MORPHOLOGY AND TOXIN STUDY OF PUFFER FISH COLLECTED FROM SABAH AND SARAWAK WATERS

Monaliza Binti Mohd Din

Master of Science (Marine Toxicology)

2013

Pusat Khidmat Maklumat Akademik

[TMVERS1TI MALAYSIA SARAWAK

P. KHIDMAT MAKLUMAT AKADEMIK

IIIII IIIIIIIM Iill II II III.

1000245954 MORPHOLOGY AND TOXIN STUDY OF PUFFER FISH COLLECTED

FROM SABAH AND SARAWAK WATERS

Monaliza Binti Mohd Din

This project is submitted in fulfillment of the requirements for the degree of Master of Science in Marine Toxicology (Marine Toxicology)

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

2013

DECLARATION

No portion of the work referred to this dissertation has been submitted in support of an

application for another degree or qualification of this or any other university or institution of

higher learning.

(MONALIZA BT MOHD DIN)

Department of Aquatic Science

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak (UNIMAS)

l

ACKNOWLEDGEMENT

First and foremost, I would like to thank ALLAH SWT for the continuous blessing that I have

received during those challenging and wonderful times that I have gone through from the

starting until the end of this project.

I would like to make my deepest appreciation and gratitude to Dr Samsur bin Mohamad for his

invaluable guidance, constructive criticism and encouragement during the course of this

research. Million of thanks are extended to Puan Che Nin bt Man from National Poison

Center, Universiti Sains Malaysia and Dr Gires Usup, Universiti Kebangsaan Malaysia for

their permission and kind assistance during sample analysis using GC-MS and LC-MS

analysis were carried out respectively.

Greatest appreciation to my beloved parents, Mohd Din bin Yaakob and Aminah bt Yusuff for

their consistent encouragement throughout this research. Grateful acknowledgment is made to

all staff and members of department aquatic science for their help and suggestions during

completion this research.

My appreciation also goes to Faculty of Resource Science and Technology for the research

facilities and scholarship of National Science Fellowship, MOSTI for the financial assistance

throughout the achievement of this research.

11

ABSTRACT

Malaysia, fatal food poisonings associated with the consumption of puffer fish have

occurred for decades, but the causative species or toxins have never been documented. To

date, there has been little study on morphological and verification of toxin profiles on puffer

fish especially on costal Sabah and Sarawak waters. Attempts was made to identify the

morphology and verify the toxin profile in Tetraodontidae species of puffer fish commonly

found in Sabah and Sarawak coastal waters Seven species puffer fish namely Lagocephalus

lunaris, Lagocephalus spadiceus, Lagocephalus sceleratus, Xenopterus naritus, Takifugu

oblongus, Tetraodon nigroviridis and Arothron stellatus were collected and morphological

identified as well as toxin profile verification using biological mouse bioassay analysis and

analytical Thin Layer Chromatography (TLC), High Performance Liquid Chromatography

(HPLC), Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography-

Mass Spectrometry (GC-MS) analysis. Most significant characteristics that drawn attention for

morphology identification of Tetraodontidae species is having beak like teeth structure

consisting of two teeth at upper jaw and lower jaw. It is interesting to note that Lagocephalus

lunaris and Lagocephalus spadiceus could be distinguish to different species by identified the

distribution patterns of small spines on the dorsal body. Based on morphological

identification, Xenopterus naritus and Takifugu oblongus were having high abundant of small

prickle on lower part bodies compared to others collected species. With respect to Arothron

stellatus, the distributions of small prickles were numerous on dorsal body with small black

spot until to the tail. To isolate unknown toxin from puffer fish, five grams of different tissues

such as muscle, skin, liver, gonad and eggs was minced and extracted with 0.1% acetic acid

(AcOH). The crude toxin was assessed and preliminary screening for tetrodotoxin (TTX)

detection by using biological mouse bioassay and analytical TLC analysis. Mouse bioassay

iii

analysis revealed that all edible tissues of all collected species were non toxic and safe for

human consumption except reproductive organ for L. lunaris, X. naritus, T. oblongus and T.

