ECOLOGY AND PHYSIOLOGY OF
PHYTOPLANKTON IN AMBON BAY, INDONESIA
by
GABRIEL ANTONIUS WAGEY
B.Sc, (Hons) Bogor Agricultural University, Indonesia, 1988 M.Sc, The University of British Columbia, Vancouver, 1995
THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
in
THE FACULTY OF GRADUATE STUDIES (Department of Earth and Ocean Sciences, Oceanography)
We accept this thesis as conforming to the required standard
THE UNIVERSITY OF BRITISH COLUMBIA
June, 2002
© Gabriel Antonius Wagey, 2002 In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.
Department of Fart.h anrT Ocean Sr.ipnr.es
The University of British Columbia Vancouver, Canada
Date July, 9 2002 ABSTRACT
This study investigated the natural phytoplankton assemblage of Ambon Bay, eastern Indonesia, over a two-year period, including the composition of the dominant microplankton and the influence of environmental factors with emphasis on the role of mangrove in Ambon Bay. A hoped-for bloom of the toxic Pyrodinium bahamense var. compressum did not materialize but other potentially harmful species from
Ambon Bay were isolated and investigated in culture to determine their growth rates under various environmental conditions and their toxin potential. This constitutes the first study of its type, not only for the Moluccan region, but Indonesia in general.
In this study, 105 phytoplankton species from Ambon Bay were identified, including several dinoflagellates new to Indonesia, such as Gymnodinium catenatum,
Alexandrium cohorticula and Fragilidium cf. mexicanum. The former two are known from S.E. Asia but the latter has not been seen since its description from Mexico.
Due to its close resemblance to the toxic genus Alexandrium it was also tested in culture for toxicity. The common, occasionally abundant, presence of G. catenatum was unexpected and raises the possibility of human health hazard due to this species in Ambon Bay. During the 1997-1998 sampling period, a strong indication of local upwelling occurred in Ambon Bay. The environmental factors that significantly influenced phytoplankton abundance were ammonium, salinity, and water temperature.
Four dinoflagellate species isolated from Ambon Bay were able to grow in culture in both natural and artificial media and, because of its importance as a shellfish poison producer in S.E. Asia, a culture of Pyrodinium was also studied.
Addition of Mangrove Soil Extract (MSE) to the culture media has significantly
enhanced the growth of Pyrodinium (a Manila Bay isolate) compared to media
without MSE. Moreover, it preferred MSE with molecular weight higher than 3000 for
increasing growth in culture.
This study has provided the first comprehensive, detailed information of
phytoplankton in eastern Indonesia and Ambon Bay in particular. It has characterized
general features of the phytoplankton community, identified dominants and
investigated possible environmental influences, particularly mangrove emphasizing
the potentially harmful species. It has significant implications for human health
related to shellfish consumption in Ambon. TABLE OF CONTENT
ABSTRACT
TABLE OF CONTENT iv
LIST OF TABLES vii
LIST OF FIGURES viii
LIST OF APPENDIX xii
ACKNOWLEDGMENTS xiii
CHAPTER 1: GENERAL INTRODUCTION 1
1.1. General Overview 1
1.1.1. Phytoplankton studies in Indonesia 1 1.1.2. Tropical phytoplankton ecology 4 1.1.3. Ambon Bay 8 1.1.4. The Banda Sea influence 12 1.1.5. Mangrove influence on phytoplankton community 16
1.2. General objectives of the study 20
CHAPTER 2: PHYTOPLANKTON ECOLOGY OF AMBON BAY 22
2.1. Introduction 22 2.2. Materials and Methods 24 2.3. Results 29
2.3.1. Biological characteristics 29 2.3.2. Physical characteristics 39 2.3.3. Chemical characteristics 43 2.3.4. Pearson's correlations of cell numbers and environmental variables 55 2.3.5. Transect line (Inner versus Outer Bays) 57 2.3.6. Canonical Correspondence Analysis 66 2.3.7. Mangrove effects 68
2.4. Discussion 70
2.4.1. Physico-Chemical Environment 70 2.4.2. Biological Environment 74 2.4.3. Mangrove effect 78
iv CHAPTER 3: ECOPHYSIOLOGICAL ASPECTS OF POTENTIALLY HARMFUL DINOFLAGELLATES FROM AMBON BAY 81
3.1. Introduction 81 3.2. Materials and Methods for the physiological studies 84
A. ISOLATION AND ESTABLISHMENT OF CULTURES 84
3.2.1. Species isolation and identification 84 3.2.2. Culture in various media and light conditions 87 3.2.3. Cultureware 88 3.2.4. Culture conditions 88 3.2.5. Chlorophyll-a, and particulate carbon and nitrogen 89
B. MANGROVE EXTRACTS 90
3.2.6. Soil Extracts 90 3.2.7. Extracts from Mangrove leaves 90 3.2.7.1. Leaf collection and extraction 90 3.2.7.2. Bioassay of leaf extracts 91 3.2.8. Molecular weight fractionation of mangrove soil extracts 92 3.2.9. Bioassay to test soil extracts 92
3.3. Results 93
A. ISOLATION AND ESTABLISHMENT OF CULTURES 93
3.3.1. Identity and sources of species isolated 93 3.3.2. Culture in various media 103
3.3.3. Light effects on growth 107
B. MANGROVE EXTRACTS 107
3.3.4. Mangrove Soil Extracts 107 3.3.5. Mangrove Leaf Extracts 112 3.3.6. Molecular Weight Fractionation of Mangrove Soil Extracts 115 3.4. Discussion 120
3.4.1. Growth and crude toxicity of the isolates 120 3.4.2. Multiple media and light experiments 124 3.4.3. Molecular Weight 133
v CHAPTER 4. GENERAL DISCUSSION 136
4.1. Future studies 143
REFERENCES 145
APPENDICES 163
vi LIST OF TABLES
Table 2.1. List of phytoplankton species identified in Ambon Bay from May 1996 to July 1998. 30
Table 2.2. Pearson's coefficients of correlation and the significant level (a) between cell number and environmental variables measured at Stations 1, 4, 5 and 7. 56
Table 3.1. Chemical composition of the media used in the experiment. 88
Table 3.2. List of dinoflagellates species used in experiments. 93
Table 3.3. Average values ± 1 SE of total carbon and nitrogen per cell, C:N ratio and Chl-a per cell for three dinoflagellates from Ambon Bay cultured in different media. 114
Table 3.4. Average growth rates (day _1) of Pbc (Pyrodinium bahamense var. compressum) and G.c (Gymnodinium catenatum) in different concentrations of mangrove leaf extracts. 116
vii LIST OF FIGURES
Figure 1.1. Map of the Eastern part of Indonesia. 3
Figure 1.2. Map of Ambon Island, and the location of inner Ambon Bay, which is connected to the Banda Sea through the outer Ambon Bay. 9
Figure 1.3. Cross-section view of the inner Ambon Bay, between Passo and Ambon city. 10
Figure 1.4. Surface current patterns in the Banda Sea during the SE and NW monsoon. 14
Figure 1.5. Two typical, mangrove-lined coastal environment favoring Pyrodin bahamense blooms. 18
Figure 2.1. Locations of sampling stations in this study 25
Figure 2.2a. Contribution of five phytoplankton groups to the total Integrated cell numbers at Station 1. 34
Figure 2.2b. Contribution of five phytoplankton groups to the total Integrated cell numbers at Station 4. 35
Figure 2.2c. Contribution of five phytoplankton groups to the total Integrated cell numbers at Station 5. 36
Figure 2.2d. Contribution of five phytoplankton groups to the total Integrated cell numbers at Station 7. 37
Figure 2.3. Monthly variations of phytoplankton abundance in Ambon Bay. 38
Figure 2.4. Shannon-Wiener's Diversity Index (FT) for phytoplankton species collected in Ambon Bay from 1996-1998. 40
Figure 2.5. Monthly temperature variations (1996-1998) in the top 20 m of Ambon Bay. 41
Figure 2.6. Monthly salinity variations (1996-1998) in the top 20 m of Ambon Bay. 42
Figure 2.7. Monthly precipitation for Ambon Bay from May 1996 to June 1998 44
viii Figure 2.8. Average monthly precipitation for Ambon Bay from 1976- 1998 45
Figure 2.9a. Nitrate concentrations in the top 20 m of Ambon Bay from May 1996-July 1997. 46
Figure 2.9b. Nitrate concentrations in the top 20 m of Ambon Bay from August 1997 - July 1998. 47
Figure 2.10a. Phosphate concentrations in the top 20 m of Ambon Bay from May 1996 - July 1997. 48
Figure 2.10b. Phosphate concentrations in the top 20 m of Ambon Bay from August 1997 - July 1998. 49
Figure 2.11. Ammonium concentrations in the top 20 m of Ambon Bay from August 1997 - July 1998. 51
Figure 2.12. Silicate concentrations in the top 20 m of Ambon Bay from August 1997 - July 1998. 52
Figure 2.13a. Chlorophyll a concentrations in the top 20 m of Ambon Bay from May 1996 - July 1997. 53
Figure 2.13b. Chlorophyll a concentrations in the top 20 m of Ambon Bay from August 1997-July 1998. 54
Figure 2.14. Vertical distribution of water temperature along the transect line from Station 1 to Station 7 during the rainy and dry seasons of Ambon Bay from August 1997 to July 1998. 58
Figure 2.15. Vertical distribution of Chl-a along the transect line from Station 1 to Station 7 during the rainy and dry seasons of Ambon Bay from August 1997 to July 1998. 59
Figure 2.16. Vertical distribution of ammonium along the transect line from Station 1 to Station 7 during the rainy and dry seasons of Ambon Bay from August 1997 to July 1998. 61
Figure 2.17. Vertical distribution of nitrate along the transect line from Station 1 to Station 7 during the rainy and dry seasons of Ambon Bay from August 1997 to July 1998. 62
Figure 2.18. Vertical distribution of phosphate along the transect line from Station 1 to Station 7 during the rainy and dry seasons of Ambon Bay from August 1997 to July 1998. 64
ix Figure 2.19. Vertical distribution of silicate along the transect line from Station 1 to Station 7 during the rainy and dry seasons of Ambon Bay from August 1997 to July 1998. 65
Figure 2.20. Distribution of phytoplankton species represented by (.) and environmental variables (arrows) from water column of Ambon Bay during 1997-1998. 66
Figure 2.21. Cell numbers at Station 1, representing the area close to mangroves and Station 7, representing the area away from mangroves. 69
Figure 3.1. Map showing the reported incidents of harmful algal blooms in Indonesia. 82
Figure 3.2. Average monthly abundance of potentially toxic dinoflagellates in Ambon Bay, during field sampling between Aug. 1997-July 1998 95
Figure 3.3. Scanning Electron Microscope (SEM) pictures of Alexandrium cohorticula used in this study. 96
Figure 3.4. Pictures of Gymnodinium catenatum isolated from Ambon Bay. 98
Figure 3.5. Scanning Electron and Light Micrographs of Fragilidium cf. mexicanum isolated from Ambon Bay. 99
Figure 3.6. Pictures of Prorocentrum gracile isolated from Ambon Bay taken with Scanning Electron Microscope (panel A), and with light microscope (panel B). 101
Figure 3.7. Scanning Electron Microscope (SEM) pictures of Pyrodinium bahamense var. compressum. 102
Figure 3.8. Growth rate of five dinoflagellate species grown in three culture media. 104
Figure 3.9. Growth of Fragilidium cf. mexicanum and P. gracile in different media. 105
Figure 3.10. Growth of Alexandrium cohorticula, G. catenatum and P. bahamense var. compressum in different media. 106
x Figure 3.11. The relationship between growth rate and Photon Flux Density (PFD) for five dinoflagellates. 108
Figure 3.12. Growth rates of Alexandrium sp. and Prorocentrum gracile cultured in media with and without the addition of Mangrove Soil Extracts (MSE). 109
Figure 3.13. Growth rates of Alexandrium cohorticula, Gymnodinium catenatum and Pyrodinium bahamense var. compressum cultured in media with and without the addition of Mangrove Soil Extracts (MSE). 110
Figure 3.14. Growth rates of G. catenatum and P. bahamense var. compressum when grown in L1 medium with the addition of mang soil extract and non-mangrove soil extract. 113
Figure 3.15. Growth rates of Pyrodinium bahamense var. compressum (A) and Gymnodinium catenatum (B) in media with the addition of different concentrations (5 and 10 ml L"1) of soil extracts from two different molecular weights (<3000 and >3000). 117
Figure 4.1. Schematic diagram of environmental factors influencing the ecology and physiology of phytoplankton in Ambon Bay, Indonesia. 137 LIST OF APPENDICES
Appendix 1. Rapid toxicity test of cultured species 163
Figure A1.1. Results of toxicity test using the MISTalert™ kit of Alexandrium cohorticula and Gymnodinium catenatum 165
Figure A1.2. Results of toxicity test using the MISTalert™ kit of Fragilidium cf. mexicanum and Pyrodinium bahamense var. compressum 166
Appendix 2. Bloom of Alexandrium affine in Ambon Bay 167
Appendix 3 Field sampling programs 172 Table A3.1. Sampling dates and environmental parameters measured 172
Appendix 4. Raw Data set 173 Table A4.1. Temperature and Salinity Data set 173 Table A4.2. Measurements of dissolved Nutrients (Stations 1 and 4) 178 Table A4.3. Measurements of dissolved Nutrients (Stations 5 and 7) 181 Table A4.4. Measurements of dissolved Nutrients (Stations 3) 184
xii ACKNOWLEDGEMENTS
I have benefited greatly from the support, encouragement and advice of many people who helped me during my PhD studies. I am deeply indebted to my supervisor, Dr. F.J.R "Max" Taylor, my committee members, Dr. Paul J. Harrison, and "Pak" Dr. Rob de Wreede for their guidance and support throughout my research projects and the completion of my thesis. I am grateful to the Eastern Indonesia University Development Project (EIUDP) for its financial support that enabled me to enter the PhD program. In particular, I am thankful to "Pak" Chris Dagg and his Staff at the offices in Burnaby, Jakarta, and Ambon. I would like to acknowledge Dr. Michael Healey for his support during my graduate studies. Part of my research project was conducted in Indonesia and two particular Institutions in Indonesia, the Indonesian Institute of Science (LIPI) and the University of Pattimura (Unpatti), have provided assistance during my fieldwork in Ambon. I wish to thank Dr. Ngurah N. Wiadnyana, Pramudji MSc, Omi, Irene and my "sampling crew" from LIPI-Ambon for their help during the field work in Ambon. I appreciate the support of Dr. Niette Huliselan and Mr. Wattimuri from the Faculty of Fisheries, Mr. Sitorus and Ms. Suriati from the Dept. of Basic Chemistry, Unpatti, to advance my research work in Ambon.
I would like to acknowledge my colleagues in Max Taylor's Lab: Juan Saldarriaga who helped me with the SEM, David Cassis for the translation, and former graduate students, Kugako Sugimoto, Linda Greenway, and Rowan Haigh, who have been supportive in different ways. I am extending my gratitude to my colleagues in Paul J. Harrison's Lab for their friendship and assistance over the years. Special thanks to Michael Lipsen and Mike Henry for teaching me how to use many application programs and lending me the camera, of course! The many discussions we had, have encouraged me to accomplish my goals. Joe Needoba generously helped me during my lab work. My warmest appreciation to my Canadian family Amo and Bett Copeland for their support and encouragement during my stay in Canada. A very special thanks to my parent-in-law for their tremendous support all along. Finally, my deepest gratitude to my wife, Venska, my brothers and sister, and my parents for their continuous love and support throughout the years. DEDICATION
/ would ti^e to dedicate this thesis to my father, Cornefis JLntonius Wagey, my mother, J-Cenriette Josephine Wagey-WuCur, and my wife, (Ravenska (Wagey-(Radjawane
'Their tove and encouragement have given me confidence and strength to achieve my goats in life CHAPTER 1: GENERAL INTRODUCTION
1.1. General Overview
I. 1.1. Phytoplankton Studies in Indonesia
Oceanographic studies in Indonesia only began to receive national attention in the early 1960s, when several oceanographic cruises started to collect samples from Indonesian waters (Nontji, 1993). During the colonial era (from the early
1600's to1945), there were a number of oceanographic cruises dating back to the
1700's; for example G.E. Rumphius (1627-1702) who published his work in
D'Amboinsche Rariteitkamer (1705) and Herbarium Amboinense (1741 -1750). A significant expedition in eastern Indonesia was the "Siboga" Expedition (1899-1900)
(Tomascik et al. 1997). Another important expedition, the Snellius I (1921-1930) is considered one of the most significant oceanographic studies in the region, and it covered the entire Indonesian archipelago. In 1984-1985, the Snellius II expedition was conducted covering, in part, eastern Indonesian waters, mainly the Banda and
Arafura Seas. Physical (Zijlstra et al. 1990; llahude et al. 1990), chemical (Wetsteyn et al. 1990; Cadee, 1988) and biological (Schaik, 1987; Gieskes et al. 1990; van
Iperen et al. 1993; Sevenboom and Wetsteyn, 1990; and Adnan, 1990) oceanographic aspects were studied during the one-year expedition of the Snellius
II. A summary of the pelagic system of the Indonesian waters was presented by
Tomascik et al. (1997). Nevertheless, despite the vast amount of marine natural resources, similar studies are difficult and very expensive to conduct today which has lead to the lack of recent oceanographic studies in Indonesia.
1 Phytoplankton studies have been part of biological oceanographic research since phytoplankton play an important role in the productivity of the oceans, including the Indonesian waters. Early phytoplankton studies in Indonesia were conducted several decades ago by foreign researchers (Allen and Cupp, 1935;
Delsman, 1939), but these reports, although commonly used as references by other researchers were published outside Indonesia, and were difficult to access by the
Indonesian researchers (Tomascik et al. 1997).
Most oceanographic studies from the early expeditions to the most current ones have focused mainly on the deep ocean, such as the Banda Sea in Eastern
Indonesia and the Indian Ocean in Western Indonesia (see Fig. 1.1). An extensive study of dinoflagellates in the adjacent Andaman Sea and Indian Ocean was reported by Taylor (1976). The eastern part of Indonesia covers the majority of the
Indonesian waters and is influenced by the Pacific as well as the Indian Ocean through the Timor Sea. Hallegraeff and Jeffrey (1984) conducted a thorough tropical phytoplankton study in the north and northwest of Australia, covering the
Gulf of Carpentaria, Arafura Sea, and Timor Sea. This eastern part is characterized by the presence of the Banda Sea and thousands of smaller islands surrounding it.
These islands make up a significant part of the tropical coastal area and are unique microenvironments. One important feature of the small islands in Indonesia is the presence of a large number of embayments. The oceanographic features of the embayment area strongly influence the phytoplankton communities and the biological processes in these waters.
2 Maluku 1 .7 000 000\
Samudra Pastfik Grots Ocsaan Pacific Ocsan %
I JF*T / ' Su .T»O,. T™i.kf Maluku , Sea 'Q Kep Sula "Buayc OS Maluku Banda Sea Kep. Kei , p uu Flores Sea Kep. Barat Daya ,, P We tar (' *i<"T*nQ p Kep. Aru r.SMKM P. 3*l»ru Kep. Ten9gara Kep. Tanimbar • 3310 i clJr~P-Timor Arafura Sea Timor Sea Source/Sumber/Bron: Alias Maluku ILanoelqfc Steunpunt Educsfee MokAkara Utrectit. 1S0S> re 1.1. Map of the Eastern part of Indonesia. It represents the Maluku, West Timor, and part of Papua Provinces. The Banda Sea is located at the center surrounded by Arafura Sea, Timor Sea, Flores Sea, and the Seram Sea. (Source: Landelijk Steunpunt Educatie Molukkers, Utrecht, 1998). 1.1.2. Tropical Phytoplankton Ecology In coastal environments, in general the role of riverine inputs is important. Cloern (1996) defined common features of shallow coastal ecosystems, which are primarily characterized by the influence of run-off from land including terrestrial inputs and exchange with the coastal ocean, and riverine inputs as a source of particle-rich fresh water and nutrients. These factors, combined with physical forcing in the coastal areas such as tidal currents, wind stress on the water surface, and horizontal and vertical density gradients, can be responsible for the temporal and spatial distribution of phytoplankton. In the tropics, the environmental conditions are mainly affected by seasonal variations driven by monsoon cycles, resulting in both rainy and dry seasons. Precipitation and subsequent river run-off are important since they may contribute to nutrient fluctuations in the water, and possibly eutrophication (Paerl, 1997). Rates of primary production in tropical coastal waters are largely limited by nitrogen supply which mainly originates from river discharge and rainfall (Howarth, 1988). Harrison et al. (1997) have found that phosphate concentration was low in the water column in the mangrove tidal creeks of the Indus River delta in Pakistan. They suggested that phosphate was adsorbed to the sediment particles and that the adsorbed phosphate was only partially biologically available. Furnas and Mitchell (1986) concluded that the phytoplankton in the central Great Barrier Reef are also nitrogen- limited and a similar condition was also observed in Albatross Bay, Northern Australia (Burford et al. 1995). However, increased nutrient inputs from river run-off and precipitation are not always the primary cause of a phytoplankton bloom 4 (Hallegraeff, 1993; Taylor et al. 1994). The abundance of some dinoflagellates such as Karenia brevis (formerly Gymnodinium breve), Alexandrium spp., and Pyrodinium bahamense var. compressum can appear to be unaffected by nutrient enrichment (Hallegraeff, 1995). Furthermore, Sakshaug and Olsen (1986) suggested that different species have different strategies for nutrient competition. The result is a species selection process that could lead to a bloom of particular species which may inhibit other phytoplankton species by higher nutrient competitiveness or allelopathy. Nutrient-limited species with high uptake capacity will benefit when the nutrient supply is patchy. Thus, generally there is a correlation between phytoplankton abundance and the increase in nutrient levels such as nitrate, ammonium, phosphate and silicate at a particular site. Another factor that could contribute to the increased nutrient level is wind mixing which may stimulate phytoplankton growth by deep water mixing or resuspending nutrients from sediments in shallow areas. Wind mixing could also cause re-suspension of benthic cysts of some dinoflagellates such as Gymnodinium catenatum (Hallegraeff and Fraga, 1998). Seasonal variations in temperature, salinity and nutrients levels are all believed to play a major part in phytoplankton species succession (Hallegraeff and Jeffrey, 1993). In the south-western part of India, Devassy and Goes (1988) found that, prior to the monsoon period, large diatoms such as Coscinodiscus sp., and Fragilaria oceanica, together with the cyanobacterium, Trichodesmium sp. (considered to be Oscillatoria sp. by some authors), were the dominant species. The seawater during this monsoon period has relatively low nutrient levels, and higher temperature and salinity. With the onset of the monsoon season, 5 characterized by an increase in precipitation and hence river run-off, there is a spontaneous increase in phytoplankton production and Skeletonema costatum becomes dominant. The dominance of Trichodesmium in the phytoplankton community succession was reported in tropical northern Australia by Revelante and Gilmartin (1982); Rothlisberg et al. (1994) and Burford et al. (1995). Trichodesmium was found to dominate the phytoplankton community in Albatross Bay, Northern Australia and bloomed during the wet season when the bay was relatively calm. As a nitrogen fixer (Carpenter, 1977), Trichodesmium is considered an important species in relation to phytoplankton succession in the tropical and subtropical waters of Australia (Revelante and Gilmartin, 1982; Burford et al. 1995). On the other side of the Pacific, in Panama Bay, Smayda (1966) reported a dominancy of diatoms over dinoflagellates, both in cell number and cell volume in the phytoplankton composition. Taylor (1987) concluded that in most coastal tropical phytoplankton communities, it is typical for diatoms to dominate the phytoplankton composition over dinoflagellates. A unique type of bloom of one tropical coastal phytoplankton species was reported in Fire Lake in the Bahamas (before it was filled by landfill), Oyster Bay in Jamaica (Seliger et al. 1969) and Bahia Fosforescente in Puerto Rico (Margalef, 1961; Seliger et al. 1971). These studies were summarized by Smayda (1980). In these locations, during blooms of the dinoflagellate Pyrodinium bahamense var. bahamense, the surrounding water turns luminescent due to high concentration of this species. Seliger etal. (1969; 1971) investigated the ecology of the blooms of P. bahamense var. bahamense, and noted that they occur close to the mangrove site. 6 Prakash (1971) suggested that the presence of mangrove trees {Rhizophora sp.) along the coast of these bays influences the persistent blooms of P. bahamense var. bahamense. In the absence of major environmental triggers, phytoplankton succession is only weakly developed. In the tropical waters of Albatross Bay Australia, Burford et al. (1995) found that due to lack of seasonal variation with respect to temperature and salinity, coupled with low nutrient concentration, phytoplankton succession was not clearly observed. A similar situation was found earlier in Ambon Bay since the nutrient level in this area is relatively low and mixing due to wind is unusual (Wenno, 1997). Although dinoflagellates are not the most dominant group among tropical coastal phytoplankton, dinoflagellates are increasingly seen as an important group in the phytoplankton community due to their high potential to cause HABs (Hallegraeff, 1993) and are major dominant of tropical oceanic water (Taylor, 1987). Sournia (1995) stated that about 300 phytoplankton species (e.g. diatoms, dinoflagellates, silicoflagellates) produces "red tides" and other harmful algal blooms. Out of the 300 taxa, only 60-80 species are actually toxic and 90% consist of flagellate species. Dinoflagellates account for 75% (45-60 taxa) of all HAB species (Smayda, 1997). Thus, dinoflagellates stand out as the main phytoplankton group contributing to potential toxic species in coastal phytoplankton communities. 7 1.1.3. Ambon Bay Geomorphologicaly, Ambon Bay is divided into two parts: the inner and the outer Ambon Bay. The inner bay is characterized by a relatively smaller size (6 km long) and is more enclosed, whereas the 25 km long outer Ambon Bay is more open system connected to the Banda Sea in the south (Fig 1.2). A narrow and shallow (300 m wide, and 15 m deep) channel connects these two parts, thus limiting the water circulation in the inner Ambon Bay. The maximum depth of inner Ambon Bay is 30 - 40 m, whereas outer Ambon Bay can reach a maximum depth of 600 m. The inner bay is within the coastal boundary layer, i.e. a zone with particular physical, biological or chemical properties that differs from the outer bay which receives more influence from the Banda Sea. The inner bay covers an area of approximately 40 km2 with a mean depth of 20 m. The deepest part is the area close to the sill reaching a maximum of 40 m (Fig. 1.3). Anderson and Sapulete (1981) noted that the sill restricts water circulation in the inner bay making it potentially susceptible to stagnation. The climate is tropical with a mean annual rainfall of approximately 600 mm, almost all of which falls during the rainy season from May-September. Greatest rainfall activity is associated with the southeast monsoon season when cooler air from Australia blows to this region. The dry season is associated with the northwest monsoon from October until March, characterized by increasing air temperature and less rainfall. There are many small rivers that carry the discharge from the surrounding catchment area into the bay. These rivers are major fresh water 8 sources during the rainy season. The mean monthly surface temperature ranges from 26° C in July to 30° C in December and salinity ranges from 29 to 34. Figure 1.2. Map of Ambon Island, and the location of inner Ambon Bay, which is connected to the Banda Sea through the outer Ambon Bay. A small channel connects the inner Ambon Bay to the outer Ambon Bay. 9 Ambon Passo 10 km Figure1.3. Cross-section view of the inner Ambon Bay, between Passo and Ambon City. Passo is located at the head of the inner Ambon Bay, and Ambon City is located on the southern part of the outer Ambon Bay. The sill is at the channel connecting the inner and outer Ambon Bay. 10 { Water circulation in the bay is dominated by tidal currents that flow from the outer bay through the narrow opening which connects the outer and inner bays. Hamzah and Wenno (1987) and Wenno (1991) determined that the current in the inner bay flows counterclockwise with a speed of 20 - 25 cm s"1 in the center of the bay. The strongest current (75 cm s"1) was observed over the sill. The wind driven current is another factor that may cause water movement in the inner bay. However, this current is apparently weaker than the tidal current. Several studies have reported the nutrient concentrations in Ambon Bay. Regardless of the lack of continuity in the data set, the variability in nutrient levels between rainy and dry season is clearly observed. Sutomo (1987) hypothesized that the increase of NO3 in this area is highly correlated to the amount of rainfall during that period. Furthermore, it seems that the influence of freshwater run-off is quite significant. From observations in 1987, Tarigan (1987) and Edward and Manik (1987) reported that the concentration of N03 and P04 in two rivers in inner Ambon Bay ranged from 14.8 -22.7 uM and 16.4 - 21.0 uJVI for nitrate and phosphate, respectively. These values are only a 'snapshot' of the nutrients that enter Ambon Bay. Therefore, more information such as nutrient concentration during rainfall and the timing of sampling need to be obtained in order to explain the variability of nutrient concentrations in this bay. A small mangrove forest is spread along the bay, especially at the eastern end of the inner bay. Sonneratia alba is the main mangrove tree in this area; other species and genera such as Rhizopora apiculata and Avicennia are also found. 