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I tnengaku membenarkan tesis (LPSI Smjanal Doktor Falsafah) ini di simpan di Perpustakaan Universiti Malaysia Sabah dengan syarat-syarat kegunaan seperti berikut:

1. Tesis adalah hakmilik Universiti Malaysia Sabah. 2. Perpustakaan Universiti Malaysia Sabah dibenarkan membuat salinan untuk tujuan pengajian sahaja. 3. Perpustakaan dibenarkan membuat salin an tesis ini sebagai bahan pertukaran antara institusi pengajian tinggi. 4. ** Sila tandakan ( / )

SULIT

TERHAD

7 I TIDAK TERHAD

~ (TANIrATANGAN PENULIS)

(Mengandungi maklumat yang berdarjah keselamatan atau kepentingan Malaysia seperti yang termaktub di dalam AKTA RAHSIA RASMI 1972)

(Mengandungi maklumat TERHAD yang telah ditentukakan oleh organisasifbadan di mana penyelidikan dijalankan)

DishhWllMelMJ CHEAL B AN

RY VUiI'9" ... A YSIA SABAH

A..lamat Tetap: g ~I./- I L-ot2.otJ~ ICfAf4fV ':)/:18/

____ 1 /tfVI J1 tV Ie r:. 11/ t:l, Rj

____ Ie E b M-I

6'700 D leu LI fIh /

rtCUikh: __ J-_' -.-:./_6_f"--,I_2-_o '_6 ___ _

A..1" AT AN: * Potong yang tidak berkenaan.

Nama Penyelia

Tarikh: --------------------------

* Jika tesis ini SULIT atau TERHAD, sila lampiran surat daripada pihak berkuasalorgansasi berkenaan dengan menyatakan sekali sebab dan tempoh tesis ini perlu dikelaskan sebagai SULIT danTERHAD.

* Tesis dimaksudkan sebagai tesis bagi Ijazah Doktor Falsafah dan Sarjana secara penyelidikan, atau disertasi bagi pengajian secara kerja kursus dan penyelidikan, atau Laporan Projek Sarjana Muda (LPSM).

DETERMINATION OF HEAVY METALS CONCENTRATION IN SQUIDS IN

KOTA KINABALU

GANESAN MENON AIL NEELAMEGAM

DISSERTATION SUBMITIED AS PARTIAL FULFILLMENT FOR THE DEGREE OF BACHELOR OF

FOOD SCIENCE WITH HONOURS

SCHOOL OF FOOD SCIENCE & NUTRITION UNIVERSm MALAYSIA SABAH

2010

DECLARATION

I hereby declare that the material in this dissertatton Is my own except for quotations, excerpts, equattons, summaries and references, which have been duly acknowledged.

20 May 2010 GANESAN MENON AIL NEELAMEGAM HN2006-4101

II

VERIFICATION

NAME GANESAN MENON AIL NEELAMEGAM

TmE DETERMINATION OF HEAVY METALS IN SQUIDS IN KOTA KINABAW

DEGREE: BACHELOR OF FOOD SCIENCE WITH HONOURS

DR. MOHO IQBAL HASHIM I SUPERVISOR

ASSOC. PROF DR. SHARIFUDIN MD. SHAARANI EXAMINER

DR. LEE JAU SHYA EXAMINER

ASSOC. PROF DR. MOHO ISMAIL ABDULLAH DEAN

MAY 2010

III

ACKNOWLEDGEMENT

This dissertation would not have been successfully written if not by the help and

support from everyone that involved directly as well as Indirectly of which is very

much appreciated and valued.

Arst of all, I would like to thank God for his blessings and for being with me all

the time especially in handling the difficult tasks which made me to complete my final

year project successfully with full of confidence.

My foremost thanks and wishes go to my supervisor, Or. Muhammad Iqbal

Hashimi. His Initial encouragement and support in this study and his continued kind

aSSistance, guidance and valuable supervision saw to the successful completion of my

dissertation. My sincere thanks to School of Food Sdence and Nutrition, Unlverlsitl

Malaysia Sabah which provided a very good learning opportunity for me during my

flnal year project.

I would like to forward my greatest appreciation to my beloved parents and all

other family members who have given me encouragement, moral support and

flnandal support all this while.

last but not least, I also would like to express my sincere thanks to all my

course mates and friends for their constant support, encouragement and helped In

accomplishing my dissertation. Thank you.

