Effects of Estuarine Acidification on Survival and Growth of the Sydney Rock Oyster Saccostrea Glomerata

Home , Bellinger River, Bermagui River, Dennis Bridge, Estuarine acidification, Lansdowne River, Mitchells Island

EFFECTS OF ESTUARINE ACIDIFICATION ON SURVIVAL AND GROWTH OF THE SYDNEY ROCK OYSTER SACCOSTREA GLOMERATA

Michael Colin Dove

Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy in The University of New South Wales

Geography Program Faculty of the Built Environment The University of New South Wales Sydney, NSW, 2052

April 2003 ACKNOWLEDGEMENTS

I would like to thank my supervisor Dr Jes Sammut for his ideas, guidance and encouragement throughout my candidature. I am indebted to Jes for his help with all stages of this thesis, for providing me with opportunities to present this research at conferences and for his friendship. I thank Dr Richard Callinan for his assistance with the histopathology and reviewing chapters of this thesis. I am also very grateful to Laurie Lardner and Ian and Rose Crisp for their invaluable advice, generosity and particular interest in this work.

Hastings and Manning River oyster growers were supportive of this research. In particular, I would like to acknowledge the following oyster growers: Laurie and Fay Lardner; Ian and Rose Crisp; Robert Herbert; Nathan Herbert; Stuart Bale; Gary Ruprecht; Peter Clift; Mark Bulley; Chris Bulley; Bruce Fairhall; Neil Ellis; and, Paul Wilson. I am very grateful to Holiday Coast Oysters and Manning River Rock Oysters for providing: the Sydney rock oysters for field and laboratory experiments; storage facilities; equipment; materials; fuel; and, access to resources without reservation. Bruce Fairhall, Paul Wilson, Mark Bulley, Laurie Lardner and Robert Herbert also supplied Sydney rock oysters for this work.

I would also like to thank the researchers who gave helpful advice during this study. Geoff Gordon, Martin Krogh, Professor Brian Bayne and Drs Alistair Poore, Peter Cooke, Anthony Roach, Ralph Elston, Ian Smith, John Nell and Mike Heasman are thanked for advice on experimental design and data analyses.

I am grateful to Bob Smith for his encouragement and help with the field investigations and Phil March for his interest in this study. The former School of Geography and the Faculty of the Built Environment, UNSW are thanked for resources and support. Thanks to Dorothy Yu and Chris Myers for laboratory assistance and help with analyses. Angelina Enno, School of Pathology, UNSW is thanked for histoprocessing of oyster samples. Allison Dove, Alan Facer, Elissa Bishop, Loren Ravenscroft, Craig Fitzgerald, Adam Wyszynski, Anya Lam and Susan Fox participated in fieldwork during this study. I would like to express thanks to Lyndal Dove for her help with printing this thesis and Greater Taree City Council for the provision of water quality equipment. Others who provided assistance and background information for this study include: Sarah Kleeman; Francis Doorman; Damian Ogburn; Mitch Tulau; Megan Burgoyne; Kavita Gosavi, Sarah Groves, Scott Mooney, Mike Horton; David Pensini; Matt Rogers; Thor Aaso; Ian Angus; Steve Filan; Professor Ian White, Professor Mike Melville, Professor Ian Burnley; and, the Neal family. In addition, I wish to thank the following organisations: NSW Fisheries; NSW Oyster Research Advisory Committee; NSW Oyster Farmers’ Association; Department of Land and Water Conservation; Hastings Council; Greater Taree City Council; and, Materialised P/L.

Finally, I thank my wife Allison Dove for her enduring patience as well as my parents and family for their continual support and encouragement throughout this work. This project was funded by an Australian Postgraduate Award Scholarship with additional grant support from Fisheries Research and Development Corporation and a studentship provided by oyster associations on the Hastings, Manning and Camden Haven Rivers.

This thesis is dedicated to my wife and daughter, Allison and Leah.

This work is copyright. Except as permitted under the Copyright Act 1968 (Cth), no part of this publication may be reproduced by any process, electronic or otherwise, without the specific written permission of the copyright owner. Neither may information be stored electronically in any form whatsoever without such permission.

II TABLE OF CONTENTS PAGE

Acknowledgements………………………………………………………………… I Table of Contents…………………………………………………………………... III List of Tables………………………………………………………………………. XII List of Figures……………………………………………………………………… XIV List of Plates……………………………………………………………………….. XVI Glossary of Terms………………………………………………………………….. XVII Abstract…………………………………………………………………………….. XX

SECTION I - INTRODUCTION AND BACKGROUND…………………….... 1

CHAPTER ONE – OYSTERS AND ACIDIFICATION: INTRODUCTION AND BACKGROUND TO THE STUDY…………………. 2

1.1. INTRODUCTION ………………………..………………..…………...... 2

1.2. ESTUARINE ACIDIFICATION………………………………………... 8

1.2.1. Past Studies on Estuarine Acidification……………………………. 8 1.2.2. Chemistry of Waters Affected by ASS…………………………….. 10 1.2.3. Impacts on Aquatic Biota…………………………………………... 11

1.3. PAST STUDIES ON ACIDIFICATION AND OYSTERS…………….. 12

1.3.1. Overseas Studies…………………………………………………… 12 1.3.2. Effects of ASS-Affected Waters on the Sydney Rock Oyster……... 14 1.3.2.1. Shell Dissolution………………………………………….. 16

1.4. OBJECTIVES AND HYPOTHESES…………………………………… 22

1.5. RESEARCH APPROACH……………………………………………….. 23

CHAPTER TWO – BIOPHYSICAL CHARACTERISTICS OF THE STUDY AREA…………………………………………………………………….. 26

2.1. INTRODUCTION……………………………………………..…………. 26

2.2. ESTUARY EVOLUTION………………………………………………... 27

2.2.1. Hastings River……………………………………………………… 27 2.2.2. Manning River……………………………………………………... 28

2.3. GEOLOGY………………………………………………………………... 31

2.3.1. Lower Hastings River……………………………………………… 31

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2.3.2. Lower Manning River……………………………………………… 31

2.4. SOILS……………………………………………………………………… 32

2.4.1. Lower Hastings River……………………………………………… 32 2.4.2. Lower Manning River……………………………………………… 33

2.5. ACID SULFATE SOILS…………………………………………………. 34

2.5.1. Formation of Iron Pyrite…………………………………………… 35 2.5.2. Oxidation of Iron Pyrite……………………………………………. 35 2.5.3. Characteristics of ASS……………………………………………... 37 2.5.4. Distribution of ASS in the Study Area…………………………….. 39

2.6. CLIMATE………………………………………………………………… 42

2.7. VEGETATION…………………………………………………………… 42

2.8. HYDROLOGY……………………………………………………………. 45

2.8.1. Rainfall……………………………………………………………... 45 2.8.2. Tidal Hydrology……………………………………………………. 45 2.8.2.1. Tidal Hydrology in the Hastings River…………………… 45 2.8.2.2. Tidal Hydrology in the Manning River…………………... 46 2.8.3. Flooding……………………………………………………………. 47 2.8.4. Artificial Drainage and Flood Mitigation………………………….. 47 2.8.4.1. Artificial Drainage and Flood Mitigation on the lower Hastings River…………………………………………….. 47 2.8.4.2. Artificial Drainage and Flood Mitigation on the lower Manning River……………………………………………. 49

2.9. LANDUSE………………………………………………………………… 49

2.10. CHAPTER SUMMARY………………………………………………….. 50

CHAPTER THREE – COMMERCIAL PRODUCTION OF THE SYDNEY ROCK OYSTER (SACCOSTREA GLOMERATA) IN NEW SOUTH WALES………………………………………………………………….. 51

3.1. INTRODUCTION………………………………………………………... 51

3.2. BIOLOGY AND ANATOMY OF THE SYDNEY ROCK OYSTER…. 51

3.2.1. Systematics and Distribution………………………………………. 51 3.2.2. Anatomy……………………………………………………………. 52 3.2.3. Biology……………………………………………………………... 52 3.2.4. Reproductive Cycle………………………………………………… 55

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3.3. HISTORY OF PRODUCTION………………………………………….. 56

3.3.1. History of the Sydney Rock Oyster Industry………………………. 56 3.3.2. History of the Hastings River Oyster Industry…………………….. 57 3.3.3. History of the Manning River Oyster Industry…………………….. 57

3.4. OYSTER PRODUCTION IN NEW SOUTH WALES………………… 58

3.4.1. Hastings River Oyster Production…………………………………. 59 3.4.2. Manning River Oyster Production…………………………………. 61

3.5. RISK FACTORS FOR OYSTER PRODUCTION…………………….. 62

3.6. DISEASE………………………………………………………………….. 63

3.6.1. QX Disease………………………………………………………… 63 3.6.2. Winter Mortality Disease…………………………………………... 64 3.6.3. Mudworm…………………………………………………………... 64

3.7. DISCUSSION……………………………………………………………... 65

3.8 CHAPTER SUMMARY………………………………………………….. 65

SECTION II - FIELD INVESTIGATIONS…………………………………….. 67

CHAPTER FOUR - SPATIO-TEMPORAL CHARACTERISTICS OF ESTUARINE ACIDIFICATION ON THE HASTINGS AND MANNING RIVERS…………………………………………………………………………… 68

4.1. INTRODUCTION………………………………………………………... 68

4.2. PAST STUDIES…………………………………………………………... 69

4.3. WATER QUALITY MONITORING OBJECTIVES………………….. 74

4.4. METHODS………………………………………………………………... 74

4.4.1. Drain Outflow Water Quality Sites………………………………… 75 4.4.2. Tidal Water Quality Sites…………………………………………... 76 4.4.3. Drain Water Quality Sites………………………………………….. 76 4.4.4. Oyster Lease Water Quality Monitoring Site……………………… 77 4.4.5. Discrete Water Quality Measurements…………………………….. 78 4.4.6. Water Sample Collection and Chemical Analysis…………………. 79 4.4.7. Continuous Water Quality Measurements…………………………. 80

4.5. RESULTS…………………………………………………………………. 80

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4.5.1. Hydrological Conditions…………………………………………… 80 4.5.1.1. Rainfall……………………………………………………. 80 4.5.1.2. Tidal Hydrology…………………………………………... 82 4.5.2. Hastings River Estuary…………………………………………….. 82 4.5.2.1. Drain Outflow Water Quality Following High Rainfall……………………………………………………. 82 4.5.2.2. Tidal Water Quality Following High Rainfall……………. 84 4.5.2.3. Metal Precipitate Mobilisation……………………………. 86 4.5.3. Manning River Estuary…………………………………………….. 87 4.5.3.1. Drain Outflow Water Quality Following High Rainfall……………………………………………………. 87 4.5.3.2. Tidal Water Quality Following High Rainfall……………. 88 4.5.3.3. Metal Precipitate Mobilisation……………………………. 92 4.5.3.4. Drain Water Quality………………………………………. 92 4.5.3.5. Oyster Lease Water Quality………………………………. 93

4.6. DISCUSSION……………………………………………………………... 96

4.6.1. Characteristics of Acidification in the Hastings River…………….. 96 4.6.2. Characteristics of Acidification in the Manning River…………….. 100

4.7. CHAPTER SUMMARY………………………………………………….. 104

CHAPTER FIVE - EXPOSURE OF OYSTERS TO ASS-AFFECTED WATERS: FIELD EXPERIMENT DESIGNS, METHODS AND WATER QUALITY RESULTS…………………………………………………………….. 105

5.1. INTRODUCTION………………………………………………………... 105

5.2. RELATED RESEARCH…………………………………………………. 106

5.3. EXPERIMENTAL DESIGN…………………………………………….. 107

5.3.1. Survival and Growth Experiment (S&GE)………………………… 107 5.3.1.1. Field Sites………………………………………………… 107 5.3.1.2. Sampling Dates…………………………………………… 109 5.3.1.3. Water Quality……………………………………………... 109 5.3.1.4. Experimental Oysters……………………………………... 110 5.3.1.5. Oyster Survival…………………………………………… 110 5.3.1.6. Instantaneous Growth Rate……………………………….. 111 5.3.2. Condition Index Experiment (CIE)………………………………… 112 5.3.2.1. Field Sites………………………………………………… 112 5.3.2.2. Experimental Oysters……………………………………... 112 5.3.2.3. Sampling Dates and Procedures…………………………...113 5.3.2.4. Condition Index…………………………………………... 113

5.4. SURVIVAL AND GROWTH WATER QUALITY CONDITIONS….. 114

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5.4.1. Rainfall……………………………………………………………... 114 5.4.2. pH and EC………………………………………………………….. 115 5.4.3. Dissolved Metals…………………………………………………… 118

5.5. CONDITION INDEX EXPERIMENT WATER QUALITY………….. 118

5.5.1. Rainfall……………………………………………………………... 118 5.5.2. pH and EC………………………………………………………….. 119

5.6. DISCUSSION……………………………………………………………... 121

5.7 CHAPTER SUMMARY………………………………………………….. 122

CHAPTER SIX - SURVIVAL AND GROWTH OF OYSTERS EXPERIMENTALLY EXPOSED TO ASS-AFFECTED WATERS IN THE MANNING RIVER ESTUARY………………………………………... 123

6.1. INTRODUCTION………………………………………………………... 123

6.2. RESULTS AND OBSERVATIONS AT EXPERIMENTAL SITES….. 123

6.2.1. Reference Sites (Sites 1 to 3)………………………………………. 123 6.2.2. Sites Exposed to ASS-Affected Waters (Sites 4 to 7)……………... 128

6.3. THE EFFECTS OF ASS-AFFECTED WATERS ON OYSTER SURVIVAL………………………………………………………………... 134

6.3.1. Results……………………………………………………………… 134

6.4. THE EFFECTS OF ASS-AFFECTED WATERS ON OYSTER GROWTH RATES……………………………………………………….. 138

6.4.1. Results: Whole Weight…………………………………………….. 138 6.4.1.1. Growth Rates During High Rainfall……………………… 138 6.4.1.2. Growth Rates From June to January……………………… 139 6.4.2. Results: Shell Height………………………………………………. 140 6.4.2.1. Growth Rates During High Rainfall……………………… 140 6.4.2.2. Growth Rates From June to January……………………… 142

6.5. OYSTER CONDITION INDEX AT THE EXPERIMENTAL SITES... 143

6.5.1. Results……………………………………………………………… 143

6.6. DISCUSSION……………………………………………………………... 145

6.6.1. Oyster Survival…………………………………………………….. 145 6.6.2. Oyster Growth……………………………………………………… 149

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6.6.3. Oyster Condition Index…………………………………………….. 150

6.7. CHAPTER SUMMARY………………………………………………….. 151

CHAPTER SEVEN – INVESTIGATION OF HASTINGS RIVER OYSTER KILLS………………………………………………………………….. 153

7.1. INTRODUCTION………………………………………………………... 153

7.2. PREVIOUS STUDIES OF HASTINGS RIVER OYSTER KILLS…… 153

7.3. LOWER HASTINGS RIVER AND LIMEBURNERS CREEK WATER QUALITY INVESTIGATION………………………………... 158

7.3.1. Methods……………………………………………………………. 158 7.3.1.1. Water Quality Sampling Sites and Dates…………………. 159 7.3.1.2. Oyster Monitoring………………………………………… 161 7.3.2. Results……………………………………………………………… 161 7.3.2.1. Rainfall……………………………………………………. 161 7.3.2.2. pH…………………………………………………………. 162 7.3.2.3. EC………………………………………………………… 164 7.3.2.4. DO and Temperature……………………………………… 164 7.3.2.5. Water Sample Analysis…………………………………… 165 7.3.2.6. Submersible Data Logger Measurements………………… 165

7.4. OYSTER KILL INVESTIGATION…………………………………….. 168

7.4.1. Date and Location………………………………………………….. 168 7.4.2. Rainfall……………………………………………………………... 168 7.4.3. Characteristics of Affected Oysters………………………………... 169 7.4.4. Histopathology Data From the Oyster Kill………………………… 172

7.5. DISCUSSION……………………………………………………………... 174

7.6. CHAPTER SUMMARY………………………………………………….. 177

SECTION III LABORATORY INVESTIGATIONS…………………………………………... 179

CHAPTER EIGHT – EXPERIMENTAL EXPOSURE OF OYSTERS TO ACIDIFIED WATER: EXPERIMENTAL DESIGNS, MATERIALS AND METHODS………………………………………………………………………... 180

8.1. INTRODUCTION………………………………………………………... 180

8.2. OYSTER VALVE MOVEMENTS……………………………………… 181

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8.3. IMPLICATIONS OF ACIDIFICATION TO OYSTER FEEDING….. 182

8.4. MEASURING OYSTER FEEDING…………………………………….. 183

8.5. FUNCTION AND STRUCTURE OF THE OYSTER GILL………….. 184

8.5.1. Function……………………………………………………………. 184 8.5.2. Structure……………………………………………………………. 184

8.6. FUNCTION AND STRUCTURE OF THE OYSTER MANTLE……... 186

8.6.1. Function……………………………………………………………. 186 8.6.2. Structure……………………………………………………………. 187

8.7. HISTOPATHOLOGY……………………………………………………. 189

8.8. EXPERIMENTAL DESIGN…………………………………………….. 190

8.8.1. Behaviour Experiment……………………………………………... 192 8.8.1.1. Behavioural Response…………………………………….. 192 8.8.1.2. Response of Oyster Soft Tissues…………………………..192 8.8.2. Feeding Experiment………………………………………………... 193

8.9. EXPERIMENTAL EXPOSURE………………………………………… 193

8.9.1. Behaviour Experiment Oysters…………………………………….. 193 8.9.2. Feeding Experiment Oysters……………………………………….. 193 8.9.3. Set-up of the Experimental Apparatus……………………………... 194 8.9.4. Source and Composition of the Test Waters………………………..196 8.9.4.1. Behaviour Experiment……………………………………. 196 8.9.4.2. Feeding Experiment………………………………………. 198

8.10. OYSTER BEHAVIOURAL RESPONSE……………………………….. 201

8.11. FEEDING RATES………………………………………………………... 202

8.11.1. Biodeposit Sampling and Analysis………………………………… 202 8.11.2. Correction for Body Size…………………………………………... 204

8.12. GROSS PATHOLOGY…………………………………………………... 205

8.13. HISTOLOGICAL METHODS AND MATERIALS…………………… 205

8.13.1. Handling and Fixation of Oysters…………………………………. 205 8.13.2. Cutting and Staining of Histological Sections…………………….. 205

8.14. CONCLUSION…………………………………………………………… 206

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CHAPTER NINE - EFFECTS OF EXPERIMENTAL EXPOSURES ON OYSTER FEEDING BEHAVIOUR AND SOFT TISSUE………………... 207

9.1. INTRODUCTION………………………………………………………... 207

9.2. OYSTERS’ BEHAVIOURAL RESPONSE TO ACIDIFIED WATER…………………………………………………………………… 207

9.3. THE EFFECT OF ASS-AFFECTED WATERS ON OYSTER FEEDING BEHAVIOUR………………………………………………… 209

9.3.1. Feeding Activity…………………………………………………….209 9.3.2. Faeces Production………………………………………………….. 210 9.3.3. Rejection Rate……………………………………………………… 210 9.3.4. Filtration Rate……………………………………………………… 211

9.4. POST EXPERIMENT OYSTER SURVIVAL………………………….. 212

9.5. OYSTER SOFT TISSUE RESPONSE TO ACIDIFIED WATER……. 212

9.5.1. Treatment 1 (pH 8.0, No Added Iron and Aluminium)……………. 213 9.5.2. Treatment 2 (pH 5.1, No Added Iron and Aluminium)……………. 213 9.5.3. Treatment 3 (pH 5.1, 7.6 mg L-1 of Aluminium)…………………... 213 9.5.4. Treatment 4 (pH 5.1, 7.7 mg L-1 of Iron)…………………………... 214 9.5.5. Treatment 5 (ASS Affected Waters Adjusted to pH 5.1)………….. 214

9.6. EFFECTS OF IRON PRECIPITATES…………………………………. 216

9.7. DISCUSSION……………………………………………………………... 222

9.7.1. Oysters’ Behavioural Response to Acidified Water……………….. 222 9.7.2. Feeding Behaviour…………………………………………………. 222 9.7.3. Response of Oyster Soft Tissue……………………………………. 224

9.8. CHAPTER SUMMARY………………………………………………….. 226

SECTION IV CONCLUDING CHAPTER……………………………………………………... 228

CHAPTER TEN – CONCLUSION……………………………………………… 229

10.1. MAJOR FINDINGS……………………………………………………… 229

10.2. IMPLICATIONS OF THIS STUDY FOR OYSTER PRODUCTION.. 235

10.3. FURTHER RESEARCH…………………………………………………. 238

X 10.4. FINAL COMMENT……………………………………………………… 240

REFERENCES

APPENDICES

XI LIST OF TABLES

TABLE No. SHORT TITLE PAGE

1.1 Estuary type, oyster production and area of high risk ASS………... 8 1.2 Studies of the response of bivalves to low pH levels………………. 17

2.1 Area of mangrove, seagrass and saltmarsh………………………… 45 2.2 Average annual rainfall data for the study area……………………. 45 2.3 Tidal prisms for selected sites on the lower Hastings River……….. 46 2.4 Tidal prisms for selected sites on the lower Manning River………. 47

3.1 Salinity and temperature ranges for the Sydney rock oyster………. 55

4.1 Studies that have investigated problems associated with ASS…….. 70 4.2 Yeo-Kal Intelligent Water Quality Analyser information…………. 79 4.3 Water quality of selected Hastings River estuary drains…………... 83 4.4 Summary of the Hastings River drain pH data…………………….. 84 4.5 Water quality of selected Manning River drains…………………... 87 4.6 Summary of the Manning River drain pH data (9/5/99)…………… 88 4.7 Variation in drain water quality……………………………………. 93

5.1 Description of Sites 1 to 7………………………………………….. 108 5.2 Sampling dates and field measurements performed……………….. 109 5.3 Summary of pH data for Sites 1 to 7 for the S&GE……………….. 115 5.4 Summary of EC data for Sites 1 to 7 for the S&GE……………….. 116 5.5 Maximum concentrations of Fe, Al and Mn at Sites 1 to 7………... 118 5.6 Water quality at CIE sites………………………………………….. 120

6.1 Summary of the three factor ANOVA results for the S&GE……… 137 6.2 Summary of the three factor ANOVA results for the CIE…………. 145

7.1 Summary of reported Hastings River oyster kills………………….. 156 7.2 pH, EC and temperature data collected by the SDL at Site A……... 168 7.3 Gross clinical signs displayed by oysters exposed to ASS- affected waters and affected by LS………………………………… 176 7.4 Definitions for a suspect case for LS in oysters and impacts caused by ASS-affected waters to oysters…………………………. 177

8.1 pH of the mantle fluid of S. glomerata…………………………….. 188 8.2 Behaviour Experiment and Feeding Experiment details…………... 191 8.3 Oyster shell heights, whole weights and soft tissue dry weights…... 194 8.4 pH, EC and temperature values of Treatments 1 to 5……………… 197 8.5 Concentrations of Fe and Al in Treatments 1 to 5…………………. 197 8.6 Treatment water pH, EC, DO and temperature…………………….. 198 8.7 Concentrations of dissolved ions measured in Treatments 6 to 8….. 199 8.8 Definitions and calculations of oyster feeding components……….. 203

XII PAGE

9.1 Summary of oysters’ behavioural response in Treatments 1 to 5….. 208 9.2 List of accumulation of Fe on oyster soft tissues…………………... 218

XIII LIST OF FIGURES

FIG. No. SHORT TITLE PAGE

1.1 SEM-EDS spectrum: oyster that has not been exposed to ASS…… 20 1.2 SEM- EDS spectrum: oyster exposed to ASS runoff for 7 days…... 20 1.3 Components of the study…………………………………………... 25

2.1 Location of the Hastings River and the Manning River…………… 26 2.2 The Hastings River catchment and landscape units………………... 29 2.3 The Manning River catchment……………………………………... 30 2.4 Hastings River ASS risk map……………………………………… 40 2.5 Manning River ASS risk map……………………………………… 41 2.6 Average monthly rainfall, temperature and evaporation data……… 43

3.1 General anatomy of the Sydney rock oyster……………………….. 53 3.2 Valve description…………………………………………………... 53 3.3 The ten largest oyster-producing estuaries in 1999/00…………….. 58 3.4 Number of bags of oysters produced in NSW……………………... 59 3.5 Hastings River oyster production…………………………………... 60 3.6 Manning River oyster production………………………………….. 61

4.1 The Hastings River estuary and sampling locations……………….. 71 4.2 The Manning River estuary and sampling locations……………….. 72 4.3 Location of water sampling sites on North Oxley Island………….. 77 4.4 Port Macquarie rainfall for the water quality study period………… 81 4.5 Taree Airport rainfall for the water quality study period…………... 81 4.6 Hastings River surface water pH, EC and drain outflow pH………. 85 4.7 Hastings/Maria River surface water pH, EC and drain outflow pH.. 85 4.8 Cattai Creek surface water pH, EC and drain outflow pH…………. 89 4.9 Lansdowne River surface water pH, EC and drain outflow pH…… 89 4.10 Ghinni Ghinni Creek surface water pH, EC and drain outflow pH... 91 4.11 Dickensons Creek surface water pH and EC………………………. 91 4.12 EC and pH at Site W (7/8/99)……………………………………… 94 4.13 pH, EC and temperature measured at Site W by the SDL…………. 95 4.14 Spatial extent of acidification on the Hastings and Manning Rivers. 98

5.1 Locations of oyster and water quality monitoring Sites 1 to 7…….. 108 5.2 S&GE rainfall and sampling dates…………………………………. 114 5.3 pH and EC at Site 2 and Site 4 (26/6/99 to 26/7/99)………………. 117 5.4 CIE rainfall and sampling dates……………………………………. 119

6.1 Site 1 summary display…………………………………………….. 125 6.2 Site 2 summary display…………………………………………….. 126 6.3 Site 3 summary display…………………………………………….. 127 6.4 Site 4 summary display…………………………………………….. 129 6.5 Site 5 summary display…………………………………………….. 130 6.6 Site 6 summary display…………………………………………….. 132 6.7 Site 7 summary display…………………………………………….. 133

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6.8 Mean survival of large oysters at experimental sites………………. 135 6.9 Mean survival of small oysters at experimental sites……………… 136 6.10 Instantaneous growth (whole weight) for day 0 to 83……………... 139 6.11 Instantaneous growth (whole weight) for day 0 to 224……………. 140 6.12 Instantaneous growth (shell height) for day 0 to 83……………….. 141 6.13 Instantaneous growth (shell height) for day 0 to 224……………… 142 6.14 Mean condition index measured at CIE sites……………………… 144

7.1 Locations of reported oyster mortalities…………………………… 155 7.2 Map of the lower Hastings River and Limeburners Creek………… 160 7.3 Rainfall and sampling dates for the study period…………………...161 7.4 EC and pH in Limeburners Creek prior to a rainfall event………… 163 7.5 EC and pH in Limeburners Creek after a rainfall event…………… 163 7.6 EC and pH in the Hastings River prior to a rainfall event…………. 164 7.7 pH, EC and temperature at Site A(I)……………………………….. 166 7.8 pH, EC and temperature at Site A(II)……………………………… 167 7.9 Port Macquarie rainfall recorded in June, July and August 2000….. 169

8.1 Cross section of the left and right gill of C. virginica……………... 185 8.2 Transitional section through a demibranch of C. virginica………... 186 8.3 Transverse section of the mantle showing the lobes……………….. 188 8.4 EC and pH conditions at Site W (4/6/99 to 12/6/99)………………. 191 8.5 Apparatus for exposure of oysters to acidified water……………… 195 8.6 TPM, POM and PIM measured in Treatments 6, 7 and 8…………..201

9.1 Mean feeding activity over a range of pH…………………………. 209 9.2 Mean faeces production over a range of pH……………………….. 210 9.3 Mean rejection rate over a range of pH……………………………. 211 9.4 Mean filtration rate over a range of pH……………………………. 212

XV LIST OF PLATES

PLATE No. SHORT TITLE PAGE

1.1 Bleached and degraded left valve of the Sydney rock oyster……… 18 1.2 Photograph of iron coated and perforated oyster shells……………. 18 1.3 Scanning electron micrographs of healthy and acid exposed oyster shells………………………………………………………… 21

2.1 Downstream view of a floodgate structure………………………… 48

3.1 Adult Hastings River Sydney rock oysters………………………… 54 3.2 Sydney rock oyster with the right valve………...…………………. 54

4.1 A blue/green acidic plume discharging from Fernbank Creek…….. 101 4.2 A red coloured acidic plume discharging from Fernbank Creek…... 101 4.3 Acidic plume discharging from the Maria River…………………... 101 4.4 Acidic plume discharging from Ghinni Ghinni Creek……………... 102 4.5 Acidic plume discharging from the Lansdowne River…………….. 102 4.6 Acidic plume discharging from Cattai Creek……………………… 102

6.1 Appearance of S&GE oysters on the 9/8/99……………………….. 124 6.2 Variation in small oyster shell morphology………………………... 131

7.1 Shell discolouration in the anterior of the left valve……………….. 171 7.2 Yellow pustules in the gonad………………………………………. 171 7.3 Yellow pustules in the labial palps………………………………… 171 7.4 Yellow pustules in the mantle……………………………………… 171 7.5 Chronic lesion in a moribund oyster from Limeburners Creek……. 173

9.1 Soft tissue responses in Behaviour Experiment oysters…………… 215 9.2 Oyster with iron flocs in the shell fluid……………………………. 217 9.3 Oyster with an extensive accumulation of iron flocs on the gills….. 217 9.4 Treatment 1 Behaviour Experiment oysters stained with PPB…….. 219 9.5 Treatment 5 Behaviour Experiment oysters stained with PPB…….. 220 9.6 Treatment 5 Behaviour Experiment oysters stained with PPB…….. 221

XVI GLOSSARY OF TERMS AND ABBREVIATIONS

Adductor muscle – translucent organ for the purpose of closing the oyster shell Aerobic – free oxygen present AHD – Australian height datum; 0 AHD = 0.46 m below mean sea level Anaerobic – free oxygen absent Anterior – the hinge end of the oyster shell ANZECC – Australian and New Zealand Environment and Conservation Council. ANZECC compiled the ‘Australian Water Quality Guidelines for Fresh and Marine Waters’ which are recommendations for managing Australian water resources in a sustainable way. APHA – American Public Health Association ASS – acid sulfate soils; refers to both actual acid sulfate soils and potential acid sulfate soils in this study Bioassay – a measure of the strength of a biologically active substance as it acts on living organisms Biodeposits – oyster faeces; comprised of true faeces and pseudofaeces CI – confidence interval CIE – Condition Index Experiment Cilia – short hair-like structures arranged in groups that beat rhythmically together to create water currents, remove particles from suspension or transport particles Condition index – the ratio of the dry soft tissue weight and the internal shell cavity capacity Davidson’s fixative – solution used for the chemical preservation of oyster soft tissue Demibranch – part of the oyster gill composed of two marginally joined lamellae DLWC – Department of Land and Water Conservation DO – dissolved oxygen (units = percentage saturation or mg L-1) EC – electrical conductivity (units = dS m-1) EDS - energy dispersive x-ray spectrometer Epithelium – the cellular tissue covering surfaces, forming glands and lining most cavities of the body ETOH - ethanol Excessive gaping – oyster valve separation beyond the range of normal feeding Faeces production – total true faeces production per unit of time Feeding activity – the rate of true faeces and pseudofaeces production Filament – a component of each lamella and are arranged in groups to form a plica Filtration rate – amount of particles cleared from a volume of water per unit of time Floc – another word for floccule Flocculation – the aggregation of suspended particles Formalin (10% sea water) – a standard fixative used to preserve oyster soft tissue for pathology GF/C – Whatman glass microfibre filters Greenspan Smart Sonde Model SD300 - brand of submersible data logger manufactured by Greenspan Technical Services Pty. Ltd., Warwick, Queensland H&E –haematoxylin and eosin Haematoxylin and eosin – general tissue thin section stain abbreviated as H&E HCl – hydrochloric acid HDPE – high-density polyethylene

XVII Haemocytes – blood cells of bivalve molluscs; haemocytes have a role in inflammation, wound repair, encapsulation and phagocytosis Haemolymph – fluid containing haemocytes Hinge – the pivot point of the left and right valve located at the anterior of the oyster Histopathology – the study of tissue changes using light microscopy and stained thin sections Holocene – the second and most recent epoch of the Quaternary period, which began approximately 10,000 years ago at the end of the Pleistocene ICPAES - Inductively Coupled Plasma Atomic Excitation Spectroscopy; used for determining the ionic composition of water samples Inflammation or Inflammatory response – the accumulation of exudate and haemocyte cells in irritated tissues to protect from further injury; may be acute or chronic Interlamellar junctions – tissue connections that join filaments at regular intervals Labial palps – large, soft flaps at the gills anterior (mouth) used to control the amount of food ingested as well as sort food before ingestion Lamella – a single arm of the demibranch, which is composed of vertical filaments LS – Limeburners syndrome Lesion – an area of tissue with impaired function due to damage by wounding or disease Mantle – a fleshy fold that covers the internal organs of a bivalve; also called a pallium Mudworm – an oyster disease caused by a spionid polychaete worm (Polydora websteri) Necrosis – death of cells in an organ or tissue caused by disease, physical or chemical injury, or interference with the blood supply NSW – New South Wales Overcatch – settlement of oyster spat on oysters PASS – potential acid sulfate soil Periostracum – a thin organic veneer covering the external surface of the shell; easily removed by abrasion Perls’ Prussian Blue – thin section stain specific for ferric iron abbreviated as PPB pH – a measure of how acidic or alkaline (basic) an aqueous solution is. It is a measure of the hydrogen ion concentration (H+). PIM – particulate inorganic matter Plica – a gill fold composed of filaments Podzol – a type of soil that commonly has a grey to white colour in the upper leached layers and a reddish brown to black B horizon POM – particulate organic matter Posterior – the valve end of the shell PPB – Perls’ Prussian Blue ppt – parts per thousand, units used for salinity Pseudofaeces – particles filtered from suspension by the gills and rejected from the pallial cavity before ingestion PVC – polyvinyl chloride Pyrite – a common mineral that occurs in ASS (iron disulfide: FeS2); the structure 2- contains S2 species QX – stands for ‘Queensland unknown’: a disease that affects the Sydney rock oyster and is caused by the protozoan parasite Marteilia sydneyi

XVIII Rejection rate – total pseudofaeces production per unit of time S&GE – Survival and Growth Experiment Salinity – the amount of salt which estuarine waters contain measured in parts per thousand (ppt). (Salinity in ppt = Electrical Conductivity in dS m-1 x 0.64) SDL – submersible data logger; used for continuous or ‘spot’ measuring of water quality variables such as pH, EC, DO and temperature. SEM – scanning electron microscopy Seston – suspended material (or particles) Sinus – wide channel containing blood (haemolymph) Sloughing – refers to the detachment of tissue layers Spat – larval bivalve molluscs SPSS – statistical software package by SPSS Inc., Chicago TPM – total particulate matter; it is measured in mg L-1 and is equivalent to the dietary abundance for oysters True faeces –particles that are filtered, ingested and move through the digestive tract Umbo – the shell above the hinge constituting the apex of the valve Valves – an oyster shell has a left valve and a right valve; in the Sydney rock oyster the left valve is cupped and the right valve is flat. Winter mortality – a disease that impacts Sydney rock oysters caused by Mikrocytos roughleyi which is proctoctistan parasite Yeo-Kal 611 Intelligent Water Quality Analyser – brand name of a hand held submersible data logger manufactured by Yeo-Kal Electronics, Brookvale, NSW.

XIX ABSTRACT

Estuarine acidification, caused by disturbance of acid sulfate soils (ASS), is a recurrent problem in eastern Australia. Affected waters are characterised by low pH and elevated concentrations of metals, principally aluminium and iron. The effects of acid and elevated metal concentrations associated with ASS, on adult Sydney rock oysters, have not been previously investigated. This study tested links between ASS-affected drainage, subsequent estuarine acidification and Sydney rock oyster production problems on the Hastings and Manning Rivers, mid north coast New South Wales. The primary objective of this thesis was to establish if estuarine acidification causes mortality and slow growth in individual Sydney rock oysters by exposing oysters to low pH, iron and aluminium using field and laboratory experiments.

Water quality data showed that estuarine acidification was spatially extensive in the Hastings and Manning Rivers following heavy rainfall and was due to mineral acids originating from drained or excavated ASS. Estuarine acidification regularly affected areas used for Sydney rock oyster production following heavy rainfall. Field experiments showed that Sydney rock oyster mortality rates were significantly higher at sites exposed to ASS-affected waters compared to locations that were isolated from ASS-affected waters. Oyster mortality increased with the time of exposure and smaller oysters (mean weight = 5 g) experienced significantly higher mortality relative to larger oysters (mean weight = 29 g). This was caused by acid-induced shell degradation resulting in perforation of the smaller oysters’ under-developed shells. Additionally, Sydney rock oyster growth rates were dramatically reduced at sites exposed to ASS- affected waters and the overall mean condition index of oysters at ASS-affected field sites was significantly lower than the overall mean condition index of oysters at non- impacted sites.

Findings from laboratory experiments showed that ASS-affected water alters oyster valve movements and significantly reduces oyster feeding rates at pH 5.5. Acidic treatments (pH 5.1) containing 7.64 mg L-1 of aluminium or ASS-affected water caused changes in the mantle and gill soft tissues following short-term exposure. Degenerative effects described in oysters in this study were also due to iron contained in ASS-affected waters. Iron precipitates accumulated on the shell, gills and mantle and were observed in the stomach, intestine, digestive tubules and rectum. This study concluded that Sydney rock oysters are unable to tolerate acidic conditions caused by ASS outflows and cannot be viably cultivated in acid-prone areas of the estuary.

SECTION I

INTRODUCTION AND BACKGROUND

1 CHAPTER ONE OYSTERS AND ACIDIFICATION: INTRODUCTION AND BACKGROUND TO THE STUDY

1.1 INTRODUCTION The Sydney rock oyster, Saccostrea glomerata (Gould 1850), is cultivated mainly in New South Wales (NSW) and southern Queensland, Australia (Nell, 1993) and is considered a commercially valuable oyster species of the world (Arakawa, 1990). The Sydney rock oyster was formerly known as S. commercialis (Anderson and Adlard, 1994).

Production of the Sydney rock oyster is the largest and oldest aquaculture industry in NSW. Production of this species of oyster has declined dramatically throughout NSW and southern Queensland over the past 25 years. In NSW, oyster production peaked in the late 1970s with more than 145,000 bags of oysters produced in one year but has since decreased to less than 50,000 bags in 1999/2000 (NSW Fisheries, 2001). Possible factors for this marked decline include: QX disease (Wesche et al., 1999), winter mortality disease (Wolf, 1967), declining water quality and in recent times, estuarine acidification (Sammut et al., 1996a; Sammut, 1998). Known factors for production declines vary amongst the estuaries in which the Sydney rock oyster is farmed. For example, winter mortality disease outbreaks have occurred in the southern half of the Sydney rock oyster’s range (Nell, 1993), whereas recurrent outbreaks of QX disease have occurred in southern Queensland, the northern estuaries of NSW and central NSW (Adlard and Ernst, 1995; Adlard and Lester, 1996).

Oysters are entirely dependent on their environment for survival and growth. Temperature and salinity are two important abiotic factors that affect the physiology, stages of development and distribution of oysters (Shumway, 1996). Other environmental factors that influence oysters include: seston (suspended particles) concentrations, light and pH (Shumway, 1996). Oyster growers are unable to control water quality and hydrological conditions in the estuary and are limited to stock movement to protect oysters from adverse environmental conditions. Because an oyster

2 is sessile, it has a limited capacity to avoid physical changes in its environment. An oyster experiences gradual and abrupt variations in environmental conditions that occur over a range of different time scales. Long-term changes in water quality have significant spatio-temporal implications for an oyster.

Sydney rock oysters inhabit estuaries which provide a well-buffered environment that, under natural conditions, only has minor fluctuations in pH (Bamber, 1987; Sammut et al., 1996a). Estuaries on the east coast of Australia generally have pH levels in the range of 6.5 to 8.5. The pH has a dominant influence over estuarine water chemistry because it controls the chemical form of metals and the characteristics of dissolved substances (Knutzen, 1981). Many marine algae, bacteria and animal species have a very low tolerance to minor pH perturbations (Knutzen, 1981).

Overseas research has shown that only 0.5 unit changes in pH can cause detrimental effects in bivalves (Loosanoff and Tommers, 1947; Kuwatani and Nishii, 1969; Bamber, 1987; 1990). In eastern Australia, oysters are mostly farmed in the intertidal zone of estuaries that are episodically, and in some reaches, chronically, affected by severe acidification. Water pH can fall to levels below pH 3 due to the outflow of acid from agricultural land on drained acid sulfate soils (ASS) (Sammut et al., 1996a). Currently, there is limited information on the tolerance of the Sydney rock oyster to acidification of this magnitude.

Most estuaries on the east coast of Australia experience acidification after an extended dry season followed by high rainfall (Sammut et al., 1995; Johnston, 1995). ASS are coastal lowland soils that contain iron pyrite (FeS2) (Dent, 1986). When ASS are drained or excavated, the exposure of pyrite to oxygen triggers oxidation and generates sulfuric acid (Dent, 1986), which is transported to nearby waterways during wet periods causing estuarine acidification (Sammut et al., 1996a). The sulfuric acid generated also mobilises iron, aluminium and manganese from the soil, and these are known to have a deleterious effect on exposed aquatic biota and habitat (Sammut et al., 1995).

Agriculture has accelerated the exposure of pyrite through extensive land drainage of coastal floodplains containing ASS (Sammut et al., 1995). This has greatly facilitated

3 the transport of ASS-affected waters to the estuary resulting in recurrent and widespread acidification of waterways following high rainfall (Sammut et al., 1996a; White et al., 1996a). A detailed description of ASS, their formation and their characteristics is provided in Chapter 2.

Estuarine acidification has severely degraded aquatic ecosystems on the east coast of Australia (Sammut et al., 1996a). The long-term impacts of acidification to the estuarine ecosystem are unknown (White et al., 1996a). Estuarine acidification threatens the biodiversity, amenity, fisheries production and the overall value of an estuary (Sammut et al., 1995). Recent studies have unequivocally demonstrated that the hydrogen ions present in ASS-affected waters rapidly damage the gills and skin of fish causing massive fish kills and resulting in the initiation of fish disease (Callinan et al., 1993; 1996; Sammut et al., 1996a; Sammut, 1998).

The effects of estuarine acidification to the Sydney rock oyster are, at present, not described in detail. Wilson and Hyne (1997) conducted the initial research and discovered that ASS leachate caused abnormal embryonic development in Sydney rock oyster larvae. Subsequent work by Dove (1997) revealed that there is a link between ASS-affected waters and oyster shell degradation, injury to the soft tissue and mortalities. However, it is unclear if unexplained production problems experienced in particular NSW estuaries are associated with ASS-affected waters. Oyster growers have reported mortalities and slow growth and believe these problems are linked to ASS- affected waters.

The Hastings River and Manning River are two important oyster producing estuaries located on the mid north coast of NSW, where declines in oyster production have been blamed on ASS-affected waters. Abandoned oyster leases in areas showing field characteristics of ASS-affected waters are common to both of these estuaries. Neither estuary is densely populated and the Manning River estuary is not affected by QX disease, which has had a profound effect on oyster populations in other estuaries (Anderson and Lester, 1992). These features make both the Hastings and the Manning River estuaries ideal locations for the field investigations for this study.

4 Oyster growers on the Hastings River initially reported an increase in mortality events and production problems and attributed them to estuarine acidification. This was shortly after a link between estuarine acidification and fish kills and disease had been established and ASS were becoming a prominent coastal issue in NSW (Sammut et al., 1996b; Callinan et al., 1996; Findlater, 1996; McKenzie, 1996). However, oyster growers had no information or evidence on which to base or substantiate their claims.

Callinan (1997a) described a new condition called ‘Limeburners syndrome’ (LS) that was the underlying cause of some, but not all of the mortalities on the Hastings River. LS appears to be unrelated in a direct sense to estuarine acidification (Callinan, 1997a). However, LS and acidification caused similar gross clinical signs, which were slow growth and mortality. Individual oysters affected by LS displayed different clinical signs to oysters exposed to acidification when examined more closely. Therefore, there were two conditions present on the Hastings River with common overall features that were easily differentiated at the individual oyster level. Hence, it was necessary for this study to define a case for impacts associated with both ASS-affected waters and oyster kills linked to LS to ensure that these are mutually exclusive.

The Manning River was used for this study, in combination with the Hastings River, to provide a study area that experiences estuarine acidification (Sonter, 1999) but has had no reported cases of LS. A study area not affected by LS was necessary for this present study because a background inflammatory condition, which was consistently present in Hastings River oysters and believed to be from LS, hampered the histopathology of acid-induced soft tissue injury in Dove’s (1997) study.

Oyster growers on the Hastings River and Manning River have associated recurrent production losses with periodic acidification of the lower estuary. They have drawn this association from a number of observations made at affected leases. These observations include: • red/ochre staining of oyster shells; • thin, brittle and pale grey shells; • shell bleaching with no evidence of growth scales or new shell growth protruding from the posterior margin of the oyster;

5 • shell perforation occurring in the left valve; • red/ochre staining of the oyster soft tissue, concentrated on the gills and often combined with discolouration of the shell fluid; • high oyster mortality relative to other sites; • slow oyster growth relative to other sites; • plumes of discoloured water passing over leases, particularly after rainfall; • red/ochre staining of trays and racks used for oyster production; and • rapid rusting of submerged galvanised metal relative to other sites.

Because the impacts of ASS-affected waters on oysters are largely unknown, this lay knowledge or observed information will be used to develop the hypotheses and objectives for this study. Sydney rock oysters located in particular areas of the estuary display a combination of signs and symptoms that strongly indicates the existence of a serious environmental problem. This present study will use a geographical approach for testing links between ASS, land drainage, estuarine acidification and problems in oysters and this requires the use of interdisciplinary methods to underpin the investigations.

The oyster growers’ observations are different to the main clinical signs of LS and other known oyster diseases. LS in non-acid areas is not associated with red/ochre staining and does not cause shell bleaching and perforation of oyster shells. Some of the observations listed above are strongly associated with field conditions that characterise ASS-affected waters (Sammut, et al., 1996a). The overriding aim of this study is to examine the impacts of acidification caused by ASS-affected waters on the survival and growth of representative, individual Sydney rock oysters. By looking at the impacts of ASS-affected waters to individual oysters it will possibly generate further hypotheses on the impacts of ASS-affected waters to oyster populations and production.

The Hastings and Manning River catchments are classified as mature, infilled barrier estuaries (Roy, 1984a), have expansive areas of ASS located in the coastal plain areas and are included in the top ten oyster producing estuaries of NSW (Table 1.1). Therefore the Hastings and Manning Rivers are ideal for this research and the information obtained from these estuaries will be applicable to all other oyster

6 producing estuaries that are affected by acidification. Table 1.1 lists the oyster producing estuaries in NSW and the estimated areas of high-risk ASS contained in the floodplain.

To date there have been very few scientific studies undertaken on the effects of acidification on bivalves and specifically the Sydney rock oyster. Overseas studies have investigated the impacts of acidified water on a small number of bivalve species and have reported on the effects of relatively small pH variations (Loosanoff and Tommers, 1947; Calabrese and Davis, 1966; Kuwatani and Nishii; 1969; Bamber, 1987; 1990). The magnitude and extent of the pH perturbation in eastern Australian estuaries resulting from ASS-induced acidification is unprecedented in these overseas studies. Problems associated with estuarine acidification on oyster production have not been sufficiently examined and oyster growers have justifiable concerns. Dove’s (1997) work, which has already established an association between acidification and oyster problems, was limited and recommended a more detailed investigation of this problem.

7 Table 1.1 Estuary type and the area of high-risk ASS in the catchments of oyster producing estuaries in NSW (Source: adapted from West et al., 1985; NSW Fisheries 2000; and, Naylor et al., 1995).

Oyster Estuary Total Oyster Area of High Risk Producing Type Production: 1999/2000 ASS in Catchment Estuary (Dozens) (Hectares)

Tweed River Drowned River Valley B/C 65,982 9700 Brunswick River Barrier Lagoon D 0 3193 Richmond River Barrier Lagoon D - 34195 Clarence River Barrier Lagoon D & C - 53043 Wooli River Barrier Lagoon D - 15455 Bellinger River Barrier Lagoon D 54,557 Nambucca River Barrier Lagoon D 128,846 - Macleay River Barrier Lagoon D 66,047 31644 Hastings River Barrier Lagoon D 233,606 21737 Camden Haven Barrier Lagoon C 155,430 Manning River Barrier Lagoon D 208,840 16884 Wallis Lake Barrier Lagoon B 2,360,870 19069 Port Stephens Drowned River Valley A & B 601,134 Hunter River Barrier Lagoon C/D 57,345 26947 Brisbane Waters Barrier Lagoon A/B 871,324 8273 Hawkesbury River Drowned River Valley A & B 1,057,542 Botany Bay Coastal Lagoon B/C 189,735 2979 Georges River Drowned River Valley B 32,667 Shoalhaven River Barrier Lagoon D 30,504 7584 Crookhaven River Barrier Lagoon C 145,539 Conjola River Barrier Lagoon A nd 4114 Clyde River Drowned River Valley B/C 540,085 Tomaga River Barrier Lagoon D nd - Tuross Lake Barrier Lagoon C 169,540 1601 Wagonga Inlet Barrier Lagoon A 241,515 Wallaga Lake Barrier Lagoon A nd Bermagui River Barrier Lagoon C/D 26,580 948 Wapengo Lake Barrier Lagoon B/C 89,264 Nelson Lagoon Barrier Lagoon C 15,000 Merrimbula Lake Barrier Lagoon A/B 308,610 Pambula River Barrier Lagoon B/C 103,163 570 Wonboyn River Barrier Lagoon C 71,754

A to D refer to the degree of maturity or infilling of the estuary. A-little infilling, D-very infilled.

1.2 ESTUARINE ACIDIFICATION 1.2.1 Past Studies on Estuarine Acidification Estuarine acidification associated with ASS is known to occur throughout Asia, in Africa and parts of Europe and North America (Klepper et al., 1992; Galle and Montoroi, 1993; Astrom and Bjorkland, 1995; Soukup and Portnoy, 1986). Brackish water aquaculture development in many tropical areas has expanded into coastal lowland areas and mangrove forests where soils and sediments have high concentrations of pyrite. This has resulted in numerous aquaculture production problems and disease outbreaks in stock associated with acidification of pond water (Sammut, 2000).

The first investigations into estuarine acidification in Australia were by Brown et al. (1983) and Hart et al. (1987). A large natural fish kill in a billabong on Magela Creek, Northern Territory, led to a water quality investigation to identify the cause. The fish kill occurred in areas affected by acidic water flowing into the billabong. The source of the acidic water was from “black soils” which had a pH of 3.5-3.9 and contained pyrite. The pyrite oxidation occurred in the dry season and monsoonal rains in the wet-season, mobilised the leached sulfuric acid, major ions and trace metals into the billabong. This created natural acidification of the billabong but had dire implications for the fish life and aquatic biota inhabiting the billabong. However, the work did not undertake a pathology study to establish a conclusive link.

In 1987, a series of large-scale fish kills on the Tweed River, in northern NSW, coinciding with low pH and dissolved oxygen levels, alerted authorities to the wide reaching effects of estuarine acidification on the east coast of Australia (Easton, 1989). Since this time, it has been demonstrated that estuarine acidification events in this region of Australia are from human induced alterations to the floodplain hydrology (White et al., 1996a). Studies on the Tweed and Richmond Rivers in Northern NSW implicated artificial drainage and flood mitigation works that facilitate pyrite oxidation and mobilise the oxidation products to nearby waterways (Lin and Melville, 1992; Sammut et al., 1996a; Sammut, 1998). In south eastern Australia, the extent of estuarine acidification is largely determined by the interim dry period and the intensity of the rainfall event (Johnston, 1995; Sammut, 1998).

Johnston (1995) and Sonter (1999) investigated the spatial and temporal variability of estuarine acidification in particular reaches of the Hastings River and Manning River, respectively. These two studies are discussed in more detail in Chapter 4. A Queensland study was conducted at East Trinity, near Cairns and adjacent to the Great Barrier Reef Marine Park, by Hicks et al. (1999). At East Trinity, mangrove swamps were drained for sugar cane production and this resulted in severe degradation of the area. Acidification of the land and adjacent waterway occurred and it was estimated that 72,000 tonnes of sulfuric acid equivalent has been released since 1976.

9 Pease et al. (1997) studied ASS and acidification of drainage on the Shoalhaven River located on the south coast of NSW. Department of Land and Water Conservation ASS mapping program has allowed areas prone to estuarine acidification to be more readily identified (Naylor et al., 1995). There have also been several Australian studies that primarily investigate the ecological implications of estuarine acidification, and these are detailed in Section 1.2.3.

1.2.2 Chemistry of Waters Affected by ASS ASS outflows are very acidic (pH 2.5 to 5) and are similar to the chemical characteristics of their source material. Elements commonly associated with ASS- affected waters include SO4, Ca, Si, Mg, Na, Al, K, Fe, Zn, Cu, B, Mn and Cl (Sammut et al., 1996a). The concentration of these elements is largely contingent on dilution of the outflow by upland flows, mixing with estuary waters and the pH dependent solubility of the particular element - this is especially the case for zinc, aluminium and iron (Sammut et al., 1996a).

3+ 2+ + Aluminium species (Al , Al[OH] , and Al[OH]2 ) are mobilised to the estuary where ASS-affected water lowers pH (Sammut et al., 1995). High concentrations of up to 90 mg L-1 were measured in the Richmond River in certain areas where the pH was less than 5.4. Driscoll et al. (1980) found that 0.1 mg L-1 of aluminium in acidified water was toxic to fish and other aquatic life. When the pH of ASS-affected waters exceeds 6, white flocs of aluminium hydroxide can sometimes be observed (Sammut et al., 1995). ASS-affected waters can appear milky, green-blue or extremely clear and is caused by low pH and high levels of aluminium (White and Melville, 1993).

Iron is another dominant element in ASS-affected waters and causes the water to appear a rusty red-brown colour. Soluble ferrous iron (Fe2+) is usually present in ASS-affected waters at pH values less than 4 and if there is an increase in pH with available oxygen, it can oxidise to form ferric iron (Fe3+). This reaction generates additional acidity (Dent, 1986; Simpson and Pedini, 1985; Sammut et al., 1995). Ferric iron is insoluble at pH values greater than 3 and forms colloidal hydroxides and oxyhydroxides which are referred to hereafter as iron precipitates or iron flocs. Iron precipitates coat streambeds

10 and benthic communities and can be transported many kilometres from their source (Sammut et al., 1996a).

1.2.3 Impacts on Aquatic Biota As mentioned previously, estuarine acidification has been responsible for large-scale fish kills in Australia (Brown et al., 1983; Easton, 1989; Callinan et al., 1993; Sammut, 1998). The subsequent investigation of these fish kills has essentially been responsible for estuarine acidification gaining the recognition it deserves in eastern Australia.

Ecological impacts of estuarine acidification reduce the environmental, recreational and commercial value of estuaries (Sammut et al., 1995). The extent of ecological impact arising from estuarine acidification is dependant on an intricate combination of factors which include, but are not limited to: the type of physical environment that is disturbed; the manner and the size of the development; the capacity of the receiving waters to neutralise or dilute the acid; the concentration and mobility of the oxidation products; the local hydrology and groundwater systems; and, the sensitivity of the receiving waters and their biota (Smith et al., 1999).

Acid and aluminium originating from ASS drainage causes severe skin and gill damage in exposed fish (Sammut et al., 1995; 1996b). Histopathology of fish gills exposed to acid and aluminium showed lamellar fusion, cell necrosis and increased mucus production that disturbed gas exchange and osmoregulation (Sammut et al., 1996b). Mortality in exposed fish was attributed to low oxygen levels in the blood and osmoregulatory stress.

Sammut et al. (1995; 1997) demonstrated that fish sub-lethally exposed to ASS-affected waters experience skin damage. An oomycete fungus, Aphanomyces invadans, infects areas of acid-induced skin damage to cause epizootic ulcerative syndrome (EUS), also known as red spot disease (Sammut et al., 1995; Callinan et al., 1996). EUS outbreaks can cause significant commercial losses to professional fisherman because the red skin ulcer makes the fish unsaleable. Fish recruitment can also be impaired by acid water because it can create a barrier to migration (Sammut et al., 1993) and destroy fish eggs,

11 which are particularly sensitive to acid exposure (Wendelaar Bonga and Dederen, 1986).

Estuarine acidification can also affect waterplant communities by: clarification, which increases light penetration; toxicity from dissolved metals; and, smothering by oxidation products (Sammut et al., 1996b). Proliferation of acid tolerant species such as the introduced Cape waterlily (Nymphaea caerulea) displaces native waterplants, supersaturates the water with dissolved oxygen and constricts waterways (Sammut et al., 1994).

Roach (1997) and Corfield (2000) studied the effects of ASS-affected waters on macrobenthic and fish communities of the Richmond River, NSW. Roach (1997) observed effects on benthic communities at sites that received acid inflows, but also noted similar changes at sites that were not affected by acid and attributed this change to inflows of freshwater. Certain sites displayed indications that they were chronically affected by acid water. The effect of lowered pH was found to be most significant when it occurred for extended durations, which prevented recolonisation and physically changed the habitat. The response of the ecosystem to acid inflows was found to be location specific. The macrobenthos responded to the chemical speciation of aluminium at certain pH ranges and this significantly affected species abundance at a site in the Tuckean Broadwater (Corfield, 2000).

1.3 PAST STUDIES ON ACIDIFICATION AND OYSTERS 1.3.1 Overseas Studies Overseas studies related to acidification and bivalves do not involve ASS, however, they provide an insight into the probable impacts of ASS-affected waters on Sydney rock oysters and a platform to base the design of experiments for this current study. As previously mentioned, overseas studies have found that detrimental impacts occur in other species of bivalves at only minor perturbations in pH (Loosanoff and Tommers, 1947; Kuwatani and Nishii, 1969; Bamber, 1987; 1990). Estuarine acidification can cause a thousand-fold increase in the level of acidity relative to normal conditions, therefore it is anticipated that similar, but more severe, impacts will occur in the Sydney rock oyster.

Loosanoff and Tommers (1947) recorded increased pumping rates (the flow of water through the gills per unit of time) at pH values between 7.0 and 6.75, but when the pH dropped below 6.5, pumping rates dramatically decreased in adult Ostrea virginica (eastern oyster). Loosanoff and Tommers (1947) also observed abnormal shell movements when pH was less than 6.5. Calabrese and Davis (1966) found growth was inhibited at pH values less than 6.75 and abnormal development occurred at pH values below 6.0 in eastern oyster (Cassostrea virginica) larvae.

Bamber (1987; 1990) investigated the effects of acidification as a consequence of inflows of large quantities of slightly acidic fresh water or industrial pollution on four bivalve species. Bamber (1987) measured feeding inhibition and a significant reduction in tissue and shell growth in young carpet shell clams (Venerupis decussata) at pH values below 7.0. Also, at pH values below 6.5, mortality dramatically increased during the experiment with smaller clams being more sensitive to the acidic conditions. Bamber (1990) investigated the effects of acidic conditions on the Pacific oyster (Crassostrea gigas), the native mussel (Mytilus edulis) and the native oyster (Ostrea edulis) and concluded that a pH less than or equal to 7 is detrimental to these bivalve molluscs. Significant mortalities were measured at: pH < 6 in C. gigas after 30 days exposure; pH 6.6 in M. edulis after 30 days exposure; and, pH 6.9 in O. edulis after 60 days exposure (Bamber, 1990). For C. gigas, inhibition of feeding, reduced shell growth and decreases in flesh weight occurred below the critical pH of 7.0 and behavioural inhibition was observed below pH 6.5 (Bamber, 1990). Bamber (1990) found feeding inhibition occurred below pH 7.2 in M. edulis and O. edulis. Feeding inhibition measured by Bamber (1987; 1990) and reduced adult pumping rates measured by Loosanoff and Tommers (1947) in other bivalve species strongly suggest that feeding in the Sydney rock oyster is likely to be impaired by exposure to ASS- affected waters.

Sunila (1986a) investigated the changes in the soft tissue in M. edulis caused by the discharge waters of a titanium dioxide plant located near Pori, Finland. Polluted water from the plant discharged into seawater in the southern part of the Gulf of Bothnia, 4.7 kilometres offshore from Pori. The discharge waters were very acidic (pH 1) and

13 contained sulphuric acid, ferro-sulphate, titanium dioxide, aluminium, mangane, vanadine, zinc, chrome, nickel and cobalt. This caused severe pollution of an area of 8 km2 and ferric hydroxide flakes were spread over a region of approximately 120 km2. Histopathology was used to evaluate health and disturbances to the mussel resulting from exposure to these discharge waters. Histopathology detected changes in the structure of the mature ova as a response to low pH. The gills of the mussels (M. edulis) examined showed no histopathological changes. On the basis of these and findings from other studies, Sunila (1986a) concluded that histopathologic analysis showed promise for the study of pollution problems in brackish waters.

1.3.2 Effects of ASS-Affected Waters on the Sydney Rock Oyster Previous research on the effects of ASS-affected waters on the health of the Sydney rock oyster is limited to studies by Wilson and Hyne (1997) and Dove (1997). Research has been conducted on ASS-induced acidification and its role in Sydney rock oyster disease outbreaks. Anderson et al. (1994), Wesche (1995) and Dove et al. (2002) have investigated QX disease outbreaks in oysters and its relationship to acidification. All of these studies concluded that a drop in pH is not a necessary factor for a QX outbreak to occur and if acidification occurs at a particular site prior to an outbreak of this disease it does not increase its severity. There has been no work to date to examine the effects of ASS-affected waters on the immune system of the Sydney rock oyster.

Wilson and Hyne (1997) investigated the toxicity of ASS-affected water, pH-adjusted seawater and aluminium to early embryonic development of Sydney rock oysters. Wilson and Hyne’s (1997) bioassay experiments showed that abnormal embryonic development resulted when larvae were exposed to treatments containing > 3.3% ASS leachate in seawater and when pH dropped below 6.75 in pH adjusted seawater containing no ASS leachate. A significant decrease of early embryonic development also occurred when aluminium was elevated above 150 µg L-1 in pH normal conditions.

Dove (1997) determined that exposure of the Sydney rock oyster to acidification caused shell dissolution and mortality. This occurred when adult oysters were exposed to naturally acidified water with an approximate pH range of 3 to 6.5 in an acidified creek over a 90-day period. No oysters were alive at the end of the experiment. Larger sized

14 oysters (mean shell height = 58.6 mm) had a higher mortality rate during the initial 40 days of the experiment. Smaller sized oysters (mean shell height = 40.8 mm) experienced dramatic mortality at day 40, which was attributed to shell dissolution and perforation. Mortality from shell dissolution is discussed in greater detail in Section 1.3.2.1.

Dove (1997) also used bioassays to expose oysters to artificially and naturally acidified water to monitor oyster behaviour and to apply histopathology. When artificially acidified water was used and salinity remained constant at 25 parts per thousand (ppt), a rapid decline in pH caused the oysters to cease feeding and close their valves. The critical pH at which most oysters closed their valves was between 3 and 4. A rapid pH drop to 5 did not cause the same response and oysters kept their valves open. Naturally acidified water caused the pH and salinity to fall to 3.8 and 8 ppt, respectively, which triggered shell closure. When oysters were exposed to pH < 5 for extended periods, excessive gaping of the valves was observed. When these oysters were subjected to a tactile stimulus they gradually closed their valves.

Dove’s (1997) histopathology investigations revealed an inflammatory response in soft tissues, particularly in the gills and mantle, in oysters exposed to artificially acidified water. However, further work is required on the histopathology of oysters exposed to acidified treatments as the results from Dove (1997) were obscured by background inflammatory cells located predominately in the mantle associated with LS (R. Callinan, NSW Fisheries, personal communication, 1997).

The work of Dove (1997) was the first study to find an association between ASS- affected waters and impacts to adult Sydney rock oysters. However, Dove’s (1997) work was limited and recommended that the effects of ASS-affected waters on oyster health be examined in greater detail. Dove’s (1997) earlier work underpins this current study.

A related investigation to the present study examined the role of acidification and oxidation products on the settling behaviour of Sydney rock oyster spat (Bishop, 2000). Bishop (2000) concluded that iron precipitation caused a decline in spat numbers and

15 impacted recruitment patterns of oyster spat. Histopathology data from Bishop’s (2000) investigation revealed that iron precipitates accumulated on the gills and soft tissue of exposed oysters. The gills appeared to be the primary target organ for iron accumulation.

Table 1.2 summarises the effects of acidification to bivalves and lists the critical pH at which these effects become manifested. From Table 1.2, increased shell dissolution is a common problem reported by these studies. Dove (1997) investigated shell dissolution in Sydney rock oysters resulting from exposure to ASS-affected waters, which is discussed in the following section.

1.3.2.1 Shell Dissolution Kuwatani and Nishii (1969), Bamber (1987; 1990) and Dove (1997) all report shell dissolution in their respective studies (Table 1.2). Dove (1997) showed that under moderate to severe acidity, bleaching and perforation degrades the shell condition of the Sydney rock oyster. Shell bleaching and perforation are the results of shell dissolution.

Shell dissolution was found to occur in other species of bivalves when decreases in pH were only very minor. Kuwatani and Nishii (1969) discovered shell dissolution starts to occur at a pH of 7.6 in the Japanese pearl oyster (Pinctada fucata). Bamber (1987; 1990) showed that shell dissolution occurs at a pH of 7.5 in carpet shell clams (V. decussata), and at pH 7.0 for the native oyster (O. edulis), the Pacific oyster (C. gigas) and the native mussel (M. edulis).

Table 1.2 shows that deleterious effects to many bivalve species start to occur in circumneutral waters. The pH values reported by these studies in some cases were several magnitudes less acid than conditions caused by ASS-affected waters. It is, therefore, not surprising that bleaching (Plate 1.1) and deterioration of the shell (Plate 1.2) occurred in Dove’s (1997) study which exposed Sydney rock oysters to ASS- affected waters that frequently had pH values less than 4.

Table 1.2 Studies of the response of bivalves to low pH levels (Source: modified from Bamber, 1990).

Species Effect Critical pH Authority

M. edulis Reduced gamete respiration 7.60 Akberali et al. (1985) Inhibition of feeding 7.20 Bamber (1990) Inhibition of shell growth 7.00 Bamber (1990) Flesh weight reduction 7.00 Bamber (1990) Increased shell dissolution 7.00 Bamber (1990) Behavioural inhibition 6.50 Bamber (1990) Increased mortality 6.50 Bamber (1990)

P. fucata Increased adult mortality 7.48 Kawatani & Nishii (1969) Increased weight loss attributed 7.66 Kawatani & Nishii (1969) to shell dissolution

C. virginica Adult reduced pumping rate 7.00 Loosanoff & Tommers (1947) Abnormal shell movement 7.00 Loosanoff & Tommers (1947) Increased larval mortality 7.00 Calabrese & Davis (1966) Inhibited larval development 6.75 Calabrese & Davis (1966) Reduced larval growth 6.75 Calabrese & Davis (1966)

M. mercenaria Increased larval mortality 6.50 Calabrese & Davis (1966) Inhibited larval development 7.00 Calabrese & Davis (1966) Reduced larval growth 6.75 Calabrese & Davis (1966)

V. decussata Inhibition of feeding 7.00 Bamber (1987) Inhibition of shell growth 7.00 Bamber (1987) Flesh weight reduction 7.00 Bamber (1987) Increased shell dissolution 7.50 Bamber (1987) Behavioural inhibition 6.00 Bamber (1987) Increased mortality 6.1-6.4 Bamber (1987)

C. gigas Inhibition of feeding 7.00 Bamber (1990) Inhibition of shell growth 7.00 Bamber (1990) Flesh weight reduction 7.00 Bamber (1990) Increased shell dissolution 7.00 Bamber (1990) Behavioural inhibition 6.50 Bamber (1990) Increased mortality 6.00 Bamber (1990)

O. edulis Inhibition of feeding 7.20 Bamber (1990) Inhibition of shell growth 6.80 Bamber (1990) Flesh weight reduction 6.80 Bamber (1990) Increased shell dissolution 6.80 Bamber (1990) Behavioural inhibition 6.50 Bamber (1990) Increased mortality 7.00 Bamber (1990)

S. glomerata Inhibited embryonic development 6.75 Wilson & Hyne (1997) Behavioural inhibition 6.00 Dove (1997) Increased shell dissolution 6.5-3.0* Dove (1997) Increased mortality 6.5-3.0* Dove (1997) Injury to soft tissue 5.7 Dove (1997)

* approximate pH range measured during field experiment

Plate 1.1 A severely bleached and degraded left valve of a Sydney rock oyster exposed to ASS-affected waters for 39 days (iron coating has been removed). Plate 3.1 shows the appearance of healthy Sydney rock oysters.

Plate 1.2 Photograph of iron coated oyster shells with perforation in the anterior of the left valve resulting from internal and external shell dissolution. The diameter of the coin = 28.4 mm.