11 Systematic oceanographic research in Ambon Bay began in the early 1970's with the establishment of the LIPI (Lembaga llmu Pengetahuan Indonesia or Indonesian Research Institute) station in Ambon. An early description of the biology of Ambon Bay was published at the beginning of the 18th century by Rumphius (Nontji, 1997). Very limited literature exists on the subject of phytoplankton of Ambon Bay and most of this information describes species composition and taxonomy at the level of genus and is limited to short term observations (three to six months) during a particular season (Nontji, 1997). To my knowledge, there have not been any detailed studies on phytoplankton ecology in relationship to mangrove ecosystems in Ambon Bay. This study is the first attempt to evaluate the status of the marine phytoplankton community and its relation to the environmental conditions in Ambon Bay. Harmful phytoplankton blooms are not novel phenomena in Ambon Bay. In 1993, three children died and more than 30 people became ill after consuming shellfish collected from Ambon Bay. Wiadnyana et al. (1995) reported that the dinoflagellate species Pyrodinium bahamense var. compressum was found in abundance and caused the shellfish in the area to be toxic for human consumption. A bloom of the non-toxic dinoflagellate species, Alexandrium affine, also occurred in Ambon Bay in November 1997 (Wagey et al. 2001). 1.1.4. The Banda Sea Influence The oceanographic conditions in Ambon Bay can be influenced by the Banda Sea, which channels into the mouth of Ambon Bay from the southwest side (see 12 Figure 1.1). The influence of the Banda Sea is partly due to the free exchange of the water mass between Ambon Bay and the open ocean, the Banda Sea. It has been suggested that the exchange of this water mass is highly influenced by the monsoonal pattern (Wyrtki 1957; 1961). In a report called "Naga" (meaning Dragon), Wyrtki (1961) hypothesized that the surface current in the Indonesian archipelago follows the monsoonal winds, the southeast (SE) monsoon (May - September) and the northwest (NW) monsoon (December - March). This monsoonal pattern resulted in upwelling and downwelling processes during the SE and NW monsoon, respectively. Figure 1.4 shows the surface current patterns in the Banda Sea during the SE and NW monsoons. Furthermore, Schaik (1990) used some molluscs as indicators to argue that during the SE monsoon, the water in the Banda Sea is highly influenced by the Pacific Ocean, whereas during the NW monsoon, the water mass from the Indian Ocean is more dominant. During the upwelling period in the Banda Sea, primary productivity ranged from 1.85 g C m"2 d"1 in the open sea to 7.0 g C m"2 d"1 in the coastal area close to Irian Jaya. During the non-upwelling period (NW monsoon), the average primary productivity was 0.91 g C m"2 d"1 throughout the Banda Sea (Gieskes et al. 1990). Wetsteyn et al. (1990) attributed the increase in primary productivity during the upwelling period to the vertical mixing mainly in the upper 150 m, which was believed to produce daily new production estimated at values between 1 - 3 g C m"2. Furthermore, Adnan (1990) found that large diatoms (> 75 um) were dominant during the upwelling period in the Banda Sea. Schaik (1987) reported that zooplankton biomass was also higher (average 1.0 g C m"2) during the SE monsoon 13 Figure 1.4. Surface current patterns in the Banda Sea during the SE and NW monsoon; o = upwelling region, • = downwelling region; * = Ambon Island. (From: Schaik, 1990, after Wyrtki, 1961). compared to the NW monsoon (0.5 g C m"2). Based on this productivity level, Dalzell and Pauly (1990) estimated that the potential yield of pelagic and demersal fisheries in the Banda Sea is 2.1 and 1.0 tons km"2 yr"1, respectively. The upwelled water from the Banda Sea influences the hydrology of the surrounding waters as the water circulates into the adjacent areas. At the time upwelling occurred in Banda Sea, cooler water was also detected in the Southern Java Sea (Wyrtki, 1962), Makassar Strait (llahude, 1971), Bali Strait (Nontji and llahude, 1975), Flores Sea (Birowo, 1979), in the Eastern Arafura Sea and the Gulf of Carpentaria (Rochford, 1966). Tarigan and Wenno (1991) and Wenno (1986) reported the presence of cooler (25.9 °C at surface) and more saline (at > 50 m depth) water in the outer Ambon Bay compared to the inner Ambon Bay during the SE monsoon. Conversely, during the NW monsoon, relatively warmer water (27.6 °C) was also found in Ambon Bay. These results indicate that there was an intrusion of water mass from the Banda Sea into Ambon Bay. Rebert and Birowo (1985) suggested that the water from Banda Sea that enters Ambon Bay from the outer bay travels through an internal wave that penetrates into the inner bay crossing the shallow sill that separates the outer bay from the inner bay. However, Wenno and Anderson (1983) and Wenno (1998, pers. comm) reported that during the upwelling period in the Banda Sea (SE monsoon) the thermocline was shallower than during the non- upwelling period causing the cooler water from the outer bay to enter the inner bay through tidal action. 15 1.1.5. Mangrove influence on phytoplankton community Ecosystems unique to tropical regions, such as mangrove forests, could influence the dynamics of phytoplankton in adjacent waters. The importance of the mangrove forest to the adjacent coastal waters has been discussed by several researchers from different parts of the world, e.g. Teixeira and Kutner (1963) in Brazil, Smayda (1966) in Panama Bay, and Carpenter and Seliger (1968) in Oyster Bay, Jamaica. The more recent reports include studies in Pakistan (Harrison et al. 1994; 1997); Thailand (Wium-Andersen, 1979); Malaysia (Tanaka and Choo, 2000); Southeast India (Kathiresan, 2000); Terminos Lagoon, Mexico (Rivera-Monroy et al. 1998); and in the Amazon, Brazil (Dittmar and Lara, 2001). Some HABs have been observed to occur at the beginning of the rainy season, and several researchers have tried to explain the relationship between hydrological conditions and HAB occurrences. Stratification due to a near-surface halocline is believed to be important in flagellate blooms since it leads to low surface macronutrients. This reduces diatom growth while the flagellates can migrate to lower, nutrient rich waters (Taylor, 1987; Taylor, 2001). On the other hand, Prakash (1975); MacLean (1989a) and Usup and Lung (1991) concluded that river run-off and other drainage from land contributes organic compounds needed for some blooms, in this case mostly flagellates. Clearly, both diatoms and flagellates can play a major role, depending on the circumstances. In the tropics, an important component in the productivity of coastal areas is the influence from the surrounding ecosystems such as seagrasses, coral reefs, and mangrove forests. Terrigenous organic matter washed from the land and carried to 16 the sea by rivers may include soils, marsh leachates, plant decomposition products, and mangrove exudates. Prakash (1971) revealed that the presence of yellowish- brown runoff from the mangrove swamps in the coastal area of Montego Bay, Jamaica, had a significant positive effect on the growth of Pyrodinium bahamense var. bahamense. Furthermore in the Pacific region, Wiadnyana et al. (1995) suggested that the existence of mangrove vegetation in the vicinity of the site where Pyrodinium blooms occur in Ambon Bay, might contribute to these blooms. In the Philippines, Gacutan et al. (1985) indicated that mangrove ecosystems that grow along the coast might contribute to the increasing number of P. bahamense var. compressum in the 1983 bloom. In the Mandovi-Zuari estuarine complex (India), Devassy and Goes (1988) noted that the introduction of humic substances into the coastal environments had a strong positive influence on the successional pattern of phytoplankton, especially on the appearance and predominance of the dinoflagellate Ceratium furca in an otherwise diatom-dominated ecosystem. Taylor (1999) proposed that there are two typical mangrove-lined coastal environments that favor the blooms of Pyrodinium bahamense (Figure 1.5). The first type of environment (Fig. 1.5A) is a semi-enclosed bay with the presence of mangroves along the coastal area of the bay and water flows circularly around the bay. In this scenario, the expected location of P. bahamense bloom is around the center of the bay. An example of this scenario is the Bahia Fosforescente in Puerto Rico (Margalef, 1961). In the second scenario (Fig 1.5B) the presence of mangroves are found along the riverbanks and along the coast. The outflow of fresh water meets the seawater and depending on the strength of the outflow water, P. 17 bahamense could most likely be found along the outer boundary of the fronts preferring the high salinity regime. Examples of this scenario can be found in Brunei and Sabah, as well as in Papua New Guinea coastal areas (Maclean, 1977; Seliger, 1989). B. Mangrove Pyrodinium Waterflow Figure 1.5. Two typical, mangrove-lined coastal environments favoring Pyrodinium bahamense blooms. Diagram A shows a semi-enclosed bay. The currents within the bay influence the actual distribution of P. bahamense. Diagram B depicts a coastal river-plume front. The dinoflagellate will be mostly concentrated on the seaside of the boundary. (Source: Taylor, 1999) Riverine input, including that from mangrove swamps, into the coastal area has been the dominant source of biologically active compounds such as humic substances (Prakash, 1971, Doblin et al. 1999). Moreover, Prakash et al. (1973) and Prakash and Rashid (1968) concluded that there was a significant increase in 18 growth rate and production of phytoplankton due to the addition of humic substances. Prakash (1971) noted that by ionic exchange, surface adsorption and chelation, humic substances form complexes with trace metals that are more stable than those of inorganic-metal complexes (Kennish, 1986). As chelators of trace elements, humic substances provide aquatic organisms with necessary inorganic constituents for growth. Chelated trace metals are usually taken up more readily by phytoplankton than the non-chelated ones (Rashid, 1985). This evidence confirms the possible effect of humic substances (possibly from the mangrove area) on phytoplankton growth. However, the specific relationship between harmful algal species and mangrove ecosystems is still poorly understood. Products of litterfall from mangrove trees are suspected to influence phytoplankton growth (Herrera -Silveira and Ramirez-Ramirez, 1996). Mangrove leaves on the ground are further processed through microbial degradation. Substances such as lignin and tannin from leaves are further degraded to produce simple phenolic compounds (Kirk, 1984; Hedges, 1988). Some phenolic compounds, such as tannic acid are found to be inhibitory to the growth of certain phytoplankton species (Herrera -Silveira and Ramirez-Ramirez, 1996). One approach to test the effect of mangroves is by conducting bioassays using phytoplankton. Cooksey and Cooksey (1978) observed that the growth of the diatom Amphora coffeaeformis showed a stimulatory response to the addition of sediment extract taken from a mangrove area. 19 1.2. General objectives of the study The objective of this thesis was to study the ecology and physiology of phytoplankton in Ambon Bay, Indonesia, and to elucidate the role of mangrove extracts on the growth rate of some dinoflagellates originating mostly from Ambon Bay. Ambon Bay was chosen as a study site for the following reasons. There have been very limited phytoplankton studies in this area (mainly conducted by LIPI). In 1993, there was an incident of PSP caused by the bloom of P. bahamense var. compressum in Ambon Bay. In this incident, Wiadnyana et al. (1995) reported the death of 3 children and dozens of people suffered from PSP symptoms. In terms of the ecophysiology of HAB species in Ambon Bay, no research is available. It is clear, however, that the increased occurrences of HABs in Ambon Bay can be a potential threat to the local community. Furthermore, the presence of two main Research Institutions in Mollucan waters, namely LIPI and University of Pattimura's Fisheries Faculty and Marine Science Center in Ambon, were an asset to this study, as they provided research equipment and facilities to conduct the ecophysiological study of some HAB species in Ambon Bay. Specifically, the objectives of this study are as follows: 1. To investigate phytoplankton composition and abundance and the effects of environmental factors on the phytoplankton assemblage in Ambon Bay. 2. To identify, culture and provide ecophysiological information on some potentially harmful dinoflagellates from Ambon Bay. 20 3. To study the influence of the mangrove ecosystem on phytoplankton growth, especially the effect of mangrove extracts on the growth rate of dinoflagellates isolated from the surrounding ecosystem. To achieve these objectives, this study was conducted both in the laboratory and in the field, in Canada and Indonesia, respectively. 21 CHAPTER 2: PHYTOPLANKTON ECOLOGY OF AMBON BAY 2.1. INTRODUCTION Indonesia is a tropical country with more than 80,000 km of coastline due to its estimated 17,500 islands. Its marine ecosystems include large areas of mangrove forests, coral reefs and seagrass beds and provide numerous unique sites for oceanographic research. However, as a country with a vast oceanic area, accessibility to some areas, and scarce funding generally have limited oceanographic research. An increase in global population, especially in tropical areas, has caused alterations in natural phenomena such as the global climate system and hydrological cycles. The coastal zone is thought to have been greatly affected by these alterations. Hence, research in coastal ecosystems is therefore critical for our understanding of the impact of these changes (Cloern, 1996). Ambon Bay is located on the southwestern part of Ambon Island stretching on a northeast-southwest axis that is approximately 31 km long and between 0.3 - 11 km wide (see Fig. 1.2). The physical description of Ambon Bay was presented in the previous chapter (see Chapter 1, Section 1.1.3). Ambon City is the capital of Maluku Province, in Eastern Indonesia. Currently, the coastal area of Ambon Bay has become an economic and social center for the local people of Ambon. The rapid development of the area along the coast of Ambon Bay and its surroundings has resulted in high sedimentation in inner Ambon Bay, especially in areas close to the rivers. Besides sediments and suspended particles from runoff, Ambon Bay also receives organic materials as domestic waste from the city. All these activities have resulted in the 22 deterioration of water quality and hence have impacted the organisms living in Ambon Bay, including the phytoplankton communities. Fluctuations in nutrient levels in the water column result in major changes in phytoplankton abundance and composition. As primary producers, the increased abundance of phytoplankton provides food for higher trophic levels. However, if the phytoplankton species that proliferate are harmful either to other organisms or to humans, this could create an environmental problem. The main objective of this chapter is to examine the interannual, and spatial variability of physical, chemical and biological parameters in Ambon Bay. Data presented are collected from different stations located throughout the inner and outer Ambon Bay. Parameters measured in this study include: 1. Water temperature, salinity and transparency 2. Dissolved nitrogen (nitrate and ammonium), silicate and phosphate concentrations 3. Chlorophyll-a 4. Phytoplankton abundance (cells L"1) This study also determines the affects of some environmental variables on phytoplankton biomass. In particular, the contribution of the mangrove forest to the phytoplankton biomass was examined. 23 2.2. MATERIALS AND METHODS Sampling was done from May 1996 to July 1998 at 5 stations in the inner and outer Ambon Bay (Figure 2.1); four stations in the inner Bay (Stations 1, 3, 4, and 5) and one station in the outer Bay (Station 7). Stations were assigned following LIPI's station numbers which have been established as reference stations. Initially, only 4 stations were established (Stations 1, 4, 5 and 7). Station 3 was added in April 1998 to complement the other existing stations for a more thorough comparative study between the inner and outer Ambon Bay. Additional stations surrounding Stations 1 and 7 were established for the comparative study of the mangrove influence on the phytoplankton community. Sampling was done using one of LIPI's research vessels. Water samples were collected with 'Van Dorn' bottles (2 L capacity). Water from the bottles was emptied into acid-cleaned 2 L plastic containers and filtered in the laboratory for Chl-a and nutrient analysis. Prior to filtration, a 50 ml aliquot of water was sampled for phytoplankton composition. A few drops of Lugol's iodine solution were used as fixative. The sample was transferred into a 50 ml glass container, sealed and kept in the dark until analyzed. The sample was then settled in a 50 ml chamber for'either direct identification and enumeration on the inverted microscope or for later analysis. Species identification was performed to the more commonly recognized phytoplankton groups, including diatoms, dinoflagellates, silicoflagellates and cyanobacteria. The phytoplankton reported here were identified to the species level however; others were recorded and enumerated only at higher taxonomic levels (e.g. Chaetoceros spp., Alexandrium spp., etc). In this study, I consider only the species 24 that contribute to > 5% to the total phytoplankton population as variables in the statistical analysis. Figure 2.1. Locations of sampling stations in this study. Insets are the maps of Indonesia (lower right) and of Ambon Island (upper left). Open circles represent sub-stations added to Stations 1 and 7. Due to the recent communal conflict in Ambon, several research facilities such as LIPI's Research and Development Center for Oceanography and Pattimura University's Marine Science Center were destroyed. As a result, some samples and equipment accumulated during my field-study period were also destroyed, since they were stored and kept in these laboratories. Consequently, it is impossible to retrieve 25 any samples, especially phytoplankton samples that would be used for further identification and enumeration. Water samples were taken every two weeks at depths of 0, 5, 10, and 20 m. A3 m-depth sample was added for Chl-a analysis. However, in case of unusual conditions (e.g. algal blooms), sampling was conducted every 2 or 3 days during the 2- week period of the phytoplankton bloom. During May 1996 - July 1997, phytoplankton samples were identified and counted from the 5 m depth. Samples from 0 and 20 m depths from the same period had been collected, but not yet been analyzed unfortunately, these samples were lost during the conflict. During the August 1997 to July 1998 period, samples were analyzed from the 0, 5 and 20 m depths. Temperature and salinity were determined by means of a reversing thermometer and a Beckman salinometer, respectively. Transparency was measured by determining the Secchi depth at each station. Chlorophyll-a was measured spectrophotometrically (Strickland and Parsons, 1972), with a phaeopigment correction (Lorenzen, 1967). Nutrient samples were frozen immediately and concentrations of nitrate, phosphate, ammonium and silicic acid were measured in the laboratory following the guidelines by IOC-UNESCO (1993), based on the methods outlined in Parsons et al. (1984). Statistical Analysis Phytoplankton abundance data were analyzed for species diversity using the following indices: The Shannon-Wiener Index (H1) = - 2,p> (log™ pi) (MacArthur, 1965) and 26 The Simpson Dominance Index (SI) = Sp2 (Simpson, 1949) ,Pielou's Evenness Index (J) = H'/Ln S (Pielou, 1966) Where: pi = proportional abundance of /'-th species; S = number of species. Data analysis of phytoplankton studies frequently involves the application of multivariate analytical techniques (Cassie, 1963; 1972; Green, 1980). In the past, some univariate procedures have been used in phytoplankton studies. However, the latter have been criticized as inefficient since most of the data analysis involves several variables that influence species distribution and abundance (Day and Quinn, 1989). The multivariate analysis of the phytoplankton community of Ambon Bay begins with the matrix of raw data, arranged with samples (or stations) as columns and species abundance as rows. This arrangement is referred as Q-type analysis (Pielou, 1969). The raw data of a phytoplankton survey may not be suitable for some statistical procedures due to a lack of normality of the data. It is conventional to transform the data using logarithms, which is especially appropriate for phytoplankton data as these can range from very low values for rare species to very high values for bloom organisms. In this study I used the transformation: Ln (cell # +1); where cell # = number of cells per liter. Furthermore, since the physical variables are represented by different units of measurement, they were standardized by dividing each variable by its standard deviation (Pimentel, 1979). 27 Many environmental variables potentially affect distribution of phytoplankton species. Canonical Correspondence Analysis (CCA) was used to explain phytoplankton distributions as correlated with environmental parameters. A thorough and illuminating account of CCA is given by ter Braak and Verdonschot (1995), ter Braak (1995) and Legendre and Legendre (1998). Briefly, CCA is a form of multivariate gradient analysis, which is able to detect a unimodal relationship between species and environmental variables. Furthermore, one can calculate the percent variance extracted by the first ordination axis that is the weighted sum of all linear combinations of environmental variables in determining the species distribution (ter Braak, 1995). The result of CCA is a two-dimensional plot that expresses the species variability pattern in relation to environmental variables. The Pearson correlation coefficient was used to analyze the statistical relationship between the measured variables during the sampling period (Zar, 1996). 28 2.3. RESULTS The present study includes measurements of some biological, physical and chemical characteristics of Ambon Bay. The effect of the mangrove ecosystem on phytoplankton was evaluated through a comparative study of phytoplankton abundance at a station close to, and far from, the mangrove area. The data were statistically analyzed using Canonical Correspondence Analysis and Pearson's correlations. 