A Journal of Biological, Physical and Social ... - Pertanika Journal - UPM [PDF]

PERTAHIHfl A Journal of Biological, Physical and Social Sciences

December, 1989

Vol. 12 No. 3

Contents Section 1 : Biological Sciences Ultrastructural I >i Hevea brasiliensis Muell.-Arg. Seeds during Imbibed Storage. - M.S. Normah and H.F. Chin. Potential Productivity of Hydroponically-grown Tomatoes in the Genting Highlands, Malaysia. - R.M. Ha/a Harun. Histological Study on Adventitious Root Formation in Stem Cuttings of Young Durian (Durio zibenthinus Murr.) Seedlings.- Hasan BM. and P.H. Dodd. Determination of Calcium in Foods by the Atomic Absorption Spectrophotometric and I itrimetru Methods E. Siong, Khor Swan Choo and Sit/ Mizura Shah id Determination of Iron in Foods by the Atomic Absorption Spectrophotometric and Colorimetric Methods.- Tee E. Siong, Khor Swan Choo and Siti Mizura Shahid. Trials on Induced Ovulation of Fugu niphobles (Jordan and Snyder) with Human Chorionic Gonadotropin S.H. Cheah and H. Satoh. Light Availability for Phytoplankton Production in Turbid Tropical Fish Ponds. -Fatimah Md. Yusoff. Preliminary Stud) on Mortality of Catfish {Clarias macrocephalus) Fn Transported 311 Plastic Sags. Ahyaudin i>. Ali, Mohamad hham Tg. Kamalden and Ad nan Abas. Aktiviti Bakterisidal • laripada Pesakit L u p u s Eritematosus Sistemik (SI E).

293 299 303

329

M. Musa,J. Mat Asan

dan Y.M. Pang. Section II : Physical Sciences Petroleum Hydrocarbon along the Coastal Areas of Port Dickson. — IMW A.T. and Ravinthar Veettu. Nickel (II) Removal from Aqueous Solutions by Adsorption on Fly-ash. - Prem Prakasl arma, Kailash Prakash Tadava and Vishwanaih Singh. The Use of Suspended Sediment Rating Curves in Malaysia : Some Preliminary Considerations.- G. Balamurugan Preservation of Oil Palm Fruits: Nonoxidative Effects of Ionizing Radiation on Palm Olein and ('rude Palm Oil

341

357 .%7

- K. En di n km u a nd T.W. U 'oodu >a rd.

377

Chlorinated Organic s in Fro pica! Hardwood Kraft Pulp and Paper Mill Effluents and their Elimination in an Activated Sludge Treatment System. - Murtedza Mohamed, M. Matayun and T.S, him. The Isolation and Identification of Two Antifungal Petrocarpans from Ulex Europaeus L. - Hasnah Mohd. Sirat and Gmeme B. Russell Delignification Pretreatmenl of Palm-Press Fibres In Chemical Method. — C.C. Tong and Y..W. Ha Preliminary Studies on the Chemical Composition of Sound and Decayed wood of Acacia mangium. - Halimahton Mai

405

Performance of Microencapsulated Fungicide in Exterior Latex Paint on Wood Substrate.- Wan Asma Ibrahim, mad Shakri Mat Seman, Nuruihuda Mohd. Nasir and Rahim Sudin, An Iterative Explicit Method for Parabolic Problems with Cylindrical Symmetry-Increased Accuracy on NonUniform Grid. -Mohd. Satteh Sahimi and Zaiton Muda.

409

Communications Chemotaxonomy of the Lauraceae: N-Methyl-2,3»&-trimethoxymorphinandien-7-one, the Major Alkaloid from Alseodaphne perakensis. — Nordin //. fMJis, Zurina Mahmud,

Laity bin din and Robert F. I

421

Section III : Social Sciences Analisis Permintaan Pelancongan di Malaysia. — Ahmad Shuib dan Noor Aziz Mohd Nor. Penyeliaan Latihan Mengajar: Implikasi dan kajian Kes Penilaian Latihan Mengajar. - Hj. Abd. Mam Hj. Salimon,

A scientific Journal published In

UNIVERSITI PERTANIAN MALAYSIA

ISSN 0126-6128

i:>:;>

ARCHIVE COPY (Please Do Not Remove) EDITORIAL BOARD

CHIN HOONG FONG (Chief Editor)

EDDIE CHIEW FOOK CHONG (Business Manager)

SULAIMAN MOHD YASSIN ANG KOKJEE KWOK CHEE YAN MAT YUSOFF ABDULLAH

ASIAH BTE MOHD ZAIN TAN HOCK SENG KAREN ANNE CROUSE BADRI KAMIS AWANG SUMANGAIA PILLAI (Secretary)

PERTANIKA is a scientific journal published thrice a year (April, August and December) by Universiti Pertanian Malaysia (University of Agriculture, Malaysia) in which papers in Bahasa Malaysia and English in any area aligned with the work done at the faculties of the University appear. Currently these include Agriculture, Forestry, Veterinary and Animal Science, Food Science and Technology, Resource Economics and Agribusiness, Engineering, Fisheries and Marine Science, Science and Environmental Studies, Extension and Continuing Education, Education and Social Studies, Human Development and Consumer Studies. PERTANIKA welcomes original reports in English or Bahasa Malaysia of research not previously or simultaneously published in any scientific or technical journal from the staff of Universiti Pertanian Malaysia and other local and overseas institutions and organisations. Contributions are reviewed by a panel of consultants whose names appear in the last issue of each volume. PERTANIKA is currently abstracted by the following: Agrindex, Biological Abstracts, Chemical Abstract, Nutrition Abstracts, Animal Breeding Abstracts, Field Crop Abstracts, Forestry Abstracts, Forest Production Abstracts, Herbal Abstracts, Horticultural Abstracts, Indian Veterinarian, Plant Breeding Abstracts, Reviews in Applied Entomology, Reviews in Plant Pathology, Soils and Fertilizers and Veterinary Bulletin. Article in triplicate should be submitted to the Chief Editor, PERTANIKA, Universiti Pertanian Malaysia, Serdang, Selangor, Malaysia. Subscription Rates(per year) Malaysia/Singapore

Overseas

Individual

$45,000

US$30.00

Institutions

$70.00

US$35.00

Overseas subscribes please add US$6.00 per issue for airmail surcharge. Cheques/Bank drafts should be made payable to UNIVERSITI PERTANIAN MALAYSIA and sent to, PERTANIKA, Universiti Pertanian Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia.

PSRTflniKfi PROF. DR. MOHD ZAMRI SAAD Deputy Director A Journal of Biological, Physical and Social Sci&aCfesatton Division Research Management Centre

Number 3, December 1989 c

u

^ersit. Putra Malaysia

Serdang, Selangor Darul Ehsan

Contents Section 1 : Biological Sciences Ultrastructural (Changes of Hevea brasiliensis Muell.-Arg. Seeds during Imbibed Storage. - M.N. Normah and M.F. Chin. Potential Productivity of Hydroponically-grown Tomatoes in the Genting Highlands, Malaysia. - fLM. Raja Harun. Histological Study on Adventitious Root Formation in Stem Cuttings of Young Durian {Durio zibenthinus Murr.) Seedlings. - Hasan BM. and RB. Dodd. Determination of Calcium in Foods by the Atomic Absorption Spectrophotometric and Titrimetric Methods. - Tee E. Siong, Khor Swan Choo and Siti Mizura Shahid. Determination of Iron in Foods by the Atomic Absorption Spectrophotometric and Colorimetric Methods. - Tee E. Siong, Khor Swan Choo and Siti Mizura Shahid. Trials on Induced Ovulation of Fugu niphobles (Jordan and Snyder) with Human Chorionic Gonadotropin S.H. Cheah and H. Satoh. Light Availability for Phytoplankton Production in Turbid Tropical Fish Ponds. -Fatimah Md. Yusoff. Preliminary Study on Mortality of Catfish (Clarias macrocephalus) Fry Transported in Plastic Bags. - Ahyaudin b. AH, Tg. Mohamad Izham Tg. Kamalden and Adnan Abas. Aktiviti Bakterisidal in vitro Fagosit daripada Pesakit Lupus Eritematosus Sistemik (SLE). - M. Musa, J. Mat Asan dan KM Pang.

285 293 299 303 313 323 329 335 341

Section II : Physical Sciences Petroleum Hydrocarbon along the Coastal Areas of Port Dickson. - Law A.T. and Ravinthar Veellu. Nickel (II) Removal from Aqueous Solutions by Adsorption on Fly-ash. - Prem Prakash Vishwakarma, Kailash Prakash Tadava and Vishwanath Singh. The Use of Suspended Sediment Rating Curves in Malaysia : Some Preliminary Considerations. - G. Balamurugan Preservation of Oil Palm Fruits: Nonoxidative Effects of Ionizing Radiation on Palm Olein and Crude Palm Oil - K. Endinkeau and T.W. Woodward. Chlorinated Organics in Tropical Hardwood Kraft Pulp and Paper Mill Effluents and their Elimination in an Activated Sludge Treatment System. - Murtedza Mohamed, M. Matayun and T.S. I Am. The Isolation and Identification of Two Antifungal Petrocarpans from Ulex Europaeus L. - Hasnah Mohd. Sirat and Graeme B. Russell. Delignification Pretreatment of Palm-Press Fibres by Chemical Method.- C.C. Tong and N.M. Hamzah. Preliminary Studies on the Chemical Composition of Sound and Decayed wood of Acacia mangium. - Halimahton Mansor. Performance of Microencapsulated Fungicide in Exterior Latex Paint on Wood Substrate.- Wan Asma Ibrahim, Ahmad Shakri Mat Seman, Nurulhuda Mohd. Nasir and Rahim Sudin. An Iterative Explicit Method for Parabolic Problems with Cylindrical Symmetry-Increased Accuracy on NonUniform Grid. -Mohd. Salleh Sahimi and Zaiton Muda.

349 357 367 377 387 395 399 405 409 413

Communications Chemotaxonomy of the Lauraceae: N-Methyl-2,3,6-trimethoxymorphinandien-7one, the Major Alkaloid from Alseodaphne perakensis. - Nordin H. iMJis, Zurina Mahmud, iMily bin din and Robert F. Tma.

421

Section III : Social Sciences Analisis Permintaan Pelancongan di Malaysia. - Ahmad Shuib dan Noor Aziz Mohd Nor. Penyeliaan Latihan Mengajar: Implikasi dari kajian Kes Penilaian Latihan Mengajar. - Hj. Abd. Main Hj. Salimon.

425 433

Members of the Editorial Board Prof. Chin Hoong Fong M.Agric.Sc, Ph.D. (Melb.) F.I.Biol. (Lond).

Faculty of Agriculture (Chief Editor)

Prof. Sulaiman bin Mohd Yassin B.Agnc.Sc. (Hons.) Malaya, M.P.A. PhD. (Cornell).

Centre for Extension and Continuing Education

(C.A.),

Assoc. Prof. Ang Kok Jee B.Sc (Madras), M.Sc (Malaya), Ph.D. (Waterloo)

Faculty of Fisheries and Marine Science

Assoc. Prof. Kwok Chee Yan B.Agric.Sc. (Malaya), M Sc. (Reading)

Faculty of Agricultural Engineering

Assoc. Prof. Mat Yusoff Abdullah B.Sc. (Malaya), M.S. (U.P.L.B.) PhD (Okla. State)

Faculty of Science and Environmental Studies

Assoc. Prof. Kamis Awang B.Sc. (For.) (Hons.) Ph.D. (A.N.U.)

Faculty of Forestry

Assoc. Prof. Asiah bte Mohd Zain B.S (Iowa), M.Sc. (Reading).

Faculty of Food Science and Technology

Eddie Chiew Fook Chong Dip.Agnc. (Malaya), B Agnc.Sc. (Hons) Malaya, M.Sc (Malaya)

Faculty of Resource Economics and Management (Business Manager)

Assoc. Prof. Tan Hock Seng, B Sc (Hons), B. Vet.Med. (Lond.), PhD M R CVS

(Guelph),

Faculty of Veterinary Medicine and Animal Science

Assoc. Prof. Karen Anne Crouse-Badri B.Sc. (Hons), (St. F.X.) M.Sc. (Dal.) Ph D. (Malaya)

Faculty of Science and Environmental Studies.

Sumangala Pillai B.Soc.Sc. (Hons)

Universiti Pertanian Malaysia Press (Secretary)

Section I Biological Sciences

Pertanika 12(3), 285-291 (1989)

Ultrastructural Changes of Hevea brasiliensis Muell.-Arg. Seeds during Imbibed Storage M.N. NORMAH* and H.F. CHIN Agronomy Department Faculty of Agriculture Universiti Pertanian Malaysia 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia Key words: Ultrastructural changes, temperature, storage. ABSTRAK Pertukaran ultrastruktur dicerap pada bijih benih Hevea yang disimpan di dalam suhu 1(PC, 22° C dan 2TC. Satu ciri kerosakan yang paling biasa ialah kesusutan membran. Pada semua suhu penyimpanan, didapati plasmalemma terlipat, tersepai dan tertarik daripada dinding sel. Dalam kajan ini pelarutan tonoplas juga dicerap. ABSTRACT Ultrastructural changes were observed in Hevea seeds stored at 1(?C, 22?C and 2TC. Membrane degeneration appeared to be the most common feature of deterioration. At all storage temperatures, the plasmalemma was observed to be increasingly folded, disintegrated or withdrawn from the cell wall. The dissolution of the tonoplast was also widely observed.

INTRODUCTION Seeds that have been classified as recalcitrant by Roberts (1973) are those that are highly susceptible to desiccation injury and not storable under conditions suitable for orthodox seeds i.e. at low moisture content and at low temperature. Hevea seeds are included in this group of seeds. Changes during deterioration in seeds have been suggested to be due to several factors. According to Delouche (1969), delayed seed germination is one of the earliest physiological signs of deterioration. The decline in respiratory rate has also been reported for various seeds such as in barley and corn under different conditions of deterioration (Woodstock and Grabe 1967). Reduction in levels of enzymes such as cytochrome oxidase, malic and alcohol dehydrogenase has been reported in non-viable maize seeds (Throneberry and Smith 1955). Impairment of carbohydrate, protein and

nucleic acid synthesis are among the biocnemical events that takes place during deterioration (French 1959; Abdul-Baki 1971; Abdul-Baki 1980). Harrington (1973) believed strongly that a major cause of ageing is protein denaturation. The diverse metabolic changes associated with seed deterioration and viability loss could result in a greater or lesser extent from disruption of membrane systems (Bewley and Black 1982). Berjak and Villiers (1970) reported that membrane aberrations increased with increasing age of maize embryos. Disintegration of cell membranes was also observed in seeds of Hevea brasiliensis (Chin etal 1981) and Theobroma cacao

(Hor 1984) killed by dehydration. Berjak (1968) and Hallam (1973) observed that mitocondria in aged corn seeds and in non-viable rye seeds were markedly swollen, had little development of cristae and there was a decrease in matric density.

* Present address: Botany Department, Faculty of Life Sciences, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor D.E., Malaysia

MN. NORMAH AND H.R CHIN

Chin et at, (1981) have shown that Hevea seeds are killed by dehydration, high temperature or freezing. Since temperature is one of the important factors in determining the viability of seeds, this study was undertaken to examine the ultrastructural changes that occur in Hevea seeds during storage at different temperatures. The imbibed storage method was employed as it was the conventional method used for storing Hevea seeds at the time this study was carried out. It is hoped that by understanding the changes that occur in the seeds during their deterioration, an improved method of storage may be devised by delaying their deterioration. MATERIAL AND METHODS

Seeds of clone RRIM 600 were obtained from Prang Besar Seed Gardens located in Telok Intan, Perak, Malaysia. Sawdust used in the study was cleaned by soaking in water for three days and changing the water everyday to remove toxic substances that might be present. It was then sterilised in an oven at 100°C for 48 hours and cooled before use. The sawdust was then mixed with distilled water to make up to 20% moisture content. Freshly harvested seeds were mixed in equal volume with moistened sawdust and seeds were spread in shallow layers inside perforated black polythene bags (30cm x 36cm) (ten holes of approximately 0.5cm in diameter). The seeds were then stored at 10°C (cold room), 22°C (air-conditioned room) and 27°C (ambient room). The storage duration was three months. After each month of storage, the ultrastructure of seeds was examined. Fresh seeds were used as a control.

further stained for 15-20 minutes in lead citrate and washed with double distilled water twice. The sections were then examined using a Phillips 400 transmission electron microscope. RESULTS

Cells from fresh seeds had clearly defined cell walls (Plate 1A). The plasmalemma adhered closely to the cell wall. The nucleus was clearly delimited by a double membrane nuclear envelope and a spherical nucleolus was located in the nucleoplasrn. Within the cytoplasm, endoplasmic reticulum and mitochondria were present and appeared intact. Ribosomes were present attached to the endoplasmic reticulum and were present also in great numbers apparently free hvthe cytoplasm (Plate IB), NumerPlate 1 Ultrastructure of radicle cells from fresh seeds

•mm

Hevea

m

is* A. Typical cell xvithprominent starch grains (S). (x4,600)

Electron Microscopy

Approximately 1 mm of the radicle of the embryonic axis about 1 mm from the tip was dissected with new razor blades while submerged under 4% glutaraldehyde solution. The tissues were then transferred immediately to fresh 4% glutaraldehyde solution and fixed for 4 hours at 4°C. The tissues were then dehydrated in acetone and embedded in resin (Epon 812Agar 100, Araldite 212). Ultra thin sections were stained for 15-20 minutes in saturated uranyl acetate and rinsed in 50% ethanol twice and with double distilled water twice. They were 286

B. Well-defined plasmalemma and cytoplasmic layer with endoplasmic reticulum and numerous ribosomes. Note small vesicles (VS) close to the plasmalemma. (x46,000)

PERTANIKA VOL, 12 NO. 3, 1989

ULTRASTRUCTURAL CHANGES OF HEVEA BRASIUENSIS MUELL.-ARG. SEEDS DURING IMBIBED STORAGE

ous starch grains and lipid bodies were present. Dictyosomes were absent but small vesicles were present close to the plasmalemma. After one month of storage at 10°C the cells were filled with large vacuoles with cytoplasm lying close to the cell wall (Plate 2A). However, in some cells, the tonoplast separating the vacuole and the cytoplasm was not clearly visible. Invagination of the plasmalemma was observed indicating derangement of the membrane (Plate 2B). Mithochondria were not well defined. Endoplasmic reticulum was present and numerous dictyosomes and small vesicles were present. Starch grains and lipid bodies were also observed. After two months of storage at 10°C the nuclei of the root cells became lobed and irregular (Plate 2Q. Endoplasmic reticulum were in short profiles and mitochondria were seen to contain fewer cristae or were more electron dense but with electron transparent areas. Lipid bodies were present but starch grains were rare. Dictyosomes were present. Cell walls were constricted at certain places especially at the plasmodesmata, and the plasmalemma appeared to be absent in certain places and in some cells, withdrawn from the cell wall. After three months of storage at 10°C, the irregular shape of the nucleus persisted. The double membrane nuclear envelope appeared to be breaking down. Mithochondria contained fewer cristae or were more electron dense and

the mithochondrial membrane was not clearly discernible. Endoplasmic reticulum was pres-

B. Cytoplasm with dictyosomes (D), small vesicles (VS) and mitochondria (M). Note the invagination of plasmalemma (1). (x 46,000)

C. Typical cells with lobed nuclei, (x 4,600) Plate 2. Ultrastucture of radicle cells from Hevea seeds stored at 1(PC after one month (A and B), two months (C) and three months (D).

A. Typical cells with large vacuoles (V). (x 2,150)

D. Cytoplasma with dictyosome (D) and degenerated mitochondria (M). Note nuclear membrane dissolution (2). (x 27,500)

PERTANIKA VOL. 12 NO. 3, 1989

287

M.N. NORMAH AND H.F. CHIN

ent in short profiles and dictyosomes were present. Invagination of the plasmalemma indicated that some deterioration had occured {Plate 2D). After one month of storage at 22°C, cytoplasm appeared more electron dense with one large vacuole and many small secondary vacuoles. Nuclei were well defined while numerous starch grains and lipid bodies were still present as in the control. Endoplasmic reticulum was present in short profiles. After two months of storage at 22°C withdrawal of the cytoplasm occurred at various places {Plate 3A). In some cells the cytoplasm appeared to be close to the cell wall and plasmalemma appeared to be absent or might have been severely broken down. Fusion of the vacuoles and breaking down of the tonoplast occurred resulting in the encroachment of the vacuolar matrix into the cytoplasm. Nuclei were not well defined. The presence of lipid bodies was evident. Starch grains, mitochondria and endoplasmic reticulum were rare. After three months of storage at 22°C, severe collapse of the cytoplasm was evident {Plate 3B). The plasmalemma was breaking down at various places. The dissolution of the tonoplast resulted in no clear definition between cytoplasm and vacuoles. The nucleus was electron dense and not well defined, as the nuclear envelope appeared to be breaking down causing the nucleoplasm to merge with the cytoplasm {Plate 3Q. Mitochondria appeared to be present but not clearly discernible. Other organelles were rare. After one month of storage at 27°C, the cells were vacuolated while the nucleus was well defined with spherical nucleolus in the nucleoplasm. Well defined mitochondria with cristae were numerous. Endoplasmic reticulum were of short profiles with ribosomes associated with them. Ribosomes were also present free in the cytoplasm. Lipid bodies and starch grains were present. However, certain parts of the plasmalemma and tonoplast appeared to be breaking down. After two months of storage at 27°C, what appeared to be tonoplast dissolution could be observed. Plasmalemma disintegration and invagination appeared at various places. Some

288

Plate 3. Ultrastructure of radicle cells from Hevea seeds stored at 2TC after two months (A) and three months (B and C).