nigroviridis where gave clinical neurotoxin symptom to the mice and lead to the death. TLC

analysis gave positive detection of TTX by producing the fluorescent yellow spots for extracts

of L. lunaris, X. naritus, T. oblongus and T. nigroviridis where Rf values were 0.78 and 0.22

with, pyridine: ethyl acetate: acetic acid: water (15: 5: 3: 6) and 1-butanol: acetic acid: water

(2: 1: 1), respectively. Meanwhile, for extracts of Lagocephalus spadiceus, Lagocephalus

sceleratus and Arothron stellatus did not produce any single one fluorescent yellow spot in

TLC analysis. Verification of toxin profile and quantitative toxin in extracted of puffer fish

was carried out using HPLC, GC-MS and LC-MS. Quantitative results from these analyses

indicate that L. lunaris, X. naritus, T. oblongus and T. nigroviridis collected from coastal

Sabah and Sarawak waters confirmed exhibit tetrodotoxin (TTX) in extracted tissues based on

verification using HPLC, LC-MS and GC-MS analysis by comparing the retention time of

sample extracts to the retention time of authentic TTX. Meanwhile, there was no detection of

TTX in L. spadiceus, L. sceleratus and A. stellatus. These finding is in agreement with the

results from mouse bioassay and TLC analysis. From this study, it was clearly demonstrated

that local puffer fish from coastal East Malaysian waters were exhibit TTX as toxin principles

and the toxin profiles could be drawn with the aim of provide the informative data as a safety

guidelines for local people in order to safeguard public health from puffer fish poisoning.

Keywords: Puffer fish, Morphology, TTX, HPLC, LC-MS, GC-MS

iv

KAJIANMORFOLOGI DAN TOKSINBAGI SPESIS IKANBUNTAL DARIPADA

PERAIRAN SABAH DAN SARA WAK

ABSTRAK

Di Malaysia, keracunan makanan yang dikaitkan oleh ikan buntal telah lama berlaku tetapi

spesis atau toksin penyebab tidak direkodkan. Sehingga hari ini, hanya terdapat sedikit kajian

terhadap morfologi dan profil toksin telah dyalankan terhadap ikan buntal terutama di

perairan timur Malaysia taitu Sabah dan Sarawak Dalam kajian ini, pelaksanaan telah

dilakukan untuk mengenalpasti morfologi dan toksin terlibat di dalam tisu ikan buntal yang

telah ditangkap di perairan pantai Sabah dan Sarawak Sebanyak tujuh spesis ikan buntal

terdiri daripada spesis Tetraodontidae iaitu LaQocephalus lunaris LagocCphalus spadiceus.

Lagocephalus sceleratus, Xenopterus naritus Taki uu oblongus, Tetraodon nigroviridis dan

Arothron stellatus telah dikenalpasti ciri-ciri morfologi dan toksin profil disahkan

menggunakan bioesei tikus, TLC, HPLC, GC-MS dan LC-MS. Hasil daripada kajian ini,

menunjukan bahawa ikan buntal mempunyai ciri-ciri morfologi yang unik kerana mempunyai

struktur gigi yang seakan paruh dimana dua gigi berada di rahang atas dan dua gigi lagi

berada di rahang bawah. Daripada hasil kenalpasti morfologi, Lagocephalus lunaris dan

Lagocephalus spadiceus boleh dibezakan dengan mengenalpasti corak taburan duri kecil

pada bahagian badan dorsal ikan tersebut. Manakala Xenopterus naritus dan Taki u

oblongus pula mempunyai taburan duri kecil yang banyak di bahagian bawah badan ikan

tersebut. Hal ini berlainan dengan Arothron stellatus di mana ikan ini mempunyai taburan

duri kecil dan bintik hitam yang banyak di bahagian dorsal badan ikan sehingga ke bahagian

ekor. Beberapa analisis telah dyalankan untuk merekodkan toksikologi ikan buntal yang

ditangkap di perairan pantai Sabah dan Sarawak. Lima gram tisu ikan yang berbeza seperti

V

isi, kulit, hati, gonad dan telur telah dicampurkan dengan 0.1% asid asetik dan telah

dianalisis dengan mengunakan bioesei tikus, TLC, HPLC, GC-MS dan LC-MS. Hasil daripada

analisis bioesei tikus menunjukan bahawa tisu pembiakan daripada Lagocephalus lunaris

