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TETRODOTOXIN (TTX) DETERMINATION OF HORSESHOE CRAB COLLECTED FROM MALUDAM SARAWAK

Ghafur Rahim Bin Mustakim

HD Bachelor of Science with Honours9469

(Aquatic Resource Science and Management)C73 2014G411

2014

Determination of Tetrodotoxin (TTX) by HPLC of Horseshoe Crab Collected from

Maludam, Sarawak.

Ghafur Rahim Bin Mustakim

This report is submitted in partial fulfilment of the requirement for the degree of Bachelor of

Science with Honours

(Aquatic Resource Science and Management)

Faculty of Resource Science and Technology

UNIVERSITI MALAYSIA SARAWAK

2014

DECLARATION

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

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

higher learning.

Ghafur Rahim Bin Mustakim

Aquatic Resource Science and Management

Department of Aquatic Science

Faculty Resource Science and Technology

Universiti Malaysia Sarawak

Acknowledgment

In the name of Allah The Most Gracious and The Most Merciful.

Alhamdulillah, thanks God for giving me the strength to complete my Final Year Project

although many obstacles experienced prior to completion of this project.

I would like to express my gratitude and appreciation to all those who gave me the possibility

to complete this report. A special thanks to my supervisor Dr. Samsur bin Mohamad, whose

help, stimulating suggestions and encouragement, helped me to complete my project. Without

great support from him it would be difficult for me to finish this project. Thanks also to Dr.

Khairul Adha Abd Rahim that helped me in statistical analysis.

Next, I would like to take this opportunity to thank my family members especially my mother

Mdm. Fatimah binti Jaafar for her endless support and encouragement.

Finally, I would like to express my appreciation all my beloved laboratory mates and my class

mate for their encouragement and support. Last but not least, thanks also to all my ‘haloqah’

mates that always pray and support me.

ii

Table of Contents

Acknowledgment i

Table of Contents ii

List of Abbreviations iv

List of Figures v

List of Tables vi

Abstract/ Abstrak vii

1.0 Introduction 1-2

2.0 Literature Review 3

2.1 Horseshoe crab 3

2.2 Horseshoe crab morphology 5

2.3 Tetrodotoxin (TTX) 8

2.4 Mechanism of TTX accumulation 9

2.5 Sign and symptom of TTX intoxication 12

2.6 TTX intoxication cases 12

2.7 Treatment of TTX intoxication 13

2.8 Toxicity assessment 13

3.0 Materials and Methods 15

3.1 Sampling site 15

3.2 Sample collection 15

3.3 Sample extraction and preparation 15

3.4 High Performance Liquid Chromatography (HPLC) 16

3.5 Data analysis 16

iii

4.0 Results and Discussion

4.1 Morphometric Measurements 19

4.2 High Performance Liquid Chromatography (HPLC)

Analyses 20

4.3 Morphometric characteristics affect to toxicity level. 25

5.0 Conclusion 26

6.0 References 27

7.0 Appendices 32

iv

List of abbreviation

ANOVA Analysis of variance

Degree Celcius

cm Centimeter

g Gram

HPLC High Performance Liquid

Chromatography

M Molar

ml Milliliter

mm Millimeter

MU Mouse Unit

PFP Puffer Fish Poisoning

PSP Paralytic Shellfish Poisoning

rpm Rotation per minutes

SD Standard Deviation

STX Saxitoxin

TLC Thin Layer Chromatography

TTX Tetrodotoxin

µl Microliter

v

List of figures Pages

Figure 1 Life cycle of horseshoe crab 4

Figure 2 The external anatomy Structure of the first leg of a

horseshoe crab 6

Figure 3 The difference in type of marginal spine and type

of telson between the four species 7

Figure 4 TTX structure 8

Figure 5 Illustration of mechanism of TTX accumulation 11

Figure 6 Sampling sites 17

Figure 7 HPLC of Standard TTX (a) with Rt 10.10 and toxin

profile of T. gigas eggs 23

Figure 8 HPLC of Standard TTX (a) with Rt 10.10 and toxin

profile of C. rotundicauda tissues. 24

vi

List of tables Pages

Table 1 Morphometric measurement of the horseshoe crab 18

Table 2 Mean and standard deviation toxicity level of

C. rotundicauda and T. gigas 20

Table 3 Amount of TTX found in C. rotundicauda from

2007-present. 21

vii

Toxicity Assessment of Horseshoe Crab in selected Sarawak Water

Ghafur Rahim Bin Mustakim

Aquatic Resource Science and Managment

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

This study was carried out to assess the toxicity and to identify the correlation between morphometric

characteristic with the level of toxicity of the horseshoe crabs collected from Maludam, Sarawak. For

morphometric study, only carapace width and weight of the horseshoe crab were measured. Based on

the characteristic analyses, two species of horseshoe crab are identified which were Carcinoscorpius

rotundicauda and Tachypleus gigas. Carapace sizes of C. rotundicauda are smaller compare to T.

