Study of Paralytic Shellfish Toxin and Tetrodotoxin in Horseshoe Crabs

Nurulhuda Bt Hashim 43643

RA 1242 S52 N974 2016

Bachelor of Science with Honours (Aquatic Resource Science and Management)

2016

Pusat UNlV

P. KHIDMAT MAKLUMAT AKADEMIK

UNIMAS

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1000272639

Study of Paralytic Shellfish Toxin and Tetrodotoxin in Horseshoe Crabs

Nurulhuda bt Hashim

(43643)

A thesis submitted in partial fulfilment of the requirement of the degree of

Bachelor of Science with Honours

(Aquatic Resource Science and Management)

Supervisor: Assoc. Prof. Dr Samsur Mohamad

Aquatic Resource Science and Management Programme

Department of Aquatic Science

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

2016

DECLARATION OF AUTHORSHIP

I, Nurulhuda bt Hashim , declare that the final year project report entitled:

Study of Paralytic Shellfish Toxin and Tetrodotoxin in Horseshoe Crabs

and the work presented in the report are both my own, and have been generated by me as

the result of my own original research. I confirm that:

" this work was done wholly or mainly while in candidature for a research degree at

this University;

" where I have made corrections based on suggestion by supervisor and examiners,

this has been clearly stated;

" where I have consulted the published work of others, this is always clearly

attributed;

" where I have quoted from the work of others, the source is always given. With the

exception of such quotations, this report is entirely my own work;

"I have acknowledged all main sources of help;

" where the thesis is based on work done by myself jointly with others, I have made

clear exactly what was done by others and what I have contributed myself;

" none of this work has been published before submission

Signed:

Aquatic Resource Science and Management Department of Aquatic Science Faculty of Resource Science and Technology Universiti Malaysia Sarawak (UNIMAS)

Date 36 Ilu 4MG

i

ACKNOWLEDGEMENT

Alhamdulillah, praise to Allah that gives me the blessing to finish my project successfully

on time. I would like to express my very great appreciation to Associate Professor Dr

Samsur Mohamad for the guide, knowledge, support, useful comments that help me

during the project duration. The advice given helps me a lot during the process

completing this project. All the time spends to meet up with me and my colleagues have

been very much appreciated.

My special thanks are extended to the lab assistants, Encik Nazri, Encik Zaidi, Madam

Lucy that gave help during my sampling activity. I would like to acknowledge assistance

from postgraduate students, Puan Noor Jawahir, Muhammad Syafiq and Mohamad Nor

Fakihin Aqsa during laboratory work.

Thanks to UNIMAS for providing me with facilities and laboratory equipment to finish

my project smoothly. Also, I would like to give my special appreciation to my parent,

Hashim bin Marin and Siti Jeleha Bt Ali for supporting me mentally and physically. Last

but not least, to the person who directly or indirectly involve in this project.

ii

Pusat Kbidmat MaklumatAkadw " UNIVERSITI MALAYSIA SARAww,.

