DETERMINATION OF TOXIN PROPERTIES AND TOXICITY STUDY OF

HORSESHOE CRAB IN KABONG, LUNDU AND MIRI SARAWAK.

Muhammad Zaid Bin Nasir

(35023)

Bachelor of Science with Honours

(Aquatic Resource and Science Management)

2015

DETERMINATION OF TOXIN PROPERTIES AND TOXICITY STUDY OF

HORSESHOE CRAB IN KABONG, LUNDU AND MIRI, SARAWAK.

Muhammad Zaid Bin Nasir

(35023)

This project report is submitted in partial fulfillment of the requirements for the Degree of

Bachelor of Science with Honours

(Aquatic Resource and Science Management)

Faculty of Resource Science and Technology

UNIVERSITI MALAYSIA SARAWAK

2015

ii

DECLARATION

I hereby declare that no portion of this dissertation has been submitted in support of an

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

of higher learning.

__________________________

MUHAMMAD ZAID BIN NASIR

Aquatic Resource and Science Management

Department of Aquatic Science

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

iii

The project entitled “Determination of toxin properties and toxicity study of horseshoe

crab in Kabong, Lundu and Miri, Sarawak” was prepared by Muhammad Zaid Bin Nasir

and submitted to the Faculty of Resource Science and Technology in partial fulfillment of

the requirements for the Degree of Bachelor of Science (Honours) in Aquatic Resource and

Science Management.

Received for examination by:

________________________

Date:

________________________

iv

ACKNOWLEDGEMENT

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

Alhamdulillah, thank to Allah s.w.t. for continuous blessing and giving me the strength to

complete my Final Year Project although many challenges and obstacles experienced from

the starting until the end of this project.

I would like to make my deepest appreciation and gratitude to my supervisor, Dr. Samsur

bin Mohamad for his invaluable guidance, stimulating suggestions, helping and also

encouragement during completing this project. Without great support from him it would be

difficult for me to finish this project. Million thanks to Mr. Benedict for helping and kind

assistance during sample analysis using the HPLC. Thanks also extended to Encik Mohd

Nor Azman from Fisheries Research Institute for permission and kind assistance during

sample analysis using LC-MS method.

Greatest appreciation to my beloved parents, Mr. Nasir bin Husin and Mdm. Zurina bte

Abu for their consistent encouragement and support throughout this project. I also would to

express my appreciation to both PhD students, Mdm. Noor Jawahir and Mr. Syafiq for the

guidance and kind assistance during this project.

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

classmate for their continuous support and encouragement especially to Nur Afifah Hanun,

Nurin Syahindah Syasya, Wan Nurain Farahah, Naquiah, Er Huey Hui and Asmadi. Last

but not least, thanks also to all my friends especially Mawar Nabilah binti Johari for

continuous advice, pray, assist and moral support to me.

v

Determination of Toxin Properties and Toxicity Study of Horseshoe Crab in Kabong, Lundu and Miri

Sarawak.

Muhammad Zaid Bin Nasir

Aquatic Resource and Science Management

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

ABSTRACT

Horseshoe crab belongs to the family of Limulidae known to possess a tetrodotoxin (TTX) which can cause

horseshoe crab poisoning and adverse effect to human health. In current study, there are two species found in

Sarawak which are Tachypleus gigas and Carcinoscorpius rotundicauda. The TTX concentration in soft

tissue and eggs from Kabong, Lundu and Miri Sarawak were analyzed and determined by application of Thin

Layer Chromatography (TLC), High Performance Liquid Chromatography and Liquid Chromatography-

Mass Spectrometry (LC-MS). Some extracted toxin for all specimens were shown to be toxic while another were non-toxic. Among the tissues, eggs were found to be highest TTX concentration (5.40 MU/g) followed

by the soft tissue (3.20 MU/g). Moreover, TTX concentrations among horseshoe crab species were different

with C. rotundicauda showed highest TTX value (13.54 MU/g) which found in eggs while the lowest value

was detected in T. gigas (3.59 MU/g). From this study, LC-MS method is the best tools to determine the TTX

and suggested being used as procedure in screening of seafood for monitoring program. Furthermore, data of

TTX levels in selected horseshoe crab from this study could be important information and uses as guideline

in order to reduce and prevent horseshoe crab poisoning cases especially in Sarawak waters.