gigas. For toxin analyses, High Performance Liquid Chromatography (HPLC) method was used.

Result obtained showed that both species of horseshoe crab found at Maludam were toxic but still

considered safe for human consumptions. C. rotundicauda toxicity level score between 0.90 MU/g to

4.67 MU/g. Meanwhile for T. gigas toxicity level score range from 0.49 MU/g - 3.01 MU/g. For

correlation study, SPSS software was used. According to correlation result, Pearson Correlation

Coefficient (p<0.05) for C. rotundicauda was 0.283 and for T.gigas was 0.021. This result

showed that morphometric measurement has weak correlation with level of toxicity in horseshoe

crab. Meaning that morphometric characteristic does not influent the toxicity level in horseshoe

crab. Present study is the first attempt to identify the relationship between morphometric

characteristic with level of toxicity.

Keywords: horseshoe crab, Carcinoscorpius rotundicauda, Tachypleus gigas, morphometric, toxicity.

ABSTRAK

Kajian ini dijalankan untuk mengetahui kadar ketoksikan dan hubung kait diantara morfometrik

dengan kadar ketoksikan belangkas yang diperolehi dari kawasan perairan Maludam, Sarawak.

Untuk kajian morfometrik, hanya lebar karapas dan jumlah berat di ukur. Berdasarkan ciri-ciri

morphologi, terdapat dua spesis belangkas yang dikenal pasti: Carcinoscorpius rotundicauda dan

Tachypleus gigas. Untuk analisa toksin, HPLC digunakan utuk mengesan Tetrodotoksin (TTX). Toksin

diekstrak daripada telur dan tisu dan dianalisa menggunakan HPLC. Keputusan analisa menunjukan

bahawa kedua-dua sepsis yang ditemui di Maludam mengandungi toksik namun masih dianggap

selamatt untuk kegunaan manusia. Tahap ketoksikan C. rotundicauda diantara 0.90 MU/g to 4.67

MU/g. Manakala untuk T. gigas kadar ketoksikan dikesan diantara 0.49MU/g-3.01MU/g. Manakala

untuk kajian saling perkaitan antara morfometrik belangkas dengan kadar ketoksikan, perisian SPSS

digunakan. Berdasarkan keputusan koefisien korelasi,untuk C. rotundicauda ialah 0.283. manakala

untuk T. gigas pula ialah 0.021. Ini menunjukkan bahawa kadar ketoksikan tidak mempunyai

perkaitan dengan kadar ketoksikan. Kajian ini merupakan yang pertama dilakukan untuk mengaitkan

hubungan antara morfometrik belangkas dengan kadar ketoksikan.

Kata kunci: Belangkas, Carcinoscorpius rotundicauda, Tachypleus gigas, morfometrik, ketoksikan.

1

Introduction

The horseshoe crab is often called “a living fossil” because the morphology of the extant

species remains quite similar to species found in the fossil record. This allow them to keep

survive in various environmental stresses for the past 150 million years (Kamaruzzaman et

al., 2012). Among four species of horseshoe crab present around the world, only one

species distributed at the coastal water of North America and another three species

distributed in Southeast Asian region (Rozihan et al., 2012). In Malaysia waters, three

species has been identified as Carcinoscorpius rotundicauda, Tachypleus gigas, and

Tachypleus tridentatus (Chatterji & Noraznawati, 2009).

In certain area of Sarawak, Sabah, and Johor, horseshoe crab are widely consumed by a

public as a meal especially eggs for the female crabs. It sold at price of RM5 to RM7 per

individual based on size and weight.