N

ABSTRACT

DETERMINAnON OF HEAVY METALS CONCENTRAnON IN SQUIDS IN KOTA KlNABALU

The species of Loligo chinensis and Lol/go duvaucel/i from Kota K1nabalu were

sampled and analyzed for seven different heavy metals include Cd, Pb, Cu, Zn, Fe, Ni

and Mn by using two different methods; wet digestion method and dry ashing

method. The differences in heavy metals concentration among different squid species

were found not significant. The concentration of heavy metals in both squid species

may reflect their similar morphology and environmental factors. In this study, both

Loligo chinensis and LOl/go duvaucelli detected to accumulate similar amount of metal

In both methods although there is difference between the metal concentration values

obtained which is not significant except for lead in wet digestion method. Comparing

metal concentrations in squids among both methods used indicates that dry ashing

method is not suitable for detennination of metals with lower melting point The high

concentration of zinc in both species and both methods reflects that zinc is needed by

the squids as a nutrient and plays important roles In the growth of squids. Comparison

with the Malaysian Food Act 1983 and Food Regulation 1985 shows that cadmium and

lead surpass the maximum limit permitted in seafood; whereas for copper and zinc,

the amount is still not exceeding the current legislative limit for squid.

v

ABSTRAK

PENENTUAN KEPEKATAN LOGAN-LOGAN BERAT DAUN $OTONG Dr KOTA KINABALU

Spesls Loligo chlnensis dan LoIlgo dwaucelll darl Kota Klnabalu d/jadlkan sebagal

sampe/ dan dlkajl untuk tujuh jenls /ogam berat yang berbeza tennasuk at Q/, Pb,

Zn, Fe, NI dan Mn dengan menggunakan dua kaedah berbeza; penghadaman basah

dan pengabuan kerlng. Perbezaan kepekatan /ogam berat dl antara spes/s sotong

berbeza dldapatl bahawa bukan perbezaan bermakna. Kepekatan /ogam berat dl

da/am kedua-dua spesis sotong menggambarkan morfoiogl dan faktor persekltaran

yang seropa. Da/am kajlan In!, kedua-dua sampe/ Loligo chlnens/s dan Lollgo

dwaucel/l dlkesan mengumpu/kan kuantltllogam berat yang seropa da/am kedua-dua

kaedah yang dlgunakan walaupun terdapat perbezaan dlantara nllal kepekatan logam

yang dlperolehl yang bukan merupakan perbezaan bennakna kecua/l untuk logam

plumbum dalam kaedah penghadaman basah. Perbandlngan antara kepekatan logam

da/am safDng me/a/ul kedua-dua kaedah yang dlgunakan menggambarkan bahawa

pengabuan kering adalah tldak sesual untuk menentukan logam yang mempunyal

takat lebur yang rendah. Kuantltl /ogam zink yang tlnggi dalam kedua-dua spes/s

satang dan da/am kedua-dua kaedah menggambarkan bahawa zink dlper/ukan oIeh

satang sebagal nutrlen dan berperanan pentlng dalam pertumbuhan satong.

Perbandlngan yang dllakukan dengan Aida Makanan Malaysia 1983 dan Peraturan­

Peraturan Makanan 1985 menunjukkan bahawa /ogam plumbum dan kMlmlum telah

meleblhl had makslmum yang dlbenarkan untuk makanan laut; manaka/a bagllogam

zlnk dan kuprom, kuantitlnya maslh di bawah had semasa yang dibenarkan untuk

satang.

