Heavy Metal Residues in the Bat Fauna of Central And

Home , Rawal lake

HEAVY METAL RESIDUES IN THE BAT FAUNA OF CENTRAL AND

NORTHERN PUNJAB

SHAGUFTA NIGHAT

07-arid-1194

Department of Zoology/Biology

Faculty of Sciences

Pir Mehr Ali Shah

Arid Agriculture University, Rawalpindi

Pakistan

2015HEAVY METAL RESIDUES IN THE BAT FAUNA OF CENTRAL AND NORTHERN PUNJAB

SHAGUFTA NIGHAT

(07-arid-1194)

A thesis submitted in the partial fulfilment

of the requirements for the degree of

Doctor of Philosophy

Zoology

Department of Zoology/Biology

Faculty of Sciences

Pir Mehr Ali Shah

Arid Agriculture University, Rawalpindi

Pakistan

2015

HEAVY METAL RESIDUES IN THE BAT FAUNA OF CENTRAL AND

NORTHERN PUNJAB

SHAGUFTA NIGHAT

(07-arid-1194)

A thesis submitted in the partial fulfilment

of the requirements for the degree of

Doctor of Philosophy

Zoology

Department of Zoology/Biology

Faculty of Sciences

Pir Mehr Ali Shah

Arid Agriculture University, Rawalpindi

Pakistan

2015

CERTIFICATION

I hereby undertake that this research is an original one, and no part of this thesis falls under plagiarism. If found otherwise, at any stage, I will be responsible for the consequences.

Name: Shagufta Nighat Signature: ______

Registration No: 07-arid-1194 Date: ______2015

Certified that the contents and form of the thesis entitled “Heavy Metal Residues in the Bat Fauna of Central and Northern Punjab” submitted by Shagufta Nighat have been found satisfactory for the requirement of the degree.

Supervisor: ______(Dr. M. Sajid Nadeem)

Co-supervisor: ______

(Dr. M.Mehmood-ul-Hassan)

Member: ______

(Prof. Dr. Mirza Azhar Beg)

Member: ______

(Dr. Tariq Mehmood)

Chairman, Department of Zoology:

Dean, Faculty of Sciences:

Director, Advanced Studies:

In the Name of Allah, the Most Gracious, The Most Merciful

I dedicate this effort to my Parents, my family and all those who blessed me with their prayers…….

CONTENTS

Page

List of Tables viii

List of Figures ix

List of Abbreviations x

Acknowledgments xi

ABSTRACT xiii

1 INTRODUCTION 1

2 REVIEW OF LITERATURE 11

2.1 BRIEF REVIEW OF SOME BAT SPECIES 23

3 MATERIALS AND METHODS 32

3.1 STUDY AREA 32

3.2 COLLECTION OF SAMPLES 34

3.3 ANALYSIS AND PROCEDURE 35

3.4 ESTIMATION OF HEAVY METALS 37

3.5 STATISTICAL ANALYSIS 38

4 RESULTS AND DISCUSSION 40

4.1 41 DISTRIBUTION OF SPECIES AT DIFFERENT SITES

4.2 VARIATION AMONG ORGANS 42

4.3 REGIONAL VARIATIONS 47

4.4 SPECIES RELATED COMPARISON 52

4.5 METAL CONCENTRATION COMPARISON IN DIFFERENT SPECIES 58

4.5.1 Scotophilus heathii 62

4.5.2 Pipistrellus javanicus 62

4.5.3 Pipestrellus tenuis 62

4.5.4 Pipestrellus ceylonicus 64

4.5.5 Pipestrellus pipistrellus 64

4.5.6 Hypsugo savii 66

4.5.7 Megaderma lyra 66

4.5.8 Rhinopoma microphylum 68

4.5.9 Taphozous nudiventris 68

4.6 GENDER RELATED VARIATIONS 70

4.7 OVERALL COMPARISON 73

CONCLUSIONS AND RECOMMENDATIONS 79

SUMMARY 81

LITERATURE CITED 84

APPENDICES 117

LIST OF TABLES

Table No. Page 3.1 Study areas of central and northern Punjab for capturing of bats 36

4.1 Distribution of bat species captured from different sites of Central 43 and Northern Punjab

4.2 Qualitative comparison of intensity of metals in liver, kidney and 48 heart of mega and micro bats

4.3 Comparisons of metal concentrations (ug/g) in the liver, heart 49

and kidney of the bats

4.4 Qualitative organ wise comparison of metal prevalence between 53 mega bats of central and northern Punjab

4.5 Qualitative organ related comparison of metal prevalence in 55

the micro bats of central and northern Punjab

4.6 Species related multiple comparisons for accumulation of Lead 58

4.7 Species related multiple comparisons for concentration of Zinc 60

4.8 Mean metal concentration (µg/g) in three organs of Scotophilus 63 heathii

4.9 Mean metal concentration (ug/g) in three organs of Pipistrellus 63 javanicus

4.10 Mean metal concentration (ug/g) in three organs of Pipistrellus 65 tenuis

4.11 Mean metal concentration (ug/g) in three organs ofPipestrellus 65 ceylonicus

4.12 Mean metal concentration (ug/g) in three organs of Pipestrellus 67 pipistrellus

4.13 Mean metal concentration (ug/g) in three organs of Hypsugo savii 67

4.14 Mean metal concentration (ug/g) in three organs of Megaderma lyra 69

4.15 Mean metal concentration (ug/g) in three organs of Rhinopoma 69 microphylum

4.16 Mean metal concentration (ug/g) in three organs of Taphozous 71 nudiventris

4.17 A Comparisons of metal concentrations (ug/g) in the two sexes of 72 mega and micro bats

4.18 Comparisons of metal concentrations in µg/g between organs of 75 mega and micro bats

LIST OF FIGURES

Figure No. Page

3.1 Map of Punjab showing the study districts. 33

4.1 Organ related comparison of metal concentration between 54 the mega bats of central and northern Punjab.

4.2 Organ related comparison of metal concentration between 56 the mega bats of central and northern Punjab.

4.3 Accumulation level of Lead in different species of micro bats. 61

4.4 Accumulation level of Zinc in different species of micro bats. 61

LIST OF ABBREVIATIONS

Dw Dry Weight

ppm Parts per million

µg/g Micro gram per gram

NA Not Assessed

ANOVA Analysis of variance

LSD Least standardized deviation

SPSS Statistical Package for Social Sciences

NARC National Agricultural Research Council

PMNH Pakistan Museum of Natural History

PMASAAUR Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi

Cd Cadmium

Cu Copper

Pb Lead

Zn Zinc

WWF World wide Fund for Nature

IARC International Agency for Research on Cancer

IUCN International Union for Conservation of Nature

NEQS National Environmental Quality Standards

DRI Dietary Reference Intakes

NRC National Research Council

ASTDR Agency for Toxic substance and Disease Registry

PCRWR Pakistan Council of Research in Water Resources,

ACKNOWLEDGEMENTS

In the name of ALLAH, The Merciful and The Compassionate, who created the universe and Bestowed the mankind with knowledge and wisdom to search for its secrets; and whose bounteous blessings enable me to complete my research work.

Cordial thanks to Professor Dr. Mazhar Qayyum, Chairman, Department of

Zoology (PMAS.AAUR) and all of the faculty members for their critical insight and valuable suggestions whenever needed.

I wish to express sincere gratitude to my hardworking and responsive research supervisor, Dr. Muhammad Sajid Nadeem ; Associate Professor, who was more than generous with his expertise, and precious time in the planning and accomplishment of present work. I am also grateful to my Praiseworthy Co-supervisor Dr. Muhammad

Mahmood-ul-Hassan, Associate Professor, Department of Zoology and Fisheries;

University of Agriculture Faisal Abad, for his demonstrative guidance and a continuous support and encouragement.

I wish to thank my committee members, Dr. Mirza Azhar Beg, and Dr. Tariq

Mehmood for their skilful guidance and valuable suggestions during the study. Special thanks to Dr. Arshad Javed, Mr. Shahid Iqbal who rendered great help during long hours of capturing bats and providing samples during the field visits. I am thankful to Mr. Sana

Ullah and Mr. Rashid for their technical assistance in the Central Research Laboratory of

PMAS-Arid Agriculture University, Rawalpindi.

Special thanks are extended to all of my friends, especially Dr. Majid Mahmood,

Dr. Zahid Sharif, Muhammad Irfan and Syed Israr Shah for their cordial cooperation and help during my work.

Lastly, I extend my admirations to my family for a moral boost, encouragement and pray for my success whenever I was in despair. I alone remain responsible for the errors that may have crept into these pages, despite of the best efforts to avoid them.

Shagufta Nighat

ABSTRACT

Bats share a significant contribution to the economy of a state and reflect the status of plant and insect populations as well as the productivity of the ecosystem on which they feed and pollinate to enhance agricultural productions by their role as pollinator and seed disperser. They also have a key role in control of insect populations

Metals are elevating in the environment due to rapid industrialization, and the activities like, combustion of fuel, processing of metals, coal mining, automobile, lead-acid batteries, and building material. Metals play an important role in the biological functions of our body, but their bio toxic effects can be harmful and can disturb the normal biological functions. The oxidative stress due to toxicity of metals can cause lethal effects to different organs and systems of the body including nervous system. It also damages the DNA. This study was carried out to check the extant of heavy metals levels viz.

Cadmium (Cd). Copper (Cu). Lead (Pb) and Zinc (Zn) in different organs of mega and micro-bats from central and northern Punjab. Levels of these cumbersomely heavy metals were determined with the help of Atomic Absorption Spectrophotometer in the liver, heart and kidney of bats. Different qualitative as well as quantitative comparisons were made including organs, locality, species and gender. No significant difference was found in regional and gender comparisons. However, the study revealed the extent of heftily ponderous metals and their trend of accumulation in different bats which may be due to their food, metabolic activity or the area from where they were captured. Heavy metals concentration was significantly different in three organs of both mega and micro- bats but was lower than the toxicity values recommended for mammals. In an overall comparison, the metals were more concentrated in kidney than liver and heart and the

pattern for different metals was as: Zinc > Lead > Copper > Cadmium. Present study may provide a baseline data which could be considered a precursor to a broad array of issues, concerned to our environment and the health of both humans and biodiversity in particular utilizing bats as bio-be speakers and pointing out the impacts of heavy metal contamination on them. As conclusion, a perpetual monitoring is required to assess concentrations of toxic metals in bat fauna of Pakistan.

Chapter 1

INTRODUCTION

Environmental pollution has become a worldwide concern as it is likely to affect the ecological system and living organisms. Exposure of animals to environmental pollution is considered to be an important component of toxicology

(NRC, 2001). Bats are currently facing the threats because of habitat loss and other environmental problems all over the world (IUCN, 2010). Bats are very sensitive to human-induced changes in the ecosystems (Estrada, et al., 1993; Estrada and

Estrada, 2001a, 2001b; Medellín et al., 2000; Moreno and Halffter, 2001; Clarke et al., 2005 a, b ; Hayes and Loeb, 2007; Kunz et al., 2007).Species with smaller ranges of habitat and having specific ecologic patterns are facing greatest risk of threat as compared to those having generalized ecological requirements and larger geographical ranges (Jones et al., 2003).

Heavy metals are the metallic elements or metalloids which have comparatively high density (4 gm/cm3 or more) and have detrimental effects even in very low concentration (Agarwal, 2002). Heavy metals are present everywhere in all types of the environmental compartments and are highly toxic because they are non-biodegradable, insoluble in water and are very persistent due to having long half-lives (Burger et al., 2007). The indiscriminate release of harmful chemicals and metallic elements in the environment by industries and other activities of man may adversely affect the quality of our air, water and food

22 23

resources and once inside the living organisms through food chain, these chemicals may accumulate in higher concentrations and induce various metabolic disorders

(Hamidullah et al., 1997)

Metals originate naturally and are integral part of the environment and are found in varying amounts in all ground and surface waters of which some are essential, required for the normal metabolic activities of aquatic organisms, while others are non-essential having no significant role in the living organisms (Coetzee et al., in 2002). Metals like mercury (Hg), Lead (Pb), Cadmium (Cd), Nickel (Ni),

Cobalt (Co) and Zinc (Zn) are quite toxic in nature to both flora and fauna

(Gerbersmann et al., 1997)

Heavy metals are largely dispersed in the environment through industrial effluents, organic wastes, burning and transport refuse and also from the power generation (Agarwal, 2002). Among the heavy metals arsenic, lead, cadmium, and mercury are considered to have serious health implications on animals. The deposition of these elements in the body of an animal can cause severe damage to intestinal tract and skeletal, central nervous and reproductive systems and mucus tissues (ATSDR, 2007). The presence of some heavy metals in ecosystems

(including seas and oceans) can have deleterious effects because: they do not degrade and have long half-lives and they may bio-accumulate in living tissues, giving rise to symptoms of toxicity (Frazier, 1979). The manufacturing of pigments, drugs, agrochemicals, plastics, batteries, electroplating and discharge of untreated effluents from different industries also cause heavy metal pollution

(Raihan et al., 1995).

The heavy metal contamination resulting from industrialization, continuously changing agricultural practices, and pollution has negatively affected the animals including bat fauna. In order to study the effects due to heavy metals toxicity on the biodiversity of the region it was felt important to select a group of animals that are very sensitive to an array of environmental stresses to which they reflect and respond in foreseeable manner. Bats are exactly a group of mammals and can be important environmental indicators (Alleva et al., 2006).

Habitat loss due to growing human population and the anthropogenic activities such as deforestation, industrial activities, and use of pesticides, loss or alteration of buildings or deliberate disturbance to their natural habitat by man are the main causes due to which bat population is declining throughout the world. Bat are very sensitive to even the minute changes in their habitat, e.g. the loss of main landscape component during their flight results in the desertion of bat roosts especially when it happens with the maternity colonies (Jones et. al., 2009).

