DETERMINATION OF TOXIN PROPERTIES AND … of Toxin Properties and... · Million thanks to Mr....
Transcript of DETERMINATION OF TOXIN PROPERTIES AND … of Toxin Properties and... · Million thanks to Mr....
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
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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
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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:
________________________
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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.
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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).
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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)
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3
4
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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
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3.7 Data Analysis 16
4.0 Results and Discussion 17
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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
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List of Tables
Table 1 The difference morphology between the four extant species
of horseshoe crab
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Table 2 Frequency of symptoms and signs among 245 patients in
Con Buri Hospital after consumption of the toxic eggs of C.
rotundicauda
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Table 3 Morphometric measurement of horseshoe crab collected
from three different locations.
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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.
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Table 5
TTX concentration of horseshoe crab collected from
Kabong, Lundu and Miri by HPLC.
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Table 6
TTX concentration of horseshoe crab collected from
different sampling site by LC-MS.
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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
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Figure 6 HPLC of standard (a) with Rt 10.11 and toxin profile (b)
of C. rotundicauda egg
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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.
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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.
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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
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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).
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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.
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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).
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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.
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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.
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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
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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).
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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.
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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).
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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
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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
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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.
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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.