A process of internal shell dissolution can also be induced by prolonged exposure to ASS-affected waters. Dove (1997) showed that oysters respond to acidified water by remaining closed for the duration of exposure. When valves are closed, the carbon dioxide produced by an oyster decreases the pH of the hemolymph (Galtsoff, 1964). Morrison (1993) reported that shell dissolution of the internal valve surface occurs in anaerobic conditions, when acids form as a result of metabolism and dissolve intracellular deposits of calcium carbonate in the mantle and the inside of the shell. Dwyer and Burnett (1996) described this process as shell decalcification and results in internal shell dissolution.

Dove (1997) noted that internal shell dissolution when combined with external shell dissolution from acidified water, eventually leads to shell breakthrough or perforation in the anterior of the left valve of the oyster (Plate 1.2). Once shell perforation has occurred the soft tissue has no protection from the shell and is exposed directly to ASS- affected waters resulting in rapid death of the oyster. Mortalities occurred in all of Dove’s (1997) oysters that experienced shell perforation.

Dove (1997) examined the ultrastructural and chemical composition of shells using scanning electron microscopy (SEM). SEM revealed that the external layer of shell, known as the periostracum, was dissolved by the acid and exposed the underlying shell matrix. Bamber (1987) observed very similar effects in the soft shell clam, V. decussata. This protective layer of the shell also includes the pigment of the shell. The loss of this pigmented layer and the effects of acid on the exposed underlying shell matrix explains the shell bleaching observed at acid sites in Dove’s (1997) study. Energy dispersive x-ray spectra (EDS) analysis (Figures 1.1 and 1.2) by Dove (1997) clearly shows the principal elements stripped from the shell whilst SEM micrographs demonstrate the associated breakdown of the periostracum and the exposure of the smooth underlying shell matrix (Plates 1.3 and 1.4).

Figure 1.1 SEM-EDS spectrum obtained from the surface layer of an oyster that has not been exposed to ASS-affected waters (Source: Dove, 1997).

Figure 1.2 SEM-EDS spectrum obtained from an oyster exposed to ASS-affected waters for 7 days (Source: Dove, 1997).

20 A. B

C. D.

Plate 1.3 Scanning electron micrographs showing: (A and B) a healthy oyster shell with a rough textured surface layer; and, (C and D) an oyster shell impacted by ASS- affected waters with a smooth surface and exposed underlying shell layers (Source: Dove, 1997).

21 1.4 OBJECTIVES AND HYPOTHESES Dove’s (1997) work focussed on investigating shell degradation, survival and soft tissue injuries from acidity and recommended that further work is required to better understand the effects of ASS-affected waters on the Sydney rock oyster. Overseas studies that have investigated the effects of acidity on bivalves relate to different species and reported relatively small changes in pH compared to what is experienced in eastern Australia.

The overriding aim of this study is to examine, using field and laboratory trials, the impacts of acidification on the survival and growth of representative, individual Sydney rock oysters. The overriding aim will be achieved by the specific objectives:

1. To identify and measure sources of acidification and describe the spatial characteristics of estuarine acidification in two oyster-producing estuaries; 2. To investigate the temporal characteristics of estuarine acidification in an area used for Sydney rock oyster production; 3. To examine survival rates of adult oysters at locations that have a high probability of exposure to ASS-affected waters compared to sites that have a low probability of exposure to ASS-affected waters; 4. To determine if smaller oysters are impacted by ASS-affected waters to a greater extent than larger oysters; 5. To measure the growth rates of adult oysters at locations that have a high probability of exposure to ASS-affected waters; 6. To further investigate production problems associated with LS which are experienced in the Hastings River; 7. To investigate gill and mantle soft tissue changes resulting from exposure to ASS-affected waters; 8. To determine the physiological response of the Sydney rock oyster, in terms of feeding behaviour, when exposed to ASS-affected waters; and 9. To examine the effects of iron precipitates on the soft tissue of the Sydney rock oyster.

22 The objectives listed above can be divided into three sections. Objectives 1 and 2 are important to this study because it is necessary to establish the range of water quality conditions that oysters are exposed to. There is limited information relating to the spatio-temporal characteristics of acidification in the Manning River and Hastings River. This information will also confirm the validity of oyster grower’s observations in relation to oysters and exposure to acidity. Objectives 3 to 5 relate to the field investigation section where Sydney rock oysters were exposed to ASS-affected waters. These two objectives utilise the water quality information obtained by undertaking objectives 1 and 2. Objective 6 is important for this study as it will provide evidence that LS is not directly related to exposure to ASS-affected waters and causes different clinical signs in affected oysters. The third group is comprised of objectives 7, 8 and 9. These three objectives relate to the laboratory investigation which was required to explore the results from the field investigation.

Objectives 3 to 9 underpin the following hypotheses:

1. Long-term exposure of the Sydney rock oyster to ASS-affected waters will cause mortalities and reduced growth rates in adult Sydney rock oysters.

2. ASS-affected waters will cause higher mortalities in young adult Sydney rock oysters compared to older market–sized Sydney rock oysters.

3. Exposure of the Sydney rock oyster to ASS-affected waters will cause changes in the gills and mantle soft tissues and will result in the accumulation of iron precipitates on the soft tissues.

4. Exposure of the Sydney rock oyster to ASS-affected waters will cause a reduction in their filtration rate.

1.5 RESEARCH APPROACH Section I of this thesis is comprised of Chapters 1, 2 and 3 (Figure 1.3). Chapter 1 provides an introduction to the study, background information on estuarine acidification and reviews on past studies on the effects of acidification on bivalves. Chapter 2

23 introduces the biophysical characteristics of the study area and provides information on the formation and characteristics of ASS. Chapter 3 provides information on the Sydney rock oyster and its production in NSW.

Section II of this thesis details the field investigations of this study. Chapter 4 contains the water quality sampling design and the laboratory analysis methods used to investigate estuarine acidification in both estuaries. Chapter 4 also presents and discusses the spatio-temporal extent of estuarine acidification on the Hastings River and Manning River estuaries. Chapter 5 contains information on the design of the field experiments used to expose oysters to acidification and presents additional water quality data from the experimental sites. This chapter also details the experimental design including the materials and methods used. Chapter 6 presents and discusses the survival and growth data collected in areas of the Manning River estuary with a high probability of exposure to estuarine acidification. Chapter 7 provides information that was obtained from an oyster kill that occurred in Limeburners Creek, a tributary of the Hastings River during the study. The probable causes for the oyster kill are discussed in this chapter. Chapter 7 also includes a water quality investigation in an area affected by oyster kills.

Section III contains the laboratory component of this study designed to investigate field results and oyster grower’s observations in further detail. Chapter 8 details the experimental design used in laboratory experiments to detect oyster behaviour and injurious effects of naturally and artificially acidified water to the gill and mantle soft tissue of the Sydney rock oyster. Chapter 8 also reports on the findings from these experiments. Similarly, Chapter 9 outlines the experimental design used in laboratory experiments intended to investigate the effects of ASS-affected waters on feeding processes of the Sydney rock oyster and presents and discusses the results of these experiments. Section IV contains Chapter 11, which is the concluding chapter for the study. Chapter 11 states the main findings from this study and their implications, draws the conclusions from this work, makes recommendations to the oyster industry based on the research and identifies areas of further study. Figure 1.3 is a flow diagram showing the four sections of the thesis and the corresponding chapters.

EFFECTS OF ESTUARINE ACIDIFICATION ON SURVIVAL AND GROWTH OF THE SYDNEY ROCK OYSTER SACCOSTREA GLOMERATA

SECTION I BACKGROUND CHAPTERS

INTRODUCTION AND BACKGROUND Chapters 1, 2 and 3: Objectives, the study area, acidification and oyster production

SECTION II FIELD INVESTIGATIONS

ESTUARY ACIDIFICATION FIELD EXPERIMENTS Chapter 4: Chapters 5, 6 and 7: Spatio-temporal characteristics of Effects of ASS-affected waters on oyster acidification in the Hastings River and survival and growth, Manning River estuaries and investigation of Hastings River oyster kills

SECTION III LABORATORY INVESTIGATIONS

LABORATORY EXPERIMENTS Chapters 8 and 9: The effects of ASS-affected waters on oyster soft tissue and feeding behaviour

SECTION IV CONCLUDING CHAPTER CONCLUSIONS Chapter 10: Findings from the study

Figure 1.3 Components and outline of the study.

25 CHAPTER TWO BIOPHYSICAL CHARACTERISTICS OF THE STUDY AREA

2.1 INTRODUCTION This chapter provides background information on the area where the field research was carried out. The information contained in Chapter 2 relates to the biophysical environment of the tidal reaches of the Hastings and Manning Rivers and the adjacent coastal plain area. Information relating to the formation and characteristics of ASS are also discussed in this chapter.

The Hastings and Manning Rivers are located on the mid north coast of NSW approximately 300 kilometres north of Sydney. In this work, the Hastings River and the Manning River including their respective tidal rivers, creeks, channels and tributaries, and the adjacent coastal plain areas were the focus of the study. Figure 2.1 shows the location of the Hastings River and the Manning River.

Figure 2.1 Location of the Hastings River and the Manning River.

The Hastings and Manning Rivers accounted for 5.6% of the state’s production of Sydney rock oysters for the year 1999/00 (NSW Fisheries, 2001); therefore research on these estuaries has management implications for the long-term viability of this area as well as other oyster producing estuaries in NSW.

2.2 ESTUARY EVOLUTION There is a strong association between the distribution of ASS, severity of acidity and estuary evolution (Lin et al, 1995; Sammut et al., 1996a). The process of soil and landform formation in the lower catchments of the Hastings and Manning Rivers is important in understanding why ASS affects oyster production.

ASS started to accumulate around 6,000 to 10,000 years ago in saline or brackish water tidal swamps and marshes of the Hastings River and Manning River estuaries following the last major sea level rise (Dent, 1986). Low energy, tidal areas of the estuary that were fed by rivers and creeks carrying sediments from coastal hills provided the conditions that allowed the formation and accumulation of ASS. These sediments became overlain by other floodplain sediment to produce the existing floodplain. ASS continue to form and accumulate in mangrove swamps, coastal lake bottoms and saltmarshes that are tidal (Sammut et al., 1996a). The organically rich sediments provide the ideal environment for bacteria to mediate the reduction of sulfate to sulfide (Dent, 1986; Lin and Melville, 1992). The formation of ASS is discussed in greater detail in Section 2.5.

2.2.1 Hastings River The Hastings River Valley includes a section of coast between Crescent Head and Tacking Point (Figure 2.2). The Hastings River is classified as a mature, infilled barrier estuary that is channelised and has a strong tidal and salinity range (Roy, 1984a). This estuary type is characterised by extensive Holocene-age coastal plains that contain significantly higher accumulations of pyrite compared to drowned river valleys (Table 1.1).

27 Cohen and Brierley (1999) report on attributes of the tidal reaches of the Hastings River. The Hastings River has a narrow coastal plain in contrast to the Manning River’s deep coastal plain. At the coastal plain a multi-channel tidal delta has developed due to the marine ingression. The channel geometry is symmetrical, greater than 250 metres wide and over 10 metres deep. The bed material of the channel is cobble lag and fine- grained silt. The floodplain is comprised of fine sands and silt. Several islands exist in the floodplain of the Hastings and Maria Rivers. Within the Hastings basin there is a network of coastal lakes in some instances connected to the estuary by wet marshes. Holocene-age (< 10,000 years old) landscapes of the Hastings River estuary consist mostly of marine, estuarine and fluvial sediments (Roy, 1984b).

2.2.2 Manning River The Manning River (Figure 2.3) is also classified as a mature, infilled barrier estuary that is channelised and has a strong tidal and salinity range (Roy, 1984a). The Manning River has a permanent ocean entrance at Harrington and a second non-permanent ocean entrance located 11 kilometres to the south at Farquhar Park.

Birrell (1987) described the tidal reaches of the Manning River. The Manning River has an extensive deltaic floodplain composed of recent (< 6,500 years old) alluvial deposits. There are several islands in the floodplain which are covered with alluvial deposits as well as soils derived from the outcrops of Permian and Carboniferous shales. There is a zone of estuarine sands along the coastline on Mitchells Island (Figure 4.2). Streams and channels are bordered by low levees with the land sloping away into the low-lying back swamps and marshes, which are characterised by poor drainage. Lower Lansdowne River, Ghinni Ghinni Creek and the southern bank of the Manning River near Taree have extensive areas of wet marshes. There are several old river channels and lagoons on the floodplain, which mark previous stream patterns. The Manning River catchment is shown in Figure 2.3.

Figure 2.2 The Hastings River catchment and landscape units (Source: Cohen and Brierley, 1999).

Figure 2.3 The Manning River Catchment (Source: Greater Taree City Council, 1996).

30 2.3 GEOLOGY The Hastings River and Manning River catchments are part of the New England Geosyncline. The New England Geosyncline lies between Port Stephens and the Queensland border and is a sedimentary basin that formed in the Palaeozoic (250 to 600 million years ago) containing Carboniferous and Permian metasediments.

The Hastings River study area falls within the Port Macquarie Block and the Manning River is part of the Hastings Block. The Lorne Basin containing the Camden Haven River catchment separates both rivers. The Hastings and Manning River catchments are comprised of high country, coastal ranges and associated valleys and the coastal plain (Cohen and Brierley, 1999; Birrell, 1987). Unconsolidated quaternary sediments bound the coastal plain and estuary in both catchments.

2.3.1 Lower Hastings River The catchment bedrock of the Hastings River is comprised of sedimentary rocks deposited in the Permian and Carboniferous period. The coastal plain is chiefly Quaternary deposits (10,000 to 1,800,000 years old) which overly the bedrock sediments. The Quaternary deposits are comprised of Pleistocene and Holocene sediments and include: fluvial deposits adjacent to the Hastings and Maria Rivers; fluvio-estuarine deposits adjacent to the channel; and, marine deposits to the east (Roy, 1984b). The Holocene sediments consist of marine sands deposited in the estuary from wave and tidal energy moving sand from the open coast to occupy the lower 3-4 kilometres of the Hastings River estuary (Roy, 1984b).

2.3.2 Lower Manning River The coastal plain of the Manning River is comprised of a narrow strip of sand dunes, which separates swamps and marshes from the ocean. It is characterised by recent Quaternary alluvial deposits of gravels, silty sands, fine sands, silts, gravels and cobbles. The most recent fluvial unit is the unaltered Holocene alluvia, comprised of very fine sands and silts, which occur in levees and along river channels (Webb, McKeown and Associates, 1997).

31 Estuarine deposits (consisting of silt and clay lagoon deposits and silty sand delta sediments) are located around the lower valley margins and in the lowlands of the Dawson River, Lansdowne River and Cattai Creek. Large areas of the low-lying swamp and marsh country have been drained and converted to pasture land for dairying (Webb, McKeown and Associates, 1997).

A barrier dune system consisting of marine deposits stretches from Wallabi Point in the south to Crowdy Head in the north and extends up to 400 m inland on Mitchells Island (Figure 4.2). Shoaling occurs at both river entrances due to near shore sands being moved into the estuary by tidal and wave action.

2.4 SOILS 2.4.1 Lower Hastings River Atkinson (1999) described the different soil landscapes in the Kempsey-Port Macquarie area. This report identifies five main soil landscape types for the coastal plain area:

1. Alluvial soil landscapes: Formed by the deposition of sediment by rivers and streams to form floodplains and alluvial plains. Typically located along the lower reaches of Pipers Creek, Hastings River and Maria River.

2. Estuarine soil landscapes: These landscapes occur where rivers and streams enter large bodies of tidal saline water. These soils are located on the tidal flats of the Hastings and Maria Rivers and extend along Connection Creek. These soils have the potential to cause an acid sulfate soil hazard.

3. Swamp soil landscapes: These soils are frequently waterlogged and soil parent material is derived from large amounts of decayed organic matter. These soils are typical of the coastal swamps located between North Shore and Crescent Head and are alluvial clay loams overlying sands.

4. Aeolian and Barrier landscapes: Formed by the accumulation of sand-sized sediment along the coast during periods of sea level change. These soils are

32 typical of the sandplains in the Limeburners Creek Nature Reserve and are characterised by deep podzols.

5. Beach Landscapes: These soils occur adjacent to sandy coastlines and lake edges and have ground surfaces and parent material deposited by wave action. These soils are deep rapidly drained podzols.

ASS may underlie alluvial soils at elevations less than ~ 5 m Australian Height Datum (AHD; which is 0.46 m below mean sea level) on the lower Hastings River floodplain. Information on ASS and their distribution in the Hastings catchment is discussed further in Section 2.5.

2.4.2 Lower Manning River DLWC Soil Landscape Series for the Manning River coastal plain were not available at the time of writing. However, similar soil landscapes to the Hastings River coastal plain exist on the Manning River coastal plain. Soil types adjacent to the lower Manning River are mostly recent alluvium overlying young marine clay deposits.

The coastal plain soils of the lower Manning River are described in Webb, McKeown and Associates (1997). The alluvial soils of the coastal plain are easily eroded and contain loosely aggregated fine-grained loams and areas of gravel. The properties of these soils can vary considerably and is largely dependant on the source of the parent material. Basalts have enriched the alluvial soils located close to the town of Wingham. These soils are considerably more fertile than the alluvial soils derived from dry sandy alluvium located on the terraces of the Lansdowne River.

In the eastern section of the coastal plain area adjacent to the dunes and beach ridges, sandy podzols and peaty podzols are found. These soils overlie sandy parent material that provides excellent drainage for these soils. Thin humic pans may be present at shallow depths where peaty podzols are found. The podzol soils of this area generally have low fertility and are acidic. It is unlikely that these podzols are the source of acid to cause estuarine acidification as they produce a weak humic acid that normally cannot

33 drive the soil pH below 4 or provide sufficient quantities of acid to impact the estuary (Walker, 1972).

ASS may underlie alluvial soils at elevations less than ~ 5 m AHD throughout the coastal plain area of the Manning River estuary. ASS in the Manning River catchment are discussed further in the next section.

2.5 ACID SULFATE SOILS To understand the process of estuarine acidification it is important to understand physical, chemical and biological processes occurring in ASS that produce acidification of waterways. This section provides information on the properties of ASS as well as the process of acid generation that leads to soil, ground, surface and estuarine waters becoming acidified.

ASS are soils and sediments that contain oxidisable and already oxidised sulfides, occurring predominately as iron pyrite (FeS2) (Melville et al., 1996). There are approximately 30,000 km2 of ASS nationally with 4,000 to 6,000 km2 occurring in NSW (White and Melville, 1996). Extensive deposits of ASS occur along the eastern and northern coastline of Australia with deposits also occurring in Western Australia, South Australia and Victoria (White and Melville, 1996).

ASS were first described in the Netherlands more than 260 years ago (Pons, 1973). However, it wasn’t until the 1930s on the west coast (Teakle and Southern, 1937) and in the 1960s on the east coast (Walker, 1963) that these soils were identified in Australia. Walker’s (1963) work was overlooked and ignored for many years even though it was published in the Australian Journal of Soil Research (Walker, 1972). Surprisingly, his warnings of environmental degradation were ignored.

Heavily engineered and modified floodplains are common to many eastern Australian estuaries, including the Hastings and Manning Rivers. Smith (1999) identified more than 50 floodgated drains in the lower Hastings River catchment. Despite investigations into fish kills in eastern Australia and increased attention to the problem, ASS are still

34 being developed, albeit with greater control. However, illegal drainage works remain a problem, and few existing drains have been remediated.

2.5.1 Formation of Iron Pyrite Iron pyrite forms in waterlogged saline sediments when there are (Dent, 1986): • anaerobic conditions, • a source of iron, • a source of dissolved sulfate, and • easily decomposed organic matter.

As bacteria breakdown organic matter under anaerobic conditions, dissolved sulfate ions are reduced to sulfides and iron III oxides to iron II (Dent, 1986). The formation of iron pyrite with iron III oxide as the source of iron may be represented by the equation (Dent, 1986):

2- 1 - Fe2O3(s)+4SO4 (aq)+8CH2O+ /2O2(aq) 2FeS2(s)+8HCO3 (aq)+4H2O (2.1)

Typical environments in which iron pyrite forms are: saline or brackish water tidal swamps; salt marshes; and mangrove forests. In these locations vegetation provides organic matter and the tidal cycle supplies sediment, renews the supply of dissolved sulfate and removes soluble by-products (Dent, 1986).

Sulfidic sediments accumulated as sea level stabilised at its present position during the last 7000 years (Dent, 1986). Therefore, ASS are expected to occur around coastal estuaries and embayments at elevations less than 5 m AHD and below the upper tidal limit at some depth in the soil profile (White and Melville, 1993).

2.5.2 Oxidation of Iron Pyrite Pyrite is oxidised to form sulfuric acid when it is exposed to oxygen. This occurs in ASS when the water table is lowered, either naturally through evaporation or transpiration processes or through human intervention when backswamps and freshwater lagoons are drained or excavated (Callinan et al., 1996). If iron pyrite in sediments remains in reduced conditions, such as below the watertable, it poses no

35 environmental risk (White et al., 1996a). The overall process of pyrite oxidation is described in the following equation (van Breemen, 1973):

15 7 2- + FeS2(s) + /4O2(g.aq) + /2H2O Fe(OH)3 + 2SO4 (aq) + 4H (aq) (2.2)

For every mole of pyrite oxidised, 4 moles of acidity are released. Pyrite oxidation proceeds in stages. The first stage produces ferrous iron, sulfate and acid and can be represented by equation 2.3:

7 2+ 2- + FeS2(s) + /2O2(g.aq) + H2O Fe (aq) + 2SO4 (aq) + 2H (aq) (2.3)

The acidity produced can be quickly neutralised by carbonates or exchangeable bases in the soil (Dent, 1986). Excess acidity reacts with the soil minerals and liberates potassium, magnesium, aluminium and silicon as well as trace elements such as copper cadmium, chromium, nickel and zinc (Hicks et al., 1999). These elements can reach toxic concentrations and severely affect the soil, groundwater and surface waters (Sammut et al., 1996a).

Pyrite can also be rapidly oxidised by ferric iron in solution. Ferric iron becomes increasingly soluble at a pH less than 4. When the pH falls to below this level, catalytic oxidation of ferrous iron by bacterium (Thiobacillus ferrooxidans) to ferric iron (equation 2.4) increases the rate of pyrite oxidation and regenerates ferrous iron (equation 2.5) enabling oxidation of pyrite in anaerobic conditions (Dent, 1986). The equations for these reactions are:

2+ 1 + T. ferrooxidans 3+ 1 Fe (aq) + /4O2(g.aq) + H Fe (aq) + /2H2O (2.4) 3+ 2+ + 2- FeS2(aq) + 14Fe (aq) + 8H2O 15Fe (aq) + 16H (aq) + 2SO4 (2.5)

Therefore, under re-flooded conditions ASS can continue to produce acid. The soluble ferrous iron produced can be transported into adjacent waterways to produce a further two moles of acid as well as reducing the dissolved oxygen content of the receiving waters. This reaction is represent by equation 2.6:

36 2+ 1 3 + Fe (aq) + /4O2(g.aq) + /2H2O FeO.OH(s) + 2H (aq) (2.6)

Aluminium becomes increasingly soluble at pH values less than 5.5 (Driscoll, 1989). Aluminium hydrolysis generates an additional 3 moles of acidity and the three stages are represented by equations 2.7 to 2.9 below:

3+ 2+ + Al (aq) + H2O Al(OH) (aq) + H (aq) (2.7) 2+ 2+ + Al(OH) (aq) + H2O Al(OH) (aq) + H (aq) (2.8) 2+ + Al(OH) (aq) + H2O Al(OH)3(aq) + H (aq) (2.9)

The oxidation of ferrous iron to ferric iron and the hydrolysis of aluminium can generate substantial amounts of acidity, often at considerable distances away from the source of pyrite in the ASS (National Working Party on Acid Sulfate Soils, 1998; White et al., 1996a). The oxidation of ferrous iron produces goethite (FeO.OH) flocs which coat benthic communities and stream banks. High concentrations of these iron flocs have been observed many kilometres from their source and their presence has significant implications for the health of aquatic ecosystems (Sammut et al., 1996a; 1996b; Bishop, 2000; Ng, 2001).

2.5.3 Characteristics of ASS The concentration of pyrite in ASS can range from 0.05% to 15% and the pH of oxidised ASS are commonly below 4. The potential for pyrite accumulation is much greater in tropical areas due to higher temperatures and a greater supply of organic matter which increases the microbial reduction of sulfate (Dent, 1986). Pyrite is unevenly distributed through the soil profile with higher concentrations commonly found near old tree roots (Dent, 1986).

Pyrite is present in estuarine muds as well as in sandy and peat soils. Generally, lower concentrations of pyrite are found in sandy textured soil as opposed to estuarine muds. However, the pyrite in sandy textured soils can still pose an acid problem due to the low acid-buffering capacity and rapid air permeability of sand. It has been estimated that for every tonne of pyrite that has undergone complete oxidation, 1.6 tonnes of sulfuric acid is produced (White et al., 1996a). This estimate does not account for additional acid

37 production from metal transformations. Hence, the total acid production rate may be higher because of the lower organic content and high porosity of coastal sand bodies.

When ASS are drained a process of soil ripening occurs. As a soil dries out, many of the physical, chemical and biological properties irreversibly change. The removal of water causes shrinkage and fissuring which increases the cohesive strength of the soil (Dent, 1986). The fissures, however, can create pathways for the movement of acidic water as well as increased oxygenation of the soil.

ASS also contain numerous secondary minerals. The mineral jarosite

(KFe3(SO4)2(OH)6) is often noticeable as a yellow mottle in ASS and is an intermediate oxidation product that can store acidity. Jarosite forms under strongly oxidising and severely acid conditions (Dent, 1986). At higher pHs jarosite may hydrolyse to goethite and release acid or may transform to haematite which appears as red mottles. Iron monosulfides are highly reactive sulfide minerals that include greigite (Fe3S4), makinawite (FeS0.94) and amorphous iron sulfide (FeS) (Sammut, 1998). Iron monosulfides are also referred to as ‘acid volatile sulfur’ (AVS). AVS is associated with ASS and has been detected in drain sediments (Hallinan, 1998). The effects of AVS on acid production and its association with toxic elements are being researched.

ASS can increase the soluble concentration of pH-dependent elements to levels toxic to biota. For example, the free aluminium produced by soil reactions can be toxic to plants and aquatic fauna (Sammut et al., 1995). Also, the toxic levels of various pH-dependent elements, such as Al, increase with increasing acidity (Lin et al., 1995).

The generation of acid in ASS is determined by the rate of oxidation of pyrite. Oxidation rates are dependent on hydrology, drainage, climatic conditions, surface vegetation, the air permeability of the overlying sediments, the amount of sulfides and soil temperature (White et al., 1996a). Climate, hydrology and drainage are critical factors in the mobilisation of oxidation products into the surrounding waterways. Drainage does not only facilitate oxidation but provides an efficient conduit to remove the oxidation products many kilometres from their source (White et al., 1996a). ASS

38 have the potential to generate sufficient quantities of acid to create a myriad of environmental problems in surrounding waterways (Sammut et al., 1996a).

2.5.4 Distribution of ASS in the Study Area DLWC Acid Sulfate Soil Maps (Figures 2.4 and 2.5) (Naylor et al., 1995) show where ASS are likely to occur in the study area. These maps indicate the probability of ASS being located at a certain distance below the ground surface. Areas are classified as high-risk and shaded red if there is a high probability of occurrence of ASS underlying that location. Major floodplain drains that are connected to the estuary are displayed on these figures as blue lines.

It is estimated that there is 16,800 ha of high-risk ASS in the lower Manning River catchment and 21,700 ha of high-risk ASS in the lower Hastings River catchment area (Naylor et al., 1995). A proportion of this area of high-risk ASS in the Hastings River catchment is located in the Kempsey local government area. There is an additional 9,600 ha and 9,300 ha of soil classified as low risk ASS in the Manning River catchment and the Hastings River catchment, respectively.

Figure 2.4 shows the distribution of high-risk acid sulfate soils for the Hastings River region. It shows that ASS sediments occur adjacent to the Hastings River, Maria River, Fernbank Creek, Limeburners Creek and the Wilson River. Areas of extensive drainage works constructed in high-risk ASS include: the lower and upper Maria River; Fernbank/Partridge Creek; and, Rawdon Island areas. All of these locations have been identified as priority areas or ‘hot spots’ that require changes to land management to improve environmental quality, due to these areas being degraded by acidification (Tulau, 1999a). Johnston (1995) reported on the chemical and physical attributes of ASS of the Maria River area as well as investigating water quality changes resulting from acidification in Maria River and Fernbank Creek. This report is discussed in detail in Chapter 4.

39 40

41 The distribution of high-risk ASS for the Manning River region is shown in Figure 2.5. ASS covers a large percentage of the Manning River’s coastal plain. Areas of extensive drainage works constructed in high-risk ASS include: Cattai-Pipeclay; Lower Lansdowne-Moto-Ghinni Ghinni Creek; North Oxley Island; and, Dickensons Creek (Figure 2.5). These locations have also been identified as priority areas or ‘hot spots’ that require changes to land management to improve environmental quality (Tulau, 1999b).

2.6 CLIMATE Climate of the study area is dominated by sub-tropical influences and is characterised by warm temperatures throughout the year. Cool northeast sea breezes relieve high temperatures during the summer months. High temperatures can be recorded if the sea breeze fails to penetrate inland. Late summer high temperatures are usually accompanied with high humidity (> 60%). Average temperatures range between 16.1 and 28.6O C during the summer months and 6.9 and 19.6O C during the winter months. Much of the coastal plain is free from frost. Figure 2.6 displays the average daily maximum and minimum temperatures and the average monthly precipitation and evaporation for Port Macquarie (Station Number 60026 and 60085) and Taree airport (Station Number 60141).

The mid north coast of NSW receives high rainfall, especially in late summer through to early autumn. Close proximity to the Great Dividing Range increases orographic rainfall events but most rainfall is convectional. Rainfall information is discussed in more detail in Section 2.8.1.

2.7 VEGETATION It is estimated that 70% of the Hastings River catchment (Cohen and Brierly, 1999) and 60% of the Manning River catchment (Greater Taree City Council, 2001) retain natural vegetation cover. However, considerable areas of vegetation on the coastal plain have been cleared for agriculture in both catchments. This has resulted in a loss of vegetation cover and alteration of dominant native species by the introduction of exotic species in the coastal zone, particularly pasture species with higher transpiration rates than native plants.

250 35

30 200 25 C) O 150 20

15 100

10 temperature ( 50 5 precipitation and evaporation (mm) 0 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

250 35

mm) 30 200 25 ation ( C) O

150 20 e ( atur 100 15

10 temper 50 5 ecipitation and evapor pr 0 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Ave. Monthly Precip. Ave. Tot. Monthly Evap.

Ave. Daily Max Temp. Ave. Daily Min. Temp.

Figure 2.6 Average monthly rainfall, temperature and evaporation data for: (A) Port Macquarie (Source: Bureau of Meteorology Station Numbers 60026 and 60085); and, (B) Taree Airport (Source: Bureau of Meteorology Station Number 60141).

The predominant vegetation community in the study area is dry sclerophyll open forests, woodland, mallee and shrublands (Cooper and Associates and Ecograph, 1999). These forests are found in shallow soils on ridges that have good drainage to maintain dry conditions. These areas are characterised by blackbutt (Eucalyptus pilularis), grey ironbark (Eucalyptus paniculata)/grey gum (Eucalyptus propinqua), tallowwood (Eucalyptus microcorys), white mahogany (Eucalyptus acmenoides) and scribbly gum.

There are a number of shallow coastal lakes bordered by reserves in the study area. The vegetation of these reserves is swamp sclerophyll, with patches of dry and wet sclerophyll forest and rainforest on elevated areas (Aikens, 1998). The vegetation that surrounds the lakes are mangrove/saltmarsh communities and swamp sclerophyll forests/sedgelands (Aikens, 1998). The main species that are found in these areas are swamp she-oak (Casuarina glauca), broad-leaved paperbark (Melaleuca quinquenervia) and swamp mahogany (Eucalyptus robusta).

Coastal heath and natural grassland dominated by kangaroo grass (Themeda australis) covers areas that are not forested, disturbed or cleared for Agriculture. Heath areas are comprised mainly of herb and sedge grasses growing in swampy conditions. Areas of improved pasture are comprised of kikuyu (Pennisetum clandestinum), paspalum (Paspalum dilatatum), broad-leaved paspalum (Paspalum wettsteinii), rhodes grass (Chloris gayana), phalaris (Phalaris sp.), rye grass (Lolium spp.) and setaria (Setaria sphacelata) (Atkinson, 1999).

The grey mangrove (Avicennia marina), river mangrove (Aegiceras corniculatum) and the milky mangrove (Exoecaria agallocha) occur in the Hastings and Manning River estuaries (West et al., 1985). On the landward side of mangrove communities in areas of tidal inundation are saltmarshes comprised of saltwater couch and succulents (Cooper and Associates and Ecograph, 1999). Many of the submerged sand beds in the Hastings and Manning Rivers are densely covered by Zostrea capricorni sea grass (West et al., 1985). Table 2.1 lists the area covered by these estuarine vegetation types.

Table 2.1 Area of mangrove, seagrass and saltmarsh in the Hastings and Manning Rivers (Source: West et al., 1985).

Estuarine Area (km2) Vegetation Hastings River Manning River

Mangrove 2.078 3.582 Seagrass 1.141 0.329 Saltmarsh 0.804 0.721

2.8 HYDROLOGY 2.8.1 Rainfall The Hastings and Manning River catchments have a dominant summer rainfall pattern with most precipitation falling between November to May and a dry season that occurs from July through to October. Average annual rainfall for selected stations throughout the study area is shown in Table 2.2.

Table 2.2 Average annual rainfall data for selected rainfall stations in the study area (Source: adapted from Bureau of Meteorology data and Aikens, 1998).

Station Average Annual Rainfall Wettest Month Dryest Month

Port Macquarie - 60026 1545.0 February September Wauchope 1289.2 February September Laurieton 1549.4 March September Moorlands - 60024 1489.5 March September Harrington - 60023 1350.8 March February Coopernook - State Forest 1535.0 March June Taree Airport - 60141 1155.6 March September Taree - 60030 1177.6 March September

2.8.2 Tidal Hydrology 2.8.2.1 Tidal Hydrology in the Hastings River Training walls at the mouth of the Hastings River provide a narrow and deep channel that allows efficient water movement in and out of the estuary keeping the entrance permanently open to the sea. Salt stratification occurs in the Hastings River when fresh

45 water, originating from catchment inflows, overlies heavier saline tidal waters. There are two astronomic tides per day with a mean spring tidal range of 1.7 m at the training walls (0.5 km upstream), 1.5 m at Settlement Point (3.3 kilometres upstream) and 1.1 m at the tidal limit in Connection Creek (46.5 kilometres upstream) (Manly Hydraulics Laboratory, 1995a). The tidal prisms for the mean spring tide for selected sites on the lower Hastings River is displayed in Table 2.3.

Table 2.3 Tidal prisms for selected sites on the lower Hastings River (Source: adapted from Webb, McKeown and Associates, 1998)

Site Tidal Prism (m3 x 106)

Estuary Entrance, Port Macquarie 17.2 Setllement Point 15.3 Dennis Bridge 6.4 Lower Maria River 3.4 Telegraph Point 0.45 Lower Limeburners Creek 0.59

2.8.2.2 Tidal Hydrology in the Manning River Training walls have been constructed at the Harrington River entrance. Water quality data collected during the present study revealed that the Manning River is regularly salt stratified, especially after periods of rainfall. There are two astronomic tides per day with a mean spring tidal range of 1.7 m at the Harrington entrance (1.1 km upstream), 1.1 m at Croki (11.5 kilometres upstream) and 1.3 m at Taree (25.4 kilometres upstream) (Manly Hydraulics Laboratory, 1995a). Tides on the Manning are largely affected by shoaling at the River entrances. Farquhar inlet temporarily closes due to heavy shoaling of marine sands, which has the effect of reducing the tidal range of the Manning River. Scouring of the river entrances after floods can increase the tidal range throughout the estuary. The tidal prisms for the mean tide following a flood event at selected sites on the lower Manning River is displayed in Table 2.4.

46 Table 2.4 Tidal prisms for selected sites on the lower Manning River when Farquhar inlet is open. (Source: adapted from Webb, McKeown and Associates, 1997)

Site Flood Tide Ebb Tide (m3 x 106)(m3 x 106)

Estuary Entrance, Harrington 10.2 9.6 Estuary Entrance, Farquhar 3.2 3.8 Taree - 3.3 Scotts Creek 0.4 0.6 Lansdowne River - 1.0

2.8.3 Flooding The lower Hastings River and Manning River are both subjected to regular flooding. Floods on the mid north coast of NSW occur mainly as a result of summer to autumn cyclonic storms. Floods can cause economic losses to oyster growers and floodplain land users, as well as contribute alluvium to the lowland soils. Floods have the potential to damage and wash away oyster growing infrastructure and stock. Flooding also causes the estuary to remain ‘fresh’ for extended periods of time, which can prevent oysters from feeding and the harvesting of oysters for market or ‘shipping’. Salt wedges, caused by density stratification, enables oysters to periodically open and feed to survive under these conditions (Nell, 1993).

2.8.4 Artificial Drainage and Flood Mitigation 2.8.4.1 Artificial Drainage and Flood Mitigation on the lower Hastings River Extensive flood mitigation works, which included the construction of drains and levees and the provision of flood and tide gates, were undertaken in the 1960s and 1970s (Smith 1999; Hastings Council, 2000). It has been estimated that > 30% of the floodgate structures on the Hastings and Maria Rivers were constructed with government funding (Smith, 1999). Floodgate structures have been constructed on numerous drains that flow into the Hastings River, Limeburners Creek, Fernbank Creek, Maria River, Wilson River, Connection Creek and Pipers Creek (see Figure 2.4). Floodgate structures are comprised of one-way flap gates that open on the ebb tide but close during the flood tide (Plate 2.1). The once brackish and tidal reaches upstream of the gates are now fresh water dominated.

Plate 2.1 Downstream view of a floodgate structure on Partridge/Fernbank Creek, Hastings River showing: (A) outflow of acidic water (pH < 4) during the ebb tide and iron precipitate coating on the concrete and the flaps of the floodgate structure; and, (B) the floodgate flaps held closed during the flood tide.

48 2.8.4.2 Artificial Drainage and Flood Mitigation on the lower Manning River In the Manning River catchment, initial drainage works were constructed on the Cundle Estate and Moto plains in 1856 to drain the backswamps to Dickensons and Ghinni Ghinni Creek (Birrell, 1987). Floodgates have the effect of isolating estuarine wetland habitats. This is likely to impact on the overall aquatic production of the Manning estuary (Middleton et al., 1985). In particular, artificial drains that flow into the Manning River, Scotts Creek, Lansdowne River, Cattai Creek, Dickensons Creek and Ghinni Ghinni Creek (Figure 2.5) are regulated by floodgate structures.

2.9 LANDUSE The original occupants of the study area were the Birpai and Ngamba people. European settlement of this region occurred in the 1820s. Currently, there are four dominant uses of land in the study area: private tenure which is comprised of rural and urban; crown land which is undeveloped and undisturbed natural vegetation; National Parks predominately located on the coastal plain and contain undisturbed natural vegetation; and, state forests.

Current land use in the study area is mainly for forestry, cattle grazing on improved pasture and tourism. Cash cropping, oyster production and fishing provides a significant contribution to this regions economy. In the last decade, large plantations of tea tree (M. alternifolia) have been established on floodplain land adjacent to the estuary.

Agricultural activities use a large proportion of the coastal plain in the study area (Hastings Council, 2000). On arrival, European settlers cleared agricultural land on the floodplains of the Manning River and Hastings River for cropping and grazing. Drains were also constructed to increase the land available for pasture to increase stocking rates since the mid 1800s (Birrell, 1987; Smith 1999). During the 1960s and 1970s, the Government funded the construction of drains, flood levees and floodgates in the study area for flood mitigation schemes (Smith 1999; Smith et al., 1999). Coastal floodplain agricultural industries are still reliant on this drainage, including tea tree plantations.

49 Tourism is a major industry on the mid north coast of NSW and causes significant seasonal population change. The mid north coast is also experiencing strong population growth of approximately 1,500 per year (Hastings, 2000). Both tourism and population growth are more concentrated on the coastal strip which places increased pressure on this area’s natural environment. It is largely the natural environment of the mid north coast that attracts visitors and residents to this area. The environmental degradation caused by ASS can be viewed as a potential threat to tourism in this area.

Therefore, the type of landuse on ASS influences the export of acidity into floodplain drains and subsequently into the estuary. Oyster growers claim that previous landuse changes and the nature of current landuse are related to oyster production problems. This claim is the focus of investigation for this present study.

2.10 CHAPTER SUMMARY The lower Hastings and Manning River catchments are characterised by a large coastal plain area that contains expansive deposits of ASS. The physical and human environmental context of the Hastings River and Manning River influences the water quality in these two estuaries. The floodplain of the lower Hastings and Manning Rivers has been heavily engineered by artificial drainage and the construction of floodgate structures on these drains. This change to the floodplain environment has altered natural processes and resulted in detrimental changes to water quality that has the potential to seriously impact the biodiversity, amenity, fisheries production and overall value of these estuaries. Both estuaries are a significant contributor to total oyster production and have an important function in the NSW oyster industry. It is for these reasons, that the Hastings and Manning Rivers were selected for this study. The following chapter provides information on the Sydney rock oyster.

50 CHAPTER THREE COMMERCIAL PRODUCTION OF THE SYDNEY ROCK OYSTER (SACCOSTREA GLOMERATA) IN NEW SOUTH WALES

3.1 INTRODUCTION The Sydney rock oyster forms the basis of the largest aquaculture industry in NSW. Cultivation has occurred for more than 130 years in NSW and Queensland, making it Australia’s oldest aquaculture industry following European settlement (White, 2002). The Sydney rock oyster industry also represents the most valuable per hectare agricultural enterprise in the state with average gross present value of production from oysters being $8,500 ha-1 (White, 2002). This compares to gross margins of $40-75 ha-1 for the beef industry, about $90 ha-1 for the dairy industry and $3,000 ha-1 for the Tea tree industry (Mullen and Kaur, 1999) which are located on the nearby coastal plain.

This chapter describes the Sydney rock oyster’s biology and anatomy and contains information on the industry that has developed, from farming this species of oyster, in NSW. This chapter details the history of the industry, methods used for production, production statistics and environmental and disease risks, that are encountered by Sydney rock oyster growers. The purpose of this chapter is to provide background information to aid the interpretation of findings from proceeding chapters.

3.2 BIOLOGY AND ANATOMY OF THE SYDNEY ROCK OYSTER 3.2.1 Systematics and Distribution The common names for S. glomerata include: the Sydney rock oyster; the commercial oyster; mangrove oyster; and, rock oyster. The Sydney rock oyster is also cultivated in New Zealand as a commercial species. The Sydney rock oyster is part of a large group of bivalve molluscs and belongs to the family Ostreidae (Rafinesque 1815) and the subfamily Crassostreinae (Torigoe 1981) (Lamprell and Healy, 1998). The Sydney rock oyster is found in the subtidal and intertidal zone, predominately in estuaries, and is distributed between the NSW and Victorian border in the south and Townsville in the north (Nell, 1993).

51 3.2.2 Anatomy The anatomy of an oyster is shown in Figure 3.1. The shell of the Sydney rock oyster is thick, hard and grows to a size of 8-10 cm but can exceed 25 cm (Thomson, 1954; Arakawa, 1990). An elastic ligament and the adductor muscle connects the two asymmetrical calcareous valves (Korringa, 1976; Eble and Scro, 1996). The left valve (Figure 3.2) is indented, the shell interior is white, shell margins are serrated and coloured dark brown to black. Both sides of the hinge are finely serrated. The right valve is flat with the outer surface being lamellated (Arakawa, 1990). Plates 3.1 and 3.2 show the appearance of the Sydney rock oyster.

The mantle or pallium is a fleshy fold of tissue that covers and protects the internal organs of the oyster (Eble and Scro, 1996). An oyster has two gills which are comprised of an inner and outer demibranch. The gills are used to acquire food and for respiration (Newell and Langdon, 1996). A detailed description of the structure of the gill and mantle and their function is provided in Chapter 8. Oysters are able to uptake calcium, dissolved nutrients and trace metals through direct absorption from seawater (Nell et al., 1983; Bevelander, 1952).

3.2.3 Biology Table 3.1 displays the ideal salinity and temperature ranges for the various stages of development. Growth of the Sydney rock oyster is interdependent on salinity, temperature and the available food levels (Holliday, 1995). Adult oysters grow best at salinities between 25 and 35 ppt (Nell and Holliday, 1988). When salinities fall below 15 ppt oysters can survive by closing their valves for periods of up to 14 days (Holliday, 1995). Density stratification can increase salinity levels to 15 ppt or greater during the final stages of the flood tide, providing oysters with the opportunity to open their valves and feed. The Sydney rock oyster’s range of osmoconformity is between 15 and 45 ppt salinity (Nell and Dunkley, 1984) and maximum filtration rates occur between 25 and 30O C (Souness and Fleet, 1979). Oysters are able to withstand large temperature variations (Table 3.1). Optimum growing temperatures for adult oysters are between 18 and 26O C (Holliday, 1995). During summer months, intertidal oysters may be exposed to high air temperatures at low tide, which can result in mortality (Potter and Hill, 1982).

Figure 3.1 General anatomy of the oyster (Source: Eble and Scro, 1996).

Figure 3.2 Valve description (A) interior view and (B) side view (Source: Carriker, 1996).

Plate 3.1 Adult Hastings River Sydney rock oysters (approximately 12 months old). The diameter of the white lid = 65 mm.

D A

Plate 3.2 Sydney rock oyster with the right valve removed showing: the mantle (M); adductor muscle (A); and digestive gland (D) in the left valve. The diameter of the white lid = 65 mm.

Table 3.1 Summary of salinity and temperature ranges for the Sydney rock oyster (Source: Holliday, 1995).

Salinity Ranges Temperature Ranges (ppt) (OC)

Growing Tolerance Growing Tolerance

Larvae 23-39 15-39 24-26 NA

Spat 20-40 0-41 14-28 11-30

Adult* 25-35 0-50 18-26 11-30

NA Data not available * Can withstand salinities < 15 ppt for periods of up to 2 weeks.

3.2.4 Reproductive Cycle Sydney rock oysters change sex during their life cycle and start out as males (Malcolm, 1987). Sex reversal generally occurs between spawning seasons when the gonad is undifferentiated (Thomson et al., 1996). As the oyster grows the proportion of females in each size class increases (Galtsoff, 1964). Spawning of the Sydney rock oyster can happen several times in the one season and generally occurs between October and April on the mid north coast of NSW. Spawning is initiated by a temperature or salinity change (Holliday, 1995) and eggs are fertilised externally in the water column.

Larvae develop into the “D” veliger swimming stage within 16 hours of fertilisation and are able to consume unicellular algae after several days. Larvae are transported passively by estuarine currents and aggregated by hydrological factors towards the entrance of the estuary (Nell, 1993). After approximately 18 days, oyster larvae develop into the pediveliger stage when they actively seek a suitable substratum on which to attach (Holliday, 1995). Once the pediveliger has attached it is referred to as “spat”.

55 3.3 HISTORY OF PRODUCTION The history of production of the Sydney rock oyster is important to this study. Production data collected since the early 1930s show that the industry is variable from year-to-year and that the NSW industry has experienced a marked decline in total production over the last 30 years (Figure 3.4) which indicates that the industry has experienced a recent change.

3.3.1 History of the Sydney Rock Oyster Industry Large shell middens along the Australian coastline indicate that native oysters were a staple food source for coastal Aboriginals. There is also evidence that the Quandamooka people of Southern Queensland cultivated oysters by using artificial reefs of oyster shell to catch spat which were then transferred to deeper water for fattening prior to being consumed (Stasko, 2000).

Substantial quantities of live wild oysters were burned so that the ashes could be used to produce lime for mortar during the colonisation of the east coast. This resulted in wild oyster stocks being severely depleted and legislation was passed in 1868 prohibiting the burning of live oysters.

One of the earliest forms of harvesting oysters was to dredge oysters from deepwater oyster beds. Around 1870, ‘claires’ or canals (6 m wide and deep enough to retain 1 m of water at low tide) were dug on the banks of Gwawley Bay, Georges River (Stasko, 2000). This was a successful method of farming oysters in France but failed in Australia due to high summer temperatures and the build up of silt causing oyster mortalities. In 1876, a Royal Commission was appointed to inquire and report on the best method of oyster cultivation and to improve the natural beds of the colony (Stasko, 2000). Over the next two decades stick, stone and shell placed on intertidal sand and mudflats were used to catch and grow oysters.

Mangrove sticks were initially set up in bundles and arranged along the shore. It was apparent that oysters at a certain level grew quicker than the oysters above and below (Stasko, 2000). The use of intertidal racking was developed to lay sticks out at the particular height that facilitated fastest growth. The method of farming using stick on

56 racks became popular because oysters were easier to handle and process and the method discouraged predators, fouling and mudworm (Stasko, 2000).

In 1888, New Zealand rock oyster spat (S. glomerata) was imported into NSW in an attempt to increase oyster stocks still largely depleted because of lime production. Following this date mudworm infestation of oyster stocks became prolific, especially in oyster dredge beds. This resulted in more oyster growers resorting to intertidal culture because it controlled the extent of mudworm infection. After 1900 demand for oysters was strong allowing the industry to grow. Production steadily increased until the late 1970s (Figure 3.4). Since this time the industry has experienced a dramatic decline in oyster production.

3.3.2 History of the Hastings River Oyster Industry John Stuart Dick founded the oyster industry on the Hastings River. Dick took out the first lease and commenced cultivation of oysters in Kooloonbung Creek in 1886. Cultivation in Kooloonbung Creek was successful and Dick expanded cultivation to the north shore of the Hastings River using sandstone and sticks to catch oyster spat and grow oysters (Moyes and Mant, 1986). Dick’s three sons, Thomas, Ernest and Charles continued the oyster industry on the Hastings River with production leases in Limeburners Creek and Top (or Big) Bay. The industry has since expanded to cover a total area of 86 ha with cultivation predominately occurring in the Hastings River, Limeburners Creek and Big Bay areas.

3.3.3 History of the Manning River Oyster Industry Initial development of the oyster industry on the Manning River prior to the 1900’s is detailed in Birrell (1987) and is summarised below. Dredging of oysters from deepwater beds in the lower estuary commenced in 1863. Over time, an increasing number of boats were dredging oysters from these naturally occurring beds. Oysters were brought to the Manning River from Sydney and spread along oyster beds near Croki in 1870 and oyster beds along the river were surveyed and offered for lease shortly after this. By 1882, 50-60 boats were operating along the river dredging for oysters. Cultivation on racks led to a decline in dredging of oysters in the Manning. By 1896 approximately 2000 bags per year were being shipped to the Sydney market from

57 leases in the Manning River. Foreshore and offshore leases now cover an area of 99 ha of the lower Manning River estuary (Webb, McKeown and Associates, 1997). Oyster leases are currently located in the Manning River, the South Passage, Scotts Creek, Pelican Bay and the Lansdowne River.

3.4 OYSTER PRODUCTION IN NEW SOUTH WALES The Sydney rock oyster is cultivated in 41 estuaries in NSW between Eden in the south and the Tweed River in the north. The total farm gate value of the NSW Sydney rock oyster industry in 2001 was $31.6 million (Nell, 2002). There were 461 permit holders working 3,507 hectares of oyster leases in 2001 (Nell, 2002). Whole oysters are sold as either “plate” (40-60 g whole weight) or “bistro” (30-40 g whole weight) oysters and smaller oysters (20-30 g whole weight) are usually shucked and sold in glass jars as “bottle” grade (Nell, 1993). Over 75% of the oysters produced are sold within NSW (Nell, 2002). The ten largest oyster-producing estuaries are listed in Figure 3.3 along with the type and total number of oysters produced in 1999/00. The Queensland Sydney rock oyster industry is significantly smaller than its NSW counterpart and had a total farm gate value of $360,100 in 2000/01 (Lobegeiger, 2002).

2500000

Bottle Grade 2000000 Bistro Grade Plate Grade 1500000

1000000 dozens of oysters 500000

0 Port River Inlet River Lake Waters River Manning Hastings Brisbane Stephens Wagonga Merimbula Hawkebury Botany Bay Clyde River Wallis Lake

Figure 3.3 The ten largest oyster-producing estuaries in 1999/00 (Source: NSW Fisheries, 2001).

Oyster production in NSW has decreased considerably over the last twenty years. Production peaked in 1976/77 where close to 150,000 bags of bottle and plate oysters were harvested. This figure has declined to around 50,000 bags of bottle and plate oysters being produced in 1999/00. Figure 3.4 highlights the changes in production that have occurred since the late 1970s.

160000 148,397.9 140000

120000

100000

80000

60000 bags of oysters

40000

20000

0 1931 1940/41 1950/51 1960/61 1970/71 1980/81 1990/91

Figure 3.4 Number of bags of bottle and plate grade oysters produced in NSW (Source: NSW Fisheries Unpublished Data).

3.4.1 Hastings River Oyster Production Production methods used to mature oysters on the Hastings River include: rack-tray culture; raft culture; rack-stick culture; and, baskets. The rack tray method produces the majority of oysters and is used extensively in Limeburners Creek and Big Bay. The main method used to collect oyster spat is by plastic slats placed into the estuary. The pediveliger larvae attach to these plastic slats after spawning occurs.

The Hastings River was the eighth largest producer of oysters in NSW for the 1999/00 financial year, contributing approximately 3% of the State’s total. The Hastings River

59 produced predominately bottle grade oysters for the 1999/00 period (Figure 3.3). In 1999/00 the total value of oyster sales was $204,825 (NSW Fisheries, 2001).

Export of single seed oysters to other estuaries is a key component of the Hastings River oyster industry and is not factored into the production figures mentioned above. The Hastings River has been the largest supplier of single seed oysters in recent years which considerably increases the total value of this industry. The Hastings River oyster industry supplied over 10 million single seed oysters in 1998/99 (46% of the State’s total) to other NSW estuaries (NSW Fisheries, 2000).

8000 6,942 7000

6000

5000

4000

3000 bags of oysters

2000

1000

0 1931 1940/41 1950/51 1960/61 1970/71 1980/81 1990/91

Figure 3.5 Hastings River oyster production (bottle and plate oysters) (Source: NSW Fisheries Unpublished Data).

Figure 3.5 highlights the variability in oyster production from year-to-year on the Hastings River. Production peaked in 1987/88 when 6,942 bags of plate and bottle oysters were produced. However, only 2,080 bags of plate and bottle oysters were harvested in the proceeding year. Recurrent episodes of mortality and slow growth have been experienced in the Hastings estuary and are investigated in this study.

60 3.4.2 Manning River Oyster Production The Manning River was the ninth largest producer of Sydney rock oysters in NSW, contributing 2.6% of the State’s total in 1999/00. Rack-tray culture is the main method used for oyster production on the Manning River, other production methods used for maturing oysters are; rack-stick culture, dredge beds, baskets and raft culture. The Manning River produces, predominately, premium grade plate oysters (Figure 3.3). In 1999/00 the total value of oyster sales was $797,756, compared to $600,053 for the 1998/99 season (NSW Fisheries, 2001; 2000).

Export of oysters to other estuaries is also a key component of the Manning River oyster industry and increases its overall value. In 1998/99 the Manning oyster industry supplied 5% (1.1 million) of the State’s single seed oysters, and 7% of trayed oysters for inter-estuary transfer (NSW Fisheries, 2000). The total worth of the Manning Oyster Industry is approximately $2 million annually (I Crisp, personal communication, 1999).

8000 6,854 7000

6000

5000

4000

3000 bags of oysters

2000

1000

0 1931 1940/41 1950/51 1960/61 1970/71 1980/81 1990/91

Figure 3.6 Manning River oyster production (bottle and plate oysters) (Source: NSW Fisheries Unpublished Data).

Oyster production figures for the Manning River indicate harvests vary considerably from year-to-year. Production peaked in the early 1960s, when over 6,500 bags of plate and bottle oysters were produced (Figure 3.6). In recent years, there has been a distinct

61 decline in the numbers of plate and bottle oysters harvested. For example, in 1991/92 4,652 bags of plate and bottle oysters were produced, compared with only 1,270 bags in 1998/99. Seasonal dips in production are, in part, now attributed to acid water discharges and associated declines in oyster health and mortalities, which is investigated in this study.

3.5 RISK FACTORS FOR OYSTER PRODUCTION There are a number of identified risks associated with the production of the Sydney rock oyster. Korringa (1976) identified hydrographic and biological conditions, predators (such as Porcupine fish, bream, toad fish and stingray), parasites and diseases and competitors as the principal risk factors for oyster production in NSW. Nell (1993) categorised heat kill, floods and pollution as environmental hazards, mudworm, winter mortality and QX as disease risks and pacific oysters, mussels and barnacles as the principal competitors.

A recent report by White (2002) has investigated production trends over the past five years and details impediments to oyster production, particularly impacts caused by human activities. This report identifies the main threats to successful oyster production as: population expansion in coastal areas; oyster diseases; the hydrological setting of production sites; geomorphic setting; and, water pollution. ASS are included in this list under geomorphic setting as a threat to growing healthy oysters (White, 2002). The key recommendation from this report is, the extension of the Coastal Lake Assessment and Management Strategy to all commercial oyster-producing estuaries in NSW be considered urgently.

Sydney rock oyster production problems were caused by the use of bis-tributyltin oxide (TBTO) for antifouling purposes. High concentrations of TBTO causes shell deformities and reduced growth rates in oysters (Nell and Chvojka, 1992). The use of TBTO was banned on vessels smaller than 25 m in 1989, which has reduced the prevalence of shell deformities and has improved growth rates (Nell, 1993). There is no evidence to suggest that TBTO is responsible for oyster production problems investigated in this study.

62 Toxic algal blooms have the potential to create significant problems for oyster production. Roughley (1926) lists three events where algal blooms have caused mortalities in oysters in NSW. In 1997, a toxic algal bloom of the diatom Rhizosolenia chunii occurred in Port Phillip Bay, in south eastern Australia, between late August and mid-October (Parry et al., 1989). The algae caused mussels (M. edulis planulatus), scallops (Pecten alba) and flat oysters (Ostrea angasi) to acquire a bitter taste and resulted in mortalities in the shellfish 3 to 8 months after the initial algal bloom (Parry et al., 1989).

High mortalities in Sydney rock oysters were reported from Womboyne Lake in March 2002. Investigations into the mortality outbreak revealed the possible involvement of a toxic dinoflagellate (R. Callinan, NSW Fisheries, personal communication, 2002).

Risk factors associated with environmental hazards are not always clearly understood due to synergistic effects of some hazards, lag effects and a lack of information on their direct and indirect impacts on oysters. Presently, the most pronounced risk factor for the production of the Sydney rock oyster is that of disease. Diseases that impact the Sydney rock oyster industry are discussed in detail in Section 3.6.

3.6 DISEASE Disease has been attributed as the primary cause of production declines since the late 1970s. QX, winter mortality and mudworm are the three principal diseases that affect the production of the Sydney rock oyster on the east coast of NSW. These diseases are discussed in the following sections.

3.6.1 QX Disease QX disease is caused by the protozoan parasite Marteilia sydneyi (Perkins and Wolf, 1976) and affects farmed and wild Sydney rock oysters. M. sydneyi is one of the most significant pathogens of the Sydney rock oyster (Wesche et al., 1999). QX disease outbreaks typically occur north of the Macleay River to Moreton Bay in Queensland, however serious and recurrent outbreaks have occurred in the Georges River (Adlard and Ernst, 1995). Recent testing has revealed that M. sydneyi is present in the Hastings

63 River (NSW Fisheries, 2003). However, M. sydneyi is not known to occur in the Manning River.

Outbreaks of this disease usually follow heavy summer rainfall when salinities are low and water temperatures are high. Methods used to control outbreaks of QX disease include raising the growing height of oyster trays to reduce exposure times and removal of oyster stocks from areas of the estuary, endemic to the disease, during the infection period (January to April) (Wesche et al., 1999). In a QX outbreak on the Clarence River, NSW, a new oyster parasite Marteilioides branchialis was observed in the gill lesions and is described in Anderson and Lester (1992).

3.6.2 Winter Mortality Disease Winter mortality is caused by the protistan parasite Mikrocytos roughleyi (Farley et al., 1988) and occurs between Port Stephens in the north and the Victorian border in the south. Outbreaks of winter mortality disease can result in significant losses (up to 80%) of mature oysters (Wolf, 1967; Koringa, 1976). Winter mortality disease has not been previously reported in the Hastings and Manning River estuaries.

The physiological effects and the mechanism of infestation by M. roughleyi are not completely understood. M. roughleyi has an incubation period of 2.5 months and is not common in oysters less than 3 years old (Wolf, cited in Farley et al., 1988). Worst outbreaks occur following periods of high salinity (Wolf, 1967). Methods used to avoid this disease include raising the growing height of oyster trays or moving oyster stocks to upstream leases, to areas of lower salinity, before the end of autumn (Nell, 1993).

3.6.3 Mudworm The third disease affecting production of the Sydney rock oyster is mudworm infestation by Polydora websteri (Skeel, 1979). P. websteri uses a chemical to bore into the shell of the oyster. A mud blister forms on the internal surface of the shell. If the blister ruptures it produces a putrid smell and makes the oyster unsuitable for sale.

Severely infected oysters suffer from poor health and mortality due to energy directed towards shell maintenance and repair instead of growth and reproduction (Larson,

64 1978). Severe infestations can result in a brittle shell (Skeel, 1979) leaving oysters vulnerable to other predators. Oyster growers can regularly remove mud by spraying oysters with water to reduce the risk of infection otherwise infected oysters can be dried in shaded areas for ten days to kill the mudworm (Nell, 1993). Mudworm has not been a major problem on the Hastings or Manning River in recent times.

3.7 DISCUSSION Oyster production in NSW has been in decline since the late 1970s. Since this time entire estuaries have been abandoned of oyster production due to recurrent QX disease outbreaks, which has contributed to this marked decline. Oyster production data (Figure 3.5 and 3.6) show that total output of oysters between estuaries is highly variable. Only six estuaries have increased their production while 18 estuaries have decreases in production over the last five years of the previous decade (White, 2002). In NSW, oysters are commonly cultivated in the most populated sections of the estuary. Presently, there is no data to determine the area of oyster production lost due to environmental degradation (White, 2002).

The key environmental requirements for a healthy Sydney rock oyster industry are well- oxygenated, clear brackish to saline waters, with pH in the range 7.00 to 8.75, with suitable tidal exchange, adequate phytoplankton and dissolved nutrient food supplies and control of upstream sources of runoff and pollution (White, 2002). The lower Hastings and Manning Rivers represent important and significant areas of Sydney rock oyster production in terms of total sales and the provision of oysters for other estuaries. The mid north coast has experienced rapid population growth in recent times which is increasing the pressure placed on the coastal environment and in particular these estuaries (Chapter 2).

3.8 CHAPTER SUMMARY The Sydney rock oyster is dependant on a healthy river system for survival. Current production trends indicate that many NSW coastal waterways are not capable of supporting healthy oyster populations which is a reflection of general water quality conditions of these regions.

65 There are a variety of poorly understood reasons for the decline in oyster production over the previous 25 years. Poor water quality conditions arising from ASS leachate is only one factor of many and requires significant research to understand its affect on overall production decreases. However, without taking the initial step of research into this problem it will remain a poorly explained reason for overall production decline. ASS-affected waters may only be a piece of the larger puzzle but information on its effects and understanding the impacts will contribute to improving environmental conditions conducive to healthy oyster production.

Many studies have clearly shown the impacts created by disturbance of ASS and associated estuarine acidification (Sammut et al., 1995; 1996a; 1996b; Sammut, 1998; Callinan et al., 1996). It is known that pollutants contained in the water will directly impact oyster health and disease susceptibility (Chu and Hale, 1994). The effects of estuarine acidification on Sydney rock oysters are not well documented or understood. It is therefore, necessary that the impacts of estuarine acidification to this oyster species be investigated.

The following chapter examines the spatial and temporal characteristics of estuarine acidification on the Hastings and Manning Rivers. This information will reveal the extent to which areas used to produce the Sydney rock oyster are impacted by acidification.

SECTION II

FIELD INVESTIGATIONS

67 CHAPTER FOUR SPATIO-TEMPORAL CHARACTERISTICS OF ESTUARINE ACIDIFICATION ON THE HASTINGS AND MANNING RIVERS

4.1 INTRODUCTION Past studies on estuarine acidification have shown that both the spatial and temporal characteristics of acidified water are important factors to consider when assessing environmental impacts (Sammut et al., 1996a; 1996b; Sammut 1998). The spatial extent of acidification can influence the magnitude of mortality events and other impacts. For example, Sammut (1998) found a close association between the magnitude of fish kills and fish disease outbreaks and the spatial extent of acidified waters on the Richmond River in northern NSW. Spatially extensive acidification exposes more fish, reduces refuge sites, restricts fish movement and often traps large numbers of fish in toxic waters. Consequently, catastrophic fish kills and large disease events occurred when acidification was widespread following high rainfall events (Sammut, 1998). The same study also showed habitat degradation, through the loss of waterplant communities and reduced fish movement and recruitment, was most likely to occur following weeks to months of persistent acidic conditions. By contrast, short-term acidification, often associated with the ebb tide pulse-flows of acid from floodgates, was less likely to trigger fish kills or disease. Both Sammut (1998) and Callinan (1997b), in a collaborative study on fish health impacts, showed that pH levels and metal concentrations were the principal determining factors. Under chronic acidic conditions moderately weak acidity was more likely to cause environmental impact than during acute, short-lived events.

Very little is known about the effects of estuarine acidification on the Sydney rock oyster. Unlike fish, oysters are unable to avoid acidified water by moving to refuge sites. Similarly, oysters are unable to move to new habitat to continue their growth under more favourable conditions. In the present study, an understanding of the environmental conditions at oyster leases is important to confirm oyster growers claims that acidification impacts oysters and to appreciate the range of water quality conditions oysters are exposed to.

The purpose of this chapter was two-fold. Firstly, to characterise estuarine acidification at the two study areas to understand the range of water quality conditions oysters are exposed to. Secondly, to obtain data and conduct field observations that enables the selection of reference and exposure sites for field experiments outlined in Chapters 5 and 6 and to design laboratory experiments detailed in Chapters 8 and 9.

The Hastings River and Manning River estuaries were chosen as the study areas due to: the known presence of acid sulfate soils in the developed floodplains; abandoned oyster leases; and, regular reports of oyster mortalities and other production problems in areas downstream of acidic drains. Furthermore, past studies have confirmed estuarine acidification occurs in both of these estuaries.

4.2 PAST STUDIES A number of studies have examined aspects of acidification on the Hastings River and Manning River systems. Most studies were conducted to determine if ASS had impacted on estuary water quality (Johnston, 1995; Sonter, 1999; Smith and Dove, 2001) but were not designed to investigate environmental impacts associated with the acidified water. Principal findings of these studies are discussed below. Similarly, many studies were either conducted on an ad hoc or short-term basis and did not account for spatial or temporal variability (Manly Hydraulics Laboratory (MHL), 1995b; Webb McKeown and Associates, 1998). Studies that investigated acidification in the study areas are summarised in Table 4.1. Figures 4.1 and 4.2 are maps of the Hastings River and Manning River estuaries, respectively, that display the locations of areas referred to in this section.

Johnston's (1995) work on the Maria River (Figure 4.1) system demonstrated that upper reaches of this tributary experiences long-term acidification. For example, his work showed that the water pH could remain below pH 5.5 for up to 6 weeks and acidic water could extend for more than 10 kilometres in the upper Maria River. Johnston’s (1995) work also showed that the persistence and the severity of acidity were dependent on the antecedent conditions, and the magnitude of rainfall events. These findings are

69 discussed further in Section 4.6.1 of this chapter, which synthesises the findings of other studies and the present research.

Table 4.1 Studies that have investigated problems associated with ASS in the Hastings River and Manning River estuaries. Refer to Figures 4.1 and 4.2 for the locations of sites.

Estuary Source Brief Details

Hastings Johnston (1995) Soil and water quality study (June to November 1994) that measured acidic River drains and tidal waters with elevated aluminium levels in the Maria River and Fernbank Creek areas

James (1997) Review of water quality studies which identified ASS discharge as a water quality issue for the Hastings River estuary

MHL (1997) Water quality data collection (September 1994 to October 1995) that measured extended periods of acidification in the upper Maria River

ERM Mitchell Soil and water quality report that measured acidic ground, surface and McCotter (1997) drain waters that had elevated aluminium and iron levels in the Fernbank Creek catchment

Smith (1999) Identified 50 floodgated drains on the Hastings River estuary

Kable (1999) Study on the breakdown of mangrove leaves which measured a reduced breakdown rate in Fernbank Creek. This was attributed to the recurrent acidic conditions at this location

Tulau (1999a) Identification of the upper and lower Maria River, Partridge Creek and Rawdon Island as ASS priority management areas

Aaso (2000) This study recommends a community-based approach incorporating economic incentives for land-based farmer cooperation as current landuse in the Maria River catchment is unsustainable

Manning Lawrie (1996) Short term spot testing of drains in the Pipeclay Canal and Lansdowne River River areas which noted very acidic drains

Webb, McKeown Short term spot testing of drains and tidal waters in the Cattai Creek, and Associates Lansdowne River and Ghinni Ghinni Creek (in August 1995 and August (1997) 1996) that identified acidic drains and tidal waters

Silcock (1998) Soil study which included spot testing of drains on North Oxley Island, measured drain pHs < 4.5

Sonter (1999) Water quality study (March to June 1999) in Pipeclay Canal and Cattai Creek which measured acidic drain and tidal waters.