Anthropogenic activities degrade the habitats of bats especially the roosts used for breeding which can alter the reproduction timing n successful breeding in seasonal breeders e.g. in bats (O, Brien, 1993).

Bats belong to class Mammalia and order Chiroptera, which is further divided into two suborders i.e. Microchiroptera and Megachiroptera, remarkable for high diversity and broad geographic distribution only second to rodents

(Simmons, 2005). According to some estimates, about 1,250 species (25 per cent of living mammals) of bats are present in the world (Mickleburgh et al., 2009;

Simmons, 2005).

Due to unique location and by having fauna and flora as a blend of

Palearctic, Ethiopian and Indo-Malayan region Pakistan has a diverse bat fauna

(i.e. species richness) but endemic species are very low in number (Roberts, 1997).

Bats constitute about 28 per cent of all of the mammals species present in Pakistan

(Roberts, 1997) but the actual number of bat species present in the country has not yet confirmed and hence is an issue of consideration (Roberts, 1997; Bates and

Harrison, 1997; Walker and Molur, 2003; Wilson and Reeder, 2005). Mahmood- ul-Hassan and Nameer (2006) and Roberts, (1997) reported four species of mega bats in Pakistan viz., Pteropus giganteus (Indian flying fox), Cynopterus sphinx

(short nosed fruit bat), Rousettus eagypiticus arabicus (egyptian fruit bat) and

Rousettus leschenaultti (fulvous fruit bat) and 46 species of micro bats, however

Walker and Molur (2003) presume the presence of some more bat species from this region if bats are extensively surveyed, explored and accurately reported.

Roberts (1997) has reported two species of fruit bat viz. Pteropus giganteus and Rousettus leschenaultia from the Pothwar region. Pteropus giganteus (Indian flying fox) has a wide distribution in Indian sub-continent (Prater, 1971). Indian flying fox is reported from different areas of Lahore and Islamabad. Changa Manga forest plantation has a large colony of Pteropus giganteus, it is also found around

Bagh-e-Jinah and governor’s house. Another colony visits Renala Khurd in March,

every year and stay there until monsoon season (Roberts, 1997). In Islamabad it is present in Saidpur village, Rawal Lake and near the Lehtrar road. (Mahmood-ul-

Hassan and Nameer, 2006).

Bats generally have very specific roosting requirements, which differ among species. Majority of micro-bats of Pakistan (46 species), roosts in caves, crevices, trees, under logs, and even in human dwellings. They may also use different types of roosts at different times, for example, a species that hibernates in a cave during the winter may use crevices in tree holes as roosts during warmer months (Behr and Helversen, 2004). Roosts are that particular sites where bats spend most of their life time (Altringham, 1996). Removal of these trees and difficulty to access to these roosts may cause removal of bat population from a particular area. Insectivorous bats are nocturnal and carry out their activities mostly at night and often be seen spiraling around street lights to capture the insects at night (Jones et al., 2003). The conservation of such natural habitats is extremely important for many wildlife species including bats (Kunz and Parson, 2009).

Bats possess an assembly of features which make them very excellent bio indicators of human-induced climatic changes and due to their cosmopolitan distribution and diversity they are considered as best suitable to estimate the changes in quality of habitat across the world (Jones et al., 2009). As bats use a wide range of sources of food, they can be used as indicators for of environmental changes. Micro-bats are insectivorous and they are present at higher trophic levels, so they would prove to be best indicators and can reflect the relationship between

environmental disturbance and trophic levels in a better way (Alleva et al., 2006).

Bats show extraordinary feeding and social behaviors, roosting and reproduction patterns because of having powered flight which is an exclusive character of this species as compared to other mammals (Patterson et al. 2003;

Simmons and Conway, 2003). Micro- bats primarily consume nocturnal insects so are believed to play a principal role in minimizing nocturnal insect population by consuming a major population (up to 100 per cent of their body mass) in a single night and also in carrying the nutrients across the land e.g. from stream corridors to trees (Pierson, 1998). They use echolocation for finding their prey in air during flight. It was recorded in a controlled experiment that a small insectivorous bat,

Myotis catch up to 1200 small fruit flies in an hour (Whitaker, 1995). Mosquitoes are among the chief dietary elements of micro- bats. A huge number and variety of arthropodes consumed by bats includes hemipterans, coleopterans, lepidopterans and trichopterans (Anthony and Kunz, 1977; Whitaker, 1993; Agosta, 2002;

Agosta and Morton, 2003). A large number of economically important insects, including corn earworm moths, tobacco budworm moths and cotton bollworm moth are predated by bats (Whitaker, 1995; Lee and McCracken, 2005), which are devastating pests of many economically important agricultural crops such as cotton, corn, and potatoes (Whitaker 2004; Cleveland et al., 2007). Some larger species of micro-bats even use to prey upon many vertebrates species including frogs, rodents, lizards, or even fish (Meyers and Helversen, 2001).

The interest in the study and conservation of bats throughout the world has

been growing because bats are regarded as threatened animals so need protection and conservation (Stebbings and Griffith, 1986). Nectar feeding bats are a boon to agriculture, medicine and fruits as they are the best and effective pollinators on earth (Kunz and Pierson, 1994; Patterson et al. 2003; Simmons and Conway,

2003).There are a large number of commercially important plants which completely rely upon bats for their pollination (Kunz and Pierson, 1994). Mega bats have a great importance in regard that they have a great role in pollination and dispersal of seeds for many plants which are very much valuable economically such as bananas, mangoes, grapes and guava and the other plants used in the fiber industry, medicine, food, and ornamental material. According to an estimate there are about 289 plant species which produce many important products, depend upon bats for their pollination (Fujita and Tuttle, 1991).

Guano, (fecal material) of insect-eating bats, is also used as fertilizer for many crops in all over the world. Guano is an effective and valued fertilizer resource and is harvested by local farmers in many parts of the world. The ecosystem of a cave greatly depends upon guano as a source of nutrients (Beck,

2010). Bats are also used as food by human. A large number of mega chiroptera as well as some micro chiroptera are being hunted in the world (Mickleburgh and

Racey, 2004). This bat hunting is related to some cultural traditions and customs e.g in the Philippines. It is believed that the meat of bats has some medicinal properties (Mildenstein, 2002).

The devastating effects of toxic metals on the environment can be a severe

hazard to the quality and stability of ecosystem (Battaglia et al., 2005). Lead poisoning in birds has been a serious problem for more than a century due to a high exposure of lead (Grinnell, 1894; Wayland et al., 1999). Cadmium is found in different things, also been reported as one of the most harmful trace elements due to high level of toxicity and persistence (Battaglia et al., 2005). Zinc is one of the essential metals, required as a key element in many biological activities, but when its concentration rises in the environment, it can generate serious toxicological problems acting as micro contaminant (Perez-Lopez et al., 2006). Heavy metals present in animals can hinder their reproductive functions and output or even can cause death (Sanpera et al., 2000).

Globally, pollution due to heavy metals is often associated with urbanization from where heavy metals could accumulate and magnify up to a toxic level and cause a serious damage to the organisms as well as to the environment

(Guven et al., 1999). Organisms mainly obtain heavy metals from contaminated food as a result of which they may accumulate as a potential chemical hazard in their bodies. By increasing public concern and awareness regarding environmental contamination due to heavy metals, there is an emergent need to monitor, manage, evaluate and remediate the ecological damages (Kushlan, 1993).

Bats have been used as ecological indicators to check the health and quality of habitat globally, as they are very well suited to reflect the environmental changes either man induced or natural (Fenton et al., 1992; Estrada et al., 1993;

Wickramasinghe et al., 2003; Kalcounis-Rueppell et al., 2007). Bats are among the

animals which have very slow reproductive rates because they breed only once in a year and a single young is produced (Prater, 1971), so the populations recover very slowly if declines. The gestation period mar vary within different species in response to environmental conditions ( Racey, 1982). It is very difficult to detect the changes in population trends in short-term studies e.g. population estimates as compared to fast reproducing animals so the long-term population changes in bat populations can be monitored easily and directly (Walsh et al., 2001).

This group of mammals has been least studied in Pakistan, so the ecology and biology of bats is not well known (Mahmood-ul-Hassan and Nameer, 2006).

Pakistan has no bat specialist at the moment that’s why here is serious deficiency of taxonomic capacity to identify bats on the basis of their morphological characters, so Roberts as well as Bates and Harrison are regarded and quoted as the main and reliable sources of knowledge on exact number of bats species within the country (Mahmood-ul-Hassan et al., 2009). Bats are not given enough consideration in education, research or environmental policies so due to lack of extensive survey and research the bat data is sparse. No legal protection has been given to bats in Pakistan, specifically in Punjab and is listed under 4th schedule of

Punjab Wildlife Act. Many species of bats are given status of Least Concern by

IUCN although their population reported decreasing by CITES (2013).

The present study was proposed and has been conducted using bats as bio indicators. The study was mainly aimed to assess the concentrations of heavy metals (Cd, Cu, Pb and Zn) and their distribution in the liver, heart and kidney of

bats , keeping in view that heavy metals contamination have negatively affected bat fauna in the central and northern Punjab due to some anthropogenic activities. It was also aimed to know whether these metals are organ, region, sex or species related, and at which level they become hazardous or toxic in the environment. Bat are used as an indicator group of environmental changes in two regions of Pakistan for the first time. This is an effort to identify the extant and levels of metal contamination in our environment. There is no published data available from

Pakistan on the potential impacts of heavy metals on bats so this study will be considered as base line data for this particular species, and future studies will be focused on, whether the levels of concentrations are species specific and will changed over time. The study was proposed to address the following objectives:

 Assessment of heavy metal levels in Mega and Micro-bats

 Estimation and comparison of metal concentration in different organs of

bats (liver, heart, kidney)

 Determination of metal concentration in bats from Central and Northern

Punjab.

Chapter 2

REVIEW OF LITERATURE

Bats are well credited in Europe and America for their ecological services

(Mickelburgh, et al., 1992; Fujita and Tuttle, 1991). The positive role of bats in the ecological set up was acknowledged in Southeast Asia in 1998, when Wildlife

Protection Ordinance providing protection of all bat species passed by the

Malaysian government (Gumal and Racey, 1999). Parsad et al., (2013) reported that P. giganteus feeds on some commercially important fruits and medicinal plants and it is obvious that by doing so they are also playing an important role in the dispersal of seeds and pollination of some important plants. Frugivorous bats also believed to improve the germination rate of seeds by ingesting it and therefore their presence in a specific habitat is important for survival of many plants which ultimately affects the forest structure and composition during the early succession stages (de Carvalho-Ricardo et al., 2014).

Kunz and Pierson reported (1994) that the bat populations appears to be decreasing almost everywhere globally, due to a range of environmental threats, which are mostly anthropogenic e.g. habitat loss, decrease in food sources, environmental pollution etc. Several species have become extinct due to environmental changes especially island species.

Generally the public have negative attitude towards bats and most of the

11 33

people do not like them considering them as dirty creatures, a source of transmitting diseases to human and animals including livestock. Misbelieve resulting from the negative perceptions of public e.g. they are associated with evil, death and are the vampires that get stuck into your hair, are among the major reasons which bring many bat species to the limits of extinction in Pakistan

(Mahmood- ul-Hassan and Nameer 2006). Bats are more difficult to study than any of the terrestrial mammals because they require a different experience and specialized equipment so they have been comparatively less studied animals

(Fenton, 1997). The general negative perception about bats stems from lack of knowledge and the ignorance of their basic role, so because of conservation communities, educational campaigns have been initiated which have often changed the views about bats which was very crucial for their conservation (Sheikh and

Molur, 2004). Many species of the bats are hunted for body fats used to cure rheumatic pains and given no protection by law in Pakistan as in other countries

(Roberts, 1997).

Mammals generally show a multitude of adaptations, which make them able to exploit various types of habitats on land, air and in the water (Kalko, 1997;

Ramirez-Pulido et al., 2005). Order Chiroptera is very important and is significant for its high range of species diversity and widespread distribution globally

(Simmons, 2005). Chiroptera makes a major portion of the mammalian taxa

(Meyer et al., 2010) and up till now they encompass about 1250 known, living species and constitute almost 25 per cent of the mammalian biodiversity worldwide

(Simmons, 2005). Pakistan is home to ten out of the total twenty six orders of

mammals (Roberts, 1997) which indicates that it has a rich diversity as compared to any other area with same climatic conditions in the world (Mahmood-ul-Hassan et al., 2009). Out of the total 195 mammalian species of our country, 63 per cent comprises of Small mammals. They are the most diverse group but are among the least studied group of chordates (Roberts, 1997).

Megachiroptera has only one family; the Pteropodidae, while the

Microchiroptera includes sixteen families (Cockrum, 1962). In Megachiroptera, inter-femoral membrane is poorly developed, second digit of the hand has a well- developed claw and retains the degree of independence from third digit, the margin of the ear forms a complete ring and no tragus is present (Roberts, 1997). Sub order

Megachiroptera has only one sub family. Three genera of Pteropinae: Rousettus,

Pteropus and Cynopterus and four species are present in Pakistan. The largest and the best known genus, Pteropus, is primarily an island taxon. This genus shows a high level of endemism so mostly found confined to a single island but a few species are found in continental areas. Micro-bats have a wider range and distribution than mega bats and are found in both, old and new world (Whitaker,

2004).

Majority of the insect-eating species of bats roosts in tree hollows in

Pakistan (Mahmood- ul-Hassan and Nameer 2006). The protection and retention of these natural hollows in both living and dead trees is extremely important, both for bats and other wildlife. Cavities in buildings are equally acceptable roosting sites for these species and blockage or removal of entrance to the roosting sites

may remove the bat population from that area (Hill and Smith, 1985).

