CHAPTER 1: INTRODUCTION

1.1 Research Background

As reported by Lim (2012), bats are volant small mammals and can be divided into two

suborders, which are Megachiroptera and Microchiroptera. Megachiroptera are known as fruit

bats and they feed on fruits or nectar from the fruit trees that they locate using their excellent

sense of smell and color vision, on the other hand, Microchiropterans are insect bats which

usually have a smaller size than Megachiropterans (Lim, 2012). The examples of common prey

insects consumed by Microchiropterans are mosquitoes, termites, cucumber beetles, ants,

leafhoppers, and roaches.

Commonly bats are nocturnal or active on night-time and can be found from all range of

habitat types such as caves, boreal forest, and mountains. Their habitat can be across the range

of the ecosystems as they exhibit a wide range of habitat associations, behaviors, and their

roosting, as well as their foraging activities. According to the past research, the foraging habitat

chosen by bats depends on their ability to fly, their shape of wings and the echolocation call.

Bats usually found in the habitat that enables the bats to conserve their energy where the

optimum condition for the environment may be provided by the habitat they inhabit

(Bartholomew, 2014). Most abundant bats species in the temperate region cover much more area

than tropics (Lim, 2012). Insectivorous bat species occupy a relatively high trophic level, which

increases their susceptibility to environmental contaminant accumulation through their diet and

ability to show the consequences of toxic pollution (Zukal et al., 2015).

 In order to determine the heavy metal concentrations, this study uses bats as the

bioindicator to review on accrual of heavy metal from the bat’ organ tissue which could reflect

the level of heavy metals pollution in their current environment. Bats species were chosen

because they fulfil the criteria of being the suitable bio-indicator of heavy metals determination

due to most of bats species are long-lived life expectancy (Arlettaz et al., 2002).

Bioaccumulation of heavy metals by living organisms is an excellent indicator of the exposure

where it has been extensively used to assess contamination levels of heavy metals in polluted

ecosystems (Bartholomew, 2014).

Duffus (2002) use the term of ‘heavy metal’ commonly used to indicate the hazardous

inorganic materials where its density value changed to 4 or 5 g/cm3. Heavy metals are natural

trace components of the aquatic environment, but their levels have increased due to industrial,

agricultural, and mining activities (Stancheva et al., 2013). Heavy metals such as copper (Cu),

cadmium (Cd) and lead (Pb) are natural elements that come from the Earth’s crust and cannot be

degraded or destroyed as well as can easily enter the human body along the food chains, causing

dangerous health issues even at very low concentrations. The other element of heavy metals that

are known in the past research are also zinc (Zn), chromium (Cr), mercury (Hg), arsenic (As)

and tungsten (W). The presence of heavy metals in the environment is not only due to human

activities alone. It is a process that can happen naturally.

As stated by Bartholomew (2014), animals being used to monitor pollution of heavy

metals because heavy metals belong to a group of the most dangerous inorganic toxic

substances. The statement was strongly agreed by Censi et al. (2006) as the heavy metal has a
great toxic effect and has the potential to accumulate in the tissue of living organisms where

metals are the most concerned environmental pollutant among other pollutants. Heavy metals

accumulation in the animals’ tissue depends on several factors such as age, homeostatic

mechanisms of the animals and their physiological state (Bartholomew, 2014). The most

common heavy metals associated with the toxic effects on wildlife are cadmium, lead, and

mercury (O’Shea & Johnson, 2009).

1.2 Problem Statement

A few studies on the investigation of the heavy metal accumulation in bats have been

done. Heavy metals in the environment can be determined using biological specimens as the

bio-indicator which gives information on the quality of the environment (Bartholomew, 2014).

This is supported by a study done by Sanchez-Chardi et al. (2007), which reported that heavy

metals accumulation and their effect can be detected by using bio-indicator. Small mammals

such as bats, is one of the samples living organisms’ samples that can be used to obtain the level

of heavy metals in the environment. According to Kunz et al. (2007), bats are sensitive to the

changes in the environment due to human-induced that cause heavy metal pollution. Therefore,

the expectation on the concentration of heavy metals in bats tissue will vary depending on the

species of the bats as well as their habitat area. Which conclude the more polluted of the area,

the higher the concentration of the heavy metals.

1.3 Objectives

The main purpose of this study is to:

i. discuss the level of concentration of heavy metals in organ tissue of bats (heart, liver,

kidney, brain, bone, fur, lungs, wing membrane) from different bat species in different

location in the past research.

ii. compare the relationships of the existence of heavy metals in the location that the bats

inhabit within Sarawak and the other country in the past research.
 CHAPTER 2: LITERATURE REVIEW

2.1 Bats

Bats are the small mammal group of the Chiroptera categorization that have two

suborders; Megachiroptera, commonly known as Megabats which is fruit bats and

Microchiroptera known as Microbats which is insect bats.