Xenopterus naritus Taki oblongus dan Tetraodon nigroviridis adalah bertoksik dan

memberikan simptom neurotoksin yang menyebabkan kematian kepada tikus tersebut. TLC

analisis juga memberikan hasil positif untuk kehadiran tetrodotoksin di dalam sampel analisis

dengan menghasilkan bintik kuning dengan nilai Rfmasing-masing. Manakala untuk ekstrak

tisu daripada Lagocephalus spadiceus, Lagocephalus sceleratus dan Arothron stellatus tidak

menghasilkan bintik kuning dalam analisis TLC. Pengesahan profil toksin di dalam ekstrak

ikan buntal telah dyalankan lebih lanjut dengan menggunakan HPLC, GC-MS dan LC-MS.

Hasil kuantitatif daripada tiga analisis ini menunjukkan bahawa ekstrak daripada

Lagoc_phalus lunaris. Xenopterus naritus. Taki u oblong-, us dan Tetraodon nigroviridis

yang telah ditangkap di perairan pantai Sabah dan Sarawak mengandungi TTX. Keputusan

analisis ini telah disahkan dengan membandingkan Rt sampel yang diuji dengan R, TTX tulen.

Sementara itu, tiada pengesanan kehadiran TTX di dalam ekstrak tisu Lagocephalus

spadiceus, Lagocephalus sceleratus dan Arothron stellatus. Hasil daripada analisis ini

mengukuhkan lagi hasil kajian daripada analisis bioesei tikus dan TLC. Kajian ini

menunjukan bahawa ikan buntal yang ditangkap daripada perairan Sabah dan Sarawak

mengandungi TTX sebagai sifat toksin dan profil toksin dapat dilakarkan untuk tujuan

menyediakan data sebagai garis panduan bagi mengelakkan keracunan ikan buntal di

kalangan orang tempatan.

Kata kunci: Ikan bunial, Morfologi, TTX, HPLC, LC-MS, GC-MS

V1

Pusat Khldmat Maklumat Akademik UNIVERSrfI MALAYSIA SARAWAK

TABLE OF CONTENT

DECLARATION

ACKNOWLEDGEMENT

ABSTRACT

ABSTRAK

TABLE OF CONTENT

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATIONS

PAGE

1

ll

111

V

VII

x

xi

xiv

1.0 INTRODUCTION

1.1 General Introduction

1.2 Objectives and Scope

1

4

2.0 LITERATURE REVIEW

2.1 Puffers 5

2.2 Feeding Habit 7

2.3 Puffer Poisoning Cases Happened in Malaysia and South-east Asia 8

2.4 Puffer Toxin 10

2.5 Tetrodotoxin (TTX) Distribution in Puffer Fish Bodies 13

2.6 Puffer Toxicity (Mouse Bioassay) 14

3.0 MORPHOLOGY IDENTIFICATION OF POTENTIAL TETRODOTOXIN BEARER IN TETRAODONTIDAE SPECIES

3.1 INTRODUCTION

3.2 MATERIALS AND METHODS

3.2.1 Field Work

15

17

17

V11

3.2.2 Laboratory Work

3.3 RESULTS

3.3.1 Morphology Description

3.3.1.1 Lagocephalus genus

3.3.1.2 Tetraodon nigroviridis

3.3.1.3 Xenopterus naritus

3.3.1.4 Arothron stellatus

3.3.1.5 Takifugu oblongus

17

21

22

22

24

28

29

30

3.4 DISCUSSION 31

3.5 CONCLUSION 34

4.0 BIOLOGICAL TOXICITY ASSESSMENT OF TETRAODONTIDAE SPECIES EXTRACTED FROM SABAH AND SARAWAK USING MOUSE BIOASSAY

4.1 INTRODUCTION 35

4.2 MATERIALS AND METHOD 37

4.2.1 Preparation of Crude Extracts and Mouse Bioassay

(Kawabata, 1978) 37

4.3 RESULTS 38

4.4 DISCUSSION 44

4.5 CONCLUSION 49

5.0 TOXIN ANALYSIS IN EXTRACTED TETRAODONTIDAE SPECIES FROM SABAH AND SARAWAK COASTAL WATERS USING THIN LAYER CHROMATOGRAPHY (TLC) AND HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