TABLE OF CONTENTS

Declaration

Acknowledgement

Table of Content

List of Abbreviation

List of Tables

List of Figures

List of Appendices

Abstract

1.0 Introduction and Objectives

2.0 Literature Review

2.1 Morphology of Horseshoe Crab

2.2 Saxitoxin (STX)

2.3 Signs and Symptoms of Paralytic Shellfish Poisoning

2.4 Treatment for STX

2.5 Mechanism of STX Action in Horseshoe Crabs

2.6 Tetrodotoxin (TTX)

2.7 Signs and Symptoms of tetrodotoxin

2.8 Treatment for TTX

3.0 Materials and Methods

3.1 Sampling Site

3.2 Field Sampling

3.3 Laboratory Analysis

3.3.1 Morphological Study

3.3.2 Toxin Extraction

3.3.3 Thin Layer Chromatography (TLC)

3.3.4 High Performance Liquid Chromatography (HPLC) STX

3.3.5 Liquid Chromatography Tandem Mass

Spectrometry (LC- MS/MS) TTX 4.0 Result and Discussion

4.1 Morphometric analysis for STX

4.2 Morphometric Analysis for TTX

1

ii

V

Vi

Vii

Viii

1

2-4

4-5

6-7

8

8

9

10

11

12

13

13-14

15

15

16

17-18

18

19-21

21-23

III

4.3 Thin Layer Chromatography Analysis 23-25

4.4 High Performance Liquid Chromatography (HPLC) for STX 26-27

4.5 Liquid Chromatography Tandem Mass 27-29 Spectrometry (LC-MS/MS) for TTX

5.0 Conclusion 30

6.0 References 31-33

7.0 Appendices 34-38

IV

List of Abbreviations

TLC Thin Layer Chromatography

HPLC High Performance Liquid Chromatography

LC-MS/MS Liquid Chromatography Tandem Mass Spectrometry

LAL Limulus Amebocyte Lysate

PSP Paralytic Shellfish Poisoning

PST Paralytic Shellfish Toxin

STX Saxitoxin

TTX Tetrodotoxin

mg Milligram

mL Millilitre

cm Centimetre

g gram

SD Standard Deviation

µm micrometre

mTon milliTon

run nanometre

°C Degree celcius

V

List of Tables

Table 1 Four clinical stage of tetrodotoxin poisoning

Table 2 Means and Standard Deviations for T. gigas and C. rotundicauda (STX)

Table 3 Means and Standard Deviations for T. gigas and C. rotundicauda (TTX)

Page

11

20

22

Table 4 Rf values of samples compared with STX Standard 24

Table 5 Rf values of samples compared with TTX Standard 24

Table 6 Toxicity Amount (mouse unit) in egg and soft tissue 28

vi

List of Figures Pages

Figure 1 Morphology of horseshoe crabs Limulus 6

Figure 2 Chemical structure of saxitoxin 7

Figure 3 The voltage-gated sodium ion channel 9

Figure 4 Molecular structure of tetrodotoxin 10

Figure 5 Sampling site in Kuala Sedili, Pasir Putih and Sadong Jaya 14 (Source: Google Map)

Figure 6 The standard Rf values for STX and TTX 16

using solvent (n-butanol: acetic acid: water)

Figure 7 a) STX standard (5 ppm), b) Chromatogram of egg sample from Pasir Putih,

c) Chromatogram of soft tissue samples from Kuala Sedili

(right: t. gigas, left: C. rotundicauda)

26

vii

List of Appendices Pages

APPENDIX 1 Measurement of horseshoe crabs at Pasir Putih, Sadong Jaya, and Kuala Sedili for STX

APPENDIX 2 Measurement of horseshoe crabs at Pasir Putih, Sadong Jaya, and Kuala Sedili for TTX

APPENDIX 3 Total length (green), carapace width (blue)

and telson length (orange) in centimetre (cm)

of Carcinorcorpius rotundicauda.

24

25

26

APPENDIX 4 TLC Rf values for STX 26

APPENDIX 5 TLC Rf values for TTX 27

APPENDIX 6 TLC Rf value compared with standard using solvent 28

n-butanol: acetic acid: water (5: 1: 2),

right side for TTX and left side for STX

APPENDIX 7 Calibration curve for STX standard in HPLC 28

VIII

Study of Paralytic Shellfish Toxin and Tetrodotoxin in Horseshoe Crabs

Nuruihuda binti Hashim

Aquatic Resource Science and Management Faculty of Resource Science and Technology

University Malaysia Sarawak

ABSTRACT

In this study, toxin analysis was assessed for paralytic shellfish toxin (PST) and tetrodotoxin (TTX) in

horseshoe crabs at 3 sampling sites, 2 sites in Sarawak (Kuching and Samarahan) and 1 site in Johor (Kuala

Sedili). For PST, toxin used for this study were saxitoxin (STX). The objectives for this study were to study and determine the toxin properties in horseshoe crab. There were total of 37 samples for PST and 32 samples for

TTX were tested for toxin analysis. Two species of horseshoe crabs were identified as Tachypleus gigas and Carcinoscorpius rotundicauda and the total length, body weight, carapace width, and telson length were measured. For toxin analysis, thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) and liquid chromatography tandem mass spectrometry (LC-MS/MS) were performed. TLC analysis for

both STX and TTX indicated no toxins presence in the horseshoe crabs. The standard STX is 0.55 and for TTX

is 0.35. Average values for STX were 0.49-0.57 and for TTX were 0.45-0.67. PST profile for HPLC indicated

no STX detected at peak compared with standard but there were other toxin presence in low concentration. For

LC-MS/MS analysis, Kuching samples was non-toxic (1.3 MU/g) and egg samples at Johor was moderately toxic (295.27 MU/g). These findings show that horseshoe crabs at Malaysia are still considered safe for human

consumption but further research must be continued.