VI

TABLES OF CONTENT

Page

TITLE I

DECLARATION II

VERIFICATION III

ACKNOWLEDGEMENT IV

ABSTRACT V

ABSTRAK VI

TABLES OF CONTENTS

UST OF TABLES XI

UST OF FIGURES XII

UST OF PHOTOGRAPHS XIV

UST OF SYMBOLS XV

UST OF APPENDIX XVI

CHAPTER 1: INTRODUCTION 1

1.1 Research Background 1

1.2 Objectives 5

1.3 Scope of Study 6

1.4 Previous studies 7

CHAPTER 2: UTERATURE REVIEW 9

2.1 Heavy metal cycle in the environment 10

2.2 Heavy metal pollution in aquatic ecosystem 10

2.3 Toxicity of heavy metals in the solution 12

2.4 Heavy metals in food 16

VII

2.5 Heavy metal as an essential minerai

2.6 Essential type of heavy metal

2.6.1 Copper

2.6.2 Zinc

2.6.3 Nickel

2.6.4 Iron

2.6.5 Manganese

2.7 Non-essential type of heavy metals

2.7.1 Lead

2.7.2 cadmium

2.8 Sample digestion for heavy metal detennination

2.8.1 Dry ashing

2.8.2 Wet Digestion

2.9 Squid

2.9.1 Squids In Sabah

2.10 FAAS Instrument

CHAPTER 3: METHODOLOGY

3.1 Sample Collection

3.2 Reagents

3.3 Apparatus

3.4 Material Preparation

3.5 Sample Preparation

3.5.1 Dry ashlng method

3.5.2 Wet digestion method

3.6 Preparation of Standard Solution

VIII

17

21

22

23

24

26

27

29

29

30

31

31

32

33

35

38

39

39

40

41

42

42

43

44

3.6.1 Standard solution for Manganese 45

3.6.2 Standard solution for Cadmium 45

3.6.3 Standard solution for Copper 46

3.6.4 Standard solution for Nickel 46

3.6.5 Standard solution for Lead 46

3.6.6 Standard solution for Zinc 47

3.6.7 Standard solution for Iron 47

3.7 Calibration and analysis using FMS 47

3.8 Water content 49

3.9 Statistical analysis 50

CHAPTER 4: RESULTS AND DISCUSSION 51

4.1 Water content 51

4.2 Calibration 51

4.3 Concentration of heavy metals 52

4.3.1 Comparison between squid species 53

4.3.2 Dry ashing method 54

4.3.3 Wet digestion method 56

4.4 Comparison between methods 60

4.4.1 Loligo chinensis 61

4.4.2 Loligo duvaucelli 63

4.5 Comparison of heavy metals concentration in squid 68 Samples with Malaysian Regulation

CHAPTERS: CONCLUSION 72

5.1 Recommendations 73

IX

REFERENCES

APPENDIX

x

74

82

UST OF TABLES

Page

Table 2.1 Oassification of metals 16

Table 2.2 The limit of heavy metal concentration In Food Act 20

Table 2.3 The limit of heavy metal concentration by USFDA 21

Table 3.1 The chemicals used with manufacturer and usage 40

Table 3.2 The apparatus used with brand and manufacturer 41

Table 3.3 Parameters for seven heavy metals before analysis 48

Table 4.1 Water content of LoIigo chinens/s and Loll9O duvaucelll 51

Table 4.2 Correlation coefficient of calibration graph for seven metals 52 analyzed

Table 4.3 Concentration of heavy metals In LoIi9O chinensis and Loi/go 53

duvaucelll by dry ashlng and wet digestion method

Table 4.4 Concentration order of heavy metals in each squid 68

samples by wet digestion method

Table 4.5 Concentration order of heavy metals In each squid samples by 68

dry ashlng method

Table 4.6 Maximum value that Is permissible by Malaysian Food Act 1983 69

and Food Regulation 1985

XI

UST OF FIGURES

Figure 2.1 Hydrology cyde of heavy metals in environment

Figure 2.2 Schematic presentations of metal reservoirs and their interaction in aquatic and terrestrial systems

Figure 2.3 Factors influendng toxicity of metals in solution

Figure 2.4 Morphology of squid

XII

Page

10

13

15

34

UST OF PHOTOGRAPHS

Photograph 2.1 Appearance of Lot/go chlnensis

Photograph 2.2 Appearance of Lo/igo duvaucelll

Photograph 3.1 Brownish smoke release from Erlenmeyer Rask

Photograph 3.2 Samples after dry ashing and wet digestion

XIV

Page

36

37

43

44

UST OF SYMBOLS

FAAS Flame Atomic Absorption Spectroscopy

Pb Lead

Cd cadmium

Cu Copper

Zn Zinc

Fe Iron

Ni Nickel

Mn Manganese

Kg Kilogram

9 Gram

J,lg/kg Microgram per kilogram

mg/kg Milligram per kilogram

J,lgg-l Microgram per gram

J,lg/mL Microgram per millimeter

nm Nanometer

% Percent

PPM Parts per million

FAO Food and Agricultural Organization

AOAC Association of Official Analytical Chemistry

H2SO4 Sulfuric Acid

HN03 Nitric Add

HO Hidrochloric add

XV

UST OF APPENDIX

Page

Appendix A Periodic Table 82

Appendix B calibration graph of copper 83

Appendix C calibration graph of zinc 84

Appendix 0 calibration graph of cadmium 85

Appendix E calibration graph of lead 86

Appendix F calibration graph of iron 87

Appendix G calibration graph of nickel 88

Appendix H calibration graph of manganese 89

Appendix I One way ANOVA 90

Appendix J Multiple Comparisons for cadmium 91

Appendix K Multiple COmparisons for lead 92

Appendix L Multiple Comparisons for copper 93

Appendix M Multiple Comparisons for zinc 94

Appendix N Multiple Comparisons for iron 95

Appendix 0 Multiple COmparisons for nickel 96

Appendix P Multiple Comparisons for manganese 97

AppendlxQ Standard solution for Nickel prepared before analysis 98

XVI

CHAPTER 1

INTRODUCTION

1.1 Research Background

Squids are the large and diverse group of marine cephalopods from the order

Teuthida. Squids live in all seas and they frequently swim In large groups called