Bats show variation in their social structure and some of the bats like to live as solitary and others especially living in caves make colonies by more than one million bats (Roberts, 1997; Anonymous, 2010). A fission-fusion social structure is also observed in many bat species. The term "fusion" refers to bats that congregate together in larger groups in one roosting area and "fission" means to breaking up and the mixing of subgroups, often ending up in separate trees with different roost mates. Literature also showed that bats make different types of sounds to communicate with others. Bat scientists and experts have become able to identify bats by listening their sound which represents some particular behavior; they exhibit right after making these sounds (Whitaker, 2004).

Altringham (1996) reported that almost all of the Pteropodiformes and

Vespertilioniformes, except most of the pteropodids, use echolocation calls to find their prey and hunting, they produce these calls through mouth or nose. An effective monitoring of these echolocation calls may be helpful to study the ecology and biology of bats for the sake of their conservation. All echo locating bats uses a variety of roosting and foraging habitats and prefer to roost near a water body, which is used as the breeding land by most of the insect species. The commonest bat roosts include old buildings (human residences), crevices, ceilings, chimneys of the old reckless buildings and abandoned wells etc. Hollows of old trees and their bark also provide them a protected refuge in the sub-urban and periurban areas (Roberts, 1997; Anonymous, 2010).

Anthropogenic activities reduce the habitat quality and specific roosts required for successful breeding; such changes may also affect the timing of reproduction and breeding success which should be investigated. Brien (1993) reported that mammals living in the tropical harsh climates usually breed seasonally. In spite of this, the changed rainfall patterns, temperature and humidity along with human induced alterations in the habitat have changed the dispersal ranges of many species of bats over the past (Meyer et al., 2010). The human induced changes are badly affecting biodiversity at all levels. Specifically due to habitat fragmentation and degradation, several species of bats come at the verge of extinction. An extended and continuous monitoring of bats is a need of time that will be helpful to know about the negative human impacts on bat diversity in the world (Jones et al., 2003).

Recently, attention has been focused on the importance of trophic transfer in the spread of contamination in the environment. Studies have shown correlations between environmental risk of contamination and trophic positions within impacted ecosystems. In the past, the majority of research dealing with trophic transfer and bioaccumulation was performed in aquatic ecosystems (Sharpe and Mackay.2000).

Recently however, studies began to also focus on the transfer of contaminants within the terrestrial environment and the risks posed to upper trophic level organisms. Connell and Markwell (1990) observed that organic contaminants present in water, air, soil, and sediments move in a predictable fashion through invertebrates, fish, plants, mammals and birds, within the food web.

Heavy metals are present in the every part of the environment, they are highly persistent with long half-lives and they are not biodegraded easily (Burger et al., 2007). Heavy metals are of both types, some are essential elements (Mn, Zn,

Cu etc.) and the metals with unknown biological function are non-essential, such as

Cd, Hg, Sn and Ag. These metals are reported to be potentially very harmful for the organisms which have high level of exposure and absorption through their diet or from air (Guven et al., 1999). Higher levels of these metal ions are very toxic to animals including humans can damage the nervous system and disturb the function of many internal organs (Lee et al., 2006). Heavy metals contamination is a major problem at local as well as at the regional level around the world, and are very influential for both structural and functional validity of the ecosystem (Qadir and

Malik, 2009). The elements required in low quantity by living beings have been named as microelements, trace elements, or micronutrients. These elements (e.g. vitamins and minerals).are found to be very imperative for all biodiversity to continue their life processes. According to Pais and Jones (1997) all types of these macro and micronutrients combined together to fulfill the specific nourishment demands of a living body. Trace elements appear in man and in food and in various environmental compartments in a broad concentration range from natural ultra- trace levels and sometimes even below to the often increased due to anthropogenic pollution ppm level (Stoeppler, 1992).

Heavy metals are non-biodegradable natural elements while some others are stable environmental trace components of both fresh and marine waters however, their concentration level have been increased due to industrial, mining domestic

and some agricultural practices (Kalay and Canli, 2000; Yousafzai and Shakoori,

2008). Exposure of animals to environmental pollution has been regarded as important component of today’s toxicology. Environmental pollution has become a worldwide concern as it is likely to affect the ecological system and living organisms (Suter, et al., 1995).

All heavy metals are not beneficial for the body and some of them are considered to be xenobiotics and are very harmful even in minor concentrations.

These include aluminum, antimony, arsenic, barium, bismuth, beryllium, cadmium, uranium, mercury, lead, bismuth, arsenic (Altringham, 1996). Most of the heavy metals are non-biodegradable while some are used as stable environmental trace elements and so are the components of both fresh and marine waters, but the amount has been increased in the environment due to domestic, industrial, mining activities as well as the agricultural practices (Kalay and Canli, 2000; Yousafzai and shakoori, 2008). In higher concentration, these elements can act in either actually or chronically toxic manner (Gulfaraz et al., 2001). Heavy metals are constantly entering into the aquatic ecosystem from natural sources such as weathering of rocks or any volcanic activity. Moreover, industrial processes and some agricultural practices (e.g. CuSO4 is used to control aquatic vegetation) have greatly increased the deployment and the magnification of these toxic metals in freshwater. Therefore, in recent past, the public awareness and concern about heavy metal pollution has increased (Qadir et al., 2008).

The presence of heavy metals has been reported from many environmental

compartments in different species of birds and mammals. Many studies have been carried out to quantify the contamination level of toxic metals and their extant in the environment (Burger and Gochfeld, 1993; Goutner et al. 2001; Kojadinovic et al. 2007; Kim and Koo, 2007; Qadir et al., 2008). These pollutants are constantly releasing from different types of anthropogenic activities and some natural processes and are increasing in the environmental compartments. Many organisms are widely used as environmental indicators in order to monitors and to provide the evidence of exposure to these contaminants and their toxic effects (Covaci et al.,

2002; Mateo and Guitart 2003; Dauwe et al., 2003, 2004; Martin-Diaz et al. 2005;

Eeva et al., 2006; Kojadinovic et al., 2007). Many birds (pheasants, egrets etc.) have been used as bio-monitors to assess the environmental contamination because they are present at higher trophic level in the food-chain and so are exposed to an extensive range of chemicals which are geographically distributed (Burger 1994,

1995; Scheifler et al. 2006; Burger et al. 2007; Deng et al. 2007; Horai et al. 2007;

Naseem, 2009). Different studies have been conducted and reported on the accumulation and contamination of heavy metal in different organs of animals

(Elliott and Scheuhammeri 1997; Mateo and Guitart 2003; Deng et al. 2007; Horai et al. 2007; Kojadinovic et al. 2007). Heavy metals are important elements which cause oxidative stress, inhibit DNA repair and may cause changes in nucleotide bases. High oxidative stress can also accelerate the mutations in birds and animals

(Bickham et al. 2000; Eeva et al. 2006).

The use of laboratory mice to evaluate risks posed by various contaminants has been customary for decades, and toxicologists have long used these laboratory

mammals as a surrogate species for man (Pascoe et.al., 1996). Only recently have efforts focused on wild mammals to understand the bioavailability and uptake of metals within a natural ecosystem. For example, small mammals may serve as intermediaries for the transfer of toxic metals to higher trophic levels (Laurinolli and Young, 1996).

Bats are considered as best suitable to measure climatic changes and quality of habitat across the world as they are diverse in distribution and retain some exclusive taxonomic and functional diversity, but unfortunately, few studies have been carried out on the suitability of bats as indicator species (Jones et al., 2009).

Bats retain an excellent ability by which they can respond to a wide array of environmental changes and disturbances overall the world, such as habitat loss and fragmentation, urbanization, agricultural intensification, deforestation, global climate changes and overhunting etc. (Meyer et al., 2010). In addition to this, many species play a key role in the ecosystem services, especially in tropical ecosystems through pollination and seed dispersers, and in controlling insect populations (von

Helversen and Winter, 2003; Kalka et al., 2008; Kelm et al., 2008; Williams-

Guillén et al., 2008; Lobova et al., 2009; Mahmood-ul-Hassan et al., 2010). Bats are the only mammals having the capability of a true and persistent flight which make it possible for them to cross physical barriers that the other mammals are not capable of doing (Fenton, 1992; Wilson and Reeder, 1993; Hutson et al., 2001).

Bats use a variety of the terrestrial habitats; their typical habitats include deserts, agricultural areas, open fields, and other urban and suburban areas. Many

of the bat species may be found to forage near freshwater bodies like streams, lakes, ponds and mostly prey upon the insects which emerge from the water.

Generally one or more species of bats will be found in the terrestrial habitat if it provides an appropriate roosting site and sufficient food stuff for them. Bats are very specific in their roosting requirements, which may differ from species to species. They mostly like to roosts in crevices, beneath logs and foliage, caves and in human residences. A report revealed that bats use different types of roosts in different seasons of the year, which are appropriate to their requirements e.g. cave hibernating species in winter may use cavities in trees for roosting during summer season (Behr and Helversen, 2004).

Bats exploit a variety of food niches and may range from the primary consumers such as seed, pollen, leaf or fruits to top predators and consume arthropods or small mammals (e.g. Megaderma lyra) and even prey on the fish

(Hill and Smith, 1985; Fenton, 1992; Beolens et al., 2009). Most of the bat species prefer to roosts near the food source as in Pteropus, insectivorous bats show changes in their diet and foraging behavior with age (Anthony and Kunz, 1977;

Rol-seth et al., 1994). It has been described that the juvenile insectivorous bats start to forage during their first flights as insect remains are found in the fecal pellets of even the youngest flying bats (Jones et al., 1995). Insectivorous bats also showed habitat change, for example the habitat used by juvenile, Myotis lucifugus changed with age, older juveniles using both cluttered and opens habitats, while the younger juveniles mostly restricted to open habitats (Adams, 1996). Older females e.g.

Rhynconycteris naso exclude the younger females from their habitats (Bradbury

and Vehrencamp, 1977). A report showed that juvenile bats are apparently less experienced than adults at capturing and handling of prey so they spend more time in hunting and processing of their food e.g. juvenile big brown bats (Eptesicus fuscus) take long time in handling the insect prey as compared to adults (Hamilton,

1996).

The indiscriminate release of harmful chemicals and heavy metals in the environment by industries and other activities of man may adversely affect the quality of our air, water and food resources. These chemicals may find their way into the living organisms through food chain where these may accumulate in higher concentrations and induce various metabolic disorders (Hamidullah et al., 1997).

Exposure refers to the coincidence of a receptor and a stressor such as when they come into contact and interact in both time and space. There is no ecological risk if there is no sufficient exposure of a body to the contaminants (Risk Assessment

Forum, 1992). The exposure of wildlife to contamination may be via three routs: oral, dermal, and inhalational as move through the environment. Oral exposure occurs by consuming contaminated food, water or soil. Dermal exposure occurs when the contaminants are directly absorbed through skin while inhalational exposure is when volatile elements or fine particles are respired into the lungs. Oral exposure may occur from multiple sources, it may be by consuming contaminated food or water (either plant or animal) (Suter et al., 1995).

Bats have an excellent capacity in responding to the wide-ranging environmental changes for example, forest disturbances, agricultural amplification

and urbanization, habitat loss, fragmentation and overhunting (Meyer et al., 2010).

Bats have enormous potential to best suited as bio indicators, as they are taxonomically stable, their population’s trends can be assessed, and the short- and long term effects can be measured. Indicator species are associated with the ecosystem and indicates the health of the environment. According to Jones et al.

(2009) a healthy population of animales reflets that, the habitat is healthy as well.

2.1. BRIEF REVIEW OF SOME BAT SPECIES

Pteropus giganteus (Indian flying fox) is widely distributed in Pakistan,

India, Sri Lanka, Bangladesh and Myanmar. It is very rare in west Rajasthann and

Sind (Prater, 1971). In Pakistan it is present in Lahore near Punjab governor’s house and Jinah garden. One or two small colonies are present near Islamabad in

Saidpur village, in the vicinity of Rawal Lake and near the Lehtrar. A large colony is also present in the area of Changa Manga forest plantation. One colony is observed to visits Renala Khurd each year in March and stays there until monsoon season (Roberts, 1997). This species is categorized as ‘Least Concern’ (IUCN.

2008)

Rousettus leschenaultii also called as fulvous fruit bat is widely distributed in South East Asia as well as in the Indian peninsula (Prater, 1971). It is a migratory species and in summer season it usually visits and colonizes Himalayan valleys up to the height of 1220 meters (Roberts, 1997). In Pakistan it is present in

Lahore and Malir (Karachi). In spite of these known colonies in Lahore and Malir this species is mainly a summer visitor to Pakistan. It has been recorded from Azad

Kashmir, Peshawar, Malakand, Sialkot, and Karachi (Roberts, 1997). This species is categorized as ‘Least Concern’ (IUCN. 2008)

Taphozous nudiventris is a large size bat. The clavicular collar of paler hairs is absent in it. A well-developed gular pouch is present in adult males consisting of a crescentric flap of skin across the orificeis. It has a dog-like face with a long and semi-naked muzzle. Nose leaf is absent in this bat. It has large eyes and wide spaced triangular ears. Adult female can be identified by a crescent shaped mark on their face (Roberts, 1997; Bates and Harrison, 1997). The species is distributed in

Mauritania, Egypt, Jordan, Turkey, Tanzania and east Burma. This species is common and is widely distributed in Pakistan. Its specimens have been collected from Multan and Bahawalpur (Punjab) (Roberts, 1997), from Salt Range (Lindsay,

1928; Roberts, 1997). It is commonly present throughout Sindh e.g. in Sukker,

Kahairpur, Jaccobabad and Thatta (Siddiqui, 1961; Roberts, 1997). No any specimen has been collected from any mountainous region of Baluchistan or

Khyber Pakhtunkhwa (Roberts, 1997; Mahmood-ul-Hassan et al., 2009). IUCN.

(2008) placed this species in the “Least Concern” category.