Keywords: Horseshoe crabs, paralytic shellfish toxin (PST), tetrodotoxin (TTX)

ABSTRAK

Dalam kajian ini, analisis toksin dinilai untuk Toksin Paralytik Kerangan (PST) dan tetrodotoksin (ITX) di

da/am belangkas di Liga tempat kajian, dua tempat kajian di Sarawak (Kuching dan Samarahan) dan satu

tempat kajian di Johor (Kuala Sedili). Untuk kajian toksin paralytic kerangan, toksin yang digunakan adalah

saxitoksin (STX). Objektif kajian ini adalah untuk mengkaji dan menentukan sifat-sifat ketoksikan di dalam

belangkas. Sebanyak 37 individu belangkas untuk PST dan 32 individu untuk 7-TX telah diuji untuk analisis

toksin. Dua spesies te/ah dikena/ pasti sebagai Tachvpleus gigas dan Carcinoscornius rotundicauda dan jumlah

panjang, berat, lebar kerapas dan panjang ekor telah diukur. Untuk kajian toksin, Kromatografi Lapisan

Nipis(TLC), Prestasi Tinggi Kromatografi Cecair (HPLC) dan Kromatografi Cecair Tandem Mass Spektrometri

(LC-MS/MS) telah dYalankan. Hasil analisis kromatografi lapisan nipis menunjukkan kedua-dua saxitoksin dan

tetrodotoksin tiada di dalam belangkas. Nilai saxitoksin asli ada/ah 0.55 dan nilai asli tetrodotoksin adalah 0.35. Purata nilai STX adalah 0.49-0.57 dan untuk TTX adalah 0.45-0.67. Profil PST untuk HPLC menunjukkan

tiada saxitok. sin (STX) dikesan di puncak apabila dibandingkan dengan nilai asli namun terdapat toksin lain

dalam konsentrasi yang rendah. Bagi analisis LC-MS/MS, sampel dari Kuching menunjukkan tiada toksin (1.3

MU/g) dan sampel dari Johor mempunyai sederhana toksin (295.27 MU/g). Kajian ini menunjukkan belangkas

di Malaysia masih dianggap selamat untuk dimakan. namun kajian selanjutnya masih perlu diteruskan.

Kata kanci; Belangkas, toksin paralytic kerangan (PST), tetrodotoksin (TTX)

1

1.0 Introduction

Horseshoe Crabs belong to Family Limulidae and phylum marine arthropods. Stormer

(1952) stated that around the world today, there are only four species of horseshoe crabs

that can be found: Limulus polyphemus, Tachypleus tridentatus, Tachypleus gigas and

Carcinoscorpius rotundicauda which three of the species, T tridentatus, T gigas and C.

rotundicauda inhabit at coastal waters of Asia. According to Kumar et al. (2015) T.

tridentatus inhabit at western and southern Japan, Taiwan, Philippines and North Borneo,

Malaysia, T. gigas lives at Bay of Bengal (North-East coast), Thailand, Malaysia,

Philippines, Borneo and Torres Straits, C. rotundicaunda can be found at Bay of Bengal,

Thailand, Malaysia, Philippines, Borneo and Torres Straits while another species, Limulus

polyphemus lives along the Atlantic coastlines of North America from Maineto to Yutan

(Berkson et al., 2002).

Horseshoe crabs are unique animals. They were not only exist almost 500 million

years ago, but they have an important role to the economic, ecological and medical

purposes (Mark et al., 2013). Horseshoe crab's blue blood contain a protein called Limulus

Amebocyte Lysate (LAL) that acts as clotting agent which also important for vertebrate

innate immunity (Mara et al., 2006). This blue blood have amebocytes which contain the

clotting enzymes that enable them to immobilize and engulf the endotoxin. Novitsky

(1984) mentioned that LAL can detect the endotoxin in pharmaceutical products. When

foreign bacteria enter into the body, the blue blood immediately form a clot to trap the

bacteria. These blue blood are helpful in finding remedies for disease that have built

immunities against the penicillin or drugs. Further research in this horseshoe crabs blue

blood is continue to be found such as detection of bacteria in the contaminated meat, fish,

and dairy products including frozen items (Walls et al., 2002).

2

Deborah et al. (2004) indicated that from the view of economic aspect, horseshoe crabs

become the source of food since 1990. Horseshoe crabs's meat and egss are the body part

which human like to consume. Since the 20th century, it also used as fertilizer, livestock

and bait for conch and leech. Even though it give benefits to the human being but most of

the public still do not aware of toxins that contain inside the horseshoe crab's body.

