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Germination of Phyllospora Comosa, Hormosira Banksii and Ma The effects of treated sewage effluent on three species of marine macroalgae A thesis submitted for the degree of Doctor of Philosophy Mary-Anne Shir B. Sc. (Hons) Sustainability Group Victoria University June 2004 STA THESIS 571.95298 SHI 30001007911011 Shir, Mary-Anne The effects of treated sewage effluent on three species of marine macroalgae Declaration This thesis is submitted in accordance with the regulations of Victoria University in fulfillment of the requirements for the degree of Doctor of Philosophy. This thesis contains no material which has been accepted for the award of any other degree or diploma in any university and no material previously published or written by another person except where duly acknowledged or referenced. Mary-Aime Shir June 2004 TABLE OF CONTENTS Declaration i Acknowledgements ii Index of figures Hi Index of tables xvii Index of appendices xxiii List of abbreviations and definitions xxvii List of publications and conference presentations xxix Abstract xxx Chapter 1 - General Introduction 1 1.1 Introduction 1 1.1.1 Effluent discharge 1 1.1.2 Toxicity testing 2 1.1.3 Algal toxicity testing 4 1.2 Aims of this study 7 Chapter 2 - Materials and Methods 8 2.1 Collection 8 2.2 Test species and toxicity test endpoints 8 2.2.1 Phyllospora comosa 8 2.2.2 Hormosira banksii 10 2.2.3 Macrocystis angustifolia 11 2.2.4 Vibriofischeri 12 2.3 Dilution water 13 2.4 Test solutions 13 2.5 Data analysis 15 2.6 Specimen preparation for light and electron microscopy 16 2.6.1 Preparation of cultures 16 2.6.2 Fixative 16 2.6.3 Tissue preparation for light microscopy 16 2.6.4 Sample preparation for scarming electron microscopy 17 2.6.5 Sample preparation for transmission electron microscopy 18 2.7 Sectioning 18 2.7.1 Light microscopy 18 2.7.2 Transmission electron microscopy 19 2.8 Microscopy 19 2.8.1 Light microscopy 19 2.8.2 Scanning electron microscopy 19 2.8.3 Transmission electron microscopy .19 Chapter 3 - The effects of sewage effluent on germination of Phyllospora comosa, Hormosira banksii and Macrocystis angustifolia 20 3.1 Introduction 20 3.1.1 Aims of this chapter 22 3.2 Results 23 3.2.1 Phyllospora comosa 23 3.2.2 Hormosira banksii 36 3.2.3 Macrocystis angustifolia 48 3.2.4 Sewage effluent and EC50 correlations 68 3.2.5 Test species sensitivity 69 3.3 Discussion 70 3.3.1 Relative sensitivity of test species 70 3.3.2 Temporal variability 73 3.3.3 Toxicity test endpoint comparisons 74 3.3.4 Treatment toxicity 75 3.3.5 Conclusion 76 Chapter 4 - The effects of sewage effluent on mortality of Phyllospora comosa and Hormosira banksii 77 4.1 Introduction 77 4.1.1 Aims of this chapter 79 4.2 Results 80 4.2.1 Phyllospora comosa 80 4.2.2 Hormosira banksii 93 4.2.3 Sewage effluent and EC50 correlations 105 4.2.4 Test species sensitivity 105 4.3 Discussion 106 4.3.1 Relative sensitivity of test species 106 4.3.2 Treatment toxicity 108 4.3.3 Conclusion 108 Chapter 5 - The effects of sewage effluent on growth of Phyllospora comosa and Hormosira banksii, and growth and reproduction of Macrocystis angustifolia 109 5.1 Introduction 109 5.1.1 Aims of this chapter 110 5.2 Results Ill 5.2.1 Phyllospora comosa Ill 5.2.2 Hormosira banksii 120 5.2.3 Macrocystis angustifolia 128 5.2.4 Sewage effluent and EC50 correlations 138 5.2.5 Test species sensitivity 138 5.3 Discussion 139 5.3.1 Relative sensitivity of test species 139 5.3.2 Temporal variability 141 5.3.3 Toxicity test endpoint comparisons 141 5.3.4 Treatment toxicity 142 5.3.5 Conclusion 143 Chapter 6 - The effects of sewage effluent on bioluminescence of Vibrio fischeri 144 6.1 Introduction 144 6.1.1 Aims of this chapter 145 6.2 Results 146 6.3 Discussion 150 6.3.1 Conclusion 151 Chapter 7 - The effects of sewage effluent on the morphology of Phyllospora comosa, Hormosira banksii and Macrocystis angustifolia 152 7.7 Introduction 152 7.1.1 Aims of this chapter 153 7.2 Results 154 7.2.1 Phyllospora comosa 154 7.2.2 Hormosira banksii 162 7.2.3 Macrocystis angustifolia 168 7.3 Discussion 171 7.3.1 Conclusion 173 Chapter 8-General Discussion 174 8.1 Conclusions 181 References 182 Appendix 1 200 Appendix 2: Tables of test NOEC, LOEC and EC50 values for Phyllospora comosa, Hormosira banksii and Macrocystic angustifolia germination bioassays and Macrocystis angustifolia germination and germination tube growth bioassays 201 Appendix 2.1 201 Appendix 2.2 202 Appendix 2.3 203 Appendix 2.4 204 Appendix 2.5 205 Appendix 2.6 206 Appendix 2.7 207 Appendix 2.8 208 Appendix 2.9 209 Appendix 2.10 210 Appendix 2.11 211 Appendix 2.12 212 Appendix 2.13 213 Appendix 2.14 214 Appendix 2.15 215 Appendix 2.16 216 Appendix 2.17 217 Appendix 2.18 218 Appendix 2.19 219 Appendix 2.20 220 Appendix 3: Tables of test NOEC, LOEC and LC50 values