use of algae in genetic ecotoxicology assessment in marine system

642 Pages·2013·9.56 MB·English

EFFECT OF SELECTED CHEMICAL
CONTAMINANTS ON GROWTH, BIOCHEMICAL
COMPOSITION, DNA DAMAGE AND
SUPEROXIDE DISMUTASE ACTIVITY IN FOUR
MARINE ALGAE

EMIENOUR MUZALINA BINTI MUSTAFA

FACULTY OF SCIENCE
UNIVERSITY OF MALAYA
KUALA LUMPUR
MALAYSIA

2013
EFFECT OF SELECTED CHEMICAL
CONTAMINANTS ON GROWTH, BIOCHEMICAL
COMPOSITION, DNA DAMAGE AND
SUPEROXIDE DISMUTASE ACTIVITY IN FOUR
MARINE ALGAE

EMIENOUR MUZALINA BINTI MUSTAFA

THESIS SUBMITTED IN FULFILMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

INSTITUTE OF BIOLOGICAL SCIENCES
FACULTY OF SCIENCE
UNIVERSITY OF MALAYA
KUALA LUMPUR
MALAYSIA

2013
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ABSTRACT

One of the important tasks of environmental monitoring is the detection of
potentially hazardous compounds such as chemical contaminants that can induce
oxidative damage in aquatic organisms. Our objective is to investigate the effect of
selected chemical contaminants (metals, textile dyes and organophosphate pesticides) on
growth (chlorophyll a, carotenoid) , biochemical composition (carbohydrate, protein and
lipid), DNA damage ([Random Amplified Polymorphic DNA (RAPD) and AP-site
(Abasic-site)] and Superoxide dismutase (SOD) enzyme activity in four tropical marine
algae, Chlorella vulgaris, Tetraselmis tetrahele, Boergesenia forbesii and Ventricaria
ventricosa after short term (four days) and long term (ten days) exposure under
laboratory conditions. Results will be used to assess the suitability of the different end-
points (biomarker) and algal species for use in development of bioassays. The algae were
exposed to different concentrations of twelve chemical contaminants including seven
metals [Cadmium (Cd); Cobalt (Co); Chromium (Cr); Copper (Cu); Iron (Fe);
Manganese (Mn) and Zinc (Zn)], three textile dyes [Supranol Br. Red 3Bur (Acidic dye);
Astrazon Red FBL (Basic dye) and Lanaset Red 2GA (Metal complex dye)] and two
organophosphate pesticides [Malathion and Dichlovos] in a range of concentrations
(0.01, 0.1, 1, 10, 100 and 500 mg/L) in Provasoli medium without EDTA using conical
flask (for microalgae: Chlorella vulgaris and Tetraselmis tetrahele) and multiwell plates
(for macroalgae: Boergesenia forbesii and Ventricaria ventricosa) with test volumes of
500mL and 10mL respectively, for 4 and 10 days under controlled conditions. The
growth rate and IC values (concentration of toxicants estimated to inhibit 50% algal
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growth relative to control) were determined on day 4 and day 10 based on Chl a content.
The biochemical composition (carbohydrate, protein and lipid) and SOD activity were
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also assessed. RAPD and AP-site content analysis was conducted on DNA extracted
from the treated samples. The results showed that, the carbohydrate, protein, lipid and
AP-site content decreased with increasing concentration of chemical contaminants. The
SOD activity in the treated algae increased with increasing chemical contaminants
concentration until the threshold, beyond which the cell lost their resistance and died.
There are changes occurring in RAPD profiles (variation in band intensity as well as gain
or loss of bands) in all treated samples compared with control. The results showed that,
of the end-points, Genomic template stability was the most sensitive for Chlorella
vulgaris, Tetraselmis tetrahele, Boergesenia forbesii and Ventricaria ventricosa.
Chlorella vulgaris, appears to be the most useful bioassay organism because it was the
most sensitive to toxicants. Finding from this study will contribute towards the
development of bioassays for the detection and monitoring of metal, textile dye and
organophosphate pesticide contamination based on DNA damage detection, growth,
biochemical composition and stress enzyme response in tropical marine microalgae and
macroalgae.