There are two toxins can be related to the horseshoe crabs, paralytic shellfish toxin

(PST) and tetrodotoxin (TTX). Paralytic shellfish poisoning occurs when the horseshoe

crabs eat the shellfish contaminated with dinoflagellate algae that produce a harmful

chemical called saxitoxin (STX).

STX is neurotoxic poisonous that produced from certain marine dinoflagellate and

cyanobacteria (Wiese et al., 2010). The characteristics of the PST are water soluble, low

molecular weight, and nitrogen containing compound. The toxins are distributed through

the food web and then accumulate in bivalve mollusc and shellfish. STX can cause

paralytic to the human called mytilotoxine. As stated in Wiese et al. (2010) study, 1 mg of

toxins from contaminated shellfish can cause lethal to a human being. In Malaysia, no

reported cases had been informed by the community.

Next, tetrodotoxin is a potent neurotoxin that can cause serious problem health to

human. According to Noguchi et al. (2011) the molecular weight of tetrodotoxin is 319 and

only 2 mg needed for the lethal dose to human. Until 2011, only one case reported and

happened at Kota Marudu, Sabah. It involved five people who had eat a meal of horseshoe

crab that resulted in one death (Razak et al., 2011). Tetrodotoxin inhibits the voltage-

dependent sodium channels at nerve and skeletal muscle giving the typical symptoms and

signs such as dizziness, vomiting, ataxia, cardiac arrhythmias and for the worst effect can

lead to death (Yuji et al., 2002).

3

There are limited studies on PST and TTX in horseshoe crabs. Thus, the aim of this

study was to increase awareness to the public about this life-threatening illness that can be

caused by this toxins and also give useful information to the local people, government and

to the general public.

Hence, the objectives of this study were:

1. to study the PST and TTX in horseshoe crabs.

2. to determine toxins properties of horseshoe crabs using thin-layer chromatography

(TLC), High-Performance Liquid Chromatography (HPLC) and liquid Chromatography-

Tandem Mass Spectrometry (LC-MS/MS).

3. to determine toxicity level between PST and TTX from selected sites.

4

Pusat Kh; ulmat Maklumat Akadem" UMVERSlTI MALAYSIA SARAww,.

2.0 LITERATURE REVIEW

2.1 Morphology of Horseshoe Crab

Horseshoe crabs are in phylum Arthropoda and in class Merostomata which has three

segments. Part of the body is called prosoma, the middle part is opisthosoma and the long

tail is called telson (Figure 1). Nancy et al. (2007) stated that horseshoe crabs can inhabit at

both estuarine and coastal areas. Horseshoe crabs play important roles in ecology to the

some animals such as shorebirds, sea turtles, silver perch, weakfish and silversides. The

body shell serves as substrates for epibionts such as barnacles and slipper limpets. Besides,

they also become predators that feed on a variety of gastropods, bivalves, and polychaetes

(Botton et al., 2003).

Next, Shuster et al. (2003) mentioned that mating period of horseshoe crab

occurred during the highest tides in late May and early June which is in full moon because

horseshoe crabs responded to optimum tidal and solar condition available during each

lunar phase. Spawning activity also the greatest at night than through the day. They spawn

in the highest numbers at intertidal shores with the waves function to deliver oxygen and

moisture. Meanwhile, the sediments at the foreshore became the incubator for the eggs.

Botton et al. (2010) emphasized that male horseshoe crabs had mating tactics as their age

increase and condition deteriorate. They change from attaching to female offshore to act as

satellites of spawning pairs.

5

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Figure 1: Morphology of horseshoe crab (taken from Briggs, 2012)

2.2 Saxitoxin (STX)

Paralytic shellfish toxin contains an active chemical called (STX) and had more

than 57 analogs that responsible for the paralytic shellfish poisoning disease (Llewellyn,

2006). Shigeru et al. (2000) stated that STX also found distributed in other aquatic

organisms such as marine snails and xanthid crabs. STX derived when a big amount of

eukaryotic dinoflagellates proliferates and lead to Harmful Algal Blooms (HAB) such as

genera Alexandriunz, and Pyrodinium in marine environment (Wiese et al., 2010). At

freshwater environment, STX can be produced from prokaryotic cyanobacteria in genera

Aphanizomenon, Cylindrospermopsis, Anabaena, Planktothrix, and Lyngbya. Besides,

STX can accumulate into the trophic levels and become the major environmental stressor

(Faber, 2012).

