UNIVERSITI PUTRA MALAYSIA

INFLUENCE OF HUMIC ACID ON WATER QUALITY AND GROWTH OF ALGAE IN FRESHWATER ECOSYSTEM

HAMID KHODA BAKHSH

FP 2001 7

INFLUENCE OF HUMIC ACID ON WATER QUALITY AND GROWTH OF ALGAE IN FRESHWATER ECOSYSTEM

By

HAMID KHODA BAKHSH

Thesis Submitted in Fulfilment of the Requirement for the Degree of Master of Science in the Faculty of Agriculture

Universiti Putra Malaysia

October 2001

DEDICATION

To my parents

Yahya Khoda Bakhsh

Mansoureh Kiamary

For their true love, favor and effort, now there is only memory of my father, he

passed away during my study in Malaysia

To my wife

Sanaz

For her attention and cooperation during my study

ii

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree Master of Science

INFLUENCE OF HUMIC ACID ON WATER QUALITY AND GROWTH OF ALGAE IN FRESHWATER ECOSYSTEM

By

HAMID KHODA BAKHSH

October 2001

Chairman : Dr. Anuar Abdul Rahim

Faculty : Agriculture

One of the approaches to improve aquaculture production is through

improvement of fertilization program for phytoplankton production and water quality

variables. There are serious problems with the use of raw organic fertilizer in ponds

and environment. Oxygen depletion, degradation of water quality, reduced light

penetration, and spread of diseases frequently occur after large doses of manure are

added to a pond at irregular intervals. This study was conducted to investigate the

influence of humic acid (HA) on primary productivity (phytoplankton production)

and water quality variables in a freshwater aquatic ecosystem.

Two experiments were carried out to evaluate effects of humic acid alone or

in combination with inorganic (urea and single super phosphate) or organic fertilizer

(cow manure) on the phytoplankton productivity. Prior to the experiments, HA

contents of cow manure (eM), chicken manure, compost and tropical peat were

estimated to determine the suitable source (quantity) for HA extraction. The results

showed that tropical peat contained 46.5% (dry weight basis) HA and 11.6-17.2 folds

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higher than the other organic fertilizers. Humic acid from the tropical peat was used

in the subsequent experiments.

An experiment was conducted at the Gharehsoo Station of Fisheries Research

Centre of Mazandaran (FRCM) in North of Islamic Republic Iran (July and August

1999) to study the effects of HA and inorganic fertilizer (urea and single super

phosphate) on growth of selected freshwater phytoplankton and water quality

variables. The experiment was carried out in eighteen 1.5 Liter transparent plastic

bottles (control , 25 ppm HA+UP, 50 ppm HA+UP, 100 ppm HA, 100 ppm HA+UP,

150 ppm HA+UP). Three species of green algae (Chlorella vulgaria, Scenedesmus

quadricauda, and Oocystis solitaria) and a species of blue-green alga (Oscillatoria

agardii) were selected for the study.

The results showed that highest population (bloom) was achieved in week 3

by 100 ppm HA. All treatments with a combination of HA and urea-phosphate led to

a blue-green algal (Oscillatoria agardii) dominancy with a low phytoplankton bloom

and low total nitrogen (TN) and total phosphorus (TP) ratio (0.7-1.03). The green

algal dominance was associated with a high TN :TP ratio (16-17) in culture media.

pH of HA treatment was within the suitable range (7.84-8.5 1) for phytoplankton

production. Light penetration correlated well with blue-green algal population.