Scotophilus heathii is distributed in Afghanistan, South China, Sri Lanka,

Thailand, Vietnam and Burma. This species is common in Pakistan and widely distributed throughout the Indus plains. It has been collected from Kohat (NWFP),

Islamabad city, Multan, Lahore and Sialkot (Punjab), Kashmoor, Sakkur,

Jacobabad, Mirpur, Dadu, Malir and Karachi (Sindh) (Lindsay, 1928; Siddiqui,

1961; Taber et al., 1967; Walton, 1974; Roberts, 1997). This species is also categorized as “Least Concern” by IUCN. (2008).

Pipistrellus pipistrellus is distributed in British Isles, West Europe,

Morocco; Greece, Turkey, Lebanon, Israel and Afghanistan, Pakistan, Burma,

China, Japan and Taiwan. It is very small in size bat that its tail length is smaller than its head and body length. Its tragus is banana shaped. The status of this bat species is unknown from Pakistan. Only one specimen was collected from Kashmir long ago in the beginning of 19th century which is kept in the British Museum however, two more specimens were collected in 1965 from Gilgit during an expedition of the Marryland University (Robers, 1997). The species has a restricted range in the Indian subcontinent (Bates and Harrison, 1997) however; it is expected to be commonly present in Pakistan But no further field studies and surveys were conducted on bats in Kashmir or Gilgit (Roberts, 1997). The species is also categorized to be ‘Common’ by IUCN, (2008).

Pipistrellus javanicus, is distributed in Afghanistan, North Pakistan, India,

South and East Tibet (China), Burma, Thailand, Vietnam, Philippines and perhaps

Australia.(Mahmood-ul-Hassan et al., 2009). The distribution within Pakistan is not reported clearly by literature; however a single specimen was collected and reported from Murree Hills (Gharial). The species placed in ‘Least Concern’ category by IUCN, (2008).

Pipistrellus ceylonicus, is distributed in India, Pakistan, Sri Lanka, Bangla

Desh, Burma, China and Vietnam (Mahmood-ul-Hassan et al., 2009). This species is common in Pakistan and especially common and abundant near Karachi and

Thatta (Sindh). It expected to be present in warmer southern latitudes of Indus plains (Mahmood-ul-Hassan et al., 2009). Taber et al. (1967) collected it from

Faisalabad (Lyallpur) and Khanewal. P. ceylonicus, is relatively of large size from all other bats of this genus. The body mass of adults’ ranges from seven to eight grams and the muzzle of this species often has glandular swellings between the eyes. Its ears, face, wings and inter femoral membranes are uniformly dark brown in color (Roberts, 1977). IUCN, (2008) categorized this species as “Least

Concern.”

Pipistrellus tenuis is distributed in Afghanistan, South China, Vietnam, and

Christmas Isle (Indian Ocean).This is the smallest of the all bats in the genus pipistrelle. This bat present in the subcontinent (Gopalakrishna and Karim, 1972).

The species has been recorded from Malakand (Roberts, 1997), Chitral (Sinha,

1980), Multan, Chaklala and Chakri, (Punjab), Sukkur (Hintona andThomas, 1926;

Siddiqui, 1961), Malir and Karachi (Walton, 1974). It is listed as ‘Least by IUCN,

(2008)

Hypsugo savii, is distributed in France, Portugal, Italy, Spain, Switzerland,

Austria, North Algeria, Spain, Turkey, Lebanon, Syria, Iran, Kazakhistan,

Turkemenistan, Uzebekistan, Afghanistan and North India (Wilson and Reeder,

2005; Mahmood-ul-Hassan et al., 2009). It is a medium sized bat with the tail length shorter than the head and body length. The status and distribution of the

species is unknown from Pakistan but literature qouted its presence in Pakistan

(Mahmood-ul-Hassan et al., 2009) bat its presence in Pakistan is not reported by

Simmons (2005), however Roberts (1997) and Walker and Molur (2003) have reported this species to be present in East Punjab, (India). The distribution and population status is unknown in the Indian subcontinent (Bates and Harrison,

1997). This species is categorized as ‘Least Concern’ (IUCN. 2008)

Megaderma lyra (False Vampire bat) is a robust species, relatively larger than the other species. The pelage is soft, fine and moderately large. It has broad wings. This species is widely distributed and can be found in many different biotypes. Roberts, (1997) reported this species from Shalimar garden Lahore,

Sialkot, Murree and Lehtrar, Islamabad. This species is distributed in

Afghanistanm South China, Thailand, Sri Lanka, Malysia, and Bangladesh. This species is categorized as ‘Least Concern/ erratically distributed’ (IUCN. 2008)

Rhinopoma microphlum is the largest among the three species of

Rhynopoma. The pelage is fine and short. The wings are short. It is very agile. This species is well adapted for the arid and desert habitats. This species is locally and erratically distributed in Pakistan. It has been recorded from Gujrat, Multan, Mailsi.

Jhellum, Sakkar, Karachi, Hyderabad, Lasbella, Malakand (Roberts, 1997). This species has been also recorded from all of the four provinces of Pakistan. The worldwide distribution of this species reported by Simmon, (2005), includes

Morroco, Senegal, Nigeria, Afghanistan, Pakistan, India, Burma, and Thailand.

This species is categorized as ‘Least Concern’ (IUCN. 2008)

The contamination due to heavy metals becomes a major problem and is of great concern at local and regional levels over the planet. The health of human depend upon the mobility of metals through environmental compartments and their exposure through which metals reach to humans and the environment (Qadir et al.

2008). According to a report the ecosystems are changing functionally and structurally at a speedy rate globally, due to human induced activities associated with the provision and processing of food and use of carbon-based energy production sources (NRC 2001, 2003; Hooper et al., 2005; Soares-Filho et al.,

2006). Presently, the human population continues to grow on an exacerbated rate and become more urbanized, so the environmental problems become even more intense as compared to past and hence the degree of change around the globe is quite obvious (Sala et al., 2000).

O’Shea and Johnson (2009) reported that the urbanization is the main cause of metal pollution. Several studies have reported varied amount of heavy metals and other toxic elements from bats, but a few of these have reported detrimental effects of these metals e.g. mercury concentrations reported from insectivorous bats, lead, cadmium and mercury are the most commonly reported heavy metals associated with toxic effects on wildlife species (Clark et al., 2005 a, b). In another study a high concentrations of cadmium in the guano of gray bats Myotis grisescens is reported from USA (Clark et al., 2005 a, b). Lead poisoning has been well-documented in both wild and captive fruit bats based on lead deposition in different organs leading to morbidity and mortality (Zook et al., 1970; Sutton and

Wilson 1983; Skerratt et al., 1998).

As reported in literature that heavy metals are non-biodegradable, they remain in the environment for a longer time and are highly toxic and eco-toxic, and are regarded as environmental contaminants (Burger et al., 2007). Several studies have documented varied amounts of heavy metal in different body tissues of different animals (Elliott and Scheuhammeri, 1997; Mateo and Guitart, 2003;

Deng, et al., 2007; Horai et al., 2007; Kojadinovic et al., 2007), e.g. in blood

(Scheifler et al., 2006), in eggs and in the feathers (Fasola et al., 1998). The accumulation levels and amount of the metals in living organisms are seems to be dependent on the species and size of individuals (Uysal et al., 1986). It also depends upon the nature of tissues or organs as well as the type of metal itself;

Many authors’ have reported the toxicity of heavy metal concentration in different tissues and organs of fish collected from different areas from where metal pollution was reported (Güven and Topcuoğlu, 1991). The results of these studies have shown that metals accumulation in the tissues of fish is dependent upon its exposure, level of concentration and duration of exposure (McGeer et al., 2000).

The overall effect of heavy metal pollution seems to be due to a tendency of increased accumulation of oxidative damage on the highly sensitive taxa however, but the general pollution in cities, some harbors, and in vicinity to an industry is high enough to leave negative impacts on wild animals. With increased global warming, there is an increasing concern which shows that metal pollution will cause further problems, which has already been confirmed in animals and humans

(Kelly et. al., 2003; Doherty et al., 2009). In addition, further studies are needed on the direct and indirect effects of pollution (such as reduced nutritional value of

foods, uptake of nutrients, and food abundance) on population dynamics and physiology.

Living organisms in every tier of the food chain are able to absorb metals from the water as it is more polluted by heavy metals as water sources constitute the major part of the biosphere (Uysal, 1986). Natural waters which are an environment for living organisms comprise a large food chain that characterizes the life itself. Phytoplankton is eaten by zooplankton and zooplankton is consumed by larger animals. With the thought of the cleaning capacity of water column’s itself; industrial wastes, household wastes and other polluting substances are released into this environment by nearly all countries. By the time being, their accumulation may have devastating effects on living organisms biologically (Kocataş, 1986).

The pollution status of heavy metals in Pakistan is of great concern and is documented by many studies; however for these metals (either essential or toxic) the health risk requires consideration of toxicity from excessive exposure of animals and plants. In many parts of the country, heavy metal contamination has been reported as a serious problem but very few studies are available concerning its health effect on the local population (PCRWR, 2010). Industrial byproducts such as chemicals, metals, dying, fertilizers, pesticides, textile, cement, petrochemical, leather, food processing and many others are among the major sources of the surface and ground water pollution in Pakistan. The discharge of industrial effluents, sewage, municipal wastes carried through drains and canals to the rivers

worsens and increases the water contamination problems in the country (Midrar-ul-

Haq et al., 2005).

Chapter 3

MATERIALS AND METHODS

3.1. STUDY AREA

Punjab (31°17΄040.6˝ N; 72°70΄97.16˝E) is a heavily populated and a developed province of Pakistan. It comprises about 56 per cent of the country's total population. Punjab is mainly a fertile plain with rivers and irrigation canals.

River Indus and its tributaries pass through Punjab from north to south. Its land is notably irrigated and canals can be found over a major part of the province.

Weather extremes are prominent, from the hot and barren south to the cool hills of the north. Most areas in Punjab experience fairly cool winters, often accompanied by rain. By mid-February the temperature begins to rise; spring time weather continues until mid-April, when the summer heats set in. The onset of the southwest monsoon is anticipated to reach Punjab by May. The official estimates the temperature above 46°C - 51°C in South Punjab. In August the oppressive heat is punctuated by the rainy season.

The northern parts of the province comprise Pothowar Plateau, which is an arid zone while the foothills of the Himalayas are found in the extreme north.

Pothwar consists of semi-arid climatic conditions. Typical humid subtropical climatic conditions are observed in this region with long and hot summer. It is large rainfed tract (Hussain and Prescott, 2006). Pothwar comprises of four districts of

31 53

Rawalpindi, Attock, Jhellum and Chakwal and some parts of Islamabad territory

(ICT).

The areas of central Punjab and northern Punjab were selected for bat sampling, regarding central Punjab as more industrialized as compared to the northern Punjab. Due to anthropogenic activities it was assumed that the population pressure may augment the environmental pollution in central Punjab as compared to the northern Punjab. The bat samples were collected from some different areas of Central Punjab e.g. Gujranwala, Faisalabad, Lahore and Shaikhupura and

Northern Punjab e.g. Jhelum, Chakwal, Attock, Rawalpindi, Islamabad and adjacent areas during the proposed period. (Fig.1). The type of industry in the

Central Punjab is mainly electrical fittings, chemicals, steel products textiles, sugar, food products, ceramics, electrical machines domestic machines, leather and the type of industry in the Northern Punjab is chemicals, engineering, poultry feed, furniture, cement and oil etc. which may be considered as the cause environmental pollution problems for the living species.

3.2. COLLECTION OF SAMPLES

Bat surveys were conducted during the study period (2010-2013) for locating roosting sites in different regions from dawn until dusk and mist nets were erected at the sampling site before sunset. The security measure were also kept assure for the field workers (students and teachers) while sampling in these areas.

Help was taken from some local people, to locate and inform about the roosting sites of bats. Different sub stations which were surveyed mist netted in the Central

Figure: 3.1. Map of Punjab showing the study districts.

Punjab were as Shalimar garden (31º35΄.333˝N, 074º22΄.863˝E), Bagh- e -Jinnah,

Badian (31°29΄.223˝ N 074°24΄.632˝ E) , Ali- pur Chathha (32º11΄.272˝ N,

074º09΄.361˝ E), Rasul Nagar (32º19΄.424 N 073º46΄.623 E), Forest Officers

Colony Gujranwala (32°5΄34˝N, 074°10΄55˝E), University of Agriculture,

Faisalabad (59°26΄31.2˝N, 073°4΄36˝E), Hiran Minar (31º97΄430.13˝N,

073º.955.197˝ E), Police lines Colony, Sheikhupura (31º42΄.44˝ N, 073º59΄.40˝ E) and the sub stations of Northern Punjab were, Saidpur Village (33°44΄17.3˝N,

073°04΄18.3˝E), Lake view (33°42΄38.3˝N, 073°07΄00.7˝E), Chak beli khan

(33°14΄85.0˝N, 33°14΄85.0˝N), Lehtrar, (33°42΄0.55.˝N, 073°25΄10.7˝E), Khokhar zar dam (32°48΄46.0˝N, 072°51΄40.9˝E), Hazro (Attock) (33°54΄45.2˝N,

072°29΄53.5˝E), NARC (33º39΄.892˝N, 073º07΄.108˝ E),Pakistan museum of natural history (PMNH) (33º43΄.194˝ N 073º03΄.631˝E), Gujar Khan

(33°15΄32.3˝N, 073°17΄25.8˝ E), Kheri Murat (33º27΄.594˝ N 072º44΄.066˝ E),

Daultana (33°11΄31.7˝N, 073°08΄42.1˝E).

Bat samples were captured from these areas using different types of nets

(mist nets and hand nets) following the capture methods of Kurta and Kunz (1989).

The length and height of mist nets varied from 2.6- 12.8 m and 2.3- 2.6 m respectively. The nets were laid in L or V shape preferably near water body just before the sun set in order to increase the capturing efficiency. Locally made hand nets were also used with the pole length of 6 feet. Capturing efforts were not always successful, so the roosting sites were visited on monthly basis. After capturing, the bats were carefully removed from the nets. GPS locations of roosts/netting stations were also taken by Garmin Etrax H. Global Position System

(GPS) (Table-1). It was tried to capture preferably, the samples of some common

species of micro and mega bats or the dead bats fallen on earth due to some hunters or accidentally and mostly samples of fruit bats were collected from the roosts nearby orchards and some home based small cultivations of fruit trees.

