Studies on Investigation of The Heavy Metal Accumulation in Bats

Sharifah Zulaikha Binti Syed Sahimi

(64138)

Proposal Final Year Project 1

(STK 3013)
Course

Lecturer: Madam Maya Asyikin bt Mohamad Arif

Bachelor of Science with Honors

Resource Chemistry
Department of Chemistry

Faculty of Resource Science and Technology

University Malaysia Sarawak
27 September 2019

1
Abstract

Wildlife such as bats are one of the small mammals that are suitable as the bio-indicator to
access the quality of the environment particularly on heavy metals concentrations. The
objective of this study are to examine the level of concentration of heavy metals in kidney and
liver tissue of bats from different species and different site in Sarawak and to determine the
relationships of the existence of the heavy metals in tissue of bats with the environment. The
sample taken from the bats will be from the kidney and liver tissue that will be extracted to be
test of the existence of heavy metals by using Inductively Coupled Plasma-Mass Spectrometry
(ICP-MS; located at the University Malaysia Sarawak). One-Way Analysis of Variance
(ANOVA) will be used as statistical analysis that shows the significant difference of the heavy
metals concentration in the bats at different species. By the end of this experiment, among of
five heavy metals in this study consists of zinc (Zn), lead (Pb) cadmium (Cd), ferum (Fe), and
arsenic (As). The heaviest metals existence in the environment will be detected and the highest
concentrated of heavy metals from one of the bat species will be compared.

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 CHAPTER 1

1.0 Introduction

As reported by Lim (2012), bats are volant small mammals and can be divided into two
suborders, which are Megachiroptera and Microchiroptera. The classification of these
mammals is single order of Chiroptera and distinguished as their ability to conduct their true
flight. They use echolocation to navigate in dark and also to detect their prey distance (Lim,
2012). He also stated that megachiroptera are 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 smaller size than Megachiropterans
(Lim, 2012). Examples of common prey insects consumed by bats are mosquitoes, termites,
cucumber beetles, ants, leaf hoppers and roaches (Braun & Grace, 2008). Insectivorous bats
are predators of some insect pests, such as cucumber beetles, June bugs, corn earworm moths,
cotton bollworm moths, tobacco budworm moths and Jerusalem crickets, which threaten the
yield of important agricultural and forest economic crops (Cleveland et al. 2006).

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
that chosen by bats are depends on their ability to fly, their shape of wings and also the
echolocation call. Bats usually found in the habitat that enable the bats to conserve their energy
where the optimum condition for the environment may be provided by the habitat they inhabit
(Bartholomew, 2014). Bats are most abundant in tropics and temperate region have less bats
species although 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 Borneo, there are eight family
of bats consist a total of 93 number of bat species that has been recorded (Bartholomew, 2014).

3
 In order to dictate the heavy metal concentrations, this study will use bats to review on
accrual of heavy metal in bats tissue able to reflect the existence of heavy metals in their current
environment. Bats was chosen because they fulfil the criteria of being the suitable bio-indicator
of heavy metals determination such as high numbers of bats species are long-lived (Arlettaz et
al., 2002) where there are some heavy metals may accumulate with age (Walker et al., 2002).
Referring to Bartholomew (2014), the 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.

According to Duffus (2002), the term ‘heavy metal’ commonly used to indicates 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 comes 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 also from zinc (Zn), chromium (Cr), mercury (Hg), arsenic (As) and
tungsten (W). The presence of heavy metal in the environment is not due to the 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 belongs to group of the most dangerous inorganic toxic
substances. According to Censi et al. (2006), heavy metal has great toxic effect and has
potential to accumulate in tissue of living organisms where metals are most concern
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). Heavy
metal pollution can affect ecological, genetical, physiological, reproductive, and behavioral
parameters, and mortality in severe cases (e.g., Vaughan et al. 1996, Van De Sijpe et al. 2004,
Pikula et al. 2010, Zocche et al. 2010, and Naidoo et al. 2013).

