STUDY OF MORPHOLOGY, STOMACH CONTENT

AND TOXIN PROPERTIES OF PUFFER FISH IN

LUNDU, SARAWAK.

Nurfakhriah Binti Alias

Bachelor of Science with Honors

(Aquatic Resource Science and Management)

2013

Faculty of Resource Science and Technology

i

Acknowledgement

First of all, I would like to express gratitude to my supervisor Dr. Samsur Mohamad for the

useful comments and knowledge given throughout the learning process for this Final Year

Project. Besides, I would like to thank laboratory assistants Encik Nazri, Encik Zaidi and

Encik Zulkifli for the helps given during my laboratory session and also during the

sampling. Also, I want to thank my course mates, Nur Jamiatul Shaharum and Sabariah

Zulkfli for the helps during the sampling and also other course mates for the

encouragements. I also would like to thank my parents, who have supported me a lot

throughout this period.

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Table of Contents

Acknowledgement............................................................................................... I

Table of Contents ................................................................................................ II

List of Abbreviations .......................................................................................... III

List of Tables ….................................................................................................. IV

List of Figures …………………………………………………………………. V

Abstract …………………………………..……………………………………. 1

1.0 Introduction …….......................................................................................... 2

2.0 Literature Review .......................................................................................

2.1 Puffer Fish .............................................................................................

2.2 Distribution of Toxic Puffer Fish ...........................................................

2.3 Feeding Habit of Puffer Fish ..................................................................

2.4 Accumulation of Tetrodotoxin (TTX) ...................................................

2.5 Puffer Fish Poisoning .............................................................................

2.6 Methods Used in Detection of Tetrodotoxin (TTX)…………………...

2.6.1 High Performance Liquid Chromatography (HPLC)……………

2.6.2 Liquid Chromatography-Mass Spectrometry (LC-MS)………….

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7

8

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10

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3.0 Materials and Methods ................................................................................

3.1 Study Area ..............................................................................................

3.2 Sampling ................................................................................................

3.3 Species Identification .............................................................................

3.4 Physical Measurement ...........................................................................

3.5 Stomach Content Analysis ....................................................................

3.6 Toxin Extraction ....................................................................................

3.7 High Performance Liquid Chromatography ..........................................

3.8.1 Phosphate Buffer ..........................................................................

3.8.2 Sodium Buffer ...............................................................................

3.8.3 Oxidizing Reagent ........................................................................

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4 Results and discussion...................................................................................