Another experiment was also carried out at the Aquatic Resource Technology

Centre, Institute of Bioscience, Universiti Putra Malaysia (Oct. and Nov. 1999) to

determine the effects of HA, CM and their combination on the growth of freshwater

algae and water quality variables (unferti lized, 100 ppm HA, Igram per liter CM,

70% HA + 30% CM). The experiment was conducted in twelve glass aquaria (60 em

x 30 cm x 30 em). The results showed that the total phytoplankton population in

ferti lized aquaria was higher than that of unfertilized aquaria. The highest bloom of

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phytoplankton was observed when a combination of HA and CM was used. Liquid

HA (100 ppm) encouraged growth of edible green algae and the highest population

of Chlorophaceae and Bacillariophyceae were observed in the mixture of HA and

CM. Cow manure treatment encouraged the growth of blue-green algae

(Cyanophyceae). Chlorophaceae population showed positive correlation with N03-N,

NH4-N:P04-P ratio. The population of periphyton increased with time and the

highest population increase was achieved with CM followed by the combination of

HA and CM, humic acid alone and tht: control (unfertilized), respectively.

Both experiments indicated that HA alone is suitable for freshwater algal

production and with better water quality variables. Besides, humic acid has the

capability of reducing the negative effect of high dosage of nitrogen (N) and

phosphorus (P) in treatments. Humic acid act as supplier and storehouse for N and P

for algae and phytoplankton in aquatic ecosystem However an inclusion of an

organic fertilizer (CM) seemed to speed up and improved the efficiency of HA on

phytoplankton population (8410 cells mL-1). The addition of liquid HA did not have

undesirable effect on water quality variables (pH, dissolved oxygen, and light

penetration).

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Abstrak tesis dipersembahkan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains

KESAN ASID HUMIK TERHADAP MUTU AIR DAN TUMBESARAN ALGA DI DALAM EKOSISTEM AIR TAWAR

Oleh

HAMID KHODA BAKHS H

o kto ber 2001

Pengel1lsi: Dr. Anuar Abdul Rahim

Fakulti: Pertanian

Satu pendekatan untuk meningkatkan pengeluaran akuakultur adalah melalui

perbaikan program pembajaan dan mutu air. Terdapat beberapa masalah serius akibat

penambahan baja organik ke kolam dan persekitaran. Pengurangan oksigen, mutu air,

pengurangan kemasukan cahaya, dan perkembangan penyakit sering berlaku setelah

banyak kuantiti baja organik ditambah ke dalam kolam Kajian ini telah dijalankan

untuk menyelidik kesan asid humik (HA) terhadap penghasilan fitoplankton dan

mutu air pada ekosistem akuakultur air tawar.

Dua eksperimen telah dijalankan untuk menilai kesan asid humik dan

kombinasinya dengan baja organik atau bukan organik terhadap produktiviti

fitoplankton. Sebelum kajian pembajaan dijalankan, kandungan asid humik di dalam

tahi lembu, tahi ayam, kompos dan gambut tropika dinilai untuk menentukan sumber

terbaik untuk pengekstratan asid humik. Keputusan kajian menunjukkan bahawa

tanah gambut tropika mengandungi 46.5% (berat kering) asid humik dan 1l.6 - 17.2

ganda lebih tinggi daripada sumber organan lain. Asid humik dari gambut tropika

digunakan untuk eksperimen-eksperimen berikutnya

Satu eksperimen telah dijalankan di Pusat Penyelidikan Perikanan Stesen

Gharehsoo di Mazandaran (FRCM) di utara Iran (Julai 1999) untuk mengkaji kesan

vi

asid humik dan baja bukan organik (urea dan fosfat ) terhadap pertumbuhan

fitoplankton air tawar dan mutu air. Eksperimen tersebut dijalankan mengguna lapan

belas botol plastik lutsinar berukuran 1.5 liter (control, 25 bsj HA+UP, 50 bsj

HA+UP, 100 bsj HA, 100 bsj HA+UP, 150 bsj HA+UP). Spesies-spesies alga hijau

(Chlorella vulgaria, Scenedesmus quadricauda, Oocystis solitaria) dan satu spesies

alga biru-hijau (Oscillatoria agardii) telah dipilih untuk dikaji. Keputusan

menunjukkan bahawa puncak perkembangan populasi tertinggi dicapai pada minggu

ke 3 dengan rawatan 100 bsj asid humik. Semua rawatan kombinasi asid humik dan

urea-fosfat membawa kepada kedominan alga biru-hijau (Oscillatoria agardii)

dengan perkembangan fitoplankton yang rendah. Kedominan alga biru-hijau berkait

rap at dengan jumlah N: jumlah P (TN:TP, 0.7-1.03) yang rendah dalam media.

