UNIVERSITI PUTRA MALAYSIA

THE DEVELOPMENT OF PHOTOSYSTEM II - HERBICIDE DETECTION METHOD USING

MICROALGAE AS A BIOSENSOR

SHAKINAZ DESA

FSAS 1997 19

THE DEVELOPMENT OF PHOTOSYSTEM II -

HERBICIDE DETECTION METHOD USING

MICROALGAE AS A BIOSENSOR

By

SHAKINAZ DESA

Thesis Submitted in Fulfillment of the Requirements for the Degree of Master of Science in the

Faculty of Science and Environmental Studies, Universiti Putra Malaysia

June 1997

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ACKNOWLEDGEMENTS

Tlns study was first suggested by Prof. Dr. Kamaruzaman bin

Ampon fonnerly of Department of Biochemistry and Microbiology in

UPM. His idea was supported by his wife, Assoc. Prof Dr. Ann Anton,

who was also an academic staff at Department of Biology, UPM. Both

are now serving at University Malaysia Sabah. This study is wisely

supervised by Assoc. Prof Dr. Nor Aripin Shamaan. I thank him very

much for giving me invaluable advice and encouragement at every step

of the way.

I wish to express my gratitude to Dr. Misri Kusnan of Department

of Biology for his brilliant ideas ,comments and tremendous support. I

am greatly indebted to Encik Ismail Omar of Department of

Biochemistry and Microbiology who has put in many long hours in

reviewing the thesis in order to produce a perfect text. My greatful

thanks also goes to Cik Rohaidah Ramli of Department of

Biochemistry and Microbiology and Puan Zaiton Basar of Department

of Biology for their assistance in the research work. Finally, I would

like to thank the staff of Graduate School, especially to Cik Fazlun for

her supportive ideas and efforts.

vi

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ............... ........................ ,................... iii LIST OF TABLES ....... .... ........ ............. ..... ...... ....... ....... ................. vii LIST OF FIGURES ......................................................................... viii LIST OF PLATES ......... ............. .......... ...... ..................... ......... ....... xi LIST OF ABBREVIATIONS ......................................................... xii ABSTRAC'r .................................................................................... xiii ABSTRAK ....................................................................................... xv

CHAPTER

I INTRODUCTION . .............................. . .. ....................... I

II LITERATURE REVIEW .................................... . . ........ 4 Herbicide . ....................................................................... 4

Introduction ......................................................... 4 Herbicide action in general ......................... .... .... 5 Tnazlne ............................................................... 7 Urea ............ ... .................. . . ..... ............................ 12

Herbicides in our environment ...... .. ....... . .... .................. 14 Risk of contamination ............. . .. ......... ..... .......... 17 Pollutant residence .. ......... ............ ................ ...... 20

Effects to the aquatic environment ... . . . ... . . ..... ......... ....... 22 Effects to humrul being .................. . ............................ .... 23 Herbicide action: inhibition ofPSII dependent electron flow .. ................. ...... ........ . .... , .......................................... 25 Herbicide binding site ....................... .............................. 27 Microalgae photosynthetic system . .................. .............. 31

Algal chlorophyll ................................................ 3] Photosynthesis: in general ....... . .......................... 33

Microalgae growth ..... . .... . ... ........... . .............................. 36 I..ag phase .............. ..... .............. . .......... .... ............ 36 Exponential phase ............................................... 36 Declining relative growth phase ....................... . . 37 Stationary phase .............................. ...... .............. 38 Deatll pllase ........ . ........................... ..................... 38 Nutrient replacing and batch culture ...... ...... .... . .. 39

iv

Fluorescence .................................................................... 40 The potential of algal chlorophyll fluorescence as biosensor ......................................................................... 42

Fluorescence potential ......................................... 45 Changes in fluorescence ...................................... 46 Requirement to develop monitoring system ....... 47

III MATERIALS AND METHODS .................................. 49 Preparation ofthe test organism .................................. 49

Selecting the test organism .................................. 57 Detennining the main test culture obligations .... 62 Culturing method ................................................ 62 Optimal age culturing ......................................... 65

Herbicide preparation .................................................. 65 Spectrofiuorometer ...................................................... 68 Satnple preparation ..................................................... 71 Statistical and data analysis ........................................ 74

IV RESULTS ..................................................................... 75 Selection oftest organism .......................................... 75

