This study analyzed heavy metal concentrations in fish species from the Perak River in Malaysia. Iron showed the highest concentration in most fish species, while cadmium showed the lowest. Concentrations decreased in the order of Fe > Zn > Pb > Cu > Cd for most species. Bioaccumulation factors for iron and zinc exceeded thresholds in several fish. Estimated daily intake of cadmium, iron, and lead exceeded safety limits for all species. However, target hazard quotients and cancer risks were below EPA limits, indicating fish from the river pose minimal health risks for local consumption.

1. i
The Accumulation Characteristics of Heavy Metals in
Commonly Found Fish of Perak River and Their
Associated Health Risk
by
Rabiatul Adawiyah Binti Meor Mohamad Zain
E12A273
A thesis submitted in fulfillment of the requirements for the degree of
Bachelor of Applied Sciences (Sustainable Science) with Honours.
Faculty Of Earth Science
UNIVERSITI MALAYSIA KELANTAN
2016

2. ii
ACKNOWLEDGMENT
First and foremost, I would like to express my highest gratitude to the Almighty
for His guidance and blessings, upon the completion of this dissertation. Also full
appreciation to my supervisor, Dr Mohammed Abdus Salam for his time, guidance,
support, advices, suggestions, comments and supervision throughout the research.
I would also like to thank all sustainable science cources lecturers, Faculty of
Earth Sciences, lab assisstants, other staffs and the faculty itself for their assisstance and
cooperation in this study.
My special thanks to Dr Wan Rasidah binti Wan Abdul Kadir and Pn Rozita binti
Ahmad as my co-supervisors at Forest Research Institute Malaysia (FRIM), who had
helped me a lot to complete my research. Not forgetting the administartion of FRIM who
giving permission for me to use the equipment in their lab to complete my research.
Highest appreciation also go to my family who encourage and give full support in
doing my research especially my parents who help me a lot during the sampling progress.
Finally thanks to all my friends for their opinions, comments, continous encouragement
and moral support in completing this dissertation. And also thanks to all those who
involve directly and indirectly in completing this dissertation. There must be hard for me
to complete this study without their motivation and ethusiasm.

3. iii
TABLE OF CONTENT
ACKNOWLEDGEMENT i
TABLE OF CONTENTS ii
LIST OF TABLES vi
LIST OF FIGURES viii
LIST OF ABBREVIATIONS x
LIST OF SYMBOLS xii
ABSTRACT xiii
ABSTRAK xiv
CHAPTER 1 INTRODUCTION
1.1 Background of study 1
1.2 Problem statement 5
1.3 Significance of research 6
1.4 Objectives 8
1.5 Scope of study 8
1.6 Hypothesis 9
CHAPTER 2 LITERATURE REVIEW
2.1 River pollution 10

4. iv
2.2 Heavy metals pollution in aquatic environment 11
2.3 Heavy metals 13
2.4 Toxicity effects to aquatic organisms 14
2.5 Toxicity effects to human 15
2.5.1 Lead 16
2.5.2 Copper 16
2.5.3 Cadmium 17
2.5.4 Iron 18
2.5.5 Zinc 18
2.6 Accumulation of heavy metals in fish 19
2.7 Previous studies 22
2.8 Method use in the determination of heavy metals
2.8.1 Atomic Absorption Spectroscopy 24
2.8.2 Inductively Couple Plasma 25
2.8.2.1 Inductively Coupled Plasma Optical Emission Spectrometry 25
2.9 Freshwater fish species for the study
2.9.1 Tinfoil barb (Barbonymus schwanenfeldii) 26
2.9.2 Crossbanded barb (Puntius bulu) 26
2.9.3 Lemon fin barb (Puntius daruphani) 27
2.9.4 Barb, spiny (Mystacoleucus marginatus) 28
2.9.5 Fowler's danio (Devario regina) 28
2.9.6 Sagor catfish (Hexanematichthys Sagor) 29
2.9.7 Striated snakehead (Channa striatus) 29

5. v
CHAPTER 3 MATERIALS AND METHODS
3.1 Study area 31
3.2 Sample collection 33
3.3 Sample preparation and preservation 37
3.4 Sample digestion and extraction 37
3.5 Chemical reagents 38
3.6 Instrumentation 39
3.7 Quality assurance and quality control 40
3.8 Statistical analysis 41
CHAPTER 4 RESULTS AND DISCUSSION
4.1 Physical characteristics of all fish species 42
4.2 Spike recovery results 44
4.3 Metals concentration in fish species 45
4.4 Heavy metal concentration profile of fish species collected from Perak river 51
4.5 Comparison with other studies 53
4.6 Bioaccumulation factors (BAF) of metals in fish species 60
4.7 Bio-concentration factors (BCF) of metals in fish species 64
4.7 Health risk assessment
4.7.1 Estimated daily intake (EDI) of heavy metals 67
4.7.2 Target hazard quotients (THQ) of heavy metals 72
4.7.3 Target cancer risk (TR) 76

6. vi
4.8 Correlation analysis
4.8.1 Correlation analysis of heavy metals in fish species collected from Perak 79
4.8.2 Correlation analysis of heavy metals among omnivore and carnivore 80
species
CHAPTER 5 CONCLUSION AND RECOMMENDATION
5.1 Conclusion 83
5.2 Recommendation 86
REFERENCES 87
APPENDIX 94

7. vii
List of Tables
NO PAGE
3.1 Physical feature of fish species of present study 34
4.1 List of samples with narrow range of weight and length 43
4.2 Spiked recovery results 44
4.3 Metal concentration in fish species of Perak rivers 49
4.4 Comparison of heavy metals concentration in (μ/g dry weight) in 58
freshwater fish species collected from different parts of the world
4.5 BAF values of metals in fish species of Perak river 61
4.6 BCF values of metals in fish species of Perak river 64
4.7: Estimated daily intake (EDI) calculated for Perak area 68
4.8 Target hazard quotients (THQ) of heavy metals due to 74
consumption of fish from Perak river
4.9 Target cancer risk (TR) of heavy metals due to consumption of fish 77
from Perak river
4.10 Pearson correlation of heavy metals in fish species of Perak river 79
4.11 Pearson correlation of heavy metals among omnivores and carnivores 82
B.1: Data of Pearson correlation of heavy metals in all fish species collected 95
from Perak river.
B.2: Data of Pearson correlation of heavy metals between 95
Hexanematichthys sagor and Channa striatus
B.3: Data of Pearson correlation of heavy metals between 96
Puntius daruphani and Barbonymus schwanenfeldii
B.4: Data of Pearson correlation of heavy metals between 96
Puntius daruphani and Puntius bulu
B.5: Data of Pearson correlation of heavy metals between 97
Puntius daruphani and Mystacoleucus marginatus

8. viii
B.6: Data of Pearson correlation of heavy metals between 97
Puntius daruphani and Devario regina
B.7: Data of Pearson correlation of heavy metals between 98
Barbonymus schwanenfeldii and Puntius bulu
B.8: Data of Pearson correlation of heavy metals between 98
Barbonymus schwanenfeldii and Mystacoleucus marginatus
B.9: Data of Pearson correlation of heavy metals between 99
Barbonymus schwanenfeldii and Devario regina
B.10: Data of Pearson correlation of heavy metals between 99
Puntius bulu and Mystacoleucus marginatus
B.11: Data of Pearson correlation of heavy metals between 100
Puntius bulu and Devario regina
B.12: Data of Pearson correlation of heavy metals between 100
Mystacoleucus marginatus and Devario regina

9. ix
List of Figures
NO PAGE
2.1 Bioaccumulation process by fishes 20
2.2 Human exposure to mercury due to fish consumption 21
3.1 Map of Perak river 32
3.2 ICP-OES used in FRIM 40
4.1 Mean concentration of heavy metals in commonly found fish species of 48
Perak river
4.2 Percentage concentration of heavy metals in fish species of Perak river 52
4.3 BAF of heavy metals in fish species 63
4.4 BCF of heavy metals in fish species 66
4.5 Estimate daily intake (EDI) of Cd, Pb, Cu, Zn, Fe through 70
consumption of fish from Perak river
4.6 Target hazard quotients (THQ) of heavy metals due to 75
consumption of fish from Perak river
4.7: Target cancer risk (TR) of heavy metals from fish consumption 78
of Perak river
A.1: Flow chart of analytical procedure for heavy metals analysis in fish 94
C.1: Measuring process 101
C.2: Weighing process 101
C.3: Dissected muscle of the fish 101
C.4: The dried sampled 102
C.5: Acid digestion process 102
C.6: Filtration process 102
C.7: Double filtration process by using syringe filter 103

10. x
C.8: Analyzing process of heavy metals by using ICP-OES at FRIM, Kepong 103
D.1: Human activities occur on the riverside of Perak river 104
D.2: Fishing activities by nearby people 104
D.3: Rakit house was built for fishing purpose 105
D.4: Boat for fishing purpose 105
D.5: Dumping waste near the riverside of Perak river 106
D.6: Fish got from the local fisher 106

11. xi
List of Abbreviations
FAO Food and Agriculture Organization
WHO World Health Organization
DOE Department of Environment
WWF World Wildlife Fund for Nature
UNEP United Nations Environment Programme
USEPA United States Environmental Protection Agency
IARC International Agency for Research on Cancer
ATSDR Agency for Toxic Substances and Disease Registry
IUCN International Union for Conservation of Nature
ICPDR International Commission for the Protection of the Danube River
MAFF Ministry of Agriculture, Fisheries and Food).UK
KAGUM Kelah Action Group of Malaysia
OEHHA Office of Environmental Health Hazard Assessment
μg microgram
dw dry weight
v/v volume/volume
Cu Copper
Cd Cadmium
Pb Lead
Zn Zinc

12. xii
Fe Iron
AAS Atomic Absorption Spectroscopy
ICP-OES Inductively Coupled Plasma-Optical Emission Spectrometer
MESS Multi element standard solution
EDI Estimated Daily Intake
THQ Target Hazard Quotients
TR Target Cancer Risk
BAF Bioaccumulation Factor
BCF Bioconcentration Factor
RfD Oral Reference Dose
CSF Oral carcinogenic slope

13. xiii
List of Symbols
% percentage
˚C degree celcius
X multiplication
> more than
< less than
E-0 exponent (x10)

14. xiv
Study on The Accumulation Characteristics of Heavy Metals In Commonly Found Fish
of Perak River And Their Associated Health Risk.
ABSTRACT
This study was conducted to assess the concentration of heavy metals such as Cd,
Cu, Zn, Fe and Pb in the muscle of fish species name as tinfoil barb (Barbonymus
schwanenfeldii), crossbanded barb (Puntius bulu), lemon fin barb (Puntius daruphani),
sagor catfish (Hexanematichthys sagor), striated snakehead (Channa striatus), barb,
spiny (Mystacoleucus marginatus) and fowler's danio (Devario regina) collected from
Perak river. Acid digestion method was applied for extraction of the heavy metals. The
heavy metals were analyzed by using inductively coupled plasma optical emission
spectrometry (ICP-OES). Among metals, Fe showed the highest concentration in all
studied species except for Hexanematichthys sagor while Cd presented the lowest
concentration for all fish species. The concentration of studied metals among the fish
were in the descending order of Fe > Zn > Pb > Cu > Cd; Fe > Zn > Cu > Cd and Zn > Fe
> Pb > Cu > Cd for Puntius daruphani, Barbonymus schwanenfeldii, Mystacoleucus
marginatus and Devario regina ; Channa striatus and Puntius bulu ; Hexanematichthys
sagor respectively. Bioaccumulation factor (BAF) of Fe was more than 100 in all studied
fish species except Barbonymus schwanenfeldii and Hexanematichthys sagor.
Bioaccumulation factor for Zn only Devario regina over the limit. Bioconcentration
factor (BCF) value of zinc was higher than 100 for all species while for lead value only
Puntius bulu species not higher than 100. Estimated daily intake (EDI) for Cd and Fe
were over the limit of oral reference dose (RfD) for all fish species. EDI for Pb showed
Puntius daruphani, Barbonymus schwanenfeldii, Hexanematichthys sagor and Devario
regina were over the RfD dose. The health risk associated with Cd, Cu, Pb, Zn and Fe
were assessed based on target hazard quotients (THQ). Potential target cancer risk (TR)
was also assessed for Cd and Pb. The results indicated that the parameters for targeted
heavy metals were below the non-carcinogenic and carcinogenic limits suggested by
United State Environmental Protection Agency (USEPA). The ultimate results indicated
that all studied species were saved to be consumed by nearby inhabitants.

15. xv
Kajian Tentang Ciri-ciri Pengumpulan Logam Berat Dalam Ikan yang Biasa
Terdapat di Sungai Perak dan Risiko Kesihatan yang Berkaitan.
ABSTRAK
Kajian ini dijalankan untuk menilai kepekatan logam berat seperti Cd, Cu, Zn, Fe
dan Pb dalam otot ikan seperti ikan Lampam (Barbonymus schwanenfeldii ), ikan
Tengalan (Puntius bulu), ikan Kerai (Puntius daruphani), ikan Duri (Hexanematichthys
sagor), ikan Haruan (Channa striatus), ikan Sia (Mystacoleucus marginatus) dan ikan
Seluang pipih (Devario regina) yang diperolehi dari sungai Perak. Penghadaman asid
telah digunakan bagi proses pengeluaran logam berat. Logam berat telah dianalisis
dengan menggunakan alat Plasma Induktif Bersama – Spektrofotometer Pelepasan Optik
(ICP-OES). Antara logam, Fe menunjukkan kepekatan tertinggi dalam semua spesies
yang dikaji kecuali spesies Hexanematichthys sagor manakala Cd menunjukkan
kepekatan yang paling rendah bagi semua spesies ikan. Kepekatan logam dikaji di
kalangan ikan itu masing-masing menunujukkan penurunan bagi Fe> Zn> Pb> Cu> Cd;
Fe> Zn> Cu> Cd dan Zn> Fe> Pb> Cu> Cd untuk spesies Puntius daruphani,
Barbonymus schwanenfeldii, Mystacoleucus marginatus dan Devario regina; Channa
striatus dan Puntius bulu; Hexanematichthys Sagor. Faktor bio (BAF) Fe adalah lebih
daripada 100 dalam semua spesies ikan yang dikaji kecuali Barbonymus schwanenfeldii
dan Hexanematichthys Sagor. Faktor bio untuk Zn hanya Devario regina melebihi 100.
Nilai faktor biopemekatan Zn adalah lebih tinggi daripada 100 untuk semua spesies
manakala bagi nilai Pb Puntius bulu spesies sahaja tidak melebihi 100. Anggaran
pengambilan harian (EDI) untuk Cd dan Fe adalah melebihi had dos rujukan (RfD) untuk
semua spesies ikan. EDI untuk Pb menunjukkan Puntius daruphani, Barbonymus
schwanenfeldii, Hexanematichthys sagor dan Devario regina adalah melebihi dos RfD.
Risiko kesihatan yang berkaitan dengan Cd, Cu, Pb, Zn dan Fe telah dinilai berdasarkan
hasil bahagi bahaya sasaran (THQ). Potensi risiko kanser sasaran (TR) juga dinilai untuk
Cd dan Pb. Keputusan menunjukkan parameter bagi logam berat yang disasarkan adalah
di bawah had bukan karsinogenik dan karsinogenik yang disyorkan oleh Agensi
perlindungan alam sekitar Amerika Syarikat (USEPA). Keputusan muktamad
menunjukkan bahawa semua spesies yang dikaji adalah selamat untuk dimakan oleh
penduduk berhampiran.