Kedominan alga hijau berhubung kait dengan TN :TP (16-17) yang tinggi pada media

kultur. pH pada rawatan asid humik adalah pada paras berkesesuaian (7.84-8.51)

untuk penghasilan phytoplankton. Penebusan cahaya berkait rapat dengan populasi

alga biru hijau.

Eksperimen juga telah dijalankan di Pusat Sumber Teknologi Akuatik,

Institut Biosains, Universiti Putra Malaysia (Oktober and November 1999) untuk

menentukan kesan asid humik, tahi lembu dan kombinasinya terhadap tumbesaran

alga air tawar dan mutu air (kawalan, asid humik, tahi Iembu (CM), HA + CM).

Eksperimen terse but dijalankan mengguna dua belas akuarium kaca (60cm x 30cm x

30cm). Keputusan menunjukkan bahawa jumlah populasi fitoplankton di dalam

akuarium yang dibaja adalah Iebih tinggi berbanding dengan tanpa pembajaan.

Perkembangan fitoplankton yang terpantas didapati apabiia kombinasi asid humik

dan tahi Iembu digunakan. Asid humik cecair (100 bsj) membantu pertumbuhan alga

hijau dan populasi tertinggi bagi Choloraphaceae dan BaciIIariophyceae didapati

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pada campuran asid humik dan tahi lembu. Baja tahi lembu menggalakkan

pertumbuhan alga biru-hijau. Chlorophaceae berkait secara terus dengan nisbah NO)­

N, NH4-N:P04• Populasi perifiton meningkat dengan peningkatan masa, dan

peningkatan populasi tertinggi dicapai dengan tahi lembu diikuti, kombinasi asid

humik dan tahi lembu, asid humik sahaja dan kawalan (tanpa baja).

Kedua-dua eksperimen menunjukkan asid humik sesuai untuk penghasilan

alga air tawar dan mutu air. Asid humik juga berupaya mengurangkan kesan negatif

kadar N dan P yang tinggi. Asid humik bertindak sebagai membekal dan penyimpan

baja lepas perlahan dengan nitrogen dan fosforus untuk alga dan microorganisma

pada sistem akuatik (8410 cells mL-1). Penambahan asid humik cair tidak memberi

kesan negatif terhadap mutu air (pH, oksigen terlarut, dan kemasukan cahaya).

viii

ACKNOWLEDGEMENTS

In the Name of ALLAH "A. W" for giving me the opportunity to study and

patience to complete this study. I would like to thank not just the few that inspired

me, but also those who have accepted my mistakes and shortcomings. I am thankful

to my supervisor and my best lecturer in Universiti Putra Malaysia (UPM), Dr.

Anuar Abdul Rahim, for his guidance throughout the course of study, Dr. Che Roos,

Dr. Abdul Hamid Yazdani, Prof Dr. Khoo Khay Chong, and Prof Dr Mohd Ghazali

Mohayidin to whom lowe a great deal for the completion of this thesis. 1 am also

thankful to the technical staff of Soil Fertility and Management Lab, GIS Lab,

Hatchery Unit, Institute of Bioscience of UPM, and MARDI, Fuzi, Noriza, Ruslan,

Mohamad, Jasni, Krishnan, and Mohamad Zabawi for their valuable practical

assistance.

Last but not the least, I am deeply indebted to my friends, Dr. Paymon

Roustaian, Mr Jahangard, Mr Reza Rafiee, Annie Christianus, Osumanu Haruna,

Prama, Fadhli, Ghada A. Elhag, Manuel Laron, Emil, Nairpot, Hanib Ali, Mr

Marnani, Dr. Reza Pourgholam, Mr Yap, Mr Chong, Hazel Matias, and Albert

Gabrael for their support and concern during the period of my graduate study.