A. Cell with withdrawn plasmalemma and dissolved tonoplast. Note lipid bodies (L) lying close to plasmalemma. (x 7,750)

B. Typical cell showing dissolution of tonoplast (x 6,000)

C. Nucleus with incomplete outer membrane. Note dissolution of tonoplast (1). (16,500).

PERTANIKA VOL. 12 NO. 3, 1989

ULTRASTRUCTURAL CHANGES OF HEVEA BRASILIENSIS MUELL.-ARG. SEEDS DURING IMBIBED STORAGE

parts of the nuclear membranes were not clearly defined and mitochondria were with less cristae or with electron dense deposits. No endoplasmic reticulum was observed but ribosomes were present in the cytoplasm. Starch grains and lipid bodies were also present. The cells at one and two months of storage were thus not very discernibly different than the control. After three months of storage at 27°C, increased vacuolation was evident (Plate 4A). Dissolution of the tonoplast was quite severe at various places causing the vacuolar content to merge into the cytoplasm. The nucleus was still intact though some parts of the envelope were discontinuous (Plate 4B). Invagination of the plasmalemma occured at various places and often the plasmalemma was discontinuous or not observed. Mithochondria contained electron dense deposits or had less cristae and appeared disintegrated (Plate 4Q. Endoplasmic reticulum was rarely observed and ribosomes were scattered throughout the cytoplasm. Lipid bodies were present but appeared fewer compared with those in the cells of the control embryos. Starch grains were rare. DISCUSSION The withdrawal of the plasmalemma observed in cells from seeds stored at 22°C and aslo from some seeds stored at 10°C is believed not due to desiccation as the seeds were stored imbibed. The moisture contents of seeds after two months of storage at 10°C and 22°C were 48.56% and 33.93% (fresh weight basis) respectively (Normah, 1987). Withdrawal of plasmalemma has been observed by Hor (1984) in cocoa seeds that had been given chilling treatment. At 10°C the temperature might be too low for Hevea seeds, while at 22°C other factors might be involved to cause the withdrawal of plasmalemma. In air-conditioned (22°C) storage, dissolution of the tonoplast resulted in the absence of compartmentalisation of the vacuolar matrix. The nuclear envelope was also dissolved causing the nuclear content to merge with the cytoplasm. Hence, it is suggested that destruction of the membrane system contributed significantly to seed death.

Plate 4, infrastructure of radicle cells from Hevea seeds stored at 2TC after three months.

A. Typical cells with disintegrating cytoplasma. (x 4,600)

B. Intact nucleus ivith part of the membrane disintegrating. Note the dissolution of tonoplast (I). (7,700).

C. Disintegrating mitochondria (M) and a starch grain (S). (35,500).

PERTANIKA VOL. 12 NO. 3, 1989

289

M.N. NORMAH AND H.F. CHIN

Endoplasmic reticulum has been suggested to be associated with synthesis of membranes for the cells and organelles. Hence, the synthesis of membranes might be affected by deterioration. Berjak (1968) observed that with ageing, short profiles of endoplasmic reticulum remained. The nucleus and nucleolus, however, appeared intact. Having an intact nucleus and possibly still functional mitochondria might have resulted in the viability of the seeds stored at 27°C (Normah 1987). Normah (1987), showed that the viability of Hevea seeds was maintained for the longest period when the seeds were stored at 27°C, follwed by 22°C and 10°C respectively. It may also be speculated that repair mechanisms similar to that suggested by Villiers and Edgcumbe (1975) were operative to a certain extent so that germination could still be maintained at around 50 percent after three months of storage at 27°C. Deterioration in the cells of seeds stored at 27°C was not uniform; some cells deteriorated more than others. With the other two storage temperatures, deterioration was observed to be quite uniform in all cells. Berjak et al (1984) reported that uniformity of cell deterioration in Avicennia propagules and the degenerative situation became equalised on further deterioration. Thus, it might be that the degenerative situation of Hevea seeds after three months storage at ambient temperature was not critical, so the repair mechanism could still be operative. Also Withers (1978) reported that damage to the endoplasmic reticulum and derived structures would appear to be less prejudicial to survival than damage to selfreplicating organelles such as mitochondria, plastids and nucleus. Features of cellular deterioration such as breaks in the structure of the plasmalemma and its withdrawal away from the cell wall; disintegrating plastids and mitochondria; condensed chromatin in the nucleus, which itself might be lobed; are among the features of aged or deteriorated orthodox seeds, including maize (Berjak and Villiers 1970), wheat (Anderson etal. 1970), rye (Hallam 1973), and also in deteriorated recalcitrant seeds such as Avicennia marina (Berjak etal. 1984) and cocoa (Hor 1984). These include ultrastructural changes associated with natural ageing, storage fungi and dehydration. 290 PERTANIKA VOL.

From this study, it appeared that Hevea seeds also showed similar features of deterioration, even though the methods of storage a n d the cause of deterioration were different. Osborne (1980) concluded that orthodox seeds that are stored imbibed maintained their viability by maintaining metabolic activities in the cells and loss of viability is due to loss of DNA integrity. With Hevea, loss of viability might not only be due to the loss of DNA integrity as has been shown by other degenerative characteristics of the cells. Recalcitrant seeds do not undergo maturation drying on parent plants and are shed at relatively high moisture content. The seeds are ready to germinate after shedding if given the right conditions for germination as has been mentioned by Berjak et al (1984) for Avicennia propagules. The same process might occur in Hevea seeds. Hence, when Hevea seeds were stored imbibed, they were given sufficient moisture and if stored at ambient temperature, they were ready to germinate. According to Berjak (1989), when Hevea seeds are stored under conditions maintaining their moisture content, they will initiate subcellular germination associated events. Though it was not quantified, the loss of stored seeds through germination was also observed in this study especially for those seeds stored at 27°C. However, at 10°C, the seeds were given the moisture but not the right temperature for germination. It appeared that at such low temperature the seeds still imbibed water and were ready to germinate as shown by the presence of dictyosomes. Dictyosomes appeared after 24 hours of imbibition and produced numerous vesicles (Berjak and Villiers 1970). When the optimum temperature was not given for a certain time, the seeds were unable to germinate and thus deteriorated. It might be that at 27°C and 22°C the appearance of dictyosomes was faster to the extent that by the end of one month, no further dictyosomes could be observed as a result of their degeneration. Berjak (1968) reported that the early degeneration of dictyosomes involves 'unstacking' of the cisternae followed by their apparent loss. Nevertheless, the cause of deterioration might not be due to a single factor for all the three temperatures of storage. At low tempera12 NO. 3, 1989

ULTRASTRUCTURAL CHANGES OF HEVEA BRASILIENSIS MVELL.-ARG. SEEDS DURING IMBIBED STORAGE

ture such as 10°C, to a certain extent, deterioration might have been due to disintegration of the organelles. It might have been also because of plasmalemmal invagination and other factors involved that were not included in this study. For 22°C and 27°C storage, the dissoluion of plasmalemma and tonoplast causing vacuolar materials to encroach into the cytoplasm and cell organelles, might have resulted in the eventually loss of organelles and cell function and finally death. The deterioration at 27°C storage was less than that of 22°C storage. However, it should be emphasised that due to complexity of the cell, there may have been other factors involved in seed deterioration and loss of viability which were not tested in the present study.

J.D. and M. BLACK 1982. Physiology and Biochemistry of Seeds in Relation to Germination. Vol. 2. Springer-Verlag, Berlin, Heidelberg, New York.

BEWLEY,

CHIN, H.F., A. MAHERAN, B.B. ANG, and H. SAMSIDAR

1981. The Effect of Moisture and Temperature on The Ultrastructure and Viability of Seeds of Hevea brasiliensis. Seed Set. & TechnoL 9: 411-422. DELOUCHE, J.C. 1969. Planting Seed Quality. Proceedings of the 1969Beltxdde Cotton Production - Mechanisa-

tion Conference, New Orleans, La., pp 16-18. FRENCH, R.C. 1959 Formation of Embryo Starch During Germination as an Indicator of Viability and Vigor in Heat Damaged Barley. Plant Physiol, 34: 500-505. HALLAM, N.D. 1973. Fine Structure of Viable and Non-viable Rye and Other Embryos. W. Heydecker (ed.) In Seed Ecology, Butterworth, London, pp. 115-144. ACKNOWLEDGEMENTS HARRINGTON, J.F. 1973. Biochemical Basis of Seed This study was carried out as part of the first longevity. Seed Sci., & TechnoL 1: 435-462. author's PhD thesis. The authors wish to thank HOR, Y.L. 1984. Storage of Cocoa (Theobroma cacao) Universiti Pertanian Malaysia for funding this Seeds and Changes Associated with their Deterioproject They are also grateful to Prang Besar ration. Ph. D. Thesis. Universiti Pertanian MalayResearch Station for the supply of seeds. sia. NORMAH, M.N. 1987. Effects of Temperature on REFERENCES Rubber (Hevea brasiliensis Muell. - Arg.) Seed ABDUL-BAKI, A.A 1971. Biochemical Differences Storage. Ph.D. Thesis. Universiti Pertanian Malaybetween Embryonic Axes from Green and Sunsia. Bleached Lima Beans. Synthesis of Carbohydrates, OSBORNE, D.J. 1980. Senescence in Seeds. In SenesProteins and Lipids./ Amer. Soc Hort. ScL 96: 266cence in Plants, K.V. Thimann (ed.) CRC Press 270 Inc. Boca Raton, Florida, pp. 14-36. ABDUL-BAKI, A.A.1980. Biochemical Aspects of Seed ROBERTS, E.H. 1973 Predicting the Storage Life of Vigor. Hort. Science 15: 765-771. Seeds. Seed Sci. & TechnoL 1: 499-514. ANDERSON, J.D., J.E. BAKER and E.K. WORTHINGTON

1970. Ultrastructural Changes of Embryos in Wheat Infected with Storage Fungi. Plant Physiol. 46: 857-859. BERJAK, P. 1968 A Lysosome-like Organelle in Root Cap of Zea mays. J. Ultrastruct Res. 23: 233-242. BERJAK, P. 1989. Storage

Behaviour of Seeds of Hevea

brasiliensis. J. nat. Rubb. Res. 4(3): 195-203.

P., and T.A. VILLIERS. 1970. Ageing in Plant Embryos. I. The Establishment of the Sequence of Development and Senescence in the Root Cap during Germination. New Phytol 69: 929-938.

BERJAK,

BERJAK, P., M. DINI and N.W. PAMMENTER.

1984.

Possible Mechanisms Underlying the Differing Dehydration Responses in Recalcitrant and Orthodox Seeds: Desiccation-associated Subcellular Changes in Propagules of Avicennia marina. Seed ScL & TechnoL, 12: 365-384.

THRONEBERRY, G.O. and F.G. SMITH. 1955. Relation

of Respiratory and Enzymatic Activity to Corn Seed Viability. Plant Physiol. 77: 584-586. VILLIERS, T.A. and D.J. EDGCUMBE. 1975. On the Cause of Seed Deterioration in Dry Storage. Seed Sci. & TechnoL 3: 761-774. WITHERS, L.A. 1978. A Fine-structural Study of the Freeze-preservation of Plant Tissue Culture. II. The Thawed State. Protoplasma 94: 235-247. WOODSTOCK, L.W. and D.F. GRABE. 1967. Relation-

ships between Seed Respiration during Imbibition and Subsequent Seedling Growth in 7jea mays L Plant Physiol. 42: 1071-1076.

PERTANIKA VOL. 12 NO. 3, 1989

(Received 8 September 1989)

291

Pertanika 12(3), 295-298 (1989)

Potential Productivity of HydroponicaUy-grown Tomatoes in the Genting Highlands, Malaysia R. M. RAJA HARUN Department of Agronomy and Horticulture Universiti Pertanian Malaysia 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia. Key words: Potential productivity, hydroponically grown tomatoes. ABSTRAK Lima penanaman percubaan telah dijalankan untuk menguji potensi pengeluaran tomato di dalam sistem hidroponik kultur air dalam di Genting Highlands,. Malaysia (1,200 m atas paras laut). Tanaman tomato dibenarkan mempunyai hanya satu batang utama yang dilatih mengikut sistem berlapis atau sistem tegak. Varieti-verieti tak berterusan (determinate) dan berterusan (indeterminate) yang mengeluarkan buah-buah yang besar, sederhana dan kecil digunakan di dalam kajian int. Tanaman yang ditanam mengikut sistem berlapis tahan selama 9 bulan; tetapi jikalau ia hanya ditanam untuk selama 6 bulan, dua kali penanaman dapat dilakukan setahun dengan memberi potensi hasil sebanyak 252-288 tan metrik/ha/tahun. Sistem tegak yang mengeluarkan ketiga-tiga saiz buah tomato mengeluarkan tiga penanaman setahun dengan anggaran hasil sebanyak 210-248 tan metrik/ha/tahun. Varieti-varieti tak berterusan mengeluarkan hasil-hasil yang berbeza; pengeluaran sebanyak tiga kali setahun akan memberi hasil sebanyak 131-216 tan metrik/ha/tahun. Hasil-hasil yang diperolehi dnripada sistem hidroponik ini adalah lima hingga 10 kali ganda lebih tinggi daripada hasil yang dapat diperolehi mengikut cam tanaman tradisional sama ada di tanah tinggi atau di tanah rendah di Malaysia. ABSTRACT Five trials were carried out to assess the potential productivity of tomatoes in a deep culture hydroponic system in Genting Highlands (1,200 m a.s.L), Malaysia. The tomatoes were maintained as single-stemmed plants using the layering and vertical methods of plant training. Determinate and indeterminate varieties producing large, medium and small-sized fruits were investigated in these trials. Under the layering system, the indeterminate plants could last for nine months but if crops were kept for only six months, two crops per year could be produced with a potential yield of 252-288 ton/ha/year. The vertical system could produce three crops per year with a range of 210-248 tonnes/ha/year for the three sizes of fruits. The determinate varieties produced a wide range of yields and, with three crops per year, a yield range of 131-216 ton/ha/ could be expected. The yields obtained in the tested hydroponic system were at least five to ten times higher than those obtained from traditional soil cultivation under highland and lowland conditions in Malaysia. INTRODUCTION Tomatoes (Lycopersicon esculentum Mill), an important vegetable crop in Malaysia, is traditionally grown in soil, in the Cameron Highlands (1,000 to 1,500 meters above sea level). Tomato cultivation using hydroponics or soilless culture techniques, widely practised in Europe, have not gained acceptance by local commercial growers. One of the reasons for this is the high initial capital costs and the fear of not

achieving sufficient productivity to off-set the high initial capital outlay. There is thus the need to assess the potential productivity of tomatoes under a hydroponic system and to monitor potential problems that may affect the productivity of the crop. The following is a report on some trials carried out to determine the potential productivity of tomato, as a crop, in a hydroponic system (Kyowa Hyponica) using a number of

R.M. RAJA HARUN

commercially available t o m a t o varieties. Determ i n a t e a n d i n d e t e r m i n a t e varieties were grown; t h e former b e i n g allowed to grow until the terminal inflorescence was p r o d u c e d while t h e latter varieties were trained as single-stemmed plants using t h e vertical or layering techniques. MATERIALS AND METHODS

TABLE 1 Composition of nutrient solution (From Lim and Wan, 1984) Major nutrients

Location

The trials were carried out in a glasshouse using the hydroponic deep culture system at the Hydroponic Unit, Universiti Pertanian Malaysia, Genting Highlands. This unit is situated at 1,200 meters above sea level and has similar climatic conditions to the Cameron Highlands where tomatoes are normally cultivated in Malaysia. The average day and night temperatures in the Genting Highlands are around 22°C and 17°C, respectively. Although temperatures vary very little throughout the year, days are more misty and windy in December and January with wind gusts of around 14-18 m/sec. In the glasshouses, however, air temperature can reach up to 35°C on bright sunny days although day temperatures of 25-30°C can be considered average. The Hydroponic System

The Kyowa Hyponica deep culture hydroponic system (previously described by Lim and Wan 1984; and Wan and Lim 1984) was the system used in the trials.This system consists of troughs (1 m x 3 m x 10 cm) laid end to end, 11 troughs per row with one meter spacing between each row of troughs. Each glasshouse of 1,000 m2 held 128 troughs each planted with 14 plants. This gave a planting density of 4.7 plants per m2 within each trough; but if paths between the rows were included, the overall planting density was 1.8 plants per m2. Nutrient Supply

The composition of the nutrient solution to the plants is as shown in Table 1. Nutrient solution was supplied to each trough from an underground tank by a submersible pump and continuously recirculated at the rate of 4 1/ min per trough until the plants were two months old. Thereafter the rate of nutrient flow was increased to 6 1/min. These comparatively fast flow rates were considered necessary to main294

tain a high level of dissolved oxygen within the 45 mm deep solution (for the first two months) and the 25 mm deep solution for the subsequent months.

Nitrogen Phosphorous Potassium Calcium Magnesium Iron

mg/1 206 62 386 136 49

Minor nutrients

mg/1

Boron Zinc Manganese Copper Molybdenum

0.54 0.05 1.30 0.01 0.01

3.82

The electrical conductivity (E.C.) and pH of the nutrient solution were monitored daily and maintained between 2.1 to 2.4 mS/sec and 5.5 to 6.5, respectively. There was little fluctuation of E.C and pH of the nutrient solution as the nutrient storage tank contained 15 m 3 of solution in addition to 15 m 3 in the troughs and pipes. Cultural Techniques There is very little local experience in the hydroponic cultivation of tomatoes. In order to assess the overall potential production in a hydroponic system, as many available tomato varieties as possible were used in order to give a better picture of the overall potential of the crop within the system. The varieties grown were supplied by various seed companies but it must be noted that the trials were not laid out to compare performance of the varieties. Seeds were sown directly in 2.5 cm gravel aggregates in perforated pots placed in the troughs and supplied with continuously recirculating nutrient solution for three weeks. Germination occurred within one week. Two weeks after germination, selection was carried out to retain only one normal seedling per pot and these were then transferred to the main cultivation troughs with PVC m u l c h i n g to prevent light from reaching the root regions. Each trough contained plants of the same variety and the troughs were randomly allocated within each row. A summary of the trials carried out is shown in Table 2.

PERTANIKA VOL. 12 NO. 3, 1989

POTENTIAL PRODUCTIVITY OF HYDROPONICALLY GROWN TOMATOES

TABLE 2 A summary of the trials

Trial No

Date of Sowing

3

5/5/86 16/2/87 16/2/87

4 5

16/2/87 16/2/87

1 2

Duration of cropping (days)

Training system

134 - 195 50-56 46-56 46-50 44-56

Layering Vertical Vertical Vertical Vertical (determinate)

No. of varieties

Plants per

Size of fruits*

variety 4 5

12 7 7

70 14 14 14 14

M S M L

L,M,S

* Size of fruit : L • Large, M = medium, S = small.

Three methods of plant training were tested. For the indeterminate varieties the plants were trained vertically, into single stemmed plants, using rafia string. This was carried out by manual removal of the side shoots. In Trial No. 1, the plants were maintained at a height of the 1.7 m frame and lowered and layered twice weekly to maintain this constant height (Walls 1977). Pruning of the lower leaves that did not subtend any fruit was regularly carried out when harvesting commenced. This method of plant training allowed the plants to be retained for up to nine months. The second training technique practised in Trials No. 2, 3 and 4 used the vertical system whereby the indeterminate varieties, also trained as single-stemmed plants, had their growing points removed upon reaching the top of the frame at 1.7 m with around 6 or 7 trusses. Under this system, the plants were removed once all the trusses had been harvested. In the third system, Trial No. 5, the plants were also trained as single stemmed plants and supported with rafia string until a final determinate inflorescence terminated further growth. To assist in fruit-set, opened flowers were sprayed with a 0.5 percent solution of sodium4-chloro-hydroxy phenoxy acetate ("Tomatlane") on alternate days. RESULTS Trial No. 1: Production under the Layering System Under this system, the crop remained in production for seven months for varieties "Firedance" and "Precodor" and around nine

months for varieties "Gross Lisse" and "Pink FR26'\ For the former two varieties, the residual effects of the fruit inducing hormone used could be discovered; the young leaves produced in the later stages were deformed, elongated and leathery with overall reduction in leaf area. As a consequence of this reduced leaf area, the fruits formed in the later trussess were unfilled, possibly indicating an insufficient supply of assimilates. Management of the crop under this layering system was laborious as the plants had to be lowered twice weekly to maintain a constant height. The lower leaves which no longer subtended the fruit had to be regularly removed. When pruning was delayed, air circulation within the canopy was reduced resulting in increased infestation of powdery mildew (Erysiphe sp.). This infestation could, however, be easily controlled by the use of Triforine ("Saprol") at the rate of 0.19 ml a.i./litre of water. With regard to insect pests, only the occasional caterpillar was observed and was removed by hand. Thus, no insecticide application was necessary for the crop. The mean yield for the varieties under this system of training (Table 3) was 15.16 kg/ plant (S.E. = 0.375) for the longer duration crop (194 days) and 9.51 kg/plant (S.E. = 0.057) for the shorter duration crop (135 days). Average fruit production per day of cropping was between 70-80 g/plant. If the crop had been retained for only six months to overcome some of the management problems i.e. 100 days of cropping after commencing harvest at around

PERTANIKA VOL. 12 NO. 3, 1989

295

R M RAJA HARUN

traditional method of cultivation in the soil. Tomato yields in the Cameron Highlands are only around 20-35 tonnes/ha/season (Nordin et aL 1986) whereas those from the lowlands are around 28.5 tonnes/ha (Haron 1987) to 30.8 tonnes/ha (Vimala 1985). These crops, unlike those under the present hydroponic system, are grown without protection and are exposed to the vagaries of weather. They are not sprayed with hormone to assist fruit set and development and are normally affected by fungal pests such Phytophthora infestans (Nordin et aL 1986). Under the present investigation a maximum yield of around 280 tonnes/ha could be expected. This is still lower than that typically obtained in the South coast of the United Kingdom which is around 328 tonnes/ha or higher in some cases (van de Vooren et aL 1986). With further selection of varieties more suited to the tropics and experience with the crop and nutrient manipulations, higher tomato yields can be anticipated. This may eventually convince the local growers to choose hydroponics as a commercial method for the cultivation of tomatoes. ACKNOWLEDGEMENTS

The author wishes to thank En. Hanib Ali for technical assistance in data collection and management of the trials. The supply of seed samples by the various seed companies is also gratefully appreciated.

298

REFERENCES HARON, A, 1987. Potensi Tomato jenis "MT 11" dan Jenis "Local White" di Tanah Gambut. TeknoL Sayur-sayuran 3: 35-38. LIM, E.S. and C.K. WAN. 1984. Vegetable Growing in the Tropics Using a Two-phase Substrate System of Soilless Culture. Proc Sixth Int. Congr. on Soilless Culture, Wageningen. PP 317-328 NORDIN, M. S,, S. A. RAHMAN, and A. ABDULLAH.

1986. Research on Chemical Control against Tomato Late Blight in Cameron Highlands. 2nd hit. Conf. Plant Protec. in the Tropics. Genting Highlands 17-20 Mar. 1988. Extended Abstracts No. P-17. van

de VOOREN, J.,

G. W. H. WELLES and

G.

HAYMAN. 1986. Glasshouse Crop Production. In, The Tomato Crop, ed. J.G. Atherton, and J. Rudich. London: Chapman and Hall. pp. 581-623 VIMALA, P. 1985. Effects of Varying Rates of Nitrogen, Phosphorous and Potassium on the Performance of Tomato on Peat. TeknoL Sayur-sayuran 1: 49-53. WALLS, I. G. 1977. Tomato Growing Today. 2nd Ed. Publ. Newton Abbot: David and Charles. 235 pp. WAN, C. K. and E.S. LIM. 1984. Growing Crops in Pots Containing Gravel Chips by the Recirculating Flow Technique under Tropical Conditions. Proc, Sixth InL Congr. on Soilless Culture. Wageningen. pp 751-762.