3.3. ANALYSIS AND PROCEDURE

After collecting and capturing, the bat samples (The whole bat) were brought to the laboratory of the Department of Zoology, PMAS-AAUR in small clean polyethylene bags, or cloth bags and preserved in refrigerator before being analyzed. Following the Walker et al. (2003) technique, date of collection, locality, sex, and species was noted for each sample. Most of the collected samples (bats) were adults and a few were sub adults so the criteria for age related comparison was not done in this study. Bats were identified up to species level on the basis of their external body measurements; they were compared following (Bates and

Harrison, 1997; Roberts, 1997; Mahmood-ul-Hassan et al., 2009). These bats were then dissected to excise liver, heart and kidney. All the samples were packed in polyethylene bag and kept frozen under 20°C until analysis. The organs were thoroughly washed with distilled water, dried and weighed. Both kidneys were pooled out for analysis. The organs were cut into pieces and a tissue of 2 gm (both mega and micro-bats) was transferred into quartz crucibles. Digestion of tissue was carried out with 1 ml of concentrated Nitric acid (HNO3) and 0.25 ml concentrated

Perchloric acid (HClO4). The free chlorine developed loosens the chemical bonds in organic compounds after gentle heating and destroy the organic matter in order to transfer the metals into the solution (Ostapezuk et al., 1984; Nighat et al., 2013).

Table 3.1: Study areas of central and northern Punjab for capturing of bats.

Coordinates No. of Region District samples Latitude Longitude Mega Micro bats -bats Central Lahore N31°32′36.47″ E74°20′18.66″ 08 Nil Punjab Gujranwala N32°8′60.00″ E74°10′60.00″ 21 13 Faisalabad N31° 25′ 4.8″ E73° 4′ 44.4″ 10 24 Sheikhupura N31°74′30.00″ E73° 95′ .52″ Nil 15

Northern Rawalpindi N33°40′38.00″ E72°51′21.00″ 16 26 Punjab Chakwal N32°56′0.63″ E73°43′14.57″ 09 14 Jhelum N33° 54′ 26″ E72° 18′ 40″ 04 16 Attock N33°43'4.749" E73'36.36.36" 11 10 Islamabad N 33° 42'0.55" E73'25.10.7" 15 35 Total 94 153

The digestion was initially preceded at low temperature and then at high temperature using hot plate until a clear straw color solution was obtained. These samples were diluted using de-ionized water and the final volume rose up to 10 ml for further processing.

3.4. ESTIMATION OF HEAVY METALS

The estimation of heavy metals was done following Sperling et al., (1999). The protocol is given here briefly; Atomic absorption spectrophotometry is a technique which gives accurate quantitative analyses of metal concentration in the samples from a variety of matrices. In order to analyze any given element, a lamp e.g. hollow cathode lamp is chosen which produce a wavelength of light that is absorbed by that element. The liquid sample is aspirated into a flame (flame, graphite furnace), where it dissolved and evaporation and atomization of the analyte occur. Air-acetylene flame is used for readily atomizable metals e.g. Zn,

Cu, Cd, Pb, Mn, Fe. A device e.g. photon multiplier can detect the amount of reduction of the light intensity due to absorption by the metal analyze, and this shows the amount of that element in the sample. Atomic Absorption

Spectrophotometer (AAS, G.B.C. 932 Plus U.K.) was used for the determinations of cadmium (Cd), copper (Cu), lead (Pb) and zinc (Zn). The precision and performance of the instrument was checked by analyzing the standard reference metal solutions before processing samples of bats under study. Samples were analyzed in triplicate to avoid any flaw obtaining exact quantity of metal. The values obtained from the sample were corrected for final digestion volume and

sample weight was taken. Metal concentration (µg/g) in each sample was calculated from the formula:

Metal conc. ( μg/g) = Absorbance of sample (ppm) x solution dilution (ml) Weight of tissue (gms)

3.5. STATISTICAL ANALYSIS

The data was arranged in a logical order, graphic presentation were made by MS Excel 2003. The data were found to have a non-normal distribution

(Shapiro-Wilk test, P < 0.05), therefore, data was log transformed to meet the assumptions of normality. Mean and standard deviation for metal concentration of each species of bats were calculated to assess the metal load within each organ.

One way ANOVA, Chi- square test and Student t-test were used for data analysis and mean values were compared by LSD (Least significant difference) and

Tuckey’s HSD (Honestly Significant Difference) test. Results are presented as mean ± Standard deviation. Values of P < 0.05 and P < 0.01 were considered as significant and highly significant when and where appropriate. All tests were performed in SPSS, version 17.0 (SPSS Inc. Chicago Illinois).

Chapter 4

RESULTS AND DISCUSSION

A total of 247 (94 mega-bats and 153 micro- bats) bats were collected during the study (2010-2013). Two species of mega bats and nine species of micro-bats were recorded during the study. Different levels of metals concentration and their distribution in mega and micro-bats were observed from two regions of

Punjab which were selected for collection of samples during this study.

Distribution and diversity of different bat species was observed from different localities and is presented in the table 4.1. Different types of comparisons were worked out to see either these metals are organ, region, gender or species specific, which confirm that heavy metals contamination, have negatively affected bat fauna in the central and northern Punjab.

The species of mega bats recorded during this study includes Pteropus giganteus and Rousettus leschenaultia belonged to family Pteropodidae. Rousettus leschenaultii was captured only from the central Punjab, so it is not used for comparing with the other species of mega bats for metal concentration. Only mean

± Sd., for this bat was calculated and is given in the Appendices. Roberts (1997) suggests that this species is a summer visitor to the Himalayan foothills up to the height of 1200 meter. The species of micro-bats recorded during this study were,

Pipestrellus pipestrellus, P. javanicus, P. tenuis, P ceylonicus, Hypsugo savii,

39 61

Rhinopoma microphyllum, Megaderma lyra, Scotophilus heathii, and Taphozous nudiventris belonging to four different families, viz Vespertilionidae,

Megdermatidae, Embellonuridae and Rhinopomatidae. Different levels of heavy metals viz. Cd, Cu, Pb and Zn concentrations were observed in different species captured. The compounds of heavy metals such as cadmium (Cd), copper (Cu), lead (Pb) and zinc (Zn) are dangerous micro-contaminants having a direct toxic effect on the aquatic organisms and through the food chain process eventually they might threaten human health. The first two metals are essential in trace quantities, but are markedly toxic, whilst lead and cadmium are non-essential element (Bryan,

1976). Bats possess multiple of characteristics that make them excellent bio indicators of man induced changes and are considered as very suitable to measure climate change and habitat quality across the world (Jones et al., 2009). Different comparisons were done to see the distribution and level of concentration in different bat species. In overall comparison, the bioaccumulation pattern of metals concentration in different organs was noted as: kidney > liver > heart and the pattern for different levels of metals concentration was as: zinc > lead >copper > cadmium. The results were reported on dry weight (dw) basis as micro gram per gram (µg/g). The study revealed that levels of heavy metals were under the permissible limits for mammals as per WHO (1989); viz. copper -71, .zinc -289, cadmium -173, and lead-25 μg/g (dw). Different comparisons showing the level of metal contamination/concentration within organ, species and regions and gender are presented in detail in tabulated form of thesis. In qualitative comparison the intensity of metal is shown in percentage but in quantitative comparison the exact concentration level or the bioaccumulation is shown in μg/g (dw).

4.1. DISTRIBUTION OF SPECIES AT DIFFERENT SITES

The distribution of different species and the diversity was recorded during the study period from different roosting sites of proposed regions. (Table-4.1).

Maximum species diversity of bats was observed from different areas of

Rawalpindi. It includes both mega- and micro-bats. The probable reason for this may be that this area has a large number of fruit orchards, on which bats visit for eating fruits (mega bats) and a large population of insects must be present there on fruiting trees, which would be beneficial or pests for plants. Micro-bats visit these areas to capture these insects which are a favorite food of these bats. After this

Chakwal, Islamabad and Faisalabad are the regions from where we captured different species of bats. Pipestrellus pipestrellus, Scotophilus heathii and Pteropus giganteus were the common species captured from more roosting sites. Hypsugo savii was captured only from Islamabad, likewise Rhinopoma microphyllum and

Megaderma lyra, were captured only from Rawalpindi. Mega bats were captured from different areas of Lahore. Small mammals are common links in the transfer of contaminants from soils and vegetation to higher trophic levels. Thus, metal concentrations in small mammals can provide information regarding potential for bioaccumulation, within the ecosystem and food chain transport of these contaminants (Johnson et.al., 1978).

4.2. VARIATIONS AMONG ORGANS

In the qualitative comparison (Table 4.2) significant difference was recorded in the per centages of all four metals viz. Cd, Cu, Pb and Zn (P ˃ 0.05) in

Table 4.1: Distribution of bat species captured from different sites of Central and Northern Punjab.

GPS Location No of Species Netting Longitude Latitude Species found stations Captured

N33°43'4.749" E73'36.36.36" Scotophilus heathii, Pipistrellus 4 pipistrellus, Pipistrellus tenuis, Attock Pteropus giganteus

N32°56′0.63″ E73°43′14.57″ Taphozous nudiventris, Scotophilus heathii, Pipistrellus ceylonicus, Chakwal Pipistrellus javanicus 6

Pipistrellus tenuis, Pipistrellus pipistrellus, Pteropus giganteus

Faisal Abad N31° 25′ 4.8″ E73° 4′ 44.4″ Pipistrellus tenuis , Taphozous nudiventris, Scotophilus heathii, Pipistrellus ceylonicus, 6

Pipistrellus pipistrellus,, Pteropus giganteus

Gujranwala N32°8′60.00″ E74°10′60.00″ Scotophilus heathii, Pipistrellus tenuis, 4 Pipistrellus pipistrellus,, Pteropus giganteus

Islamabad N 33° 42'0.55" E73'25.10.7" Hypsugo savii, Scotophilus heathii, 6 Taphozous nudiventris, Pipistrellus ceylonicus, Pipistrellus pipistrellus, Pteropus giganteus

Jhellum N33° 54′ 26″ E72° 18′ 40″ Taphozous nudiventris, Scotophilus 4 heathii, Pipistrellus javanicus, Pteropus giganteus

Lahore N31°32′36.47″ E74°20′18.66″ Pteropus giganteus, Rousettus 2 leschenaultia

Lehtrar N 33° 42'0.55" E73'25.10.7" Scotophilus heathii, Pipistrellus tenuis, 3 Pipistrellus pipistrellus,

Rawalpindi N33°40′38.00″ E72°51′21.00″ Megaderma lyra, Taphozous 7 nudiventris, Rhinopoma microphlum, Pipistrellus javanicus, Pipistrellus tenuis, Pipistrellus pipistrellus,, Pteropus giganteus

Shaikhupura N31°74′30.00″ E73° 95′ .52″ Taphozous nudiventris, Scotophilus 3 heathii, Pipistrellus pipistrellus,

liver and heart of mega bat but it was non-significant for kidney, while in micro- bats all the four metals showed significant difference in all three tissue samples

(liver, heart and kidney). Concentrations of Cd (cadmium), Cu (copper), Pb (lead) and Zn (zinc) in different body tissues of mega and micro-bats collected from central and northern region of Punjab, Pakistan (Table 4.3).

When computed the level of metal concentration i.e. quantitative comparison between different organs of both mega and micro-bats all the four metals were significantly different among tissues (P ≤ 0.05, Table-4.2). The concentration levels given for different organs are estimated by atomic absorption spectroscopy method, which shows that this particular organ has this particular amount of metal, and are given in results in µg/g. Zinc was found highest in the liver tissues of both mega and micro-bats as 4.422 ± 2.61µg/g and 4.163 ±

2.38µg/g respectively. The bio accumulation pattern of metals concentration in different organs was as: kidney >liver > heart, and for different metals was as: zinc

> lead >copper > cadmium. Spear (1981) reported that zinc mostly deposited in bones, liver and kidney in animal’s body although zinc is present in all tissues of organisms.

Results of the present study revealed that Concentration of cadmium was highest (2.28±1.99 µg/g) in the kidney of mega bats and in the heart of micro-bats

(0.49 ± 0.65 µg/g). Copper showed highest level of concentration in the heart tissues of both mega bats (2.85 ± 1.54 µg/g) and micro-bats (0.96 ± 1.15 µg/g).

Lead was highest in the liver tissues of mega bats (3.61 ± 5.27µg/g) while in the

kidney tissue samples of micro-bats (1.72 ± 1.32µg/g). Zinc was with highest concentration among all the four metals in both of mega as well as micro-bats, it showed high concentration in the liver samples of mega bats (4.42 ± 2.61 µg/g) as well as in liver of micro-bats (4.16 ± 2.38 µg/g). This study reveals that levels of heavy metals were below the tolerable limits for mammals recommended by WHO

(1989).

Uysal et al. (1986) reported that the accumulation of the metals in living organisms are dependent upon the species, the size of individuals, tissues and organs as well as the type of metal itself. Bats eat a variety of food from flower to insects, fish and small mammals, 70 per cent of the bats are insectivorous. Arellano et al. (1999) reported that differences in the pattern of heavy metals distribution might be a result of the differences in feeding habits, habitats, ecological needs and metabolism of the animals. Méndez and Alvarez-Castañeda (2000) gave comparative analysis in the liver of two species of ichthyophagous bats and reported the heavy metal load as (μg/g in dry weight) 133 (Zn), 27.4 (Cu), 1.25

(Pb). 6.5 (Cd) for Myotis vivesi and 57 (Zn) 13.1 (Cu), 0.57 (Pb), 8.0 (Cd) for

Noctilio leporinus. In present study the concentrations of zinc and lead were high in the liver of micro-bats while cadmium and copper were lower but these values are lower than the above values except lead which was 1.66 (μg/g).