Keywords: Horseshoe crab, tetrodotoxin (TTX), thin layer chromatography (TLC), high performance liquid

chromatogrpahy (HPLC), liquid chromatography-mass spectrometry (LC-MS).

ABSTRAK

Belangkas adalah tergolong daripada keluarga Limulidae yang dipercayai mengandungi tetrodotoksin (TTX)

yang boleh menyebabkan keracunan belangkas dan memberi kesan sampingan kepada kesihatan

manusia.Dalam kajian semasa, terdapat hanya dua species belangkas ditemui di perairan Sarawak iaitu T.

gigas and C. rotundicauda. Kepekatan dan kandungan TTX dalam tisu lembut dan telur belangkas daripada

Kabong, Lundu dan Miri Sarawak telah dianalisis dan dikenalpasti melalui penggunaan sistem TLC, sistem

HPLC dan sistem LC-MS. Sebahagian daripada toksin yang dikeluarkan menunjukkan keputusan toksik

manakala sebahagian yang lain tidak toksik. Antara bahagian tisu, telur dikenalpasti mengandungi TTX

yang sangat tinggi (5.40 MU/g) diikuti oleh tisu lembut (3.20 MU/g). Tambahan itu, kandungan TTX antara spesis belangkas adalah berbeza di mana C .rotundicauda menunjukkan nilai TTX yang tertinggi (13.54

MU/g) yang ditemui dalam telur manakala nilai terendah dikesan dalam spesies T. gigas (3.59) MU/g).

Daripada kajian ini, penggunaan LC-MS adalah aplikasi terbaik untuk mengenalpasti TTX dan digalakkan

untuk digunakan sebagai prosedur dalam pemeriksaan makanan laut untuk aktiviti pemantauan. Selain itu,

data mengenai kandungan TTX di dalam belangkas terpilih daripada kajian ini boleh dijadikan maklumat

penting dan digunakan sebagai panduan dalam mengurangkan dan mencegah kes keracunan belangkas

terutamanya di perairan Sarawak.

Kata Kunci: Belangkas, tetrodotoksin (TTX), kromatografi lapisan nipis (TLC), kromatografi cecair

berprestasi tinggi (HPLC), cecair kromatografi- spectrometri jisim (LC-MS).

vi

Table of Contents

Declaration i

Acknowledgement iii

Abstract iv

Table of Contents v

List of Tables vii

List of Figures viii

List of Abbreviations ix

1.0 Introduction 1

2.0 Literature Review

2.1 Horseshoe Crab and Its Importance

2.2 Taxonomy

2.3 Morphology of Horseshoe Crab

2.4 Tetrodotoxin (TTX)

3

3

4

4

7

2.5 Poisoning Case Due to Consumption of Horseshoe Crab 9

3.0 Materials and Method

3.1 Sampling Site

3.2 Samples Collection

3.3 Sample Extraction and Preparation

3.4 Thin Layer Chromatography (TLC)

3.5 High Performance Liquid Chromatography (HPLC)

3.6 Liquid Chromatography – Mass Spectrometry

11

11

12

12

13

15

15

3.7 Data Analysis 16

4.0 Results and Discussion 17

vii

4.1 Morphometric Measurement Based on Different Location

4.2 Toxin Analysis

4.2.1 Thin Layer Chromatography (TLC)

4.2.2 High Performance Liquid Chromatography (HPLC)

4.2.3 Liquid Chromatography – Mass Spectrometry (LC-MS)

5.0 Conclusion and Recommendation

6.0 References

7.0 Appendices

17

21

21

24

29

35

36

39

viii

List of Tables

Table 1 The difference morphology between the four extant species

of horseshoe crab

6

Table 2 Frequency of symptoms and signs among 245 patients in

Con Buri Hospital after consumption of the toxic eggs of C.

rotundicauda

10

Table 3 Morphometric measurement of horseshoe crab collected

from three different locations.

18

Table 4

Rf value of extracted crude toxin of horseshoe crab

collected from Kabong, Lundu and Miri 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.