PERTANIKA VOL. 12 NO. 3, 1989

(Received 2 November,

1987)

Pertanika 12(3), 299-302 (1989)

Histological Study on Adventitious Root Formation in Stem Cuttings of Young Durian (Durio zibenthinus Murr.) Seedlings HASAN B.M. and P.B. DODD Department of Agronomy and Horticulture Faculty of Agriculture Universiti Pertanian Malaysia 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia. Keywords: Histological study, stem cutting, durian, adventitious roots. ABSTRAK Keratan yang diperolehi dari anakpokok durian (Durio zibethinus Murr.) berakar dengan mudah. Kajian histologi yang dijalankan ke atas zon perakaran keratan-keratan ini mendapati bahawa dinding sel-sel penghasil adalah nipis. Keadaan ini amat sesuai bagi sel-sel ini untuk berubah kembali dengan mudah kepada bentuk yang tidak khusus dan akhirnya bersedia membentuk primordia akar. Kajian ini juga menunjukkan bahaxoa ranting-ranting dari stok muda belum lagi membentuk sebarang halangan mekanikal yang dapat menyusahkan pembentukan akar 'adventisV. ABSTRACT Cuttings obtained from young durian (Durio zibenthinus Murr.) seedlings rooted very readily. A histological study carried out on the rooting zone of these cuttings showed that cell walls of the derivative cells appeared relatively thin which probably is desirable for these cells to readily revert back to the undifferentiated state and ultimately become root primordia. It also showed that the stem had yet to develop any form of mechanical barrier that may restrict the development of adventitious roots. INTRODUCTION Following the differentiation of derivative The formation of an adventitious root begins tissues, active meristematic foci appear within with the differentiation of derivative cells in the them. Inside these foci, the root initials, conical stem to form meristematic foci. Inside each in shape, begin to form. Then differentiation meristematic focus the root initial is formed of phloem and xylem of the root initial takes which later develops into a root primordium place and subsequently becomes structurally and finally emerges as a rootlet. associated with the closest vascular bundle of In most plants, parenchyma cells are the the parent stem. About this time, a root growones capable of reverting to meristematic activing tip is formed which aids the embryo rootlet ity and it is generally these cells which start to to emerge through the bark and epidermis divide to form root initials (Tukey 1979). (Tarasenko 1965). There are cases, however, where root initials In case of rooting, plants are often classiarise from the cambium and medulla fied as 'easy-to-root' (ETR) or as 'difficult-to(Tarasenko 1965., Bose et al 1975). Likewise, root' (DTR) species. There are situations, not all parenchyma cells become meristematic however, when cuttings of DTR species root and produce roots. Usually in the stem, pareneasily, such as, when collected from juvenile or chyma cells near or just outside the phloem, in rejuvenated stocks. The main difference bethe phloem rays or in the interfascicular region tween the two types is that, in ETR species and between vascular bundles, most often produce perhaps juvenile stocks of DTR species, the thinroot initials (Tukey 1979). walled derivative cells show signs of recent

HASAN B. M. AND P.B. DODD

division and they contain cytoplasm and organelles of which nuclei are clearly visible. In contrast, the derivative cells of DTR plants are usually found between fibres, elongated periclinally and are in the process of being transformed into thick-walled sclereids lacking living contents (Beakbane 1969). Furthermore, in ETR plants, the root initials arise in the secondary phloem usually in association with rays. Soon, rays make contact at their distal ends with living cells by means of cytoplasmic strands. In cuttings of DTR plants, the root initials attach to the ends of fibres, sclereids or other elements without living protoplast (Beakbane 1969). Other possible factors that may delay the formation of root initials are i) the presence of a greater extent of tissue sclerification approaching an uninterrupted layer of sclerenchyma in the pericycle, and ii) when derivative tissues are less conspicuous (Bose et al 1975). MATERIALS AND METHODS

The stock plants used in this study were unknown seedlings obtained from Malaysia and grown at Wye College. They were grown individually in 25 cm black plastic pots containing a peat based compost, kept in a heated glasshouse maintained at a minimum temperature of 25°C, 12-hour day length at an intensity of 150 to 160 |iE/nrr,/second. These seedlings were used as stock plants when they were about two years of age. Between 8 to 10 cm long, healthy shoots were detached from the stock plants using a sharp scalpel. Following their detachment, leaves were removed leaving only two or three near the shoot tip. If the remaining leaves were too large they were trimmed to about one half their length. Bases of these cuttings were individually dipped for 15 seconds in a solution of 50% alcohol containing 5000 ppm indolebutyric acid (IBA), to a depth of 1.0 cm and surface-dried for five to ten minutes. Prior to planting, cuttings were quickly dipped in a solution of 0.4% 'nimrod' fungicide up to about 4 to 5 cm from the base, to reduce rotting. Various cutting pieces, including unrooted stem segments and calli from cuttings were collected for histological studies to study the anatomical changes that take place during the 300

rooting process. These samples were collected at weekly intervals. Samples were kept in vials containing excess FAA and fixed under vacuum for at least 24 hours. The subsequent processes that is dehydration in a series of solutions with varying proportions of water, alcohol and tertiary butanol, infiltration with wax, embedding in wax, and mounting were done in the normal way as described by Purvis et al (1966). Transverse sections 15 |im thick were made using a sledge microtome and temporarily mounted on a glass slide with Haupt's adhesive. Prior to staining with Safranin 0 and counter staining with fast green, wax from the sections was removed by immersing the slides completely in lcJw-sulphur xylene until the wax had fully dissolved. The subsequent staining procedures that is rehydration in descending strengths of alcohol, staining with Safranin 0, dehydration in ascending strengths of alcohol, immersion in a 50:50 mixture of alcohol/clove oil, counter staining with fast green, and permanent mounting in euparal, were done in the usual way as described by Purvis et al (1966). From each section, representative 'fields of view' showing the rooting process were examined and photomicrographs of these 'fields' were taken with an Orthomat microscope camera, attached to a Leitz microscope. RESULTS AND DISCUSSION

Anatomically, softwood cuttings obtained from very juvenile stock plants have yet to develop any form of mechanical barriers, such as thick layers of cork tissue outside the cortex, or a sclerenchymatic sheath peripheral to the primary phloem. Cell wall of the parenchymatous cells, especially the cortex, appeared relatively thin (Plates I to V). The relationship between stem anatomy and adventitious rooting may have an important bearing on the capacity of these stems to form adventitious roots. From the histological examinations made on the samples taken immediately after cuttings were made (week 0) (Plate I) and one week after propagation (Plate II), no pre-formed root initials could be traced. However, after the cuttings had been propagated for two weeks, root primordia became conspicuous (Plate III)

PERTANIKAVOL 12 NO. 3, 1989

ADVENTITIOUS ROOT FORMATION IN STEM CUTTINGS OF YOUNG DURIAN SEEDLINGS

and by the third week, some roots emerged ' (Plate IV); others were already fully initiated but had yet to develop into roots.

,

Hate IV Adventitious root (x 40)

Plate I Transverse section of the stew immediately after cutting was made (x ^D)

These roots were believed to be initiated from the living cells in the pericylic zone, near the fibre groups and in association with ray parechynia and cortical cells. When callus was present, root could also be initiated from the callus cells (Plates V and VI).

mi Plate II One week after cutting was made (x 40) Plate V (x 40)

Plate III Root fmmordium from cells in thepericydic zone (x 100) .

yj ROO1 inmai (arrow) from the callus cells formed ai ih€ base of softwood stem cutting

PERTANIKA VOL. 12 NO. 3, 1989

SOI

HASAN B. M. AND P.B. DODD

CONCLUSION The information from this study forms a useful understanding for further studies on the rooting of cuttings from less juvenile materials.

BERKBANE A.B. 1969. Relationship between Structure and Adventitions Rooting. Comb. Pror. Int. Plant Prop. Soc. 19: 192-201. T.K.,

T.P.

MUKHERJEE,

and

T.

ROY.

1975. Standardization of Propagation from Cutting under Mist. I. Effect of Type of Wood and

302

PURVIS M.J., D.C. COLLIER, and D. WALLS.

1966.

Laboratory Techniques in Botany, 2nd edn, London: Butterworth 439 pp. TARASENKO, M.T.

REFERENCES

BOSE,

Size of Cutting on Root formation. Punjab Hortic /., 15: 139-143.

1965. Morphological and Ana-

tomical Features of the differentiation of Tissues and Organs in Vegetative Propagation of Higher Plants. Izvestiya Timir. SeVskok. Akad. 3: 58-70. TUKEY, H.B. 1979. Back to the Basics of Rooting. Comb. Proc. Int. Plant Prop. Soc. 29: 422-428.

PERTANIKA VOL. 12 NO. 3, 1989

(Received 2 September, 1988)

Pertanika 12(3), 303-311 (1989)

Determination of Calcium in Foods by the Atomic Absorption Spectrophotometric and Titrimetric Methods TEE E. SIONG, KHOR SWAN CHOO and SITI MIZURA SHAHID Division of Human Nutrition Institute for Medical Research 50588 Kuala Lumpur, Malaysia. Key words: Calcium in foods, atomic absorption spectrophotometry, potassium permanganate titration. ABSTRAK Laporan ini membentangkan hasilsatu kajian perbandingan penentuan kandungan kalsium dalam pelbagai jenis makanan dengan kaedah-kaedah spektrofotometri penyerapan atom (AAS) dan titratan dengan kalium permanganat. Larutan abu telah disediakan bagi setiap sampel makanan (dianalisis secara duplikat). Satu alikuot larutan tersebut telah dianalisis dengan kaedah AAS, manakala satu lagi dengan kaedah titratan. Sejumlah 132 jenis makanan yang terdiri daripada 8 kumpulan makanan telah dikaji. Nilai min bagi analisis duplikat setiap makanan dengan kedua-dua kaedah itu telah dibentangkan mengikut kumpulan makanan. Hasil yang diperolehi dengan kaedah AAS dan titrimetri didapati mempunyai keselarian dan korelasi yang baik (r = 0.998). Ini telah disahkan dengan ujian "paired t"yang menunjukkan bahawa bagi 6 kumpulan makanan yang dikaji, perbezaan kandungan kalsium yang diberi oleh kedua-dua kaedah tidak bererti (p < 0.05). Walaupun begitu perbezaan bagi 2 kumpulan yang lain iaitu kekacang dan sayuran didapati bererti dan angka statistik t yang diperolehi kecil. Kedua-dua kaedah didapati memberi perbezaan min hasil bilas yang tidak bererti dan menghampiri 100. Didapati juga perbezaan yang tidak bererti bagi varians kaedah-kaedah itu. Hasil kajian ini telah menunjukkan bahawa kedua-dua kaedah dapat digunakan dengan memuaskan bagi analisis zat ini. Walaupun demikian, pilihan sesuatu kaedah juga bergantung kepada beberapa faktor yang lain, termasuk adanya alat dan kepakaran yang diperlukan. ABSTRACT This report presents results of a comparative study of the determination of calcium in a wide variety of foods using the atomic absorption spectrophotometric (AAS) and potassium permanganate tifration methods. Ash solution for each food sample (determined in duplicate) was prepared and an aliquot subjected to AAS analysis, while another aliquot was determined by the titrimetric method. A total of 132 foods, belonging to 8 food groups were studied. Mean values for duplicate analysis of each food determined by the two methods were tabulated according to food groups. Results obtained by the AAS and titrimetric methods showed good general agreement, and a high correlation coefficient (r = 0.998) was obtained. This was confirmed by paired t-test which showed that for 6 of the food groups studied, there was no statistically significant difference (p < 0.05) in calcium concentrations determined by the two methods. For the remaining 2 groups, legumes and vegetables, a significant difference in results was obtained. However, in both cases, the t-statistic calculated was small. Both methods were found to give mean percent recovery values which were not significantly different and close to 100. There was also no significant difference in variances given by the two methods. Results of the study therefore have shown that either method can be used satisfactorily for the analysis of this nutrient. The choice of method, however, also depends on various other factors, including availability of required instrument and expertise. INTRODUCTION Calcium has been documented in studies of nutrient composition of local foods since the

early part of the century. One of the earliest reports was that of Morris and Oliveiro (1933) who documented the content of this mineral in

TEE E. SIONG, RHOR SWAN CHOO AND SITI MIZURA SHAH1D

between the two analytical methods. This could some 60 types of foods. In that study, calcium be of assistance to laboratory workers intending was precipitated as calcium oxalate, converted to use either methods, such as in situations to calcium oxide, weighed and reported as such. mentioned above. The study was carried out Some years later, Leong and Morris (1947) used together with a comparative study of the detera different procedure for determining this mination of iron using the AAS and colorimetric mineral. Calcium was again precipitated as methods (Tee ei al 1989). oxalates, but instead of using the more cumbersome gravimetric procedure, calcium present MATERIALS AND METHODS was next titrated with potassium permanganate Samples of foods from various food groups were and results expressed as milligram calcium. purchased from local markets and retail stores Subsequent reports on nutrient analyses of local for analysis. Wherever applicable, refuse in each foods had used this titrimetric method for deterfood item was removed and its proportion in mining calcium. the food determined. The edible portions were The potassium permanganate titration meblended and aliquots taken for analysis. thod (after precipitation of calcium as oxalate) An amount of 5-15 g of the homogenized has remained the method of choice for detersample was dried in an air oven at 105°c for S mination of calcium in foods for many laborahours. The dried sample was next charred until tories, including this Division. In recent years, it ceased to smoke. The charred sample was the atomic absorption spectrophotometric then ashed in a muffle furnace at 550°C until (AAS) method has been introduced. This, and a whitish or greyish ash was obtained. The ash the titrimetric methods, are recognized methods was treated with concentrated hydrochloric acid, for determination of calcium in foods, and are transfered to a volumetric flask and made up cited in Pearson's Chemical Analysis of Foods to 50 ml. For each food studied, two ash solu(Egan ei al 1981). Both methods are currently in use by laboratories in the country earning tions were prepared, i.e. duplicate analysis was out studies into nutrient composition of foods. carried out. An aliquot of each ash solution was used for the determination of calcium by the The choice of either the AAS or titrimetric method has relied on various factors, includAAS method and another aliquot by the titriing availability of the required instrument as metric method. well as expertise. For various reasons, it would For the AAS method, a Varian Atomic be important to determine it the AAS and titriAbsorption Spectrophotometer model 175 with metric methods give comparable results. Differan air-acetylene flame, and wavelength set to ent laboratories participating in a joint pro422.7 nm was used. Calcium carbonate was used gramme for the analysis of calcium using the as standard to prepare a calibration curve with two different methods would need to deterat least 4 concentrations of calcium within the mine if the results obtained are comparable. analytical range. To eliminate phosphorus interBefore switching over to a newly purchased ference in the determination, lanthanum was atomic absorption spectrophotometer, a laboadded to the test ash solution and standard ratory would need to find out if the results to solutions so that the final solutions contained be obtained would be comparable to those 1% La. Concentration of calcium in test solupreviously obtained with the titrimetric method. tions was calculated from the standard curve On the other hand, in a laboratory using the prepared. For each ash solution, at least three AAS method, it may be necessary to switch to readings were obtained and the average cal the titrimetric method if the spectrophotoculated. meter breaks down for a considerable length in the titrimetric method, an aliquot of of time. the ash solution was reacted with ammonium This report presents results of a comparaoxalate solution to precipitate out the calcium. tive study of the determination of calcium in a After centrifugation and decanting the superwide variety of foods using the AAS and titrinatant liquid, the precipitate was redissolved in metric methods. It is hoped that the results in4X sulphuric acid. Calcium in solution was dicate clearly significant differences, if any, titrated against 0.0IN potassium permanganate, 304 PERTANIKAVOL. 12 NO. 3, 1989

DETERMINATION OF CALCIUM IN FOODS BYTHE AAS AND TITRIMETRIC METHODS

with the solution kept at about 75-85°C throughout the titration. For each ash solution prepared, at least two titrations were carried out to determine the average titre. Standard solutions of calcium carbonate were similarly titrated and the titre used for calculation of calcium in the test ash solutions. Recovery studies were performed by adding a known amount (about 50% of the estimated calcium content of the food) of calcium stock standard to the food. Preparation of ash solution and analysis of calcium using the AAS and titrimetric methods were carried out as described above. Details of the AAS and potassium permanganate titration methods used are described in the laboratory manual in use in this Division (Tee et al 1987). All results were expressed as per 100 g edible portion of the food. Mean values for duplicate analysis of each food determined by the two methods were calculated and results tabulated according to food groups. For each food group, the paired t-test was carried out using the ABSTAT statistical programme to determine if the two methods gave significantly different results. Correlation coefficient was calculated using the same programme. Analytical process standard deviations of the two methods were compared using the F-ratios method (Wernimont 1985).

TABLE 1 Calcium in cereals and products as determined by the atomic absorption spectrophotometri and titrimetric methods mg Ca/100 g edible portion English/local name

AAS method

Titrimetric method

Bread, coconut

17.7

Bread, ryemeal Bread, white Bread, wholemeal Noodle laksa, thick, dry Noodle laksa, thick, wet Oats, processed, tinned Oats, rolled Rice, broken Rice bran, coarse Rice bran, fine Rice noodle (Loh-see-fun) Wheat flour, high protein Wheat flour, wholemeal Wheat germ

49.0 43.9

15.9 50.3 42.3 38.9

10.3 5.1

9.2 4.0

49.6 39.7

47.6 38.7

7.5

7.1

50.4 45.2

51.3 51.1

4.6

3.7

26.9 45.5 55.2

24.8 42.9 53.3

Each value is the mean of duplicate analysis TABLE 2 Calcium in legumes and products as determined by the atomic absorption spectrophotometric and titrimetric methods mg ca/100 g edible portion AAS method

Titrimetric method

42.4 132.9 30.6

40.5 127.6 24.8

75.1

70.8

179.9 183.7

160.8 156.9

224.7

191.3

262.7 curd (Tau-hoo-fa) 67.3 curd (Tau-hoofok) 82.1 6.3 milk, packet milk, unsweetened 14.4 25.0 noodles

239.3 66.3 77.0

RESULTS AND DISCUSSION

A wide variety of foods from various food groups were studied, to determine if different food matrixes would affect the results obtained. A total of 132 foods, belonging to 8 food groups were studied. Mean values for duplicate analysis of each food determined by the AAS and titrimetric methods were tabulated according to food groups (Tables 1 to 8). In all the tables, the English names of the foods are given, and arranged in alphabetical order. Where these names may be ambigious or unclear, or when the English names are not known, the local names of the foods have been included. The scientific names of the foods are also tabulated where appropriate. There was generally good agreement in the results obtained by the two methods (Tables 1 to 8). This is clearly seen in the scatter dia-

38.2

Baked beans, canned Chickpea/Common gram Dhal, Mysore Soya bean, fermente (Tempeh) Soya bean cake (Tau-Kua), spiced Soya bean cake (Tau-kua) Soya bean curd sheets (Fucok)

Soya bean curd, strands (Fucok)

Soya Soya Soya Soya Soya

bean bean bean bean bean

5.1

12.5 25.7

Each value is the mean of duplicate analysis

PERTANIKA VOL. 12 NO. 3, 1989

305

TEE E. SIONG, KHOR SWAN CHOO AND SITI MIZURA SHAHID

TABLE 3 Calcium in nuts and seeds as determined by the atomic absorption spectrophptometric and titrimetric methods mg Ca/100 g edible portion Scientific name

English/local name Almond Arecanut shavings Brazil nut Candlenut Cashew nut Chestnut, Chinese Coconut cream Coconut flesh, old Coconut flesh, young Coconut milk Coconut water Lotus seed Peanut butter Sesame seed/Gingelly seed Walnut, dried Watermelon seed, black, dried

Prunus

amygdalus

Areca catechu Beriholktia excelsa Aleurites

moluccana

Anacardium

occidentak

Castanea spp. Cocos nucifera Cocos nucifera Cocos nucifera Cocos nucifera Cocos nucifera Nelumbo nucifera Arachis hypogea Sesamum indicum

Juglans regia CUruUus vulgaris

AAS method 243.1 39.6 173.9 152.6 37.0 15.8 6.6 9.1 20.5 8.0 15.2 131.6 45.7 55.3 131.9 56.7

Titrimetric method 221.9 43.6 200.9 148.5 35.1 16.0 6.8 9.4

19.5 8.8

13.2 117.4 45.4 53.0 118.5 53.8

Each value is the mean of duplicate analysis

TABLE 4 Calcium in vegetables as determined by the atomic absorption spectrophotometric and titrimetric methods mg Ca/100 g edible portion English/local name

Scientific name

Asparagus, canned Asparagus, fresh Drumstick, fresh pods Gourd, bottle/Calabash Kadok, leaves Leek Mushrooms, grey oyster, fresh Peas, garden, fresh Purslane Radish, Chinese, pickled Rhubarb/Pie plant, petioles Seaweed, agar (Agar-agar) Spinach, Ceylon

-

Spinach (Bayam pasir)

-

Asparagus offidnalis Asparagus offidnalis Moringa oleifera Lagenaria

vulgaris

Piper sarmentosum Allium Pisum

porrum sativum

Portulaca oleracea Raphanus Rheum

sativus rhaponticum

Basella rubra

Tree tomato

Cyphomandra betacea

Yam bean

Pachyrrhizus erosus

Each value is the mean of duplicate analysis

306

PERTANIKA VOL. 12 NO. 3, 1989

AAS method

Titrimetric method 13.7 12.3 22.4 14.42 219.5 15.8

14.7 13.9 23.8 15.8 246.1 16.2 1.0 62.5 76.1 94.9 268.9 510.2 116.2 318.4 11.2

58.7 72.4 98.8 253.0 502.1 112.5 287.2 11.6

12.4

12.5

2.2

DETERMINATION OF CALCIUM IN FOODS BYTHE AAS AND TITRIMETRIC METHODS

TABLE 5 Calcium in fruits as determined by the atomic absorption spectrophotometric and titrimetric methods mg Ca/100 g edible portion

English/local name Avocado Banana (Pisang kelat) Binjai

Cashew apple Custard apple Date, dried Durian cake Grapefruit Jering Kundang Lychee Mango (Bacang gelok)

Nutmeg, fresh Persimmon, dried Prunes, dried Pulasan Soursop Strawberry

Titrimetric method

AAS method

Scientific name Persea americana Musa sapientium Mangifera caesia Anacardium occidental Annona squamosa Phoenix dactylifera Durio zibethinus Citrus paradisi Pithecellotrium lobatum Bouea macrophylla Litchi chinensis Mangifera foetida Myristica fragrans Diospyros kaki Prunus spp. Nephelium mutabile Annona muricata Fragaria grandijlora

12.1

13.8 5.7 6.9 2.0 16.4 47.6 9.4 28.5 31.3 4.9 5.1

6.8 6.7 2.0

15.5 42.2 11.1 26.8 38.1 5.2 5.1

16.0 26.8 43.1 56.3

15.5 24.1 36.1 52.4

7.8 12.0 12.0

7.1

10.6 11.9

TABLE 6 Calcium in meat and eggs as determined by the atomic absorption spectrophotometric and titrimetric methods mg Ca/100 g edible portion AAS method Beef extract Beef rendang, canned Chicken feet, deboned Chicken gizzard Chicken heart Chicken intestines Duck egg, salted, yolk Duck egg, yolk Mutton curry, canned Ox maw Turtle egg, white Turtle egg, yolk

40.4 31.1 25.1 7.4 6.0 7.7 184.1 151.3 16.1 10.7 19.6 165.2

Titrimetric method 43.2 26.6 23.3 7.2 6.0 5.7 188.7 139.7 15.6 9.7 21.0 157.9

Each value is the mean of duplicate analysis

PERTANIKA VOL. 12 NO. 3, 1989

307

TEE E. SIONG, KHOR SWAN CHOO AND SITI MIZURA SHAHID

TABLE 7 Calcium in fish and fish products as determined by the atomic absorption spectrpohotometric and titrimetric methods mg Ca/100 g edible portion

Scientific name

English/local name Anchovy, cleaned, dried Anchovy, whole, dried Cuttlefish, dried Fish balls Fish bladder, dried Fish bladder, fried Fish curry, canned Fish roe Fish sauce (Budu) Hairtail scad, dried Live crab/Swimming crab Oyster sauce Oyster Prawn paste (Hay-ko) Sea crab/Blue crab Shark's fin, dried Shrimp, fermented (Cincalok) Threadfin, dried Yellow banded trevally, dried

Stolephonts commersonii

StoUphorus row mersonii Sepia officinalis

Megalaspis cordyla

Ostrea spp. Ostrea spp.

