangeand multiple F test, Biometrics, v. 11, p. 124. 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
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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. 4665. 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. 4047. 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. 744748. 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 extendedspectrum--lactamaseproducing 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.
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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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Lasbela University Journal of Science and Technology (2013) Vol-2 MARINE SCIENCES
ISSN 2306-8256
RESEARCH ARTICLE
Antagonistic potential of marine isolate DK6-SH8 against fish pathogens

Muhammad Naseem Khan1, 2, Meng Li1, Zulfiqar A Mirani2, Jingxue Wang1&Hong Lin1
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 vulnicus. 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,
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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 shrimps 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://eztaxone.ezbiocloud.net/) and identify within cultured database of systematically identified and verified bacteria. The number of percent pairwise similarity was obtained
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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 L 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 1 2 3 4 5 6 7 8 9 10 Name Vibrio kanaloae Vibrio pomeroyi Vibrio splendidus Vibrio artabrorum Vibrio gigantis Vibrio celticus Vibrio atlanticus Vibrio tasmaniensis Vibrio crassostreae Vibrio cyclitrophicus Strain LMG 20539(T) LMG 20537(T) ATCC33125(T) Vb 11.8(T) CAIM 25(T) Rd 8.15(T) Vb 11.11(T) LMG 21574(T) CAIM 1405(T) P-2P44(T) Pairwise Similarity (%) 99.65 98.75 98.70 98.68 98.54 98.54 98.34 98.33 98.33 97.98 Diff/Total nt 5/1438 18/1436 18/1381 19/1441 21/1442 21/1438 24/1442 24/1441 24/1441 29/1434 Accession AJ316193 AJ491290 X74724 EF599164 EF094888 EF599162 EF599163 AJ514912 EF094887 U57919 Authors Thompson et al (2003a) Thompson et al (2003a) Baumann et al (1980) Dieguez et al (2011) Le Roux et al (2005) Beaz-Hidalgo et al (2010) Dieguez et al (2011) Thompson et al (2003b) Faury et al (2004) Hedlund and Staley (2001)
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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 vulnicus,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.
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
Table 2: Antagonistic activity of DK6-SH8 against fish pathogens

Microorganism Vibrio anguillarum Vibrio alginolyticus Vibrio campbellii Vibrio harveyi Vibrio parahaemolyticus Vibrio tubiashii Vibrio vulnicus Source LMG 4437T; cod, Norway LMG 4408T LMG 11216T; USA LMG 7890T; USA (1982) LMG 2850T; Japan LMG 10936T; USA LMG 13545T; USA IN : invisible Zone of inhibition in mm SEM* 22 + 0.34 19 + 0.58 13 + 0.34 14 + 0.89 IN 23 + 0.34 20 + 0.58
* Mean of triplicates + Standard Error of Mean
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, Gramnegative 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 DK6SH8 against fish pathogens suggest that stain Vibrio kanaloae DK6-SH8 could be a
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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 fastgrowing 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.
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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
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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. 6569. 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,
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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 Muzafar1, Abida Peer Muhammad2, Illahi Bakhsh Marghazani3, Sajid Ali Khosa3, Nasrullah3and Ahmed Nawaz Khosa3
1 2
Sardar Bahadur Khan Women University, Quetta, Balochistan Health, 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
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such as milk, cream, butter, yogurt, ghee, sour milk (Webb et al., 1974; Hassan, 2005). Goat has been referred as the poor mans 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 mammals 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 physicochemical 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
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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
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(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 pH Electrical conductivity (ms) Titratable acidity Specific Gravity Goat 6.24 7.80 0.10 1.02 Sheep 6.65 7.20 0.11 1.04 Camel 5.47 3.18 0.14 1.03
Table 2: Chemical properties of goat, sheep and camel milk

Parameters Total solids % Casein % Ash % Goats 11.6 4.4 0.4 Sheep 20.7 11.11 1.06 Camels 13.5 6.3 0.6
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CONCLUSION Based on the result of present study, it is concluded that sheep milk has higher physicochemical 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. Physicochemical 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. 203206. 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 PhysicoChemical 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 ACRs 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.
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Vol 2.

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

ENVIRONMENTAL SCIENCES RESEARCH ARTICLE