4.1 Species Identification of Puffer Fish Samples........................................

4.2 Morphological Assessment......................................................................

4.3 Physical Measurement.............................................................................

4.4 Meristic Count........................................................................................

4.5 Stomach Content Analysis ......................................................................

4.6 Toxicity Analysis ....................................................................................

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30

5 Conclusion .................................................................................................... 32

6 References .....................................................................................................

7 Appendices ………………………………………………………………...

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37

iii

List of Abbreviations

AcOH Acetic acid

AR Anal ray

BW Body weight

DR Dorsal ray

FR Fin ray

GPS Global positioning system

HL Head length

HPLC High Performance Liquid Chromatography

MU Mouse unit

NaOH Sodium hydroxide

PR Pectoral ray

SL Standard length

STX Saxitoxin

TL Total length

TTX Tetrodotoxin

iv

List of Tables

Tables

Description

Page

1 Taxonomic groups of spotted green and yellow puffer fish 17

2 The average of BW, TL, SL and HL of X. naritus 19

3 The average of BW, TL, SL and HL of T. nigroviridis 20

4 The range of AR, FR, DR and PR of X. naritus and T. nigroviridis 20

5 Prey items found in stomachs of T. nigroviridis 27

6 Prey items found in stomachs of X. naritus 28

7 Toxicity level of T. nigroviridis and X. naritus collected from Lundu 30

v

List of Figures

Figure

Description Page

1

Location of sampling area, Rambungan River and Sampadi Island

13

2

Leaves fragments 22

3 Prawn 22

4 Bivalves

23

5

Gastropod Species 1 23

6 Shell fragments of gastropod species 2

24

7 Oligochaete 24

8 Crab’s leg 25

9 Exoskeleton of prawn 25

10 Fish scales 26

11 Fish bones 26

1

Study of Morphology, Stomach Content and Toxin Properties of Puffer Fish in

Lundu, Sarawak

Nurfakhriah Alias

Aquatic Resource Science and Management Programme

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

Abstract

Morphological characteristics, stomach content and toxicity properties of puffer fish were studied. A sample

of 28 individuals of Tetraodon nigroviridis and 21 individuals of Xenopterus naritus were collected from

three sampling sites in Sarawak waters using three layered net. These two puffer fish species showed the

difference in their morphological characteristic (e.g., skin colour). Analysis showed that morphometric

measurement of T. nigroviridis collected from Rambungan River and Sampadi Island (BW: 47.04±11.60 g;

TL: 11.1±1.2 cm; SL: 8.0±0.7 cm; HL: 3.4± 1.3 cm) and (BW: 93.32±35.14 g; TL: 13.7±1.6 cm; SL:

10.5±1.2 cm; HL: 4.0±0.4 cm) respectively were different in their range of size. Analysis also showed the

difference in the range of size of X. naritus (BW: 335.00 ±35.36 g; TL: 23.3 ±2.8 cm; SL: 18.7 ±1.8 cm; HL:

4.8 ± 0.4 cm), (BW: 317.78±102.82 g: TL: 18.3 ±2.1cm; SL: 18.3 ±1.8 cm; HL: 5.5 ±0.7 cm) and

(BW:278.00 g; TL: 20.1 cm; SL: 16.3 cm; HL: 4.1 cm) collected from Rambungan River, Sampadi Island

and coastal area of Lundu respectively. Meristic count analysis of T. nigroviridis and X. naritus (AR: 11-14;

FR: 8-1I; DR: 13-15; PR: 20-23) and (AR: 24-28; FR: 10-12; DR: 30-35; PR: 16-18) respectively showed the

difference in the range of the rays counted. Stomach content analysis showed that T. nigroviridis was

carnivorous fish and X. naritus was omnivorous fish. The concentration of TTX detected in liver of X.

naritus was 39.39µg/g by using HPLC.

Key words: Tetraodon nigroviridis, Xenopterus naritus, morphological characteristics, morphometric

measurement, stomach content analysis, HPLC

Abstrak

Ciri-ciri morfologi, kandungan perut dan toksin bagi ikan buntal telah dikaji. Sebanyak 28 ekor Tetraodon

nigroviridis dan 21 ekor Xenopterus naritus telah ditangkap di tiga tempat persampelan di Sarawak

menggunakan pukat tiga lapis. Dua spesis buntal ini menunjukkan perbezaan dari segi ciri-ciri morfologi

(cth: warna kulit). Analisa menunjukkan perbezaan ukuran morfometrik T. nigroviridis antara Sungai

Rambungan dan Pulau Sampadi (BW: 47.04±11.60 g; TL: 11.1±1.2 cm; SL: 8.0±0.7 cm; HL: 3.4± 1.3 cm)

and (BW: 93.32±35.14 g; TL: 13.7±1.6 cm; SL: 10.5±1.2 cm; HL: 4.0±0.4 cm). Analisa juga menunjukkan

linkungan saiz X. naritus (BW: 335.00 ±35.36 g; TL: 23.3 ±2.8 cm; SL: 18.7 ±1.8 cm; HL: 4.8 ± 0.4 cm),