I am indebted to Fisheries Research Center of Tehran and Mazandaran (FRCM)

especially Dr. Rostamy and the staff of the Biology Section, Mr. Rostamian, Mr.

Roohy and Ms. Tahamy and the staff of Hydrobiology Section, Mrs. Vahedy and Mr.

Nasrollahzadah, and also I am grateful to Fisheries Office of Golestan, Dr. Hoseiny,

and staff specially of Fisheries Research Station (FRS) of Gharahsoo, Mr. Aghily,

Mr. Bandany and Mr Eiry.

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I certify that an Examination Committee met on 19th October 2001 to conduct the final examination of Hamid Khoda Bakhsh on him Master of Science thesis entitled "Influence of Humic Acid on Water Quality and Growth of Algae in Freshwater Ecosystem" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 198 1. The committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows:

AHMAD HUSNI HANIF, Ph.D. Department of Land Management Faculty of Agriculture, Universiti Putra Malaysia. (Chairman)

ANUAR ABDUL RAHIM, Ph.D. Department of Land Management Faculty of Agriculture, Universiti Putra Malaysia. (Member)

CHE ROOS SAAD, Ph.D. Aquatic Resources Technology Center, Faculty of Technology Agriculture, Universiti Putra Malaysia. (Member)

ABDUL HAMID YAZDANI JAHROMI, Ph.D. Department of Aquaculture, Ministry of lahad Sazandegi Iran, Tehran (Member)

cr.u� M�����ALI MOHA YIDIN, Ph.D Professor, Deputy Dean of Graduate school, Universit Putra Malaysia

Date: 30 .OV !lm

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This thesis submitted to the senate of Universiti Putra Malaysia has been accepted as fulfilment for the requirement for the degree of Master of Sciences.

xi

AINI IDERIS Ph.D, Professor Dean of Graduate School, Universiti Putra Malaysia

Date: t 0 JAN 2002

DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged . I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

HAMID KHODA BAKHSH

Date: 3 0 NCV Z001

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TABLE OF CONTENTS Page

1)�J)I��1rI()�...... . . . . ... . . . . . ...... . . ....... ...... . . . . .••.•.. . . . . .•••••••••••.••••.• 11 �]J�1r�<:1r.. . . . . . . .. . . . . ........... ........ ..... . . . . . . ... . ... . . . . .. . .•. . ...•... .... . . . III ��1r�....... . . . . . ... . . .......... . . . .. . ............ . . . .. . ..••••••••.. •...••••••..... VI ACKNOWLEDGEMENTS. . .......... ............ ................... ..... ......... IX APPROVAL SHEETS......... . .................................................. ... x DECLAAATION FORM........................................................... XII LIST OF TABLES....... ... .... ... . . . . . .......................... ... .. . . . . ... . . ...... xv LIST OF FIGURES... . ....... ............. .................................... ...... XVIII LIST OF ABBREVIATIONS...... ............ .............................. . ..... xx

CHAPTER

I INTRODUCTION................................................... .. . 1

II �I1r��1r(]� �VI�W.............. ................... ... ...... .... 5 History of Aquaculture ................................................. '" 5 Water Quality... ..................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 7

Dissolved Oxygen ........................ . ...................... '" 8 pH and Alkalinity ..... . .. . ...................... ................... 9 Temperature ................ . ... .................................... 11 Macrophytes....... .......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 12

Fertilizer........................... . ........................................ . 13 Organic Fertilizer................................................... 13 Inorganic Fertilizer.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Phosphorus.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 16 Nitrogen . .............. . ... . ................. . ................ . .... . . 17 Nitrogen and Phosphorus Ratio (N:P)... ...... . . . . . . . . . . . . . . . . . . 17 Liquid Fertilizers .............. '" . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . '" 19

Phytoplankton ................. . ......... ..... . ....... . . '" . . . . . . " . . . . .. . . . . 20 Green and Blue-green Algae............ ...... . . . . . . . . . . . . . . . . . . .. 2 1