16. 1
CHAPTER 1
INTRODUCTION
1.1 Background of the study
Fish is defined as an important main protein source of food for human
(Ebrahimpour et al., 2011). According to the Food and Agriculture Organization
(FAO) report on 2010, the world per capita food fish supplies increased from an
average of 18.1 kilograms (live weight equivalent) in 2009 to an amount
estimated 18.8 kilograms in 2011 (Medeiros et al., 2014). The consumption of
food fish in Malaysia has increased by 150% since 1961. The average Malaysian
consumes about 52 kilograms of seafood per year with an expected increase of its
consumption in 2020 to be at 1.68 billion kilograms (FAO, 2013).
In recent years, production of freshwater fish is increased in Malaysia. The
commonly species are keli, patin and tilapia which can also be processed into
convenience products rather than being sold fresh. The convenience foods are
gaining popularity among Malaysian and also worldwide consumer. By 2015,
world total demand for fish and fishery products is projected to expand by 20
million tons to 183 million tons (Mohamad et al., 2010).

17. 2
The consumption of fish has increased due to importance to health as it
provides healthy, low cholesterol sources of protein and other nutrients
(Kamaruzzaman et al., 2010) such as the presence of Omega-3 fatty acids, fats,
amino acids and vitamins. It also contains several minerals, including calcium,
iron, cadmium, lead, copper, and zinc (Sofia, 2005). The content of essential
metals in fish can give positive benefits of the Omega-3 and protein in fish
(Ebrahimpour et al., 2011).
Fishes are most important organisms in the aquatic food chain that
commonly situated at the top level. Normal metabolism of fishes can accumulate
metals from food, water, or sediment (Zhao et al., 2012) and sensitive to heavy
metals contamination (Akan et al., 2012). Thus fishes are often act as an
important biological indicator to investigate metal levels in their living
environments and to assess ecological and health risks posed by anthropogenic
waste discharges (Zhao et al., 2012). Most of the freshwater fishes are restrained
to specific microhabitat within inter connected river or stream system. Once the
system becomes contaminated by heavy metals, fish species either shifts to less
polluted segment of river or stream system or die off which at last disturb the food
chains (Akan et al., 2012).
Heavy metals discharged into aquatic environment might damage the
species diversity as well as ecosystems, due to their toxicity, long persistence, and
accumulative behavior (Ebrahimpour et al., 2011) and finally integrated by
human as one of the main consumers resulting in health risks (Rahman et al.,
2012). Fish that occupy high levels in the aquatic food chain are known for their

18. 3
ability to accumulate heavy metals in their body parts (Taweel et al., 2013),
including muscle. Therefore for this study, muscles were selected as a primary
site of metal uptake and since fishes are essential component of human diet, they
need to wisely monitor to ensure that unnecessary high level of heavy metals are
not being transferred to human population through consumption of fish.
Highly increasing of contamination of water, soil and food that contribute
to heavy metal pollution has attracted huge concern among the worldwide. This
pollution not only poses threat to public water suppliers but also gives threat to an
ecological and human health risk through the consumption of aquatic products.
The studies on the heavy metal pollution in fish become important for
characterizing its associated health risks (Islam et al., 2014)a
and to identify the
process of the accumulation of those heavy metals in fish body. In recent decades,
much attention has been given to the investigation of trace metals elements in
food products due to the increasing concern about the health risks of food
consumption (Guerin et al., 2011).
After decades of rapid urbanization, population growth and
industrialization, developing countries are now home to many of the world’s most
critical air, water and solid waste problems. Early studies have identified the
increase in the pollution of particular heavy metals in freshwater systems around
the world, specifically in rivers. The pollution has mainly been caused by
industrial processes and industrial waste (Hashim et al., 2014). Other than that,
deforestation, domestic or animal farming sewage, sand mining and agriculture
act as the main source of heavy metal pollution (DOE, 2002). The Department of

19. 4
Environment reported higher concentrations of heavy metals in the waters off the
west coast of Peninsular Malaysia compared to other areas because of the
extensive land use and industrialization.
The sources of drinking water for humans mostly come from the rivers. At
the same time, rivers are considered a sink for waste and certainly riverine aquatic
ecosystems suffers the consequences of the domestic and industrial activities
occurring in its watershed (Taweel et al., 2013). Many harmful substances are
washed into the aquatic environment due to the bad drainage systems from areas
surrounding. This situation lead to the huge monitoring and analysis of many
pond, lake, and river throughout the world especially on the developed countries
with increasing of industrial and urbanization activities.
Perak river is the second longest river in Peninsular Malaysia, it starts
from the north-western corner of the state, flows south to Teluk Intan, where it
bends westward and into the Straits of Malacca. The river divides the state into
two nearly equal halves and thus forms its natural backbone. The industrial
activities mostly occurred at the midstream and downstream of Perak river. This
presents a concern in terms of the health of local aquatic ecosystems and the
people inhabiting the area.
In present study, the accumulation characteristics of copper, cadmium,
lead, iron, and zinc were investigated in commonly found freshwater fish of Perak
River. There are seven types of freshwater fish species selected for the analysis of
heavy metals concentrations for present study. The accumulation factors were

20. 5
investigated considering the concentration of sediments and water of Perak river
from other studies as fishes can accumulate metals influenced by food, water, or
sediment. Their associated health risk to the people surroundings or consumed the
fish of Perak River were investigated. The samples are collected from upstream to
the downstream of due to the availability of fish species of Perak River.
1.2 Problem Statement
Nowadays the consumption of fishes as one of the main menu intake for
Malaysian people is exceedingly high without realizing the content of heavy
metals in the fish. The consumption of aquatic animals with accumulation of
heavy metals may cause serious threats to human health (Kamaruzzaman et al.,
2011). Thus it is significant to determine the accumulation characteristics of
heavy metals in fish and its associated health risk problems.
The processes and pathways of pollutants from one trophic level to
another can be describes by biomagnification and bioaccumulation of heavy
metals in living organisms (Akan et al., 2012). Those trophic level also included
the participation of human as a consumer in a food chain. According to this, it is
necessary to identify the concentrations of level of heavy metals in commonly
consumed fish in order to evaluate the possible risk of the fish consumption.

21. 6
Anthropogenic activities had contributed to the massive heavy metals
pollution. Developing countries now home for the world’s most critical air, water
and solid waste problems due to of rapid urbanization, growth of population, and
industrialization. Early studies had acknowledged the rise of particular heavy
metal pollutions in freshwater system around the world especially rivers that
arise mainly from industrial processes and industrial waste (Hashim et al., 2014).
Therefore, it is important to determine the relationships of those anthropogenic
activities with the levels of heavy metals in freshwater fishes in order to access
the human health risk assessment and also the level of the water quality of the
river.
1.3 Significance of research
Food safety at present is a major concern to the environmental scientists.
The increasing demand of food safety research has accelerated regarding the risk
related with food consumption of aquatic organisms that contaminated by heavy
metals (Islam et al., 2014)b
. For characterizing health risks assessment, studies on
heavy metal pollution in fish are important. Studies have shown that the main
contributors to metal contamination in freshwater environments are from urban
and industrial developments (Islam et al., 2014)a
.

22. 7
Thus, it was necessary for us to conducting the research on the
accumulation of heavy metals in freshwater fish that commonly and mostly
consumed by Malaysian people. Indirectly the pollution status of Perak River also
was indicated by conducting the study. The heavy metals were dumped to the
rivers basin by domestic, industrial and mining activities which absorbed and
deposited into the sediments. Therefore, it was important to investigate the
accumulation characteristic of heavy metals in the organs of fish as this would
serve an essential function in order to identify the save part of the fish to be
consumed on our daily diet. It prevented the consumers from consuming
contaminated fish species.
The present study gave the scenarios on how the heavy metals accumulate
in fish’s muscle tissues through various biochemical processes such as
bioaccumulation, bioconcentration and biomagnification processes. The analysis
of possible heavy metals that accumulated in fish may elucidate the effects of
heavy metals to human health risk by comparing the concentrations of selected
heavy metals.

23. 8
1.4 Objectives
i) To assess the accumulation characteristics of heavy metals in seven types of
freshwater fish species such as tinfoil barb (Barbonymus schwanenfeldii),
crossbanded barb (Puntius bulu), lemon fin barb (Puntius daruphani), sagor
catfish (Hexanematichthys sagor), striated snakehead (Channa striatus), barb,
spiny (Mystacoleucus marginatus) and fowler's danio (Devario regina) collected
from Perak River.
ii) To elucidate the effect of heavy metals to human health risk through fish
consumption for the residence of Perak.
1.5 Scope of study
This study will investigate the accumulation characteristics of copper,
cadmium, lead, iron and zinc were in commonly found and consumed freshwater
fish of Perak River. The study also will identify the health risk assessment that
associated with the studied heavy metals in the fish species of Perak River
through the calculation of Estimated Daily Intake (EDI) of heavy metals, Target
Hazard Quotients (THQ) and Target Cancer Risk (TR) of heavy metals.

24. 9
1.6 Hypothesis
The consumption of fishes with highly contaminated heavy metals for
long term exposure may cause serious health problem to the consumers. It was
hypothesized that heavy metals composition in fish body was influenced by
bioaccumulation and bioconcentration factor. The accumulations of various
metals in fish body are in different amounts. These differences result from
different affinity of metals to fish tissues, different uptake, deposition and
excretion rates. Thus, the interaction of heavy metals with fish can be identified
through the analysis. In this present study, only muscle tissue was chosen to be
analyzed as it was the only part that been consumed by Malaysia people. Health
risk assessment can be analyzed through the procedure mentioned in the
methodology.
The significance of heavy metals in fish can be analyzed through Pearson
correlation analysis. If the levels of the concentration of heavy metals in selected
fish exceed the standard value recommended by FAO/WHO 1984, Malaysian
Food Regulation 1985 and other international limits, the human health was
severely affected. Apart from that, an effective measure should be conducted by
government for the analysis of heavy metals in fish of Perak River. At the same
time, the pollution status of Perak River will be determined. This present study
aimed to provide an essentials information on the assessment of commonly
consumed fish species of Perak River considering human health.

25. 10
CHAPTER 2
LITERATURE REVIEW
2.1 River pollution
Rivers were used as a source of drinking water for humans and be
submerged for waste. Undoubtedly, aquatic environment become suffered due to
the consequences arisen from the domestic and industrial activities occurring in
its watershed. A river system had been drains for the areas surrounding it and this
lead to the disposal of various types of harmful substances into the aquatic
environment. Many pond, lake, and river systems in the developed world have
been subjected to regular monitoring of their contaminant levels (Taweel et al.,
2013). Pollution was one of the largest threats to our rivers. The reduction of river
water quality was a clear indicator of the decline in the environmental health of a
river basin.
The sources of pollution come from domestic and industrial sewerage,
effluents from livestock farms, manufacturing and agro-based industries,
suspended solids from mining, housing and road construction, logging and
clearing of forest and heavy metals from factories. Urbanization significantly
contributed to the increase in water pollution problems. Inefficient waste disposal
systems and lack of proper management lead to waste and sewage ending up in
rivers. Rivers contaminated by sewage contain high levels of organic pollutants,

26. 11
and become breeding grounds for harmful bacteria and viruses that lead to arising
of various diseases and loss of reproductive ability of fish and other aquatic
organisms (WWF Malaysia, 2014).
Pollution of the aquatic environment causes by inorganic chemicals had
been considered a major threat to the aquatic organisms including fishes (Akan et
al., 2012). According to United Nations Environment Programme (UNEP), in
developing countries, rivers downstream from major cities are little unclear than
open sewers. It also reported that 1.2 billion people are being affected by polluted
water and that dirty water contributes to 15 million child deaths every year
(UNEP, 2007). Rivers pollution leads to the degradation of biodiversity. The
contamination of heavy metals creates huge problem towards those species
particularly the fishes which survive in polluted rivers and this problem may
contribute to the health damage of human which is the main consumers of fishes
(Taweel et al., 2012).
2.2 Heavy metals pollution in aquatic environment.
A pollutant is any substance in the environment which causes harmful
effects to the environment, reducing the quality of life and may ultimately
cause death (Duruibe et al., 2007). Heavy metals were environmental priority
pollutants and becoming one of the most serious environmental problems because
of their persistence, toxicity, non-biodegradability, and ability to be integrated
into food chain. Over the past century, rapid industrialization had resulting the

27. 12
discharged of heavy metals containing industrial effluents into the world’s rivers
and lakes that consequently accumulated in marine species and sediments
(Rahman et al., 2014).
The manufacturing sector was considered as the major contributor of
metal pollution in the environment. Metal finishing processes such as
electroplating, etching, and preparation of metal components for various
industries had been identified as a major source of wastes containing high
concentrations of heavy metals. These kind of industries had contributed more
than 69,000 m³ per annum of sludge containing heavy metals in several states of
Peninsular Malaysia in 1992 (Shazili et al., 2006). These metals could reach food
chains through various biochemical processes such as bioconcentration,
bioaccumulation and biomagnification in various trophic level that eventually
threaten the health of the humans that consumed aquatic organism such fish.
(Kamaruzzaman et al. 2011)
Increasing of industrialization and extensive agricultural activities
contributed to the prominent levels of metals in water body. Contaminations of
aquatic ecosystems and nearby areas with metals have been receiving worldwide
attention, especially in developing countries. Metals and metalloids from natural
and anthropogenic sources enter the aquatic environment continuously where they
pose a serious threat to human and ecological health, due to their toxicity, long
persistence, bioaccumulation, and biomagnification in the food chain (Islam et al.,
2014)b
.