2.3.1. Biological characteristics The composition of phytoplankton species in Ambon Bay was analyzed from 4 stations (Stations 1, 4, 5 and 7). A limitation exists in the phytoplankton analysis. Phytoplankton abundance was only counted from large phytoplankton groups mainly diatoms, dinoflagellates, and cyanobacteria. The other groups such as picoplankton and other flagellates were not included. In general, phytoplankton in Ambon Bay can be categorized into 5 groups, namely the centric diatoms, pennate diatoms, photosynthetic dinoflagellates, heterotrophic dinoflagellates and cyanobacteria. The average cell concentration during non-bloom conditions was around 2 - 3x104 cells L"1, whereas during the blooms, cell numbers were as high as 2x106 cells L"1. The results of the phytoplankton analysis are presented as a species list (Table 2.1) and as cell numbers from each group (Figure 2.2 a-d). During the study period of 1996 to 1998, 105 phytoplankton species were identified in Ambon Bay (Table 2.1). The diatoms accounted for 78 species (63.4%), 29 followed by dinoflagellates with 44 species (35.8 %), silicoflagellates consisted of 2 species (1.6%), and cyanobacteria comprised only 1 species. Table 2.1. List of phytoplankton species identified in Ambon Bay from May 1996 to July 1998. No. Species name Diatoms 1 Actinoptychus senarius Ehrenberg 2 Asterionellopsis glacialis (Castracane) Round 3 Asteromphalus flabellatus (Brebisson) Greville 4 Bacteriastrum comosum Pavillard 5 Bacteriastrum elongatum Cleve 6 Bacteriastrum hyalinum Lauder 7 Bacteriastrum furcatum Shadbolt 8 Bellerochea horologicalis von Stosch 9 Odontella mobiliense (Bailey) Grunow 10 Cerataulina pelagica (Cleve) Hendey 11 Cerataulina dentata Hasle 12 Chaetoceros curvisetus Cleve 13 Chaetoceros lorenzianus Grunow 14 Chaetoceros coarctatus Lauder 15 Chaetoceros compressum Lauder 16 Chaetoceros affinis Lauder 17 Chaetoceros laevis Leuduger-Fortmorel 18 Chaetoceros atlanticus Cleve 19 Chaetoceros aequatorialis Cleve 20 Chaetoceros diversus Cleve 21 Chaetoceros peruvianus Brightwell 22 Chaetoceros decipiens Cleve 23 Chaetoceros spp. 24 Corethron criophilum Castracane 25 Coscinodiscus centralis Ehrenberg 26 Coscinodiscus granii Gough 27 Coscinodiscus radiatus Ehrenberg 28 Coscinodiscus spp. 29 Cylindrotheca closterium (Ehrenberg) Lewin & Reinmann 30 Dactyliosolen fragilissimus (Bergon) Hasle 31 Dytilum sol Grunow 32 Ephemera planamembranacea (Hendey) Paddock 33 Eucampia cornuta (Cleve) Grunow 34 Fragillaria oceanica (Cleve) Hasle 35 Guinardia flaccida (Castracane) H. Peragallo 36 Guinardia striata (Stolterforth) Hasle comb. 37 Hemiaulus hauckii Grunow in van Heurck 38 Hemiaulus membranaceus Cleve 30 39 Hemiaulus spp. 40 Lauderia annulata Cleve 41 Leptocylindrus danicus Cleve 42 Leptocylindrus mediterraneus (H. Peragallo) Hasle 43 Leptocylindrus spp. 44 Lioloma delicatulum (Cupp) Hasle comb. 45 Lithodesmium undulatum Ehrenberg 46 Melosira nummuloides CA. Agardh 47 Navicula spp. 48 Nitzschia bicapitata Cleve 49 Nitzschia longissima (Brebisson, in Kutzing) 50 Pseudo-nitzchia pungens (Grunow ex Cleve) Hasle 51 Pseudo-nitzchia delicatissima (Cleve) Heiden 52 Pseudo-nitzschia spp 53 Pleurosigma spp. 54 Planktoniela sol (Wallich) Schutt 55 Rhizosolenia accuminata H. Peragallo 56 Rhizosolenia imbricata Brightwell 57 Rhizosolenia decipiens Sundstrom 58 Rhizosolenia robusta Norman in Pritchard 59 Rhizosolenia styliformis Brightwell 60 Rhizosolenia bergonii H. Peragallo 61 Rhizosolenia spp 62 Proboscia alata (Brightwell) Sundstrom 63 Pseudosolenia calcar-alvis (Schultze) Sundstrom 64 Skeletonema costatum (Greville) Cleve 65 Stephanophyxis sp. 66 Thalassionema nitzschisoides (Grunow) Mereschkowsky 67 Thalassionema sp. 68 Thalassiosira anguste-lineata (A. Schmidt) G. Fryxell & Hasle 69 Thalassiosira sp. -1 70 Thalassiosira sp -2 71 Thalassiosira punctigera (Castracane) Hasle 72 Thalassiosira decipiens (Grunow) Jorgensen 73 Lioloma elongatum (Grunow) Hasle 74 Thalassiothrix mediteranea (Grunow in van Heurck) 75 Thalassionema javanicum (Grunow in van Heurck) Hasle 76 Rhizosolenia setigera Brightwell Silicoflagellates 77 Dictyocha speculum Ehrenberg 78 Dictyocha fibula Ehrenberg Dinoflagellates 79 Alexandrium cohorticula (Balech) Balech 80 Alexandrium affine (Inoe & Fukuyo) Balech 81 Alexandrium sp. 82 Akashiwo sanguinea (Hirasaka) G. Hansen & Moestrup 83 Ceratium furca (Ehrenberg) Claparede & Lachmann 84 Ceratium fusus (Ehrenberg) Dujardin 85 Ceratium breve (Ostenfeld & Schmidt) 31 86 Ceratium massiliense (Gourret) Jorgensen 87 Ceratium praelongum (Lemmermmann) Kofoid 88 Ceratium tripos (O.F. Muller) Nitzsch 89 Ceratium symetricum Pavillard 90 Dinophysis caudata Saville-Kent 91 Dinophysis argus (Stein) Abe 92 Dinophysis hastata Stein 93 Dinophysis miles Stein 94 Dinophysis brevisulcus (Tai & Skogsberg) 95 Fragilidium cf. mexicanum (Balech) Fragilidium cf. mexicanum (Balech) cysts 96 Gonyaulax polygramma Stein 97 Gonyaulax spinifera (Claparede & Lachmann) Diesing 98 Gymnodinium abbreviatum Kofoid and Swezy 99 Gymnodinium sphaericum 100 Gymnodinium catenatum Graham 101 Gymnodinium sp. 102 Gyrodinium spirale (Bergh) Kofoid and Swezy 103 Gyrodinium sp. 104 Noctiluca scintillans (Macartney) Kofoid and Swezy 105 Oxyphysis oxytoxoides Kofoid 106 Oxytoxum parvum Schiller 107 Oxytoxum variabile Schiller 108 Oxytoxum sp. 109 Ornithocercus splendidus Schutt 110 Prorocentrum cf. gracile Ehrenberg 111 Prorocentrum micans Ehrenberg 112 Protoperidinium depressum (Bailey) Balech 113 Protoperidinium ovatum Pouchet 114 Protoperidinium corniculum (Kofoid and Michener) 115 Protoperidinium conicum (Gran) Balech 116 Protoperidinium pellucidum Bergh 117 Protoperidinium sp. 118 Pyrocystis fusiformis Kofoid 119 Scripsiella trochoidea (Stein) Loeblich III 120 Goniodoma polyedrichum (Pouchet) Jorgensen 121 Triceratium sp 122 Pyrophacus steinii (Shiller) Wall and Dale 123 Athecate Dinos Dinoflagellate cysts (others) 124 Cochlodinium polykrikoides Margalef Cyanobacteria 125 Trichodesmium thiebautii (Gomont ex Gomont) Geitler 32 The contribution of each group to the total phytoplankton composition is presented in Fig. 2.2 (a-d). During the 1996-1997 sampling period, centric diatoms comprised, on average 40% of the total phytoplankton population, but decreased in 1997-1998 due to the increase in pennate diatoms, as well as photosynthetic dinoflagellates. These increments were observed at all stations where there were blooms of pennate diatoms (October and December 1997) and photosynthetic dinoflagellates (November 1997). Further analysis of species contribution to cell densities revealed that at all stations there were two dominant diatom species, Thalassionema nitzschioides and Planktoniella sol. These two species were common within the diatom group throughout the sampling period. However, another diatom, Dactyliosolen fragilissimus also contributed to the dominance of diatoms as it bloomed once in October 1997. A similar pattern was observed for the dinoflagellates. Within this group, Gymnodinium catenatum was found most frequently. Nevertheless, Alexandrium affine was the species that proliferated rapidly in early November 1997 contributing to the first recorded A. affine bloom in Ambon Bay (Wagey et al. 2001). An analysis of the vertical distribution of phytoplankton in the water column revealed that > 60% of the phytoplankton biomass was distributed in the upper 5 m. In general, the vertical distribution of the phytoplankton biomass at all stations showed a similar pattern (Fig. 2.3). The diversity of phytoplankton composition was measured by using the species diversity index (H1) (MacArthur, 1965), for stations 1, 4, 5 and 7. 33 Q) % cP CD co E 0 ro O) o S c «> O —• "Si^ CO ro _ ro ,o % ^ o "Si. E >; O LU c co 0 _ cn .c o V >, cu co o ro ^ ^ . CO o> ~o Q- ^ c ll c ro " •— ^ w m T- o & ii E O Q & I g §-co J o cn C CD t ^ el •s 8 .a o S a. >> £ P. -C -O £= Q. i- O §> -S a £ o ® v ^1 I < ° CD II .1*8 o ' £ K Q or r .y i- § -o UJ Oro .E X CN CN 0 g> LL (%) Jaquinu nao 34 0 _ ro • i o 2 co ra ^- ro oC J0z o 2 - .E o! a >i| o .n -Q i: Q. <- O c o «> M- .Q X ° 0 II .y o 3 0 o E ^ O .E X CN CN ;0 ZJ CD (%) jaquinu |po 0 LU 'ro J= co co E 1 0 ro & § H 2 ui °- c |> co g ~ co ro g-1 55 ro g, co ™ o *X - Sffi !«= £c -EQ C>U! o UJ 3 '-^ c oo cu _ 05 c ^ s ° -cro ^ o £ .22 C II •pro" - •- ^ w — •<- o ro ii. E o ro Q —' cu cu >> CO 2§ co CO CO T~ S 2 §"«> J o g CU D> T- RO *>; s C 0 >«- o .c 2 < « g O a. > .c o -S o. t, (D O .o^c* 111 a cu 2 9> -o JJ «c o « H- L I 0VCK o o o o o * %' o oo CD CM o > (%) jeqwnu \\ao c <4-> cu C/> c co CO — CO UJ to £ Q CU CD O I I- o cc I- o • 1 CO CO g. CO ^ iS CO CO CN "o o _CQD CO £ E 0 3 >< -o~ UJ c co cu T. _ Ol r a: 05 B 0 CJ T (O CU g CcnD o CO co a> T3 CL O cu c 11 z CO c (O Q o CO "CD CL O CD b H CU >.C0 £ CL o cn ~ cx> ft CO to t- (%) jeqiunu \\ao Q. •CaD o CM CO E m •ac ro CD O <0 T3 TC5D a> o 1 neo) jeqwnu o t.l liao) Jeqtunu |jso (S0L x , CO T3 ro>i no oc E < CD cO ro •a c ro c o c ro a. o S, sz Q. (/> C o ro > c o 00 CM iC D 3 cn (sOLX^naoJjsqiunuiiso S0L xt.n ||83)j9qwnu ||eo 38 During 1996-1997, there was a large range in species diversity from 0.3 to 3.5 at all four stations (Fig. 2.4). The species diversity index was relatively low between June to early August of 1996 (except for Stn.7), suggesting the existence of dominant species during that period. From August 1996 to February 1997, the diversity index was relatively constant with a value about 2.0. In contrast to 1996, a high species diversity index was obtained from June to August 1997, followed by a low index from September to December 1997, indicating the re-appearance of dominant species during the phytoplankton bloom. During the end of October, Stations 4, 5 and 7 had a low diversity index between 0.5 -1.5, whereas Station 1 had a relatively high species diversity of around 2.5. In January 1998, species diversity increased to between 2.0 - 3.0. These values were relatively constant up to July 1998. 2.3.2. Physical Characteristics Figure 2.5 shows the annual temperature variation in Ambon Bay from 4 sampling stations. Temperature in Ambon Bay during the 1996-1998 sampling period ranged from 23.5 - 30.5° C. The temperature is highly influenced by the monsoonal pattern. An anomaly of water temperature in Ambon Bay was observed during September - December 1997 when an intrusion of cooler water occurred in inner Ambon Bay and caused the range of the water temperature there to drop markedly to 23 - 24° C. This phenomenon was associated with a water replacement event in the inner Ambon Bay. 39 Figure 2.4. Shannon-Wiener's Diversity Index (H') for phytoplankton species collected in Ambon Bay from 1996 - 1998 40 —r i i rn—I—F rn I l l I I I i r r r r i i i i i—i— MJJ ASONDJ FMAMJ J ASONDJ FMAMJ J 1996 1997 1998 RS DS RS DS RS 23 24 25 26 27 28 29 30 31 Figure 2.5. Monthly temperature variations (1996-1998) in the top 20 m of Ambon Bay. (See Fig. 2.1 for sampling stations) RS = Rainy season DS = Dry Season 41 E Q. CD Q —T—i—fii—i i i—r—r-!—i—i—i—i—i—i—r—n—r—i i i i i MJ J ASONDJ FMAMJ J ASONDJ FMAMJ J 1996 1997 1998 RS DS RS DS RS r^r^ i i i i i i i i 17 19 21 23 25 27 29 31 33 35 Figure 2.6. Monthly salinity variations (1996-1998) in the top 20 m of Ambon Bay. (See Fig. 2.1 for sampling stations). RS = Rainy Season DS = Dry Season 42 In general, the salinity distribution in the water column was relatively uniform with only small fluctuations (Figure 2.6). It ranged from 16 in the surface layer on a rainy day to 35 during a sunny day. The typical salinity range in Ambon Bay is about 31-33. River runoff is the main source of fresh water in Ambon Bay. Small rivers and creeks empty into Ambon Bay, carrying suspended materials, mainly sediment from the surrounding mountains and, as a result, during the rainy season the water in Ambon Bay turns brownish. The monthly precipitation in Ambon Bay is shown in Figure 2.7. From May to August 1996, the amount of rainfall was slightly higher than the average value over 22 years (1976-1998) in the same months, which is about 250 mm (see Fig. 2.8). In the next annual rainy season (May - August, 1997), the rainfall was below the average monthly value. In 1997, July had the highest precipitation compared to the other months. Meanwhile, between August and September 1997, precipitation was minimal. 2.3.3. Chemical Characteristics The concentrations of major nutrients, namely nitrate (N03), phosphate (P04), ammonium (NH4), and silicate (Si04) were measured in Ambon Bay. During May 1996 - July 1997 and August 1997 - July 1998, nitrate and phosphate were higher in the bottom layer (15-20 m, Fig. 2.9 a-b; Fig 2.10 a-b) especially at Stations 1,4 and 5, which are located in the inner Bay. In the outer bay, nitrate showed a similar pattern but had a lower concentration compared to the inner bay. P04 at the surface was very low and, sometimes reaching an undetectable quantity (< 0.1 )j,M) and 43 cu c>o "co c cu cDo) 1_ CU > co szcu -I—' CO 0) CO o c = .2 CO X> CO co a3 CD cNn- —c •<- o E I is-xT CQ TJ c •£ xoi >!2 E 2 < CL £ fo c Q 2 >^ ro <"> -«—' T— a. || cu c a. UJ >.to x: T- c + o ,, to £. ro D5 XI ro ,_ cu 2 < LU 00 csi cu CD i 1 r ooooooooooooooo owoioowoiooinowoio (LUUl) ||B^U!By 46 0.5 2.5 4.5 6.5 8.5 10.5 12.5 14.5 16.5 Figure 2.9b. Nitrate concentrations in the top 20 m of Ambon Bay from August 1997 - July 1998. (See Fig. 2.1 for sampling stations). " i V i i —i n—II 1 1 n i i MJJASONDJ FMAMJ 1996 1997 ri i i i i i i i i i i i i i i i i i i LI HM 0.2 0.6 1.0 1.4 1.8 2.2 2.6 3.0 3.4 3.8 Figure 2.10a. Phosphate concentrations in the top 20 m of Ambon Bay from May 1996 - July 1997. (See Fig. 2.1 for sampling stations). 48 0.2 0.6 1.0 1.4 1.8 2.2 2.6 3.0 3.4 3.8 Figure 2.10b. Phosphate concentrations in the top 20 m of Ambon Bay from August 1997 - July 1998 (See Fig. 2.1 for sampling stations). 49 increased with increasing depth. During the months of May-June 1996, the concentration of P04 was very high (e.g. 2.3 uM) at all stations. At Station 5, a high P04 concentration was present for the same months in 1998 (Fig. 2.10b). Measurements for ammonium and silicate were done during the 1997-1998 sampling period (Fig. 2.11 and 2.12, respectively). From August to November 1997, the ammonium level was extremely low (< 0.5 uM). Ammonium started to increase in December 1997 and, at Station 5, a significant increase to 14.9 uM was observed in the bottom layer. At Station 1, ammonium level was high during December 1997 to February 1998 reaching a concentration up to 17.2 uM. During the dry season, the silicate concentration was around 10 uM. However, a marked increase was observed during rainy season due to the high silicate concentration in the river water, which increased the silicate concentration up to 170 uM. Chlorophyll-a varied from 0.5 - 8.0 mg m"3. Most of Chlorophyll-a values ranged from 0.5 - 1 mg m"3. However, during the 1996-1997 sampling period, Chlorophyll-a reached an average value of 6 mg m"3 between the months of May - August due to phytoplankton blooms. During the bloom period, Station 1 had the highest Chl-a concentration (7.6 mg m"3) compared to the other stations (Fig. 2.13a) During October to December 1997, there were periods of phytoplankton blooms in Ambon Bay. Figure 2.13b depicts the Chl-a distribution at all stations from August 1997 - July 1998. In November 1997, based on Chl-a analysis, Station 4 had the highest Chl-a concentration, up to 8 mg m"3. High Chl-a was also observed at Station 5. In October 1997, prior to the bloom at Stations 4 and 5, Station 7 50 ASO N D J FMAMJJ 1997 1998 I I I I I I I I I I | | JJ! uM O 20 40 60 80 100 120 140 160 Figure 2.12. Silicate concentrations in the top 20 m of Ambon Bay from August 1997 - July 1998. (See Fig. 2.1 for sampling stations). showed an increased in Chl-a concentration reaching 5 mg m"3. In December 1997, a bloom of T. nitzschioides re-occurred at Station 4 with Chl-a concentrations up to 3 mg 2.3.4. Pearson's correlations of cell numbers and environmental variables Pearson's correlation was used to analyze the correlations between cell abundance and different parameters at all stations for the 1997 to 1998 data (Table 2.2). The results for Station 1 showed that cell number was negatively correlated with NH4 and Si(OH)4 to a significant degree (p< 0.05 and p<0.001 for NH4 and Si(OH)4, respectively). Conversely, salinity had a significantly positive correlation with cell number (p<0.01). At Station 4, cell number was significantly positively correlated with Chl-a and salinity (p<0.05) and significantly negatively correlated with NH4, temperature and Si(OH)4 (p<0.01 for NH4 and Si(OH)4; p<0.05 for temperature). Station 5 showed increased cell number with increased salinity and decreased NH4 (p<0.01 and p<0.05 for salinity and NH4, respectively). Station 7, unlike the other 3 stations, is located in the outer bay area and showed the following correlations between different parameters. As observed in the other 3 stations, NH4 level and salinity had a significant correlation with cell number. Increased salinity and decreased NH4 were significantly correlated with higher cell number count (p<0.01 and p<0.005 for salinity and NH4, respectively). The values from stations 1,4,5 and 7 were then averaged to show the overall correlation of cell number with the environmental parameters mentioned above. The result from Table 2.2 demonstrated that NH4 and salinity are highly significantly 55 a? 0.00 8 0.00 2 0.32 9 0.72 5 0.00 2 -0.55 1 -0.61 3 o -0.21 9 -0.08 0 -062 3 T3 ro o CO 0 2? cO> = 0.68 3 0.58 3 0.02 0 0.92 8 0.48 3 0.16 6 0.46 2 -0.09 2 -0.12 4 ^ T- ro -0.15 8 2 *- w £! § ll Trans 8 §*™ c < W 0.58 5 0.00 4 0.46 2 0.03 1 0.55 5 0.00 7 0.54 5 0.00 9 BI E 2 0.55 4 0.00 7 J 23 Sal ,—. ro o. a Q g 0.03 2 0.04 4 0.12 4 0.09 3 — . CD 0.07 5 -0.45 8 -0.43 2 -0.33 8 -0.36 7 -0.38 7 > ' II Temp — C CL — ro E 00 T- CO LO 0 o •<* CO o O - —I 0.00 9 0.04 8 LP d d 0.00 1 0.01 7 -0.54 4 -0.42 6 i -0.65 9 -0.50 4 v-" CN .55 CM I CO W |j 0 .2O c "—o ro 0.85 3 0.89 8 0.06 3 0.78 2 0.35 8 0.29 4 -0.04 2 -0.02 9 -0.20 6 c co o -0.23 4 ro c ro o O TJ CM J2 3 CO 0 CO 0 0.13 8 0.54 0 0.34 7 0.11 4 0.11 7 0.60 4 0.06 6 0.77 1 b ro 0.48 1 > -0.15 8 o 0 o o ° E T3 0 CO 0 co O) rc o c 0.48 8 0.02 1 0.48 6 0.02 2 0.22 8 0.30 7 0.34 9 0.11 1 1_ CD 0.18 0 0.42 3 0 —i > CO Chl-a CL ro 0ro co 1c %c CO LO o r0o co LO O jp >£ o o c CD Sfc ifc o cn CO CD CD CD CD d d c CD v v ro > CO 8 0 c 8 a 8 a CM CM LO IS- 0 * c St n 1 St n 4 combine d cc CO CO Al l station s 56 correlated with cell number (p<0.05). The other parameters that showed significant correlation with cell number are Si(OH)4 (p<0.01). 2.3.5. Transect line (Inner versus Outer Bays) The position of stations 1,3,5 and 7 formed a transect line which runs from the inner Bay (Stations 1 and 3) to the sill (Station 5), and terminates at the outer Bay (Station 7). Measurements of some parameters at these four stations were profiled in Figures 2.14 to 2.19, to evaluate the seasonal changes of the parameters from the inner to the outer bay. Temperature during the rainy season was relatively higher in the upper 5 m (Fig. 2.14) of the Bay. The vertical distribution of temperature tends to be more uniform during the rainy season (about 27-29 °C). Unlike the rainy season, the dry season was relatively more stratified across the transect line (about 26 °C to almost 30 °C) (Fig. 2.14). Chl-a distribution exhibited a distinct pattern at Stations 1 and 3 compared to Stations 5 and 7 (Fig. 2.15). During the rainy season, high Chl-a content (>1.5 mg m"3) was found between 5 to 10 m at Stations 1 and 3. Moving towards the outer Station, Chl-a decreased to 1.2 mg m"3 at Station 5 and 1 mg m"3 at Station 7 (at 5 to 10 m; see Fig. 2.15). In general, Chl-a was lower at the very top layer (0-5 m) compared to the 5- 10 m depth, especially for Stations 1 and 3 in the inner bay. However, high Chl-a was found in the top layer (0-2 m) at Stations 1 and 3.The outer bay Station (Station7) had the lowest Chl-a content and a more uniform distribution with increasing depth. 57 lure 2.14. Vertical distribution of water temperature along the transect line from Station 1 to Station 7 during the rainy and dry seasons of Ambon Bay from August 1997 to July 1998 (see Fig. 2.1 for station locations). Stn 1 Stn 3 Stn 5 Stn 7 mg m"3 0.6 0.8 1 1.2 1.4 1.6 1. Dry Season Stn 1 Stn 3 Stn 5 Stn 7 mg nr- 0.7 0.9 1.1 1.3 1.5 1.7 1.9 2.1 2.3 2.5 2.7 Figure 2.15. Vertical distribution of Chl-a along the transect line from Station 1 to Station 7 during the rainy and dry seasons of Ambon Bay from August 1997 to July 1998 (see Fig. 2.1 for station locations). 59 Chl-a distribution varied seasonally with depth. Unlike the rainy season, all stations during the dry season had the higher Chl-a concentration in the top layer (0-5 m) with the maximum value of 2.1 mg m"3 at Stations 3 and 5. The bottom layers (5 to 20 m) showed a slight depletion (1.3 to 1.1 mg m"3) of Chl-a content from the inner stations to the outer station. During rainy season, the distribution of ammonium with depth was relatively more uniform at all stations compared to the dry season, except for a higher concentration of NH4 in the top layer (about 1-3 m) at Stations 1 and 3. The dry season had a more stratified distribution of NH4 compared to the rainy season (Fig. 2.16). Below the 5 m layer, there was a well-defined patch of high NH4 concentration in the inner bay stations. Especially at Station 3, at about 10 m, NH4 concentration rose to 7.2 uM. In the top layer (about 0-3 m) at Stations 1 and 3, NH4 concentration was also relatively high (~ 5.7 uM). Furthermore, a similarly higher concentration of NH4 was found again at 15 - 20 m from Station 1 to Station 7. The outer bay Station (Stn 7) had a uniform distribution of NH4 from the top layer to 15 m (5.1 uM). A slight increased of NH4to about 5.4 fiMwas observed between15 to 20 m. Concentrations of NO3 during the rainy season were relatively higher at Station 1 compared to the other stations (Fig. 2.17). The level of NO3 decreased from the inner to the outer bay stations, with the lowest level of NO3 (about 0.5 to 1.5 uM) at Station 7. However, NO3 increased with increasing depth at all stations. The highest level of N03 existed in the bottom layer at 15-20 m, especially at Station 1. The top layer had the lowest N03 concentration observed at all stations. 60 Stn 1 Stn 3 Stn 5 Stn 7 Dry Season Stn 1 Stn 3 Stn 5 Stn 7 uM 5 5.8 6.6 7.4 Figure 2.16. Vertical distribution of ammonium along the transect line from Station 1 to Station 7 during the rainy and dry seasons of Ambon Bay from August 1997 to July 1998 (see Fig. 2.1 for station locations). 61 ure 2.17. Vertical distribution of nitrate along the transect line from Station 1 to Station 7 during the rainy and dry seasons of Ambon Bay from August 1997 to July 1998 (see Fig. 2.1 for station locations). A more obvious stratification of N03 occurred during the dry season (Fig. 2.17). N03 concentration clearly increased with depth. Throughout all stations the top layer (0-5 m) was almost depleted of N03. A slight increase in N03 was found at 5-10 m and the highest NO3 value (3.75 uM) was observed at 20 m depth. The difference in N03 level between the inner stations and the outer station was clearly observed between 15-20 m. Phosphate had a similar pattern during the rainy and dry season (Fig. 2.18). The upper layer (0-5 m) had the lowest P04 concentration at all stations. For Stations 1 and 3, the highest P04 level was found at 20 m depth, whereas the outer stations (Stations 5 and 7) had a lower P04 concentration at 20 m. However, a high P04 concentration at the outer station was located at about 10 m depth. At this depth, the P04 level for Station 5 reached a maximum value of 0.55 uJvl during both rainy and dry seasons. The Si(OH)4 distribution during the dry season was similar to the rainy season with a slightly lower concentration range (6-12 uM, Fig. 2.19). From 0-5 m, higher Si(OH)4 concentrations were measured at Stations 1 and 3 compared to Stations 5 and 7. During the rainy season, the lowest Si(OH)4 concentration was also found between 5-10 m as during the dry season. The highest Si(OH)4 level was again observed in the bottom layer (15-20 m), especially at Stations 1 and 3 during both seasons. 63 Stn 1 Stn 3 Stn 5 Stn 7 uM 0.2 0.3 0.4 0.5 0.6 0.7 Figure 2.18. Vertical distribution of phosphate along the transect line from Station 1 to Station 7 during the rainy and dry seasons of Ambon Bay from August 1997 to July 1998 (see Fig. 2.1 for station locations). 64 Stn 1 Stn 3 Stn 5 Stn 7 j | I I | I [ I [ | | | UM I I L_ I J . I J I I I I 0 10 20 30 40 50 Dry Season Stn 1 Stn 3 Stn 5 Stn 7 4 6 8 10 12 14 16 18 Figure 2.19. Vertical distribution of silicate along the transect line from Station 1 to Station 7 during the rainy and dry seasons of Ambon Bay from August 1997 to July 1998 (see Fig. 2.1 for station locations). 65 2.3.6. Canonical Correspondence Analysis The result of Canonical Correspondence Analysis (CCA) is presented in Figure 2.20, based on the data collected during thel997-1998 sampling program. The data from the 1996-1997 sampling program were not included due to the limited numbers of environmental factors that were measured. The CCA graph depicts a distinct effect of environmental factors on the distribution and composition of phytoplankton. The two main factors affecting CM Leptmed + Gymnabb Alexl* High precipitation P04 *Si03 Aste.sp • O I -2 . 5 + 3.5 Figure 2.20. Distribution of phytoplankton species represented by (•) and environmental variables (arrows) from water column of Ambon Bay during 1997-1998 relative to the first two canonical axes with average influence of environmental parameters at the center of the ordination plane. Length of arrows and their angles to the axes give a qualitative indication of their relative importance to the ordination. 66 phytoplankton are the physical properties, mainly temperature, salinity and transparency, and the chemical properties, which are the nutrient concentrations. The physical properties can be interpreted as low precipitation, whereas the chemical properties could be interpreted as high precipitation. The connection between these two regimes represents a "precipitation line" which is influenced by seasonal variations. Based on CCA, the important nutrients revealed in this study are N03, P04 and Si(OH)4, which are closely located in the upper right quadrant of the ordination plane. Interestingly, NH4 had an effect distinct from the other nutrient regimes indicating a strong influence of NH4 on the distribution of phytoplankton. The species distribution on the CCA ordination graph indicated a strong influence of some environmental variables on the distribution of some phytoplankton species. For instance, the diatoms Dactyliosolen fragilissimus, Thalassiosira sp. and Asterionellopsis glacialis showed a close association with concentrations of Si(OH)4 and N03, which are the required nutrients for cell growth. There was a close association between Goniodoma polyedricum and salinity. Species showing a close association with the positive region of the second axis (i.e. the Y-axis) in the ordination plane indicate a strong influence of nutrient level on the distribution of these species. The species responsible for phytoplankton blooms in Ambon Bay such as Thalassionema nitzschioides, Alexandrium affine, Gymnodinium catenatum and Planktoniella sol are located at the center of the ordination plane showing that both physical and chemical factors are major players in their distribution. However, there is a slight tendency for these species to be more influenced by the physical properties of the water than nutrient concentrations. 67 The diatoms Chaetoceroscurvisetusand Pseudo-nitzschiapungensare also located at the center of the graph with the tendency of being more strongly influenced by the level of NH4. 2.3.7. Mangrove effects To determine the possible effect of mangroves on phytoplankton biomass, a preliminary study was conducted to compare phytoplankton abundance from the station located closest to the mangrove (Station 1) and the one located away from mangrove (Station 7). Based on 6 observations, phytoplankton abundance at Station 1 (close to the mangrove) is higher compared to Station 7 (furthest from the mangrove) (Fig. 2.21), except at one sampling date (June 24, 1998). Statistical analysis using Student's t-test revealed that the difference in biomass was statistically significant for 4 out of 6 measurements (p<0.05, see Fig. 2.21). Thus, in general, phytoplankton abundance at the station located closer to a mangrove area tended to be higher than at the station further away. 68 04/29/98 05/14/98 05/28/98 06/10/98 06/24/98 07/24/98 Date of sampling Figure 2.21. Cell numbers at Station 1, representing the area close to mangroves and Station 7, representing the area away from mangroves (Error bars: ± 1SE, n = 3). Points without error bars are those where error range fell within the point. "fr = significantly different (p<0.05) 69 2.4. DISCUSSION 2.4.1. Physico-Chemical Environment In southeast Asia, precipitation is driven by the monsoonal weather pattern (Wyrtki, 1957, 1961). In the eastern part of Indonesia, precipitation has been greatly influenced by the southeast (SE) and northwest (NW) monsoons which determine the hydrological parameters in this region (Zijlstra et al. 1990; Tomascik et al. 1997). The precipitation in Ambon Bay affects the seasonal fluctuations of water temperature and salinity in the inner Ambon Bay. Wenno (1986) concluded that the upwelling and downwelling events associated with the monsoon patterns in the Banda Sea play a major role in determining the physical properties of the water including water circulation in the outer Ambon Bay. During the rainy season, temperature and salinity were lower compared to the dry season. Based on the seasonal patterns in Ambon Bay, the period between August to December constitutes the dry season. However, during October to December 1997, the temperature profile clearly indicated the presence of an intrusion of cooler water into inner Ambon Bay, which likely contributes to the water replacement process in the inner Ambon Bay. According to Anderson and Sapulete (1981, and further elaborated in Wenno and Anderson, 1983), it is estimated to take 4.5 months to replace 50% of the water in the inner Ambon Bay. The most likely timing of this water replacement is during the southeast monsoon period. The physical and chemical characteristics of Ambon Bay water, especially temperature, precipitation and nutrient levels during August - November 1997, revealed the occurrence of a water-replacement process in the inner 70 Ambon Bay. This was supported by the results of temperature measurements which showed a significant decrease of temperature, and also by a lack of precipitation in both inner and outer bays. At the same time, nutrient concentrations of NO3, P04 and NH4, increased indicating a possibility of an active re-suspension mechanism. These variations in temperature and nutrient could be related to the mixing and advection processes causing nutrients to be re-suspended and hence resulting in phytoplankton blooms. Tarigan and Wenno (1991) reported that this phenomenon was linked to the upwelling process in the Banda Sea. They concluded that the upwelling process in the Banda Sea could result in an intrusion of the upwelled water into Ambon Bay. Decreased temperature and increased nutrient level from August-November 1997 coincided with the fact that during that year there was a strong El Nino (97-98). However, whether the El Nino phenomenon has a direct impact on the upwelling process in the Banda Sea is still unknown. The phenomenon observed in the inner Ambon Bay is similar to the upwelling phenomenon in other coastal areas (Beers et al. 1971; Huntsman and Barber, 1977; Lara-lara et al. 1980). I will call this period 'the local upwelling period' in Ambon Bay. The Chl-a concentration in Ambon Bay was high during the rainy season (May to August) in 1996, but was low during the rainy season in 1997. This difference could be related to the unusually low amount of rain in Ambon Bay during the usually rainy months in 1997. However, phytoplankton biomass (Chl-a) increased significantly in October 1997 and remained high until December 1997. This phenomenon is unusual since the October to December period is regarded as part of the dry season in Ambon Bay. The increase in Chl-a during October and December 1997coincides with the 71 significant drop in water temperature as described above. A correlation between high precipitation and high Chl-a was again observed during the early rainy season in 1998, which was indicated by the increase in Chl-a during May - June 1998. Thus, there appears to be a high correlation between Chl-a content and precipitation in Ambon Bay. Precipitation is believed to increase nutrient input into the water. Paerl (1997) and Paerl et al. (1990) reported that atmospheric deposition through rainfall could increase the amount of dissolved inorganic nitrogen (DIN) by more than 70% for N03 and 27% for NH4. Macronutrients in Ambon Bay such as nitrate (N03), ammonium (NH4) and phosphate (P04) are also influenced by precipitation. The concentrations of these nutrients are relatively low (< 0.5 uJvl) and sometimes undetectable at the upper layers, but they tend to increase toward the bottom with concentrations as high as 4 u.M for P04, 14 u,M for NH4, and 18 u.M for N03. The reason for the patchy nutrient distribution could be the result of the utilization of these nutrients in the upper layer of the water column (mainly in the upper 5 m) by phytoplankton. The increased concentration in the deeper water column could be due to several reasons such as: the regeneration of nutrients by microorganisms, mainly NH4 (Dagg et al. 1980), input from the sediments, for N03 and P04 (Howarth, 1988, Cloern, 1996), and the lack of utilization of phytoplankton in the bottom layer. In Ambon Bay, fresh water is mainly contributed by river runoff. The river runoff in Ambon carries high amounts of sediment composed mainly of silt and small particles. Qualitatively, this could be observed during rainy days as the surface water in the inner Ambon Bay turned brownish green due to high amount of sediment 72 emptied by the rivers to the bay. Nutrients from rainfall contain high amounts of nitrate and ammonium, each reaching a value of 9.4 LIM and 30 \xM, respectively (measurement on April 26, 1998). The high nutrient level occurred during the first few days of heavy rainfall, whereas a prolonged rainy period would cause the nutrient level to drop significantly. In Inner Ambon Bay, rain is not only a source of nutrient input, but it also plays an important role in releasing nutrients through rivers or small creeks in the mangrove forest to the area surrounding Station 1. This is supported by the increase in Chl-a in the area around Station 1 during the rainy season. The silicate profile in Ambon Bay could be influenced by nearby anthropogenic activities such as agriculture and housing developments. Such activities lead to erosion and high sedimentation, especially in the inner Ambon Bay. Silicate measurements were done during the 1997 to 1998 sampling period and the concentration of silicate clearly indicated a process of silicate utilization by diatoms. During October to December 1997, there were 2 diatom blooms of the species Dactyliosolen fragilissimus and Thalassionema nitzschioides. These blooms caused a substantial draw-down of silicate during that period. Ammonium seems to be preferred over nitrate by phytoplankton. The vertical distribution of ammonium and Chl-a showed a significant reduction in ammonium during October to November 1997, the same time during which Chl-a reached its highest concentration. The high cell numbers during the same period also supports this finding. 