shoals. Squids have soft body which are in elongated tubular shape, short compact

heads and two flns at the tail end. They also have chromatophores embedded In their

skin and the ability to expel Ink and rapidly change the colours and patterns of their

bodies, often to blend with their surroundings If threatened. Most of the squids range

in size from less than 30 centimetres to nearly 4S centimetres In length, including the

arms (Oarke, 1966).

There are 30 families and approximately 460 species of squid collectively

include cuttlefish known to exist in oceans around the world (Sugiyama et al., 1989).

In Malaysia common squids are known with local name as 'Sotong biasa', 'Sotong

cumlt' or 'Cumlt-cumit' and its scientific name Is Loligo spp (ISSCAAP, 2004). Squids

are consumed as one of the main seafood CUisine among all categories of people

around the wood. Calamari is a famous Mediterranean cuisine in which squids are

butter-coated and deep fried. In Malaysian CUisine, squid Is used In stir-fries, rice, and

noodle dishes. In some parts of South East Asian countries, the squids are dried and

shredded and eaten as seasoned snacks.

Nowadays, health-conscious about heavy metals content in seafoods has

increased significantly. Trace elements and metals are continuously released In the

aquatic environment via natural and anthropogenic Influx. The determination of these

analytes in squid has interest because of the importance of many of these elements to

human health. This interest arises from two areas of concem, nutritional and

toxicological. Nutritional because trace metals such as ca, Fe, Mg, Zn, Cu, Co and AI

are necessary for maintenance of optimum health and toxicological since certain

metals such as Pb, Cd, As and Hg are detrimental to optimum health. Furthermore, it

is an important aspect of environmental analysis because seafood is used as

bloindicator organisms to assess bioavailability contaminant concentrations in coastal

waters. Molluscs have long been known to naturally accumulate metals to high

concentrations. Although metals in gastropod and bivalve molluscs have been the

subject of considerable research, metal metabolism in cephalopod molluscs have been

relatively poorly investigated. In addition, cephalopods are quantitatively important in

many food webs. Cephalopods have been reported to accumulate metals including Cu,

Zn and Cd to concentrations far exceeding that of surrounding seawater (Stephen and

Julian, 2002).

The term 'heavy metals' can be refer as a group of metallic elements with

atomic weights greater than 40 and are characterized by similar electronic distribution

in their extemal shell. These include alkali earth metals (e.g., caldum, magnesium),

alkali metals (e.g., sodium, potaSSium), lanthanides and actinides (e.g., uranium). It Is

also known as trace metal. In aquatic system, the heavy metals of greatest concem

are copper, zinc, cadmium, mercury and lead. These elements are toxic to organisms

above speciflc threshold concentration but many of them (e.g., copper and zinc) are

essential for metabolism at lower concentrations. Toxic heavy are usually present at

2

low concentration in aquatic ecosystem, but deposits of anthropogenic origin have

raised the heavy metal concentration, creating environmental problems in coastal

zones, lakes and rivers (Iyenger et a/., 1998).

In Malaysia, rapid Industrial development Is undergoing and there have been

inddences of toxic pollution from industry. Metal contamination In the aquatic

environment arises from industrial processes (e.g., mining, smelting, finishing and

plating of metals, paint and dye manufacture) and from pipes and tanks in domestic

systems, discharging a variety of toxic metals such as cadmium, copper, zinc and lead

into the environment (Moore and Ramamoorthy, 1984). In the aquatic environment,

the concentration, transport, transformation, and disposition of a chemical or material

are primarily controlled by the following:

• The physical and chemical properties of the compound

• The physical, chemical and biological properties of the eoosystem

• The sources and rates of input of the chemical into the environment.

Pollution by heavy metals in coastal environment has become a global phenomenon

because of its tOxidty, persistence for several decades in the aquatic environment,

bloaccumulatlon and biomagniflcatlons In the food chain (Valls and Lorenzo, 2002).

Nowadays, squids are an important seafood resource for human consumption.