5.1 INTRODUCTION

5.2 MATERIALS AND METHODS

5.2.1 Preparation of Partially Purified Toxin

5.2.2 Thin Layer Chromatography (TLC)

51

53

53

53

viii

5.2.3 High Performance Liquid Chromatography (HPLC) 54

5.2.4 Data Analysis 54

5.3 RESULTS 55

5.3.1 Thin Layer Chromatography (TLC) 55

5.3.2 High Performance Liquid Chromatography (HPLC) 57

5.4 DISCUSSION 78

5.5 CONCLUSION 85

6.0 TOXIN VERIFICATION IN EXTRACTED TETRAODONTIDAE SPECIES COLLECTED FROM SABAH AND SARAWAK COASTAL WATERS USING GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC-MS) AND LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY (LC-MS)

6.1 INTRODUCTION 87

6.2 MATERIALS AND METHODS 90

6.2.1 Preparation of Filtered Toxin 90

6.2.2 Chemicals 90

6.2.3 Gas Chromatography-Mass Spectrometry (GC-MS) 90

6.2.2 Liquid Chromatography- Mass Spectrometry (LC-MS) 92

6.3 RESULTS 94

6.3.1 Gas Chromatography-Mass Spectrometry (GC-MS) 94

6.3.2 Liquid Chromatography- Mass Spectrometry (LC-MS) 100

6.4 DISCUSSION 102

6.5 CONCLUSION 104

7.0 CONCLUSION AND RECOMMENDATION 105

REFERENCES 108

APPENDIX 125

ix

LIST OF TABLE

Table No. Title Page

Table 3.1 List of Tetraodontidae found in Sabah and Sarawak coastal waters 21

Table 4.1 Anatomic distribution of toxicity in six puffer species collected from Sabah 41 and Sarawak coastal waters.

Table 4.2 Anatomic distribution of toxicity in Lagocephalus genus puffer collected 42 from Sabah coastal waters.

Table 4.3 Anatomic distribution of toxicity in the major puffer species collected from 43 Sarawak coastal waters.

Table 5.1 Rf value of extracted crude toxin from Sabah and Sarawak coastal puffers 56 with respect to Rf value authentic TTX (0.78 and 0.22) with pyridine: ethyl acetate: acetic acid: water and 1-butanol: acetic acid: water respectively.

Table 5.2 Concentration of TTX in extracted Lagocephalus lunaris collected from 60 coastal Bako, Kudat and Tawau waters.

Table 5.3 Concentration of TTX in extracted Tetraodon nigroviridis collected from 61 coastal Bako, Sampadi and Sematan waters.

Table 5.4 Concentration of TTX in extracted Xenapterus naritus collected from 62 coastal Batang Sadong and Sematan waters.

Table 5.5 Concentration of TTX in extracted Takifugu oblongus collected from Bako 63 coastal waters.

Table 6.1 Concentration of TTX in three species of puffer fish analyzes using GC- 96 MS.

X

LIST OF FIGURE

Figure No.

Figure 2.1

Figure 3.1 (a)

Title

Chemical structure of tetrodotoxin.

Page

Figure 3.1 (b)

Figure 3.1 (c)

Figure 3.2

Figure 3.3

Figure 3.4

Figure 3.5

Figure 3.6

Figure 3.7

Figure 3.8

Figure 5.1

Figure 5.2

Figure 5.3

12

Selected sampling site for puffer fish sampling in Sabah and 18 Sarawak waters.

Location of sampling site in Kuching, Sarawak 19

Location of Sampling site at Sabah which consists of Kudat and 20 Tawau divisions

Comparison of Lagocephalus genus collected from the coastal 23 waters of Sabah and Sarawak.

Lateral view and body colour division of T. nigroviridis with the 25 larger of shape of nasal organ (a) and beak like teeth structure (b).

Body colour comparison between adult (a) and juvenile of T. 26 nigroviridis (b).

Dorsal view of two dominant spot types patterns in T. nigroviridis. 27

Yellow puffer fish, Xenopterus naritus with golden colouration 28 torpedo shape fish (a) and morphological illustration of Xenopterus naritus (b).