73 2.4.2. Biological Environment In discussing the biological environment, it is necessary to note that phytoplankton abundance from May 1996 to July 1997 is based on results from 5 m depth samples, whereas from August 1997 to July 1998, the phytoplankton abundance was expressed as average values for the entire water column (0-20 m). There appears to be a difference in species composition during the local "upwelling" period (i.e. SE monsoon from May - Sept) compared to the non-upwelling period (NW monsoon from Dec- March) in Ambon Bay. There were three successive blooms starting with the diatom Dyctiliosolen fragilissimus. In October 1997, D. fragilissimus reached a maximum abundance of 1.12 x 106 cells L"1 at Station 7 and 0. 6 x 106 cells L"1 at Station 5. In early November 1997 the dinoflagellate species, Alexandrium affine bloomed at Stations 4 and 5 with a density of up to 4.5 x106 cells L" 1. A detailed account of the A. affine bloom in Ambon Bay can be found in Wagey et al. 2001 (see Appendix 2). In December 1997, the third species to bloom was Thalassionema nitzschioides found in high abundance at Station 4, reaching a density of 0.7x106 cells L"1. During the bloom period, these three species were also found in relatively high abundance at other stations in Ambon Bay. This pattern of succession from diatoms to dinoflagellates has been described by Margalef (1978) as a typical bloom pattern found in phytoplankton ecology. At high nutrient concentrations the diatoms, which are smaller in size compared to dinoflagellates, will take advantage of the high nutrient level since diatoms have a higher uptake rate, hence higher population growth rate. If high nutrient conditions persist, dinoflagellates will usually dominate the phytoplankton composition. This was 74 clearly indicated in the species succession from diatoms to dinoflagellates. Prior to the successive blooms in 1997, at 5 m, the diatom Pseudo-nitzschia pungens dominated at all stations and the group Chaetoceros spp. was the co-dominant species. It is informative to compare species composition in other areas with the composition in Ambon Bay. Historically, Delsman (1939) reported similar species of phytoplankton in the Java Sea to the species in Ambon Bay. Delsman reported that species from the genera Rhizosolenia, Chaetoceros and Coscinodiscus were dominant in the phytoplankton samples from the Java Sea. Studies of phytoplankton composition in the eastern part of Indonesia such as the Arafura and Timor Seas (Hallegraeff and Jeffery, 1984) and the Banda Sea (Adnan, 1990) showed a similarity at the genus level between the two areas. The Banda Sea might have a significant effect on the distribution of phytoplankton species found in Ambon Bay. Van Iperen et al. (1993) reported that Thalassionema nitzschioides is the dominant diatom found in the surface sediments of the Banda Sea. Another diatom species that was also important in the Banda Sea is Planktoniela sol. Thus, it appears that the same phytoplankton species contributed to the phytoplankton composition in both the Banda Sea and Ambon Bay. 7". nitzschioides was the dominant species in Ambon Bay throughout the study period, reaching a density > 2 x 106 cells L"1 in October 1997 during the bloom. Planktoniela sol was also important in the composition of phytoplankton in Ambon Bay since this species was present continuously during 1996-1998 and reached a relatively high density (1.6 x 104 cell L"1) in November 1997. Planktoniella sol is a typical tropical species, and is relatively large in size; therefore this species is an important 75 constituent of standing crop in this region. Thus, it appears that there could be an influence from other surrounding waters, such as the Banda Sea, on the composition, distribution and the bloom of phytoplankton in Ambon Bay. However, this suggestion should be supported by further studies to determine whether the phytoplankton mentioned above were transported by currents from the Banda Sea to Ambon Bay during the SE monsoon season. The Canonical Correspondence Analysis (CCA) graph showed in general the effect of various environmental factors on the distribution of some important phytoplankton species in Ambon Bay. Nutrient concentrations in Ambon Bay showed a correlation with the distribution of diatoms, whereas dinoflagellates, although influenced by the nutrients, are mostly associated with physical factors like temperature, salinity and transparency. The results of the CCA could be interpreted that physical and chemical parameters in Ambon Bay are controlled mostly by the amount of precipitation in this area, as shown in the "precipitation line" on the CCA analysis (Fig. 2.20). The variation of any parameters explained in the "precipitation line" could be affected by the monsoonal pattern, which in fact could lead to processes such as advection and upwelling. This could be seen in the variation of nearly all environmental parameters measured in this study, which resembles the monsoonal pattern in this area. The concentration of Si(OH)4 and P04 showed a negative association with temperature and transparency in the CCA graph. During the rainy season, a decrease in temperature and transparency leads to an increase in Si(OH)4 and P04 in Ambon Bay. This result was supported by the correlation coefficients showing that Si(OH)4 and 76 P04 had a negatively significant correlation with temperature and transparency. This was the observation during 1997-1998 sampling period. The line connecting the variables of temperature and transparency in the lower left quadrant and Si(OH)4, P04 and N03 in the upper right quadrant (see Fig. 2.20) could represent the monsoonal pattern in Ambon Bay. The area of high temperature and transparency represents the dry season (less precipitation), whereas the area of high silicate and phosphate represents the rainy season. The CCA graph also indicated that NH4 is an important parameter that determined phytoplankton ecology in Ambon Bay during the study period. This is supported by the high NH4 concentration in Ambon Bay during 1997-1998. It has also been established that NH4 is often preferentially taken up compared to N03 during phytoplankton blooms (McCarthy et al. 1977; Varela, 1997). The results from the CCA supported the results from Pearson's correlation coefficients, which revealed that ammonium and salinity are two factors that are significantly correlated with cell densities at each station. This finding might indicate the influence of water from outside Ambon Bay, mainly the Banda Sea that entered the bay and through advection and tidal current moved into the inner Ambon Bay. This higher salinity water could also be associated with local upwelling in Ambon Bay that occurred during September - December 1997. It appears that the result of CCA is in agreement with the correlation coefficients calculated using Pearson's correlation. Thus, both Pearson's correlations and CCA provided information that is consistent with the influence of environmental variables on the distribution and composition of phytoplankton species in Ambon Bay. 77 2.4.3. Mangrove effect The contribution of mangrove forests to estuaries is well established (Moran et al. 1991; Harrison etal. 1994; Herrera-Silveira etal. 1996; Bano etal. 1997; Dittmar and Lara, 2001). A detailed explanation of the influence of mangrove ecosystem on phytoplankton communities was provided in Chapter 1 (section 1.1.5). Mangroves serve as a source of nutrients, both macro and micronutrients and provide a rich feeding ground for fish, molluscs, and crustaceans in the surrounding area. Furthermore, mangrove functions as a nursery site for fish and other organism living in, or nearby, the mangrove forest. A comparative study between sampling stations in the inner bay (close to the mangrove area) and the outer bay indicated that a higher biomass of phytoplankton was found in the inner bay stations, compared to the outer bay station. Among the inner bay stations (Stations 1, 4 and 5), Station 1 was located closest to the mangrove site. However, in terms of phytoplankton biomass, Station 4 had a higher biomass compared to Station 1. This may be the result of the circulation of the tidal current in the inner bay, discussed by Wenno (1991). The tides in the inner bay form a loop from Station 5 to Station 1 and decreases in the vicinity of Station 4. This tidal flow could result in a higher accumulation of phytoplankton biomass at Station 4 compared to Stations 1 and 5. The nutrients released from Station 1 would be transported via the tidal current and could be used by phytoplankton in Station 4. Thus, it is possible that mangroves contribute to the higher biomass of the waters nearby this ecosystem. Several reports support the role of mangrove as a "nutrient exporter" to the surrounding ecosystem (Harrison et al. 1994). A study of mangrove influenced sites in the Berry Islands, Bahamas reported that 78 mangrove-derived humic substances were exported offshore at least 1 km from the mangrove site and account for about 10% of the total dissolved organic materials found in the surrounding waters (Moran et al. 1991). Berry Island is influenced by the open ocean currents, which could result in a rapid export of humic substances. Inner Ambon Bay is a relatively closed embayment. Thus nutrient export from the mangrove site to the adjacent water could occur over a relatively longer period. From a study in the Caete mangrove forest in Brazil, Dittmar and Lara (2001) clearly showed that mangroves provide nutrients to the coastal environment. Similar results were reported by Tanaka and Choo (2000) for Malaysian mangrove habitats. In these studies they reported that the concentrations of nitrogen and organic matter in the surrounding seawater were mostly controlled by biological activities such as nutrient uptake, but the phosphate level in the water was affected by physical and chemical processes involving mangrove-sediment and bottom water interactions. Nixon et al. (1984) argued that mangrove ecosystems function more as a "nutrient sink" instead of a "nutrient exporter". Kristensen (1998) found that sediments in mangrove forests served as sink for mostly nitrogen (N03, NO2, and NH4), but later released these into the water through tidal mechanisms. In Ambon Bay, a relatively large mangrove forest is located at Station 1. This area is the head of the inner Ambon Bay and thus could be a nutrient source for phytoplankton in Ambon Bay, especially in the inner bay. Water circulation through tidal exchange could bring nutrients that were released by mangrove forests to the vicinity of Station 4, following which they may be exported to the outer bay. Tanaka and Choo (2000) found that 50% of dissolved inorganic nitrogen (DIN) exported from 79 mangrove forests in Western Malaysia is in the form of ammonium; a similar mechanism is believed to occur in Ambon Bay. A relatively high ammonium concentration is produced through decomposition in the mangrove forest, but if it is not used, ammonium could then accumulate in the sediment. During upwelling in the Banda Sea (during the southeast monsoon), nutrients may be mixed from the sediments into the surface due to "local upwelling" in Ambon Bay and this may lead to a phytoplankton bloom in this area as was observed in this study. 80 CHAPTER 3: ECOPHYSIOLOGICAL ASPECTS OF POTENTIALLY HARMFUL DINOFLAGELLATES FROM AMBON BAY 3.1. INTRODUCTION Recurrent blooms of harmful algae have been major problems throughout Southeast Asia (Azanza and Taylor, 2001; Taylor, 1995; Taylor, 1987b). These blooms have resulted in major economical losses and sometimes loss of human life (Hallegraeff, 1993; Bricelj and Shumway, 1998). In this region, the majority of people rely on fisheries and mariculture products as their daily protein source. With the rapid population increase in this region, there is a need for better management and mitigation to predict and monitor HAB incidents. However, many parts of Southeast Asia still lack an understanding of these natural phenomena, and hence the ability to reduce the incidence of toxic blooms. The first harmful phytoplankton bloom report was the cyanobacterium, Trichodesmium sp. in the Arafura and Banda Seas, in the tropical Pacific Ocean, described by Baas Becking in 1951 when a bloom decayed on exposed coral (Wood, 1965). HAB awareness in Indonesia received more attention in 1984 after several blooms occurred in East Flores, Ujung Pandang, and Sebatik Island (see map in Fig. 3.1) (Adnan, 1984). The incidents include reports of Paralytic Shellfish Poisoning (PSP)-like symptoms in people living in coastal areas after consuming marine products. The dinoflagellate Pyrodinium bahamense var. compressum was believed to be the causative species of the incident (Adnan, 1984). The same species has also been reported to cause HABs in other locations in Indonesia such as Kao Bay, Halmahera (Wiadnyana, 1994) and Riau Province in Sumatera (Praseno and Adnan, 81 1994) and to cause PSP problems in other Southeast Asian waters (MacLean, 1989a and b; Seliger, 1989; Jaafaretal. 1989; Ting and Wong, 1989; Jara, 1993; Cheong, 1993; Azanza and Taylor, 2001). Although there have been few reported HAB incidents in Ambon Bay, the recent 1993 bloom of P. bahamense var. compressum resulted in the death of 3 children and made dozens of people sick. These incidents were believed to be due to consuming shellfish (Wiadnyana, 1995) Wagey et al. (2001) reported a bloom of the non-toxic dinoflagellate Alexandrium affine in Ambon Bay. In order to deal with HABs, it is important to understand the ecological and physiological aspects of harmful phytoplankton species, although Smayda (1997) asserted that there is not much difference with regard to the ecophysiological profiles between harmful and non-harmful species. INDONESIA 300 km Figure 3.1. Map showing the reported incidents of harmful algal blooms in Indonesia. 1= Riau; 2= Ujung Pandang; 3= Sebatik island; 4= Kao Bay; 5 = East Flores 82 This study is the first attempt to understand and gain better knowledge of phytoplankton blooms in Ambon Bay and to examine the possible influence of the surrounding ecosystems on these phytoplankton. To enhance the understanding of the bloom dynamics of phytoplankton species, information at the species level is required. De Madariaga and Joint (1992) stated that knowledge of the physiological state of phytoplankton is a key factor in understanding the dynamics of marine phytoplankton. Several physiological studies of phytoplankton were conducted as early as the 1930's (Wood, 1965). However, almost all these studies were limited to phytoplankton species in the temperate zone. There has been limited research on the physiology of tropical phytoplankton, even though such studies would provide information on the harmful phytoplankton species. Although similar species have been found in the temperate and the tropical regions, it has been reported that genetic variation exists within the same species collected from different or even the same locations (Brand, 1989). Moreover, Brand (1989) stated that one must consider the genetic variation within a single cultured species. Therefore it is worthwhile to study individual species isolated from different geographical locations. My studies in Ambon Bay indicate a possible correlation between proximity to the mangrove ecosystem (Station 1) and phytoplankton abundance (Fig. 2.21, in the previous chapter). The relationship between mangrove soil extracts and dinoflagellate growth rates is the main topic examined in this chapter. This chapter will also discuss the growth rates of dinoflagellates in different culture media. 83 3.2. MATERIALS AND METHODS FOR THE PHYSIOLOGICAL STUDIES A. Isolation and Establishment of Cultures 3.2.1. Species Isolation and Identification Species were isolated from Ambon Bay in 1998, during the field work. Water samples were taken from Station 1 and Station 4 (see Figure 2.1 for stations' location). Surface water was collected from the inner Ambon Bay in a 20 L carboy that had been rinsed and then filled to capacity. The carboy was placed in an icebox covered with a black plastic bag and immediately brought to the laboratory for species isolation. In the laboratory, the content of the carboy was siphoned through a 100 um mesh and reverse filtered to remove the fractions that were smaller than 10 u.m. This method concentrated the 10 - 100 u.m fractions ten fold, which enabled a more efficient scanning process using a dissecting microscope (10-80 X). Dinoflagellate chains or single cells were collected using a fine micro-pipette (Guillard and Keller, 1984), washed at least twice with filtered seawater, and allowed to grow in a 4-well Nunclon® multidish. This process was conducted repetitively to obtain maximum number of isolates. Several obstacles found during the isolation process included: 1. Contamination by other eukaryotic autotrophs and/or cyanobacteria. Guillard and Keller (1984) mentioned that these contaminants are difficult to avoid even when a clean technique was applied. The maintenance medium used for species isolation was enriched with macronutrients (46.67 mg/L N03 and 5.0 mg/l ^-glycerophosphate), metals (2.50 mg/L Fe, 0.54 mg/L Mn, 0.073 mg/L Zn, 0.016 mg/L Co, and 1.73 x10"3 mg/L Se), EDTA (3.64 mg/L) and vitamins (0.2 mg/L 84 3 3 thiamine, 2 x10" mg/L biotin, 4 x 10" mg/L B12). This recipe is the standard medium for cell culture at the NEPCC (Northeast Pacific Culture Collection, University of British Columbia, Canada). A series of several washes using the medium was done to reduce the small coccoid green-colored algae (2-5 jim) and a small dose of Na- penicillin G (up to 500 (j.g/ml) was added to control cyanobacteria. 2. Temperature in the incubator. Due to the instability of the electricity supply in the building in Ambon, there were several power outages that resulted in the crashing of isolated cells. Cells could die due to increased temperature and lack of light during power outages, even only for a few hours. The isolates were brought to Canada (Dept. Earth and Ocean Sciences, UBC) as stock cultures and placed in an incubator, with a 14:10 light: dark cycle. Temperature was maintained at 27 ± 0.5 °C, and light at 150 urnol photon m"2 s"1. Stock cultures were kept in 250 mL Erlenmeyer flasks covered with 50 mL (borosilicate Pyrex®) beakers. Cultures were visually inspected daily for growth condition. Non-axenic clones for all 5 species have been routinely maintained for over 4 years. Scanning Electron Microscopy- Sample preservation was done by fixinglO ml of culture with 2.5 % glutaraldehyde for 30 min. To obtain cells without mucilage, the fixed culture solution was filtered through a 13-mm diameter and 8-u.m pore size Nuclepore membrane filter using a filter apparatus. Cells were then washed (by way of filtration) with fresh culture medium (seawater-based) and distilled water for 15 minute, respectively. 85 The filter apparatus was dissembled and the filters were placed on a petri dish. Further fixation of the cell was done by adding two drops of 2% Os04 on top of each filter. To increase the absorption of Os04 by the cells, filters containing the cells were microwaved for 1 min at approximately 210 Watts. Samples were dehydrated in an ethanol series (30, 50, 70, 90 and 3 x 100% ethanol) for 15 min per concentration, critical point dried with C02 and sputter-coated with gold. The morphology of the cell was examined under a Hitachi S-4700 Scanning Electron Microscope. Calcofluor White for fluorescent light microscopy To stain the theca of Fragilidium cf. mexicanum, a fluorescent stain, Calcofluor White M2R was used in this study. A stock solution of 10 mg ml"1 Calcoflour White M2R was diluted with distilled water to a working solution of 10 u.g ml'1. A drop of cultured-sample of Fragilidium cf. mexicanum was placed on a glass slide and covered with cover slip. To stain the sample, a drop of 10 u.g ml"1 Calcofluor was added along the margin of the cover slip on one end. At the same time, excessive solution was drawn from the other end of the cover slip using kimwipes®. Subsequently, samples were washed using distilled water by applying the same technique as for the Calcofour. After 3-5 minutes, samples were observed under UV light on a fluorescent microscope (excitation: 340 - 400 nm; emission: 400 -440 nm). 86 3.2.2. Culture in various media and light conditions Cultures were grown in three different media without silicate to investigate the growth rates of the five dinoflagellates. The complete recipes of the media used in this experiment are given in Table 3.1. The three media used were HESNW (Harrison Enrichment Solution, Natural Water), ESAW (Enrichment Solution, Artificial Water) and L1. The first two recipes are media developed by Harrison et al. (1980) from a formulation by Provasoli et al. (1957), whereas L1 medium was developed by Guillard and Hargraves (1993). HESNW now contains double the concentration of Fe compared to ESAW, but vitamin concentrations in HESNW and ESAW are the same. As a phosphate source, sodium glycerophosphate was added to HESNW, whereas ESAW and L1 used NaH2P04 as their phosphate source. L1 media contains additional metals such as nickel, vanadium and chromium, which are absent in the other two media. Photon Flux Density (PFD) ranged from 10, 20, 40, 100, 150, 200 and 400 2 1 pmol photon m" s" and was supplied by Vita-Lite® fluorescent tubes. The 0.5 lk values (PFD where growth rate (u,) = 0.5 p.max) were calculated for each species. 87 Table 3.1. Chemical composition of the media used in the experiment. 1J Enrichment Component HESNW L1 ESAW"' (uM) (uM) (uM) 549.09 883 549.09 NaN03 NaHzPCvHpO - 36.3 21.79 - Na2-glycerophosphate 21.79 - 14.86 11.7 14.86 Na2-EDTA.2H?0 3 1 11.7 5.92 x 10"1 FeCI3.6H20 5.92 x 10" - - Fe(NH4)2(S04)2.6H20 5.97 9.0 x 10"1 - MnCI2.4H20 - 2.42 - 2.42 MnS04.4H20 3 1 1 8.0 x 10"" 2.54 x 10" ZnS04.7H20 2.54 x 10' - 5.0 x 10"' CoCI2.6H20 1 1 - 5.69 x 10"1 CoS04.7H20 5.69 x 10" - 1.0 x 10" - CuS04.5H20 1 1 9.0 x 10"" 5.2 x 10" Na2Mo04.2H20 5.2 x 10' 61.46 H3BO3 61.46 - 1.0 x 10"" 1.0 x 10"" 1.0 x 10"^ Na2Se03 - 1.0 x 10"" - NiS04.6H20 - 1.0 x 10" - NaV04 - 1.0 x 10"3 K?Cr04 3 2.97 x 10"1 3.0 x 10"' 2.97 x 10"1 Thiamine HCI • 5 J 1.47 x 10"J 2.1 x 10"a 1.47 x 10" Biotin 35 J 4.09 x 10"J 3.7 x 10"1U 4.09 x 10 D1B122 L_ v Guillard and Hargraves (1993) 2> Harrison etal. (1980) 3.2.3. Cultureware All glassware was soaked in water, scrubbed with a brush (no soap), and rinsed three times in hot tap water. It was then acid-washed in 10% HCI (overnight), soaked in distilled water, air-dried, and autoclaved prior to use. 3.2.4. Culture conditions All isolates were grown in an environmental chamber at 27 ± 0.5 °C, and 14:10 L: D cycle (supplied by Vita-Lite® fluorescent tubes). All media except ESAW 88 were prepared using filtered seawater from Bamfield Marine Station (salinity 31-33 %o) as the medium base. Borosilicate glass test tubes (50 ml) were filled with 40 ml of culture medium and a 1-2 ml inoculum of each species was added to each tube. Growth was monitored by in vivo fluorescence using a Turner Designs Model 10-AU fluorometer. Cultures were swirled gently every day and fluorescence measurements were taken after the cultures acclimated for eight generations (2-3 weeks). All experiments were conducted in triplicate. Growth rate (fj.) was calculated during the log-phase using the formula: u = In^/FcMtrtn), where F-i = fluorescence at time ti Fo = fluorescence at time to 3.2.5. Chlorophyll-a, and particulate carbon and nitrogen Samples for particulate carbon and nitrogen were filtered through pre- combusted Whatman GF/F filters and frozen at -20 °C in a desiccator. After drying at ca. 60 °C for 24 h, samples were prepared for analysis with a Carlo Erba NA-1500 Element Analyzer as described by Verardo et al. (1990). Samples for Chl-a were filtered through a pre-combusted Whatman GF/F filters and stored at -20 °C in a desiccator until analysis. Chlorophyll-a was extracted with 90% acetone, and stored in the dark at 4 °C for 20 - 24 h. Chlorophyll-a concentrations were measured using in vitro fluorometry with a Turner Designs ™ Model 10 fluorometer as outlined by Parsons et al. (1984). 