Heavy metals are non-biodegradable natural resources while some are stable environmental trace components of fresh and marine waters, but their levels have increased now due to domestic, industrial, mining and many agricultural

practices (Kalay and Canli 2000; Yousafzai and Shakoori, 2008). The water contamination with a wide range of pollutants became a matter of great concern over the last decades in the world (Al-weher, 2008). According to Uysal et al.

(1986); the accumulation levels of the metals in living organisms are dependent to different things viz. species, the size of individuals, tissues and organs as well as the type of metal. If the concentration levels of heavy metals or these micro contaminants increase beyond a certain level, they can act either rapidly or chronically toxic manner (Gulfaraz et al., 2001). In a research conducted on rats in

Florida, male rats showed focal testicular necrosis, reduced spermatogenesis, and reduced fertility associated with about8.5 pprn cadmium in the kidneys (Kotsonis and Klaassen, 1977).

The compounds of zinc, copper, lead and cadmium appear to be dangerous contaminants having a direct toxic effect on the aquatic organisms and through the food chain process eventually threaten the human health (Bryan, 1976). The first two metals are essential in trace quantities for enzymatic activity and many biological processes otherwise they are markedly toxic while the non-essential metals such as cadmium, mercury and lead are toxic even at low concentrations.

Investigations on toxicity make possible to reveals the effects of sub-lethal concentrations on growth, behavior, physiology, and biology of organisms, and also to determine their adaptive capabilities to forecast the possible consequences of toxic effect of metals (Carlson, 1971; Galvez et al., 1998).

Analytical recoveries determined by Walker et al. (2007) in England from

kidney samples of brown long-eared bats were 112 ± 5.22 per cent (n=23) for Pb and 96.2 ± 4.01 per cent (n=22) for Cd, these values are much higher from the values recovered in this study from the kidney of micro-bats ( Pb was 1.72± 0.12

µg/g and Cd was 0.37 ± 0.04 µg/g) Streit and Nagel (1993) reported, in adults of the insectivorous bat Pipistrellus pipistrellus, levels of lead in the liver between

2.95 and 38.5 mg/g dry mass, and copper levels between 15.7 and 32.0 mg/g dry mass. When these values were compared with values of liver in this study and they were as,1.66 µg/g for lead and 0.24 µg/g for copper which were found to be within the limits given in above values. Kawai et al. (1976), investigated cadmium levels in bats at Judges Cave and they have reported that the values were high enough to damage the population.

Investigations on toxicity make it possible to evaluate effects of sub lethal concentrations on growth, behavior, physiology, and biology of organisms, to determine their adaptation and to forecast possible consequences of toxic effect

(Carlson, 1971; Galvez et al., 1998).

4.3. REGIONAL VARIATIONS

Non-significant difference was noted in the level of metal concentrations

(µg/g) between regions sampled for mega bats and micro-bats (Fig. 4.1 and 4.2) but a significant difference was observed in the intensity of metal given in per cent age

(qualitative comparison) for zinc in the heart tissue of mega bats and cadmium and

Table 4.2: Qualitative comparison (per cent age) of metals in liver, kidney and heart of mega and micro-bats.

Family Organ Cadmium Copper Lead Zinc P value* (n=153) (n=153) (n=153) (n=153) Mega Bats Liver ( per 100 100 92.5 98.8 0.002

cent)

Heart ( per 94.6 97.8 100 96.8 0.037

cent)

Kidney ( per 92.6 95.7 94.7 93.6 0.810

cent)

Micro-bats Liver ( per 28 88 82 95 0.000

cent)

Heart ( per 15.7 78.4 80.4 97.4 0.000

cent)

Kidney ( per 25.4 85.6 77 98.6 0.000

cent)

*Pearson Chi square 2 sided significance

Table 4.3: Comparisons of metal concentrations (µg/g) in the liver, heart and kidney of the bats (mean ± standard deviation).

Cadmium Copper Lead Zinc P value

Mega bats Liver 0.781±1.11a 1.792 ± 1.14b 3.613 ± 5.27c 4.422±2.61c 0.000

Heart 0.696±0.62a 2.855 ± 1.54b 2.289 ± 1.87c 3.761±2.67d 0.000

Kidney 2.287±1.99a 1.666 ± 1.19b 2.158 ± 1.38ab 3.503 ± 2.74c 0.000

Micro Liver 0.236±0.25a 0.754 ± 1.04a 1.659 ± 1.51b 4.163 ± 2.38c 0.000 Bats Heart 0.490 ± 0.65a 0.958 ±1.15ab 1.292 ± 0.72b 4.075 ± 2.05c 0.012

Kidney 0.378 ± 0.29a 0.827 ± 0.91a 1.721 ± 1.32b 3.983 ± 2.78c 0.000

Similar super scripts in each row shows non-significant difference (P > 0.05)

copper in the liver samples of micro-bats in the samples taken from central and northern regions of Punjab (Table 4.4 and 4.5). It was hypothesized that Central

Punjab is more industrialized than the Northern Punjab hence more metal pollution was expected from this area. However due to urbanization, perhaps Northern

Punjab is now equally industrialized in population and pollution therefore no significant difference in metal levels observed but proportionally there was difference in metal concentrations in different bat species. Another probability is that the samples were collected from the areas having no heavy industry in the vicinity. The distribution disparity pattern for some metals was observed in the samples of mega bats collected from these regions.

It was observed that zinc was proportionally high in the liver, heart and the kidney of the bat samples from northern Punjab. It revealed that zinc is rising in northern Punjab due to industrial activities, such as mining coal, waste combustion and steel processing etc. Zn is used in dye casting, automobile and rubber industries. Although Zn is essential for human health but it can be dangerous and can damage the pancreas due to high levels and it can also disturb the protein metabolism (Goyer, 1997).

Lead was observed proportionally high in the samples of liver and kidney collected from central Punjab. Lead can damage almost every system in human body. Lead is used in building constructions, lead-acid batteries, bullets and shots and is a part of solder, paints. Lead coated water pipes are used in our houses. Lead is also present in imported canned foods, ceramic dishware and cigarette smoke.

Copper was high in all of the bat samples of the northern region while it was

relatively high in the kidney samples collected from the Central region. Copper is commonly used in everyday objects including, water pipes, locks, door handles, and electrical objects. Copper leaches out from copper containers in which acidic foods are stored and occasionally copper piping is a potential source of copper in the diet if water is slightly acidic and has been allowed to remain in contact with the piping for a longer time. Malik et al. (2010) reported that in Pakistan, surface and ground water contamination with Cu does not show any significant problem, and the total concentration of Cu is in the range of 8.88–357.40mg/kg in the industrial area the capital city of Pakistan (Islamabad). Cadmium occurred in the comparable proportion in liver and heart but it occurred in higher proportion in the kidney samples of the bats from Central Punjab. Cd exposure may result from commercial applications including electroplating and manufacture of batteries and fertilizers and it can cause pulmonary, renal and cardiac disorders in humans and kidney and liver disorders in birds Cd and its compounds are carcinogenic to humans and are classified as Group 1 by International Agency for Research on

Cancer, as Cd and its compounds cause lung cancer, Cd toxicity can also leads to the kidney, pulmonary and skeletal damages and a positive associations have been observed for cancers in the kidney and prostate (Goyer, 1997). In Pakistan, high Cd concentration in drinking water may come from effluents discharge of marbles, steel industries as well as from mining and metal plating. The concentration of Cd in waste water of Central Punjab was observed above the safe limit set by NEQS which ranges from 0.18 to 0.37mg/L (Mahmood and Malik, 2014). In another study, variation of Cd ranges from 0.19 – 0.62mg/L in the northern regions of

Pakistan, (Khan et al., 2009). Natural and anthropogenic sources contribute to the

levels of Cd and sediment concentrations of Cd range from 0.03 to 1mg/kg in marine sediments and as high as 5mg/kg in river and lake sediments on worldwide level (IARC, 2012.). In the soil of Islamabad, capital city of Pakistan, the dust road along Islamabad the expressway cd concentrations ranges from 5.8–6.1 and 4.5–

6.8mg/kg, it has been found that these values are much higher than many of the cities around the world (Sezgin et al., 2004; Faiz et al., 2009).

In the liver samples of micro-bats collected from both regions of Punjab, all the four metals from central were found to be relatively more heavily loaded than the samples of the northern Punjab. In the heart samples from central region, zinc, lead and copper were relatively high with the exception of the cadmium in the samples of northern region. As for case of kidney, zinc and lead were greater in the samples of northern region while copper and cadmium were proportionally high in the kidney samples of central Punjab.

Punjab is the most industrialized and heavily populated province of

Pakistan, both in terms of extent and type of industry. Different industries producing textiles, sports goods, heavy machinery, electrical appliances, surgical instruments, cement, vehicles, sugar mill plants, aircraft, agriculture machinery, and processed foods are present in the Punjab. The urbanization and industrialization for human welfare and development has led to increase the disposal of heavy metals, radio nuclides, and various types of pollutants in the environment (www.Wikipedia, 2013).

Table 4.4: Qualitative organ wise comparison of metal prevalence of mega bats between central and northern Punjab.

Organ Metal Central Punjab Northern Punjab P value

(n = 38) (n = 56)

Liver Cadmium (per cent) 100 100 1.00

Copper (per cent) 100 100 1.00

Lead (per cent) 97.3 89.3 0.14

Zinc (per cent) 97.3 98.2 0.64

Heart Cadmium (per cent) 94.7 94.6 0.68

Copper (per cent) 94.7 100 0.16

Lead (per cent) 100 100 1.00

Zinc (per cent) 92.1 100 0.06

Kidney Cadmium (per cent) 94.7 91.1 0.41

Copper (per cent) 97.4 94.6 0.47

Lead (per cent) 97.4 92.8 0.32

Zinc (per cent) 92.1 94.6 0.46

*Pearson Chi square 2 sided significance

Figure: 4.1.Organ related comparison of metal concentration between the mega bats of central and northern Punjab.

Table 4.5: Qualitative organ related comparison of metal prevalence in the micro-bats of central and northern Punjab.

Organ Metal Central Punjab Northern Punjab P value (n = 52) (n = 101) Liver Cadmium (per 38.5 22.8 0.033* cent) Copper (per 94.2 83.1 0.042* cent) Lead (per cent) 90.4 83.1 0.385

Zinc (per cent) 98.1 94 0.241

Heart Cadmium (per 11.5 17.8 0.221 cent) Copper (per 84.6 74.3 0.103 cent) Lead (per cent) 86.5 77.2 0.122

Zinc (per cent) 98 97.1 0.581

Kidney Cadmium (per 30.7 22.8 0.189 cent) Copper (per 86.5 85.2 0.512 cent) Lead (per cent) 71.1 80.2 0.145

Zinc (per cent) 98.1 99 0.566

*Fisher's Exact test 1 sided significance

Figure: 4.2.Organ related comparison of metal concentration between the micro-bats of central and northern Punjab.

Environmental challenges of Pakistan are primarily associated with an imbalanced economic and social development in recent decades. All major cities of Pakistan face haphazard, unplanned expansion due to a shift of population from rural to urban areas which worsen the situation to cope up with this challenge. Since the municipal authorities or other utility service providers have limited resources, haphazard urban congestion is the prime reason for deterioration of natural resources like air, water and soil quality (WWF. 2008; PCRWR. 2010).

4.4. SPECIES RELATED COMPARISON

As there are nine species of micro-bats captured, so a species wise comparison in the micro-bats was made but it was not worked out for mega bats because there was only one species collected from the two regions. The metal concentration within species was calculated as an overall comparison in all the metals was pooled and the metal load in a particular species was determined. In another comparison the mean metal concentration was determined in which the load in each organ of a species was recovered and presented here. The results showed that the concentration levels of lead and zinc were significantly different among different species of micro-bats (Table- 4.6; 4.7, Fig. 4.3; 4.4) but there was no difference in concentrations of cadmium and copper in an overall comparison of micro-bats, lead concentration was highest in Megaderma lyra (3.03±2.31 μg/g) and lowest in Taphozous nudiventris (1.12 ± 0.85 μg/g (Fig-4.2)). Zinc with highest mean metal concentration was note in Rhinopoma microphyllum (5.75 ± 3.08 µg/g) and was lowest in Pipestrellus javanicus (3.06 ±1.98 µg/g (Fig-4.3).

The environmental factors affect the uptake and accumulation of metals in fish. (Jezierska and Witeska, 2001). Various species of fish from the same water body may accumulate different amounts of metals so interspecies differences in metal accumulation may be related to living and feeding habits (Kidwell et.al.

1995). This study revealed that the bio accumulation of metals differs within species, the possible reason for that may be due to the feeding pattern or due to the metabolic profile of metals. O’Shea, et al., (2001) reported that insectivorous bats accumulate the elements after feeding on insects that had spent the aquatic phase of their life cycle in contaminated water. Lead is reported to bio accumulates in the skeleton and wet tissue of mammals and it reduces the reproductive capacity of the animals. Several studies have reported the lead poisoning and potential Pb- mediated effects on reproduction in bats (Clark, 1979; Sutton and Wilson, 1983;

Hariono et al., 1993). Walker et al. (2007) reported differences of metal concentration in the kidneys among different species viz, pipistrelles, brown-long eared or whiskered bats and in Natterer’s bats. The levels of concentration of Pb in barbastelle bats were observed as 2.17 μg/g and Cd concentrations ranged between

0.28 and 2.13 μg/g.in the kidney. Heavy metals include both essential elements and the metals with unknown biological function. Zinc was the second most abundant metal followed by iron in the list of essential metals. Zinc is reported as an essential micronutrient which catalyzes the enzymatic activity, make protein structure, and regulates the gene expression in animals (DRI, 2001). Zinc is present in all the tissues of all organisms; in general, zinc-specific sites of accumulation in animals are bone, liver and kidney (Spear, 1981). Zn deficiency has been recognized for many years but it can be toxic when exposures exceed physiolog-

Table 4.6: Species related multiple comparisons for accumulation of Lead (µg/g) by Tukey’s HSD.