The species are all known around the world apart from Antarctica as the representation

of the second-largest mammal order (Zukal et al., 2015). Just as Dietz and Kiefer (2016) stated,

there are a total of 1200 species that discovered in the world in different countries. The study

supported by a study done by Jones et al. (2009), whereby in terms of biodiversity, bats are the

second largest order in the class Mammalia after rats and squirrels (Rodentia) and have the

highest diversity in both tropical and subtropical regions. There are about 16 families of

insectivorous bats worldwide (Jalaweh, 2004), and only seven families can be found in Borneo.

Jalaweh (2004) also recorded these families are Emballenuridae, Megadermatidae, Nysteridae,

Rhinolophidae, Hipposideridae, Vespertilionidae, and Molossidae Eight species of insectivorous

bats have been recorded from Wind Cave, Bau and they were located on the ceiling and also

inside bell holes in the darker part of the caves.

The bats usually live in tropical areas, especially the warmer and milder area. For

instance, insectivorous bats that occupy high tropic level compared to other living organisms

such as insects or birds were good indicators owing to the relationship between the contaminant

and environmental disturbance and trophic levels (Jones et al., 2009). Typically, bats can make

their home in a wide range of site. These include the caves and trees as well as buildings. Caves
have been long known to support large colonies of bats (Rahman et al., 2011). In earliest

research, bats do often to prove to coexist with humans in urban, industrial, and agricultural

landscapes. Therefore, they can potentially be exposing themselves to increased pollution levels.

2.2 Dietary of Bats

Bats are primary predators of night-flying insects where they can consume up to 1,200

mosquito sized insects in an hour and 3,000 insects in a single night (Bartholomew, 2014).

Usually, the bats with small body size have relatively long-life spans compared to the similar

size of non-volant small mammals. In agreement with Hernout et al. (2013) research, bats are

commonly known as long-lived mammals with proven longevity of 41 years in records of

Brandt’s bat (Myotis brandtii) that found in Siberia. The discovery supported by Bartholomew

(2014) that stated the oldest bats could live up to 40 years as well as the most average age of bats

can live between five to six years, and they have high daily food intake.

As bats have rich dietary diversity as Park et al. (2015) mention in his research,

approximately 70% of all bats are predominately insectivorous, nectarivores and frugivorous

species are known to be essential pollinators and seed dispersers for many wild and cultivated

plants. When the bat has highly intake of food consumption, they usually hunt at night and

consume between 40% to 100% of their body mass due to their high metabolic rates (Hernout et

al., 2013). To conclude, the longer the bats’ life, the higher on the daily food intake.
2.3 Heavy Metals

In Mansour et al. (2016) study, they reported that heavy metals are natural components of

the Earth’s crust and cannot be degraded or destroyed. Generally, the heavy metals are known

within in chemical group of periodic tables, and the elements have the different density to

differentiate the property of the element. Thus, Zukal et al. (2015) refer the ‘heavy metals’ term

as any metallic element with specific density higher than 5 g/cm3 as well as an element that is

toxic and poisonous even at low concentration. Heavy metals can be separated into two, essential

elements and non-essential elements. The essential elements are usually functioning in living

organism mechanism, which are cobalt (Co), copper (Cu), iron (Fe), manganese (Mn) and zinc

(Zn). While for non-essential elements, it cannot perform physiologically in living organisms due

to toxicity such as arsenic (As), cadmium (Cd), lead (Pb), mercury (Hg) and vanadium (V).

These elements are dangerous and can invade the living organism through food chains. When the

agricultural soil polluted with heavy metals, the plant will ingest the heavy metals that

consequently accumulate in their tissue. Food chains can be the main reason for these heavy

metals’ contamination. As Mina (2017) stated, an easy example of the exposure of heavy metals

comes from when any mammals were drinking contaminated water, by eating an infected of

heavy metals’ prey and even by contacting any contaminated soil as well as through inhalation in

the contaminated area.

The essential elements can give effect toxicity when obtaining it in high concentrations.

Heavy metals such as arsenic (As), cadmium (Cd) and lead (Pb), are the most hazardous heavy

metals that can be affected on living organism including humans even in low concentrations

(Mina, 2017). Heavy metals are both extremely toxic and ubiquitous in natural environments as
stated Hsu et al. (2006), and they are present in soil, surface water and plants, can be quickly

mobilized by human activities specifically in agricultural, mining and dumping industrial waste

especially in natural habitats such as in forests, rivers, lakes, and ocean. For example, the mining

industrial waste area, the research by Zocche et al. (2010) discovers the alkalinity, pH and

hardness were interrelated and gave effect to the bioavailability and the toxicity of several

elements like aluminum (Al), cadmium (Cd), copper (Cu), chromium (Cr), lead (Pb), nickel (Ni),

arsenic (As) and zinc (Zn).