Arothron stellatus or known as `buntal pasir bintang' locally in 29 Sarawak (a) and morphological illustrations of A. stellatus (b).

Morphological of T. oblongus with brownish green bands on the 30 dorsal side reaching to the lateral side (a) and in puffed-up condition with small pickles at the belly are clear shown (b).

Illustration of retention factors (Rf) value calculation. 53

HPLC of authentic TTX (a) with Rt 13.71 and toxin profile of 66 toxic tissues extracted from muscle (b), skin (c), liver (d), gonad (e) and egg (f) of L. lunaris collected from Bako coastal water.

HPLC of authentic TTX (a) with Rt 13.40 and toxin profile of 67 toxic tissues extracted from muscle (b), skin (c) and liver (d) of L. lunaris collected from Kudat coastal water.

xi

Figure 5.4 HPLC of authentic TTX (a) with Rt 13.40 and toxin profile of 68 toxic tissues extracted from muscle (b), skin (c) and liver (d) of L. lunaris collected from Tawau coastal water.

Figure 5.5 HPLC of authentic TTX (a) with R, 13.7 and toxin profile of toxic 69 tissues extracted from muscle (b), skin (c), liver (d), gonad (e) and egg (f) of T. nigroviridis collected from Sampadi coastal water.

Figure 5.6 HPLC of authentic TTX (a) with Rt 13.7 and toxin profile of toxic 70 tissues extracted from muscle (b), skin (c), liver (d), gonad (e) and egg (f) of T. nigroviridis collected from Sematan coastal water.

Figure 5.7 HPLC of authentic TTX (a) with Rt 13.4 and toxin profile of toxic 71 tissues extracted from muscle (b) and liver (c) of T. nigroviridis collected from Bako coastal water.

Figure 5.8

Figure 5.9

Figure 5.10

Figure 5.11

Figure54.12

Figure 5.13

Figure 6.1

HPLC of authentic TTX (d) with Rt 14.7 and toxin profile of toxic 72 tissues extracted from skin (e), gonad (f) and egg (g) of T. nigroviridis collected from Bako coastal water.

HPLC of authentic TTX (a) with & 13.7 and toxin profile of toxic 73 tissues extracted from gonad (b) and egg (c) of X. naritus collected from Batang Sadong coastal water.

HPLC of authentic TTX (a) with Rt 13.5 and toxin profile of toxic 74 tissues extracted from gonad (b) and egg (c) of X. naritus collected from Sematan coastal water.

HPLC of authentic TTX (a) with Rt 13.7 and toxin profile of toxic 75 tissues extracted from skin (b) and liver (c) of T. oblongus collected from Bako coastal water.

HPLC of authentic TTX (d) with Rt 12.1 and toxin profile of toxic 76 tissues extracted from muscle (e), gonad (f) and egg (g) of T. oblongus collected from Bako coastal water.

HPLC of authentic TTX (a) with Rt 13.7 and no detection of toxin 77 profile (b) tissues extracted from L. spadiceus, L. sceleratus and A. stellatus captured from east coast Malaysian water.

Calibration curve of tetrodotoxin. 91

Figure 6.2 The GC-MS chromatograms (upper) and mass spectrum (lower) of 97 the C9-base-(TMS)3 derivatives from extracts of L. lunaris; (a) muscle, (b) skin and (c) liver displayed a parent peak at m/z 407, a base peak at m/z 392, and a fragment peak at m/z 376.

xii

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Figure 6.3 The GC-MS chromatograms (upper) and mass spectrum (lower) of 98 the C9-base-(TMS)3 derivatives from extracts of T. nigroviridis; (a) muscle, (b) skin and (c) liver displayed a parent peak at m/z 407, a base peak at m/z 392, and a fragment peak at m/z 376.

Figure 6.4 The GC-MS chromatograms (upper) and mass spectrum (lower) of 99 the C9-base-(TMS)3 derivatives from extracts ofX. naritus; (a) skin and (b) liver displayed a parent peak at m/z 407, a base peak at m/z 392, and a fragment peak at m/z 376.

Figure 6.5 LC-MS analysis for toxin profile of TTX standard (a). The LC-MS 101 chromatogram of extracted muscle T. oblongus (b) and reference selected ion m/z 320 mass for both standard TTX and extracted of muscle T. oblongus collected from Bako coastal waters (c).