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1.1 Problem Statement

A number of studies on the investigation of the heavy metal accumulation in bats has
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 the study done by Sanchez-Chardi et al. (2007), which stated the
heavy metals accumulation and its effect are able to be detected by using bio-indicator. Small
mammals such as bats, is one of the sample living organisms that can be used to obtained the
level of heavy metals in the environment. According to Kunz et al. (2007), bats are sensitive
to changes in the environment due to human-induced where one of the changes is the 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. Whereas, the more
polluted of the area, the higher the concentration of the heavy metals.

1.2 Objectives

The main purpose of this study are:

1. To examine the level of concentration of heavy metals in kidney tissue of bats from
different bat species and different site in Sarawak.
2. To determine the relationships of the existence of heavy metals in bats tissue with the
environment.

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 CHAPTER 2

2.0 Literature Review

2.1 Bats Background

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). As
stated by Barclay and Harder (2003), bats with its small body size have relatively long life
spans compared to similarly size non-volant small mammals. The oldest bats, for example, live
up to 40 years, and most have an average age of between five and six years (Gaisler et al. 2003).
Due to their relatively long life (e.g. up to 40 years old) and their high daily food intake (e.g.
up to 0.5 g/gbw/d on a wet basis measured experimentally) (Anthony & Kunz, 1977; Podlutsky
et al., 2005), bats can be particularly prone to chemical exposure, especially to contaminants
such as metals, that accumulate through the food chain (Hickey et al., 2001). Bats can be
divided into two suborders, which are Megachiroptera and Microchiroptera (Lim, 2012).

A B
Figure 1: A. Microchiroptera B. Megachiroptera

(Reproduced with permission from Wilson, 2019).

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 Microchiroptera are insectivorous bats. There are about 16 families of insectivorous
bats worldwide (Jalaweh, 2004) and only seven families can be found in Borneo. Jalaweh
(2004) also stated that 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 (Norhayani et al., 2002). In
Borneo, there are 92 species of bats of which 75 are insectivorous bats (Jalaweh, 2004). Bats
occur on every continent except Antarctica and represent the second largest mammal order,
comprising around 20% of mammal species, with the greatest diversity in the tropics (Zukal et
al., 2015). This is supported study done by Jones et al. (2002), where he stated that in terms of
biodiversity, bats are second largest order in the class Mammalia after rats and squirrels
(Rodentia), and have the highest diversity in both tropical and subtropical regions.

Bats usually found in the habitat that enable them to conserve their energy where the
optimum condition for the environment may be provided by the habitat that they inhabit
(Bartholomew, 2014). 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
contaminant and environmental disturbance and tropic levels (Alleva et al., 2006). Commonly
bats can make their home in a wide range of site. These includes the caves and trees as well as
buildings. Caves have been long known to support large colonies of bats (Rahman et al., 2011).
As Bartonička & Zukal, (2003) stated, bats often coexist with humans in urban, industrial, and
agricultural landscapes, thereby potentially exposing themselves to increased pollution levels.

Just as Walker et al. (2002) studied, these issue makes the bats faces high risk of
accumulation to the heavy metals pollutants in their organs. Bats are also sensitive to human-
induced changes to ecosystems (Jones et al., 2009). When the bats inhabit the human area, they
called as synanthropes. This allowed the bats to spread their regions because their suitable
natural shelters are limited and the threat of heavy metals contamination will increase due to
the proximity of human activities. Thus, the quality of the habitats can be detailed whether the
places are being contaminated with heavy metals or not (Bartholomew, 2014). In polluted
areas, bats accumulate metals through the food chain and long-term exposure to elevated levels
can result in a variety of pathological conditions or even death (Zukal et al., 2015). Overall, it
may be said the condition of the heavy metals’ concentration in the environment was discussed
in the past research whereas the relationship between the heavy metals in bats tissue depending

7
where the bats inhabit or live to detect whether the heavy metals uptake on it surrounding or
the area.