Polynemus

indicus

Sela roides leptolepis

AAS method 547.5 1238.1 103.1 58.5 19.3 18.0 338.0 13.1 390.6 95.6 232.8 24.8 180.9 286.1 168.7 418.1 450.3

29.3 157.4

Titrimetric method 500.5 1255.2 95.7 56.5 19.0 20.6 320.8 12.6 383.9 98.1 226.3 16.9 174.5 325.9 167.6 425.5 475.3 34.8 150.4

Each value is the mean of duplicate analysis

TABLE 8 Calcium in miscellaneous foods as determined by the atomic absorption spectr photometric and titrimetric methods mg Ca/100 g edible portion

English/local name Anise seed, dried Cardamon Choocolate, raisin Cinnamon Coffee mixture, powder Cumin seeds, black Cumin seeds, white Curry powder Fenugreek seeds Galangal Honey

Scientific name Pimpinella anisum Ehitaria cardamomum Cinnamomum zeylanicu m Nigella saliva Cuminum cyminum Trigonella foenum-graecum Ijmguas galanga -

AAS method 950.6 1769.7 182.6 600.9

167.5 816.8 1165.1 576.2 179.8 12.8

Titrimetric method 1004.8 1704.0 178.4 534.0 180.2 818.1 1093.3 560.1 174.8 9.5 8.6

7.0 Continued

PKRTANIKA VOL. 12 NO. 3, 1989

on next page

DETERMINATION OF CALCIUM IN FOODS BYTHE AAS AND TITRIMETRIC METHODS

TABIH 8: Continued

mg Ca/100 g edible portion AAS method

Scientific name

English/local name _

Jam, egg (Seri kaya)

Jam, pineapple

1.7

8.0 3.3

133.2 501.9 7.9 761.3 120.4

124.0 488.1 7.7 711.1 122.1

8.4

Jelly crystals

Malted milk powder Marmalade Milk-based diet supplement, powder Pepper, powder, white Sugar cane juice Tamarind paste (Asamjawa) Treacle, black Yeast, dried, brewer's Yeast, granules, tinned

Titrimetric method

Piper nigrum Saccharum offirinarum Tamarindus indica Saccharomyces cerevisiae Saccharomyces cerevisiae

6.5

6.1

101.7 517.4 400.7 68.6

89.2 487.6 420.2 65.5

Each value is the moan of duplicate analysis

600 -

100

200

300

400

500

600

Atomic absorption spactrophotometrtc method

Fig. 1: Calcium concentration determined by the AAS and Titrimetric methods (mg Calcium per 100 g edible portion).

PERTANIKA VOL. 12 NO. 3, 1989

309

TEE E. SIONG, KHOR SWAN CHOO AND SITI MIZURA SHAHID

gram, plotting 126 pairs of results obtained (Figure 1). T h e remaining 6 pairs were omitted from the plot as they were much higher than the majority of the values obtained. A good correlation coefficient (r = 0.998) was obtained for all 132 pairs of results obtained. Results of paired t-test for all food groups

The pooled standard deviation obtained for all the 132 foods studied was 14.5 for the AAS method and 15.8 for the titrimetric method, Comparing the variance obtained for all foods, the observed F-ratio was calculated to be 1.17. There was thus no statistically significant difference (p < 0.05) in the variances given by the

studied (Table 9) showed that for 6 food groups, there was no statistically significant difference (p < 0.05) in calcium concentration determined by the AAS and titrimetric methods. For the remaining 2 groups, legumes and vegetables, a significant difference in results was obtained. However, in both cases, particularly for vegetables, the t-statistic calculated was small, just above the significance level. Recovery of added calcium to the foods was determined in 14 separate studies. Results obtained (Table 10) showed that mean percent recpvery values for both methods were close to 100, with small coefficient of variation. There was no statistically significant difference between the two mean recovery values (p < 0.05).

^°

methods.

CONCLUSIONS In this study, the AAS and the potassium permanganate titration methods did not give significantly different calcium concentrations for a wide variety of foods. Both methods gave good recovery values, and no significant difference in process variablility was observed. Either method can, therefore, be used satisfactorily for this analysis. There are, however, advantages and disadvantages for both methods, The titration method tends to be more tedious and more prone to errors due to the number of steps involved in preparing the solution for titration. This include adjustment

TABLE 9 Summary statistics of paired t-test of calcium concentration of various foods determined by the atomic absorption spectrophotometric and titrimetric methods Food group

Cereals and products Legumes and products Nuts and seeds Vegetables Fruits Meat and eggs Fish and fish products Miscellaneous

n

Calculated t-statistic

15 13 16 16 18 12 19 23

0.939 3.094 0.749 2.312 1.312 1.316 0.134 1.862

Statistical sign ificancel

N.S 2

Ss N.S.

S. N.S. N.S. N.S. N.S.

1

at p < 0.05 not statistically significant * statistically significant

2

TABLE 10 Recovery values obtained by the atomic absorption spectrophotometric and colorimetric methods AAS method

Titrimetric method

14

14

Mean ± SD

96.9 ± 9.3%

93.5 ±

Coefficient of variation

9.6

7.3

Number of determinations

310

PERTANIKA VOL. 12 NO. 3, 1989

DETERMINATION OF CALCIUM IN FOODS BYTHE AAS AND TITRIMETRIC METHODS

of pH of ash solution, precipitation of calcium as oxalate, and collection and cleaning of the precipitate. The titration itself has to be carefully performed, keeping the test solution at a temperature of 75-85°C. The procedure is, however, relatively much cheaper, requiring no expensive instrument. In the hands of an experienced worker, this method can provide reliable results. The AAS method, on the other hand, requires the purchase of an expensive spectrophotometer. It has also to be borne in mind that maintaining the instrument to ensure optimal performance is a difficult task. It is however, a relatively simpler procedure. The ash solution can be used directly for spraying in the spectrophotometer, after the instrument has been appropriately set up. It would be the method of choice, provided the required budget is available. ACKNOWLEDGEMENTS We would like to acknowledge the assistance of Ms Chin Suan Kee of this Division and. Ms Ng Yin Kwan of Tunku Abdul Rahman College, Kuala Lumpur, in carrying out some of the analyses. We thank Dr. M. Jegathesan, Director

of the Institute for Medical Research for permission to publish the results of this study. REFERENCES EGAN, H., R.S. KIRK and R. SAWYER.

1981. Pearson's

Chemical Analysis of Foods. 8th Edn. London: Churchill Livingstone, pp. 26-28. LEONG, P.C. and J.P. MORRIS.

1947.

Calcium and

Oxalic Acid of Vegetables. Med. J. Mai 1 : 289 - 297. MORRIS, J.P. and CJ. OLIVEIRO. 1933. Calcium in Tropical Foods. Mai Med. J. 8 : 236 - 238. TEE, E.S., S. SITI MIZURA, R. KULADEVAN, S.I. YOUNG,

S.C. KHORand S.K. CHIN (editors).

1987.

Lab-

oratory Procedures in Nutrient Analysis of Foods. Division of Human Nutrition, Institute for Medical Research, Kuala Lumpur, pp. 82 - 84 and 92 - 96. TEE, E.S., S.C. KHOR and

S. SITI MIZURA.

1989.

Determination of Iron in foods by the Atomic Absorption Spectrophotometric and Colorimetric Methods. Pertanika. 12(3): 313-322. WERNIMONT, C.T. 1985. Use of Statistics to Develop and Evaluate Analytical Methods. Association of Official Analytical Chemists: Virginia.

PERTANIKA VOL. 12 NO. 3, 1989

(Received 6 June, 1989)

311

Pertanika 12(3), 315-322 (1989)

Determination of Iron in Foods by the Atomic Absorption Spectrophotometric and Colorimetric Methods TEE E SIONG, KHOR SWAN CHOO and SITI MIZURA SHAHID Division of Human Nutrition, Institute for Medical Research, 50588 Kuala Lumpur, Malaysia Key words: Iron in foods, atomic absorption spectrophotometry, colorimetry. ABSTRAK Satu kajian perbandingan penentuan kandungan zat best dalam pelbagai jenis makanan telah dijalankan dengan menggunakan kaedah penyerapan atom spektrofotometer (AAS) dan kaedah metri warna melalui tindak balas dengan fenantrolin. Sejumlah 156 jenis makanan, yang terdiri daripada 8 kumpulan makanan telah dikajL Bagi setiap makanan (dianalisis secara duplikat), larutan abu telah disediakan dan satu alikuot telah dianalisis dengan kaedah AASy manakala satu lagi dengan kaedah fenantrolin. Nilai min bagi analisis duplikat setiap makanan telah dibentangkan mengikut kumpulan makanan. Keputusan daripada kaedah AAS dan fenantrolin menunjukkan keselarian yang baik dengan koefisien korelasi 0.987. Analisis statistik dengan menggunakan ujian "berpasangan t" menunjukkan perbezaan keputusan dari kedua-dua kaedah adalah tidak bererti (p < 0.05) bagi 5 kumpulan makanan. Walaupun perbezaan yang bererti telah diperhatikan bagi 3 kumpulan makanan yang lain, angka statistik-t yang diperolehi hanya melebihi sedikit sahaja di atas paras bererti. Hasil bilas yang diperolehi bagi kedua-dua kaedah adalah memuaskan dan tidak mempunyai perbezaan yang bererti. Akan tetapi, varians bagi kaedah fenantrolin lebih tinggi sedikit. Hasil kajian menunjukkan bahawa kedua-dua kaedah tersebut dapat digunakan dengan memuaskan untuk analisis zat galian ini. ABSTRACT A comparative study of the determination of iron in a wide variety of foods was carried out using the atomic absorption spectrophotometric (AAS) and phenanthroline colorimetric methods. A total of 156 foods, belonging to 8 food groups were studied. For each food (determined in duplicate), ash solution was prepared and an aliquot subjected to AAS analysis, while another aliquot was determined by the phenanthroline method. Mean values for duplicate analysis of each food determined by the two methods were tabulated according to food groups. Results obtained by the AAS and phenanthroline methods showed good general agreement, with a correlation coefficient of 0.987. Statistical analysis using paired t-test showed that for 5 food groups, there was no significant difference (p < 0.05) in results given by the two methods. Although a significant difference was observed for the remaining 3 groups, the t-statistic calculated was just above the significance level. Recovery values given by the two methods were satisfactory, and were not significantly different. Variance for the phenanthroline method was, however, slightly higher. Results of the study suggest that both methods can be used satisfactorily far the analysis of this mineral. INTRODUCTION

Iron deficiency anaemia has long been recognized, and is still an important nutritional deficiency problem in the country, afflicting particularly the vulnerable groups. (Tee, 1985). Thus, there has always been an interest in identifying local foods rich in iron.

Early methods for the determination of iron in foods had relied on the gravimetric procedure. Morris and Rosedale (1935) reported the precipitation of iron in foods with ammonium nitrosophenyl hydroxylamine ("cupferron"), followed by separation and weighing of the mineral as ferric oxide. Subsequently, there was

TEE E. SIONG, KHOR SWAN CHOO AND SITI MIZURA SHAHID

a switch over to colorimetric procedures for the determination of this mineral. Simpson et al (1951) estimated iron based on colour development with thioglycolic acid. Two years later, Leong (1953) reported the use of ortho-phenanthroline for colour development. This colour reagent, as well as bipyridyl, continued to be used by subsequent investigators (Tee et al 1987). Determination of iron using atomic absorption spectrophotometry (AAS) has been introduced in recent years. The choice of either the AAS or colorimetric method has relied on various factors, including availability of the required instrument as well as expertise. For various reasons, it would be important to determine if the AAS and colorimetric methods give comparable results. Different laboratories participating in a joint programme for the analysis of iron using the two different methods would need to determine if the results obtained are comparable. Before switching over to a newly purchased atomic absorption spectrophotometer, a laboratory would need to find out if the results to be obtained would be comparable to those previously obtained with the colorimetric method. On the other hand, in a laboratory using the AAS method, it may be necessary to switch to the colorimetric method if the spectrophotometer breaks down for a considerable length of time. This report presents results of a comparative study of the determination of iron in a wide variety of foods using the AAS and colorimetric methods. It is hoped that the results would indicate clearly if significant differences are given by the two analytical procedures. This could be of assistance to laboratory workers intending to use either methods, such as in situations mentioned above. The study was carried out together with a comparative study of the determination of calcium using the AAS and titrimetric methods (Tee et al 1989). MATERIALS AND METHODS

Samples of foods from various food groups were purchased from local markets and retail stores for analysis. Wherever applicable, refuse in each food item was removed and its proportion in 314

the food determined. The edible portions were blended and aliquots taken for analysis. An amount of 5-15 g of the homogenized sample was dried in an air oven at 105°C for 3 hours. The dried sample was next charred until it ceased to smoke. The charred sample was then ashed in a muffle furnace at 550°C until a whitish or greyish ash was obtained. The ash was treated with concentrated hydrochloric acid, transfered to a volumetric flask and made up to 50 ml. For each food studied, two ash solutions were prepared, i.e. duplicate analysis was carried out. An aliquot of each ash solution was used for the determination of iron by the AAS method and another aliquot by the colorimetric method. For the AAS method, a Varian Atomic Absorption Spectrophotometer model 175 with an air- acetylene flame was used. Wavelength was set to 248.3 nm for solutions with iron concentrations ranging from 2.5 to 10 |ig/ml, or 327.0 nm for concentrations ranging from 25 to 100 |ig/ml. Ferric nitrate solution for atomic absorption spectrophotometry (BDH) was used as standard. A calibration curve with at least 4 concentrations of iron within the analytical range was prepared. Concentrations of iron in test solutions was calculated from the standard curve prepared. For each ash solution, at least three readings were obtained and the average calculated. In the colorimetric procedure, an aliquot of the ash solution was reacted with 1,10-phenanthroline hydrochloride and the resulting red complex read in a uv-vis spectrophotometer at 510 nm. For each ash solution, two tubes were prepared for reaction with ihe colour reagent and the average absorbance reading used for calcultion. A standard curve was prepared using ferric nitrate solution and used for calculation of iron in the test ash solutions. Analytical grade iron wire and ferrous ammonium sulphate have also been found to be suitable for use. The latter tend to be unstable and turn yellow on keeping. Recovery studies were performed by adding a known amount (about 50% of the estimated iron content of the food) of iron stock standard to the food. Preparation of ash solution and analysis of iron using the AAS and

PERTANIKA VOL. 12 NO. 3, 1989

DETERMINATION OF IRON IN FOODS BYTHE AAS AND COLORIMETRIC METHODS

colorimetric methods were carried out as described above. Details of the AAS and colorimetric methods used are described in Tee et al (1987). All results were expressed as per 100 g edible portion of the food. Mean values for duplicate analysis of each food determined by the two methods were calculated and results tabulated according to food groups. For each food group, the paired t-test was carried out using the ABSTAT statistical programme to determine if the two methods gave significantly different results. Correlation coefficient was calculated using the same programme. Analytical process standard deviations of the two methods were compared using the F-ratios method (Wernimont 1985). RESULTS AND DISCUSSION

A wide variety of foods from various food groups were studied, to determine if the nature of the foods affected the results obtained. A total of 156 foods, belonging to 8 food groups were studied. Mean values for duplicate analysis of each food determined by the AAS and phenanthroline colorimetric methods were tabulated according to food groups (Tables 1 to 8). In all

the tables, the English names of the foods are given, and arranged in alphabetical order. Where these names may be ambiguous or unclear, or when the English names are not known, the local names of the foods are included. The scientific names of the foods are also tabulated where appropriate. There was generally good agreement in the results obtained by the two methods, as can be seen from Tables 1 to 8, and the scatter diagram plotting 150 pairs of results obtained (Fig. 7). The remaining 6 pairs were omitted from the plot as they were much higher than the majority of the values obtained. A good correlation coefficient of 0.987 was obtained for all 156 pairs of results. Results of paired t-test for all food groups studied (Table 9) showed that for 5 food groups, there was statistically no significant difference (p < 0.05) in iron content determined by the AAS and colorimetric methods. For the remaining 3 groups, a significant difference in results was obtained. However, in all these cases, the t-statistic calculated was small, just above the significance level. Determination of spiked iron in the foods was carried out in 10 separate studies. Table 10

TABLE 1 Iron in cereals and products as determined by the atomic absorption spectrophotometric and colorimetric methods mg Fe/100 g edible portion English/local name

AAS method

Bread, coconut Bread, ryemeal Bread, white Bread, wholemeal Noodle laksa, thick, dry Noodle laksa, thick, wet Oats, processed Oats, rolled Rice, broken Rice bran, coarse

1.21 3.59 2.40 3.33 2.53 0.25 2.66 2.01 1.64 12.38 0.28 3.37 4.10 8.44

Rice noodle (Lok-see-fun)

Wheat flour, high protein Wheat flour, wholemeal Wheat germ

Colorimetric method 1.39 3.43 2.33 3.18 2.42 0.27 4.16 3.49 2.54 17.96 0.39 5.10 8.14 8.67

Each value is the mean of duplicate analysis PERTANIKA VOL. 12 NO. 9, 1989

315

TEE E. SIONG, KHOR SWAN CHOO AND SITI MIZURA SHAHID

TABLE 2 Iron in legumes and products as determined by the atomic absorption spectrophotometric and colorimetric methods mg Fe/100 g edible portion Colorimetric method

AAS method Baked beans, canned Chickpea/Common gram Dhal, Mysore Soya bean, fermented (Tempeh) Soya bean cake (Tau-kua), spiced Soya bean cake (Tau-kua) Soya bean curd, sheets (Fucok) Soya bean curd, strands (Fucok) Soya bean curd (Tau-hoo-fa) Soya bean curd (Tau-hoo-pok) Soya bean milk, packet Soya bean milk, unsweetened Soya bean noodles

2.11 4.99 5.46 3.09 5.32 2.27 7.06 9.01 0.28 3.30 0.17 0.34 1.37

2.11 5.14 5.85 2.48 4.46 2.53 8.23 10.02 0.42 3.48 0.23 0.34 1.38

Each value is the mean of duplicate analysis

TABLE 3 Iron in nuts and seeds as determined by the atomic absorption spectrophotometric and colorimetric methods mg Fe/100 g edible portion English/local name Almond Arecanut shavings Brazil nut Candlenut Cashew nut Chestnut, Chinese Coconut flesh, old Coconut flesh, young Coconut milk Coconut water Lotus seed Peanut butter Sesame seed/Gingelly seed Walnut, dried Watermelon seed, black, dried

Scientific name Prunus

amygdalus

Areca catechu Bertholletia excelsa Aleurites

moluccana

Anacardium

occidentals

Castanea spp. Cocos nucifera Cocos nucifera

Colorimetric method

3.00 5.44 1.34 1.73 5.95 0.91 1.21 0.49

3.34 7.39 2.34 3.63 6.62 0.96 1.44 0.51 1.18 0.05 2.03 1.86 4.62 2.55 7.86

Cocos nucifera

LOO

Cocos nucifera

0.03 2.88 1.94 5.05 2.63 5.98

Nelumbo nucifera Arachis hypogea Sesamum

indicum

Juglans regia Citrullus

vulgaris

Each value is the mean of duplicate analysis

316

AAS method

PERTANIKA VOL. 12 NO. 3, 1989

DETERMINATION OF IRON IN FOODS BYTHE AAS AND COLORIMETRIC METHODS

TABLE 4 Iron in vegetables as determined by the atomic absorption spectrophotometric and colorimetric methods mg Fe/100 g edible portion

English/local name

Scientific name

Asam gelugor, shoots

Garcinia atroviridis

Asparagus, canned Asparagus, fresh Broccoli

Asparagus officinalis

Cemperai

Champereia grijfithii

Chilli, small Chives, Chinese Coriander leaves Cucumber, hairy Drumstick, fresh pods Garlic, bulbs Garlic, plants Gourd, bottle/Calabash Kadok, leaves Leek Mushrooms, grey oyster, fresh Mustard leaves, Chinese (Sazvi) Mustard leaves, Indian (Kai-coy) Parsley Peas, garden, fresh Salted vegetable Seaweed, agar (Agar-agar) Seaweed, dried Spinach, Ceylon Spinach, red Spinach (Bayam duri) Waterchestnut Winter melon / Wax gourd Wolfberry leaves

Capsicum annuum

Yam bean (Sengkuang)

AAS method

Asparagus officinalis Brassica oleracea

Allium odorum Coriandrum sativum Cucumis spp. Moringa oleifera Allium sativum Allium sativum Lagenaria vulgaris Piper sarmentosum Allium porrum Brassica juncea Brassica juncea Petroselinum crispum Pisum sativum Basella rubra Amaranthus gangeticus Amaranthus spinosus Scirpus tuberosus Benincasa hispida Lycium chinense Pachyrrhizus erosus

0.88 7.06 0.53 0.47 1.96 0.68 0.62 3.86 0.15 0.27 0.48 0.31 0.22 2.26 0.33 0.84 1.35 1.46 9.90 0.75 1.97 5.33 22.94 0.88 2.64 1.69 0.32 0.14 3.11 0.26

Colorimetric method 0.99 6.91 0.59 0.71 1.90 1.24 0.70 2.99 0.15 0.27 0.84 0.42 0.27 1.73 0.28 0.98 1.32 1.45 10.25 0.78 2.05 5.28 21.71 1.07 2.44 1.05 0.39 0.21 2.70 0.34

Each value is the mean of duplicate analysis

PERTANIKA VOL. 12 NO. 3, 1989

317

TEE E. SIONG, KHOR SWAN CHOO AND SITI MIZURA SHAHID

TABLE 5 Iron in fruits as determined by the atomic absorption spectrophotometric and colorimetric methods mg Fe/100 g edible portion AAS method

Colorimetric method

Persea americana

0.49

0.57

Musa sapientium

0.38

0.53

English/local name

Scientific name

Avocado Banana (Pisang kelat) Binjai

Mangifera caesia

0.30

0.30

Cashew apple

Anacardium occidental

0.23

0.27

Custard apple

Annona squamosa

0.36

0.36

Date, dried

Phoenix dactylifera

0.80

0.76

Duku

iMnsium domesticum

0.23

0.27

Fruit cocktail in syrup, canned

-

0.58

0.24

Grapefruit

Citrus paradisi

0.26

0.22

Jering

Pithecellobium lobatum

0.74

0.70

Lime musk (Limau kasturi)

Citrus microcarpa

0.15

0.17

Lychee

Litchi chinensis

0,20

0.21

Mango (Bacang gelok)

Mangifera foetida

0.22

0.22

Mango (Kwini)

Mangifera odorata

0.31

0.27

Nutmeg, fresh

Myristica fragrans

0.22

0.37

Olive

Olea europaea

1.12

1.12

Orange, Mandarin

Citrus reticulata

0.20

0.20

Pear, green

Pyrus communis

0.20

0.23

Persimmon, dried

Diospyros kaki

0.99

1.10

Pineapple syrup, canned

Ananas comosa

0.28

0.29

Prunes, dried

Prunus spp.