Till now, there are several cases reported regarding intoxication due to consumption of

horsesoe crab meal. And study done showed that, there are present of tetrodotoxin (TTX)

and saxitoxin (STX) in the horseshoe crab tissue or/and eggs. TTXs are causative agent for

puffer fish poisoning (PFP), whereas STXs responsible for paralytic shellfish poisoning

(PSP) (Meunier et al., 2009). But, toxic analysis done showed that TTX are the major toxin

found in the horseshoe crab (Noguchi & Arakawa, 2008).

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The horseshoe crab Carcinoscorpius rotundicauda is one of such TTX-bearers. Study done

at Thailand, Bangladesh and Cambodia proved that so far only this species consist of high

level of TTX that can caused intoxication when consumed (Kungusawan et al., 1987; Tanu

& Noguchi, 1999; Ngy et al., 2007). In certain intoxication cases happened, T. gigas is

mistaken as C. rotundicauda, resulting poisoning incident in Thailand (Miyazawa &

Noguchi, 2001). Previous study stated that the present of TTX in the horseshoe crab came

from the outside resources. And high probability, it come from the bacteria that accumulate

into the horseshoe crab tissue or eggs via the food chain (Noguchi & Arakawa, 2008).

Unfortunately, in Malaysia their are still lack of information obtain regarding horseshoe

crab toxicity even it been consumed by local people. For this reason, this study must be

continue to know the level of TTX in horsrshoe crab especially in Sarawak coastal.

Perhaps, by finish this study the level of TTX can be identified for the safety among the

local people that consumed horseshoe crab.

The objective of this study were:

1. To identify toxin properties of horseshoe crab by using High Performance Liquid

Chromatography (HPLC) methods.

2. To determine toxicity of toxin in horseshoe crab tissues and eggs.

3. To determine the correlation between horseshoe crab morphometric characteristic and

level of toxicity.

3

2.0 Literature Review

2.1 Horseshoe crab

Horseshoe crabs are marine arthropods that belong to class merestomata. There are benthic

organism that prefer calm seas and sea for their biogenic activities (Samsur & Nur Izzatie,

2011). So that, its diet more preferred to another benthic organisms such as bivalves,

gastropods, polychaetes and worms (Kamaruzzaman et al., 2012). Horseshoe crab play

important role in ecology, since it provide essential food for migrating birds (Gillings et

al., 2007). Commercially, horseshoe crab highly demands in pharmaceutical industry

because it blueblood can be used for detecting bacteria and toxins (John et al., 2012).

Demographic data showed that the global distribution pattern of horseshoe crab Atlantic

horseshoe crab, L. polyphemus most commonly found in Gulf of Mexico, Southeast Asian

horseshoe crab, T. gigas can be found in the shores of the bay of Bengal particularly along

the coast of India to Indo-Chhia, NorthVietnam, Borneo and Celepes, T. tridentatus can be

found along the Northern shores of Japan up to South Vietnam and along the Western

islands of the Philippines and mangrove horseshoe crab, C. rotundicauda can be found

along northern shores of the bay of Bengal to the Southern coast of the Philippines

(Elizabeth, 2001; Chatterji et al., 1992). Among four extant species, only two species can

be found in Sarawak, T. gigas and C. rotundicauda (Izzatie, 2010).

Normally, horseshoe crab will migrate from deeper to shallow water for breeding

purposed. And it occurs during high tides of the full and new moon season (Jennifer,

2010). Males utilize modified prosomal appendages to attach to females. Females deposit

4

eggs 7–20 cm below the sand surface where it fertilized externally by the males (Jennifer,

2010). The eggs are left to develop into a complete life cycle as shown in figure 1.

Figure 1: Life cycle of horseshoe crab (Gerhart, 2007)

2.2 Morphology of horseshoe crab

The physical characteristics of horseshoe crab are not too complex since there are no

significant changes from their ancestor. Generally for all 4 species exists, the physical

characteristic just look same with the present of telson, carapace and abdomen as shown in

Figure 2. The most obvious characteristic of the prosoma are the two compound eyes,

located near the front, and the numerous legs underneath. The abdomen, also called the

opisthosoma, attaches to the prosoma by a hinge joint. The book gills, which are used for

oxygen exchange, dominate the underside of the abdomen. A hard shell, called the

carapace, covers each part of the horseshoe crab (Gerhart, 2007).