Peat and Humic Substances........................ ... ....... . . . . . ... . . ... 24 Humic acid and Carbon... ..................... . ....... . .......... 28 Humic acid and Nitrogen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 28 Humic acid and Phosphorus .................................... '" 29 Humic Substances, Plant and Algae............................. 30 Humic Substances, Pollution, Heavy Metals and Toxicity... 35

III HUMIC ACID CONTENT OF SELECTED ORGANIC MATERIALS....... ...................................................... 40 Introduction ................... . .......................................... '" 40 Materials and Methods..... .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 4 1

Statistical Analysis................................................ 42 Results and Discussion. ........... .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Conclusion .............................. . ................ . ............... '" 46

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IV EFFECT OF HUMIC ACID ON GROWTH OF SELECTED FRESHWATER PHYTOPLANKTON AND WATER �(])l�I1r1{. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .• .•••••••.•.•.•.. lJ7 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Materials and Methods... ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Results.. ......... . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1

Phytoplankton.................................................... 51 Chlorophyll a ...................... . .................... . ... . .. .. 59 Water Quality. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1

Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 Phytoplankton Population and Humic acid. . . . . . . . . . . . . . . . . . . 66 Phytoplankton Composition and TN :TP .................... 67 Phytoplankton Population and Nutrient Concentration... 69 Humic acid, Blue-green Algae and Nutrient Fixation...... 70 Chlorophyll a and Water Quality.............................. 72

Conclusion.............................. ..................... .... ......... 75

V EFFECT OF HUMIC ACID AND COW MANURE ON WATER �UALITY AND GROWTH OF ALGAE IN FRESHWATER ECOSySTEM. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 Materials and Methods.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Results...... ......................................................... . ...... 8 1

Phytoplankton.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 1 Chlorophyll a......................... ... . . . . . . . . . . . . . . . . . . . . . . . . . 90 Water Quality............... ........................... . . . . . . . . . . 94

Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 04 Phytoplankton and Nutrient Input.............................. 1 04 Phytoplankton Population and Chlorophyll a... ......... . . . . 1 07 Water Quality Variables............ ...... . . . . . . . . . . . . . . . . . . . . . . . 1 07

Conclusion.................................................................. 113

VI GENERAL DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 General Discussion.................................... . ............. . ..... 115

Phytoplankton Community and N:P Ratio.................... 117 Water Quality............ .................................... ..... 119

Conclusions................................................................ 1 20 Recommendation........................... ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 1

REFERENCES. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

APPENDICES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

BIODATA OF THE AUTHOR.. . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . .. 1 68

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LIST OF TABLES

Table Page

1 Optimum range of water chemistry for aquaculture....... ..... ... . . . . 8

2 Factors influence the increase and decrease of DO levels in pond.... 9

3 Some organic fertilizers commonly used in pond culture of fish and crustaceans.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

4 Physicochemical factors potentially controlling cyanobacterial bloom formation ...... '" . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

5 The composition (% dry weight) of C, H, N, 0, in humic acid from different sources...... .. . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

6 The benefits of humic substances in agriculture and environment... 3 1

7 Significant effects of humic substances on crop production.......... 32

8 Humic acid content of four sources of organic materials............. 42

9 Major compounds in plant tissues (% dry weight)......... . . . . . . . . . . . . 43

1 0 Percentage of humic acid obtained from Malaysian tropical peat soil... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

1 1 Different levels of humic acid and inorganic fertilizers applied in the experiment. . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

1 2 Phytoplankton density in culture media of different treatments during the experiment..................... ................................. 53

13 Trend and respective maximum value of chlorophyll a in different treatment during the experiment.. ... ........ ....... ... ................... 59

14 Total nitrogen and total phosphorus ratio in six different treatments.............. ........... . ................. ..... ................... 65

15 The average N:P ratio composition in aquatic organisms...... ...... 69

16 Total nitrogen and total phosphorus range in control and 1 00 ppm HA during the experiment................................................. 70