These heavy metals elements are provenly lethal and sub-lethal as well as bio-toxically

harmful to any living organism. The examples of toxic effect are mutagenic or teratogenic,

neurotoxic, and carcinogenic. As lethal the toxic effects are, some of it when consuming above

the bio-recommended limits, it may cause the rapid or immediate death of an individual. The

toxic effect may into acute or chronic to any living organisms (Duruibe et al., 2007).

2.4 Bats as Bio-Indicator

Bioindicators are known as any living organisms such as plants, animals and even

microbes. In researchers’ reference, these living organisms are used to assess the health of the

environment, constituting, and also become a valuable tool to detect any changes in the

environment, either positive or negative (Parmar et al., 2016).

In this research, bats are an excellent small mammal which suitable as bio-indicator to

monitor heavy metal toxicity that presents in the environment. Bartholomew (2014) prove it in

his discovery on bats was the mammals used as the bio-indicator for pesticide pollution as well

as mercury exposure by using its guano. Even in drastic ecological process or environmental
changes, the bats still showed a broad geographic range effect and can be diverse in any tropic.

Bats as bioindicators will give many data such as taxonomy relatively stable which explain the

different level of sample on the populations, individuals and feeding rates. Furthermore, bats are

exceptional bioindicators due to their fast metabolism where the heavy metals can be detected

even at low concentration. Hence, with several data collected from the bats, as an excellent

bioindicator, the responses of any changes in wide range trophic levels will be able to show the

effect of environmental degradation on specific ecological processes.

2.5 Heavy Metals Accumulation in Bats

Every living thing is having exposure to mother nature. As for the bats, they are almost

similar to human beings, that may expose to heavy metals through three generally main

pathways: direct ingestion, inhalation through mouth and nose as well as dermal absorption

through skin exposure (Mansour et al., 2016). The accumulation of heavy metals in bats is the

effect of human activities such as mining and foraging, which turn out dangerous environment

contamination. Referring to O’shea (2009) research, the consequences of environmental

contaminations on bats might take place through direct exposure, or indirectly, due to ecological

restructuring that may alter food supply, physical habitat, availability, and quality of water,

among other factors. Each of the effect on the contaminants resulting in many potential high

toxicants exposure that pose many threats for any living things in the contaminant area.

As for the bats, these issues are strictly focusing on the selection of their roosting site.

The important in every roosting site that the bats inhabit are their needs to drink water and easy
access for their food-chain. If the roosting site is strict to the contaminated area, as hypothesized

by Walker et al. (2007), it is supposed that the accumulation of toxic metals by the bats likely to

reflect differences in dietary exposure and the degradation of the habitats present for example, in

the coal mining areas. This hypothesis was supported by Zocche et al. (2010) in his research that

stated the possible explanations for the different level of heavy metal accumulation in bats could

be the diet type, the distance and the local taken by these bats to feeding. An insectivorous bat

species is highly taking insects for their diet. The bats can accumulate heavy metals elements

when feeding on insect inhabiting contaminated waters (Zocche et al., 2010). Therefore, in the

research, the heavy metals can transfer or accumulate from sediments/water/soil/plants or other

sources to insect larvae and adults, and finally to the bats themselves (Hernout et al., 2013).

Zocche et al. (2010) acknowledge this research and explained more about how the insect

can be related to the heavy metal accumulation in bats. The research concludes the

contamination of terrestrial insects with heavy metals were ingested from feeding on the plants in

mining area that resulting in a possible exposure of bats (Zocche et al., 2010). The factors of

accumulation heavy metals in bats consideration are not only from the water and food source but

Bayat et al. (2014) mention the other factor on heavy metals accumulation in bats can also be

from the air by flying activities. When bats exposed themselves in the air, it explains on how the

bat metabolism are known to significantly influence on the accumulation of toxicant from the air

towards in bat tissues (Bayat et al., 2014).

The exposure of the contaminants environment is explicitly focusing on the heavy metal

pollutant, especially several elements such as aluminum (Al), cadmium (Cd), copper (Cu),
chromium (Cr), lead (Pb), nickel (Ni), arsenic (As) and zinc (Zn). The highest toxicant among

those elements is arsenic (As), cadmium (Cd) and lead (Pb) which is very dangerous. Mansour et

al. (2016) confirm that not only the pathways of heavy metals different within the food chain but

they may also differ depending upon the type of toxicants. The level of toxicant of the heavy

metals accumulates in bats can be different, reflecting on the exposure or consumption of the

bats. However, even in low concentration from arsenic (As), cadmium (Cd) or lead (Pb), it will

affect high toxicity to health and would be fatal. These effects include the bats and this matter

already recorded in Mansour et al. (2016) research when the bats feed in the insects that may

accumulate heavy metals such as cadmium (Cd) and copper (Cu) in their tissue, and the bats

showed an acute or chronic effect on their health.