Preliminary test .................................................. 75 Cell density ......................................................... 78 Growth curve ..... , ................................................ 80 Chlorophyll concentration .................................. 84

Basic C. vulgaris obligation detetmination ................. 90 Culturing method selection ................................. 90 Optimal age detennination ................................. 92

Experiment using Atrazine .......................................... 97 Experiment using Simazine ........................................ 100 Experiment using Vinron ................... ........................ 1 06 Comparative study of atrazine, simazine and diuron ...................................................................... 109

V DISCUSSION ............................................................ 112 Selection ofChlorelia vulgaris ........ .. .................... 112

Suitability of test organism .............................. 112 Efficiency ........................................................ 115 Stability ........................................................... 116

v

Reliability on the fluorescence method ................... 119 Herbicide concentration and variable fluorescence

detemunatlon ... ........................................................ 12 1 Teclmical advantages and disadvantages ................ 125

VI CONCLUSION .. .............................. . ......................... 128 Suggestion .... . . . . ... . ... ... . .. . . . . . . ......... . ......... ....... . . . . . . .... 129

REFERENCES ............................................................................. 132

APPENDIX A Bold Basal media 147

BIOGRAPHICAL SKETCH 148

vi

LIST OF TABLES

Table Page

] Some examples ofberbicide and their specific inhibition 6

2 Comparison of environmental zone ................ ..... .... ... ..... 21

3 Test organisms and origin ................................................ 51

4 Basic information on the test herbicide ............................ 67

5 The growth rate oftest organism ............ .... ..... .......... ...... 84

6 The chlorophyll concentration per cell (�glcell) ....... . . . . . . 87

7 The relative fluorescence for atrazine at day 21 and 28, and the statistic comparison with day 14 ........... .............. 99

8 The relative fluorescence for simazine at day 21 and 28, and the statistic comparison with day 14 .......... ..... .... ...... 104

9 TIle relative fluorescence for diUl'on at day 21 and 28, and the statistic comparison with day 14 ...... ................... 108

vii

LIST OF FIGURES

Figure Page

1 General structure of triazine .......................................... 8

2 Structure of diuron or DCMU ..... .......... ...... .... .............. 13

3 The basic process of how pesticides can enter a waterbody .. ............ .......... ..... .......... ..... ............ ...... ......... 16

4 The process of herbicide accumulation along a food chain ................................................................................ 24

5 Binding sites of herbicides to a DIID2 complex of the reaction centre of PSI I .......................................... ......... 29

6 Formula structure of Chi a ............................................. 32

7 Steps of filling P680+ electron hole ............................... 35

8 The characteristic pattern of growth .............. ................. 37

9 Summary of the process used in the determination of the fluorescence method .. .......... ................................. ..... 50

10 The process of culturing microalgae ............................... 56

1 1 The process to determine chlorophyll concentration ....... 6 1

12 The procedure of detecting herbicide ............................... 73

13 The relationship between relative fluorescence and atrazine concentration ...................................................... 76

14 The relationship between cell density and age of culture .................................. ............................................. 79

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Page

15 Growth curve of Chlorella vulgaris ........... ............ .... 8 1

16 Growth curve of Scenedesmus quadriqauda ... ...... ....... 82

17 Growth curve of Chlorococcum sp ...... .... ................. .... 83

18 The relationship between relative fluorescence and chlorophyll concentration of Hibiscus sp. leaves ........ 85

19 The relationship between chlorophyll concentration and age of culture .......................... .... ,. .... .......... ........... 86

20 The relationship between chlorophyll fluorescence and age of culture ... .............................. ................ ............ ... 89

2 1 Growth curve of Chlorella vulgariS in two culturing nleth.ods .......................................................................... 91

22 The relationship between relative fluorescence and chlorophyll content and age of culture ......... ......... ......... 93

23 The relationship between relative fluorescence with different chlorophyll concentration over atrazine concentration .. 95

24 The relationship between relative fluorescence of atrazine treated sample and chlorophyll concentration .............. 96

25 The relationship between relative fluorescence and atrazine concentration ................................................................ 98

26 The relationship between relative fluorescence and pure atrazine ............... ................... ..................... ........... ....... 101

27 The relationship between relative fluorescence and simazine concentration ............ ............................................. ....... 102