2.2 Bats Dietary

Across species, bats have a diverse array of diets; whilst approximately 70% of all bats
are predominately insectivorous, nectivorous and frugivorous species are known to be
important pollinators and seed dispersers for a large number of wild and cultivated plants (Kunz
et al. 2011). Different species of bats have different diet composition of food and the source of
the food might have any presence of heavy metals in their tissue. Just as Bartholomew (2014)
stated, the heavy metals in different bats species also shows the differences of contaminations.

The food requirements of bats usually well served by open grasslands or edges of
forests where insects are abundant and some moths, mantis, lacewing as well as other insects
have ‘ears’ which have the ability to detect the echolocation calls of the bats and trigger escapes
responses (Jalaweh, 2004). This is related to the bat behaviours, which only come out at night
too hunt for food (Altringham, 1996) and eat the insects. In Indiana, USA, it has been estimated
that a single colony of 150 big brown bats (Eptesicus fuscus) feed on nearly 1.3 million insect
pests each year (Mansour et al., 2016). Referring to Hazebroek and Morshidi (2000), an
individual of bat is estimated to eat up to 5g of insects per night. This study proven in Muda
and Hamid (1994) studies done in Malaysia that stated 12 orders of insect were found in the
stomach content, feces analysis and food remains in roosting sites of bats.

While most bats species feed on insects, they might be exposed to heavy metals through
their feeding behavior when they consume insects which spend their aquatic life phase for long
period in contaminated water (Hoenerhoff & Williams, 2004). Insectivorous bat species, easily
contaminated mainly through bioaccumulation through the food-chain (Hsu et al., 2006). This
is due to the heavy metals that are transferred from sediments/water/soil/plants or other sources
to insect larvae and adults, and finally to the bats themselves (Hernout et al. 2013). Meanwhile,
the primary means of heavy metal contamination in fruit bats is likely to be through
atmospheric pollution, with secondary contamination from contact with contaminated foliage
whilst searching for and eating food (Zukal et al., 2015). As stated by Hernout et al. (2013),
heavy metals are naturally present in the environment makes any conclusion concerning risk
and adverse effects to bats difficult. Therefore, the possibility of bats to be exposed to heavy

8
metals where it increasingly and can be detected for long period of time due to their
bioaccumulation process.

2.3 Heavy Metals Surrounding Bats

In Mansour, Soliman and Soliman (2016) study, they reported that heavy metals are
natural components of the Earth’s crust and cannot be degraded or destroyed and can easily
enter the living organisms along the food chains, causing detrimental health effects even at
very low concentrations. This study supported by Censi et al. (2006), that stated heavy metal
has great toxic effect and has potential to accumulate in the tissue of living organisms where
metals are most concern environmental pollutant among other pollutants. According to Ikem
et al. (2003), contamination of the heavy metals in water, soils sediments and biota was the
major concern due to their toxicity, persistence and bioaccumulative nature. Bats have the same
ecosystems as the other animas that exposed to many environmental factors of heavy metals.
One of the factors would be the consumption of heavy metals in their dietary that has been
contaminated with heavy metals naturally from proximity of human activities.

The 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). Previous study that was carried out by Varsha
and Bakre (2012) shown that soil and water samples show the background concentration of
lead, cadmium, chromium, copper and zinc. Since bats are able to consume huge number of
preys each night, there is a high probability of the bats to be exposed to heavy metals when
consume the prey in the contaminated soils and water bodies for a long time (Hoenerhoff &
Williams, 2004). To conclude, both the source water and soil may affect the food resources of
bats. Emissions of heavy metals into the environment occur via a wide range of processes and
pathways, including air pollution through combustion, extraction and processing; surface water
pollution via runoff and releases from storage and transport; into soils and consequently into
ground waters, insects and crops (Zukal et al., 2015).

