OCCURRENCE OF THE CYANOBACTERIAL NEUROTOXIN, ANATOXIN-A, IN NEW YORK STATE WATERS by Xingye Yang A dissertation submitted in partial fulfillment of the requirements for the Doctor of Philosophy Degree State University of New York College of Environmental Science and Forestry Syracuse, New York January 2007 Approved: Faculty of Chemistry ---------------------------------------------- ------------------------------------------------ Gregory L. Boyer, Major Professor William Shields, Chairperson, Examination Committee ------------------------------------------------ ------------------------------------------------- John P. Hassett, Faculty Chair Dudley J. Raynal, Dean, Instruction and Graduate Studies UMI Number: 3290535 Copyright 2008 by Yang, Xingye All rights reserved. UMI Microform 3290535 Copyright 2008 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 Acknowledgements I would like to express my sincerest gratitude to Dr. Gregory L. Boyer, my major professor and academic advisor for his guidance, support, and assistance over the past years. He has provided me with invaluable knowledge and skills. I thank Dr. David J. Kieber for his advice and instrument support. I acknowledge Dr. John P. Hassett for his support on both my research and my career. I appreciate Dr. Francis X. Webster for his help on chemical characterization. I thank Dr. James P. Nakas for advice on my career development. Thanks are also due to Dr. William Shields for serving as chairman of this examination committee. I appreciate critical reviews and comments on the thesis from all the examiners on this committee. I would like to thank Dr. Israel Cabasso and Dr. Paul Caluwe for their help and advice on my study. I would also like to thank past and present members of the Boyer lab. To Michael F. Satchwell for his help whenever I need any; to Steve Ragonese, Amber Hotto, Elizabeth Konopko, Dr. Guozhang Zou, John Usher, Karen Haward, and Nick Smith. Special thanks to Dave Kiemle for his help on LCMS and NMR analysis and to Emily White and other members at Dr. Kieber’s lab for help on photochemistry, and to Dr. Hui Zhao, Suoding Li, Xinwei Wang, Ju Feng, and all my colleagues and friends. They make my life and study at SUNY-ESF memorable. This study was supported by the National Oceanic and Atmospheric Agency Coastal Oceans Program through their MERHAB-LGL project # NA160 P2788 and New York Sea Grant Award NA16R61645. This thesis is dedicated to my parents, whose endless love and support always motivate me. Without them, none of my accomplishments could have come true. i Abstract YANG, XINGYE. Occurrence and distribution of a cyanobacterial neurotoxin, anatoxin- a, in New York State waters. Typed and bound thesis, 232 pages, 19 Tables, 63 Figures, 2006. Cyanobacterial blooms are a serious environmental and health problem throughout the world. In New York State, cyanobacterial toxins such as the hepatotoxic microcystins and the neurotoxic anatoxin-a have been detected in various lakes including the lower Great Lakes. Compared to microcystins, the occurrence and distribution of anatoxin-a in New York State is poorly studied. This study provided the first systematic monitoring for anatoxin-a in New York State waters. Anatoxin-a is a bicyclic alkaloid (MW 165) which has potent nicotinic agonistic activity and has caused several animal fatalities around the world and in New York State. It may be produced by several genera of cyanobacteria including Anabaena, Aphanizomenon, Planktothrix and Microcystis. Anatoxin-a is mainly retained within cells but can be released into the surrounding water column during cell senescence and lysis. To better understand its behavior in natural waters, the stability of anatoxin-a was examined under natural and artificial light and different pH conditions. Under the influence of light and/or pH, anatoxin-a readily degraded with a first order or a pseudo- first order kinetics. Anatoxin-a has a half-life of 4-10 hours under natural and artificial solar radiation, and several days up to several months in the absence of light. Several degradation products were formed during photolysis of anatoxin-a. The primary degradation product of anatoxin-a under artificial light was identified by NMR as ii tricycloanatoxin-a and the degradation was proposed to proceed via an intramolecular rearrangement process. In New York State, anatoxin-a was periodically detected at concentrations above an cautionary level of 0.1 μg L-1 in the western basin of Lake Erie, the embayments along the southern shoreline of Lake Ontario, nearshore sites on Lake Champlain, and in other smaller inland lakes such as Onondaga Lake, Lake Neatahwanta and