28 The relationship between relative fluorescence and pure sitnazine .................... . .................. . . ............................... 105

ix

Page

29 The relationship between relative fluorescence and diuron concentration ................ ,............................................. 107

30 The relationship between relative fluorescence and pure diuron .................. ...................... ,',............................... 110

31 The relationship between relative fluorescence and herbicide concentration ................................................... ............ 111

x

LIST OF PLATES

Plate

1 A fish pond located below the palm oil plantation

Page

at Semenyih, Selangor ..... ... . .................. ........................ 18

2 A corn plantation situated above the fishing pond level at UPM's pond area .... ................................................... 19

3 Chlorella vulgaris with cup shaped chloroplast .......... . 53

4 Scenedesmus quadriqauda in form of colony spesies 54

5 Selenastrum sp. in cresent moon shaped, while Chlorococcum sp. shows a shape chloroplast ...... ......... 55

6 A haemocytometer is used to count cells ... .... . ............. .. 58

7 A batch culture allow the culture to grow without replenishing with fresh nutrient .......... ............................ 63

8 A nutrient replaced culture ............................................ 64

9 PSII inhibitor herbicide: atrazine, simazine and diuron. 66

lOA spectrofluorometer connected to a personal computer with Perkin-Elmer Fluorescence Data Manager prograr.nme ..................................................................... 69

xi

LIST OF ABBREVIATIONS

BBM Bold Basal media

ChI Chlorophyll

EPA Environmental Protection Agency

GC Gas chromatography

PSII Photosystem II

Q qumone

UPM Universiti Putra Malaysia

xii

Abstract of thesis submitted to the Senate of Universiti Putra Malaysia in fulfilment of the requirements for the degree of Master of Science.

THE DEVELOPMENT OF PHOTOSYSTEM II - HERBICIDE DETECTION METHOD USING MICROALGAE AS A

BIOSENSOR

BY

SHAKINAZ BT DESA

JUNE 1997

Chairman Assoc. Prof. Dr. Nor Aripin bin Shamaan

Faculty Science and Environmental Studies

Herbicides have been found to be significant pollutants of water

bodies. In order to control and monitor the pollution, a convenient,

sensitive and reliable method is needed. The main objective of this

study is to develop a new method to measure the fluorescence yield of

microalgae in order to detect photosystem II (PSII) specific herbicides

in polluted water. TIle system was based on a simple mixing of

microalgae with PSII specific herbicides. The measured signal was in

the form of fluorescence intensity. The study involved the screening of

test organisms, detennining the best condition of the culture and

obtaining a standard curve for each of the PSII herbicides. Only three

herbicides were studied; atrazine, simazine and diuron. Chlorella

vulgaris was found as the most suitable test organism compared with

Chlorococcum sp., Scenedesmus quadriqauda and Selenastrum sp ..

Nutrient replacement culturing method was selected as the best

condition to grow C. vulgaris. A standard curve was determined for

each of the herbicides. C. vulgaris was able to signal atrazine's

concentration above O.04/lM according to the standard curve equation.

Simazine can be signalled at above O.05flM from its equation, while

diuron can be signalled at above than O.09/lM. However, -this basic

study needs more infonnation and improvement to be done in the

future, so that its full potential can be realised.

XlV

Abstrak tesis diserahkan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan Ijazah Master Sains.

PEMBENTUKAN KAEDAH MENGESAN RACUN RUMPAI FOTOSISTEM II MENGGUNAKAN MIKROALGA SEBAGAI

BIOSENSOR

Oleh

SHAKINAZ BT DESA

JUN 1997

Pengerusi Prof. Madya Dr. Nor Aripin bin Shamaan

Fakulti Sains dan Pengajian Alam Sekitar

Racun rumpai merupakan salah satu bahan cemar utama di dalam

badan air. Bagi mengawal dan mengesan pencemaran, kaedah yang

mudall, sensitif dan dipercayai adalah diperlukan. Objektif utama

kajian ialah untuk membentuk kaedah bam bagi mengukur floresen

mikroaIga dalam mengesan racun mmpai fotosistem II (FSII) yang

spesiftk di dalam air tercemar. Sistem illi berasaskan pencampuran

mikroalga dan racun rumpai FSII spesifik. Isyarat diukur di dalam

xv

bentuk keamatan floresen. Kajian ini melibatkan pemilihan organisma

ujian, kajian pengkulturan terbaik dan mendapatkan keluk piawai bagi

setiap faeun rumpai ujian. Tiga raeun rumpai yang dikaji; atrazin,

simazin dan diuron. Chlorella vulgaris didapati merupakan organisma

ujian yang paling sesuai berbanding Chlorococcum sp., Scenedesmus

quadriqauda dan Selenastrum sp .. Kaedah penggantian nutrien dipilih

sebagai kaedah pengkulturan terbaik bagi menumbuhkan C. vulgaris.