Tulau (1999b) Identification of Cattai-Pipeclay, lower Lansdowne-Moto-Ghinni Ghinni Creek as ASS priority management areas

Smith and Dove Water quality study (February 1999 to August 2001) of drains on North Oxley (2001) Island which measured persistent acidic conditions

Figure 4.1 The Hastings River estuary and water quality sampling locations. Numbers 1 to 16 show the locations of drains listed in Table 4.3.

see Figure 4.3

Figure 4.2 The Manning River estuary and water quality sampling locations. Numbers 1 to 15 show the locations of drains listed in Table 4.5.

Sonter (1999) found that acidified water in Cattai Creek (Figure 4.2) was largely restricted to the upper water column during drier periods due to salt stratification. During wet weather, stratification was broken down and the entire water column was acidified. Studies on the Richmond River in northern NSW showed similar processes (Sammut et al., 1994) indicating that water quality in acid impacted areas can be both vertically and laterally variable. This is an important finding for the experimental design of water quality sampling, as the heterogeneity of water quality should be considered in order to accurately measure acidification (Sammut et al., 1996c).

Smith and Dove (2001), in a related study, conducted intensive water quality sampling in an acidified drain that outflows directly upstream of an oyster lease in Scotts Creek, Manning River (Figures 4.2 and 4.3). Sequential discrete sampling (Sammut et al., 1996c) was used to assess drain water quality and the frequency, severity and duration of acidic outflows. This provided information on the temporal characteristics of acidification at this site and data from this study is presented in this chapter.

An interesting field observation made by the author and oyster farmers which is also reported in the literature (Johnston, 1995; Sammut et al.; 1996a; 1996c; Sammut 1998; Corfield, 2000), is the large iron precipitation events that occur following major acidification events. These iron precipitate events are spectacular in nature because massive iron floccules (flocs) are transported over large distances in red/orange plumes of partially or fully neutralised water resulting in smothered bed sediments, structures and aquatic plants. Sammut et al. (1996a) found that these iron precipitate events could occur many kilometres downstream from their source and could degrade habitat by smothering surfaces. Other work has shown that iron precipitates can clog the gills of crustaceans (Simpson and Pedini, 1985; Govinnage-Wijesekera, 2001) and impact the survival and growth of bivalves (Winter, 1972). Bishop (2000) showed that iron flocs at non-acid impacted sites on the Hastings River affected settlement and survival of Sydney rock oyster spat.

The present study investigates the transport and dispersion of iron flocs in the study areas given their potential to impact on oysters at sites that may not be directly affected

73 by low pH or dissolved metals. These areas are also characterised by abandoned oyster leases and iron staining along the bank. Clearly, iron precipitate contamination of estuarine waters is important to the current investigation. The implications of suspended iron flocs for the Sydney rock oyster are examined in Chapter 8 of this study.

4.3 WATER QUALITY MONITORING OBJECTIVES Past studies on acidification in both estuaries were specific water quality surveys that provide useful but limited information on the overall water quality of oyster producing areas. The present study builds on the past water quality surveys by extending the range of water quality sampling and monitoring drain and estuary locations in the oyster producing areas of both estuaries. The present study also examines other water quality variables, such as metals, that are known to impact on gilled organisms. The data from the present and past studies is synthesised to describe water quality degradation associated with ASS on both estuary systems.

The specific objectives of the present study are: • to examine the pH and chemical composition of ASS-affected waters entering both estuaries via floodplain drains; • to characterise acidification in both study areas, and • to obtain water quality data that will be useful for the selection of experimental field sites and design of the laboratory experiments used in the latter chapters of this study.

4.4 METHODS This section describes the methods used to characterise acidification in the Hastings River and Manning River estuaries. The methods used to collect spatial and temporal data overlap and are specified in the following sections.

The spatial study on the Hastings River and Manning River was conducted to determine if there was an association between the spatial distribution of acidity and abandoned leases and the recurrent poor growth reported at some leases. The study was not resourced to conduct continuous monitoring of sites on the Hastings River. Data from other studies (e.g. Johnston, 1995; MHL, 1997) on the Hastings River were used to

74 build on the understanding of the temporal character of acidification events on the Hastings River.

4.4.1 Drain Outflow Water Quality Sites Spot or discrete in situ measurements (Sammut et al., 1996c) of pH, electrical conductivity (EC), water depth, dissolved oxygen (DO) and temperature were made at the confluence of floodplain drains (displayed on Figures 2.4 and 2.5) and the main channel of the estuary following high rainfall. A common feature of drains in the study areas was a floodgate structure that had a set of one-way flaps that allowed drain water to outflow into the estuary during the ebb tide (Smith, 1999; Smith et al., 1999). Drains that had floodgate structures were sampled on the downstream (estuary) side, where accessible. Water samples for elemental analysis were collected when drain outflow waters were very acidic (~ pH < 4).

Before drain sampling commenced a field inspection of drains was carried out to locate major drains and to establish if they contained acidic water or displayed indicators of acid water (e.g. red iron staining, concrete corrosion of structures, water logged soils, acid scalded pastures, etc.) (Smith, 1999). Drains identified as potential sources of acid outflow were sampled on four occasions on the Hastings River (18-19/6/99, 29- 30/11/99, 1-2/12/00 and 12-13/2/01) and two occasions on the Manning River (27/5/98 and 9/5/99). Validation for using these sampling dates is provided in Section 4.4.3.

All drain sampling sites were identified using the coding system described in Smith (1999). This system assigned each drain an individual code based on the initials of the tributary name, followed by the distance upstream along the centre channel line from either the Hastings River or Manning River ocean entrance and concludes with the letter ‘L’ or ‘R’ to denote whether the drain is situated on the left or right bank. For example a drain located on the left bank of the Maria River, 45 kilometres from the Hastings River entrance would be coded as: MR45.0L.

A submersible data logger (SDL) was placed below a floodgate in the Manning River and floated at a depth of 0.15 m from the surface to measure drain outflows. The location of the SDL was at Site W displayed on Figure 4.3. Data collection commenced

75 on the 19/2/99 and concluded on the 18/8/00. The data set is not complete due to malfunctions in the SDL unit during this period.

4.4.2 Tidal Water Quality Sites Spot or discrete in-situ measurements of pH, EC, water depth, DO and temperature were conducted along the centreline of main channels of the Hastings River and Manning River estuaries. Sampling in both estuaries was conducted on the same dates as drain outflow water quality was measured to establish the impact on the receiving tidal waters (Sammut et al., 1996c). Hastings River tidal water sampling sites are shown in Figure 4.1 and Manning River tidal water sampling sites are shown in Figure 4.2.

Tidal water sampling sites were coded using the initials of the tributary name, followed by the distance upstream from the Hastings River or Manning River ocean entrance, in a similar way to the drain identification detailed in Section 4.4.1.

SDLs were used to characterise tidal water quality at Sites W and R in the Manning River, which are displayed on Figures 4.2 and 4.3. The SDL at Site R (Figure 4.2) was suspended 0.15 m above the channel bed and the SDL at Site W (Figures 4.2 and 4.3) was floated at a depth of 0.15 m.

4.4.3 Drain Water Quality Sites Spot or discrete in situ measurements of pH, EC, water depth, DO, temperature and oxidative reduction potential (ORP) were collected in an acidified drain, located on North Oxley Island (Figures 4.2 and 4.3) from the 19/2/99 to the 18/8/00. Figure 4.3 displays the three sampling sites (X, Y, and Z) located within the drain. Site X was located on the drain side of the floodgate. Site Y was in the drain approximately 80 m upstream from the floodgate. Site Z was situated at Weeks Lane at the confluence of four lateral drains and the main drain. The sampling dates at these sites were: 19 Feb 1999; 9 Mar 1999; 14 April 1999; 5 May 1999; 4 Jun 1999; 5 Jul 1999; 3 Aug 1999; 24 Aug 1999; 10 Sep 1999; 30 Sep 1999; 5 Nov 1999; 14 Dec 1999; 26 Jan 2000; 10 Mar 2000; 31 Mar 2000; 19 Apr 2000; 7 May 2000; 30 May 2000; 20 Jun 2000; 10 Jul 2000; and, 18 Aug 2000.

76 To Manning lateral drains Weeks Lane River X Scotts Ck drain N W Z Y floodgate

0.5 km

Figure 4.3 Oyster lease sampling site (Site W) in Scotts Creek and drain sampling sites (Sites X, Y and Z) on North Oxley Island.

The drain was approximately one kilometre in length and had a structure that comprised of 4 floodgates (3 m x 3 m). The catchment area for the drain was approximately 150 ha of former backswamp (Smith and Dove, 2001). The soils of this area are characterised by recent alluvium overlying recent marine clay deposits and have been mapped as high-risk of ASS (Figure 2.5) (Naylor et al., 1995). Silcock (1998) conducted soil and water quality sampling at this site. This study detected ASS at 0.2 to 0.4 m below the ground surface in the area adjacent to Weeks Lane (Figure 4.3). The study site had several visual indicators of acidification including: vegetation scalds; jarosite in spoil heaps; iron precipitates on vegetation and drain walls; and, corrosion of concrete structures. This drain was selected for water quality sampling because the outflow was directly adjacent to an abandoned oyster lease (Site W, Figure 4.3).

4.4.4 Oyster Lease Water Quality Monitoring Site Spot or discrete in situ measurements of pH, EC, water depth, DO, temperature and ORP were collected at the abandoned oyster lease (Number 79-182) named Site W (Figure 4.3) for this study. Site W was located directly downstream of the outflow from the North Oxley Island drain (Figure 4.3) discussed in Section 4.4.5. Water quality sampling dates at Site W were the same as the drain water quality sampling dates at Sites X, Y and Z and are specified in Section 4.4.4. The SDL at Site W provided continuous water quality measurements of drain outflow conditions.

4.4.5 Discrete Water Quality Measurements Past studies on estuarine acidification recommended the use of discrete water sampling locations to represent sources of acidified water (eg. drains), areas upstream of acid outflow points to represent non-acidic freshwater inflows, and then a series of locations downstream to well-buffered waters to determine the spatial extent of acidification (Sammut et al., 1996c).

Discrete water quality measurements were made in situ and sampling was conducted approximately two weeks after a high rainfall event in the catchment. Sampling was conducted in this manner due to the time taken for acidified groundwater to be mobilised into drainage networks and then into the estuary (Sammut et al., 1996c). Sampling was performed during the last quarter of the ebb tide, as this is when drain outflow into the estuary occurs (White et al., 1996b).

A Yeo-Kal Intelligent Water Quality Analyser (Model 611) regularly calibrated with certified, standard solutions was used for all discrete field measurements of water temperature, pH, EC, DO and ORP. Specifications of the Yeo-Kal Intelligent Water Quality Analyser (Model 611) are listed in Table 4.2. A TPS Field Meter with an Ionode (IJ 42) KCl-filled intermediate junction pH probe was commonly used to assess the accuracy of surface pH measurements performed with the Yeo-Kal Intelligent Water Quality Analyser.

Acidified waters are regularly stratified because of density differences between fresh water, acidic water and saline, pH neutral water (Sammut et al., 1994; Sammut et al., 1996a; Johnston, 1995; Sonter, 1999). Therefore, a surface and bed measurement was performed at each site where water depth was greater than 0.3 m. Surface measurements were made at a depth of 0.1 m and bed measurements were made at 0.2 m above the bottom substrate (Sammut et al., 1996c). A Palin Test Photometer (Model 5000) was used to measure alkalinity of particular water samples. Alkalinity was measured in the field to avoid changes in the water sample due to the highly reactive nature of ASS-affected waters (Sammut et al., 1996c). EC and pH were the principal

78 variables reported on and the results of other measured variables were selectively used in the study.

Table 4.2 Yeo-Kal Intelligent Water Quality Analyser (Model 611) information (Source: adapted from Yeo-Kal Electronics, 1996)

Parameter Sensor Type Units Range Resolution

Temperature PN junction in stainless sleeve O C 0-50 0.1

Electrical Four electrode cell mS cm 0-80 0.1 Conductivity µS cm 0-8000 1.0

Salinity Four electrode cell ppt 0-60 0.1

Dissolved Polargraphic Sensor with % Sat. 0-200% 1% Oxygen teflon membrane mg L-1 0-20 0.1

pH Combination silver/silver pH 0-14 0.01 chloride type with sintered teflon junction

Oxidation Combination bare metal mV -900-900 2 Reduction electrode common reference Potential junction with pH probe

Depth Dual active silicon strain gauge m 0-100 0.1

4.4.6 Water Sample Collection and Chemical Analysis Water samples requiring chemical analysis in the laboratory were collected in acid washed, 0.5 L plastic containers and stored in a chilled ice-box before being frozen for storage. Surface water samples were ‘gulp’ sampled and bed water samples were collected with a train of three biological-oxygen-demand bottles in series (Boyd, 1979).

Water samples were prepared for laboratory analysis by filtering through 0.22 µm cellulose nitrate filter paper and then analysed using Inductively Coupled Plasma Atomic Excitation Spectroscopy (ICPAES) (Model Perkin Elmer Optima 3000 DV) for Na, K, Mg, Ca, Si, Zn, Cu, B, S, Al, Fe, and Mn. Sulphate was determined using a modified version of the Turbidimetric Method outlined in APHA (1998). The Potentiometric Method (APHA, 1998) was used to determine chloride concentration of

79 water samples. These dissolved species were targeted for analysis because they provide information on the nature and toxicity of the ASS-affected waters (Sammut et al., 1996a).

4.4.7 Continuous Water Quality Measurements Sammut et al. (1996a) showed that automatic water quality sampling, using SDLs, was a useful way of demonstrating temporal variation in key water quality variables. Automatic and logged sampling provides a more continuous record of water quality that shows: tidal variation; the effects of rainfall and lag effects between flooding and tidal water acidification; and, recovery of water quality to background conditions (Sammut et al., 1996c).

Long-term, continuous time-series water quality measurements of pH, EC and temperature were collected on either a Greenspan Technical Services Smart Sonde (Model SD300) SDL or a Yeo-Kal Intelligent Water Quality Analyser (Model 611) SDL. Both of these instruments were installed in the estuary for periods of 14 to 21 days. On removal from the estuary, the stored data were downloaded, the instrument was checked for calibration drift, the probes were inspected and cleaned and the relevant probes were recalibrated using standard, certified solutions prior to redeployment.

4.5 RESULTS 4.5.1 Hydrological Conditions 4.5.1.1 Rainfall Rainfall is a principal factor in the mobilisation of oxidation products from the floodplain and into the estuary (Sammut et al., 1996a; White et al, 1996b). Rainfall data for the Hastings River estuary for the study period are displayed in Figure 4.4 and were obtained from the Bureau of Meteorology Station (Number 60026) located at Hill Street, Port Macquarie (Figure 4.1). Rainfall data for the Manning River estuary for the study period are displayed in Figure 4.5 and were obtained from the Bureau of Meteorology Station (Number 60141) located at Taree Airport (Figure 4.2). Station Numbers 60026 and 60141 were considered the most representative rainfall stations for the study areas.

180 drain outflow 160 and tidal water sampling dates 140

120

100

60 precipitation (mm) 40

0 1/02/99 1/04/99 1/06/99 1/08/99 1/10/99 1/12/99 1/02/00 1/04/00 1/06/00 1/08/00 1/10/00 1/12/00 1/02/01

Figure 4.4 Rainfall recorded at Port Macquarie for the period 1/2/99 to the 16/2/01 (Source: Bureau of Meteorology Station Number 60026) and tidal water and drain outflow sampling dates.

oyster lease and drain 250 sampling dates

200 drain outflow and tidal water sampling dates

150

100 precipitation (mm)

0 1/02/98 1/06/98 29/09/98 27/01/99 27/05/99 24/09/99 22/01/00 21/05/00

Figure 4.5 Rainfall recorded at Taree Airport for the period 1/2/98 to the 31/9/00 (Source: Bureau of Meteorology Station Number 60141) and water sampling dates.

4.5.1.2 Tidal Hydrology Tidal attenuation increases with distance upstream from the ocean entrance in both the Hastings River and Manning River (see Section 2.8.2) (MHL, 1995a). Acidified tributaries of both estuaries commonly experience poor dilution and mixing conditions due to tidal attenuation (Johnston, 1995; Sonter, 1999). This has implications for the severity and duration of estuarine acidification (Sammut et al., 1996a). For example, the channel width and depth of the Maria River channel decreases with increasing distance upstream thereby reducing the dilution capacity of this reach. Tidal attenuation is also higher in this reach due to its distance upstream and the more confined channel hydraulics (MHL, 1995b). Shoaling at the Manning River ocean entrances restricts tidal exchange in this estuary (Webb, McKeown and Associates, 1997), which has implications for tidal flushing and neutralisation of ASS-affected waters.

4.5.2 Hastings River Estuary 4.5.2.1 Drain Outflow Water Quality Following High Rainfall There are in excess of 50 locations where drains through ASS in the lower Hastings River catchment outflow into the estuary (Smith, 1999). The extensive drainage network on the Hastings River floodplain is shown on Figure 2.4. Table 4.3 presents pH, EC, iron, aluminium, manganese, silicon, zinc and Cl:SO4 data for selected drains that outflow into tributaries of the Hastings River. The concentrations of iron, aluminium, manganese, silicon and zinc are presented in Table 4.3 because these dissolved species are commonly measured in ASS-affected waters at elevated levels (Sammut et al., 1996a; Sonter, 1999).

The results of analyses for selected drain outflow waters indicate that strongly acidic water, which contains elevated concentrations of metals discharges into the Hastings River estuary after rainfall events (Table 4.3). The pH data of drains discharging into the Hastings and Maria Rivers collected during the four sampling occasions are summarised in Table 4.3 and displayed on Figures 4.6 and 4.7, respectively. Drain water quality measurements collected on the four sampling occasions are included in Appendices A and B.

82 All drains listed in Table 4.3, with the exception of HR16.8R have a low pH combined with a Cl:SO4 ratio of less than 4 indicating that the outflow water has originated from oxidised pyrite contained in the drained floodplain soils. A Cl:SO4 ratio of less than 4 and pH values less than 4 indicate mineral acidity rather than naturally occurring humic acids due to the release of sulfate during pyrite oxidation (Mulvey, 1993). Sulfate, released from pyrite oxidation, reduces the Cl:SO4 although secondary acidification from the oxidation of iron and hydrolysis of aluminium may also drive the pH down (Sammut et al., 1996b).

Table 4.3 Water quality of selected Hastings River estuary drain outflows. For location of drains refer to Figure 4.1.

Drain Drain Date pH EC Fe Al Mn Si Zn Cl:SO4 No. ID (dS m-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)

Connection Creek 1 CC38.4R 13/02/01 3.19 0.9 7.75 2.27 0.38 6.39 0.10 1.1 2 CC44.1R 13/02/01 3.58 1.0 36.70 1.37 0.53 6.52 0.10 0.5

Fernbank Creek 3 FC11.6L 13/02/01 3.28 1.3 35.90 1.84 0.40 10.30 0.06 1.3

Hastings River 4 HR08.1R 13/02/01 4.10 1.1 12.20 0.46 0.16 4.60 0.03 3.8 5 HR16.0R 12/02/01 2.81 5.9 48.10 9.53 1.45 15.80 0.23 1.6 6 HR16.5R 18/06/99 3.37 2.7 4.03 8.34 1.22 12.15 ND 2.8 7 HR16.8R 12/02/01 3.48 8.8 33.90 19.40 2.30 17.90 0.47 4.4

Maria River 8 MR21.7L 01/12/00 3.19 1.6 1.27 7.37 0.58 8.98 0.11 2.4 9 MR23.0L 30/11/99 3.64 3.5 2.54 2.73 0.66 5.28 ND 3.6 10 MR24.2R 12/02/01 3.83 3.2 0.98 2.19 0.34 6.44 0.05 3.0 11 MR33.8R(A) 13/02/01 3.06 1.9 8.02 5.34 0.56 5.84 0.13 1.0 12 MR33.8R(B) 02/12/00 2.77 2.4 11.40 4.43 0.57 2.18 0.12 1.1 13 MR34.1R 02/12/00 3.20 2.4 3.22 20.70 1.47 3.65 0.25 0.9 14 MR35.5R 02/12/00 2.91 5.3 15.30 2.08 0.31 5.51 0.05 2.6

Pipers Creek 15 PC34.5L 12/02/01 3.47 1.1 2.80 1.58 0.36 4.36 0.06 2.1 16 PC34.7L 18/06/99 4.29 0.7 0.32 1.60 0.13 10.10 ND 2.2

ND = Not detected

83 The drain coded as HR16.8R has extremely high concentrations of aluminium (19.40 mg L-1), iron (33.90 mg L-1), manganese (2.30 mg L-1) and zinc (0.47 mg L-1). These concentrations exceed the threshold values of the Australian and New Zealand Environment and Conservation Council (ANZECC) guidelines for the maintenance of water quality for biological systems (ANZECC, 2000). This drain outflows into the main channel of the Hastings River at a point where the channel is wide and deep and drainage density is low. Therefore acidic flows would be quickly neutralised by estuarine waters when ECs are high or diluted when the river has low ECs.

The drainage density and drainage volume are high in the upper reaches of the Maria River (> 20 km upstream from the Hastings River entrance) and Connection Creek. The artificial drains in these areas enable more efficient export of large quantities of fresh, acidic water containing elevated concentrations of iron and aluminium into the main channel. A summary of the drain pH data measured on the four sampling occasions is presented in Table 4.4.

Table 4.4 Summary of the Hastings River drain outflow pH data.

18-19/6/99 29-30/11/1999 1-2/12/2000 12-13/02/2001

n 38313539 Min. pH 3.35 3.16 2.72 2.81 Max. pH 8.00 7.50 7.20 7.24 Median pH 6.01 6.15 6.15 5.87 Standard Dev. pH 1.13 1.33 1.45 1.42

4.5.2.2 Tidal Water Quality Following High Rainfall The pH and EC data collected in the Hastings River and Maria River were plotted against distance upstream from the Hastings River entrance (Figures 4.6 and 4.7). The pH measured at major drain outflow locations in Hastings River and Maria River was also plotted on Figures 4.6 and 4.7.

84 Fernbank Creek pH drain outflow pH EC 9 45 8 40 7 35

6 30 ) -1 5 25 pH 4 20

3 15 EC (dS m 2 10 1 5 0 0 6 7 8 9 10 11 12 13 14 15 16 17 18 distance upstream from the ocean entrance (km)

Figure 4.6 Hastings River surface water pH, EC and drain outflow pH measured on the 18 and 19/6/99.

Maria River pH drain outflow pH EC 9 45 Maria Anabranch 8 Pipers Creek 40 7 35

6 30 ) -1 5 25 pH 4 20

3 15 EC (dS m 2 10 1 5 0 0 0 1020304050 distance upstream from the ocean entrance (km)

Figure 4.7 Hastings/Maria River surface water pH, EC and drain outflow pH measured on the 18 and 19/6/99.

85 Water pH and EC decrease with increasing distance upstream in the Hastings River channel (Figure 4.6). This decrease in pH and EC is attributable to neutral, poorly- buffered, freshwater inflows from the upper catchment in combination with acidic outflows from floodplain drains 15-17 kilometres upstream from the Hastings River entrance (Figure 4.6). The pH drops below neutral in the surface waters at the centre of the main channel close to the drain outflow locations.

Figure 4.7 shows very low EC values present in the Maria River system following the June rainfall event due to the dominance of floodwaters in the system. The decrease of pH in the main channel of the Maria River is pronounced and is caused by acidic water discharging from the numerous drains on the Maria River floodplain (Figure 2.4). Additional tidal water data collected on the four sampling occasions are listed in Appendix C.

4.5.2.3 Metal Precipitate Mobilisation The extent of iron and aluminium precipitate mobilisation was observed during each sampling date and on other occasions following rainfall. The primary sources of iron flocs entering the main channel of the Hastings River were from Fernbank Creek, Maria River, drains located on Rawdon Island (Figure 4.1) and drain HR08.1R (Table 4.3). Aluminium flocs were commonly observed in Fernbank Creek outflows.

Iron flocs contaminated the near-shore downstream reach of the Hastings River for a distance of 0.2 km at Rawdon Island (Figure 4.1) and 2.5 km downstream of Fernbank Creek after high rainfall (Figure 4.14). Similarly, iron flocs originating from drains in the Maria River smothered the northern bank of the Hastings River for a distance that was 6.1 kilometres from the Hastings River ocean entrance on the 20/3/01 (the Hastings River Maria River confluence is located 9.5 kilometres upstream from the ocean entrance). This occurred after a flood event where 187 mm of rainfall was recorded in 10 days.

Iron flocs are mobilised distances greater than 15 kilometres from their source. Similar observations have been made on the Richmond River in northern NSW, where plumes of neutral but iron contaminated water affects the main channel of the river for 3 km

86 downstream of the source (Sammut et al., 1996a). Plates showing the extent of metal precipitate mobilisation are provided in Section 4.6.1.

4.5.3 Manning River Estuary 4.5.3.1 Drain Outflow Water Quality Following High Rainfall Figure 2.5 displays drains that intersect high-risk ASS in the lower Manning River catchment. pH, EC, iron, aluminium, manganese, silicon, alkalinity and Cl:SO4 data for selected drains in the Manning River estuary sampled on the 27/5/98 and 9/5/99 are listed in Table 4.5. Table 4.5 shows that low pHs and elevated metal concentrations are present in drain outflow water on the two sampling dates which were conducted following high rainfall. Drains sampled on the 27/5/98, typically have higher EC values yet the pH and metal concentrations of all listed drain outflows on this date exceed recommended guidelines as stipulated in ANZECC (2000). The pH data of drains sampled on the 9/5/99 are summarised in Table 4.6.

Table 4.5 Water quality of selected Manning River drain outflows measured on the 27/5/98 and 9/5/99. For the locations of drains refer to Figure 4.2.

Drain Drain Date pH EC Fe Al Mn Si Cl:SO4 ALK No. ID (dS m-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)

Cattai Creek 1 CC15.7L 27/05/98 4.11 15.2 0.14 1.53 ND 2.55 6.82 13 2 CC16.1L 09/05/99 3.55 1.1 4.90 1.05 0.24 2.65 6.57 NS 3 CC16.5L 09/05/99 3.04 1.8 9.82 17.71 2.13 12.83 2.10 NS 4 CC16.9L 27/05/98 3.87 10.9 ND 2.64 0.06 1.85 24.32 8

Ghinni Ghinni Creek 5 GG15.0R 09/05/99 3.46 1.5 4.02 1.74 0.85 6.47 5.17 NS 6 GG15.4R 27/05/98 3.58 21.2 0.32 5.11 0.57 3.10 9.74 21 7 GG15.5R* 27/05/98 3.21 22.3 2.17 11.28 1.41 6.76 7.79 8 8 GG15.8L 09/05/99 3.31 11 2.71 2.84 2.57 12.00 4.21 NS 9 GG16.6R* 27/05/98 3.45 15.4 2.82 32.57 4.03 16.07 5.19 8

Lansdowne River 10 LR13.1L 09/05/99 3.22 10.2 4.74 2.14 1.10 9.05 2.97 NS 11 LR15.1R 09/05/99 3.2 3.6 38.40 3.38 1.00 8.29 3.05 NS 12 LR15.4L 09/05/99 3.07 7.6 3.09 0.76 0.43 5.91 3.31 NS 13 LR15.9R 09/05/99 2.97 9.3 4.83 5.35 1.06 10.67 1.47 NS 14 LR16.1L* 27/05/98 3.47 15 2.87 9.62 0.99 7.87 6.74 8 15 LR16.6L* 27/05/98 4.91 15.3 3.37 7.04 1.18 12.36 7.44 0

* = Drain measurement collected upstream from floodgate ND = Not detected NS = Not sampled

The Cl:SO4 ratio (Table 4.3) indicates that the drain outflow waters on the 9/5/99 have interacted with oxidised sediments in the floodplain. Low pH drain outflows containing elevated metal concentrations are originating from the Cattai-Pipeclay area and the lower Lansdowne-Moto-Ghinni Ghinni Creek area. These two areas are listed as ASS priority management areas on the lower Manning River floodplain in Tulau (1999b). The water quality data collected from the Manning River estuary and floodplain drains on the 27/5/99 and 9/5/99 were used in the process of designating ASS priority management areas on the Lower Manning River Floodplain in Tulau (1999b).

Table 4.6 Summary of the Manning River drain outflow pH data measured on 9/5/99.

9/05/99

n28 Min. pH 3.12 Max. pH 6.90 Median pH 5.99 Standard Dev. pH 1.06

4.5.3.2 Tidal Water Quality Following High Rainfall The pH and EC data collected in Cattai Creek and Lansdowne River are displayed in Figures 4.8 and 4.9, respectively. The pHs of drain outflows in Cattai Creek, and Lansdowne River are also plotted on these figures as drain outflow pH.

Figure 4.8 shows poor water quality conditions in Cattai Creek on the 9/5/99. The surface water is increasingly acidic and fresh upstream from the junction with the Manning River. The four drains discharging into Cattai Creek in the upstream section of this transect contain acidic water which has a pronounced influence on the channel pH and EC values. The drain coded as CC16.5L has a pH of 3.04 and very high concentrations of aluminium (17.71 mg L-1), iron (9.82 mg L-1) and manganese (2.13 mg L-1) (Table 4.5). Appendix D lists the water quality data of drains discharging into Cattai Creek on the 27/5/98 and 9/5/98.

pH drain outflow pH EC 8 8

6 6 ) -1

pH 4 4 CC16.5L EC (dS m 2 2

0 0 9 101112131415161718

distance upstream from the ocean entrance (km)

Figure 4.8 Cattai Creek surface water pH, EC and drain outflow pH measured on the 9/5/99.

pH drain outflow pH EC

8 20

6 15 ) -1

pH 4 10 EC (dS m

2 5

0 0 10 12 14 16 18 20

distance upstream from the ocean entrance (km)

Figure 4.9 Lansdowne River surface water pH, EC and drain outflow pH measured on the 9/5/99.

Surface water pH and EC as well as drain pH data for the Lansdowne River on the 9/5/99 are shown in Figure 4.9. The surface water is increasingly acidic and fresh upstream from the junction with the Manning River to approximately 15 kilometres from the Manning River entrance. The minimum pH and EC measured in the Lansdowne on the 9/5/99 was 6.01 and 8.7 dS m-1, respectively. Figure 4.9 indicates that floodplain drain outflows, 15-16 kilometres upstream from the Manning River entrance, reduces the pH and EC of surface waters in Lansdowne River. The drains are very acidic (pH < 4.0) and contain elevated concentrations of aluminium, iron and manganese (Table 4.5 and Appendix D).

The pH and EC data collected in Ghinni Ghinni Creek and Dickensons Creek are displayed in Figures 4.10 and 4.11, respectively. The pHs of drain outflows into Ghinni Ghinni Creek are also plotted on Figure 4.10 as drain outflow pH. Ghinni Ghinni Creek connects the Lansdowne River to the Manning River creating Jones Island (Figure 4.2). The rapid increase in pH to normal estuarine conditions at the > 21 km point (Figure 4.10) is due to the mixing and neutralising effects of the main channel tidal waters. The appearance of the water in Ghinni Ghinni Creek on this date was a milky green/blue colour from the presence of suspended aluminium flocs, which are precipitated by the neutralisation process.

Dickensons Creek was another tributary of the Manning River affected by acidification on the 9/5/99 (Figure 4.11). The surface waters of the entire creek were acidified below pH 6.16. The minimum pH and EC measured in Dickensons Creek on the 9/5/99 was 3.95 and 9.4 dS m-1, respectively. Dickensons Creek water also had a milky green/blue appearance from elevated concentrations of suspended aluminium flocs.

Fish and eels were observed at the surface gulping air, swimming slowly and behaving erratically in both Ghinni Ghinni Creek and Dickensons Creek on the 9/5/99. The DO levels in surface and bed waters in Ghinni Ghinni Creek were as low as 18.2% and 5.2% saturation, respectively, on this date. Sammut (1998) found that this behaviour was related to blood hypoxia in fish with damaged gills, either from aluminium and acid induced lesions, or the accumulation of metal flocs in the lamellar spaces of the gills.

90 Additional water quality data collected from acidified Manning River tributaries on the 27/5/98 and 9/5/99 are presented in Appendix E.

pH drain outflow pH EC 8 32 7 28 6 24 )

5 20 -1

pH 4 16 3 12 EC (dS m 2 8 1 4 0 0 14 15 16 17 18 19 20 21 22 23 distance upstream from the ocean entrance (km)

Figure 4.10 Ghinni Ghinni Creek surface water pH, EC and drain outflow pH measured on the 9/5/99.

pH EC 7 28

6 24

5 20 ) -1 4 16 pH

3 12 (dS m EC 2 8

1 4

0 0 21 22 23 24 25 distance upstream from the ocean entrance (km)

Figure 4.11 Dickensons Creek surface water pH and EC measured on the 9/5/99.

4.5.3.3 Metal Precipitate Mobilisation The extent of iron and aluminium precipitate mobilisation was observed during both sampling dates and on other occasions following high rainfall. The main sources of iron and aluminium flocs entering the main channel of the Manning River were from Cattai Creek, Lansdowne River, Ghinni Ghinni Creek and a drain located on North Oxley Island (Figures 4.2 and 4.3). Flocs from Cattai Creek were observed extending for distances in excess of 1 kilometre downstream of its confluence with the Manning River. Flocs from the Lansdowne River extended more than 700 metres and flocs from Ghinni Ghinni Creek extended more than 500 metres from the confluence of each system and the Manning River. Plates showing the extent of metal precipitate mobilisation are provided in Section 4.6.2.

4.5.3.4 Drain Water Quality Water quality at Sites X, Y and Z within the drain located on North Oxley Island (Figure 4.3) were influenced by antecedent climatic conditions, in particular rainfall, and estuarine conditions at the floodgate structure (Site W, see Figure 4.3). The floodgate structure leaked which allowed the inflow of saline tidal water during the flood tide. This caused density and pH stratification of the drain in periods of low rainfall. An example of water quality conditions in the drain during a dry period is listed in Table 4.7 on the 4/6/99. The Cl:SO4 ratio on the 4/6/99 indicates the influence of oxidised pyrite in the catchment soils affecting water quality. High intensity rainfall events, for example the one that occurred in April 1999 where 172 mm fell over 5 days (Figure 4.5), caused displacement of saline water from the drain, a reduction in EC levels and moderately acidic and well-mixed conditions in the drain.

Very acidic conditions were measured in the drain on the 5/7/99 (Table 4.7). Acidic conditions extended the entire length of the drain with only a 0.01 pH unit variation detected in surface waters. On this date high aluminium and iron concentrations and a low Cl:SO4 ratio were recorded.

92 Table 4.7 Variation in drain water quality.

Date Parameter SITE WXY Z

14/04/99 pH Surface 5.45 5.44 5.46 5.61 Bed 5.45 5.43 5.49 5.50

EC (dS m-1) Surface 2.2 2.2 2.2 2.2 Bed 2.2 2.1 2.1 3.3

Al (mg L-1) Surface 0.02 0.03 nd 0.02

Fe (mg L-1) Surface 0.04 0.03 nd 0.02

Cl:SO4 Surface 2.16 1.40 nd 0.91

4/06/99 pH Surface 5.09 3.61 3.61 3.48 Bed 5.72 5.78 5.70 5.49

EC (dS m-1) Surface 23.6 11.5 13.0 4.9 Bed 27.2 27.4 26.9 24.6

Al (mg L-1) Surface 2.04 3.11 nd 9.25

Fe (mg L-1) Surface 0.32 0.54 nd 2.81

Cl:SO4 Surface 2.17 1.33 nd 1.46

5/07/99 pH Surface 3.71 3.70 3.71 3.71 Bed 3.71 3.70 5.62 3.97

EC (dS m-1) Surface 3.6 3.5 3.6 2.8 Bed 3.6 3.5 4.2 5.5

Al (mg L-1) Surface 5.09 5.46 nd 7.77

Fe (mg L-1) Surface 0.28 2.47 nd 3.15

Cl:SO4 Surface 0.12 0.16 nd 0.07

nd = no data

4.5.3.5 Oyster Lease Water Quality Drain water quality had a pronounced influence over the tidal water quality conditions at Site W, particularly in terms of pH, EC, iron and aluminium levels (Table 4.7). Continuous long-term sampling at Site W revealed highly variable water quality conditions dependant on the tide, drain conditions and antecedent climatic conditions. Figure 4.12 shows that the duration of each acid pulse is limited to approximately 6

93 hours and pHs decreased at the start of the ebb tide. Figure 4.13 displays the pH, EC and temperature data measured by the SDL at Site W. This figure shows that over the study period low pH water frequently outflows from the drain and acidified the oyster lease. The SDL measured pHs at or below 5.5 on 114 days during the study period.

start of ebb tide influence

10 20 9 18 8 16

7 14 ) 6 12 -1

pH 5 10 4 8 3 6 EC (dS m pH 2 4 EC 1 start of flood tide influence 2 0 0 21:36 2:24 7:12 12:00 16:48 21:36 2:24 Time Figure 4.12 EC and pH at Site W on the 7/8/99.

Acid outflows (pH < 5.5) persisted, on average, for 10 days but ranged between 1 to 20 days. The duration of acid outflows were dependant on antecedent rainfall intensity. When larger rainfall events (> 50 mm) occurred, for example on the 5/7/99, the initial outflow would be circumneutral and would rapidly degenerate into low pH outflows about 10 days after the rainfall. This is due to rainwater runoff diluting the acidified drain water initially, then as groundwater base-flows commenced, the drain pH would rapidly decrease at all drain monitoring sites (Sites X, Y and Z) and at Site W. Water quality measured at Sites W, X, Y and Z during the study period are listed in Appendix F.

94 10 40 10 40 A. B. C) C) O pH O 8 temp. 8 pH 30 . ( p 30

6 6 20 20 pH pH and tem and temp. (

) temp. ) -1 4 4 -1

10 10 2 EC 2 EC EC (dS m EC (dS m 0 0 0 0 19/02/99 01/03/99 11/03/99 21/03/99 31/03/99 03/05/99 23/05/99 12/06/99 02/07/99 22/07/99

10 40 10 40 C. D. C) C) pH O 8 pH O 8 . ( 30 . ( 30 p p

6 6 20 20 pH pH and tem and tem SDL temp. ) ) -1 4 -1 4 malfunction temp. EC 10 10 2 2 EC EC (dS m EC (dS m 0 0 0 0 29/07/99 18/08/99 07/09/99 27/09/99 17/10/99 06/11/99 30/10/99 19/11/99 09/12/99 29/12/99 18/01/00

Figure 4.13 EC (blue), pH (red) and temperature (black) data measured at Site W for the period (A) 19/2/99 to 6/4/99 (B) 5/5/99 to 31/7/99 (C) 1/8/99 to 31/10/99, and (D) 1/11/99 to 31/1/00.

95 EC at Site W also varied considerably over the study period. It ranged from 0 to 45 dS m-1. EC at Site W was dependant on: the EC levels in Scotts Creek; the EC levels in the drain; tidal stage; and, antecedent rainfall. High ECs were measured during prolonged dry periods and low ECs were recorded immediately following rainfall. EC/salinity at Site W influenced the pH (Figure 4.12) due to the buffering capacity of the saline tidal water. As EC increased on the flood tide, pH also increased at Site W (Figure 4.12). This was primarily due to the flood tide placing pressure on the four floodgates to reduce the drain outflow and the buffering action of the saline estuary water reducing the acidity of the ebb tide drain outflow.

The EC data collected by the SDL indicated prolonged periods of low EC following rainfall (Figure 4.13). ECs were reduced to levels below the tolerance of the Sydney rock oyster for extended periods. The lower salinity tolerance for adult oysters is 15 ppt (23.4 dS m-1) for 14 days (Holliday, 1995). Conditions of reduced EC levels for extended periods occurred following the large rainfall event in mid July 1999 (186 mm of rainfall in 7 days) and during late October and early November 1999 following smaller recurrent rainfall events (~130 mm rainfall in 20 days). On these occasions, EC levels remained below 23.4 dS m-1 for 46 and 22 days, respectively.

During the periods mentioned above (186 mm of rainfall in 7 days and ~130 mm rainfall in 20 days), EC may have briefly exceeded 23.4 dS m-1 but was not detected by the SDL as it only monitored water quality conditions at a one-hour sampling interval in the first case and at a two-hour sampling interval in the second case.

4.6 DISCUSSION 4.6.1 Characteristics of Acidification in the Hastings River Water quality data collected from the Hastings River estuary after rainfall events clearly show poor water quality conditions present in the Maria River, Connection Creek and Fernbank Creek. Water discharging from floodplain drains is acidified and has elevated concentrations of toxic metals that exceed the acceptable limits for aquatic ecosystems (ANZECC, 2000; Sammut et al., 1995; 1996a). Floodgates and artificial drainage systems that increase acid production and export, as well as attenuate or completely restrict tides that would otherwise buffer water pH in the drains, facilitate widespread

96 acidification throughout the estuary. The spatial extent of acidification of Hastings River tidal waters measured on the 18/6/99 is displayed in Figure 4.14. This figure also shows the extent of iron precipitate coating of the stream banks observed on this date.

Sammut et al. (1996a) and Johnston (1995) showed that floodgates can store acidified water in floodplain drains for prolonged periods. The stored acid can be released during low tide when the hydraulic head of the drains is higher than the tidal reach; the floodgate opens when this occurs and closes on high tide when the pressure of the tidal water forces the gates to close. Under dry weather conditions, when tidal waters are well buffered, the low tide pulses of acidic water are neutralised close to the outflow point, but iron flocs can nevertheless travel for many kilometres downstream. By contrast, wet weather can deplete the acid neutralising capacity of tidal waters and increase acid outflows from floodgates leading to extensive acidification of tidal reaches (Sammut et al., 1996b). Acid may also move up and down the river during wet weather due to plug-flow displacement from the drains. Density differences between acid water and tidal water can result in an upstream and downstream movement of an acid plug (Sammut et al., 1996a).

This widespread estuarine acidification after rainfall events in the Hastings River estuary has profound implications for the aquatic ecosystems in these river and creek systems. The extent of the acid encompasses parts of the river system where recurrent oyster losses and poor production occurs. Many oyster leases in the Maria River are not used to produce oysters. Ten out of nineteen aquaculture leases were surrendered in the lower Maria River (NSW Fisheries Unpublished Data) and the other nine oyster lease areas were poorly maintained and stocked with few or no oysters during the entire study period. Similarly, oyster leases in the area downstream of Fernbank Creek on the southern bank of the Hastings River were mostly abandoned and the remaining infrastructure was coated with iron flocs. This strongly suggests that both of these areas are incapable of supporting oyster production.

Figure 4.14 Spatial extent of acidification and iron precipitate coating in the Hastings River and Manning River estuaries measured on the 18/6/99 and 9/5/99, respectively.

It has been estimated that Fernbank Creek discharges, on average, 400 tonnes of sulfuric acid into the Hastings River per year (White, 1998). Johnston (1995) estimated the ASS in the Maria River catchment are capable of producing approximately 1.8 x 109 m3 of dilute sulphuric acid at pH 3.5. Sammut et al., (1996a) estimated that ASS are capable of producing 200 kg of sulfuric acid per hectare per year and can leach acid into the groundwater for hundreds of years.

The temporal persistence of acid events in Maria River and Fernbank Creek is dependant upon the intensity of the rainfall event and the duration of the interim dry (or low rainfall) period (Johnston, 1995; MHL, 1997; ERM Mitchell McCotter, 1997). Johnston (1995) reported that acidic conditions (< pH 5.5) persist for 4 to 6 weeks in the upper reaches of the Maria River. MHL (1997) measured a pH of approximately 6.6 continuing from October 1994 to January 1995 in the Maria River at Green Valley and in Connection Creek (32.2 and 42.5 km upstream from the Hastings River entrance respectively). Hastings Council data shows Partridge/Fernbank Creek drain discharging acidic water of pH < 3.5 into Fernbank Creek every day at low tide for a period greater than two months.

This study and previous studies (ERM Mitchell McCotter, 1997; Johnston, 1995) have measured extremely high concentrations of iron and aluminium. Johnston (1995) measured an aluminium concentration of 3.06 mg L-1 and ERM Mitchell McCotter (1997) measured 20 mg L-1 of aluminium and 14 mg L-1 of iron at the Partridge Creek drain discharging into Fernbank Creek. The maximum concentration of iron and aluminium measured at Fernbank Creek during this study was 35.90 mg L-1 and 13.84 mg L-1, respectively.

Plumes of acidic water were observed emanating from Fernbank Creek and moving upstream along the southern bank of the Hastings River for a distance of approximately 2.5 kilometres after a rain event in July 1998. Milky blue/green water from high concentrations of suspended aluminium (Plate 4.1) and red coloured water from high concentrations of suspended iron (Plate 4.2) is discernable from the saline and well-

99 buffered Hastings River water. Plumes of acidic water originating from the Maria River typically extended along the northern bank of the Hastings River and were characterised by high concentrations of suspended iron flocs. Plate 4.3 shows a plume of fresh, acidic water containing suspended iron flocs mixing with saline, well-buffered Hastings River water at the confluence of these two river systems.

4.6.2 Characteristics of Acidification in the Manning River Water quality data collected from the Manning River estuary after high rainfall events clearly shows poor water quality conditions present in the Cattai Creek, Lansdowne River, Ghinni Ghinni Creek and Dickensons Creek systems. Inflows of acidified water from these tributaries have caused acidification of the main channel of the Manning River. Water discharging from floodplain drains is acidified and has elevated concentrations of toxic metals. This situation existed on the Hastings River and has numerous ramifications for the aquatic ecosystems in acidified areas of the Manning River. The spatial extent of acidification and iron precipitate coating of the stream banks in the Manning River tidal waters measured and observed on the 9/5/99 is displayed in Figure 4.14.

Acidic conditions in Ghinni Ghinni Creek persist for long periods after rainfall because acidic water becomes trapped in this area by fast flowing currents in the Manning River and Lansdowne River. Fish and other aquatic fauna also become trapped in this system when the Lansdowne River is acidified and acidic water enters Ghinni Ghinni Creek from Dickensons Creek.

Plumes of acidic water impact the main channel of the Manning River after large rainfall events. Aerial photographs taken of the Manning River after rainfall in July and August 1998 further highlight the extent of the acidification problem. Plate 4.4 shows an acid plume originating from Ghinni Ghinni Creek and Dickensons Creek discharging into the Manning River. Plate 4.5 shows an acidic plume at the confluence of the Lansdowne River and the Manning River and Plate 4.6 shows an acidic plume discharging from Cattai Creek into the Manning River passing through numerous oyster leases.

100 Plate 4.1 A milky blue/green coloured acidic plume from Fernbank Creek affecting the Hastings River.

Plate 4.2 A red coloured acidic plume (due to high concentrations of suspended iron) from Fernbank Creek affecting the Hastings River.

Plate 4.3 Acidic plume characterised by high concentrations of suspended iron from the Maria River affecting the main channel of the Hastings River.

101 Plate 4.4 Acidified water originating from Ghinni Ghinni Creek and Dickensons Creek affecting the Manning River.

Plate 4.5 Acidified water originating from the Lansdowne River affecting the Manning River.

Plate 4.6 Acidified water originating from Cattai Creek affecting the Manning River and passing through a number of oyster leases.

102 As was the situation in the Hastings River, the extent of the acid encompasses parts of the river system where recurrent oyster losses and poor production occurs. Oyster leases in, and at the mouth of, Cattai Creek have largely been abandoned and the remaining infrastructure is coated with iron flocs. Many of the oyster leases in the Lansdowne River are also abandoned or stocked with very low number of oysters.

The frequency of acidification at an abandoned oyster lease in Scotts Creek was directly related to the occurrence of medium to high rainfall events in the local catchment area. The severity and duration of acidic conditions at this site was chiefly dependent on the drain water quality, the amount of rainfall in the local catchment and antecedent dry conditions. This is consistent with the findings of other studies conducted on the mid north and north coasts of NSW (Sammut et al., 1996a; Johnstone, 1995; Sonter, 1999) Recurrent and/or high rainfall events can cause deterioration of drain water and acidic (< pH 5.5) conditions in tidal waters for extended periods.

Water quality data showed that rainfall intensity determined the severity of acidification at all monitoring sites in the drain. The pH and EC levels in the drain had a major influence over the pH and EC levels at Site W and in the estuary during the ebbing tide. The floodgates created highly variable conditions at this oyster lease that rapidly changed at different tidal stages.

At this particular site, the mobilisation of ASS-affected waters has profound implications for water quality conditions in the receiving tidal waters. There are more than 25 oyster leases located throughout the Scotts Creek system. Scotts Creek is recognised as an important area by Manning River oyster growers as it is used to condition oysters before market.

Acidification reported in the estuaries of eastern Australia is comparable to acid rain- impacted freshwater systems, which have a pH range of 4.2 to 5.5 (Baker and Schofield, 1982). It is important to note that the concentrations of aluminium measured in ASS- affected waters in this and other studies (Sammut et al., 1996a; Johnston, 1995; Sonter, 1999) are alarmingly high compared to levels reported in acidic freshwater systems overseas (Driscoll et al., 1980; Baker and Schofield, 1982). Sammut et al. (1994),

103 Sammut et al. (1996b) and Sammut (1998) described the impacts of elevated concentrations of aluminium to fish in acidic estuarine waters in eastern Australian. However, there is limited information on the short and long-term consequences of increased aluminium levels for other types of aquatic fauna inhabiting ASS-affected waters (Roach, 1997; Corfield, 2000).

4.7 CHAPTER SUMMARY Water quality monitoring on the Hastings River and Manning River estuaries confirmed that estuarine acidification is extensive and severe following high rainfall events. Areas designated for the production of oysters are impacted, particularly leases downstream of major floodgates and acidified tributaries. There are numerous drains that discharge severely acidified water contaminated with high concentrations of iron and aluminium on both estuaries following high rainfall. Iron flocs were observed to extend for distances more than 10 kilometres from their source in the Hastings River and contaminated partially acidified and pH neutral waters in both estuaries. The mobilisation of floodplain oxidation products is efficiently distributed throughout areas of the estuary, to maximise the impact, including leased areas designated for oyster production. Drain water quality was found to be unacceptable in relation to the specifications outlined in current guidelines (ANZECC, 2000) for the protection of aquatic ecosystems. Drain waters have the capability to cause a variety of short and long-term impacts to the estuarine ecosystem.

The water quality data relating to estuarine acidification of the Hastings River and Manning River presented in this chapter provided crucial information that enables an understanding of the water quality conditions oysters are exposed to following high rainfall and for the design of the field observation experiments discussed in the following chapters as well as laboratory experiments, which are discussed in Chapters 8 and 9. The Manning River was selected for the field observation experiments detailed in the following chapters due to: a number of suitable experimental sites that were exposed to ASS-affected waters; two related studies (Sonter, 1999; Smith and Dove, 2001) conducted at or near the experimental sites; and, no occurrence of LS or QX disease which could interfere with the interpretation of the results.

104 CHAPTER FIVE EXPOSURE OF OYSTERS TO ASS-AFFECTED WATERS: FIELD EXPERIMENT DESIGNS, METHODS AND WATER QUALITY RESULTS

5.1 INTRODUCTION A number of studies have shown that marine and estuarine bivalves have a low tolerance to small changes in pH (Bamber, 1987; 1990; Kuwatani and Nishii, 1969; Knutzen, 1981; Wilson and Hyne, 1997; Dove, 1997). This chapter aims to investigate the acute and chronic effects of acidic water from ASS outflows on the survival and growth of the Sydney rock oyster under field conditions.

Information obtained from the water quality investigation showed that areas of the Manning River used for oyster production experience acidification following high rainfall. Field observation experiments were designed to examine the effects of extended periods of acidification on oysters. The experimental design and methods used to expose oysters to acidification under field conditions are detailed in this chapter. Oyster growers have reported that ASS-affected waters increase mortality and reduce growth in oysters. Therefore, this experimental work focused on the measurement of oyster survival and growth at sites impacted by ASS-affected waters.

Two field observation experiments were conducted in the Manning River estuary. The first investigated the effects of ASS-affected waters on survival and growth of oysters and was named the Survival and Growth Experiment (S&GE). The second focussed on the effects of ASS-affected waters on condition index of oysters and was named the Condition Index Experiment (CIE). Experimental oysters were placed at four sites that were impacted by acidification following high rainfall. Additional oysters were placed at three reference sites that had a very low probability of exposure to ASS-affected waters. All sites were selected on the basis of information obtained during the water quality investigations detailed in Chapter 4. Both experiments examined the impacts of ASS-affected waters to oysters under actual estuarine conditions. Additional water

105 quality data was collected during the S&GE and the CIE and are presented in this chapter (Sections 5.4 and 5.5).

5.2 RELATED RESEARCH Dove (1997) is the only study that has investigated the effect of ASS outflows on survival of developed Sydney rock oysters. In Dove’s (1997) study, a small-scale field experiment was used to expose small and large adult oysters to ASS outflows in Fernbank Creek (Figure 4.1), an acidified tributary of the Hastings River. The aim of that experiment was to examine the Sydney rock oyster’s tolerance to naturally acidified water and the experiment lasted for 90 days.

Prolonged exposure to acidified water containing elevated concentrations of iron and aluminium resulted in high mortalities after approximately 40 days. The smaller oysters experienced higher mortality rates compared to the larger oysters. This was attributed to shell perforation where the shell of the oyster is dissolved by acidic conditions to the extent where it exposes the soft tissue of the oyster.

Based on the findings from Wilson and Hyne’s (1997) study on the effects of ASS- affected waters on Sydney rock oyster larvae (see Section 1.3.1), it was theorised that ASS-affected waters would impact the settlement and recruitment of the Sydney rock oyster. Bishop’s (2000) related study used field experiments to determine the settlement and recruitment patterns of Sydney rock oyster spat in acid impacted areas of the Hastings River. The results of the study showed that sites located near ASS outflow sources had a greater rate of decline in spat numbers. Bishop (2000) postulated that mortality was caused by iron flocs, as there were no significant acidification events. The study also tested the hypothesis that iron coated substratum inhibited the settlement of larvae. However, no clear differences emerged between iron coated and non-iron coated substratum at the experimental sites. Bishop (2000) believes this result may have been confounded by the development of biofilms, which removed the compositional differences between the two substrates. Laboratory experiments confirmed that iron flocs accumulated on the gill and mantle of six-month old Sydney rock oysters.

106 Bishop (2000) concluded that there needs to be further investigation of: the effects of continual acid outflow on oyster spat; the influence of the substratum composition and acid water on settlement and recruitment of spat; and, the impacts of high concentrations of iron and aluminium to the gills and soft tissue of oyster spat.

5.3 EXPERIMENTAL DESIGN This section details the experimental design, sampling techniques, field measurements and laboratory analyses used for the S&GE and the CIE. The S&GE and CIE were designed to investigate the effects of acute and chronic exposure to ASS-affected waters to satisfy objectives 3, 4 and 5 of this study. Both experiments used oyster leases that experienced acidification after high rainfall and oyster leases that had a very low probability of exposure to ASS-affected waters. The latter oyster leases were selected as reference or comparison sites. The selection of all experimental sites was based on the information obtained from investigating the characteristics of acidification on the Manning River detailed in Chapter 4.

5.3.1 Survival and Growth Experiment (S&GE) 5.3.1.1 Field Sites Details of the seven field sites used for the S&GE are listed in Table 5.1 and their location is displayed on Figure 5.1. All sites were located within existing or former oyster leases, with the exception of Site 5, which was situated in Cattai Creek (Figure 5.1). Sites 1, 2 and 3 were oyster leases that had a very low probability of being exposed to ASS-affected waters because they were distant from major acid outflows and plumes (Figure 4.14 and Plates 4.4, 4.5 and 4.6). Sites 4, 5, 6 and 7 were locations that had a very high probability of exposure to ASS–affected waters after rainfall. These particular sites were situated in tributaries and the main channel of the Manning River that experienced acidification (Figure 4.14).

107 Table 5.1 Description of Sites 1 to 7.

Site Location Oyster Distance Comments Number Lease Upstream* Number (km)

1 Manning River 85-210 5.1 Very Low probability of ASS discharge exposure 2 Manning River 84-176 11.9 Very Low probability of ASS discharge exposure 3 Manning River 69-137 6.3 Very Low probability of ASS discharge exposure 4 Scotts Creek 79-182 14.3 Located 5 m from a large floodgate structure 5 Cattai Creek - 15.1 Extended periods of acidic and fresh conditions 6 Lansdowne River 85-220(1) 13.5 Area of declining oyster productivity 7 Manning River 59-174 9.3 Situated downstream from the mouth of Cattai Creek

* From the Manning River entrance at Harrington

Figure 5.1 Locations of oyster and water quality monitoring Sites 1 to 7 on the Manning River.

108 5.3.1.2 Sampling Dates Water quality measurements and a count of surviving oysters were performed every fortnight, for a 14-week period commencing on the 1/6/99. To extend the data collection period an additional three site visits were conducted on the 1/10/99, 15/11/99 and 10/1/00. The measurements performed on each sampling date are listed in Table 5.2.

Table 5.2 Sampling dates and field measurements performed.

Sampling Measurements Dates

01/06/99 Water quality, surviving oysters and whole oyster weight 15/06/99 Water quality, surviving oysters and shell dimensions 28/06/99 Water quality, surviving oysters and whole oyster weight 12/07/99 Water quality, surviving oysters and shell dimensions 26/07/99 Water quality, surviving oysters and whole oyster weight 09/08/99 Water quality, surviving oysters and shell dimensions 23/08/99 Water quality, surviving oysters and whole oyster weight 06/09/99 Water quality, surviving oysters and shell dimensions 01/10/99 Water quality, surviving oysters and whole oyster weight 15/11/99 Water quality, surviving oysters, whole oyster weight and shell dimensions 10/01/00 Water quality, surviving oysters, whole oyster weight and shell dimensions

5.3.1.3 Water Quality On every sampling date, discrete in situ water quality measurements close to the experimental oysters were performed at the seven sites using a Yeo-Kal 611 Intelligent Water Quality Analyser and a Palintest Photometer (Model 5000). A surface water sample was collected for ICPAES and chloride analysis. Water sampling and analysis techniques were conducted using the methodology outlined in Sections 4.4.6 to 4.4.8.

A Greenspan Technical Services Smart Sonde (Model SD300) and a second Yeo-Kal 611 Intelligent Water Quality Analyser were installed at Sites 2 and 4, respectively. The SDLs provided measurements of pH, EC and temperature, at intervals of either 1 or 2 hours, to characterise differences in water quality conditions between a reference site (Site 2) and a site exposed to ASS-affected waters (Site 4).

109 5.3.1.4 Experimental Oysters Experimental Sydney rock oysters were sourced from the Manning River and inspected to ensure that they did not display clinical signs of LS (Chapter 7). Experimental oysters were acclimated at Site 2 for 14 days before being randomly deployed to the seven sites on the 1/6/99. Oysters were single seed stock that was removed from PVC (polyvinyl chloride) catching slats. Two size classes of adult oysters were used for the S&GE. One size class was comprised of oysters that were a marketable size (i.e. 20 to 60 g) and had a mean weight (± 95% confidence interval (CI)) of 29.1 ± 0.4 g. These oysters were referred to as “large” oysters for this experiment. The second size class was comprised of oysters that were 1 to 2 years in age and had a mean weight (± 95% CI) of 5.1 ± 0.1 g. These oysters were referred to as “small” oysters. At all sites, four replicates of 50 large oysters and 50 small oysters were placed in polyethylene mesh baskets, attached to 7 mm nylon rope and suspended approximately 0.3 m below water level. Four foam floats were used for buoyancy at each site.

On each sampling date, all oysters were collected by boat, washed and counted for surviving oysters before whole weight measurements of individual oysters were made using an electronic weight balance. All of the surviving experimental oysters were weighed on the dates listed in Table 5.2. Two mesh baskets, one containing 50 large oysters and the other containing 50 small oysters at the start of the experiment, at each site were selected to measure oyster shell height (Figure 3.2) on the dates listed in Table 5.2 using digital vernier callipers. To reduce the influence of over-catch and epibiont accumulation during this experiment all oysters were cleaned on the 15/11/99 and replaced at their original sites.

5.3.1.5 Oyster Survival Dove (1997) found that large and small oysters are able to withstand pulse flows of severely acid water (pH < 4) discharging from a floodplain drain for a period of approximately 40 days before high mortalities were measured. Oyster mortality (as a percentage of live animals) for the S&GE was measured bi-monthly at each experimental site. Oyster survival was calculated using the formula:

Survival = (No. of living shell/[No. of living shell + No. of dead shell]) x 100

110

To investigate differences among the impacted sites and the reference sites, a three factor analysis of variance (ANOVA) was used. The factors were Acid (fixed factor), Size (fixed factor) and Site (nested within Acid – random factor). The ANOVA was performed on the oyster survival data from two sampling dates (23/8/99 and 10/1/00) during the S&GE. The 23/8/99 (day 83) was after a period of high rainfall where 379 mm of precipitation was recorded from the start of the experiment (1/6/99) at the Bureau of Meteorology Station Number 60141. The 10/1/00 (day 224) was the final sampling date for the S&GE.

5.3.1.6 Instantaneous Growth Rate Instantaneous growth was expressed as the percent increase in whole weight or shell height per day (Rheault and Rice, 1996; Toro et al., 1995). Growth rate was also determined for the same two periods detailed in Section 5.3.1.5 during this experiment (i.e. from the 1/6/99 to 23/8/99 (day 0 to 83), which corresponded to a period of high rainfall and from 1/6/99 to 10/1/00 (day 0 to 224), which represents the growth rate for the entire experiment). Instantaneous growth was calculated using the formula (Ricker, 1975; Rheault and Rice, 1996):

% increase per day = (ln[Wt/Wo]/t) x 100

Where Wo is the initial mean wet weight in grams (or shell height in mm) and Wt is the mean weight (or height) at time “t” in days.

The instantaneous growth rate (based on oyster whole weight) was calculated using the mean weight of the total number of oysters alive at each site on the 23/8/99 and 10/1/00. Pooling of the oyster whole weights was necessary due to the high mortality at particular sites exposed to ASS-affected waters. The mean instantaneous growth rates were plotted for the two time intervals (day 0 to 83 and day 0 to 224). Bootstrap was used to approximate the 95% CIs of the plotted means.

111 5.3.2 Condition Index Experiment (CIE) Condition index is an effective measure of the nutritive status of bivalves (Crosby and Gale, 1990). Condition index can be used as an ecophysiological indicator to characterise the “health” of oysters (Lucas and Beninger, 1985) and has been used in studies involving the Sydney rock oyster and pollutants in the marine environment (Avery et al., 1996). Condition index has also been used to assess the meat condition of Sydney rock oysters to compare its quality against Pacific oysters (C. gigas) (Mason and Nell, 1995) and to assess the performance of triploid oysters versus diploid oysters in five estuaries (Hand and Nell, 1999).

Mason and Nell’s (1995) study provided a baseline of condition index for the Sydney rock oyster in different growing environments within Port Stephens. Hand and Nell (1999) provide further condition index information for oysters grown in the Hastings River, Georges River, Hunter River, Tilligerry Creek and Lake Pambula. However, there is still a lack of information regarding oyster condition throughout NSW over different seasons.

Condition index was used for this study to investigate if oyster quality and health are affected by acidification caused by ASS outflows. An experiment designed to measure oyster condition index was undertaken after it was determined that exposure to ASS- affected waters reduced oyster growth rates in the Manning River. Condition index was not measured during the SG&E because it required the sampled oysters to be sacrificed.

5.3.2.1 Field Sites The same sites used for the SG&E were selected for this experiment due to the existing water quality information for the individual sites collected during the S&GE. However, Site 5 was not used because of the high mortality rate measured at this location.

5.3.2.2 Experimental Oysters Five hundred and fifty single seed Sydney rock oysters were collected from Sites 1, 2 and 3 for the CIE. The mean weight (± 95% CI) of these oysters was 19.7 ± 0.6 g. Oysters were acclimated at Site 2 for 30 days before being randomly deployed to Sites

112 1, 2, 3, 4, 6 and 7 (Figure 5.1) on the 1/2/00 in mesh baskets using the same system described in Section 5.3.1.4.

5.3.2.3 Sampling Dates and Procedures Seventy-two oysters were used to establish the initial condition index of the oysters on the 1/2/00. Twelve oysters were then collected from each of the six sites on the 6/4/00, 9/6/00, 7/8/00, 23/10/00 and 10/1/01. Water samples and water quality measurements were collected at each site visit using the methodology detailed in Section 5.3.1.3 and Sections 4.4.6 to 4.4.8.

5.3.2.4 Condition Index Within six hours of collection, oysters were cleaned of fouling and commensal organisms, washed, blotted dry and their whole weight was measured using a weight balance. Oysters were opened from the hinge and the soft tissue was removed from the valves with a scalpel by cutting the adductor muscle. Shells were washed in deionised water, blotted dry and weighed. The soft tissue was also washed in deionised water to remove any shell debris and dried for 48 hours at 80O C. The dried soft tissue was placed in desiccators to cool and then weighed to determine the dry soft tissue weight.

The gravimetric method recommended by Crosby and Gale (1990) was used to calculate condition index. The following condition index formula was used:

CI = dry soft tissue weight (g) x 1000 / internal shell cavity capacity (g)

Where:

internal shell cavity capacity = whole weight (g) – shell weight (g) (Lawrence and Scott, 1982).

To investigate differences in oyster’s condition indices at acid-impacted sites and reference sites, a three factor ANOVA was used. The factors were Acid (fixed factor), Date (random factor) and Site (nested within Acid – random factor).

113 5.4 SURVIVAL AND GROWTH WATER QUALITY CONDITIONS Water quality information collected at the seven experimental sites between the dates 1/6/99 and 10/1/00 is presented in this section. Rainfall information is also included in this section because of its influence on the water quality at the seven sites.

5.4.1 Rainfall Rainfall on the lower Manning River floodplain was a key factor in the mobilisation of acidified water as well as other oxidation products into particular areas of the estuary (Chapter 4). Daily rainfall during the S&GE is displayed in Figure 5.2.

During the S&GE, there was a high rainfall event where a total of 186 mm of rainfall was recorded between the 11/7/99 and the 17/7/99 (Figure 5.2). Areas of the upper Manning River catchment also received considerable quantities of rainfall during the same period. This resulted in extensive flooding of the lower Manning River floodplain. The driest period was at the beginning of spring in 1999. The first five months of 1999 were particularly wet and a total of 657 mm of rainfall was recorded during this period. This amount of rainfall was sufficient to cause acidification of Sites 4, 5, 6 and 7 before experimental oysters were placed at these sites on the 1/6/99.

sampling dates 100 90 80 70 60 50 40 30 precipitaion (mm) 20 10 0 Jul-99 Oct-99 Jun-99 Jan-00 Aug-99 Sep-99 Dec-99 Nov-99 May-99

Figure 5.2 S&GE rainfall (Source: Bureau of Meteorology, Station Number 60141) and sampling dates.

114

5.4.2 pH and EC Rainfall influenced the EC levels at Sites 1, 2 and 3 and both EC levels and the pHs at Sites 4, 5, 6 and 7. Sites 1, 2 and 3 were characterised by higher ECs with the measured mean EC values being greater than 18.7 dS m-1 for these sites. Sites 4, 5, 6 and 7 had mean EC values less than 16.4 dS m-1. Graphs of pH and EC measured on each sampling date are presented with the oyster data in Chapter 6 (refer to Figures 6.1 to 6.7).

A summary of the pH and EC data collected at the seven sites on the 11 site visits during the S&GE is included in Tables 5.3 and 5.4, respectively. Table 5.3 highlights the variation in pH that was measured at the seven sites. Sites 1, 2 and 3 had higher pHs on the ebb and flood tides than Sites 4, 5, 6 and 7.

The EC data displayed in Table 5.4 shows that Sites 1, 2 and 3 have higher EC values than Sites 4, 5, 6 and 7. Experimental sites located further upstream (Table 5.1 and Figure 5.1) have reduced EC values. Appendix G contains additional water quality data collected during each site visit. Alkalinity levels measured at Sites 4, 5, 6 and 7 were commonly lower than Sites 1, 2 and 3. SDLs were used to gather additional pH, EC and temperature data at Sites 2 and 4. A display of the pH and EC data between the 26/6/99 and 27/7/99 is included in Figure 5.3.

Table 5.3 Summary of pH data for Sites 1 to 7 for the S&GE (calculated from data collected on the 11 sampling visits).