(BW: 317.78±102.82 g: TL: 18.3 ±2.1cm; SL: 18.3 ±1.8 cm; HL: 5.5 ±0.7 cm) and (BW:278.00 g; TL: 20.1

cm; SL: 16.3 cm; HL: 4.1 cm) yang ditangkap daripada Sungai Rambungan, Pulau Sampadi dan kawasan

pantai Lundu masing-masing menunjukan perbezaan. Analisa kiraan meristik T. nigrovirids dan X. naritus

AR: 11-14; FR: 8-1I; DR: 13-15; PR: 20-23) and (AR: 24-28; FR: 10-12; DR: 30-35; PR: 16-18) masing-

masing menunjukkan perbezaan dari segi lingkungan jari-jari sirip. Analisa kandungan perut menunjukkan

T. nigroviridis adalah ikan karnivor manakala X. naritus adalah ikan omnivor. Kepekatan TTX yang telah

dikenal pasti dalam hati X. naritus ialah 39.39µg/ menggunakan HPLC.

Kata kunci: Tetraodon nigroviridis, Xenopterus naritus, ciri-ciri morfometrik, ukuran morfometrik, analisa

kandungan perut, HPLC

2

Introduction

This study was conducted in Rambungan River and coastal area of Lundu including

coastal area of Sampadi Island, Sarawak. In previous year, Sarawak Forestry Corporation

(SFC) has issued a statement that Sampadi Island would be gazetted as marine protected

area (MPA) (The Star, 2012). According to Executive Order 13158, MPA is any means

area of the marine environment that has been reserved by Federal, State, territorial, tribal,

or local laws or regulations to provide lasting protection for part or all of the natural and

cultural resources therein. Sampadi Island is being considered to be included as one of

MPAs in Malaysia regarding to the high marine conservation value possessed by the

surrounding water bodies of the island.

Fishing is one of the main activities carried out by native communities living in

coastal area of Lundu. Puffer fish is one of target species by fishermen. The fish are caught

using traditional tools such as scoop net and modern equipments like trammel net and gill

net which are more efficient and commercially used by fishermen now days (Chuan &

Lim, 2004). The puffer fish caught will be sold in market and later are consumed by local

communities as one of their delicacy. A few poisoning cases had been reported in Sarawak

after consuming puffer fish. The poisoning is caused by tetrodotoxin (TTX) contained in

body tissues of the puffer fish. Despite this, little information is available about the TTX in

puffer fish species present in Sarawak waters.

Puffer fish is also recognized as blowfish and globefish (Sabrah et al., 2006). Puffer

fish is named so based on its ability to inflate its body where the inflation causes its

stomach to expand into peritoneal cavity surrounding the axial musculature. For example, a

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marine puffer fish known as Diodon holocanthus will looks extremely spherical when

inflating its stomach.

A number of puffer fish species contain complex neurotoxin known as tetrodotoxin

(TTX). Improperly prepared puffer fish based meal has caused a series of food poisoning

related death cases in Southeast Asia (Chen et al., 2011). While in Malaysia, food

poisoning cases caused by ingestion of puffer fish are selectively reported. The puffer fish

poisoning are believed to be caused by TTX contained in the fish. TTX is highly potential

to disturb nervous system when entering human body system even at low concentration

(Arakawa, 2010). The puffer fish liver which is believed as the most toxic tissue in puffer

fish possesses a specific TTX uptake mechanism (Arakawa, 2010; Ikeda et al., 2010;

Matsumoto et al., 2007).

Local people of Sarawak including the native communities of Lundu area consume

puffer fish as one of their delicacies. Therefore, this study is important to assess the

biological data and to determine the toxin properties of puffer fish in the area in order to

provide public awareness about the neurotoxin contained in the puffer fish. In recent years,

only little data of biological and toxin properties have been documented for puffer fish in

Lundu coastal area. In previous study carried out by Muthukrishnan (2010), toxicity

analysis of puffer fish samples collected from Sampadi River was carried out using TLC.