Species M. P. P. P. P. R. H. S. T. P pipistrellus Javanicus tenius ceylonicus microphylum savii heathii nudinentris value Lyra

Mean 3.03a 1.89b 1.86bc 1.75bc 1.60bc 1.42bc 1.41bc 1.35bc 1.12c 0.000

SD 2.31 1.85 0.39 0.61 0.97 0.84 0.75 0.81 0.85

Similar super scripts in each row shows non-significant difference (P > 0.05)

4 3.5 3 2.5 2 1.5

1 Lead conc. (µg/g) conc. Lead 0.5 0

Micro bat species

Figure: 4.3. Accumulation level of Lead in different species of micro-bats.

Table 4.7: Species related multiple comparisons for concentration of Zinc (µg/g) by Tukey’s HSD, (mean ± standard deviation).

Species R. P. P. H. T. M. S. P. P. P value microphylum ceylonicus pipistrellus savii nudinentris lyra heathii tenius Javanicus

0.001 Mean 5.75a 5.24ab 4.26ab 4.21ab 3.99b 3.97b 3.88b 3.38b 3.06b

SD 3.08 2.39 3.12 4.03 2.21 1.74 1.79 3.30 1.98

Similar super scripts in each row shows non-significant difference (P >0.05)

7 6 5 4 3 2

1 Zinc conc (µg/g) conc Zinc 0

Micro bat species

Figure: 4.4. Accumulation level of Zinc in different species of micro-bats.

cal needs (Solomons and Ruz, 1998). The acute gastrointestinal effects and headaches, impaired immune function, changes in lipoprotein and cholesterol levels, reduced copper status, and zinc iron interactions (DRI, 2006) The largest natural emission of zinc to water results from erosion. Natural inputs to air are mainly due to igneous emissions and forest fires. Anthropogenic and natural sources are of a similar magnitude. Khan et al., (2011)

4.5. METAL CONCENTRATION COMPARISON IN DIFFERENT

SPECIES

4.5.1 Scotophilus heathii:

In this species Zinc accumulation was (3.78± 1.93 µg/g) followed by lead

(1.09 ± 0.90 µg/g) and copper (1.04± 1.66). Cadmium was lowest (0.03 ± 0.09

µg/g) among all metals. The same pattern was observed in the heart and kidney i.e.

Zn > Pb > Cu > Cd. Zn was found with maximum metal load in all the three organs and cadmium with the lowest (Table-4.8).

4.5.2. Pipistrellus javanicus:

The mean metal load observed in this species of bats was comparatively low, however distribution and bioaccumulation was different within liver, kidney and heart. In this species maximum concentration of metals was observed in heart as compared to other two organs. Cadmium was not found in the liver and heart of

Table 4.8: Mean metal concentration (µg/g) in three organs of Scotophilus heathii.

Liver Heart Kidney

Cadmium 0.03 ± 0.09 0.03 ± 0.09 0.10 ± 0.22

Copper 1.04 ± 1.66 0.56 ± 0.80 1.05 ± 1.36

Lead 1.09 ± 0.90 0.85 ± 0.86 1.12 ± 1.00

Zinc 3.78 ± 1.93 4.05 ± 1.98 3.46 ± 1.66

Table 4.9: Mean metal concentration (µg/g) in three organs of Pipistrellus javanicus.

Liver Heart Kidney

Cadmium NA NA 0.26 ± 0.24

Copper 0.34 ± 0.10 0.96 ± 0.48 0.53 ± 0.35

Lead 1.77 ± 0.53 1.84 ± 0.59 2.10 ± 0.19

Zinc 2.78 ± 0.64 4.06 ± 2.29 2.02 ± 2.07

this species. Zn was the most accumulated metal in all of the three organs but its highest load was in the heart (4.06 ± 2.29 µg/g ; Table 4.9).

4.5.3. Pipestrellus tenuis:

In the P. tenuis the order of mean metal concentration was again as liver ˃ kidney ˃ heart. Zn was found with highest load and cadmium with the lowest all three organs studied. Cadmium usually concentrates in the internal organs of animals rather than in muscle or fats. Cd accumulates typically higher in kidney than in liver, and also higher in liver than in muscles. Cadmium is a non-essential heavy metal with toxic effects; it may accumulate in humans from food chain and its deposition usually increases with age (WHO, 1992). Zinc was highest (4.55

±3.38 µg/g) in liver and lowest (2.14 ± 1.01µg/g) in the heart (Table 4.10). Wild animals may drink water from different water sources and consume soils that differ in contaminant levels and by the relative proportion of daily food consumption attributable to each food type and the contaminant concentration in each food type.

(Al-weher, 2008).

4.5.4. Pipestrellus ceylonicus:

This species was observed heavily loaded with the metals. All the three organs showed higher mean values for zinc .In the liver mean concentration of zinc was 5.91 ±2.65 (µg/g), in kidney 5.23 ±2.87 (µg/g) and in heart, 4.59±1.40 (µg/g).

Cd was lowest in all the three organs (Table- 4.11). PCRWR, (2010) reported that in Pakistan, main contributors to the surface and ground water pollution are the byproducts of various industries such as taxtile, metal, dying chemicals, fertilizers,

Table 4.10: Mean metal concentration (µg/g) in three organs of Pipistrellus tenuis.

Liver Heart Kidney

Cadmium 0.10 ± 0.17 0.47± 0.96 0.12 ± 0.17

Copper 0.60 ± 0.37 1.27 ± 1.09 0.63 ± 0.51

Lead 1.41 ± 0.77 1.40 ± 0.82 1.74 ± 0.94

Zinc 4.55 ± 3.38 2.14 ± 1.01 3.44 ± 4.43

Table 4.11: Mean metal concentration (µg/g) in three organs of Pipestrellus ceylonicus.

Liver Heart Kidney

Cadmium 0.03 ± 0.07 0.04 ± 0.11 0.04 ± 0.16

Copper 0.29 ± 0.27 0.37 ± 0.55 0.28 ± 0.36

Lead 0.70 ± 1.09 0.67 ± 0.80 1.46 ± 1.22

Zinc 5.91 ± 2.65 4.59 ±1.40 5.23 ± 2.87

pesticides, cement, petrochemical, energy and power, leather, sugar processing, construction, engineering, food processing, mining, and others.

4.5.5. Pipestrellus pipistrellus:

In this species the highest mean concentration of Zinc was found in kidney

(4.79 ± 4.22 µg/g), and lowest in liver (3.05 ± 2.75 µg/g). Pb accumulated more in liver (2.44 ±2.96 µg/g) than other two organs. Copper and cadmium load was higher in kidney, followed heart and liver. Like other species Cd was lowest in the entire organs. Many authors reported that kidney and liver are mostly the target organ to accumulate toxicants like heavy metals but here in this species, heart showed more mean concentration than liver (Table -4.12).

4.5.6. Hypsugo savii:

H. savii was collected only from Islamabad area. Cd was not detected in heart and kidney of this species however a small concentration is recorded from liver. Zn accumulated almost equally in liver, heart and kidney. Small concentration of Pb and Cu was also found in all of the three organs. Kidneys were found with lpwest metal load in this species (Table-4.13).

4.5.7. Megaderma lyra:

Liver was again heavily loaded with metals in this bat species followed by

Table 4.12: Mean metal concentration (µg/g) in three organs of Pipestrellus pipistrellus.

Liver Heart Kidney

Cadmium 0.11 ± 0.22 0.13 ± 0.39 0.16 ± 0.35

Copper 0.51 ± 0.68 0.80 ±1.55 0.46 ± 0.44

Lead 2.44 ± 2.96 1.32 ± 0.70 1.60 ± 0.76

Zinc 3.05 ± 2.75 3.88 ± 2.22 4.79 ± 4.22

Table 4.13: Mean metal concentration (µg/g) in three organs of Hypsugo savii.

Liver Heart Kidney

Cadmium 0.01 ± 0.01 ND ND

Copper 0.75 ± 0.67 0.32 ± 0.53 0.10 ± 0.07

Lead 1.70 ± 0.92 0.57 ± 0.72 0.36 ± 0.44

Zinc 3.04 ± 0.94 3.46 ± 1.70 3.43 ± 1.13

heart and kidney. In this bat the pattern of accumulation of heavy metals was found as Kidney ˃ liver ˃ heart. In liver Zn accumulation was 4.22± 2.18 (µg/g), lead was

2.18± 0.43 (µg/g) and cadmium 0.42± 0.52 (µg/g). It was observed that cadmium accumulated more than copper in liver of this species. Heart has highest value for zinc 3.41± 1.74 (µg/g) followed by Pb, Cu and Cd. In the kidney mean metal load was observed as Pb (4.71± 3.60 µg/g), Zn (4.27± 1.39 µg/g) then Cu and Cd. In

Lead is reported to bio accumulates in the skeleton and wet tissue of mammals and it reduces the reproductive capacity of the animals. Several studies have reported the lead poisoning and potential Pb-mediated effects on reproduction in bats (Clark, 1979; Sutton and Wilson, 1983; Hariono et al., 1993).

4.5.8. Rhinopoma microphylum:

Few samples of this species were captured from different areas of

Rawalpindi. Liver deposited greater load of matals i.e. zinc 6.11± 3.26 µg/g, lead

1.32± 1.06 µg/g, copper 0.22±0.29 µg/g and cadmium 0.10 ± 0.11 µg/g. Heart and kidney have a high mean concentration of zinc followed by lead, copper and cadmium (Table 4.15). Zinc is present in all the tissues of all organisms; but zinc- specific sites for accumulation in animals are bones, liver and kidney (Spear,1981).

The acute gastrointestinal effects and headaches, impaired immune function, changes in lipoprotein and cholesterol levels, reduced copper status, and

Table 4.14: Mean metal concentration (ug/g) in three organs of Megaderma lyra.

Liver Heart Kidney

Cadmium 0.42 ± 0.52 0.09 ± 0.14 0.07 ± 0.06

Copper 0.31 ± 0.25 1.01 ± 0.75 0.46 ± 0.34

Lead 2.18 ± 0.43 2.21 ± 0.18 4.71 ± 3.60

Zinc 4.22 ± 2.18 3.41 ± 1.74 4.27 ± 1.39

Table 4.15: Mean metal concentration (ug/g) in three organs of Rhinopoma microphylum

Liver Heart Kidney

Cadmium 0.10 ± 0.11 0.03 ± 0.09 0.05 ± 0.12

Copper 0.22 ± 0.29 0.46 ± 0.47 0.62 ± 0.64

Lead 1.32 ± 1.06 0.96 ± 0.60 0.82 ± 1.15

Zinc 6.11 ± 3.26 6.11 ± 3.26 4.10 ± 2.24

zinc iron interactions all are due to higher zinc accumulation (DRI. 2006). Khan et al., (2011) from Pakistan reported, Zn as a significant element of automobile components, its presence in the roadside soil showed that vehicular traffic is the major anthropogenic source of pollution.

4.5.9. Taphozous nudiventris:

Zinc was found greater in heart (4.32± 2.17 µg/g) followed by lead and copper. Similarly Zn was high (3.45± 2.17µg/g) in the liver; than lead (0.89±

0.85(µg/g) and copper; (0.68 ±0.50 µg/g) Concentration of Cd was lowest (0.04±

0.09µg/g) in liver. The pattern of metal concentration was as Zn˃ Cu ˃ Pb ˃ Cd in the kidney samples of this bat (Table 4.16).

4.6. SEX RELATED VARIATIONS

Dimorphism is present in bats which are morphologically shown by different sexes as male and female. During the study it was observed that female bats (mega bats) were slightly larger in size as compared to males. Mean metal concentration was 2.41 ± 2.12 µg/g in the male and 2.43 ± 2.16 µg/g in the female samples of mega bats.

However mean metal concentration in micro-bats was observed as

2.03±2.22 µg/g in males and 2.18±2.20 µg/g in females. No significant difference was observed in the metal contamination between male and female of mega and micro-bats (Table.4.17).

Table 4.16: Mean metal concentration (ug/g) in three organs of Taphozous

nudiventris

Liver Heart Kidney

Cadmium 0.04 ± 0.09 0.03 ± 0.12 0.06 ± 0.13

Copper 0.68 ± 0.50 1.05 ± 1.44 0.89 ± 0.74

Lead 0.89 ± 0.85 0.86 ± 0.75 0.80 ± 0.90

Zinc 3.45 ± 2.17 4.32 ± 2.17 4.01 ± 2.55

Table 4.17: Comparisons of metal concentrations (µg/g) in the two sexes of mega and micro-bats (mean ± standard deviation).

(n) Male Female P Value

Mega Bats 66 ♂ 28 ♀ 2.41 ± 2.12 2.43 ± 2.16 0.847

Micro-bats 74 ♂ 79 ♀ 2.03 ± 2.22 2.18 ± 2.20 0.231

However, difference in level of metal accumulation was observed in both sexes. Female bats showed a slight higher metal accumulation in them as compared to the male bats. The accumulation level of different metals in different tissues of both sexes may be influenced by a combination of factors, such as dietary preferences, physiological metabolism in relation to stage in the reproductive cycle or foraging behavior (Alquezar et al. 2006). Khan (1995) and Komarnicki (2000) reported the gender differences with respect to bioaccumulation pattern of nonessential metals in several species of mammals including human. They reported that Zn tends to deposit and increases in the soft tissues of females of some mammals such as mole.