22

Table 5

TTX concentration of horseshoe crab collected from

Kabong, Lundu and Miri by HPLC.

25

Table 6

TTX concentration of horseshoe crab collected from

different sampling site by LC-MS.

32

ix

List of Figures

Figure 1 The external anatomy of a horseshoe crab 5

Figure 2 Structure of the first leg 6

Figure 3 Sampling Site ; (1) Kabong; (2) Lundu; (3) Miri 11

Figure 4 Illustration of retention factors (Rf) calculation 14

Figure 5 HPLC of standard (a) with Rt 10.11 and toxin profile (b)

of T.gigas soft tissue

26

Figure 6 HPLC of standard (a) with Rt 10.11 and toxin profile (b)

of C. rotundicauda egg

27

Figure 7 Calibration curve for HPLC 28

Figure 8 Full scan total ion current (TIC) chromatography for ion

spray LC-MS analysis for toxin profile of (a) TTX standard

and (b) selected ion mass chromatograms of standard TTX

is [M+H] = 162.

30

Figure 9 Full scan total ion current (TIC) chromatography for ion

spray LC-MS analysis for toxin profile of egg (a) species

C.rotundicauda and (b) species T.gigas.

31

x

List of Abbreviations

TTX : Tetrodotoxin

TLC : Thin Layer Chromatography

HPLC : High Performance Liquid Chromatography

mm : Millimetre

ml : Millilitre

cm : Centimetre

µm : Micrometre

g : Gram

˚C : Celcius

M : Molar

mM : Mili-Molar

rpm : Round Per Minute

KOH : Potassium Hydroxide

NaOH : Sodium Hydroxide

UV : Ultraviolet

1

1.0 Introduction

Horseshoe crabs are well known as “living fossils” with geological history covering

hundreds of millions of years and an ancestry reaching back 455 million years to the

doorstep of the Cambrian (Tanacredi et al., 2007). The morphology of the extant species is

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 (John et al., 2012). Among

the four species of horseshoe crab present in the world, three species are distributed in

Southeast Asian region and the other one species is distributed at the coastal water of North

America. In Malaysia, three species had been identified which are Tachypleus gigas,

Carcinoscorpius rotundicauda and Tachypleus tridentatus (Chatterji & Noraznawati,

2009).

Horseshoe crab is also very important in economical and ecological aspect. Early

settlers to the New World reported use of horseshoe crabs by Native Americans for food,

tool and to enrich soils for growing crops (Kreamer & Michels, 2009). The blood of

horseshoe crab is important to provide Limulus amebocyte lysate (LAL) that clots in the

presence of minute quantities of bacterial endotoxin and used to ensure that

pharmaceuticals and surgical implants are free from bacteria contamination (Tanacredi et

al., 2007).

In Southeast Asian countries, horseshoe crabs are often catches and consumed as

food. People do not realize the presence of toxic in the horseshoe crab. Food poisoning due

to the consumption of horseshoe crabs have occurred sporadically in Thailand (Dao et al.,

2009). About 100 people were poisoned resulting of 5 deaths in 1995 (Kanchanapongkul,

2008). Two species, Tachypleus gigas and Carcinoscorpius rotundicauda are known to

inhabit Thailand, which responsible for all of the cases of poisoning (Dao et al., 2009).

2

Analyses of toxins in Carcinoscorpius rotundicauda performed by several research

groups have revealed that the species possesses paralytic shellfish poisoning (PSP) toxins

and TTX which indicating that these toxins caused the cases of poisoning. These findings

show that the frequency of occurrence of Carcinoscorpius rotundicauda with a high level

of TTX (Dao et al., 2009). TTX is a major toxin in the eggs (Kungsuwan et al., 1987;

Kanchanapongkul, 2008). It have potent neurotoxin which can cause death with no

effective antidote been found yet.

Many studies about the presence of TTX in the horseshoe crab had been conducted

from the outside of Malaysia. In Malaysia, there are still lack of information obtain

regarding the toxicity of horseshoe crab although it had been consumed by many of the

local people. For this reason, this study must be continued to know the level of TTX in

horseshoe crab especially in Sarawak coastal area. There are some preliminary studies

regarding to the toxicity of horseshoe crab in Sarawak. Perhaps, after finish this study, the

level of TTX can be identified for the safety among the local people who consumed

horseshoe crab as food resources.

