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

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.

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

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

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

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

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

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

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

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

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

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

xiii

List of Symbols

% percentage

˚C degree celcius

X multiplication

> more than

< less than

E-0 exponent (x10)

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.

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.

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).

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

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

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

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.

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.

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.

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.

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.

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,

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

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.

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

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).

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).

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.

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).

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.

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).

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.

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)

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).

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.

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).

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.

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

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).

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

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

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).

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.

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"

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.

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

35

Duri Sagor catfish Hexanematichthys

sagor

7

Haruan Striated

snakehead

Channa striatus 4

Sia Barb, spiny Mystacoleucus

marginatus

10

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

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.

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

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.

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.

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.

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.

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)

Om

niv

ore

s

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)

Car

niv

ore

s

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)

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

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

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

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

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

Co

nc

(µg

/g)

Species

Cd Cu Fe Zn Pb

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

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.

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

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

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.

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

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

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.

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.

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)

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)

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

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

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.

63

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

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

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.

66

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

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).

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

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.

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)

Spe

cie

s

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)

Spe

cie

s

Pb

b)

RfD

RfD

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)

Spe

cie

s

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)

Spe

cie

s

Zn

d)

RfD

40

RfD

300

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)

Spe

cie

s

Fe

e) RfD

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

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.

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

THQ

Val

ue

s

Fish Species

Fe Zn Pb Cd Cu

1

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.

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.

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

TR v

alu

es

Fish Species

Pb Cd

10-6 to 10-4

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)

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

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.

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

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.

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.

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.

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.

87

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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.

95

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).

96

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).

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).

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).

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).

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).

101

APPENDIX-C

Figure C.1: Measuring process

Figure C.2: Weighing process

Figure C.3: Dissected muscle of the fish

102

Figure C.4: The dried sampled

Figure C.5: Acid digestion process

Figure C.6: Filtration process

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.

104

APPENDIX-D

Figure D.1: Human activities occur on the riverside of Perak river.

Figure D.2: Fishing activities by nearby people.

105

Figure D.3: Rakit house was built for fishing purpose.

Figure D.4: Boat for fishing purpose

106

Figure D.5: Dumping waste near the riverside of Perak river.

Figure D.6: Fish got from the local fisher.

Thk

Fish g