Heavy metals and other toxins from industrial waste, however, can not only affect water
quality but accumulate in the invertebrate community, which then forms food for bats (Sijpe et
al., 2004; Jones et al., 2009). In term of water pollution, heavy metals such as cadmium (Cd),
copper (Cu), lead (Pb), mercury (Hg), and zinc (Zn) are essential trace metals to living
organisms (Li et al., 2002). Their excessive release in the environment refers mainly to human

9
anthropogenic activities which include mining, waste disposal, fertilizers, pesticides containing
metals such as Cu, Zn and Mn, traffic polution (exhaust gases, tire wear, and brake lining wear),
industrial effluents, domestic emissions, and atmospheric depositions (Frickel & Elliott 2008).
Releasing of large quantities of metals (e.g., arsenic (As), cadmium (Cd), chromium (Cr), lead
(Pb), and mercury (Hg) into the environment leads to their distribution between environmental
media and accumulation in food chains; inducing adverse human health effects at the cellular
level (Fulladosa et al. 2002), as well as higher levels (Stewart et al. 2003).

According to Zhuang et al. (2009), bioaccumulation and biomagnification in the food

chains is the main problem related to the persistence of heavy metals. Eleven elements (e.g.,
arsenic, cadmium, cobalt, chromium, copper, mercury, manganese, nickel, lead, tin and
thallium) are recognized as being of greatest wildlife concern (Beyersmann & Hartwig 2008).
As stated by Mansour et al. (2016), lead, mercury, arsenic and cadmium, which are heavy
metals, are considered amongst the most hazardous, however, light metals such as aluminum
and selenium can be toxic to living organisms at higher concentrations. They also mention that
the others are essential elements for a range of normal functions in animals at trace doses, for
example manganese, nickel, cobalt, copper, iron, and zinc, but it still can be toxic at elevated
concentrations (Mansour et al., 2016).

Heavy metals are both extremely toxic and ubiquitous in natural environments and they
occur in soil, surface water and plants, and it is readily mobilized by human activities such as
mining and dumping industrial waste in natural habitats such as forests, rivers, lakes, and ocean
(Larison et al., 2000). In mining industrial waste area, a study mention by O’Shea (2001), the
alkalinity, pH and hard- ness are interrelated and affect the bioavailability and toxicity of
several elements like aluminum (Al), cadmium (Cd), copper (Cu), chromium (Cr), lead (Pb),
nickel (Ni), arsenic (Ar) and zinc (Zn). There are caves and abandoned mines that provide
roosting habitat for bat species that depend on subterranean conditions (Tobin et al., 2018) at
Bau, Sarawak. The caves such as Wind Cave and Fairy Cave in that area are filled with bats
species that exposed with the contamination of heavy metals effected from the abandoned gold
mines. Disturbances at natural caves such as commercial development, recreation or land use
changes can affect use by bats, causing a shift in use to abandoned mines, which can provide
alternative roost sites if they supply suitable habitat (Tobin et al., 2018). Open pit mines may
be filled with water, becoming lakes, with potentially toxic levels of heavy metals made
available to animals through drinking or through contaminated food chains (O’Shea, 2001).

10
Moreover, in mining areas, insectivorous bats can be used as bio-indicators of human health
risks (Jones et al., 2009).

Figure 2: The comparison between the damage index (DI) and damage frequency (DF)
detected in the bat species from the control area and mining area using the Comet assay
(Zocche et al., 2010)

In comparison, few studies have considered the exposure and potential impacts on bats
of toxic heavy metals, such as mercury (Hg), lead (Pb) and cadmium (Cd), even though these
elements are readily transferred through insectivore food chains (Ma & Talmage, 2001). Sub-
lethal concentrations of Cd, Pb, Hg, arsenic and essential trace elements in the body organs,
fur and guano of various species have also been reported but most studies are from North
America and Australia (Clark & Shore, 2001; Hickey et al., 2001; O’Shea et al., 2001).
Conclusively, the exposure of the heavy metals in bats can happen through the consumption of
the contaminated drink source as well as inhalation and ingestion of the food sources.