1.07

1.03

Pulasan

Nephelium mutabile

0.12

0.17

Rambai

Baccaurea motleynana

0.21

0.19

Soursop

Annona muricata

0.32

0.37

Strawberry

Fragaria grandiflora

0.21

0.20

Water apple

Eugenia aquea

0.17

0.17

Each value is the mean of duplicate analysis

318

PERTANIKA VOL. 12 NO, 3, 1989

DETERMINATION OF IRON IN FOODS BY THE AAS AND COLORIMETRIC METHODS

TABLE 6 Iron in meat and eggs as determined by the atomic absorption spectrophotometric and colorimetric methods mg Fe/100 g edible portion AAS method

Colorimetric method

10.66 4.20 2.82 0.82 1.43 2.05 0.58 1.67 0.69 0.84 8.43 3.48 3.67 0.73 0.36 2.17

Beef extract Beef liver, rendang, canned Chicken curry, canned Chicken feet, deboned Chicken gizzard Chicken heart Chicken intestines Corned beef Duck Duck, roasted Duck egg, salted, yolk Duck egg, yolk Mutton curry, canned Ox maw Turtle egg, white Turtle egg, yolk

12.89 4.46 3.11 0.85 1.79 2.69 1.11 1.72 1.36 1.28 8.27 3.50 3.82 0.87 0.33 2.65

Each value is the mean of duplicate analysis

TABLE 7 Iron in fish and fish products as determined by the atomic absorption spectrophotometric and colorimetric methods mg Fe/100 g edible portion English/local name

Scientific name

AAS method

ColorimetVic method

Anchovy, cleaned Cuttlefish, dried Fish balls Fish bladder, dried Fish bladder, fried Fish curry, canned

Stolephorus commersonii Sepia offecinalis

3.29 2.29 0.55 2.05 2.63 3.37

3.03 2.90 0.50 1.89 2.14 3.59

1.38 3.23 3.33 0.83 0.75 4.99 21.56 0.50 3.04 1.22 1.07 2.05

1.80 3.17 3.27 0.95 0.50 4.96 22.31 0.61 2.52 1.07 0.96 1.68

Fish roe Fish sauce (Budu) Hairtail scad, dried Live crab/Swimming crab Oyster sauce Oyster Prawn paste (Hay-ko) Sea crab/Blue crab Shark's fin, dried Shrimp, fermented (Cincalok) Threadfin, dried Yellow banded trevally, dried

— Megalaspis cordyla Ostrea spp. Ostrea spp. _ Polynemus indicus Selaroides leptolepis

Each value is the mean of duplicate analysis PERTANIKA VOL. 12 NO. 3, 1989

319

TEE E. SIONG, KHOR SWAN CHOO AND SITI MIZURA SHAHID

TABLE 8 Iron in miscellaneous foods as determined by the atomic absorption spectrophotometric and colorimetric methods mg Fe/100 g edible portion AAS method

Scientific name

English/local name

Pimpinella anisum Elettaria cardamomum Cinnamomum zeylanicum Coriandrum sativum Nigella saliva Cuminum cyminum Trigonella foenum-graecum Languas galanga -

Anise seed, dried Cardamon Cinnamon Coffee mixture, powder Corianda seeds Cumin seeds, black Cumin seeds, white Curry powder Fenugreek seeds Galangal Honey Jam, egg (Seri kaya) Jam, pineapple Jelly crystals Malted milk powder Marmalade Milk-based diet supplement, powder Pepper, powder, white Sugar, brown Sugar, coconut palm (Gula Melaka) Sugar cane juice Tamarind paste (Asam Jawa) Treacle, black Yeast, dried

Piper nigrum Saccharum officinarum Tamarindus indica Sacchawmyces cerevisiae

100.53 20.56 2.17 4.47 67.39 42.37 42.94 35.82 16.74 0.23 0.62 0.35 1.16 0.16 17.25 0.09 3.46 4.59 2.76 0.86 0.06 2.95 14.07 32.81

Colorimetric method 86.19 19.85 2.53 4.75 51.15 29.91 27.46 35.03 17.56 0.47 0.54 0.48 1.13 0.22 17.77 0.12 3.47 5.26 2.68 0.73 0.15 3.44 16.94 27.40

Each value is the mean of duplicate analysis TABLE 9 Paired t-test of iron concentrations determined by the atomic absorption spectrophotometric and colorimetric methods Food group

Cereals and products Legumes and products Nuts and seeds Vegetables Fruits Meat and eggs Fish and fish products Miscellaneous 1 1 s

Calculated t-statistic 14 13 15 30 26 16 18 24

2.352 0.973 2.033 0.827 0.376 2.765 0.191 2.112

at p < 0.05 uatistically significant not statistically significant

320

PERTANIKA VOL. 12 NO. 3, 1989

Statistical significance1 S/2 N.S3 N.S. N.S. N.S. S. N.S. S.

DETERMINATION OF IRON IN FOODS BYTHE AAS AND COLORIMETRIC METHODS

TABLE 10 Recovery values obtained by the atomic absorption spectrophotometric and colorimetric methods

Number of determinations Mean ± SD

Coefficient of variation

AAS method

Colorimetric method

10 84.2 ± 11.1

10 90.7 ± 11,2% 12.3

25

y • 0.0957 + 1.0469x

5

10

15

20

25

Atomic absorption spoctrophotomctric method

Fig. 1 Iron concentration determined by the AAS and colorimetric methods (mg iron per 100 g edible portion). n = 150 (results for 6 foods not included)

shows that mean % recovery obtained by the color imetric method was closer to 100 than that for the AAS method. Statistically however, there was no significant difference (p < 0.05) in mean recovery values given by the two methods. Pooled standard deviation calculated for all the 156 foods studied was 0.39 for the AAS method and 0.63 for the colorimetric method. Comparing the variance obtained for all foods, the observed F-ratio was calculated to be 2.66. Variance for the colorimetric method was thus

significantly higher than that for the AAS method (p < 0.05). CONCLUSIONS Results of this comparative study do not show significantly different iron concentrations for a wide variety of foods between the AAS and the o-phenanthroline colorimetric methods. Both methods were found to give satisfactory recovery values. Variance for the colorimetric method was however slightly higher than that for the AAS method. Either method can, therefore, be

PERTANIKA VOL. 12 NO. 3, 1989

321

TEE E. SIONG, KHOR SWAN CHOO AND SITI MIZURA SHAHID

used satisfactorily for this analysis. There are, however, other considerations in the choice of a method for use. In the colorimetric method, several steps are required in preparing solutions for reading in a spectrophotometer. All glassware, chemicals, and water used should be iron-free to prevent contamination to the test solutions. Fortunately, the red colour complex formed is stable for a number of hours. The procedure is also relatively much cheaper, requiring only a low-cost spectrophotometer operating in the visible range. In the hands of a careful worker, the method can perform satisfactorily. The AAS method, on the other hand, requires the purchase of a high-cost spectrophotometer which is also rather expensive to operate and maintain. It is however, a relatively simpler procedure. The ash solution can be used directly for spraying in the spectrophotometer, after the instrument has been appropriately set up. It would be the method of choice, provided the required budget is available. Compared to a similar comparative study on calcium determination in foods (Tee et aLt 1989), it has been observed that there were more variations between iron content given by the AAS and colorimetric methods. Correlation coefficient for this study was sligthly lower and recovery values were also lower with larger coefficient of variation. Process variation for the colorimetric method was also found to be higher. These relate to the observation that iron determination is rather prone to contamination from the environment. Various precautions, requiring greater degree of care and skill in the laboratory worker, have to be taken to minimise this.

322

ACKNOWLEDGEMENTS We would like to acknowledge the assistance of Ms Chin Suan Kee of this Division and Ms Ong Ching Ching, Tunku Abdul Rahman College, Kuala Lumpur, in carrying out some of the analyses. We thank Dr. M. Jegathesan, Director of the Institute for Medical Research for permission to publish the results of this study. REFERENCES P.C. 1953. The Nutritive Value of Coconut Toddy. Br. j . Nutr. 7 : 253-259.

LEONG,

MORRIS, J.P. and J.L. ROSEDALE.

1935.

The Mi-

neral Content of Some Tropical Foods. Mai Med. / 10 : 1-6. SIMPSON, LA., A.Y. CHOW and C.C. SOH. . 195L

A

"Study of the Nutritional Value of Some Varieties of Malaysian rice. Bulletin New Series No. 5, Institute for Medical Research, Kuala Lumpur. TEE, E.S. 1985. Nutritional Anaemias: Spectrum and Perspectives with Relevance to Malaysia. Specialist and Reproductive Research Centre, National Population and Family Development Board, Kuala Lumpur. TEE, E.S., S. Sm MIZURA, R. KULADEVAN, S.I. YOUNG,

S.C. KHORand S.K. CHIN (editors).

1987. Lab-

oratory Procedures in Nutrient Analysis of Foods. Division of Human Nutrition, Institute for Medical Research, Kuala Lumpur, pp. 85-88 & 92-96. TEE, E.S., S.C. KHOR and S. SITI MIZURA.

1989.

De-

termination of Calcium in Foods by the Atomic Absorption Spectrophotometric and Titrimetric Methods. Pertanika 2(3): 303-311. WERNIMONT, G.T. 1985. Use of Statistics to Develop and Evaluate Analytical Methods. Association of Official Analytical Chemists, Virginia.

PERTANIKA VOL. 12 NO. 3, 1989

(Received 6 June, 1989)

Pertanikal2(3), 323-328(1989)

Trials on Induced Ovulation of Fugu niphobles (Jordan and Snyder) with Human Chorionic Gonadotropin S.H. CHEAH Department of Fisheries Biology and Aquaculture, Faculty of Fisheries and Marine Sciences, Universiti Pertanian Malaysia, 43400 UPM Serdang, Selangor Darul Fhsan, Malaysia. H. SATOH Fisheries Laboratory, Faculty of Agriculture, University of Tokyo, Bentenjima, Shizuoka Prefecture 431-02, fapan. Key words : Induced ovulation, hormone, Fugu niphobles. ABSTRAK Kesan penggunaan HCG (Human Chorionic Gonadotropin) untuk ovulasi Fugu niphobles telah dikaji. Ikan betina yang buncit abdomennya dibahagikan kepada empat kumpulan 1, 2, 3 dan 4, dan dirawat dengan hormon HCG pada takaran masing-masing, 0, 100, 200 dan 300 I.U.. Selepas ikan betina dikumpulkan mengikut kumpulan, ikan-ikan disuntik dan ikan yang didapati tidak berovulasi disuntik semula selepas 72 jam. Takaran suntikan dikira selepas berat badan betina diambil kira dan takaran diberikan sebagai LU./g berat badan. Data kejayaan ovulasi untuk rawatan 0, 1, 2, 3 dan 4 I.U./g berat badan masing-masing, 40%, 83%, 70%, 90% dan 80%. Ini menunjukkan bahawa ikan dalam kumpulan kawalan iaitu kumpulan yang tidak menerima suntikan hormon dapat berovulasi dalam keadaan kurungan dan penggunaan HCG dapat meninggikan kadar kejayaan ovulasi. ABSTRACT The effect of administration of HCG (Human Chorionic Gonadotropin) on ovulation o/Fugu niphobles was studied. The female fish which exhibited distended abdomen were randomly assigned to four groups and the treatments for groups 1, 2, 3 and 4 were 0, 100, 200 and 300 I.U. of HCG per fish respectively. The fish were injected immediately after the groups were established and unovulated females were reinjected after 72 hours. The dosage for each fish was calculated based on the body weight and expressed as I. U./g body weight. The ovulatory success in fish administered with 0, 1, 2, 3 and 4 I.U. ofHCG/g body weight was 40%, 83%, 70%, 90% and 80% respectively. This indicated that fish in the control group could also ovulate in captivity and that administration of HCG would enable a higher degree of ovulatory success. INTRODUCTION Fugu niphobles locally known as Kusafugu in Japan is toxic and the poison Tetrodotoxin is mainly found in the ovary, liver, blood and skin. In recent years, research on the origins and metabolism of the poison has been actively pursued in Japan. The changes in the amount of the poison during egg development, growth and maturation are important. The fry of this

fish could be used as food for the more economically valuable Torafugu {Fugu rubripes) to reduce cannabalism during fry and fingerling rearing. The earliest reports on breeding of this fish were those by Uno (1955) and Fujita et al (1966). The natural breeding season for this fish in Japan ranges from May to early July. It ordinarily spawns only 1-5 days following each

S.H. CHEAH AND H. SATOH

full or new moon, at rising tide. The males shed milt everyday during one or three series of high tides in succession whereas the females appear to discharge sticky eggs completely in one spawning period (Uno, 1955). Fujita^a/. (1966) successfully ovulated F. niphobles using Synahorin at a dose of 10 RU per fish and it was also reported that ovulation occurred 24 to 48 hours after the administration of Synahorin. As it is difficult to collect naturally spawned eggs or fry from the sea, there is a need to be able to spawn this fish in captivity and also be able to culture the fry and fingerlings. In order to pursue the problem of establishing a consistent technique for breeding F. niphobles, an experiment was conducted to determine the effect of administration of HCG (Human Chorionic Gonadotropin, commercial namePuberogen) at different dosages on ovulation. The results of the ovulatory response and egg incubation experiments are discussed. MATERIALS AND METHODS

kept together. Fish that did not ovulate in the hormone treated groups after three days were reinjected with a second and final dose of the hormone according to the specific treatment dosages. In the first trial, the fish were checked for ovulatory response once a day at 1500 hours but in the subsequent trials, the fish were checked twice a day at 1000 hours and 1500 hours. When a gentle pressure on the abdomen resulted in the release of eggs, the females were anaesthetized and weighed. The weight of eggs that were stripped from each female was also determined. The data for ovulatory success and failure were subjected to the KolmogorovSmirnov test. < A small fraction of about 100 to 200 stripped eggs were then put into three 1 1 beakers containing seawater, A semen-water mixture was then poured into the beakers to fertilize the eggs. The beakers were given a fresh change of seawater after 15 minutes and all the labelled beakers were put into a water bath which had a water temperature of 24°C. The water in the beakers were given an 80% change every day. In good eggs, hatching occurred about 5-6 days after fertilization. The hatching rate was determined by counting the number of larvae and number of unhatched eggs.

Fugu niphobles (Jordan and Snyder) that were collected from fishermen at Ehima, Atsumi Peninsula, Aichi Prefecture, Japan in the months of June and July 1985, were packed in 30 litre plastic bags under oxygen and transported back to the University of Tokyo hatchery at BentenRESULTS AND DISCUSSION jima, Shizuoka Prefecture. At the hatchery, the broodfish were immediately separated by sex The ovulatory response of non hormone and and the females that had ovulated before the hormone administered fish is summarised and start of the experiment were separately grouped, presented in Table 1. The results showed that then anaesthetized with 1 % urethane, weighed, 40% offish in the control group (non hormone stripped and their eggs fertilized followed by treated) ovulated under tank conditions and egg incubation. that ovulation occurred about 48 hours after The unovulated females which exhibited the commencement of the experiments. Ovudistended abdomen were randomly divided into lation and opposition had already occurred in four groups (1, 2, 3 and 4) and immediately fish number 1 and as such the weight of eggs anaesthetized, weighed and then injected with collected from it was only 1.2 g. HCG (Puberogen) intramuscularly at the dosThe ovulatory success in fish that received ages of 0, 100, 200 and 300 I.U. per fish respec1 I.U. of HCG/g body weight was 83%. The tively. The amount of HCG administered per number of fish that ovulated 24 and 72 hours fish was then computed based on the body after the first injection were 2 and 1 respecweight of the fish and then expressed in terms tively while 2 fish ovulated 24 hours after the of I.U. of HCG/g body weight of fish. The fish second injection. In the study reported by Fujita were then released into 2.0 ton tanks containet al (1966) on induced spawning of this fish, ing 1.5 m3 of water. The tanks had a flow through 10 Rabbit Units (RU) of Synahorin were insystem and fish within the same treatment were jected per fish in the first trial and the fish 324

PERTANIKA VOL. 12 NO. 3, 1989

INDUCED OVULATION OF FUGU NIPH0B1JLS WITH HUMAN CHORIONIC GONADOTROPIN

TABLE 1 The ovulatory response of Fugu niphobles after administration of Human Chorionic Gonadotropin (HCG). Fish Number

Total Length (cm)

Body Weight (g)

1 2

8 9 10

11.5 11.0 11.8 13.0 11.2 14.0 11.4 12.6 14.2 14.0

63.0 51.5 64.9 84.8 54.5 90.3 46.1 62.9 94.9 84.0

1

11 12 13 14 15 16

11.8 12.2 13.1 13.4 12.6 13.0

78.4 71.7 82.5 89.8 75.6 77.7

2

17 18 19 20 21 22 23 24 25 26

10.5 11.8 13.4 12.2 11.6 14.9 14.4 11.3 11.2 13.3

41.7 58.1 84.6 61.9 53.7 117.5 96.0 54.9 49.0 83.8

3

27 28 29 30 31 32 33 34 35 36 37

13.3 12.7 11.8 13.3 11.8 13.9 13.2 12.3 13.3 14.5 12.2

96.4 77.0 69.3 96.4 60.4 116.9 75.2 64.1 78.4 89.0 62.5

4

38 39 40 41 42

13.0 12.1 13.0 13.3 12.7

81.5 71.4 73.5 74.5 83.8

HCG Hormone (I.U./g) 0

• 5

Note : S 51 52 F

-

Ovulatory Ovxilatory Ovulatory Ovulatory

Ovulatory response

S S S S F

Response Time (Hours)

Weight of eggs (g)

48 48 48 48

1.2 13.9 14.9 15.9

24 24 72 24 24

19.2 17.5 23.5 26.9 20.0

24 48 72 24 24 48 48

0.3 18.0 24.2 12.1 1.2 32.1 17.3

24 24 24 24 24 48 48 24 48 48

2L1 18.3 19.3 21.1 10.7 30.5 24.8 12.0 19.2 20.5

24 24 48 48

15.4 11.4 14.4 19.8

F F

F F F SI SI SI S2 S2 F SI SI SI S2 S2 S2 S2 F F F SI SI SI SI SI SI SI S2 S2 S2 F SI SI SI S2 F

success without hormone administration success after the first injection success after the second injection failure PERTANIKA VOL. 12 NO. 3, 1989

325

S.H. CHEAH AND H. SATOH

ovulated after 24 hours. However, in their second trial, some fish that did not have well enlarged abdomens 24 hours after the first injection required a second injection for successful ovulation. The ovulatory success in fish which were administered with 2 I.U. of HCG/g body weight was 70%. Three fish ovulated between 24-72 hours after the first injection while four fish ovulated between 24 and 48 hours after the second injection. The highest percentage of ovulatory success i.e. 90% was recorded from fish that had been administered with 3 I.U. of HCG/g body weight. Seventy percent of the fish that ovulated did so after a single injection of HCG (Puberogen). However, three fish could only ovulate after the administration of the second injection. When the ovulatory success and failure rates were subjected to the KolmogorovSmirnov test, there were no significant differences in the success rates for the different dosage rates (P > 0.05). In Limanda yokohamae, ovulation was successful when the females were injected with HCG (Puberogen) at the rate of 3.4 to 8.6 I.U. /g body weight (Hirose et al 1979 in Lam

occurred prior to stripping a n d fertilization thus causing zero hatching of egg samples collected from fish numbers 1, 18, 19, 23, 34, 39, a n d 4 1 . In future studies, the fish should be checked at more frequent intervals especially in the evening and at night. T h e r e was also zero or low hatching rates in fish n u m b e r s 3, 4, 12, 27, 3 1 , and 38. T h e plastic bag that was used to pack them under oxygen leaked very badly a n d as such the stressful conditions in the low oxygenated water may have caused some damage to the eggs. When Fugu niphobles were injected with Synahorin at a dosage of 10 RU per fish, the hatching rates ranged from 9 L 3 to 9 8 . 6 % whereas those in the control fish were 94.6% (Fujita et al 1966). As there was a time lapse of at least 1 day from the time the broodstock was caught till the time of h o r m o n e administration in the present study, the onset of atresia may have commenced a n d as such the time interval between capture and h o r m o n e admini-

stration should be preferably reduced. A comparative study on the use of Synahorin (pituitary gland of horse) versus HCG would be beneficial considering the high hatching rates obtained when Synahorin was administered. 1982) but in the case of Plecoglossus altivelis In Macquaria ambigua, all females injected higher dosages ranging from 12.7 to 25.0 I.U./ with a spawning threshold dosage of 500 LU. g body weight were required for successful per kg of HCG or dosages of 1000 and 2000 ovulation (Hirose et al 1977 in Lam 1982). I.U. per kg of HCG spawned completely. HowT h e ovulatory success in fish that were ever the mean hatching rate of 87.4% of eggs administered with 4 I.U. of H C G / g body weight spawned by females injected with 500 LU. per was 80%. As the sample size was rather small, kg (0.5 I.U./g) of HCG was significantly higher this result should be viewed with caution. (P < 0.01) than the mean hatching rate of 44.2% T h e effect of the dosage of HCG adminisin fish that were injected with 2000 I.U. per kg tered to Fugu niphobles on hatching rates is of HCG. Thus the use of HCG at the two dospresented in Table 2. In this study it was obages above the spawning threshold level resulted served that normal developing eggs turned grey in apparent deterioration of egg quality in M. usually after 3 days of incubation and hatched ambigua (Rowland 1983). out between 5 to 7 days at 24°C. Generally this During the preparation of the fish groups study was unable to demonstrate any correlafor HCG administration, some females were tion between the dosage of h o r m o n e adminisobserved to have ovulated. The eggs were then tered a n d the hatching rate. Egg samples from stripped, fertilized and incubated. However the fish n u m b e r s 14, 20, 35 and 40 had high hatcheggs became opaque, white or light brown and ing rates indicating that hatching failure was did not develop the characteristic grey colour not d u e to the stripping, fertilization n o r incuof developing eggs by day 3 at 24°C. Generally bation techniques but was probably due to the there is a certain length of time after ovulation timing of egg stripping. In this study, the fish when the eggs remain viable in the ovarian 'were checked for ovulation only twice a day and cavity. In this case, it was inferred that the eggs it was possible that overripening of eggs had that were collected from females which had 326

