THE POTENTIAL OF ORNAMENTAL PLANT, SANSEVIERIA …

THE POTENTIAL OF ORNAMENTAL PLANT,

SANSEVIERIA TRIFASCIATA TO INHIBIT THE

GROWTH OF ALEXANDRIUM TAMIYAVANICHII, A

TOXIC DINOFLAGELLATE

BY

IMA AMIRAH BINTI MOHD SUBERI

A thesis submitted in fulfilment of the requirement for the

degree of Master of Science (Biotechnology)

Kuliyyah of Science

International Islamic University Malaysia

JULY 2017

ii

ABSTRACT

Harmful algal bloom (HAB) is a phenomenon that can cause harm to human health

such as food poisoning and loss in aquaculture industries. However, in Malaysia,

research on mitigation of HAB is very limited. This study aims to determine the

potential of ornamental plant Sansevieria trifasciata in inhibiting the growth of HAB

species, Alexandrium tamiyavanichii. The crude extracts and active fractions of this

plant were tested on A. tamiyavanichii at different concentrations (0.001, 0.01, 0.1,

0.5, 1, 1.5, 2 and 2.5 mg/mL) for 24 hours. Toxicity test were conducted on artemia

using different concentrations of crude extracts (10, 50, 100 mg/mL) and active

fractions (10, 50, 100 and 500 mg/mL). Phytochemical screening was done to

determine the constituents by using standard procedures. Results obtained showed that

all concentration with different extracted method tested inhibited the growth of A.

tamiyavanichii. As the concentration increase, the pH of medium will decrease and

cause the removal efficiency to increase. The best removal efficiency was showed by

methanol fraction from ethanol fresh plant extract which was 93.86% at concentration

0.1 mg/mL and pH 7.46. Artemia showed 100% mortality at concentration 50 mg/mL

for ethanol fresh plant extract. For ethanol dried plant extract and distilled water fresh

plant extract, 100% mortality of artemia was at 50 mg/mL while distilled water dried

plant extract was at 100 mg/mL. All fractions showed 100% mortality of artemia at

concentration 500 mg/mL. In phytochemical screening tests, compounds detected in

ethanol fresh and dried plant extracts were alkaloid, tannins, glycoside, reducing sugar

and terpenoids whereas in distilled water fresh and dried plant extract detected

reducing sugar and terpenoids. For active fractions, all showed the presence of

alkaloid, tannins, glycoside, reducing sugar and terpenoids. The compounds detected

might have the function in inhibiting the targeted algae. Further study needed in order

to determine the bioactivity and its effect on HABs’ mitigation. This study showed

that S. trifasciata has the potential to be used for mitigation of HAB particularly A.

tamiyavanichii which help to minimize the impact of HAB in aquaculture field and

human or animal health.

iii

خلاصة البحث

إن الطحالب الدضرة الدنتشرة ىي أحد الظواىر الدؤذية للصحة البشرية لأثرىا في التسمم الغذائي والخسائر في صناعات الإستزراع الدائي. وعلى الرغم من ذلك فإن الأبحاث الدتركزة على التخفيف من انتشار

محدود جدا. تهدف ىذه الدراسة إلى التعرف على إمكانيات نبتة الزينة الطحالب الدضرة في ماليزيا ( في تثبيط نمو Sansevieria trifasciataالدسماة الذلب الثلاثية الأحزمة أو سانسيفييرا تريفاشياتا )

(. تم Alexandrium tamiyavanichiiالطحالب الدضرة من نوع أليكساندريوم تاميافانيتشي )ت والأجزاء النشطة منها ضد طحالب الأليكساندريوم تاميافانيتشي على تراكيز مختلفة اختبار الدستخلصا

ساعة. تم 24مغ/مل( لددة 2.5، و 2، و 1.5، و 1، و 0.5، و 0.1، و 0.01، و 0.001)، و 10القيام باختبار السمية ضد الأرتيميا )الروبيان الدلحي( للمستخلصات الخامة على تراكيز مختلفة )

مغ/مل(. تم 500، و 100، و 50، و 10مغ/مل( وللأجزاء النشطة على التراكيز ) 100 ، و50القيام بالدسح النباتي الكيميائي لتحديد الدكونات باتباع الطرق القياسية. أظهرت النتائج أن جميع التراكيز