28. 13
2.3 Heavy metals
Heavy metals can be defined as any metallic element that has a relatively
high density and is toxic or poisonous even at low concentration (Duruibe et al.,
2007). Metals occur naturally in the earth's crust, and in the environment that can
diverge between different regions resulting in spatial variations of background
concentrations. The distribution of metals in the environment was managed by the
properties of the metal and influenced by environmental factors. Approximately
30 metals and metalloids were potentially toxic to humans from 92 metals that
naturally occurred. Heavy metals was the metallic elements having an atomic
weight higher than 40.04 g/mol (the atomic mass of Ca) (Morais & Garcia, 2010).
The term ‘heavy metal’ had been used to describe metals that are
environmental pollutants. Some metals were essential when taken up by
organisms but their excessive presence will reverse the effect so that benefit
becomes toxicity (Sofia, 2005). Metals were very toxic because ions or
compound form were soluble in water where the fish live and may be easily
absorbed into the fish and bind to structural proteins and enzymes. In humans,
some metals could cause severe physiological and health effects (Taweel et al.,
2011).
Heavy metals could accumulate to toxic concentrations and caused
ecological damage under certain environmental conditions (Damodharan &
Reddy, 2013). Heavy metal pollution was a serious and massive
environmental problem due to their toxicity. It entered the environment through

29. 14
various natural methods and human activities. As a resulted, it was being
accumulated in fish and other aquatic organisms. Fish were the final organism in
the aquatic food chain and a significant food source for human being (Taweel et
al., 2011).
2.4 Toxicity effects of heavy metals to aquatic organisms.
Heavy metal pollution in the environment had become concerned due to
the continuously increasing rate of contaminated water, soil and food in many
regions of the world. Studies had shown that urban and industrial developments
are the main contributor to metal contamination in freshwater environments
(Islam et al., 2014)a
. Heavy metal can be combined into food chains and absorbed
by aquatic organisms to a level that might affects their physiological state.
Heavy metals considered as an effective pollutants which have drastic
environmental impact on all organisms. Trace metals such as Zn, Cu and Fe play
a biochemical role in the life processes of all aquatic plants and animals which
were essential in the aquatic environment in trace amounts (Akan et al., 2012).
For normal metabolism of fish, the essential metals from water, food or
sediment must be consumed but unconsciously similar to the route of essential
metals, non-essential metals also being taken up by fish and accumulate in their
tissues (Kamaruzzaman et al., 2010).

30. 15
Concentration of metals becomes toxic to the fish when its level exceeds
the limit. This threshold limit not only changes from metal to metal but also from
one species to another. Toxic effects of metals become more definite when
various metabolic activities inside organism body fail to detoxify (Akan et al.,
2012).
2.5 Toxicity effects of heavy metals to human
Pollutants such as heavy metals, pesticides and herbicides pose health
hazards to human beings and aquatic life. Consumption of fish, prawn or other
aquatic life that have accumulated heavy metal pollutants result in disturbed
reproduction rates and life spans. Pesticide and herbicide contamination may lead
to death or chronic long term illness in humans as well as impair the fertility and
development of both humans and aquatic life (WWF Malaysia, 2014). Thus,
researched towards trace metal pollution in fish were important to evaluate the
health risks (Islam et al., 2014)a
.
The content of toxic heavy metals in fish can work against their beneficial
effects. Several adverse effects of heavy metals to human health including serious
threats like renal failure, liver damage, cardiovascular diseases and even death
have been known for long time (El-Moselhy et al., 2014). Some metals were
essential to human health. Metals were naturally occurring elements that become
contaminants when human activities increase their concentrations above normal
levels in the environment (Taweel et al., 2011).

31. 16
2.5.1 Lead
Lead (Pb) was one of the oldest metals known to man that had been used
in various manufacturing and industrial activities. These anthropogenic activities
were responsible for most of the lead pollution which the inputs greatly exceed
those from natural sources. Most of the lead occurred in the environment is in
inorganic form (Sofia, 2005). Lead may cause renal failure and liver damage.
Besides, long term exposure to lead will result in coma, mental retardation and
even death (Rahman et al., 2012). Contaminated air, water, soil, food, and
consumer products are some of the routes of exposure to lead. Lead poisoning is
normally ranked as the most common environmental health hazard (Ahmed et al.,
2014).
2.5.2 Copper
Copper (Cu) was an essential metal for all living organisms and was found
in all body tissues. It was widely distributed in nature in free state and in sulfides,
arsenides, chlorides and carbonates. Widespread use of copper in industrial,
agricultural, and also domestic activities made copper become one of the most
common environmental pollutants. Approximately 17,000 metric tons of solid
copper wastes were deposited annually into the oceans. Excessive storage of
copper in the liver can cause Wilson’s disease, an inborn error of metabolism,
also called hepatolenticular degeneration.

32. 17
Wilson’s disease heightens the urinary excretion of copper considerably.
Main excretion route of copper was via the bile and only a few percent of the
absorbed amount is found in urine (Sofia, 2005).
2.5.3 Cadmium
Cadmium (Cd) injures the kidney and cause symptoms of chronic toxicity,
including impaired kidney function, poor reproductive capacity, hypertension,
tumors and hepatic dysfunction (Rahman et al., 2012). Cadmium had not been
found to occur naturally in its pure state and its concentration seems to be directly
proportional to zinc and lead concentrations. Use of cadmium in agriculture and
industry had been identified as a major source of large distribution into the
environment and food. The major route of exposure to cadmium for the non-
smoking general population was through the consumption of food which was the
contribution from other pathways to total uptake was small. Cadmium had been
classified as Class 1 by The International Agency for Research on Cancer (IARC)
which is ‘The agent (mixture) is carcinogenic to humans’. Lead and cadmium
were the most commonly disseminated environmental metal poisons and each of
these persistent contaminants had been blamed for major poisoning incidents
(Ahmed et al., 2014).

33. 18
2.5.4 Iron
Iron (Fe) was an integral part of many proteins and enzymes that maintain
a good health (Institute of Medicine, 2001). Iron was an essential component of
proteins for human which involved in oxygen transport (Adu, 2010). Iron was the
fourth most abundant metal in the earth’s crust and the most abundant transition
metal. Iron can easily change valence and form complexes with oxygen. Iron
mediated reactions support the respiration of nearly all aerobic organisms.
However, unless properly protected, iron catalyzes the formation of radicals that
can damage biological molecules, cells, tissues, and entire organisms. Exposure to
excess iron that typically from multiple blood transfusions over many years can
lead to numerous pathological consequences (Ponka et al., 2007).
2.5.5 Zinc
Zinc (Zn) was one of the most common elements in the Earth's crust. Zn
was found in the air, soil and water and also presented in all foods. In its pure
elemental (or metallic) form, Zn was a bluish-white, shiny metal. Metallic Zn had
many uses in industry. A common used for Zn was to coat steel and iron as well
as other metals to prevent rust and corrosion. Zn compounds that may be found at
hazardous waste sites were zinc chloride, zinc oxide, zinc sulfate, and zinc
sulfide. Most zinc ore found naturally in the environment was in the form of zinc
sulfide. Zinc enters the air, water, and soil as a result of both natural processes and
human activities.

34. 19
Most zinc enters the environment as the result of mining, purifying of
zinc, lead, and cadmium ores, steel production, coal burning, and burning of
wastes. Zinc was an essential element needed by our body in small amounts but
mostly were exposed to zinc compounds in food (ATSDR, 2005).
2.6 Accumulation of heavy metals in fish
The ability of fishes to accumulate heavy metal has been proved to be
affected by many factors such as ecological needs, swimming patterns, metabolic
activities, and living environments. Among these factors, living environment is
often considered to be more important because aquatic systems are quite complex
and metal contaminants are not uniformly dispersed. Skin or gill tissue of pelagic
fishes are usually being detected of high levels of trace metals in water since
metals can be absorbed on fish skin or accumulated through breathing by gill
(Zhao et al., 2012).
Metal accumulated in fish in various pathways such as ingestion of food,
suspended particulate matter and metal ion exchange through gills and skin.
Heavy metals enter into fish through five routes which are food, suspended
particle, gills, intake of water and integuments. Metals get absorbed into blood
and transported to various organs for either storage or excretion from these
pathways (Akan et al., 2012). Diffusion process enabled transport or absorption in
gills and surface mucus are the mechanism of uptake from water (figure 2.1).

35. 20
The amount ingested by the organisms, the way in which the metals are
distributed among the different tissues and the extent to which the metal is
retained in each tissue type are reflected through bioaccumulation process
(Nwamaka & State, 2013).
Figure 2.1: Bioaccumulation process by fishes. (Aboriginal Affairs and Northern
Development Canada (AANDC), 2011)
Level of trace metals in different organs of fish is used as an indicator of
metal pollution in an ecosystem, which is considered as an important tool to
highlight the role of high level of metals in aquatic organisms. Concentration of
heavy metals in different tissues or organs of fishes is directly influenced by
contamination in aquatic environment, uptake, regulation and elimination inside
the fish body.

36. 21
The way of exposure of the organisms such as through diet or their
elevated level in surrounding environment also influenced the accumulation
process of metals in different organs and tissues. Therefore, heavy metals show
different accumulation pattern in organs (Akan et al., 2012).
Figure 2.2: Human exposure to mercury due to fish consumption. (Utah
Department of Environmental Quality, 2014)

37. 22
2.7 Previous studies
Kamaruzzaman et al., (2010) were conducted a study on the levels of
some heavy metals in fishes from Pahang River estuary Malaysia. The aim of the
study was to assess the concentration of lead, copper and zinc in six different fish
species. It can be served as an indicator to indicate the pollution in the Pahang
River estuary. The study had shown that all catfishes (Arius sp.) presented the
highest metals content. Tissue analysis revealed that the stomach accumulated the
highest level of those selected metals. According to the results, metals
concentration in the edible part of the examined fish which was muscle were in
the safety permission levels for human food consumption (Kamaruzzaman et al.,
2010).
Taweel et al., (2013) were investigated a study on the assessment of heavy
metals in tilapia fish (Oreochromis niloticus) from the Langat River and
Engineering Lake in Bangi, Malaysia. The evaluation of the health risk from
tilapia consumption also was investigated. Concentrations of the heavy metals
such as copper(Cu), cadmium(Cd), zinc(Zn), lead(Pb) and nickel(Ni) were
determined in the liver, gills and muscles of tilapia fish. There were differences in
the concentrations of the studied heavy metals between different organs and
between sites. The health risks evaluation were assessed based on the target
hazard quotients. A health risk analysis of the heavy metals measured in the fish
muscle samples indicated that the fish can be classified at one of the safest levels
for the general population and that there are no possible risks pertaining to tilapia
fish consumption (Taweel et al., 2013).

38. 23
Islam et al., (2014) had conducted a study on metal speciation in sediment
and their bioaccumulation in fish species of three urban rivers in Bangladesh. Six
trace metals which were chromium (Cr), nickel (Ni), copper (Cu), arsenic (As),
cadmium (Cd) and lead (Pb) were measured in sediments and soft tissues of three
commonly consumed fish species (Channa punctatus, Heteropneustes fossilis,
and Trichogaster fasciata) collected from three urban rivers around Dhaka City,
Bangladesh. The abundance of total metals in sediments varied in the decreasing
order of Cr>Ni>Pb>Cu>As>Cd. Sequential extraction tests showed that the
studied metals were mostly associated with the residual fraction followed by the
organically bound phase. The rank of biota sediment accumulation factor for fish
species were in the descending order of Cu >As >Pb >Ni >Cr >Cd. Based on the
resulted, metal concentrations in fish exceeded the international permissible
standards suggesting that these species were not safe for human consumption
(Islam et al., 2014)a
.
According to (Rahman et al., 2012), concentrations of eight heavy metals
(Pb, Cd, Ni, Cr, Cu, Zn, Mn, and As) in the muscles of ten species of fish
collected from Bangshi River at Savar in Bangladesh were measured in two
different seasons. The concentrations of the studied heavy metals, except Pb in
Corica soborna, were found to be below the safe limits suggested by various
authorities and thus gave no indication of pollution. The present study also
showed that, Zn was the most and Cd was the least accumulated metal in the
studied fish muscles.

39. 24
From the human health point of view, this study showed that there was no
possible health risk to consumers due to intake of studied fishes under the current
consumption rate (Rahman et al., 2012).
2.8 Method use in the determination of heavy metals
2.8.1 Atomic Absorption Spectroscopy
The energy sources of an Atomic Absorption Spectroscopy (AAS) emitted
resonance line radiation. The instrument’s detector measured the level of
absorption from a sample, which was fed as an aerosol and vaporized. Analytic
concentration was determined from this. The most advanced instruments had
more than one channel for simultaneous determination of several elements
(Galbraith Laboratories, 2011).
In direct aspiration atomic absorption spectroscopy a sample is aspirated
and atomized in a flame. A light beam from a hollow cathode lamp, whose
cathode is made of the element to be determined, is directed through the flame
into a monochromatic, and onto a detector that measures the amount of light
absorbed. Absorption depends upon the presence of free unexcited ground state
atoms in the flame. Since the wavelength of the light beam is characteristic of
only the metal being determined, the light energy absorbed by the flame is a
measure of the concentration of that metal in the sample (Horvath, 2009).

40. 25
2.8.2 Inductively Couple Plasm
Inductively Coupled Plasma (ICP) had two types which were Optical
Emission Spectrometry (OES) and Mass Spectrometry (MS). ICP used very high
temperature plasma, sustained with a radiofrequency electric current, that
efficiently desolvates, vaporizes, excites and ionizes atoms. ICP was coupled with
MS or OES which ICP as the method of ionization, with MS or OES as the
method of identification and detection of ions. Both methods were highly
sensitive and since all atoms in a sample were excited at once, they could be
detected simultaneously (Thermo scientific, 2014).
2.8.2.1 Inductively Coupled Plasma Optical Emission Spectrometry
Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) is
an elemental analysis technique that derives its analytical data from the emission
spectra of elements excited within a high-temperature plasma. The purpose of the
ICP-OES optical system is to separate element-specific wavelengths of light,
emitted from the excited sample and to focus the resolved light onto the detector
as efficiently as possible. The spectrometer is comprised of two sections, the fore-
optics and the polychromatic. When the light exits the polychromatic, it is focused
on to the detector.

41. 26
2.9 Freshwater fish species for the study
2.9.1 Tinfoil barb (Barbonymus schwanenfeldii)
Tinfoil barb (Barbonymus schwanedfeldii) or also known as Striped-tail
Tinfoil Bard, Red Tin-foil is a freshwater fish species that classified in
Cypriniformes (Carps) (Fishbase). This fish belongs to a genus of carp-like fishes,
having downward-pointing “whiskers” at each end of its upper lip. Tinfoil barbs
are large, peaceful fish. They are fast swimmers, preferring to swim in shoals and
are a schooling species. It is distinguishable from other similar species of the
genus by the red dorsal fin which terminates in a black tip. The body is laterally-
compressed and silver or silver-gold. Their common name (tinfoil barb) is derived
from the “tin-plated” look of the scales. Tinfoil barbs are benthopelagic (fish
having neutral buoyancy), which allows them to float. It migrates back to their
natal spawning ground to reproduce. They are an egg-scattering species, so they
do not care for their eggs. Eggs hatch in 16 hours and fry (new born) are free
swimming within another two days (IUCN, 2011).
2.9.2 Crossbanded barb (Puntius bulu)
The Tengalan / Crossbanded barb has similar body shape to the lampam /
Tinfoil barb that also classified in Cypriniformes. The species can be found in
remote rivers, usually occupying deep pools. It is a difficult fish to catch on rod
and line. Worms are the best bait, mounted on a small hook to match its dainty
bite. It scientific name is Puntius bulu. The distinctive features are slightly curved

42. 27
dorsal fin, and faint vertical black stripes along the flanks. Scales are smaller. It
usually found at deep stretches of large rivers (KAGUM, 2013). This species
commonly found in Southeast Asia; Sumatra, Borneo and Indochina.
Crossbanded barb also occurs at midwater to bottom depths in large lowland
rivers and lakes. Moves into the flooded forests when water levels are high,
feeding mainly on submerged plants as well as on some filamentous algae and
insects which occur on the plants. Feeds also on crustaceans and small fishes that
can be conclude as omnivores. Formerly common, but has become very rare
recently (Fishbase, 2015).
2.9.3 Lemon fin barb (Puntius daruphani)
The Lemon fin barb is also known as the Yellow fin barb, Pale barb or
Golden belly barb. They are a Potamodromous fish which is defined as a
migratory fish which migrates wholly within freshwater. It is an omnivore. This is
a less common species of Puntius. The Kerai is a powerful fish, occupying the fast
main current of the river. It can grow to about 5 kg and is thus a formidable
quarry for the angler. It salient features is it possesses the typical compressed
Puntius body, but longer than the lampam’s, with pale white scales laced with a
brassy sheen. Fins are light grey. The prefer habitat usually at the main current of
large rivers and feeds just below the surface, in the fast water. Commonly diets
are worms and crustaceans. This species distributed at the upland waters of
Pahang and Perak rivers (KAGUM , 2014).