Bats are indeed expected to consume relatively high and prolonged exposure of the heavy

metals’ contamination as well as susceptible to the environmental changes or pollutants.

Unquestionably, the bats are more likely to be known as bio-indicators animals rather than be the

species to be hunted for food. These actions are known in some countries due to the culture of

eating exotic animals. People should have an awareness of the dangers of having these exotic

animals as meals. The effect probably can be fatal on the toxic accumulate in these animals as

well as the unknown exposure of the animals through their nesting or roosting sites.

2.6 Analysis of Heavy Metals in Tissue

Heavy metals can be analyzed in numerous samples such as liver, kidney, whole-body or

carcasses of the bats as the bio-indicator agent. There are numerous studies or research regarding

this analysis to prove how much the bio-indicator animal can accumulate heavy metals in their
surroundings. Moreover, a persistent sampling of previous researchers, analyze liver and kidneys

to get more information on the element contaminations in the organs of the bats. Aside from the

liver and kidney, the other organs that can be analyze were intestine, heart, lungs as well as

muscle. Some of the previous research also analyze skin and fur for further investigation on the

accumulation of heavy metals in bats. The heavy metals may accumulate differently in different

targeted organs, About Hernout et al. (2015) research, the information on level exposure for

heavy metals are restrictedly to the studies with limited sample sizes, areas of study and the

tissue animals’ type. The research explains on how the wild mammals have positive correlations

to determine the concentrations of metals in any of the organs of the body such as hair, blood as

well as the concentrations in kidneys, liver and muscle (Hernout et al., 2015).

Several existing studies indicate that the kidney tissue from the bats was the target organs

for the determination of heavy metals. The heavy metal concentrations within this particular

tissue were highly correlated (Zukal et al., 2015). Therefore, the researchers can use a more

significant and unique comprehensive scale of the dataset on metal concentrations (Cd, Cu, Pb,

and Zn) in different bat organs and tissues (kidney, liver, stomach, bones, and fur) to establish

the toxicological pressure of metal contamination in the study area on bats. Some of the

researchers such as Zukal et al. (2015) discover lead accumulates in bones, liver, and kidneys He

also found arsenic in liver, kidneys and brain as well as cadmium in kidneys and liver; and

mercury in fur and liver (Zukal et al., 2015). Most of the researchers targeted kidney tissue due

to it is the organ that has the main storage site of metals pollutants, especially Cd (Bartholomew,

2014). Generally, Bartholomew stated that the concentration of cadmium commonly found

against the kidney tissues because of the presence of metal-binding with proteins. On the past
research, McGeer et al. (2000) determine on the kidney tissue actively regulate the essential

heavy metals in both kidneys to reflect the exposure and accumulation of the heavy metals for a

prolonged time.

Moreover, referring to Hernout et al. (2016), he observed a significant positive

relationship between concentrations of metals could be found in fur as well as in the other

internal tissues studied (stomach, kidney, liver, and bones). Table 1 shows the non-essential

metals such as Cd and Pb could be toxic at low concentrations, essential metals, such as copper

and Zn, are securely controlled at constant concentrations in tissues of mammals. Hence, mostly

the result would be within a narrow range.

The positive relationships between concentrations in bones, kidneys and liver and fur

suggest that bats’ fur samples can be used as a biomarker of chronic metal exposure for bats,

particularly for non-essential metals. The positive relationships between ingested food (stomach

and stomach content) and hair contamination indicate that concentrations in fur can be used as a

biomarker of recent sources of metal exposure, measured on many individuals. The strong

positive relationship between metal concentration, particularly for Pb, followed by Cd, Cu and

Zn, in fur and stomach contents confirm the assumptions that food is an exposure route of metal

contamination for bats.

What Hernout et al. (2016) trying to prove is on how fur sampling can be highly suited as

a non-invasive sampling technique as the hair is easily accessible, easily transportable and the
storage as well as the collection of the samples do not require controlled conditions. The

advantage of this sampling method is that the collection does not require any sacrifice of the

animals (Hernout et al., 2016). The exposure of the heavy metals on the bats’ surroundings for

sure is proven that the heavy metals can also be detected at the bats’ physical (body).
 CHAPTER 3: MATERIALS AND METHOD

3.1 Study Species and Study Area

Each of the sampling data was compiled from different countries and different areas. The

sampling data were taken to complete the study’s objective to compare and discuss the

accumulation of heavy metals in bats’ data in Sarawak with other countries. First, the samples

from Mina et al. (2019) studied, the researcher used bat carcasses and collected it from a

different area of north and central Portugal. The researcher collected a total of 56 individuals of

four species for their study (Hypsugo savii, Nyctalus leisleri, Pipistrellus pipistrellus,

and Pipistrellus pygmaeus). Another study recorded the sample located on two different sites;

Stockamollan (55° 38’N, 13° 27’E) and Sodra Asum (55° 38’N, 13° 47’E) (Akerblom et al.,