فضت حموضة وبمختلف طرق الاستخلاص الدختَبرة قد أثبتت نمو الأليكساندريوم تاميافانيتشي. انخالوسط الاختباري بازدياد التركيز وأزالت أيضا احتمال ارتفاعها. تمت ملاحظة أن أفضل كفاءة للإزالة

% على 93.83كانت بالجزء الديثانولي من الاستخلاص الإيثانولي للنبات الطازج، حيث كانت بنسبة % على التركيز 100 . أظهرت الأرتيميا معدل موت بلغ7.46مغ/مل وحموضة معدلذا 0.1تركيز مغ/مل لدستخلصات الديثانول للنبات الطازج. أما بالنسبة لدستخلصات الإيثانول للنبات المجفف 50

مغ/مل، أما 50% على تركيز 100ومستخلصات الداء الدقطر للنبات النضر، كان معدل الدوت كل الأجزاء معدل موت مغ/مل. أظهرت 100مستخلصات الداء الدقطر للنبات المجفف كان على تركيز

مغ/مل. في الاختبارات النباتية الكيميائية، تم العثور على 500% على تركيز 100الأرتيميا بنسبة قلويدات، وتانينات، وقليكوزيدات، وسكر مختزل، وتيربينويدات. أما مستخلصات الداء الدكرر للنبات

ما بالنسبة للأجزاء النشطة فقد أظهرت كلها الطازج والمجفف فقد ظهر فيها سكر مختزل وتيربينويدات. أوجود قلويدات، وتانينات، وقليكوزيدات، وسكر مختزل، وتيربينويدات. القدرة التثبيطية لنمو الطحالب قد تمكن في ىذه الدركبات. ىناك حاجة إلى الدزيد من الدراسات لتحديد النشاط الحيوي وتأثيره التثبيطي

راسة أن لدى الذلب الثلاثي الأحزمة الإمكانية في استعمالو كمثبت لنمو على نمو الطحالب. أظهرت الدالطحالب الدنتشرة الدضرة، وبالتحديد نوع الأليكساندريوم تاميافانيتشي، مدا قد يساعد في التقليل من أثر

الطحالب الدنتشرة الدضرة في حقول الاستزراع الدائي وعلى الصحة البشرية والحيوانية.

iv

APPROVAL PAGE

I certify that I have supervised and read this study and that in my opinion, it conforms

to acceptable standards of scholarly presentation and fully adequate, in scope and

quality, as a thesis for the degree of Master of Science (Biotechnology).

.……………………………............

Normawaty Mohammad Noor

Supervisor

.……………………………............

Deny Susanty

Co-Supervisor

…………………………….............

Yukinori Mukai

Co-Supervisor

I certify that I have read this study and that in my opinion it conforms to acceptable

standards of scholarly presentation and fully adequate, in scope and quality, as a thesis

for the degree of Master of Science (Biotechnology).

…………………………….............

Shahbudin bin Saad

Examiner

…………………………….............

Zainudin bin Bachok

External examiner

This thesis was submitted to the Department of Biotechnology and is accepted as a

fulfilment of the requirement for the degree of Master of Science (Biotechnology).

…………………………….............

Suhaila Mohd Omar

Head,Department of Biotechnology

This thesis was submitted to the Kuliyyah of Science and is accepted as a fulfillment

of the requirement for the degree of Master of Science (Biotechnology).

…………………………………….

Kamaruzzaman bin Yunus

Dean, Kuliyyah of Science

v

DECLARATION PAGE

I hereby declare that this thesis is the result of my own investigations, except where

otherwise stated. I also declare that it has not been previously or concurrently

submitted as a whole for any other degrees at IIUM or other institutions.

Ima Amirah binti Mohd Suberi

Signature………………………………… Date…………………………..

vi

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION OF

FAIR USE OF UNPUBLISHED RESEARCH

THE POTENTIAL OF ORNAMENTAL PLANT,

SANSEVIERIA TRIFASCIATA TO INHIBIT THE GROWTH

OF ALEXANDRIUM TAMIYAVANICHII, A TOXIC

DINOFLAGELLATE

I declare that the copyright holder of this thesis is jointly owned by the student

and IIUM.

Copyright © 2017 by Ima Amirah binti Mohd Suberi and International Islamic University

Malaysia. All rights reserved.

No part of this unpublished research may be reproduced, stored in a retrieval

system, or transmitted, in any form or by any means, electronics, mechanical,

photocopying, recording or otherwise without prior written permission of the

copyright holder except as provided below.