EBB TIDE FLOOD TIDE Site Median Min Max Site Median Min Max pH pH pH pH pH pH

1 8.00 7.53 8.78 1 8.12 7.41 8.67 2 7.92 7.21 8.65 2 7.89 7.42 8.40 3 7.95 7.59 9.04 3 8.10 7.64 8.67 4 5.92 3.51 7.81 4 7.58 5.72 8.34 5 5.47 4.45 7.36 5 5.64 4.46 7.33 6 6.84 5.51 7.01 6 6.95 6.19 7.75 7 6.65 5.27 7.90 7 7.48 5.89 8.00

115 Table 5.4 Summary of EC data for Sites 1 to 7 for the S&GE (calculated from data collected on the 11 sampling visits).

EBB TIDE FLOOD TIDE Site Median Min Max Site Median Min Max EC EC EC EC EC EC (dS m-1)(dS m-1)(dS m-1)(dS m-1)(dS m-1)(dS m-1)

1 30.4 14.5 44.4 1 41.1 20.2 50.5 2 21.5 5.9 31.4 2 23.0 8.7 33.4 3 27.9 13.8 40.0 3 38.6 16.2 51.9 4 15.9 1.6 27.9 4 19.8 6.2 32.3 5 3.3 0.3 26.8 5 4.1 1.1 28.1 6 12.9 2.3 21.7 6 18.4 3.9 25.1 7 16.0 1.9 28.3 7 23.3 1.9 34.1

Figure 5.3 highlights the large variability in pH at Site 4 (which corresponds to Site W in Chapter 4) due to the influence of the floodgate and acidified drainage water. Section 4.5.3.5 discusses the water quality data measured at Site 4 during this experiment in greater detail. Site 2 experienced slightly decreased pHs because of the reduced buffering capacity in the river caused by the massive influx of fresh flood waters during this period. This caused a decrease in the pH at Site 2 to pH 6.5. This was the minimum pH recorded by the SDL at this site during the S&GE. The water quality data collected at the seven sites over the experimental period shows the varying degree by which each site is impacted by ASS outflows.

116

9 45 8 40 pH 7 35 6 30 ) -1 5 25 pH 4 20

3 15 EC (dS m EC 2 10 1 5 0 0 9/06/99 19/06/99 29/06/99 9/07/99 19/07/99 29/07/99

9 45 8 40 pH 7 35 6 30 ) -1 5 25 pH 4 20

3 15 EC (dS m EC 2 10 1 5 0 0 9/06/99 19/06/99 29/06/99 9/07/99 19/07/99 29/07/99

Figure 5.3 pH (bold line) and EC (thin line) at Site 2 (A) and Site 4 (B) for the period: 26/6/99 to 26/7/99.

117 5.4.3 Dissolved Metals Table 5.5 lists the maximum concentrations of iron, aluminium and manganese measured at the seven experimental sites. Elevated levels of iron, aluminium and manganese were measured at Site 4 after the large rainfall event in mid-July. These maximum-recorded values are considerably greater than the values measured at other sites. This is mainly due to the close proximity of this site to the acid outflow source (see Figure 4.3) which reduces the amount of dilution and neutralisation of the outflow before it impacts oysters at this particular site. Red and pearly-white flocs were commonly observed in the water during the initial three months of the experiment at Sites 4, 5, 6 and 7. This indicates high concentrations of suspended iron and aluminium.

Table 5.5 Maximum concentrations of Fe, Al and Mn at each site measured on the ebb tide during the S&GE.

EBB TIDE Site Fe Al Mn (mg L-1)(mg L-1)(mg L-1)

1 0.030.070.16 2 0.02 0.1 0.08 3 0.040.060.19 4 25.95 9.95 8.34 5 0.722.020.45 6 BD 0.08 0.61 7 BD 0.14 0.22

n = 11, BD = below detection limits

5.5 CONDITION INDEX EXPERIMENT WATER QUALITY 5.5.1 Rainfall Daily rainfall during the CIE is displayed in Figure 5.4. A total of 1,059 mm of rainfall was measured during this experiment (Figure 5.4). March was the wettest month with 373 mm of rainfall being recorded in this month. Two high rainfall events of 211 mm and 65 mm were recorded in a 24-hour period in March. The driest month was September with a total of 9 mm of rainfall being recorded in the entire month. Less rainfall was recorded during CIE compared to the S&GE.

118

250 sampling dates

200

150

100 precipitation (mm) 50

0 01-Jul-00 01-Apr-00 01-Oct-00 01-Jan-01 01-Jun-00 01-Jan-00 01-Feb-00 01-Mar-00 01-Dec-00 01-Aug-00 01-Sep-00 01-Nov-00 01-May-00

Figure 5.4 CIE rainfall (Source: Bureau of Meteorology Station Number 60141) and sampling dates.

5.5.2 pH and EC Water quality monitoring during the CIE was not carried out as frequently as for the S&GE. A different rainfall pattern was evident from Figures 5.2 and 5.4 between the two experiments. The data from the SDL located at Site 4 shows that recurrent rainfall events throughout July (Figure 5.2) caused pHs to fall below 5 and resulted in highly variable EC levels at this site (Figures 5.3 and 4.13). Table 5.6 displays the pH, EC, temperature and alkalinity data measured during the CIE. The water quality data measured during the CIE are tabulated in Appendix H.

119 Table 5.6 Water quality at Sites 1, 2, 3, 4, 6 and 7 measured during the condition index experiment.

Sampling Site pH EC Temp Alkalinity Date (dS m-1)(OC) (mg L-1)

01/02/00 1 8.18 44.6 22.88 103 2 8.02 32.7 23.47 76 3 8.29 51.7 22.73 73 4 7.93 28.7 23.21 76 6 7.46 23.9 23.74 65 7 7.96 33.4 23.41 76

06/04/00 1 8.02 34.2 22 76 2 7.84 19.7 22.26 73 3 8.08 34.6 22.2 80 4 7.84 20.9 22.49 76 6 6.88 13 22.35 60 7 7.36 20 22.43 60

09/06/00 1 8.12 45.4 14.06 95 2 8.05 34.4 13.15 80 3 8.18 45.8 14.39 85 4 7.93 34.6 12.33 65 6 7.65 35.1 12.59 73 7 6.76 30.6 11.45 47

07/08/00 1 7.93 34 16.37 73 2 7.75 25 16.06 85 3 7.79 36.7 16.55 76 4 5.64 21.7 17.18 47 6 5.32 24.2 14.21 NS 7 6.97 10.8 14.13 30

23/10/00 1 8.19 48.2 20.01 76 2 7.67 35 26.69 73 3 8.31 46.4 21.09 65 4 7.84 33.8 21.59 90 6NSNS NS NS 7 8.05 37.5 21.14 73

12/01/01 1 7.99 40.5 23.41 80 2 7.73 33.3 27.12 100 3 7.91 39 23.96 95 4 7.81 35.2 24.42 90 6 7.58 37.4 24.74 90 7 7.82 36.3 24.07 80

NS = not sampled

The lowest pH measured during the CIE was 5.32 at Site 6 on the 7/8/00 (Table 5.6). This is the only date that moderately acidified waters were detected at the experimental sites, with the exception of Site 4. However, high concentrations of suspended iron

120 flocs were observed causing the water to appear a deep red/burgundy colour on the 6/4/00, 9/6/00 and 7/8/00 at Sites 4, 6 and 7. On these dates the alkalinity levels measured at Sites 4, 6 and 7 were lower than Sites 1, 2 and 3, with the exception of Site 4 on the 6/4/00 (Table 5.6). Temperature has a significant influence on oyster growth and temperatures at all sites were typically low throughout the winter months.

5.6 DISCUSSION The water quality monitoring at the seven experimental sites has measured extended periods of acidic conditions at sites exposed to ASS-affected waters (Sites 4, 5, 6 and 7). Sites that had a low probability of exposure to ASS-affected waters (Sites 1, 2 and 3) had circumneutral pHs for the entire experimental period. The results of the water quality monitoring show that the conditions required for the S&GE and CIE were met in order to investigate differences that occur between acidified and non-acidified sites.

The water quality monitoring has measured large differences in water quality conditions at oyster lease sites that are impacted by ASS-affected waters and those which are not. Firstly, pH and EC levels at locations impacted by ASS-affected waters fluctuate dramatically and were dependant upon antecedent climatic conditions, predominately rainfall. Secondly, elevated concentrations of iron, aluminium and manganese were measured at the sites exposed to ASS-affected waters and were not detected at the sites where pHs were circumneutral. Thirdly, suspended iron flocs were commonly observed at all of the sites impacted by ASS-affected waters, particularly after rainfall. All of these differences are consistent with the findings relating to estuarine acidification presented in Chapter 4.

Lease sites that are located close to the acid outflow sources, especially in areas of the estuary that are well-flushed (e.g. Site 4), experience widely fluctuating pHs, EC levels and dissolved metal concentrations. Water quality for such leases was dependent on the tidal stage and the nature of the drain outflow water. Drain outflow water quality on the mid north coast of NSW was largely dependant on antecedent rainfall (Johnston, 1995; Smith and Dove, 2001).

121

5.7 CHAPTER SUMMARY The experimental design and methods detailed in the first sections of this chapter enabled oysters to be exposed to ASS-affected waters under actual estuarine conditions using a typical oyster cultivation technique. The following chapter presents and discusses the data obtained from the S&GE and CIE.

The water quality findings show favourable conditions to investigate differences in survival and growth rates between the seven experimental sites. The water quality data shows that oysters located at Sites 4, 5, 6 and 7 were exposed to prolonged periods of acidification, particularly during the ebb tide, caused by ASS outflows during the experimental period. The water quality data also shows that oysters at sites 1, 2 and 3 experienced circumneutral pHs during the same period. This water quality data will be very useful in examining associations between estuarine acidification, oyster mortalities, slow growth and oyster quality. Oyster survival, growth rates and health at Sites 1 to 7 are discussed in detail in the following chapter.

122 CHAPTER SIX SURVIVAL AND GROWTH OF OYSTERS EXPERIMENTALLY EXPOSED TO ASS-AFFECTED WATERS IN THE MANNING RIVER ESTUARY

6.1 INTRODUCTION The overriding objective of this study was to investigate associations between acidification, oyster mortalities and slow growth. It was hypothesised that: ASS- affected waters cause mortalities and reduce growth rates in Sydney rock oysters; and, ASS-affected waters impact small oysters more than large Sydney rock oysters.

The previous chapter presented water quality findings at seven sites on the Manning River selected on the basis of their probability of exposure to ASS-affected waters. There were notable differences in pH, EC levels and concentrations of dissolved metals at the four sites exposed to ASS-affected waters compared to the three reference sites.

The experimental design and water quality information relevant to the S&GE and CIE were also presented in Chapter 5. This chapter contains the results and discussion from the S&GE and CIE which were designed to test for an association between estuarine acidification and oyster production problems. Section 6.2 presents an overview of water quality and oyster data obtained from the seven experimental sites during the S&GE.

6.2 RESULTS AND OBSERVATIONS AT EXPERIMENTAL SITES Figures 6.1 to 6.7 contain summary display graphs, which show large oyster survival, small oyster survival and whole weight data measured during the S&GE. EC, pH and rainfall data obtained during this experiment are displayed on each figure. Sections 6.2.1 to 6.2.5 discuss the data measured at the experimental sites.

6.2.1 Reference Sites (Sites 1 to 3) Sites 1 to 3 were characterised by high oyster survival with very little variation between and amongst large and small oysters at all three sites (Figures 6.1 to 6.3). Weight loss in large oysters was measured on the 26/7/99 at Sites 1, 2 and 3. This can be attributed to low ECs caused by flooding and subsequent reduced feeding opportunities for

123 oysters. Small oyster weight gain at Sites 1, 2 and 3 was negligible or non-existent between 12/7/99 and 26/7/99 (Figures 6.1 to 6.3). During this period water temperatures were low and affected by the mid-July flood. The lowest EC levels detected at Sites 1, 2 and 3 during the S&GE were measured on the 26/7/99 and were also due to the mid-July flood during which freshwater inputs dominated the estuary flows. The pH values measured at Sites 1, 2 and 3 on this date were all > 8. The lowest pH levels at Sites 1, 2 and 3 were measured before the flood and did not fall below circumneutral conditions. Aluminium and iron concentrations did not exceed the ANZECC (2000) guidelines at Sites 1, 2 and 3 (Table 5.5).

Plate 6.1 Appearance of S&GE oysters on the 9/8/99: (A) oysters from Site 1 with healthy shells; and, (B) oysters from Site 4 with iron-coated shells.

124 small oysters 100 large oysters 80

% surviving 40

20 A. 0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00 50 B.

) large oysters 30 ght (g

e wei 20 whol small oysters 10

0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

10 50

9 EC

40 C) pH O 8 . ( mp 7 30 pH ) & te 6 20 -1 5 temp. 10 4 C. EC (dS m

3 0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

90 D.

60 (mm)

30 precip.

0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

Figure 6.1 Site 1 summary display: (A) mean percentage survival (± 95% CIs); (B) mean whole weight (± 95% CIs); (C) pH, EC and temperature; and, (D) rainfall.

125 small oysters 100 large oysters 80

% surviving 40

20 A. 0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

50 B. 40 ) (g 30

large oysters 20 whole weight 10 small oysters 0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

10 35

9 pH 30 C) 8 25 O

7 20 temp. pH ) & temp. (

6 15 -1

5 10 EC (dS m 4 C. EC 5

3 0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

D. 60 (mm)

precip. 30

0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

Figure 6.2 Site 2 summary display: (A) mean percentage survival (± 95% CIs); (B) mean whole weight (± 95% CIs); (C) pH, EC and temperature; and, (D) rainfall.

126 large oysters 100 small oysters 80 g 60 rvivin su

% 40

20 A. 0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

50 B. 40

) large oysters 30

20 whole weight (g

10 small oysters 0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

10 50

40 C) O

8 pH . ( mp 7 30 pH ) & te 6 20 -1 5 temp. EC 10 EC (dS m 4 C. 3 0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

D. 60 (mm)

precip. 30

0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

Figure 6.3 Site 3 summary display: (A) mean percentage survival (± 95% CIs); (B) mean whole weight (± 95% CIs); (C) pH, EC and temperature; and, (D) rainfall.

127 6.2.2 Sites Exposed to ASS-Affected Waters (Sites 4 to 7) The S&GE was conducted whilst the temporal characteristics of acidification were being measured at Site 4 (W). There was a considerable difference between large and small oyster survival at Site 4 after the 26/7/99 (Figure 6.4). Small oysters experienced an increased mortality rate after this date. More than 70% of small oysters removed from baskets at Site 4 after the 9/8/99 had shell perforation (Plate 6.2). The water quality data collected at this site shows a high variability in both pH and EC induced by rainfall events (Section 4.5.3.5 and Figure 5.3). On the 9/8/99, 9.95 mg L-1 of dissolved aluminium and 25.95 mg L-1 of dissolved iron were measured at this site (Table 5.5).

Site 5 was characterised by dramatic mortality in large and small oysters after 12/7/99 (Figure 6.5), which corresponded to the date when shell perforation in small oysters was first noted. More than 85% of dead small oysters had shell perforation on the 9/8/99. At Site 5, all of the small oysters that had a flat section at the anterior of the left valve, caused by the PVC catching slat, exhibited shell perforation. Small oysters that had developed a rounded anterior section in their left valve had a much lower incidence of shell perforation. Plate 6.2 shows this difference in shell morphology. Whole weights in large and small oysters decreased from the start of the experiment at Site 5. Oysters were not weighed after the 26/7/99 due to high mortality at this site. Low pH and EC levels were measured at Site 5 during all site visits (Tables 5.3 and 5.4). The highest values of pH and EC were recorded on the 1/10/99 following a short dry period, which allowed brackish estuary water to neutralise the acidity at this site. However, by this date there were very few oysters alive at Site 5.

Rainfall in late October through to the end of the experiment caused pH and EC to drop 2.6 units and 25 dS m-1, respectively at Site 5. The maximum dissolved aluminium and iron concentrations measured at Site 5 were 2.02 mg L-1 and 0.72 mg L-1, respectively (Table 5.5). However, suspended iron was observed in the water during all site visits and formed a thick coating on all oysters and baskets located at this site.

128 100 large oysters

60 shell perforation small oysters first noted

% surviving 40

20 A. 0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

50 B. 40 )

30 ght (g i e large oysters e w 20 hol w 10

small oysters 0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

10 50 C. 9

40 C)

pH O

8 . ( mp 7 30 pH ) & te 6 temp. 20 -1 5 10 4 EC (dS m EC 3 0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

90 D.

60 . (mm) p

preci 30

0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

Figure 6.4 Site 4 summary display: (A) mean percentage survival (± 95% CIs); (B) mean whole weight (± 95% CIs); (C) pH, EC and temperature; and, (D) rainfall.

129 100

80 small oysters Shell 60 perforation first noted

% surviving 40 large oysters 20 A. 0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

50 B.

40 ) (g 30 large oysters 20 whole weight 10 small oysters 0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

10 50 C. 9

40 C) O

8 pH . ( mp 7 30

pH temp. ) & te 6 20 -1 5 10 4 EC (dS m EC 3 0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

90 D.

60 . (mm) p

preci 30

0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

Figure 6.5 Site 5 summary display: (A) mean percentage survival (± 95% CIs); (B) mean whole weight (± 95% CIs); (C) pH, EC and temperature; and, (D) rainfall.

130 rounded flat anterior anterior left valve left valve

Plate 6.2 Variation in the shell morphology in the anterior of the left valve of small oysters. Oysters that have a flat section on their left valve (right hand side) also display shell perforation.

Small oysters at Site 6 experienced high mortality in comparison to large oysters during the period 12/7/99 through to the 6/9/99 (Figure 6.6). All small oysters with flat sections (from PVC catching slats) in the rear of their shell (Plate 6.2) were dead at Site 6 after 26/7/99. All of these oysters had evident shell perforation. Weight loss was measured in large oysters during the first three months of the experiment at Site 6 (Figure 6.6). Low pH and EC values were recorded at this site in the period before the flood event due to high rainfall throughout June and July. Low EC values persisted at Site 6 over most of the experimental period. No dissolved iron or aluminium was detected by ICPAES analysis at this site. However, iron flocs were observed in high concentrations in the water and it formed a thick coating on both the oysters and baskets. ICPAES sample preparation removed these colloidal species of iron and aluminium from the water samples.

Site 7 was the only experimental oyster lease exposed to ASS-affected waters and located in the main channel of the Manning River (Figure 5.1). There was also a large difference in the mortality rate between large and small oysters at this site (Figure 6.7). Shell perforation was noticed in large and small oysters at Site 7 after the 9/8/99. Whole weight loss was measured in large oysters between the dates 28/6/99 to 26/7/99.

131 100 large oysters

60 shell perforation small oysters

% surviving 40 first noted

20 A. 0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

50 B.

40 ) (g 30

small oysters 20 whole weight 10 small oysters 0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

10 50 C. 9

40 C) 8 O

7 pH 30 pH ) & temp. ( 6 20 -1 5 temp. EC 10 4 EC (dS m

3 0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

90 D.

60 (mm)

precip. 30

0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

Figure 6.6 Site 6 summary display: (A) mean percentage survival (± 95% CIs); (B) mean whole weight (± 95% CIs); (C) pH, EC and temperature; and, (D) rainfall.

132 100 large oysters 80 small oysters

%surviving 40 shell perforation first noted 20 A. 0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

50 B.

40 ) (g 30

large oysters 20 whole weight 10 small oysters 0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

10 50 C. 9

40 C)

pH O 8

7 30 pH ) & temp. ( 6 20 -1 5 temp. 10 4 EC (dS m EC 3 0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

90 D.

60 (mm)

precip. 30

0 20/05/99 9/07/99 28/08/99 17/10/99 6/12/99 25/01/00

Figure 6.7 Site 7 summary display: (A) mean percentage survival (± 95% CIs); (B) mean whole weight (± 95% CIs); (C) pH, EC and temperature; and, (D) rainfall.

133

The pH and EC at Site 7 was strongly influenced by rainfall in the Cattai Creek catchment (Sonter, 1999). The pH and EC varied considerably at this site ranging from 5.27 to 7.90 and 1.9 to 33.8 dS m-1, respectively (Tables 5.3 and 5.4). This variation can be attributed to the strong combined influence of Cattai Creek and the Manning River at this location. Dissolved aluminium and iron were measured at this site only in low concentrations. However, suspended iron flocs were commonly observed at this site and formed a thick coating on the oysters and baskets, especially following high rainfall.

6.3 THE EFFECTS OF ASS-AFFECTED WATERS ON OYSTER SURVIVAL 6.3.1 Results Large and small oysters’ mean survival data measured on each sampling date at the seven sites are plotted on Figures 6.8 and 6.9, respectively. Oyster survival at Sites 1, 2 and 3 were very similar on all sampling dates (Figures 6.1, 6.2, 6.3, 6.8 and 6.9). Both large and small oysters at these three sites had a high survival percentage. The mean (± 95% CI) survival percentage for large and small oysters located at Sites 1, 2 and 3 for the period between 1/6/99 and 10/1/00 was 95.3 ± 2.0% and 96.7 ± 1.3%, respectively.

Figures 6.1(A) to 6.7(A) display the difference in oyster survival between large oysters and small oysters at each individual site. The increase in the mortality rate of large and small oysters at Sites 4, 6 and 7 after the 26/7/99 can be seen on Figures 6.8 and 6.9. Mortalities at Site 5 in large oysters started to occur after the 12/7/99 sampling and small oysters at Site 5 experienced high mortality after the 26/7/99 sampling (Figures 6.5 and 6.8). The increase in mortality at Site 5 is dramatic in comparison to the other acidified sites. At Sites 4, 6 and 7, large oysters had a better survival rate than small oysters at the same site between the 28/6/99 and 10/1/00.

The results of the three factor ANOVA for the comparison of acid sites to the reference sites on the 23/8/99 and 10/1/00 are listed in Table 6.1. The survival data measured at all sites on the 23/8/99 and the 10/1/00 are listed in Appendix I.

134 100

60 1 2

% surviving 3 40 4

5 20 6

7 0 May-99 Jul-99 Aug-99 Oct-99 Dec-99 Jan-00

Figure 6.8 Mean survival (± 95% CIs, n=4) of large oysters at experimental sites.

135 100

80 1

60 3

4 % surviving 40 5

20 7

0 May-99 Jul-99 Aug-99 Oct-99 Dec-99 Jan-00

Figure 6.9 Mean survival (± 95% CIs, n=4) of small oysters at experimental sites.

136 Table 4.6 Summary of the three factor analysis of variance results for the comparison of acidified sites to the reference sites on the 23/8/99 and 10/1/00.

Source of df 23/08/99 10/01/00 Variation Mean Square F p Mean Square F p

Acid 1 10624.381 2.192 0.199 17222.625 2.272 0.187 Site (Acid) 5 4699.867 13.599 0.006 6005.275 25.958 0.001 Size 1 757.786 1.516 0.443 1944.643 1.038 0.495 Acid x Size 1 493.714 1.429 0.286 1807.149 7.812 0.038 Size x Site(Acid) 5 345.600 3.389 0.012 231.342 3.527 0.009 Error 42 101.976 65.595

The results listed in Table 6.1 indicate that there was a significant difference in the mean survival rates among experimental sites on the 23/8/99 and 10/1/00. There was no significant difference between the mean survival rate of small oysters and mean survival rate of large oysters on the 23/8/99 and 10/1/00 across all sites.

There was no interaction between the factors Acid and Size on the 23/8/99 but there was an interaction between these two factors on the 10/1/00. There was an interaction between the factors Site (Acid) and Size on both the 23/8/99 and the 10/1/00.

Post hoc analysis using a Tukey HSD test of the interaction between the factors Size and Site(Acid) was performed for the dates 23/8/99 and 10/1/00 to examine differences between sites for large and small oysters and between large and small oysters at different sites. The results of the post hoc analysis are displayed in Appendix I and confirmed that small oysters at ASS-affected sites experienced significantly higher mortalities than large oysters at the same sites on the 10/1/00. However, there was not a significant difference between large and small oyster survival at the sites isolated from ASS-affected waters on this same date.

Therefore, from the results listed in Table 6.1 and displayed graphically in Figures 6.8 to 6.9 and Appendix I, at the conclusion of the experiment small oyster survival was significantly lower than large oyster survival at the sites exposed to ASS-affected waters. Also, sites isolated from ASS-affected waters had significantly higher survival

137 percentages of large and small oysters compared to sites exposed to ASS-affected waters with the exception of Site 7.

6.4 THE EFFECTS OF ASS-AFFECTED WATERS ON OYSTER GROWTH RATES The growth rate of oysters at Site 5 was not calculated because of the high mortality rate experienced at this site during the S&GE. Growth rates were calculated using oyster whole weight and shell height data during a period of high rainfall (day 0-83) and over the entire experiment (day 0 to 224). The percent per day weight increases for oysters are displayed in Figures 6.10 and 6.11 and the percent per day shell height increases are displayed in Figures 6.12 and 6.13.

6.4.1 Results: Whole Weight 6.4.1.1 Growth Rates During High Rainfall Figure 6.10 displays the percent per day mean weight increase calculated from whole weight of large and small oysters during a period of high rainfall at the start S&GE. Site 5 is not included in Figure 6.10 due to the high oyster mortality at this site influencing the growth rate calculation.

Percent per day mean weight increase for small oysters located at Sites 1, 2 and 3 were substantially greater than at Sites 4, 6 and 7 (Figure 6.10). Small oysters at Sites 4 and 6 lost weight during this period and therefore returned a negative result. Additionally, small oysters at Sites 1, 2 and 3 gained considerably more weight than large oysters located at the same sites (Figure 6.10).

Sites 1 and 3 were the only locations at which large oysters experienced mean weight increases during the high rainfall period (Figure 6.10). Sites 1 and 3 are the closest to the Manning River ocean entrance at Harrington allowing faster recovery from ‘fresh’ or low EC conditions following rainfall compared to all other sites. The average weight of large oysters located at Sites 2, 4, 6 and 7 decreased during this period. Water quality measurements collected at Site 2 did not detect acidic water, however the recorded EC values were low during this period because of recurrent rainfall events in the Manning River catchment. Site 2 is located 11.9 kilometres upstream (Figure 5.1 and Table 5.1),

138 which would mean that recovery from fresh conditions would take longer than at Sites 1 and 3 because of tidal attenuation and proximity to freshwater base flows.

large oysters small oysters

0.8

0.6

0.4

0.2

-0.2 % per day weight increase -0.4

-0.6 01234567 site

Figure 6.10 Instantaneous growth for small and large oysters presented as percentage increase per day in weight for each site during the first 83 days of the experiment (mean growth rates plotted with ~ 95% CIs).

6.4.1.2 Growth Rates From June to January Figure 6.11 displays the mean instantaneous growth rates of large and small oysters from the start of the S&GE to the end of the S&GE (i.e. 1 June 1999 to 10 January 2000). The mean instantaneous growth rates of large and small oysters at all sites were positive in this period. Large and small oysters at Sites 1, 2 and 3 gained more weight than the large and small oysters at Sites 4, 6 and 7. At all sites, small oysters had a faster mean growth rate than the large oysters located at the same sites (Figure 6.11). Sites 1 and 3 had the best growth performance in small oysters over the entire experiment (day 0 to 224), as was the case during the high rainfall period. As stated previously, out of all of the seven experimental sites, Sites 1 and 3 are situated the closest to the Manning River ocean entrance. Figure 6.11 indicates that sites exposed to ASS-affected waters experience good growth in periods of lower rainfall. This is more

139 evident in the growth data recorded from large oysters at the experiment sites displayed in Figure 6.11. In this instance, the difference between sites exposed to ASS-affected waters and those sites isolated from ASS-affected waters is much less apparent when compared to the period of high rainfall. However, all of the oysters exposed to ASS- affected waters at Sites 4, 6 and 7 during this experiment had a mean instantaneous growth rate value that was lower than was measured in oysters of the same size at the reference sites (Sites 1, 2 and 3).

large oysters small oysters

0.7

0.6

0.5

0.4

0.3

0.2

0.1 % per day weight increase 0

-0.1 01234567 site

Figure 6.11 Instantaneous growth for small and large oysters presented as percentage increase per day in weight for each site measured over the entire experiment (224 days) (mean growth rates plotted with ~ 95% CIs).

6.4.2 Results: Shell Height 6.4.2.1 Growth Rates During High Rainfall Figure 6.12 displays the percent per day mean shell height increase for small and large oysters at Sites 1, 2, 3, 4, 6 and 7 during a period of high rainfall (day 0 to day 83). Percent per day mean shell height increase for small oysters located at Sites 1, 2 and 3 were substantially greater than at Sites 4 and 6 (Figure 6.12). Oysters at Site 7 had a similar mean shell height increase as was measured in oysters at Sites 1, 2 and 3. Small

140 oysters at Sites 4 and 6 had a reduction in shell height during this period and therefore returned a negative result. Furthermore, small oysters at Sites 1, 2 and 3 had considerably greater shell height increases than large oysters located at the same sites.

Site 3 was the only location where large oysters experienced mean shell height increases during the high rainfall period (Figure 6.12). The mean shell height of large oysters at all other sites decreased during this period. Water quality measurements at Sites 1 and 2 did not detect acidic water, however the recorded EC values were low, particularly at Site 2, during this period because of freshwater inflows from the recurrent rainfall events. It should be noted that during the high rainfall period, water temperatures were at their lowest which reduces the rate of shell growth. In addition, oysters were being handled on a fortnightly basis throughout this period, which can cause shell breakages and damage before the oysters were measured.

large oysters small oysters

0.25

0.2

0.15

0.1

0.05

-0.05 % per day height increase -0.1

-0.15 01234567 site

Figure 6.12 Instantaneous growth for small and large oysters presented as percentage increase per day in weight for each site during the first 83 days of the experiment (mean growth rates plotted with ~ 95% CIs).

141 6.4.2.2 Growth Rates From June to January The mean percent per day shell height increase at all sites during the entire experimental period were positive and are displayed in Figure 6.13. Large and small oysters at the reference sites had faster mean growth rate in terms of shell height than the sites exposed to ASS-affected waters, with the exception of Site 1. Small oysters had a faster mean growth rate, also in terms of shell height, than large oysters located at the same sites. Sites 1 and 3 had the best growth performance for small oysters over the period of high rainfall as well as the entire experiment. Figure 6.13 shows that sites exposed to ASS-affected waters produce good growth in periods of low rainfall. This can be seen in the growth data from large oysters displayed in Figure 6.13. In this instance, the differences between Sites 4, 6 and 7 and Sites 1, 2 and 3 are much less apparent. The water quality data showed that low rainfall corresponds to an improvement in water quality. However, small oysters at sites exposed to ASS-affected waters (Sites 4, 6 and 7) had a lower growth rate value compared to the sites isolated from ASS-affected waters (Sites 1, 2 and 3) (Figure 6.13).

large oysters small oysters

0.25

0.2

0.15

0.1

0.05

% per day height increase 0

-0.05 01234567 site

Figure 6.13 Instantaneous growth for small oysters presented as percentage increase per day in shell height for each site measured over the entire experiment (224 days) (mean growth rates plotted with ~ 95% CIs).

142

6.5 OYSTER CONDITION INDEX AT THE EXPERIMENTAL SITES 6.5.1 Results The initial mean (± 95% CIs, n = 71) condition index of oysters at the start of the CIE (on the 1/2/00) was 109.7 ± 4.9. Site 6 experienced high oyster mortality rates and all of the experimental oysters were dead at this site by the 9/6/00. Experimental oysters were missing at Site 1 on the 12/01/01 which prevented sampling on this date. The mean condition index measured at each site on the six sampling dates is displayed in Figure 6.14. The oyster condition index data is included in Appendix J.

There was less rainfall recorded during the CIE than recorded in the same period during the previous year (see Figures 5.2 and 5.4). Therefore, it is likely that all of the experimental sites did not receive the same volume of ASS outflow during the CIE as was experienced during the S&GE.

The mean condition index of oysters from Site 6 on the 6/4/00 was greater than all other sites (Figure 6.14). The pH and EC at Site 6 on this date was 6.88 and 13 dS m-1, respectively. A decrease in the mean condition index was measured from the start of the experiment to the first sampling date on the 6/4/00 at all sites. High rainfall occurred during mid-March where more than 240 mm of rainfall was measured over 9 days in the lower Manning River catchment. It is probable that the oysters spawned between the 1/2/00 and the 6/4/00 which would account for the decline in condition index. During this 60-day period there were small rainfall events (Figure 5.4) and water temperatures were low. From the 7/8/00 to the 23/10/00 mean condition index increased at all sites. Likewise, all sites recorded increases in condition index for the period 23/10/00 to 12/1/01, with the exception of Site 2. The decrease in condition index at Site 2 may be attributed to early spawning by the oysters. All sites show a rapid increase in condition between 7/8/00 and the 23/10/00, probably due to gonad development. Sites 2 and 3 had the most pronounced increases with the condition index at Site 3 increasing from 80.9 to 140.9 and the condition index at Site 2 increasing from 77.9 to 129.7 during this period.

143 180 1

160 2

140 3

4 120

7 100 6

80 Condition Index

0 25/01/00 15/03/00 4/05/00 23/06/00 12/08/00 1/10/00 20/11/00 9/01/01

Figure 6.14 Mean condition index (95% CIs, n = 12) measured at Sites 1, 2, 3, 4, 6, and 7.

144 Table 6.2 summarises the results of the three factor ANOVA analysis. The mean condition indices measured at the reference sites were not significantly higher than the condition indices measured at sites exposed to ASS-affected waters. However, there was a significant difference between the mean condition indices measured on the five sampling dates. The ANOVA results indicate that there was an interaction between the two factors Site(Acid) and Date and also Acid and Date.

Table 6.2 Summary of the three factor analysis of variance results for the comparison of condition index at acidified sites and reference sites.

Source of df Mean Square F p Variation

Acid 1 5484.035 0.677 0.443 Date 4 41781.080 8.396 0.033 Acid x Date 4 4886.695 3.429 0.039 Site(Acid) 4 3548.349 2.549 0.094 Site(Acid) x Date 12 1373.557 3.862 0.000 Error 260 355.656

6.6 DISCUSSION 6.6.1 Oyster Survival Small and large oysters at sites exposed to ASS-affected waters experienced significantly higher mortality than small and large oysters at sites that were not exposed to ASS-affected waters at the conclusion of the experiment (10/1/00). ASS-affected waters also impacted the survival of small oysters significantly more than large oysters at the same sites on this date.

ASS-affected waters had a pronounced influence on the water quality, especially in terms of pH and EC at exposed sites (Chapters 4 and 5). Water quality varied between each site and was largely related to the hydrological and geomorphic setting of the oyster lease (White, 2002). Oyster leases located in tidally attenuated areas of the estuary suffered the highest mortality rates due to the acidic and fresh conditions persisting for prolonged periods. Tidally attenuated areas typically have reduced brackish water mixing and capacity to neutralise the acidity.

145

Sites that are well flushed and exposed to ASS-affected waters had dramatic and wide- ranging variations in pH and EC. This provided oysters with short periods of more favourable water quality conditions, mostly during the final stages of the flood tide. The flood tide waters not only neutralised the acidity but also increased the salinity which gave oysters an opportunity to actively feed. These conditions mostly occur at the final stages of the flood tide when more saline waters migrate upstream. Density and pH stratification is a common feature of estuarine waters that are impacted by ASS- affected waters (Sammut et al., 1994; Sammut et al., 1996c) and was known to occur near the sites used in the S&GE (Sonter, 1999). Therefore, oysters situated at sites exposed to ASS-affected waters commonly experienced acidic conditions after rainfall and during the ebb tide, and more saline and pH neutral conditions as the flood tide displaced and neutralised the acidic water (Tables 5.3 and 5.4 and Figure 4.12).

Natural backswamp areas of the study area have been extensively modified (Smith et al., 1999; Tulau, 1999b) which changes the natural hydrological characteristics of the coastal floodplain (Sammut et al., 1996a; White, 2002). Results from the S&GE suggest that the geomorphic location of sites selected to grow oysters was an important factor in terms of survival of oysters. Oyster growing areas situated in tributaries that have an extensive backswamp system that has had drainage modifications are more susceptible to problems associated with estuarine acidification. Cattai Creek and Lansdowne River catchments contain extensive backswamp areas (Birrell, 1987; Naylor et al., 1995). Cattai Creek and the Lansdowne River both receive the drainage waters of heavily modified floodplains (Sonter, 1999; Tulau, 1999b) and the experimental sites located in or near these systems experienced low oyster survival rates during the S&GE.

The proximity of the ASS outflow source in relation to the oyster lease controlled the amount of dilution that occurs before the oysters are exposed. Sites located close to ASS outflows (i.e. near floodgate structures of acidified floodplain drains) receive a stronger concentration of ASS oxidation products compared to sites distant from the ASS outflow. In a similar way, sites that are downstream of numerous ASS outflows receive an increased concentration of acid and oxidation products (Sammut et al.,

146 1996a). Both of these factors help to explain the large variation in oyster mortality measured at the four sites exposed to ASS-affected waters.

The primary cause of mortality in small oysters was exposure to acidified water that entered through a perforated left valve. Oysters are able to protect their soft tissue from the direct effects of acid by closing their valves (Dove, 1997). Once the shell is breached through a combination of internal and external shell dissolution, acidified water directly impacts on the oyster tissue. Small oysters are more susceptible to shell perforation because their shells have not fully developed and are therefore thinner. The fourth objective of this study was to determine if ASS-affected water impacts smaller oysters more than larger oysters. Small oysters had a significantly higher mortality rate than larger oysters at the same sites and this high mortality rate was directly related to the prevalence of shell perforation in smaller oysters. However, dead oysters of both sizes with no shell perforation were consistently found at all of the sites exposed to ASS-affected waters.

Death in oysters that did not experience shell perforation was attributed to the acidic conditions and elevated concentrations of iron and aluminium. It is highly likely that these factors have a synergistic impact on oyster survival. Bamber (1987; 1990) investigated the effects of acidity alone on mortality of several bivalve species. Bamber (1987; 1990) showed that mortality in bivalves increased with the time of exposure and decreased in larger animals. Both of these findings are consistent with the results obtained from the S&GE. Bamber (1987; 1990) found the critical pH for significant mortality after 30 days exposure ranged from 6.6 for M. edulis down to 6.0 for C. gigas. The minimum pH values measured during the S&GE were dramatically lower than these levels. However, pH was not continually suppressed over the entire experimental period and fluctuated considerable during the tidal cycle (Figure 4.12). The time taken for dramatic mortality in small Sydney rock oysters in this present study was greater than 30 days and ranged between 42 days and 70 days.

Winter (1972) demonstrated that iron at neutral pH levels significantly reduced the survival of M. edulis. This study showed that ferric hydroxide flocs at concentrations above 1.0 mg L-1 caused a mortality of 75% over 3 months and 0.4 mg L-1 caused a

147 mortality of 40% over 5 months. The control animals used in Winter’s (1972) laboratory experiments experienced a mortality of 20% during the 5 months. During the first month of the experiment mussels exposed to the highest concentration of ferric hydroxide flocs (4.0 mg L-1) experienced a lower mortality rate compared to mussels exposed to both 1.0 and 2.0 mg L-1 of iron. Winter (1972) attributed this to most of the iron at the high concentration being rejected as pseudofaeces so only a small amount of iron entered the digestive tract. Cruz (1969) showed that when iron was absorbed in large quantities by the digestive tract it caused mortality and internal lesions in fish. The rejection and ingestion of iron is investigated in further detail in Chapters 8 and 9 as very high concentrations of iron were measured and observed during the field investigations of this study. Aluminium concentrations increase as pH decreases and is more toxic between pH 5.0 and 5.5 than at pH 3 (Driscoll et al., 1980; Baker and Schofield, 1982; Driscoll, 1989; Sammut et al., 1995). The concentrations of aluminium at sites exposed to ASS-affected waters were elevated and aluminium cannot be discounted as a factor for the high mortality rates measured at these locations. Chapter 8 of this study investigates the effects of aluminium and iron at pH 5.1 on oyster soft tissue to experimentally elucidate the possible causes for mortalities in oysters resulting from exposure to ASS-affected waters.

The reference sites, which were isolated from ASS-affected waters, had a mean mortality rate less than 5% in small and large oysters over the entire S&GE. For comparison, Wisely et al. (1979) measured a mortality rate of 10% for oysters (ranging in size from 29-40 g) cultured subtidally on the Manning River between the months November to February.

In conclusion, sites exposed to ASS-affected waters experienced low pH levels, reduced EC levels and increased concentrations of dissolved and suspended metals, namely iron and aluminium. Increased mortality rates in large and small oysters were measured at sites that recurrently experienced these conditions following high rainfall. Sites that are well-flushed and distant from ASS outflows experienced low mortality rates when compared to sites in well-flushed areas that are close to the ASS outflows. It is recommended to avoid areas that are acidified after high rainfall or relocate oysters, if practicable, in acid-prone areas in the event of high rainfall. Cultivation of smaller

148 oysters in areas affected by ASS outflows is strongly discouraged. The investigation of large and small adult oyster survival at locations that are exposed to ASS-affected waters has satisfied objectives 3 and 4 of this study.

6.6.2 Oyster Growth There is an association between reduced oyster growth rates and sites that are exposed to ASS-affected waters. Small and large oysters at sites exposed to ASS-affected waters had reduced growth rates when compared to the same size oysters at the experimental sites not exposed to ASS-affected waters.

All of the sites exposed to ASS-affected waters during the S&GE showed a reduced growth rate (in terms of weight gain and shell height increases) compared to sites isolated from ASS-affected waters. Minor or negative growth rates in small and large oysters were measured during periods of high rainfall, which also corresponded to reduced EC levels and/or poor water quality conditions at these sites. During this period, sites that were isolated from ASS-affected waters had a marginally better growth rate in large oysters and strong growth in small oysters even though water temperatures were low. The variation that existed between sites impacted by ASS-affected waters and sites not impacted was most evident when growth rates were calculated for the entire experiment. In this instance growth rates at sites that experienced acidification following rainfall showed a reduced growth rate in terms of whole weight and shell height. This difference becomes even more apparent when comparing the data for small oysters at the sites impacted and the three reference sites.

These results are likely to be influenced by the variation in EC between sites impacted by ASS-affected waters and sites that are not. All of the sites isolated from ASS- affected waters had higher median EC readings due to the fact that they were isolated from fresh floodplain inflows into the estuary. Extended periods of low EC is known to affect the growth of oysters (Bayne and Newell, 1983) and cause stress (Sunila, 1986a).

In experiments using acidic seawater, shell growth was reduced at pH values less than 7.0 in V. decussata, O. edulis, C. gigas and M. edulis (Bamber, 1987; 1990). Bamber (1987; 1990) also measured stepped reductions with pH in the length, area and weight

149 of bivalve shells following 30 days exposure. Bamber’s (1987; 1990) results indicate that the whole weight loss measured in Sydney rock oysters under acidic conditions during this present study can be from shell dissolution combined with flesh weight reductions. The following section discusses flesh weight reductions due to acidic conditions and high iron concentrations at pH neutral conditions because it is directly relevant to oyster condition index.

Therefore, reduced growth rates in oysters were chiefly attributed to low pH and EC levels allowing for fewer feeding opportunities for oysters at sites exposed to ASS- affected waters. Shell dissolution (Bamber, 1987; 1990) and high concentrations of iron (Winter, 1972) are also believed to contribute to the poor growth performance measured in oysters at sites exposed to ASS-affected waters. Additionally, geomorphic and hydrological factors mentioned in Section 6.6.1, which increase the probability of survival, are also likely to enhance growth rates based on the results obtained from this experiment.

6.6.3 Oyster Condition Index The condition index data highlights the variability that exists between different oyster growing areas of the Manning River. The combined mean condition indices for the entire experiment were lower at sites exposed to ASS-affected waters relative to the reference sites. Throughout the autumn and winter months oyster condition at all sites were similar. Differences in condition index were not apparent until spring where oysters at Sites 2 and 3 had rapid increases in condition as their gonads developed. In the middle of summer, oysters at Sites 3, 2, 4 and 7 had comparable condition indices. Late winter and early spring was characterised by low rainfall and there were small recurrent rainfall events throughout late spring and early summer. Water quality data indicates that conditions were good at all sites during this period.

On the 6/4/00, the mean oyster condition index was high at Site 6 compared to other sites. The decrease in condition at all other sites between the 1/2/00 and the 6/4/00 was attributed to spawning in the oysters. A very high mortality rate was recorded at Site 6 on the 6/4/00 and no oysters remained after the 9/6/00. It is likely that the large rainfall event in early March (Figure 5.4) caused a decline in water quality conditions at Site 6

150 (see Section 5.5). High concentrations of colloidal iron were observed at this site on the 6/4/00 suggesting the mobilisation of ASS oxidation products into the Lansdowne River.

Dead oysters removed from Site 6 did not show any evidence of shell perforation, however oyster shells were smothered in iron flocs and the soft tissue appeared an ochre red colour. High concentrations of colloidal iron were observed during the S&GE at this site. Iron exists as either Fe2+ (ferrous which is the reduced form) or Fe3+ (ferric which is the oxidised form) and at pH values greater than 3, ferric iron is insoluble and forms colloidal hydroxides and oxyhydroxides (Hounslow, 1995).

Winter’s (1972) findings highlight the detrimental nature of ferric hydroxide flakes at neutral pH levels on the mussel M. edulis. This study found that a diet with elevated concentrations of iron (0.4 to 4.0 mg L-1) caused high mortalities and reduced growth in laboratory experiments that lasted for 5 months. Mussels that were fed a diet consisting of standard food (1.59 mg L-1) and a low concentration of iron (1.0 mg L-1) for 3 months experienced a 38% decrease in their dry soft tissue weight and had a lower soft tissue dry weight than mussels that were deprived of standard food and ferric hydroxide flakes (Winter, 1972). Also, very slight reductions in pH alone can reduce the soft tissue condition of bivalves. Bamber (1987; 1990) showed that tissue growth was reduced at pH values less than 7.0 in V. decussata, O. edulis, C. gigas and M. edulis. For example, the shell area and flesh weight of C. gigas decreased as pH was decreased from 8 to 5.5 (Bamber, 1990). Pronounced flesh weight reductions were also measured in V. decussata (Bamber, 1987). These findings are relevant to the current study and help explain the overall lower condition index values measured in oysters at the sites exposed to ASS runoff. The effect of acidic treatments containing elevated iron and aluminium levels on oyster soft tissue is examined in Chapter 8 of this study.

6.7 CHAPTER SUMMARY The results of the field observation experiments clearly demonstrate that ASS-affected waters have a detrimental impact Sydney rock oysters and are the cause of reduced survival and growth. ASS-affected waters were also the cause of reductions in oyster condition index. All of the leases exposed to ASS-affected waters experienced reduced

151 survival rates and growth inhibition during stages of the study period characterised by high rainfall in comparison to the sites isolated from estuarine acidification. A significant finding of the S&GE is the extent that small oysters are impacted by ASS- affected waters, which supports the observations of Dove (1997) and is consistent with Bamber’s (1987; 1990) findings.

It would be impossible for an oyster grower to cultivate small oysters successfully at any of the experimental sites exposed to ASS-affected waters in wet periods. Additionally, small single seed oysters that have a flat section in their shell caused by PVC catching slats are particularly susceptible to mortality in acidified conditions due to the decreased CaCO3 reserve.

The field investigations in this study indicate that oyster growers must manage stock movement to avoid areas that are episodically and chronically affected by acid outflows. Additionally, these findings indicate that land and water management strategies on the Hastings and Manning River systems and local environmental decision making should take acidification into consideration.

These experiments have provided considerable evidence that supports the observations of oyster growers and helps to explain the production problems that they experience in certain areas of the Hastings River and Manning River. However, due to the complex nature of this problem, more information on the mechanisms that cause mortality and reasons for reduced growth rates are needed. In light of this, the laboratory investigations were designed to better understand oyster behaviour, examine the effects of acidification on oyster feeding processes and soft tissue. The following chapter investigates oyster kills that occurred on the Hastings River that displayed no clinical signs of exposure to ASS-affected waters.

152 CHAPTER SEVEN INVESTIGATION OF HASTINGS RIVER OYSTER KILLS

7.1 INTRODUCTION For over a decade, oyster growers in areas of the Hastings River have experienced heavy oyster mortalities that were only recently attributed to a new condition known as Limeburners Syndrome (LS) (Callinan, 1997a). Areas affected by mortalities include Limeburners Creek, Big Bay and the Maria River (Figures 7.1 and 4.1) (Steen, 1996). LS-induced oyster kills cause large financial losses to oyster growers and are seasonally recurrent (Callinan, 1997a; Steen, 1996). Dove (1997) found that LS lesions in Hastings River oysters confounded the interpretation of histopathological data and oyster growers suspect that LS is an acid-induced condition, however no direct link has been established. This study has chiefly investigated associations between estuarine acidification and oyster mortalities, slow growth and poor health. However, it is important for the present study to distinguish oyster production problems caused by LS from production problems that are caused by the exposure of oysters to ASS-affected waters.

This chapter contains information obtained from investigating Hastings River oyster kills that have different clinical signs to acid induced mortalities. This investigation into Hastings River oyster kills has two components. The first was a long-term water quality study in areas where oyster kills have been reported. This was conducted to examine if acid was a necessary factor for LS outbreaks. The second involved defining a case for an oyster kill (Baldock and Reantaso, 2002). An oyster kill, which was detected in August 2000, was described and the case was based on the clinical signs and gross pathology of affected oysters. Investigation of an oyster kill was conducted to differentiate between LS and acid effects.

7.2 PREVIOUS STUDIES OF HASTINGS RIVER OYSTER KILLS Hastings River oyster kills have been detected prior to this study and were investigated (Callinan, 1997a; Steen, 1996; Adlard, 1993; Desmarchelier, 1993). This section summarises these reports of oyster kills and poor oyster production on the Hastings River.

153

Callinan’s (1997a) and Steen’s (1996) related study investigated oyster production problems in Limeburners Creek and Big Bay. Steen (1996) conducted continuous water quality monitoring in Limeburners Creek and Big Bay from June to mid August 1996. Steen’s (1996) water quality data showed that no acidification was detected at the monitoring sites. However, this water quality study was brief (2.5 months), limited to two sites and may not have measured water quality under climatic conditions that induce acidification.

A broad summary of previous studies that recorded oyster kills in the Hastings River is detailed in Steen (1996). The main observations from previous studies are detailed below and in Table 7.1.

Adlard (1993) investigated the cause of reduced growth rates in Hastings River Sydney rock oysters. Adlard (1993) concluded that the mortalities were not a result of winter mortality or QX disease. Histology of oysters found spherical bodies of unknown origin. A small number of oysters were also tested for the aetiological agent responsible for Brown Ring Disease (Vibrio sp.) due to similar clinical signs in the affected oysters from the Hastings River (Desmarchelier, 1993). However, the bacteria responsible for Brown Ring Disease were not detected (Desmarchelier, 1993).

Steen (1996) identified the leases where oyster mortalities have been detected in Limeburners Creek between 1992 and 1995 (Figure 7.1). Steen (1996) found that most oyster mortalities were reported from oyster leases immediately downstream and upstream of the Limeburners Creek road bridge (Figure 7.1). This particular area represents the most intensively farmed location on the Hastings River estuary which may account for the high number of reported oyster kills at this site.

154

Figure 7.1 Locations of reported oyster mortalities (Source: adapted from Steen, 1996).

155 Table 7.1 Summary of reported Hastings River oyster kills (Source: adapted from Steen, 1996).

Reference Location Date Mortality Comments/Observations

Manton (1993)a Limeburners Creek January, 40-50% - bottle and plate size oysters affected (Immediately 1993 - yellow mark on the shell downstream from - affected Leases: 71-025 and 59-347 the road bridge)

Langton (1993)b Limeburners Creek Sept. to ~28% - affected older oysters (3 years old) (Immediately Dec., 1993 - associated with reduced growth in surviving oysters upstream from - yellow starchy deposit on the lip of the valve and in the road bridge) - yellow starchy deposit in the anterior of shell - two year old oysters exhibited signs of stunted growth - oysters less than 18 months old on the same lease unaffected - no known parasites found in affected oysters - affected lease: 83-193 a Manton S.D. (1993). Unpublished Report on Oyster Mortalities on the Hastings River, 15/1/93. NSW Fisheries. b Langton, D. (1993). Unpublished Report on Oyster Mortalities on the Hastings River as Observed on Lease 83-193. Port Macquarie Oyster Farmers Association Inc.

Callinan’s (1997a) study showed that outbreaks of LS generally were detected between September and December in dry periods, which were characterised by high salinities, and on leases with low flow conditions. Callinan’s (1997a) histopathology data did not identify a causative agent but showed a severe but non-specific inflammatory response in affected oysters. The mortalities were associated with a yellow algal growth on the external shell surface of affected oysters and the algae were identified as a common diatom (Melosira sp.) not known to be toxic (Callinan, 1997a). A surface-water algal material (commonly referred to as ‘scum’ by oyster growers) was reported to be in contact with the oysters during mortality outbreaks and analysis of the algae revealed a mix of non-toxic species (Callinan, 1997a). Callinan (1997a) also states that there was no substantial evidence before the study to suggest oyster mortalities were from exposure to estuarine acidification and there was no clear relationship between dredging and oyster mortality. Oyster growers were concerned that channel dredging was a risk factor.

The most recent investigation into Hastings River oyster production problems was conducted by Lake (1997). Lake’s (1997) epidemiological study suggested putative risk factors for production problems occurring in the Hastings River but did not test causal links. The aim of the study was to identify plausible associations and potential

156 risk factors for oyster production. The study defined a case for poor oyster production on the Hastings River, identified putative risk factors for production using farmer interviews and identified areas of the estuary most affected by poor production.

The study proposed that production problems were potentially caused by several interrelated problems with the ultimate mitigating factor being location (Lake, 1997). Lake (1997) also separated production problems into ‘oyster kills’, which relates to mortality of oysters and ‘oyster degeneratus’, which relates to slow growth in affected oysters. Putative risk factors for oyster kills and oyster degeneratus include: climatic variation; lease location; cultivation methods and practices; and, surrounding landuse activities.

Lake (1997) suggests five hypotheses that require further investigation which are: 1. Present landuse in Limeburners Creek has inhibited oyster production through altered environmental processes; 2. Excessive drainage work in the Maria River has meant it is currently unable to sustain oyster production due to acidic drain outflows; 3. Siltation in Big Bay has reduced the estuarine habitat suitable for oyster production; 4. Freshwater influxes in disturbed acidic landscapes will result in oyster kill; and, 5. The ad hoc nature of cooking methodology results in unnecessary oyster kill.

Findings from Dove (1997), water quality investigations (Chapter 4) and field observation experiments (Chapter 6) conducted in this present study support hypotheses 2 and 4.

Recent testing by Macquarie University, NSW and Queensland Museum for M. sydneyi, has discovered that it is present in the Hastings River estuary (NSW Fisheries, 2003). Polymerase chain reaction (PCR) testing of oysters from the Hastings River has found low levels of M. sydneyi organisms in the gills. M. sydneyi was also discovered to occur in Merimbula, Wagonga, Tuross, Clyde River, Port Stephens and Wallis Lake which are all Sydney rock oyster producing estuaries of

157 NSW. During the present study, the clinical signs of QX disease were not evident but no testing for the pathogen was conducted.

These studies and oyster growers’ lay knowledge suggest that an unidentified agent (or agents) is episodically impacting the Hastings River Sydney rock oyster industry and causes oyster kills in combination with reduced growth and poor health in affected oysters. The affected oysters display clinical signs which are different to oysters that have been exposed to acidification. Problems generally occur between September and December in dry periods that result in high salinity conditions in Limeburners Creek and the Hastings River (Callinan, 1997a). Observations from two previous oyster kills (Table 7.1) reveal the presence of a yellow material on the soft tissue of affected oysters and mortality was chiefly identified in older oysters (Manton, 1993; Langton, 1993). An area of the Hastings River estuary that has regularly experienced these problems is near the Limeburners Creek road bridge (Figure 7.1).

7.3 LOWER HASTINGS RIVER AND LIMEBURNERS CREEK WATER QUALITY INVESTIGATION 7.3.1 Methods It is unknown whether acid is either a necessary or sufficient factor for LS outbreaks and oyster growers believe that acid increases the susceptibility of oysters to LS. Steen (1996) measured water quality at one site in Limeburners Creek and Big Bay for a period of 2.5 months and did not measure acidic conditions. No outbreaks of LS were detected during Steen’s (1996) study. A purpose of this chapter was to examine the role of acidity in LS outbreaks. Therefore, a more extensive and longer-duration water quality investigation was conducted in areas where LS outbreaks were reported to establish if acid was a necessary factor for LS outbreaks. Sampling focussed on measuring pH and EC in particular, and included iron, aluminium and other species due to their potential to impact oyster health (Chapter 6). Water quality sampling was combined with frequent inspections of experimental sentinel oysters to detect an oyster kill outbreak that could then be intensively sampled. Results of the water quality investigation are presented in Section 7.4.

158 7.3.1.1 Water Quality Sampling Sites and Dates Figure 7.2 displays the locations of water quality sampling sites. Sites 1 to 4 were along the centreline of the main channel of the Hastings River and extended 2.5 km upstream from Settlement Point. Sites 5 to 19 were situated in Limeburners Creek and were along the centreline of the main channel. At all sites an in situ measurement of pH, EC, DO and temperature was taken on the surface and bed. Surface and bed water samples were collected from Sites 1, 4, 12 and 19 for laboratory analysis. The sampling procedure and methodology detailed in Chapter 4 (Section 4.4) was used for these measurements. Sites A, B and C (Figure 7.2) were used as oyster monitoring sites. Water quality samplings Sites 1 to 7 (Figure 7.2) do not correspond to the sampling sites of the same numbers displayed on Figure 5.1 and discussed in Chapters 5 and 6.

The sampling period was from 17 Nov 1997 to 30 Mar 1999. Sampling was conducted on the following dates: 17 Nov 1997; 4 Dec 1997; 20 Mar 98; 25 Mar 1998; 27 Mar 98; 2 Apr 1998; 17 Apr 1998; 27 Apr 1998; 4 May 1898; 15 May 1998; 2 Jun 1998; 5 Jun 1998; 21 Jul 1998; 31 Jul 1998; 10 Aug 1998; 17 Aug 1998; 31 Aug 1998; 15 Sep 1998; 1 Oct 1998; 16 Oct 1998; 9 Nov 1998; 7 Dec 1998; 25 Jan 1999; 4 Feb 1999; 4 Mar 1999; and, 30 Mar 1999 (Figure 7.3). Sampling was conducted at this frequency to ensure a continuum of data in the hope that an outbreak of LS would occur.

A Greenspan Technical Services SDL measuring pH, EC and temperature was deployed at Site A (Figure 7.2) to provide long-term, continuous time series data at a depth of 0.5 m for the periods: 6 Nov 1997 to 2 Dec 1997; 4 Dec 1997 to 28 Dec 1997; 9 Apr 1998 to 25 May 1998; and, 11 Aug 1998 to 31 Aug 1998.

159

Figure 7.2 Map of the lower Hastings River and Limeburners Creek showing water quality monitoring sites.

160 7.3.1.2 Oyster Monitoring Approximately 10,000 oysters were placed at three oyster lease sites that had reported production problems. Sites A and B were located in Limeburners Creek and Site C was in Big Bay (Figure 7.2). Oysters were inspected fortnightly between the 6/11/97 to 31/7/98 to detect an oyster kill. Approximately 3,000 oysters were placed into 10 covered, plastic trays and randomly distributed on intertidal racks at Sites A, B and C.

7.3.2 Results 7.3.2.1 Rainfall Precipitation data for this section were obtained from the Bureau of Meteorology (Station Number 60026) and was collected at Hill Street, Port Macquarie (Figure 4.1). This rainfall station was considered the most representative rainfall station for the study area. Rainfall for the period 1/10/97 to 31/3/99 is displayed in Figure 7.3. Total rainfall for the study period was 2,725 mm and the total rainfall for 1998 was 1,845 mm. The annual total average rainfall for Port Macquarie is 1,518 mm.

200 sampling dates 180 160 140 120 100 80 60 precipitation (mm) 40 20 0 Jul-98 Apr-98 Oct-98 Oct-97 Jan-99 Jan-98 Jun-98 Feb-99 Mar-99 Feb-98 Mar-98 Dec-98 Dec-97 Aug-98 Sep-98 Nov-98 Nov-97 May-98

Figure 7.3 Rainfall (Source: Bureau of Meteorology Station Number 60026) and sampling dates for the study period.

161 7.3.2.2 pH The minimum pH measured in the surface waters during the study was 6.31 at Site 19 on the 5/6/98. The minimum pH for bed waters was 6.57 at Site 17 measured on the same date. This occurred immediately after a large rainfall event where 150 mm of rainfall was recorded in two days (Figure 7.3). This also resulted in EC levels in Limeburners Creek being depressed in both surface and bed waters. Figure 7.4 displays the pH and EC conditions during a period of low rainfall where both the pH and EC values are high and consistent with distance upstream. However, immediately after rainfall both EC and pH decrease with increasing distance upstream in Limeburners Creek (Figure 7.5).

The minimum pH measured in surface waters of the main channel of the Hastings River was 7.05 on the 15/9/98. Figure 7.6 shows the difference in pH between surface and bed waters resulting from density stratification. The water quality data collected during this study showed that estuarine acidification caused by ASS outflows did not occur in the lower Limeburners Creek and lower Hastings River

areas during the data collection period. Cl:SO4 ratios were greater than 5 and the minimum pH values measured were greater than 6 on all occasions during the study period which indicates that the estuarine waters had not interacted with FeS2 contained in ASS (Mulvey, 1993). However, pH was reduced to a level below neutral conditions (pH 7) after high rainfall (Figure 7.5). This pH decrease was attributed to inflows of large quantities of humic acids that originate in the adjoining Limeburners Creek Nature Reserve (Figure 7.2). Following high rainfall, natural drainage courses in the nature reserve allow humic acids to enter Limeburners Creek which results in suppressed pH levels for short durations. The pH data for all of the sampling sites and dates are listed in Appendix K.

162 pH (surface) pH (bed) EC (surface) EC (bed) 9 60

8.5 50

8 40 ) -1

pH 7.5 30 EC (dS m 7 20

6.5 10

6 0 3456789 distance upstream from the ocean entrance (km) Figure 7.4 EC and pH in Limeburners Creek surface water and bed water prior to high rainfall on the 4/12/97. Distance upstream from the ocean entrance was calculated using the method detailed in Section 4.4.1.

pH (surface) pH (bed) EC (surface) EC (bed)

9 60

8.5 50

8 40 ) -1

pH 7.5 30 EC (dS m 7 20

6.5 10

6 0 3456789 distance upstream from the ocean entrance (km) Figure 7.5 EC and pH in Limeburners Creek surface water and bed water after high rainfall on the 5/6/98. Distance upstream from the ocean entrance was calculated using the method detailed in Section 4.4.1.

163 pH (surface) pH (bed) EC (surface) EC (bed)

9 60

8.5 50

8 40 ) -1

pH 7.5 30 EC (dS m 7 20

6.5 10

6 0 33.544.555.56 distance upstream from the ocean entrance (km) Figure 7.6 EC and pH stratification in the Hastings River prior to high rainfall on the 17/8/98. Distance upstream from the ocean entrance was calculated using the method detailed in Section 4.4.1.

7.3.2.3 EC EC at all sites was influenced by rainfall and runoff. The minimum EC level measured in Limeburners Creek surface and bed waters occurred on the 5/6/98 (Figure 7.5) and the minimum EC level measured in the Hastings River occurred on the 17/8/98 (Figure 7.6) for surface waters and 2/6/98 for bed waters. Maximum EC for all sites occurred on the 7/12/98. Stratification resulting from EC differences between surface and bed waters was typical in the main channel of the Hastings River and is displayed graphically in Figure 7.6. Stratification was not as pronounced in Limeburners Creek as it was in the Hastings River. The EC data for all of the sampling sites and dates are listed in Appendix K.

7.3.2.4 DO and Temperature The lowest DO measurement was 20.8% saturation in the bed waters at Site 19 on the 31/8/98. The maximum was at Site 8 on the 31/3/99 when the bed waters had a DO saturation of 142.1%. Water temperatures at the 19 sample sites ranged from a minimum of 10.63O C at Site 16 on the 5/6/98 to 29.58O C at Sites 18 and 19 on the

164 4/3/99. The minimum temperature was measured in the bed waters and the maximum temperature was measured in the surface waters. The DO and temperature data for all of the sampling sites are listed in Appendix K.

7.3.2.5 Water Sample Analysis Analysis of the surface and bed water samples did not detect any unusually high

concentrations of dissolved ions (Fe, Al, Ca, Mn, K, Mg, SO4, As, Cu, Si and Zn) in Limeburners Creek or the Hastings River. Appendix L lists the concentrations of dissolved ions measured in surface and bed waters at Sites 1, 4, 12 and 19.

7.3.2.6 Submersible Data Logger Measurements The pH, EC and temperature data collected by the SDL located at Site A are displayed graphically in Figures 7.7 and 7.8 and are summarised in Table 7.2. The median pH, EC and temperature value at Site A was 8.07, 44.25 dS m-1 and 22.03O C, respectively (Table 7.2).

Figures 7.7 and 7.8 show the variation in pH and EC at Site A. Figure 7.7 (A) and (B) are typical displays of dry conditions in Limeburners Creek catchment. In these two figures, EC is close to oceanic levels and the pH range was 7.5 to 8.5. Figure 7.7 (C) and (D), as well as Figure 7.8 (A), (C) and (D), show reduced EC levels due to the influx of fresh water following rainfall. This also caused a decrease in pH to below 7 in two circumstances during data collection. There is rapid recovery of EC levels in Limeburners Creek after short dry periods and is due to the close proximity of this area to the ocean entrance and the small catchment area of Limeburners Creek. Diurnal variation in temperature was also evident in Figures 7.7 and 7.8. Fresh water inflows do not have a large influence on the temperature variation in Limeburners Creek. These data show that estuarine acidification resulting from the disturbance of ASS was not a problem in lower Limeburners Creek during the study period.

165 A. B.

10 60 10 60 EC

EC C) C) O 9 50 9 50 O

8 40 8 40 pH pH 7 30

pH ) & temp. ( 7 30 pH ) & temp. ( -1 -1 6 20 6 20 temp. temp. 5 10 5 10 EC (dS m EC (dS m 4 0 4 0 27/09/97 7/10/97 17/10/97 27/10/97 6/11/97 1/12/97 11/12/97 21/12/97 31/12/97

C. D.

60 10 60 EC C) C) EC O O 50 9 50 pH 40 8 40 pH probe 30 malfunction 7 30 pH ) & temp. ( ) & temp. ( -1 -1 20 6 20 temp. temp. 10 5 10 EC (dS m EC (dS m 0 4 0 5/04/98 15/04/98 25/04/98 5/05/98 15/05/98 25/05/98 4/06/98 8/08/98 13/08/98 18/08/98 23/08/98 28/08/98 2/09/98

Figure 7.7 pH (red), EC (blue) and temperature (black) at Site A, Limeburners Creek: (A) 6/10/97 to 2/11/97; (B) 4/12/97 to 28/12/97; (C) 9/4/98 to 25/5/98; and (D) 11/8/98 to 31/8/98.

28 A. B. 10 60 10 60 EC C) C) 9 50 O

9 50 O pH 8 40 8 40 pH 7 30 ) & temp. ( 7 30 pH pH ) & temp. ( -1 -1 6 20 6 20 temp. temp. 5 10 5 10 EC (dS m EC EC (dS m 4 0 4 0 27/09/98 2/10/98 7/10/98 12/10/98 17/10/98 22/10/98 31/08/98 7/09/98 14/09/98 21/09/98 28/09/98 5/10/98

C. D.

10 60 10 60 EC EC C) C) O 9 50 O 9 50 pH 8 40 8 40 pH 7 30 7 30 ) & temp. ( pH pH ) & temp. ( -1 -1 temp. 6 temp. 20 6 20 5 10 5 10 EC (dS m EC (dS m 4 0 4 0

4/11/98 11/11/98 18/11/98 25/11/98 2/12/98 9/12/98 31/01/99 7/02/99 14/02/99 21/02/99 28/02/99 7/03/99 14/03/99

Figure 7.8 pH (red), EC (blue) and temperature (black) at Site A, Limeburners Creek: (A) 3/9/98 to 30/9/98; (B) 1/10/98 to 16/10/98; (C) 9/11/98 to 5/12/98; and (D) 4/2/99 to 13/3/99.

29 Table 7.2 Summary of pH, EC and temperature data collected by the SDL at Site A.

pH EC Temperature (dS m-1)(OC) (n = 10,396) (n = 13,692) (n = 13,687)

Minimum 6.86 3.30 15.32 Maximum 9.00 56.25 29.64 Median 8.07 44.25 22.03 Mean - 39.72 22.00 Standard Dev. 0.32 12.26 2.58

7.4 OYSTER KILL INVESTIGATION 7.4.1 Date and Location The oyster kill investigated during this study was identified in late August 2000 in the lower Limeburners Creek area. The location is shown on Figure 7.1 (labelled as 2000). Figure 7.1 also displays the locations of oyster kills which were detected between the years of 1992 and 1995 (Steen, 1996). Two oyster kills were detected in Limeburners Creek in June and December 1999 during the present study (labelled as 1999 on Figure 7.1).