43. 28
2.9.4 Barb, spiny (Mystacoleucus marginatus)
Barb, spiny (Mystacoleucus marginatus) or synonym with Barbus
marginatus is classified in Cypriniformes. This very common species is widely
distributed throughout Southeast Asia. It occurs in large numbers and is not
thought to have significant threats at present, and is assessed as Least Concern.
The species is widely distributed throughout Southeast Asia. It is a very common
species in all suitable habitats in its range. Population status in Java is unknown,
where rivers are impacted by high levels of pollution. This species found in a
wide range of habitats, from lowland rivers and marshlands to montane streams,
well adaptive in impounded rivers. It can be an indicator of degraded streams if it
becomes dominant and thought to breed when water levels rise. The species is
occasionally seen in markets (Rainboth 1996), but of low fishery interest, due to
its procumbent dorsal spine that makes it difficult for fishers to remove it from
nets. This species occasionally seen in the aquarium trade (IUCN, 2015).
2.9.5 Fowler's danio (Devario regina)
The queen danio (Devario regina) is a tropical fish belonging to the
minnow family (Cyprinidae). Originating in India, Myanmar, Thailand,
northwestern Malaya, and the Mekong River basin, this fish is sometimes found
in community tanks by fish-keeping hobbyists. It grows to a maximum length of
3.1 inches (7.8 cm). In the wild, the queen danio is a rheophilic species found in
fast-moving rivers with sandy bottoms in a tropical climate, and prefer water with

44. 29
an ideal temperature range of 73-77°F (23-25°C). Its diet consists of annelid
worms, small crustaceans, and insects. The queen danio is oviparous (an egg
layer). (Seriously Fish, 2015).
2.9.6 Sagor catfish (Hexanematichthys sagor)
Sagor catfish is classified in Actinopterygii (ray-finned fishes). This
species distributed to Indo-West Pacific: Pakistan, west and east coast of India,
Bangladesh, Myanmar, Thailand, Malaysia, Singapore, Indo-Australian
Archipelago (but not occurring in Papua New Guinea or Australia). It usually
found along the coastline, mainly around estuaries in brackish environment. It
ascends into fresh water of the upper tidal zone. Sagor catfish is carnivore that
feeds on invertebrates and small fishes. This species is an important food fish that
marketed fresh to be consumed (Fishbase, 2015).
2.9.7 Striated snakehead (Channa striatus)
Channa striata has been considered the most widely introduced species of
snakehead. Haruan / Striated Snakehead is one of the most widely distributed
species in this country, found in rivers, lakes, swamps, even ditches. It prefers still
waters with ample aquatic vegetation; normal environment for frogs and small
fishes which serve as its favourite food. It is thus definitely a carnivore, preferring
to lie in ambush amidst the vegetation. Traditionally, the favoured baits for the

45. 30
haruan are dressed frogs which are cast and retrieved or live fish placed among
aquatic vegetation. It body elongated with a round cross-section. Head is
compressed while dorsal and anal fins are very elongated, almost reaching the tail
fin. The body and head covered in small scales. It usually found in swampy or
stillwater, with plenty of structure and weeds. This species is distributed
throughout all states of the Peninsula Malaysia, Sunda land, Sulawesi, Moluccas,
Singapore, India, Indochina and China. It is carnivore feeding on worms, prawns,
frogs, and especially other fishes (KAGUM, 2015).

46. 31
CHAPTER 3
MATERIALS AND METHODS
3.1 Study area
Sungai Perak is the 'River of Life' for Perak State in Malaysia. It flows
over 400 km in a 15,000 km2 catchment that covers 70% of the state lands. As the
second longest river in Peninsular Malaysia, it starts from the north-western
corner of the state, flows south to Teluk Intan, where it bends westward and into
the Straits of Malacca. The river divides the state into two nearly equal halves and
thus forms its natural backbone (ICPDR, 2011). The source of Perak River is in
the mountainous Perak-Kelantan-Thailand border of the Belum Forest Reserve.
Some of the branches of the river are the Bidor River and Kinta River. One of the
streams that flow into the Perak River is known locally as the Sungai Kangsar.
Figure 3.1 showed the map of Perak river. The samples were collected
along the river according to the availability of the fish from the midstream to the
downstream of Perak river.

47. 32
Figure 3.1: Map of Perak river.
Kuala Kangsar
N 4° 46' 26.1",
E 100° 56' 44.2"
Teluk Intan
N 4° 0' 49.2"
E 101° 1' 04.1"
Kota Setia
N 4° 1' 25"
E 100° 52' 11.5"

48. 33
3.2 Sample collection
Fish samples were collected along the Perak River from midstream to the
downstream due to the availability of the fish to be collected. Sampling was
carried out during the dry season on August 2015. There were seven species of
fishes collected in the present study which were tinfoil barb (Barbonymus
schwanenfeldii), crossbanded barb (Puntius bulu), lemon fin barb (Puntius
daruphani), sagor catfish (Hexanematichthys sagor), striated snakehead (Channa
striatus), barb, spiny (Mystacoleucus marginatus) and fowler's danio (Devario
regina). The fish were collected by the fisherman and some species were
purchased at the wet market. The detailed of the freshwater species were
mentioned in Table 3.1.

49. 34
Table 3.1: Physical feature of fish species of present study
Local
Name
Common
Name
Scientific Name N Picture of species (taken by myself) Picture of species (sources from
google)
Tengalan Crossbanded
barb
Puntius bulu 3
Kerai Lemon fin
barb
Puntius daruphani 3

50. 35
Duri Sagor catfish Hexanematichthys
sagor
7
Haruan Striated
snakehead
Channa striatus 4
Sia Barb, spiny Mystacoleucus
marginatus
10

51. 36
Immediately after collection, fish samples were washed thoroughly using deionized water and placed in icebox before transferring
them to the laboratory (Kamaruzzaman et al., 2010).
Lampam
Sungai
Tinfoil barb Barbonymus
schwanenfeldii
3
Seluang
Pipih
Fowler's
danio
Devario regina 20

52. 37
3.3 Sample preparation and preservation
The length and weight of the fish samples were measured and noted once
arrived at the laboratory. Fish samples were washed by running tap water and
thawed at room temperature prior to analysis (Taweel et al., 2013). Whole fish
will be dissected on a clean bench shortly with stainless steel knife which had
been sterile with acetone and hot distilled water (Kamaruzzaman et al., 2010).
The muscles tissues was removed, placed in glass bottles and frozen for metal
analysis. The samples were stored in clean glass bottle separately at -20 ºC for 24
hours. All samples were dried separately at 120 ºC for 24 hours in dry oven. Then,
each sample was blended homogenously until the sample turned into powdered
form and then packed in polyethylene bag and sealed separately before acid
digestion processes.
3.4 Sample digestion and extraction
0.5 g dried samples were weighed and put into 50 ml beaker. 6 ml of
(65%) concentrated nitric acid (HNO3) was added into the beaker then, followed
by adding 2ml of (30%) hydrogen peroxide (H2O2) into the beaker. All the
beakers were placed on the hot plate separately at 60˚C for 2 hours until become
dryness and this to ensure complete digestion of all organic matters. 3 % of
diluted nitric acid (HNO3) was dropped into the beakers placed on the hot plate
after 20 minutes (Taweel et al., 2013). The digested solutions were left to cool
down in ambient temperature.

53. 38
After cooling, the digested solution were filtered through 0.45 μm
Whatman filter paper into falcon tubes and doubled rinsed with deionized water to
ensure that the entire digest were transferred into the tube. The filtrated samples
were made up to 30 ml by adding Mili-Q deionized water for the dilution. The lab
works were done in environmental lab of University Malaysia Kelantan.
Clear solutions with no residues were obtained at this stage for samples as
well as the blank and ready for analysis (Jalal et al., 2013). Blanks were used
simultaneously in each batch of analysis to verify the analytical quality (Taweel et
al., 2013). The assessments of concentrations of the following five heavy metals:
copper (Cu), cadmium (Cd), lead (Pb), iron (Fe) and zinc (Zn) were calibrated
from prepared diluting stock solutions of 1000 mg/L of each element. The
determinations of Cu, Cd, Pb, Fe and Zn in the fish muscle tissue were carried out
using inductively-coupled plasma spectrometry (ICP-OES), model (Varian 725-
ES, Australia). The concentrations of heavy metals were expressed as μg/g dry
weight (dw).
3.5 Chemical reagents
All reagents that used were analytical grade reagent. Double-deionized
water was used for all dilutions. Nitric acid (HNO3) of 65% and hydrogen
peroxide (H2O2) of 70% used in the present study was ultra-pure quality (Merck,
Darmstadt, Germany). The element standard solutions from Merck that used for
the calibrations were prepared by diluting stock solutions of 1000mg/L of each

54. 39
element. All of the glass ware and plastics were soaked overnight in 10% (v/v)
nitric acid, rinsed with distilled and deionized water and dried before being used
(Taweel et al., 2013).
3.6 Instrumentation
All target heavy metals was analyzed by inductively-coupled plasma
optical emission spectrometry (ICP-OES) model (Varian 725-ES, Australia) at
Forest Research Institute Malaysia (FRIM), Kepong for metal speciation. ICP-
OES is an analytical technique used for element determinations. Inductively
Coupled Plasma-Optical Emission Spectrometer (ICP-OES) is capable of
measuring concentrations of multi-elements at ppm level simultaneously. With
the Auto-sampler, which can handle as much as 180 samples, the ICP-OES can
analyze samples in number and speed unrivalled by traditional atomic absorption
spectrometer. Figure 3.2 showed the instrument ICP-OES used to analyze the
target heavy metals.

55. 40
Figure 3.2: Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-
OES) used in FRIM, Malaysia.
3.7 Quality assurance & Quality control
Procedural blank was carried out to obtain accurate result during heavy
metals analysis in each species of fish which lead to meaningful data for the
present study. In present study, blank sample was prepared by addition 6 ml of
concentrated nitric acid and 2 ml of hydrogen peroxide only. Blank sample was
important to avoid cross contamination to occur during the sample digestion.
Results obtained by blank samples were reliable and proved no contamination
occurred during the experimental progress. Quality data was produced during this
study.

56. 41
Matrix Spike was prepared for every batch of digestion method to increase
the confidence in the accuracy and validity of the sample test results. The
analyzed samples were spiked and run in ICP-OES and the concentrations of the
metal contents were determined from the calibration curve. The amounts of
spiked metals recovered were used to calculate the percentage recoveries (𝑅 %) as
follows:
R % =
𝐶1−𝐶2
𝐶3
where 𝐶1 is the spiked sample result, 𝐶2 is the un-spiked sample result, and 𝐶3 is
the concentration of the multi element standard solution (MESS). Determination
of metals concentrations was carried out in triplicate per sample of fish tissues.
Dilution factors of the collected data were corrected by calculations and the
values were presented in the units of µg/g.
3.8 Statistical Analysis
Statistical analysis was carried out by using Microsoft EXCEL 2010 and
SPSS statistical package program version 20. Pearson correlations were
performed to evaluate significance relationship of heavy metals between different
species. Health risk analysis was conducted by comparing the standard values
suggested.

57. 42
CHAPTER 4
RESULTS AND DISCUSSIONS
4.1 Physical characteristics of all fish species
The previous studies had mentioned that the level of bioaccumulation is
based on the role of age, species, and trophic transfer. The concentrations of
metals may vary with the age and bodyweight within the same species (Islam et
al., 2014)b
. In addition, feeding habits of the species can be considered as one of
factor that influenced the concentration levels of metals (Islam et al., 2014)a
. The
characteristics of each sampled of fish species for present study was summarized
in Table 4.1.

58. 43
Table 4.1: List of samples with narrow range of weight and length
Feeding
Habit
Local
Name
Common
Name
Scientific Name Habitat N Mean
Weight
(g)
Mean
Length
(cm)
Omnivores
Tengalan Crossbanded
barb
Puntius bulu Freshwater,
benthopelagic,
3 423
(480-380)
35
(38-32)
Kerai Lemon fin
barb
Puntius daruphani Freshwater,
benthopelagic
3 230
(300-190)
27
(30-25)
Sia Barb, spiny Mystacoleucus
marginatus
Freshwater,
benthopelagic
10 35
(75-15)
16
(20-13)
Lampam
sungai
Tinfoil barb Puntius
schwanenfeldii
Freshwater,
benthopelagic
3 110
(210-110)
21
(24-19)
Seluang
pipih
Fowler's
danio
Devario regina Freshwater,
benthopelagic
20 0.5
(10-0.25)
12
(15-9)
Carnivores
Duri Sagor catfish Hexanematichthys
sagor
Brackish,
demersal
7 48
(80-20)
18
(20-12)
Haruan Striated
snakehead
Channa striatus Freshwater,
brackish,
benthopelagic
4 38
(75-20)
18
(20-16)

59. 44
4.2 Spike recovery results
Spike recovery analysis is conducted to evaluate the method of validation
of the test procedure. The spiked resulted were showed in Table 4.2. Validation of
analytical procedure of spike matrix was tested using standard multi-element. The
rate ranged from 98.5% (Cu) to 119% (Cd). The result indicated that the acid
digestion method used for the fish samples and the ICP-OES analysis were
reliable and consistent. The recoveries values were within the range of 90% –
120% which comply with the Standard Operating Procedure (SOP) reported by
USEPA, (2011) and Commission Decision, (2002).
Table 4.2: Spiked recovery results
Heavy Metals Spiked Conc. (µg/g) Unspiked Conc.
(µg/g)
Recovery (%)
Cd 0.521 0.283 119
Cu 1.349 1.152 98.5
Fe 94.532 92.452 102.97
Zn 84.511 84.285 113
Pb 5.032 4.869 108.67