1. Any material contained in or derived from this unpublished research

may only be used by others in their writing with due

acknowledgement.

2. IIUM or its library will have the right to make and transmit copies

(print or electronic) for institutional and academic purposes.

3. The IIUM library will have the right to make, store in a retrieval

system and supply copies of this unpublished research if requested

by other universities and research libraries.

By signing this form, I acknowledged that I have read and understand the IIUM

Intellectual Property Right and Commercialization policy,

Affirmed by Ima Amirah binti Mohd Suberi

………………………… …………………………

Signature Date

vii

ACKNOWLEDGEMENTS

I would like to express my gratitude towards my main supervisor, Assoc. Prof. Dr.

Normawaty Mohammad Noor who always support, advised and give a hand

throughout my study. Also lots of thanks to my co-supervisors, Assoc. Prof. Dr. Deny

Susanti and Assoc. Prof. Dr. Yukinori Mukai for their useful advises and opinion.

Not to forget my friend, Sister Nor Salamah binti Mohamad Hidayat who also

help me during my difficulties. To my family especially my husband that always

understands and supports me until I manage to complete my study.

Lastly, I would like to thank Kuliyyah of Science, IIUM for the facilities

provided. This study also partially supported by FGRS grant from Ministry of Higher

Education, Malaysia.

viii

TABLE OF CONTENTS

Abstract……………………………………………………………………………... ii

Abstract in Arabic…………………………………………………………………... iii

Approval Page………………………………………………………………............. iv

Declaration Page…………………………………………………………………..... v

Copyright Page……………………………………………………………………… vi

Acknowledgements……………………………………………….………………… vii

List of tables………………………………………………………………………… x

List of figures…………………………………………………………..…………… xi

List of abbreviation………………………………………………………….……… xvii

List of symbols……………………………………………………………………… xviii

CHAPTER 1: INTRODUCTION…………………………………………………

1.1 Introduction…………………………………………………………….

1.2 Objectives………………………………………………………………

1.3 Hypothesis……………………………………………………………...

1.4 Significant of study…………………………………………………….

19

19

21

21

22

CHAPTER 2: LITERATURE REVIEW…………………………………………

2.1 Harmful algal bloom…………………………………………………...

2.2 HABs problem in Malaysia…………………………………………….

2.3 Alexandrium tamiyavanichii…………………………………………...

2.4 Mitigation of HAB …………………………………………………………...

2.4.1 Sansevieria trifasciata…………………………………………...

2.5 Phytochemical screening……………………………………………….

2.6 Toxicity test…………………………………………………………….

23

23

25

27

28

30

32

33

CHAPTER 3: METHODOLOGY………………………………………………...

3.1 Establishment of A. tamiyavanichii culture……………………………

3.2 Extraction of Sansevieria trifasciata……………………………………….

3.3 Fractionation process…………………………………………………..


3.5 Test of crude extracts and active fractions on A. tamiyavanichii……….

3.6 Toxicity test on brine shrimp (artemia)………………………………...

34

34

35

36

37

40

41

CHAPTER 4: RESULTS AND FINDINGS……………………………………...


4.2 Test of crude extracts and active fractions on A. tamiyavanichii……….

4.2.1 Ethanol fresh plant extract………………………………………

4.2.2 Ethanol dried plant extract………………………………………

4.2.3 Distilled water fresh plant extract……………………………….

4.2.4 Distilled water dried plant extract……………………………….

4.2.5 DCM fraction from ethanol fresh plant extract………………….

4.2.6 Methanol fraction from ethanol fresh plant extract……………...

43

43

44

44

49

54

59

64

69

ix

4.2.7 DCM fraction from ethanol dried plant extract………………….

4.2.8 Methanol fraction from ethanol dried plant extract……………..

4.3 Toxicity test on brine shrimp (artemia)………………………………...

4.4 Summary of results…………………………………………………….

4.5 Morphology observations……………………………………………...

74

79

84

87

88

CHAPTER 5: DISCUSSION……………………………………………………...


5.2 Test of crude extracts and active fractions on A. tamiyavanichii………

5.3 Toxicity test…………………………………………………………….

5.4 Conclusion……………………………………………………………..

89

89

91

96

97

REFERENCES…………………………………………………………………….. 99

x

LIST OF TABLES

Table No. Page No.

4.1 Phytochemical screening on crude extracts and active fractions (fresh

and dried plant sample).