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 2.4 Bat as Bio-indicator

Concern over the loss of ecosystem services, such as pollination and insect pest control,
has heighted awareness of how reliant humans are upon high quality, functioning ecosystems
and the use of bio-indicators (biological processes, species, or communities) has been
suggested as one way of assessing changes in environmental quality over time as a result of
anthropogenic impacts (e.g. Carignan & Villard 2002; Holt and Miller 2010). The potential
role for bats as bio-indicator species has been highlighted by Jones et al. (2009) and the result
of his exploration of these ideas edited in his research, Mammalian Biology. Jones et al. (2009)
stated the outline a range of characteristics that make bats suitable bio-indicator species are
including their position at high trophic levels, widespread distribution and relative taxonomic
stability.

Bio-indicators are important especially in the ecosystem due to the sensitivity of the
living organisms in their environment. The indicators of the environmental usually react in any
expected signs to prove any specific environmental disturbances. Bats are one of the group of
small mammals which is suitable as bio-indicator to monitor heavy metal toxicity that present
in the environment. This is because, they have adequate lifespan for the malignant agents for
example heavy metals to manifest in them (Ieradi et al., 2003). Bats have relatively long life
span and their metabolism is intensive so there is high probability of the heavy metals to
accumulate in them due to their feeding habits and also the presence of the heavy metals in
their habitat as well. They also vulnerable especially to the disturbance and the pollutions
because of their dependently on their food resources and habitat. 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 contaminant and environmental
disturbance and tropic levels (Alleva et al., 2006).

An excellent bio-indicator showed a wide range of the effects of the environmental

changes or drastic ecological process. Therefore, bats being important bio-indicator of
environment since bats had been used as bio-indicator of habitat quality (Wickramasinghe et
al., 2003; Kalcounis-Rueppell et al., 2007). In addition, bats were used as bio-indicator because
bats also were the first mammals used as the bio-indicator for pesticide pollution as well as
mercury exposure by using its guano (Bartholomew, 2014). Increasing in tropic level indicates
the increase in dietary accumulation and metabolic activities of insectivorous bats makes them
able to show the impacts of pollutants (Jones et al., 2009). Dietary of bats are rich which include

12
insects, fruits, leaves, nectar, pollen, seeds, fish and other vertebrate (Patterson et al., 2003).
Other characteristics of bats are they show taxonomic stability, population that are able to be
monitored, have both short and long term effects towards their population that can be measured
and their wide distribution also make them become suitable to be used as bio-indicator (Jones
et al., 2009).

According to Bartholomew (2014), the intensive metabolisms of the bats also make
them important bio-indicator where the heavy metals can be detected even at very low
concentration. Bats were expected to consume relatively high and prolonged exposure of
chemicals contamination since bats are long-lived species with respect their small body size
which may cause the accumulation of contaminations such as heavy metals (Hickey et al.,
2001). To sum up, bats are very sensitive to environmental anthropogenic alteration–which
threatens the survival of many species (Hutsonet al., 2001), so they may successfully be
employed as bio-indicators (Jones et al., 2009).