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ABSTRAK

Salah satu tugas penting dalam pemonitoran alam sekitar ialah untuk mengesan
bahan-bahan berpotensi berbahaya seperti bahan pencemar kimia yang boleh
mengakibatkan kerosakan oksidatif pada organisma akuatik. Tujuan kami ialah untuk
menyiasat kesan bahan pencemar kimia (logam, bahan pewarna pencelup kain dan bahan
ubat pembunuh serangga organofosfat) ke atas pertumbuhan (klorofil a, karotenoid),
komposisi biokimia (karbohidrat, protein dan lemak), kerosakan DNA [Amplifikasi
Polimorfik DNA Secara Rawak (RAPD) dan Tapak-AP (Tapak Abasik)] dan aktiviti
enzim Superoxide dismutase (SOD) di dalam empat alga marine tropika, Chlorella
vulgaris, Tetraselmis tetrahele, Boergesenia forbesii and Ventricaria ventricosa selepas
didedahkan untuk jangka pendek (empat hari) dan jangka panjang (sepuluh hari) di
dalam makmal. Keputusan akan digunakan untuk menilai kesesuaian titik-akhir
(petunjuk biologi) berbeza, dan spesis alga untuk digunakan dalam pembangunan ujian
biologi. Alga didedahkan kepada dua belas jenis bahan pencemaran kimia termasuk
tujuh logam [Cadmium (Cd); Cobalt (Co); Chromium (Cr); Kuprum (Cu); Ferum (Fe);
Manganese (Mn) dan Zink (Zn)], tiga bahan pewarna pencelup kain [Supranol Br. Red
3Bur (Acidic dye); Astrazon Red FBL (Basic dye) dan Lanaset Red 2GA (Metal
complex dye)] dan dua bahan ubat pembunuh serangga organofosfat [Malathion dan
Dichlovos] dalam julat kepekatan berbeza (0.01, 0.1, 1, 10, 100 and 500 mg/L) di dalam
medium Provasoli tanpa EDTA menggunakan kelalang konikal (untuk mikroalga:
Chlorella vulgaris dan Tetraselmis tetrahele) dan piring pelbagai lubang (untuk
makroalga: Boergesenia forbesii dan Ventricaria ventricosa) dengan isipadu ujian,
500mL and 10mL setiap satu, selama empat dan sepuluh hari di dalam keadaan terkawal.
Kadar pertumbuhan, nilai IC (kepekatan bahan toksik dianggarkan merencat 50%
50
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pertumbuhan alga dibandingkan dengan kontrol) telah ditentukan pada hari keempat dan
kesepuluh berdasarkan kandungan klorofil a. Komposisi biokimia (karbohidrat, protein
dan lemak) dan aktiviti enzim SOD juga dinilai. Analisis RAPD dan kandungan Tapak-
AP dijalankan ke atas DNA yang telah diekstrak daripada sampel yang telah dirawat.
Keputusan menunjukkan bahawa kandungan karbohidrat, protein, lemak dan tapak-AP
menurun mengikut peningkatan kepekatan bahan pencemar kimia. Aktiviti SOD di
dalam alga yang telah dirawat meningkat dengan peningkatan kepekatan bahan pencemar
kimia sehingga peringkat ambang, melepasi keadaan dimana, sel kehilangan pertahanan
dan mati. Terdapat perubahan ditunjukkan pada profil RAPD (variasi di dalam intensiti
band, dan juga perolehan atau kehilangan band) di dalam sampel yang telah dirawat
dibandingkan dengan sampel kontrol. Keputusan menunjukkan, diantara titik-akhir
(petunjuk-bio), Kestabilan Templat Genomik adalah paling sensitif untuk Chlorella
vulgaris, Tetraselmis tetrahele, Boergesenia forbesii dan Ventricaria ventricosa.
Chlorella vulgaris, muncul sebagai organisma ujian biologi paling berguna kerana ia
paling sensitif kepada bahan toksik. Penemuan daripada kajian ini akan menyumbangkan
kearah pembangunan ujian biologi untuk mengesan dan memonitor pencemaran logam,
bahan pewarna pencelup kain dan bahan ubat pembunuh serangga organofosfat
berdasarkan pengesanan kerosakan DNA, pertumbuhan, komposisi biokimia dan
tindakan balik enzim yang tertekan, didalam mikroalga dan makroalga tropika marin.

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ACKNOWLEDGEMENTS

First and foremost, I would like to express my sincerest gratitude and
appreciation to my supervisor Prof. Dr. Phang Siew Moi for her guidance and
encouragement throughout the period of study.
I would like to express my heartfelt gratitude to my parents, En. Mustafa and Pn.
Rohana and my siblings, Kamal, Tiar, Sikin, Shahrul, Fahmie and Eba for their
continuous moral support and encouragement to me all the time in striving towards this
achievement.
I have been blessed with wonderful friends and thank them for their valuable
help and friendship and all the enjoyable moments we have shared Lastly, I would like
to express my thanks to the Ministry of Science,Technology and Innovation Malaysia (R
& D Grant No: 03-01-01-001BTK/ER/020), National Science Fellowship and Institute of
Biological Sciences, University Malaya (Vot F: F0222/2004B) for financial support to
carry out my research work.

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