43

4.2 Summary results of crude extracts. 87

4.3 Summary results of active fractions. 87

5.1 Comparisons of phytochemical compounds detected in S. trifasciata

in the study with other.

91

xi

LIST OF FIGURES

Figure No. Page No.

2.1 Alexandrium tamiyavanichii 27

2.2 Sansevieria trifasciata 31

3.1 Cultures of algae in the lab 34

3.2 (a) S. trifasciata plant, (c) plants were cut into small pieces, (d)

dried plant were blended and (e) extraction process rotary

evaporator

36

3.3 (a) VLC column and (b) extract with silica gel 37

3.4 Phytochemical screening process 40

4.1 Removal efficiency and cell density of 0.001 mg/mL ethanol

fresh plant extract on A. tamiyavanichii

46



46

4.3 Removal efficiency and cell density of 0.1 mg/mL ethanol fresh

plant extract on A. tamiyavanichii

46



47

4.5 Removal efficiency and cell density of 1 mg/mL ethanol fresh


47



47

4.7 Removal efficiency and cell density of 2 mg/mL ethanol fresh

plant extract on A. tamiyavanichi

48



48

4.9 Removal efficiency of ethanol fresh plant extract on A.

tamiyavanichii

48

xii


dried plant extract on A. tamiyavanichii

51



51

4.12 Removal efficiency and cell density of 0.1 mg/mL ethanol dried


51



52

4.14 Removal efficiency and cell density of 1 mg/mL ethanol dried


52



52

4.16 Removal efficiency and cell density of 2 mg/mL ethanol dried


53



53

4.18 Removal efficiency and cell density of ethanol dried plant

extract on A. tamiyavanichii

53

4.19 Removal efficiency and cell density of 0.001 mg/mL distilled

water fresh plant extract on A. tamiyavanichii

56



56



56



57

4.23 Removal efficiency and cell density of 1 mg/mL distilled water


57

4.24 Removal efficiency and cell densityof 1.5 mg/mL distilled


57



58

4.26 Removal efficiency and cell density of 2.5 mg/mL distilled 58

xiii


4.27 Removal efficiency and cell density of distilled water fresh


58


water dried plant extract on A.tamiyavanichii

61


water dried plant extract on A. tamiyavanichii

61



61



62



62



62



63



63

4.36 Removal efficiency and cell density of distilled water dried


63

4.37 Removal efficiency and cell density of 0.001 mg/mL DCM

fraction from ethanol fresh plant extract on A. tamiyavanichii

66



66



66



67

4.41 Removal efficiency and cell density of 1 mg/mL DCM fraction

from ethanol fresh plant extract on A. tamiyavanichii

67



67

xiv


from ethanol fresh plant extract on A. tamiyavanichii

68



68

4.45 Removal efficiency and cell density of DCM fraction from

ethanol fresh plant extract on A. tamiyavanichii

68

4.46 Removal efficiency of 0.001 mg/mL methanol fraction from


71



71



71



72

4.50 Removal efficiency of 1 mg/mL methanol fraction from ethanol


72



72



73



73

4.54 Removal efficiency of methanol fraction from ethanol fresh


73


fraction from ethanol dried plant extract on A. tamiyavanichii

76



76



76



77

4.59 Removal efficiency and cell density of 1 mg/mL DCM fraction 77

xv

from ethanol dried plant extract on A. tamiyavanichii



77


from ethanol dried plant extract on A. tamiyavanichii

78



78

4.63 Removal efficiency and cell density of DCM fraction from

ethanol dried plant extract on A. tamiyavanichii

78



81



81



81



82



82



82



83



83

4.72 Removal efficiency of methanol fraction from ethanol dried


83

4.73 LC50 of ethanol fresh plant extract on artemia 85

4.74 LC50 of ethanol dried plant extract on artemia 85

4.75 LC50 of distilled water fresh plant extract on artemia 85

4.76 LC50 of distilled water dried plant extract on artemia 85

xvi

4.77 LC50 of DCM fraction from ethanol fresh plant extract on

artemia

86

4.78 LC50 of DCM fraction from ethanol dried plant extract on

artemia

86

4.79 LC50 of methanol fraction from ethanol fresh plant extract on

artemia

86

4.80 LC50 of methanol fraction from ethanol dried plant extract on

artemia

86

4.81 a: the ruptured cell, b and c: untreated cell

88

xvii

LIST OF ABBREVIATION

HAB Harmful algal bloom

PSP Paralytic shellfish poisoning

DSP Diarrheic shellfish poisoning

CFP Ciguatera fish poisoning

ASP Amnesic shellfish poisoning

sp. Species

L:D Light:dark

µm Micrometer

g Gram

VLC Vacuum liquid chromatography

mg Milligram

HCL Hydrochloric acid

mL Mililitre

mg/mL Milligram per mililitre

RE Removal efficiency