2.5 Analysis of Heavy Metals in Bats

Heavy metals have been analyzed in numerous samples such as liver, kidney, whole
body/carcasses of the living organisms as the suitable bio-indicator. In a recent paper, Zukal et
al. (2015) have provided an overview of heavy metal research on wild bat populations to date;
pointed out major gaps in our present knowledge; and suggested future directions and
approaches for the study of heavy metal contamination and its possible direct adverse effects
on bats. The wild mammals have a positive correlations for determination concentrations of
metals in hair and blood (Vermeulen et al., 2009) and concentrations in kidneys, liver as well
as muscle (Marcheselli et al., 2010). Apart from that, animal tissues such as kidney that used
to determine the presence of elemental composition is a good indicator of environmental
pollution (Długaszek & Kopczynski, 2013). Few studies have considered the exposure and
potential impacts of toxic heavy metals such as Hg, Pb and Cd (Ma & Talmage, 2001) on bats.
These types of bio-indicator specimens were often used as the sample size that large enough
for any chemical analysis. What one author refers to as a whole body or carcass may not match
the definition of other authors (King et al. 2003) in various studies. Information on levels of
exposure for metals are, however, restricted to studies with limited sample sizes, areas of study,
tissue types and metals studied (Carravieiri & Scheifler, 2013).

13
 Several existing studies indicate that the kidney tissue from the bats was the target
organs for the determination of heavy metals. Based on the study conducted by Bartholomew
(2014), presence of few species of bats that consume the heavy metals in their diet had
accumulated the heavy metals in their particular tissues including kidney tissues. This study
also proven that kidney tissue is one of the important target organs for the main storage site of
metals pollutants especially Cd (Bartholomew, 2014). Generally, concentration of cadmium is
always come up against the kidney tissue because of the presence of metal binding with
proteins. Kidney tissues are also actively regulate the essential heavy metals which make the
concentrations of heavy metals in both tissues reflect the exposure and accumulation of the
heavy metals for a prolonged time (McGeer et al., 2000). Persistent sampling of liver and
kidneys has the limitation or more information of the element contaminations, to be exact, the
heavy metals accumulate differently in different targeted organs. According to Zukal et al.
(2015), lead, for example, accumulates in bones, liver, and kidneys; arsenic in liver, kidneys,
and brain; cadmium in kidneys and liver; and mercury in fur and liver. They also mention this
could be the reason it is impossible to compare levels between different organs, heavy metal
concentrations within particular tissue types are highly correlated (Zukal et al., 2015).

The data in Hernout et al. (2016) used to explore the correlations between the
concentrations of individual metals in different tissues, different metals in the same tissue, and
concentrations of metals in tissues and soils where the bats were sampled as well as for metal
analysis. Therefore, the researchers can use a bigger and unique comprehensive scale of 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 study
area on bats. In another study reported by Hernout et al. (2016), a strong positive correlations
were found between concentrations in tissues for a given metal (liver and kidneys for Cd, Cu
and Pb; stomach and fur and fur and bones for Pb), suggesting recent as well as long term
exposure to these contaminants. Just as he stated in his study also, the non-essential metals
such as Cd and Pb, could be toxic at low concentrations, essential metals, such as copper and
Zn, are tightly regulated at constant concentrations in tissues of mammals and, therefore,
mostly present within a narrow range.

14
 Figure 3: The table explains the relationships between bat fur and internal organs
such as kidneys, liver, stomach and stomach content as well as bones and soil concentrations
(Hernout et al., 2016)

Laboratory studies show that exposure of bats to metals can elicit a range of effects
including tremors, spasms, general slowness, lack of control in body movement, effects at the
physiological and histological levels (e.g. oxidative stress, DNA damage, tissue damage
including inclusion bodies, neurochemical alterations), and possibly mortality following
exposure to lead, cadmium, and zinc (Nam et al., 2012). The direct comparison between heavy
metals contamination from geographically distant areas in the results, is impossible, therefore,
the comparison of contaminated and the reference from localities on a regional scale remains
useful (Zoche et al. 2011; Naidoo et al. 2013). The exposure of the heavy metals on the bats’
surroundings for sure proven that the heavy metals can be detected also at the bats’ physical
(body).