No atypical oyster mortality was detected in the experimental oysters located at Sites A, B and C (Figure 7.2) between the 6/11/97 and the 31/7/98. All experimental oysters had to be removed on the 31/7/98 because of excessive overcatch. This prevented oyster mortality monitoring during the time when cases of LS are commonly reported (September to December). However, water quality monitoring continued until the 30/3/99 in the circumstance that an outbreak in cultivated oysters was identified. An oyster kill was detected in late August 2000 in Limeburners Creek and oysters from this event were examined grossly and using histopathology. The location of this oyster kill is shown in Figure 7.1 (labelled as 2000).

7.4.2 Rainfall Figure 7.9 shows that in the two-month period before the oyster kill no heavy rain events were measured at the Bureau of Meteorology Station Number 60026 located at

168 Hill Street, Port Macquarie (Figure 4.1). The largest rainfall was in mid July when 33 mm was recorded. All other daily rainfall events were less than 25 mm. Late winter and early spring is a typically dry period on the mid north coast of NSW.

Salinity data obtained from the NSW Shellfish Quality Assurance Program showed that the dry conditions during this time resulted in high salinities in Limeburners Creek due to negligible influence from freshwater catchment inflows. Salinity levels measured on the 13/7/00, 27/7/00, 23/8/00 and 31/8/00, recorded by the NSW Shellfish Quality Assurance Program, were all above 30 ppt.

15 precipitation (mm) 10

0 June-00 July-00 August-00

Figure 7.9 Rainfall recorded in June, July and August 2000 (Source: Bureau of Meteorology, Station 60026 – Hill Street, Port Macquarie).

7.4.3 Characteristics of Affected Oysters This section describes the pattern of oyster mortality and gross pathology of moribund Sydney rock oysters sampled during the oyster kill, which was detected in August 2000.

169 Moribund animals from the oyster kill were sampled on the 1/9/00 and preserved for histopathology using the methodology detailed in Section 8.13.

Moribund oysters were randomly sampled from amongst adjacent oyster leases at the locations labelled as ‘2000’ on Figure 7.1. Oysters cultivated by both rack-tray and pontoon methods were collected. Oysters cultivated using the rack-tray method suffered higher mortalities than the oysters cultivated using pontoons.

Moribund oysters could be identified by their shell appearance. The lip of the right valve of the affected oysters indicated growth had ceased and was very brittle, deformed and showed signs of mantle recession on the inward side. There were distinct patches of dead oysters centrifugally spread from a focus. The patches of dead oysters were diffuse across several leases in this area of Limeburners Creek (Figure 7.1). Moribund oysters were collected from the perimeter of the patches of dead oysters.

Plates 7.1 to 7.4 are typical examples of moribund oysters collected from Limeburners Creek following an oyster kill on the 1/9/00 (Plates 7.1 and 7.2) and the 1/6/99 (Plates 7.3 and 7.4), which are included for comparative purposes. Gross pathology examination of moribund oysters detected pustules in the soft tissues of oysters. ‘Pustules’ were small abscesses filled with puss and were: yellow to orange in colour; an irregular shape; had a soft and pasty consistency; and, ranged in size from 1 mm to 7 mm. Pustules were observed on and in the gills, mantle, labial palps, digestive gland, adductor muscle and gonad (Plates 7.1 to 7.4). Yellow/orange to brown pustules were also evident on the internal surface of the oyster shell and stained the internal shell. The most common location of the stain was in the anterior of the left valve (Plates 7.1 and 7.2). In some instances oysters had created a new shell layer over the pustule to isolate it within the shell.

170 Plate 7.1 Shell discolouration and shell deposits in the anterior of the left valve (specimen collected on the 1/9/00).

Plate 7.2 Yellow pustules in the gonad (large arrow) and the valves of affected oysters were brittle (small arrow) (specimen collected on the 1/9/00).

Plate 7.3 Yellow pustules in the labial palps (specimen collected on the 1/6/99).

Plate 7.4 Example of an oyster in poor condition with yellow pustules in the mantle (specimen collected on the 1/6/99).

171

The general condition of moribund oysters’ soft tissue ranged from very poor to a normal appearance. In particular moribund animals: the soft tissue was grey and watery to the extent of being translucent; the mantle soft tissue was thin, weak and easily torn; and, oysters were very easy to open. Mudworm (P. websteri), indicated by a tubular blister located under the gills and mantle, had also infested a high proportion of oysters sampled.

7.4.4 Histopathology Data From the Oyster Kill Oysters sampled from this oyster kill were examined using histopathology. Lesions in moribund oysters were characterised by foci of inflammatory cells located in the mantle and digestive gland. Myocardial necrosis associated with accumulations of haemocytes was observed in the heart. Histopathological diagnosis on representative oysters from this kill reported that the animals had experienced limited food availability or other factor that prevented ingestion of a normal ration (R. Elston, Aqua Technics, USA, personal communication, 2002). An example of a chronic lesion in the mantle epithelium of a moribund oyster collected from Limeburners Creek on the 1/9/00 is displayed in Plate 7.5.

An active infectious process was observed in one oyster based on intense multifocal haemocytosis. This oyster was sent to the Department of Microbiology and Parasitology to be tested for QX disease using in-situ hybridisation. The test indicated that the oyster was not infected with M. sydneyi. Several morphologically equivalent microcell type organisms were observed in the digestive gland, which suggested that the infection could be due to Mikrocytos roughleyi (R. Callinan, NSW Fisheries, personal communication, 2000; R. Elston, Aqua Technics, USA, personal communication, 2002).

172

Plate 7.5 Example of a chronic lesion in the mantle epithelium of a moribund oyster collected from Limeburners Creek on the 1/9/00. H&E stain, x 160.

173 7.5 DISCUSSION Water quality investigations in the lower Hastings River and Limeburners Creek did not measure estuarine acidification caused by ASS outflows at the 19 monitoring sites during the study period. Regular drain water quality monitoring was also conducted throughout the study and did not detect any acidified water in drains connected to Limeburners Creek. Circumneutral pH values were measured in Limeburners Creek and Hastings River after high rainfall. EC suppression was generally for short periods of time and brackish estuary conditions rapidly returned after the rainfall had ceased. The water quality data measured during this investigation did not reveal any water quality variable tested that could be identified as a potential problem for oyster production in this area.

Although this lower region of the Hastings River estuary is not impacted by estuarine acidification, water quality data from Chapter 4 of this study showed that leases located further upstream were being affected by acidification. The data from Chapter 4 and this chapter indicate that acidic conditions are not commonplace in the lower Hastings River and Limeburners Creek areas due the close proximity to the ocean entrance which increases tidal mixing and is the source of strongly-buffered oceanic water.

Evidence obtained from this and previous studies (Callinan, 1997a; Steen, 1996; Lake 1997) point to an unidentified agent that is causing production problems in areas of the Hastings River and there is need for further and more detailed investigation. Information obtained from oyster growers regarding oyster mortalities, poor health and slow growth reveal that the clinical signs are seasonally recurrent and coincide with high salinities in Limeburners Creek and the lower Hastings River (Callinan, 1997a; Steen, 1996; Lake, 1997). Low pH conditions are unlikely to occur under these conditions.

Clinical signs of affected oysters from this investigation were: • a deformed, weak and friable shell void of any evidence of growth; • grey, watery soft tissue containing yellow pustules in the gills, mantle, labial palp, adductor muscle and digestive gland;

174 • yellow/orange staining of the internal shell surface particularly in the anterior of the left valve; and, • older oysters displayed the above clinical signs more frequently than younger oysters located on the same lease.

These clinical signs are similar to the observations of Manton (1993), Langton (1993) and Callinan (1997a). The clinical signs displayed by affected oysters, the nature of the oyster kills and the associated slow growth and poor health, so far appear unique to the Hastings River. The evidence collected from this study is insufficient to suggest a cause or causes for the mortality.

Farley (1968) observed shell abnormalities in C. virginica that were infected by Haplosporidium nelsoni. Abnormalities included raised yellow-brown conchiolin deposits on the internal surfaces of valves and fouled inner margin of the left valve caused by ‘mantle recession’. The creamy yellow fluid in the shell deposits contained moribund H. nelsoni plasmodia, haemocytes and cellular debris (Farley, 1968). Farley (1968) referred to oysters displaying these clinical signs as in ‘remission’ of the disease. This report identified mantle recession, poor condition, pale digestive gland, pustules, epithelial exudates, conchiolin shell deposits, weak adductor muscle and mortality as gross signs of H. nelsoni infection. A difference between Farley’s (1968) study and investigations into LS was that the parasite was visible using histology in oysters infected by H. nelsoni. Farley (1968) was able to see the parasites using histology, however this has not been the case with oysters affected by LS (R. Callinan, NSW Fisheries, personal communication, 2003).

Similar responses, including mantle recession, pale digestive gland, pustules, diapedesis, weak adductor muscle and mortality were reported in Sydney rock oysters affected by Australian winter disease which is also known as winter mortality disease (Roughley, 1926; Wolf, 1967; Farley et al., 1988). M. roughleyi is the protistan parasite responsible for winter mortality disease and causes focal lesions on oyster tissues as well as heavy mortalities in larger sized oysters (Wolf, 1967; Lester and Adlard, 1996). Outbreaks are seasonally recurrent and are associated with low water temperatures and high salinities (Roughley, 1926; Wolf, 1967; Farley et al., 1988).

175

Water quality data presented in this chapter did not detect acidification caused by the oxidation of pyrite in Limeburners Creek or the lower Hastings River. This strongly suggests that acid is not a necessary factor for LS. The gross clinical signs of mortalities, slow growth and shell deformities are the only similarities that LS bears with production problems caused by exposure to ASS-affected waters (Table 7.3).

Table 7.3 Gross clinical signs displayed by oysters exposed to ASS-affected waters and affected by LS. X indicates an observation based on data from the present study.

slow growth mortality poor condition pustules weak adductor muscle mantle recession shell deposits shell deformities shell degradation shell bleaching shell perforation iron coating on shell iron on soft tissue

Oyster impacted by ASS-affected waters XXX----XXXXXX LS in oysters XXXXXXXX - ----

Based on the information obtained from this present study and previous investigations into LS, a case definition can be developed for oysters impacted by ASS-affected waters and for LS oysters. Baldock and Reantaso (2002) describe a case definition as:

‘a set of standard criteria for deciding whether an individual unit of interest has a particular disease or other outcome of interest.’ (Baldock and Reantaso, 2002, page 4).

Because the case definitions in this study are based on clinical signs and gross pathology of oysters, a broad suspect case was developed to increase the sensitivity of the definition (Baldock and Reantaso, 2002). From the information presented in Table 7.3, differences are most evident on the external shell surface for ASS affected oysters and within the shell for oysters affected by LS. Table 7.4 contains the definition for a

176 suspect case based on field observations for oysters impacted by ASS-affected water and oysters affected by LS.

Table 7.4 Definitions for a suspect case based on field observations for LS in oysters and oysters impacted by ASS-affected waters.

Outcome Study Unit Case Definition of Interest

Oyster impacted Animal An oyster with visible iron precipitates on the shell by ASS-affected surface or displays evidence of shell degradation. waters

LS in oysters Animal An oyster that has visible pustules in the soft tissues or stained patches on the internal surfaces of the shell.

There is need for further study of LS-induced oyster production problems to identify the causes and reduce or prevent future oyster kills. This study has enabled future studies to concentrate on the two separate oyster production problems that occur on the Hastings River.

7.6 CHAPTER SUMMARY This chapter measured water quality in the lower Hastings River and Limeburners Creek and examined an oyster kill that was detected in Limeburners Creek in August 2000, in order to investigate production problems. Water quality investigations conducted on the lower Hastings River and Limeburners Creek areas suggest that acidification of these areas is unlikely. Sampling of moribund oysters on the 1/9/00 confirmed that there were a number of similarities between other oyster kills that have been detected in this area prior to this current study that have been attributed to LS. An exact cause or causes for oyster kills and oyster degeneratus has not been identified in previous investigations or by the present study. There are a number of similarities between LS outbreaks and winter mortality disease outbreaks. The recurrent nature and the widespread extent of this problem is of particular concern for the Hastings River oyster industry as it threatens not only their livelihood but also the viability of oyster

177 production in particular parts of this estuary. Further study of this problem is necessary to identify the exact cause or causes.

Section III follows this chapter and details the laboratory investigations conducted for this present study. The laboratory investigations were essential to the study to investigate effects of acidified waters that are difficult and impractical to measure in the field and to enable more accurate interpretation of the field observation experiment data. Therefore, the laboratory experiments presented in Section III were designed to provide additional, and more detailed, information on the impacts of ASS-affected waters to representative, individual Sydney rock oysters.

178

SECTION III

LABORATORY INVESTIGATIONS

179 CHAPTER EIGHT EXPERIMENTAL EXPOSURE OF OYSTERS TO ACIDIFIED WATER: EXPERIMENTAL DESIGNS, MATERIALS AND METHODS

8.1 INTRODUCTION Findings in Chapter 4 indicate that oysters are exposed to water quality conditions at harmful levels in estuaries impacted by ASS-affected waters. The results of the S&GE and the CIE (Chapter 6) showed that oysters exposed to recurrent sub-lethal acidic conditions over prolonged periods experienced reduced survival and growth rates. Water quality testing was conducted during the field experiments, however it was impossible to determine the exact conditions oysters were exposed to due to the large variability in water quality conditions at all field sites over time. Experimental work was required because of the variability of water quality at field sites and the inability to test for the separate and combined effects of metals under field conditions.

The purpose of this chapter is to outline the design, methods and materials required to investigate the physiological and histopathological effects of ASS-affected waters on the Sydney rock oyster. This was done in order to achieve objectives 7 to 9 of this study and test the hypotheses that: • exposure of the Sydney rock oyster to ASS-affected waters will cause a reduction in their filtration rate; and, • exposure of the Sydney rock oyster to ASS-affected waters will cause changes in the gills and mantle soft tissues and will result in the accumulation of iron precipitates on the soft tissues.

Filtration rate is an oyster feeding trait and was examined in this study because it is affected by contaminants (Widdows and Staff, 1997). Overseas research that investigated the effects of acidification on bivalves demonstrated, using laboratory experiments, that exposure to pH values < 7 reduces feeding activity (Bamber 1987; 1990) and pumping rates (Loosanoff and Tommers 1947). The evidence from field investigations and the abovementioned overseas studies suggest that it is highly likely that estuarine acidification will affect the feeding behaviour of Sydney rock oysters.

180

Histopathology was used as a diagnostic tool in this present study to assess the impacts of acidified waters on the gill and mantle soft tissue. This was conducted for two reasons: firstly, to explain poor oyster health and examine iron accumulation at field sites exposed to ASS-affected waters; and secondly, to provide further information relating to changes in filtration rate caused by ASS-affected waters.

The following sections provide background information on: oyster valve movements under acidic conditions; methods to assess feeding processes in oysters; and, the structure and function of the oyster gill and mantle. Following this background information, the experimental design and methods used for two laboratory experiments are presented. The results from this experimental work are presented and discussed in the following chapter.

8.2 OYSTER VALVE MOVEMENTS It was necessary to understand oysters’ behavioural response before feeding measurements were undertaken. This was to ensure that oysters opened their valves and produced biodeposits in the experimental apparatus. Other studies on the effects of acidic water on bivalves reported that acidic conditions elicit abnormal valve responses, including excessive gaping and behavioural inhibition (Bamber, 1987; 1990; Loosanoff and Tommers, 1947). Using behaviour descriptions from these studies, Dove (1997) noted the following valve responses in Sydney rock oysters exposed to artificially acidified treatments: • excessive gaping – valve separation beyond the range of normal feeding; • clomping – shell adductions (used to eject water and to remove faecal material); • no activity – oyster valves remain closed and inactive for long periods; and, • open valves – valves are separated a normal distance and the oyster is exposing the mantle and gills to the test water. Valve activity is an obvious indicator of oyster behaviour and can be closely observed using laboratory experiments.

181 8.3 IMPLICATIONS OF ACIDIFICATION TO OYSTER FEEDING Two studies have investigated the effect of acidification on feeding activity in V. decussata, O. edulis, C. gigas, M. edulis (Bamber, 1987; 1990) and one study investigated the effect of acidification on pumping rates in O. virginica (Loosanoff and Tommers, 1947). Loosanoff and Tommers (1947) recorded increased pumping rates at pH values between 7.0 and 6.75, but when the pH dropped below 6.5 pumping rates dramatically decreased in adult O. virginica. Loosanoff and Tommers (1947) also observed abnormal shell movements when pH was less than 6.5.

Bamber (1987) measured feeding inhibition and a significant reduction in tissue and shell growth for the species V. decussata at or below pH 7.0. Bamber (1990) investigated the effects of acidic conditions on feeding activity in C. gigas, M. edulis and O. edulis. For C. gigas, suppression of feeding activity occurred below pH 7.0 and behavioural inhibition was observed below pH 6.5. Feeding activity was reduced at or below pH 7.2 for O. edulis and M. edulis. Feeding activity is discussed further in the following section.

In the abovementioned studies (Loosanoff and Tommers, 1947; Bamber, 1987; 1990), acidification was not caused by ASS, but was the consequence of inflows of large quantities of slightly acidic fresh water or industrial pollution. Also, these studies used artificially acidified test waters.

The literature reports that deleterious effects in numerous bivalve species occur at a pH less than or equal to 7.0 (Bamber; 1987; 1990; Akberali et al., 1985; Kuwatani and Nishii, 1969; Loosanoff and Tommers, 1947; Calabrese and Davis, 1966). This is an alarming finding for Australian oyster growers who farm in locations that experience chronic acidification often at much lower pH levels. The pH perturbation in an estuary affected by ASS outflows can be in the order of 4 pH units (Chapter 4). Given that pH is measured on a logarithmic scale, this represents a ten thousand-fold increase in the hydronium concentration relative to normal estuarine pH levels. Hence, it is very important to understand the effects of acidification on the Sydney rock oysters’ feeding behaviour.

182 8.4 MEASURING OYSTER FEEDING Bamber (1987; 1990) quantified oyster feeding activity in acidified bioassays by collecting, drying and weighing all true faeces and pseudofaeces produced by oysters in a specified period when exposed to a range of pH levels. Pumping rate was measured by Loosanoff and Tommers (1947) and is defined as the volume of water pumped through the mantle cavity per unit of time (Iglesias et al., 1998). Direct measurements of pumping rates are difficult to perform and can inhibit pumping (Iglesias et al., 1998; Newell and Langdon, 1996). Clearance rate is a measure of oyster feeding and is favoured over the direct measurement of pumping rates (Iglesias et al., 1998).

Clearance rate provides a rapid, sensitive and effective measure of water quality and can be used for the biomonitoring and toxicity assessment of effluents and industrial discharges (Widdows and Staff, 1997). Clearance rate (L h-1) and filtration rate (mg h-1) are essentially the same measure using different units and is the volume of water cleared of particles per unit time multiplied by the particle concentration (Iglesias et al., 1998). Pseudofaeces are made up of mucous-coated material rejected from the palps and the marginal food groove of the gill (Newell and Langdon, 1996). The mantle moves this material using cilia to the ventral free edge, adjacent to the labial palps. Ciliary action or ‘clomping’ (rapid closure and opening of the valves) ejects the material as ‘pseudofaeces’ from within the valves (Newell and Langdon, 1996).

The Sydney rock oyster is a suspension feeder, meaning that particulate food is captured from water drawn into the shell by the gill. Feeding and absorption rates in bivalves can be determined through measurements of suspended particles and biodeposit production using the biodeposition method (Iglesias et al., 1998). Iglesias et al. (1998) details and discusses the biodeposition method as well as certain considerations required before applying this method for quantifying food processing rates in bivalves. Iglesias et al. (1998) identifies two assumptions that underpin the biodeposition methodology. The first is that the organic matter to inorganic matter ratio is similar for both the available “food” and the actual material filtered by oysters. The second is that both the pseudofaeces and true faeces are based on oysters filtering the same source of total particulate matter (TPM).

183 The biodeposition method can be used in this present study to quantify food processing rates in Sydney rock oysters when exposed to ASS-affected waters, however, the two abovementioned assumptions must be addressed. In order to address the first assumption natural silt collected from the surface of the deposited sediment in the estuary was used as the diet in all treatments. To address the second assumption, a flow-through experimental apparatus was designed to deliver constant TPM levels and regular testing of TPM was performed throughout all treatments to ensure a constant constitution. The flow-through experimental apparatus was based on Widdows (1985) apparatus for the measurement of clearance rate, which allows quantification of the composition of suspended particles as well as true faeces and pseudofaeces of individual animals.

8.5 FUNCTION AND STRUCTURE OF THE OYSTER GILL 8.5.1 Function The descriptions of the gill and mantle function, structure and composition are based on information for C. virginica due to the many similarities that are common to this species and S. glomerata. The gills collect food particles and, together with the mantle to a lesser extent, are used for gas exchange (Newell and Langdon, 1996). The gills achieve this by creating a water current and filtering suspended food particles which are then sorted and separated from the other materials in suspension (Galtsoff, 1964). The gills are also used to disperse and separate sex cells during spawning (Galtsoff, 1964). The labial palps are located at the anterior of the gills. The function of the labial palps is to control the amount of food ingested as well as sort food before ingestion.

8.5.2 Structure There are two gills and each gill is comprised of an inner demibranch and outer demibranch with each having ascending and descending lamellae (Figure 8.1) (Newell and Langdon, 1996). Each lamella is composed of numerous filaments, which are joined to each other by interlocking ciliary junctions (Newell and Langdon, 1996). Filaments are arranged in plicae and in one plica there are ordinary filaments, transitional filaments and principal filaments (Figure 8.2). Each type of gill filament has a simple epithelium made up of cells that either have or do not have cilia (Eble and

184 Scro, 1996). A sinus runs through the filament and two fibrous protein skeletal rods lie under the epithelium on the frontal and lateral surfaces (Newell and Langdon, 1996).

Chemical or physical injury to oyster gills caused by ASS-affected waters is likely to have implications for feeding and respiration. The effects of ASS-affected waters on the soft tissue of oysters are previously undescribed. This chapter aims to investigate the response of the gill and mantle soft tissues resulting from short-term exposure to artificially and naturally acidified water that closely resembles acidic estuarine conditions measured in Chapter 4.

Figure 8.1 Cross section of the soft tissue of C. virginica showing the components of the left and right gill (Source: Newell and Langdon, 1996).

185

Figure 8.2 Transitional histological section through a demibranch of C. virginica showing the ordinary, transitional and principal filaments that comprise a plica (Source: Newell and Langdon, 1996, modified from Galtsoff, 1964).

8.6 FUNCTION AND STRUCTURE OF THE OYSTER MANTLE 8.6.1 Function The mantle, or pallium, is a fleshy fold of tissue that covers the internal organs (Figure 8.1) (Galtsoff, 1964; Eble and Scro, 1996). The main role of the mantle is shell formation (Galtsoff, 1964). The mantle is involved in other functions which include (Galtsoff, 1964): receiving and conveying sensory stimuli to the nervous system; shedding and dispersing eggs during spawning; respiration by providing direct exchange

186 of gases between the surface tissues of the oyster and the surrounding water; storage of reserve materials such as glycogen and lipids; and, secretion of mucus. The mantle also aids in excretion by discarding blood cells containing waste products (Galtsoff, 1964).

The mantle lays down the inorganic (mainly calcium carbonate) and the organic (conchiolin) components of the shells (Morrison, 1993). The shell surface of the mantle is permeable to these ions and they are transported through the epithelium of the shell surface into the extrapallial space for shell formation (Morrison, 1993). The ion exchange is likely to take place in two directions whether shell is being laid down or dissolved (Morrison, 1993). Shell dissolution occurs in anaerobic conditions, when acids form as a result of metabolism and dissolve intracellular deposits of calcium carbonate and the inside of the shell (Morrison, 1993). This explains internal shell dissolution described in Chapter 1.

8.6.2 Structure The mantle mostly consists of vesicular cells and is covered by a columnar epithelium (Galtsoff, 1964; Morrison, 1993). Small muscle fibres occur beneath the epithelium and also run across the mantle. The mantle has a shell surface and pallial surface epithelia with connective tissue in between (Figure 8.3) (Eble and Scro, 1996). Epithelial cells on the pallial surface are columnar and have a thick basement membrane, whereas the shell surface epithelial cells are shorter and have a thinner basement membrane (Morrison, 1993).

Measurements of the mantle liquid of the Sydney rock oyster after removal from the estuary were performed for this present study. The median pH of the mantle liquid of the Sydney rock oyster was 6.64 (Table 8.1). Galtsoff (1964) found that the pH of the mantle liquid for C. virginica was 6.7. The results obtained from the Sydney rock oyster in this study are comparable to Galtsoff’s (1964) findings. Galtsoff (1964) also measured a 0.56 unit decrease in pH in this species when the oyster was stored in anaerobic conditions for six days. Knowledge of mantle liquid pH is important to this study as it controls shell dissolution (Chapter 1) and influences the nature of metal species.

187

Figure 8.3 Transverse section of the mantle margin showing the outer lobe, the middle lobe and the inner lobe (Source: Galtsoff, 1964).

Table 8.1 pH of the mantle fluid of S. glomerata.

Median 6.64 Max. 6.89 Min. 6.22 Standard Dev. 0.17

n = 18

188 8.7 HISTOPATHOLOGY Histopathology is a tool to show tissue changes using light microscopy and stained thin sections. Histopathology can be used to evaluate the health of an organism by examining morphological changes in the cells and tissues arising from certain known pollutants or conditions that induce stress in the organism (Sunila, 1986a; Goldberg, 1980). Histopathological studies have been used to investigate the short- and long-term effects of local pollution on bivalves in several studies (Sunila, 1986a; 1986b; Yevich and Barszcz, 1977; Goldberg, 1980). The sessile nature of bivalves makes them ideal for this application (Sunila, 1986a).

Little is known about the soft tissue response caused by pollutants in brackish water environments (Sunila, 1986a). Additionally, prolonged periods of low salinity, as experienced in estuarine environments following high rainfall, can cause stress to the oyster. There are no detailed histopathological studies of the effects of ASS-affected waters on the Sydney rock oyster (Dove, 1997). Dove (1997) observed changes in the gill and mantle potentially due to exposure to acid, therefore these organs will be the focus of investigation in this present study.

The gill of M. edulis has been the focus of several histopathological examinations to determine the effects of known pollutants conducted by Sunila (1986b; 1987; 1988). Sammut (1998) used histopathology to describe degenerative gill and skin changes in fish (Sillago ciliata) caused by exposure to acidified treatments with and without added aluminium as well as naturally acidified waters. This study found damage to the skin and gills was rapid and mortality was caused largely through disruption to osmoregulation and gas exchange. Work by Sammut (1998) and Callinan (1997b) showed that acid-induced skin damage was a factor in EUS outbreaks and other mycotic skin diseases.

To overcome stresses caused by low salinity, oysters will be exposed using experimental treatments where salinity levels can be controlled. The field observation experiment detailed in Chapters 5 and 6 was unable to separate the effects of low pH and salinity effectively. Therefore, experimental exposures combined with

189 histopathological investigation will provide further information to help understand the effects of ASS-affected waters on oysters.

8.8 EXPERIMENTAL DESIGN Two laboratory experiments were conducted to expose oysters to artificially and naturally acidified treatments. The purpose of the first experiment was to investigate behavioural and soft tissue response whilst the second was used to examine oysters’ feeding rates. The first experiment is referred to as the ‘Behaviour Experiment’ and the second as the ‘Feeding Experiment’. A total of eight treatments were used in the two experiments and oysters were exposed to pH levels ranging from 5.1 to 8.0. Table 8.2 lists the type of treatment water, purpose and target pH and EC used in the Behaviour and Feeding Experiments. As stated previously, it was necessary to establish oysters’ behavioural response to acidified treatments before the Feeding Experiment could be undertaken to ensure oysters remain open and feed in weak acid treatments.

The laboratory experiments were designed to resemble realistic environmental conditions. An important consideration in the design of the two experiments was selecting pH and EC levels that resembled field conditions. Dove’s (1997) study did not measure estuarine acidification in areas of the estuary used for oyster production. Therefore, Dove (1997) did not have actual estuarine water quality data to establish the lowest pH that could occur at a salinity where the oyster valves were open and the animal was feeding and exposing their soft tissue.

Data from the SDL (Figure 4.13) situated at Site W (Figure 4.3) were used as a guide to select the minimum pH and maximum EC levels for both experiments to ensure that laboratory conditions were similar to estuarine waters affected by ASS. Sydney rock oysters typically open their valves to feed at salinities greater than 15 ppt (23.4 dS m-1) (Holliday, 1995). Between the 4/6/99 and the 12/6/99 low pH values (pH 4.41) were measured at EC levels that exceeded 23.4 dS m-1 at Site W (Figure 8.4). Based on this information, a minimum pH of 5 and a maximum EC of 31 dS m-1 were used for the two laboratory experiments. pH 5.1 is a moderately weak acid but shown to be toxic to + fish with added aluminium due to the presence of Al(OH)2 and Al(OH)3 (Sammut, 1998), which were also shown to be toxic by Driscoll et al. (1980).

190 Table 8.2 Behaviour Experiment and Feeding Experiment details.

Exp. Purpose Treatment Treatment Water Target No. of Number pH / EC (dS m-1) Oysters

Behaviour behaviour and soft 1 seawater + deionised water 8 / 29 24 Experiment tissue repsonse

2 seawater + deionised water 5.1 / 29 24 + 0.1 M HCl

3 seawater + deionised water 5.1 / 29 24 + Al + 0.1 M HCl

4 seawater + deionised water 5.1 / 29 24 + Fe + 0.1 M HCl

5 seawater + ASS-affected water 5.1 / 29 24 + 0.1 M HCl

Feeding feeding 6 seawater + deionised water 8 / 29 18 Experiment measurements + natural silt

7 seawater + ASS-affected water 6.5 / 29 18 + natural silt + 0.1 M HCl

8 seawater + ASS-affected water 5.5 / 29 18 + natural silt + 0.1 M HCl

8 40

7 pH 35 valves 6 open 30

5 25 ) -1

pH 4 20 valves 3 closed 15 EC (dS m

2 10

1 5 EC 0 0 3/06/99 5/06/99 7/06/99 9/06/99 11/06/99 13/06/99

Figure 8.4 EC (thin line) and pH (bold line) conditions at Site W between the 4/6/99 and the 12/6/99. The EC value of 23.4 dS m-1 is indicated by the horizontal black line.

191 8.8.1 Behaviour Experiment 8.8.1.1 Behavioural Response Behaviour was observed and recorded to ensure that oysters had open valves and were feeding at pH 5.1 at the selected EC level (29 dS m-1). Five treatments (numbered 1 to 5 and detailed in Table 8.2) were conducted and twenty-four oysters were observed for behavioural responses during each treatment. The duration of exposure was six-hours, commencing from the moment an oyster opened its valves. Six hours was chosen for the duration of exposure because it was the duration that oysters were exposed to acidic conditions in one tidal cycle in the estuary (Figure 4.12). Abnormal oyster behaviour, including excessive gaping, clomping or no activity, was also recorded during Treatments 1 to 5.

8.8.1.2 Response of Oyster Soft Tissues The experimental work was also designed to examine short-term, sub-lethal effects of weak acidity (pH 5.1) on the gills and the mantle soft tissues of Sydney rock oysters. Histopathology was used to examine the response in the soft tissues. Aluminium and iron were added to Treatments 3 and 4, respectively and ASS-affected water was added to Treatment 5. The pH, iron and aluminium concentrations used in these treatments were based on actual field data obtained during this present study from oyster leases (Chapter 4).

Dove (1997) determined that exposure to acidic conditions caused degenerative changes in the gill soft tissue of Sydney rock oysters including interstitial inflammation and necrosis in the gills. However, Dove’s (1997) histopathology data were confounded by a background soft tissue condition present in the experimental oysters. This condition was due to LS (R. Callinan, NSW Fisheries, personal communication, 1997), which has previously been described in oysters sourced from the Hastings River estuary (Callinan, 1997a). To avoid this problem in the present study, all oysters were sourced from the Manning River after it was established that there were no clinical signs of LS (Sections 7.4.4 and 7.5) in these oysters.

The duration of exposure in all treatments for the Behaviour Experiment was six-hours, commencing from the moment an oyster opened its valves. Of the 24 oysters used in

192 Treatments 1 to 5, 12 oysters were preserved for histopathology and 12 oysters were returned to Site 2 (Figure 5.1) for monitoring of post-experiment survival.

8.8.2 Feeding Experiment To determine if filtration rates are influenced by acidification, Sydney rock oysters were exposed to ASS-affected waters under laboratory conditions. The Feeding Experiment was used to measure oyster feeding rates whilst controlling food levels and other physico-chemical water quality variables, chiefly pH. The treatment waters used in the experimental exposures were based on water quality conditions measured at leases impacted by ASS-affected waters in Chapter 4 and displayed in Figure 8.4.

The Feeding Experiment used three treatments referred to as Treatment 6, Treatment 7 and Treatment 8. Treatment 6 contained no ASS-affected water and the pH was maintained at 7.96 for the entire experiment. ASS-affected waters were used to acidify the test waters to pH 6.5 in Treatment 7 and pH 5.5 in Treatment 8. Details relating to the experimental apparatus used to expose oysters to acidified water are presented in Section 8.9.3.

8.9 EXPERIMENTAL EXPOSURE 8.9.1 Behaviour Experiment Oysters One hundred and fifty Sydney rock oysters were randomly collected from oyster leases in the Manning River isolated from areas impacted by ASS-affected waters (i.e. Sites 1, 2 and 3 shown in Figure 5.1). Oysters were acclimated at Site 2, a non-acid impacted site, from the 1/2/00 for a minimum of 30 days before transfer to the laboratory. The mean shell height (± 95% CI) of all of the oysters used in the Behaviour Experiment was 51.89 ± 0.72 mm. A Greenspan Smart Sonde SDL was installed at Site 2 and recorded pH, EC and temperature at this site during the acclimation period to ensure that oysters were not exposed to acidic conditions prior to the experimental work.

8.9.2 Feeding Experiment Oysters Eighty Sydney rock oysters of mean height 57.94 ± 1.39 (95% CI) mm were acclimated for a minimum period of 30 days at Site 2 (Figure 5.1) from the 1/10/00. Once again, the Greenspan Technical Services Smart Sonde was installed at Site 2 during this period

193 to ensure that oysters were not exposed to acidification prior to the Feeding Experiment. Eighteen oysters were used in each of the three treatments. Physical attributes of the oysters collected for the Feeding Experiment are summarised in Table 8.3.

Table 8.3 Shell heights, whole weights and soft tissue dry weights of experimental oysters (all values listed are the means ± 95% CI, n =18).

Treatment Shell Height Whole Weight Soft Tissue Dry Weight (mm) (g) (g)

6 58.77 ± 2.16 18.875 ± 2.059 0.658 ± 0.097 7 54.74 ± 2.25 17.581 ± 2.131 0.752 ± 0.112 8 59.66 ± 2.47 21.297 ± 1.633 0.901 ± 0.148

8.9.3 Set-up of the Experimental Apparatus A flow-through system was used to maintain a stable pH in the test trays and to encourage oysters to feed by controlling flow. The flow-through system used recirculated water and was designed to expose oysters to eight different test waters (Treatments 1 to 8). The aquarium is illustrated in Figure 8.5 and the design was based on Widdow’s (1985) apparatus for measurement of clearance rate.

The apparatus used in this present study consisted of a 60 L header tank which gravity fed ten 2.9 L trays (120 mm x 300 mm x 80 mm). A baffle was placed in each tray to reduce turbulence in the trays. A flow rate of 0.5 L min-1 was delivered to each tray for the duration of each treatment. This flow rate was selected for reasons detailed below. The header tank and the eight trays overflowed into a 200 L reservoir where pH, EC, DO and temperature were continuously monitored using a Yeo-Kal 611 Intelligent Water Quality Analyser. Water was intermittently pumped to the header tank from the reservoir using two 2,000 L h-1 pumps. A third pump ran continuously to stir the reservoir water and to prevent sedimentation. All components of the experimental apparatus were made from food-grade or stabilised plastic to prevent any reaction with the acidic test water.

194 header tank

baffle

control tray

tray 1

tap 2

control tray

reservoir

stirrer SDL pump pump

Figure 8.5 Apparatus for exposure of oysters to acidified water (modified from Widdows, 1985).

195 For the Behaviour Experiment, six trays each contained four oysters and the control trays were used to collect water samples for chemical analyses. However, oysters were placed individually into trays 2 to 6 for the Feeding Experiment and trays 1 and 8 (Figure 8.5) were used as control trays to collect water samples for measurement of suspended particles.

It was important to ensure that each tray had equivalent and consistent flow rates during the entire Feeding Experiment (Iglesias et al., 1998). A pilot experiment was conducted before the Feeding Experiment to determine an appropriate flow rate to ensure: biodeposits were not being resuspended by water current flows; sedimentation of suspended particles was not occurring on the tray bottoms; and, that the ratio of particulate organic matter (POM) to particulate inorganic matter (PIM) did not vary during and between treatments (Iglesias et al., 1998).

8.9.4 Source and Composition of Test Waters The main constituents of the treatment water used in Treatments 1 to 8 are listed in Table 8.2. Seawater was used in all treatments and was collected offshore from Port Macquarie (31O 25’ 30” S, 152O 55’ 20” E). ASS-affected waters were mixed with seawater in Treatments 5, 7 and 8 (Table 8.2) and were collected from Fernbank Creek (Figure 4.1) immediately before the start of these exposures. The pH was stabilised in Treatments 2, 3, 4, 5, 7 and 8 using 0.1 M Analar hydrochloric acid (HCl). A Yeo-Kal 611 Intelligent Water Quality Analyser was used for measurements of pH, EC, DO and temperature for both experiments. The methods used to determine other variables of water quality in the treatment waters and the measured levels of each variable are detailed in the respective sections below.

8.9.4.1 Behaviour Experiment Three artificially acidified test waters and one naturally acidified test water were used to investigate the effects of acidified water on oyster soft tissue and behaviour. Oysters were also exposed to pH 8.0, which was a mixture of seawater and deionised water. This was done to ensure that the oyster sampling and handling procedure did not cause lesions in the soft tissue and that oyster behaviour was not a reaction to the aquarium

196 environment. A summary of the five treatments that includes pH, EC, DO and temperature conditions is provided in Table 8.4.

Table 8.4 pH, EC and temperature values (means are ± 95% CI) of Treatments 1 to 5.

Treatment Treatment Water Time Mean pH pH Range Mean EC Mean Temp Number (h) (dS m-1)(OC)

1 Seawater + Deionised H2O 6 8.02 ± 0.009 7.99 - 8.12 29.3 ± 0.01 26.76 ± 0.045

2 Seawater + Deionised H2O + 0.1 M HCl 6 5.11 ± 0.005 5.05 - 5.18 29.3 ± 0.02 24.86 ± 0.064

3 Seawater + Deionised H2O + Al + 0.1 M HCl 6 5.12 ± 0.007 5.04 - 5.18 29.3 ± 0.02 25.42 ± 0.073

4 Seawater + Deionised H2O + Fe + 0.1 M HCl 6 5.08 ± 0.007 5.01 - 5.16 30.8 ± 0.01 22.28 ± 0.035 5 Seawater + ASS-Affected Water + 0.1 M HCl 6 5.09 ± 0.008 5.01 - 5.18 28.9 (no shift) 20.85 ± 0.038

A stock solution of aluminium chloride was added to Treatment 3 and a stock solution of iron chloride was added to Treatment 4. The iron and aluminium chloride were AR grade and were used because sulfate derivatives of divalent cations were found to evoke an inflammatory response in mussels (Sunila, 1988). HCl was used instead of H2SO4 to acidify treatments to avoid unstable aluminium-sulfate complexes that can decouple changing the aluminium species present in the treatment water (Sammut, 1998). Also, HCl has been used to acidify treatments in a number of other studies (Loosanoff and Tommers, 1947; Kuwatani and Nishii, 1969; Calabrese and Davis, 1966; Allan and Maguire, 1992). The concentrations of dissolved and suspended iron and aluminium for each experiment are listed in Table 8.5. Total metal concentrations were determined using the Nitric Acid Digestion method detailed in APHA (1998).

Table 8.5 Concentrations of dissolved and suspended iron and aluminium measured in Treatments 1 to 5.

Treatment Treatment Water Dissolved Suspended Number Fe Al Fe Al (mg L-1)(mg L-1)(mg L-1)(mg L-1)

1 Seawater + Deionised H2O ND ND ND ND

2 Seawater + Deionised H2O + 0.1 M HCl ND ND ND ND

3 Seawater + Deionised H2O + Al + 0.1 M HCl ND 1.4 ND 6.24

4 Seawater + Deionised H2O + Fe + 0.1 M HCl ND ND 7.71 ND 5 Seawater + ASS-Affected Water + 0.1 M HCl 0.201 0.292 13.25 5.86

ND = Not detectable (Fe not detectable when < 0.04 mg L-1, Al not detectable when < 0.02 mg L-1)

197

8.9.4.2 Feeding Experiment The selected pH values for the three treatments were 7.96 (Treatment 6), 6.5 (Treatment 7) and 5.5 (Treatment 8). A pH value of 6.5 was selected for Treatment 7 as this was the critical level at which Loosanoff and Tommers (1947) measured dramatic changes in pumping rates in O. virginica. Table 8.2 lists the treatments and includes the target pH and provides details relating to the treatment water. Treatment water was obtained by mixing seawater with deionised water (Treatment 6) or ASS-affected water (Treatments 7 and 8). Treatment water was pre-filtered to 11 µm before the Feeding Experiment.

The diet in the Feeding Experiment consisted of natural silt which was collected from the intertidal mud flats adjacent to Site 2 (Figure 5.1). Natural silt collection, storage and filtration were conducted according to the methodology outlined in Bayne et al. (1999a). Silt was scraped from surface sediments to a depth of 2-3 mm and stored at 4O C prior to each treatment. Silt was sieved through 140 µm and 11 µm nylon mesh, left to stand for 60 minutes and then decanted into the reservoir of the experimental apparatus.

Regular measurements of pH, EC, DO and temperature were performed throughout Treatments 6, 7 and 8. The mean pH, EC, DO and temperature values measured during each treatment are listed in Table 8.6. Table 8.6 indicates that pH, EC, DO and temperature were stable and similar in the three treatments.

Table 8.6 Treatment water pH, EC, DO and temperature (values displayed are means ± 95% CI).

Treatment n pH EC DO Temp. Number (dS m-1) (% Sat.) (OC)

6 69 7.96 ± 0.017 29.2 ± 0.02 88.7 ± 0.14 25.63 ± 0.153 7 89 6.50 ± 0.002 29.3 ± 0.02 88.1 ± 0.67 25.55 ± 0.097 8 102 5.50 ± 0.003 29.3 ± 0.02 85.6 ± 1.51 26.22 ± 0.172

198

A water sample was collected before each treatment commenced and analysed to determine the concentration of aluminium, iron, manganese, zinc and silicon. Table 8.7 lists the concentrations of these metals measured in Treatments 6, 7 and 8. Analysis of samples from all of the treatments did not show elevated concentrations of dissolved iron. However, iron flocs were visible on the GFC filters in Treatments 7 and 8 and the experimental water in Treatment 8 appeared orange suggesting that iron was precipitating out of solution. An elevated concentration of dissolved aluminium was measured in Treatment 8 compared to Treatments 6 and 7 (Table 8.7).

Table 8.7 Concentrations of dissolved Al, Fe, Mn, Zn and Si measured in Treatments 6 to 8.

Treatment Al Fe Mn Zn Si Number (mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)

6 ND ND 0.02 0.02 0.22 7 ND 0.01 0.20 0.05 3.20 8 0.11 0.03 0.15 0.03 3.92

ND = Not detectable

During Treatments 6 to 8, water samples were collected from trays 1 and 8 for quantitative measurement of seston concentration or TPM using the gravimetric method (Iglesias et al., 1998). TPM is measured in mg L-1 and is the dried suspended matter, which is equivalent to dietary abundance for oysters (Hawkins et al., 1996). The organic component of TPM represents the dietary or “food” quality (Hawkins et al., 1996; Bayne et al., 1987).

The samples were filtered onto pre-ashed and pre-weighed glass microfibre filters (Whatman GFC, Catalogue Number 1822 047). These filters were ashed at 450O C for 4 to 6 hours, weighed and placed in a desiccator before use. A one-litre aliquot of the treatment water was filtered through the glass microfibre filter and the filter was rinsed with 10 ml of 0.9% ammonium formate to remove any salts (Bayne et al., 1999b). Deionised water was also filtered onto pre-ashed and pre-weighed glass microfibre

199 filters to ensure that filters were not contaminated during drying or weighing (Widdows, 1985).

The filters were oven dried for a minimum of 12 hours at 80O C to a constant weight before weighing. The final step involved ashing the filters at 560O C for 4 to 6 hours before being placed in a desiccator to cool and then weighed for the final time (B. Bayne, personal communication, 2000). This was done to calculate the concentration of PIM and POM in the water samples. Forceps were used when handling filters.

The Feeding Experiment diets for Treatments 6, 7 and 8 are displayed in Figure 8.6. The mean TPM concentration for Treatments 6 (pH 7.96) and 7 (pH 6.5) are similar, however the organic content is slightly greater in Treatment 7. Treatment 8 (pH 5.5) had a greater mean TPM concentration and this was attributed to ASS oxidation products (iron and aluminium) in the treatment water being in a suspended state. This is reflected by the high PIM value. Attempts were made to remove iron precipitates from the treatment water by filtration and allowing flocs to settle in a sedimentation tank before the ASS-affected water was added to Treatments 7 and 8 to achieve similar compositions of suspended particles for all treatments. However, this was not effective in removing the dissolved iron from the treatment water as can be seen in Figure 8.6.

200 9 pH 5.5 pH 6.5 pH 7.96 8

5 -1 8 mg L 4 8

3 7 6 2 6 1 7 7 6 0 8 TPM POM PIM

Figure 8.6 Mean TPM, POM and PIM measured in Treatments 6 (pH 7.96), 7 (pH 6.5) and 8 (pH 5.5) (means are ± 95% CIs). Numbers in the columns denote the treatment number.

The increased concentrations of TPM and PIM measured in Treatment 8 have implications for the oyster diet and the calculation of filtration rates. The two assumptions of the biodeposition method were (Iglesias, 1998): • the organic matter to inorganic matter ratio must be similar for both the available “food” and the actual material filtered by the oysters, and • the pseudofaeces and true faeces are based on the oysters filtering the same source of total particulate matter (TPM). These assumptions were addressed for Treatments 6 and 7, but not for Treatment 8. Therefore this difference in available food levels experienced in Treatment 8 was taken into consideration in the interpretation of the filtration rate results.

8.10 OYSTER BEHAVIOURAL RESPONSE The experimental apparatus allowed close inspection of oysters. Observations of individual oysters for the four behavioural traits detailed in Section 8.2 were performed in all treatments. The observations and descriptions of oyster behaviour for Treatments 1 to 5 are presented in the following chapter.

201

8.11 FEEDING RATES 8.11.1 Biodeposit Sampling and Analysis Trays 2 to 7 were used for measurements of oyster true faeces and pseudofaeces and trays 1 and 8 were used as controls (Figure 8.5). Water samples were collected from the control trays to measure the concentration of suspended particles. One oyster was placed into each of the 6 trays and the time taken for oysters to open their valves was measured. Oysters were then left undisturbed for a period of 2 to 3 hours before a measurement of true faeces and pseudofaeces was performed.

A wide-mouth pipette was used to sample the true faeces and pseudofaeces from the trays. All of the pseudofaeces and true faeces produced in a one hour time period were collected from the trays and filtered onto pre-ashed and pre-weighed Whatman GFC filters. A second measurement of pseudofaeces and true faeces was performed immediately after the first measurement for the same period of time to obtain an average value for the weight of biodeposits produced. This entire procedure was repeated three times to expose 18 oysters to each treatment.

The filtered samples of oyster true faeces and pseudofaeces were analysed using the same methodology as suspended particles. After the biodeposits were filtered through the GFC filter, it was rinsed with 10 ml of 0.9% ammonium formate and was oven dried for no less than 12 hours at 80O C before being re-weighed (Bayne et al., 1999b). The filters were then ashed at 560O C for 4 to 6 hours, placed in a desiccator to cool and then weighed for the final time (B. Bayne, personal communication, 2000). This was done to calculate the organic component of the true faeces and pseudofaeces.

Once all measurements of biodeposits had been conducted, the oysters were weighed and the dimensions of height, length and width were recorded with digital vernier callipers. The mean shell height and whole weights for all of the Feeding Experiment oysters are listed in Table 8.3. The oysters were shucked and the soft tissue of individual oysters were dried at 80O C for 12 hours before being placed in a desiccator to cool and then weighed to determine soft tissue dry weight.

202 The variables measured during the three treatments were: total suspended particulate matter; particulate organic matter; total faeces; faeces organic matter; total pseudofaeces; and, pseudofaeces organic matter. These data were then used to calculate rejection rate, faeces production, feeding activity and filtration rate. The calculations used to determine each of these components are listed in Table 8.8. The weight of true faeces and pseudofaeces produced by each oyster in the three treatments is provided in Appendix M.

Table 8.8 Definitions and calculations of oyster feeding behaviour components (Source: Bayne et al., 1999a).

Measured Variable Derived Variable Description/Calculation

Total suspended TPM (mg L-1): Suspended matter dried particulate matter at 80O C

Particulate organic POM (mg L-1): TPM ashed at 560O C for 4 h matter Particulate inorganic PIM (mg L-1): TPM-POM matter Particulate organic OC (fraction): POM/TPM content

Total faeces Faeces prodn (mg h-1): Faeces dried at 80O C

Faeces organic FOM (mg h-1) : Faeces ashed at 560O C for 4 h matter Faeces inorganic FIM (mg h-1) : Faeces prodn-FOM matter Faeces organic FOC (fraction): FOM/Faeces prodn content

Total Rejection rate, RR (mg h-1): Pseudofaeces pseudofaeces dried at 80O C

Pseudofaeces organic PsOM (mg h-1): Pseudofaeces matter ashed at 560O C Pseudofaeces PsIM (mg h-1): RR-PsOM inorganic matter Pseudofaeces PsOC (fraction): PsOM/RR organic content Filtration rate FR (mg h-1): (FIM+PsIM)x(TPM/PIM) Clearance rate* CR (L h-1): (FIM+PsIM)/PIM Feeding activity** Feeding activity (mg h-1): Faeces prodn+RR

* Estimate ** Bamber (1987;1990) N.B. Drying time for suspended particulates and biodeposits was > 12 hours

203 8.11.2 Correction for Body Size Body size of the experimental oysters is an important variable affecting most physiological responses (Widdows, 1985). There were slight differences in the dry body mass of oysters used in the three treatments (Table 8.3). The Feeding Experiment was designed to test for the variance of feeding behaviour amongst individuals that is not weight dependent but due to exposure to ASS-affected waters. The variations in dry body mass can be removed by correcting feeding rates to a standard body size using the allometric equation (Bayne and Newell, 1983):

Y = aXb or log10 Y = log10 a + b log10 X

Where Y = measured feeding variable, X = dry body mass in grams, and a is the intercept. The slope, b is the allometric exponent in the equation which describes the physiological rate as a function of body size (Bayne et al., 1999a). Mean dry body mass (± 95% CI, n = 54) of the experimental oysters was 0.77 ± 0.07 g. This mean body mass was used as the standard body mass in place of a standard 1 g animal and the corrections for weight differences were calculated using the following equation (Widdows, 1985; Widdows and Staff, 1997):

Log Yc = log Yo – (b log Xo – b log Xc)

Where Yc is the corrected value for a standard body mass (Xc) and Yo and Xo are the individual’s measured rate and body mass, respectively. The weight-exponent was taken from Bayne’s et al. (1999b) study that measured clearance rate in Sydney rock oysters and estimated b as 0.641.

Single factor ANOVA was used to test for differences between Treatments 6, 7 and 8 for weight-corrected feeding activity, faeces production, rejection rate and filtration rate data. SPSS Version 11.0.0 (SPSS Inc.) statistical software package was used to perform each single factor ANOVA. Post hoc pairwise comparisons of the results were made using the Least Significant Difference test.

204 8.12 GROSS PATHOLOGY Oysters removed from the Behaviour Experiment were observed for any gross changes in their soft tissue appearance following the six hours of exposure. In particular, any visible accumulation of iron or aluminium in their shell liquid or on their soft tissue was noted.

8.13 HISTOLOGICAL METHODS AND MATERIALS 8.13.1 Handling and Fixation of Oysters Twelve oysters were removed from each treatment of the Behaviour Experiment after six hours of exposure to the treatment water. The time of exposure commenced from the first instance that individual oysters opened their valves. After oysters were removed from the aquarium, the soft tissue of the animal was immediately excised from the valves. To do this, oysters were opened from the hinge and a sterile scalpel was used to cut away the adductor muscle from the right and left valves. The soft tissue was rinsed in deionised water to remove shell fragments and three incisions were made into the digestive gland to allow penetration of the fixative. Oysters infected by mudworm were discarded.

Formalin (10% sea water) was used to preserve oyster soft tissue for histopathology (Howard and Smith, 1983). Howard and Smith (1983) describe Formalin (10% sea water) as a good general fixative for bivalves. The ingredients for this fixative are provided in Appendix N. Formalin (10% sea water) was used for this study in preference to Davidson’s fixative because acid fixatives interfere with iron (Howard and Smith, 1983).

Oysters were placed in formalin (10% sea water) fixative for 24 to 48 hours, at room temperature, before being stored in 70% ETOH solution (Howard and Smith, 1983). Additional information on the preparation, processing and staining of sections is provided below and in Appendix N.

8.13.2 Cutting and Staining of Histological Sections Two transverse tissue cross sections were taken, the first through the intestine, digestive diverticula, stomach and labial palps and the second through the adductor muscle,

205 kidney and gills. Processing of the specimens involved replacing water in the tissue with wax at 60O C under vacuum to give the tissue enough stability to be cut. The tissue was processed in a Shandon Hypercentre XP Tissue Processing System. Tissue was then embedded into molten Paraplast wax using a Tissue-Tek Embedding Console System.

Sections were cut at 5 µm using Feather S35 Microtome Blades on a Microm HM 330 Microtome. Once cut sections were floated on a water bath of boiled deionised water. Sections were picked up on acid washed glass slides and dried overnight at 58 OC. All oysters were stained with haematoxylin and eosin (H&E). Oysters from Treatments 1, 4 and 5 were also stained with Perls’ Prussian Blue (PPB). The staining procedure for both stains is listed in Appendix N. Angelina Enno (School of Pathology, University of New South Wales) performed the histopathology processing. Oyster sections stained with H&E stain were examined for changes to the gills and mantle resulting from exposure to the test waters using light microscopy. PPB is a stain specific for ferric iron (Howard and Smith, 1983) and these sections were examined for iron accumulation also using light microscopy.

8.14 CONCLUSION The experimental design for the Behaviour Experiment and the Feeding Experiment presented in this chapter will enable exposure of oysters to naturally and artificially acidified treatments in order to observe oyster’s valve movements, sample oysters for histopathology and collect and quantify biodeposits to determine the feeding behaviour of oysters. The next chapter presents and discusses the findings of both the Behaviour Experiment and the Feeding Experiment.

206 CHAPTER NINE EFFECTS OF EXPERIMENTAL EXPOSURES ON OYSTER FEEDING BEHAVIOUR AND SOFT TISSUE

9.1 INTRODUCTION This chapter presents and discusses the findings from the two laboratory experiments described in the previous chapter. A number of studies have effectively used laboratory experiments to identify and understand the direct impacts of acidified water to different bivalve species (Loosanoff and Tommers, 1947; Calabrese and Davis, 1966; Kuwatani and Nishii, 1969; Bamber, 1987; 1990; Wilson and Hyne, 1997). Laboratory experiments enable precise control of water quality and close monitoring of individual animals. Laboratory experiments were used in this present study in order to obtain information to help explain the high mortalities and slow growth measured at field sites recurrently impacted by ASS-affected waters (Chapter 6).

Therefore, the purpose of this chapter is to demonstrate that exposure of Sydney rock oysters to acidic water causes: altered valve movements; reductions in feeding behaviour; and, changes in the gill and mantle soft tissues. This chapter commences by describing oysters’ behavioural response to the acidic (pH 5.1) treatments of the Behaviour Experiment. The results obtained from the Feeding Experiment are then presented and are followed by the histopathology data to identify soft tissue changes resulting from the acidic treatments and exposure to elevated concentrations of iron. The chapter concludes with a discussion of the main findings arising from both the Behaviour and Feeding Experiments and a chapter summary.

9.2 OYSTERS’ BEHAVIOURAL RESPONSE TO ACIDIFIED WATER Observations of behavioural response exhibited by oysters were performed in all treatments of the Behaviour Experiment and are described in this section. Details of the treatment water used in the Behaviour Experiment are listed in Tables 8.2, 8.4 and 8.5.

The observed behavioural traits in the five treatments were: • open valves – observed in all treatments,

207 • excessive gaping – observed in Treatments 3 and 5, • clomping – observed in Treatment 5, • no activity – observed in Treatments 4 and 5. The proportion of oysters displaying each behavioural trait described above in each treatment is listed in Table 9.1.

Table 9.1 Summary of oysters’ behavioural response in Treatments 1 to 5.

Behaviour Trait Mean Open Excessive Clomping No Treatment Treatment Water pH Valves Gaping Activity Number

1 Seawater + Deionised H2O 8.0 24/24 - - -

2 Seawater + Deionised H2O + 0.1 M HCl 5.1 24/24 - - -

3 Seawater + Deionised H2O + Al + 0.1 M HCl 5.1 24/24 7/24 - -

4 Seawater + Deionised H2O + Fe + 0.1 M HCl 5.1 22/24 - - 2/24 5 Seawater + ASS-Affected Waters + 0.1 M HCl 5.1 19/24 1/24 8/24 5/24

Proportion displaying behaviour trait

Five oysters in Treatment 5 and two oysters in Treatment 4 were inactive for the entire exposure period (Table 9.1). In Treatments 1, 2 and 3, all oysters opened their valves and produced true faeces and pseudofaeces. Excessive gaping was only observed in the acidified test waters. Clomping occurred in Treatment 5 and was attributed to the high concentrations of suspended particles (Table 8.5) in the treatment water. The time taken for individual oysters to open their valves in each treatment varied between 1 and 272 minutes.

Bamber (1987; 1990) found that oysters were slow to respond to stimuli in acidified treatments. Oysters were prodded every hour to assess their response to a stimulus. Oysters in Treatments 2 to 5 were slower to react after prodding, especially in the latter stages (i.e. 4 to 6 hours of exposure). The results obtained from the Behaviour Experiment show that oysters actively feed at pH 5.1. This was an essential prerequisite for the Feeding Experiment. The results from the Feeding Experiment are presented in the following section.

208 9.3 THE EFFECT OF ASS-AFFECTED WATERS ON OYSTER FEEDING BEHAVIOUR 9.3.1 Feeding Activity Feeding activity is the amount of true faeces and pseudofaeces produced by individual oysters over a designated period of time (Bamber, 1987; 1990). The mean feeding activity data from the Feeding Experiment are displayed in Figure 9.1. Figure 9.1 shows an increasing decline in feeding activity as pH is reduced. The feeding activity at pH 5.5 (Treatment 8) was significantly lower than the feeding activity measured at pH 6.5 and 7.96 (Treatment 7 and 6, respectively) (Figure 9.1).

The results of the single factor ANOVA for feeding activity, faeces production, rejection rates and filtration rates are tabulated in Appendix O. Also included in Appendix O are the results from the Least Significant Difference post hoc multiple comparisons.

16 b

) 12 -1 b

6 a

feeding activity (mg h 4

0 5678 pH

Figure 9.1 Mean (± 95% CIs, n = 18) feeding activity over a range of pH. Means sharing letters are not significantly different (P > 0.05).

209

9.3.2 Faeces Production Faeces production is the amount of true faeces produced by an individual oyster per hour (Table 8.8) (Bayne et al., 1999a). The mean faeces production data from the Feeding Experiment are displayed in Figure 9.2. This figure shows that faeces production decreases as pH is reduced. The mean faeces production at pH 5.5 was significantly lower than at pH 6.5 and 7.96.

7 b

6 ) -1 5 b

(mg h 4 n

3 a 2 faeces prod 1

0 5678 pH

Figure 9.2 Mean (± 95% CIs, n = 18) feaces prodn over a range of pH. Means sharing letters are not significantly different (P > 0.05).

9.3.3 Rejection Rate Rejection rate is the amount of pseudofaeces production by individual oysters in an hour and was quantified by collecting and measuring the total amount of material egested (Bayne et al., 1999a). The mean rejection rate data from the Feeding Experiment are displayed in Figure 9.3. Figure 9.3 shows that the rejection rate also decreases as pH

210 decreases. As was the case in the previous feeding traits, the rejection rate at pH 5.5 is significantly lower than at pH 6.5 and 7.96.

10 b 9 8 ) -1 7 b 6 5 4 a 3 rejection rate (mg h 2 1 0 5678 pH

Figure 9.3 Mean (± 95% CIs, n = 18) rejection rate over a range of pH. Means sharing letters are not significantly different (P > 0.05).

9.3.4 Filtration Rate The mean filtration rate data obtained from the Feeding Experiment are displayed in Figure 9.4. A significant reduction in the filtration rate was measured at pH 5.5 compared to pH 6.5 and 7.96. Filtration rate is dependent on the TPM to PIM ratio. Figure 8.6 shows that the TPM to PIM ratio was lower in Treatment 8 (pH 5.5) compared to Treatment 6 (pH 8.0) and 7 (pH 6.5).

211 b 16

) 12 b -1 10

filtration rate (mg h 4 a

0 5678 pH

Figure 9.4 Mean (± 95% CIs, n = 18) filtration rate over a range of pH. Means sharing letters are not significantly different (P > 0.05).

9.4 POST EXPERIMENT OYSTER SURVIVAL Twelve oysters from Treatments 1 to 5 of the Behaviour Experiment were replaced in the estuary at the conclusion of each treatment and monitored for survival over a four- week period at Site 2 (Figure 5.1). Monitoring of post experiment oyster survival was undertaken to determine if short-term exposure to the five treatments were lethal. No mortalities were recorded in the oysters from the five treatments four weeks after exposure suggesting that farmed oysters can recover from short-term acid exposure. This finding has implications for modified management practices for farmed oysters in acid-impacted estuaries.

9.5 OYSTER SOFT TISSUE RESPONSE TO ACIDIFIED WATER This section details the results of the short-term effects of exposure of Sydney rock oysters to: acidified water; acidified water containing aluminium or iron; and, ASS- affected waters. Histopathology was used to determine if acidified treatments induced cell changes in the mantle and gills. The tissue and cell changes observed in Treatments

212 2 to 5 of the Behaviour Experiment were compared to the 12 oysters from Treatment 1 to ensure that the changes were a result of exposure to the treatment waters. The histopathology data for Treatments 1 to 5 are detailed in the following sections. Examples of the soft tissue responses are shown in Plate 9.1.

9.5.1 Treatment 1 (pH 8.0, No Added Iron and Aluminium) Histopathology examination did not reveal any significant aggregations of haemocytes in the gills or mantle soft tissues of oysters from this treatment. However, there was focal necrosis of the frontal and lateral cells of the ordinary filaments in particular oysters. Two oysters had very mild, focal accumulations of haemocytes located in the gills. This response was not typical of the other oysters from Treatment 1. Due to the limited histological information available for the Sydney rock oyster, data from Treatments 2 to 5 were compared to the data derived from Treatment 1.

9.5.2 Treatment 2 (pH 5.1, No Added Iron and Aluminium) Oysters from Treatment 2 typically had increased haemocyte activity in the gills when compared to oyster sections from Treatment 1. There were mild, focal aggregations of haemocytes located in the interlamellar junctions and the haemolymph sinuses of plicae and ordinary filaments of the gill of particular oysters (Plate 9.1A). Frontal and lateral cell necrosis was observed to a greater extent in Treatment 2 oysters than was observed in Treatment 1, however, it could not be determined if this was due to the acidity. No significant findings were observed in the mantle soft tissue of the 12 oysters from Treatment 2.

9.5.3 Treatment 3 (pH 5.1, 7.6 mg L-1 of Aluminium) Oysters from Treatment 3 had extensive haemocyte activity throughout the gills. Large accumulations of haemocytes were observed in the interlamellar junctions and haemolymph sinuses of plicae and ordinary filaments of the gill. There were gill lesions present in all oysters from this treatment. The most common lesion was in the haemolymph sinuses of plicae. Rupturing of this sinus caused infiltrations of haemocytes into the adjacent water tube (this occurred in 11 oysters) (Plate 9.1C). There were infiltrations of haemocytes into the pallial cavity through necrotic frontal and lateral cells of ordinary filaments (Plate 9.1B). This response was observed in six

213 oysters. Haemocytes were commonly observed in the junctions between adjacent filaments, congesting the gills. There was also necrosis and sloughing of mantle epithelial cells predominately on the pallial surface in oysters from Treatment 3.

9.5.4 Treatment 4 (pH 5.1, 7.7 mg L-1 of Iron) There were mild to moderate, focal aggregations of haemocytes located in the interlamellar junctions and the haemolymph sinuses of plicae and ordinary filaments of oysters from Treatment 4. There was necrosis and sloughing of the mantle epithelial cells on the pallial surface in two oysters. Corresponding thin sections stained with PPB showed iron at the sites where mantle necrosis and sloughing was occurring. The degree of haemocyte activity throughout the gills in this treatment was comparable to that observed in Treatment 2.

9.5.5 Treatment 5 (ASS-Affected Waters Adjusted to pH 5.1) Treatment 5 contained 13.5 mg L-1 of dissolved and suspended iron and 6.2 mg L-1 of dissolved and suspended aluminium, which was from the added ASS-affected water. Oysters from Treatment 5 had mild to moderate haemocyte activity throughout the gills. Moderate aggregations of haemocytes were observed in the interlamellar junctions and haemolymph sinuses of plicae and ordinary filaments. Focal necrosis and sloughing of the mantle epithelial cells on the pallial surface was observed in the thin sections as well (Plate 9.1D).

As was observed in the previous treatments, there was necrosis of the frontal cells and lateral cells of particular gill filaments. Haemocytes in the sinuses of the ordinary filaments were escaping into the pallial cavity through necrotic frontal cells and lateral cells of these filaments. This was observed in 5 oysters from Treatment 5. Corresponding thin sections stained with PPB showed iron had accumulated at sites of necrosis and sloughing of mantle epithelial cells. The soft tissue response in Treatment 5 was not as severe as was observed in oysters from Treatment 3 even though the aluminium levels were similar.

214 A. B.

C. D.

Plate 9.1 Soft tissue responses in Behaviour Experiment oysters: (A) haemocyte infiltration into the haemolymph sinuses of the plica and filaments (Treatment 2, x 40); (B) haemocyte infiltration into the haemolymph sinuses of the plica and filaments with rupture of the ordinary filaments (Treatment 3, x 40); (C) rupture of the haemolymph sinus of plica (Treatment 3, x 40); and, (D) necrosis and sloughing of mantle epithelial cells (Treatment 5, x 160).

215 9.6 EFFECTS OF IRON PRECIPITATES The water used for Treatments 4 and 5 appeared orange and suspended iron flocs were clearly visible. Water samples were collected during these treatments and analysed using ICPAES for the concentration of dissolved and suspended iron. The results from ICPAES analysis (Table 8.5) show that there was: 7.71 mg L-1 of suspended iron in Treatment 4; and, 0.201 mg L-1 and 13.25 mg L-1 of dissolved and suspended iron, respectively, in Treatment 5. These levels of iron were commonly measured at acidified field sites during the present study (Chapter 4) and are consistent with levels measured in other studies investigating estuarine acidification in eastern Australia (Sammut et al., 1996a; Sammut, 1998; Sonter, 1999).

All oysters removed from Treatments 4 and 5 displayed gross signs of iron flocs in the shell fluid (Plate 9.2) and on the gill surface (Plate 9.3). Twelve oysters removed from Treatments 1, 4 and 5 were fixed in formalin (10% sea water), processed for histopathology and stained with PPB to investigate the extent of iron precipitate accumulation on the soft tissues. PPB stains iron blue, nuclei appear red and the background appears a pale red colour. Table 9.2 lists the presence and extent of iron accumulation on and in the soft tissues of Behaviour Experiment oysters.

No iron was observed in any of the histopathology sections from oysters removed from Treatment 1 (Table 9.2 and Plate 9.4). However, histopathology data revealed that oysters removed from Treatments 4 and 5 had extensive accumulations of iron on and in their soft tissues (Table 9.2).

Iron precipitates were observed: on the gill epithelium; on the mantle epithelium; in the stomach; in the intestine; and, in the rectum of oysters removed from Treatment 4 (Table 9.2). Similarly, iron precipitates were observed: on the gill epithelium (Plate 9.5A); on the mantle epithelium and in the pallial cavity (Plate 9.5B and 9.5C); in the stomach (Plate 9.5D); in the intestine (Plate 9.6A); in the digestive gland ducts (Plate 9.6B); in the digestive tubules (Plate 9.6C); and, in the rectum (Plate 9.6D) of oysters removed from Treatment 5.

216

Plate 9.2 Oyster with the right valve removed showing iron flocs in the shell fluid. Treatment 4 oyster after 6 hours of exposure to acidified water with added iron chloride.