Xlll

LIST OF ABBREVIATION

FRI Fisheries Research Institute

GC Gas Chromatography

GC-MS Gas Chromatography Mass Spectrometry

HPLC High Performance Liquid Chromatography

LC Liquid Chromatography

LC-MS Liquid Chromatography Mass Spectrometry

MLD Minimum Lethal Dose

MS Mass Spectrometry

MU Mouse Unit

Rf Retention factor

Rt Retention time

SPE Solid-Phase Extraction

TLC Thin Layer Chromatography

TMS Trimethylsilyl

TTX Tetrodotoxin

ZIC-HILIC Zwitterionic- Hydrophilic Interaction Chromatography

xiv

CHAPTER I

INTRODUCTION

1.1 General Introduction

The family name, Tetraodontidae, refers to the teeth, fused into two upper and two lower

plates, which are used for crushing the shells of crustaceans and molluscs (King, 2001; Torda

et al., 1973). This fish are variously called "puffer fish" or "blowfish" due to ability inflate

themselves with water or air when threatened, making it difficult for a predator to swallow

them (Froese & Pauly, 2008). Puffer fish has a rounded body and a broad blunt snout (Mat

Piah, 2011). According to Beumer (1978), although this form allows a certain measure of

maneuverability, puffer fish do not appear to have the capacity for sustained rapid

acceleration. The broad snout and short, powerful jaws provide strong biting and crushing

power but do not provide rapid closing speed (Torda et al., 1973; Beumer, 1978; King, 2001;

Krumme et al., 2007).

Puffer fish poisoning is probably the most common fish poisoning along the coasts of Asia

(Chew et al., 1983). Most of the poisoning cases have been caused by marine species and

presences of tetrodotoxin (TTX) as the principal toxin (Laobhripatr et al., 1990). TTX is a

lethal marine toxin with no known antidote (Hwang & Noguchi, 2007). In addition to puffer

fish, TTX is also contained in other aquatic animals, such as octopus, gastropod, xanthid crab,

and horseshoe crab, which are thought to bioaccumulate TTX through the food chain, starting

from bacteria that naturally produce TTX in marine environments (Noguchi et al., 2006).

I

However, the present of paralytic shellfish toxins (PSTs) in puffer fishes also been reported in

numerous of studies (Kungsuwan et al., 1997; Sato et al., 1997; Ngy et al., 2008). TTX and

PSTs are potent neurotoxins of low molecular weight, which inhibit nerve and muscle

conduction by selectively blocking sodium channels (Narahashi, 2001).

Meanwhile, in Asian-pacific countries, small sized puffer fish from brackish water or

freshwater also possess paralytic toxins and occasionally cause food poisoning and fatalities in

human, such as in Thailand and Bangladesh (Laobhripatr et al., 1990; Mahmud et al., 2003a).

Several finding found that the toxic principles of puffer fish are different depending on the

species and their habitat (Arakawa et al., 2010). For example, Tetraodon nigroviridis, T.

steindachneri, and T. ocellatus collected from Thailand and Taiwan possess TTX (Mahmud et

al., 1999a; Lin et al., 2002), whereas the main toxins of T. leiurus, T. survatii from Thailand,

T. cutcutia, Chelonodon patoca from Bangladesh, and Colomesus asellus from Brazil are

PSTs (Oliveira et al., 2006). In fact, the presence of both TTX and PSTs within the same

species is also documented for Thailand freshwater puffer fish T. fangi (Sato et al., 1997).

Similarly, some marine puffer fishes possess PSTs as their main toxin (Landsberg et al.,

2006).

In Malaysia, at least 16 species of puffer fishes have been recorded and several studies related

of puffer fish on toxicology data were carried out in the country (Scott, 1959). In early 1970s,

three species of puffer fishes, Lagocephalus lunaris lunaris, L. lunaris spadiceus and Arothron

stellatus collected from west Malaysia waters were found to be toxic and caused mouse

lethality (Berry & Hassan, 1973). However, details toxin properties verification was not

2

carried out during these studies. The first documented poisoning case in Malaysia was

reported occurred in Sabah, where four intoxications happened in 1985 (Lyn, 1985) and two

years later, nine fatal cases were reported (Kan et al., 1987) as both cases are resulting from

consumption of puffer fish. However, until now the identities of the causative organism

remain unknown, and no details study was carried out in Sabah waters.