2017). The sites are from southern Sweden, and the study species was in total 17 individuals

with 14 species of bats recorded. Akerblom et al. (2017) taken the fur of the bats as samples

from all the female, the male also juveniles. Next, the sample from Rahman et al. (2018)

research, recorded only one species Eonycteris spelaea taken from two caves; Soto-rodmukh

(24° 38’58.44” N and 93° 01’19.11” E) and Boro-rodmukh (24° 38’53.53” N and 93° 01’20.84”

E). Both caves were in Bhuban Hills, in the southeast of Barak Valley, India, with six

individuals only. In Bartholomew’s (2014) research, the bats’ sample taken in four different

areas in Sarawak; Lambir Hills National Park (NP), Niah NP, Gunung Mulu NP, and Tanjung

Datu NP. The species was recorded in five families: Hipposideridae, Pteropodidae,

Megadermatidae, Nycteridae and Emballonuridae with total of 163 number of individuals of the

bats’ species.
3.2 Sample Collection and Preparations

Overall, of all the sampling collection was done in the long-term plan to collect all the

individual of the bats' samples. The sample was done during an ecological monitoring program

to collect all the carcasses left in the study site between August and October of 2006 to 2014

(Mina et al., 2019). All the samples were frozen at -20°C after the collection due to the

protection of a freshly dead bat, which only dead for one or two days in the landscape, no signs

of scavenging animals, and no-fly larva infestation on the bats' own body. Mina et al. (2019)

divided collection to internal organs (liver, heart, bone, and brain) and external organs (wing

membrane and fur) as the biological samples to be analyzed. Akerblom et al. (2017), the fur

collection was taken from 22 July and 23 July 2013 on annual surveillance for the common of

bat lyssavirus type 2. The sample collection was recorded as a non-invasive approach as the fur

samples were clipped from the back of the bats. Only then, after the fur sampling, the bats were

released (Akerblom et al., 2017). The fur samples were kept frozen at -18°C in a plastic tube

and sent to the laboratory for further analyses.

Meanwhile, Rahman et al. (2018) did the bat sampling collections in September 2015

and immediately released pregnant bats and juvenile individuals. Only adult male individuals of

bats were taken and were dissected into 24 parts of four different organs such as the liver,

kidney, and lungs from six individuals of the bats (Rahman et al., 2018). In Bartholomew's

(2014) research, the sample collections were carried out from 30 January until 13 April 2014

and kept in the freezer. The bats were soak in chloroform before any dissection to collect

kidney tissues as well as liver tissues and placed them in a tube containing absolute alcohol

(Bartholomew, 2014). Generally, morphological measurements of bats were taken by using

calipers or rulers to take the measurements of head and body length (HB), tail length (T),

hindfoot length (HF), ear length (E), forearm length (FA), total length (TL), ventral tail length

(TVL), and wingspan measurement while portable spring scale will be used to measure the

weight of the bats (Bartholomew, 2014). The bats were identified based on the selected

morphological measurements. The measurements taken were using a Vernier caliper

(Mitutoyo), and bats were weighted using a Pesola spring balance for morphometric studies.

3.3 Bats’ Tissue Digestion and Analyses

In the past research, when the bats were dissected, the post-mortem examination was

carried out. The bat’s organs were harvested and placed accordingly in a zip-lock bag. The

samples of organ bats were well arranged labeled as internal and external organs (kidney, liver,

lung, brain, heart, bone, fur, and wing membrane). All the samples were collected and returned

to the laboratory for further analyses. For external organs, fur and wing membrane must be

washed using detergents such as Trixton.X-100, 2:3 ratio of acetone and distilled water to make

sure removing any unwanted substance or external contamination (Mina et al., 2019). In the

general analyses, to obtain the desired amount of weight (100 µg dw), the samples were

combined where the identical organ of the bats’ individuals was dried separately in an oven for

48 hours at 50°C (Ruiz et al., 2019). The dried samples were ground by using pestle and mortar

(Bartholomew, 2014) or ground for chemical analysis.

Next, the digestion of all the bats’ internal and external organ samples was done by

adding 3 mL of concentrated nitric acid (HNO3) and 2 mL of hydrochloric acid (HCl)

(Bartholomew, 2014) with a microwave digestion system (Anton Paar Microwave Sample
preparation System, Multiwave 3000) (Ruiz et al., 2019). The mixed solution then allowed to

cool in room temperature and proceed with the filtration of 0.45 µm cellulose filter. After that,

the filtered solutions were placed in a volumetric flask diluted to 50 mL per sample with de-

ionized water. Further analysis was carried with an analytical chemistry instrument.