17 Correlation of some biological and chemical water variabless...... 73

1 8 Fertilizer composition of the study.... ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

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1 9 Population o f phytoplankton community during four weeks of experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,. . . . . . . . . . . . . . . . . . . . . . . . . 86

20 Algal composition in 4 different treatments during the experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

2 1 Trend and respective maximum value of chlorophyll a i n four different treatments during the experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

22 Correlation of some biological and chemical water variables during the experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

23 Zooplankton observed m four treatments during the experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . '" . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

24 Trend and respective maximum value of dissolved oxygen in different treatment during the experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

25 N03-N, NH4-N and available phosphorus ratio in four different treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 02

26 Organic carbon (%) in sediment (soil) of each treatment at the end of experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 03

27 Concentration of nitrogen (N) and phosphorus (P) in different sources used for pond fertilization regime (% dry weight). . . . . . . . . . . 1 05

28 Fertil izer composition, phytoplankton population, TN:TP and N03-N+ NH4-N: P04-P ratio of the study. 1 1 8

29 Total phytoplankton population in culture media of different treatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

30 Total phytoplankton population of green and blue-green algae in culture media of different treatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

31 a Trend and respective maximum value of phytoplankton population in different treatment during the experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 56

3 1 b Total phytoplankton population in culture media of different treatments during the experiment. . . . . . . . . . . . " . . . . . . . . . . . . . . . . . . . . . . . . . 156

32 Phytoplankton biomass (mg mL-1) in culture media of different treatments during the experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 57

33 Chlorophyll a concentration in six treatments during experiment. . . 158

34 pH in culture media of different treatment during the experiment. . . . . . . . . . . . . . . . . . . . . . . . .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

xvi

35 Light loss in culture media of different treatment during the experiment ............... . . . ..... . .. . . . . . . . . . . . . . ..... . . .. . . . . . . ... . . . .. . . . . 159

36 Total nitrogen and total phosphorus in culture media of different treatments . . . . ................ ... ... . . . . . . .. . . . .. . . .... . . ... . . . . . . . . . ..... . . 159

37 Total nitrogen (N) and total phosphorus (P) concentration in six treatments during three weeks of experiment . . . . . . . . . . . . . . . . . . . . . . . . . 160

38 Total population of four phytoplankton groups in different culture media during the experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

39 Population of benthic and periphyton algae per surface (cm2) in different treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1 61

40 Chlorophyll a concentration in four treatments during the experiment . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 62

41 Chlorophyll a concentration of benthic algae during the experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 62

42 Temperature and rainfall data at the experiment site (UPM) during October and November 1999 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 63

43 pH of water in different treatments during the experiment . . . . . . . . . . . . 1 63

44 Comparisons of midday dissolved oxygen in different treatments . . 1 64

45 Comparisons of dissolved oxygen (midday and midnight) in different treatments . . . " . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 1 64

46 Comparisons of light penetration in different treatments . . . . . . . . . . . . . 1 65

47 Comparisons of nitrogen (N03-N) in different treatments . . . . . . . . . .. 1 66

48 Comparisons of total ammonium (NH4-N) in different treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 66

49 Comparisons of phosphorus (P04-P) in different treatments . . . . . . .. 1 67

50 N03 -N, NH4-N and phosphate (P04-P) concentration ratio ill four treatments during the experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 67

xvii

LIST OF FIGURES

Figures

1 A typical food chain, from nutrients to large predator fish (bass) in

Page

a fishing pond.. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

2 Model structure of humic and fulvic acid... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

3 The covalent coupling of the metabolic to humic substance. . . . . . . . . . . 36

4 Total phytoplankton population of green and blue-green algae in different treatments... .. . . . . . . . .. ...... ...... ............. . . ....... ....... .. 52

5a Composition of phytoplankton community (population) and TN :TP ratio in treatments ..... . . . . ........ .... ............ . . . . . . . . .. . . . . .