In addition, the sex of horseshoe crab can be differentiate by the present of pedipals/ first

legs looks like “boxing glove” as shown in Figure 3. For species identification, there are

two common ways used to recognize the different horseshoe crab species based on their

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morphology characteristics. Firstly based on type and size of marginal spines and secondly

based on shape of telson cross section either round or triangle as shown in Figure 4.

Commonly, sizes of carapace width for female are larger than a male horseshoe crab.

Large size in females important in order to tow males during the spawning season and a

large body can carry more eggs (Botton & Loveland, 1992).

6

7

Figure 3: The differences in type of marginal spine and type of telson between the four species

( Sekiguchi & Shuster, 2009)

Limulus Polyphemus

Carcinoscorpius rotundicauda

Tachypleus tridentatus

Tachypleus gigas

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2.3 Tetrodotoxin (TTX)

Tetrodotoxin also known as puffer toxin derived it name from the Tetraodontidae family of

the puffer fish. TTX was isolated in form of crystaline for the first time on early 1950s

(Chau et al., 2011). And TTX is a water soluble heterocyclic guani-dine (Jirasak, 2008) It

can be found in both terrestrial and marine organism. Recent study found that more than 10

variance TTX was extracted and analyzed from the different animal (Miyazawa &

Noguchi, 2001). TTX is heat-resistant toxin. Meaning that, it cannot be degraded during

cooking process (Naguchi & Ebesu, 2001). “The chemical properties of TTX are dictated

by its unique charge behavior and the labile nature of the orthoester group centered about

the C10 carbon atom” (Moczydlowski, 2013) as shown in figure 5. TTX act as an inhibitor

on neurotransmitter through their blocking effect on voltage sensitive sodium channel that

caused paralysis of the muscle concerned (Hashimoto, 2001).

Figure 4: Structure of tetrodotoxin (Moczydlowski, 2013)

Commonly, in puffer fish TTX will concentrated on liver and ovary for a marine species.

For fresh water and breakish water species, TTX concentrated on skin layer (Noguchi &

Arakawa, 2008). In horseshoe crab study, TTX widely found concentrated in soft tissue for

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male and in egg for female. TTX in horseshoe crab believed come from it diets (Tanu &

Noguchi, 1999).

Based on previous study, stated that from all three horseshoe crab species existed in

Southeast Asia, one species has be confirmed contain TTX. It was C. rotundicauda. In

Vietnam, researcher found that from 12 C. rotundicauda tissue specimen extracted, 10

specimens showed positive result towards TTX (Ha et al., 2009). Also the same result

obtained in Cambodia show that C. rotindicauda were the main species that contribute in

TTX intoxication (Ngy et al., 2007).

2.4 Mechanism of TTX accumulation in marine organism

Based on the study done by Noguchi and Arakawa (2008), the source of toxin in marine

organism can be either endogenous or exogenous. Endogenous referred to the organism

that produced its own toxin without influenced by other organism. Meanwhile exogenous

referred to the organism that contain toxin from outside sources and influenced by other

organism. In many cases studied shown that almost marine organisms such as puffer fish

commonly obtain their toxic via exogenous resources.

Major way how TTX accumulated in marine animal was derived from a food chain. It has

been proved by Noguchi and Arakawa (2008). From their experiment found that puffer fish

that has been cultured with non TTX diet were non- toxic. But it suddenly becomes toxic

when it was supplied with foods that contain TTX. This experiment proved that, TTX

accumulated in marine organism via the food chain consisting of several steps and starting

with marine bacteria as a primary source of TTX as illustrated in figure 5.

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Another possible way TTX accumulate in marine organism is via direct interaction with

TTX producing bacteria. Recent study found that more than 12 different species of TTX

producing bacteria found in puffer fish (Moczydlowski, 2013). And main bacteria that

produce TTX in horseshoe crab come from Vibrio spp (Kungsuwan et al., 1988). These

bacteria will acts as parasite or create symbiotic relationship directly with marine organism

and accumulated inside their body without via food chain. But the amount of TTX produce

through this mechanism only contribute small amount of total TTX in the marine organism

compared to biomagnification mechanism through the food chain (Noguchi and Arakawa,

2008).

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Vibrio alginolyticus

Shewanella alga, s. putrefaciens TTX producing marine bacteria

Alteromonas tetraodonic etc

: Food chain

: Parasitism / symbiosis

: Decomposition

Figure 5: Illustration of mechanism of TTX accumulation

(Edited from: Noguchi & Arakawa, 2008)

TTX dissolved in seawater or

adsorbed on a precipitated with

dead planktonic cells etc.