89 B. Mangrove Extracts 3.2.6. Soil Extracts Mangrove soils were taken from a mangrove area in inner Ambon Bay in August 1998. Surface soils were taken from under Sonneratia alba, Rhizophora apiculata, and Bruguiera gymnorhiza trees, located in the mangrove area. In the laboratory, these soils were air-dried and sifted to remove plant and larger materials. Immediately after sieving, the soils were placed in plastic bags and stored frozen. Dried soil samples were transported to Canada, and in the laboratory the soil was first sieved through a 1 cm mesh, then onto a 2-4 mm mesh filter. A volume of tested media that was double the soil volume was mixed in a flask and autoclaved at 121°C for 45 min. The slurry was allowed to sit for 2 days. The supernatant was filtered through a Whatman® No. 1 filter paper. The liquid portion was collected and autoclaved again for 20 min for sterilization. The soil extract was then stored in the refrigerator and in a dark container. As a control, unfertilized topsoil was obtained from the local forest area at the Pacific Spirit Park located at UBC campus and was prepared following the same procedure as used for the mangrove soil. 3.2.7. Extracts from Mangrove Leaves 3.2.7.1. Leaf collection and extraction Litter fall under the mangrove trees was collected for one week in July 1998. The litter trap was a 10 mm nylon mesh cloth stretched over a 1 m x 1 m wooden frame. Two traps were suspended below the canopy from branches of the mangrove at a height above the highest tide level to prevent inundation. One trap was placed 90 under a Sonneratia alba tree, and another under a Rhizophora apiculata tree. These two species comprise the majority of mangrove trees in the area. Litter was sorted into leaves and twigs and washed with distilled water. To extract material from the leaves, 100 grams of chopped leaves were added to 200 ml of solvent, and left overnight at room temperature. The solvent was either distilled water or ethanol/water (ethanol concentrations were 10 and 70%). Extraction was repeated twice, and the combined extracts were filtered through a No.1 Whatman® filter paper to remove large particles of leaves. Solvent solutions were removed using roto-evaporation and the extract was frozen until required. It should be noted however, that although majority of the solvent was removed through evaporation, a very small fraction of the solvent could still remain as residue. 3.2.7.2. Bioassay of leaf extracts Two species were used in this experiment: Gymnodinium catenatum and Pyrodinium bahamense var compressum. The base medium used in this experiment was Enrichment Solution Artificial Water (ESAW). Cells were grown in 50 ml borosilicate glass test tubes with 40 ml of medium. The concentrations of mangrove extract were: 5, 10, 25, and 50 u.g/ml medium, and 0 (ig/ml medium as a control. To test the effect of ethanol on cell growth, a low concentration (0.2%) of ethanol was applied in the medium. Cultures were monitored and given a gentle swirl every day. All experiments were conducted in triplicate. Growth measurements were taken every two or three days, by in vivo fluorescence. 91 3.2.8. Molecular weight fractionation of mangrove soil extracts Soil extraction was conducted by a similar procedure to that discussed in Section 2.1.1. The supernatant of soil extract was separated into two molecular- weight fractions by means of Amicon YM 3 (retention limit 3 kD) membranes in an Amicon ™ pressure filtration cell at a pressure of 450 kPa. Unlike the Sephadex™ chromatography method that could cause loss of some fractions of the sample after filtration (Thurman et al. 1982), this method was chosen for molecular size fractionation (Gjessing and Lee, 1967) to reduce sample loss. Ultrafiltration was accomplished by a Sorvall ™ Model RC5C superspeed chamber within an insulated chamber at 20° C. The filtrate containing compounds < 3 kD and the sediments remaining on the filter membrane (substances > 3 kD) were collected. Two species were used in this experiment; Gymnodinium catenatum and Pyrodinium bahamense var. compressum. 3.2.9. Bioassay to test soil extracts ESAW was used as the base medium in the bioassay experiment. Cells were grown in 50 ml borosilicate glass test tubes with 40 ml medium. To each tube, 5 and 10 ml of mangrove soil extract/L seawater were added. The control received no mangrove soil extract. Growth rate data were subjected to statistical analysis using 1-way ANOVA to test the significance of mean growth rates (u.) with different treatments. 92 3.3. RESULTS A. ISOLATION AND ESTABLISHMENT OF CULTURES 3.3.1. Identity and Sources of Species Isolated Four dinoflagellate species were successfully isolated for further use in cultured experiments. The species were, Alexandrium cohorticula Balech, Gymnodinium catenatum Graham, Fragilidium cf. mexicanum Balech and Prorocentrum cf. gracile Schiitt. A strain of the fifth species used in this study, Pyrodinium bahamense var compressum (Bohm) Steidinger, Tester and Taylor, was obtained from Manila Bay, courtesy of Dr. Rhodora Azanza (University of Philippines, Manila). Table 3.2 summarizes the locations and the isolators of each species. Table 3.2. List of dinoflagellates species used in experiments. Species Source Location Isolator 1. Alexandrium cohorticula Ambon Bay (May, 1998) G. A. Wagey 2. Gymnodinium catenatum Ambon Bay (June, 1998) G.A. Wagey 3. Fragilidium cf. mexicanum Ambon Bay (May, 1998) G.A. Wagey 4. Prorocentrum cf. gracile Ambon Bay (May, 1998) G.A. Wagey 5. Pyrodinium bahamense var compressum Manila Bay (1996) R. Azanza Average monthly abundance of dinoflagellates is presented in Figure 3.2 for the potentially toxic species found in Ambon Bay during the field work (see section 2.2. in the previous chapter for sampling methods). These species include: Alexandrium cohorticula, Gymnodinium catenatum, and Fragilidium cf. mexicanum 93 Alexandrium cohorticula Balech Cells of Alexandrium cohorticula (Figure 3.3) have nearly round shape with diameter ranging from 25 to 40 u,m. This species was always observed in chains, sometimes of up to 16 cells per chain. The average monthly abundance of Alexandrium cohorticula in Ambon Bay was relatively low, less than 1000 cells L"1 (Fig. 3.2A) compared to Gymnodinium catenatum, and Fragilidium cf. mexicanum This species had a relatively high abundance (800 cells L"1) in August 1997. It was absent from September to November 1997, then it occurred in relatively low abundance (100-400 cells L"1) during the remaining months of the sampling period. Thus, no clear seasonal pattern was observed for this species. The identification of this species was made using SEM and calcofluor staining methods, indicated a typical A. cohorticula characteristics. Balech (1995) noted that this species is similar to Alexandrium tamiyavanichi. However, there are several differences between A. cohorticula grown in my culture and A. tamiyavanichi. These include: 1. The location of the comma shaped slot in the APC (Apical Pore Complex). For A. cohorticula, the comma is located slightly closer to the left margin, giving room to the APC pore. In A. tamiyavanichi, the comma is located more towards the center of the APC. 2. The shape of A. cohorticula, is more oval than of A. tamiyavanichi. A. tamiyavanichi has a cone-shaped epitheca compared to A. cohorticula, which has a more regularly rounded epitheca. 94 1.0 A. cohorticula 0.8 o 0.6 x = 0.4 co O 0.2 0.0 40 G. catenatum B 30 ? 20 S 10 1.2 - 6.92 x irr Fragilidium cf. 1.0 - mexicanum to mm O 0.8 - X Li 0.6 - "co O 0.4 - 0.2 - 0.0 - I I Aug Sept Oct Nov Dec Jan Feb Mar Apr May June July 1997 1998 Figure 3.2. Average monthly abundance of potentially toxic dinoflagellates in Ambon Bay during field sampling between August 1997 - July 1998. A = A. cohorticula; B= G. catenatum; C= Fragilidium cf. mexicanum. Figure 3.3. Scanning Electron Micrographs of Alexandrium cohorticula isolated from Ambon Bay (panel A and C). Panel B shows the APC (apical pore complex) and the first apical (1') using light microscopy. Pv = ventral pore. 3. The left sulcal list of A. cohorticula forms a well-developed projection compared to A. tamiyavanichi. Gymnodinium catenatum Graham The cells of Gymnodinium catenatum (Figure 3.4) were always united in long chains, sometimes more than 32 cells per chain. Average cell length and width varies from 35 to 50 and 25 to 45 u.m, respectively. The abundance of G. catenatum appeared to be high in Ambon Bay, reaching almost 40 000 cells L"1during September 1997(Fig. 3.2B). It had a relatively steady abundance, averaging 20 000 cells L"1 from Oct. 1997 to April 1998. In the following months (May-June 1998) there was a marked decrease to a level of around 1000 cells L"1, which coincided with the period of rainy season in Ambon Bay. Fragilidium cf. mexicanum Balech Cells are single (non-chains), oval, longer than wide, and slightly dorso- ventraly flattened. It is the largest of all the studied isolates (Figure 3.5). The cells are relatively large, ranging from 47 - 84 u.m long and 40 - 70 p/n wide, respectively. The theca surface is smooth, with only slight ridges at the plate margins. The cingulum is quite distinct with girdle displacement of 3A to 1. Cingular ridges are low. The sulcus is narrow and sharply excavated with reduced sulcal lists. The ventral pore (Pv) is small but well defined, easier to see with light microscopy than scanning electron microscopy, and located in a highly unusual lower part of 1', not on the marginal side like most Alexandrium species, in which the ventral pore is located on the upper right margin. 97 B C Figure 3.4. Pictures of Gymnodinium catenatum isolated from Ambon Bay. Panel A and B show the pictures taken with Scanning Electron Microscope (SEM). Panel C shows a chain of G. catenatum from natural sample, taken with light microscope. Ab is the acrobase on the apical part running from the sulcus (S). Figure 3.5. Scanning Electron and Light Micrographs of Fragilidium cf. mexicanum isolated from Ambon Bay. A = Ventral view; B = Plate structure drawing (by: F.J.R.Taylor); C = First apical plate and S.a; D= Apical Pore Complex (APC); E = Sulcal plates; F = Apical plate structure with APC. P.v = ventral pore, S.a = anterior plate, 1' = firstapica l plate; s.d.a = right anterior lateral plate, s.m.a. = anterior median plate, s.m.p = posterior median plate, s.s.a. = left anterior lateral plate. Bars = 10 urn, unless stated otherwise. The average monthly abundance of Fragilidium cf. mexicanum (Fig. 3.2C) was relatively low compared to G. catenatum in Ambon Bay, except for the month of November 1997, when a marked increase of cell number was observed, exceeding 600,000 cells L"1. Increased cell numbers also occurred in the beginning of the rainy season from April to June 1998, but decreased in July 1998. Prorocentrum cf. gracile Schutt The fourth species is referred here as Prorocentrum cf. gracile (Figure 3.6). This species is elongated but has a rather round margins on both sides, not as elongated as the type which occurs in the temperate waters. Its length and width vary from 25 to 50 and 16 to 30 u.m, respectively. The surface of the cells is reticulated and the pores are sometimes in rows, although are usually singly around the margin of the valves. According to Schutt (1895), this species is normally slender and elongated, and is twice as long as its width (Steidinger, 1997). This species also resembles Prorocentrum triestinum but is larger and wider. In view of the above limitations, the taxon was tentatively referred to P. cf. gracile. Pyrodinium bahamense var. compressum (Bohm) Steidinger, Tester and Taylor The diameter of P. bahamense var. compressum ranges from 42 to 60 u.m (Figure 3.7). This isolate was obtained from Manila Bay, Philippines, and was reported by Estudillo and Gonzales (1984). Chains of up to 8 cells were seen in the culture flasks. However, normally they are in 2 - 4 cells per chain. In the field more than 8 cells per chain are common. 100 Figure 3.7. Scanning Electron Microscope (SEM) pictures of Pyrodinium bahamense var. compressum. Panel A shows 2 cells from the culture used in this study (isolates provided by Dr. Rhodora Azanza). Panel B shows a clean cell from field sample collected from Port Moresby, Papua New Guinea (taken by Dr F.J.R. Taylor). Scale Bars = 30 um 3.3.2. Culture in various media The growth rate of the dinoflagellates in the test media is shown in Figure 3.8, and the growth curves for each species are presented in Figures 3.9 and 3.10. Growth rates of the five species ranged from 0.16 to 0.48 d"1. P. bahamense had the lowest average growth rate, and Prorocentrum cf. gracile the highest average growth rate. After reaching the log phase in cell growth, the standard error of the growth curves became larger, indicating more variable growth amongst the three replicates. All species were able to grow in artificial medium (ESAW). A. cohorticula, G. catenatum, P. bahamense, and Pr. cf. gracile grew in all media tested, whereas Fragilidium cf. mexicanum did not grow in L1 medium. Pr. cf. gracile showed a preference for L1 medium, while the other species did not exhibit a clear preference among the media tested. A one-way ANOVA, was performed to test for significant differences among the growth rates of each species in the different media. For A. cohorticula and Fragilidium cf. mexicanum, the test demonstrated a significant difference in growth of these two species in HESNW compared to ESAW media (p < 0.05). A. cohorticula and Fragilidium cf. mexicanum grew statistically faster in HESNW compared to in ESAW. Pr. cf. gracile showed a significantly higher growth in L1 compared to the HESNW medium (p < 0.05). The other two species, P. bahamense and Gymnodinium catenatum did not show a significant difference in growth rate among the three media tested. 103 0.60 a b • ESAW 0.50 • L1 a b 0.40 ro I DHESNW CD ro 0.30 ] X X I 0.20 1 o CD 0.10 0.00 Frag Pgr Ac Gc Pbc Species Figure 3.8. Growth rate of five dinoflagellate species grown in three culture media. Frag = Fragilidium cf. mexicanum; Pgr = Prorocentrum cf. gracile; Ac = Alexandrium cohorticula Gc = Gymnodinium catenatum; Pbc = Pyrodinium bahamense var. compressum ESAW = Enrichment Solution Artificial Water; L1 and HESNW = Enriched Solution Natural Waters (see text for explanation on different media used). Error bars = 1 SE, n = 3. a and b indicate significantly different values (p<0.05) 104 18 Fragilidium cf. mexicanum 16 14 ESAW 12 HESNW 10 8 f-ii 6 4 2 I CO -4—' 'c CD 10 12 > cu o 40 c cu Prorocentrum cf. gracile o CO 35 cu -•-ESAW o 30 13 -•-HESNW 25 20 -| 15 10 10 12 14 Days Figure 3.9. Growth of Fragilidium cf. mexicanum and P. cf. grac/Ve in different media. Error bars = ± 1 SE, n = 3. See text for explanation of media (ESAW, L1 and HESNW) used. 105 35 Alexandrium cohorticula 30 -•- ESAW 25 -•-L1 -•- HESNW 20 -! 15 10 5 0 10 12 14 18 Gymnodinium catenatum 16 -| 14 ESAW 12 -•-L1 -•- HESNW 10 8 cz 6 ZJ 4 CU > 2 JP. cu 1_ o0 18 c 0 (/o) 0 40 o Pyrodinium bahamense var 3 35 compressum 30 -•-ESAW 25 -•-HESNW 20 15 10 Figure 3.10. Growth Alexandrium cohorticula, G. catenatum and P. bahamense var compressum in different media. Error bars = ± 1 SE, n = 3. See text for explanation of media (ESAW, L1 and HESNW) used. Thus, certain species demonstrated a significant difference in growth in a particular medium while others did not show a preference towards any specific medium. 3.3.3. Light effects on growth Growth rates of five species were measured under seven different photon flux densities (PFD) (Fig. 3.11). In general, all species had a similar increase in growth rate between 40 to 100 u.mol photon m"2 s"1. These species did not show growth inhibition at high PFD, except for P. gracile which exhibited declining growth rate at the highest PFD of 400 umol photon m"2 s"1. 1 The values of u.max and 0.5lk varied between 0.26 - 0.39 day" and 45 - 67 umol photon m"2 s~1, respectively. The 0.5lk values however, do not vary significantly among species (p<0.005). B. MANGROVE EXTRACTS 3.3.4. Mangrove Soil Extract A comparison of growth rates of dinoflagellates from Ambon Bay with or without the addition of Mangrove Soil Extract (MSE) in different culture media is presented in Figures 3.12 and 3.13. Fragilidium cf. mexicanum did not grow in L1 medium in the absence of MSE. The addition of MSE resulted in the growth of this species. In contrast, the addition of MSE inhibited the growth of Prorocentrum cf. gracile in ESAW. Alexandrium cohorticula grown in ESAW and L1 media showed no significant difference in growth rate in the absence or presence of MSE. However, A. cohorticula showed higher growth in the absence of MSE in HESNW medium. 107 0.5 0.4 0.3 0.2 • Alexandrium sp. 0.1 OA. cohorticula Prorocentrum gracile >> 53 ~T3 04- 9- o 100 200 300 400 500 Photon Flux Density (u.mol photon m"1 s"1) Figure 3.11. The relationship between growth rate and Photon Flux Density (PFD) for five dinoflagellates. In panel A, the species include: Alexandrium sp., Alexandrium cohorticula, and Prorocentrum gracile. In panel B, the species are Pyrodinium bahamense var. compressum and Gymnodinium catenatum. Error bars : ± 1SE n = 3; R2 = Coefficient of determination Fragilidium cf. mexicanum 0.50 • Without-MSE • MSE 0.40 fa 0.30 T T -*C—.D co 0.20 H o 0.10 0.00 ESAW L1 HES Prorocentrum gracile 0.60 ESAW Figure 3.12. Growth rates of F. cf. mexicanum and Prorocentrum gracile cultured in media with and without the addition of Mangrove Soil Extracts (MSE) No growth observed for P. gracile in ESAW + MSE medium. Error bars = + 1 SE ; n = 3 ** = p < 0.005 109 Alexandrium cohorticula ESAW HES Gymnodinium catenatum 0.50 _I_ • Without-MSE 0.40 El MSE '>. 0.30 0.20 3 o c5 0.10 o.oo ESAW L1 HES Pyrodinium bahamense var. compressum 0.30 ESAW L1 HES Figure 3.13. Growth rates Alexandrium cohorticula, Gymnodinium catenatum and Pyrodinium bahamense var. compressum cultured in media with and without the addition of Mangrove Soil Extracts (MSE). Error bars = + 1 SE ; n = 3 * = p<0.05; ** =p<0.005 Gymnodinium catenatum and Pyrodinium bahamense var. compressum generally increased their growth rate upon the addition of MSE. Student's t-test was conducted to determine any significant differences in growth rate for each species in the absence or presence of MSE. Prorocentrum cf. gracile showed a significant enhancement in growth rate when MSE was added to HESNW medium compared to the growth in HESNW without MSE (p<0.005). In contrast to the positive effect of MSE in HESNW, the addition of MSE was inhibitory to the growth of this species in ESAW, whereas in L1 medium, no significant difference was observed in the growth of Pr. cf. gracile in the absence or presence of MSE. Alexandrium cohorticula showed a significantly faster growth in HESNW medium when no MSE was added compared to when MSE was added (p<0.05). In the other two media, Alexandrium cohorticula showed no significant growth preference in the absence or presence of MSE. Pyrodinium bahamense var. compressum grew significantly faster in ESAW with MSE than in ESAW without MSE. In L1 and HESNW media, no difference was observed in the growth of Pyrodinium bahamense var. compressum whether MSE was added or not. Gymnodinium catenatum showed no significant difference in growth rate with or without MSE in all three media tested. In ESAW and HESNW, Fragilidium cf. mexicanum demonstrated no significant difference in growth in the absence or presence of MSE, whereas in L1 medium without MSE, this species did not grow. To distinguish the effect of "mangrove" soil extract more specifically, the addition of "non-mangrove" soil extract, in this case non-fertilized topsoil (collected from UBC) was introduced into L1 medium. For G. catenatum and P. bahamense, 111 their growth rate in L1 medium with the addition of "mangrove" soil extract was compared to their growth rate in "non-mangrove" soil extract, as source of organic materials from a non-mangrove origin (Fig. 3.14). G. catenatum showed a significantly higher growth rate in L1 medium in the presence of "non-mangrove" soil extract (compared to L1 in the presence of "mangrove" soil extract (p<0.05)). In contrast, P. bahamense demonstrated a significantly higher growth rate due to the addition of "mangrove" soil extract into L1 medium. Thus, mangrove soil extract can augment growth of some dinoflagellates species while others prefer soil extract from a "non-mangrove area". Several physiological parameters listed in Table 3.3 were measured for A cohorticula, G. catenatum and P. bahamense. Although there was clearly an interspecific variability among values for each species, the cells grew well in each medium. Cell sizes varied with A. cohorticula being the smallest and P. bahamense the largest. A. cohorticula showed higher values for all parameters when grown in medium without MSE. There were no distinct differences in growth rate for G. catenatum and P. bahamense var. compressum when grown in medium with or without of MSE. 3.3.5. Mangrove Leaf Extracts Based on the increased growth rate of G. catenatum and P. bahamense in media with MSE, further bioassay analyses were conducted to examine the effect of mangrove leaf extract on the growth of these species. 112 0.60 • Non-Mangrove SE 0.50 • Mangrove SE 0.40 ro 0.30 o c5 0.20 0.10 0.00 G. catenatum P. bahamense var. compressum Figure 3.14. Growth rates of G. catenatum and P. bahamense var. compressum when grown in L1 medium with the addition of mangrove soil extract and non-mangrove soil extract. Error bars = ± 1 SE; n = 3. * = p < 0.05 113 co d d d d 8 o CM r- co co -r^ ro co co LU CO : CD CJ> co o d I d d > I LO cn co d LO CM CM CO ^J* = CO CO CM 0 O LU T- O CoO d d o o z cn co CO CM CJ co K CD CO r-- •>-' d LU co co h~ LO CO CO T- d d d d £ ,E 8 d d CM o cn co CD CM d LU LU LU CO CO CO O LU LU £ CO CO - ^ 5 . CB CO c roCO S o -c 3 (1) ro to Q. 3 to 3 JS co .§ E .53 I? o ro •3 S CD i & 8 Mangroves leaves of Sonneratia alba and Rhizophora apiculata, which dominate the Ambon Bay mangrove forest were extracted in ethanol and water. G. catenatum and P. bahamense showed no growth when leaf extract (extracted in ethanol) was added to different media. There was no indication of cells survival within this treatment. This is supported by the result that when pure ethanol was added to the medium (0.2%), there was no cell growth in the presence of ethanol for both species. G. catenatum grew in media with water extracted materials of mangrove leaves, whereas P. bahamense showed growth only in 10 u.g mL"1 of R. apiculata leaf extract (in 40 ml medium). P. bahamense did not grow in other concentrations of R. apiculata and S. alba extracts. G. catenatum grew more rapidly in R. apiculata extract compared to extracts of S. alba. However, in general, the growth rates of G. catenatum were lower than the control (see Table 3.4). Thus, G. catenatum seemed to tolerate the leaf extract better compared to P. bahamense var. compressum. 3.3.6. Molecular Weight Fractionation of Mangrove Soil Extracts Mangrove soils were fractionated into smaller and larger than 3000 molecular weights (MW). The growth rates of P. bahamense and G. catenatum in ESAW medium were different when grown in culture media containing mangrove soil extracts with different molecular weights. The bioassay for P. bahamense var. compressum with the addition of 10 ml L"1 soil extract showed that this species has significantly higher growth in MSE with compounds greater than 3000 MW (p< 0.005; Fig. 3.15A). 115 Table 3.4. Average growth rates (day1) of Pbc (Pyrodinium bahamense var. compressum) and G.c (Gymnodinium catenatum) in different concentrations of mangrove leaf extracts (water solvent). S.a = Sonneratia alba; R.a = Rhizophora apiculata. (n = 3); DNG = did not grow Mangrove leaf extracts (u.g ml") Mangrove Dinoflagellate 0 5 10 25 50 Species species Pbc 0.16 DNG DNG DNG DNG S.a G.c 0.25 0.21 0.17 0.17 0.16 Pbc 0.16 DNG 0.14 DNG DNG R.a G.c 0.25 0.24 0.28 0.17 0.22 116 0.30 ** • oooo 0.25 1 JL • >3000 0.20 0.15 0.10 0.05 0.00 0.60 B • OOOO 0.50 • >3000 0.40 0.30 JL 0.20 ** 0.10 0.00 5 ml L"1 10mlL"1 Control Non-fractionated Figure 3.15. Growth rates Pyrodinium bahamense var. compressum (A) and Gymnodinium catenatum (B) in media with the addition of different concentrations (5 and 10 ml L"1) of soil extracts from two different molecular weights (< 3000 and > 3000). The control represents no addition of mangrove soil extracts; whereas the non-fractionated represents the addition of mangrove soil extracts without fractionation. Error bars indicate 1 SE; n = 3. ** =p<0.005 117 A significant difference in the growth rate of P. bahamense was also observed when comparing the growth without the addition of MSE (control) to growth in media with the addition of MSE with > 3000 MW. No significant difference in growth was demonstrated between the control group (no addition of MSE) and the addition of MSE with < 3000 MW. The effect on cell growth of fractionated MSE and non- fractionated soil extract were also statistically analyzed. For P. bahamense, the addition of 10 ml L"1 MSE with <3000 MW showed a significantly lower growth rate compared to the addition of MSE without fractionation. There was no significant difference in growth rate between MSE with > 3000 MW and non-fractionated MSE. The experiment using 5 ml L"1 of mangrove soil extract to compare the growth rate between MSE with < 3000 MW and MSE with > 3000 MW, could not be analyzed statistically since only one of the three replicate treatments with MSE with < 3000 MW survived. The bioassay performed with G. catenatum showed the opposite result compared to P. bahamense. At 5 ml L"1 of MSE, G. catenatum has a significantly higher growth in MSE with < 3000 MW than in MSE with > 3000 MW (p<0.005; Fig. 3.15B). The same tendency was observed for 10 ml L"1 MSE, however the result was not statistically significant. The growth rate of G. catenatum in 5 and 10 ml L"1 MSE with > 3000 MW was significantly lower than the non-fractionated MSE (p< 0.005), but it was not significantly different compared to the control. Addition of 5 ml L"1 MSE with < 3000 MW did not demonstrate any significant difference in growth rate compared to the control and to the non-fractionated MSE. A significantly lower growth rate of G. catenatum was observed in 10 ml L"1 MSE with < 3000 MW, 118 compared to non-fractionated MSE (p<0.005), but no significant differences observed compared to the control. 3.4. DISCUSSION 3.4.1. Growth and crude toxicity of the Isolates Alexandrium cohorticula This is the first study to report the existence of this species in Ambon Bay. Growth rates (u) of Alexandrium cohorticula varied from 0.20 - 0.35 d"1. Alexandrium cohorticula has been found in the waters of Japan and Thailand (Ogata et al. 1990; Kodama et al. 1988). Although there were no comparable values of growth rates for this species, both Ogata et al. (1990) and Kodama et al. (1988) reported that they have successfully grown A. cohorticula in culture. The optimum growth temperature of A. cohorticula is around 25 °C, indicating the tropical nature of this species. There is very limited information on A. cohorticula, perhaps because this species has not been commonly found in water samples, although it has a wide distribution, including the tropical and sub-tropical regions (Balech, 1995). F.J.R. Taylor (unpubl.) clearly identified it A. cohorticula in Phuket, Andaman Sea in 1973. This species is known to produce a toxin that is similar in nature to the toxin produced by other Alexandrium species (Kodama et al. 1988), and could potentially cause PSP (Hallegraeff, 1993). The crude toxicity result of this study (see Appendix 1) indicated that the strain of A. cohorticula from Ambon Bay is a toxin producer. Although the abundance of A. cohorticula was relatively low during the study period, it could potentially still be harmful to humans. According to Andersen (1996), cell concentrations of P. bahamense var. compressum as low as 200 cells L"1, with a long enough exposure of the shellfish to the dinoflagellates, could cause Paralytic Shellfish Poisoning (PSP) in humans. 