60. 45
4.3 Metals concentration in fish species
It is well known that heavy metals can be accumulated in fish tissues
through different organ (Islam et al., 2014)b
For this study, muscle tissue from
seven different types of fish species has been chosen. Concentration of five heavy
metals (Cd, Cu, Pb, Fe and Zn) in the muscles of seven fish species are listed in
Table 4.3 and Figure 4.1 respectively. Among metals, Fe showed the highest
concentration in every species except for Hexanematichthys sagor while Cd
presented the lowest values for all types of fish species. The lowest results of Cd
were obtained by (Taweel et al., 2013) that conducted the research on the
assessment of heavy metals in tilapia fish from Langat river. Tilapia is an
omnivore which was same with Puntius daruphani, Barbonymus schwanenfeldii,
Mystacoleucus marginatus, Puntius bulu and Devario regina.
As a whole, the mean concentrations of heavy metals in four types of fish
species which are Puntius daruphani, Barbonymus schwanenfeldii,
Mystacoleucus marginatus, and Devario regina showed the descending order of
Fe > Zn > Pb > Cu > Cd. For Channa striatus and Puntius bulu species the
concentration of copper is higher than lead while for Hexanematichthys sagor
species, the concentration levels of heavy metals in zinc is higher than iron.
Previous studies reported that chronic exposure to Cu and Zn is related
with Parkinson’s disease and these elements might act alone or together over time
to induce the disease (Rahman et al., 2012). Fishes are known to have a high
threshold level of zinc. In this study Devario regina exhibited a tendency to

61. 46
accumulated high concentration of zinc compared to other species (84.285 ±
9.312 μg/g) while the lowest concentration of zinc (18.268 ± 1.525 μg/g) was
found in Puntius bulu as compared to other species. The amount of zinc
concentration determined in all fish samples were far below the permissible limit
recommended by Malaysian Food Regulation and FAO/WHO. But according to
MAFF 2000, the zinc concentration of Channa striatus and Devario regina were
over the permissible limit which is 50 μg/g (Table 4.3).
Copper was detected in all examined fish samples with range from 0.306
μg/g to 1.916 μg/g. The highest level of copper (1.916 ± 0.156 μg/g) was found in
Channa striatus while the lowest concentration of copper (0.306 ± 0.051 μg/g)
was found in Hexanematichthys sagor species as compared to other species.
Copper is an essential part of several enzymes and is vital for the synthesis of
hemoglobin. However, high intake of copper may cause adverse effects to health
problem (Rahman et al., 2012). Based on the permissible limit recommended by
Malaysia Food Regulation (30 μg/g), WHO/FAO (10 μg/g), China (50 μg/g) and
MAFF (20 μg/g) the concentrations of copper in seven types of fish species of
Perak river still far below the limit and safe to be consumed.
Lead is a non-essential element and it is well known for the causes of
neurotoxicity, nephrotoxicity, and many others adverse health effects (García et
al., 2010). The detailed of lead concentration detected for each fish species were
listed in Table 4.3. Among individual fish species Devario regina contained the
highest levels of Pb concentrations (4.869 ± 1.461 μg/g) and it exceeded all the
permissible limits recommended. The lowest concentration of lead was found in

62. 47
Puntius bulu (0.378 ± 0.11μg/g). However, lead concentration in Barbonymus
schwanenfeldii (3.963 ± 0.08 μg/g) and Hexanematichthys sagor (2.421 ± 0.835
μg/g) were also higher than permissible limits. For Puntius daruphani species, the
concentration of lead (1.752 ± 0.35 μg/g) exceeded the permissible limit
recommended by FAO/WHO 1984 and China National Standard but still below
the permissible limit of Malaysian Food Regulation 1985 and MAFF 2000 (Table
4.3).
Cadmium contributes the highest concentration in Puntius bulu which was
approximately (0.34 ± 0.132 μg/g) and this exceed all the permissible limits
recommended except the value by Malaysian Food Regulation which is 1 μg/g .
Almost all fish species obtained high levels of cadmium concentrations which
was exceed the permissible limits by FAO/WHO 1984, China National Standard
and MAFF 2000 but still in the range of permissible limits recommended by
Malaysian Food Regulation 1985 (Table 4.3). The lowest level of cadmium
(0.246-0.276 μg/g) was found in Hexanematichthys sagor species. Ingestion of
small amounts of contaminated fish that contain cadmium over long periods of
time may lead to some form of cadmium intoxication (Sofia, 2005) and its
accumulation in our body is very difficult to be excreted.
The concentration of hazardous metal like iron found to be highest in
Devario regina (140.798 ± 11.359 μg/g) species compared to other fish species
while the lowest concentration was found in Puntius bulu (32.75 ± 3.009 μg/g).
Devario regina can be categorized as small type of fish. It small in size was one
of the factor of the accumulation of trace metals. All fish species collected from

63. 48
Perak rivers have the concentration of iron below the permissible limits
recommended by FAO/WHO 1984 (Table 4.3). Iron is an essential nutrient for the
human body. But excess iron may lead for certain cancers and eventually to death
(Anderson & Fitzgerald, 2010).
Figure 4.1: Mean concentration of heavy metals in commonly found fish species
of Perak river.
0
20
40
60
80
100
120
140
160
Conc(µg/g)
Species
Cd Cu Fe Zn Pb

64. 49
Table 4.3: Metal concentration in fish species of Perak rivers.
Fish Species Heavy metals (μg/g)
Cd Cu Fe Zn Pb
Puntius
daruphani
Average 0.282 ± 0.089 1.428 ± 0.331 52.248 ± 1.96 33.568 ± 4.093 1.752 ± 0.35
Range 0.18 - 0.342 1.086-1.746 51.09-54.51 29.184-37.29 1.38-2.076
Barbonymus
schwanenfeldii
Average 0.294 ± 0.084 1.344 ± 0.219 38.244 ± 8.351 24.598 ± 4.604 3.963 ± 0.08
Range 0.234-0.39 1.104-1.536 32.43-34.488 19.284-27.108 3.9-4.02
Puntius bulu Average 0.34 ± 0.132 1.56 ± 0.67 31.712 ± 1.739 18.268 ± 1.525 0.378 ± 0.11
Range 0.27-0.492 0.786-1.94 29.712-32.55 16.68-18.402 0.3-0.45
Hexanematichthys
sagor
Average 0.262 ± 0.015 0.306 ± 0.051 32.75 ± 3.009 36.434 ± 3.157 2.421 ± 0.835
Range 0.246-0.276 0.06-0.342 29.328-34.986 33.498-39.774 1.83-3.012
Channa striatus Average 0.27 ± 0.091 1.916 ± 0.156 140.798 ± 11.359 58.916 ± 2.841 1.434 ± 0.288
Range 0.168-0.342 1.746-2.052 127.704-148.008 55.668-60.138 0.588-1.638
Mystacoleucus
marginatus
Average 0.284 ± 0.043 1.178 ± 0.178 88.114 ± 2.101 31.342 ± 1.065 1.26 ± 0.551
Range 0.234-0.312 0.972-1.29 86.556-90.504 30.408-32.502 0.87-3.546
Devario regina Average 0.283 ± 01 1.152 ± 0.098 92.452 ± 3.137 84.285 ± 9.312 4.869 ± 1.461
Range 0.162-0.42 0.69-1.266 52.596-96.066 73.206-98.22 3.132-6.09
Permissible Limit MFR (1985) 1 30 - 100 2
FAO/WHO 0.2 10 300 150 1.5

65. 50
(1984)
China 0.1 50 - - 0.5
England /
MAFF (2000)
0.2 20 - 50 2
*MFR (Malaysian Food and Regulation) 1985
*FAO/WHO (Food and Agriculture Organization / World Health Organization) 1984
*China National Standards Management Department (2001)
*MAFF (Ministry of Agriculture, Fisheries and Food).UK: Center for Environment, Fisheries and Aquaculture Science; 2000.

66. 51
4.4 Heavy metal concentration profile of fish species collected from Perak river.
Iron had shown the highest percentage of composition compared to other
heavy metals in this study. The highest percentage of accumulation of Fe (70 %)
was shown in Mystacoleucus marginatus (Figure 4.2). Percentage of Fe in
Mystacoleucus marginatus was two times higher than composition of
Hexanematichthys sagor (40 %) species. This two species showed the difference
in percentage because of the different of feeding habits of the fishes were
different. All species showed the safe level of concentrations of Fe that in range
of the permissible limits. Excessive intake of Fe may cause toxicity in human
body. Industrial wastes and agricultural runoff were the factor of the iron been
released into the river.
Zinc composition in Hexanematichthys sagor was 50 % and it was 25 %
in Mystacoleucus marginatus which was one time higher. Different habitat of this
species influenced the accumulation of zinc in their muscle tissues. The
composition of zinc in all species were below the permissible limits
recommended by WHO/FAO 1984 and Malaysian Food Regulation but for
Devario regina long term exposure may lead to high accumulation of zinc over
the permissible limits.
Percentage of concentration of lead was slightly higher in Barbonymus
schwanenfeldii species which was approximately 10% as compared to other
species collected along Perak river. Lead is a cumulative poison. In fact, it is
considered a non-essential element which is similar to calcium in metabolism

67. 52
processes. Percentage composition of copper in Puntius bulu was three times
higher than composition of copper in Hexanematichthys sagor.
Cadmium composition showed the lowest accumulation compared to other
trace metals. Cadmium is classified as one of the major ecotoxic metals that can
cause harmful effects on physiological processes of living things. The
composition trend of cadmium was quite similar to copper. The highest
composition of cadmium obtained in Puntius bulu and the lowest composition
was found in Hexanematichthys sagor.
Figure 4.2: Percentage concentration of heavy metals in fish species of Perak river.
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Species
Fe Zn Pb Cu Cd

68. 53
4.5 Comparison with other studies
The study on the assessment of heavy metals in tilapia fish from Langat
River and Engineering Lake in Bangi, Malaysia had been conducted by Taweel et
al., (2013), figure 4.4. Zinc concentration was the highest followed by copper,
lead and cadmium show the lowest concentration of heavy metals in tilapia fish.
Both Langat River and Engineering Lake showed the same pattern of the
accumulation of heavy metals in tilapia fish (Oreochromis niloticus). This pattern
of heavy metals concentration was similar to the present study. Tilapia is one of
the most common fish consumed by Malaysia people. Although it was not
included in this present study, considering the feeding habit which is also
omnivore, the resulted from this present study was comparable to Taweel et al,
(2013).
According to Islam et al., (2014)b
, the concentration of copper, cadmium
and lead were slightly different between Channa striata species and Channa
punctata species from Paira river, Bangladesh although there were from the same
genus. Comparing with the present study on Perak river, the concentration of
copper, cadmium and lead in Channa striata species was higher than Channa
striata species and Channa punctata species from Paira river, figure 4.4. This
resulted indicated that, source of pollution in Perak river was higher than Paira
river.

69. 54
Ahmad et al., (2015), found that lead concentration was not detected in
Mystacoleucus argenteus from Sungai Lembing area but compare with present
study, lead concentration was found in Mystacoleucus marginatus with value 1.26
µg/g. However, the concentration of cadmium (0.47 µg/g) in Mystacoleucus
argenteus from Sungai Lembing was higher than Mystacoleucus marginatus from
Perak river which is 0.28 µg/g. Although both species were from same family, the
accumulations of heavy metals were different between the species depending on
the bioaccumulation level of the species and also the environment or habitat of the
species.
As comparing with the study from Sungai Lembing, Barbonymus
schwanenfeldii from Perak river show higher concentration of heavy metals in
cadmium and lead compared to Barbonymus schwanenfeldii collected from
Sungai Lembing abandoned mining site, Pahang. However for Channa striata
species, Channa striata from Perak river showed high concentrations of cadmium
but low concentration of lead compared to Channa striata from Sungai Lembing,
Pahang. This resulted show that different level of toxicity found from Perak river
and Sungai Lembing due to the different sources of pollution.
Kwok et al., (2014), had conducted a research on the concentration of
heavy metals in Channa asiatica species from Pearl River Estuary,China. Channa
asiatica classified as same family with Channa striata. As comparing to present
study, Channa asiatica show high concentration of cadmium, copper and lead
than Channa striata from Perak river. However low concentration of zinc found
in Channa asiatica (40.3 µg/g) compared to Channa striata (58.93 µ/g) from

70. 55
Perak river. Based on the concentration resulted accumulated in fish species,it can
be conclude that Pearl river estuary was more polluted than Perak river. This
resulted quite different with the study on the Pearl River Delta conducted by
Leung et al., (2014).
According to Leung et al., (2014), the concentration of cadmium, copper,
lead and zinc in Channa asiatica species was quite lower than recorded by Kwok
et al., (2014) and present study on Perak river. This might be because of the
different environment between estuary and delta. According to Kwok et al.,(2014)
the fish species were collected from Mai Po ramsar site which the environment
was more muddy and brackish rather than Pearl river delta. Clarias fucus species
from Pearl river delta was compared with Devario regina species from present
study as both species from the same genus of Carp. The concentration of
cadmium, zinc and lead was high in Devario regina species compared to Clarias
fucus species. But the concentration of copper was high in Clarias fucus species
compared to Devario regina species.
Jalal et al., (2013), conducted study on the bioaccumulation of selected
heavy metals in freshwater haruan fish (Channa striatus) collected from Pahang
river basin, Malaysia. As comparing with present study from Perak river, the
concentration of copper and lead in Channa striatus from Pahang river basin was
higher than Channa striatus species of Perak river. Whereas the concentration of
zinc in Channa striatus from Perak river show high value (58.92 µg/g) compared
with the value of Channa striatus collected from Pahang river basin which is 1.82
µg/g. This resulted was comparable with Kwok et al, (2014). Zinc was widely

71. 56
used in industrial activities. This indicated that more industrial activities occurred
along the riverside of Perak river compared to Pahang river basin.
As comparing with regional study from Kelantan river, Barbonymus
schwanenfeldii showed low concentration of cadmium and lead compared to
Barbonymus schwanenfeldii of present study from Perak river. According to
Rohasliney et al., (2014), Tachysurus maculatus collected from Kelantan river
also showed low concentration of cadmium and lead compared to
Hexanematichthys sagor species collected from Perak river. Both of this species
were from the same family, which were catfish and the family of Ariidae. Same
pattern also recorded for Puntioplites bulu species. The concentration of cadmium
and lead was high in Puntius bulu from Perak river compared to the species from
Kelantan river. This concluded that, Perak river was more polluted than Kelantan
river due to the accumulation of heavy metals in fish species collected from both
area.
The concentration of Puntius daruphani and Puntius bulu species from
present study on Perak river was compared to the study on Puntius ticto species
collected from Bangshi river, conducted by Rahman et al., (2012) as they were
from the same genus and family. According to Rahman et al., (2012), Puntius
ticto showed high concentration of cadmium, copper, zinc and lead compared to
Puntius daruphani and Puntius bulu species collected from Perak Perak. This
indicate that the accumulation of heavy metals were different among the species
although they were from the same genus. Besides, the resulted showed that the
level of pollution of Bangshi river were higher than Perak river.