Plate 9.3 Oyster with the left valve cut-away showing extensive accumulation of iron flocs on the gills. Treatment 5 oyster after 6 hours exposure to ASS-affected waters.

217

Table 9.2 List of iron accumulation on the gills, on the mantle, in the stomach, in the digestive gland tubules and in the rectum of Sydney rock oysters.

Iron Accumulation Exp. Oyster Iron Gills intestine Stomach Digestive Gland Rectum Mantle Present Tubules

11No------2No------3No------4No------5No------6No------7No------8No------9No------10No------11No------12No------

41Yes D - B - A C 2Yes D A B - A C 3Yes D - A - - B 4Yes D B A - A C 5Yes D C A - A D 6Yes D D - - A C 7YesD----C 8YesD----C 9Yes D B A - - C 10 Yes C - A - - C 11 Yes D B B - - C 12 Yes D C B - A C

51Yes D - A - - A 2Yes D D C C - B 3Yes D - B B - A 4Yes D - - A - C 5YesCC - - CC 6Yes C D B C - A 7Yes D D C - - B 8Yes D B B - - B 9Yes D - B - - C 10 Yes D - A - A C 11 Yes C B A - - C 12 Yes C A A - A B

A = very minor accumulation C = moderate accumulation B = minor accumulation D = extensive accumulation

218 A. B.

C. D.

Plate 9.4 Treatment 1 Behaviour Experiment oysters stained with PPB showing: (A) a gill plica (x 40); (B) the intestine (x 40); (C) digestive tubules (x 40); and, (D) the rectum (x 40).

219 A. B.

C. D.

Plate 9.5 Treatment 5 Behaviour Experiment oysters stained with PPB showing: (A) iron on a gill plica (x 40); (B and C) iron on the mantle and in the pallial cavity (x 40); and, (D) iron in the stomach (x 15).

220 A. B.

C. D.

Plate 9.6 Treatment 5 Behaviour Experiment oysters stained with PPB showing: (A) iron in the intestine (x 15); (B and C) iron in the secretory-absorptive cells of digestive gland tubules (x 160); and, (D) iron in the rectum (x 40).

221 9.7 DISCUSSION 9.7.1 Oysters’ Behavioural Response to Acidified Water Observations of oysters from Treatments 1 to 5 indicate that oysters attempt to feed under acidic conditions when salinity conditions are satisfactory. This directly exposes oyster soft tissue to conditions that are potentially injurious. Additionally, this finding permitted feeding rates to be quantified as oysters produced true faeces and pseudofaeces under acidic conditions. Exposure to acidified water (pH 5.1) containing aluminium or ASS-affected waters caused abnormal valve movements in a small proportion of experimental oysters.

9.7.2 Feeding Behaviour The results from the Feeding Experiment have revealed that oyster feeding rates are inhibited by ASS-affected waters. As the concentration of ASS-affected water is increased, which also increases the acidity, oyster feeding rates are reduced.

Feeding activity, faeces production, rejection rate and filtration rate (Figures 9.1 to 9.4) were significantly reduced at pH 5.5. The large difference in all feeding traits (feeding activity, faeces production, rejection rate and filtration rate) between Treatments 6 and 7 and Treatment 8 can be attributed to the presence of ASS-affected waters. Treatment 8 had a higher concentration of TPM and PIM compared to Treatments 6 and 7. This was attributed to the presence of oxidation products contained in ASS-affected waters, namely iron and aluminium. However, it cannot be established whether the significant reduction in feeding at pH 5.5 was due to a reduction in pH alone or from the influence of the oxidation products contained in ASS-affected waters. The results obtained from Treatments 6 and 7 indicate that the reduction in feeding behaviour traits is likely to be predominantly influenced by pH as the experimental conditions were similar in all other respects apart from pH.

The experimental conditions used in Treatments 7 and 8 were frequently measured in the estuary following high rainfall at oyster lease sites in the Hastings River and the Manning River (Chapter 4). Also, ASS-affected water in this pH range commonly contains high concentrations of ASS oxidation products, such as iron flocs.

222 The findings from the Feeding Experiment are consistent with overseas studies investigating feeding and pumping in other species of bivalves exposed to acid (Bamber, 1987; 1990; Loosanoff and Tommers, 1947). The results from this study support Loosanoff and Tommers’ (1947) finding that pumping rate dramatically decreases at pH values below 6.5 in O. virginica. The findings from the Feeding Experiment further reinforce the deleterious impacts caused by slight reductions in pH to bivalves. This is a significant finding and helps to explain poor growth rates measured in the field studies detailed in Chapter 6.

Results from the Feeding Experiment also highlighted the change in the dietary abundance of food available to oysters in ASS-affected waters. High concentrations of colloidal iron and aluminium alter the ratio between the inorganic component and the organic component of ASS-affected waters. This difference can be seen in Figure 8.6 where PIM greatly exceeds POM at pH 5.5. This equates to a low organic content, or a small proportion of food, and a large proportion of non-utilisable matter within the available seston (Hawkins et al., 1996; Bayne et al., 1987). Therefore, the nutritional quality of ASS-affected waters is low when quality is expressed as organic content per unit volume of diet. During this present study TPM and PIM was 28 and 21.5 mg L-1, respectively, at an acidified oyster lease located downstream from Fernbank Creek.

Attempts were made to remove the iron from the treatment waters in Treatments 7 and 8 to address the assumptions that underpin the biodeposition methodology. This was unsuccessful in the case of Treatment 8, however it highlights the realistic environmental problem caused by high concentrations of suspended iron to bivalves. Furthermore, the histopathology data presented in Section 9.6 clearly showed the accumulation of iron flocs on the gills and mantle and in the digestive gland and rectum. Winter (1972) demonstrated that, after adding 4 mg L-1 of iron as ferric hydroxide, 94% of the iron was expelled as pseudofaeces. However, this was at neutral pH values and low concentrations of iron are accumulated, over time, to high concentrations in the oyster soft tissue (George et al., 1976).

The results from Treatment 8 strongly suggest that a combination of low pH and iron is impairing feeding in oysters. The effect of high concentrations of iron on oyster

223 physiology is worthy of further investigation and is currently being investigated by the author. The author acknowledges that further investigation is still required to elucidate the effects of low pH alone from the other constituents of ASS-affected waters on oyster feeding.

9.7.3 Response of Oyster Soft Tissues This study identified changes in the gill and mantle soft tissues resulting from exposure to acidic waters relative to oysters exposed to pH neutral waters. Changes were most noticeable in acidic treatments containing added iron, aluminium or ASS-affected waters. Lesions in the gills were observed after only 6 hours of exposure to acidified treatments containing aluminium. The extensive inflammatory response and gill lesions observed in Treatment 3 suggests that the presence of aluminium in combination with low pH causes a more intense response in the gill and mantle soft tissues than water of a low pH with no added aluminium (Treatment 2). It is likely that this was only the initial stage of changes in the soft tissue as exposure time was only for a short duration and oysters were immediately fixed in formalin after exposure. Sammut (1998) showed that gill lesions in sub-lethally exposed fish can repair within 24-48 hours of exposure but in moribund fish, lesions became more severe before death occurred in the recovery waters.

Comparison of corresponding thin sections stained with H&E and PPB revealed aggregations of inflammatory cells were not only associated with iron accumulation. Further research is required to examine the effects of suspended iron precipitates at neutral and alkaline pH levels. Winter (1972) found that exposure of the mussel M. edulis to iron did not result in acute toxicity but caused high mortality and decreases in body weight. The mobilisation of suspended iron precipitates can be several kilometres from the ASS outflow location (Chapter 4). Iron flocs were observed grossly in oysters removed from the S&GE (Chapter 6). Based on the results from the laboratory investigations, it is highly probable that the high concentrations of iron at acidified field sites were contributing to the high mortality rates and negative growth rates measured during the S&GE.

224 Other studies have confirmed that iron is not toxic to bivalves at neutral pH levels. Sunila (1988) found that ferric iron did not cause a toxic reaction in the gills of M. edulis. Also, it has been established that not all of the iron that enters the gut is absorbed. George et al. (1976) estimated that 30% of the iron presented to the gut is not absorbed and is passed via the faeces in the mussel M. edulis. An interesting finding from the Behaviour Experiment was that iron chloride contained in Treatment 4 was not observed in digestive tubules, however iron in the treatment containing ASS-affected water was observed in the secretory-absorptive cells of digestive tubules. Iron transformations in ASS-affected waters are likely to be different than in the artificial test waters due to the reaction of iron with other pyrite oxidation products and other elements present in the natural waters. The resulting iron chemical species are therefore, likely to be different to those in the artificial test waters. It is also likely that the species of aluminium contained in Treatment 3 was different to the aluminium in Treatment 5 for the same reasons. This would account for the more intense response in soft tissues in Treatment 3 oysters. Further work should model iron and aluminium speciation in the treatment waters to account for metal transformations.

Soft tissue responses observed in oysters from Treatment 4 were likely to be induced by the combination of acidity and iron as opposed to the iron alone. Although there is no evidence of direct iron toxicity in Sydney rock oysters, it is very probable that iron impairs gill function by congesting the ciliary junctions thereby affecting feeding processes and gas exchange.

Elevated iron concentrations are harmful to fish (Cruz, 1969). Cruz (1969) investigated the pathological action of iron by injecting iron salt (at 200 mg of FeSO4 by kg of body weight) into the digestive tract of fish. The survival time of the inoculated fish ranged from 2.5 h to 90 h. Cruz (1969) found internal lesions in various organs when iron was absorbed in large quantities by the digestive tract. Common responses of the fish were: destructive gill lesions; congestion of the gills, liver and kidney; and, liver necrosis. This is a relevant finding for this current study, as intracellular iron was commonly observed in the epithelium of digestive tubules in oysters exposed to ASS-affected waters. The long-term implications of iron accumulation in the digestive tract by the Sydney rock oyster are unknown.

225

Further research is required to separate the effects of acidity on other organs and the role of iron and aluminium at various pH values, including pH neutral treatments. Also, histopathological investigation of the response of oysters exposed to acute and chronic acidification under estuarine conditions would aid in identifying causes of the high oyster mortality measured under field conditions.

9.8 CHAPTER SUMMARY This chapter demonstrated that exposure of Sydney rock oysters to acidified water alters their valve movements, inhibits their feeding behaviour and causes changes to their gill and mantle soft tissues. Feeding activity, true faeces production, rejection rate and filtration rate were dramatically reduced in weakly acidified treatments (pH 5.5) that contained ASS-affected water. Therefore, the hypothesis that ASS-affected water reduces filtration rates in Sydney rock oysters was supported. It could not be established if the reduction in feeding was a result of the acidity alone or due to the presence of oxidation products contained in the ASS-affected waters. However, the data from the Feeding Experiment and other studies (Bamber, 1987; 1990; Loosanoff and Tommers, 1947) strongly suggests that the reduction in pH caused by the addition of ASS-affected water is the main factor that inhibits oyster feeding.

This chapter has confirmed that acidified water containing aluminium causes a degenerative soft tissue response in the gills and, to a lesser extent, the mantle of the Sydney rock oyster after only a short period of exposure. Injuries to the gills of oysters were a result of the combined effect of low pH and aluminium. Histopathology has revealed that iron is extensively accumulated on the gill and mantle and in the intestine, stomach digestive tubules and rectum of oysters exposed to ASS-affected waters.

ASS outflows dramatically alter the biochemical composition of suspended particles in the estuarine waters that it affects. ASS-affected waters bind phosphorus (Simpson and Pedini, 1985) and increase the concentrations of iron and aluminium (Sammut et al., 1996a; 1996b). The chemical and physical nature of suspended particles in areas of the estuary impacted by ASS-affected waters is different to the properties of suspended particles that are present under normal estuarine conditions.

226

All experimental exposures resembled estuarine water quality conditions that were measured following rainfall at sites used for production of oysters (Chapter 4). The results from these laboratory investigations aid in the explanation of decreased growth performance measured at sites recurrently exposed to ASS-affected waters and presented in Chapter 6. Also, this chapter highlights the deleterious effects of high concentrations of iron precipitates contained in ASS-affected waters to oyster health.

The final Section (IV) containing Chapter 10 follows and details the major findings of this present study, the implications of this work for the oyster industry and discusses areas of future research.

227

SECTION IV

CONCLUDING CHAPTER

228 CHAPTER TEN CONCLUSION

10.1 MAJOR FINDINGS The overriding objective of this study was to determine whether acidified outflows from ASS caused mortality and reduced growth in exposed Sydney rock oysters. A geographical approach involving interdisciplinary methods underpinned the investigations due to the complex interactions between soil, water and biological processes. The overriding objective was successfully achieved and the following hypotheses were supported:

1. Long-term exposure of the Sydney rock oyster to ASS-affected waters will cause mortalities and reduced growth rates in adult oysters.

2. ASS-affected waters will increase mortalities in young adult Sydney rock oysters compared to older market–sized Sydney rock oysters.

3. Exposure of the Sydney rock oyster to ASS-affected waters will cause changes in the gills and mantle soft tissues and will result in the accumulation of iron precipitates on the soft tissues.

4. Exposure of the Sydney rock oyster to ASS-affected waters will cause a reduction in their filtration rate thereby affecting survival and growth.

The first two specific objectives of this study were: to identify and measure sources of acidification and describe the spatial characteristics of estuarine acidification in two oyster-producing estuaries; and, to investigate the temporal characteristics of estuarine acidification in an area used for Sydney rock oyster production. Water quality monitoring of the Hastings River and Manning River estuarine waters showed that acidity was indeed due to mineral acids from ASS, and acidification was spatially extensive following high rainfall. The severity of acidification measured in particular areas of these estuaries were comparable to levels reported in previous studies

229 (Johnston, 1995; MHL, 1997; ERM Mitchell McCotter, 1997; Sonter, 1999; Silcock, 1998; Webb, McKeown and Associates, 1997) as well as other estuaries in eastern Australia (Sammut et al., 1996a; Sammut, 1998; Pease et al., 1997; Hicks et al., 1999).

The temporal characteristics of estuarine acidification on the Hastings River and the Manning River were dependent on the intensity and recurrence of the antecedent rainfall, which has also been reported in other studies (Sammut et al., 1996a; Johnston, 1995; Sonter, 1999; MHL, 1997). The temporal persistence of acidity in areas used for oyster production in both estuaries was sufficient to cause serious production problems, namely mortality and slow growth, to the extent that it was impossible to commercially produce oysters resulting in abandonment of these areas.

Oxidation products, namely iron and aluminium precipitates were mobilised great distances downstream from their source during estuarine acidification. Iron flocs were observed more than 15 kilometres from their source on the Hastings River coastal plains and were present in waters that had circumneutral pH values (pH ~ 7.5). Coatings of iron precipitates were commonly observed in areas recurrently impacted by estuarine acidification and smothered oysters, oyster racks and trays, mangroves and the benthos of both the Hastings River and the Manning River. Iron precipitate mobilisation and coating were also reported in the Richmond River system, in northern NSW, following heavy rainfall (Sammut et al., 1996a; Roach, 1997; Corfield, 2000). The presence of iron precipitate coatings in the intertidal zone was an accurate indicator of areas that were prone to acidification and corresponded to areas where oyster production had ceased.

The results from a field observation experiment conducted on the Manning River showed that oysters recurrently exposed to ASS-affected waters experience high mortality rates compared to oysters located in areas that were not exposed to ASS- affected waters and had circumneutral pH levels. Mortalities at sites exposed to ASS- affected waters increased with the time of exposure and were significantly greater in smaller oysters. This finding supports the second hypothesis and was consistent with Bamber’s (1987; 1990) findings. This finding was not surprising given the higher order

230 magnitude of acid events on the Hastings and Manning Rivers compared to the conditions reported in Bamber’s (1987; 1990) work.

Mortalities resulted from the dramatic change in water quality conditions induced by ASS-affected waters. ASS-affected waters have a relatively low EC and pH compared to brackish water and does not efficiently mix with brackish water due to density differences (Sammut et al., 1994; Sammut et al., 1996c; Johnston, 1995). This results in ASS-affected waters existing as a ‘plug’ of water that is repeatedly moved forward and backward in the estuary by tides for long periods of time before the plug is broken down and neutralised (Sammut et al., 1994; 1996a). The widely fluctuating pH levels combined with low ECs and high concentrations of iron and aluminium were beyond the tolerance threshold of the Sydney rock oyster and caused high mortalities after 42 days of repeated exposure. For comparison, laboratory experiments conducted by Bamber (1990) subjected C. gigas continually to pH 6.0 and reported significant mortalities after 30 days.

Information obtained from the field investigations also indicated that hydrologic characteristics and geomorphic location of an oyster lease controls the extent of impact caused by ASS-affected waters. Poorly-flushed sites exposed to ASS-affected waters experienced higher mortality rates compared to sites that were well-flushed. This was attributed to the additional stress caused by extended periods of weak acidity and reduced EC levels induced by ASS-affected waters and exacerbated by tidal attenuation.

The primary cause of mortality in smaller oysters was from shell perforation. In the present study shell perforation occurred rapidly in small single seed oysters that had a flat section in the anterior of the left valve. Shell degradation and dissolution was reported in the literature in studies investigating the effects of lowered pH on bivalves and the process was found to commence at neutral pH values (Bamber, 1987; 1990; Kuwatani and Nishii, 1969). Therefore, the severe extent of oyster shell degradation, dissolution and perforation reported in Dove (1997) and observed in this present study was not surprising considering the strength and duration of acid exposure.

231 As stated earlier, sites exposed to ASS-affected waters also had elevated iron concentrations. Investigation of the effects of iron precipitates to oysters conducted as part of this present study implicated elevated levels of iron contained in ASS-affected waters as a major factor for poor oyster health, slow growth, mortalities and feeding inhibition. Elevated iron concentrations were responsible for mortalities and slow growth in mussels (Winter, 1972).

Sites exposed to ASS-affected waters experienced reduced growth, in terms of whole weight gain and shell height increase, when compared to sites that were isolated from ASS-affected waters. The differences in growth rates were greater in small oysters at the sites that were impacted by acidification compared to sites isolated from the ASS- affected waters. Growth rate reduction was attributed to the poor water quality conditions caused by ASS outflows. Low pH has been shown to reduce bivalve flesh weights and dissolve their shells (Bamber, 1987; 1990) which has implications for oyster development and growth. Water quality data showed that all of the sites isolated from ASS-affected waters had a higher median EC level due to the fact that they were isolated from fresh and acidic floodplain inflows. Growth rates improved at sites impacted by ASS-affected waters in dryer conditions. Dryer conditions also corresponded with a dramatic improvement in water quality conditions at sites exposed to ASS-affected waters.

Poor growth rates at sites exposed to ASS-affected waters were attributed to a number of factors that are likely to have a synergistic relationship. Findings from the laboratory investigation of this study have yielded plausible theories as to why growth rates are reduced in the presence of ASS-affected waters. Possible reasons why growth rates are reduced in areas exposed to ASS-affected waters are discussed further below.

Shell dissolution directly impacts upon oyster whole weight measurements as well as shell height measurements. Sydney rock oysters removed from acidified sites during the present study commonly had no ‘lip’ or protrusion of new shell growth from the posterior edge of their shell. However oysters removed from the reference sites consistently showed evidence of new shell growth in this section of the shell.

232 This study investigated oysters behaviour at a pH and EC level that was measured in oyster producing areas impacted by ASS-affected waters. This was done to determine if oysters had open valves so that feeding traits of oysters could be measured. Reduced EC levels also reduce the feeding opportunities for Sydney rock oysters and the lower salinity tolerance limit for the Sydney rock oyster is approximately 15 ppt (Holliday, 1995). Oysters are able to withstand salinities below this level for short periods, however their shell is likely to be closed which prevents feeding processes. Shell closure by oysters at acidified sites was an effective measure to protect soft tissue from acidity, iron and aluminium as the lowest pH levels were measured in the field when salinity was less than 15 ppt. However, laboratory exposures showed when acidic conditions (pH 5.5) were accompanied with salinities above 15 ppt Sydney rock oysters experienced significant reductions in their filtration rate.

This study discovered that ASS-affected waters change the dietary abundance of food available to oysters. This is mainly caused by the elevated concentrations of colloidal iron and aluminium, which dramatically alters the ratio between the inorganic component and the organic component of seston. Therefore, there is a low organic content, or a small proportion of food, and a large proportion of non-utilisable matter within the available seston. The nutritional quality of water impacted by ASS outflows is low when quality is expressed as organic content per unit volume of diet. This is likely to influence oyster growth rates when they experience long-term exposure to ASS-affected waters, as was the case in the S&GE conducted as part of this research. Further research is required to separate the effects of acidity and the effects ASS oxidation products, in particular iron and aluminium, on oyster feeding and other aspects of oyster physiology.

The large reduction in feeding behaviour at pH 5.5 was attributed to damage in the gills of oysters by acid, aluminium and the accumulation of iron on the gill surfaces. This is likely to be a key factor in the reduced filtration rates in Sydney rock oysters exposed to ASS-affected waters. One of the major functions of the oyster gill is to facilitate feeding. Short-term exposure (6 hours) to ASS-affected waters caused changes in the gill and mantle soft tissues which could interfere with feeding processes. Gill lesions were observed in oysters exposed to acidified treatments containing added aluminium

233 and ASS-affected waters. The severity of the lesions observed in the gills is likely to be exacerbated by extended periods of exposure.

Gross pathology of oysters revealed extensive aggregations of iron and aluminium flocs on the gills and mantle. Oyster thin sections stained with Perls’ Prussian Blue, showed that iron was accumulating on the gill, on the mantle, in the stomach, in the intestine, in the digestive tubules and in the rectum of oysters experimentally exposed to ASS- affected waters. Accumulation of iron occurred after short-term exposure to ASS- affected waters. There was no evidence from the laboratory experiments conducted for this study that suggested iron precipitates cause a toxic response to exposed oysters. This is consistent with the findings of Winter (1972) and Sunila (1988) in experiments involving the mussel M. edulis.

Condition of oysters was found to decrease with decreasing pH in Bamber’s (1987; 1990) studies. To the best of my knowledge this was the first study to investigate the effect of reduced pH levels on condition index of oysters under field conditions. This study found that condition index was significantly reduced at sites exposed to ASS- affected waters when compared to sites that were not exposed to ASS-affected waters. It was also found that Sydney rock oysters with highly developed gonads, which were exposed to ASS-affected waters, had a very high mortality rate. This was attributed to the acidic and fresh conditions causing additional stress on the oyster when it was already in a vulnerable state. Further investigation of oyster condition index response to ASS-affected waters is recommended by this study, especially in light of Iglesias et al.’s (1996) findings that condition index in the mussel, M. galloprovincialis, was correlated to clearance rate. The ability for oyster growers to be able to fatten oysters prior to marketing is a critical stage of oyster production and can be impacted by ASS-affected waters. This experiment provided useful baseline information on oyster condition index in the Manning River and provides a platform for future investigations.

This study documented findings from oyster kills resulting from LS. The purpose of investigating LS-induced oyster kills was to demonstrate that oyster production problems caused by LS had different clinical signs to oysters affected by acid exposure. Although there were general similarities between oysters exposed to estuarine

234 acidification and affected by LS, the findings and observations from oyster kills attributed to LS reinforced that there were two separate causes of oyster production problems in the Hastings River estuary. An intensive, long-term water quality investigation under a wide range of estuarine conditions did not detect the presence of acidified water originating from ASS in Limeburners Creek. This study concluded that acidification was not a necessary factor for oyster kills associated with LS. This present study did not identify the cause or causes of the oyster kill and strongly urges further research of this recurrent and serious problem.

In summary, the findings from this present study closely resemble those from other studies which have investigated the impacts of low pH on bivalves (Bamber, 1987; 1990; Loosanoff and Tommers, 1947; Kuwatani and Nishii, 1969; Dove, 1997). A unique aspect of this research was that many of the impacts caused by acid were greater in magnitude than previously reported and shown to occur under actual field conditions. This was attributed to the additional components that are contained in ASS-affected waters, specifically iron and aluminium. The study unequivocally demonstrated that Sydney rock oysters cannot tolerate weakly acidic conditions caused by ASS outflows and the impacts of ASS-affected waters extend into pH neutral areas of the estuary due to the mobilisation of ASS oxidation products.

10.2 IMPLICATIONS OF THIS STUDY FOR OYSTER PRODUCTION The continued existence of an oyster population depends on the survival, growth and reproduction of individuals within that population (Bayne et al., 1979). This, and past studies (Dove, 1997; Wilson and Hyne, 1997; Bishop, 2000) have demonstrated that ASS-affected waters were detrimental to survival, growth and reproduction of individual Sydney rock oysters. Therefore, it is highly likely that problems at the individual oyster level caused by ASS-affected waters translate to the population level and contributes to decreases in oyster production in impacted estuaries. This study has provided the diagnostic information for the oyster industry to rapidly assess problems associated with ASS-affected waters using the clinical signs detailed in this work. The histopathology findings are more relevant to professional diagnosis and can be used to refine the case definition.

235 Many leases in acidified tributaries of the Hastings River and Manning River estuaries have been abandoned and surrendered before oyster growers were able to identify causes of poor production. This represents a large loss of once productive estuary and also a loss of livelihood for the affected oyster growers.

Even though this study focussed on the negative impacts of ASS-affected waters on individual oysters, there were findings from this study that relate to management of oyster stock to reduce the risks associated with estuarine acidification. Oyster growers on the Hastings and Manning Rivers now realise the risks and impracticality of attempting to persevere with oyster production in acid-prone areas.

This study confirmed the observations and reports of oyster growers that ASS pose a serious threat to oyster production and when these soils are disturbed they create a production hazard which is at a considerable distance from the source of pollution. Prior to this study the mortalities and slow growth experienced by oyster growers were largely inexplicable or blamed on unrelated factors. Now that it is known that estuarine acidification impacts oyster stocks, the Sydney rock oyster industry is able to seek and achieve a greater commitment from local and state governments to ameliorate, manage and prevent further acidification of estuarine waters.

This study has measured growth and survival rates at oyster lease sites recurrently exposed to ASS-affected waters. This data enables an accurate assessment of the economic loss to the state’s oyster industry due to estuarine acidification. This is important in light of the declines experienced by the industry over the last 30 years.

The findings of this study were regularly and widely disseminated amongst estuarine stakeholders. Oyster growers, land holders, local councils and state government agencies were briefed on the problems caused by estuarine acidification to oysters through a broad variety of forums during the course of this study. This study has raised awareness amongst estuarine stakeholders and has demonstrated the need for improved environmental goals to preserve and maintain the health of estuarine waters.

236 During the present study the Hastings Council adopted a Local Environment Plan (LEP) to improve planning processes in the light of potential problems from ASS. This policy will be useful as a proactive measure but reactive strategies to address existing acid problems are also urgently needed. Compliance with these plans must be achieved for the LEP to be successful and to prevent further unnecessary degradation of the estuarine ecosystem.

There is also the need for a more representative measure of the severity of ASS-affected water to aquatic habitats other than pH alone. It would be beneficial to measure other constituents of ASS-affected waters, such as iron and aluminium concentration, due to their contribution of ‘downstream’ or ‘off-site’ acid production and environmental impacts.

Until ASS outflows from ‘hotspot’ areas are significantly reduced, oyster farmers are forced to avoid areas impacted by estuarine acidification or quickly relocate stock during wetter periods in acid-prone areas. During dry periods the risks of problems associated with acidification are reduced but growers must have the mechanism in place to hastily remove stock to sites that are characterised by circumneutral pH levels should high rainfall eventuate. To protect smaller oyster stock, it is important to restrict cultivation to areas of the estuary isolated from ASS-affected waters. In most cases, movement of oyster stick culture is restricted by the impracticality of moving large numbers of oysters and availability of alternative leases. During wet years stock movement would be highly impractical.

The information from this research enables efficient assessment of other oyster growing regions in eastern Australia to determine if acidification is a potential threat. The research also equips oyster growers with the knowledge to easily identify the key indicators of acidification and areas susceptible to acidification. Hastings River and Manning River oyster growers were always very active in identifying floodplain development on ASS that had the potential to degrade the estuarine environment and consequently impact their livelihood.

237 The drain and estuarine water quality sampling on both the Hastings and Manning Rivers provides a benchmark to evaluate acid outflow management techniques. It will also enable the detection of unauthorised works if there is a sudden downward pH shift in the drain outflow.

This study represents the most comprehensive work on the effects of ASS-affected waters on not only the Sydney rock oyster but any bivalve species. It is hoped that this study will: increase the awareness of the environmental impacts of ASS-affected waters; strengthen the resolve to reduce the magnitude and frequency of acidic outflows into the estuary; and, prevent unmanaged disturbance of soils containing iron sulfides.

Acidification of estuaries resulting from the disturbance of acid sulfate soils is a significant problem to all floodplain and estuarine stakeholders. Likewise, a solution to this issue will only be facilitated by a multilateral approach. Oyster growers fear estuarine acidification to the extent where they have invested considerable money and resources to not only investigate but also find solutions to this problem. The threat of ASS outflows to the estuarine ecosystem and their livelihood has resulted in oyster growers forming an effective and focussed push against coastal developments that contribute to this problem.

10.3 FURTHER RESEARCH This work concentrated on the direct impacts of ASS-affected waters on individual oysters. ASS-affected waters are likely to impact other components of the estuarine ecosystem that cause a range of indirect effects on oysters.

Recurrently acidified tributaries experience extraordinary changes in water quality. A long-term change in water quality conditions is commonly accompanied by habitat modification. Acidification and high concentrations of toxic elements are deleterious to aquatic flora and fauna and therefore has the potential to dramatically reduce the productivity of acidified systems. The long-term impacts of acidification to the estuarine ecosystem are unknown (White et al., 1996a) and more research needs to be directed into this area.

238 The elevated concentrations of iron and aluminium in ASS-affected waters react with phosphate in solution to form insoluble mineral compounds, which precipitate out of the water column (Simpson and Pedini, 1985). The implication of this process is that phosphate is not available for algal growth, which can alter the phytoplankton biomass (Simpson and Pedini, 1985). Additionally, acidified water containing elevated concentrations of aluminium is toxic to phytoplankton (Folsom et al., 1986). Acid rain studies have shown that acidification alters the species composition of zooplankton and phytoplankton (Geelen and Leuven, 1986; Mills et al., 1987) and it is probable that this also occurs in acidified tributaries of estuaries. Oysters rely on both dissolved phosphate and phytoplankton for nutrition (Nell et al., 1983). ASS-affected waters are likely to have a pronounced effect on oysters’ food sources that has not been investigated or quantified. Therefore, further research is required to better understand the long- and short-term ecological impacts of ASS outflows to the entire estuarine ecosystem.

Further research is needed on the long-term oyster soft tissue response to exposure to ASS-affected waters as well as the effect of iron and aluminium at other pH values. This study focussed on whether ASS-affected waters cause a soft tissue response and now that has been established, detailed description of lesions in the soft tissues is required. The laboratory experiments conducted as part of this present study were focussed and more work is required to be able to ascertain the toxicity of ASS-affected waters to the Sydney rock oyster over a greater range of pH values and exposure times. ASS-affected water would impact other endemic bivalve species in the same way as it impacts Sydney rock oysters. It is probable that elevated concentrations of iron precipitates will have detrimental impacts to all aquatic gilled organisms exposed to ASS-affected waters. This study did not have the scope to investigate longer-term impacts of estuarine acidification to the Sydney rock oyster.

Oyster feeding behaviour was one trait that was impacted by ASS-affected waters. It is very likely that ASS-affected waters impair other physiological processes of the Sydney rock oyster. This has implications for the growth of populations of oysters and oyster production. More research is required to gain a better understanding of the effects of

239 ASS-affected waters on other physiological processes and to separate the effects of acidity from other components of ASS outflows.

As already stated, the information generated from this study will enable an accurate assessment of the economic consequences of estuarine acidification to the oyster industry. The oyster industry is facing challenges from a number of disease and environmental risks. The economic consequences of estuarine acidification do not stop with the estuary based fishery industries. There would be serious implications for the economies of many coastal towns if these industries were no longer viable in estuaries along the east coast of Australia.

This study has identified some of the direct impacts caused by ASS-affected waters on individual Sydney rock oysters. The author acknowledges that there are numerous other detrimental effects of ASS-affected waters to oysters that this study did not have the scope to investigate providing opportunity for future studies.

Information on the identification of problems associated with ASS-affected waters to oyster stock has, and continues to be presented to the Sydney rock oyster industry. Prior to and during this present study Hastings and Manning River oyster growers were very effective in bringing to the attention of government agencies and local councils ASS related problems before and as they occurred. It is highly likely that many other estuaries will benefit from this knowledge gained by Hastings and Manning River oyster growers to identify and warn of ASS related problems in the future.

10.4 FINAL COMMENT Estuarine acidification associated with the disturbance of ASS is undoubtedly a major environmental concern for the eastern Australian Sydney rock oyster industry. Other environmental issues facing the industry including diseases, product safety and declining water quality issues compound the problems caused by ASS-affected waters. There is also increasing pressure placed on eastern Australian estuaries by continued development of their floodplains in order to accommodate rapidly expanding coastal communities.

240 The impacts of estuarine acidification are not limited to the oyster and other estuary- based fishery industries. These industries are purely an indicator of environmental degradation caused by ASS outflows. The problem of estuarine acidification has far reaching implications for every estuarine stakeholder. Numerous coastal towns along Australia’s east coast have a strong dependence on a healthy river system to attract tourism to their region. Tourism is a significant industry for much of Australia’s east coast. Estuarine acidification is slowly receiving greater recognition with an increase in awareness amongst the stakeholders. The exact remedy of the problem is complex and varies for different locations. Further research is necessary but is only one component of the overall solution. Research, sound and improved management of the land and water resources and stakeholder cooperation must all be combined if we are to create a healthier and more productive estuarine ecosystem.

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Appendix A. Listing of field and analytical water quality data following rainfall for Hastings River estuary drains.

HASTINGS RIVER ESTUARY DRAIN WATER QUALITY

Drain Date pH EC DO Temp Cl:SO4 Lab. Fe Al Ca Mn K Mg S SO4 As Cu Si Zn ID (dS m-1)(mg L-1)(oC) pH (mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)

BC19.1L 18/06/99 5.89 11.1 2.4 8.72 8.8 5.64 0.04 0.02 72.96 0.56 67.08 226.00 128.53 385.59 <0.35 <0.005 4.89 <0.02 CC38.4R 19/06/99 5.05 0.3 4.2 11.27 3.7 4.28 0.84 0.13 5.40 0.06 2.60 8.75 11.40 34.20 <0.35 <0.005 3.64 <0.02 CC38.4R 30/11/99 3.66 1.3 3.9 20.74 3.7 3.49 3.11 0.38 16.10 0.13 6.17 30.20 38.40 115.20 <0.09 0.007 1.280 <0.04 CC38.4R 13/02/01 3.19 0.9 1.8 23.38 1.1 3.19 7.75 2.27 15.70 0.38 9.14 27.10 55.90 167.70 <0.12 <0.004 6.39 0.10 CC39.1R 13/02/01 3.91 0.9 1.3 23.84 1.0 3.77 0.98 0.47 32.10 0.82 6.34 26.80 64.40 193.20 <0.12 <0.004 4.99 0.06 CC44.1R 13/02/01 3.58 1.0 0.6 22.50 0.5 3.37 36.70 1.37 18.90 0.53 7.21 24.00 65.10 195.30 <0.12 <0.004 6.52 0.10 CC44.1R 02/12/00 3.98 2.8 4.1 21.51 2.6 3.72 1.42 0.52 44.10 0.31 17.70 81.80 81.70 245.10 <0.14 <0.004 5.39 0.06 CC44.8R 19/06/99 4.96 0.3 2.3 12.64 4.2 4.69 0.03 0.14 8.94 0.11 2.43 8.20 13.22 39.66 <0.35 <0.005 3.30 <0.02 FC11.6L 26/05/98 3.00 0.9 11.6 8.18 2.5 3.59 1.31 2.31 4.46 <0.002 2.76 10.66 22.10 66.30 <0.40 <0.004 1.24 <0.01 FC11.6L 19/06/99 4.18 0.3 2.3 10.83 3.8 3.76 2.38 0.69 4.54 0.13 2.79 6.93 18.09 54.26 <0.35 <0.005 7.62 <0.02 FC11.6L 30/11/99 3.48 1.1 2.7 21.83 4.3 3.20 6.30 0.15 4.38 0.11 2.19 12.40 21.10 63.30 <0.09 <0.006 0.651 <0.04 FC11.6L 13/02/01 3.28 1.3 2.1 22.49 1.3 3.17 35.90 1.84 11.70 0.40 6.66 26.50 60.20 180.60 <0.12 <0.004 10.30 0.06 FC11.6L 02/12/00 3.09 1.4 3.6 21.32 2.1 3.05 6.58 1.06 11.80 0.26 6.54 26.10 48.30 144.90 <0.14 0.01 5.81 0.07 HR16.0R 18/06/99 3.37 1.5 10.5 9.02 2.3 3.28 5.45 8.69 19.15 1.01 13.61 32.42 80.96 242.87 <0.35 <0.005 12.40 <0.02 HR16.0R 12/02/01 2.81 5.9 2.5 22.56 1.6 2.77 48.10 9.53 44.00 1.45 28.30 97.90 193.00 579.00 <0.12 <0.004 15.80 0.23 HR16.5R 18/06/99 3.37 2.7 10.7 9.75 2.8 3.32 4.03 8.34 28.48 1.22 18.83 56.05 88.66 265.98 <0.35 <0.005 12.15 <0.02 HR16.5R 12/02/01 3.23 8.5 3.1 22.74 7.0 3.22 5.33 6.10 52.20 1.03 40.90 131.00 142.00 426.00 <0.12 0.01 11.70 0.12 HR16.8R 18/06/99 3.35 2.2 10.9 9.42 2.6 3.21 4.80 3.64 28.77 1.04 18.24 51.22 91.29 273.87 <0.35 <0.005 12.17 <0.02 HR16.8R 12/02/01 3.48 8.8 3.0 22.37 4.4 3.25 33.90 19.40 77.20 2.30 47.70 175.00 212.00 636.00 <0.12 0.01 17.90 0.47 HR8.1R 13/02/01 4.10 1.1 4.5 20.23 3.8 3.90 12.20 0.46 9.01 0.16 6.43 21.20 31.70 95.10 <0.12 <0.004 4.60 0.03 HR8.1R 19/06/99 6.13 5.0 0.3 9.79 7.8 5.11 5.00 0.13 31.55 0.07 29.71 93.64 67.51 202.54 <0.35 <0.005 4.41 <0.02 MA29.6L 18/06/99 5.99 0.5 2.8 9.71 26.8 6.22 1.28 0.09 4.79 0.02 5.81 10.74 7.33 21.98 <0.35 <0.005 4.11 <0.02 MR19.2R 19/06/99 5.62 0.1 8.5 10.94 17.0 6.02 0.49 0.25 2.80 0.02 2.41 5.50 4.73 14.20 <0.35 <0.005 2.79 <0.02 MR21.7L 12/02/01 3.53 0.9 6.3 24.70 0.6 3.46 1.46 3.00 12.90 0.38 6.65 21.40 43.80 131.40 <0.12 <0.004 7.74 0.09 MR21.7L 18/06/99 4.21 0.2 6.9 10.00 2.9 4.29 0.31 1.17 4.99 0.12 2.78 6.66 15.19 45.57 <0.35 <0.005 5.72 <0.02 MR21.7L 01/12/00 3.19 1.6 - 25.07 2.4 3.40 1.27 7.37 30.50 0.58 18.00 64.80 96.10 288.30 <0.14 0.01 8.98 0.11 MR23.0L 19/06/99 3.87 0.3 2.5 10.38 1.4 3.80 0.23 2.65 7.46 0.49 3.94 8.75 28.01 84.02 <0.35 <0.005 8.44 <0.02

Appendix A. (Continued)

HASTINGS RIVER ESTUARY DRAIN WATER QUALITY

Drain Date pH EC DO Temp Cl:SO4 Lab.FeAlCaMnKMgSSO4 As Cu Si Zn ID (dS m-1)(mg L-1)(oC) pH (mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)

MR23.0L 30/11/99 3.64 3.5 5.4 20.30 3.6 3.48 2.54 2.73 21.30 0.66 13.10 58.20 67.60 202.80 <0.09 0.010 5.280 <0.04 MR23.0L 01/12/00 3.72 2.2 - 24.60 2.4 3.79 0.70 3.08 34.00 0.62 13.70 52.80 81.10 243.30 <0.14 <0.004 10.00 0.12 MR23.0L 12/02/01 4.04 1.2 2.6 24.13 4.5 3.89 0.63 2.44 16.40 0.49 7.69 28.00 43.60 130.80 <0.12 <0.004 7.82 0.09 MR24.2R 12/02/01 3.83 3.2 4.8 23.53 3.0 3.71 0.98 2.19 24.10 0.34 15.40 57.90 72.90 218.70 <0.12 <0.004 6.44 0.05 MR30.8R 19/06/99 4.58 2.4 7.0 10.61 2.2 4.24 1.87 0.74 50.08 0.15 9.55 48.02 79.13 237.39 <0.35 <0.005 8.72 <0.02 MR32.8L 18/06/99 5.99 0.5 8.3 8.44 7.6 5.60 0.58 0.13 5.22 0.03 4.39 9.50 8.91 26.73 <0.35 <0.005 3.10 <0.02 MR33.8R(A 19/06/99 4.58 0.1 3.8 9.91 4.5 4.48 0.84 0.26 2.33 0.04 1.02 3.97 5.90 17.71 <0.35 <0.005 1.88 <0.02 MR33.8R(A 13/02/01 3.06 1.9 2.6 23.80 1.0 3.00 8.02 5.34 22.40 0.56 7.64 35.90 78.50 235.50 <0.12 0.01 5.84 0.13 MR33.8R(A 02/12/00 3.22 3.1 2.4 21.05 2.5 3.42 2.22 1.86 24.40 0.33 9.63 44.10 72.80 218.40 <0.14 0.01 2.44 0.06 MR33.8R(B 30/11/99 3.66 0.5 2.8 21.08 2.0 3.34 2.76 0.21 3.92 0.10 2.84 7.45 15.10 45.30 <0.09 0.02 0.13 <0.04 MR33.8R(B 13/02/01 2.91 1.6 3.2 24.71 1.4 3.00 8.95 5.77 16.90 0.53 5.47 29.20 75.40 226.20 <0.12 <0.004 5.36 0.13 MR33.8R(B 02/12/00 2.77 2.4 2.9 22.15 1.1 2.89 11.40 4.43 19.30 0.57 5.77 31.50 80.60 241.80 <0.14 <0.004 2.18 0.12 MR34.1R 19/06/99 3.99 0.5 6.4 10.88 1.8 3.93 0.37 4.50 14.91 0.26 2.54 13.35 29.45 88.35 <0.35 <0.005 2.20 <0.02 MR34.1R 30/11/99 3.71 0.8 3.8 19.29 1.0 3.52 1.30 5.81 16.50 0.47 2.33 23.60 42.10 126.30 <0.09 <0.006 0.104 <0.04 MR34.1R 13/02/01 3.38 1.6 1.7 23.14 0.9 3.28 4.20 8.82 37.20 0.86 8.81 40.70 89.60 268.80 <0.12 <0.004 5.70 0.18 MR34.1R 02/12/00 3.20 2.4 2.8 20.42 0.9 3.25 3.22 20.70 53.10 1.47 10.70 64.40 144.00 432.00 <0.14 0.01 3.65 0.25 MR35.5R 19/06/99 4.68 0.2 2.7 11.06 4.5 4.39 0.01 0.26 3.56 0.05 2.60 4.56 7.12 21.35 <0.35 <0.005 1.78 <0.02 MR35.5R 30/11/99 3.41 2.0 1.5 20.74 1.0 3.11 24.40 1.47 26.20 0.36 7.80 51.50 82.10 246.30 <0.09 <0.006 2.70 <0.04 MR35.5R 13/02/01 4.43 2.3 0.8 22.80 4.1 4.08 22.30 0.33 19.30 0.27 14.30 43.40 47.10 141.30 <0.12 <0.004 5.19 0.03 MR35.5R 02/12/00 2.91 5.3 1.2 20.47 2.6 2.97 15.30 2.08 35.70 0.31 17.30 83.50 118.00 354.00 <0.14 <0.004 5.51 0.05 MR41.0R 18/06/99 4.91 0.2 2.4 11.50 4.2 4.86 0.33 0.42 4.56 0.10 1.55 6.70 9.70 29.10 <0.35 <0.005 4.44 <0.02 MR41.1R 18/06/99 3.94 2.6 1.9 10.04 3.5 4.68 0.60 0.52 4.45 0.11 1.59 6.81 10.19 30.57 <0.35 <0.005 4.64 <0.02 MR41.5L 18/06/99 4.37 0.4 4.6 10.11 2.4 4.05 0.16 1.07 8.29 0.21 1.91 12.05 21.58 64.73 <0.35 <0.005 4.89 <0.02 PC34.5L 18/06/99 3.86 0.4 6.6 8.47 2.2 3.70 0.18 1.21 6.83 0.17 2.81 10.31 23.81 71.42 <0.35 <0.005 4.42 <0.02 PC34.5L 12/02/01 3.47 1.1 1.4 23.80 2.1 3.37 2.80 1.58 12.40 0.36 7.71 21.30 35.50 106.50 <0.12 <0.004 4.36 0.06 PC34.6R 18/06/99 5.26 0.4 4.4 10.00 3.3 4.91 0.01 0.08 6.68 0.07 3.08 8.61 14.70 44.10 <0.35 <0.005 6.93 <0.02 PC34.7L 18/06/99 4.29 0.7 2.4 9.49 2.2 4.26 0.32 1.60 10.15 0.13 4.07 15.16 31.46 94.38 <0.35 <0.005 10.10 <0.02

Appendix B. Field data for Hastings River estuary drains.

HASTINGS RIVER ESTUARY DRAIN WATER QUALITY (PHYSICO-CHEMICAL PARAMETERS)

18-19/6/99 29-30/11/1999 1-2/12/2000 12-13/02/2001 Drain pH EC DO Temp pH EC DO Temp pH EC DO Temp pH EC DO Temp ID (dS m-1) (% Sat.) (oC) (dS m-1) (% Sat.) (oC) (dS m-1) (% Sat.) (oC) (dS m-1) (% Sat.) (oC)

CC38.4R 5.05 0.3 4.2 11.27 3.66 1.3 3.9 20.74 5.40 3.8 3.4 21.58 3.19 0.9 1.8 23.38 CC38.8R 6.14 0.5 6.3 11.36 6.11 1.2 5.8 20.66 6.20 4.1 3.1 20.74 6.23 2.2 1.7 23.34 CC39.1R 6.12 0.3 4.3 10.49 6.15 0.7 3.5 20.84 6.67 0.9 0.7 21.91 3.91 0.9 1.3 23.84 CC41.0R 6.06 0.5 5.8 11.24 6.40 1.0 0.1 17.46 6.64 1.6 0.0 19.85 6.45 2.1 0.2 23.08 CC42.5R 6.15 0.6 5.6 11.02 6.02 2.2 3.1 21.26 6.34 3.6 4.0 21.12 6.53 3.2 0.2 22.45 CC44.1R 5.73 0.4 0.3 10.95 4.11 1.9 5.0 20.74 3.98 2.8 4.1 21.51 3.58 1.0 0.6 22.50 CC44.8R 4.96 0.3 2.3 12.64 3.93 1.0 0.7 20.07 3.84 1.2 1.3 22.80 6.35 0.4 0.2 24.19 CC46.4R 5.56 0.5 1.4 11.36 6.24 3.6 3.5 21.66 5.55 8.7 1.4 23.01 6.15 0.9 0.7 23.79 FC10.1R 6.61 22.4 6.5 9.51 ------FC10.2R 6.74 9.9 7.7 9.49 ------FC10.4R 5.68 6.4 6.3 8.82 ------FC11.1R 6.02 1.6 6.9 8.52 ------FC11.3R 6.19 16.7 0.7 10.87 6.21 31.6 2.3 21.90 7.05 16.5 1.1 19.77 6.74 10.6 1.6 20.99 HR4.0R 7.64 26.0 6.4 9.58 7.09 40.7 - 20.77 7.24 39.6 0.9 23.81 HR7.7R 6.13 5.0 0.3 9.79 4.73 0.4 3.0 20.89 4.30 0.7 - 17.75 4.10 1.1 4.5 20.23 HR12.1R 5.85 17.7 7.1 11.91 6.94 28.2 6.8 18.23 6.05 21.0 - 23.21 5.25 15.5 6.4 23.42 HR16.0R 3.37 1.5 10.5 9.02 3.25 3.1 8.0 17.40 2.72 6.3 - 21.97 2.81 5.9 2.5 22.56 HR16.5R 3.37 2.7 10.7 9.75 3.55 11.4 6.1 17.48 4.75 11.8 - 21.88 3.23 8.5 3.1 22.74 HR16.6R - - - - 6.38 22.3 3.4 19.82 5.93 14.5 - 24.35 4.54 5.6 5.5 23.27 HR16.8R 3.35 2.2 10.9 9.42 6.61 27.8 0.9 22.36 6.13 13.0 - 23.91 3.48 8.8 3.0 22.37 LC5.7R 7.52 16.6 5.2 11.21 - - - - 7.09 37.0 - 18.25 - - - - LC5.9R 8.00 47.6 5.9 17.12 - - - - 7.20 38.1 - 20.07 - - - - MA29.6L 5.99 0.5 2.8 9.71 6.48 0.5 6.7 22.86 6.15 4.2 - 25.84 5.51 5.1 2.8 24.12 MR10.3L 7.19 20.0 10.2 12.00 - - - - 6.92 14.1 - 24.70 6.88 7.3 4.3 24.09 MR19.2R 5.62 0.1 8.5 10.94 7.50 7.4 7.4 22.29 6.62 8.6 3.8 21.28 6.63 1.8 5.7 24.23 MR20.1L ------6.59 8.7 - 22.41 5.87 7.9 3.1 23.21 MR21.4L - - - - 6.95 9.0 7.7 16.22 - - - - 6.52 1.5 1.1 21.96 MR21.7L 4.21 0.2 6.9 10.00 6.09 6.4 6.9 21.29 3.19 1.6 - 25.07 3.53 0.9 6.3 24.70 MR22.3R 6.08 1.1 2.2 11.39 6.73 9.6 3.1 19.94 7.01 3.2 1.9 19.69 6.77 1.7 3.5 23.17 MR22.4R 6.27 0.7 5.9 10.02 6.73 14.5 0.6 18.89 7.08 5.1 0.6 18.90 6.89 1.1 0.9 21.80 MR23.0R 6.13 0.9 9.2 10.42 - - - - 6.90 2.5 1.4 20.62 6.49 3.8 4.3 23.66 MR23.3R 5.92* 1.3* 7.4* 10.54* - - - - 6.53 5.8 3.3 19.10 6.45 5.6 3.6 23.26 MR23.6R 6.03 0.4 7.4 12.24 6.68 7.5 0.1 21.31 6.62 2.4 2.1 20.73 6.08 7.7 4.0 22.35 MR23.8R 6.38 0.9 2.5 11.36 7.19 2.9 8.4 21.40 6.82 3.3 4.7 21.86 4.98 4.4 6.9 23.64 MR24.2R 5.31 1.2 7.3 10.39 6.69 2.0 7.0 19.88 - - - - 3.83 3.2 4.8 23.53 MR30.8R 4.58 2.4 7.0 10.61 6.25 0.6 7.0 23.48 ------MR32.8L 5.99 0.5 8.3 8.44 4.23 0.8 3.5 20.14 5.14 1.4 - 23.55 6.35 3.2 3.3 23.34 MR33.8R(A) 4.58 0.1 3.8 9.91 4.05 0.6 3.1 19.42 3.22 3.1 2.4 21.05 3.06 1.9 2.6 23.80 MR33.8R(B) - - - - 3.66 0.5 2.8 21.08 2.77 2.4 2.9 22.15 2.91 1.6 3.2 24.71 MR39.5R 6.03 0.6 2.4 10.03 ------PC34.5L 3.86 0.4 6.6 8.47 3.16 1.6 2.3 19.49 3.01 1.3 - 24.54 3.47 1.1 1.4 23.80 PC34.6R 5.26 0.4 4.4 10.00 5.98 0.7 5.4 19.30 5.59 3.1 - 22.82 6.17 1.4 2.9 23.71 PC34.7L 4.29 0.7 2.4 9.49 5.75 1.0 3.0 18.22 3.89 1.3 - 22.73 4.07 1.1 1.1 24.17 TA28.7R 6.3* 0.3* 6.7* 11.06* ------WR29.3L ------5.76 3.0 4.9 23.73 WR29.5L ------6.76 0.3 5.7 23.42 WR29.7L ------5.27 1.1 8.5 26.09 WR30.6L ------6.30 0.6 2.1 22.95

* = Drain measurement collected upstream from floodgate

Appendix C. Estuary pH and EC transect data for the Hastings River following rainfall. HASTINGS RIVER ESTUARY CHANNEL TRANSECT DATA

18/06/99 29/11/99 Channel pH EC Channel pH EC ID (dS m-1) ID (dS m-1)

surface bed surface bed surface bed surface bed

HR5.6 7.92 8.12 33.2 49.7 HR5.6 7.98 8.26 29.0 51.0 HR6.8 7.91 8.05 30.1 40.6 HR6.8 7.98 8.16 25.3 43.6 HR9.5 7.83 8.08 28.3 42.1 HR9.5 7.89 8.07 22.8 41.0 HR11.37.747.9524.734.7 HR11.37.847.9821.835.7 HR12.87.707.9119.833.1 HR12.87.778.0218.639.3 HR15.5 7.69 7.69 11.8 21.3 HR15.5 7.76 7.88 7.0 30.1 HR17.5 6.92 6.99 9.1 11.4 HR17.5 7.67 7.66 4.5 23.8 MR11.57.057.0814.715.9 MR11.57.617.6827.828.4 MR13.07.077.0310.510.6 MR13.07.437.5022.923.0 MR15.0 7.02 6.92 4.7 5.6 MR15.0 7.44 7.42 15.3 15.6 MR17.0 6.90 6.78 1.2 1.3 MR17.0 7.42 7.40 13.6 13.9 MR19.4 6.66 6.50 0.3 0.4 MR19.4 7.37 7.28 6.5 8.2 MR21.1 6.50 6.42 0.2 0.2 MR21.1 7.41 7.22 4.1 4.8 MR23.0 6.38 6.30 0.3 0.3 MR23.0 6.80 6.81 2.0 2.7 MR25.3 6.45 6.38 0.2 0.2 MR25.3 7.10 7.06 1.0 1.0 MR27.0 6.34 6.31 0.3 0.3 MR27.0 7.13 7.08 0.6 0.8 MR30.0 5.92 5.88 0.3 0.3 MR29.1 6.72 6.72 0.5 0.5 MR31.0 5.87 5.81 0.3 0.3 MR31.0 6.49 6.44 0.6 0.6 MR33.3 5.96 5.85 0.3 0.3 MR33.3 6.28 6.08 0.6 0.7 MR35.5 5.80 5.81 0.3 0.3 MR35.0 5.74 5.80 0.9 0.9 MR37.3 5.79 5.76 0.3 0.3 MR37.3 5.77 5.80 1.0 1.0 MR38.0 5.90 5.89 0.4 0.4 MR38.7 5.55 5.53 0.8 0.8 MR40.0 5.01 4.97 0.2 0.2 MR41.6 4.97 4.92 0.2 0.2

1/12/00 12/02/01 Channel pH EC Channel pH EC ID (dS m-1)(ID dS m-1)

surface bed surface bed surface bed surface bed 12/02/01 HR5.6 7.74 7.98 17 43.6 HR4.9 7.71 8.15 20.4 44.7 HR6.8 7.67 8.01 12 47.4 HR6.8 7.7 8.21 18.5 48.6 HR9.5 7.6 7.98 16.7 41.1 HR8.0 7.59 8.25 16.5 49.3 HR11.3 7.42 7.75 8 29.4 HR9.5 7.58 8.11 14.7 36 HR12.8 7.44 7.9 8.2 38.6 HR11.3 7.43 7.96 8.7 29.7 HR15.5 7.32 7.38 2.7 9.9 HR12.8 7.35 7.92 5.3 26.5 HR17.5 7.32 7.47 1 19.1 HR15.5 7.27 7.29 1 2.2 MR11.5 7.08 7.1 11.4 12.3 HR17.5 7.26 7.24 0.3 0.3 MR13.0 6.98 6.97 7.8 8.2 MR11.5 6.87 6.92 4.5 5.6 MR15.0 6.85 6.83 4.6 4.7 MR13.0 6.72 6.71 1.7 1.7 MR17.0 6.86 6.82 2.8 2.8 MR15.0 6.63 6.62 0.9 0.9 MR19.4 6.85 6.79 1.9 1.9 MR17.0 6.56 6.55 0.6 0.6 MR21.1 6.78 6.75 1.8 1.8 MR19.4 6.51 6.5 0.5 0.5 MR23.0 6.5 6.56 1.6 1.7 MR21.1 6.51 6.49 0.4 0.4 MR25.3 6.56 6.58 1.3 1.3 MR23.0 6.4 6.41 0.5 0.5 MR27.0 6.49 6.51 1.7 1.7 MR25.3 6.45 6.47 0.4 0.4 MR29.1 6.15 6.14 4.1 4.1 MR27.0 6.39 6.38 0.6 0.6 MR31.0 5.95 5.94 4.3 4.3 MR29.1 5.8 5.81 1.1 1.1 MR33.3 5.88 5.77 4.2 4.3 MR30.0 5.58 5.58 1.1 1.1 MR35.0 5.95 5.93 4.2 4.2 MR32.0 5.35 5.35 1 1.1 MR37.3 6.01 6.02 3.4 3.7 MR33.3 5.3 5.1 1 1.2 MR38.7 6.01 6 2.5 2.5 MR35.0 5.43 5.41 1.2 1.2 MR39.0 6 5.99 2.5 2.5 MR37.3 5.49 5.48 0.8 0.9 MR39.0 5.65 5.64 0.5 0.5

Appendix D. Listing of field and analytical water quality data following high rainfall for Manning River estuary drains.

MANNING RIVER ESTUARY DRAIN WATER QUALITY

Drain Date pH EC DO Temp. Alk. Cl:SO4 LAB pH Fe Al Ca Mn K Mg S SO4 As Cu Si Zn -1 o -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 ID (dS m ) (% Sat.) ( C) (mg L )(mg L)(mg L)(mg L)(mg L)(mg L)(mg L)(mg L)(mg L)(mg L)(mg L)(mg L)(mg L)

CC15.7L 27/05/98 4.11 15.2 75.1 9.94 13 6.8 4.42 0.14 1.53 30.18 <0.002 29.37 88.12 105.16 315.47 <0.40 <0.004 2.55 <0.01 CC16.1L 09/05/99 3.55 1.1 49.9 16.52 NS 6.6 3.71 4.90 1.05 7.26 0.24 6.38 18.82 29.20 87.61 <0.35 <0.005 2.65 <0.02 CC16.1L 27/05/98 4.93 18.8 99.4 6.44 21 32.3 4.95 <0.01 0.27 38.30 <0.002 43.36 108.12 79.97 239.91 <0.40 <0.004 0.96 <0.01 CC16.5L 09/05/99 3.04 1.8 67.0 18.28 NS 2.1 3.22 9.82 17.71 20.81 2.13 7.89 40.75 114.27 342.81 <0.35 <0.005 12.83 <0.02 CC16.9L 27/05/98 3.87 10.9 108.0 5.18 8 24.3 4.31 <0.01 2.64 18.46 0.06 19.13 51.16 43.72 131.16 <0.40 <0.004 1.85 <0.01 DC22.9L 27/05/98 6.39 26.1 84.9 8.95 42 30.9 6.51 <0.01 <0.05 54.97 <0.002 58.41 150.81 95.19 285.57 <0.40 <0.004 0.49 <0.01 DC23.4L 27/05/98 6.22 25.2 87.4 8.62 38 12.6 6.43 <0.01 <0.05 121.08 0.27 113.16 328.64 222.16 666.48 <0.40 <0.004 1.23 <0.01 DC24.3R 27/05/98 5.75 23.5 96.8 7.05 26 25.4 5.92 <0.01 <0.05 58.19 0.10 56.10 156.51 103.68 311.04 <0.40 <0.004 1.09 <0.01 GG15.0R 09/05/99 3.46 1.5 33.2 18.36 NS 5.2 3.52 4.02 1.74 13.04 0.85 11.55 23.47 47.53 142.59 <0.35 <0.005 6.47 <0.02 GG15.4R 27/05/98 3.58 21.2 101.8 4.05 21 9.7 3.89 0.32 5.11 57.94 0.57 47.22 148.82 157.32 471.96 <0.40 <0.004 3.10 <0.01 GG15.5R* 27/05/98 3.21 22.3 110.5 4.56 8 7.8 3.56 2.17 11.28 134.84 1.41 105.29 340.24 287.35 862.06 <0.40 <0.004 6.76 <0.01 GG15.8L 09/05/99 3.31 11.0 46.5 18.98 NS 4.2 4.07 2.71 2.84 94.30 2.57 63.80 225.94 239.19 717.58 <0.35 <0.005 12.00 <0.02 GG16.6R* 27/05/98 3.45 15.4 121.5 7.94 8 5.2 3.76 2.82 32.57 151.32 4.03 78.27 306.58 329.72 989.16 <0.40 <0.004 16.07 <0.01 LR13.1L 09/05/99 3.22 10.2 27.4 16.42 NS 3.0 3.51 4.74 2.14 41.24 1.10 24.10 92.64 186.10 558.30 <0.35 <0.005 9.05 <0.02 LR15.1R 09/05/99 3.20 3.6 42.1 17.27 NS 3.1 3.32 38.40 3.38 15.13 1.00 7.86 33.48 98.39 295.17 <0.35 <0.005 8.29 <0.02 LR15.4L 09/05/99 3.07 7.6 143.5 24.19 NS 3.3 3.72 3.09 0.76 36.30 0.43 22.94 71.23 125.44 376.31 <0.35 <0.005 5.91 <0.02 LR15.4L 27/05/98 6.22 21.3 86.1 8.70 21 12.1 6.39 <0.01 <0.05 40.32 <0.002 40.21 112.74 98.48 295.45 <0.40 <0.004 0.23 <0.01 LR15.9R 09/05/99 2.97 9.3 64.6 18.92 NS 1.5 3.41 4.83 5.35 38.06 1.06 26.95 94.63 175.90 527.69 <0.35 <0.005 10.67 <0.02 LR16.1L 09/05/99 3.05 2.9 84.2 20.42 NS 7.3 3.39 2.07 2.34 14.28 0.59 12.14 24.56 43.16 129.48 <0.35 <0.005 4.14 <0.02 LR16.1L* 27/05/98 3.47 15.0 107.5 8.13 8 6.7 3.69 2.87 9.62 55.13 0.99 37.22 133.15 127.51 382.54 <0.40 <0.004 7.87 <0.01 LR16.6L 09/05/99 3.07 5.9 35.0 18.17 NS 5.5 3.41 2.63 4.06 29.57 0.98 25.11 61.10 81.86 245.59 <0.35 <0.005 7.69 <0.02 LR16.6L* 27/05/98 4.91 15.3 95.9 9.14 0 7.4 4.92 3.37 7.04 70.37 1.18 52.78 177.80 153.23 459.70 <0.40 <0.004 12.36 <0.01 LR18.7L 09/05/99 6.51 6.1 63.3 19.58 NS 6.1 6.30 0.01 <0.05 57.49 0.34 51.93 158.14 124.21 372.64 <0.35 <0.005 2.79 <0.02 SC14.3R* 27/05/98 6.40 25.6 102.8 9.38 30 12.6 5.45 <0.01 0.01 76.23 0.72 60.98 184.81 174.02 522.06 <0.40 <0.004 1.91 <0.01

* = Drain measurement collected upstream from floodgate

Appendix E. Manning River estuary pH and EC data for surface and bed waters.

MANNING RIVER ESTUARY CHANNEL TRANSECT DATA Channel Date pH EC ID (dS m-1)

surface bed surface bed

CC9.5 27/05/98 6.74 NS 28.0 NS CC11.6 27/05/98 6.20 6.92 20.6 30.1 CC14.0 27/05/98 5.23 6.41 15.9 31.3 CC15.1 27/05/98 4.89 5.02 17.2 18.0 DC22.5 27/05/98 6.30 NS 25.8 NS DC23.6 27/05/98 6.02 NS 24.6 NS DC25.0 27/05/98 4.74 NS 18.6 NS GG15.8 27/05/98 5.59 5.85 21.3 21.6 GG22.7 27/05/98 7.45 7.62 25.9 40.9 CC10.2 09/05/99 4.97 7.18 3.9 30.1 CC11.0 09/05/99 5.00 7.29 3.2 33.1 CC11.5 09/05/99 4.51 6.28 1.8 27.1 CC12.7 09/05/99 4.50 6.37 1.9 28.6 CC13.5 09/05/99 4.59 6.33 1.8 28.5 CC14.2 09/05/99 4.62 6.37 1.4 28.6 CC14.7 09/05/99 5.00 6.29 2.3 27.5 CC16.5 09/05/99 3.51 NS 0.7 NS CC9.4 09/05/99 5.02 7.78 4.2 37.3 DC21.5 09/05/99 6.16 6.24 17.2 19.8 DC22.0 09/05/99 4.95 7.05 13.0 32.5 DC22.8 09/05/99 4.46 6.71 13.1 32.1 DC23.5 09/05/99 3.95 6.38 12.1 29.5 DC23.9 09/05/99 4.16 5.99 11.5 22.8 DC24.5 09/05/99 4.04 5.02 9.4 16.7 GG14.2 09/05/99 6.36 6.61 12.7 24.0 GG15.1 09/05/99 6.00 6.16 11.3 16.1 GG16.1 09/05/99 5.88 6.27 13.7 21.0 GG16.7 09/05/99 6.09 6.16 14.0 15.6 GG16.8 09/05/99 6.15 6.15 13.4 14.4 GG18.6 09/05/99 6.35 6.42 16.9 17.0 GG19.7 09/05/99 6.48 6.55 17.6 19.1 GG21.1 09/05/99 5.31 6.12 15.7 18.2 GG21.5 09/05/99 6.16 6.24 17.2 19.8 GG22.7 09/05/99 7.55 7.61 16.2 33.2 LR10.5 09/05/99 6.51 7.60 18.2 32.1 LR11.2 09/05/99 6.43 7.63 17.2 35.4 LR12.5 09/05/99 6.46 7.62 15.4 36.1 LR14.0 09/05/99 6.23 7.60 11.4 37.7 LR15.0 09/05/99 6.01 7.53 10.1 36.5 LR15.7 09/05/99 6.05 7.43 8.7 35.5 LR16.4 09/05/99 6.26 7.24 10.3 34.7 LR18.2 09/05/99 6.52 7.19 9.9 34.1 LR18.9 09/05/99 6.45 7.19 9.7 33.1 LR19.9 09/05/99 6.97 7.01 9.0 31.6

NS = not sampled

Appendix F. Water quality data for Sites W, X, Y and Z for the period 19/2/99 to 18/8/00.