In the case of determining the concentration of heavy metals, past research has slightly

different ways to determine the element on each of the collections of the bat individuals they

found. To compare the heavy metal analysis in past researcher’s results, in Mina et al. (2019)

research, the taken result was the concentration of arsenic (As), cadmium (Cd), copper (Cu),

manganese (Mn), lead (Pb) and zinc (Zn). Different from Akerblom et al. (2017), he stated on

determining only mercury (Hg) concentration due to the case of bat lyssavirus case. As for

Rahman et al. (2018) research, the heavy metal concentration result taken were zinc (Zn),

copper (Cu), manganese (Mn) and iron (Fe). These elements can be group based on the type of

elements; the essential elements (Cu, Mn, Fe, Zn), the non-essentials elements (Cd, Pb), and the

non-essential metalloid (As).

The heavy metals concentrations from the sample mix solutions were analyzed using

Inductively Coupled Plasma Mass Spectrometry (ICP-MS) (Elan 6100 DRC + from

PerkinElmer-Sciex) (Ruiz et at., 2019). All concentrations of the heavy metals were prepared by

using quantitative standards. The detection limits of the standards were around 1 ng/L (1 ppt)

and below, including the blank solution (3:2 nitric acid (HNO3) with hydrochloric acid (HCl))

(Ruiz et al., 2019). By testing a certified reference material, DOLT-3 (Dogfish Liver Certified

Reference Material for Trace Metals from National Research Council Canada), and blanks, the
accuracy and precision of the extraction and analytical process were evaluated (Mina et al.,

2019). Mina et al. (2019) also use the calibration of the instrument (ICP-MS) in a 5-point

calibration curve per element. In general, any of the lower detection in the metal concentration

of statistical analyses, the analyses were replaced by the value of the detection of the limit on

heavy metal concentrations. All the result of the metal concentrations was expressed in µg/g

(ppm) on a dry weight (d.w.) basis (Ruiz et al., 2019).

 CHAPTER 4: RESULT AND DISCUSSION

4.1 General Result

Overall, all the concentration of heavy metals was recorded and varied among all the

bats’ species and families in each study site. In each of the bats’ organ, eight heavy metals such

as of arsenic (As), cadmium (Cd), copper (Cu), lead (Pb), mercury (Hg), manganese (Mn), iron

(Fe), and zinc (Zn) were analyzed. There is a significant difference between the concentrations

obtained in different sampling locations. Figure 2, as well as Table 2, Table 3, and Table 4, show

the highest and the lowest heavy metal concentrations present in the bat's organs from a different

site.

Depending on the site, the organ tissues show that the highest concentration is

varied. As shown in Figure 2, the North and Centre of Portugal has the highest

accumulation of metal zinc (Zn) which is 239.33 ppm (µg/g dw). Generally, in Table 2,

bone organ represents the lowest level concentration (0.01 µg/g dw) of cadmium (Cd)

in all the organs or tissues than the other heavy metals' element. Meanwhile, Table 3

shows that the Hg concentration is the highest, which is in 2.3 ppm (µg/g) accumulated

in fur organ at Sodra Asum (Akerblom et al., 2017). Rahman et al. (2018) recorded on

the highest reading heavy metal is iron (Fe) in the liver organ (98.4 ± 0.22 µg/g dw) at

Bhuban Hills in Table 3. Table 4 shows the highest concentration of heavy metals

in Cynopterus Horsfield's kidney was 2.1108 ± 0.0428 ppm (µg/g dw). 

Overall, the analysis of all the past studies confirms that different study sites

showed different heavy metals accumulation in each of the bat's organ species. Despite
the differences, the pattern of the concentration in the different organs of bats for each

of the species taken was related. These explain how the interaction between the bats'

species or families can be familiar, although in different study sites. Overall, the highest

heavy metal concentrations recorded in the result can be concluded as mainly from the

essential elements; Zn, Cu, Mn, and Fe, while the lowest is from the non-essential

elements or metalloids as As, Pb, Cd, and Hg. In all the bats' tissues on the results from

the different study areas, Table 2 shows the sequence of Zn > Cu > Mn > Pb > As > Cd.

Table 3 shows the sequence of Fe > Zn > Cu > Mn (Bhuban Hills, India only). While

Table 4 shows sequence of Fe > Zn > Cd > As. The comparison on each of the heavy

metals accumulate in the bats' organ, or body parts have been done separately.

4.2 Analysis of Heavy Metals Concentration in Fruit Bats

The heavy metals concentration is varied over the surrounding, especially from

the contaminated area. From the result, it was discovered that there is 13 type of bats

species from four different study site. Only one species that have the same sample

(Eonycteris spelaea) from Bhuban Hills and Sarawak study site in Table 3 and Table 4.

Each of the species in the study site was detected from morphological measurements.

These measurements can help to detect all the bats’ species to gain more information on

each of the bat’s individuals, such as species, gender, age, and to detect how the bats

inhabit the study sites. 