TTX in sediment

Small zooplankton

Detritus feeder

Flatworm

Ribbonworm

Arrowworm

Xanthid crab

Small gastropods

Skeleton shrimp

Pufferfish

Large gastropods

Horseshoe crab

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2.5 Signs and Symptoms of TTX intoxication

Signs and symptoms of TTX poisoning depend on the amount of toxin consumed, age and

healthy status of the victims (Noguchi & Ebesu, 2011). Commonly, the higher the amount

of toxin consumed, the more obvious sign of poisoned can be observed. The symptom can

be appeared shortly within a few hours after consumption (Razak et al., 2011). The

victim’s symptoms of TTX intoxication include oral numbness, muscular weakness,

nausea, vomiting, and sensory deficit. In serious cases it will followed by flaccid paralysis

and caused death because of the respiratory failure (Moczydlowski, 2013). As discussed

by Bradford and Joseph (2008), TTX intoxication has four staged of progression. Stage

one includes oral parenthesis, stage two motor paralysis, stage three muscular

incoordination and stage four respiratory paralysis.

2.6 TTX intoxication cases

Many cases reported due to human intoxication by TTX come from puffer fish. Lack of

intoxication cases information reported regarding horseshoe crab ingestion, especially in

Malaysia. And in some cases patients refuse to go to the hospital. So that, no proper

clinical data were recorded.

A study done in Madagascar found that toxin level at 16 mouse unit/g can cause ill and

death (Ravaonindrina et al., 2001). And the lethal dose for human estimated about

10,000MU (Noguchi & Ebesu, 2001)

In Thailand between 1994 until 2006, about 280 cases reported due to TTX intoxication.

Main sources of this intoxication come from ingestion of horseshoe crab eggs. Believed

13

come from C. rotundicauda species. From 245 medical recorded, 100 patient in stage 1, 74

were in stage 2, 3 were in stage 3 and 68 were in stage 4 (Jirasak, 2008). In Cambodia on

2006, poisoning cases was reported due to ingestion of horseshoe crab. And study done by

Ngy et al. (2007) found that C. rotundicauda contain high level of TTX and dangerous for

meal.

In Malaysia there are only several serious cases reported due to TTX intoxication by the

horseshoe crab. Until 2011 only one death reported. It was happened at Kota Marudu,

Sabah. The cases involved five people who had ingested a meal of horseshoe crab that

resulted in one death (Razak et al., 2011).

2.7 Treatment of TTX intoxication

Until now, there are no antidotes or antitoxins found to neutralize TTX. In a serious TTX

intoxication cases, the victims only can be help using artificial respiration treatment, by

means it just can slowdown the death process without any significant treatment. Recently a

monoclonal anti-TTX antibody was investigated (Kawatsu et al., 1997), but it can be used

only for research purposed as a chemical reagent and further study done shown that it have

no contribution for clinical purposes (Noguchi & Arakawa, 2008).

2.8 Toxicity assessment

There are several approaches that can be used for detection of TTX. A few common

methods used including the mouse bioassay, thin-layer chromatography, high performance

liquid chromatography (HPLC), Gas Chromatography-Mass Spectrometry (GC-MS) and

liquid chromatography-mass spectrometry (LC-MS).

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Mouse bioassay has historically been the universally applied tool to assess the toxicity

level in monitoring programs. The lethal potency is expressed in mouse unit (MU). One

MU (mouse unit) is defined as the amount of toxin required to kill a 20g male mouse

within 30 min after single intraperitoneal injection (Arakawa et al., 2010). This bioassay,

however, shows low precision and requires a continuous supply of mice of a specific size.

In addition, the mouse assay unable to provide any information on toxin composition, and

cannot distinguish TTX from other neurotoxins such as paralytic shellfish poison (PSP)

(Asakawa et al., 2012). Thin layer chromatography (TLC) is useful means for TTX

detection, but they are not suitable for TTX determination (Asakawa et al., 2012).

Therefore HPLC has been explored for both qualitative and quantitative analysis of TTX

and its standard. Nagashima et al. (1987) reported that reversed phase ion pairing HPLC

method has also been the system of choice by many researchers that conduct research on

TTX. Because HPLC are the fastest and efficient analysis of TTX and its standard.