6

According to Mons et al. (2010) chemical structure of STX form a group related

tetrahydropurine that make up four subgroup; i) carbamate carbamate (STX, neoSTX and

gonyautoxins (GNTX 1-4); ii) N-sulfo-carbamoyl (GNTX5-6, C 1-4); iii) decarbamoyl (dc-)

(dcSTX, dcneoSTX, dcGNTXI-4); and iv) deoxydecarbamoyl (do-) (doSTX, doneoSTX

and doGNTXI) components with the 299 g/mol in molecular weight (Figure 2). Because

of STX are soluble in water, methanol and ethanol, selective muscular, very potent and

heat-stable neurotoxin and remain stable in slightly acidic environment, but a small amount

of STX can give adverse effects (Faber, 2012).

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HHH STX HH OS03 GNTX2 H OSO3 H GNTX3 OH HH nooSTX OH H OSO3 GNTX1 OH OS03 H GNTX4

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N-eiiloeerbenoyi deawbenayi deoxydaastamoyl toadne tooaine toxins GNTX5(B1) dcSTX doSTX

cl dcGNTX2 C2 dCG NTX3

GNTX6(B2) dcneoSTX doneoSTX C3 dcGNTX1 doGNTX1 C4 dcGNTX4

Figure 2. Chemical structures of STX (Source: Mons et a!., 2001)

7

2.3 Signs and Symptoms of Paralytic Shellfish Poisoning

Paralytic shellfish poisoning disease caused by dinoflagellate that had been

identified which were Pyrodinium bahamense and Gymnodinium catenatum (Stephen et

al., 2013). Toxin accumulated inside the horseshoe crabs later will be consumed by the

human. In mild cases, symptoms of having poisoning are tickling sensation or

unresponsiveness around lips almost 30 minutes. It shows the absorption of PSP toxins

through the buccal mucous membranes and later the ticking sensation can be feeling at the

face and neck. Other severe symptoms after ingestion of the horseshoe crabs contaminated

with PSP toxins were difficult in breathing, vomiting and may cause cardiovascular failure

due to the muscle paralysis (Maria et al., 2014). Acres (1978) pointed out that in severe

poisoning cases, the muscular paralysis become deeper and the pulse show no alarming

abnormality. Within 2-24 hours of paralysis respiratory, it leads to death.

2.4 Treatment for STX

Since the treatment for PSP disease is almost similar to puffer fish poisoning, if no

vomiting had occurred, gastric lavage should be given to remove the sources of unabsorbed

toxins. Kanchanapongkul (2001) stated, there were 25 cases at Thailand and all patients

had been given general supportive care and closely observed for respiratory failure. During

the progression of paralysis period, intubation which assist by ventilation for the breathing

so the air pushed into the lungs to deliver breath to the patient, and mechanical ventilation

were used. Kao (1972) mentioned that activated charcoal can also be used to treat the

patients. Apart from using the activated charcoal, recovering from the poisoning required

receiving fluid therapy, blood pH monitoring by taking the patient blood and assisted

ventilation. Insufficient data were available whether the STX can change the pH of the

blood.

8

2.5 Mechanism of STX Action in Horseshoe Crabs

Sodium channel was unique because it became molecular target for the neurotoxin

which altered the channel function by binding to the several different receptor sites

(Catterall, 2007). According to Maria et al. (2014) STX have high affinity for binding site

1 of the voltage-gated sodium ion channel (Figure 3), thus inhibits temporary permeability

of sodium ions by binding tightly to a cell surface receptor site and prolong the gating of

potassium channels in heart muscle cells. The blockage of channel in human prevents

depolarization of the membrane and prevents subsequent impulse-generation in the

peripheral nerves and skeletal muscles, leading to a prolonged relaxed state; paralysis

(Kao, 1972). Excitation is not occurred because the calcium voltage-gated channels do not

open thus the potassium and chloride conductance remains unaltered.

Figure 3: The voltage-gated sodium ion channel taken from (Catterall et. al, 2007)

9

2.6 Tetrodotoxin (TTX)

Tetrodotoxin or the puffer toxin continuously had been found at various type of

organism at marine and freshwater environment (Chen and Chung, 2014). According to

Ngy et al. (2007) tetrodotoxin had been detected in horseshoe crab Carcinoscorpius

rotundicauda at Cambodia. The TTX-producing bacteria were identified from the

following genera: Actinomyces, Aeromonas, Alteromonas, Bacillus, Pseudomonas,

and Vibrio. Noguchi et al. (2006) mentioned TTX accumulated by the species through the

dietary transfer that enter into the food chain involving the invertebrates and fish.