120 Gymnodinium catenatum The growth rate (u.) of Gymnodinum catenatum in this study varied from 0.25 - 0.39 d"1. A thorough review of Gymnodinum catenatum in culture has been provided by Blackburn et al. (1989). Using samples from Tasmanian waters (Australia), they worked out the complete life cycle of this species, which has been reported to cause PSP problems in Tasmania (Hallegraeff and Sumner, 1986). Blackburn et al. (1989) reported the u. values for this species was between 0.16-0.28 The common, sometimes abundant presence of G. catenatum in Ambon Bay provides an interesting extension of the distribution of this species, especially in Southeast Asia (Hallegraeff and Fraga, 1998). Fukuyo et al. (1993) reported the presence of G. catenatum in the Philippines, and Godhe et al. (1996) reported it in western Indian coastal waters. To my knowledge this is one of the first reports on the presence of Gymnodinum catenatum in Indonesian waters. Identifying G. catenatum requires good scanning electron microscopy since the features of this species are very similar to Gyrodinium impudicum, a non-toxic dinoflagellate (Fraga et al. 1995), which also differs in size. The result of the MISTalert™ toxicity test of G. catenatum (see Appendix 1) indicated that there was toxin production within the cells grown in culture. Samples have been sent to Marine Biotoxin Program, Northwest Fisheries Science Center, NOAA in Seattle for more detailed information but the results were not available at the time of writing. 121 Fragilidium cf. mexicanum Fragilidium cf. mexicanum was recorded in high abundance in Ambon Bay in November 1997, at the same time when A. affine was blooming (Wagey et al. 2001). Steidinger (1997) noted that the characteristic of the first apical plate of Fragilidium is similar to Alexandrium or Goniodoma and subject to different interpretations. In fact the whole tabulation is indistinguishable from Alexandrium and Goniodoma (F.J.R. Taylor, pers. com. 2002) The isolates from Ambon Bay have narrow anterior part, which differs from the strain used by Balech (1988) to describe F. mexicanum. This is the first study to report the presence of F. cf. mexicanum in Ambon Bay, which highly resembles F. mexicanum found in Mexican coastal waters. Since this species highly resembles Alexandrium species, toxicity test was performed. The results using the MISTalert™ kit (Jellet Technology) showed that Fragilidium cf. mexicanum in culture did not produce toxins. Prorocentrum cf. gracile Prorocentrum cf. gracile was the fourth possibly toxic species isolated from Ambon Bay. I found that this species was relatively easy to culture. It has the highest growth rate of all the species studied (the highest was u, = 0.53 day"1). P. cf. gracile appears to have a worldwide distribution, but because the tropical forms differ in morphology from the temperate ones, it may be a different species. Chiu et al. (1994) indicated that the tropical form of P. cf. gracile contributed up to 40% of the total dinoflagellate abundance in Hong Kong waters. This form was also found off 122 Mexican Pacific coast (Hernandez-Becerril et al. 2000) and in Chile (Alvial and Garcia, 1986). Botes et al. (2000) noted that P. gracile could potentially be harmful to abalone farming in the south coast of South Africa. The more elongate, temperate form (the type) is common in European and western Canadian waters. Pyrodinium bahamense var. compressum A bloom of Pyrodinium bahamense var. compressum has caused fatalities in Ambon Bay (Wiadnyana, 1995), yet very little is known in terms of its ecophysiology. One of the initial purposes of this study was to find Pyrodinium bahamense bloom in Ambon Bay, and to isolate the cells to test a mangrove stimulation hypothesis. However, during the field work, a bloom of Pyrodinium bahamense did not occur. F.J.R.Taylor (pers.com; 2002.) has noted that during field research, the absence of such toxic bloom focusing on particular species is an unusually common phenomenon. Thus, isolates of Pyrodinium bahamense var compressum from Manila Bay (provided by Dr. R. Azanza) were used as a substitute to study the ecophysiology of this species. When first transferred from the original medium (f/2 medium; Azanza, pers. com., 1998) to ESAW and HESNW media in this study, P. bahamense var. compressum from Manila Bay grew very slowly. Aberrant cells were seen and limited movement was observed initially. After two months, the cells seemed to adapt and started to divide more rapidly. From that point onward, the cells have been able to grow steadily in conditions set for this study. 123 The growth rates (u) of P. bahamense var. compressum varied from 0.16 - 0.22 d"1. These values were slightly lower than those reported by Usup (1995), who reported a maximum growth rate of 0.4 division day"1, which is equivalent to p = 0.27 day"1. Different media (higher selenium and vitamin concentrations) and culture conditions used in this experiment might explain the difference in growth rates. In nature, during the peak of a P. bahamense var. compressum bloom in Papua New Guinea, MacLean (1977) reported a maximum division rate of 0.3 day"1. This is equivalent to p. = 0.21 day ~1. The cell size measured in this study ranged from 40 - 55 um in length, and 42 - 60 um in diameter. Steidinger et al. (1980) reported relatively smaller cell size of 33 - 47 um and 37 - 52 um in length and diameter, respectively when describing P. bahamense var. compressum from Brunei and Sabah. 3.4.2. Multiple media and light experiments The results of this study showed that all species tested have the ability to grow long term under the present culture conditions. I have been able to keep four species in culture for 4 years. This is important, especially for certain species such as P. bahamense var compressum, which will allow more studies to be done on this species by using cultures. Other species like G. catenatum and A. cohorticula might also be of interest to elucidate some comparative studies since other researchers have been working on these two species in cultures as well. To my knowledge this study is the first to report the presence of Fragilidium cf. mexicanum and P. cf. gracile in Ambon Bay. The ability to grow species in culture has significant 124 implications, such as gaining further information on new isolated species from remote areas that can then be grown and studied anywhere. Tropical dinoflagellate species are not exposed to the same seasonal variability compared to the temperate species. The only seasonal change is the onset and cessation of the rainy season, although rain may also occur during the dry season. At the same time, the photoperiod is almost constant regardless of the season, and the cells are adapted to high light intensities in nature. In Ambon Bay, high turbidity due to sedimentation usually occurs during rainy days. On a sunny day, even during the rainy season, turbidity can be low, high irradiance persists and the photic zone could reach the bottom layer (about 25 m) of the inner Ambon Bay. The results of growth rate studies and PFD indicated that cells from all five species grew optimally at an irradiance of 150 - 200 umol photons m"2s"1. Prezelin and Sweeney (1978) indicated that, in general, dinoflagellates would decrease their growth rate when grown at light intensities below 50- 100 umol photons m~2 s'\ The physiological changes associated with this low light response include a reduced cell volume, decrease in cell division, increase in pigment synthesis, and decrease in photosynthesis. However, Chan (1978) argued that this generalization should be made with some exceptions. He reported that Amphidinium carterae was effectively saturated at light intensities as low as 50 umol photons m"2 s~\ although it maintained the same growth rate (1 doubling per day) up to a light intensity of 250 umol photons rrr2s-1. Chan (1978), supported by Popovich and Gayoso (1999), also reported that some diatoms could grow in low light at 32-80 umol photons m"2 s"1. The results of 125 the 0.51k also support the relatively higher light requirement for dinoflagellates. The 0.51k values from this study (45 - 67 pmol photons m"2 s"1) demonstrated that dinoflagellates isolated from Ambon Bay have a relatively high 0.5lk, which reflects their preference for high light intensities. Thompson et al. (1991) tested several diatom species isolated from the temperate regions and found the 0.5lk values were from 16 to 28 pmol photons m"2 s"1. The results of light intensity experiments provide new information on the physiology of these species. Usup (1995) reported that P. bahamense var compressum survived low irradiance when grown in modified ES. Although Usup (1995) did not test the growth rate under high light intensities, the results of this study showed that P. bahamense could also sustain high light intensities. This has ecological implications, as most tropical species would be expected to tolerate high irradiance. The optimal growth rate of Gambierdiscus toxicus, a tropical benthic dinoflagellate, occurs when the light intensity reaches 10-11% of full solar irradiance (Bomber et al. 1988). The total irradiance penetrating the ocean surface (at full sunlight) is around 1500 pmol photons m"2s"1. Thus the optimal PFD (Photon Flux Density) for G. toxicus is around 150 umol photons m"2s"1. This value is comparable to the finding in my study. Guillard and Keller (1984) stated that dinoflagellates reach optimum growth at 10% of full sunlight. A similar result was shown by Morton et al. (1992) who reported that the maximal growth occurred at around 4500 u.W cm"' (approximately 150 u/nol photons m"2s"1) for several tropical benthic dinoflagellates associated with Ciguatera poisoning, such as Gambierdiscus toxicus, Prorocentrum 126 lima, P. mexicanum, Amphidinium klebsii, Ostreopsis siamensis and O. heptagona. Nielsen (1992) reported that the optimum growth for the dinoflagellate Gyrodinium aureolum was at irradiances between 170 - 260 umol photons m"2 s"1. In culture, Prezelin (1987) showed a higher adaptation of several dinoflagellates species such as Gonyaulax polyedra, Peridinium cinctum, Ceratium furca and Glenodinium sp. to light intensities as high as 800 umol photons m"2 s"1. The significant difference in light requirement between dinoflagellates and diatoms indicates that diatoms are able to acclimate to low light when they are below the photic zone due to sinking or when the irradiance was not high due to seasonal variability (i.e. during winter or fall). Dinoflagellates, on the other hand, have the advantage of swimming vertically in the water column and hence could benefit from the sunlight when they are at the surface. Furthermore, Prezelin (1987) stated that vertical migration in dinoflagellates is a mechanism related to the photosynthetic process, involving a biological clock and photosynthetic capabilities of the cells. Therefore, this mechanism might vary amongst species, but in general it can be stated that dinoflagellates tolerate higher light intensity than diatoms. MANGROVE EXTRACTS In Ambon Bay, the mangrove forest located in the inner Ambon Bay may influence the surrounding waters by supplying organic matter and nutrients to phytoplankton (see Chapter 1). This hypothesis was investigated by examining the growth rates of some dinoflagellates isolated from Ambon Bay and Manila Bay. 127 The results suggested that the mangrove soil extracts (MSE) was not inhibitory to the growth of dinoflagellates, except for Prorocentrum gracile. Prorocentrum gracile did not grow when MSE was added to an artificial medium (ESAW). Soil extract has been reported to increase cell yield when added to culture media since the classical work by Erd-Schreiber in the late 1890's (Provasoli et al. 1957; McLaughlin, 1979; Gedziorowska and Plinski, 1986; Carlsson et al. 1993; Guillard and Hargraves, 1993). Prakash and Rashid, (1968) and Prakash et al. 1973 attributed the addition of soil extract in culture media to the chelating action of humic substances that are present in the soil. Chelation by soil extract could stimulate metal complexation, which could increase the availability of some trace metals (e.g. iron) to phytoplankton (Ingle and Martin, 1971; Matsunaga et al. 1984), and could also reduce or eliminate the inhibitory effect of toxic metals, such as copper and zinc (Graneli et al. 1986). This may also be true for mangrove sediment. The growth of P. bahamense var. compressum with MSE was statistically higher compared to media without MSE (p<0.05). Thus, MSE could have a stimulant effect on the growth of this species. The results from this study support those of Usup (1995) who showed a preference of P. bahamense var. compressum for soil extract in the medium, although in his experiment, Usup did not use soil extract from mangrove areas nor different culture media. Other studies on the growth rate of P. bahamense have also shown a stimulatory effect upon the addition of soil extracts regardless of the culture media (McLaughlin and Zahl, 1961; Oshima et al. 1985). In the present study, the growth rates of Gymnodinium catenatum in media with MSE were not statistically different from non-MSE. 128 Other species also did not show a positive growth in the presence of MSE in the media. As previously stated, P. gracile did not grow in ESAW in the presence of MSE. Alexandrium cohorticula showed a higher growth rate in the HESNW medium without MSE compared to medium with MSE. This type of response was reported by Braarud and Rossavik (1951) who showed that soil extract did not stimulate growth of some Prorocentrum species such as P. micans, P. adriaticum and P. arcuatum. Explanations of the observed responses include the regulation of trace metal supply via chelation, and possible phytotoxicity of soil extracts against P. gracile and A. cohorticula. The latter possibility was thought to be more logical since soil extracts may contain humic substances, which could form complexes with metals and become toxic to some species, but harmless to others (Nielsen and Ekelund, 1993). Moreover, Harbison (1986) reported that mangrove sediment could function as an entrapment for toxic metals, and if the concentrations of these metals are high, it could inhibit the growth of phytoplankton. This was supported by Anderson and Morrel (1978) who proved that growth of A. tamarensis was inhibited with the presence of copper at the concentration > 10~97 M in the medium. Most dinoflagelates are sensitive to copper and zinc, however, at lower concentrations these metals are required for cell growth. The addition of chelators could sustain the growth of the cells with increased concentrations of copper or zinc. Since the other species did not show growth inhibition in the presence of MSE, there is a possibility that the reduced growth shown by P. gracile and A. cohorticula was perhaps due to overchelation, which decreased the growth of this species due to the unavailability of trace metals as free ionic form. Temple et al. 129 (1992) and later Muggli and Harrison (1996) showed compelling results that some phytoplankton subjected to higher EDTA concentration (as synthetic chelator) experienced declining growth rates, up to 30% for oceanic diatoms. . Unlike A. cohorticula, Fragilidium cf. mexicanum showed an increased tendency for higher growth rates in media with added MSE. In general, studies have shown that several Alexandrium species prefer soil extracts (Prakash and Rashid, 1968; Martin and Martin, 1975; Mahoney etal. 1988; 1995). Further analysis on P. bahamense var compressum and G. catenatum was performed to investigate whether these two species showed a preference of MSE compared to soil extracts from a non-mangrove area (non-MSE). P. bahamense showed a high preference for MSE over no-MSE, suggesting that this species can utilize substances present in MSE. Harbison (1986) indicated that mangrove soils could function as a protective sink (i.e. trap) for trace metals, which are normally released through runoff in most urban areas including inner Ambon Bay that receives the influence from the population who live in Ambon City and its surroundings. Furthermore, Harbison (1984) found a significant amount of trace metals in soil samples from mangrove substrate compared to non-mangrove substrates. This finding suggests that P. bahamense var compressum could utilize some of the trace metals present in mangrove soil. Ingle and Martin (1971) and Yamouchi (1984) showed positive effects of trace metals on phytoplankton growth, when they formed complexes with organic ligands. Trace metals such as iron and manganese are important for photosynthetic processes and nutrient assimilation. However, some trace metals (e.g. copper, cadmium and zinc) are known to be toxic to 130 phytoplankton. Berland et al. (1976) reported that the growth rates of Amphidinium carterae and Prorocentrum minimum were inhibited when copper was added at concentrations between 10 - 50 ug L'1. At lower concentrations, copper, as well as zinc, were required for the cell growth. The requirement for, and sensitivity to, trace metals varies among phytoplankton species (Huntsman and Sunda, 1980). For Gymnodinium sanguineum (now referred to as Akashiwo sanguinea), Doucette and Harrison (1989; 1990) found that this species required a high iron concentration to attain optimum growth. Alexandrium tamarensis showed growth inhibition with increased concentrations of copper (Anderson and Morel, 1978; Morel et al. 1978). Taylor (1987) concluded that dinoflagellates are more sensitive to copper than diatoms. In contrast, G. catenatum prefers the addition of non-MSE in the medium. The reason is perhaps due to the relatively lower amount of trace metals in non-MSE compared to MSE that could be tolerated by this species. Current theory adopts the hypothesis proposed by Provasoli et al. (1957), later developed by Prakash and Rashid (1968) and Prakash et al. (1973), that the organic material (e.g. humic substances) contained in soil extracts act as a natural chelator. In the presence of trace metals as free ions, these chelators will bind with trace metals making the trace metals bioavailable. However, with the addition of MSE, assuming a relatively high trace metal concentration in MSE (Harbison, 1986), it is possible that the chelator bound trace metals could no longer stimulate the growth of G. catenatum. As a result, there was significant reduction in growth rates between the additions of MSE and non-MSE. Sunda and Huntsman (1985) hypothesized that the ratio of trace 131 metals, as free ionic concentration, and the amount of natural chelators present in seawater could affect the phytoplankton species composition in a region. Graneli et al. (1986) stated that higher amounts of chelators over trace metals could shift the dominance of species towards dinoflagellates, whereas increased trace metals could benefit diatoms. Other parts of the mangrove forest could also contribute to its surrounding ecosystem. Litter fall from mangrove trees plays an important role in providing organic material to the mangrove ecosystem. However, in this study it seemed that mangrove leaves did not directly enhance the growth of phytoplankton. From the experiment using ethanol as the extraction solvent, no growth was observed. This could be related to the presence of ethanol in the extracts. Maas et al. (2000) supported the results of this study by indicating that Alexandrium minutum failed to grow when ethanol (0.2%) was added in the media. The presence of even a small amount of ethanol in the media was inhibitory to the growth of dinoflagellates in the present study. When ethanol was not used as the extraction solvent of mangrove leafs, G. catenatum showed a low growth rate and P. bahamense did not grow at all. Leaves from mangrove trees contain high concentrations of tannin and lignin. Both compounds are known to inhibit growth of microorganisms (Doig and Martin, 1974; Neilson et al. 1986; Gonzales-Farias and Mee, 1988; Basak et al. 1998; Ashton et al. 1999) and may have suppressed the growth of dinoflagellates used in this experiment. Thus, mangrove leaf extracts inhibit the growth of some phytoplankton 132 species, whereas soil extracts showed a stimulatory effect on some dinoflagellate species. 3.4.3. Molecular Weight MSE was fractionated based on molecular weight and was used to further examine which fraction of MSE enhances the growth rates of P. bahamense var. compressum and G. catenatum. Harbison (1986) found that mangrove soil with a particle size < 63 um contained 83% organic matter. The compounds that are usually found in MSE are similar to those in marine sediments, which might have high molecular weight compounds (Rashid, 1985; Stevenson, 1982). The results from molecular weight fractionation support the hypothesis that the availability of various DOM (Dissolved Organic Matter) to phytoplankton is a function of the molecular weight of the material. P. bahamense var. compressum prefers the high molecular weight (HMW) over the low molecular weight (LMW) components in MSE, indicating that P. bahamense var. compressum is able to take up the compounds in HMW to enhance their growth. Prakash and Rashid (1968, 1969) and Prakash (1971) reported that Pyrodinium bahamense var. bahamense isolated from Oyster Bay, Jamaica exhibit a maximal increase in growth when the medium was enriched with humic acids containing a lower molecular weight (< 700 MW) fraction. In contrast to their result, this study found that the higher molecular weight fraction is most responsible for the increased growth rate of P. bahamense var. compressum. The explanations for this discrepancy could be several. First, the 133 source of the extracts that was applied in their study, which utilized seawater containing mangrove leaf leachates, compared to mangrove soil. Rashid and King (1969) reported that higher molecular weight compounds are found in soil of marine sediments compared to water. Second, it could be related to the methodology that was used to fractionate the extract. In their method, Prakash and Rashid (1968, 1969) and Prakash (1971), used the evaporation method which could have resulted in the loss of compounds such as humic substances, which constitutes the majority of DOM. This method caused problems in extracting organic fractions in the past (Aiken et al. 1985; Buffle, 1990; Ishiwatari, 1992). With the invention of ultrafiltration using membranes with different molecular weight cutoffs, no sample is lost during the fractionation process (Gjessing and Lee, 1967; Gjessing, 1970; Wershaw and Aiken, 1985). A third possibility, but less likely, is that Pyrodinium bahamense isolated from Jamaica, which is a different variety than that on the Pacific side (Steidinger et al. 1980), might differ genetically in this respect. However, Balech (1985) maintained that the two are basically the same. Some flagellate species, such as Dunaliela tertiolecta, Amphidinum carterae and Prorocentrum micans could directly take up high molecular weight compounds (Klut, 1987). Carlsson and Graneli (1998) reported that in utilizing organic matter, these species might take large molecules directly through a process called pinocytosis, in which the cell membrane is invaginated to form vesicles that will enclose the liquid containing high molecular weight compounds (Kivik and Vesk, 1974; Klut et al. 1987). However, there is no evident yet that P. bahamense var. compressum utilizes this mechanism. 134 One important aspect of this study includes isolation of the compound from mangrove soil that is responsible for phytoplankton growth inhibition or stimulation. It was not the intention of this study to determine the exact compound that is responsible for the increased growth rates on each molecular weight fraction and this should be explored in future studies. 135 CHAPTER 4: GENERAL DISCUSSION This study has enhanced the understanding of the phytoplankton ecophysiology of Ambon Bay, especially the inner Ambon Bay. In general, the result of this study can be illustrated in the schematic diagram in Figure 4.1. Inner Ambon Bay is a dynamic area due to its link to the Banda Sea through water exchange with the outer Ambon Bay, the input of fresh water (mainly from precipitation and river runoff) and also due to the influence it receives from the mangrove ecosystem. Inner Ambon Bay is influenced by hydrographical aspects of the outer Ambon Bay. Factors such as tidal current and seasonal upwelling in Ambon Bay influenced the nutrient concentrations, temperature, salinity, and phytoplankton community structure. The effect of Ambon City also signifies the anthropogenic component released into Ambon Bay, which could result in increased nutrient loading in Ambon Bay. The hydrographic and nutrient variations in Ambon Bay fluctuate seasonally following the monsoonal pattern, demonstrated during the course of this study (1996- 1998). The average water temperatures from 1996 -1997 were 26° C during the rainy season and 29° C during the dry season. At the end of the rainy season to the beginning of dry season in 1997 a water temperature anomaly was observed in Ambon Bay. A marked decrease in water temperature occurred during August 1997 until February 1998. This is the opposite temperature change that takes place during the dry season. This phenomenon was reported previously by Tarigan and Wenno (1991), who suggested the presence of cooler water entering Ambon Bay from the Banda Sea. The intrusion of cooler water into Ambon Bay indicates an 136 O >. O) _o g CO >* sz CL Tc3 ro >^ _CJg) o o CD CD c o c CD >- 13 O >- CO CD CO 137 upwelling in the Banda Sea (llahude, 1990). Coincidently, 1997-1998 was the period associated with one of the strongest El-Nino phenomenon in history. The impact of the water temperature anomaly in Ambon Bay was prominent from September- December 1997 indicating a possible local upwelling in Ambon Bay. Through the local upwelling process, there were indications that nutrients from deeper layers, especially from the outer bay were brought to the surface. This situation is favorable for phytoplankton growth and may trigger phytoplankton blooms. The results of this study showed that in 1996-1997, during which an upwelling was not observed, the environmental variables affecting the phytoplankton community structure, such as nutrient concentrations, temperature, and salinity, reflect the typical seasonal pattern. This is supported by the relatively higher and lower phytoplankton biomass (mg Chl- a m"3) during the rainy and dry seasons, respectively (Figure 2.13a). On the contrary, during the dry season in 1997, an unusually high phytoplankton biomass was seen indicating phytoplankton bloom conditions (see Figure 2.13b). There were a series of phytoplankton blooms detected in Ambon Bay. The first bloom observed during the 1997-1998 period was Dactyliosolen fragilissimus in September 1997, followed by Thalassionema nitzschioides and then Alexandrium affine in November 1997. The results of this study suggest that the advection process could play a major role in bringing the phytoplankton cells to the inner bay. In the inner bay, phytoplankton cells were then distributed by the tidal current. 