mm Milimetre

etOH Ethanol

MeOH Methanol

DCM Dichloromethane

dH2O Distilled water

Cells/mL Cells per mililitre

xviii

LIST OF SYMBOLS

% Percentage

ºC Degree celcius

19

CHAPTER 1

INTRODUCTION

1.1 INTRODUCTION

Harmful algal bloom (HAB) is a phenomenon occurred when there is single cell algae

of microscopic or macroscopic phytoplankton grow rapidly in huge number. It also

called as red tides as they composed of red pigments content dinoflagellates and cause

water change to red colour when bloom. However, the bloom colour is depending on

the algae type present and also their pigmentation. Over the last decade, this event had

been increasingly reported in various countries including Malaysia.

First HAB event in Malaysia was reported in year 1976 (Roy 1977). Bloom of

harmful algae caused big impact especially to our aquaculture field and economy. In

the year 2005 to 2006, there were bloom events in Penang that caused massive fish

kill and huge loss estimated not less than RM20 millions (Lim et al., 2012). The

causative organism responsible for this incident still remained unknown. Then, bloom

occurred in Sebatu, Melaka involved Alexandrium tamiyavanichii and caused three

people were hospitalised after consuming cultured green mussels that obtained from

newly established mussel’s farm (Lim et al., 2012). Recently, bloom of this species

was reported in Kuantan port and several people were hospitalised after consuming

contaminated shellfish. Others HAB reports that caused loss to economy were by

Cochlodinium polykrikoides at Kota Kinabalu, Sabah (Anton et al., 2008) and Perak

(Harun, Mohammad-Noor and Shahbudin, 2013). This indicates that HAB event in

20

Malaysia should be given great intention due to its high impact to aquaculture and

human health.

Several ways of prevention and mitigation have been reported to inhibit HABs.

One of the methods is by applying clay sprayed to affected water. This method has

been applied in Japan, Australia, South Korea and China (Pan et al., 2011). Harmful

algal cells in the affected water will be removed through flocculation and

sedimentation when clay is used (Pan et al., 2011). Other studies on HAB inhibition

used modified poly aluminium chloride-treated clay, acid treated clay (Pan et al.,

2011) and barley straw. Study conducted by Zhou et al., (2008) showed that man-

made diallyl trisulfide and garlic solution have similar algicidal affect towards algal

bloom. Both treatments introduced towards Alexandrium tamarense could inhibit the

growth at certain concentration.

In addition, research by Wei-dong et al., (2009) showed that extract of Chinese

fir has the potential to inhibit the growth of Alexandrium tamarense. Other studies

showed that Heterosigma akashiwo and Alexandrium tamarense effectively be

inhibited using seaweed such as Ulva pertusa, Corallina pilulifera and Sargassum

thunbergi (Renjun et al., 2007). All of these macroalgae released some

allelochemicals which able to inhibit the growth of both microalgae. Use of

allelopathy for HABs control became popular among researchers nowadays due to its

low cost, effectiveness and environmental friendly. However, in Malaysia, mitigation

of HABs is still in initial stage.

Therefore, in this study an attempt was made to use extract from ornamental

plant, Sansevieria trifasciata which commonly known as mother-in-law’s tongue or so

called snake plant to mitigate HAB particularly Alexandrium tamiyavanichii. There

21

are approximately 70 distinct species of snake plant that have been identified (Alan

and Thomas, 1988). It is a succulent plant and originally found in tropical central

Africa and drier part of eastern and southern Africa. The leaves are flat and nearby

horizontal in shape (Carlquist and Schneider, 2007) with length about 70 to 100 cm

and 5 to 6 cm wide (Gautam et al., 2012). Previous studies showed that the species has

potential in medicinal, fodder, soil conservation and fibre (Khalumba et al., 2005).

Result from this study will be used in the management and mitigation of HAB event

in Malaysia.

1.2 OBJECTIVES

1. To isolate locally Alexandrium tamiyavanichii and establish into clonal culture.

2. To produce ethanol and water crude extract from fresh and dried plant sample of

Sansevieria trifasciata.