72. 57
Ahmad et al., (2009) found that the concentration of Puntius bulu species
collected from Lake Chini, Pahang was highest for zinc and lowest for cadmium.
This resulted was comparable with present study from Perak river and Langat
river (Taweel et al.,2013). As comparing with present study, the concentration of
cadmium, copper and zinc in Puntius bulu collected from Chini Lake were lower
while the concentration of lead was higher. The movement of the water in the lake
which is immobile might be the causes of high heavy metals accumulate and
dense into the lake compared to the river that freely flows.
Islam et al., (2014)a
was conducted research on three different river from
Dhaka area, Bangladesh. Channa punctatus species was collected to be compared
with Channa striata species from present study. The concentration of cadmium
and lead in Channa punctatus species in all three studied river was lower
compared to Channa striata species from present study on Perak river. Whereas
the concentration of copper in Channa punctatus species from Buriganga river
and Shitalakha river was higher compared to Channa striata species from Perak
river. However, the concentration of copper in Channa punctatus species from
Turag river was slightly different and comparable with Channa striata species
from Perak river with the value range from 1.1 µg/g to 2.2 µg/g.

73. 58
Table 4.4: Comparison of heavy metals concentration in (μ/g dry weight) in freshwater fish species collected from different parts
of the world.
Area Species Cd Cu Fe Zn Pb Reference
Langat River &
Engineering Lake,
Bangi, Malaysia
Oreochromis niloticus 0.05-0.03 1.69-1.01 - 26.13-20.58 0.26-0.99 (Taweel et
al., 2013)
Pahang River,
Basin, Malaysia
Channa striatus - 2.24 - 1.82 0.01 (Jalal et
al.,2013)
Chini Lake,
Peninsular
Malaysia
Puntius bulu 0.14 0.28 - 2.73 0.98 (Ahmad et
al., 2009)
Sungai Lembing,
Pahang, Malaysia
Mystacoleucus
argenteus
Barbonymus
schwanenfeldii
Channa striata
0.47
0.13
0.08
- - - NA
0.19
1.44
(Ahmad et
al.,2015)

74. 59
Kelantan River,
Malaysia
Barbonymus
schwanenfeldii
Puntioplites bulu
Tachysurus maculatus
0.03
0.038
0.053
- - - 0.10
0.069
0.156
(Hashim et
al.,2014)
Paira River,
Bangladesh
Channa striata
Channa punctate
0.009- 0.03
0.008-0.04
0.2–2.2
0.4-1.6
- - 0.4-1.0
0.2-1.1
(Islam et al,.
2014)b
Turag River,
Bangladesh
Buriganga River,
Bangladesh
Shitalakha River,
Bangladesh.
Channa punctatus
Channa punctatus
Channa punctatus
0.007-0.013
0.022-0.053
0.02-0.043
1.1-2.2
2.3-5.9
2.3-4.7
- - 0.052-0.72
0.78-1.5
0.13-0.79
(Islam et
al.,2014)a
Pearl River
Estuary, China
Channa asiatica 50.9 3.54 - 40.3 6.32 (Kwok et al.,
2014)
Bangshi River,
Bangladesh
Puntius ticto 0.46 38.11 - 174.61 7.22 (Rahman et
al., 2012)
Pearl River Delta,
China
Clarias fuscus
Channa asiatiea
0.02
0.04
1.4
1.02
- 27.8
25.8
0.37
0.24
(Leung et al.,
2014)

75. 60
4.6 Bioaccumulation factors (BAF) of metals in fish species
Average metal concentrations in fish species and sediments from present
study were used for calculating the BAF. The BAF is an index of the ability of a
fish species to accumulate a particular metal with respect to its concentration in
sediment. It was calculated by the following equation (Kwok et al., 2014):
BAF =
𝐶 𝑓𝑖𝑠ℎ
𝐶 𝑠𝑒𝑑𝑖𝑚𝑒𝑛𝑡
x 100%
where C fish is the metal concentration in fish (µg/g dw), and C sediment is the
metal concentration in sediment (µg/g [dw]). For this study, the concentration of
Cd, Cu, Pb, Fe and Zn of sediment was adapted by other final year project student
that conducted a research on the same area of Perak river (Farah, 2015). BAF
values higher than 100% indicate the bioaccumulation of the contaminant in the
sample.
Metals contained in sediment can be bioaccumulated in fish tissues (Islam
et al., 2014)ᵇ. The accumulation of metals in muscle tissues of fish could have a
direct impact on health throughout the food chain. Table 4.6 listed the BAF values
calculated among different fish species and metals.
The ranking order of mean BAF values of metals for Puntius daruphani
and Mystacoleucus marginatus were Fe >Zn > Cd > Cu > Pb ; for
Hexanematichthys sagor and Devario regina were Fe > Zn > Pb > Cd > Cu. The
ranking order for Barbonymus schwanenfeldii , Puntius Bulu and Channa striatus

76. 61
was Fe > Pb > Zn > Cu > Cd ; Fe > Zn > Cd > Cu > Pb and Fe > Zn > Cd > Cu >
Pb respectively (Table 4.5). These differences can be explained by their ingestion
of sediment as well as the feeding habit behavior of the fishes. At some area,
concentration levels of heavy metals might be high but accumulation is lower than
expected due to metal complexation (Islam et al., 2014)a
.
Table 4.5: BAF values of metals in fish species of Perak river.
Fish Species (BAF)
Cd Cu Fe Zn Pb
Puntius
daruphani
13.85 ± 1.01 13.05 ± 2.72 172.99 ± 91.33 70.17 ± 19.47 7.83 ± 1.73
Barbonymus
schwanenfeldii
7.48 ± 1.66 7.38 ± 0.39 97.58 ± 19.96 31.92 ± 7.78 15.23 ± 0.88
Puntius bulu 17.35 ± 6.04 14.86 ± 7.54 103.15 ± 50.77 37.6 ± 7 1.59 ± 0.76
Hexanematichthys
sagor
7.01 ± 1.6 3.03 ± 0.64 90.27 ± 24.25 78.65 ± 23.97 9.07 ± 2.78
Channa striatus 6.87 ± 1.82 10.73 ± 2.09 361.57 ± 57 75.81 ± 5.73 3.78 ± 1.27
Mystacoleucus
marginatus
7.36 ± 1.77 6.48 ± 0.40 226.1 ± 28.97 40.42 ± 4.15 6.45 ± 3.17
Devario regina 11.94 ± 2.17 10.55 ± 0.82 306.11 ± 161.3 173.14 ±
10.15
22.22 ± 5.74
Bold indicate BAF > 100

77. 62
Bioaccumulation of Fe values was more than 100 (BAF > 100) in all
studied fish species except Barbonymus schwanenfeldii and Hexanematichthys
sagor. Thus suggesting a greater rate of Fe accumulation in those fish species.
Iron was an essential component in human diet which involved in oxygen
transport (Adu, 2010). This showed that the accumulation of iron was easily
occurred. Zinc in Devario regina species also showed BAF > 100 while other
species below hundreds. This finding was quite different from the study of Kwok
et al., (2014) which showed that the BAF of Cd > 100. According to Kwok, heavy
metals bioaccumulation could occur with the increasing of age or size of fish
species.

78. 63
Figure 4.3: BAF of heavy metals in fish species. Red line indicated the BAF value > 100

79. 64
4.7 Bio-concentration factor (BCF) of metals in fish species.
Bio-concentration factor (BCF) was used to evaluate bioaccumulation of
metal element in organisms and it can also be used for field investigation data
(USEPA, 1991). It is calculated by this following equation:
BCF =
𝐶 𝑓𝑖𝑠ℎ
𝐶 𝑤𝑎𝑡𝑒𝑟
where, Cfish is the concentration of the metal element in the muscle of organisms;
Cwater is the concentration of the metal element in water environment. In this
study, the concentration of water for Fe, Zn and Pb were adapted from other final
year project student which was also conducted in Perak river (Loh, 2015).
Table 4.6: BCF values of metals in fish species of Perak river.
Fish Species BCFs
Fe Zn Pb
Puntius daruphani 38.09 ± 4.59 264.15 ± 56.37 223.33 ± 1.67
Barbonymus schwanenfeldii 18.6 ± 10.96 255.33 ± 75.79 267.24 ± 22.63
Puntius bulu 23.02 ± 1.36 154.04 ± 65.51 44.08 ± 9.31
Hexanematichthys sagor 8.08 ± 1.46 280.19 ± 8.43 239.88 ± 15.73
Channa striatus 64.09 ± 22.17 541.64 ± 87.32 185.33 ± 68.82
Mystacoleucus marginatus 40.85 ± 16.43 325.19 ± 72.11 130.83 ± 48.32
Devario regina 67.38 ± 7.82 524.05 ± 176.47 435.75 ± 124.1
Bold indicate BCF > 100

80. 65
Bio-concentration factor (BCF) is used to evaluate the ability of the
aquatic organism to accumulate chemicals from the water environment. If BCF
>1, it indicates that the organism has a potential to accumulate the chemical but is
generally not considered to be significant unless the BCF exceeds 100 or more
(Tao et al., 2012 ; USEPA, 2011).
In this study, BCF value of zinc was higher than 100 for all species while
for lead value only Puntius bulu species not higher than 100. However, iron value
of BCF showed all fish species not higher than 100. According to Tao et al.,
(2012), the BCF values were not consistent between different fish species because
fish are more broadly distributed and may migrate between lake areas in response
to different environments in the lake. The average BCF of each species for the
entire lake was evaluated without considering spatial variation due to their high
mobility. In addition, the feeding habit or predation behavior may influence the
bio-concentration of heavy metals. Comparing the BCF values resulted from
Taihu Lake, BCF values of lead was higher than zinc and this was slightly
different from the present study in Perak river. The BCF values resulted from
Perak river in zinc was higher than in lead in mostly all types of studied fish
species. This indicated that the hypothesis mentioned early was accepted.

81. 66
Figure 4.4: BCF of heavy metals in fish species. Red line indicated the BCF value > 100

82. 67
4.7 Health risk assessment
For appraising the health risk associated with heavy metal contamination
of fishes inhabited in the Perak River, estimated daily intake (EDI), target hazard
quotients (THQ) and target cancer risk (TR) were estimated.
4.7.1 Estimated daily intake (EDI) of heavy metals
The estimated daily intake (EDI) of heavy metals by human is evaluated
according to the mean concentration and the consumption amount of metals in
aquatic organism. The EDI of metals was determined by the following equation
(Taweel et al., 2013):
= metal concentration (μg/g w.w.) x consumption rate (g/d)
body weight (kg)
where metal concentration in fish was obtained on bases of wet weight, body
weight for adults is considered to be 64 kg in respective to Malaysian country and
were derived from numerous local Malaysia studies (Taweel et al., 2013). The
consumption rate was 160 g/d/person for Malaysian adults (Ahmad & Sarah,
2015; Taweel et al., 2013). The oral reference dose (RfD) was used to evaluate
the EDIs of metals in fishes. The oral reference dose (RfD) for Fe, Cu, Cd, Pb and
Zn suggested by former studies and US-EPA was 70, 40, 0.5, 4 and 300
μg/kg/day, respectively (Zhao et al., 2012).

83. 68
There are many approaches of human exposure to heavy metals such as
breathing and dermal exposure. However, food consumption is one of the most
important approaches (Zhao et al., 2012).The estimated daily intake of heavy
metals by human was evaluated according to the mean concentration and the
consumption amount of metals in aquatic organism. Table 4.7 showed the EDIs of
Fe, Cu, Cd, Zn and Pb through consumption of fishes from Perak rivers.
Table 4.7: Estimated daily intake (EDI) calculated for Perak area.
Fish Species Estimated daily intake (μg/kg/day)
Cd Cu Fe Zn Pb
Puntius daruphani 0.71 3.57 130.62 83.92 4.38
Barbonymus
schwanenfeldii
0.74 3.36 95.61 61.5 9.91
Puntius bulu 0.85 3.9 79.28 45.67 0.95
Hexanematichthys
sagor
0.66 0.77 81.88 91.09 6.05
Channa striatus 0.66 4.79 352 147.29 3.59
Mystacoleucus
marginatus
0.71 2.95 220.29 78.36 3.15
Devario regina 0.71 2.88 231.13 210.71 12.17
RfD 0.5 40 70 300 4

84. 69
Dietary intake of iron and cadmium exceeded the RfD through
consumption of fish which was mainly certified to the fact that high
concentrations of Cd and Fe were accumulated in seven types of fishes from
Perak rivers. Cadmium is reported to be commonly present in rivers resulted from
industrial activities and are disadvantageous for aquatic organisms (Kumar &
Singh, 2010). Iron is an essential nutrient for the human body but excessive
consumption may lead to detrimental effects. Since, estimated daily intake of Cd
and Fe were in high level, the consumption of studied fish species of Perak river
should be avoided to prevent health risk effects to the consumers.
The intake of lead in four types of fish species which were Puntius
daruphani, Barbonymus schwanenfeldii, Hexanematichthys sagor and Devario
regina also exceeded the RfD levels. According to these results, excessive
consumption of Puntius daruphani, Barbonymus schwanenfeldii,
Hexanematichthys sagor and Devario regina should be avoided to prevent
harmful effects caused by Pb accumulation as lead poisoning is normally ranked
as the most common environmental health hazard (Ahmed et al., 2014). Other
metals such as Cu and Zn showed the average value of EDIs in all types of fish
species that were below than the RfDs level. This indicating that normal
consumption of fishes from Perak river would not result of health risk from this
two type of metals.