138 Fresh water input, mainly from river runoff, affects the temperature, salinity and nutrient level in the inner Ambon Bay. Specifically in this study, it is demonstrated that precipitation and river runoff influenced the seasonal variations of phytoplankton composition and abundance (see Fig. 2.2 and Fig.2.5). It is depicted in the Canonical Correspondence Analysis (CCA) that fresh water input determines the variations of major nutrients (e.g. nitrate, phosphate and silicate) as well as temperature and salinity. In Ambon Bay, the SE monsoon is associated with the rainy season, whereas the NW monsoon is associated with the dry season. The CCA result reflects the effect of monsoon season in determining the variations of nutrients, temperature and salinity, which in turn affect the phytoplankton composition and abundance in inner Ambon Bay (Fig. 2.20). Apparently, this effect was seen mostly at the beginning of the rainy season due to the high nutrient input from river runoff and precipitation. A crude toxicity test indicated that some isolated species from Ambon Bay are toxin producer, namely A. cohorticula and G. catenatum. This is the first study to report that these two species exist in Ambon Bay and could potentially be harmful. To assess the influence of mangrove forest on phytoplankton abundance in Ambon Bay, a "transect line" which is composed of sampling stations located in the vicinity of mangrove forest and stations located further away from mangrove area was established. Comparison of phytoplankton biomass (Figure 2.15) among these stations revealed that stations located closer to the mangrove site had higher phytoplankton biomass compared to the station furthest from the mangrove site. This 139 trend suggested that mangrove forest can influence phytoplankton composition and abundance in the inner Bay. A report by Fleming et al. (1990) on the influence of mangrove detritus in an estuarine ecosystem revealed that mangroves as carbon supplier have a "localized" or limited range of influence. They stated that organic materials from mangroves could be used by zooplankton within a radius of 2 km from the mangrove site. Thus, the grazing of zooplankton on phytoplankton could be reduced, leading to increased phytoplankton abundance in areas closer to the mangroves. Another report also suggested the importance of studying the residence time of water in a specific area in relation to the flushing event during the monsoon rainy season on biological cycles such as grazing of zooplankton (Harrison et al. 1997). Thus, depending on the period of the flushing event, export of carbon from the mangrove ecosystem could be directly utilized by zooplankton in the adjacent area. To further determine which component of the mangrove forest is important in affecting phytoplankton growth, mangrove soil extract (MSE) and mangrove litter (leaves) were added to the media of some important species (e.g. the HAB producing species such as, P bahamense var. compressum and Gymnodinium catenatum) in culture. It has been suggested that mangrove litter fall (in the form of mangrove leachates) contributes to phytoplankton yield of the surrounding waters (Prakash, 1975). In this study, I found that non-biodegradable mangrove leaves are inhibitory to the growth of phytoplankton species tested in this study. Mangrove soil extract can be inhibitory or stimulatory for phytoplankton growth, depending on the species of phytoplankton and the type of media. Species such as Pyrodinium 140 bahamense var. compressum that bloomed in Ambon in 1996 showed a tendency for higher growth in the presence of mangrove soil extract. Thus, soil extract from the mangrove forest could increase the growth rate of certain phytoplankton species in the surrounding waters, which combined with other favorable factors such as local upwelling could result in phytoplankton bloom. To analyze whether different fractions of MSE can influence phytoplankton growth, MSE was fractionated into fractions higher and lower than 3000 MW. The bioassay using the two different fractions were done on the species P. bahamense var. compressum and G. catenatum. P. bahamense showed higher growth in media with MSE >3000 MW, whereas G. catenatum prefers MSE<3000 MW. This study suggests that each species has a different growth substance requirement, which could be in the higher or lower fraction of MSE. This experiment by itself does not reveal the compounds or chemical substance in each fraction that is responsible for phytoplankton growth. However, it is a preliminary study to demonstrate that different fractions (based on MW) of MSE can enhance the growth of certain phytoplankton species. Further investigation is required to determine the compounds in the different fraction of MSE that could be responsible for phytoplankton growth. Thus, in Ambon Bay, contribution from the mangrove forest could influence phytoplankton composition and abundance in the inner Bay. However, further research needs to be conducted to explore the connection between the mangrove forest and phytoplankton abundance in the surrounding area. The result of this study may apply only to an enclosed coastal area (embayment area) where mangrove forest contributions are more pronounced due to the limited water circulation. 141 Therefore, studies on the contribution of the mangrove forest to phytoplankton ecology should be elaborated in other areas with different coastal characteristics. The results of this study can be regarded as advancing the existing knowledge of the ecology and physiology of phytoplankton in Ambon Bay. The significance of this study is as follows: 1. This is the first study in Indonesia to include both field ecological and physiological aspects of coastal phytoplankton. It provides a basis for similar ecophysiological phytoplankton studies in other regions of Indonesia or elsewhere. 2. This study has revealed which important environmental factors influence phytoplankton composition and abundance in Ambon Bay. This information should be beneficial in predicting phytoplankton blooms in the region, including harmful species. 3. Knowledge of HAB species in Indonesia is still limited. In this study some HAB species from Ambon Bay were successfully isolated and grown in culture. The culture technique can be applied to other HAB species from other tropical regions, which can provide important information regarding the ecophysiology of new HAB species. Two species, A. cohorticula and G. catenatum isolated from Ambon Bay produced toxin in culture. Thus, they should be considered as potentially harmful to human health in Ambon. 4. During fieldwork in 1997- 1998, a strong El-Nino phenomenon was observed almost worldwide. The El-Nino affect was also seen in Ambon Bay, indicating 142 a possible close link between physical, chemical, and ecological processes in the Banda Sea and in Ambon Bay. 4.1. Future Studies 1. The present study is essentially a preliminary one and much remains to be determined concerning phytoplankton in outer and inner Ambon Bay. For example, the smaller nano- and picoplanktonic fractions require further study. A more detailed, critical taxonomic study, especially of harmful species, is needed to supplement the information provided in this thesis. It would be informative to study the connection between oceanographic processes in the Banda Sea and Ambon Bay, and examine whether they affect the phytoplankton ecology in both areas. These data (e.g. species composition) should be compared to the results from Snellius II expeditions conducted almost 20 years ago. 2. The study on the impact of MSE should be taken further by identifying the compounds contained in the higher and lower molecular weight fraction in relation to phytoplankton growth. Further biochemical analysis, including chemical isolation, should be done on the MSE fraction and tested on the growth of some phytoplankton species in culture. 3. A more thorough toxicity analysis is being done to confirm the toxicity test from this study. Samples from the culture in this study have been sent for further toxicity analysis by the Marine Biotoxin Program, Northwest Fisheries Science Center, NOAA in Seattle, but the results were not available at the 143 time of writing. 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Zijlstra, J.J. & Baars, M.A., 1990. Productivity and fisheries potential of the Banda Sea ecosystem. In: K. Sherman, L.M. Alexander & B.D. Gold (eds). Large marine ecosystems: patterns, processes and yields: AAAS, Washington, D.C. pp: 54-65. 162 Appendix 1. Rapid Toxicity test of cultured species used in this study We tested the toxicity of the cells in culture using the MISTalert™ kit. Methods: The toxicity of four dinoflagellates (Alexandrium cohorticula, Gymnodinium catenatum, Fragilidium cf. mexicanum, and Pyrodinium bahamense var. compressum) isolated from Ambon Bay was examined. One ml of cultured cells was used to perform cell count. Another 5 ml of cultured cells were harvested and centrifuged at 3700 g for 20 min. at 4 °C. The supernatants were aspirated with a vacuum pipette leaving a small and tight phytoplankton pellet ready to be analyzed. Samples were diluted 1 in 5 with phytoplankton buffer provided by the manufacturer, prior to application to the test strip. A total of 100 pl_ of the diluted sample was applied to the MIST alert ™ cassette. The result was read after 20 min. Duplicate samples were applied for each species. The whole procedure was performed less than 30 min. Results: Figure A1.1 shows the result of Alexandrium cohorticula (Ac) and Gymnodinium catenatum (Gc). For Alexandrium cohorticula (Ac), only the C line appeared intensely on the cassette. The T line was not present in Ac. This result is similar to T=0% of C in the look up table (left panel), which clearly indicated the presence of toxins in Ac. For Gymnodinium catenatum (Gc), the C and T line were present on the cassette. The intensity of the T line compared to C line is less clear to 163 determine exactly the category of Gc in the look up table. The result of Gc could be interpreted as T=50% of C or T=75% of C, which could suggest that Gc may or may not contain toxin. Figure A1.2 shows the toxicity result of Fragilidium cf. mexicanum and Pyrodinium bahamense var. compressum. Fragilidium cf. mexicanum contained both C and T lines. The intensity of both lines appears to be very similar, indicating that this species is not toxic (T=100% of C). Pyrodinium bahamense var. compressum also shows the C and T line. The comparison of the intensity of the T and C lines is difficult to determine. Based on the look up chart it could be interpreted as T=100% of C or T=75% of C, which indicated that no toxin is present in Pbc. This method is a simple, rapid and qualitative test to determine the presence or absence of saxitoxins and their analogs (Laycock et al. 2001). The purpose of this method is not to determine the amount of toxin present in the cells, which should be determined by further quantitative and analytical tests. Reference: Laycock, M.V., J.F. Jellet, E.R. Belland, P.C. Bishop, B.L. Theriault, A.L. Russell- Tattrie, M.A. Quilliam, A.D. Cembella, and R.C. Richards. 2001. MIST Alert™: A rapid test for Paralytic Shellfish Poisoning toxins. In: Harmful Algal Blooms 2000. Proc. 9th Int. Conf. Harmful Algal Blooms. Hallegraeff, G.M., S.I Blackburn, C.J. Bolch, and R.J. Lewis (Eds.) IOC-UNESCO, Paris, pp: 254-256. 164 SH eg CD T3 3 e 3 CN CN u CJ -Js H2 cu" Ge l O ° ^> a j Ac l < — ^ — co £ o > m H g (D ca H CO PH O o . "3 o i3 3 " CO S S 3 U E •*H» S- >H C3 a 11 a 2 CO • — •B - CD o M> S a C aj "CO PH 3 co CD • TH +3 O a S X •In O S PH O *" CJ 4-» J3 a C+H a o O o GO co • pH 'fi Ik 3 o CO -5: I o 0 o cj CJ CO CO a CO CO CO a a a a < 'x 'x OJ c 'x "x 'x p o o o DO H H 1 o H E— H o o z ,o *Hf CM tO ii ° fl - -2 5 J4 SI u o ? CD CM O £ O OH CO S—' co CO O s -3 oo CU k < .2 ft, H o 5 c ~ CJ OQ fl CM DH O 99 > 3 CO H =: cu — 5 •M M <0 2 to 3 -M co CQ •5 £ ~ 13 S S CO CqO PccHj jO -C OEH o u y. o - CU M C s co CM O 5 3 o "M C ft «3 ? c 3 & _§ OO OJ o ^ H 8 CN s $ co co CO CO CO C C C C < o *X C i- C "x "x X o c O - H O - Eo - o o 166 Appendix 2. Bloom of Alexandrium affine in Ambon Bay. This part has been published in: Wagey, G.A., F.J.R. Taylor, and P.J. Harrison. 2001. Bloom of the dinoflagellate Alexandrium affine (Inoue and Fukuyo) Balech, in tropical Ambon Bay, Indonesia. In: Harmful Algal Blooms 2000. Proc. 9th Int. Conf. Harmful Algal Blooms. Hallegraeff, G.M., S.I Blackburn, C.J. Bolch, and R.J. Lewis (Eds.) IOC- UNESCO, Paris, pp: 120-123. 167 BLOOM OF THE DINOFLAGELLATE ALEXANDRIUM AFFINE (INOUE AND FUKUYO) BALECH, IN TROPICAL AMBON BAY, INDONESIA Gabriel A. Wagey*, F.J.R. Taylor*, and P.J. Harrison Dept. of Earth and Ocean Sciences, Oceanography. Ur of British Columbia, 1461-6270 University Blvd. Vancouver, BC. V6T 1Z4. CANADA. 128'08°E 128'14°E ABSTRACT A bloom of Alexandrium affine was observed for the first time in Ambon Bay, Indonesia during November 1997. The water became reddish brown and covered a portion of the inner part of Ambon Bay. The average abundance of A. affine was 2 x 10" cells l'1. This outbreak of A. affine was unique since this is the first occurrence of an Alexandrium bloom reported in Ambon Bay. The influence of river runoff and the amount of rainfall prior to the bloom are suspected to be important factors in maintaining the nutrient level, especially nitrate and ammonium, in inner Ambon Bay. INTRODUCTION Figure 1. Map of Ambon Bay and sampling stations. Insets: Ambon Island and map of Indonesia The genus Alexandrium has more than 30 species bloom of Alexandrium affine from October - November and many can produce toxins [1]. In Southeast Asia, the 1997. The results presented here are part of the Ambon species A. tamiyavanichi is considered the only known Bay phytoplankton study conducted from August 1996 - Alexandrium species to produce toxin [1]. Alexandrium July 1998. This study is critical in understanding the affine has been known to inhabit a wide range of dynamics of HABs, especially in a small tropical geographical areas and has been found in temperate embayment like inner Ambon Bay. waters, as well as in the tropics [1]. In 1974, 1975 and 1977 Fukuyo et al. [2, 3] reported a bloom of METHODS Protogonyaulax affinis in several parts of Japan. However, there were no reports on the toxicity of this As part of a continuous two week sampling species at the time of those blooms. schedule from August 1996 - August 1998, water samples The first reported case of a HAB in Ambon Bay were collected at stations in the inner and outer parts of was in 1994, caused by a paralytic shellfish poisoning Ambon Bay. When the bloom of A. affine was reported, species, Pyrodinium bahamense var. compressum. sampling was conducted every day for the first 5 days and During that outbreak, three children died and several then every 2 days until November 18, 1997. Three stations more people had to be hospitalised after consuming were chosen to represent the inner Ambon Bay (Figure 1), shellfish from the bay [4]. since the bloom only occurred in this area. Ambon Bay is a small embayment in Ambon Water samples were collected at three different Island in the eastern part of Indonesia (Fig. 1). Ambon depths (0, 5 and 20 m) with a 2.5 L hand-held Van Dorn Bay is divided into two parts, the inner and outer bays, bottle. Temperature and salinity were determined by which are separated by a shallow sill at the narrow means of a thermometer and a Beckman salinometer. channel (300 m wide and 15 m deep) between the two Chlorophyll-a was measured spectrophotometrically [8], bays. with a phaeopigment correction [9]. Nutrient samples Several studies have reported nutrient were frozen immediately and concentrations of nitrate, concentrations in Ambon Bay, although the reports are phosphate, ammonium, and silicic acid were measured in lacking in continuity. Sutomo [5] measured Chl-a in the laboratory following the IOC Methods [10, 11]. A few Ambon Bay and hypothesized that the increase in N0 3 drops of Lugol's iodine solution were used as a fixative and in this area was highly correlated with the amount of subsamples of phytoplankton were settled in a 50 ml rainfall during the rainy season (April - September). chamber and counted on an inverted microscope. For more Other studies [6, 7] reported that in 1987 the precise identification of A. affine, scanning electron concentration of N0 and P0 in two rivers emptying 3 4 microscopy was used. In this study we consider only the into the inner Ambon Bay ranged from 14.8 - 22.7 uM dominant species that were found in the water samples. and 16.4 - 21.0 \sM for nitrate and phosphate, Upon receiving a bloom report from the local respectively. Previously, there have been no reports on people, a water sample was taken from the location near the NH and Si0 concentrations for Ambon Bay. 4 4 "LIPI" Marine Station (see Figure 1) on November 5, 1997. In this paper we will discuss the variations of Subsequent sampling was conducted on November 7, 8, 10, some environmental parameters associated with the 14 and 18 to monitor the bloom as it progressed in Ambon 168 Bay. Earlier sampling data from October 15, 1997 were stand. The highest concentration of A. affine was 4.4 x 106 used to compare to the conditions before the bloom. cells L"' (0 and 5 m), recorded at this station on November 8, 1997. The presence of another Alexandrium was also high at this station reaching more than 1 million cells L"'. This potentially new species has several characteristics such as; non chain-forming, produces temporary cysts and belongs to the Gessnerium group. Nutrients at this station were relatively high in N03 and P04 in the bottom layer. Silicic acid was the highest at this station compared to the other stations. Station 4 is located on the westside of the inner Ambon Bay. This area is relatively calm during the dry season (south-west monsoon). The bloom of A. affine was spotted at Stn. 4 due to the circulation pattern in the inner bay being counter-clockwise, resulting in the accumulation of cells in the western part of the inner Bay. The highest abundance of A. affine (0 and 5 m) at this 6 1 Figure 2. Micrograph of Alexandrium affine from station was found on Nov. 8, 1997 (4.35 xlO cells L" ). Ambon Bay However, there was an increased abundance on Nov. 7, 1997. The profiles of Chl-a and NO3 at this station have a similar pattern to that at Station 1, which is in agreement RESULTS with the cell abundance pattern. The concentrations of th NH4 and Si0 were similar at all depths. On November 5 1997, a very noticeable 4 Station 5, is at the mouth of the inner bay and connects the phytoplankton bloom occurred in Ambon Bay, inner to the outer bay, and thus has the strongest influence Indonesia. The water turned reddish brown, which from currents. The highest abundance of A. affine was on attracted the attention of the local people since red tides Nov. 7, 1997. However, this station has the lowest number have not been common in this area. This water of A. affine compared to Station 1 and 4. NO3 and P04 discoloration covered the inner portion of Ambon Bay, were relatively similar at all depths indicating fairly strong which is about 500 m wide and 2 km long. The bloom mixing in this area. continued for at least two weeks (until mid November 1997) before it disappeared. From surface visual observations, the outbreak was patchy and moved DISCUSSION counter-clockwise with the current. An intensive examination of this event, including microscopic The Alexandrium affine bloom in Ambon Bay occurred in analysis determined that the cause of the water a relatively short period (1-2 weeks). The highest cell discoloration was a dinoflagellate Alexandrium affine abundance was found in the 5 m depth layer, reaching (Inoue and Fukuyo) Balech (Figure 2). almost 5 millions cells L . The bloom appeared to start in the area close to station 5 and moved counter-clockwise Figure 3 shows the cell abundance (dominant with the current to station 1 and accumulated at station 4. species) and Chl-a concentrations from each station. Other species also found in relatively high abundance The majority of the phytoplankton cells were found at 5 during the bloom were Thalassionema nitzschioides, m. At station 5, on Nov. 7, cells were also found at the Planktoniela sol, Trichodesmium sp., and Alexandrium sp. surface layer. This probably coincides with the The high phytoplankton abundance in Ambon Bay is rather initiation process of the bloom. typical of a coastal system, where it has a high uptake of In general, the characteristics of the nutrient NO3 derived from run-off and mixing processes. It is also profile (Figure 4) are as follows; a clear reduction of obvious that as the phytoplankton abundance started to nitrate during the developmental stage of the bloom, decrease, the concentration of ammonium (NH ) started to followed by the increase of ammonium a few days after 4 increase. Part of the ammonium input is believed to be due the peak in A. affine. There was a sharp decline of to grazing activity by zooplankton. The influence of silicic acid on Nov. 7, indicating possible water domestic sewage from the population surrounding the exchange between the outer and inner bay. Phosphate inner Ambon Bay is also critical in determining the level of concentrations during the bloom were less variable, but ammonium in the bay. showed a slight decrease during the bloom. Phosphate concentrations ranged from 0.2 |iM at the surface to 0.9 The co-existence of some large diatoms (e.g. U.M at the bottom. Planktoniela sol) during the A. affine bloom is believed to There were no reports of PSP poisoning of contribute to the draw down of silicic acid on Nov. 7. This humans after the outbreak of A. affine in Ambon and its condition, however, changes rapidly when water from the surrounding areas. outer bay, through tidal circulation, replaces a portion of the water in the inner bay, resulting in an increased of silicic acid concentration in the inner bay. The water from Inter-station variation the outer bay is highly influenced by the open ocean and Station 1 is located at the head of the inner contains a relatively high concentration of silicic acid, up Ambon Bay, and marked by a fairly large mangrove to 60 uM (Wagey, unpublished data), 169 The influence of river run-off and rainfall are Acknowledgment: The authors wish to acknowledge the suspected to be important since both nitrate and supports by "Lm" Marine Station, especially Dr. Ngurah ammonium concentrations from rainfall were as high Wiadnyana and his coleagues for the assistance during as 50 u.M. The connection between the inner bay samplings in Ambon. GAW was supported by the Eastern and the open ocean through the outer bay and the Indonesia University Development Project (ERTDP), and the influence of several small rivers that discharge large Pattimura University, Ambon. amounts of domestic sewage and land run-off during the rainy season, may result in an estuarine r12 ecosystem which is typical of Ambon Bay. 10 When an outbreak of toxic phytoplankton occurs, the consumption of shellfish and some CO pelagic fish such as clupeoids, becomes dangerous 8 E to humans since these organisms might accumulate toxic compounds from the algae. Therefore, for 6 management purpose, rapid action to disseminate the - a (m g information about red tide blooms is critical. The - 4 major constraint in Indonesia for rapid dissemination Ch i of news of a red tide outbreak is basically due to the 2 vast geographical area and the limited awareness of the people of the red tide phenomenon. The red tide - 0 in Ambon Bay occurred in a relatively small area. In less than a day, the local people were informed about the outbreak and some precautionary actions 12 5 i r were taken to avoid shellfish consumption until further investigations were made. 10 _i oi CO REFERENCES 8 E ° 3 1. E. Balech. The genus Alexandtium Halim 6 (Dinoflagellata). (Sherkin Island, Ireland). 151 pp. - a (m g (1995) 4 2. Y. Fukuyo, K. Yoshida, and H. Inoue. In: Toxic Ch i Dinoflagellates, D.M. Anderson, A.W. White, and D.G. 2 Baden (Eds). (Elsevier, New York), pp 27-32 (1985) 3. Y. Fukuyo, H. Takano, M. Chihara, and K. Matsuoka. 0 (Eds). Red Tide Organisms in Japan. An Illustrated Taxonomic Guide. (Uchida Rukakuho, Tokyo). 430 pp. (1990) r12 4. N.N. Wiadnyana, T. Sidabutar, K, Matsuoka, T. Ochi, Station 5 10 M. Kodama and Y. Fukuyo. International Conference « 4 on Toxic Phytoplankton. July 12-16, 1995. Sendai, CO Japan. (1997) 8 E 5. Sutomo, Proceeding of Ambon Bay Symposium (in 6 Indonesian), pp: 24-33 (1987) • a (m g 6. Z. Tarigan. LIPI- Reports 1987 (in Indonesian), pp: 4 102-105.(1987) Chi - 7. Edward and J.M Manik. LIPI- Reports 1987 (in 2 Indonesian), pp: 112-116 (1987) 8. J.D.H. Strickland, and T.R. Parsons, A practical - 0 11/8 11/10 11/14 11/18 handbook of seawater analysis. Fisheries Research 10/15 Sampling dates Board of Canad. Ottawa. 310 pp. (1972) 9. CS. Lorenzen, Limnol Oceanogr. 12:343-346.(1967) • Thalassionema nitzschioides H Alexandrium affine 10. IOC-UNESCO. Nutrient Analysis in Tropical Marine D Alexandrium sp Waters. Manuals and Guides 28. UNESCO. (1993) Figure 3. Cell abundance of dominant species (bar graph) 11. T.R. Parsons, Y. Maita, and CM. Lalli. A Manual of during the bloom of A. affine. The line graph represents the Chemical and Biological Methods for Seawater Chlorophyll-a concentrations at 0 m (—O—), 5 m (—•— Analysis. (Pergamon Press, Oxford) (1984) and 20 m (—•—) at stations 1,4, and 5. 170 Stn 5 -• Om -• 5 m -± 20 m 60 n 40 O oo 20 3? 1.0 o CL 10/15 11/7 11/8 11/10 11/14 11/18 10/15 11/7 11/8 11/10 11/14 11/1 £ Figure 4. Nitrate, ammonium, silicic acid and phosphate concentrations at station 1 and station 5 in the Inner Ambon Bay, measured during the bloom of A. affine between Oct. 15 - Nov. 18, 1997. Ammonium concentrations for station 1 were not determined. Results for station 4 were not shown since they had similar values to Station 5. 171 Appendix 3. Field Sampling Programs Table A3.1. Sampling dates and environmental parameters measured during the field program in Ambon Bay, Indonesia, from May 3 1996 to July 24 1998. Year Date Stations Phytoplankton Nutrients Chl-a & T&S Sub of Samplings 5m only 0, 5, 20 m N03 P04 NH4 Si(OH)4 phaeopfg stations 1996 3-May-96 Stn 1,4,5,7 15-May-96 Stn 1,4,5,7 4-June-96 Stn 1,4,5,7 17-June-96 Stn 1,4,5,7 9-July-96 Stn 1,4,5,7 23-July-96 Stn 1,4,5,7 1O-Aug-96 Stn 1,4,5,7 20-Aug-96 Stn 1,4,5,7 10-Sept-96 Stn 1,4,5,7 24-Sept-96 Stn 1,4,5,7 8-Oct-96 Stn 1,4,5,7 21-Oct-96 Stn 1,4,5,7 3-Nov-96 Stn 1,4,5,7 29-Nov-96 Stn 1,4,5,7 19-Dec-96 Stn 1,4,5,7 31-Dec-96 Stn 1,4,5,7 1997 17-Jan-97 Stn 1,4,5,7 3-Feb-97 Stn 1,4,5,7 30-Mar-97 Stn 1,4,5,7 7-June-97 Stn 1,4,5,7 9-July-97 Stn 1,4,5,7 9-Aug-97 Stn 1,4,5,7,3 25-Aug-97 Stn 1,4,5,7,3 9-Sept-97 Stn 1,4,5,7,3 23-Sept-97 Stn 1,4,5,7,3 15-Oct-97 Stn 1,4,5,7,3 7-Nov-97 Stn 1,4,5,7,3 25-Nov-97 Stn 1,4,5,7,3 2-Dec-97 Stn 1,4,5,7,3 10-Dec-97 Stn 1,4,5,7,3 22-Dec-97 Stn 1,4,5,7,3 1998 22-Jan-98 Stn 1,4,5,7,3 5-Feb-98 Stn 1,4,5,7,3 19-Feb-98 Stn 1,4,5,7,3 5-Mar-98 Stn 1,4,5,7,3 19-Mar-98 Stn 1,4,5,7,3 6-April-98 Stn 1,4,5,7,3 29-April-98 Stn 1,4,5,7,3 14-May-98 Stn 1,4,5,7,3 28-May-98 Stn 1,4,5,7,3 10-June-98 Stn 1,4,5,7,3 24-June-98 Stn 1,4,5,7,3 8-July-98 Stn 1,4,5,7,3 24-July-98 Stn 1,4,5,7,3 172 Appendix 4. Raw Data Set Table A4.1. Temperature and Salinity Raw data set StnJ Stn 4 Stn 5 Sin 7 Stn 3 Date Temp. Salinity Temp. Salinity Temp. Salinity Temp. Salinity Temp. Salinity of Sampling Depth (m) °C 3 May 96 0 3 5 28.20 30.12 28.10 30.85 27.60 31.00 27.50 30.40 10 15 20 27.50 30.85 27.30 31.07 27.10 30.90 27.20 31.10 15May96 0 29.10 29.94 30.40 29.40 30.70 29.07 30.20 29.20 3 29.10 30.82 29.90 29.73 30.30 30.21 29.40 30.13 5 28.90 30.63 29.60 29.98 30.20 30.23 29.10 30.19 10 28.50 30.77 29.30 30.76 30.50 28.93 29.90 30.40 15 28.60 30.85 29.30 30.85 30.60 29.57 29.30 30.53 20 28.40 30.86 29.10 30.95 28.50 30.56 28.80 30.58 4 June 96 0 27.50 29.61 27.10 24.41 27.30 26.61 27.00 28.70 3 28.10 30.73 27.40 29.00 28.10 30.58 28.00 31.25 5 27.90 30.91 27.70 30.41 27.80 30.92 27.50 31.35 10 27.90 31.12 28.10 30.92 28.10 31.06 27.80 31.38 15 28.00 31.94 28.10 31.02 28.00 31.19 28.00 31.41 20 27.90 31.95 27.90 31.17 27.80 31.23 27.90 31.43 17 June 96 0 27.90 30.90 28.00 28.95 27.50 21.98 26.70 19.77 3 28.50 30.93 27.80 30.39 27.50 30.57 27.10 31.32 5 28.30 31.05 23.00 31.07 27.50 30.93 26.90 30.94 10 27.40 31.18 27.80 31.91 27.40 31.82 26.90 31.15 15 27.50 31.51 27.40 31.92 27.40 31.86 26.80 31.42 20 27.80 31.60 27.40 31.98 27.40 31.97 26.80 31.50 9 July 96 0 26.00 13.68 26.30 14.13 26.30 11.46 26.20 15.17 3 26.30 27.94 26.90 30.51 25.90 30.07 26.20 31.32 5 26.50 31.30 26.50 31.33 26.20 31.37 26.30 31.36 10 26.50 31.45 26.70 31.37 26.10 31.45 26.50 31.43 15 26.50 31.48 26.70 31.46 26.40 31.49 26.40 31.48 20 26.30 33.49 26.40 31.49 26.30 31.54 26.10 31.61 23July96 0 28.10 27.06 28.40 29.02 27.60 26.67 26.40 27.10 3 28.00 29.02 28.40 30.00 27.00 31.23 26.10 31.41 5 28.00 31.04 27.00 31.07 26.70 31.44 26.30 31.40 10 27.00 31.07 27.50 31.10 27.00 31.62 26.20 31.50 15 27.00 31.12 27.30 31.23 27.00 31.68 25.90 31.52 20 27.00 31.20 27.00 31.30 26.70 31.88 26.10 31.56 10 Aug 96 0 26.40 21.65 27.00 26.14 27.20 32.81 29.50 33.60 3 27.10 33.28 27.30 31.92 27.20 32.53 27.00 33.59 5 26.90 33.86 27.00 33.61 26.90 33.74 29.00 34.64 10 26.60 34.15 26.80 34.55 26.70 33.99 26.90 35.17 15 26.70 34.19 26.70 34.72 26.80 34.08 26.90 35.31 20 26.40 34.61 26.60 34.77 26.60 34.67 28.50 35.90 20 Aug 96 0 28.00 31.10 27.40 31.02 27.50 27.37 26.30 32.52 3 27.20 31.64 27.40 31.38 26.90 32.63 26.20 33.68 5 27.20 32.07 27.00 32.09 26.80 34.05 26.50 33.57 10 26.60 33.73 26.70 33.65 26.50 34.08 26.50 34.00 15 26.60 34.06 26.60 33.92 26.50 34.27 26.30 34.06 20 26.50 34.50 26.60 34.17 26.30 34.29 26.40 34.19 10 Sept 96 0 27.50 25.06 27.15 26:02 26.35 24.97 26.65 31.91 3 27.40 31.00 27.20 31.07 26.70 30.51 26.75 31.90 5 27.40 32.10 27.30 31.21 27.30 30.87 26.90 32.06 10 26.80 32.15 26.90 31.35 26.80 30.96 26.90 32.71 15 26.60 32.37 26.70 31.39 26.70 32.07 26.80 32.90 20 26.55_ 32.40 26.60 32.08 26.60 32.81 26.60 33.29 173 Table A4.1. Continued. Temperature and Salinity rawdata set Stn 1 Stn 4 Stn 5 Stn 7 Stn 3 Date Temp. Salinity Temp. Salinity Temp. Salinity Temp. Salinity Temp. Salinity of Sampling Depth (m) °C °C C C C 24 Sept 96 0 29.70 28.00 29.60 32.03 29.40 30.10 28.80 30.31 3 28.70 30.21 28.10 32.57 28.30 31.62 28.80 30.52 5 27.90 30.40 27.80 32.59 28.50 31.73 28.80 30.68 10 26.20 30.75 27.30 32.68 28.20 32.28 28.70 30.74 15 27.20 30.83 27.30 32.73 27.20 32.35 28.60 30.91 20 26.85 30.88 27.20 32.77 27.10 32.66 28.50 30.95 8 Oct 96 0 29.40 29.69 29.10 30.38 28.70 29.94 29.00 31.00 3 29.15 30.27 28.60 32.19 29.10 32.17 29.15 31.14 5 26.10 31.54 28.40 32.21 28.90 32.19 29.20 31.23 10 27.10 31.57 27.80 32.43 26.90 32.73 28.80 32.29 15 27.50 31.65 27.70 32.47 27.15 32.83 29.00 32.73 20 27.10 32.00 26.90 32.59 26.80 32.89 29.00 32.81 21 Oct 96 0 29.50 29.14 29.90 29.91 29.90 30.01 28.70 30.06 3 29.70 29.73 29.70 30.05 29.40 30.05 28.40 30.25 5 29.20 30.05 28.90 30.09 29.10 30.09 28.60 30.43 10 28.00 30.21 28.20 30.13 27.80 30.53 28.00 30.52 15 27.80 30.43 28.10 30.17 27.10 30.58 28.20 30.68 20 27.50 30.97 27.50 30.20 27,50 31.03 28.30 30.72 3 Nov 96 0 27.90 20.05 29.20 25.06 28.60 27.06 28.40 30.00 3 29.25 30.00 29.70 30.01 28.50 30.03 28.20 30.05 5 28.75 30.00 28.90 30.05 28.20 31.02 28.10 30.13 10 27.90 31.60 27.80 31.02 27.20 31.06 27.80 31.02 15 27.70 31.70 27.75 31.09 27.10 31.10 27.80 31.09 20 27.40 31.85 27.65 31.43 27.40 31.15 27.80 31.10 29 Nov 96 0 30.50 32.76 30.10 34.02 29.70 34.39 29.10 34.05 3 29.50 33.18 29.35 35.13 29.50 35.02 28.80 35.00 5 29.20 33.50 29.30 35.17 29.20 35.06 28.70 35.02 10 28.30 34.07 28.70 35.24 28.50 35.13 28.40 35.01 15 27.90 34.64 26.40 35.27 27.80 35.17 28.30 35.20 20 27.25 35.07 26.40 35.30 27.60 35.20 28.20 35.20 19Dec96 0 29.00 27.31 28.33 29.90 28.11 28.73 27.61 30.90 3 5 28.89 32.69 28.28 31.90 28.17 31.48 27.56 32.51 10 15 20 28.50 34.74 27.67 33.19 27.89 34.06 27.11 33.91 31 Dec 96 0 28.40 33.51 28.60 33.60 28.40 34.50 28.60 34.53 3 28.80 28.60 28.40 28.70 5 28.50 33.97 23.30 34.19 28.50 34.56 28.80 34.61 10 . 