Food pollution are caused by heavy metal pollution which they can exist naturally and

also through anthropogenic. The contamination of heavy metals In squids will cause

an effect on human body and will cause disease and death. Marine organisms

accumulate most trace elements to concentration of many times higher than those

3

present In seawater. The toxldty of the heavy metal can be seen In some issues. The

best known example of deaths attributable to contaminants Ingested in seafood is the

so-called Mlnamata disease caused by mercury released from the Chlsso Corporation

plant, which was first reported in Japan In 1956 (Pradyumna, 2005). Other trace

metals also have been the cause of environmental poisoning outbreaks. An unusual

disease, known as "Itai-ital" disease broke out In the Jlntsu River basin In Japan (louiS

et aI., 1996). The symptoms of the disease Indude yellow discoloration of the teeth,

loss of the sense of smell, a reduction In red blood cell numbers, lumbar and leg pains

and skeletal bones. It is now believe that the primary cause of the ltal-ltai syndrome

was the discharge of cadmium-rich effluents from zinc mine to the river and watering

system.

4

1.2 Objectives

The main objectives of this research are : -

1) To determine and compare the level of heavy metal concentration; lead (Pb),

nickel (Ni), cadmium (Cd), copper (Cu), Iron (Fe), Manganese (Mn) and zinc

(Zn) In Loligo chlnensis and Lotigo duvaucelli and compare two sample

preparation methods; dry-ashing method and wet digestion method.

2) To compare the level of lead, nickel, cadmium, copper, iron, manganese and

zinc in squids sample with Malaysian Food Act 1983 and Food Regulation 1985.

5

1.3 Smpe of study

Major scope of this study is to determine the amount of heavy metals; lead (Pb),

nickel (Ni), cadmium (Cd), copper (Cu), chromium (Cr),and zinc (Zn) in two type squid

sample that has been collected which are Lol/go chinensis and Lol/go duvaucel/ from

Kota Kinabalu, Sabah. This study also carried out to determine whether squids caught

in sea areas of Kota Klnabalu are safe for consumption based on maximum level of

metals concentration allowed in Malaysian Food Act 1983 and Food Regulation 1985.

This study use Flame Atomic Absorption Spectrometry (FAAS) to detect the

concentration level in squid sample. Two types of different sample preparation

methods are used in this study, dry ashing method and wet digestion method. These

two sample preparation methods are widely used In the determination process of

heavy metals in food. So this study will determine which sample preparation methods

are more suitable for detection of lead (Pb), nickel (Ni), cadmium (Cd), copper (Cu),

chromium (Cr), and zinc (Zn) in seafood especially squids.

6

1.4 Previous study on detennination of heavy metals In squids

For many years, there are widely studies about the heavy metals in the squids using

many different variables. However, the species and location of squids that had been

researched is limited. The first widely documented Instance of public health Impact

resulting from aquatic contamination by heavy metals occurred at Minamata Bay In

Japan, commencing in 1953 (louis, 1996).

A study conducted on heavy metals In squids in Tanjung Karang, Selangor

shows that Lollgo edu/is contains zinc at highest concentration followed by iron and

copper (Tuldmat, 2006). The order of heavy metal concentration from highest to

lowest Is Zn > Fe > Cu > Ni > Pb > Cd. The study also reveals that heavy metal

content in squids at T Is lower than the permitted maximum limit by Food Regulation

1985.

Another study conducted on heavy metals In seafoods in Kuala Kemaman,

Terengganu shows that Lol/go ch/nensis contains zinc at highest concentration

followed by copper and iron (Tuldmat, 2002). The order of heavy metal concentration

from highest to lowest is Zn > CU > Fe > Cd > Pb > NI. The study also reveals that

heavy metal content In squids at Kuala Kemaman is lower than the permitted

maximum limit by Food Regulation 1985.

There are also wortdwide researchers studied about heavy metal concentration

in squids from various species. Prafulla (2001), studied about concentration of heavy

metals in the sqUid, LoI/go duvaucell and Doryteuthls slbogae caught from the

Southwest Coast of India. The mean values (highest at the three stations) of highly

hazardous metals In the muscle of the two species, were: Hg < 0.05, 0.07; Cd is 0.55,

7

0.89; Pb is 0.99, 0.89; Cr is 0.72, < 0.45 and Ni is 0.45, 0.19 ppm, all within the

international safety limits. However, elevated levels of some of the metals, particular1y

Cd, Zn and Cu were sometimes observed. The study also shows that The metal levels

In the squids varied greatly between the species and In the same species (L

duvaucell) from different regions. The present study shows that the average

concentrations of all metals are significantly lower in of both the squids from all the

regions and are far below the legal limits. They do not seem to cause any health

hazard by consuming the edible parts of squids.

8

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