PERTANIKA VOL. 12 NO. 3, 1989

INDUCED OVULATION OF FUGUNIPHOBIJCS WITH HUMAN CHORIONIC GONADOTROPIN

TABLE 2 The relationship between the dosage of HCG administered and hatching rate of Fugu niphobles HCG Hormone Dosage (I.U./g) 0

Fish number

1 2 3

1

2

3

Mean hatching rate (%)

0 34.4 0 0

0 26.7 0 0

0 32.0 0 0

0 31.0 0 0

Hatching rate of eggs (%) in replicate

1

11 12 13 14 15

14.3 0 11.8 82.8 62.8

3.2 21.3 8.6 88.5 60.4

6.4 3.9 14.5 84.7 65.9

8.0 8.4 11.6 85.3 63.0

2

17 18 19 20 22 23

0 0 0 91.3 73.7 0

2.4 0 0 89.8 74.9 0

1.6 0 0 82.5 58.7 0

1.3 0 0 87.9 69.1 0

3

27 28 29 30 31 32 33 34 35 36

4.2 1.3 47.2 4.2 0 41.9 77.1 0 79.2 80.2

2.1 1.6 39.0 2.1 0 42.4 66.7 0 91.7 47.1

7.4 1.9 49.1 7.4 0 23.0 78.9 0 95.9 36.6

4.6 1.6 45.1 4.6 0 35.8 74.2 0 88.9 54.6

4

38 39 40 41

0 0 60.9 0

0 0 71.1 0

0 0 81.2 0

0 0 71.1 0

ovulated prior to the commencement of the trials had exceeded this critical period of time for successful fertilization and as a result egg samples from 17 fish had zero hatching with the exception of egg samples from 1 fish. CONCLUSION Even though administration of Human Chorionic Gonadotropin (HCG) to female Fugu niphobles increased the ovulatory success rates, there were no significant differences in the success rates which were the result of administering different dosage rates (P > 0.05).

ACKNOWLEDGEMENTS The authors would like to express their appreciation to the J a p a n International Cooperation Agency for funding this experiment and for providing financial support to the senior author during his stay in Japan. T h e senior author would also like to express his appreciation to Universiti Pertanian Malaysia, Serdang, Selangor, Malaysia for permitting him to be at Japan. The authors would like to thank Dr. Y.K. Law for making a brief English summary of the report by Dr. Y. Fujita, T. Komiyama and N. Yokata. Finally, the authors would like to thank

PERTANIKA VOL. 12 NO. 3, 1989

327

S.H. CHEAH AND H. SATOH

Assoc. Prof. Dr. KJ. Ang for his comments on the manuscript. REFERENCES FUJITA, Y., T. KOMIYAMA and N. YOKATA.

1966.

Arti-

ficial Egg Taking and High Density Hatching Method of Fugu niphobles. The Aquiculture 14(1) :

31-36. (In Japanese). LAM, TJ. 1982. Applications of Endocrinology to Fish Culture. Can.}, Fish. AquaL Sci. 39 : 111-137.

328

ROWLAND, SJ.

1983. The hormone-induced Ovu-

lation and Spawning of the Australian Freshwater Fish Golden Perch, Macquaria ambiqua (Richardson) (Percichthyidae). Aquaculture 35 : 221-238. UNO, Y. 1955. Spawning Habit and Early Development of a Puffer, Fugu (Torafugu) nipholes (Jordan et Snyder)./ Tokyo Univ. Fish. 41(2): 169183.

PERTANIKA VOL. 12 NO. 3, 1989

(Received 23 June, 1987)

Pertanika 12(3), 329-333 (1989)

Light Availability for Phytoplankton Production in Turbid Tropical Fish Ponds FATIMAH MD. YUSOFF Department of Fisheries Biology and Aquaculture, Faculty of Fisheries and Marine Sciences, Universiti Pertanian Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia. Key words: Light, phytoplankton production, turbid, tropical fish ponds. ABSTRAK Produktiviti primer bagi air hijau telah ditentukan di lapisan yang berbeza dalam kolam ikan yang keruh semasa musim hujan (bulan Met, November dan Disember 1986), dengan menggunakan teknik botol teranggelap. Zon 1 % cahaya didapati kurang daripada 0.75 m dalam, apabila nilai pekali pemupusan cahaya melebihi 5. Produktiviti pada tiap-tiap kedalaman berbeza -beza secara bererti iaitu pada jarak kedalaman 0.25 m apabila nilai pekali pemupusan berjulat daripada 5.09 ke 8.39, dan berbeza dengan bererti pada jarak 0.05 m kedalaman iaitu pekali pemupusan berjulat daripada 3.74 ke 4.47. Produktiviti tidak berbeza dengan bererti apabila nilai pekali pemupusan 1.67. ABSTRACT Primary productivity of green vmter was determined at different depths using light-dark bottle technique in turbid fish ponds during raining season (May, November and December 1986). The 1 % light zone was less than 0.75 m when attenuation coefficients were more than 5. Productivity at various depths was significantly different at intervals of 0.25 m when light attenuation coefficients ranged between 5.09 to 8.39, and at depth intervals of 0.50 m when attenuation coefficients ranged between 3.74 to 4.47. Productivity did not differ significantly when attenuation coefficient was 1.67. INTRODUCTION

The productivity of pond water is a function of light energy, temperature, and supply of nutrients. In the wet tropics, water from wellweathered drainage basins usually contains very low nutrients necessary for phytoplanktonic carbon fixation. However, their shortage in fish ponds can be overcome by adding inorganic or organic fertilizers. Like most countries in the tropics, Peninsular Malaysia experiences high rainfall ranging from 20C0-3500 mm/yr. Improper land used practices coupled with heavy rainfall have contributed to highly turbid streams and rivers in Malaysia. From the air, rivers can be easily mistaken for winding laterite roads. This turbid silt-laden water forms the water source for most fish ponds in the country. It is not uncommon

to observe fish ponds in Malaysia with turbidity ranging from 200 to over 2000 mg/L. Adverse effects of high turbidity on fish population are well known (Ellis 1936, Cordone and Kelly 1961, Chutter 1969). Turbidity also decreases light penetration and thereby reduces the rate of photosynthesis. Thus, availability of light can exert a major control on the phytoplankton activity in turbid fish ponds. This experiment demonstrates the effects of light availability on the phytoplanktonic productivity in turbid fish ponds. MATERIALS AND METHODS

Green water was prepared in a 500 liter transparent fibre-glass tank by adding Conway's nutrient medium and vitamins (Bl and B12)at the rate of 1 ml and 0.1 ml per liter water

FATIMAH MD. YUSOFK

respectively. The algae culture (90% Chlorelia sp.) was then added at the rate of about 1 liter per 15 liters of water. After 7 days, the green water was used for the determination of productivity at different water depths in fish ponds. Light and dark bottles were filled with well mixed green water using a Van Dorn water sampler. The bottles were immediately stored in light proof wooden boxes. In the ponds, each pair of light and dark bottles were incubated at the centre of each pond at 10 cm, 25 cm, 50 cm and 75 cm depths for about two hours (between 1100 hrs to 1300 hrs). Three fish ponds located in the Agriculture University of Malaysia were used in this experiment. Light availability for photosynthesis in the waveband 400-700 nm in the ponds was measured using a light meter. A LICOR model LI-888 integrating quantum meter was connected through a LICOR sensor-selector to a LICOR quantum sensor (LI-190 SB) and a spherical underwater quantum sensor (LI-193 SB). Under water light was measured at 10 cm, 25 cm, 50 cm and 75 cm depths at the centre of each pond. Data from the LICOR sensor in air and the LICOR sensor underwater were used to obtain percentages of total surface 400-700 nm photon flux density (PFD) which remained at each depth. The attenuation coefficient (b), which represents a composite for all wavelengths, was calculated following the equation of McNabb ei al (1988):

where y is the percentages of surface light at depth, z is depth, b is the attenuation coefficient and a is the y intercept. Initial oxygen content of the green water was determined using the Winkler method (American Public Health Association 1985). After the incubation period of 2 hours, the oxygen content of light and dark bottles was determined. Phytoplanktonic productivity was calculated according to the following equations (Wetzel and Likens 1979, Cole 1983):

330

Fig 1 Total monthly rainfall (mm) at the University of Agriculture Malaysia, during 1986 and 1987.

RESULTS

In y = a + bz

Gross productivity (mg C / m V h r (LB -DBx 1000x0.375 PQx t

Net productivity (mg C / m V h r LB- IB x 1000 x 0.375 PQx t where LB is concentration of oxygen in light bottle (mg/L), DB is concentration of oxygen in dark bottle, IB is concentration of oxygen in initial bottle, PQ is photosynthetic quotient (assumed to be 1.2), t is hours of incubation, and 0.375 is molecular weight ratio of carbon to oxygen gas. The experiment was repeated six times during the rainy period (May, November and December 1986, Figure 1). Statistical analysis using ANOVA with Duncan's Multiple Range Test was performed on the algal productivity data to determine significant differences between different depths at P < 0.05.

Figure 2 shows the relationship between the percentage of surface light and the pond depths. When the attenuation coefficient was high (b = 10.25), the surface light decreased rapidly attaining 1% at a depth of about 0.35 m. As attenuation coefficient lessened, the 1% light zone extended deeper to 0.73 m and 1.90 m at b = 5.00 and b = 1.70 respectively. Table 1 shows that when ponds were very turbid with mean attenuation coefficients ranging from 5.09 to 8.39, phytoplanktonic productivity at depth intervals of 0,25 m differed significantly. Gross productivity at 0.1 m was significantly higher than productivity at 0.25 m. At 0.25 m, it was higher than at 0.50 m, and at 0.5 m it was higher than at 0.75 m. When values of attenuation coefficients were between

PERTANIKA VOL. 12 NO. 3, 1989

LIGHT AVAILABILITY FOR PHYTOPLANKTON PRODUCTION IN TURBID TROPICAL. FISH PONDS

3.74 to 4.47, productivity differed significantly at depth intervals of about 0.5 m (Table 1). When the extinction coefficient was 1.67, productivity did not differ throughout pond depth.

1-31-2• \ \

1 -1 PERCENT OF SURFACE LIGHT.

20

0

40

60

80

\

100 0 9t \*

0-8-

E

\ \

\

25-

0-7-

111

HIH =

—"

\

1.0-

I /

wV

\

^v \

\ .

#

0-6

/ /

0 5-

\

#

\

0-4-

\

•

0 3-

*\ *V

* N

0-2•

•

v

^

*

0 101

0-3 0-5

1

3

5

10

30 50 2

~

3

0-25-

4 LIGHT

5

6

7

8

ATTENUATION

9

10

11

COEFFICIENT

w

0*50-

Fig 3

075-

at 0.75 m depths (*).

100

Fig 2

Relationship between light attenuation coefficient and relative gross productivity at 0.25 m (•) and

Percentage of incident light that would remain after passing through depths of water expressed on linear {upper) and a logarithmic (lower) scale.

Figure 3 shows the relationship between attenuation coefficients and the relative gross primary productivity (relative productivity value was proportion of the algal productivity at a specific depth compared to the productivity near the surface, i.e 10 cm depth). At high attenuation coefficient values, changes in light availability produced little change in relative productivity. At low light attenuation coefficients, a little increase in turbidity would greatly decrease gross productivity {Figure 3). DISCUSSION Phytoplankton in fish pond is important because it forms a very important source of food for zooplankton, aquatic insects and herbivo-

rous/omnivorous fishes. Zooplankton and aquatic insects, in turn, serve as food for omnivorous/carnivorous fishes. One of the main factors governing phytoplankton production in pond water is subsurface light. Underwater light availability for photosynthesis is mainly controlled by suspended particles and dissolved coloured compounds (Wetzel 1983). The higher the concentrations of suspended particles or dissolved compounds, the lower is the light transmitted and thus available to the photosynthetic organisms at lower depths. Turbidity caused by soil particles does not only affect underwater light penetration, but also causes an erroneous relationship between Secchi disk transparency and chlorophyll concentrations. Several workers have proposed the use of empirical relationships between Secchi disk depth and chlorophyll a to predict chlorophyll levels from changes in transparency (Carlson 1977). However, the Secchi disk transparency provides little information about

PERTANIKA VOL. 12 NO. 3, 1989

331

FATIMAH MD. YUSOFF.

TABLE 1 Mean light attenuation coefficients, and gross and net productivity at different depths, on different days in turbid fish ponds (n = 3). GPP is gross primary productivity and NPP is net primary productivity. Dates

Light attenuation

Depth

GPP

NPP

coefficients

(cm)

(mgC/m3/hr)

(mg C / m 3 / h r )

Range

Mean

5/5/86

6.52-10.25

8.39

11/23/86

2.96-7.75

5.16

11/30/86

4,23-6.05

5.09

12/7/86

3.37-5.95

4.47

12/14/86

3.00-4.59

3.74

12/21/86

1.20-2.50

1.67

10 25 50 75 10 25 50 75 10 25 50 25 10 25 50 75 10 25 50 75 10 25 50

75

608.4a 413.5b 90.8c 31.5d 738.3a —

575.1b 155.3 C 999.8 a

_ 487.8 b 85.5 C

957.7a 989.P 521.8b 306.9" 814.P 668.8*» 479.2bc 312.9C 929.0a 988.3 a _ 988.3*

525.6a 344.5 b 26.6C 0.0d 613.8a — 469.2 b 48.4C 895.0a 395.4b 20.0c 655.8a 677.4a 142.0b 80.4b 526.6a 432.3ab 249.1** 121.2C 768.5a 798.P _ 853.0a

Mean productivity values for the same date within the same column followed by a different letter are significantly different at P < 0.05.

algal biomass in turbid ponds if the relationship disregards the effects of substances other than algae which attenuate subsurface light. This is because transparency does not simply depend on vertical attenuation of light due to chlorophyll, but also on absorption and scattering of light by suspended particles. In this study, in turbid ponds with attenuation coefficients of more than 5, particulate suspensoids drastically reduced the light transmission to less than 1% of total surface light in less than 0.75 m depth (Figure 2). Other studies have shown that the sunlight zone for photosynthesis extends to the depth where 1% of 332

surface light remains (Cole 1983). Thus, primary productivity was limited to less than 1 m depth when ponds in this study were very turbid. McNabb etal (1988) reported that 1% of surface 400-700 nm photon flux density was present at 2.0 m depth when the attenuation coefficient was 2.06 in Wonogiri Reservoir in Java. Since light penetration in turbid ponds is limited, the bottom layer of turbid ponds is likely to be heterotrophic, succumbing to accumulation of toxic decomposition products such as ammonia and under deoxygenated conditions, hydrogen sulphide. However, turbid periods usually occur during wet seasons when

PERTANIKA VOL. 12 NO. 3,1989

LIGHT AVAILABILITY FOR PHYTOPLANKTON PRODUCTION IN TURBID TROPICAL FISH PONDS

formation of stagnant deoxygenated bottom water does not develop due to complete mixing of water column in shallow ponds. Besides giving rise to heterotrophic layers in ponds, particulate suspensoids also lessen the phytoplanktonic carbon fixation capacity in deeper layers. Phytoplankton populations in 0.75 m fixed less carbon than those at 0.5 m layer, and those at 0.5 m fixed less than at 0.25 m. Primary production differences between water layers were found to be significant (Table 1). This phenomenon occurred in extremely turbid conditions when attenuation coefficients were more than 5.0 (Table 1). Attenuation coefficients of more than 2.0 resulted in significant difference in phytoplanktonic production between bottom and surface layers (Table 1). Phytoplanktonic production in subsurface layers decreased rapidly with increasing light attenuation coefficients until a saturation point was reached. At this point, increased coefficients resulted only in samll decreases in production (Figure 3). Once light is limited in an aquatic system, other means of increasing phytoplankton production, such as fertilizing ponds, proves futile. Therefore, 1 m deep fish ponds should not have attenuation coefficients of more than 2.0 in order for all layers of water to be photosynthetically productive. This study showed that turbidity caused by silt and soil materials greatly influenced availability of light for photosynthesis at lower depths in ponds. Since subsurface light is a very important factor for production of algae used as food in ponds, turbidity should be controlled in water used for fish grow-out ponds. It is

recommended that water for fish ponds should go through baffles which slow the water flow thus allowing solids to settle out. It would be even better for the water to go through limestones heaps, especially for soft water, because the limestone stack would not only filter the silt out, but would also increase the alkalinity of the water. REFERENCES AMERICAN PUBLIC HEALTH ASSOCIATION. 1985. Stan-

dard Methods for the Examination of Water and Wastewater. 16th edition. Washington D.C. APHA. 1134 pp. CARLSON, R.E. 1977. A Trophic State Index for Lakes. Limnol Oceanogr. 22: 361-369. CHUTTER, F.M 1969. The Effects of Silt and Sand on the Invertebrate Fauna of Streams and Rivers. Hydrobiology 34: 57-76. COLE, A.G. 1983. Textbook of Limnology. St. Louis: The C.V. Mosby Company, 401 pp. CORDONE, A.J.and D.W. KELLY.

1961. The Influ-

ences of Inorganic Sediment on the Aquatic Life of Streams. Calif. Fish Game 47: 189-228. ELLIS, M.M. 1936. Erosion Silt as a Factor in Aquatic Environment. Ecology 17(1): 29-42. MCNABB, C D . , T.R. BATTERSON, H.M. EIDMAN, and

KOMAR SUMANTADINATA. 1988. Carbon Limitation in Fertilized Fish Ponds in Java. 19th Annual Conference World Aquaculture Society. Honolulu. WETZEL, R.G. 1983. Limnology. New York: Saunders College Publishing. 110. WETZEL, R.G. and G.E. LIKENS.

1979.

Limnological

Analyses. Philadelphia: W.B. Saunders Company. 357 pp.

PERTANIKA VOL. 12 NO. 3, 1989

(Received 13 December, 1988)

333

Pertanika 12(3), 335-340 (1989)

Preliminary Study on Mortality of Catfish (Clarias macrocephalus) Fry Transported in Plastic Bags. *AHYAUDIN B. ALI, *TG. MOHAMAD IZHAM TG. KAMALDEN AND **ADNAN ABAS ^School of Biological Sciences **School of Medical Sciences Universiti Sains Malaysia 11800 Penangy Malaysia. Key words: Catfish, Clarias macrocephalus, mortality, transportation, plastic bag. ABSTRAK Anak ikan keli (Clarias macrocephalus,) diangkut di dalam beg plastik dengan menggunakan air bersih dan oksigen tulen pada nisbah 1:3. Kenaikan suhu dan amonia semasa pengangkutan mempunyai hubung kait yang rapat dengan kepadatan ikan dan jangka masa mengangkut. Oksigen terlarut tidak menjadi masalah, malahan kepekatannya meningkat apabila gas oksigen mula terlarut ke dalam air semasa mengangkut. Kadar kematian semasa pengangkutan rendah, tetapi meningkat selepas pengangkutan selesai. Walau bagaimanapun, kadar kematian dapat dikurangkan dengan memasukkan garam (NaCl) ke dalam air. ABSTRACT Catfish (Clarias macrocephalus) fry were transported in plastic bags using clean water and pure oxygen at a ratio of 1:3. The rise in temperature and total ammonia during transportation was related to transportation time and packing density. Though dissolved oxygen increased when pure oxygen began to dissolve in the water during transportation it did not pose a problem. Low mortalities were observed among the less densely packed fish but post-transporatation mortalities were fairly high. Mortalities, however, could be reduced with the addition of salt (NaCl) to the water.

The plastic bag method generally involves INTRODUCTION packing a certain number or weight of fish in In Southeast Asia, fish are widely transported in a plastic bag filled with clean water and pure sealed plastic bags with air replaced by pure oxygen at the ratio of 1:3 by volume (Sampson, oxygen (Sampson 1987). The convenience and 1987). Salt (NaCl) may be added to the water associated low cost has resulted in its widespread to reduce mortalities (Piper etal 1982; Nikinmaa use. Even though the plastic bag method of et al. 1983). However, there does not appear to transporting fish is easy and economical (Frose, be rules and it appears to be trial and error 1986), the hauling stress experienced usually based rather than scientifically determined results in poor fry survival (Sampson 1987). (Sampson 1987). This study investigates the The senior author has experienced a 98% mortality of catfish {Claiias macrocephalus) fry trans- effects of different packing densities, transportation time, and salt concentrations on mortalported in plastic bags. The fry died within 1 to ity of catfish fry during and after transporta2 days after arrival and a post-mortem indicated tion. transportation stress as the most probable mortality cause. This major problem slows down MATERIALS AND METHODS catfish farming in Perlis, Kedah, Penang, and The experiments were conducted using plastic Northern Perak where the low fry production bags (71 cm x 56 cm), dechlorinated water and from local hatcheries is further aggravated by catfish fry with mean size of 146 mg ± 1 2 mg high losses during transportation.