Site Surf. (s) Date Temp. EC DO % pH ORP Site Surf. (s) Date Temp. EC DO % pH ORP or Bed (b) (OC) (dS m-1) (% Sat.) (mV) or Bed (b) (OC) (dS m-1) (% Sat.) (mV)

W s 19/02/99 24.37 10.8 115.0 6.47 - W s 05/07/99 14.78 3.6 9.6 3.71 - W b 19/02/99 24.57 13.7 107.9 7.00 - W b 05/07/99 14.79 3.6 22.2 3.71 - X s 19/02/99 24.69 10.3 110.2 6.23 - X s 05/07/99 14.77 3.5 27.7 3.70 - X b 19/02/99 26.14 16.4 45.7 7.07 - X b 05/07/99 14.79 3.5 21.9 3.70 - Y s 19/02/99 25.30 11.0 102.6 6.45 - Y s 05/07/99 14.87 3.6 52.1 3.71 - Y b 19/02/99 25.92 15.5 65.0 7.06 - Y b 05/07/99 14.86 4.2 1.3 5.62 - Z s 19/02/99 25.85 8.7 106.1 3.98 - Z s 05/07/99 13.75 2.8 15.0 3.71 - Z b 19/02/99 24.81 12.0 91.9 6.66 - Z b 05/07/99 14.56 5.5 17.4 3.97 -

W s 09/03/99 24.55 16.7 102.2 7.41 - W s 03/08/99 12.71 4.2 67.1 4.26 - W b 09/03/99 24.58 16.8 101.8 7.59 - W b 03/08/99 12.67 4.2 66.7 4.25 - X s 09/03/99 24.43 14.6 76.1 5.90 - X s 03/08/99 12.66 4.2 67.1 4.27 - X b 09/03/99 24.50 15.7 84.4 6.36 - X b 03/08/99 12.63 4.2 69.2 4.24 - Y s 09/03/99 24.09 7.8 55.0 3.41 - Y s 03/08/99 12.71 4.1 88.4 4.42 - Y b 09/03/99 24.32 14.5 63.4 5.92 - Y b 03/08/99 12.47 5.2 68.1 5.42 - Z s 09/03/99 23.56 4.4 46.2 3.44 - Z s 03/08/99 12.09 3.7 83.5 4.22 - Z b 09/03/99 22.84 10.3 - 5.87 - Z b 03/08/99 12.48 4.9 77.0 4.18 -

W s 14/04/99 22.70 1.8 19.9 5.45 - W s 24/08/99 18.74 20.5 98.5 6.50 - W b 14/04/99 22.72 1.8 0.0 5.43 - W b 24/08/99 18.60 20.8 98.7 6.49 - X s 14/04/99 22.72 1.8 4.1 5.44 - X s 24/08/99 18.87 20.3 97.0 6.30 - X b 14/04/99 22.71 1.7 0.0 5.43 - X b 24/08/99 18.34 21.3 104.0 6.42 - Y s 14/04/99 22.73 1.8 17.6 5.46 - Y s 24/08/99 19.37 19.5 99.7 5.98 - Y b 14/04/99 22.75 1.7 0.0 5.49 - Y b 24/08/99 18.22 21.6 112.8 6.34 - Z s 14/04/99 23.56 1.8 71.8 5.61 - Z s 24/08/99 19.20 8.4 103.2 3.32 - Z b 14/04/99 22.54 2.7 36.2 5.50 - Z b 24/08/99 19.52 20.5 - 4.71 -

W s 05/05/99 19.47 22.9 97.5 6.38 - W s 10/09/99 18.13 19.2 83.2 5.49 - W b 05/05/99 19.71 23.6 100.3 6.95 - W b 10/09/99 18.23 19.8 83.2 5.55 - X s 05/05/99 18.63 19.8 93.7 5.49 - X s 10/09/99 18.06 18.2 82.8 5.19 - X b 05/05/99 18.97 20.8 93.5 5.76 - X b 10/09/99 18.10 19.0 83.4 5.25 - Y s 05/05/99 17.81 14.4 84.6 3.75 - Y s 10/09/99 17.97 17.5 83.8 5.04 - Y b 05/05/99 19.03 20.4 75.6 5.99 - Y b 10/09/99 18.68 24.4 87.5 6.34 - Z s 05/05/99 17.00 12.8 74.4 3.40 - Z s 10/09/99 16.89 8.3 99.8 3.16 - Z b 05/05/99 17.51 19.2 33.9 4.43 - Z b 10/09/99 18.42 18.1 9.0 4.93 -

W s 04/06/99 18.51 23.6 81.7 5.09 - W s 30/09/99 18.91 22.6 87.6 7.54 210 W b 04/06/99 18.61 27.2 76.3 5.72 - W b 30/09/99 19.03 23.7 78.1 7.37 208 X s 04/06/99 17.02 11.5 84.6 3.61 - X s 30/09/99 18.77 22.7 88.1 7.54 205 X b 04/06/99 18.36 27.4 85.7 5.78 - X b 30/09/99 20.01 25.1 56.1 7.00 206 Y s 04/06/99 17.15 13.0 79.3 3.61 - Y s 30/09/99 20.52 25.3 59.1 6.94 210 Y b 04/06/99 18.86 26.9 67.1 5.70 - Y b 30/09/99 21.71 27.9 50.4 6.91 186 Z s 04/06/99 16.50 4.9 83.7 3.48 - Z s 30/09/99 20.14 19.9 78.8 3.85 357 Z b 04/06/99 18.87 24.6 45.8 5.49 - Z b 30/09/99 18.89 20.3 29.3 5.68 -17

Appendix F. (Continued).

Site Surf. (s) Date Temp. EC DO % pH ORP Site Surf. (s) Date Temp. EC DO % pH ORP or Bed (b) (OC) (dS m-1) (% Sat.) (mV) or Bed (b) (OC) (dS m-1) (% Sat.) (mV)

W s 05/11/99 24.06 15.6 112.0 7.06 161 W s 07/05/00 19.62 22.3 90.6 6.55 102 W b 05/11/99 24.10 15.6 113.7 7.08 160 W b 07/05/00 20.31 24.7 78.4 7.41 114 X s 05/11/99 24.02 15.6 104.7 6.87 169 X s 07/05/00 16.47 15.8 115.9 4.21 329 X b 05/11/99 24.17 15.5 114.6 6.93 167 X b 07/05/00 19.96 23.8 84.8 6.71 229 Y s 05/11/99 24.23 14.9 107.1 7.09 126 Y s 07/05/00 17.61 16.5 127.6 5.20 274 Y b 05/11/99 23.40 15.9 106.5 7.20 122 Y b 07/05/00 20.43 23.3 86.7 6.56 202 Z s 05/11/99 25.30 7.1 97.1 3.16 457 Z s 07/05/00 19.89 15.4 88.1 3.84 457 Z b 05/11/99 23.97 8.0 95.6 3.81 438 Z b 07/05/00 22.34 19.8 72.1 5.70 317

W s 14/12/99 22.35 14.4 71.5 7.47 121 W s 30/05/00 12.76 30.6 99.4 7.98 80 W b 14/12/99 22.53 14.6 72.6 7.57 117 W b 30/05/00 13.05 32.0 98.4 8.08 82 X s 14/12/99 22.49 14.6 73.8 7.63 116 X s 30/05/00 12.30 29.3 98.7 7.84 85 X b 14/12/99 22.58 14.7 74.4 7.65 116 X b 30/05/00 14.16 34.0 102.7 8.23 81 Y s 14/12/99 22.36 13.6 64.6 6.94 109 Y s 30/05/00 12.45 29.6 100.1 7.85 79 Y b 14/12/99 23.00 14.5 56.0 7.12 90 Y b 30/05/00 14.33 31.8 105.5 8.30 81 Z s 14/12/99 21.80 9.5 60.7 5.75 127 Z s 30/05/00 13.46 27.4 80.9 7.25 66 Z b 14/12/99 20.98 9.5 60.2 5.65 123 Z b 30/05/00 14.05 29.9 68.3 7.30 68

W s 26/01/00 22.42 24.7 59.3 7.36 96 W s 20/06/00 15.64 27.8 97.5 5.77 180 W b 26/01/00 22.44 24.7 59.5 7.35 95 W b 20/06/00 15.59 28.3 97.6 5.83 174 X s 26/01/00 22.44 24.6 62.2 7.35 93 X s 20/06/00 15.93 23.1 90.7 4.95 203 X b 26/01/00 22.44 24.6 59.2 7.35 92 X b 20/06/00 15.51 29.7 101.7 6.02 193 Y s 26/01/00 22.42 24.4 51.6 7.24 82 Y s 20/06/00 16.09 17.1 69.6 4.86 208 Y b 26/01/00 23.55 25.1 47.9 7.27 67 Y b 20/06/00 16.03 32.3 131.3 6.62 191 Z s 26/01/00 21.34 20.5 34.0 6.80 124 Z s 20/06/00 17.11 17.8 138.9 3.14 464 Z b 26/01/00 21.22 20.3 28.6 6.78 93 Z b 20/06/00 14.78 27.0 65.7 5.52 318

W s 10/03/00 21.41 1.1 0.4 6.14 22 W s 10/07/00 16.26 27.5 123.8 5.80 144 W b 10/03/00 21.42 1.1 -0.1 6.10 18 W b 10/07/00 16.23 27.5 124.3 5.87 145 X s 10/03/00 21.41 1.1 23.8 6.13 26 X s 10/07/00 16.35 26.9 128.1 5.59 170 X b 10/03/00 21.41 1.1 11.9 6.11 21 X b 10/07/00 16.34 27.5 126.8 5.79 165 Y s 10/03/00 21.42 1.2 -0.2 6.15 31 Y s 10/07/00 16.45 23.9 119.4 5.12 290 Y b 10/03/00 21.38 1.1 24.3 6.10 34 Y b 10/07/00 16.00 30.2 103.4 6.09 263 Z s 10/03/00 21.63 1.1 3.5 6.23 12 Z s 10/07/00 17.11 17.0 148.6 2.94 489 Z b 10/03/00 21.79 0.8 2.8 6.09 34 Z b 10/07/00 16.10 26.3 182.9 4.95 356

W s 31/03/00 23.65 3.6 44.4 6.22 82 W s 18/08/00 14.88 31.6 99.1 6.60 85 W b 31/03/00 23.38 3.6 36.4 6.19 86 W b 18/08/00 15.18 31.7 101.8 7.06 78 X s 31/03/00 24.93 3.1 57.9 6.30 99 X s 18/08/00 14.79 31.0 98.8 6.78 74 X b 31/03/00 23.36 3.5 45.6 6.22 96 X b 18/08/00 15.28 32.7 99.6 6.98 77 Y s 31/03/00 24.55 3.3 78.3 6.57 123 Y s 18/08/00 14.82 31.2 98.1 6.90 75 Y b 31/03/00 23.01 4.3 1.3 6.00 22 Y b 18/08/00 15.72 34.4 102.6 7.47 76 Z s 31/03/00 28.26 4.4 4.1 5.99 28 Z s 18/08/00 16.02 24.1 142.3 4.91 231 Z b 31/03/00 28.11 7.0 29.4 3.79 410 Z b 18/08/00 14.20 28.2 94.0 5.48 203

W s 19/04/00 22.02 18.2 - 6.55 96 W b 19/04/00 22.26 20.7 - 6.98 108 X s 19/04/00 21.74 15.5 - 6.01 128 X b 19/04/00 22.26 20.8 - 6.93 127 Y s 19/04/00 21.61 15.3 - 6.09 134 Y b 19/04/00 22.15 17.2 - 6.99 -94 Z s 19/04/00 22.34 12.5 - 3.91 407 Z b 19/04/00 22.82 16.2 - 5.79 132

Appendix G. S&GE water quality data for Sites 1 to 7.

SITE: 1

DATE pH DO EC Temp Alkalinity NO2 - N PO4 - P NH3 - N NO3 -N Cl:SO4 Al As Ca Cu Fe K Mg Mn Na S Si Zn (% Sat.) (dS m-1)(OC) (mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1) (mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)

01/06/99 8.04 121.9 44.4 19.91 73 0.000 0.26 0.19 0.085 11.4 <0.05 <0.35 298.12 <0.005 <0.007 341.81 862.23 <0.001 sat. 603.16 1.29 <0.02 15/06/99 7.41 101.2 41.4 14.52 85 0.000 0.12 0.63 0.040 11.5 <0.05 <0.35 241.15 <0.005 <0.007 274.89 692.64 <0.001 sat. 483.72 1.27 <0.02 28/06/99 8.25 95.3 21.0 18.05 76 0.000 0.46 0.15 0.076 38.3 <0.07 <0.17 110.69 0.01 <0.01 114.52 318.77 0.03 sat. 194.11 2.70 <0.16 12/07/99 7.69 102.1 24.9 17.31 65 0.004 0.54 0.38 0.040 2.6 <0.07 <0.17 137.43 0.05 <0.01 155.46 403.19 0.05 148.54 286.95 2.34 <0.16 26/07/99 8.74 129.9 14.5 15.77 65 0.014 0.28 0.30 0.120 10.0 <0.04 <0.11 105.49 <0.007 <0.17 131.79 307.19 <0.002 sat. 247.25 3.56 <0.02 09/08/99 8.80 136.7 32.3 17.96 73 0.004 0.16 0.03 0.250 11.0 <0.04 <0.11 127.89 <0.007 <0.17 181.34 389.72 0.04 sat. 316.37 1.59 <0.02 23/08/99 8.02 117.8 30.4 18.67 60 0.003 0.06 0.36 0.055 13.5 <0.04 <0.11 145.63 <0.007 <0.17 190.43 444.92 0.00 sat. 371.09 2.22 <0.02 06/09/99 7.68 68.8 43.2 18.66 65 0.000 0.12 0.25 0.180 11.0 <0.04 <0.11 201.47 <0.007 <0.17 256.67 561.25 0.06 sat. 527.53 1.65 <0.02 01/10/99 8.00 95.1 30.9 18.69 80 0.000 0.00 0.11 0.200 11.4 <0.04 <0.11 193.75 <0.007 <0.17 238.42 574.23 0.03 sat. 529.37 1.77 <0.02 15/11/99 7.53 45.2 17.0 20.26 76 0.000 0.10 0.04 0.470 18.3 0.07 <0.12 112.26 <0.06 0.03 119.22 344.04 0.01 sat. 264.77 4.83 <0.11 10/01/00 7.93 78.0 34.1 22.62 80 0.003 0.10 0.03 0.190 2.6 <0.02 <0.09 302.000 <0.006 <0.04 414.000 506.000 0.159 sat. 998.000 1.200 <0.04

SITE: 2

01/06/99 7.92 115.9 31.4 19.14 73 0.003 0.26 0.26 0.085 12.5 <0.05 <0.35 178.97 <0.005 <0.007 196.40 519.49 0.01 sat. 361.72 2.33 <0.02 15/06/99 - - - - 80 0.004 0.16 0.11 0.110 14.5 <0.05 <0.35 158.82 <0.005 <0.007 179.72 462.73 0.02 sat. 308.21 1.84 <0.02 28/06/99 7.29 91.2 13.4 16.59 76 0.007 1.09 0.23 0.080 6.2 <0.07 <0.17 102.60 0.01 <0.01 105.65 291.62 0.04 sat. 175.51 3.19 <0.16 12/07/99 7.21 119.1 16.2 17.22 56 0.025 0.40 0.71 0.072 2.9 <0.07 <0.17 82.51 0.02 <0.01 85.34 237.00 0.05 sat. 161.71 3.41 <0.16 26/07/99 8.27 129.2 7.8 14.99 52 0.011 0.38 0.16 0.012 6.0 0.10 <0.11 83.39 0.01 <0.17 100.39 228.95 0.02 sat. 175.57 6.45 <0.02 09/08/99 8.65 136.7 24.4 17.38 76 0.004 0.14 0.02 0.080 7.8 <0.04 <0.11 211.23 0.01 <0.17 289.63 632.80 0.08 sat. 521.63 4.41 <0.02 23/08/99 7.99 123.5 21.5 18.36 76 0.001 0.20 0.19 0.000 12.7 <0.04 <0.11 137.20 <0.007 <0.17 172.45 400.30 0.03 sat. 340.24 2.72 <0.02 06/09/99 7.63 113.5 21.5 20.49 65 0.000 0.22 0.47 0.350 7.0 <0.04 <0.11 255.15 <0.007 <0.17 324.30 732.09 0.08 sat. 693.84 3.09 <0.02 01/10/99 7.93 106.0 15.9 20.46 65 0.003 0.44 0.06 0.350 9.2 <0.04 <0.11 101.55 <0.007 <0.17 115.82 295.30 0.02 sat. 352.30 3.31 <0.02 15/11/99 7.53 87.5 5.9 20.18 60 0.003 0.14 0.63 1.000 26.4 0.06 <0.12 48.34 <0.06 0.02 46.07 139.05 0.06 1069.93 99.66 6.24 0.04 10/01/00 7.67 77.0 29.1 23.00 90 0.004 0.14 0.04 0.220 2.8 0.040 <0.09 159.000 <0.006 <0.04 222.000 360.000 0.072 2350.000 690.000 0.979 <0.04

Appendix G. (Continued).

SITE: 3

01/06/99 7.89 114.2 40.0 19.31 65 0.027 0.28 0.17 0.018 13.5 <0.05 <0.35 215.35 <0.005 <0.007 247.17 629.44 <0.001 sat. 434.03 1.37 <0.02 15/06/99 8.14 141.4 39.7 15.66 80 0.006 0.07 0.44 0.059 18.8 <0.05 <0.35 147.39 <0.005 <0.007 171.42 430.11 0.01 sat. 329.92 0.81 <0.02 28/06/99 8.12 94.9 19.7 17.53 65 0.003 0.16 0.11 0.068 14.6 <0.07 <0.17 105.27 0.00 <0.01 109.94 301.80 0.04 sat. 176.68 2.72 <0.16 12/07/99 7.95 140.4 25.7 18.00 73 0.004 0.18 0.11 0.072 1.8 <0.07 <0.17 155.12 0.01 <0.01 168.62 453.78 0.05 sat. 299.71 3.16 <0.16 26/07/99 8.61 126.6 13.8 15.62 65 0.016 0.38 0.11 0.150 6.4 0.06 <0.11 139.90 <0.007 <0.17 187.66 406.44 <0.002 sat. 321.20 6.61 <0.02 09/08/99 9.04 174.1 35.4 19.48 73 0.003 0.14 0.02 0.080 16.1 <0.04 <0.11 164.83 <0.007 <0.17 230.40 479.16 <0.002 sat. 393.59 1.67 <0.02 23/08/99 7.93 109.7 28.0 18.03 80 0.000 0.14 0.11 0.170 8.7 <0.04 <0.11 262.52 <0.007 <0.17 350.52 768.11 0.04 sat. 651.25 3.19 <0.02 06/09/99 7.64 78.9 38.6 18.82 60 0.000 0.20 0.05 0.063 9.5 <0.04 <0.11 244.88 <0.007 <0.17 319.04 711.07 <0.002 sat. 640.66 1.97 <0.02 01/10/99 7.78 71.1 32.0 18.43 65 0.000 0.10 0.03 0.072 9.3 <0.04 <0.11 204.85 <0.007 <0.17 260.25 578.89 0.19 sat. 583.20 1.53 <0.02 15/11/99 7.59 62.3 14.2 18.83 80 0.001 0.12 0.00 0.500 19.2 0.03 <0.12 92.53 <0.06 0.02 98.51 283.22 0.04 0.00 206.02 4.58 <0.11 10/01/00 7.88 75.6 34.7 22.75 73 0.006 0.12 0.03 0.150 8.9 <0.02 <0.09 267.000 <0.006 0.042 402.000 470.000 0.121 2530.000 543.000 1.010 <0.04

SITE: 4

05/05/99 6.38 97.5 22.9 19.47 - - - - - 3.5 <0.05 <0.35 132.36 <0.005 <0.007 116.44 351.58 1.03 sat. 287.02 1.04 <0.02 01/06/99 7.51 99.2 29.5 18.62 85 0.006 0.30 0.19 0.085 7.2 <0.05 <0.35 209.71 <0.005 <0.007 230.36 615.81 0.02 sat. 420.67 2.83 <0.02 04/06/99 5.09 81.7 23.6 18.51 - - - - - 2.2 2.04 <0.35 133.17 <0.005 0.32 103.48 333.85 1.67 sat. 383.95 8.53 <0.02 15/06/99 7.58 96.2 32.3 13.75 76 0.000 0.20 0.57 0.043 11.4 <0.05 <0.35 132.86 <0.005 <0.007 151.06 385.51 0.03 sat. 259.36 1.46 <0.02 28/06/99 5.52 46.9 1.6 15.86 21 0.007 0.54 0.51 0.089 0.4 0.15 <0.17 39.79 0.01 <0.01 19.70 53.47 1.17 256.32 74.97 0.45 <0.16 05/07/99 3.71 9.6 3.6 14.78 - - - - - 0.1 5.09 <0.17 70.28 0.01 0.28 20.51 96.51 2.61 445.53 157.82 11.77 0.09 12/07/99 5.28 43.3 1.8 17.10 0 0.006 0.38 1.00 0.032 0.3 <0.07 <0.17 46.78 0.01 <0.01 26.73 75.49 1.41 422.81 100.19 1.09 0.05 26/07/99 4.03 66.0 5.2 14.59 8 0.032 0.34 0.45 0.028 2.1 9.71 <0.11 169.12 0.04 3.95 70.59 275.43 8.34 1327.57 480.36 26.64 0.26 03/08/99 4.26 67.1 4.2 12.71 26 4.9 2.41 <0.12 76.16 <0.06 1.75 22.33 104.82 2.67 527.13 225.32 8.01 0.03 09/08/99 3.51 101.4 10.7 17.84 26 0.001 0.26 0.06 0.055 3.4 9.95 <0.11 216.45 <0.007 25.95 137.58 446.73 6.00 sat. 659.12 29.30 0.22 23/08/99 7.63 113.2 22.6 17.86 65 0.001 1.30 0.00 0.000 8.9 <0.04 <0.11 222.76 <0.007 <0.17 280.51 622.97 0.11 sat. 539.58 4.08 <0.02 24/08/99 6.49 98.7 20.8 18.60 - - - - - 6.1 <0.04 <0.11 155.68 <0.007 <0.17 165.76 428.62 0.61 sat. 600.31 2.84 <0.02 06/09/99 4.93 110.1 15.9 20.68 13 0.000 0.07 0.03 - 10.2 1.60 <0.11 123.31 <0.007 1.04 101.20 297.03 1.95 sat. 445.87 5.61 <0.02 10/09/99 5.49 83.2 19.2 18.13 42 - - - - 11.1 0.15 <0.12 134.62 <0.06 0.17 116.07 376.24 1.60 sat. 382.23 1.64 <0.11 30/09/99 7.54 87.6 22.6 18.91 73 - - - - 14.5 0.04 <0.12 165.48 <0.06 <0.01 172.27 498.56 0.31 sat. 400.74 2.25 0.10 01/10/99 7.39 61.2 18.9 19.91 60 0.001 0.30 0.06 0.130 11.0 <0.04 <0.11 91.24 <0.007 <0.17 97.91 257.92 0.17 sat. 310.57 2.24 <0.02 05/11/99 7.06 112.0 15.6 24.06 80 - - - - 19.4 0.04 <0.12 114.40 <0.06 <0.01 116.58 341.60 0.05 sat. 248.05 5.47 <0.11 15/11/99 6.70 77.2 6.8 20.22 42 0.001 0.06 0.00 0.600 17.2 0.06 <0.12 62.26 <0.06 0.02 51.17 157.72 0.74 1124.95 152.92 4.55 <0.11 14/12/99 7.47 71.5 14.4 22.35 - - - - - 8.1 <0.02 <0.09 197.000 <0.006 <0.04 263.000 409.000 0.114 2430.00 397.000 1.140 <0.04 10/01/00 7.81 78.1 27.9 23.21 76 0.001 0.16 0.03 0.110 2.3 <0.02 <0.09 174.000 <0.006 <0.04 237.000 374.000 0.003 2330.00 776.000 1.580 <0.04 26/01/00 7.36 59.3 24.7 22.42 76 - - - - 8.3 <0.02 <0.09 165.000 <0.006 0.060 215.000 371.000 0.005 sat. 377.000 0.919 <0.04

Appendix G. (Continued).

SITE: 5

01/06/99 5.21 71.5 4.1 15.48 8 0.003 0.06 0.10 0.009 14.3 0.24 <0.35 28.62 <0.005 0.72 30.29 82.55 0.23 700.98 67.65 3.97 <0.02 15/06/99 4.93 62.9 3.5 12.23 65 0.001 0.02 0.30 0.015 24.0 0.51 <0.35 13.15 <0.005 0.62 16.94 38.09 0.11 338.58 30.71 2.36 <0.02 28/06/99 5.54 33.5 1.4 15.23 0 0.003 0.12 0.06 0.043 0.6 <0.07 <0.17 11.17 0.01 <0.01 11.66 28.36 0.14 243.60 20.78 0.53 <0.16 12/07/99 4.73 69.9 1.4 15.73 8 0.001 0.08 0.10 0.059 0.5 0.08 <0.17 5.56 0.01 <0.01 6.20 14.03 0.11 108.47 12.30 1.37 <0.16 26/07/99 4.45 98.3 0.3 14.13 0 0.007 0.46 0.03 0.006 2.9 2.02 <0.11 9.45 0.06 0.42 6.45 18.39 0.45 128.41 32.95 4.27 0.08 09/08/99 5.47 104.5 3.3 16.46 26 0.000 0.26 0.26 0.160 18.1 0.36 <0.11 16.44 0.01 0.47 11.63 44.34 0.24 333.93 52.41 1.36 <0.02 23/08/99 5.85 85.9 3.9 17.40 13 0.001 3.20 0.00 0.032 15.3 <0.04 <0.11 22.98 <0.007 <0.17 20.75 69.22 0.14 570.89 86.44 0.54 <0.02 06/09/99 6.06 96.3 6.7 18.45 13 0.000 0.10 0.02 0.170 26.1 <0.04 <0.11 19.61 <0.007 <0.17 17.42 58.33 0.12 515.68 87.81 0.27 <0.02 01/10/99 7.36 79.1 26.8 19.87 56 0.000 0.12 0.05 0.120 9.4 <0.04 <0.11 169.58 <0.007 <0.17 202.17 493.60 0.13 sat. 495.73 1.52 <0.02 15/11/99 4.73 37.7 1.4 18.95 8 0.001 0.08 0.06 0.089 20.3 1.09 <0.12 11.89 <0.06 0.09 11.14 33.43 0.29 247.44 35.64 5.40 0.19 10/01/00 6.49 60.5 21.4 22.01 56 0.000 0.02 0.22 0.120 9.7 0.047 <0.09 146.000 <0.006 <0.04 198.000 356.000 0.261 2020.000 293.000 0.951 <0.04

SITE: 6

01/06/99 6.93 81.3 17.4 18.62 52 0.011 0.12 0.23 0.063 10.6 <0.05 <0.35 121.01 <0.005 <0.007 121.46 348.35 0.23 sat. 246.86 3.85 <0.02 15/06/99 7.01 84.4 21.7 14.73 26 0.006 0.12 0.34 0.240 7.9 <0.05 <0.35 143.22 <0.005 <0.007 149.50 412.01 0.16 sat. 293.86 3.90 <0.02 28/06/99 6.30 66.8 4.2 15.13 8 0.009 0.22 0.63 0.059 0.8 <0.07 <0.17 29.48 0.01 <0.01 27.79 78.25 0.15 652.03 48.76 1.14 <0.16 12/07/99 5.51 82.8 6.5 15.98 21 0.000 0.08 0.00 0.047 9.7 <0.07 <0.17 45.69 0.00 <0.01 41.59 123.92 0.38 987.99 92.37 1.18 <0.16 26/07/99 6.39 116.2 2.3 14.56 21 0.006 0.64 0.14 0.068 5.9 <0.04 <0.11 16.37 0.02 <0.17 14.25 41.79 0.23 355.28 49.86 0.90 <0.02 09/08/99 6.89 103.5 11.2 16.77 52 0.004 0.14 0.30 0.110 6.2 <0.04 <0.11 136.46 0.01 <0.17 155.56 381.26 0.61 sat. 343.40 3.24 <0.02 23/08/99 6.84 108.9 19.3 17.83 52 0.003 0.26 0.06 0.700 13.6 <0.04 <0.11 108.15 <0.007 <0.17 123.45 318.68 0.20 sat. 294.39 1.38 <0.02 06/09/99 6.23 90.9 12.9 20.66 30 0.000 0.12 0.06 0.170 7.9 <0.04 <0.11 153.33 <0.007 <0.17 173.72 438.85 0.50 sat. 411.69 1.36 <0.02 01/10/99 6.99 76.7 15.7 19.87 60 0.004 0.07 0.05 0.170 9.9 <0.04 <0.11 134.19 <0.007 <0.17 152.44 387.54 0.22 sat. 358.68 3.98 <0.02 15/11/99 6.25 49.9 5.8 21.44 26 0.001 0.14 0.06 0.580 16.5 0.08 <0.12 42.56 <0.06 <0.01 39.22 121.17 0.41 905.05 103.03 3.01 <0.11 10/01/00 6.97 56.3 14.5 22.65 60 0.004 0.12 0.57 0.250 11.8 <0.02 <0.09 86.000 <0.006 <0.04 106.000 233.000 0.139 1590.000 185.000 0.642 <0.04

Appendix G. (Continued).

SITE: 7

01/06/99 7.78 111.5 33.8 18.59 65 0.001 0.12 0.26 0.059 14.1 <0.05 <0.35 238.45 <0.005 <0.007 261.72 694.85 0.03 sat. 472.92 2.42 <0.02 15/06/99 6.65 92.4 16.0 13.15 52 0.003 0.04 0.29 0.140 9.6 <0.05 <0.35 114.56 <0.005 <0.007 119.35 336.79 0.14 sat. 228.41 2.98 <0.02 28/06/99 6.35 52.3 2.7 16.24 8 0.009 0.20 0.14 0.330 14.6 <0.07 <0.17 20.60 0.01 <0.01 21.98 57.47 0.10 504.08 31.77 0.54 <0.16 12/07/99 5.27 81.0 5.0 16.09 21 0.000 0.08 0.06 0.024 0.8 <0.07 <0.17 28.53 0.01 <0.01 28.22 81.43 0.21 669.29 58.72 2.23 <0.16 26/07/99 5.64 119.7 1.9 14.93 0 0.003 0.22 0.06 0.015 3.8 0.14 <0.11 19.69 0.01 <0.17 18.55 54.30 0.22 449.63 54.63 0.44 <0.02 09/08/99 7.30 145.3 8.0 19.31 42 0.004 0.26 0.00 0.130 8.6 <0.04 <0.11 76.38 <0.007 <0.17 87.72 222.48 0.22 sat. 201.27 1.55 <0.02 23/08/99 7.90 115.5 25.6 17.60 80 0.003 0.20 0.12 0.094 8.5 <0.04 <0.11 248.12 <0.007 <0.17 320.11 729.43 0.08 sat. 623.75 3.26 <0.02 06/09/99 7.33 121.9 23.2 21.01 60 0.000 0.24 0.04 0.260 8.0 <0.04 <0.11 216.96 <0.007 <0.17 265.87 599.36 0.11 sat. 576.54 1.59 <0.02 01/10/99 7.63 75.1 24.8 19.43 73 0.001 0.12 0.03 0.240 9.6 <0.04 <0.11 166.43 <0.007 <0.17 198.75 476.33 0.05 sat. 465.63 1.96 <0.02 15/11/99 6.11 63.8 4.4 18.64 26 0.000 0.06 0.00 0.380 21.2 0.14 <0.12 28.43 <0.06 0.00 26.84 85.16 0.22 662.47 69.64 3.23 0.07 10/01/00 7.65 75.4 28.3 22.31 85 0.003 0.10 0.14 0.160 5.6 <0.02 <0.09 195.000 <0.006 <0.04 290.000 400.000 0.019 2320.000 419.000 1.720 <0.04

Appendix H. Tables of field and analytical water quality data collected during the CIE.

DATE SITE pH EC DO Temp Alkalinity NH3 - N NO2 - N NO3 -N PO4 - P (dS m-1) (% Sat.) (OC) (mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)

01/02/00 1 8.18 44.6 102.6 22.88 103 0.61 0.004 0.14 0.42

01/02/00 2 8.02 32.7 94.4 23.47 76 0 0.003 0.28 0.2

01/02/00 3 8.29 51.7 117.4 22.73 73 0.09 0.003 0.42 0.14

01/02/00 4 7.93 28.7 93.3 23.21 76 0.13 0 0.63 0.22

01/02/00 6 7.46 23.9 87 23.74 65 0.2 0 0.072 0.2

01/02/00 7 7.96 33.4 89.7 23.41 76 0.29 0 0.27 0.18

10/03/00 4 6.14 1.1 0.4 21.41 13 NS NS NS NS

31/03/00 4 6.22 3.6 44.4 23.65 73 NS NS NS NS

06/04/00 1 8.02 34.2 89.4 22 76 0.19 0.009 0.17 0.26

06/04/00 2 7.84 19.7 80.9 22.26 73 0.16 0.011 0.072 0.2

06/04/00 3 8.08 34.6 94.8 22.2 80 0.16 0.006 0.063 0.72

06/04/00 4 7.84 20.9 76.7 22.49 76 0.08 0.007 0.53 0.32

06/04/00 6 6.88 13 56.5 22.35 60 0.3 0.007 0.055 0.18

06/04/00 7 7.36 20 73.7 22.43 60 0.28 0.006 0.1 0.24

19/04/00 4 6.55 18.2 NS 22.02 NS NS NS NS NS

07/05/00 4 6.55 22.3 90.6 19.62 NS NS NS NS NS

30/05/00 4 7.98 30.6 99.4 12.76 NS NS NS NS NS

09/06/00 1 8.12 45.4 82.4 14.06 95 0.19 0.004 0.18 0.22

09/06/00 2 8.05 34.4 95.6 13.15 80 0.2 0.003 0.12 0.18

09/06/00 3 8.18 45.8 84.4 14.39 85 0.3 0.004 0.072 0.2

09/06/00 4 7.93 34.6 88.1 12.33 65 0.11 0.001 0.032 0.24

09/06/00 6 7.65 35.1 84.7 12.59 73 1 0 0.1 0.14

09/06/00 7 6.76 30.6 76.2 11.45 47 0.13 0.014 0.047 0.08

20/06/00 4 5.77 27.8 97.5 15.64 NS NS NS NS NS

10/07/00 4 5.8 27.5 123.8 16.26 52 NS NS NS NS

07/08/00 1 7.93 34 85.2 16.37 73 0.08 0 0.14 0.07

07/08/00 2 7.75 25 90.4 16.06 85 0 0.001 0.1 0.16

07/08/00 3 7.79 36.7 88.7 16.55 76 0 0.001 0.1 0.12

07/08/00 4 5.64 21.7 72.4 17.18 47 0.07 0.006 0.021 0.08

07/08/00 6 5.32 24.2 67.7 14.21 NS NS NS NS NS

07/08/00 7 6.97 10.8 49.8 14.13 30 0 0.001 0.021 0.07

18/08/00 4 6.6 31.6 99.1 14.88 NS NS NS NS NS

23/10/00 1 8.19 48.2 NS 20.01 76 0.44 0 0.047 0.1

23/10/00 2 7.67 35 NS 26.69 73 0.37 0.006 0.021 0.4

23/10/00 3 8.31 46.4 NS 21.09 65 0.11 0 0.063 0.1

23/10/00 4 7.84 33.8 NS 21.59 90 0.23 0.001 0.032 0.07

23/10/006NSNSNSNSNS NSNSNSNS

23/10/00 7 8.05 37.5 NS 21.14 73 0.16 0 0.089 0.06

12/01/01 1 7.99 40.5 67.4 23.41 80 0.42 0.003 0.11 0.3

12/01/01 2 7.73 33.3 76.5 27.12 100 0.06 0.007 0.055 0.26

12/01/01 3 7.91 39 73.9 23.96 95 0.05 0.003 0.12 0.14

12/01/01 4 7.81 35.2 68.4 24.42 90 0.1 0.006 0.04 0.32

12/01/01 6 7.58 37.4 62 24.74 90 0 0.003 0.036 0.06

12/01/01 7 7.82 36.3 63 24.07 80 0.02 0.004 0.032 0.28

NS = Not Sampled

Appendix H. (Continued)

DATE SITE Cl:SO4 Al As Ca Cu Fe K Mg Mn Na S Si Zn (mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)

01/02/00 1 10.38 0.131 <0.09 327.000 <0.006 <0.04 561.000 503.000 0.141 2670.000 653.000 1.310 <0.04

01/02/00 2 8.63 <0.02 <0.09 280.000 <0.006 <0.04 460.000 477.000 0.037 sat. 565.000 1.700 <0.04

01/02/00 3 9.97 <0.02 <0.09 354.000 <0.006 <0.04 577.000 528.000 <0.001 sat. 727.000 1.010 <0.04

01/02/00 4 7.65 <0.02 <0.09 224.000 <0.006 <0.04 314.000 422.000 0.004 2390.000 484.000 1.710 <0.04

01/02/00 6 4.93 <0.02 <0.09 231.000 <0.006 <0.04 351.000 431.000 0.090 2390.000 519.000 2.370 <0.04

01/02/00 7 6.03 <0.02 <0.09 296.000 <0.006 <0.04 492.000 477.000 0.008 2580.000 634.000 1.540 <0.04

10/03/00 4 7.25 0.185 <0.09 10.800 <0.006 0.271 7.220 19.000 0.424 112.000 23.600 0.317 <0.04

31/03/00 4 5.36 <0.02 <0.09 27.400 <0.006 <0.04 15.800 60.800 0.615 382.000 57.900 0.877 <0.04

06/04/00 1 8.77 <0.02 <0.09 241.000 <0.006 <0.04 358.000 436.000 <0.001 2400.000 530.000 1.880 <0.04

06/04/00 2 11.44 <0.02 <0.09 86.900 <0.006 <0.04 94.500 228.000 <0.001 1560.000 204.000 2.710 <0.04

06/04/00 3 8.82 <0.02 <0.09 182.000 <0.006 <0.04 257.000 375.000 <0.001 2220.000 463.000 1.670 <0.04

06/04/00 4 5.61 <0.02 <0.09 189.000 <0.006 <0.04 266.000 378.000 <0.001 2230.000 427.000 3.060 <0.04

06/04/00 6 7.70 <0.02 <0.09 86.300 <0.006 <0.04 91.700 224.000 0.172 1540.000 190.000 2.230 <0.04

06/04/00 7 11.40 <0.02 <0.09 81.200 <0.006 <0.04 88.500 214.000 0.003 1490.000 201.000 1.740 <0.04

19/04/00 4 5.14 <0.02 <0.09 196.000 <0.006 <0.04 233.000 392.000 1.330 2350.000 417.000 2.180 <0.04

07/05/004NSNSNSNSNSNSNSNSNSNSNSNSNS

30/05/00 4 7.37 <0.009 <0.14 226.00 0.02 0.01 203.00 694.00 0.07 5840.00 495.00 2.16 0.22

09/06/00 1 5.10 <0.009 <0.14 451.00 <0.004 0.06 431.00 1410.00 0.01 12000.00 1040.00 0.94 0.01

09/06/00 2 7.24 <0.009 <0.14 247.00 0.00 0.01 229.00 776.00 0.02 6660.00 578.00 1.77 <0.007

09/06/00 3 8.83 <0.009 <0.14 283.00 <0.004 <0.005 276.00 899.00 0.01 7730.00 675.00 0.85 <0.007

09/06/00 4 7.12 <0.009 <0.14 245.00 <0.004 0.01 229.00 763.00 0.01 6560.00 566.00 1.02 0.01

09/06/00 6 8.87 <0.009 <0.14 257.00 <0.004 <0.005 241.00 802.00 0.05 6870.00 563.00 1.78 <0.007

09/06/00 7 10.05 <0.009 <0.14 162.00 <0.004 0.01 149.00 509.00 0.20 4380.00 363.00 1.06 0.02

20/06/00 4 6.02 <0.009 <0.14 226.00 0.02 0.01 184.00 650.00 1.46 5320.00 535.00 0.87 0.24

10/07/00 4 6.04 <0.009 <0.14 234.00 0.01 0.01 189.00 669.00 1.49 5450.00 549.00 0.97 0.14

07/08/00 1 4.82 <0.009 <0.14 141.00 0.00 0.03 131.00 452.00 0.02 4100.00 525.00 0.76 <0.007

07/08/00 2 5.78 <0.009 <0.14 258.00 0.01 0.01 241.00 813.00 0.01 6870.00 577.00 2.21 <0.007

07/08/00 3 9.26 <0.009 <0.14 243.00 <0.004 0.01 234.00 774.00 0.02 6600.00 544.00 1.16 <0.007

07/08/00 4 6.75 0.08 <0.14 193.00 0.01 0.37 143.00 528.00 1.70 4120.00 457.00 0.80 0.08

07/08/006NSNSNSNSNSNSNSNSNSNSNSNSNS

07/08/00 7 6.97 <0.009 <0.14 80.50 0.01 0.10 78.20 267.00 0.31 2150.00 192.00 0.45 0.04

18/08/00 4 6.82 0.07 <0.14 217.00 0.01 0.01 193.00 661.00 0.42 5600.00 544.00 0.91 0.04

23/10/00 1 8.12 <0.009 <0.14 356.00 <0.004 0.01 347.00 1120.00 0.01 9600.00 786.00 0.70 <0.007

23/10/00 2 6.06 <0.009 <0.14 337.00 0.00 0.01 322.00 1050.00 0.00 8920.00 743.00 1.51 0.01

23/10/00 3 9.35 <0.009 <0.14 317.00 0.00 0.01 309.00 1000.00 0.01 8470.00 692.00 0.57 0.01

23/10/00 4 8.76 <0.009 <0.14 240.00 0.01 0.01 225.00 746.00 0.05 6390.00 530.00 1.34 0.01

23/10/006NSNSNSNSNSNSNSNSNSNSNSNSNS

23/10/00 7 6.77 <0.009 <0.14 347.00 0.00 0.01 329.00 1080.00 0.02 9120.00 764.00 0.89 0.01

12/01/01 1 8.35 <0.009 <0.14 304.00 0.01 0.01 292.00 959.00 0.01 8110.00 675.00 1.38 <0.007

12/01/01 2 8.80 <0.009 <0.14 238.00 0.01 0.01 222.00 743.00 0.01 6260.00 525.00 2.19 <0.007

12/01/01 3 9.05 <0.009 <0.14 291.00 0.01 <0.005 280.00 910.00 0.01 7570.00 625.00 1.18 0.01

12/01/01 4 9.38 <0.009 <0.14 253.00 0.00 0.01 239.00 786.00 0.01 6670.00 551.00 1.93 0.01

12/01/01 6 7.53 <0.009 <0.14 310.00 <0.004 <0.005 296.00 972.00 0.04 8150.00 675.00 0.97 <0.007

12/01/01 7 8.41 <0.009 <0.14 290.00 0.00 <0.005 274.00 907.00 0.02 7690.00 642.00 1.71 <0.007

NS = Not Sampled

Appendix I. Oyster survival data measured on the 23/8/99 and 10/1/00 at Sites 1 to 7.

ACID SITE SIZE % SURV(23/8/99) % SURV(10/1/00) CODES R 1 L 94 94 R = Reference R 1 L 100 98 A = Acid R 1 L 100 92 L = Mature oysters R 1 L 100 98 S = Juvenile oysters R 1 S 100 98 R 1 S 100 96 R1S 98 98 R1S 96 94 R 2 L 100 100 R 2 L 100 100 R2L 92 90 R2L 98 96 R 2 S 100 98 R2S 94 92 R2S 98 98 R 2 S 100 98 R 3 L 100 94 R 3 L 100 94 R3L 98 98 R3L 98 90 R3S 98 98 R3S 94 94 R 3 S 100 100 R3S 96 96 A4L 92 78 A4L 96 90 A4L 90 82 A4L 96 96 A4S 88 78 A4S 78 64 A4S 84 72 A4S 76 58 A5L 68 52 A5L 18 12 A5L 4 0 A5L 0 0 A5S 22 8 A5S 34 0 A5S 46 0 A5S 8 4 A 6 L 100 94 A6L 84 82 A6L 90 84 A6L 94 88 A6S 58 48 A6S 54 48 A6S 52 38 A6S 62 46 A 7 L 100 98 A7L 96 92 A7L 98 96 A7L 98 98 A7S 94 90 A7S 94 82 A7S 92 84 A7S 82 76

Appendix I. (Continued)

large oysters small oysters

a,b a,b a,b a,b a a,b a,b,c a,b,c,d c,d,e 100 b,c,d e

f 60 g g

% surviving 40

0 1234567 site Percentage survival on the 23/8/99 at the seven sites (post hoc analysis results for the factor Size x Site(Acid) are displayed as letters indicating means which are not significantly different, p > 0.05).

large oysters small oysters

a a a a a a a 100 b b b c 80

60 d e

% surviving 40

20 f

0 1234567 site Percentage survival on the 10/1/00 at the seven sites (post hoc analysis results for the factor Size x Site(Acid) are displayed as letters indicating means which are not significantly different, p > 0.05).

Appendix J. Oyster condition index measured at Sites 1, 2, 3, 4, 6 and 7.

6/04/00 9/06/00 7/08/00 23/10/00 12/01/01 Site Condition Site Condition Site Condition Site Condition Site Condition Index Index Index Index Index

1 59.38 1 63.64 1 74.78 1 103.97 2 123.67 1 49.79 1 95.03 1 90.69 1 68.17 2 148.48 1 55.42 1 63.01 1 79.35 1 116.51 2 121.08 1 62.57 1 64.77 1 88.48 1 113.28 2 122.34 1 69.34 1 62.17 1 81.14 1 117.70 2 151.97 1 62.25 1 73.09 1 91.55 1 97.91 2 154.87 1 59.97 1 66.59 1 116.29 1 110.01 2 111.56 1 51.04 1 77.34 1 102.30 1 112.85 2 105.92 1 56.42 1 69.73 1 61.79 1 105.92 2 121.60 1 60.97 2 76.43 1 80.05 1 100.68 2 125.98 1 41.07 2 84.42 1 113.21 1 114.17 3 165.49 1 77.68 2 63.78 1 85.69 1 81.53 3 170.10 2 54.39 2 66.46 2 77.81 2 113.01 3 171.53 2 55.82 2 83.03 2 81.74 2 136.99 3 155.78 2 31.76 2 72.66 2 78.27 2 141.99 3 153.30 2 70.72 2 83.93 2 85.79 2 123.01 3 153.03 2 56.90 2 58.60 2 85.04 2 130.33 4 152.57 2 57.12 2 81.70 2 83.18 2 154.17 4 124.66 2 58.51 2 83.52 2 76.25 2 125.71 4 82.67 2 62.69 2 78.37 2 83.55 2 114.64 4 157.25 2 76.18 2 99.73 2 72.87 2 161.64 4 90.53 2 55.02 3 52.83 2 70.34 2 107.29 4 159.67 3 47.89 3 63.71 2 62.01 2 143.36 4 122.92 3 65.32 3 66.69 3 85.59 2 104.16 4 158.78 3 48.08 3 61.99 3 81.76 3 204.51 4 147.33 3 62.99 3 82.15 3 86.09 3 127.60 7 66.15 3 52.31 3 58.29 3 102.23 3 119.20 7 117.92 3 64.31 3 60.06 3 98.94 3 91.21 7 130.27 3 59.27 3 51.42 3 66.25 3 146.76 7 115.96 3 48.60 3 50.42 3 83.71 3 129.32 7 128.07 3 59.62 3 59.27 3 70.09 3 169.22 7 139.15 3 64.96 3 72.25 3 64.05 3 127.13 7 116.84 3 55.06 3 45.24 3 82.17 3 162.92 7 121.03 4 25.69 4 61.69 3 77.26 3 128.72 7 115.77 4 62.03 4 53.80 4 72.08 3 143.48 7 112.27 4 66.33 4 101.80 4 92.56 4 59.00 4 34.71 4 85.51 4 57.55 4 96.29 4 44.93 4 108.27 4 110.66 4 140.15 4 71.04 4 57.33 4 98.01 4 91.66 4 78.00 4 97.16 4 59.35 4 91.04 4 58.73 4 86.72 4 82.03 4 85.62 4 47.10 4 109.74 4 90.54 4 63.15 4 43.20 4 79.39 4 108.71 4 104.29 4 40.15 4 104.36 4 64.26 4 113.14 4 51.15 7 49.71 4 74.83 4 118.07 7 46.95 7 69.67 4 60.05 4 98.65 7 34.93 7 55.38 7 73.66 7 65.90 7 43.34 7 72.08 7 57.78 7 82.40 7 27.49 7 70.80 7 86.12 7 69.79 7 44.73 7 65.88 7 52.99 7 75.16 7 56.15 7 79.46 7 60.37 7 98.93 7 42.57 7 62.94 7 42.27 7 72.95 7 45.24 7 53.06 7 57.38 7 203.33 7 46.70 7 140.71 7 77.22 7 75.55 7 62.01 7 55.30 7 68.51 7 46.18 7 41.65 7 85.27 7 74.26 7 49.49 7 52.01 6 105.85 7 66.67 7 99.53 6 82.80 6 111.96 7 56.75 7 45.30 6 84.69 6 83.48 6 99.26 6 70.90 6 43.83 6 110.16 6 63.47 6 74.54 6 103.56 6 90.71 6 118.63 6 94.84 6 109.73 6 87.14 6 74.33 6 59.01 6 81.52 6 60.51 6 69.38

Appendix K. Lower Hastings River and Limeburners Creek pH, EC, DO and temperature data.

SITE: 1 SITE: 2

pH Elec. Cond. Dissolved Oxygen Temperature pH Elec. Cond. Dissolved Oxygen Temperature (mS cm-1) (% Saturation) (oC) (mS cm-1) (% Saturation) (oC)

Date surface bed surface bed surface bed surface bed Date surface bed surface bed surface bed surface bed

17/11/97 8.17 8.22 52.1 53.2 81.0 80.6 17.40 17.00 17/11/97 8.14 8.19 52.8 53.2 81.4 79.7 17.20 17.00 04/12/97 8.22 8.26 50.3 53.4 55.0 60.8 22.10 22.00 04/12/97 NS NS NS NS NS NS NS NS 20/03/98 NS NS NS NS NS NS NS NS 20/03/98 8.08 8.13 51.8 52.7 NS NS 24.91 24.22 25/03/98 8.13 8.15 52.0 52.4 NS NS 22.17 21.78 25/03/98 8.15 8.16 52.3 52.0 NS NS 22.01 21.68 27/03/98 NS NS NS NS NS NS NS NS 27/03/98 NS NS NS NS NS NS NS NS 02/04/98 8.14 8.22 50.1 51.9 NS NS 23.53 23.51 02/04/98 8.12 8.22 48.9 51.7 NS NS 23.53 23.47 17/04/98 8.22 8.07 54.0 49.1 NS NS 22.85 22.57 17/04/98 8.22 8.04 54.1 48.2 NS NS 22.74 22.65 27/04/98 8.20 8.21 46.7 47.1 NS NS 22.11 22.10 27/04/98 8.12 8.29 44.4 48.8 NS NS 22.09 22.22 04/05/98 7.53 8.29 13.4 43.6 NS NS 20.44 21.51 04/05/98 7.45 8.15 13.1 33.7 NS NS 20.36 20.90 15/05/98 7.73 7.88 37.1 47.0 81.2 83.3 14.71 16.14 15/05/98 7.61 7.86 33.3 48.1 79.2 78.1 14.29 16.27 02/06/98 7.82 7.89 36.4 25.4 88.5 89.3 13.48 15.66 02/06/98 7.81 7.90 36.6 43.1 87.1 87.4 13.54 15.81 05/06/98 7.71 7.87 29.0 39.7 94.6 90.5 12.89 14.94 05/06/98 7.62 7.89 27.5 43.5 94.8 88.5 12.60 16.06 21/07/98 8.20 8.26 44.1 48.1 85.4 83.7 17.31 18.14 21/07/98 8.20 8.26 43.5 48.6 83.7 82.5 17.18 18.25 31/07/98 8.21 8.20 46.0 45.7 87.1 86.9 15.20 15.18 31/07/98 8.19 8.21 44.4 45.6 87.2 85.7 14.95 15.17 10/08/98 8.14 8.17 49.0 49.9 91.5 96.7 16.72 17.08 10/08/98 8.12 8.17 47.9 49.6 92.2 93.3 16.62 16.61 17/08/98 7.28 8.14 9.5 38.6 108.5 93.1 15.69 16.79 17/08/98 7.65 7.95 6.6 36.4 108.9 92.5 15.67 16.73 31/08/98 7.46 7.91 24.8 46.6 98.9 95.9 17.60 17.86 31/08/98 7.49 7.90 24.1 46.8 107.4 95.1 17.59 17.81 15/09/98 7.05 7.87 13.1 46.2 98.9 89.6 19.31 18.49 15/09/98 7.25 7.77 11.8 44.9 98.3 87.4 19.27 18.53 01/10/98 NS NS NS NS NS NS NS NS 01/10/98 NS NS NS NS NS NS NS NS 16/10/98 7.75 7.76 54.5 54.9 94.8 96.3 19.83 19.55 16/10/98 7.73 7.79 52.3 55.0 94.3 95.6 20.88 19.62 09/11/98 8.08 8.11 52.3 53.0 78.4 82.4 20.27 19.83 09/11/98 8.03 8.11 51.3 52.5 76.9 80.1 20.69 19.86 07/12/98 7.92 7.94 62.7 63.2 96.8 99.9 24.45 24.81 07/12/98 7.90 7.93 62.6 64.5 92.6 91.7 24.88 23.41 25/01/99 8.07 8.15 46.7 49.1 89.0 109.3 26.43 25.51 25/01/99 8.10 8.15 47.4 49.2 103.5 99.2 26.41 25.70 04/02/99 8.16 8.39 27.9 48.5 115.7 126.0 25.92 25.15 04/02/99 8.17 8.42 27.2 52.2 115.1 123.7 25.98 24.88 04/03/99 8.16 8.52 20.0 44.0 126.5 113.4 25.61 24.13 04/03/99 8.03 8.43 17.9 35.1 124.4 124.5 25.69 25.38 30/03/99 7.80 7.82 43.8 46.1 91.8 98.4 24.31 24.36 30/03/99 7.82 7.83 37.8 44.7 129.4 110.5 24.35 24.47

Summary: Summary:

N of cases 2323232318182323 N of cases 2323232317172323 Minimum 7.05 7.76 9.5 25.4 55.0 60.8 12.89 14.94 Minimum 7.25 7.77 6.6 33.7 76.9 78.1 12.60 15.17 Maximum 8.22 8.52 62.7 63.2 126.5 126.0 26.43 25.51 Maximum 8.22 8.43 62.6 64.5 129.4 124.5 26.41 25.70 Median 8.08 8.15 46.0 48.1 91.7 91.8 20.27 19.83 Median 8.03 8.13 44.4 48.6 94.3 91.7 20.69 19.86 Mean 7.92 8.10 39.8 47.7 92.4 93.1 20.01 20.18 Mean 7.91 8.08 38.7 47.8 97.4 93.9 20.15 20.29 Standard Dev. 0.33 0.20 15.2 7.1 15.4 14.2 4.12 3.50 Standard Dev. 0.28 0.19 15.7 6.8 15.4 13.9 4.33 3.52

NS = Not sampled NS = Not sampled

Appendix K. (Continued)

SITE: 3 SITE: 4

pH Elec. Cond. Dissolved Oxygen Temperature pH Elec. Cond. Dissolved Oxygen Temperature (mS cm-1) (% Saturation) (oC) (mS cm-1) (% Saturation) (oC)

Date surface bed surface bed surface bed surface bed Date surface bed surface bed surface bed surface bed

17/11/97 8.14 8.15 53.0 53.2 77.6 77.6 17.20 17.00 17/11/97 NS NS NS NS NS NS NS NS 04/12/97 8.11 8.23 51.7 53.8 58.4 64.1 21.80 21.80 04/12/97 NS NS NS NS NS NS NS NS 20/03/98 NS NS NS NS NS NS NS NS 20/03/98 8.01 8.01 49.8 50.0 NS NS 25.12 24.76 25/03/98 8.12 8.16 51.3 49.4 NS NS 22.76 21.85 25/03/98 8.08 8.15 50.4 52.4 NS NS 23.28 21.88 27/03/98 NS NS NS NS NS NS NS NS 27/03/98 NS NS NS NS NS NS NS NS 02/04/98 8.10 8.23 48.9 52.1 NS NS 23.62 23.42 02/04/98 8.10 8.11 48.4 49.7 NS NS 23.59 23.62 17/04/98 8.21 8.04 51.2 48.2 NS NS 22.63 22.57 17/04/98 8.18 7.99 52.5 46.5 NS NS 22.49 22.98 27/04/98 8.13 8.25 44.2 50.1 NS NS 22.11 21.82 27/04/98 8.10 8.24 42.6 49.4 NS NS 22.18 21.80 04/05/98 NS NS NS NS NS NS NS NS 04/05/98 NS NS NS NS NS NS NS NS 15/05/98 7.57 7.87 32.6 49.9 81.4 80.1 14.19 16.76 15/05/98 7.59 7.84 33.3 45.0 79.2 81.7 14.14 15.71 02/06/98 7.80 7.90 35.8 43.6 87.8 86.6 13.30 16.06 02/06/98 7.80 7.92 38.7 48.3 88.7 85.5 12.97 16.21 05/06/98 7.60 7.90 27.4 41.2 93.7 90.4 12.58 15.73 05/06/98 7.58 7.90 26.6 40.5 95.0 89.7 12.48 15.79 21/07/98 8.19 8.26 42.9 50.1 85.3 82.5 17.10 18.41 21/07/98 8.16 8.19 42.0 44.1 85.3 84.7 16.94 17.17 31/07/98 8.09 8.18 38.3 46.9 90.5 83.9 14.36 15.48 31/07/98 8.13 8.20 39.6 44.9 88.8 83.4 14.42 15.40 10/08/98 8.10 8.16 46.5 48.7 93.4 92.8 16.65 16.59 10/08/98 8.10 8.19 43.6 49.1 93.5 91.8 16.91 16.55 17/08/98 7.38 8.15 5.6 41.4 107.3 90.4 15.65 17.10 17/08/98 7.81 8.08 3.8 32.7 93.3 106.7 16.50 15.61 31/08/98 7.32 7.89 20.6 47.3 105.3 95.1 17.77 17.86 31/08/98 7.51 7.97 19.3 47.5 104.0 92.4 18.25 17.88 15/09/98 7.20 7.94 10.5 46.9 98.1 88.9 19.39 18.48 15/09/98 7.06 7.90 9.5 48.9 97.2 85.3 19.24 18.42 01/10/98 NS NS NS NS NS NS NS NS 01/10/98 NS NS NS NS NS NS NS NS 16/10/98 7.72 7.78 50.9 54.1 87.9 94.1 20.64 19.93 16/10/98 7.73 7.74 49.9 50.1 91.9 93.7 20.63 20.57 09/11/98 8.05 8.12 51.5 53.0 79.0 82.6 20.54 19.89 09/11/98 8.06 8.08 52.6 52.9 84.5 86.1 20.05 19.85 07/12/98 7.86 7.90 62.5 64.7 93.6 95.4 25.01 23.27 07/12/98 7.82 7.86 62.5 64.8 94.2 90.9 24.98 23.18 25/01/99 8.03 8.14 43.9 49.1 99.4 94.0 27.04 25.48 25/01/99 8.02 8.12 43.4 47.2 107.1 96.4 27.17 25.99 04/02/99 8.14 8.42 23.4 52.4 116.3 123.0 26.10 24.87 04/02/99 8.07 8.39 19.2 48.8 115.8 121.2 26.14 25.01 04/03/99 8.12 8.54 19.2 49.7 126.5 115.0 25.41 23.96 04/03/99 8.05 8.46 19.4 41.5 116.0 114.7 25.34 24.01 30/03/99 7.78 7.83 31.9 44.8 114.7 100.1 24.28 24.19 30/03/99 7.79 7.84 38.7 46.0 108.1 95.3 24.27 24.11

Summary: Summary:

N of cases 2222222218182222 N of cases 2121212116162121 Minimum 7.20 7.78 5.6 41.2 58.4 64.1 12.58 15.48 Minimum 7.06 7.74 3.8 32.7 79.2 81.7 12.48 15.40 Maximum 8.21 8.54 62.5 64.7 126.5 123.0 27.04 25.48 Maximum 8.18 8.46 62.5 64.8 116.0 121.2 27.17 25.99 Median 8.07 8.15 43.4 49.6 93.5 90.4 20.59 19.91 Median 8.02 8.08 42.0 48.3 93.9 91.4 20.63 20.57 Mean 7.90 8.09 38.4 49.6 94.2 90.9 20.01 20.11 Mean 7.89 8.06 37.4 47.6 96.4 93.7 20.34 20.31 Standard Dev. 0.31 0.20 15.2 5.0 16.1 13.2 4.43 3.30 Standard Dev. 0.28 0.19 15.6 6.0 10.9 11.3 4.61 3.69

NS = Not sampled NS = Not sampled

Appendix K. (Continued)

SITE: 5 SITE: 6

pH Elec. Cond. Dissolved Oxygen Temperature pH Elec. Cond. Dissolved Oxygen Temperature (mS cm-1) (% Saturation) (oC) (mS cm-1) (% Saturation) (oC)

Date surface bed surface bed surface bed surface bed Date surface bed surface bed surface bed surface bed

17/11/97 8.22 8.25 50.1 53.1 83.6 84.9 17.20 17.20 17/11/97 8.23 8.26 52.7 52.8 85.0 85.0 17.40 17.40 04/12/97 8.23 8.24 53.8 49.8 62.5 64.6 21.40 21.40 04/12/97 8.24 8.24 53.8 53.8 63.0 66.5 21.40 21.40 20/03/98 8.10 8.13 52.3 52.8 NS NS 24.71 24.30 20/03/98 8.08 8.12 49.6 50.8 NS NS 25.48 24.91 25/03/98 8.08 8.08 51.8 51.7 NS NS 22.55 22.56 25/03/98 NS NS 50.1 50.0 NS NS 23.56 23.56 27/03/98 8.21 8.21 52.6 52.9 NS NS 24.40 24.40 27/03/98 8.21 8.21 52.9 52.8 NS NS 24.42 24.40 02/04/98 8.12 8.13 49.6 49.7 NS NS 23.67 23.67 02/04/98 8.10 8.14 49.2 50.0 NS NS 23.74 23.70 17/04/98 NS NS NS NS NS NS NS NS 17/04/98 8.08 8.16 49.3 51.1 NS NS 22.67 23.08 27/04/98 8.21 8.23 48.2 48.6 NS NS 22.28 22.35 27/04/98 NS NS NS NS NS NS NS NS 04/05/98 7.61 7.63 11.8 20.8 NS NS 20.71 20.42 04/05/98 7.57 7.45 8.3 12.5 NS NS 20.53 20.35 15/05/98 7.67 7.70 36.0 37.1 78.6 77.4 14.84 14.66 15/05/98 7.60 7.59 33.8 33.9 76.3 74.2 14.38 14.36 02/06/98 7.72 7.74 36.0 36.6 82.9 82.2 13.61 13.77 02/06/98 7.67 7.67 34.0 34.0 82.7 82.7 13.27 13.27 05/06/98 7.34 7.35 17.5 20.1 94.6 92.8 12.30 12.43 05/06/98 7.22 7.19 13.8 13.9 102.7 101.9 11.99 11.97 21/07/98 8.10 8.12 43.0 43.7 81.1 81.4 17.94 18.01 21/07/98 8.05 8.08 41.7 41.9 80.1 80.8 17.84 17.86 31/07/98 8.15 8.17 43.0 44.4 88.5 87.7 14.89 15.04 31/07/98 8.16 8.16 43.1 43.3 90.1 90.1 14.94 14.96 10/08/98 8.11 8.12 47.9 48.1 97.3 97.5 17.09 17.10 10/08/98 NS NS NS NS NS NS NS NS 17/08/98 7.69 7.70 19.7 25.5 111.2 102.3 15.93 15.75 17/08/98 7.52 7.56 20.2 22.4 109.9 103.2 15.51 15.61 31/08/98 NS NS NS NS NS NS NS NS 31/08/98 7.10 7.22 16.3 22.2 103.8 104.6 17.32 16.64 15/09/98 NS NS NS NS NS NS NS NS 15/09/98 7.00 7.18 14.1 21.8 97.8 100.2 19.34 18.84 01/10/98 7.14 7.30 12.0 22.0 89.9 75.3 27.33 23.84 01/10/98 7.41 7.44 22.2 24.0 87.3 88.6 24.94 24.66 16/10/98 NS NS 49.8 52.1 91.9 93.6 20.63 20.21 16/10/98 7.59 7.60 45.8 46.0 83.1 90.9 21.37 21.23 09/11/98 8.07 8.08 45.1 52.8 78.5 81.1 19.99 19.82 09/11/98 8.03 8.04 52.6 52.5 84.7 84.8 19.97 19.97 07/12/98 7.97 7.97 62.1 62.1 103.3 105.8 25.33 25.32 07/12/98 7.97 7.97 61.9 61.9 101.8 106.7 25.45 25.45 25/01/99 8.07 8.10 44.5 48.0 94.6 109.4 27.03 25.88 25/01/99 8.00 8.01 46.8 46.4 81.5 94.5 26.71 26.72 04/02/99 8.33 8.34 41.9 42.2 128.7 131.5 26.39 26.39 04/02/99 8.33 8.36 47.5 48.3 109.2 112.5 26.43 26.12 04/03/99 8.40 8.49 35.4 38.5 134.5 135.3 26.43 26.41 04/03/99 8.30 8.30 32.2 32.2 131.1 129.1 26.72 26.72 30/03/99 7.78 7.79 40.8 42.6 108.9 106.1 24.29 24.26 30/03/99 7.74 7.78 39.0 40.9 123.2 111.0 24.35 24.28

Summary: Summary:

N of cases 2222232317172323 N of cases 2323242418182424 Minimum 7.14 7.30 11.8 20.1 62.5 64.6 12.30 12.43 Minimum 7.00 7.18 8.3 12.5 63.0 66.5 11.99 11.97 Maximum 8.40 8.49 62.1 62.1 134.5 135.3 27.33 26.41 Maximum 8.33 8.36 61.9 61.9 131.1 129.1 26.72 26.72 Median 8.09 8.11 44.5 48.0 91.9 92.8 21.40 21.40 Median 8.00 8.01 44.5 44.7 88.7 92.7 21.39 21.32 Mean 7.97 7.99 41.1 43.3 94.7 94.6 20.91 20.66 Mean 7.83 7.86 38.8 40.0 94.1 94.9 20.82 20.73 Standard Dev. 0.32 0.31 13.7 11.6 18.3 19.0 4.63 4.37 Standard Dev. 0.40 0.38 15.3 14.1 17.2 15.3 4.58 4.56

NS = Not sampled NS = Not sampled

Appendix K. (Continued)

SITE: 7 SITE: 8

pH Elec. Cond. Dissolved Oxygen Temperature pH Elec. Cond. Dissolved Oxygen Temperature (mS cm-1) (% Saturation) (oC) (mS cm-1) (% Saturation) (oC)

Date surface bed surface bed surface bed surface bed Date surface bed surface bed surface bed surface bed

17/11/97 8.25 8.28 52.8 52.8 86.8 85.6 17.60 17.60 17/11/97 8.26 8.28 52.8 52.8 85.3 84.9 17.60 17.60 04/12/97 8.25 8.25 52.7 53.7 60.0 62.6 21.40 21.50 04/12/97 8.25 8.26 52.4 53.6 59.4 62.0 21.50 21.50 20/03/98 7.95 8.08 50.0 51.4 NS NS 25.77 25.05 20/03/98 8.02 8.06 49.9 50.8 NS NS 25.74 25.31 25/03/98 7.95 7.96 50.0 50.0 NS NS 23.78 23.78 25/03/98 7.95 7.95 50.1 50.1 NS NS 23.84 23.83 27/03/98 8.21 8.21 52.8 52.6 NS NS 24.26 24.22 27/03/98 8.20 8.21 52.7 52.7 NS NS 24.19 24.21 02/04/98 8.07 8.08 48.0 48.6 NS NS 23.34 23.40 02/04/98 8.06 8.09 47.6 48.5 NS NS 23.31 23.46 17/04/98 8.12 8.14 50.3 50.9 NS NS 23.05 22.98 17/04/98 8.12 8.13 49.2 50.4 NS NS 23.38 22.97 27/04/98 8.11 8.11 44.6 44.6 NS NS 22.06 22.06 27/04/98 8.09 8.09 44.0 44.0 NS NS 22.02 22.03 04/05/98 7.73 7.53 5.2 5.5 NS NS 20.62 20.38 04/05/98 7.29 7.24 4.9 4.8 NS NS 20.40 20.33 15/05/98 7.55 7.55 32.6 32.7 77.0 76.6 14.26 14.38 15/05/98 7.53 7.52 31.4 31.8 76.2 75.3 14.17 14.13 02/06/98 7.64 7.64 32.9 32.9 81.5 82.6 13.18 13.17 02/06/98 7.61 7.61 31.8 32.0 82.8 82.5 13.03 13.05 05/06/98 7.16 7.13 11.4 11.4 101.7 101.2 11.80 11.79 05/06/98 7.08 7.04 10.2 10.7 101.0 99.6 11.70 11.80 21/07/98 8.06 8.06 41.6 41.5 80.6 81.3 17.86 17.85 21/07/98 8.04 8.05 41.1 41.2 80.6 81.2 17.83 17.82 31/07/98 8.05 8.11 40.7 43.0 89.1 88.5 14.29 14.85 31/07/98 8.01 8.08 41.1 34.5 88.4 90.4 14.41 14.58 10/08/98 8.08 8.11 47.7 47.7 95.1 96.0 16.97 16.98 10/08/98 8.09 8.09 47.5 47.5 98.7 98.7 16.97 16.93 17/08/98 7.48 7.49 19.2 20.2 111.8 109.3 15.51 15.50 17/08/98 7.46 7.47 18.4 19.9 110.8 108.7 15.46 15.51 31/08/98 7.08 7.08 11.7 15.7 103.2 103.2 17.18 16.61 31/08/98 7.00 7.00 10.3 12.1 100.9 101.2 16.87 16.85 15/09/98 6.88 NS 8.1 NS 94.0 NS 20.33 NS 15/09/98 6.88 6.87 5.8 6.9 94.1 93.6 20.05 20.41 01/10/98 7.56 7.58 26.7 27.1 92.0 92.3 24.19 24.11 01/10/98 7.62 7.64 28.0 28.4 94.5 95.1 23.97 23.89 16/10/98 7.52 7.59 45.6 ND 87.7 94.8 22.37 21.52 16/10/98 7.56 7.57 45.3 45.2 88.5 91.9 21.75 21.78 09/11/98 7.97 8.06 49.6 51.8 82.8 81.2 21.25 20.32 09/11/98 8.00 8.05 50.0 51.3 81.4 81.3 21.05 20.55 07/12/98 7.95 7.96 61.7 61.7 99.1 103.0 25.68 25.68 07/12/98 7.95 7.96 61.5 61.5 105.2 105.5 25.84 25.82 25/01/99 7.96 7.97 47.6 47.8 78.5 90.5 26.82 26.77 25/01/99 7.92 7.94 47.9 48.0 80.4 89.7 26.89 26.87 04/02/99 8.35 8.35 44.1 44.2 104.5 92.7 26.33 26.38 04/02/99 8.33 8.37 41.5 44.2 104.4 114.9 26.51 26.25 04/03/99 8.27 8.28 31.6 31.6 132.9 130.3 26.84 26.85 04/03/99 8.23 8.25 31.2 31.4 129.5 128.6 27.03 26.99 30/03/99 7.68 7.70 37.3 37.5 126.0 126.4 24.38 24.45 30/03/99 7.68 7.69 37.0 37.2 123.1 142.1 24.41 24.42

