Investigation of the Toxicology and Public Health Aspects of the Marine
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Investigation of the Toxicology and Public Health Aspects of the Marine Cyanobacterium, Lyngbya majuscula Thesis submitted by Nicholas John Osborne Bachelor of Science, Faculty of Science, University of Adelaide Bachelor of Science (Hons.), School of Medicine, The Flinders University of South Australia Master of Agricultural Science, School of Land and Food, University of Queensland A thesis submitted for the degree of Doctor of Philosophy m the School of Population Health, Faculty of Health Sciences, University of Queensland 2004 THE{iSKaa!DF2:;2KSUIfflUBaARt Declaration of Originality I declare that the work presented in this thesis, to the best of my knowledge and belief is original and my own work, except as acknowledged in the text, and that the material has not been submitted, either in whole or in part for a degree at this or any other university. Nicholas John Osborne 2004 Abstract Lyngbya majuscula is a filamentous marine cyanobacterium with a worldwide distribution in temperate and tropical regions to a depth of 30m. Over 70 chemicals have been isolated and characterised from this organism, many of which are biologically active. Dramatic responses have been elicited after human exposure to lyngbyatoxin A (LA) and debromoaplysiatoxin (DAT), toxins extracted from L. majuscula. These chemicals have been found to cause irritation at concentrations as low as 100 pmol and exposure of humans to this cyanobacterium in the enviroimient is associated with irritant contact dermatitis, as well as eye and respiratory irritation. Previously, L. majuscula has been reported as implicated in negative health outcomes only in Hawaii and Okinawa. Recently large blooms of L. majuscula have occurred with increasing repetition in the Moreton Bay region as well as other areas along the eastern Australian coastline. A broad study of this organism and its potential effect on human health was undertaken. LA and DAT and were found in samples of L. majuscula collected from Eastern Moreton Bay and North Deception Bay, Queensland, Australia, respectively. Samples of L. majuscula obtained from West Maui, Hawaii and the freshwater L. wollei from Florida contained LA. A quantitative measure of the irritant effects of the chemicals found in L. majuscula was made using a mouse ear-swelling test. The relative toxicities of two purified toxins, LA and DAT, were examined. These were found to produce swelling to a similar extent. The time course of inflammation and histopathological results were also similar for the two purified toxins. Less than 1 |ig per ear of either toxin or a mixture (1:1) of the two toxins caused a measurable increase in ear thickness. When toxins were combined (1:1) there was an additive, not synergistic effect. Increases in ear thickness occurred within 15 minutes. Crude extracts of L. majuscula from Moreton Bay were also applied to mice ears. The effect of crude extracts from Eastern Moreton Bay were not fully explained by the measured LA content, suggesting other toxin(s) and/or modulating factors were present. This effect was not found with L. majuscula containing DAT from North Deception Bay. Some samples of L. majuscula containing no measurable quantities of LA or DAT were found to exert an inflammatory response. This response had a different time course to response to LA or DAT. In an effort to understand the potential exposure of humans to the toxins of Z. majuscula the spatial and temporal distribution of LA and DAT were made in Eastern Moreton Bay and Northern Deception Bay. Not all samples of L. majuscula contained LA or DAT. More than ten-fold differences in DAT concentration were found at a single time at North Deception Bay (0.93-11.17 mg/kg freeze dry weight). LA was predominantly found at Eastem Moreton Bay while DAT was found only at North Deception Bay. Highest concentrations of toxin occurred when bloom size and density were also at their maximum (131.9 mg/kg LA and 43.0 mg/kg DAT). In an attempt to predict where L. majuscula biomass and toxins were maximal, and hence greatest chance of human exposure, a variety of physical and biotic parameters were obtained. Bloom intensity occurred when water temperature was maximal. Low precipitation periods were noted to occur before blooms. Total and dissolved reactive phosphorus were present at bloom initiation and peaks of chlorophyll-a in the water column were found at the peaks of Z,. majuscula bloom intensity. In an attempt to understand the extent of human exposure to the toxic effects of L. majuscula several epidemiological studies were completed. To assess the potential affects a survey of the health of ocean users in the North Deception Bay area, a residential area close to L. majuscula blooms, was undertaken. A postal survey was mailed to 5000 residents and a response rate of 27.4% was achieved. High numbers of people (78.2%) responding to the survey reported marine recreational water activity in Moreton Bay. Of those having marine