The accuracy of toxin properties data of puffer fish in the river will be improved as during

this study, HPLC will be used for toxicity analysis instead of TLC. This is because HPLC

is able to provide more accurate data on toxin analysis compared to TLC. Other than type

of species, the location of where the samples are collected is another factor that influences

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the ratio of toxicity level among the puffer fish samples (Ngy at al., 2008; Rodriguez et al.,

2012). Therefore, sampling will be conducted in two different locations; Sampadi Island

and Sampadi River to determine the difference of toxin properties of puffer fish between

these two locations.

The objectives of this study are:

1. To record data of puffer fish species present in Lundu District.

2. To analyze stomach content in order to determine the diet composition of puffer

fish population.

3. To determine the toxin properties of puffer fish in Lundu District using High

Performance Liquid Chromatography (HPLC).

5

2.0 Literature Review

2.1 Puffer Fish

Puffer fish belong to order Tetraodontiformers. ‘Tetra’ means four, ‘odontos’

means teeth and ‘formes’ means shape. Therefore, puffer fish species belong to this order

possess four teeth. Tetraodontiformers are divided into two families; family Diodontidae

and family Tetraodontidae. A total of 189 species and 28 genera of puffer fish belong to

family Tetraodontidae (Veeruraj, 2011). Puffer fish from family Diodontidae can be

identified due to spines on its body while the one from family Tetraodontidae possesses

smooth and sharpnose. Diodontidae can be distinguished from Tetraodontidae by the

presence of single tooth in each jaw of Diodontidae members.

Puffer fish inhabits marine water including tropical area and subtropical area of

Atlantic, Indian and Pacific Ocean, brackish water and freshwater area (Che Awin, 2008).

Most of puffer fish species live in tropical seas and they are normally found in shallow area

near shore waters (Mehmet, 2011) such as coral reef area. Puffer known as Lagocephalus

lunaris is a marine puffer fish species while puffer Tetraodon nigroviridis is a marine-

brackish species where this species can be found in river mouth area. For freshwater

environment, puffer species that can be found is T. leiurus.

According to Monaliza and Mohamad (2011), there are 12 species of marine-

brackish puffers have been identified in Sabah and Sarawak. Certain of the puffer fish

species do migration where they migrate into more brackish or freshwater habitats.

Common puffer fish species that can be found in Malaysian water are Lagocephalus

wheeleri, L. sceleratus and L. lunaris lunaris. While L. wheeleri, L. sceleratus and

6

Xenopterus naritus are puffer fish species that are consumed by a part of Malaysian

communities. Puffer fish, Lagocephalus sceleratus inhabits in ocean in the depths ranging

from 18 to 100 m and coral reef area (Mehmet, 2011).

Xenopterus naritus is widely present in Asian countries like China, Vietnam, Thailand,

Indonesia and Malaysia (Chuan & Lim, 2004). In Malaysian waters, the puffer fish

species is abundantly present in State of Sarawak only. X. naritus could be found living in

coastal water, estuaries and upper river area where the water salinity reaching zero. X.

naritus could be found in coastal water and mangroves area where the fish is abundantly

occur in estuaries of Batang Sadong, Batang Lupar and Batang Saribas in Sarawak.

According to toxicity study carried out by Monaliza and Mohamad (2011), X. naritus

is a non toxic puffer fish. According to Chuan & Lim (2004), the most promising period

for high catch for this species would be in March and October every year especially in the

middle of the months which king tide occurs and at the end of the months which springtide

occurs. The fish is a local delicacy where the flesh is directly cooking, salted or dried and

fermented. Other than flesh, the fish eggs also been dried. Tetraodon nigroviridis is widely

distributed in freshwater and brackishwater in tropical Asia from Sri Lanka to Indonesia,

northward to Vietnam (Matsunuma et al., 2011).