3. To produce active fractions from ethanol crude extract of fresh and dried plant

sample of Sansevieria trifasciata.

4. To determine the effect of crude extracts and active fractions on Alexandrium

tamiyavanichii and artemia.

5. To determine the constituents of phytochemical in Sansevieria trifasciata.

1.3 HYPOTHESIS

1. Clonal culture from locally Alexandrium tamiyavanichii will be successfully

isolated and cultured.

22

2. Two fractions will be obtained with different polarities which are dichloromethane

and methanol fractions.

3. Crude extracts and active fractions will inhibit the growth of Alexandrium

tamiyavanichii but there will be no effects on artemia.

4. Several important phytochemical constituents will be detected in the plant.

1.4 SIGNIFICANT OF STUDY

Lots of cases involving harmful algal bloom have been globally reported including

Malaysia. HAB events were known to cause tremendous loss in economic, disturbing

to aquaculture field and harmful to human and animal health. Therefore, urgent

actions needed in order to minimize the effects. So, due to positive result obtained

from preliminary study using S. trifasciata on C. polykrikoides, extracts and active

fractions of this plant were used in this research for A. tamiyavanichii mitigation.

Plant extracts and fractions were used in this study due to environmental friendly

compared to method that use chemical.

23

CHAPTER 2

LITERATURE REVIEW

2.1 HARMFUL ALGAL BLOOM

Harmful algal bloom (HAB) is a phenomenon that occurs throughout seawater and

freshwater cause by many factors including increasing of nutrient level which

contributed mostly from human activities such as aquaculture. The bloom of algae

normally is characterized by a high cell density of a single species. There are two

types of algal blooms i.e; toxic and non-toxic blooms. Toxic HAB is caused by

species of algae that has the capability to produce toxin and could harm human health

and aquaculture industry. Non-toxic HAB is caused by high cell density of a species

and cause problem to aquaculture industry.

Harmful algal bloom is caused by a group of phytoplankton (microalgae) such

as dinoflagellates, diatoms and blue green algae. Dinoflagellate is a free swimming

microalga. Examples of dinoflagellate that can cause harmful algal bloom are

Alexandrium spp., Pyrodinium bahamense, and C. polykrikoides. Then, diatom is a

dominant microalgae in the marine water and the most studied HAB species is

Pseudonitzschia spp.. For blue green algae, most of the species are freshwater. Among

species that have been identified as HAB species are Anabaena spp., Aphazinomenon

sp., Cylindrospermum sp., Microcystis, Oscillatoria, Nostoc, Hapalosiphon and

Nodularia (Gires, Lew and Asmat, 2002).

24

There are several seafood toxicity that globally report which are paralytic

shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), ciguatera fish

poisoning (CFP), amnesic shellfish poisoning (ASP) and cyanobacteria toxins. For

PSP, toxin producers involved are P. bahamense var. compressum, A. tamiyavanichii

and A. minutum whereas bivalve mollusks are the primary vector. Main symptoms

showed by people that affected by PSP are paralyzed and death due to respiratory

failure (Gires, Lew and Asmat, 2002). Dinoflagellates Dinophysis, P. lima and P.

micans are toxin producers that responsible for DSP while mussel is the primary

vector. Diarrhea is the main symptom showed by people affected by this seafood

toxification (Gires, Lew and Asmat, 2002).

Benthic dinoflagellates involves in CFP as toxic producers are G. toxicus,

Ostreopsis spp. and Coolia spp. Marine fishes are the primary vectors responsible for

CFP and main symptoms showed by affected people also paralyzing and in extreme

cases death (Gires, Lew and Asmat, 2002). For ASP, it is known to be caused by

diatoms such as Pseudo-nitzschia. This algal produced domoic acid and contaminate

anchovies (Gires, Lew and Asmat, 2002). The final one is cyanobacteria toxins such

as anatoxin-a, microcystin and nodularin that caused by cyanobacteria or bluegreen

algae. All these toxins are harmful and can cause death. For anatoxins-a, species

involved are Anabaena spp., Aphazinomenon sp. and Cylindrospermum sp..

Microcystin toxin is produced by species of Microcystis, Oscillatoria, Anabaena,

Nostoc and Hapalosiphon while nodularins is produced by several species of

Nodularia (Gires, Leaw and Asmat, 2002).

According to Sunda et al., (2006), most of the red tide events documented

were caused by dinoflagellates which in bloom condition will discolour the effected

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