In contrast, for Xenopterus naritus or yellow puffer particularly abundant in Batang Saribas,

Seri Aman, Sarawak are considerably safe for human consuming with toxicity level below 10

mouse unit (MU) and it been marketable in local fish market (Bojo et al., 2006). However, the

details study on toxin properties was not clearly documented. The local people believed that

the fish is safe to eat if properly prepared, nobody knows exactly about the toxin properties

and the people are not advised about the risk of its consumption. It also has been reported that

X. naritus vigorously marketed in all Division of Sarawak and several cases of mild poisoning

was reported among local fisherman (pers. comm. ). Recently, three people from Kuching were

poisoned by puffer fish after eating the fish bought from fish monger in Kuching area (pers.

comm. ) and the species identified as X. naritus and T. nigroviridis (Mohamad, unpublished).

Previous studies showed that no conclusive evidence or complete scientific records that

available on toxicological information of puffer fish species that been widely consumed in

Sabah and Sarawak. Therefore, this study emphasis on identifying the causative species that

involved by assessing their toxicity level and toxin properties or principal toxin in order to

clarify the puffer fish poisoning problems. In order to achieve the whole objectives, this study

was proposed to examine the toxicity level of puffer fishes in different tissues collected from

3

selected area of Sabah and Sarawak waters. The samples were asses for tetrodotoxin using

biological mouse bioassay and thin layer chromatography (TLC) and the crude toxins was

quantified using high performance liquid chromatography (HPLC), liquid chromatography

mass spectrometry (LC-MS) and gas chromatography mass spectrometry (GC-MS). Both

toxicity level and composition data will be useful to be guideline for puffer fish consumer in

this area. Perhaps, the data obtained from this study will be used as an informative data by the

authority for improving the methodology of consumer protection.

1.2 Objectives and Scope

There is a need to study the toxicity and toxin properties of puffer fish collected from Sabah

and Sarawak waters since the fish species are known varied and it also depends on the

locations. The toxicities level of different tissues and principle toxin of identified species will

be investigated from both states.

Hence the primary objectives of the research were to:

1. To examine the morphology characteristics of common edible puffer species collected from

Sabah and Sarawak waters.

2. To assess the toxicity level and toxin components of study puffer fish using mouse bioassay

analysis, Thin Layer Chromatography (TLC), High Performance Liquid Chromatography

(HPLC), Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography-

Mass Spectrometry (GC-MS) analysis.

4

Pusat Khidmat MaktumatAkademik UNIVERSITI MALAYSIA SARAWAK

CHAPTER II

LITERATURE REVIEW

2.1 Puffers

Puffer fish sometimes possess a strong toxin which responsible for puffer poisoning and most

of the cases poisoning has been reported to be caused by marine species of the family

Tetraodontidae (Noguchi & Ebesu, 2001). This family is belong to Tetraodontiformers order

which is comes from the Greek word `tetra' meaning four and `odontos' meaning teeth (Atack,

2006).

Therefore, members fall under this order are all having two fused teeth in each jaw which

made up a structure like `beak' (Mohsin & Ambak, 1983). Their beak can function to break

the hard shells of their prey such as gastropods and crabs. This special feature can be use to

differentiate between Diodontidae and Tetraodontidae family. Porcupine fishes (Diodontidae)

is similar to the puffer fishes based on their physical morphology but Diodontidae can be

distinguished by having a single (unsutured) tooth in each jaw and very large spine covered

their body (Shipp, 1988). Meanwhile, puffer fishes have scale less naked body as well and

most of the species are partially covered by the small spines in their dorsal body such as

Lagocephalus lunaris and many other species have small fleshy fin on the dorsal surfaces

(Shipp, 1988).

Generally, puffers have large heads with large eye on the both sides. Pointed nose is also one

of the features of puffers where they have two nostrils on each side (Ahmad & Kibria, 1998).

The puffers fish are easily recognized by the absent of pelvic fins (Shipp, 1988). Their body

5

shape is short with sub cylindrical form and due to this shape, puffers are not fast swimmer.