Satu keluk piawai diperolehi untuk setiap raeun rumpai setiap satu. C.

vulgaris berupaya mengesan kepekatan atrazin di atas O.04J.lM

mengikut persamaan keluk piawai. Simazin boleh dikesan pada

kepekatan di atas O.05J.lM daripada persamaannya. Manakala diuron

dapat dikesan di atas O.09�lM. Bagaimanapu� kajian asas ini

memerlukan lebih banyak maklumat dan pembaikan di masa hadapan

bagi membolehkan penggunaan potensi sepenuhnya.

xvi

CHAPTER I

INTRODUCTION

Current methodologies for pesticide detection, particularly herbicides, are

not ideally suited for large scale monitoring programme. These include gas

chromatography (GC) and high penomumce liquid chromatography (HPLC).

These techniques need large sample volumes, extensive extraction and cle�Ul

up procedures for analyses. lbe chemical analysis of a water sample is

expensive and very time-consuming. Thus, the number of analyzed samples is

limited.

Other techniques include flow cytogenic analysis usmg whole cell

clastogenicity (Biradar and Rayburn, 1 995); photoacoustic spectroscopy on

excised leaf (Fuks et aI., 1992); leaf disc buoyancy (LDB) method (Saltzman

and Heuer, 1985); oxygen electrod method (Saka and Chisaka, 1 982);

treatment of activated carbon� reverse osmosis and deionisation (Delavaud et

aI., 1992); measuring aJJeviation of phytotoxic injury (Yanase et aI., 1990)

and soil extraction method by enzyme immunoassay and gas chromatography

(Del Valle and Nelson, 1994).

A good analytical method must be accurate, precise, sensitive and

selective. Therefore, we need a method that is capable to measure low

concentrations of herbicides, low cost, fast result, easy handling and able to

monitor large scale progranune. The purpose of this study is to develop a

method that utilises natural phenomenon as an early warning system.

The method should be able to detect herbicide at below the level of

pennissible concentration.

In tins work, the basic principle is based on the measurement of changes

in the yield of variable chlorophyll a - fluorescence of PSIL The method

involves a test organism and a spectrofluorometer. Aquatic organisms are

used for toxicology tests because they experience the changes in the aquatic

environment.

The changes of yield of the in vivo chlorophyll a - fluorescence of

photosystem II are correlated quantitatively with the concentration of

herbicides. Therefore, it can be used to set up a low price monitoring system

and to be a promising basis for a detection device. Other studies that share the

same principle were done by Jansen et a1. (1993), Yanase and Andoh (1992),

Duke et at. (1991), Voss et al. (1984), Richard et al. (1983) and Bohme et al.

(1981).

2

As microalgae grow in aquatic environment, they are exposed to

environmental changes. These disturbance, however, are adapted by

microalgae in various way. For example, some increase the cell density

(Trifonova, 1993), produce spore or resting stage (Nixdorf and Hoeg, 1993)

or by migrating (Vincent, 1990). However, there are microalgae that could

not survive with the changes. But, these microalgae can serve as a simple

plant cell model in the herbicidal sites test systems (Fedtke, 1993).

The inhibition of herbicide towards the photosynthetic electron transfer

can be detected by measurement of chlorophyll fluorescence (Gleiter and

Renger, 1993; Percival and Baker, 1991; Habash et aI., 1985). Fluorescence

is a reradiated light energy fraction which cannot be used to drive

photosynthetic electron transfer. The inhibition results in an increased

fluorescence intensity at constant excitation intensity (Schrotter et aI., 1994).

There are three objectives in this study. Firstly, to select the best test

organism. Secondly, to determine the selected test organism obligation and

thirdly to obtain a relationship between the fluorescence yield and herbicide

concentration.