Therefore, the study objectives are to:

1) To identify toxin properties of horseshoe crab by using Thin Layer Chromatography

(TLC) and High Performance Liquid Chromatography (HPLC) and Liquid

Chromatography – Mass Spectrometry (LC-MS) methods;

2) To determine the toxicity level in horseshoe crab tissues and eggs.

3

2.0 Literature Review

2.1 Horseshoe Crab and Its Importance

Horseshoe crabs are categorized as benthic communities which prefer calm seas or

estuaries with muddy sandy bottoms for their biogenic activities. They migrate from the

deep water to the shore for breeding purposes (Chatterji & Noraznawati, 2009). Horseshoe

crab provides essential food resource for the migratory birds (Gillings et al., 2007). Their

blood is used to provide Limulus amebocyte lysate (LAL) for biomedical use which apply

in pharmaceuticals and surgical implants (Kreamer & Michels, 2009).

Only four species of the horseshoe crab exist in the world (Kanchanapongkul,

2006). It is interesting to know that among these four species, Limulus Polyphemus and

Tachypleus tridentatus occur in north-south-north direction, whereas, Carcinoscorpius

rotundicauda and Tachypleus gigas occur in east-west-east direction. The interesting facts

are three species of the horseshoe crab, C. rotundicauda, T. gigas and T. tridentatus are

found along the coast of Malaysia (Chatterji & Noraznawati, 2009). Among the four

species, only two can be found in Sarawak, T. gigas and C. rotundicauda (John et al.,

2012).

For breeding purpose, horseshoe crab will migrate to shallow water from the deeper

water. This will occur during the new moon season and full moon season (Jeniffer et al.,

2010). They fertilize externally (Hajeb et al., 2009). Males use modified prosomal

appendages to attach to female. Females deposit egg 7-20 cm below the surface of the sand

(Jeniffer et al., 2010).

4

2.2 Taxonomy

Horseshoe Crab is organism from Kingdom of Animalia. It is categorized under

phylum Arthropod as it is an invertebrate animal which having an external skeleton or

exoskeleton and in subphylum Chelicerata. As horseshoe crab possess of appendages

which are mouthparts at their proximal end, this marine organism had been classified under

Class Merostomata. The Xiphosura is the order of this organism as it includes a large

number of extinct lineages. There are only four extant species of horseshoe crab in the

family of Limulidae.

2.3 Morphology of horseshoe crab

The horseshoe crab’s body is divided into three sections which are telson, carapace

and abdomen as shown in Figure 1. The front section is called the prosoma. The most

obvious characteristic of the prosoma are the two compound eyes which located near the

front and the numerous legs underneath. The middle section is abdomen also as

opisthosoma which attaches to the prosoma by a hinge joint. A hard shell, called carapace

help to covers each part of the horseshoe crab (Gerhart, 2007). Telson is the tail of the

horseshoe crab. This part looks dangerous. These crabs mainly use it for digging and to

help turn itself back over if they get flipped over on the beach.

Horseshoe crab use book gills to get oxygen from the water. If these primitive gills

stay moist, horseshoe crabs can remain out of water up to four days. Horseshoe crabs have

no jaws or teeth. To chew its food, the crab must stimulate walking movements.

5

Figure 1: The external anatomy of a horseshoe crab (Dery, 2005).

For species identification, there are many ways used to recognize the different

horseshoe crab species based on their morphology characteristics. Firstly based on their

shape of telson cross section either round or triangle and secondly based on type and size

of marginal spines. The other differences are shown by Sekiguchi and Nakamura (1979) in

the Table 1.

Basically, the sizes of carapace width for female are larger than male horseshoe

crab. Large sizes in females are important in order to tow males during the spawning

season and a large body can carry more eggs (Botton & Loveland, 1992). In addition, the

present of pedipals/first leg looks like “boxing glove” as shown in Figure 2 can

differentiate the sex of the horseshoe crab.