According to Berra (2001), puffer fishes propel themselves by fluttering the dorsal and anal

fins which originate far posterior. Atack (2006) stated that, puffer fish have ability to puff up

themselves twice their original size, a defense mechanism designed to scare away their enemy.

This mechanism increased their diameter especially in the species that possess spines or

prickles on their side (Moyle & Cech, 2000).

There are 185 species and 28 genera of puffer fish in the family Tetraodontidae (Oliveira et

al., 2006). According to Sabrah et al. (2006) and Froese and Pauly (2007), these fishes

typically occur in tropical seas and estuarine environments. Thresher (1984) also mentioned

that puffers are found in both temperate and inshore tropical seas especially grass flats and

mangrove swamps area. Common three species of Tetraodontidae have been found at

freshwater western Borneo included Tetraodon nigroviridis, Tetraodon leiurus and Tetraodon

palembangensis (Robert, 1989).

Puffer fish are very common and often caught in large numbers by trawlers or line fishing in

east Malaysia waters. They are known as `Ikan Buntal' and certain species such as

Lagocephalus sceleratus, Lagocephalus lunaris, Xenopterus narilus and Tetraodon

nigroviridis are consumed in some part of Sabah and Sarawak (Atack, 2006). Lagocephalus

wheeleri is consumed in some parts of the country and puffer fish poisoning is quite frequent

including a few fatalities (Kan et al., 1987; Kanchanapongkul, 2001; Zaki et al., 2005).

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2.2 Feeding Habit

Puffer fish usually feed on invertebrates, plants, prawns, gastropods, and bivalves (Dawes,

2005). Puffer uses their powerful jaws and teeth to graze their food (Marshall, 2000). Based

on the puffer diet, the poisoning process was believed to begin at the bottom of the food chain.

According to Noguchi and Arakawa (2008), TTX is produced primarily by marine bacteria,

and puffer fish accumulate TTX via the food chain that begins with these bacteria. This

process was followed by small omnivores, carnivores or scavengers and by organisms higher

up the food chain that would cause in the accumulation of higher concentration of TTX. It is

hypothesized that non-toxic puffer fish can be produced if they are cultured with TTX-free

diets in net cages at sea or aquaria (Noguchi et al., 2006).

Noguchi (2006) found that stomach contents such as starfish, small gastropod and skeleton

shrimp known to possess TTX had been detected. These puffer fish seem to be attracted by

TTX, and select TTX-bearing organisms as food, resulting in accumulating TTX as a possible

defense mechanism. Noguchi and Arakawa (2008) suggested, the amount of TTX produced by

TTX-producer in puffer fish appear to be too little to account for the TTX accumulation in

puffer fish, therefore bioconcentration likely has a large role in the accumulation of TTX in

puffer fish.

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2.3 Puffer Poisoning Cases Happened in Malaysia and South-east Asia

Bernama News (2008) reported there were two cases of food poisoning due to consumption of

puffer fish happened in Kulai and Batu Pahat, Johor. The incident from the consumption of

puffer fish caused death of two out of 36 victims in separate incidents in Johor. Due to those

cases, the Johor Health Department had issued a ban on the sale of puffer fish. The Health

Ministry advised the public not to buy or eat the puffer fish or its eggs if they are not properly

handled or cleaned during the cutting process (Bernama News, 2008). Bernama News (2008)

also mentioned the victim was experienced the symptoms of puffer poisoning such as

numbness in mouth, tongue, hands, legs, face, vomiting, diarrhea, dizziness, abdominal pain,

difficulty in speaking.

The China Post (2009) reported Malaysian fisherman died after eating puffer fish on 17cn

February. This incident happened at Dungun, Kuala Terengganu. Five fishermen cook puffer

fish when they had ran out of food supply after being out to the sea for several days. One

fisherman aged 43 years old died several hours later after consuming puffer fish eggs. The

other four fishermen were hospitalized for further treatment. All fishermen reported feel dizzy,

head spun, drifted off after one hour after their meal and become worse. This incident

happened because they do not know the fish contained TTX. Health Officer from Dungun,

Terengganu said that in year 2008, there were hundreds of people reported suffered from food

poisoning after consuming puffer fish.

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