When comparing the past research, heavy metals indeed a presence in bats

organs. The result also showed that heavy metals accumulation in the organs indeed

presents even in low concentrations that can be detected from sites. Among the four
sites, the result shows that the highest reading concentration of heavy metals (Zn) is at

the North and Centre of Portugal (Figure 2). There are two possibilities why the

concentration is higher as compared to the other study sites. One of the possibilities was

the diversity of the physical requirements of the bat’s species, which includes the

availability of the food source and the roosting site area. The other possibilities are

because the samples were a fresh bats carcass, and the research took eight years to

collect the carcasses. It has proven to be accurate by Mina et al. (2019) collected the bat

carcasses from 2006 to 2014. The bats’ species are indeed a small mammal that requires

plenty of tissue samples to detect the heavy metal accumulation in the bat’s tissue. Each

of the study sites has recorded a different number of bats individuals taken for sample

collection with different time durations. The number of individuals of the bats can also

help to gain information on how polluted the environment in the study sites due to the

bats are excellent bio-indicator. The higher the heavy metals concentration in the bats’

tissue, the higher possibilities on the study area contaminated with heavy metals

pollution. This statement can be supported, as the typical heavy metal pollution in the

environment is Cr, Mn, Ni, Zn, Cd, and Pb (Ali et al., 2019). However, some of the

heavy metals indeed can be hazardous depending on the consumption limits and the

dose, as well as the duration of the exposure.

Generally, this study mainly uses the relationship between the exposure of heavy

metals in biological samples with the environment. Ali et al. (2019) agree with the

importance of the metal and metalloids element in our surroundings due to the essence of

it for biological life. Each of the table results showed the relationship between the bats’
organ and the concentration of the heavy metals in a different site (Table 2, Table 3,

Table 4). The element concentration varies from organ to organ and is dependent on body

size, tissue structure, and targeted organ (Rahman et al., 2018). Focusing on Table 2, it

was shown on the highest heavy metal concentration reading is mainly from the fur bats,

239.33 ppm (µg/g dw), and the wing membrane with the mean of 92.82 ppm (µg/g dw).

Both readings are from Zn concentration.

Meanwhile, the lowest reading was recorded in 0.01 ppm (µg/g dw) in the bats’

bone tissue. The other elements, such as Cu, Mn, Pb, and As, also showed the mean

concentration reading even in low concentration. This can be explained due to the

performance of ICP-MS, which can run the analysis for any detection limit of the

standard in 1 ppt (ng/L) and below. Next, Table 3 represents two different study sites,

mainly from Sweden and India. In Sweden, Akerblom et al. (2017) did the collection of

the bats’ in two places Stockamollan and Sodra Asum. In this research, Akerblom et al.

(2017) only focusing the Hg element accumulates in only one of the bats’ organs, which

is fur. The highest reading on Hg concentration is 2.3 ppm (µg/g dw) in Sodra Asum. The

result from Sweden was taken to compare because not all from the past research using the

Hg element for analysis. The reason behind this research was that there is no clear

duration pattern in bats identified for Hg in the past research, but only for smaller

samples have been done in average total sample sizes. It was supported whereby there are

no other time-trend data for Hg in bats elsewhere, so far as we were aware (Walker et al.,

2007). That is why the research did the collection in annual surveillance for bat lyssavirus

type 2. 
 Subsequently, Table 3 also shows the concentration of heavy metals reading from

Bhuban Hills in India. In Rahman et al. (2018) research, we only can compare the heavy

metals on Cu, Mn, Zn, and Fe accumulate only in three of the bats’ organs, which in

kidney, liver, lungs. The highest concentration was 98.4 ppm (µg/g dw) of Fe in the liver

tissues, while the lowest is in 0.6 ppm (µg/g dw) of Mn also in liver bats tissues. Rahman

et al. (2018) focusing these four elements on narrowing down the list of the essential

trace element for one species (Eonycteris spleaea) only in his research. In this research,

Cu was the main element to be focusing on the study sites due to the species, specifically

on the variation of diet feeding habits. This is due to Rahman et al. (2018) opinion on the

food that may contain some essential trace elements in polluted surroundings. 

From this point, the comparison of past studies continues to Sarawak in Table 4.

The research in Sarawak has four different sample collection. In Table 4, each of the

different species of bats, only the kidney organ was taken to be analyses. The highest

concentration of Fe recorded is 2.1108 ppm (µg/g dw) in Cynopterus horsfield species.

While the lowest would be 0.0301 ppm (µg/g dw) also Fe concentration in Balionycteris

maculata, the other heavy metals element such as As Cd and Zn also record in the table.

In Bartholomew’s (2014) research, the element As was focused due to the possibility of

the dangerous heavy metal accumulation in the bio-indicator tissues through high

consumption. 