85. 70
0 0.2 0.4 0.6 0.8 1
Puntius daruphani
Barbonymus Schwanenfeldii
Puntius Bulu
Hexanematichthys Sagor
Channa striatus
Mystacoleucus Marginatus
Devario regina
EDI of Cd (µg/kg/day)
Species
Cd
a)
0 5 10 15
Puntius daruphani
Barbonymus Schwanenfeldii
Puntius Bulu
Hexanematichthys Sagor
Channa striatus
Mystacoleucus Marginatus
Devario regina
EDI of Pb (µg/kg/day)
Species
Pb
b)
RfD
RfD

86. 71
0 1 2 3 4 5 6
Puntius daruphani
Barbonymus Schwanenfeldii
Puntius Bulu
Hexanematichthys Sagor
Channa striatus
Mystacoleucus Marginatus
Devario regina
EDI of Cu (µg/kg/day)
Species
Cu
c)
0 50 100 150 200 250
Puntius daruphani
Barbonymus Schwanenfeldii
Puntius Bulu
Hexanematichthys Sagor
Channa striatus
Mystacoleucus Marginatus
Devario regina
EDI of Zn (µg/kg/day)
Species
Zn
d)
RfD
40
RfD
300

87. 72
Figure 4.5: Estimate daily intake (EDI) of Cd, Pb, Cu, Zn, Fe through consumption of
fish from Perak river.
4.7.2 Target hazard quotients (THQ) of heavy metals
Current non-cancer risk assessment methods are typically based on the
employment of the target hazard quotient (THQ), a ratio between the estimated
dose of a contaminant and the reference dose below which there will not be any
appreciable risk. The THQ will be determined with the method described by US
EPA (US-EPA, 2000). Target hazard quotients (THQ):
= EF x ED x FIR x C x (10ˉ³)
RfD x WAB x TA
0 100 200 300 400
Puntius daruphani
Barbonymus Schwanenfeldii
Puntius Bulu
Hexanematichthys Sagor
Channa striatus
Mystacoleucus Marginatus
Devario regina
EDI of Fe (µ/kg/day)
Species
Fe
e) RfD

88. 73
where EF is exposure frequency (365 d/year); ED is the exposure duration (70
years), equivalent to the average lifetime; FIR is the food ingestion rate (160
g/d/person for Malaysian adults); C is the metal concentration in the muscle of
fish (μg/g); WAB is the average body weight that is 64 kg in respective to
Malaysia which were derived from numerous local Malaysia studies (Taweel et
al., 2013), and TA is the averaging exposure time for non-carcinogens (365 d/year
x ED) (Zhao et al., 2012). The same oral reference dose was used as mentioned in
the calculation of EDI.
All fish species discussed in this study were commonly consumed and
commercial products by surrounding residents. Thus, average heavy metal
concentrations of fishes were used for calculation of THQ for the surrounding
residents. THQs of the five studied heavy metals from consuming seven different
types of fish in Perak river were listed in Table 4.8 and shown in Figure 4.6

89. 74
Table 4.8: Target hazard quotients (THQ) of heavy metals due to consumption of
fish from Perak river.
Fish Species Target hazard quotients (THQ) (x10ˉ³)
Fe Zn Pb Cd Cu Total (THQ)
Puntius
daruphani
1.87 0.28 1.1 1.41 0.09 4.74
Barbonymus
schwanenfeldii
1.37 0.21 2.48 1.47 0.08 5.6
Puntius bulu 1.13 0.15 0.24 1.7 0.1 3.31
Hexanematichthys
sagor
1.17 0.3 1.51 1.31 0.02 4.31
Channa striatus 5.03 0.49 0.9 1.35 0.12 7.88
Mystacoleucus
marginatus
3.15 0.26 0.79 1.42 0.07 5.69
Devario regina 3.3 0.7 3.04 1.42 0.07 8.53
According to figure 4.6 there was no THQ value over 1 through the
consumption of fishes from Perak river. This indicated that health risk associated
with heavy metals exposure were insignificant. Total THQ (TTHQ) was also
included in this study because human are often exposed to more than one
pollutant and suffer combined or interactive effects. The total THQ was treated as
the arithmetic sum of the individual metal THQ values. The consumption of iron
showed the highest of THQ value while copper showed the lowest value of THQ.

90. 75
Total THQ values from all the studied metals also lower than 1 for all
types of fish species and this indicated that the fish species of Perak river were
safe to be consumed.
A study done by Islam et al., (2014)b
in the determination of heavy metals
in fish in Bangladesh and health implications reported that the THQ of Cu, Cd
and Pb were below 1. The same study by Taweel et al., (2013) also showed lower
THQ values of Cd, Cu, Pb and Zn. The finding of this present studied was in-line
with other mentioned study outside from Malaysia. This indicates that consuming
fish from the study sites does not pose a health risk to the inhabitants.
Figure 4.6: Target hazard quotients (THQ) of heavy metals due to consumption
of fish from Perak river.
0
0.001
0.002
0.003
0.004
0.005
0.006
THQValues
Fish Species
Fe Zn Pb Cd Cu
1

91. 76
4.7.3 Target cancer risk (TR)
For carcinogens, risks were estimated as the incremental probability of an
individual to develop cancer over a lifetime, as a result of exposure to that
potential carcinogen (i.e., incremental or excess individual lifetime cancer risk
USEPA 1989). Acceptable risk levels for carcinogens range from 10ˉ4
(risk of
developing cancer over a human lifetime is 1 in 10,000) to 10ˉ6
(risk of
developing cancer over a human lifetime is 1 in 1,000,000). Target risk were
calculated using this equation:
= EF x ED x FIR x C x CSF x (10ˉ³)
WAB x TA
where the CSF is the oral carcinogenic slope factor. The CSF for Pb was used
from (USEPA, 2010) database which was 8.5 x 10ˉ³ (µg/g/day) while Cd was
used from (OEHHA, 2011) which was 0.6 (µg/g/day). These two metals were
calculated since these elements may promote both non-carcinogenic and
carcinogenic effects depending on the exposure dose. The TR value calculated
was shown in Table 4.9 and Figure 4.7 respectively.

92. 77
Table 4.9: Target cancer risk of heavy metals due to consumption of fish from
Perak river.
Fish Species
Target cancer risk (TR)
Pb Cd
Puntius daruphani 3.72E-05 4.23E-04
Barbonymus schwanenfeldii 8.42E-05 4.41E-04
Puntius bulu 8.03E-06 5.1E-04
Hexanematichthys sagor 5.14E-05 3.93E-04
Channa striatus 3.05E-05 4.05E-04
Mystacoleucus marginatus 2.68E-05 4.26E-04
Devario regina 1.03E-04 4.25E-04
Target risk values for lead range from (8.03E-06 to 1.03E-04) whereas
(5.1E-04 to 3.93E-04) for cadmium. In general, the excess cancer risks lower than
10−6
are considered to be negligible, cancer risks above 10−4
are considered
unacceptable (USEPA 1989, 2010) and risks lying between 10−6
and 10−4
are
generally considered an acceptable range (Islam et al. 2014)b
. In fish species TR
values for lead and cadmium were between 10–6
to 10-4
and regarded as
negligible.

93. 78
Therefore, based on the results of the present study, the potential health
risk for the inhabitants due to metal exposure through consumption of fish should
not be neglected. (Islam et al., 2014)b
recorded that the TR value of lead was
lower than 10-6
and considered negligible.
Figure 4.7: Target cancer risk of heavy metals from fish consumption of Perak
river.
0.00E+00
1.00E-04
2.00E-04
3.00E-04
4.00E-04
5.00E-04
6.00E-04
TRvalues
Fish Species
Pb Cd
10-6
to 10-4

94. 79
4.8 Correlation analysis
4.8.1 Correlation analysis of heavy metals in fish species collected from Perak
river.
Fish species collected from Perak area shown positive significant
correlation between Fe with Zn whereas relationship between Fe with Pb, Cd and
Cu in all fish species from Perak river showed no significant relationship. Zn with
Pb in all species were correlated significantly whereas Zn with Cd and Cu were
not correlated significantly. Correlation between Pb with Cd and Cu in all species
showed negative correlation and not significant. Cd with Cu in all species from
Perak river showed no significant relationship (P > 0.01). This indicated that Fe
and Zn were discharged the most in water bodies that contribute to the heavy
metals pollution. Iron and zinc were released and discharged into the river
through the construction, mining activities and mostly from metal industry.
Table 4.10: Pearson correlation of heavy metals in fish species of Perak river.
Fe Zn Pb Cd Cu
Fe 1
Zn 0.658** 1
Pb 0.184 0.616** 1
Cd -0.204 -0.281 -0.056 1
Cu 0.420 0.037 -0.100 0.059 1
ʽʽ*ʼʼ shows correlation is significant at (p<0.01)

95. 80
4.8.2 Correlation analysis of heavy metals among omnivore and carnivore
All seven types of fish species collected from Perak river were
differentiate into omnivores and carnivores according to their feeding habit. The
correlation coefficient has been calculated within the omnivore and carnivore.
The correlation between Puntius daruphani and Barbonymus schwanenfeldii
show high negative relationship of Fe with Pb (Table 4.11). This indicate that the
accumulation capacity of Fe within this two species were different although the
feeding habit were same. Different environment or habitat might influence the
ability to accumulate the metals.
Strong significant correlation of Fe with Zn and Pb was shown between
Puntius daruphani and Puntius bulu. The significant relationship of Cd with Cu
occurred between Puntius daruphani and Mystacoleucus marginatus compared to
other elements. The correlation between Puntius daruphani and Devario regina
show strong positive relationship of Fe with Zn and Pb. This showed that the
binding capacity of the targeted metals were strong within this two species.
Significant correlation of Zn with Pb also found between Puntius daruphani and
Devario regina.
Strong positive significant relationship of Zn with Pb was shown between
Barbonymus schwanenfeldii and Puntius bulu compared to other elements.
Negative correlation of Fe with Pb was occurred between Barbonymus
schwanenfeldii and Mystacoleucus marginatus. Strong positive significant

96. 81
relationship of Fe with Zn was reported between Barbonymus schwanenfeldii and
Devario regina.
Strong positive significant relationship of Fe with Zn also reported
between Puntius bulu and Mystacoleucus marginatus. Whereas between Puntius
bulu and Devario regina showed positive correlation of Fe with Zn and Pb. Same
feeding habit among these species had resulted same accumulation of trace
metals. The relationship of Mystacoleucus marginatus and Devario regina
showed strong significant of Zn with Pb and Cd with Cu.
Strong relationship was occurred among all species for Fe with Zn and Pb.
Whereas, among all omnivores, it’s showed no significant correlation (p > 0.05)
of Fe with Cd and Cu , Zn with Cd and Cu and Pb with Cd and Cu (Table 4.11).
This indicated that the muscles of the fish were not available to bind the heavy
metals and accumulated into the fish body.
Strong positive correlation (p < 0.05) of Fe with Zn and Cu was shown
between Hexanematichthys sagor and Channa striatus which having carnivores
feeding habit. Strong positive significant also occurred between Zn and Cu among
this two species. Whereas strong negative significant shown between Fe with Pb
and Zn with Pb within this species (Table 4.11).
No significant correlation was found between Fe with Cd, Zn with Cd and
Pb with Cd and Cu among these two carnivores. This showed that the carnivores
also having lower capacity to accumulate cadmium and copper.

97. 82
Table 4.11: Pearson correlation of heavy metals among omnivores and carnivores.
ʽʽ**ʼʼ shows correlation is significant at (p<0.01), ʽʽ*ʼʼ shows correlation is significant at (p<0.05)
a)Puntius daruphani ; b)Barbonymus Schwanenfeldii ; c)Puntius Bulu ; d)Mystacoleucus Marginatus ; e)Devario regina ;
f)Hexanematichthys sagor ; g)Channa stria
Heavy
Metals
Omnivores Carnivore
a) - b) a) – c) a) – d) a) – e) b) – c) b) - d) b) – e) c) – d) c) – e) d) – e) f) - g)
Fe : Zn 0.423 0.975** -0.355 0.993** -0.064 0.653 0.962** 0.993** 0.997** 0.729 0.955**
Fe : Pb -0.929** 0.820* 0.144 0.902* -0.218 -0.773* 0.538 0.744 0.943** 0.411 -0.826*
Fe : Cd -0.305 -0.228 0.048 -0.049 -0.353 -0.203 -0.265 -0.306 -0.350 -0.330 -0.011
Fe : Cu -0.122 -0.197 -0.484 -0.573 -0.453 -0.551 -0.685 -0.444 -0.481 -0.060 0.971**
Zn : Pb -0.716 0.764 -0.394 0.916* 0.933** -0.483 0.733 0.706 0.954** 0.844* -0.816*
Zn : Cd 0.067 -0.250 -0.125 -0.038 0.171 0.324 -0.076 -0.205 -0.324 -0.043 0.045
Zn : Cu 0.724 -0.296 -0.098 -0.593 -0.173 0.108 -0.484 -0.504 -0.475 -0.066 0.964**
Pb : Cd 0.160 -0.129 -0.411 0.102 0.129 -0.104 0.367 -0.294 -0.222 -0.108 0.400
Pb : Cu 0.008 -0.271 -0.390 -0.472 -0.062 0.147 0.136 -0.571 -0.492 -0.348 -0.705
Cd : Cu 0.662 -0.437 0.812* 0.759 -0.635 0.497 0.448 -0.571 -0.553 0.887* 0.193

98. 83
CHAPTER 5
CONCLUSIONS AND RECOMMENDATIONS
5.1 Conclusion
The study was conducted to investigate the accumulation characteristics of
heavy metals concentration in seven types of freshwater fish species collected
from Perak river and their associated health risk. Health risk assessment was
assessing through the estimation of Estimate Daily Intake (EDI), Target Hazard
Quotient (THQ) and Target Cancer Risk (TR). The accumulation was determined
through the Biota-sediment accumulation factor and bio-concentration factor by
fish species.
The resulted indicated that the studied of heavy metal concentrations such
as Cu, Cd, Pb, Zn and Fe were significantly different between fish species and
between different places. It is well documented that it very difficult to compare
the metal concentration between species because of many factors as the
environments, feeding habits, habitats of the fish and also level of water pollution
(Kamaruzzaman et al., 2010). The previous studies had mentioned that the level
of bioaccumulation is based on the role of age, species, bodyweight and trophic
transfer. In present study it showed that different fish species obtained different
concentrations of heavy metals in their body.

99. 84
Devario regina had obtained the highest concentration of zinc compared
to other species although it was small type of fish. However, for Puntius bulu
species which was big in size and bodyweight, it was resulted that the
concentration of lead, iron and zinc were in the lowest amount. This clearly
showed that size and bodyweight had influenced the accumulation of trace metals
into the fish body.
Different environments may vary in the different sources of pollution.
Hexanematichthys sagor species had shown the lowest concentration of copper
and cadmium compared to other species. Hexanematichthys sagor species was
collected at the mud area different from other species and this is clearly approved
the hypothesis that different environments influenced the accumulation of heavy
metals differently.
In view of human health risk, the THQ values indicated no non-
carcinogenic risk from the consumption of all seven types of fish from Perak
river. Whereas for EDI, Fe and Cd in all seven types of fish species studied
exceeded the oral reference dose (RfD) suggested by USEPA. For Pb, only three
species which were Puntius bulu, Channa striatus and Mystacoleucus marginatus
did not exceed the RfD while Cu and Zn showed that the THQ were in range with
RfD for all fish studied species. Target cancer risk (TR) was evaluated for Pb and
Cd only as it’s may pose both non-carcinogenic and carcinogenic effects.

100. 85
The BAF among seven types of fish species showed slightly different in
values. This can be explained by their ingestion of sediments as well as feeding
habits of the different species in different sampling site. Heavy metal
bioaccumulation could occur with increasing size/ age of species (Kwok et al.,
2014). Bioaccumulations of individual metals among the sampling sites were not
similar in pattern due to environment-specific phenomenon. It was considered that
ingested sediments in the digestive tract of fish acted as acid ambient, which
accelerated the bioaccumulation of greater metal concentrations than were
expected (Islam et al., 2014)b
.
For bio-concentration factor, only three heavy metals (Zn, Fe and Pb)
were calculated according to the availability of water concentration information.
The average of BCF for each species was evaluated without considering spatial
variation similar with the study conducted by Tao et al., (2012) at Taihu Lake,
China.
In conclusion, the concentration of heavy metals resulted in this study still
in range and not hugely over the permissible limit recommended but long term
consumption may lead to the harmful effects of certain heavy metals to the human
health of Perak residents.