6

Table 1: The difference morphology between the four extant species of horseshoe crab (Sekiguchi &

Nakamura, 1979).

Figure 2: Structure of the first leg a) female b) male (Gerhart, 2007).

a b

7

2.4 Tetrodotoxin (TTX)

Tetrodotoxin (TTX), a pufferfish toxin named after its order name

Tetraodontiformers, is the principle of puffer fish poisoning. This toxin is one of the most

potent nonproteinaceous toxins as well as the best known marine natural toxins (Asakawa

et al., 2012). It can be found in both terrestrial and marine organism. TTX is a heat stable

toxin in neutral to weakly acidic solutions means it does not decompose even by cooking at

high temperature (Arakawa et al., 2010). TTX was isolated for the first time as a

crystalline prism from toxic pufferfish ovaries by Yokoo.

For freshwater and brackish water species, TTX concentrated on skin layer

(Noguchi & Arakawa, 2008). In horseshoe crab study, TTX widely found concentrated in

soft tissue for male and in egg for female (Tanu & Noguchi, 1999). The causative species

is C. rotundicauda (Kanchanapongkul, 2008).

TTX is believed not come from the crab itself but it is from the prey that they ate.

Diet feeding for horseshoe crab is arthropods, mollusks and detritus that may contain TTX

carrying bacteria (Tanu & Noguchi, 1999). This toxin has been detected in many aquatic

organism including the other vertebrates and invertebrates. A few intestinal bacteria of

TTX-bearing animals were found to produce TTX. This situation suggested that the TTX

was being passed along the food web (Asakawa et al., 2012).

TTX blocks sodium ion channel of the nerve cell membrane which resulting

ataxia, diarrhea, respiratory insufficiency, vomiting, paralysis and even rapid death in

seriously intoxicated humans (Chulanetra et al., 2011). The type and variety of symptoms

depend on the amount of toxin ingested, age and health of the victim (Noguchi & Ebesu,

2001). TTX act on both the central and peripheral nervous system. Sensory neurons are

affected first and then motor neurons at a higher dose of TTX (Wan et al., 2007).

8

Nowadays, there is no effective antidote or specific treatment for TTX in order to

remove the toxin from the human body. The victim can be help by using artificial

respiration treatment. This treatment helps to slowdown the death process without any

significant treatment. Since there is no antidotes for TTX poisoning, treatment is mainly

supporting therapy, normal saline infusion, mechanical ventilation for oxygen supply,

gastric emptying procedure, treatment with dopamine and normal saline infusion for

distending intravascular volume (Noguchi & Arakawa, 2008).

Noguchi and Arakawa (2008) explained that the TTX is formed by associated or

parasitic bacteria that are right accumulated inside the puffer body and not obtained via the

food chain. There are low number amount of TTX produced by bacteria to account for the

accumulation in puffer fish.

9

2.5 Poisoning Case Due to Consumption of Horseshoe Crab

There are many cases reported regarding to the poisoning of TTX to the human in

the world especially from the puffer fish. But in Malaysia, there was lack cases reported

regarding horseshoe crab ingestion. This is maybe due to lack information from the

patients who refuse to go to the hospital for medical check-up purpose. Therefore, they are

no proper clinical data was recorded.

Before 1994, there were only six cases of horseshoe crab poisoning reported in

Thailand. Since 1994, horseshoe crab poisoning cases were increased in Chon Buri which

is located on the eastern coast of Thailand. The causative species is C. rotundicauda.

Between the period of January 1994 to December 2006, 280 cases of TTX poisoning

following ingestion of the toxic eggs of the horseshoe crab C. rotundicauda were admitted

to the Chon Buri Hospital (Kanchanapongkul, 2008). Table 2 shows the frequency of

symptoms and signs among 245 patients after consumption of the toxic eggs C.

rotundicauda.

From all the three species, C. rotundicauda is the toxin species, while T. gigas and

T. tridentatus are non toxic (Kungsuwun et al., 1987). Tanu and Noguchi (1999) said that

the horseshoe crab, C. rotundicauda in Bangladesh also has high level of tetradotoxin. In

Cambodia, the toxicity studies in horseshoe crab were continued. Cambodian horseshoe

crab, C. rotundicauda also contain high toxicity level and not suitable to eat (Ngy et al.,

2007).