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2.2 Distribution of Toxic Puffer Fish

Marine puffer fish from family Tetraodontidae may contain highly toxic

neurotoxin, tetrodotoxin (TTX) (Ikeda et al., 2010; Islam et al., 2011; Ngy et al., 2008) or

saxitoxin (STX) (Nakashima et al., 2004) in their body tissues. In Tetraodontidae family

members, complex TTX is highly recorded in their livers and ovaries (Arakawa, 2010).

Tetraodontidae family members that are regarded as toxic puffer fish includes

Lagocephalus sceleratus (Rodriguez et al., 2011), Tetraodon nigroviridis, T.

steindachneri, and T. ocellatus (Ngy et al., 2008). L. lunaris, L. spadiceus and Arothron

stellatus found in Malaysian waters are toxic puffer fish that can cause food poisoning after

consumption (Monaliza & Mohamad, 2011). Toxicity analysis conducted on L. lunaris

shows that this species contain toxin in their muscle tissues (Rodriguez et al., 2011).

In recent years, STX has been discovered in several marine and freshwater puffer

fish species along with TTX (Abbott et al., 2009). Thus, certain puffer fish species may

possess TTX as their major toxin while STX as their minor toxin and vice versa.

According to Mehmet (2011), TTX can be found in liver, gonads, intestines and skin

tissues of puffer fish and is highly potential to cause the individuals that consume the

tissues to death.

For Japanese marine puffer fish recognized as Takifugu pardalis, T. poecilonotus

and T. vermicularis, STX is their minor toxin while in puffer known as Arothron

firmamentum, STX is its major toxin where the toxin is mainly accumulated in its ovary,

but its skin only contain TTX (Nakashima et al., 2004). Study carried out by Noordin

(2011) recorded that Carinotetraodon salivator contain low toxin level in its tissues.

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2.3 Feeding Habit of Puffer Fish

Studies conducted by Mohamad and Isa (2012) recorded that diet composition of

puffer fish is mainly composed of crustaceans, therefore puffer fish is regarded as

carnivorous fish. Other than crustaceans, puffer fish communities also consume mollusks

and small bivalves (Abbot et al., 2009). The bivalves are believed to accumulate toxin in

their muscles after been exposed to toxin-containing plankton such as Alexandrium

(Nakashima et al., 2004). According to study carried out by Mehmet in 2011,

Legocephalus sceleratus is a carnivorous puffer fish species where the puffer fish species

fed on shrimps, fishes, crabs, squids and cuttlefish. In other words, L. sceleratus feeds on

cephalopods, crustaceans and fishes.

Ikeda et al. (2010) suggest that toxin accumulation in puffer fish is originated from

their food chains. Matsumoto et al. (2010) also proposed that TTX accumulation in puffer

fish occur via bioaccumulation since cultured marine puffer fish are found not toxic

whereas non-toxic puffer fish turn into toxic after been fed on artificial TTX contained

diets during culture period (Matsumoto et al., 2010). The toxin is possibly absorbed

through the intestine and later distributed into tissues in various part of the puffer fish body

through blood circulation (Matsumoto et al., 2007).

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2.4 Accumulation of Tetrodotoxin (TTX)

TTX is predominantly accumulated in liver and ovary of puffer fish that caused

these organs to possess high toxicity level (Ikeda et al., 2010; Matsumoto et al., 2007;

Nakashima et al., 2004; Ngy et al., 2008), however, the toxin levels are vary among

different puffer fish species (Islam et al., 2011). A patient will suffer the most severe

poisoning when he or she ingests puffer fish liver (Rodriguez et al., 2012). Thus, the liver

can be regarded as the most toxic organ in puffer fish.

During maturation period, the rate of TTX-binding plasma activity increase and

TTX is transported from liver to ovary (Ikeda et al., 2010). As a result, TTX level increase.

TTX is transferred into skin tissues through circulatory system (Arakawa et al., 2010).