Non-invasive sampling approaches such as sampling of fur, feather or claw are

invaluable in field-based monitoring studies to understand levels of pollution in wildlife
species, particularly for protected species, such as bats (D'Hav_e et al., 2006). Hair sampling
is highly suited as a non-invasive sampling technique as hair is easily accessible, stable, and its
storage does not require restricted conditions (Appenzeller & Tsatsakis, 2012). This is be
supported by Beernaert et al. (2007) study, as he stated also about the hair is particularly useful
for monitoring metals as the sulfhydryl group of the keratin present in the hair matrix has the
ability to bind metal cations. He also mention the organism can excrete metals in hair via their
continuous contact with the bloodstream during growth (Beernaert et al., 2007). Hair analysis
also can provide reliable information on exposure to non-essential metals (e.g. Cd and Pb) as
these are accumulated in storage organs and are then deposited in the hair as well as in the
nails, teeth, urine, faeces and sweat (Kales & Christiani, 2005).

Unlike bone and stomach content analysis, which can provide measures of long and
short-term exposure to metals, respectively, hair reflects exposure during the period in which

15
the hair is growing (Kales & Christiani, 2005). The hair analysis can indicate metal
bioavailability from the environment (McLean et al., 2009; Pereira et al., 2004; Marcheselli et
al., 2010). These methods are less damaging and stress inducing than invasive approaches such
as blood sampling or the taking of biopsies, and allow the monitoring of both levels of pollution
and the fitness of a given population over time, without significantly affecting the fitness of
individuals (D'Hav_e et al., 2006). Thus, the determination of heavy metals in bats as bio-
indicator will be analysed from different internal organs that can be detect whether the habitat
that the bats inhabit are polluted with heavy metals or less polluted. Whereas, there is a
significant different of heavy metals concentrations in different tissue of bats. As conclusion,
the more polluted of the habitat, the higher the concentrations of the heavy metals.

16
 CHAPTER 3

3.1 Study Sites

The study site is the area in the project boundary that was planned in the studies where
the sampling can be carried out. The sampling of bats will be done at Wind Cave Nature
Reserve (WCNR) (1° 24.915’ N and 110° 08.109’ E) in Bau district of Sarawak and in
University Malaysia Sarawak (UNIMAS) (1°27'34.79"N and 110° 26' 23.39" E). WCNR
reserve covers 6.16 hectares and includes Wind Cave (WC) itself and the surrounding forest
(Rahman et al., 2011). It was located about 5 km southwest of Bau and 30 km from Kuching.
Meanwhile at UNIMAS, have vast site of 810 hectares (Universiti Malysia Sarawak, 2019).

Figure 4: Location of the study sites in Sarawak

17
3.2 Sample Collection

Field sampling will be performed using ground methods which are mist-netting, harp-
trapping and hand netting (Abdullah, 2003; Hall et al., 2002). The nets placement in the field
will be great effect in capture success. All the morphological measurements of bats will taken
by using calipers to take the measurements of head and body length (HB), tail length (T), hind
foot length (HF), ear length (E), forearm length (FA), total length (TL), tail ventral length
(TVL), and wing span measurement while portable spring scale will be used to measure the
weight of the bats (Bartholomew, 2014). Species name and the area captured will be recorded
for all the captured bats in a table of samples. The bats should be euthanized by using
chloroform before isolation of organs will conducted. Eight to 12 mist nets and three harp traps
will be set in various locations supposed to be in the flyways of bats, such as above streams,
narrow pathways in the forest, trails, forest edge and the cave openings (Faisal et al., 2007;
Mohd-Azlan et al., 2008).

Bats will be identified based on selected morphological measurements of the bats that
taken using a vernier calliper (Mitutoyo), and bats will be weighted using a Pesola spring
balance for morphometric studies (Abdullah, 2003). Adult individuals will be selected to
maximize the chance of detecting concentrations (above the limit of detection LOD), since Cd
and Pb accumulation can increase with age (e.g. in bones for Pb) (Rudy, 2009). The bats that
are located in areas with extreme concentrations of heavy metals that has high as well as low
concentrations will be included to give a complete spatial coverage of the sample collection’s
study sites.