101. 86
5.2 Recommendation
Present study was conducted from limited sample which was collected
only one time in one season. Seasonal variation based on sample collected in
different season will help to explore more intensive information related to metal
accumulation and health risk of associated from fish consumption from Perak
river. Considering the season might result on clearer feature of the metal
accumulation by fish. Previous studied (Islam et al., 2014)a
had resulted in
different concentration of heavy metals in the same species of fish between
different season.
The accumulation characteristics of different organ will help to identify
the pathway of heavy metals into the fish. Present study was conducted
considering only the fish muscle. So, organ segment analysis will help to give the
metal pathways to fish body in Perak river. Taweel et al., (2013) found that there
were differences in the concentrations of heavy metals between different organs
and site.
Water and sediment samples should be collected together in an area that is
polluted and other areas where the level of pollution is variable. This is to enable
the investigation on the absorption of metals and the comparison of
concentrations between different sampling areas. The study and research on the
Perak river should be conducted continuously to assess the level of the pollution.
Based on the research documented, it was still few research been done on Perak
river compared to Pahang river.

102. 87
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109. 94
APPENDIX-A
Figure A .1: Flow chart of analytical procedure for heavy metals analysis in fish.
Length and weight of the sample were measured once arrived at the laboratory.
Whole fish were dissected using stainless still knife that had been sterile. The muscles
tissues was removed and placed in glass bottles .
The samples were stored in clean glass bottle separately at -20 ºC for 24 hours.
The samples were dried separately at 120 ºC for 24 hours by using dry oven.
The samples were blended homogenously until the sample turned into powdered form.
Samples then were packed in polyethylene bag and sealed separately.
0.5 g of dried samples were weighed into 50 ml beaker, 6 mL nitric acid (65%) and 2
ml hydrogen peroxide (30%) were added to each samples to be digested on a hot plate
for 2h.
3 % of diluted nitric acid were dropped into the beakers on the hot plate after 20 min.
After cooling down, the digested solution were filtered using 0.45 μm Whatman filter
paper.
The filtrate were made up to 30 ml by adding Mili-Q deionized water.
The sample ready to be analyzed by ICP-OES.

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APPENDIX-B
Table B.1: Data of Pearson correlation of heavy metals in all fish species
collected from Perak river.
Correlations
Fe Zn Pb Cd Cu
Fe
Pearson Correlation 1 .658
**
.184 -.204 .420
Sig. (2-tailed) .001 .425 .376 .058
N 21 21 21 21 21
Zn
Pearson Correlation .658
**
1 .616
**
-.281 .037
Sig. (2-tailed) .001 .003 .218 .873
N 21 21 21 21 21
Pb
Pearson Correlation .184 .616
**
1 -.056 -.100
Sig. (2-tailed) .425 .003 .810 .667
N 21 21 21 21 21
Cd
Pearson Correlation -.204 -.281 -.056 1 .059
Sig. (2-tailed) .376 .218 .810 .798
N 21 21 21 21 21
Cu
Pearson Correlation .420 .037 -.100 .059 1
Sig. (2-tailed) .058 .873 .667 .798
N 21 21 21 21 21
**. Correlation is significant at the 0.01 level (2-tailed).
Table B.2: Data of Pearson correlation of heavy metals between
Hexanematichthys sagor and Channa striatus
Correlations
Fe Zn Pb Cd Cu
Fe
Pearson Correlation 1 .955
**
-.826
*
-.011 .971
**
Sig. (2-tailed) .003 .043 .983 .001
N 6 6 6 6 6
Zn
Pearson Correlation .955
**
1 -.816
*
.045 .964
**
Sig. (2-tailed) .003 .048 .933 .002
N 6 6 6 6 6
Pb
Pearson Correlation -.826
*
-.816
*
1 .400 -.705
Sig. (2-tailed) .043 .048 .431 .117
N 6 6 6 6 6
Cd
Pearson Correlation -.011 .045 .400 1 .193
Sig. (2-tailed) .983 .933 .431 .714
N 6 6 6 6 6
Cu
Pearson Correlation .971
**
.964
**
-.705 .193 1
Sig. (2-tailed) .001 .002 .117 .714
N 6 6 6 6 6
**. Correlation is significant at the 0.01 level (2-tailed).
*. Correlation is significant at the 0.05 level (2-tailed).

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Table B.3: Data of Pearson correlation of heavy metals between Puntius daruphani and
Barbonymus schwanenfeldii
Correlations
Fe Zn Pb Cd Cu
Fe
Pearson Correlation 1 .423 -.929
**
-.305 -.122
Sig. (2-tailed) .403 .007 .557 .817
N 6 6 6 6 6
Zn
Pearson Correlation .423 1 -.716 .067 .186
Sig. (2-tailed) .403 .109 .900 .724
N 6 6 6 6 6
Pb
Pearson Correlation -.929
**
-.716 1 .160 .008
Sig. (2-tailed) .007 .109 .762 .988
N 6 6 6 6 6
Cd
Pearson Correlation -.305 .067 .160 1 .662
Sig. (2-tailed) .557 .900 .762 .152
N 6 6 6 6 6
Cu
Pearson Correlation -.122 .186 .008 .662 1
Sig. (2-tailed) .817 .724 .988 .152
N 6 6 6 6 6
**. Correlation is significant at the 0.01 level (2-tailed).
Table B.4: Data of Pearson correlation of heavy metals between Puntius daruphani and
Puntius bulu
Correlations
Fe Zn Pb Cd Cu
Fe
Pearson Correlation 1 .975
**
.820
*
-.228 -.197
Sig. (2-tailed) .001 .046 .664 .708
N 6 6 6 6 6
Zn
Pearson Correlation .975
**
1 .764 -.250 -.296
Sig. (2-tailed) .001 .077 .633 .568
N 6 6 6 6 6
Pb
Pearson Correlation .820
*
.764 1 -.129 -.271
Sig. (2-tailed) .046 .077 .808 .604
N 6 6 6 6 6
Cd
Pearson Correlation -.228 -.250 -.129 1 -.437
Sig. (2-tailed) .664 .633 .808 .387
N 6 6 6 6 6
Cu
Pearson Correlation -.197 -.296 -.271 -.437 1
Sig. (2-tailed) .708 .568 .604 .387
N 6 6 6 6 6
**. Correlation is significant at the 0.01 level (2-tailed).
*. Correlation is significant at the 0.05 level (2-tailed).

112. 97
Table B.5: Data of Pearson correlation of heavy metals between Puntius daruphani and
Mystacoleucus marginatus
Correlations
Fe Zn Pb Cd Cu
Fe
Pearson Correlation 1 -.355 .144 .048 -.484
Sig. (2-tailed) .489 .786 .928 .331
N 6 6 6 6 6
Zn
Pearson Correlation -.355 1 -.394 -.125 -.098
Sig. (2-tailed) .489 .440 .813 .853
N 6 6 6 6 6
Pb
Pearson Correlation .144 -.394 1 -.411 -.390
Sig. (2-tailed) .786 .440 .418 .444
N 6 6 6 6 6
Cd
Pearson Correlation .048 -.125 -.411 1 .812
*
Sig. (2-tailed) .928 .813 .418 .050
N 6 6 6 6 6
Cu
Pearson Correlation -.484 -.098 -.390 .812
*
1
Sig. (2-tailed) .331 .853 .444 .050
N 6 6 6 6 6
*. Correlation is significant at the 0.05 level (2-tailed).
Table B.6: Data of Pearson correlation of heavy metals between Puntius daruphani and
Devario regina
Correlations
Fe Zn Pb Cd Cu
Fe
Pearson Correlation 1 .993
**
.902
*
-.049 -.573
Sig. (2-tailed) .000 .014 .926 .235
N 6 6 6 6 6
Zn
Pearson Correlation .993
**
1 .916
*
-.038 -.593
Sig. (2-tailed) .000 .010 .943 .215
N 6 6 6 6 6
Pb
Pearson Correlation .902
*
.916
*
1 .102 -.472
Sig. (2-tailed) .014 .010 .848 .344
N 6 6 6 6 6
Cd
Pearson Correlation -.049 -.038 .102 1 .759
Sig. (2-tailed) .926 .943 .848 .080
N 6 6 6 6 6
Cu
Pearson Correlation -.573 -.593 -.472 .759 1
Sig. (2-tailed) .235 .215 .344 .080
N 6 6 6 6 6
**. Correlation is significant at the 0.01 level (2-tailed).
*. Correlation is significant at the 0.05 level (2-tailed).

113. 98
Table B.7: Data of Pearson correlation of heavy metals between Barbonymus
schwanenfeldii and Puntius bulu
Correlations
Fe Zn Pb Cd Cu
Fe
Pearson Correlation 1 -.064 -.218 -.353 -.453
Sig. (2-tailed) .904 .678 .492 .367
N 6 6 6 6 6
Zn
Pearson Correlation -.064 1 .933
**
.171 -.173
Sig. (2-tailed) .904 .006 .747 .743
N 6 6 6 6 6
Pb
Pearson Correlation -.218 .933
**
1 .129 -.062
Sig. (2-tailed) .678 .006 .807 .907
N 6 6 6 6 6
Cd
Pearson Correlation -.353 .171 .129 1 -.635
Sig. (2-tailed) .492 .747 .807 .175
N 6 6 6 6 6
Cu
Pearson Correlation -.453 -.173 -.062 -.635 1
Sig. (2-tailed) .367 .743 .907 .175
N 6 6 6 6 6
**. Correlation is significant at the 0.01 level (2-tailed).
Table B.8: Data of Pearson correlation of heavy metals between Barbonymus
schwanenfeldii and Mystacoleucus marginatus
Correlations
Fe Zn Pb Cd Cu
Fe
Pearson Correlation 1 .962
**
.538 -.265 -.685
Sig. (2-tailed) .002 .271 .612 .134
N 6 6 6 6 6
Zn
Pearson Correlation .962
**
1 .733 -.076 -.484
Sig. (2-tailed) .002 .097 .887 .331
N 6 6 6 6 6
Pb
Pearson Correlation .538 .733 1 .367 .136
Sig. (2-tailed) .271 .097 .474 .798
N 6 6 6 6 6
Cd
Pearson Correlation -.265 -.076 .367 1 .448
Sig. (2-tailed) .612 .887 .474 .373
N 6 6 6 6 6
Cu
Pearson Correlation -.685 -.484 .136 .448 1
Sig. (2-tailed) .134 .331 .798 .373
N 6 6 6 6 6
**. Correlation is significant at the 0.01 level (2-tailed).

114. 99
Table B.9: Data of Pearson correlation of heavy metals between Barbonymus
schwanenfeldii and Devario regina
Correlations
Fe Zn Pb Cd Cu
Fe
Pearson Correlation 1 .962
**
.538 -.265 -.685
Sig. (2-tailed) .002 .271 .612 .134
N 6 6 6 6 6
Zn
Pearson Correlation .962
**
1 .733 -.076 -.484
Sig. (2-tailed) .002 .097 .887 .331
N 6 6 6 6 6
Pb
Pearson Correlation .538 .733 1 .367 .136
Sig. (2-tailed) .271 .097 .474 .798
N 6 6 6 6 6
Cd
Pearson Correlation -.265 -.076 .367 1 .448
Sig. (2-tailed) .612 .887 .474 .373
N 6 6 6 6 6
Cu
Pearson Correlation -.685 -.484 .136 .448 1
Sig. (2-tailed) .134 .331 .798 .373
N 6 6 6 6 6
**. Correlation is significant at the 0.01 level (2-tailed).
Table B.10: Data of Pearson correlation of heavy metals between Puntius bulu and
Mystacoleucus marginatus
Correlations
Fe Zn Pb Cd Cu
Fe
Pearson Correlation 1 .993
**
.744
*
-.306 -.444
Sig. (1-tailed) .000 .045 .277 .189
N 6 6 6 6 6
Zn
Pearson Correlation .993
**
1 .706 -.205 -.504
Sig. (1-tailed) .000 .058 .349 .154
N 6 6 6 6 6
Pb
Pearson Correlation .744
*
.706 1 -.294 -.571
Sig. (1-tailed) .045 .058 .286 .118
N 6 6 6 6 6
Cd
Pearson Correlation -.306 -.205 -.294 1 -.571
Sig. (1-tailed) .277 .349 .286 .118
N 6 6 6 6 6
Cu
Pearson Correlation -.444 -.504 -.571 -.571 1
Sig. (1-tailed) .189 .154 .118 .118
N 6 6 6 6 6
**. Correlation is significant at the 0.01 level (1-tailed).
*. Correlation is significant at the 0.05 level (1-tailed).

115. 100
Table B.11: Data of Pearson correlation of heavy metals between Puntius bulu and
Devario regina
Correlations
Fe Zn Pb Cd Cu
Fe
Pearson Correlation 1 .997
**
.943
**
-.350 -.481
Sig. (2-tailed) .000 .005 .496 .334
N 6 6 6 6 6
Zn
Pearson Correlation .997
**
1 .954
**
-.324 -.475
Sig. (2-tailed) .000 .003 .531 .341
N 6 6 6 6 6
Pb
Pearson Correlation .943
**
.954
**
1 -.222 -.492
Sig. (2-tailed) .005 .003 .673 .322
N 6 6 6 6 6
Cd
Pearson Correlation -.350 -.324 -.222 1 -.553
Sig. (2-tailed) .496 .531 .673 .255
N 6 6 6 6 6
Cu
Pearson Correlation -.481 -.475 -.492 -.553 1
Sig. (2-tailed) .334 .341 .322 .255
N 6 6 6 6 6
**. Correlation is significant at the 0.01 level (2-tailed).
Table B.12: Data of Pearson correlation of heavy metals between Mystacoleucus
marginatus and Devario regina
Correlations
Fe Zn Pb Cd Cu
Fe
Pearson Correlation 1 .729 .411 -.330 -.060
Sig. (2-tailed) .100 .418 .523 .909
N 6 6 6 6 6
Zn
Pearson Correlation .729 1 .844
*
-.043 -.066
Sig. (2-tailed) .100 .034 .935 .901
N 6 6 6 6 6
Pb
Pearson Correlation .411 .844
*
1 -.108 -.348
Sig. (2-tailed) .418 .034 .838 .499
N 6 6 6 6 6
Cd
Pearson Correlation -.330 -.043 -.108 1 .887
*
Sig. (2-tailed) .523 .935 .838 .019
N 6 6 6 6 6
Cu
Pearson Correlation -.060 -.066 -.348 .887
*
1
Sig. (2-tailed) .909 .901 .499 .019
N 6 6 6 6 6
*. Correlation is significant at the 0.05 level (2-tailed).

116. 101
APPENDIX-C
Figure C.1: Measuring process
Figure C.2: Weighing process
Figure C.3: Dissected muscle of the fish

117. 102
Figure C.4: The dried sampled
Figure C.5: Acid digestion process
Figure C.6: Filtration process

118. 103
Figure C.7: Double filtration process by using syringe filter (to avoid bubble occurred in
the diluted samples)
Figure C.8: Analyzing process of heavy metals by using ICP-OES at FRIM, Kepong.

119. 104
APPENDIX-D
Figure D.1: Human activities occur on the riverside of Perak river.
Figure D.2: Fishing activities by nearby people.

120. 105
Figure D.3: Rakit house was built for fishing purpose.
Figure D.4: Boat for fishing purpose

121. 106
Figure D.5: Dumping waste near the riverside of Perak river.
Figure D.6: Fish got from the local fisher.
Thk
Fish g