3

Introduction

CHAPTER II

IjITRRA TlJRE REVIEW

Herbicide

Herbicides are chemicals with the potential to kill and destroy

plants (Cobb, 1992). Herbicide can inhibit photosynthesis by

interfering with :

1 . the reproduction, development, structure, and integrity of chloroplasts,

2. biosynt11etic pat11ways that are involved in 11le production of output

products such as starch, amino acids synthesis,

3. photochemical induction pathways involved 10 the conversion of

radiation energy to chemical energy (Moreland and Hilton, 1976).

Herbicides may be classified in several ways, according to t11e

chemical compound, physiological characteristics or its selectivity

(Percival and Baker, 199 1). Herbicide can be classified into two major

groups : organic and inorganic. Examples of organic herbicides are

phenol and its derivatives, carboxylic acid and its derivatives,

4

pyridazine and pyrimidine, triazine, phenylurea, organophosphorus and

organoarsenic compounds . Examples of inorganic herbicides are

borate, sodium azide, sodium chlorate, copper sulfate and calcium

cyanamide.

Herbicide action bt general

Generally, the purpose of herbicides is to kill plant by inhibiting

photosynthetic electron transport (Palmeira et aI., 1995; Cobb, 1992).

Two biochemical mechanisms have been demonstrated to be of

primary importance in herbicidal action; they are firstly, inhibition of

PSII electron transport and secondly, diversion of electron transfer

through PSI (Fuerst and Norman, 1991). There are other mechanism of

herbicides actions in plants. They are summarized in Table 1.

There are also herbicides that enhance the development of auxin,

e.g., pheooxyalkanoic acid, benzoic acid, pyridine, and quinoline

carboxilic acid (Cobb, 1992). Excessive development of auxin may

cause tIle potassium ion accumulation in guard cells. Resulting in the

opening of the stomata and an increase in the photosynthesis rate.

Subsequently, cells die of excessive food production. Herhicide also

5

affect the increase of cell division and differentiation of cambium

tissues in higher plants.

Table 1

Some examples of herbicide and their specific hlhibition.

Inhibition

Photosyntllesis PSI

PS2

Pigment biosynthesis

Lipid syntllesis

Amino acid chain branching biosynthesis

Glutamine synthesis

Herbicide

Bipyridinium, Paraquat, Diquat Urea, Triazines, Triazinones, Uracil

Pyridazinone,

Metflurazone.

Cyclohexanadiones, Thiocarbamate

Sulphonilurea, Imidazolinone, Sulphonanilides.

Oxitin, Phosphinotrisine,

Reference

Dodge, 1988

Cobb, 1992

Britton et al. 1989.

Cobb, 1992

Dodge, 1988

Cobb, 1992

6

Most of the chemical compounds that inhibit photosynthesis

electron transport do so by binding to a set of overlapping sites on the

photo system II (PSII) reaction centre (Tietjen et aI., 1991; Sandmann

and Boger, 1986; Lavergne, 1982; Pfister et al., 1974). The phytotoxic

symptoms arise from the interaction of the herbicide molecule with

PSII. These chemical compounds include urea derivatives and

triazines.

Urea derivatives and triazines were introduced between 1950 and

1960. Diuron was the frrst herbicide known to specifically inhibit

photosynthetic electron flow (Wessels and Van der Veen, 1956). The

site of inhibition at the acceptor site of PSII was proposed, based on

the effect on chlorophyll fluorescence yield by Duysens and Sweers

(1963). Since then, diuron, atrazine and their photo affinity labelling

analogues have been used extensively in photosynthesis research.

Triazine

Triazine herbicides were first introduced in 1955 by Geigy.

Triazines are a commercially important class of herbicides. Triazine is

an extreme volatile crystalline solid which melts at 86°C and boils at

7

114 DC at 1 attn pressure. It is easily soluble in ether and ethanol at

Triazine is highly refracting rhombohedral crystals. The density is

approximately 1.38 glcm3• Triazine can be purified without

appreciable Joss by repeated distillation over metallic sodium (Smolin

and Rapoport, 1959).

Cyanuric chloric (2,4,6-trichloro-l ,3,5,-triazine) is the base

compound to foon all 1,3,5-triazine herbicide. General chemical

structure of triazine is shown in Figure 1.

Figure 1: General structure of triazine.

8