Muscle also contain toxin but in much lower level (Ikeda et al., 2010; Rodriguez et al.,

2011). In female puffer fish, toxicity level is high in liver during ordinary period and high

in ovary in maturation period (Ikeda et al., 2010). This demonstrated that TTX originated

from food chains is transported mainly into liver and skin tissues of puffer fish during

ordinary period while the toxin is predominantly accumulated in their ovary during

maturation period.

The toxin contained in puffer fish tissues that is mainly composed of TTX is

believed to be used by the puffer fish communities not only to protect themselves but also

their eggs from predators. This is because puffer fish communities possess the highest TTX

content during spawning season where their gonads size also increase during the spawning

season (Yu & Yu, 2002). Puffer fish called L. sceleratus scores the highest toxicity level

during spawning season. Meanwhile, Yu et al. (2004) suggested the toxin level in puffer

10

fish largely depends on TTX-producing bacteria living symbiotically in the host bodies.

However, there is critism on the ability of the bacteria in producing TTX.

According to Matsumoto et al. (2007) the accumulation of TTX in puffer fish liver

involves a specific mechanism known as carrier-mediated transport system where the

endogenous and exogenous compounds will be accumulated in liver. The accumulation

and excretion of TTX and STX involve binding protein (Nakashima et al, 2004). The

binding protein are known as puffer fish STX and TTX-binding protein (PSTBP) that play

critical role in plasma protein binding of TTX and STX in marine puffer fish from

Tetraodontidae family (Matsumoto et al., 2010).

2.5 Puffer Fish Poisoning

Food poisoning cases due to ingestion of toxic puffer fish has been widely reported

in coastal countries of East and South-East Asia, including in Japan, China, Taiwan (Islam

et al., 2011), Bangladesh (Islam et al., 2011; Rodriguez et al., 2011), Thailand (Rodriguez

et al., 2011) and Cambodia (Ngy et al., 2008) while in Malaysia puffer fish poisoning are

selectively occur. Limb weakness and progressive muscular paralysis are among the

symptoms exhibited by individuals suffering puffer fish poisoning and the poisoning may

lead them to death resulting from respiratory paralysis (Islam et al., 2011).

According to O’Leary et al., (2004), the poisoning resulted from the consumption

of the puffer fish is caused by the tetrodotoxin (TTX) contained in the puffer fish tissue

and TTX poisoning is an important issue in South-eastern Asia. Tetrodotoxin (TTX) is

heat-stable, water-soluble and a non-protein organic compound which is aminoperhy-

11

droquinazoline. The quinazoline derivative is regarded as one of the strongest paralytic

toxins today and TTX is named after the order from which puffer fish normally grouped

into, Tetraodontiformes (Mehmet, 2011).

According to Matsumoto et al. (2007), TTX is able to block voltage-gated sodium

channel after entering organism bodies where the individuals will suffer severe food

poisoning, leading to fatality. The ability of TTX as a blocker is associated with its

characteristic of high affinity to the sodium channels, and the blocking action will inhibits

depolarization and propagation of action potential, paralysing the peripheral nervous

system (Islam et al., 2011). Thus, TTX is a disturbance in sodium conductance and

neuronal transmission in skeletal muscle.

2.6 Methods used in detection of tetrodotoxin (TTX)

Methods that can be used in TTX detection include High Performance Liquid

Chromatography (HPLC) and Liquid chromatography–mass spectrometry (LC-MS).

2.6.1 High Performance Liquid Chromatography (HPLC)

HPLC is a quantitative method to detect tetrodotoxins (TTXs) in samples including

gastropods and puffer fish (Chen & Chou, 1998). According to O’ Leary et al. (2004),

HPLC system comprises of pump with a manual injector, analytical column and

fluorescence detector. The detector is set with an excitation wavelength lex of 380 nm, and

an emission wavelength lem of 505 nm. The fluorescent detection is followed by post-

column degradation. Phosphate buffer which is composed of heptasulfonic acid acts as

12

mobile phase in the system which is pumped through the HPLC column at 0.3 ml/min.