AHYAUDIN B. ALI, TG. MOHAMAD IZHAM TG. KAMALDEN AND ADNAN ABAS

and 20 mm ± 1 mm long (total length), the size sold by most hatcheries. The plastic bags were thoroughly washed to remove any trace of chemicals present, and prior to the experiments, the fry were acclimatised for one week and starved for 12 h to clear their guts. In experiment A, fry at four different packing densities (5, 10, 20, and 30 ind./l - each density replicated 5 times), were subjected to four different transportation times (0.25, 4, 8, and 12 h). Dissolved oxygen (D.O.) and water temperature were measured at specified time intervals using a Yellow Spring Instrument (Model 57) D.O. meter with a thermistor. Final total ammonia concentrations in the 8 and 12 h treatments for the 10, 20, and-30 ind./l densities were measured using the indophenol method (Boyd 1979), while the initial and final pH values were also recorded using a Hanna (HI 8314) pH meter. After the experiments,the fish were kept in aquaria (18 cm x 32 cm x 22 cm) filled with 3 1 dechlorinated water and reared for one week to determine post-transportation mortality. During rearing, the water was fully aerated and changed every 2 days, and the fish were fed to satiation with Tubifex worms at 5% body weight 3 times daily. All mortalities were recorded. In experiment B, fry (from the same group as in experiment A) at 5 and 30 ind./l densities were packed in plastic bags containing water at four different salt concentrations (0, 10, 15, and 20 mg/1 - each concentration was replicated 5 times), and subjected to transportation times of 8 and 12 h, respectively. After the experiments, all fish were kept in aquaria filled with dechlorinated water for one week to determine post-transportation mortality. The water management and feeding offish were as in experiment A. RESULTS Experiment A

The temperature increased gradually during transportation (Figure 1A). However, for each packing density, the rise was small and the minimum and maximum values recorded were 26 and 32°C, repectively. There was also a gradual increase in D.O. (Figure 1A) and the 336

minimum and maximum concentrations were .12.0 and 16.7 mg/1, respectively. For post-transportation rearing, the temperature was relatively constant but there were some fluctuations in D.O. especially for the 20 and 30 ind./l densities (Figure IB). The final total ammonia-N concentrations for the 8 and 12 h transportation increased in relation to the packing densities (Figure 2). The concentrations for the 8 h transportation increased from 0.29 mg/1 for 10 and 20 ind./l densities to 0.48 mg/1 for 30 ind./l density, respectively. For the 12 h transportation the increase was from 0.20 mg/1 for the 10 ind./l to 0.29 and 0.36 mg/1 for the 20 and 30 ind./l densities, respectively. In all treatments the pH levels remained essentially circum-neutral, and declined slightly at the end of the study (Figure 2). No mortalities were recorded during transportation for 5 and 10 ind./l densities but there were fairly low mortalities for 20 and 30 ind./l densities (Figure 3). Post transportation mortalities after one week of rearing were high for all the densities and all transportation times. For the 5 and 10 ind./l densities, the mortality rates increased with longer transportation times, whereas, the opposite occurred for the 20 and 30 ind.l/ densities (Figure 3). Experiment B

The D.O. and temperature were stable during transportation as well as during post-transportation rearing. The average D.O. and temperature ranged from 11.6 to 15.3 mg/1 and 29.8 to 33.3°C, respectively, during transportation. During post-transportation the average D.O. and temperature ranged from 2.0 to 6.6 mg/1 and 25.5 to 28.6 C, respectively. There were no mortalities during transportation. The addition of salt, especially at 20 mg/ 1, during transportation reduced post-transportation rearing mortalities for both the 5 ind.l/ and 30 ind./l densities; however, lower salt concentrations were less effective (Table 1). During post-transportation rearing, mortalities occurred randomly whether in the first 3 or the last 4 days of rearing. If there were high mortalities in the first three days then the last 4 days would have lower mortalities and vice versa (Table 1).

PERTANIKA VOL. 12 NO. 3, 1989

MORTALITY OF CATFISH (C. MACROCEPHALUS) FRY TRANSPORTED IN PIASTIC BAGS

8 8

11 A 6

28

4

0

I

24

V

e d

22

14

i

m P e ;

'*

20

12

14 (°C)

o X

y e n (mg/l)

12 10

h

10

I i r f -t r i i i T hrh 0.25 4 8 12 0.25 4 8 12 0.25 4 8 12

HI T r -r I i f 0.25 4 8 12

8

Time interval (h)

5 ind./l

D i

6

X

8

10 ind./

V\

20 ind./l

35

30 ind./l

30 /

s •

25

I v

20 15

y

T e m P • u

3 10

9

;

5

(mg/l) 1 2 3 4 5 6

1 2 3 4 5 6

1 2 3 4 5 6

1 2 3 4 5

6.

No. of days Fig. I Temperature (line) and dissolved oxygen (bar) measured at different time intervals during transportation (A) and daily during post-transportation rearing (B).

PERTANIKA VOL. 12 NO. 3, 1989

337

AHYAUDIN B. ALI..TG.. MOHAMAD IZHAM TG. KAMALDEN AND ADNAN ABAS

-0.6

10 r

-0.5

Ammonia Nitrogen PH

(mg/l)

10

20

30

.

10

20

30

Fry density (individuals/liter) Fig. 2 Final total ammonia-N concentrations (line) and pH levels (bar) for fry transported at two different time intervals and three different packing densities. 10

B

A 5 ind./l

C

10 ind./l

- 120

D

- 6 - 20 ind./l

-=^- 30 ind.l/

-

6 transport mortality

- 110 - 100 -90 -80 - 7 0 Post transport mortality

-50 -40 -30 2r 20

0.25 4

8

12

.0.25 4

8

12

.0.25 4

8

12

.0.25 4

8 12

Time interval (h)

Fig. 3. Total mortality rates (%), during transportation (bar) and post-transportation rearing of seven days (line), for fry transported at four different time intervals and packing densities.

338

PERTANIKA VOL. 12 NO. 3, 1989

MORTALITY OF CATFISH (C. MACROCEPHALUS) FRY TRANSPORTED IN PLASTIC BAGS

TABLE 1 Effects of different salinities (NaCl) on post-transportation mortality (%) of catfish (Clarias macrocephalus) fry packed at 5 and 30 ind./l densities and transported at two different transport times.

5 individuals/1 8 h

Salinity (mg/1)

0

30 individuals/1 12 h

8 h

12 h

day3

day7

total

day3

day7

total

day3

day7

total

day3

day?

total

32.0

8.0

40.0

0

36.0

36.0

44.7

33.3

78.0

18.7

46.7

65.4

10

0

44.0

44.0

32.0

32.0

64.0

16.0

46.7

52.7

4.0

61.3

65.3

15

16.0

80.0

96.0

12.0

12.0

24.0

58.7

20.0

78.7

71.3

13.3

84.6

20

4.0

32.0

36.0

12.0

8.0

20.0

8.7

30.7

38.7

5.3

19.3

24.6

DISCUSSION

A rise in temperature during transportation is unavoidable. Even though catfish can tolerate temperature extremes in their natural environment (Ali and Ahmad 1988), the water temperature in the plastic bag must be allowed to equilibriate slowly with the water temperature of the place to be stocked to avoid shock that might kill the fry (Sampson 1987). Dissolved oxygen is not a problem since the pure oxygen used would dissolve with longer transportation time and lead to an increase in D.O. towards the end of transportation. If pure oxygen is not available, hydrogen peroxide may be used as an oxygen source (Taylor and Ross 1988) or atmospheric air can be used if the transportation time does not exceed 20 h (Frose 1986). The main problem faced during transprotation is the change in water quality, primarily of pH and ammonia (Sampson 1987). The increase in carbon dioxide during transportation would increase carbonic acid and result in lower pH (Boyd 1979). The severity of the drop is related to packing density (Siraj et al 1985), transportation time and the buffering capacity of the water (Sampson 1987). Concurrently, an increase in total ammonia concentrations as a result of excretion also occurred. The increase is also related to packing density (Siraj et al 1985) but can be minimised by properly starving the fish prior to transportation. Properly starved fish would not regurgitate food, thus, lessening water fouling problem (Sampson 1987), while, ammonia toxicity during transportation can be reduced by the

low pH and low water temperature (Colt and Tchobanoglosus 1976; Robinette 1983; Sampson 1987). In this study, the fairly high total ammonia-N detected is probably due to the short starvation period of 12 h, and ammonia at these levels can affect fish stamina (Robinette 1983) possibly leading to higher post-transportation mortality. Stress is an important factor affecting fish mortalities. Thus, different levels of stress could also be responsible for the higher ammonia-N levels for fish transported at 8 h compared to those transported at 12 h. During transportation, no mortalities occurred in the less densely packed bags but occurred in the 20 and 30 ind./l bags indicating the greater stress of higher packing densities. However, the transportation stress for the 5 and 10 ind./l densities finally manifested itself during post-transportation rearing with high fry mortalities, although, for the 20 and 30 ind./l densities, the mortalities were lower because most of the stressed fish had already died during transportation. Nikinmaa et al (1983) and Frose (1986) suggested adding salt to water during transportation to reduce post-transportation mortalities. The role of salt is primarily to reduce physiological stress by reducing the osmotic work needed to maintain stable ion levels (Nikinmaa et al 1983). Depending on species, the suggested concentrrations range from 1 to 10 g/ 1 (Frose 1986). Adult catfish have been successfully transported in water containing 3 g/ 1 salt. However, no suggested concentration for fry is available. This study indicated that adding 20 mg/1 salt to water during transportation

PERTANIKA VOL. 12 NO. 3, 1989

339

AHYAUDIN B. ALI, TG. MOHAMAD IZHAM TG. KAMALDEN AND ADNAN ABAS

could lower post-transportation mortalities among catfish fry packed at both 5 ind./l and 30 ind./l densities. However, lower salt concentrations have less noticeable effects. CONCLUSION

The use of plastic bags to transport fish should be encouraged. However,further research should be done to "fine tune" the technique to make it more reliable. Since it is difficult to maintain optimum water quality condition during transportation, fish should be treated with care prior to, during and after transportation in order to reduce losses. Adding salt to water during transportation can reduce mortalities, but rough handling of fish should be avoided at all times (Frose 1986). Proper conditioning before transportation would help to identify weaker fish for removal, thus, reducing mortality during and after transportation (Sampson 1987). Adequate starvation before transportation also helps to reduce the problem of water fouling and ammonia toxicity (Sampson 1987). Packing procedure suggested by Frose (1986) is recommended. ACKNOWLEDGEMENT

This study was funded by a R & D research grant. We thank the School of Biological Sciences, Universiti Sains Malaysia for providing ancillary facilities. REFERENCES

BOYD, C.E. 1979. Water Quality in Warm Water Fish Ponds! Agricultural Experimental Station. Auburn University, Auburn, Alabama, USA. COLT, J. and TCHOBANOGLOSUS.

1976.

Evaluation

of the Short Term Toxicity of Nitrogenous Compounds to Channel Catfish, Ictalurus punctatus. Aquaculture 8: 209-224. FROSE, R. 1986. How to Transport Live Fish in Plastic Bags INFOFJSH Market Dig. 4: 35-36. NIKINMAA, M., A. Soivio, T. NAKARI, a n d

S.

LiNDGREN. 1983, Hauling Stress in Brown Trout (Salmo trutta): The Physiological Responses to Transport in Fresh Water or Salt Water, and Recovery in Natural Brackish Water. Aquaculture 34: 93-99. PIPER, R.G., I.B., MCELWAIN, L.E. O R M E , J.P. MCCRAKEN, L.G. FOWLER, and J.R. LEONARD.

1982. Fish Hatchery Management. U.S. Fish. Wildl. Serv., Washington D.C., USA. ROBINETTE, H.R.

1983.

Ammonia, Nitrate, in

Water Quality in Channel Catfish Ponds, C.S. Tuker (ed.), pp. 28:24. South. Coop. Ser., Bull. 290, Mississippi State Univ., Mississippi, USA. SAMPSON, D. 1987. Fish in Plastic Bags. Aquaculture NewsNo. 3, p. 7 - Inst. Aqua. Newsheet, Stirling Univ., UK. SIRAJ, S.S., S.H. CHEAH and Z.A. AlZAM.

1985.

Ef-

fects of Packing Densities in Plastic Bags on Survival of Larvae and Fry of Helostoma Temmincki (C&V). Pertanika 8: 387-390. TAYLOR, N.I. and L.G, Ross.

1988.

The Use of

Hydrogen Peroxide as a Source of Oxygen for Transportation of Live Fish. Aquaculture 70: 8392.

A.B. and M. AHMAD. 1988. Water quality in Rice Fields and Sump Ponds and its Relationship to Phytoplankton Growth in Rice Field Fish Culture System. Trap. EcoL 29: 63-70.

ALI,

340

PERTANIKA VOL. 12 NO. 3, 1989

(Received 23 March, 1989)

Pertanika 12(3), 341-347 (1989)

Aktiviti Bakterisidal in vitro Fagosit daripada Pesakit Lupus Eritematosus Sistemik (SLE) M. MUSA, J. MAT ASAN dan Y.M. PANG Jabatan Imunologi, Pusat Pengajian Sains Perubatan, Universiti Sains Malaysia, 11800 Minden, Pulau Pinang, Malaysia. Kata Punca: Neutrofil, monosit, in zritro, pesakit Lupus Eritematosus Sistemik. ABSTRAK Di dalam kajian ini, neutrofil dan monosit daripada darah periferi pesakit SLE telah diasingkan dan diuji aktiviti bakterisidalnya terhadap Staphylococcus aureus berbanding dengan sel daripada kontrol normal. Keputusan ujian ini menunjukkan bahawa neutrofil dan monosit daripada pesakit SLE masih mempunyai aktiviti bakterisidal yang normal, walaupun sebelum ini pernah dilaporkan terdapat kecacatan pada fungsi fagositosis dan kemotaksis. Fagosit daripada pesakit ini dapat membunuh bakteria dengan berkesan. Fungsi serumnya sebagai opsonin di dalam aktiviti bakterisidal juga didapati normal ABSTRACT Neutrophils and monocytes of the peripheral blood of patients with SLE were isolated and tested for their bactericidal activity against Staphylococcus aureus, compared to cells obtained from normal controls. Despite having abnormalities in phagocytic and chemotactic functions as previously reported, the results of this study showed that neutrophils and monocytes from patients with SLE exhibited normal bactericidal activity. Phagocytes of these patients were able to kill the bacteria very effectively. Their serum function as opsonin in the bactericidal activity was also normal. PENGENALAN

Lupus Eritematosus Sistemik (SLE), sejenis penyakit inflamatori sistemik wujud dengan adanya manifestasi autoimun. Gangguan autoimun ini umumnya berkaitan dengan kehadiran antibodi antinukleus (ANA) dan antibodi anti-ds DNA di dalam serum pesakit. Oleh itu, patogenesis penyakit SLE dikatakan hasil daripada kesan buruk yang disebabkan oleh pembentukkan kompleks imun berkenaan (Chia et al 1979; Zvaifler 1981). Punca pembentukan antibodi tersebut masih tidak jelas diketahui. Selain daripada kecacatan tersebut, fungsi imuniti sel pesakit juga dilaporkan tergangu berdasarkan gerak balas imuniti sel. Seterusnya setengah-setengah fungsi sel leukosit daripada pesakit SLE seperti kemotaksis dan fagositosis dilaporkan juga merosot (Ward 1974; Svensson 1980). Kecacatan aktiviti fagositosis mungkin ada kaitan dengan kekurangan aviditi penerima Fc (Katayama^a/. 1983) dan

juga kecacatan fungsi penerima komplemen sel berkenaan (Hurst, Nuki dan Wallington 1984). Seterusnya Salmon dan rakannya (1984) mendapati bahawa kecacatan pada fungsi fagositosis adalah disebabkan oleh proses penelanan bukan pada perlekatan melalui penerima Fc. Walau bagaimanapun, semua kajian berkenaan hanya mengukur proses fagositosis atau penelanan partikel, oleh itu aktiviti bakterisidal atau pembunuhan bakteria yang merupakan fungsi utama fagosit di dalam pertahanan perumah menentang jangkitan masih perlu dikaji. Di samping itu, selain daripada aktiviti sel, fungsi serum sebagai opsonin yang mempunyai peranan penting di dalam aktiviti bakterisidal harus juga diperhatikan. Kajian ini dijalankan untuk menentukan sama ada terdapat kecacatan aktiviti bakterisidal pada fagosit (kecacatan intrinsik) daripada SLE dan seterusnya meneliti jika ada kecacatan pada faktor serum pesakit SLE itu juga.

M. MUSA, J. MAT ASAN DAN Y.M. PANG

Penentuan Aktiviti Bakterisidal Neutrofil dan Monosit Serum Pesakit SLE dan Kontrol Normal Penentuan aktiviti bakterisidal neutrofil dan. Darah periferi daripada 6 orang pesakit SLE monosit dilakukan di dalam kolam-96 plat diperolehi daripada Hospital Besar, Pulau mikrotiter (Linbro). Di dalam kolam plat terPinang iaitu berdasarkan kriteria diagnosis sebut, neutrofil (0.1 ml) atau ampaian sel untuk SLE meliputi ciri klinikal dan laporan mononukleus (0.1 ml) yang telah disediakan makmal. Keenam-enam pesakit menunjukkan daripada pesakit SLE atau kontrol normal dierciri klinikal meliputi anemia, ruam dan lesi kulit, amkan selama 1 jam pada suhu 37°C/5% CO2, Empat daripadanya mengalami gangguan sisudara. Selepas eraman, supernatan dibuang dan tem renal. Kesemuanya memberikan ujian ANA sel dibasuh dengan medium kultur RPMI. yang positif dengan titer yang tinggi. Kesemua Monosit yang melekat kepada plat selepas pesakit baru saja menerima rawatan prednisoeraman dan basuhan seterusnya digunakan lon dan berada di dalam wad ketika sampel untuk tujuan eksperimen. Cerakinan bakteridarah mereka diambil. Drug ini dipercayai sidal dilakukan secara bersilang dan setiap satu merencat gerak balas imun menerusi tindakansecara triplikat. Sel pesakit SLE atau kontrol nya ke atas limfosit. Empat daripada pesakit normal ditambahkan dengan ampaian bakteria juga merupakan kes berulang dan telah (50 ul/kolam) yang sebelumnya telah disediamempunyai riwayat SLE sebelumnya. Darah kan sama ada dengan serum kontrol normal daripada pendcrma Tabung Darah digunakan atau serum pesakit SLE. Plat mikrotiter kesebagai kontrol normal. Setiap eksperiman, mudiannya diemparkan selaju 800 g pada suhu darah kontrol diambil daripada pendermayang 4°C selama 5 minit. Kawalan untuk pertumpadan umur dengan pesakit. Serum daripada buhan bakteria disertakan juga iaitu tanpa darah yang dibiarkan beku diasingkan dan terus kehadiran sel. Plat kemudiannya dieramkan digunakan untuk tujuan eksperimen. pada suhu 37°C/5% CO2, udara. Bilangan hidup bakteria dilakukan pada 0 dan 60 minit Bakteria masa eraman. Ini dilakukan dengan menamBakteria Staphylococctis aureus diperolehi daribah larutan 1% Triton x 100 (200 ui/kolam) pada stok Jabatan Mikrobiologi Perubatan, untuk memecahkan fagosit dan selepas diUniversiti Sains Malaysia. Ampaian bakteria (fasa baurkan, sampel (10 ul) diambil lalu disebarlog) disediakan di dalam medium kultur RPMI 5 kan di atas piring agar nutrien, dan dieramkan (Sigma) pada kepekatan 2 x 10 sel/ml. Serum semalaman. Peratus pembunuhan bakteria didaripada pesakit SLE atau kontrol normal tentukan dengan membandingkan bilangan (kepekatan akhir 10%) ditambah kepada hidup bakteria yang dieramkan bersama sel ampaian bakteria. dengan bilangan hidup bakteria kawalan (tanpa Fagosit Pesakit SIJi dan Kontrol Normal sel) selepas eraman. Darah periferi berantikoagulan daripada pesakit SLE dan kontrol normal diperolehi seKEPUTUSAN perti di atas. Neutrofil dan monosit diasingkan dengan kaedah Histopaque 1.119/1.077 Aktiviti Bakterisidal Neutrofil SIJL Berbanding (Sigma). Neutrofil dipungut daripada lapisan dengan Kontrol Normal interfasa 1.119 dan 1.077, manakala sel Untuk meneliti adanya kecacatan intrinsik mononukleus yang mengandungi monosit pada fungsi bakterisidal neutrofil daripada dipungut daripada lapisan interfasa 1.077 dan pesakit SLE, aktiviti bakterisidal neutrofil plasma. Sel tersebut dibasuh dengan larutan daripada pesakit SLE dan neutrofil kontrol garam seimbang Hanks (HBSS) (Sigma). normal dengan kehadiran serum kontrol Ampaian neutrofil yang mempunyai viabiliti normal sebagai opsonin dibandingkan. Hasil lebih daripada 95% dibuat di dalam medium ujian ini ditunjukkan di dalam GambarajahL kultur RPMI pada kepekatan 2 x 10(i sel/ml. Neutrofil SLE mempunyai aktiviti bakterisidal Ampaian sel mononukleus pula disediakan pada yang berkesan (membunuh lebih daripada kepekatan 4 x 10fi sel/ml. 50%) setanding dengan neutrofil daripada 342 PERTANIKAVOi 12 NO. 3, 1989 BAHAN DAN KAEDAH

AKTIVm BAKTERISIDAL IN VITROFAGOSYT DARIPADA PESAKJT LUPUS ERITEMATOSUS SISTEMIK (SLE)

100

SLE

KONTROL

Gambarajah 1. Peratus pembunuhan bakteria oleh neutrofil daripada pesakit SUi atau kontrol normal dengan kehadiran serum opsonin kontrol normal. Setiap corak pada histogram mewakili seorang pesakit dan kontrol normal dalam setiap eksperimen.

kontrol normal. Keputusan ini menunjukkan tiada kecacatan pada fungsi bakterisidal neutrofil daripada pesakit SLE. Aktiviti Bakterisidal Monosit SLIi Berbanding dengan Kontrol Normal

Penelitian yang serupa juga dilakukan terhadap fungsi bakterisidal monosit. Walaupun

peratus pembunuhan bakteria oleh monosit berbeza dengan yang ditunjukkan oleh neutrofil, monosit daripada pesakit SLE tidak menunjukkan kecacatan (lebih 50% pembunuhan) pada fungsi bakterisidal jika dibandingkan dengan kontrol normal masingmasing di dalam setiap eksperimen yang dijalankan {Gambarajah 2). Setiap eksperimen

100

SLE

KONTROL

Gambarajah 2. Peratus pembunuhan bakteria oleh monosit daripada pesakit SW atau kontrol normal dengan kehadiran serum opsonin kontrol normal. Setiap corak pada histogram mewakili seorang pesakit dan kontrol normal dalam setiap eksperimen. PERTANIKA VOL. 12 NO. 3, 1989

S4S

M. MUSA,J. MAT ASAN DAN Y.M. PANG

mewakili seorang pesakit SLE berserta kontrol normal masing-masing. Fungsi Serum SLE Berbanding dengan Serum Kontrol Normal untuk Aktiviti Bakterisidal

Untuk menentukan sama ada serum pesakit SLE mempengaruhi atau mengganggu aktiviti bakterisidal fagosit daripada kontrol normal,

aktiviti bakterisidal neutrofil dan monosit daripada kontrol normal dengan kehadiran sama ada serum pesakit SLE atau serum kontrol normal sebagai opsonin dibandingkan. Gambarajah 3 menunjukkan aktiviti bakterisidal neutrofil yang berkesan sama ada dengan kehadiran serum daripada pesakit SLE atau serum kontrol normal. Keputusan yang serupa

100

SLE

KONTROL

Gambarajah 3. Peratus pembunuhan bakteria okh neutrofil daripada kontrol normal dengan kehadiran serum opsonin sama ada daripada pesakit SIJi atau kontrol normal. Setiap corak pada histogram mewakili seorang pesakit dan kontrol normal dalam setiap eksperimen.