Summary: Summary:

N of cases 2625262419182625 N of cases 2626262619192626 Minimum 6.88 7.08 5.2 5.5 60.0 62.6 11.80 11.79 Minimum 6.88 6.87 4.9 4.8 59.4 62.0 11.70 11.80 Maximum 8.35 8.35 61.7 61.7 132.9 130.3 26.84 26.85 Maximum 8.33 8.37 61.5 61.5 129.5 142.1 27.03 26.99 Median 7.96 8.06 44.4 44.4 92.0 92.5 21.73 21.52 Median 7.98 8.01 42.8 44.1 94.1 93.6 21.63 21.64 Mean 7.84 7.89 38.3 39.9 93.9 94.3 20.81 20.73 Mean 7.82 7.83 37.8 38.1 94.0 96.2 20.77 20.73 Standard Dev. 0.39 0.36 15.7 14.8 17.3 16.6 4.53 4.56 Standard Dev. 0.41 0.43 16.0 15.9 16.7 18.7 4.58 4.52

NS = Not sampled NS = Not sampled

Appendix K. (Continued)

SITE: 9 SITE: 10

pH Elec. Cond. Dissolved Oxygen Temperature pH Elec. Cond. Dissolved Oxygen Temperature (mS cm-1) (% Saturation) (oC) (mS cm-1) (% Saturation) (oC)

Date surface bed surface bed surface bed surface bed Date surface bed surface bed surface bed surface bed

17/11/97 8.24 8.27 52.6 52.7 86.1 85.0 17.80 17.80 17/11/97 8.25 8.27 51.3 52.8 87.7 86.5 18.00 18.00 04/12/97 8.25 8.22 53.1 52.8 58.5 62.1 21.60 21.90 04/12/97 NS NS NS NS 50.9 61.8 21.80 21.80 20/03/98 NS NS NS NS NS NS NS NS 20/03/98 7.98 8.03 49.6 49.8 NS NS 26.07 25.59 25/03/98 7.95 7.95 49.8 49.8 NS NS 23.85 23.85 25/03/98 7.94 7.97 49.9 50.3 NS NS 23.87 23.70 27/03/98 8.20 8.21 52.5 52.5 NS NS 24.19 24.19 27/03/98 8.20 8.20 52.4 52.4 NS NS 24.17 24.17 02/04/98 8.04 8.06 47.8 48.2 NS NS 23.39 23.42 02/04/98 8.06 8.06 48.0 47.9 NS NS 23.42 23.41 17/04/98 8.10 8.12 49.2 50.1 NS NS 23.15 22.95 17/04/98 8.05 8.09 48.3 49.3 NS NS 23.31 23.02 27/04/98 8.08 8.08 43.7 43.6 NS NS 22.01 22.01 27/04/98 8.06 8.13 43.2 44.2 NS NS 22.00 22.07 04/05/98 7.22 7.19 4.5 5.0 NS NS 20.38 20.39 04/05/98 7.17 7.16 4.7 4.6 NS NS 20.47 20.43 15/05/98 7.51 7.49 31.3 31.1 77.0 76.0 14.11 14.14 15/05/98 7.48 7.49 30.4 31.0 77.3 76.3 14.16 14.04 02/06/98 7.60 7.60 31.7 31.6 82.5 82.1 13.04 13.03 02/06/98 7.57 7.60 30.7 31.6 82.6 82.6 12.92 13.09 05/06/98 7.02 7.01 9.4 9.2 98.3 98.3 11.65 11.59 05/06/98 6.99 6.89 8.1 9.3 97.0 98.1 11.47 11.72 21/07/98 8.03 8.05 41.2 41.2 79.7 80.7 17.87 17.84 21/07/98 8.00 8.07 40.7 41.9 79.0 79.8 17.78 17.97 31/07/98 8.08 8.09 40.9 41.3 89.1 89.2 14.34 14.40 31/07/98 8.04 8.05 40.2 40.3 89.9 90.0 14.05 14.07 10/08/98 8.06 8.09 47.4 47.4 94.4 95.6 16.88 16.97 10/08/98 8.12 8.12 47.3 47.5 98.3 100.1 17.16 17.20 17/08/98 7.43 7.44 18.1 18.5 110.0 109.3 15.48 15.44 17/08/98 7.37 7.37 15.2 17.4 110.0 109.5 15.50 15.44 31/08/98 7.01 7.00 9.9 10.2 106.7 102.2 16.61 16.49 31/08/98 7.02 7.00 8.7 9.8 104.0 102.9 16.67 16.40 15/09/98 6.86 6.84 5.4 7.0 91.8 93.5 20.21 19.97 15/09/98 6.85 6.85 4.9 5.4 92.1 93.0 20.17 19.89 01/10/98 7.67 7.69 29.0 31.0 96.6 96.9 23.84 23.50 01/10/98 7.70 7.66 33.2 14.9 97.5 99.4 23.10 22.88 16/10/98 7.53 7.54 45.4 45.4 87.9 90.4 22.08 22.07 16/10/98 7.45 7.46 45.7 45.7 88.7 89.6 22.49 22.50 09/11/98 8.00 8.02 50.2 50.4 76.9 79.9 21.05 20.92 09/11/98 7.96 7.97 49.5 49.2 78.1 80.5 21.34 21.34 07/12/98 7.92 7.95 61.3 61.4 103.6 105.0 26.01 25.90 07/12/98 7.92 7.94 61.0 61.1 104.8 104.8 26.24 26.14 25/01/99 7.90 7.91 47.8 47.8 83.1 87.8 26.90 26.83 25/01/99 7.88 7.89 47.7 47.7 84.5 88.5 26.87 26.84 04/02/99 8.32 8.34 41.0 42.6 97.5 107.6 26.37 26.22 04/02/99 8.28 8.34 37.8 41.5 90.8 122.4 26.91 26.39 04/03/99 8.21 8.23 31.0 31.2 129.4 128.6 27.09 27.09 04/03/99 8.16 8.26 30.4 31.8 129.7 129.3 27.23 27.21 30/03/99 7.66 7.68 36.1 36.5 131.5 130.0 24.38 24.39 30/03/99 7.65 7.66 35.2 35.2 120.7 126.5 24.35 24.36

Summary: Summary:

N of cases 2525252519192525 N of cases 2525252519192626 Minimum 6.86 6.84 4.5 5.0 58.5 62.1 11.65 11.59 Minimum 6.85 6.85 4.7 4.6 50.9 61.8 11.47 11.72 Maximum 8.32 8.34 61.3 61.4 131.5 130.0 27.09 27.09 Maximum 8.28 8.34 61.0 61.1 129.7 129.3 27.23 27.21 Median 7.95 7.95 41.2 42.6 91.8 93.5 21.60 21.90 Median 7.94 7.97 40.7 41.9 90.8 93.0 21.90 21.94 Mean 7.80 7.80 37.2 37.5 93.7 94.7 20.57 20.53 Mean 7.77 7.78 36.6 36.5 92.8 95.9 20.83 20.76 Standard Dev. 0.42 0.44 16.3 16.2 17.7 16.9 4.57 4.55 Standard Dev. 0.42 0.45 16.4 16.8 17.3 17.6 4.67 4.58

NS = Not sampled NS = Not sampled

Appendix K. (Continued)

SITE: 11 SITE: 12

pH Elec. Cond. Dissolved Oxygen Temperature pH Elec. Cond. Dissolved Oxygen Temperature (mS cm-1) (% Saturation) (oC) (mS cm-1) (% Saturation) (oC)

Date surface bed surface bed surface bed surface bed Date surface bed surface bed surface bed surface bed

17/11/97 8.22 8.25 52.2 52.2 82.7 85.4 18.20 18.20 17/11/97 8.20 8.22 51.1 50.4 84.9 85.0 18.60 18.60 04/12/97 8.23 8.24 53.3 52.0 50.9 61.8 21.80 21.80 04/12/97 8.20 8.21 52.2 52.4 61.2 63.3 22.20 22.10 20/03/98 NS NS NS NS NS NS NS NS 20/03/98 7.97 7.97 49.1 49.1 NS NS 26.23 26.17 25/03/98 7.90 7.90 49.1 49.1 NS NS 24.18 24.16 25/03/98 7.89 7.89 49.0 49.0 NS NS 24.21 24.21 27/03/98 8.19 8.20 52.1 52.3 NS NS 24.05 24.12 27/03/98 8.13 8.13 51.6 51.3 NS NS 23.74 23.74 02/04/98 7.99 8.04 45.1 47.1 NS NS 23.19 23.34 02/04/98 8.00 8.01 45.5 46.1 NS NS 23.21 23.24 17/04/98 8.06 8.06 47.0 48.3 NS NS 23.37 22.90 17/04/98 8.03 8.05 44.2 48.0 NS NS 23.59 22.86 27/04/98 8.01 8.03 41.9 42.1 NS NS 22.00 22.01 27/04/98 7.98 7.99 40.9 41.5 NS NS 22.03 21.94 04/05/98 7.28 7.23 4.1 4.1 NS NS 20.43 20.41 04/05/98 7.08 7.07 3.8 3.8 NS NS 20.35 20.33 15/05/98 7.43 7.45 28.8 30.0 76.2 75.3 13.99 13.93 15/05/98 7.33 7.34 27.4 28.2 76.8 77.0 13.84 13.78 02/06/98 7.49 7.54 28.9 30.2 82.2 82.0 12.72 12.88 02/06/98 7.43 7.44 27.8 28.7 80.7 80.7 12.60 12.83 05/06/98 7.02 6.94 6.0 7.3 96.7 96.7 11.22 11.37 05/06/98 6.96 6.93 5.3 5.7 95.0 95.3 11.35 11.15 21/07/98 7.97 8.02 40.1 40.7 79.0 79.5 17.76 17.80 21/07/98 7.96 7.98 39.6 40.2 79.1 79.4 17.73 17.88 31/07/98 8.03 8.03 39.5 39.5 91.7 91.1 13.95 13.95 31/07/98 8.02 8.03 38.1 38.2 89.9 90.0 13.77 13.76 10/08/98 8.02 8.02 46.8 46.8 93.0 93.2 16.72 16.72 10/08/98 8.00 8.01 46.6 30.5 94.6 95.5 16.74 16.76 17/08/98 7.36 7.37 15.7 17.2 111.8 109.5 15.37 15.42 17/08/98 7.36 7.33 14.4 16.0 110.0 109.0 15.39 15.36 31/08/98 6.93 6.93 6.5 7.2 101.0 101.4 17.19 16.40 31/08/98 6.88 6.88 5.8 6.6 104.6 102.7 16.45 15.95 15/09/98 6.84 6.83 4.5 5.2 91.4 93.2 19.92 19.86 15/09/98 6.83 6.79 3.6 4.6 91.4 92.9 19.96 19.68 01/10/98 7.63 7.74 25.6 37.3 94.6 97.6 24.51 22.37 01/10/98 7.72 7.75 38.9 41.5 100.6 100.9 22.08 21.73 16/10/98 7.38 7.41 45.8 45.6 84.0 89.1 22.86 22.95 16/10/98 7.35 7.36 45.2 45.2 88.9 89.7 23.14 23.12 09/11/98 7.85 7.92 47.7 48.5 74.6 76.1 21.93 21.73 09/11/98 7.84 7.85 47.9 47.7 73.7 73.0 21.96 21.95 07/12/98 7.86 7.88 60.5 60.5 95.4 103.0 26.70 26.66 07/12/98 7.84 7.85 60.2 60.3 101.1 101.2 26.92 26.82 25/01/99 7.85 7.86 47.5 47.5 75.6 92.9 27.00 26.99 25/01/99 7.83 7.84 47.4 47.5 79.9 82.5 26.97 26.90 04/02/99 8.19 8.25 33.7 37.7 87.7 114.3 26.56 26.13 04/02/99 8.12 8.17 31.0 32.9 99.2 100.8 26.73 26.52 04/03/99 8.05 8.14 29.4 30.4 124.2 124.0 27.36 27.25 04/03/99 8.03 8.05 29.1 29.3 124.3 123.2 27.45 27.41 30/03/99 7.64 7.64 34.4 34.3 121.3 123.7 24.35 24.34 30/03/99 7.60 7.61 33.4 33.4 119.8 121.8 24.29 24.30

Summary: Summary:

N of cases 2525252519192525 N of cases 2626262619192626 Minimum 6.84 6.83 4.1 4.1 50.9 61.8 11.22 11.37 Minimum 6.83 6.79 3.6 3.8 61.2 63.3 11.35 11.15 Maximum 8.23 8.25 60.5 60.5 124.2 124.0 27.36 27.25 Maximum 8.20 8.22 60.2 60.3 124.3 123.2 27.45 27.41 Median 7.86 7.90 40.10 40.70 91.40 93.20 21.93 21.80 Median 7.87 7.87 40.25 40.85 91.40 92.90 22.06 21.95 Mean 7.74 7.76 35.4 36.5 90.2 94.2 20.69 20.55 Mean 7.71 7.72 35.7 35.7 92.4 92.8 20.83 20.73 Standard Dev. 0.42 0.43 16.8 16.4 17.1 16.3 4.76 4.68 Standard Dev. 0.42 0.44 16.7 16.4 15.9 15.6 4.80 4.78

NS = Not sampled NS = Not sampled

Appendix K. (Continued)

SITE: 13 SITE: 14

pH Elec. Cond. Dissolved Oxygen Temperature pH Elec. Cond. Dissolved Oxygen Temperature (mS cm-1) (% Saturation) (oC) (mS cm-1) (% Saturation) (oC)

Date surface bed surface bed surface bed surface bed Date surface bed surface bed surface bed surface bed

17/11/97 NS NS 50.8 50.9 83.3 83.7 19.10 19.00 17/11/97 NS NS NS NS NS NS NS NS 04/12/97 8.12 8.06 50.5 51.4 60.3 66.3 23.10 22.50 04/12/97 NS NS NS NS NS NS NS NS 20/03/98 NS NS NS NS NS NS 26.55 26.35 20/03/98 7.87 7.92 49.0 49.0 NS NS 26.55 26.35 25/03/98 7.89 7.89 48.9 48.9 NS NS 24.28 24.28 25/03/98 7.89 7.89 48.9 48.9 NS NS 24.30 24.30 27/03/98 8.11 8.11 51.4 51.3 NS NS 23.64 23.62 27/03/98 8.10 8.11 51.1 51.2 NS NS 23.51 23.53 02/04/98 7.97 7.99 43.0 40.1 NS NS 22.91 23.14 02/04/98 NS NS NS NS NS NS NS NS 17/04/98 8.00 8.00 43.3 46.5 NS NS 23.34 22.80 17/04/98 NS NS NS NS NS NS NS NS 27/04/98 7.96 7.96 40.2 40.2 NS NS 22.08 22.09 27/04/98 NS NS NS NS NS NS NS NS 04/05/98 7.11 7.06 3.5 3.7 NS NS 20.38 20.33 04/05/98 NS NS NS NS NS NS NS NS 15/05/98 7.36 7.29 26.3 26.6 76.4 76.1 13.71 13.69 15/05/98 NS NS NS NS NS NS NS NS 02/06/98 7.39 7.39 26.8 26.8 81.1 81.1 12.58 12.54 02/06/98 7.36 7.36 26.1 26.4 81.9 81.1 12.59 12.59 05/06/98 6.89 6.85 4.1 4.2 94.7 94.8 11.03 11.03 05/06/98 6.91 6.88 3.6 3.6 95.2 95.3 10.99 10.99 21/07/98 7.92 7.94 39.4 39.4 78.3 79.1 17.71 17.70 21/07/98 7.94 7.95 39.2 39.2 79.8 80.2 17.70 17.70 31/07/98 7.93 7.97 36.3 37.2 88.5 88.7 13.67 13.70 31/07/98 NS NS NS NS NS NS NS NS 10/08/98 7.93 7.94 46.4 46.4 94.7 94.8 16.68 16.67 10/08/98 NS NS NS NS NS NS NS NS 17/08/98 7.25 7.25 13.1 13.3 110.6 110.2 15.28 15.27 17/08/98 7.22 7.22 12.2 12.6 111.9 109.7 15.27 15.27 31/08/98 6.73 6.64 4.1 6.1 102.4 100.4 15.28 16.52 31/08/98 6.72 6.74 3.1 3.8 99.2 100.7 15.94 15.00 15/09/98 6.82 6.79 3.2 4.1 91.8 92.9 19.88 19.61 15/09/98 6.81 6.81 2.5 2.5 93.4 93.4 19.55 19.45 01/10/98 7.68 7.76 39.5 45.6 99.4 100.5 22.08 21.12 01/10/98 7.72 7.74 47.6 48.4 104.2 107.5 20.78 20.64 16/10/98 7.24 7.26 44.8 44.8 86.4 86.4 23.10 23.03 16/10/98 7.22 7.23 44.7 44.7 83.1 84.8 23.13 23.13 09/11/98 7.76 7.82 46.8 47.6 70.2 70.2 22.15 22.03 09/11/98 7.80 7.79 47.4 47.4 71.1 71.7 22.03 22.08 07/12/98 7.82 7.83 59.9 60.0 99.6 100.7 27.16 27.12 07/12/98 7.77 7.80 59.8 59.9 100.2 101.9 27.27 27.20 25/01/99 7.81 7.79 47.3 47.4 74.3 76.9 27.16 26.97 25/01/99 7.78 7.78 47.4 47.4 77.2 79.4 27.19 27.03 04/02/99 8.05 8.09 28.9 30.2 89.9 108.8 26.79 26.64 04/02/99 8.00 8.02 27.7 28.3 96.5 97.3 26.95 26.77 04/03/99 7.99 8.00 29.0 29.0 132.7 128.8 27.58 27.57 04/03/99 8.01 8.01 28.8 28.9 123.6 123.4 27.67 27.63 30/03/99 7.57 7.57 32.3 32.4 125.0 126.2 24.27 24.27 30/03/99 7.56 7.56 31.8 31.8 123.1 123.4 24.34 24.34

Summary: Summary:

N of cases 2424252519192626 N of cases 1717171714141717 Minimum 6.73 6.64 3.2 3.7 60.3 66.3 11.03 11.03 Minimum 6.72 6.74 2.5 2.5 71.1 71.7 10.99 10.99 Maximum 8.12 8.11 59.9 60.0 132.7 128.8 27.58 27.57 Maximum 8.10 8.11 59.8 59.9 123.6 123.4 27.67 27.63 Median 7.82 7.83 39.50 40.10 89.90 92.90 22.12 22.06 Median 7.77 7.78 39.20 39.20 95.85 96.30 23.13 23.13 Mean 7.64 7.64 34.4 35.0 91.6 93.0 20.83 20.75 Mean 7.57 7.58 33.6 33.8 95.7 96.4 21.52 21.41 Standard Dev. 0.43 0.45 17.0 16.9 17.9 17.3 4.94 4.84 Standard Dev. 0.45 0.45 18.7 18.6 16.3 16.0 5.36 5.39

NS = Not sampled NS = Not sampled

Appendix K. (Continued)

SITE: 15 SITE: 16

pH Elec. Cond. Dissolved Oxygen Temperature pH Elec. Cond. Dissolved Oxygen Temperature (mS cm-1) (% Saturation) (oC) (mS cm-1) (% Saturation) (oC)

Date surface bed surface bed surface bed surface bed Date surface bed surface bed surface bed surface bed

17/11/97 8.17 8.19 49.1 49.9 78.4 82.3 19.50 19.50 17/11/97 8.13 8.15 47.6 48.5 79.5 85.2 20.30 20.00 04/12/97 8.11 8.13 50.6 50.6 62.9 63.3 22.90 22.90 04/12/97 8.11 8.10 50.6 50.6 59.2 59.9 23.00 23.00 20/03/98 7.83 7.90 49.2 49.1 NS NS 26.51 26.36 20/03/98 7.80 7.86 49.2 49.1 NS NS 26.43 26.40 25/03/98 7.89 7.90 48.9 48.9 NS NS 24.34 24.32 25/03/98 7.87 7.87 48.6 48.6 NS NS 24.33 24.32 27/03/98 8.10 8.11 51.0 51.0 NS NS 23.64 23.56 27/03/98 8.09 8.10 51.0 51.0 NS NS 23.39 23.40 02/04/98 7.94 7.94 43.1 43.3 NS NS 22.80 22.82 02/04/98 7.92 7.92 41.9 41.9 NS NS 22.62 22.62 17/04/98 7.95 7.96 44.2 44.7 NS NS 22.80 22.74 17/04/98 7.91 7.91 43.3 43.5 NS NS 22.85 22.68 27/04/98 7.95 7.96 39.2 39.7 NS NS 22.01 22.13 27/04/98 7.90 7.91 37.7 37.5 NS NS 21.86 21.86 04/05/98 6.99 6.95 2.6 2.7 NS NS 20.38 20.36 04/05/98 6.91 6.88 2.2 2.2 NS NS 20.43 20.42 15/05/98 7.28 7.26 24.7 25.3 76.7 76.9 13.63 13.71 15/05/98 7.27 7.22 23.7 23.8 76.8 76.9 13.64 13.67 02/06/98 7.32 7.32 25.2 25.7 81.1 80.9 12.78 12.70 02/06/98 7.26 7.26 24.4 24.5 80.7 80.5 12.95 12.94 05/06/98 6.77 6.73 3.1 3.5 95.4 95.1 11.02 11.20 05/06/98 6.84 6.81 2.5 2.5 96.0 95.6 10.64 10.63 21/07/98 7.89 7.93 38.8 39.2 79.9 81.1 17.79 17.79 21/07/98 7.82 7.86 37.7 37.8 80.3 80.7 17.84 17.79 31/07/98 NS NS NS NS NS NS NS NS 31/07/98 NS NS NS NS NS NS NS NS 10/08/98 7.88 7.92 46.1 46.3 94.8 97.5 16.67 16.88 10/08/98 7.80 7.85 45.5 45.9 91.7 92.5 16.57 16.58 17/08/98 7.18 7.18 11.1 11.8 110.2 108.8 15.26 15.27 17/08/98 7.18 7.15 9.9 11.2 109.3 106.6 15.28 15.35 31/08/98 6.67 6.69 2.6 3.1 99.6 100.5 15.27 14.81 31/08/98 6.77 6.76 1.5 2.5 100.9 100.0 15.93 14.98 15/09/98 6.82 6.79 2.1 2.4 94.0 93.7 19.58 19.80 15/09/98 6.82 6.79 1.7 1.8 93.1 93.5 19.58 19.55 01/10/98 7.63 7.65 48.2 50.5 104.6 106.0 20.69 20.27 01/10/98 7.55 7.56 48.7 51.6 106.6 107.9 20.64 20.00 16/10/98 7.15 7.16 44.3 44.3 81.6 82.5 23.41 23.41 16/10/98 7.13 7.13 44.0 44.0 80.7 83.2 23.86 23.69 09/11/98 7.72 7.73 47.1 45.7 66.8 65.0 22.17 22.17 09/11/98 7.67 7.69 46.8 46.8 67.8 65.1 22.27 22.25 07/12/98 7.80 7.83 59.7 60.0 99.8 99.9 27.36 27.07 07/12/98 7.78 7.80 59.2 59.4 98.7 102.2 27.79 27.62 25/01/99 7.76 7.76 47.2 47.3 76.8 87.7 27.62 26.93 25/01/99 7.75 7.76 47.2 47.2 82.5 86.7 27.62 27.16 04/02/99 7.99 8.01 26.4 27.3 94.0 108.5 27.21 26.87 04/02/99 7.84 7.90 24.5 26.2 93.5 94.8 26.98 26.94 04/03/99 8.03 8.01 28.8 28.8 129.4 126.8 27.99 27.69 04/03/99 8.01 8.02 28.7 29.0 130.5 125.1 27.42 27.31 30/03/99 7.57 7.57 31.0 30.7 123.5 126.8 24.41 24.41 30/03/99 7.57 7.56 30.3 30.4 134.3 133.8 24.44 24.48

Summary: Summary:

N of cases 2525252518182525 N of cases 2525252518182525 Minimum 6.67 6.69 2.1 2.4 62.9 63.3 11.02 11.20 Minimum 6.77 6.76 1.5 1.8 59.2 59.9 10.64 10.63 Maximum 8.17 8.19 59.7 60.0 129.4 126.8 27.99 27.69 Maximum 8.13 8.15 59.2 59.4 134.3 133.8 27.79 27.62 Median 7.80 7.83 43.10 43.30 94.00 94.40 22.17 22.17 Median 7.78 7.80 41.90 41.90 92.40 93.00 22.27 22.25 Mean 7.62 7.62 34.6 34.9 91.6 93.5 21.11 21.03 Mean 7.59 7.59 33.9 34.3 92.3 92.8 21.15 21.03 Standard Dev. 0.46 0.47 17.9 17.8 18.0 18.0 4.88 4.78 Standard Dev. 0.43 0.45 18.0 18.0 19.5 18.6 4.84 4.84

NS = Not sampled NS = Not sampled

Appendix K. (Continued)

SITE: 17 SITE: 18

pH Elec. Cond. Dissolved Oxygen Temperature pH Elec. Cond. Dissolved Oxygen Temperature (mS cm-1) (% Saturation) (oC) (mS cm-1) (% Saturation) (oC)

Date surface bed surface bed surface bed surface bed Date surface bed surface bed surface bed surface bed

17/11/97 8.09 8.12 46.8 49.6 81.2 81.7 20.50 19.50 17/11/97 8.13 8.11 47.5 49.5 81.0 81.2 20.30 19.50 04/12/97 8.10 8.09 49.7 50.3 58.7 61.6 23.10 23.10 04/12/97 NS NS NS NS NS NS NS NS 20/03/98 NS NS NS NS NS NS NS NS 20/03/98 NS NS NS NS NS NS NS NS 25/03/98 7.85 7.85 48.3 48.3 NS NS 24.37 24.37 25/03/98 7.84 7.89 48.5 49.1 NS NS 24.59 24.40 27/03/98 8.00 8.07 50.8 51.3 NS NS 23.26 23.42 27/03/98 8.03 8.10 50.2 50.8 NS NS 23.14 23.08 02/04/98 7.92 7.91 41.6 41.6 NS NS 22.59 22.58 02/04/98 7.88 7.80 37.5 48.7 NS NS 22.34 23.44 17/04/98 7.87 7.87 42.3 42.4 NS NS 22.66 22.61 17/04/98 7.78 7.89 39.0 46.6 NS NS 22.88 22.41 27/04/98 7.86 7.95 35.4 39.4 NS NS 21.80 21.71 27/04/98 7.97 8.09 40.5 44.7 NS NS 21.96 21.17 04/05/98 6.89 6.85 2.1 2.0 NS NS 20.78 20.71 04/05/98 6.87 6.72 2.1 6.1 NS NS 21.56 20.26 15/05/98 7.20 7.17 20.7 21.7 78.8 78.0 13.61 13.58 15/05/98 7.28 7.58 22.5 37.6 77.4 70.3 13.63 14.33 02/06/98 7.23 7.27 23.6 25.3 81.0 80.4 13.05 12.95 02/06/98 7.24 7.64 23.9 35.8 82.7 78.3 12.84 13.58 05/06/98 6.74 6.57 1.2 2.7 93.8 93.9 10.43 10.63 05/06/98 6.70 6.72 2.2 32.9 92.3 44.8 10.88 13.44 21/07/98 7.72 7.82 36.6 37.0 80.0 80.2 17.84 17.80 21/07/98 7.78 8.03 36.1 45.1 80.4 74.3 17.89 17.80 31/07/98 NS NS NS NS NS NS NS NS 31/07/98 NS NS NS NS NS NS NS NS 10/08/98 7.72 7.75 45.3 45.4 90.0 91.3 16.51 16.47 10/08/98 7.63 7.69 45.7 46.1 89.8 89.5 16.37 16.01 17/08/98 7.42 7.23 8.8 13.9 102.6 104.5 15.35 15.42 17/08/98 7.42 7.76 10.1 29.4 108.2 107.2 15.13 15.84 31/08/98 6.84 6.75 1.9 2.8 99.9 100.2 16.19 15.66 31/08/98 6.90 6.97 3.4 13.3 102.0 82.1 15.96 16.82 15/09/98 6.87 6.73 0.9 2.0 92.7 92.9 19.82 19.66 15/09/98 6.81 6.79 1.4 2.6 92.2 92.8 19.58 18.74 01/10/98 7.40 7.41 48.6 52.8 100.9 105.3 20.60 19.70 01/10/98 7.19 7.19 51.4 53.9 110.1 109.8 20.10 19.51 16/10/98 7.11 7.11 43.7 43.7 84.9 86.8 24.27 24.20 16/10/98 7.03 7.07 43.9 43.7 80.1 83.7 24.55 24.27 09/11/98 7.64 7.65 46.4 46.3 65.5 63.9 22.28 22.27 09/11/98 7.54 7.55 43.6 44.7 62.5 59.3 22.32 22.32 07/12/98 7.74 7.81 58.5 59.9 100.8 100.1 28.41 27.15 07/12/98 7.68 7.88 59.0 61.4 99.2 98.9 28.00 25.87 25/01/99 7.72 7.74 47.3 46.9 80.4 90.9 27.68 27.17 25/01/99 7.71 7.72 47.1 47.6 78.5 74.9 27.51 27.06 04/02/99 7.79 7.87 23.6 26.5 95.1 96.1 27.09 26.83 04/02/99 7.78 8.02 24.2 29.3 93.0 89.7 27.03 26.12 04/03/99 7.98 7.99 28.0 28.2 130.8 128.7 27.73 27.74 04/03/99 7.91 8.03 26.4 31.4 131.6 118.6 29.58 24.85 30/03/99 7.58 7.55 29.1 31.1 137.0 131.2 24.48 24.37 30/03/99 7.51 7.67 30.1 36.8 134.8 114.4 24.81 23.49

Summary: Summary:

N of cases 2424242418182424 N of cases 2323232317172323 Minimum 6.74 6.57 0.9 2.0 58.7 61.6 10.43 10.63 Minimum 6.70 6.72 1.4 2.6 62.5 44.8 10.88 13.44 Maximum 8.10 8.12 58.5 59.9 137.0 131.2 28.41 27.74 Maximum 8.13 8.11 59.0 61.4 134.8 118.6 29.58 27.06 Median 7.72 7.75 39.10 40.50 91.35 92.10 22.04 21.99 Median 7.63 7.72 37.50 44.70 92.20 83.70 21.96 21.17 Mean 7.55 7.55 32.6 33.8 91.9 92.7 21.02 20.82 Mean 7.50 7.60 32.0 38.6 93.9 86.5 21.00 20.62 Standard Dev. 0.42 0.48 18.2 18.0 19.4 18.3 4.89 4.79 Standard Dev. 0.42 0.46 17.9 14.8 19.1 19.6 5.10 4.19

NS = Not sampled NS = Not sampled

Appendix K. (Continued)

SITE: 19

pH Elec. Cond. Dissolved Oxygen Temperature (mS cm-1) (% Saturation) (oC)

Date surface bed surface bed surface bed surface bed

17/11/97 NS NS NS NS NS NS NS NS 04/12/97 NS NS NS NS NS NS NS NS 20/03/98 7.80 7.80 49.0 49.0 NS NS 27.78 26.57 25/03/98 7.86 7.87 48.3 48.8 NS NS 24.46 24.45 27/03/98 8.00 8.03 50.1 50.1 NS NS 23.24 23.17 02/04/98 7.83 7.81 36.5 48.7 NS NS 22.57 23.51 17/04/98 7.75 7.89 37.3 46.4 NS NS 22.91 22.52 27/04/98 7.98 8.08 40.0 45.3 NS NS 21.95 21.20 04/05/98 6.83 7.43 2.0 3.2 NS NS 21.39 20.96 15/05/98 7.16 7.53 23.6 38.9 75.0 64.9 13.35 14.54 02/06/98 7.24 7.62 23.5 37.4 80.1 75.8 12.63 13.79 05/06/98 6.31 6.87 1.6 26.5 93.3 94.6 11.03 12.90 21/07/98 7.71 8.11 37.1 47.6 79.7 69.8 18.02 18.04 31/07/98 7.72 7.69 26.3 30.6 92.1 90.8 13.06 12.97 10/08/98 7.49 7.56 44.7 45.4 88.2 87.2 16.48 15.73 17/08/98 7.07 7.85 7.7 31.8 106.7 97.1 15.37 15.89 31/08/98 6.68 6.70 3.1 27.0 102.4 20.8 15.93 17.41 15/09/98 6.78 6.73 1.3 38.8 92.7 31.4 19.65 18.78 01/10/98 6.90 6.92 53.2 54.1 111.4 111.6 19.57 19.41 16/10/98 6.92 7.01 43.4 44.1 82.2 74.4 25.00 22.43 09/11/98 7.54 7.53 43.7 46.1 65.4 61.7 22.61 23.02 07/12/98 7.68 7.81 59.3 60.6 94.5 97.9 27.74 26.56 25/01/99 7.66 7.69 45.8 47.8 77.1 85.3 27.75 26.95 04/02/99 7.78 7.80 23.6 24.3 89.9 95.6 27.00 27.01 04/03/99 7.98 8.04 27.6 29.2 133.8 128.6 29.58 27.09 30/03/99 7.58 7.67 30.0 37.5 143.9 122.3 24.80 23.22

Summary:

N of cases 2424242417172424 Minimum 6.31 6.70 1.3 3.2 65.4 20.8 11.03 12.90 Maximum 8.00 8.11 59.3 60.6 143.9 128.6 29.58 27.09 Median 7.62 7.69 36.80 44.70 92.10 87.20 22.26 21.82 Mean 7.43 7.59 31.6 40.0 94.6 82.9 20.99 20.76 Standard Dev. 0.48 0.43 17.8 12.3 20.4 28.3 5.46 4.72

NS = Not sampled

Appendix L. List of analytical data for surface and bed waters for Sites 1, 4, 12 and 19 located in Limeburners Creek and the lower Hastings River.

SITE: 1

Date Measurement Alkalinity Cl:SO4 Lab. Fe Al Ca Mn K Mg S SO4 As Cu Si Zn (mg L-1) pH (mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)

25/03/98 surface 85 12.0 7.81 <0.01 <0.05 347.67 <0.002 400.48 986.12 720.45 2161.34 <0.40 <0.004 <0.03 <0.01 02/04/98 surface 65 12.7 7.93 <0.01 <0.05 171.55 <0.002 178.50 500.80 507.66 1522.97 <0.40 <0.004 0.01 <0.01 02/04/98 bed 56 37.3 7.95 <0.01 <0.05 121.53 <0.002 148.94 351.38 277.88 833.64 <0.40 <0.004 <0.03 <0.01 17/04/98 surface 52 15.1 7.80 <0.01 <0.05 187.47 <0.002 209.63 560.89 499.34 1498.01 <0.40 <0.004 0.18 <0.01 17/04/98 bed 65 14.5 7.89 <0.01 <0.05 207.28 <0.002 220.46 596.52 571.14 1713.42 <0.40 <0.004 0.01 <0.01 27/04/98 surface 56 25.9 7.93 <0.01 <0.05 99.31 <0.002 101.06 291.11 313.73 941.18 <0.40 <0.004 <0.03 <0.01 27/04/98 bed 56 21.6 7.83 <0.01 <0.05 138.75 <0.002 153.52 418.90 443.11 1329.33 <0.40 <0.004 0.08 <0.01 04/05/98 surface 38 24.0 7.78 <0.01 <0.05 14.89 <0.002 14.40 44.52 61.94 185.81 <0.40 <0.004 <0.03 <0.01 04/05/98 bed 38 23.3 6.79 <0.01 <0.05 20.61 <0.002 22.91 67.52 80.26 240.77 <0.40 <0.004 <0.03 <0.01 20/05/98 surface 52 28.1 7.54 <0.01 <0.05 98.47 <0.002 98.36 287.31 191.95 575.84 <0.40 <0.004 0.16 <0.01 20/05/98 bed 56 NS 7.72 <0.01 <0.05 NS <0.002 NS NS NS NS <0.40 <0.004 <0.03 <0.01 02/06/98 surface 60 12.5 7.71 <0.01 <0.05 287.41 <0.002 330.29 829.59 597.85 1793.55 <0.40 <0.004 0.87 <0.01 02/06/98 bed 73 9.4 7.71 <0.01 <0.05 298.38 <0.002 345.69 861.42 620.93 1862.80 <0.40 <0.004 0.76 <0.01 05/06/98 surface 65 7.3 7.47 <0.005 <0.03 265.99 0.002 271.87 796.27 503.28 1509.85 <0.15 0.018 1.36 <0.02 05/06/98 bed 56 7.9 7.60 <0.005 <0.03 283.83 0.001 291.21 849.08 500.36 1501.07 <0.15 0.006 0.77 <0.02 21/07/98 surface 80 9.1 7.98 <0.005 <0.03 469.44 0.001 508.09 1379.88 686.25 2058.75 <0.15 0.004 0.86 <0.02 31/07/98 surface 65 10.6 7.86 <0.005 <0.03 379.20 0.000 402.73 1114.03 647.95 1943.84 <0.15 0.003 0.75 <0.02 31/07/98 bed 60 10.4 7.90 <0.005 <0.03 379.42 0.000 403.91 1109.51 659.26 1977.78 <0.15 0.003 0.66 <0.02 10/08/98 surface 73 13.6 7.98 <0.005 <0.03 413.86 0.001 446.80 1206.11 641.02 1923.05 <0.15 0.006 0.42 <0.02 10/08/98 bed 76 12.8 7.94 <0.005 <0.03 385.67 0.000 410.86 1123.13 613.65 1840.94 <0.15 0.004 0.65 <0.02 17/08/98 surface 52 8.5 6.87 0.005 <0.03 103.89 0.001 93.96 295.67 128.21 384.64 <0.15 0.005 2.56 <0.02 17/08/98 bed 52 9.5 7.00 0.005 <0.03 140.62 0.001 133.62 412.15 221.66 664.97 <0.15 0.004 2.06 <0.02 31/08/98 surface 56 9.3 7.23 0.005 <0.03 182.85 0.001 179.04 538.65 298.94 896.83 <0.15 0.001 1.93 <0.02 31/08/98 bed 65 7.5 7.45 <0.005 <0.03 255.91 0.002 260.59 756.47 413.34 1240.01 <0.15 0.002 1.50 <0.02 16/10/98 surface 73 16.5 8.08 <0.005 <0.03 436.60 0.003 480.01 1279.14 650.63 1951.88 <0.15 0.003 0.28 <0.02 16/10/98 bed 60 16.9 8.09 <0.005 <0.03 492.57 0.004 545.57 1431.25 654.29 1962.86 <0.15 0.004 0.21 <0.02 07/12/98 surface NS 8.5 7.89 <0.007 <0.05 285.87 <0.001 341.14 833.31 557.86 1673.58 <0.35 <0.005 0.53 <0.02 07/12/98 bed NS 8.1 7.92 <0.007 <0.05 336.89 <0.001 402.07 974.50 657.90 1973.70 <0.35 <0.005 0.51 <0.02 04/01/99 surface NS 8.1 7.81 <0.007 <0.05 197.28 <0.001 223.68 567.72 386.93 1160.80 <0.35 <0.005 2.33 <0.02 04/01/99 bed NS 7.7 7.81 <0.007 <0.05 215.84 <0.001 246.85 624.41 418.91 1256.73 <0.35 <0.005 2.11 <0.02 25/01/99 surface NS 8.8 7.96 <0.007 <0.05 265.37 <0.001 319.72 751.05 513.85 1541.55 <0.35 <0.005 0.93 <0.02 25/01/99 bed NS 9.2 7.98 <0.007 <0.05 264.38 0.01 319.23 755.76 503.57 1510.71 <0.35 <0.005 0.82 <0.02 04/03/99 surface NS 11.4 7.71 <0.007 <0.05 84.20 0.01 92.31 243.42 157.20 471.61 <0.35 <0.005 3.54 <0.02 30/03/99 surface NS 5.3 8.21 <0.007 <0.05 251.19 <0.001 275.38 737.34 519.84 1559.52 <0.35 <0.005 1.51 <0.02 30/03/99 bed NS 10.2 8.25 <0.007 <0.05 199.36 <0.001 228.85 584.72 379.26 1137.78 <0.35 <0.005 1.81 <0.02

NS = Not sampled

Appendix L. (Continued)

SITE: 4

Date Measurement Alkalinity Cl:SO4 Lab. Fe Al Ca Mn K Mg S SO4 As Cu Si Zn (mg L-1) pH (mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)

25/03/98 surface 76 10.1 7.93 <0.01 <0.05 179.91 <0.002 189.54 524.25 553.88 1661.65 <0.40 <0.004 0.02 <0.01 02/04/98 surface 56 8.7 7.96 <0.01 <0.05 205.47 <0.002 218.51 594.59 644.65 1933.95 <0.40 <0.004 0.18 <0.01 02/04/98 bed 52 14.9 7.95 <0.01 <0.05 200.86 <0.002 212.65 577.82 528.86 1586.58 <0.40 <0.004 0.08 <0.01 17/04/98 surface 60 11.0 7.82 <0.01 <0.05 268.00 <0.002 287.47 763.42 651.15 1953.46 <0.40 <0.004 0.38 <0.01 17/04/98 bed 52 9.5 7.85 <0.01 <0.05 214.71 <0.002 224.42 616.81 631.87 1895.62 <0.40 <0.004 0.44 <0.01 27/04/98 surface 60 15.5 7.71 <0.01 <0.05 130.71 <0.002 134.30 389.98 407.77 1223.32 <0.40 <0.004 0.26 <0.01 27/04/98 bed 60 28.8 7.79 <0.01 <0.05 74.22 <0.002 84.87 222.06 266.36 799.08 <0.40 <0.004 <0.03 <0.01 20/05/98 surface 56 11.1 7.70 <0.01 <0.05 189.23 <0.002 215.56 554.63 422.72 1268.17 <0.40 <0.004 0.43 <0.01 20/05/98 bed 56 53.3 7.68 0.04 0.12 40.27 <0.002 35.53 114.15 91.88 275.65 <0.40 <0.004 0.41 <0.01 02/06/98 surface 73 10.1 7.70 <0.01 <0.05 266.75 <0.002 302.53 768.22 565.07 1695.21 <0.40 <0.004 1.02 <0.01 02/06/98 bed 65 11.6 7.69 <0.01 <0.05 287.65 <0.002 329.78 827.44 601.77 1805.30 <0.40 <0.004 0.77 <0.01 21/07/98 surface 65 9.2 7.80 <0.005 <0.03 405.24 0.000 433.68 1193.77 658.00 1973.99 <0.15 0.003 1.13 <0.02 31/07/98 surface 73 10.8 8.10 <0.005 <0.03 322.86 0.001 336.28 946.97 551.65 1654.95 <0.15 0.008 0.71 <0.02 31/07/98 bed 76 11.1 7.81 <0.005 <0.03 329.00 0.000 344.10 965.26 570.08 1710.23 <0.15 0.006 0.96 <0.02 10/08/98 surface 76 12.5 7.84 <0.005 <0.03 361.00 0.001 380.71 1048.42 567.42 1702.26 0.031 0.006 0.85 <0.02 10/08/98 bed 60 12.6 7.95 <0.005 <0.03 416.32 0.001 447.04 1210.24 651.92 1955.77 <0.15 0.002 0.37 <0.02 17/08/98 surface 21 8.5 6.48 <0.005 <0.03 26.88 0.001 21.48 67.97 46.48 139.45 0.029 0.004 2.74 <0.02 17/08/98 bed 52 9.0 6.90 0.005 <0.03 97.49 0.001 89.58 283.16 167.38 502.14 0.014 0.008 2.35 <0.02 31/08/98 surface 56 8.9 7.41 <0.005 <0.03 155.65 0.001 153.16 459.48 256.12 768.36 0.025 0.006 2.06 <0.02 31/08/98 bed 56 7.6 7.35 0.007 <0.03 219.84 0.001 221.30 648.10 352.10 1056.31 <0.15 0.007 1.80 <0.02 15/09/98 surface 26 4.6 6.82 0.091 <0.03 66.13 0.004 60.26 192.30 113.79 341.38 0.023 0.006 2.83 <0.02 16/10/98 surface 52 16.9 8.07 <0.005 <0.03 421.20 0.007 458.91 1235.47 576.64 1729.92 <0.15 0.003 0.55 <0.02 16/10/98 bed 65 17.0 8.06 <0.005 <0.03 427.60 0.007 469.93 1251.11 597.83 1793.48 <0.15 0.004 0.57 <0.02 07/12/98 surface NS 8.1 7.86 <0.007 0.01 303.73 0.01 359.27 864.90 583.72 1751.15 <0.35 <0.005 1.06 <0.02 07/12/98 bed NS 7.9 7.98 0.01 <0.05 356.21 <0.001 433.05 1022.37 688.19 2064.56 <0.35 <0.005 0.37 <0.02 04/01/99 surface NS 8.7 7.76 <0.007 <0.05 91.32 <0.001 103.46 266.15 216.97 650.92 <0.35 <0.005 2.50 <0.02 04/01/99 bed NS 2.9 7.61 0.01 <0.05 296.12 <0.001 349.18 851.61 552.96 1658.88 <0.35 <0.005 1.17 <0.02 25/01/99 surface NS 10.4 7.94 <0.007 0.01 165.33 <0.001 202.17 485.56 311.82 935.46 <0.35 <0.005 0.86 <0.02 25/01/99 bed NS 10.1 7.61 <0.007 <0.05 218.97 <0.001 269.15 637.37 387.68 1163.03 <0.35 <0.005 0.90 <0.02 04/03/99 surface NS 9.2 7.73 <0.007 <0.05 123.76 <0.001 135.16 361.60 237.67 713.00 <0.35 <0.005 3.06 <0.02 30/03/99 surface NS 7.3 8.25 <0.007 0.03 250.83 0.01 275.72 735.65 499.62 1498.86 <0.35 <0.005 2.30 <0.02 30/03/99 bed NS 7.6 8.22 <0.007 0.02 246.85 <0.001 277.53 711.16 491.83 1475.49 <0.35 <0.005 1.74 <0.02

NS = Not sampled

Appendix L. (Continued)

SITE: 12

Date Measurement ALK Cl:SO4 Lab. Fe Al Ca Mn K Mg S SO4 As Cu Si Zn (mg L-1) pH (mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)

25/03/98 surface 56 12.0 7.95 <0.01 <0.05 374.91 <0.002 432.62 1059.07 749.03 2247.09 <0.40 <0.004 0.18 <0.01 02/04/98 surface 65 20.3 7.93 <0.01 <0.05 55.03 <0.002 64.17 164.36 237.33 711.98 <0.40 <0.004 <0.03 <0.01 02/04/98 bed 56 18.1 7.91 <0.01 <0.05 134.92 <0.002 163.64 392.76 364.75 1094.25 <0.40 <0.004 0.01 <0.01 17/04/98 surface 56 12.7 7.81 <0.01 <0.05 132.58 <0.002 138.29 386.72 432.27 1296.81 <0.40 <0.004 0.20 <0.01 17/04/98 bed 47 16.7 7.92 <0.01 <0.05 140.86 <0.002 146.91 411.14 448.23 1344.68 <0.40 <0.004 0.07 <0.01 27/04/98 surface 56 35.4 7.71 <0.01 <0.05 57.80 <0.002 57.94 172.23 205.66 616.98 <0.40 <0.004 <0.03 <0.01 27/04/98 bed 56 20.8 7.71 <0.01 <0.05 86.48 <0.002 85.23 254.84 308.42 925.27 <0.40 <0.004 0.08 <0.01 04/05/98 surface 13 36.3 6.63 <0.01 <0.05 12.16 <0.002 12.93 34.99 27.41 82.24 <0.40 <0.004 <0.03 <0.01 04/05/98 bed 8 12.8 6.37 <0.01 <0.05 20.40 <0.002 19.69 57.14 43.38 130.13 <0.40 <0.004 0.16 <0.01 15/05/98 surface 73 21.1 7.39 <0.01 <0.05 100.36 <0.002 103.21 299.47 229.13 687.38 <0.40 <0.004 0.81 <0.01 15/05/98 bed 52 55.7 7.44 <0.01 <0.05 25.97 <0.002 26.05 77.02 72.30 216.90 <0.40 <0.004 <0.03 <0.01 02/06/98 surface 65 9.1 7.45 <0.01 <0.05 209.29 <0.002 228.84 610.92 448.84 1346.51 <0.40 <0.004 0.95 <0.01 02/06/98 bed 52 8.8 7.32 <0.01 <0.05 218.36 <0.002 240.02 637.08 465.81 1397.43 <0.40 <0.004 0.92 <0.01 21/07/98 surface 65 9.0 7.74 <0.005 <0.03 413.91 0.001 441.97 1222.33 608.21 1824.64 <0.15 0.011 0.28 <0.02 31/07/98 surface 76 9.5 7.54 <0.005 <0.03 339.50 0.001 355.63 995.29 561.97 1685.91 <0.15 0.003 0.82 <0.02 31/07/98 bed 65 9.9 7.53 <0.005 <0.03 367.43 0.001 388.22 1076.61 557.99 1673.98 <0.15 0.003 0.83 <0.02 10/08/98 surface 73 12.0 7.90 <0.005 <0.03 400.35 0.001 426.15 1169.48 617.98 1853.93 <0.15 0.002 0.52 <0.02 10/08/98 bed 76 12.4 7.89 <0.005 <0.03 389.73 0.001 414.28 1139.40 622.59 1867.76 <0.15 0.001 0.55 <0.02 17/08/98 surface 52 12.6 6.99 0.048 <0.03 121.89 0.001 119.24 357.09 171.72 515.15 <0.15 0.010 1.02 <0.02 17/08/98 bed 52 9.8 6.96 0.051 <0.03 116.76 0.001 113.47 342.68 194.78 584.34 <0.15 0.006 1.02 <0.02 31/08/98 surface 42 10.6 6.91 0.259 <0.03 42.31 0.001 38.71 119.05 69.01 207.02 <0.15 0.006 1.27 <0.02 31/08/98 bed 38 9.2 6.83 0.309 <0.03 45.92 0.001 42.08 128.67 78.28 234.83 <0.15 0.004 1.33 <0.02 15/09/98 surface 13 3.5 6.73 0.331 <0.03 23.54 0.001 21.71 61.99 37.52 112.55 <0.15 0.006 1.53 <0.02 16/10/98 surface 52 14.4 7.75 <0.005 <0.03 366.75 0.009 391.62 1073.33 560.09 1680.28 <0.15 0.005 0.85 <0.02 16/10/98 bed 73 16.2 7.78 <0.005 <0.03 369.34 0.009 391.77 1080.78 518.23 1554.68 <0.15 0.004 0.80 <0.02 07/12/98 surface NS 6.6 7.80 <0.007 <0.05 294.19 <0.001 336.14 839.10 581.63 1744.88 <0.35 <0.005 1.07 <0.02 07/12/98 bed NS 7.2 7.77 <0.007 <0.05 295.34 <0.001 341.02 855.08 578.85 1736.54 <0.35 <0.005 1.09 <0.02 04/01/99 surface NS 8.6 7.56 <0.007 <0.05 177.07 <0.001 204.61 508.03 339.21 1017.64 <0.35 <0.005 1.39 <0.02 04/01/99 bed NS 8.8 7.72 <0.007 <0.05 213.63 <0.001 247.10 612.69 408.22 1224.65 <0.35 <0.005 1.49 <0.02 25/01/99 surface NS 8.4 7.82 <0.007 <0.05 296.52 <0.001 361.60 859.61 559.86 1679.58 <0.35 <0.005 0.99 <0.02 25/01/99 bed NS 8.2 7.80 <0.007 <0.05 321.59 <0.001 387.92 924.23 597.85 1793.54 <0.35 <0.005 1.07 <0.02 04/03/99 surface NS 10.4 7.72 0.01 <0.05 147.20 <0.001 168.91 431.34 283.93 851.80 <0.35 <0.005 1.32 <0.02 30/03/99 surface NS 12.5 8.14 <0.007 <0.05 139.86 0.01 158.01 406.96 256.32 768.97 <0.35 <0.005 1.41 <0.02 30/03/99 bed NS 8.7 8.11 0.01 <0.05 203.79 0.01 224.35 578.86 374.20 1122.59 <0.35 <0.005 2.05 <0.02

NS = Not sampled

Appendix L. (Continued)

SITE: 19

Date Measurement Alkalinity Cl:SO4 Lab. Fe Al Ca Mn K Mg S SO4 As Cu Si Zn (mg L-1) pH (mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)(mg L-1)

25/03/98 surface 76 11.1 7.65 <0.01 <0.05 353.57 <0.002 398.31 999.31 745.70 2237.11 <0.40 <0.004 0.63 <0.01 02/04/98 surface 47 10.9 7.67 <0.01 <0.05 74.98 <0.002 75.85 224.90 295.99 887.96 <0.40 <0.004 <0.03 <0.01 02/04/98 bed 52 11.6 7.68 <0.01 <0.05 210.65 <0.002 231.93 605.05 579.84 1739.51 <0.40 <0.004 0.30 <0.01 17/04/98 surface 60 29.8 7.75 <0.01 <0.05 75.42 <0.002 76.04 222.00 196.92 590.76 <0.40 <0.004 0.04 <0.01 17/04/98 bed 60 15.6 7.66 <0.01 <0.05 134.55 <0.002 136.04 393.92 398.52 1195.56 <0.40 <0.004 0.15 <0.01 27/04/98 surface 47 25.2 7.61 <0.01 <0.05 72.82 <0.002 72.11 215.53 262.82 788.45 <0.40 <0.004 0.01 <0.01 27/04/98 bed 52 22.1 7.71 <0.01 <0.05 75.75 <0.002 79.78 234.00 302.99 908.97 <0.40 <0.004 <0.03 <0.01 04/05/98 surface 26 11.7 6.84 0.08 <0.05 13.62 <0.002 13.35 34.94 30.19 90.57 <0.40 <0.004 0.25 <0.01 04/05/98 bed 60 8.9 7.69 <0.01 <0.05 113.44 <0.002 173.64 493.93 487.16 1461.48 <0.40 <0.004 0.46 <0.01 15/05/98 surface 47 16.7 7.37 <0.01 <0.05 66.27 <0.002 62.38 194.02 179.54 538.62 <0.40 <0.004 0.31 <0.01 15/05/98 bed 47 21.6 7.31 <0.01 <0.05 71.60 <0.002 70.25 209.71 174.53 523.58 <0.40 <0.004 0.15 <0.01 02/06/98 surface 56 8.8 7.27 <0.01 <0.05 157.32 <0.002 163.71 451.40 325.51 976.52 <0.40 <0.004 1.19 <0.01 02/06/98 bed 76 9.4 7.30 <0.01 <0.05 287.20 <0.002 328.97 829.33 589.90 1769.71 <0.40 <0.004 0.82 <0.01 05/06/98 surface 26 14.4 6.74 0.166 0.044 14.41 0.002 13.32 34.44 22.18 66.53 <0.15 0.009 2.04 <0.02 05/06/98 bed 90 8.0 7.43 0.004 <0.03 327.93 0.001 338.34 981.72 590.31 1770.93 <0.15 0.002 1.19 <0.02 21/07/98 surface 60 8.7 7.80 0.003 <0.03 327.90 0.001 340.01 969.80 572.84 1718.53 <0.15 0.017 0.28 <0.02 31/07/98 surface 65 9.4 7.31 0.056 <0.03 204.88 0.001 203.69 601.79 369.73 1109.18 <0.15 0.011 1.38 <0.02 31/07/98 bed 65 8.0 7.48 0.018 <0.03 256.12 0.000 260.58 754.78 464.32 1392.96 <0.15 0.005 0.98 <0.02 10/08/98 surface 60 11.2 7.69 <0.005 <0.03 366.57 0.001 388.33 1071.79 608.63 1825.88 <0.15 0.006 0.56 <0.02 10/08/98 bed 65 12.3 7.77 <0.005 <0.03 354.02 0.000 371.40 1033.26 590.39 1771.17 <0.15 0.006 0.56 <0.02 17/08/98 surface 38 10.2 6.88 0.065 <0.03 74.36 0.000 73.16 218.47 131.58 394.73 <0.15 0.007 1.28 <0.02 17/08/98 bed 65 11.9 7.49 0.002 <0.03 360.01 0.000 375.80 1046.09 485.63 1456.88 <0.15 0.003 0.78 <0.02 31/08/98 surface 30 9.3 6.78 0.334 <0.03 33.23 0.001 29.69 89.19 42.23 126.70 <0.15 0.005 1.31 <0.02 31/08/98 bed 76 9.8 7.11 0.025 <0.03 187.58 0.001 182.18 547.28 299.28 897.84 <0.15 0.001 1.66 <0.02 15/09/98 surface 21 8.6 6.73 0.402 0.037 12.14 0.001 10.87 27.22 15.18 45.54 <0.15 0.003 1.34 <0.02 16/10/98 surface 52 12.6 7.47 0.001 <0.03 349.98 0.014 362.61 1021.41 525.97 1577.91 <0.15 0.005 0.97 <0.02 16/10/98 bed 52 14.9 7.53 <0.005 <0.03 437.23 0.013 464.76 1272.93 523.95 1571.84 <0.15 0.004 0.87 <0.02 07/12/98 surface NS 6.3 7.62 0.01 <0.05 263.32 0.01 284.72 755.32 533.02 1599.06 <0.35 <0.005 1.25 <0.02 07/12/98 bed NS 6.7 7.66 0.00 <0.05 271.03 <0.001 302.01 776.90 524.73 1574.19 <0.35 <0.005 1.33 <0.02 04/01/99 surface NS 9.3 7.55 0.01 <0.05 149.32 <0.001 166.53 435.39 303.16 909.47 <0.35 <0.005 1.61 <0.02 04/01/99 bed NS 8.4 7.48 0.01 <0.05 152.78 <0.001 168.96 440.31 310.58 931.75 <0.35 <0.005 1.70 <0.02 25/01/99 surface NS 12.8 7.69 0.00 <0.05 143.98 <0.001 159.28 408.49 284.71 854.14 <0.35 <0.005 2.50 <0.02 25/01/99 bed NS 7.4 7.72 0.00 <0.05 323.90 <0.001 384.92 926.15 613.77 1841.30 <0.35 <0.005 1.18 <0.02 04/03/99 surface NS 10.8 7.61 0.02 <0.05 146.14 <0.001 169.84 430.57 273.32 819.96 <0.35 <0.005 1.28 <0.02 30/03/99 surface NS 9.7 7.94 0.01 <0.05 168.50 0.01 186.04 482.80 301.31 903.92 <0.35 <0.005 2.03 <0.02 30/03/99 bed NS 9.4 8.09 0.00 <0.05 173.09 0.01 191.95 498.35 320.40 961.20 <0.35 <0.005 1.99 <0.02

NS = Not sampled

Appendix M. Feeding Experiment data.

Treat. Shell Shell Shell Whole Soft Tissue Mean True Mean Rejection Feeding Height Length Width Weight Dry Mass Faeces Production Rate Activity (mm) (mm) (mm) (g) (g) (mg h-1)(mg h-1)(mg h-1)

6 53.73 40.16 15.87 18.948 0.861 6.2 13.0 19.1 6 54.48 33.33 13.94 15.732 0.487 7.7 12.2 19.9 6 61.17 50.69 14.68 22.473 0.790 8.6 13.2 21.8 6 46.65 35.45 14.17 12.596 0.837 10.7 8.8 19.4 6 56.55 39.26 13.02 16.167 0.501 7.2 11.2 18.4 6 49.41 44.95 14.87 15.986 0.786 3.5 7.0 10.5 6 51.25 39.16 13.98 13.832 0.830 6.8 3.6 10.3 6 61.98 44.18 19.15 19.696 0.630 2.4 4.0 6.4 6 59.80 41.69 13.20 17.157 1.088 2.8 6.8 9.6 6 54.30 35.76 15.56 15.159 0.464 5.3 2.9 8.1 6 54.71 41.94 14.00 16.908 0.390 1.8 6.2 8.0 6 63.76 45.67 16.39 24.936 0.498 1.7 2.8 4.6 6 53.74 40.45 18.77 20.487 0.903 2.6 1.5 4.1 6 53.88 44.15 15.59 20.509 0.796 1.9 1.9 3.8 6 47.74 37.46 12.14 11.311 0.413 2.3 1.9 4.2 6 56.84 34.23 11.88 12.270 0.345 1.7 1.9 3.6 6 56.75 36.38 16.59 16.462 0.630 2.3 2.3 4.6 6 49.79 41.42 15.07 16.403 0.586 2.2 2.4 4.6 7 60.84 44.88 16.54 21.466 0.598 3.5 6.0 9.5 7 68.49 43.79 15.73 24.851 1.041 3.0 7.0 10.0 7 54.79 37.96 14.16 15.918 0.477 4.0 3.0 7.0 7 46.03 39.75 14.01 13.900 0.594 2.5 6.0 8.5 7 67.17 44.36 14.48 23.394 1.446 1.0 5.0 6.0 7 71.52 52.61 18.98 26.483 0.884 4.5 4.0 8.5 7 60.72 43.05 14.49 18.420 0.818 6.0 5.5 11.5 7 57.64 40.48 15.47 20.044 0.690 1.5 7.5 9.0 7 58.19 41.55 18.08 17.612 0.849 2.0 3.5 5.5 7 69.99 49.73 15.13 25.924 0.510 3.0 2.5 5.5 7 53.71 33.99 14.84 13.960 0.659 6.0 7.5 13.5 7 59.70 48.12 16.02 24.717 0.499 2.5 9.5 12.0 7 63.09 42.86 13.29 21.047 0.513 2.6 1.6 4.2 7 55.10 41.37 17.69 16.276 0.976 4.9 2.7 7.5 7 61.23 48.96 21.71 27.470 0.799 7.4 7.4 14.8 7 52.31 35.57 14.99 11.926 0.612 2.3 5.2 7.5 7 60.02 36.69 12.41 15.817 0.712 0.9 1.9 2.9 7 60.35 39.67 17.47 18.841 0.857 0.8 0.5 1.3 8 70.87 39.79 17.92 29.617 1.365 1.4 2.4 3.7 8 57.25 42.92 17.90 19.815 0.802 0.8 2.1 2.9 8 67.89 44.13 17.34 25.534 0.775 2.0 2.5 4.5 8 56.28 40.82 16.70 17.491 0.910 1.3 3.8 5.1 8 57.65 35.50 17.52 23.088 0.848 4.5 5.1 9.6 8 59.96 40.66 14.40 18.569 0.773 2.2 3.3 5.4 8 55.91 41.72 16.18 20.709 1.017 2.6 3.0 5.7 8 50.24 37.95 15.50 18.373 0.721 2.4 3.3 5.7 8 60.84 44.57 16.73 18.485 0.846 1.6 2.8 4.4 8 68.10 38.56 18.29 25.871 1.888 1.6 2.4 4.0 8 63.61 40.65 16.59 20.084 0.818 1.3 2.0 3.3 8 57.79 38.31 19.31 18.583 0.644 1.1 2.0 3.2 8 59.54 40.18 22.21 22.805 0.821 0.9 1.7 2.6 8 54.13 40.14 16.19 16.344 0.539 2.5 2.4 5.0 8 55.44 39.60 20.34 18.660 0.544 0.5 0.8 1.4 8 62.86 40.06 20.85 24.297 0.996 1.3 2.6 3.9 8 55.38 40.09 17.32 20.277 0.720 1.1 2.9 4.0 8 60.17 45.13 18.94 24.386 1.196 1.4 1.6 3.0

Appendix N. Histopathology details.

(A) Fixation of Oyster Soft Tissue Formalin (10% sea water) (Lillie, 1965; C.A. Farley, personal communication, cited in Howard and Smith, 1983) for oysters comprises:

1. 10 ml 37-40% formaldehyde 2. 90 ml filtered ambient sea water

(B) Preparation, Processing and Staining of Sections The School of Pathology, UNSW, provided the notes below on the preparation, processing and staining of sections for histopathology.

HAEMATOXYLIN AND EOSIN STAIN 1. Dewax Tissue Sections 2. Stain in Harris’ Haematoxylin 4 minutes 3. Wash in Water 4. Differentiate in Acid Alcohol 1 dip 5. Immediately Wash in Water 6. Blue Sections in Scott’s Blue 10 dips (ensure sections are blue) 7. Wash in Water 8. Stain in Eosin 3 minutes 9. Blot Excess Stain 10. Dehydrate, Clear and Mount.

Results: Nuclei- Blue/Black Cytoplasm- Pink Muscle Fibres- Deep Pink/Red Collagen- Pale Pink/Red Red Blood Cells- Orange/Red Fibrin- Deep Pink

Appendix N. (Continued).

IRON STAIN 1. Bring Sections to Distilled Water 2. Mix Equal Parts of 2% HCl and 2% Potassium Ferrocyanide solutions and Filter 3. Incubate Sections 30 minutes 4. Wash in Water 5. Counterstain with 1% Neutral Red 5 minutes 6. Wash in Water 7. Dehydrate, Clear and Mount

Results: Haemosiderin (ferric iron salts)- Blue Nuclei- Red Background- Pale Red.

Appendix O. ANOVA and multiple comparison results from the Feeding Experiment

Table 1 One-way ANOVA of feeding activity by treatment and results of Least Significant Difference post hoc analyses.

Source of Variation SS df MS F P-value

Between Groups 486.2621 2 243.131 10.03945 0.000211 Within Groups 1235.095 51 24.21756

Total 1721.357 53

Multiple Comparisons

Dependent Variable: FA LSD

Mean Difference 95% Confidence Interval (I) EXP (J) EXP (I-J) Std. Error Sig. Lower Bound Upper Bound 1 2 2.788 1.6404 .095 -.505 6.081 3 7.284* 1.6404 .000 3.991 10.577 2 1 -2.788 1.6404 .095 -6.081 .505 3 4.496* 1.6404 .008 1.203 7.789 3 1 -7.284* 1.6404 .000 -10.577 -3.991 2 -4.496* 1.6404 .008 -7.789 -1.203 *. The mean difference is significant at the .05 level.

Table 2 One-way ANOVA of faeces production by treatment and results of Least Significant Difference post hoc analyses.

Source of Variation SS df MS F P-value

Between Groups 95.47346 2 47.73673 10.27687 0.000178 Within Groups 236.8983 51 4.645065

Total 332.3718 53

Multiple Comparisons

Dependent Variable: FP LSD

Mean Difference 95% Confidence Interval (I) EXP (J) EXP (I-J) Std. Error Sig. Lower Bound Upper Bound 1 2 1.400 .7184 .057 -.042 2.842 3 3.247* .7184 .000 1.805 4.689 2 1 -1.400 .7184 .057 -2.842 .042 3 1.847* .7184 .013 .405 3.289 3 1 -3.247* .7184 .000 -4.689 -1.805 2 -1.847* .7184 .013 -3.289 -.405 *. The mean difference is significant at the .05 level.

Appendix 0. (Continued)

Table 3 One-way ANOVA of rejection rate by treatment and results of Least Significant Difference post hoc analyses.

Source of Variation SS df MS F P-value

Between Groups 151.464 2 75.73202 7.111553 0.001887 Within Groups 543.1068 51 10.64915

Total 694.5708 53

Multiple Comparisons

Dependent Variable: RR LSD

Mean Difference 95% Confidence Interval (I) EXP (J) EXP (I-J) Std. Error Sig. Lower Bound Upper Bound 1 2 1.388 1.0878 .208 -.796 3.572 3 4.037* 1.0878 .001 1.853 6.221 2 1 -1.388 1.0878 .208 -3.572 .796 3 2.649* 1.0878 .018 .465 4.833 3 1 -4.037* 1.0878 .001 -6.221 -1.853 2 -2.649* 1.0878 .018 -4.833 -.465 *. The mean difference is significant at the .05 level.

Table 4 One-way ANOVA of filtration rate by treatment and results of Least Significant Difference post hoc analyses.

Source of Variation SS df MS F P-value

Between Groups 758.3511 2 379.1755 13.71156 0.00002 Within Groups 1410.339 51 27.65372

Total 2168.691 53

Multiple Comparisons

Dependent Variable: FR LSD

Mean Difference 95% Confidence Interval (I) EXP (J) EXP (I-J) Std. Error Sig. Lower Bound Upper Bound 1 2 2.714 1.7529 .128 -.805 6.233 3 8.951* 1.7529 .000 5.432 12.470 2 1 -2.714 1.7529 .128 -6.233 .805 3 6.237* 1.7529 .001 2.718 9.756 3 1 -8.951* 1.7529 .000 -12.470 -5.432 2 -6.237* 1.7529 .001 -9.756 -2.718 *. The mean difference is significant at the .05 level.

N.B. for all multiple comparisons tables in Appendix N: Treatment 6 = 1, Treatment 7 = 2 and Treatment 8 = 3.