Data is collected using software such as Maxima Data collection software.

Meanwhile, sodium hydroxide (NaOH) solution is pumped at 0.3 ml/min through a

second HPLC pump into a mixing device. Then, the solution will be mixed with the mobile

phase eluant. This mix then passed through Teflon tubing which was immersed in a water

bath maintained at 95 8C. After passing through the Teflon tubing, the mix then entered

the fluorescence detector.

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3.0 Materials and Methods

3.1 Study Area

Figure 1 Location of sampling area, Rambungan River and Sampadi Island

3.2 Sampling

Puffer fish samples were randomly collected from Rambungan River (N01° 39.78’

E110° 07.54’) and coastal area of Sampadi Island (N01° 42.0’ E110° 03.50’) and coastal

water of Lundu, Sarawak using three layered net with mesh size of 4 cm for its inner layer

and 14 cm for its outer layer. The samples collected were kept in cooler box filled with ice

before being dissected. The puffer fish were dissected and transported back to laboratory

for further analysis. In laboratory, the samples were kept in freezer at temperature of -

20°C.

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3.3 Species Identification

The morphological characteristic including colour and shape of their bodies,

numbers of tooth and spines distribution were observed using naked eyes. The distributions

of spots or stripes on their bodies were described. Species of the samples were identified.

3.4 Physical Measurement

Total length (TL), standard length (SL) and head length (HL) for each puffer fish

was measured to nearest 0.1 centimetre using measuring board (Simon & Mazlan, 2008).

TL of the samples was measured from tip of the snout to the tip of caudal fin. SL of the

samples was measured from anterior tip of the snout to the posterior end until the last

fleshy part excluding the caudal peduncle in a straight line. HL was measured from tip of

the snout to the posterior edge of the opercular bone. The puffer fish sampled were sorted

into specific range of size. The body weight (BW) of each puffer fish samples was

recorded using electronic balance to nearest 0.01 gram (Mehmet, 2011). The number of

anal fin, caudal fin, dorsal fin and pectoral fin of each sample were counted. The data were

recorded in a field sample data label sheet prepared earlier.

3.5 Stomach Content Analysis

Stomach content analysis of the puffer fish samples were carried out according to

methods used by Hyslop (1985). The stomachs were removed from the fish bodies and

placed in specimen bottles. The stomachs were preserved using 10% formalin to avoid any

further digestion activity by digestive enzyme contained in the stomachs. The preservation

also can prevent the contents of the stomach from decomposed. Further examination of the

stomach contents were carried out in laboratory. In laboratory, the stomachs were

15

transferred into 0.45 µm sieve and rinsed with running tap water. The items found in

stomach of the samples were observed under stereo microscope. The items examined were

identified.

3.6 Toxin Extraction

The puffer fish samples were dissected into gonad, liver, muscle and skin.

Approximately 5 grams each of gonad, liver and muscle were minced. Each sample was

mixed in same volume of 0.1% acetic acid (AcOH). Then, the mixture was heated in

boiling water bath at 100 ºC for 10 minutes. The slurry was centrifuged at 10 000 g for 15

minutes with 22 ºC using centrifuge KUBOTA 6200. Supernatant was collected from the

sample by filtration through 0.45µm milipore filter paper. Finally, the supernatant was

transfer into vial bottle and kept in freezer at temperature of ­ 20 ºC.

3.7 High Performance Liquid Chromatography (HPLC)

HPLC was conducted using Waters 600 Controller HPLC system using a

Symmetry® C18 5µM (4.6 × 150 mm) column for TTX analysis.

3.7.1 Phosphate Buffer

In preparation of 60 Mm ammonium phosphate (Ph 5) buffer solution, 300 ml

Mili-Q water and 205 µ l phosphoric acid were mixed. 2M ammonium was added into the

solution to adjust the solution to pH 5. Approximately 1.0 g heptanesulfonic acid (HSA)

was added into the mixture followed by addition of Mili-Q water till the volume reach 500

ml. Then, 10 ml of acetonitrile was added. Finally, the mixture was filtered using 0.45 µm

Milipore filter paper.