10

Table 2: Frequency of symptoms and signs among 245 patients in Con Buri Hospital after consumption of

the toxic eggs of C. rotundicauda (Kanchanapongkul ,2008).

Symptoms and signs N (%)

Circumoral, lingual numbness 240 (98)

Hands and feet numbness 232 (94.7)

Weakness 146 (59.6)

Dizziness, vertigo 133 (54.3)

Nausea, vomiting 129 (52.6)

Transient hypertension 97 (39.6)

Respiratory paralysis 68 ( 27.7)

Fixed dilated pupils 36 (14.7)

Ophthalmoplegia 30 (12.2)

Hypotension (BP <90/60 mmHg) 14 (5.7)

Polyuria 1 (0.4)

*N = Frequency / number of patients

11

3.0 Materials and Methods

3.1 Sampling Sites

Samples of horseshoe crab had been collected at Kabong, Lundu and Miri, Sarawak

(Figure 3) along the intertidal zone randomly. The coordinates of the selected area had

been recorded by using Global Positioning System (GARMIN, 62S).

Figure 3: Sampling Site: (1) Kabong ; (2) Lundu ; (3) Miri (Source: Google Maps, 2015)

1

2

U

1

2

1

3

1

3

1

1 2 3

South China

Sea

Sarawak

12

3.2 Samples Collection

Sixty three individuals of adult horseshoe crabs (male and female) had been

collected by fisherman. The samples were kept alive and transported to Ecotoxicology

Laboratory, Faculty of Resource Science and Technology, UNIMAS for further analysis.

For each sample, the measurement of total carapace length (from the tip to the tip of the

telson), carapace width and body weights for both sexes were recorded. The total carapace

length and the carapace width were measured to the nearest centimeter (cm) using a

measuring board (Wildco, Model 118), meanwhile the body weight was measured using a

single pan electronic balance (Adventurer, ARA 520) to the nearest gram (g).

3.3 Sample Extraction and Preparation

The specimens were individually dissected into the soft tissues and eggs (female)

from live specimens and kept at -20˚C for prior analysis. Toxin was extracted from

horseshoe crab tissues according to Diener et al. (2007). Each tissue was minced by using

mortar and pastel and 3 g of tissue were extracted with 9 ml of 0.03 M acetic acid using an

ultrasonic probe for 1 minute. Then, the homogenate sample was heated in a water bath for

10 minutes at 100˚ C. The homogenate sample was cooled in ice cube. After that, the

homogenate was centrifuged at 8000 rpm for 30 minutes. The sample extract was filtered

through a 0.45 µm nylon membrane filter and the filtrate was analyzed using HPLC based

on method recommended by Yotsu et al. (1989). The extraction steps were repeated for

egg extraction.

13

3.4 Thin Layer Chromatography (TLC)

The toxin extracted was used for toxin identification and this process was used

Thin-Layer Chromatography (TLC). Silica gel-60 F₂₅₄ pre-coated plate was used. The TLC

plate was sliced into 4 cm width and 5 cm long. Then, 0.5 cm line was draw below and

above by using the pencil. The spot was marked along the line below with at least 1 cm

gaps in between. Figure 4 showed the diagram of TLC plate. The extraction of crude toxin

was spotted at the marked spot drawn by a pencil below the line. The TLC plate was dried

up with the hair drier. The solutions were prepared in the proportion of pyridine: ethyl

acetate: acetic acid: water (15:5:3:4) and butanol: acetic acid: water (12:3:5) (Noguchi &

Mahmud, 2001). The plate was placed in the butanol- acetic acid- water solvent.

After the solvent reach the line drawn at the above part of TLC plate, the plate was

taken out from the solvent. 10% KOH was sprayed at the plate. The plate was dried with

dryer until the plate completely dry. Then, the spot was visualized under the UV light.

Pencil was used to mark the spot. The steps were repeated by using solvent pyridine- ethyl

acetate- acetic acid- water. The Rf values were calculated by using the Rf formulae. The Rf

value were compared with the standard. Ascent Scientific (TTX standard 319.27 HPLC

grade) standard was used.