recreational water activity, 34.6% reported at least one symptom, with skin itching the most reported symptom (22.7%) while fever was the least (0.4%). Younger participants had greater water exposure and symptoms than older participants. Participants with greater exposures were more likely to have skin and eye symptoms, suggesting agents in the marine enviroimient contributing to symptoms. Of those entering Moreton Bay waters 29 (2.7%) reported severe skin symptoms, 12 of who attended health professionals. Six (0.6%) reported the classic symptoms of recreational water exposure to L. majuscula, severe skin symptoms in the inguinal region. Participants with knowledge of L. majuscula reported less skin, gastrointestinal and fever and headache symptoms. Anecdotal evidence reported an outbreak of symptoms similar to those expected with exposure to L. majuscula on Fraser Island during the late 1990s. Examination of first aid records from Fraser Island revealed an outbreak of symptoms in January and February 1998. This coincided with the presence of a bloom of L. majuscula. The other four years examined had no L. majuscula blooms and the number of Z. majuscula symptoms was much reduced. Acknowledgements I would like to acknowledge several people without whose continued help and support this thesis would not be possible. Firstly, I would like to thank my principal supervisor Glen Shaw whose wealth of knowledge and enthusiasm on this topic are boundless. I would like to thank my associate supervisor Penny Webb who has patiently enlightened me in the study of epidemiology. I would like to acknowledge the support and helpfulness of all at the National Research Centre for Environmental Toxicology. I would like to acknowledge Judy O'Neil, Bill Dennison and Marine Botany, University of Queensland for their continued advice and comments, as well as resources and support. Geoff Eaglesham and Queensland Health contributed much to this thesis by assisting with measurement and characterisation of toxins. Healthy Waterways contributed many resources enabling completion of this thesis. Ian Marshall and Gerard Neville inspired me to investigate public health aspects of cyanobacteria more fully. The Bribie Island Environmental Society members assisted with the pilot surveys. The Queensland Parks Service contributed time and effort collecting samples and supplying information for this thesis. John Bums of the Cyanolab, Florida contributed by supplying samples and information on Lyngbya wollei. Professor R. E. Moore assisted by supplying standards to measure Lyngbya toxins. I would like to thank Ursula Keuper-Bennett for supplying samples of Lyngbya majuscula from Maui, Hawaii. Simon Forsythe of the School of Population Health assisted by contributing computer programs for data analysis. I would like to thank the Bureau of Meteorology for supplying weather data. Miles Yates and the Environmental Protection Agency contributed data on the presence of Lyngbya in Moreton Bay. Graham Webb of the Environmental Protection Agency and the Caboolture Shire Council contributed faecal coliform data. I would also like to thank the Environmental Protection Agency for use of their high volume air sampler. Finally, I would like to thank my partner Claire Pietersen for her continued support, encouragement and remaining patient, on two continents, while I completed this thesis. Table of Contents Declaration of Originality 2 Abstract 4 Acknowledgements 8 Table of Contents 10 List of Abbreviations 14 List of Tables 16 List of Figures 20 1. Introduction 24 2. Literature Review 26 2.1 Introduction 26 2.2 Toxic Cyanobacteria 27 2.2.1 Hepatotoxins - Microcystins, Nodularins and Cylindrospermopsin 27 2.2.2 Neurotoxins 29 2.2.3 Dermatoxins 30 2.2.4 Lipopolysaccharide Endotoxins 30 2.2.5 Allergic Reactions 30 2.3 Lyngbya majuscula 31 2.4 Toxicology of Z. majuscula 35 2.4.1 Toxicity of the Lyngbya toxin, debromoaplysiatoxin 37 2.4.2 Toxicity of the Lyngbya toxins, Lyngbyatoxin A, B and C 40 2.5 Factors Effecting Toxicity 42 2.6 Human Health Effects Associated with Exposure to L. majuscula 45 2.7 Human Toxicity from Consumption of Z. majuscula 48 2.8 Toxicity of Z. majuscula in Other Organisms 50 2.9 Differential Toxicology oiLyngbya majuscula 51 2.10 Ecological Implications of Z. majuscula in Marine and Terrestrial Environments 52 3. Dermal Toxicology of Lyngbya majuscula 54 3.1 Introduction- Toxicology 54 3.2 Aim 56 3.3 Methods - Toxicology 57 3.3.1 General 57 3.3.2 Mice 58 3.3.3 Mouse Ear Swelling Test (MEST) 58 3.3.4 Statistical Analysis 59 3.3.5 Histopathology 60 3.4 Results-Toxicology 61 3.4.1 Mouse Ear Swelling Test 61 3.4.2 Crude Extracts of Zj'wg&ja 64 3.4.3 Other Potential Toxins 67 3.4.4 Histopathogy 68 3.5.1 Mouse Ear Swelling Test 76 3.5.2 Vehicle 77 3.5.3 Weight Loss 78 3.5.4 Similarities in Effects of LA and DAT 78 3.5.5 Differential Effects between Crude Extracts and Purified Toxins 82 3.5.6 Hypothesis for Dififerences In Oedema 88 3.5.7 Time Course of Inflammatory Response 91 3.5.8 Histopathology 92 3.5.9 Recovery of Mouse Ears .* 96 3.5.10 Conclusions 97 4.