5b Composition of phytoplankton community (population) and TN :TP ratio in treatments ..... ... . . ......... .. . . ............ ... . . ..... . . .

6 Composition of phytoplankton biomass and chlorophyll a In

55

56

treatments... . . ...... . . .. . . .. ... . . . .. . ....... . . . .... .. .. . . .. .. ........... . . . . . . 57

7 Composition of phytoplankton biomass and TN:TP ratio in treatments.. . . . . . . . . .... . . .. . . . ... . . . . . . .... .. . ..... . ....... .... . ...... . . . . ... . 58

8 Concentration of chlorophyll a in different treatments during the experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

9 pH in culture media of different treatments during the experiment. . . . . ... ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

10 Light loss (%) in culture media of different treatments ... . .......... .. 63

1 1 Concentration of (a) total nitrogen, and (b) total phosphorus in different treatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

12 Total population of four phytoplankton groups in different treatments...... . . . . .. .. . . . . .. ... . . . . . . .... .. .. . . . ... . . . .......... .... . . ... . . . 82

13 Phytoplankton population and N03-NH4:P04 ratio in four treatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

14 Phytoplankton composition and N03-NH4:P04 ratio in four treatments . . . . ... . . . . ..... .. . . . .. . . . .. . . . . . .. .. . ............. ..... . ... . . ..... . . 85

15 Population of benthic and periphyton algae in different treatments during the experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

xviii

1 6 Concentration of chlorophyll a (planktonic) in different treatments during the experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1

1 7 Concentration of chlorophyll a (benthic algae) in different treatments during the experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

1 8 pH in different treatments during the experiment. . . . . . . . . . . . . . . . 95

1 9 Dissolved oxygen concentration ( 10 cm deep) in aquaria during the experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

20 Dissolved oxygen in midday and midnight (29th day) in different treatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

21 Light penetration at 20 cm depth in aquaria of different treatments during the experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 00

22 Changes in (a) N03-N, (b) NH4-N and (c) P04-N concentration in different treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 1

23 Changes of chlorophyll a and light penetration in different treatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 0

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LIST OF ABBREVIATIONS

ANOVA= Analysis of variance

00= Dissolved oxygen

DOC= Dissolved organic carbon

DOM= Dissolved organic matter

Chl. a= Chlorophyll a

L.P= light penetration

FW= Freshwater

HA= Humic acid

CM= Cow manure

HA + CM= Humic acid and Cow manure

se= Standard Error

ppm= Part per million

ppt= Part per thousand

TN= Total nitrogen

TP= Total phosphorus

TN:TP= Total nitrogen and total phosphorus ratio

UP= Urea and single supper phosphate

DNMRT= Duncan New Multiple Range Test

xx

CHAPTER I

INTRODUCTION

At present aquaculture is practiced in almost every country of the world. The

extent of aquaculture depends on several factors such as availability of expertise,

technology, water, land, government policy and others (Wheaton, 1 977).

One approach to improve aquaculture production is through improvement of

baSic aquaculture practices such as the management of water quality, selection of fast

growing and disease resistant species, reliable and adequate seed supply, formulation

of better artificial feed, enhancement of the production of natural food, post-harvest

handling and others (Landau, 1 992). Although there are commercial intensive fish

farms, the majority still practices semi-intensive farming which involved fertilization

of ponds using cow manure, inorganic fertilizers (such as urea and phosphate) and little

feeding.

It is very difficult to persuade fish farmers to change from semi-intensive

farming to intensive farming due to their limited knowledge, labour and capital. One

practical way to enhance production is through the improvement of existing semi­

intensive farming. One of the problems commonly encountered in semi-intensive

farming is the use of huge amount of cow manure, which lead to the degradation of

water quality (oxygen depletion and reduced light penetration), spread of diseases

(bacterial and parasitic), lower hygiene and production (Lin et a/., 1 997).