Lake Agawam. Anatoxin-a exceeded 1 μg L-1 in Lake Ontario, Lake Champlain, Onondaga Lake and Lake Agawam. In these lakes, the locations where anatoxin-a was observed are primarily shallower waters and are used extensively for recreational purposes. Exposure of humans or pets to cyanobacterial blooms in these areas during summer and fall is possible. The detection of anatoxin-a in waters in proximity to human habitats at concentrations higher than the cautionary level indicates the health risk associated with anatoxin-a is real. Author Name Xingye Yang Candidate for the degree of Doctor of Philosophy Date ___________ Major Professor Gregory L. Boyer Faculty of Chemistry State University of New York College of Environmental Science and Forestry, Syracuse, New York Signature of Major Professor ________________________________________ iii Table of Contents ACKNOWLEDGEMENTS i ABSTRACT ii TABLE OF CONTENTS iv LIST OF FIGURES vii LIST OF TABLES xii CHAPTER 1. INTRODUCTION AND STATEMENT OF RESEARCH HYPOTHESIS 1 CHAPTER 2. LITERATURE REVIEW 7 2.1. Cyanobacteria and cyanobacterial blooms 7 2.2. Cyanobacterial toxins 16 2.2.1. Introduction to cyanobacterial toxins 16 2.2.2. The neurotoxic cyanobacterial toxin, anatoxin-a 27 2.2.3. Detection of anatoxin-a in natural waters 38 2.3. The New York State lakes 39 2.4. References 49 CHAPTER 3. MATERIALS AND METHODS 71 3.1. Preparation of anatoxin-a standards 71 3.1.1. Preparation of anatoxin-a standards for toxin analysis 71 3.1.2. Synthesis of two non-toxic degradation products of anatoxin-a 72 3.2. Analysis of anatoxin-a, dihydroanatoxin-a and epoxyanatoxin-a 78 3.3. Sampling in New York State lakes and processing of water samples for toxin analysis 81 3.3.1. Sampling in the lower Great Lakes 81 3.3.2. Sampling in Lake Champlain 84 3.3.3. Sampling in other New York State lakes 84 3.3.4. Extraction of anatoxin-a from water samples 87 3.4. Stability studies of anatoxin-a 88 iv 3.4.1 Effects of temperature and pH on anatoxin-a in the dark 88 3.4.2. Effects of pH, natural and simulated solar radiation on the stability of anatoxin-a 88 3.4.3. Photolysis of anatoxin-a in natural lake water 94 3.4.4. Characterization of degradation product(s) of anatoxin-a 95 3.5. References 95 CHAPTER 4. EXPERIMENTAL RESULTS 97 4.1. Stability of anatoxin-a under different environmental factors 97 4.1.1. Introduction 97 4.1.2. Materials and methods 98 4.1.3. Results 101 4.1.4. Discussion 121 4.1.5. References 127 4.2. Occurrence and distribution of anatoxin-a in the lower Great Lakes 129 4.2.1. Introduction 129 4.2.2. Materials and methods 130 4.2.3. Results 132 4.2.4. Discussion 150 4.2.5. Acknowledgement 155 4.2.6. References 155 4.3. Identification, occurrence and spatial distribution of anatoxin-a in Lake Champlain, New York 160 4.3.1. Introduction 160 4.3.2. Materials and methods 163 4.3.3. Results 165 4.3.4. Discussion 172 4.3.5. Acknowledgments 178 4.3.6. References 178 4.4. Occurrence and distribution of anatoxin-a in other New York State lakes 183 v 4.4.1. Introduction 183 4.4.2. Materials and methods 185 4.4.3. Results 187 4.4.4. Discussion 205 4.4.5. Acknowledgements 209 4.4.6. References 210 CHAPTER 5. DISCUSSION 214 APPENDIX 219 VITAE 232 vi List of Figures Figure 1.1. Chemical structure of anatoxin-a (protonated form). 2 Figure 2.1. Three common bloom-forming cyanobacteria. Anabaena flos- aquae, Microcystis aeruginosa and Oscillatoria sp. 14 Figure 2.2. Warning flyer released by Vermont Department of Health regarding blue-green algal blooms in Lake Champlain and the potential health risks 17 Figure 2.3. Structures of microcystins 19 Figure 2.4. Structures of the alkaloid cyanobacterial toxins 20 Figure 2.5. Conformation of anatoxin-a and structures of the two known non- toxic degradation products, epoxyanatoxin-a and dihydroanatoxin-a 30 Figure 2.6. Mechanism of anatoxin-a poisoning 31 Figure 2.7. Sections of the 125 MHz 13C NMR spectrum of anatoxin-a 34 Figure 2.8. A plausible biosynthetic scheme for the neurotoxic anatoxin-a and its precursor 11-carboxy anatoxin-a 35 Figure 2.9. New York State water bodies 42 Figure 2.10. The Great Lakes Basin 43 Figure 2.11. A view of the lower Great Lakes from SeaWiFS satellite showing an algal bloom in the western basin of Lake Erie and a lakewide algal bloom in Lake Ontario 46 Figure 2.12. Map of Lake Champlain Basin 47 Figure 3.1. Anatoxin-a standard curves by HPLC-FD 73 Figure 3.2. Typical standard curves for anatoxin-a analysis by LCMS 74 Figure 3.3. Sample HPLC-FD chromatograms of anatoxin-a (ATX) and its degradation products 75 Figure 3.4. Typical sample LCMS chromatograms and mass spectra for anatoxin-a and l-phenylalanine 76 Figure 3.5.