28.60 28.60 28.30 28.65 15 28.40 28.30 28.10 28.60 20 28.00 34.73 28.00 34.85 27.80 35.03 28.60 34.82 17 Jan 97 0 27.70 33.50 27.00 34.02 27.50 34.45 27.20 31.06 3 5 27.70 33.56 27.10 34.14 27.45 34.49 26.80 34.00 10 27.60 33.59 26.90 35.07 27.35 34.58 26.70 34.81 15 27.55 34.00 27.00 35.09 27.30 35.03 26.60 35.03 20 27.10 34.52 26.70 35.12 26.90 35.07 26.00 35.17 3 Feb 97 0 30.50 30.00 30.00 30.00 29.20 31.06 29.30 30.06 3 5 29.30 31.06 29.30 31.05 28.80 32.00 29.20 32.09 10 15 20 29.00 34.62 28.80 32.43 28.40 33.61 29.40 33.52 30 Mar 97 0 28.30 30.81 29.90 33.29 29.20 32.96 29.20 33.91 3 29.90 32.53 29.80 33.47 29.20 33.85 29.10 33.96 5 29.60 33.07 29.60 33.65 29.10 33.91 29.10 34.00 10 29.30 34.17 29.20 33.72 29.00 34.07 29.10 34.12 15 29.00 34.48 28.90 34.12 29.00 34.13 29.00 34.18 20 28.60 34.83 28.70 34.20 28.90 34.28 29.00 34.21 174 Table A4.1. Continued. Temperature and Salinity rawdata set StnJ Stn 4 Stn 5 Stn 7 Stn 3 Date Temp. Salinity Temp. Salinity Temp. Salinity Temp. Salinity Temp. Salinity of Sampling Depth (m) °C 7 June 97 6 27.60 34.21 27.50 30.97 27.45 31.42 27.60 32.15 3 27.50 27.30 27.40 27.50 5 27.45 34.29 27.30 32.06 27.35 32.67 27.55 32.53 10 27.45 27.10 27.20 27.45 15 27.20 26.80 27.10 27.35 20 26.90 34.56 26.90 33.71 26.90 33.29 27.30 33.90 9 July 97 0 27.60 32.82 27.60 29.86 27.55 34.49 27.70 34.10 3 27.60 27.55 27.50 27.65 5 27.45 33.33 27.50 30.92 27.45 34.52 27.60 34.15 10 27.40 27.40 27.40 27.50 15 27.40 27.30 27.20 27.40 20 26.10 34.48 27.10 33.20 27.05 34.57 27.10 34.40 9 Aug 97 0 27.30 32.90 26.80 29.61 27.00 33.14 26.90 32.95 27.40 32.85 3 26.90 33.43 26.90 31.00 26.90 33.32 26.90 32.97 27.00 33.12 5 26.80 33.82 26.90 32.33 26.90 33.73 26.90 33.03 26.90 33.15 10 26.70 34.17 26.70 32.73 26.80 33.95 26.80 34.06 26.80 33.72 15 26.40 34.19 26.40 33.44 26.70 34.07 26.70 34.09 26.60 34.00 20 26.30 34.32 26.30 33.71 26.50 34.21 26.30 34.12 26.30 34.07 25 Aug 97 0 24.30 33.76 25.20 34.58 25.20 32.97 24.40 35.00 25.10 33.86 3 24.60 33.92 25.15 35.07 24.90 33.53 24.40 35.12 24.90 33.95 5 24.50 34.28 24.25 35.26 24.30 33.67 24.40 35.27 24.50 34.00 10 24.10 34.66 24.20 35.32 24.20 35.08 24.40 35.32 24.10 34.22 15 23.25 34.70 24.00 35.35 24.10 35.12 24.30 35.37 23.85 34.57 20 23.45 35.02 24.00 35.41 24.20 35.20 24.40 35.42 24.10 35.00 9 Sept 97 0 26.40 32.51 27.40 32.07 26.60 33.60 26.00 33.68 26.50 33.70 3 26.10 33.67 26.80 34.01 24.10 33.75 26.00 34.12 26.10 33.95 5 25 50 33.82 2S.80 34.04 25.80 34.00 25.90 35.18 25.40 34.02 10 25.20 33.93 24.80 35.09 24.60 34.10 25.80 35.20 25.20 34.15 15 24.60 34.04 24.70 34.18 24.40 35.15 25.40 35.25 25.00 34.28 20 24.80 33.96 24.60 35.25 24.10 35.22 25.30 35.26 24.30 34.75 23 Sept 97 0 25.70 34.00 26.10 34.31 25.90 33.64 24.50 34.62 26.20 33.83 3 25.20 34.21 25.30 34.68 25.40 33.98 24.30 34.71 25.30 34.17 5 24.50 34.53 25.00 35.08 24.50 34.20 24.40 35.03 24.80 34.36 10 24.00 34.90 24.40 35.12 24.10 34.63 24.10 35.07 24.30 34.82 15 24.00 35.00 24.30 35.17 24.00 35.05 23.90 35.09 24.10 35.10 20 24.10 35.00 24.30 35.21 23.80 35.09 23.90 35.12 24.00 35.14 15 Oct 97 0 27.40 32.47 28.00 31.02 27.50 34.13 26.40 34.23 27.50 33.56 3 26.00 32.83 27.40 32.83 26.20 34.19 25.50 34.37 26.20 33.62 5 25.30 33.26 26.10 33.12 26.20 34.26 25.10 35.02 24.60 33.93 10 24.80 33.71 25.00 33.66 24.50 35.00 25.10 35.18 24.60 34.23 15 24.30 34.00 24.90 33.69 24.40 35.00 24.80 35.27 24.30 34.27 20 24.30 34.00 24.30 35.00 24.40 35.00 24.70 35.30 24.20 34.29 7 Nov 97 0 27.80 32.61 27.40 32.89 26.90 34.20 27.30 32.91 3 27.70 33.40 25.90 33.00 25.40 34.30 26.20 33.67 5 26.80 33.73 25.80 33.17 25.20 34.35 25.60 33.68 10 25.00 34.22 25.20 33.38 24.90 34.38 25.00 33.97 15 24.90 34.47 24.80 33.61 24.80 34.47 24.70 34.14 20 24.50 34.50 24.70 33.72 24.60 34.56 24.60 34.36 25 Nov 97 0 30.50 33.29 29.60 34.17 29.70 33.19 27.90 35.41 29.80 33.30 3 27.90 34.16 27.30 34.88 27.70 33.92 27.70 35.57 27.70 33.52 5 27.70 34.19 26.70 35.43 26.20 34.37 27.70 35.60 27.10 33.67 10 26.30 34.20 25.80 35.47 24.80 34.81 27.50 35.62 25.60 33.81 15 25.20 34.23 25.30 36.00 24.60 35.05 27.20 35.66 24.30 34.14 20 24.20 34.27 25.40 36.00 24.70 35.09 27.20 35.68 24.60 34.17 2 Dec 97 0 30.30 34.27 30.00 33.65 29.90 35.00 28.50 35.07 3 29.20 35.00 29.50 35.00 29.00 35.00 27.70 35.13 5 27.70 35.04 28.40 35.02 28.10 35.05 27.70 35.15 10 26.70 35.13 27.10 35.19 27.20 35.07 27.60 35.21 15 26.00 35.14 26.40 35.22 27.00 35.14 27.40 35.27 20 26.00 35.16 25.90 35.34 26.50 35.20 27.30 36.00 175 Table A4.1. Continued. Temperature and Salinity rawdata set Stn 1 Stn 4 Stn 5 Stn 7 Stn 3 Date Temp. Salinity Temp. Salinity Temp. Salinity Temp. Salinity Temp. Salinity °C 3C 10 Dec 97 0 30.10 34.13 29.90 34.68 29.80 33.72 28.50 33.72 30.00 33.27 3 28.80 34.17 29.30 34.75 28.60 34.06 28.50 34.12 28.80 34.18 5 28.90 35.00 27.30 35.03 27.80 35.61 28.50 34.27 28.80 34.23 10 27.50 35.03 26.70 35.08 25.60 35.18 28.50 34.44 27.60 34.59 15 26.00 35.05 25.80 35.14 25.30 34.21 28.40 34.51 25.70 34.90 20 25.90 35.14 24.50 35.21 25.40 34.25 28.30 34.57 25.60 rW 35.00 99 Q7 n 29.90 33.82 29.80 33.78 28.90 33.71 30.10 32.42 3 29.10 34.73 28.90 34.57 29.60 33.89 28.70 33.75 29.70 33.79 5 29.00 34.82 28.40 34.61 28.60 33.86 28.60 33.83 28.40 33.83 10 27.90 34.96 27.90 35.04 28.30 34.27 28.50 34.12 27.20 34.55 15 26.20 35.02 35.07 26.80 27.00 34.55 28.50 34.15 25.90 34.62 20 25.80 OH. I o 22 Jan 98 zo.ou 0 29.30 32.86 28.60 33.72 28.70 33.91 27.90 34.92 28.70 33.00 3 29.20 33.97 28.50 33.93 28.60 33.94 27.90 34.99 28.60 33.70 5 28.40 34.06 28.40 33.95 28.50 34.50 27.90 35.02 28.60 34.18 10 27.80 34.09 27.80 34.21 27.90 34.54 27.80 35.06 27.90 34.65 15 27.60 34.13 27.10 34.27 27.80 34.83 27.70 35.12 27.20 34.67 20 25.20 34.17 26.60 34.29 25.00 35.00 27.10 35.17 24.70 35.05 5 Foh Qft n 29.40 34.00 29.20 35.42 29.70 33.46 3 30.00 33.56 29.30 34.62 29.30 34.00 29.10 35.57 29.70 33.81 5 30.00 33.63 29.30 34.81 29.20 34.13 29.10 35.65 29.60 33.90 10 29.10 34.00 28.90 34.83 29.00 34.17 28.90 35.68 28.60 34.55 15 28.60 34.12 28.50 28.60 34.86 34.22 28.80 35.74 28.70 34.67 20 28.80 34.18 28.00 27.70 34.90 34.28 28.80 35.76 28.30 34.70 19 Feb 98 0 30.60 33.64 30.30 34.85 30.20 32.84 29.80 33.67 30.20 33.65 3 30.30 33.73 30.00 34.91 30.10 34.02 29.70 34.18 30.10 34.00 5 29.90 33.95 30.10 35.06 30.10 34.03 29.70 35.02 29.90 34.02 10 29.70 34.01 29.40 33.08 29.50 34.19 29.50 35.10 29.80 34.21 15 29.70 34.08 28.90 35.12 29.50 34.22 29.40 35.16 29.00 34.62 20 29.70 34.13 28.00 35.19 29.30 34.25 29.00 35.20 28.70 34.92 5 March 98 0 30.10 31.67 29.60 33.79 29.40 35.11 29.00 35.10 29.70 35.27 3 29.70 32.52 29.30 35.25 29.20 35.25 28.90 35.26 29.40 35.81 5 29.50 32.91 29.30 36.00 29.20 35.78 28.90 35.42 29.30 36.25 10 29.20 34.61 29.20 36.02 29.20 35.92 28.70 35.75 29.10 36.43 15 29.10 35.10 28.90 36.08 29.10 36.03 28.70 35.87 28.90 36.52 20 28.70 35.18 27.00 36.10 28.60 36.09 28.80 36.00 28.10 36.73 19 March 98 0 30.30 30.68 30.20 32.62 29.60 33.41 29.40 33.78 29.90 30.80 3 30.00 31.53 29.80 33.64 29.60 33.52 29.30 33.83 29.60 31.10 5 29.80 31.57 29.60 33.85 29.30 33.83 29.30 33.91 29.30 31.29 10 29.50 32.47 29.20 34.03 29.40 34.17 29.20 33.93 29.60 31.48 15 29.00 33.10 29.00 34.10 28.80 34.18 29.20 33.97 29.20 32.00 20 27.40 33.18 27.90 34.20 28.50 34.22 29.20 34.00 28.50 32.26 6 April 98 0 30.40 29.69 30.10 31.32 29.90 32.14 29.60 34.72 30.50 33.51 3 30.40 32.46 30.30 32.78 29.60 33.56 29.50 34.77 30.40 33.78 5 30.10 33.04 30.10 32.82 29.00 33.91 29.50 34.81 29.90 33.95 10 29.80 34.12 29.60 33.57 28.70 34.00 29.40 34.95 29,60 34.02 15 29.50 34.21 29.20 34.01 28.30 35.09 29.00 35.00 29.00 34.16 20 34.29 27.90 28.70 34.05 28.10 35.25 28.80 35.00 28.50 34.29 29 April 98 0 30.90 32.74 31.00 32.86 30.50 33.82 29.80 34.80 30.90 31.94 3 30.10 33.83 30,80 33.02 30.20 34.29 29.60 34.83 30.60 32.17 5 29.80 33.92 30.50 33.03 29.70 34.70 29.50 34.87 30.10 32.53 10 29.00 34.58 29.70 34.39 29.40 35.06 29.40 34.91 29.50 33.19 15 29.30 35.02 29.30 34.40 28.80 35.19 29.30 35.22 29.30 33.42 20 29.00 35.10 28.30 34.45 29.10 35.21 29.40 35.27 29.10 34.00 14 May 98 0 30.60 26.14 31.40 30.02 30.60 33.02 28.50 33.14 30.50 30.00 3 30.00 32.70 29.90 32.07 29.40 33.06 27.80 34.25 29.80 32.04 5 29.00 33.63 29.40 32.15 29.10 33.15 27.60 34.41 29.10 33.10 10 28.20 33.77 28.70 32.56 28.70 33.24 27.60 34.46 28.30 33.25 15 28.00 34.00 28.40 33.00 28.30 34.00 27.40 34.50 28.00 33.49 20 27.60 34.19 27.40 33.29 27.80 34.10 27.30 34.53 27.20 33.77 176 Table A4.1. Continued. Temperature and Salinity rawdata set Stn 1 Stn 4 Stn 5 Stn 7 Stn 3 Date Temp. Salinity Temp. Salinity Temp. Salinity Temp. Salinity Temp. Salinity of Sampling Depth (m) °C "C °C °C °C 28 May 98 0 29.70 33.54 30.00 31.71 29.40 32.70 27.70 34.52 29.30 31.00 3 29.10 33.72 29.50 32.00 29.10 32.81 27.50 34.73 29.30 32.50 5 28.80 34.00 29.30 33.51 28.60 33.25 27.50 34.89 29.00 33.17 10 28.00 34.05 27.90 34.00 28.20 34.02 27.40 34.95 28.20 33.56 15 27.90 34.23 27.50 34.07 27.80 34.16 27.20 35.00 27.80 34.00 20 27.90 34.57 27.50 33.72 27.30 34.27 27.00 35.07 27.50 34.00 10 June 98 0 28.70 29.00 29.80 25.00 27.70 19.06 28.20 32.64 27.80 22.00 3 28.64 33.01 29.30 31.50 27.70 31.68 27.95 33.00 29.00 30.00 5 28.50 33.70 29.20 32.00 27.80 32.00 27.80 33.21 28.00 31.54 10 27.50 34.05 27.70 33.22 27.30 33.72 27.60 33.29 27.70 31.78 15 27.40 34.12 27.40 33.39 27.20 34.15 27.40 33.43 27.50 32.24 20 27.20 34.15 27.40 33.58 27.40 34.21 27.30 33.47 27.30 34.00 24 June 98 0 29.10 28.13 29.20 30.47 28.10 26.61 28.00 29.63 29.40 27.91 3 28.60 32.16 29.00 31.97 27.80 31.87 27.60 32.44 29.00 32.11 5 27.90 32.21 28.60 32.23 27.60 32.58 27.50 32.29 28.10 32.35 10 27.50 32.35 27.60 32.25 27.60 32.60 27.50 32.56 27.65 32.38 15 27.30 32.42 27.40 32.32 27.50 32.67 27.60 32.45 27.50 32.42 20 27.20 32.68 27.40 32.57 27.40 32.70 27.60 32.74 27.50 32.57 8 July 98 0 27.30 11.42 28.80 25.41 28.50 24.81 27.60 29.00 28.30 25.17 3 28.90 31.47 28.10 31.93 28.30 31.62 28.20 32.23 28.10 31.95 5 28.20 32.08 28.00 32.12 27.70 32.06 28.10 32.25 27.80 32.13 10 27.70 32.17 27.50 32.30 27.40 32.17 27.80 32.37 27.30 32.37 15 27.30 32.32 27.10 32.45 27.10 32.58 27.70 32.39 27.10 32.35 20 26.90 32.41 26.70 32.47 26.70 32.69 27.60 32.40 26.80 32.45 24 July 98 0 28.00 33.57 29.60 29.72 28.80 31.00 27.30 32.00 29.10 32.00 3 27.20 33.89 28.40 32.05 27.40 33.00 27.10 32.74 27.80 33.50 5 26.90 34.00 27.40 33.11 27.10 33.52 27.00 33.18 27.00 34.05 10 26.80 34.10 26.80 33.52 27.40 33.57 27.00 33.52 26.60 34.10 15 26.70 34.25 26.70 33.60 26.70 34.00 26.70 33.57 26.50 33.50 20 26.50 34.30 26.50 33.71 26.60 34.05 26.30 34.00 26.40 34.00 177 Table A4.2. Measurements of dissolved nutrients (Stations 1 and 4) Stn 1 Stn 4 Date Depth N03 P04 NH4 Si(OH)4 N03 P04 NH4 Si(OH)4 of Sampling (m) (uM) (uM) (uM) (uM) (uM) (P.M) (u.M) (uM) 96/05/03 0 5 0.27 0.26 0.55 1.03 10 20 0.14 4.86 0.49 1.24 96/05/15 0 5 0.52 1.34 2.92 0.93 10 20 0.33 4.24 1.39 1.88 96/06/04 0 5 0.95 2.26 0.58 1.42 10 20 2.3 2.89 3.36 1.42 96/06/17 0 5 2.48 3.41 3.90 1.82 10 20 4.03 3.64 5.00 1.59 96/07/09 0 5 1.54 0.21 0.29 0.38 10 20 3.88 0.27 2.36 1.41 96/07/23 0 5 1.92 1.91 2.53 0.95 10 20 2.66 1.18 3.39 2.53 96/08/10 0 5 0.07 1.33 1.95 1.16 10 20 7.91 1.81 3.06 0.63 96/08/20 0 0.25 0.32 0.05 0.16 5 1.00 0.27 0.00 0.16 10 4.89 0.43 1.90 0.32 20 15.81 0.80 9.13 0.59 96/09/10 0 0.92 0.34 0.22 0.29 5 13.61 0.74 0.07 0.39 10 3.38 0.44 0.81 0.44 20 0.44 0.39 5.11 0.44 96/09/24 0 0.43 0.05 0.51 0.00 5 0.36 0.26 0.20 0.10 10 1.02 0.20 0.16 0.00 20 13.70 0.77 4.53 0.15 96/10/08 0 1.40 0.35 0.00 0.25 5 0.08 0.45 0.00 0.25 10 0.28 0.25 0.00 0.25 20 7.01 0.55 4.32 0.60 96/10/21 0 0.45 0.00 0.25 0.00 5 0.35 0.15 0.54 0.00 10 0.20 0.00 0.30 0.10 20 11.36 0.54 0.30 0.10 96/11/03 0 0.11 0.00 0.11 0.00 5 0.00 0.00 0.00 0.00 10 1.24 0.00 0.05 0.00 20 5.99 0.19 3.51 0.38 96/11/29 0 0.43 0.00 0.13 0.00 5 0.22 0.07 0.04 0.00 10 0.13 0.00 0.04 0.00 20 8.25 0.29 4.45 0.67 96/12/19 0 0.53 1.01 5 0.69 0.68 10 0.71 0.77 20 1.15 0.97 96/12/31 0 1.73 1.27 5 0.39 0.98 0.43 0.88 10 20 0.67 1.40 0.46 1.14 178 Table A4.2. Continued Measurement of dissolved nutrients (Stations 1 and 4) Stn 1 Stn 4 Date Depth N03 P04 NH4 Si(OH)4 N03 P04 NH4 Si(OH)4 of Sampling (m) (UM) (UM) (UM) (UM) (UM) (JJM) 97/01/17 0 5 0.28 0.36 0.41 0.31 10 20 1.42 0.64 1.26 0.92 97/02/03 0 5 0.40 0.37 0.22 0.39 10 20 1.55 0.32 0.07 0.34 97/03/30 0 0.11 0.00 0.93 0.00 5 0.00 0.00 0.25 0.15 10 0.11 0.00 1.13 0.10 20 8.26 0.77 1.42 0.10 97/06/07 0 0.00 0.25 6.92 52.81 0.36 0.00 1.81 4.43 5 0.00 0.35 3.89 6.30 0.00 0.00 5.45 4.15 10 0.00 0.50 3.20 7.42 0.05 0.00 3.93 4.25 20 3.80 0.50 3.37 10.62 0.16 0.00 2.67 3.88 97/07/09 0 1.60 0.32 0.03 24.99 0.63 0.39 1.25 39.03 5 0.75 0.21 0.03 7.52 0.44 0.25 0.26 10.05 10 1.70 0.27 0.03 7.15 3.05 0.49 1.77 9.24 20 2.49 0.43 0.79 7.06 4.78 0.54 0.50 12.50 97/08/09 0 1.04 0.05 0.03 13.15 1.08 0.44 0.95 64.65 5 2.80 0.15 0.03 6.67 0.96 0.15 0.03 12.50 10 3.00 0.30 1.21 7.33 2.24 0.20 0.03 8.27 20 6.40 0.30 0.03 13.53 2.96 0.69 0.03 11.37 97/08/25 0 2.36 0.16 4.51 32.08 0.29 0.05 2.39 68.81 5 0.00 0.36 2.83 8.84 0.24 0.41 3.07 10.23 10 4.52 0.46 2.71 8.75 2.26 0.51 2.71 7.54 20 10.73 0.93 2.35 20.27 6.64 2.51 2.63 13.49 97/09/09 0 0.14 0.00 2.43 36.93 0.09 0.96 1.56 58.64 5 0.01 0.28 0.92 4.60 0.01 0.51 1.75 8.17 10 1.59 0.62 4.39 4.98 2.46 0.40 1.71 4.89 20 4.26 0.62 1.32 11.37 4.96 1.46 1.64 11.65 97/09/23 0 0.17 0.00 4.83 15.79 0.09 0.00 0.28 13.06 5 3.84 0.00 0.88 14.56 0.17 0.00 0.32 12.97 10 12.40 0.00 0.96 17.20 9.17 0.00 0.00 12.78 20 13.36 0.40 0.00 18.61 7.59 0.56 0.00 8.27 97/10/15 0 1.24 0.15 0.00 53.37 0.99 0.15 0.00 28.00 5 0.93 0.10 0.28 11.09 1.37 0.20 0.70 12.78 10 7.54 0.55 0.40 14.47 3.24 0.35 0.92 19.36 20 18.21 1.71 0.64 29.60 16.22 1.06 1.91 20.95 97/11/07 0 0.50 0.20 1.48 3.48 0.06 0.15 1.24 3.48 5 0.56 0.10 1.08 3.10 0.00 0.10 1.08 1.97 10 0.18 0.25 1.40 3.01 0.81 0.30 3.15 2.72 20 1.87 0.40 2.63 6.30 1.43 0.35 1.40 4.51 97/11/25 0 0.26 0.20 2.35 18.42 0.11 0.15 0.12 28.28 5 0.16 0.25 0.96 4.42 0.21 0.20 2.87 4.32 10 0.21 0.20 2.87 4.13 0.01 0.30 3.55 8.27 20 11.43 0.61 2.47 20.39 3.91 0.56 1.79 17.38 97/12/02 0 2.62 0.00 1.16 22.36 0.69 0.00 2.31 13.81 5 0.92 0.00 4.55 2.91 0.77 0.00 4.43 5.36 10 2.00 0.00 0.28 4.04 1.00 0.00 0.76 6.01 20 15.19 0.00 0.88 6.86 7.71 0.00 1.95 10.52 97/12/10 0 0.37 0.35 4.47 3.10 0.68 0.10 5.75 20.01 5 0.50 0.10 8.15 3.10 0.43 0.25 3.71 5.07 10 0.75 0.25 4.43 3.10 0.87 0.20 5.19 5.54 20 10.10 0.80 7.95 3.57 10.91 0.95 5.23 19.83 97/12/22 0 0.00 0.05 10.98 7.42 0.05 0.15 8.23 1.22 5 0.15 0.15 10.46 0.28 0.00 0.15 3.78 0.28 10 0.05 0.15 9.65 0.19 0.00 0.25 6.48 1.41 20 7.12 0.45 9.80 18.61 5.92 0.74 7.66 12.03 179 Table A4.2. Continued Measurement of dissolved nutrients (Stations 1 and 4) Stn 1 Stn 4 Date Depth N03 P04 NH4 Si(OH)4 N03 P04 NH4 Si(OH)4 of Sampling (m) (uM) (HM) (HM) (HM) (uM) (HM) (uM) (uM) 98/01/22 0 1.19 0.15 17.19 10.24 0.79 0.00 8.66 7.80 5 1.39 0.15 5.90 13.72 1.19 0.10 3.71 7.70 10 1.09 0.30 6.81 14.19 0.25 0.10 5.33 5.92 20 18.42 1.79 4.85 37.40 1.59 0.20 5.71 6.39 98/02/05 0 1.15 0.15 6.57 19.92 0.86 0.00 6.38 1.78 5 0.96 0.05 4.57 6.20 0.96 0.30 0.71 1.78 10 0.62 0.05 8.66 1.88 0.72 0.15 4.62 3.10 20 1.39 0.15 4.76 2.25 0.86 0.10 4.43 2.44 98/02/19 0 1.25 0.20 9.26 21.21 0.90 0.10 6.19 12.75 5 0.70 0.00 14.14 5.12 0.80 0.00 5.08 15.91 10 0.85 0.20 6.43 3.91 0.80 0.05 7.34 4.28 20 1.25 0.10 4.01 4.19 2.59 0.20 7.42 10.79 98/03/05 0 0.47 0.00 6.97 23.40 0.26 0.05 6.19 10.34 5 0.31 0.10 6.35 11.93 0.57 0.00 1.39 9.77 10 0.36 0.15 6.35 9.11 0.47 0.05 6.35 10.99 20 0.93 0.00 4.06 13.15 4.77 0.55 6.19 34.30 98/03/19 0 0.55 0.41 6.66 34.12 0.50 0.25 6.09 21.06 5 0.40 0.30 3.33 14.63 0.00 0.10 1.71 13.25 10 0.20 0.41 6.57 12.69 0.15 0.10 5.04 12.88 20 9.76 0.86 3.76 31.91 2.95 0.61 6.95 24.28 98/04/06 0 2.28 0.24 7.12 60.85 0.25 0.15 13.47 15.78 5 0.30. 0.05 5.80 9.80 0.05 0.10 3.64 8.16 10 0.20 0.05 7.30 9.71 0.05 0.05 6.52 7.71 20 0.50 0.15 10.41 15.69 0.10 0.15 8.56 20.68 98/04/29 0 1.29 0.20 11.75 20.30 0.15 0.25 10.83 22.27 5 0.10 0.10 5.79 8.55 0.05 0.20 3.83 9.68 10 0.30 0.25 13.55 9.02 0.10 0.10 5.19 6.58 20 1.39 1.47 7.39 12.59 1.74 0.89 3.51 19.64 98/05/14 0 8.95 0.00 7.02 190.10 1.17 0.21 5.40 34.67 5 1.09 0.21 6.60 11.84 1.25 0.10 5.40 6.95 10 7.40 0.05 6.85 19.64 1.57 0.10 7.31 8.93 20 9.15 0.57 6.93 31.10 5.93 0.47 6.39 26.22 98/05/28 0 0.45 0.06 14.29 17.42 0.30 0.18 8.51 34.01 5 0.55 0.06 9.46 12.54 0.10 0.12 11.36 17.52 10 9.10 0.53 8.10 21.85 1.94 0.00 7.83 19.18 20 12.98 0.88 5.51 31.25 3.88 0.35 10.28 26.64 98/06/10 0 4.57 0.38 8.72 38.06 1.80 0.32 8.55 81.75 5 1.59 0.27 8.78 3.48 0.41 0.38 8.55 3.48 10 3.23 0.21 10.17 5.64 1.02 0.38 8.37 5.17 20 10.70 0.59 3.72 23.96 3.55 0.80 4.94 20.67 98/06/24 0 1.66 0.11 6.63 53.28 1.00 0.05 6.16 82.51 5 4.52 0.32 7.32 10.81 0.09 0.21 2.44 6.01 10 6.55 0.42 6.57 11.93 0.46 0.27 6.34 10.99 20 10.78 0.85 5.58 26.97 3.49 0.48 6.51 17.20 98/07/08 0 0.13 0.10 8.31 47.74 0.09 0.00 8.37 58.82 5 0.09 0.26 8.14 5.36 0.05 0.15 1.63 7.14 10 0.42 0.26 6.98 7.80 1.75 0.26 5.35 9.40 20 13.15 0.41 5.06 29.79 6.84 0.46 8.72 18.14 98/07/24 0 0.38 0.00 1.86 2.98 0.00 0.11 0.00 61.34 5 6.02 0.38 1.86 623 0.00 0.00 0.00 1.21 10 7.26 0.27 0.58 11.44 0.75 0.22 0.17 2.98 20 14.39 0.43 0.00 23.33 4.27 0.32 0.00 13.85 180 Table A4.3. Measurements of dissolved nutrients (Stations 5 and 7) Stn 5 Stn 7 Date Depth N03 P04 NH4 Si(OH)4 M03 P04 NH4 Si(OH)4 of Sampling (m) (uM) (uM) (11M) (^M) (jiM) (jiM) (uM) (uM) 96/05/03 0 5 0.22 1.03 0.38 0.88 10 20 0.09 1.03 0.3 1.76 96/05/15 0 5 1.3 0.16 10 20 3.91 1.24 96/06/04 0 5 0.17 1.98 0.2 2.32 10 20 2.64 2.66 2.26 2.15 96/06/17 0 5 3.93 2.59 1.92 2.45 10 20 3.92 2.5 4.28 1.14 96/07/09 0 5 2.07 0.21 2.47 0.27 10 20 2.45 0.21 2.85 0.43 96/07/23 0 5 3.98 1.14 1.98 1.68 10 20 3.01 1.36 0.99 0.91 96/08/10 0 5 3.76 1.06 0.9 1.26 10 20 6.09 1.45 3.85 1.45 96/08/20 0 0.10 0.16 0.85 0.32 5 0.00 0.32 1.65 0.32 10 1.05 0.00 1.95 0.32 20 1.90 0.43 3.54 0.37 96/09/10 0 1.29 0.29 0.63 0.39 5 0.92 0.34 0.18 0.29 10 1.03 0.44 0.26 0.39 20 2.69 0.29 0.92 0.34 96/09/24 0 0.24 0.15 0.43 0.10 5 0.71 0.26 0.79 0.51 10 1.54 0.15 0.67 0.15 20 3.94 0.26 0.67 0.10 96/10/08 0 0.04 0.25 0.24 0.45 5 0.68 0.30 0.28 0.50 10 1.44 0.50 0.04 0.40 20 2.20 0.50 0.80 0.25 96/10/21 0 0.88 0.10 0.54 0.05 5 0.74 0.15 0.54 0.15 10 2.00 0.15 0.74 0.10 20 3.22 0.20 1.66 0.20 96/11/03 0 0.78 0.00 0.21 0.00 5 0.31 0.00 0.21 0.00 10 0.83 0.00 0.31 0.00 20 4.44 0.57 0.47 0.00 96/11/29 0 0.30 0.00 0.09 0.00 5 0.09 0.00 0.09 0.00 10 0.52 0.00 0.86 0.00 20 8.04 0.96 0.43 0.00 96/12/19 0 0.23 0.21 5 0.38 0.22 0.76 0.57 10 20 1.45 0.32 0.66 0.71 96/12/31 0 5 0.5 0.47 0.51 0.97 10 20 0.8 0.51 0.37 0.58 181 Table A4.3 Continued Measuremer I OT UISsuive i J 1 IU11 Id UO V Stn 5 Stn 7 P04 NH4 Si(OH)4 N03 P04 NH4 Si(OH)4 N03 Date Depth OM) OM) OM) (uM) OM) of Sampling (m) OM) OM) OM) 97/01/17 0 1.03 0.31 5 0.36 0.49 10 0.56 20 0.85 0.53 0.55 97/02/03 0 0.06 0.00 5 0.08 0.72 10 0.01 0.00 20 0.00 0.15 0.16 0.30 97/03/30 0 0.88 0.20 5 0.45 0.25 0.04 0.40 10 0.10 0.00 0.52 0.45 20 0.10 0.20 0.32 0.40 12.86 0.25 0.00 0.36 7.40 97/06/07 0 0.85 0.29 0.32 5 0.15 0.34 0.73 3.44 0.00 0.00 0.03 3.69 10 0.37 0.29 0.96 2.90 0.11 0.00 0.03 2.96 20 1.10 0.44 0.79 4.35 0.64 0.45 0.00 5.26 60.86 0.20 0.15 0.03 6.11 97/07/09 0 0.36 0.00 0.09 5 0.20 0.05 0.03 3.17 • 0.51 0.15 0.03 8.27 10 0.16 0.20 0.50 4.71 0.43 0.15 0.03 3.95 20 6.34 0.41 0.03 9.06 0.79 0.15 0.03 6.01 39.28 1.20 0.00 0.29 23.96 97/08/09 0 0.60 0.15 0.36 5 1.28 0.00 1.08 20.48 1.16 0.15 0.62 7.99 10 1.80 0.20 2.12 13.53 0.84 0.30 1.73 7.23 20 3.00 0.25 1.73 8.74 1.76 0.34 0.03 7.89 37.01 1.88 0.26 4.23 28.92 97/08/25 0 0.58 0.16 2.51 5 3.18 0.93 1.87 13.39 1.97 0.67 4.39 10.23 10 4.14 0.62 2.79 9.95 2.07 0.36 3.63 9.58 20 6.83 0.72 4.99 11.72 1.88 0.26 2.87 7.91 48.02 0.01 0.23 0.96 21.42 97/09/09 0 0.01 0.40 0.20 5 0.09 0.17 0.00 10.81 0.01 0.00 4.15 6.48 0.27 10 3.74 0.90 0.00 24.53 0.00 1.32 5.45 1.37 20 10.40 1.69 0.00 9.40 0.34 0.00 5.07 8.17 1.05 0.00 0.00 10.34 97/09/23 0 0.09 0.00 0.00 5 3.05 0.00 0.00 9.30 1.05 0.00 0.00 8.46 10 8.29 0.06 0.00 10.52 1.74 0.00 2.23 7.14 1.66 20 6.90 0.17 1.67 11.18 0.00 0.00 7.05 22.93 1.43 0.20 0.00 8.64 97/10/15 0 1.12 0.20 0.53 5 0.68 0.40 0.00 14.85 1.31 0.20 0.00 10.05 10 7.17 0.65 0.98 14.28 1.37 0.20 0.00 8.93 20 9.92 1.01 1.20 19.92 2.62 0.25 0.00 9.21 3.10 4.04 97/11/07 0 0.12 0.25 1.28 5 0.00 0.35 5.55 2.44 4.23 10 0.06 0.60 3.55 3.19 3.66 20 0.99 0.70 1.44 4.70 3.10 24.43 0.01 0.15 4.23 8.17 97/11/25 0 0.01 0.15 3.95 5 0.21 0.10 2.15 5.45 0.01 0.20 6.03 5.83 10 3.56 0.46 2.99 11.93 0.01 0.20 4.07 4.13 20 5.22 0.51 2.51 18.79 0.31 0.20 3.75 3.95 14.38 1.16 0.00 0.64 14.66 97/12/02 0 1.00 0.00 1.32 5 0.85 0.00 1.08 4.89 1.54 0.00 1.00 5.54 10 1.08 0.00 0.52 6.95 1.62 0.00 2.95 3.76 20 4.32 0.00 2.51 11.18 1.93 0.00 0.80 4.13 19.45 0.75 0.25 6.31 5.83 97/12/10 0 0.31 0.30 5.63 5 0.56 0.30 6.31 11.93 0.62 0.30 4.39 2.82 0.68 0.35 6.15 1.41 10 7.11 0.40 5.99 13.15 0.56 1.40 5.11 1.88 20 8.42 0.90 5.75 0.00 4.13 2.02 0.15 8.09 3.57 97/12/22 0 0.15 0.05 8.23 5 0.29 0.10 10.08 0.75 0.39 0.10 8.94 3.57 10 0.63 0.05 11.84 3.01 0.39 0.10 6.34 3.29 20 5.15 0.15 10.93 10.71 0.58 0.10 7.76 3.38 182 Table A4.3. Continued Measurement of dissolved nutrients (Stations 5 and 7) Stn 5 Stn 7 NH4 Si(OH)4 Date Depth N03 P04 NH4 Si(OH)4 N03 P04 (UM) (UM) (UM) OiM) of Sampling (m) (LIM) (UM) 0>M) (UM) 5.14 4.42 98/01/22 0 0.35 0.15 7.52 9.87 0.20 0.15 5 0.15 0.15 7.57 8.36 0.40 0.15 9.52 4.32 3.48 10 1.14 0.45 8.33 7.99 0.40 0.05 6.62 20 13.21 1.14 8.57 31.20 1.44 0.10 6.85 4.98 2.52 3.29 98/02/05 0 0.62 0.15 3.43 2.25 0.62 0.35 5 2.11 0.10 5.04 2.16 0.29 0.15 1.90 3.29 10 0.48 0.15 4.47 2.25 0.38 0.45 1.57 2.91 2.72 20 2.16 0.00 3.28 5.64 0.38 0.25 4.04 7.79 3.63 98/02/19 0 0.75 0.10 6.19 9.58 1.00 0.00 5 0.65 0.00 5.61 9.95 0.55 0.15 6.11 3.63 10 0.80 0.05 4.30 5.12 0.50 0.10 6.48 3.63 3.16 20 1.85 0.10 2.99 8.47 0.75 0.20 6.80 4.51 3.48 98/03/05 0 0.42 0.00 5.70 10.05 0.26 0.05 5 0.31 0.05 5.24 9.77 0.31 0.00 6.93 3.76 3.66 10 0.26 0.00 10.04 9.49 0.36 0.00 4.34 20 0.83 0.00 7.71 9.77 0.52 0.00 6.97 4.13 9.84 98/03/19 0 0.15 0.05 8.43 19.77 0.30 0.00 4.90 5 0.40 0.05 6.90 14.63 0.25 0.10 5.66 10.40 10 0.10 0.20 6.33 13.15 0.15 0.05 5.71 7.73 20 1.50 0.41 7.28 18.30 0.25 0.05 7.28 8.56 7.80 98/04/06 0 0.05 0.05 8.50 34.64 0.00 0.00 8.56 5 0.35 0.10 9.21 9.34 0.05 0.10 10.83 5.35 10 0.00 0.05 7.48 7.44 0.35 0.19 9.93 4.72 20 0.00 0.19 10.29 17.32 0.15 0.15 8.02 4.63 5.87 9.30 98/04/29 0 0.40 0.05 4.63 8.83 0.20 0.15 5 0.20 0.20 8.03 7.05 0.05 0.30 7.43 6.95 10 0.90 0.25 9.99 5.17 0.10 0.10 7.19 5.36 20 0.75 0.25 3.39 0.00 0.05 0.15 7.27 3.48 15.97 98/05/14 0 2.28 0.05 6.02 34.30 1.97 0.16 4.07 5 1.37 0.05 6.60 8.08 2.64 0.10 6.48 8.08 10 1.93 0.26 4.73 8.27 2.48 0.16 6.14 8.36 20 2.32 0.05 5.52 7.99 2.04 0.16 6.77 6.11 14.84 98/05/28 0 0.40 0.06 12.45 20.56 3.03 0.00 8.17 5 0.55 0.18 6.40 13.37 5.02 0.18 7.35 12.91 10 0.85 0.12 10.96 14.66 4.23 0.00 10.41 10.60 20 3.98 0.12 6.40 14.66 2.44 0.12 10.82 11.25 7.56 7.61 98/06/10 0 8.66 0.43 5.99 177.51 0.98 0.43 4.13 5 2.37 0.43 7.32 10.71 1.59 0.16 8.43 10 1.96 3.80 6.74 6.11 1.39 0.54 7.44 4.51 4.13 20 1.92 0.27 8.60 5.83 1.95 0.05 7.15 29.04 98/06/24 0 5.31 0.11 6.34 53.00 1.62 0.16 7.09 5 0.58 0.05 7.96 6.11 0.42 0.21 7.62 2.82 10 0.00 0.21 6.68 4.98 0.42 0.16 6.74 4.04 20 1.33 0.11 7.32 5.92 0.34 0.16 7.32 3.29 10.52 98/07/08 0 0.25 0.26 9.13 41.63 0.00 0.31 7.15 5 0.17 0.10 6.92 6.39 0.00 0.15 8.20 8.08 10 1.79 0.26 6.80 9.77 0.00 0.36 9.42 6.58 20 6.64 0.57 8.37 16.73 0.67 0.31 9.59 7.33 0.00 4.19 98/07/24 0 0.00 0.00 0.00 44.71 0.00 0.00 5 0.17 0.00 0.29 5.95 0.00 0.00 0.00 2.70 10 0.25 0.00 0.00 8.28 0.00 0.00 0.05 2.24 20 1.29 0.05 0.00 5.12 1.58 0.16 0.00 6.51 183 Table A4.4. Measurements of dissolved nutrients (Station 3) Stn 3 Date Depth N03 P04 NH4 Si(OH)4 of Sampling (m) uM uM uM uM 97/08/09 0 0.80 0.15 0.03 24.43 5 0.68 0.20 0.03 16.35 10 1.76 0.20 0.03 7.33 20 2.96 0.30 0.03 12.50 97/08/25 0 C 0 10 20 97/09/09 0 c D 10 20 97/09/23 0 c 0 10 20 97/10/15 0 c 0 10 20 97/11/07 0 0.18 0.40 2.19 3.85 5 0.00 0.50 2.31 2.44 10 0.00 0.60 0.92 3.10 20 2.55 0.70 3.59 5.07 97/11/25 0 0.31 0.20 1.91 4.23 5 0.31 0.20 2.87 3.66 10 1.51 0.20 4.83 4.04 20 4.71 0.51 2.43 3.66 97/12/02 0 C 0 10 20 97/12/10 0 0.06 0.20 6.95 21.33 5 0.18 0.20 5.67 9.21 10 0.81 0.30 3.07 6.95 20 8.48 0.90 7.87 13.62 97/12/22 0 0.10 0.05 9.70 4.70 5 0.43 0.05 6.34 3.66 10 0.05 0.15 11.60 4.04 20 7.70 0.45 13.87 14.85 98/01/22 0 0.74 0.10 16.57 7.89 5 0.50 0.25 10.81 8.17 10 1.14 0.10 6.57 7.33 20 20.56 2.09 9.57 39.65 98/02/05 0 0.57 0.10 3.43 3.76 5 0.72 0.15 4.76 3.01 10 1.05 0.15 4.19 1.97 20 1.05 0.10 2.71 1.31 98/02/19 0 0.60 0.05 6.56 9.40 5 0.90 0.05 7.21 8.93 10 0.90 0.00 8.40 8.56 20 1.30 0.20 10.54 5.30 98/03/05 0 0.05 0.00 5.16 12.22 5 0.36 0.00 4.92 10.34 10 0.47 0.05 7.25 8.93 20 2.33 0.40 6.89 13.81 98/03/19 0 0.30 0.05 5.28 18.03 5 0.35 0.15 3.19 14.90 10 0.00 0.10 8.62 16.28 20 2.55 0.51 4.04 16.83 Table A4.4. Continued Measurement of dissolved nutrients (Stations 3) stn 3 Date Depth N03 P04 NH4 Si(OH)4 of Sampling (m) UM uM UM UM 98/04/06 0 0.30 0.10 10.83 32.83 5 0.30 0.00 6.52 8.16 10 0.05 0.05 7.36 8.44 20 0.05 0.05 7.24 12.06 98/04/29 0 0.10 0.20 11.99 23.49 5 0.20 0.20 7.15 8.46 10 0.05 0.25 7.07 7.14 20 0.20 0.25 6.39 8.74 98/05/14 0 1.17 0.26 9.96 38.34 5 1.13 0.05 6.73 6.95 10 2.56 0.31 4.77 11.93 20 4.98 0.31 8.01 35.61 98/05/28 0 0.35 0.18 10.96 31.07 5 2.09 0.06 11.98 18.07 10 6.41 0.12 8.44 13.46 20 0.00 0.35 11.23 24.25 98/06/10 0 8.25 0.54 10.58 120.09 5 0.57 0.16 10.23 3.95 10 1.02 0.32 9.01 4.60 20 3.43 0.64 8.02 13.44 98/06/24 0 1.79 0.32 6.45 63.71 5 0.79 0.11 7.03 7.14 10 2.41 0.27 6.34 9.02 20 3.98 0.48 7.85 13.34 98/07/08 0 0.38 0.26 6.22 46.98 5 0.09 0.21 7.03 7.61 10 2.82 0.26 8.90 10.90 20 5.89 0.31 8.78 18.51 98/07/24 0 0.00 0.00 0.00 24.54 5 0.25 0.05 0.00 2.98 10 8.54 0.49 0.93 13.02 20 3.98 0.22 0.23 11.72