100

50 SLE

KONTROL

Gambarajah 4. Peratus pembunuhan bakteria okh monosit daripada kontrol normal dengan kehadiran serum opsonin sama ada danpada pesakit SIJi atau kontrol normal. Setiap corak pada histogram mewakili seorang pesakit dan kontrol normal dalam setiap eksperimen.

344

PERTANIKA VOL. 12 NO. 3, 1989

AKTIVITI BAKTERISIDAL IN V777?O FAGOSIT DARIPADA PESAKIT LUPUS ERITEMATOSUS SISTEMIK (SLE)

juga diperolehi untuk aktiviti bakterisidal monosit pada eksperimen masing-masing

autoar.tibodi (Zvaifler 1981), kehadiran kompleks imun beredar di dalam serum (Koffler et (Gambarajah 4). ai 1971) dan kerentanan terhadap jangkitan (Staples et al. 1974). Kerentanan terhadap Penentuan Aktiviti Bakterisidal Neutrofil SLE jangkitan kemungkinan ada kaitannya dengan dengan Kehadiran Serumnya kecacatan fungsi fagosit. Landry (1977) telah Untuk memastikan perjumpaan sebelum ini menunjukkan kaitan di antara kecacatan fagoiaitu tiada kecacatan aktiviti bakterisidal sama sitosis imun in vitro dengan kekurangan gerak ada pada sel, fungsi serum atau kedua-duanya balas keimunan sel. Kecacatan pada gerak balas untuk fagosit SLE, aktiviti bakterisidal neumetabolisme oksidatif fagosit SLE juga ditrofil SLE dengan kehadiran serumnya sendiri laporkan (Wengerdan Bole 1972). Kecacatan dilakukan. Jika ada kecacatan pada kedua-dua pada sel telah dikaitkan dengan kecacatan pada faktor tersebut kesan gandaan akan dapat fungsi penerima Fc dan komponen komplediperhatikan. Sebagai kontrol, aktiviti bakterisimen (Hurst, Nuki dan Wallington, 1984). dal neutrofil kontrol normal dengan kehadiran Kecacatan fagositosis menerusi penerima serum SLE atau serum kontrol normal dan komplemen dikaitkan dengan bilangan dan neutrofil SLE dengan kehadiran serum kontrol fungsi penerima tersebut, sedangkan kecacatan normal juga dilakukan. Gambarajah 5 menun- fagositosis menerusi penerima Fc dikaitkan jukkan neutrofil daripada peakit SLE yang dengan fungsi penerima itu sendiri kerana dieramkan dengan kehadiran serumnya pertambahan penerima Fc pada monosit darimempunyai aktiviti bakterisidal yang berkesan pada SLE pernah dilaporkan (Hurst et al. 1984; berbanding dengan semua kontrol yang diserFries et al 1984). Kecacatan pada fungsi pentakan. Keputusan ini mengesahkan lagi bahawa erima dikatakan berlaku pada proses penelanan tiada kecacatan pada aktiviti bakterisidal sama tetapi tidak pada perlekatan (Salmon et al ada pada sel atau serum daripada pesakit SLE. 1984). Meski pun banyak kajian telah dibuat terhadap aktiviti fagositosis atau lebih jelas lagi penelanan oleh sel daripada pesakit SLE, proses PERBINCANGAN pembunuhan selepas penelanan berlaku masih Beberapa kecacatan imun telah dilaporkan tidak diketahui. untuk penyakit SLE termasuk pembentukan 1UU " A

A A

99 "

• A

% p E M B U N U H A N

98-

I A

•

• a

D

97*

* •

96-

95-

m B

94 •

SERUM SLE

SERUM KONTROL SLE

SERUM SLE

SCRUM KONTROL

KONTROL

Gambarajah 5. Peratus pembunuhan bakteria oleh neutrofil daripada pesakit SIJi dan kontrol normal dengan kehadiran serum opsonin sama ada daripada pesakit SIJi atau kontrol normal Setiap corak pada histogram mewakili seorang pesakit dan kontrol normal dalam setiap eksperimen. PERTANIKA VOL. 12 NO. 3, 1989

S45

M. MUSA, J. MAT ASAN DAN Y M PANG

Aktiviti pembunuhan bakteria oleh fagosit merangkumi beberapa peringkat termasuk kemotaksis, perlekatan dan penelanan, degranulasi dan pembunuhan intrasel. Kajian kami cuba mengukur hasil akhir daripada proses keseluruhan pembunuhan iaitu aktiviti bakterisidal. Data kami menunjukkan neutrofil dan monosit daripada pesakit SLE mempunyai aktiviti bakterisidal in vitro yang berkesan (peratus pembunuhan bakteria sentiasa melebihi 50%). Walaupun sampel darah untuk kajian terpaksa diperolehi daripada pesakit SLE yang baru menerima rawatan drug imuno supresif, data kajian menunjukkan ia tidak mempunyai kesan langsung ke atas fungsi fogosit. Fogosit telah diketahui penting untuk pertahanan tidak spesifik perumah. Kesan drug imunosupresif mungkin bertindak ke atas pengeluaran autoantibodi oleh limfosit di dalam pesakit autoimun termasuk SLE. Seterusnya serum daripada pesakit SLE juga tidak mengganggu aktiviti bakterisidal oleh fagosit kontrol normal dan mempunyai fungsi opsonin yang normal. Sebelum ini Hurst dan rakannya (1984) juga menunjukkan bahawa serum daripada pesakit SLE yang mempunyai kecacatan fagositosis tidak merencat fagositosis oleh monosit normal. Kajian kami hanya mengukur 'hasil akhir' daripada aktiviti keseluruhan bakterisidal. Oleh itu adalah menarik jika pengukuran pembunuhan intrasel selepas proses fagositosis tamat dapat ditentukan. Meskipun proses fagositosis atau penelanan fagosit SLE mempunyai kecacatan seperti yang dilaporkan, ini tidak bermakna tiada langsung bakteria dapat ditelan. Proses pembunuhan bakteria oleh fagosit umumnya berlaku di dalam fagosom iaitu selepas penelanan, tetapi agen pembunuhan dapat juga dikeluarkan ke ekstrasel dan pembunuhan boleh terjadi di permukaan membran sel (Okamura, Ishibashi dan Takano 1979). Kesimpulan daripada kajian ini mencadangkan fagosit daripada pesakit SLE masih mempunyai aktiviti bakterisidal yang berkesan meskipun terdapat kecacatan pada sesetengah peringkat pada proses pembunuhan bakteria. Kaitan di antara aktiviti bakterisidal fogosit pesakit SLE dengan kerentanan terhadap infeksi masih perlu diselidik. 346

PENGHARGAAN

Kerja penyelidikan ini telah dibiayai oleh geran Penyelidikan Jangka Pendek, Universiti Sains Malaysia, Pulau Pinang. Kami juga melahirkan rasa terima kasih kepada pihak Hospital Besar Pulau Pinang di atas kerjasama mereka. RUJUKAN CHIA, D., E.V. BERETT,J. YAMAGATA, D. KNUTSON, C. RESTIVO & D. FURST.

1979.

Quantitative and

Characterization of Soluble Immune Complexes Precipitated from Sera by Polyethylon Glycol (PEG). Clin. Exp. Immunol 37: 299. FRIES, L.F., W.W. MULLINS, K.R. CHO, P.H. PLOTZ &

M.M. FRANK. 1984. Monocyte Receptors for the Fc Portion of IgG are Increased in Systemic Lupus erythematosus. J. Immunol 132: 695. HURST, N.P., G. NUKI 8C T. WALLINGTON.

1984.

Evidence for Intrinsic Cellular Defects of 'Comploment' Receptor-mediated Monocytes Phagocytosis in Patients with Systemic Lupus erythematosus (SLE). Clin. Exp. Immunol. 55: 303. KATAYAMA, S., D. CHIA, D.W. KHUTSON 8C E.V.

BARNETT. 1983. Decreased Fc Receptor Avidity and Degradative Function of Monocytes from Patients with Systemic Lupus erythematosus. / Immunol 131; 1217. KAVAI, M., K. LUKACS, I. SONKONLY, K. PALOCZI & G.Y.

SZEGEDI. 1979. Circulating Immune Complexes and Monocyte Fc Function in Autoimmune Disease. Ann. Rheum. (Dis). 38: 79. KOFFLER, D., V. AGNELLO, R. THOBURN & H.G.

KUNKEL, 1971. Systemic Lupus erythematosus: Prototype of Immune Complexe Nephritis in Man. / Exp. Med. (suppl). 134: 1695. LANDRY, M. 1977. Phagocyte Function and Cell Mediated.Immunity in Systemic Lupus erythematosus. Arch. Dermatol 113: 147. OKAMURA N., S. ISHIBASHI & T. TAKANO.

1979.

Evi-

dence for Bactricidal Activity of Polymorphonuclear Leukocytes without Phagocytosis./ Biochem. 86: 469. SALMON, J.E., R.P. KIMBERLY, A. GIBOFSKY, & M.

FOTINO. 1984. Defective Mononuclear Phagocyte Function in Systemic Lupus erythematosus: Dissociation of Fc Receptor-Ligand Binding and Internalization. / Immunol 133: 2525. STAPLES, P.J., D.N. GERDING, J.I. DECKER 8C R.S.

GORDAN. 1974. Incidence of Infection in Systemic Lupus erythematosus. Arthrit. Rheum. 17: 1.

PERTANIKA VOL. 12 NO. 3, 1989

AKTIVITI BAKTERISIDAL IN VITROFAGOSIT DARIPADA PESAKIT LUPUS ERITEMATOSUS SISTEMIK (SLE)

SVENSSON, B. 1980. Monocyte in vitro Function in Systemic Lupus erythematosus. I. A Clinical and Immunological Study. Scand. J. Rheum. (Suppl). 31: 29.

WENGER, M.E.

& G.G.

BOLE.

1972.

Nitroblue

Tetrazolium Test in Systemic Lupus. N. Kngl J. Mod. 287: 1150.

from Patients with Connective Tissue Diseases on Red Cell Binding and Phagocytosis by Monocytes. Immunology 33: 109.

ZVAIFLER, NJ. 1981. Aetiology and Pathogenesis of Systemic Lupus Erythematosus. In Textbook of Rheumatology, ed W.N. Kelly, E.D. Harris, S. Ruddy, & C.B. Sledge, pp. 1079-1079. Philadelphia: W. B. Saunders.

WARD, P.A. 1974. Leukotaxis and Leukotactic Disorders. Am. J. Pathol 77: 520.

(Diterima 10 Oktober, 1989)

TEMPLE, A. & G. LOEWI.

1977.

The Effect of Sera

PERTANIKA VOL. 12 NO. 3, 1989

347

Section II Physical Sciences

•

Pertanika 12(3), 349-355 (1989)

Petroleum Hydrocarbon along the Coastal Areas of Port Dickson LAW A.T. AND RAVINTHAR VEELLU Department of Marine Science and Fishing Technology Faculty of Fisheries and Marine Science Universiti Pertanian Malaysia 43400 UPM, Serdang, Selangor Darul FJisan, Malaysia Key words : Petroleum hydrocarbon, Port Dickson, Malaysia. ABSTRAK Kandungan hidrokarbon petroleum di dalam air dan pasir di sepanjang pantai Port Dickson telah dikaji di antara bulan Disember 1984 dan bulan November 1985. Min danjulat kepekatan hidrokarbon petroleum di dalam air masing-masing adalah 32.24 ppb dan 2.52- 73.34 ppb. Manakala julat kandungan hidrokarbon petroleum di dalam pasir adalah di antara 2.1 dan 70.4 mg/kgpasir kering Didapati bahawa turun naik kepekatan hidrokarbon petroleum yang ketara didapati di setiap stesen antara tarikh pensampelan dalam tempoh kajian ini diadakan. Kajian ini menunjukkan bahawa kawasan pantai Port Dickson telah dicemari oleh hidrokarbon petroleum tetapi peringkatnya masih agak rendah. ABSTRACT The petroleum hydrocarbon content in water and sand along the coast of Port Dickson was studied between December 1984 and November 1985. The mean and range of petroleum hydrocarbon in water were 32.24 ppb and 2.52-73.34 ppb respectively, while for the sand, the range was between 2.1 and 70.4 mg/kg dry sand. A pronounced fluctuation of hydrocarbon level in water with sampling date was detected at the sampling stations during the sampling period. The results indicated that the coastal area of Port Dickson carried some degree of hydrocarbon pollution. INTRODUCTION Port Dickson is a well known recreational beach been conducted. The objective of the present situated in western Peninsular Malaysia. It faces study was to assess the level of oil in the coastal the Straight of Malacca, one of the busiest routes waters and in the sand along the beach of Port for oil tankers in the world. It has been estiDickson. mated that 4000 oil tankers carrying about 200 MATERIALS AND METHODS million metric tons of crude oil pass through this straits anually (Ridzwan etal. 1983). Tanker Study Area accidents and release of oily bilge water in the A total of 5 sampling stations were established straits have created a potential risk of oil polalong the shoreline of Port Dickson (Fig. 1). lution in this area. Additionally, there are two The stations were visited 10 times between oil refineries located in Port Dickson. These December 1984 and November 1985. will make the coastal area of Port Dickson more Sample Collection prone to oil pollution. In the Straits of Malacca, Water samples were collected at half a metre except for a study of oil pollution which was depth with a 3-litre glass round bottom flask. carried out from January, 1979 to June 1980, Sand samples along the beach at each station under an IOC/WMO Marine Pollution (Petrowere collected randomly by using a 30 cm x 30 leum) Monitoring Pilot Project (MAPMOPP) cm quadrat. The top 1 cm of the sand was in the Indian Region ( Sen Grupta 1980), no scooped out with a stainless steel spatula. Four other studies of hydrocarbon distribution have

IAW AT. AND RAV1NTHAR VEELLU

subsamples were taken, and wrapped in aluminium foil. The samples were kept cool and brought back to the laboratory. They were kept at -20°C until analysis.

i;

101*50'E

PORT DICKSON Bus Station

- 2* 30

L

1 km i

TT# v 9 ' Sirusa Straits of Malacca

\ JPM marine station ^Sirusa Inn

IV

V•

-2*25 N

Blue Lagoon 'K Linggi

Fig. 1 Map showing the sampling stationsC) Hydrocarbon

Analysis.

Water. Immediately after taking a water sample, 2 liters of it were extracted thrice with dichloromethane (40 ml:40 ml:20 ml). The combined dichloromethane extract was freed from the residual water by treatment with 10 g anhydrous sodium sulfate and the polar organic substances were removed with 3 g silica gel (60120 mesh). It was then evaporated to dryness in a rotary evaporator. The fluorescence of the residues, dissolved in 10 ml n-hexane, was measured with a Kontron fluorometer (SFM 23) with 1-cm quartz cell. The fluorescent intensity was measured at 374 nm with excitation at 310 nm (Parsons et al 1984). Esso Tapis A crude oil was used for the calibration. The detection limit of hydrocarbon in water was 0.5 ppb. Triplicate experiments were conducted 350

on each sample, a 93% recovery was achieved from synthetic sea water containing 10 ppb of ESSO Tapis A crude oil. The standard deviation for the recovery test was ± 0.6 ppb. Sand. Ten grams of the sand sample was weighed and transferred into a 500 ml glass separating funnel. 100 ml of distilled water was added then followed with 50 ml of n-hexane. The mixture was shaken for 5 minutes and the hexane extract was separated. The sand-water mixture that was left behind was extracted twice with 25 ml n-hexane. The combined hexane extract was dehydrated with anhydrous Sodium Sulfate and freed from polar organic substances with silica gel. The hydrocarbon content in the combined hexane was determined by fluorescence method. The recovery of Esso Tapis A crude oil, 10 \lg, in oil free marine sediment was 85%. Gas-liquid Chromatographic Analysis. After

determination of hydrocarbon contents in water and sand by the flourescence method, the samples of each station (taken between 6.12.84 and 26.1.85) were pooled and the volume was reduced to 0.1 ml by using pure nitrogen gas at room temperature. Fractional analysis of the hydrocarbon residues in the cencentrated sample was performed with a Hewlet-Packard 5840A gas chromatography equipped with a flame ionization detector. ESSO Tapis A crude oil (20 mg/ml) was used as the reference. The conditons of the chromatography were the same as described previously (Law 1984). The attenuation setting for the analysis was at 2. The results of the oil residues fraction analysis in water and sand are presented in Figure 2. RESULTS Table 1 shows the petroleum levels in water and in sand collected along the shoreline of Port Dickson. The mean and range of hydrocarbon in water at stations 1,2,3, 4, and 5 were 19.2 ppb (8.53-31.72 ppb), 28.51 ppb (9.91-54.59 ppb), 28,29 ppb (2.52-62.77 ppb), 38.05 ppb (9.59-73.34 ppb), and 47.15 ppb (13.79 - 61.97 ppb) respectively. The mean and range of hydrocarbon content in sand at stations 1, 2, 4, and 5 were 17.72 mg/kg dry sand (2.28 - 29.33 mg/kg dry sand). 34.92 mg/

PERTANIKA VOL. 12 NO. 3, 1989

PETROLEUM HYDROCARBON ALONG THE COASTAL AREAS OF PORT DICKSON

ESSO Tapis A crude oil

kg dry sand (4.23 - 67.19 mg/kg dry sand), 36.20 mg/kg dry sand (1.79 - 70.36 mg/kg dry sand), and 23.14 mg/kg dry sand (5.28 - 50.46 mg/kg dry sand) respectively. DISCUSSION

Fig. 2

The overall mean and range of the hydrocarbon levels in water of the present study are 32.24 ppb and 2.52-73.34 ppb respectively. A comparison of the hydrocarbon levels in water from the present study with values reported in other parts of the world are presented in Table 2. The results indicate that the hydrocarbon level in Port Dickson coastal waters was lower than that found in the Sarawak and Kuantan coastal waters (Mohamed ei at 1988 ; Law and Zulkifli 1987), and much lower than that detected in. the coastal waters off Kuala Terengganu (Law and Rahimi 1986). The level was also lower than that found in the Egyptian Rea Sea, 36.8 ppb (Hanna 1983), the Southern Baltic Sea, 55.3 ppb (Law and Andrulewicz 1983), and about 10 times lower than that of the Boston Harbour waters. 292 ppb (Ahmad ei al 1974). However, the level was about two thousand times higher than that detected in the Pacific Ocean (Cretney and Wong 1971). The results indicate that the coastal waters off Port Dickson carry some degree of petroleum hydrocarbon pollution. A pronounced fluctuation of hydrocarbon level in water with sampling date was observed at all the sampling stations (Table 1). There was an approximate tenfold difference in level in which a maximum was detected on 26th June 1985 and a minimum on 19th December 1984. The results indicate that during the Southwest monsoon (data obtained between June and September), a higher level of petroleum hydrocarbon is found compared to the level detected during the Northeast monsoon (data obtained between November and January). This may be obviated by the strong wind action across the Straits of Malacca which drives the surface film of oil towards the Malaysian coastal waters. Further studies are being conducted to eluciGas chromatograpic spectra of oil residues in water date this expectation. No significant difference of hydrocarbon (W) and in sand (S) of the sampling stations. levels in water was detected among the stations at the same date of sampling of the following dates 28/12/84, 6/6/85, 28/8/85 and 19/9/ PERTANIKA VOL. 12 NO. 3, 1989

351

00 Or

TABLE 1 Petroleum Hydrocarbon contents in water and sand along the coast of Port Dickson (in ESSO Tapis A Crude Oil equivalents).

NO

Station Sampling Date

I

II

Water1

Sand2

Water

III

Sand

Water

Sand

6 12 83 31 72+ 1.64 29.33 ±21.04 22.67 ± 1.79 67.19 ±78.89 (HT)4 (HT) 19.12.84 8.53 ±1.95 10.89 ±5.27 3.25 ±0.11 58.39 ±48.0 (HT) (HT) — "*~ -' 28.12.84 12.92 + 1.20 2.28 ±0.61 9.91 ±0.40 4.23 ±2.58 (LT) (LT) 26,1.85 23.60 ±0.17 28.36 ±17.06 20.64 ±3.45 8.86 ±7.46 (LT) (LT) _ 54.59 ±8.37 26.6.85 (HT) 13.79 + 5.35 19.10 ±9.51 28.8.85 (LT) (LT) _ 52.81 ±15,78 51,21 ±2.10 19.9.85 (HT) (HT) _ _ _ _ 49.57 ±5.45 5.10.85 (HT) 30.10.85 _ _ 60.02 ± 3.08 (HT) 20.11.85 46.71 ±4.24 61.97 ±18.62 (LT) (LT) -

, ' • '

. . .

Mean 19.20 (Range) ;8.53-31.72

17.72 2.28-29.33

1 ppb: mean ± S.D. - mg/kg dry sand 9 not determined 4 High tide during sampling *' Low tide during sampling

28.51 3.25-54.59

47.15 34.92 4.23-67,19 13.79-61.97

Water

Sand

Water

Sand

. 15.11 ± 3.00 70.36 ± 39.57 38.59 ± 2.53 50.46 ± 37.54 (LT) (LT)S : 2.52 ±0.92 35.02 ±34.04 10.81 ±3.68 23.40 ±16.66 (LT) • (HT) !*.. 10.24 ±0.80 1.79 ±1.44 9.59 ±4.36 5.28 ±0.57 (IT) (HT) * 21.94±0.58 37.62±34.13 14.87 ±0.98 13.42 ±4.88 (HT) ./• (IT) '.