16

3.7.2 Sodium Buffer

In preparation of 4N sodium hydroxide (NaOH) buffer solution, approximately

74.32 g of solid form sodium was dissolved in 500 ml Mili-Q water.

3.7.3 Oxidizing Reagent

. 60 mM ammonium phosphate (Ph 5), an iron­pairing reagent was used as mobile

phase at flow rate 0.8 mL/min. The eluent from the column was mixed with an equal

volume of 4 N NaOH. Then, the mixture was heated in a reaction coil at 110ºC. The toxin

was detected by using a fluorescence detector (Waters 2475 Multi Fluorescense Detector)

at 505 nm emission with 381 nm excitation. The concentration of TTX was calculated

using formula below:

(a/b) × 5

Where, a = peak area of sample

b = peak area of TTX standard

The amount of TTX calculated was in unit of MU/g for 10 µl mixture injected into

HPLC system.

17

4.0 Results and Discussion

A total of 49 puffer fish specimens belong to family tetraodontidae comprising of

28 individuals of Tetraodon nigroviridis and 21 individuals of Xenopterus naritus were

sampled for this particular study. All collected specimens were analyzed in stomach

content analysis.

4.1 Species Identification

A total of 21 individuals of Xenopterus naritus were identified and this species is

also known as yellow puffer fish. X. naritus was identified according its prominent

yellowish colouration especially towards the lower part of the body (Chuan & Lim, 2004).

The remaining 28 samples collected were identified as Tetraodon nigroviridis or also

known as green-spotted puffer fish. The identification of T. nigroviridis was made

according to identification key feature by Mohsin and Ambak (1983) where tetaodontidae

fish that have dorsal fin with less than 20 rays and possesses black spots belong to genus

Tetraodon.

Table 1: Taxonomic groups of spotted green and yellow puffer fish

Common Name Classification

Spotted green puffer fish Order: Tetraodontiformes

Family: Tetraodontidae

Genus: Tetraodon

Species: Tetraodon nigroviridis

Yellow puffer fish Order: Tetraodontiformes

Family: Tetraodontidae

Genus: Xenopterus

Species: Xenopterus naritus

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Both Xenopterus naritus and Tetraodon nigroviridis belong to same order and

family which are tetraodontiformes and tetraodontidae respectively as shown in Table 1.

Common name of T. nigroviridis is spotted green puffer fish while X. naritus is known as

yellow puffer fish. These two species are believed to be given name based on their

morphological characteristic where T. nigroviridis has spots on its body and the body is

mainly covered with green colour skin while the skin of X. naritus is mainly yellow in

colour.

4.2 Morphological Assessment

X. naritus possesses yellowish skin colour especially towards the lower part of its

body. T. nigroviridis possesses yellow-green skin colour of upper part of its body while

off-white to grey underneath. Besides, T. nigroviridis possesses small dark brown spots on

upper part of its body. T. nigroviridis has rounded body in cross section. The head and

body is covered with spinules. The back and side of its body has dark greenish yellow with

many black spots while its body is white in colour. Its dorsa, anal and pectoral fins are pale

while its caudal fin is pale with several transverse dark lines.

Tetraododon nigroviridis has about similar morphological feature with

Tetraododon fluviatilis. Therefore, it is quite difficult to differentiate between these two

species. However, T. fluviatilis could be differentiated with T. nigroviridis by the presence

of one black spot which is relatively about the size of the eye, surrounded by a white area

behind the pectoral fin. According to key feature given by Mohsin and Ambak (1983), T.

fluviatilis has black caudal fin with white edges. However, T. nigroviridis has pale caudal

fin with dark lines.