Generally, the sex differences due to the concentration difference for deposition of essential elements viz. Co, Fe, Zn, and Mo may be associated with differences in the metabolic profile of metals involved in the activity of sexual hormones, the uptake of metals, nutritional requirements or interactions between elements (Goyer, 1997; Chmielnicka, 2002; Lopes et al., 2002). In a study on bats in 1979, Clark reported that lead in mammals has been found at higher levels in females as compared to males except for the big brown bat where concentrations in males were significantly higher than females by about 1.5 times. Males evidenced with significantly high mean values for Pb and Co in liver at a polluted site of

Spain but no significant sex-dependent variation was detected for kidneys, males and females showed a similar pattern of Cd accumulation in livers (Pankakoski et al., 1993; Clark et al., 1992: Komarnicki, 2000). In another study no significant difference of any metal on renal concentrations due to sex or age was observed in

the brown long-eared bats in south west England (Walker et. al., 2007).

In pipistrelle bats, however, adults had significantly higher kidney Cd concentrations than the juveniles but there was no variation in the renal Pb concentration. It was also reported that the renal Hg and Pb concentrations have not been found to increase with age in various other terrestrial mammals (Massie et al.,

1993; Shore and Rattner, 2001; López Alonso et al., 2003).

4.7. OVERALL COMPARISON

In living organism heavy metals get accumulated when they are taken up and stored at a faster rate as compared to the rate at which they are broken down and metabolized or excreted out of the body. The heavy metals are poisonous as they bio accumulate, meaning that the level of the toxicant increased in biological organism over time as compared to the environment.

To study and check the overall levels of toxic metals or the net load, all of the four metals (Cd, Cu, Pb, Zn) studied were pooled within organs of both mega bats and micro-bats. The mean metal concentration in the liver of mega bats was

2.43 ± 0.2.37µg/g. It was observed as 2.40 ± 0.10 µg/g in the kidney and 2.41 ±

0.11 µg/g in the heart. In overall comparison for three organs of mega bats the pattern of metal accumulation was as liver > heart > kidney. The heavy metal load in micro-bats was observed as 2.76 ± 0.15 µg/g in the liver, 2.11 ± 0.11 µg/g, in the kidney and 1.79 ± 0.07 µg/g, in the heart. The pattern of metal accumulation in

Table 4.18: Comparisons of metal concentrations in µg/g between organs of mega and micro-bats (mean ± standard deviation).

Liver Kidney Heart P Value

Mega Bats 2.43 ± 0.2.37 2.40 ± 0.10 2.41 ± 0.11 0.989

Micro-bats 2.76 ± 0.15a 2.11 ± 0.11b 1.79 ± 0.07c 0.000

micro-bats was as liver > kidney > heart. The result showed that there was no significant difference between metal concentration in the liver, heart and kidney of mega bats but, there was a highly significant difference in the organs of micro-bats.

The study also revealed that the target organs for heavy metal contamination are liver and kidney because the bioaccumulation trend for metals was observed as; Liver > kidney > heart. Liver was observed with high metal load as compared to other two organs in both mega and micro-bats (2.43 ± 0.12 µg/g in mega and 2.76 ± 0.15 µg/g in micro-bats (Table - 4.18). Heart showed the minimum load for the metal contamination in both of the mega and micro-bats.

Liver accumulates high concentrations of metals, irrespectively of the uptake route and liver is considered a good monitor of water pollution with metals. Metal concentrations in the kidneys rise slower than in liver, and usually reach slightly lower values, except for such metals as cadmium and zinc that show very high affinity to kidneys, therefore the kidneys may be considered a good indicator of pollution too. During depuration, kidney metal levels (Jezierska and Witeska,

2001).

Medvedev (1995) reported that kidney and liver are the principal target organs for metals in mammals. Uysal et al. (1986) reported that the accumulation levels of the metals depend on species, the size of individuals, tissues or organs as well as the type of metal to which exposure is occurred. The accumulation level and toxicity of heavy metals is generally found to be species specific and the metal itself and might be related to their feeding habits and the bio-concentration

capacity. It is well recognized that heavy metal uptake occurs mainly from water, food and sediments (Huang, 2003).

However, the efficiency and level of metal uptake from contaminated water and food may differ in relation to ecological needs and metabolism of the body of the animal. Streit and Nagel (1993) reported levels of lead in the liver ranged between 2.95 and 38.5 μg/g for cadmium between 0.044 and 1.53 μg/g and copper levels between 15.7 and 32.0 μg/g in the insectivorous bat Pipistrellus pipistrellus, however, in present study lead concentration was 1.658 μg/g, cadmium 0.236 μg/g and copper was 0.754 μg/g in the liver of micro-bats. Streit and Nagel believed that the hunting area was strongly affected by metal contamination so the values reported are much higher. Thompson (2007) reported that generally, concentrations of Pb above10 mg/g on a wet weight basis in the kidney and liver are diagnostic for

Pb toxicity in animals.

Bats have been used to monitors levels of different metals e.g. copper in cases of water contamination resulting from oil drilling and mining. The bats accumulated these contaminants after feeding on insects that have spent the aquatic stage of their life cycle in contaminated water. Fresh fruits have comparatively low copper concentrations (O’Shea , et al., 2001).

Lead accumulation is reported in wild mammals, but few reports reveal the toxic effects of the metal on the wild or on non-laboratory species. Lead is identified to be very toxic to plants, animals and microorganisms. The bio

accumulation of Pb by fish may create detrimental effects on fisheries resources and could constitute a considerably health hazard, to man. Lead reduces reproductive capacity while the consumption of the lead polluted fishes by man interferes with hemoglobin synthesis (WHO, 1989).

Numerous authors have reported the lethal effects of zinc and copper in different species of mammals. Copper is documented as a serious cause of liver toxicity in many domestic animals e.g. dogs, cats, horses, cattle, sheep and goats.

(Kelly, et al.,1993). Copper is often not considered as a threat to humans except when present in at abnormally high values, where it causes anaemia, disorder of bones and connective tissues and liver damage but copper toxicity has been reported in various wildlife species that may be hazardous for the animals and showing copper contamination.

The main sources of zinc are mining, zinc production facilities, iron and steel production, corrosion of galvanized structures, coal and fuel combustion, waste disposal and incineration and the use of zinc-containing fertilizers and pesticides. Ramsay (1965) explained that the heavy metals exposure estimates should be considered as the main reason for differences found in contaminant concentrations of different foodstuff exploited by the animals.

CONCLUSIONS AND RECOMMENDATIONS

This study confirms the presence of heavy metals (Cd. Cu, Pb, Zn) in the liver, heart and kidney of mega and micro-bats but the concentration is not up to the levels associated with the adverse effects in mammals. There was no significant difference (P>0.05) in the concentration of heavy metals of bats collected from central and northern Punjab. Results revealed that accumulation of zinc and lead in the body tissues has increased with increasing metal concentrations in the environment.

Bats are generally disliked by the people, they perceived as they are dirty creatures and are thought to be responsible for transmitting diseases as they stuck in hair or by entering in human ears. Many dead specimens were found especially of mega-bats which showed that the orchardists killed them because these consume fruits, conservation management should focus on this aspect, which might result in the decrease of bat population. It will take time and effort to change the present thinking and to make people “bat-friendly”. The best way to overcome this problem is, a campaigns should be started at school and college level giving education to children by fun and different activities for bat awareness and conservation after which they should start admitting the positive role of bats and will become admirers and devotees for their conservation.

Although heavy metals are important to man in many respects as used in the manufacture of many important house hold products but their bio toxic effects

79 101

cannot be denied when unduly exposed. It is recommended therefore that every industry and factory should be given a plan for the disposal of effluents by legislation which they must follow, otherwise they should be penalized. Legal and adequate protection should be given to the breeding colonies of bats as protected in other countries, particularly for the threatened species in our country.

Since bats are the least studied animals in Pakistan, so more research and studies are recommended to check the extant of toxicants in the food of micro and mega bats which is one of the causes of species decline. As conclusion, environmental pollution is increasing by increasing the industrialization. Bat data is very sparse in the country; this study will be used as a baseline data to compare metal contamination in different bat species. A continuous monitoring of bat population and their exposure to environmental toxicants in the biosphere is required to assess the critical habitats and the causes of species decline which is then correctly be interpreted.

SUMMARY

An analytical study was carried out to measure the proportion, pattern of distribution of heavy metals (Cd, Cu, Pb, Zn) as well as their extant within the species in different organs of bats as these species proved to be the excellent bioindicators for human-induced changes, climate and habitat quality changes, globally.

Heavy metals are one of the major sources of pollutants in the environment and are discharged largely into the air, as a result of various industrial activities as well as traffic and energy production. Organisms’ exposure has been increased to pollution due to industrialization as a result of the public demand for an improved life quality.

During the study bat surveys were conducted for locating roosting sites and samples were collected from different regions of central and northern Punjab. The exact location of these regions was taken using GPS. Two species of mega bats viz.

Pteropus giganteus and Rousettus leschenaulti (Pteropodidae).were captured from both central and northern Punjab. Rousettus leschenaultii was captured only from the central Punjab, so it is not used to compare with the other species of mega bats for metal concentration. Nine species of micro-bats were recorded during this study, Pipestrellus pipestrellus, P. javanicus, P. tenuis, P ceylonicus, Hypsugo savii, Rhinopoma microphyllum, Megaderma lyra, Scotophilus heathii, and

Taphozous nudiventris belonging to four different families, viz Vespertilionidae,

Megdermatidae, Embellonuridae and Rhinopomatidae.

81 82

A total of 94 samples of mega bats and 153 samples of micro-bats were collected during the study. Bats were captured and brought to the laboratory of

Zoology Department of PMAS-AAUR. Bats were dissected to excise liver, heart and kidney. After preliminary requisite the samples were analyzed on Atomic

Absorption Spectrophotometer to find out concentrations levels of heavy metals.

The data was statistically computed. All the values obtained for heavy metal load in the specific organs are expressed in micro gram per gram (µg/g) of dry weight.

The mean metal concentration in mega bats resulted with significant difference among three organs. Zinc with mean concentration 4.42±0.27 μg/g was highest in the liver, followed by 3.76±0.27 μg/g in heart and 3.50 ± 0.29 μg/g in the kidney. Lead was 3.61 ± 0.56 μg/g in liver, following heart with mean concentration 2.29 ± 0.1 μg/g and 2.16 ± 0.14 μg/g in the kidney. Copper was observed with mean metal concentration as 1.79 ± 0.12 μg/g in the liver, following

2.85 ± 0.16 μg/g in heart and 1.66 ± 0.12 μg/g in the kidney. Cadmium was observed minimum in mean concentration in all of the four metals analyzed. The bio accumulation pattern resulted was relatively greater 2.29±0.21 μg/g in the kidney, then 0.78±0.11 μg/g in the liver, and 0.69±0.06 μg/g in the heart and of mega bats. In mega bats non-significant difference was observed when compared qualitatively for liver, heart and kidney. In region wise and gender related comparisons there was non-significant difference in the mean metal concentration however proportional concentration differences were observed during this study. In region wise qualitative comparison, zinc and lead were observed in 97.3 per cent of samples collected from central Punjab but zinc was 98.2 per cent and lead was 89.3

per cent recovered from northern Punjab.

In the samples of micro-bats, region wise, organ wise, gender related and species wise comparisons were worked out. Metal concentrations were found to differ significantly within the three organs of micro-bats. Zinc showed mean metal concentration as 4.16 ± 0.19 μg/g in the liver, 4.07 ± 0.16 μg/g in heart and 3.98 ±

0.22 μg/g in the kidney. Lead was greater1.72 ± 0.12 μg/g in the kidney followed by liver, 1.66 ± 0.13μg/g and in heart it was 1.29 ± 0.65 μg/g. Copper accumulated more in the heart as 0.96 ±0.10 μg/g then 0.83 ± 0.07 μg/g in the kidney and then

0.75 ± 0.09 μg/g, in the liver. Cadmium concentration was 0.24±0.03 μg/g in liver,

0.49 ± 0.13μg/g in the heart and 0.38 ± 0.04 μg/g in kidney. There was non- significant difference in the metal concentration of samples of micro-bats collected from both regions but in the qualitative comparison micro-bats showed significant difference for cadmium and copper. Gender related comparison also showed non- significant difference for both sexes of micro-bats. Species wise comparison was also worked out for micro-bats. Lead and zinc were significantly different among different species of micro-bats. Lead was highest in mean concentration in

Megaderma lyra (3.03±0.54 μg/g) and lowest in Taphozous nudiventris (1.12 ±0.09

μg/g) when the overall concentration of metals was compared in different species.

Zinc concentration was observed greater in Rhinopoma microphyllum and was lowest in Pipestrellus javanicus (3.06 ± 0.47μg/g), there was no significant difference in concentrations of cadmium and copper in different species.

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APPENDICES

Table_ Mean metal concentration (µg/g) in three organs of Rousettus leschenaultia

Liver Heart Kidney

Cadmium 0.29 ± 0.31 0.25 ± 0.57 0.58 ±0.91

Copper 0.43 ±0.32 0.24 ± 0.15 0.30 ± 0.34

Lead 0.89 ± 0.90 1.26 ±1.06 1.35 ±1.06

Zinc 2.96 ± 1.21 1.50± 0.59 1.51±0.82

Liver: Zn > Pb > Cu > Cd

Heart: Zn > Pb > Cd > Cu

Kidney: Zn > Pb > Cd > Cu

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2.5

2 Liver 1.5 Heart Kidney 1

0.5

0 Cadmium Copper Lead Zinc

Figure: Mean metal concentration in three organs of Rousettus leschenaultii

119

Photographs of Some Field Visits

Lake View Psrk

120

Bagh- e – Jinnah, Lahore

Lake view Park

121

FFish Hatchery, Faisal Abad

Chak Beli Khan

122

NARC. Islamadab

Lake View Park

123

Lehtarar

Fish Hatchery, Faisal Abad

124

Fish Hatchery, Faisal Abad

Laboratory, Zool, Dept.PMAS_AAUR

125

FFaisal Abad

Lehtarar