3.3 Bats’ Tissue Analysis

The bats will be dissected, and the post-mortem examination will be carried out as well
as the body organs will be excised for various studies. All the captured bats will be collected
by hand and with the nets and returned to the laboratory. Before analysis, the samples will be
sorted and cleaned of debris and dust, and rinsed with 1 N HCl and deionized water to remove
surface contamination (Hsu, Selvaraj, & Agoramoorthy, 2005). Then the cleaned, whole body
samples then dried to constant weight at 60 ∘C for 72 h. Next, kidney tissues of each bats species
will be pooled separately and weighed. For the analysis of samples, dried material will digested
in a mixture of concentrated HNO3 –H2O2 (5:2 ml) in closed Teflon beakers at 80∘C for 8 h.

18
Then, the tissue samples will placed in oven at 110 ∘C for 48 hours. The dried tissue samples
will be ground by using pestle and mortar.

The concentrations of heavy metals (Cd, Pb, Cu and Zn) are selected for analysis since
they are the most documented metals in several studies determining concentrations in bat
tissues and risks of metals to bats (Carravieiri & Scheifler, 2013). The heavy metals that
determined from the sample solutions will be analyzed using Inductively Coupled Plasma-
Mass Spectrometry (ICP-MS; located at the University Malaysia Sarawak). Preceding to
analyses by ICP-MS, the dried samples will undergo digestion that made up to a fixed volume
of 10 ml with Millipore water to obtain a final digest containing 10% acid. The calibration
standards will be prepared in the same acid matrix and the quantification will performed by
internal standardization where the analyte signals and the internal standard signals were
compared. The calibration curve will be used to convert the analyte signal into concentration
values. This method determines concentrations accurately and corrects for drift (changes in
sensitivity over time) and matrix effects (sample-related changes in sensitivity) (Hsu et al.,
2005).

All concentrations are expressed in mg/g (ppm) on a dry-weight basis using weights
obtained from oven-dried specimens. All specimens will run in patches that included known
standards, method blanks, and spiked specimens. Simultaneous performance of analytical
blanks determinations and standard reference material (Bovine liver BCR 185 for animal
samples) confirmed that the accuracy of methods was within acceptable limits. The accuracy
of the most of the metals was within ±10%. To be able to compare the results with previous
studies (Lüftl et al., 2003; Pikula et al., 2010), the assumption of the concentrations expressed
in dry weight were four times higher than wet weight values (Clark & Shore, 2001).

3.3 Statistical Analysis

This type of statistical will be conducted to verify the result obtained. Statistical analysis will
be performed using one-way ANOVA using SPSS version 14.0 to check for significant
difference between metal concentrations in the relationships in kidney tissue of bats from
different species at different study sites. All the measurement and analyze will be carried out
in truplicates. Thus, the Microsoft- Excel and Paleontological Statistics (PAST) version 2.1
will be used to conduct the statistical analysis. The results were expressed as mean ± standard
deviation (SD) in ppm.

19
CHAPTER 4

4.1 Expected Outcome

At the end of this study, the level of concentration of heavy metals in kidney and liver tissue
of bats from different species will be determine. Besides, the relationship of the existence of
the heavy metals in the bats tissue will be ascertain.

20
 CHAPTER 5

5.0 WORK PLAN

𝐏𝐫𝐨𝐣𝐞𝐜𝐭 2019 2020

⁄ 𝐌𝐨𝐧𝐭𝐡𝐬
𝐀𝐜𝐭𝐢𝐯𝐢𝐭𝐢𝐞𝐬 Sept Oct Nov Dec Jan Feb Mar Apr May

Literature
Review

Proposal writing

Proposal
presentation

Sample
preparation

Sample
collection

Sample analysis

Data analysis

Thesis writing

Preparation for
final
presentation
Final
Presentation

21
 CHAPTER 6

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