for Phyllospora comosa and Hormosira banksii mortality bioassays 221 Appendix 3.1 221 Appendix 3.2 222 Appendix 3.3 223 Appendix 3.4 224 Appendix 3.5 225 Appendix 3.6 226 Appendix 3.7 227 Appendix 3.8 228 Appendix 3.9 229 Appendix 3.10 230 Acknowledgements This thesis would not have been possible without the encouragement and assistance of many people. I am sincerely grateful to the following: Dr Trevor Burridge for instigating my interest in marine ecotoxicology and motivating me throughout my candidature. My fellow research students who included Stuart Campbell, Juanita Bite and Sue Pascoe for their support and encouragement. Thanks also to all the laboratory technicians over the years who offered constant assistance. Jermy Stauber and fellow staff of CSIRO who provided fiinding for part of my research and gave invaluable feedback on manuscripts. Victoria University for financial support through an Australian Postgraduate Award Scholarship. Timothy Lavery for all his patience, love and support. My family for all their understanding. List of Figures page Figure 2.1 Collection site of macroalgal species. 9 Figure 3.1 Germination (Mean ± Standard Error) of Phyllospora comosa zygotes exposed to copper (a) and chromium (b) for 48 hours. 23 Figure 3.2 Response over time of Phyllospora comosa zygotes exposed to copper (a) and chromium (b) for 48 hours. EC50 ± 95% confidence intervals. 24 Figure 3.3 Germination (Mean ± Standard Error) of Phyllospora comosa zygotes exposed to modified and unmodified primary treated effluent. 26 Figure 3.4 Response over time of Phyllospora comosa zygotes exposed to unmodified (a) and modified (b) primary treated effluent for 48 hours. EC50 + 95% confidence intervals. 27 Figure 3.5 Germination (Mean ± Standard Error) of Phyllospora comosa zygotes exposed to modified and unmodified secondary treated effluent. 29 Figure 3.6 Response over time of Phyllospora comosa zygotes exposed to unmodified (a) and modified (b) secondary treated effluent for 48 hours. EC50 ± 95% confidence intervals. 30 Figure 3.7 Germination (Mean ± Standard Error) of Phyllospora comosa zygotes exposed to modified and unmodified chlorinated secondary treated effluent. 32 Figure 3.8 Response over time of Phyllospora comosa zygotes exposed to unmodified (a) and modified (b) chlorinated secondary treated effluent for 48 hours. EC50 ± 95% confidence intervals. 33 Figure 3.9 Germination (Mean ± Standard Error) of Phyllospora comosa zygotes exposed to varying salinity. 34 Figure 3.10 Response over time of Phyllospora comosa zygotes exposed to treatments of reduced salinity for 48 hours. EC50 ± 95% confidence intervals. 35 Figure 3.11 Germination (Mean ± Standard Error) of Hormosira banksii zygotes exposed to copper (a) and chromium (b) for 48 hours. 36 Figure 3.12 Response over time of Hormosira banksii zygotes exposed to copper (a) and chromium (b) for 48 hours. EC50 ± 95% confidence intervals. 37 Figure 3.13 Germination (Mean ± Standard Error) of Hormosira banksii zygotes exposed to modified and unmodified primary treated effluent. 39 Figure 3.14 Response over time of Hormosira banksii zygotes exposed to unmodified (a) and modified (b) primary treated effluent for 48 hours. EC50 ± 95%) confidence intervals. 40 Figure 3.15 Germination (Mean ± Standard Error) of Hormosira banksii zygotes exposed to modified and unmodified secondary treated effluent. 41 Figure 3.16 Response over time of Hormosira banksii zygotes exposed to unmodified (a) and modified (b) secondary treated effluent for 48 hours. EC50 ± 95% confidence intervals. 42 Figure 3.17 Germination (Mean ± Standard Error) of Hormosira banksii zygotes exposed to modified and unmodified chlorinated secondary treated effluent. 44 Figure 3.18 Response over time of Hormosira banksii zygotes exposed to unmodified (a) and modified (b) chlorinated secondary treated effluent for 48 hours. EC50 ± 95%) confidence intervals. 45 IV Figure 3.19 Germination (Mean ± Standard Error) of Hormosira banksii zygotes exposed to varying salinity. 46 Figure 3.20 Response over time of Hormosira banksii zygotes exposed to treatments of reduced salinity for 48 hours. EC50 + 95% confidence intervals. 47 Figure 3.21 Germination (± 95% confidence interval) of Macrocystis angustifolia spores exposed to copper (a) and chromium (b) for 48 hours. 49 Figure 3.22 Germination (a) and growth (b) response over time of Macrocystis angustifolia spores exposed to copper for 48 hours. EC50 ± 95% confidence intervals. 50 Figure 3.23 Germination (a) and growth (b) response over time of Macrocystis angustifolia spores exposed to chromium for 48 hours. EC50 ± 95% confidence intervals. 51 Figure 3.24 Germination (± 95%) confidence interval) of Macrocystis angustifolia spores exposed to modified and unmodified primary treated effluent.
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