According to Edward (2013), the structure of TTX is (CI IH17N3O8i Mr = 319.27

g/mol) consist of positively charged cyclic guanidinium and sometimes drawn as

zwitterion due to the acidity of the three Nitrogen atoms ( blue colour), a pyridine ring (red

colour) and hydroxyl group which help to stabilize the TTX-sodium channel binding at

aqueous surface (Figure 4). Tetrotoxinic acid formed by the catalyzed hydrolysis of three

carbon-oxygen bond that resulting to the variety of chemical intermediates which included

a hydroxyl-lactone species that convert the carboxylate.

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CHZCH

Figure 4: Molecular Structure of tetrodotoxin taken from (Vaishali et. al, 2014)

10

2.7 Signs and Symptoms of Tetrodotoxin

According to Attaya et al. (1987) the symptoms for tetrodotoxin was similar as

paralytic shellfish poisoning. (Kanchanapongkul, 2008) mentioned that there were four

clinical staging of tetrodotoxin poisoning in horseshoe crabs (Table 1). The tetrodotoxin

poisoning occurred at Chon Buri, Thailand by ingestion of the toxic eggs from horseshoe

crabs. Total of 280 cases from 1994-2006 were recorded at Thailand with 245 available

medical records. Four clinical staging of tetrodotoxin poisoning (Table 1), the highest

patient recorded was in stage 1 and the lowest patient in stage 3. The most frequencies sign

and symptoms for the TTX poisoning were lingual, hands and feet numbness. For clinical

analysis it was important to collect the urine and blood samples from affected patients. The

stage of TTX poisoning depend on the amount of TTX ingested, time duration after

ingested and the general body health status (Bentur et al., 2008).

Table 1: Four clinical staging of tetrodotoxin poisoning (extracted from Kanchanapongkul (2008)

Stages of poisoning Signs and symptoms

Stage 1 Perioral and lingual numbness or

paresthesia, nausea, vomiting

Stage 2 Numbness progresses markedly, motor

paralysis of extremities

Stage 3. Progressive motor paralysis, bulbar muscle

paralysis. Patient is conscious

Stage 4 Respiratory failure, hypoxia,

unconsciousness and hypotension may

occur, fixed and dilated pupils

11

2.8 Treatment for TTX

Treatment for the tetrodotoxin poisoning was depending on the clinical staging but

currently no antidote available for TTX poisoning. According to Kanchanapongkul (2008)

patients from stage 1 and 2 were discharged within 24-48 hours. For patients in stage 4,

ventilation and intubation were given and some of the patients were transferred to the

intensive care unit (ICU). There were also some severe cases that needed respiratory

support for the period of 48-72 hours for the TTX was excreted completely. For early

stage, Clark et al. (1999) suggested that activated charcoal can be used to absorb the toxin

through the stomach. Other than activated charcoal, gastric lavage was removal the small

volumes of liquid by using the passage of a tube via the mouth or nose down into the

stomach to reduce the severity of poisoning. This procedure should be done within the 60

minutes after the ingestion of TTX. Besides, treatment for TTX poisoning was using

intravenous fluids to maintain the fluid-electrolyte in the body and hemodialysis was also

another option for TTX poisoning treatment (Nakashima et al., 2007).

12

3.0 Materials and Methods

3.1 Sampling Sites

The study sites ( Figure 5) selected were located at Pasir Putih, Kuching (1°39'49"N

110°44'300"E), Sadong Jaya, Samarahan (1°30.1'37.8"N 110°44'24.3"E) and Kuala

Sedili, Johor (1 °50'21 "N 104°8'59"E). Samples were collected in the cooler box containing

ice and transported back to the laboratory for toxin analysis.

3.2 Field Sampling

The horseshoe crab samples from Pasir Putih were caught by fisherman on 3'' March

2014 and samples from Sadong Jaya was collected on 4 March 2014 and samples from

Kuala Sedili on 8 November 2013. All the samples proceeded for laboratory analysis.

13

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Figure 5: Sampling sites in Kuala Sedili, Pasir Putih and Sadong Jaya (Source: Google Map)

14