Since there is no precise guideline on the application of cow manure ('f organic

fertilizer into a pond (unaerated), there is a strong tendency to over-fertilize a pond that

leads to excessive organic matter, high biological oxygen demand, low dissolved

oxygen (particularly in the morning), reduced light penetration necessary for

photosynthesis, and the formation of anaerobic conditions at the bottom of the pond

(Lin et al., 1 997). For these reasons, some farmers exclusively use inorganic fertilizers

as they are more convenient. In fish ponds, chemical fertilizers stimulate

phytoplankton production which indirectly increases fish yield. They contain nitrogen

(N), phosphorous (P) and potassium (K) that are needed by phytoplankton (Boyd,

1 979). Even though the use of inorganic fertilizers is more convenient, the availability

of resulted natural food is limited.

Commercial pond managers use a combination of organic and inorganic

fertilizers. A combined application of both fertilizers stimulates pond productivity

largely through autotrophic pathways and also through heterotrophic pathways (Green

et al., 1 989). The quantity of zooplankton, benthos and detritus can be indirectly

increased through such fertilization regime. With fertilization, the fish and prawn

production does not have to start at the bottom of the food chain but can be started at a

level closer to the fish and prawn with less energy loss (Schroeder, 1 978; Geiger,

1983). Many research have shown the benefits of fertilizers on fish and prawn

production (Ling, 1967; Wohlfarth and Schroeder, 1979; Teichert-Coddington et al.,

1990). Mortimer ( 1954) reported that the carp production in fertilized ponds was 2 to

1 0 times higher than those of unfertilized ponds, while tilapia production in fertilized

ponds (5, 135-9000 kg ha-l) was 2 to 4 times higher than in unfertilized ponds (2,240 kg

ha-l).

Studies have indicated that the use of humic acid (organic acid) can improve the

production of terrestrial plants (Sladky, 1959; Stevenson, 1 982; Steinberg and Baltes,

1 984; Levinsky, 1 998). Some studies have also shown that humic acid can increase the

productivity of phytoplankton (prakash and Rashid, 1 968; Prakash et al., 1 973; Lee

2

and Bartlett, 1 976; Graneli and Moreira, 1990; Vrana and Votruba, 1 995; Faust, 1 999)

in environment and laboratory experiments. Most of these reports have involved

marine phytoplankton growth. Prakash and Rashid ( 1968) indicated that the

biologically active and ingredients of humic substances stimulate growth of marine

phytoplankton. However, little work has been done on the influence of humic

substances on the growth of freshwater algal species, a major purpose of this

investigation. It is necessary to know whether humic acid as natural organic fertilizer

can improve production of selected phytoplankton in the freshwater pond.

According to Schroeder ( 1978) and Lin et al. ( 1997) it is well recognized that

pond fertilization with animal manure stimulates production of bacteria,

phytoplankton, zooplankton, and benthos. The nutrient availability and efficiency of

animal manures to phytoplankton production have long been treated as a black box in

pond dynamics. There are also some negative aspects of using animal manures such as

oxygen depletion, unsuitable for high-yield culture, cost of gathering and transportation

(ICAAE, 1 996a). Therefore, results of this study can be compared to effectiveness of

humic acid and another organic fertilizer (cow manure) on phytoplankton population

and water quality variables in an aquatic ecosystem.

As noted, no work has been reported on the influence of tropical peat humic acid

on algal growth and also influence of humic acid on water quality variables. There is

no information available regarding the toxic efficacy of this source of humic

substances.

3

Objectives

The aim of this study was to investigate the potential use of humic acid in

improving productivity (phytoplankton) and provide more insight on water quality

variables in a freshwater ecosystem. In this context, the specific objectives of this

study were as follows:

1 . To evaluate the different sources of humic acid content.

2. To evaluate the effect of humic acid and its combination with inorganic

fertilizer on freshwater phytoplankton population and water quality variables.

3 . To evaluate the productivity of phytoplankton and water quality variables

in aquatic ecosystem fertilize with cow manure and liquid humic acid.

4