           Based on the result obtained in Table 2, Table 3, and Table 4, it can be conclude
that the highest heavy metals level was more to essential elements (Zn, Cu, Mn, Fe) with

Zn being the highest. There are several reasons for this. For instance, the bat might

consume food containing a high amount of Zn. This is proven to be accurate as the fruit

bat’s main diet is fruit and nectar. Besides that, Zn, Cu, and Fe are generally known as the

trace elements required in any physiological function in living things. The concern on

these natural heavy metals is when the amount of heavy metals is excessive, which can

cause several effects. Zn element is needed for protein and enzyme synthesis that

naturally present in some significant organs such as liver, heart, pancreas, and kidney

while ingestion and inhalation (Rahman et al., 2018). The excessive heavy metals can

happen due to the fruit tree applied with chemical fertilizer or even in the combustion of

fossil fuels in every human agricultural activity. 

           On the other hand, Cu is known in any of the living tissues due to the significant

role of central nervous system growth, an essential element for cellular respiration, and

conserving the tissue strength of the living body (Rahman et al., 2018). Nevertheless, Cu

also can be anthropogenic due to human activity such as industrial productions. With any

of the human activity near to the bat roosting sites, the bats’ species need to adjust with

the urban surrounding for them to survive. This is a significant concern because Ali et al.

(2019) have recorded the finding of during mining, smelting, or other industrial

processes, the heavy metals released into the atmosphere by dry and wet deposition. 

As revealed on the result obtained, the non-essentials elements (Cd, Pb, Hg) and

non-essential metalloid (As) overall, have a low concentration reading. However, the
polluted area’s concern will work continuously under surveillance on each of the study

sites. This is because, as mention before, As, Cd and Pb elements are the most competent

research due to it is the most hazardous heavy metal elements. In can be concluded that

these heavy metals (As, Cd, Pb, Hg) can cause lethal and sub-lethal to any living things

(Mina et al., 2017). 

4.3 Sample Tissue Indicator of Heavy Metal Accumulation in Fruit Bats

Comparing the heavy metals’ concentration in fruit bats’ tissues can be

concluded that the external organ such as fur and wing membrane has the highest

reading compared to other internal organs (brain, liver, heart, bone, kidney, lungs). This

result is supported by Hernout et al. (2016) on his founding of the heavy metals

concentrations in bat’s fur were similar to bat’s stomach content as the biomarker for

any metal exposure. It can be explained by the individual factor variables such as age,

gender, diet, and habitat area, as well as the inter-species factor of the bats, were known

to be the primary reason for the bats’ exposure in their external organ. The fur and wing

membrane can easily be bioaccumulated heavy metals during the bats growing phase

and hunting sessions due to the expectation on the higher-level concentration of heavy

metals in older hair in bats’ fur (Mina et al., 2019). Hence after comparing the past

research obtained in the results, the use of the fur and wing membrane is strongly

suggested by monitoring any potential heavy metals accumulations or exposure to the

bats’ species as the bioindicator animal. As revealed by Mina et al. (2019), the research

on external tissue indeed can be an excellent representative for heavy metals

concentrations in the internal organs of bats’ species. Therefore, the sampling of

monitoring heavy metal exposure in any of the study sites can be done by non-lethal

sampling.

CHAPTER 5: CONCLUSION AND RECOMMENDATION

5.1 Conclusion

In conclusion, the heavy metals concentration readings were detected in the bats’ tissue

in each of the study sites. Each of the study sites has a distinctive duration to collect all

the bat species. The study also shows that different species of bats with different study

sites show the various level of heavy metals concentration in the bats’ tissues. The Zn,

Mn, Fe, and Cu have the highest reading concentration generally than the non-essential

elements and metalloids such as As, Pb, Cd, and Hg. The concentration reading also

can provide information on how polluted heavy metals in the specific area of study

sites. It also gives more details on how easily the bats’ species exposed themselves in

any contaminated area by choosing the roosting site, dietary choices and how long the

age of the bats consume in heavy metals exposure. These are indeed a bio-indicator

characteristic, and bat species are genuinely the best bio-indicator animal chosen. At the

end of the discussion, although there are a few relationships found in external and

internal organs with the accumulation of the heavy metals, overall, the best biological

sample result showed in the fur organ and wing membrane. Thus, we can conclude that

the external organ (fur, wing membrane) are excellent non-lethal sampling to

biomonitoring the heavy metal exposure in any study sites.

6.2 Recommendation

Fur and wing samples are known to be used in the heavy metals’ determination

in bats sampling. It can be done in a non-lethal way. This explains how well each of the

bats’ individuals is treated when dissecting the skin (fur) from the bat individual

without cutting any muscles of it using a sanitized dissecting tool. The bats should be

monitored while waiting for it to recovered. The monitoring is including a shelter and

feeding time for the bats. Then, after the bats’ recovery, it will be released to the same

place it had been captured. In this way, the number of bats population can be increased

by reducing any of the bats to be killed due to obtaining the bioaccumulation of heavy

metals concentration in bats to monitor the anthropogenic phases.