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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.
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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
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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
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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).
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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
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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.